Volume 1, Issue 1May 2011
INAUGURAL ISSUE
ISSN: 2159-8967www.AFABjournal.com
2 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
Sooyoun Ahn Arkansas State University, USA
Walid Q. AlaliUniversity of Georgia, USA
Kenneth M. Bischoff NCAUR, USDA-ARS, USA
Claudia S. Dunkley University of Georgia, USA
Lawrence GoodridgeColorado State University, USA
Leluo GuanUniversity of Alberta, Canada
Joshua GurtlerERRC, USDA-ARS, USA
Yong D. HangCornell University, USA
Divya JaroniSouthern University, USA
Weihong Jiang Shanghai Institute for Biol. Sciences, P.R. China
Michael JohnsonUniversity of Arkansas, USA
Timothy KellyEast Carolina University, USA
William R. KenealyMascoma Corporation, USA
Hae-Yeong Kim Kyung Hee University, South Korea
W.K. KimUniversity of Manitoba, Canada
M.B. KirkhamKansas State University, USA
Todd KostmanUniversity of Wisconsin, Oshkosh, USA
Y.M. Kwon University of Arkansas, USA
Maria Luz Sanz MuriasInstituto de Quimica Organic General, Spain
Melanie R. MormileMissouri University of Science and Tech., USA
Rama NannapaneniMississippi State University, USA
Jack A. Neal, Jr.University of Houston, USA
Benedict OkekeAuburn University at Montgomery, USA
John PattersonPurdue University, USA
Toni Poole FFSRU, USDA-ARS, USA
Marcos RostagnoLBRU, USDA-ARS, USA
Roni ShapiraHebrew University of Jerusalem, Israel
Kalidas ShettyUniversity of Massachusetts, USA
EDITORIAL BOARD
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 3
EDITOR-IN-CHIEFSteven C. RickeUniversity of Arkansas, USA
EDITORSTodd R. CallawayFFSRU, USADA-ARS, USA
Cesar CompadreUniversity of Arkansas for Medical Sciences, USA
Philip G. CrandallUniversity of Arkansas, USA
EDITORIAL STAFFMANAGING EDITOR
Ellen J. Van LooFayetteville Arkansas, USA
TECHNICAL EDITORJessica C. ShabaturaEureka Springs Arkansas, USA
ONLINE EDITION EDITORC.S. ShabaturaEureka Springs Arkansas, USA
ABOUT THIS PUBLICATION
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4 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
TABLE OF CONTENTS
Using Mortality Compost in Vegetable Production: A comparison Between Summer and Winter Composting and its Use in Cabbage ProductionC. S. Dunkley, D. L. Cunningham, C. W. Ritz, K. D. Dunkley, and A. Hinton
6
Determination of Antifungal Activity of Pseudomonas sp. A3 Against Fusarium oxysporum by High Performance Liquid Chromatography (HPLC)P. Velusamy, H. S. Ko, and K. Y. Kim
15
Multi Food Functionalities of Kalmi Shak (Ipomoea aquatica) Grown in BangladeshH.U. Shekhar, M. Goto, J. Watanabe, I. Konishide-Mikami, Md. L. Bari and Y. Takano-Ishikawa
24
Using Hydrogen- Limited Anaerobic Continuous Culture to Isolate Low Hydrogen
Threshold Ruminal Acetogenic BacteriaP. Boccazzi, and J. A. Patterson
33
Effect of Plant-based Protein Meal Use in Poultry Feed on Colonization and Shedding of Salmonella Heidelberg in Broiler BirdsW. Q. Alali, C. L. Hofacre, G. F. Mathis, and A. B. Batal
45
Optimization of Fermentative Production of Keratinase From Bacillus Subtilis NCIM 2724 S. M. Harde, I. B. Bajaj, and R. S. Singhal
54
ARTICLES
An Overview of Stress Response Proteomes in Listeria monocytogenesK. A. Soni, R. Nannapaneni, and T. Tasara
66
REVIEWS
Instructions for Authors86
EXTRAS
The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors.
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 5
elcome to the inaugu-
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INTRODUCTION TO THE INAUGURAL ISSUE
LETTER FROM THE EDITOR
WVolume 1, Issue 1
May 2011
INAUGURAL ISSUE
6 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
www.afabjournal.comCopyright © 2011
Agriculture, Food and Analytical Bacteriology
ABSTRACT
A study was conducted to determine the effectiveness of composting to breakdown the carcasses of
daily poultry mortality and in the process destroy pathogenic microorganisms that may be present. The
study was conducted during the summer and repeated in the winter to determine whether the time of year
would affect the temperature profile or the length of time required for the process to be completed. Daily
mortalities were collected from a nearby producer and layered in a compost bin each day for four days.
Samples were collected from the litter before it was placed in the bin. Compost samples were collected
every other day for a week after the bin was compiled and then once per week until the process was com-
pleted. The samples were evaluated for microbial content. Temperature was taken and recorded at random
points in the bins on a daily basis. Upon completion of the composting process, the material was used as
a soil amendment in two vegetable plots while a third plot without compost material served as the control.
Soil samples were collected from each of the plots prior to application of the compost material. Cabbage
seedlings were then planted in each of the plots. Vegetative samples and soil samples were collected and
evaluated for microbial presence prior to planting and at week, 1, 3, 7, and again at reaping.
The summer compost had the highest temperature of 156°F on d 9 during the primary phase while the
winter compost had the highest temperature of 156°F on d 42 during the secondary phase of the compost.
The summer compost samples were Salmonella enterica (SE) negative from d 2 of the trial but mixed bac-
terial colonies remained for the duration of the study. The vegetative samples in plot 1 had coliform levels
of 3.48 log10/gm at wk10 but the levels was not considered significantly different from the other two plots
(p<0.05). The results show that while winter composting can effectively breakdown poultry carcasses and
attain high temperatures, summer compost is more efficient and had consistently higher temperatures.
Keywords: poultry, winter compost, summer compost, daily mortality
Received: August 31, 2010, Accepted: October 19, 2010. Released Online Advance Publication: May, 2011. Correspondence: Claudia S. Dunkley, [email protected]: +1 -229-386-3363 Fax: +1-229-86-3239
Using Mortality Compost in Vegetable Production: A Comparison Between sSummer and Winter Composting and its Use
in Cabbage Production
C. S. Dunkley1, D. L. Cunningham2, C. W. Ritz2, K. D. Dunkley3, and A. Hinton4
1Department of Poultry Science, University of Georgia,Tifton, Georgia 31793-04782Department of Poultry Science, University of Georgia, Athens, Georgia 30602-2772
3School of Science and Math, Abraham Baldwin Agricultural College, Tifton, Georgia, 317934USDA ARS, Russell Research Center, Athens Georgia, 30605
Agric. Food Anal. Bacteriol. 1: 6-14, 2011
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 7
InTRoduCTIon
Composting is considered a positive alternative
method of processing dead birds in an environmen-
tally safe manner (Ritz and Worley, 2005). It is a natural
biological decomposition process that takes place
under aerobic and thermophilic conditions (Wilkin-
son, 2007), furthermore, it is an aerobic process that
destroys rather than creates odor causing volatile
compounds (Grewal et al., 2005). Composting poul-
try mortality in the USA began in the 1980’s and the
process has been described as the above ground
burial of dead animals in mounds of supplemental
carbon such as sawdust, litter, straw or wood shav-
ings (Kalbasi et al., 2005). The composting process
generally follows two phases; the primary phase is
considered the heating or developing phase and the
secondary phase is considered the curing or matu-
ration phase (Wilkinson, 2007). The process reduces
the carcasses to nutrient rich humus which can be
used as a soil amendment. Composting poultry
mortalities can also be considered a value added
product because instead of burying the birds (which
could result in environmental pollution), the product
can be sold as an organic fertilizer.
Even though composting is an effective way
to dispose of daily mortalities, food safety concerns
from the general public have limited the use of the
mortality composted materials. There is consider-
able concern regarding the potential for contamina-
tion of agricultural products for human and animal
consumption with pathogens that may be present in
animal carcasses and manure from compost (Jones,
1999; Keener et al., 2000). Composting has, however,
been established as a pathogen reduction technol-
ogy (Wilkinson, 2007). Research has shown that the
process will control nearly all pathogenic viruses,
bacteria, fungi, protozoa (including cysts) and hel-
minth ova to acceptable levels. Endospore forming
bacteria such as Bacillus anthracis and prions such
as bovine spongiform encephalopathy are excep-
tions (Kalbasi et al., 2005). The inactivation of patho-
genic micro-organisms in the compost is dependent
upon several mechanisms during composting. Ex-
posure to heat, microbial antagonism, production of
organic acids and ammonia, competition for nutri-
ents, physical composition of composting material
and bedding type are all factors that will determine
the fate of the microbes (Epstein, 1997; Hess et al.,
2004; Turner, 2002). Temperature is considered the
most important factor in pathogen inactivation. Not
to be ignored, however, is the effect of time since
inactivation is a function of both temperature and
the length of time the pathogens are exposed to the
high temperatures. Haug (1993) reported that patho-
gen exposure at temperatures of 131°F to 140°F for
a couple of days were enough to kill a vast majority
of enteric pathogens. Other reports have shown a
99.9% elimination of total coliform and Escherichia
coli (E. coli) organisms from beef feedlot manures in
the first seven days of composting with the average
temperature being 92.3°F to 106.7°F (Larney et al.,
2003).
The majority of cases of food borne illnesses ob-
served in human are from the consumption of animal
food products, although a variety of pathogens have
been recovered from vegetables (Beuchat, 1996).
The most likely source of pathogens observed in
vegetables would occur from the cross-contamina-
tion of the produce from animal manure or improper
composted manure that has been used to amend
the soil. Presently consumer demand for organically
produced vegetables is on the increase; these are
usually fertilized using animal manure. However, re-
cent outbreaks of Salmonella and E. coli infections
have demonstrated the need to properly compost
animal wastes prior to use in vegetable production.
Islam et al. (2004) used composted poultry manure
and composted dairy cattle manure to amend soil
which was later planted with carrots and radish; they
observed that Salmonella Typhimurium survived for
up to 231 days after the soil was amended. In the
study conducted by Islam et al. (2004), the compost
process and the irrigation water that was used in the
study was also inoculated with S. Typhimurium.
The use of mortality compost has been limited
due to fear of contamination and/or recontamina-
tion of the compost by pathogenic microorganisms.
It has been used to amend pasture land but is not
approved to be applied to land that is used to pro-
8 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
duce food directly for human consumption. Senne
et al. (1994) investigated the survival of Highly Patho-
genic Avian Influenza Virus (HPAIV) and the adeno-
virus in composted poultry carcasses and observed
that HPAIV was completely inactivated after 10 days
of composting. The adenovirus was inactivated after
a further 10 days of composting. Conner et al. (1991)
failed to recover enteric microorganisms infused into
poultry carcasses after 14 days of primary composting
or after 14 days of secondary composting. Presently
there are limited scientific reports on the safety of
poultry mortality composting and even less on the
use of this compost on soils used for growing food
for human consumption. While work such as that con-
ducted by Islam et al. (2004) provides us an indication
of the potential length of time some pathogenic mi-
croorganisms can survive in the soil, it is still unclear
as to the survival or the re-emergence of these micro-
organisms from the composted product after it has
been incorporated or top-dressed to production soils.
With this in mind the objectives of this study were to
evaluate the temperature profile of both a summer
and winter compost, determine and characterize mi-
crobes present in the compost bin during the process
of carcass composting and to evaluate the presence
of “post compost” microorganisms in the soil and on
cabbage plants fertilized using composted materials.
MATeRIAlS And MeThodS
The study was conducted in the summer (2008) and
winter (2009) in two phases. Phase I involved the
composting of the daily mortality and Phase II in-
volved the application of the composted product to
soil in which cabbage seedlings were planted.
Phase I
For the first phase of the project, the daily mortality
was collected from a nearby four-house commercial
broiler farm. Daily mortality was collected for four
days averaging thirty-eight birds each day. Cloacal
swabs were taken from six birds from each day’s col-
lection and the swabs were sent to the lab for micro-
bial analysis. Litter obtained from a local broiler farm
was used as the carbon source for the compost. A re-
modeled swine barn was used as the compost facility
which consisted of three bins; one primary bin, one
secondary bin and a storage bin. The compost pile
was layered as described in Ritz and Worley (2005).
Primary Phase
During the primary phase of the trial the temper-
ature was taken using a 48” compost thermometer.
The temperature was taken from the core of the pile
and two other random areas within the bin. The am-
bient temperature was also taken on a daily basis to
determine an effect on the bin temperature. The tem-
peratures taken each day were averaged and used as
the temperature for that particular day. Samples were
randomly collected in triplicates every other day from
the primary bin to evaluate the presence or absence
of SE or E. coli in the bin. The samples were placed
in plastic bags and taken immediately to the lab for
evaluation. The duration of the primary phase would
be dependent on the time/day the temperature of
the pile dropped below 129°F. The compost pile was
turned for a second heating phase when the temper-
ature dropped below 129°F.
Secondary Phase
The pile was turned by flipping the contents of the
primary bin into the secondary bin; this was done us-
ing a “skid loader”. Again, the temperature was taken
daily from the core of the pile and two other random
areas in the pile using a 48” compost thermometer.
Compost samples were collected in triplicates ran-
domly from the pile every three days and immediately
transported to the lab for evaluation. The end of the
secondary phase would be indicated by the drop in
the temperature below 100ºF. At the end of the sec-
ondary phase the composted material remained in
the bin until it was transported to the vegetable plot
to be used in the second phase of the study.
Phase II
Phase II of the study involved the application of
the composted material to plots of soil. Three 3’ X
20’ plots were designated as treatment plots were
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 9
during this phase; in plot 1 the soil was top-dressed
with the composted carcasses. In plot 2 the compost
material was incorporated in the soil. Plot 3 was not
treated with the composted material and was con-
sidered the control plot. Three soil samples were col-
lected from each of the experimental plots prior to
allocation of treatments and evaluated for microbial
content. Compost samples were collected from three
areas in the finished composted pile and were evalu-
ated for microbial content prior to application to the
experimental plots. Cabbage seedlings were planted
at a distance of 12” X 8” in each plot. Nine seedlings
were randomly selected and evaluated for microbial
content prior to planting. Three vegetative and soil
samples were randomly collected from each of the
three experimental plots after the first, third and sev-
enth week of planting. Three soil and three vegeta-
tive samples were also collected after the tenth week
of planting. All the samples that were collected were
evaluated for microbial content.
Compost analysis
Twenty-five g compost material was weighed and
transferred to a sterile Tekmar bags (Seward Labora-
tory Systems, Inc., Bohemia, NY 11716) then blended
in 100 ml buffered peptone water using a Seward
Stomacher Laboratory blender on high for 2 minutes.
Serial dilutions of the suspension were made in 0.1%
peptone (w/v). Escherichia coli and total coliforms
were enumerated by transferring 1 ml from serial di-
lutions onto 3M Petrifilm (3M Microbiology, St. Paul,
MN,55144) and incubating at 35°C for 24-48 hr. E. coli
were identified as blue colonies with trapped gas, and
total coliforms were determined by counting red col-
onies with trapped gas in addition to blue colonies.
Presence of salmonellae was determined by pre-
enrichment of the remaining stomached compost
material in buffered peptone water by incubation at
35°C for 24 hr. After incubation, 0.1 ml of the suspen-
sion was transferred to 9.9 ml Rappaport-Vassiliadis
broth (Becton-Dickinson and Co. , Sparks, MD 21030)
and 0.5 ml was transferred to 9.5 ml Tetrathionate
broth with brilliant green-iodine (Becton-Dickinson
and Co.) and incubated for 24 hr. at 42°C. A 10 mm
loopful from each broth sample was plated onto XLT4
agar (Becton-Dickinson and Co.) and Brilliant green
sulfa agar (3). Plates were then incubated 24 – 48 hr.
at 35°C. Suspect Salmonella colonies were biochemi-
cally tested using Triple Sugar Iron and Lysine Iron
agar (Becton-Dickinson and Co.) and serology was
performed using Difco Bacto Poly O antiserum (Bec-
ton-Dickinson and Co.).
Vegetative analysis
Vegetative material was weighed and 2 X volume
of buffered peptone water was added. The samples
were then stomached for 2 min. on high and soaked
at room temperature for 1.5 hr. before diluting for
E.coli determination. Procedures for E. coli, total co-
liforms and Salmonella enrichment were performed
as was done with the compost samples.
Statistical analysis
Statistical analyses were performed using Graph-
Pad InStat® version 3.05 for Windows 95 (GraphPad
Software, San Diego, CA, USA). One-way Analysis of
Variance (ANOVA) with Tukey-Kramer Multiple Com-
parison tests was performed to determine significant
differences in group means. The P value for all ANO-
VA tests was < 0.05.
ReSulTS And dISCuSSIon
Temperature Profile
Summer Trial
Temperature changes were monitored on a daily
basis during the primary and secondary phases of the
experiment. Monitoring the compost temperature
is essential in timing the turning of the pile (Malone,
2006) and temperature is considered the most impor-
tant factor in pathogen inactivation (Wilkinson, 2007).
During the summer compost (Fig. 1), it was observed
that the temperature in the compost bin increased to
121° F on d 1 of the study and continued an upward
trend with a high temperature of 156° F on d 9. The
10 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
0
20
40
60
80
100
120
140
160
180
0 10 20 30 40 50 60 70 80
Daily Summer Temp Summer Ambient Temp
Daily Winter Temp Winter Ambient Temp
temperature in the bin remained elevated above
130° F from d 2 until d 17. Pathogen inactivation
is a function of both temperature and time. Haug
(1993) stated that exposure to an average tempera-
ture of 131 to 140°F for two days is usually enough
to kill a vast majority of enteric pathogens. After d
17 the temperature began a decline and on d 18
the temperature was 128° F. Ritz and Worley (2005)
stated that when oxygen becomes limited in the pile
the temperature would begin to fall. This would be
about seven to twenty one days after the compost
is capped when they recommended that the pile be
turned. In the summer study the pile was turned for
a second heating phase on d 19. The compost pile
quickly reheated to a high of 147° F (Fig. 1) on day
24 (five days after the pile was turned). Similar el-
evations in temperature were observed by Murphy
and Carr (1981) after they turned the compost pile
when the heat in the compost dropped below 125°
F. We observed the pile was not maintaining the
heat during this secondary heating as evidenced by
the fluctuation of the heat in the pile and the low
temperature at the base of the pile. For these rea-
sons the pile was turned again on d 46 when the
temperature had fallen to 129° F (Fig. 1). During this
second turn we observed that the pile was extremely
wet at the bottom of the pile and concluded that
this resulted in the falling temperature within the
pile. Ritz and Worley (2005) stated that the desirable
moisture levels in the compost should be 40 to 60%
and that too much water could make the compost
pile soggy and anaerobic. Litter material was incor-
porated in the compost during this second turn and
it was observed that the temperature within the pile
rose steadily to a high of 155° F on the fifth day af-
ter the pile was turned. The temperature in the pile
gradually declined but remained above 140° F un-
til d 66 after which it continued a steady downward
trend to 129° F on d 71. The temperature dropped
below 100° F after d 80 and upon examination of the
pile no carcass materials were observed. With the
temperature below 100°F (84°F) and the absence of
Figure 1: Temperature profile for daily mortality compost in the summer and winter trials and the ambient temperature during both summer and winter trials.
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 11
mal-odors from the composted material, d 80 was
marked as the end of the second stage. Mukhtar et
al. (2004) stated that the end of the second phase is
marked by a reduction of the internal temperature
77 to 86° F, a reduction in the bulk density and the
lack of an unpleasant odor when the pile is turned.
Winter Trial
Ambient temperature should have little or no
effect on the compost temperature. However, we
were interested in the effects of the external tem-
perature on the length of time for each phase of the
compost process, as well as the temperature pro-
file of the pile. The winter compost trial began in
November and the temperature was monitored for
eighty days. The temperature in the pile rose from
76°F on d 0 to 131°F on d 3. The pile was turned on
d 35 when the temperature was 129°F. The highest
temperature during the primary phase of the win-
ter compost was 136°F on d 4 compared to a high
of 156°F on d 9 in the summer compost (Fig. 1).
The ambient temperature on d 4 during the winter
trial was 73°F compared to 93°F during the summer
trial. The 20° difference in the ambient temperature
does not necessarily mean there will be a higher
compost temperature during the summer trial es-
pecially because a high temperature of 156°F was
recorded during the winter compost in the second-
ary phase when the ambient temperature was 43°F
which was the coldest day during the trial. Because
the summer compost had two turns we could not
justifiably compare the duration of the primary
phase in the summer compost to the primary phase
in the winter compost. However, the summer com-
post had higher temperatures on average during
the primary and secondary phases when compared
to the primary phase during the winter compost
(Table 1). When comparing the two trials the high-
est temperature observed during the secondary
phase was 156°F in the winter compost compared
to a high of 155°F during the summer trial (Table
1). Gonzalez and Sanchez (2005) observed temper-
atures above 140°F during their summer trials but
did not see temperatures above 140°F during their
winter trials.
Microbial Profile
Cloacal swabs were collected from six birds on
each of the days bird carcasses were collected.
The results from the summer compost show that
none of the birds were SE positive. However five
of the six birds which were sampled on day 1 and 2
were E. coli positive and all the birds tested posi-
tive for coli-form. Only three birds were E. coli posi-
tive on days 3 and 4 but the numbers on day four
were too numerous to count. Similar results were
observed in the winter compost with none of the
birds testing positive for SE, all coli-form positive
and some of the birds testing positive for E. coli,
these result however, were too numerous to count.
The compost piles from both the summer and win-
ter trials were sampled and analyzed for microbial
content (Table 2). Even though none of the sample
birds tested positive for Salmonella, the summer
compost tested positive for SE on day 1 of the trial
and E. coli positive on day 1 and 2 but was negative
for both of these microbes for the duration of the
trial. The winter compost tested negative for both
SE and E. coli for the duration of the trial. Both the
Table 1. Temperature profile comparison between summer and the winter trials.
Summer
Compost1
Winter
Compost 2
Primary Phase avg. temp. 141.14°F 130.9°F
Secondary Phase avg. temp. 145.19°F 137°F
Primary Phase high temp. 156°F 136°F
Secondary Phase high temp. 155°F 156°F
1 Represents the temperatures in the summer com-post. (This data references the temperature based on the second time the bin was turned during the summer trial).
2 Represents the temperature in the winter compost.
12 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
summer and winter samples were positive for co-
liform for the duration of the trial. Temperature is
considered the most important factor in pathogen
inactivation (Wilkinson, 2007) but the length of time
exposure to high temperature also functions in the
inactivation of pathogens (Haug, 1993). In the sum-
mer trial SE was not observed in the compost on d
2 when the temperature in the pile was 137°F nei-
ther was E. coli isolated on d 3 when the tempera-
ture was 137°F. Haug (1993) stated that two days is
usually enough to kill the vast majority of enteric
pathogens. Grewal et al. (2005) detected E. coli,
SE and Listeria in manure on d 0 of a trial but after
3 days of composting at 55°F they did not detect
any of these pathogens.
Even though SE and E. coli were not observed
in the compost at the end of Phase 1 of the trial,
we wanted to determine if there was any evidence
of re-growth after the temperatures returned to
more favorable levels. Under certain conditions,
enteric pathogens have been known to re-grow in
composted organic material when the temperature
declines to sub lethal levels (Wilkinson, 2007). Also,
there is the potential of Salmonella or E. coli colo-
nies to survive the thermophilic conditions of the
compost in clumped materials in the pile. Millner
et al. (1987) showed that SE was not suppressed
in compost taken from 158°F compost pile zones.
When Johannessen et al. (2004) investigated the
influence of manure on the hygienic quality of let-
tuce, E. coli O157:H7 was isolated from the ma-
nure that was used to fertilize and also from the
soil one week after fertilizing. However, they did
not isolate the E. coli from lettuce that was grown
in the soil. In this study neither SE nor E. coli was
isolated from the cabbage samples throughout
the trial (Table 3). However, coliforms were iso-
lated from treatment plots 1 and 2 during each
of the weeks that samples were collected. There
were numerical differences between the treatment
plots but the differences were not considered sig-
nificant (p<0.05). Coliforms were isolated from the
control plot in weeks 1 and 3 of the trial but were
not detected thereafter, the levels were not sig-
nificantly different from the other two treatments
(p<0.05). Islam et al. (2004) and Natvig et al. (2002)
observed that pathogens could be transferred
from manure to the surface of vegetables by way
of contaminated soil. In this trial neither SE nor E.
coli was detected in the soil samples for the dura-
tion of the trial but coliform was isolated from all
three plots (data not shown). This result was an ex-
pected because coliform bacteria are commonly
found in the environment.
Day 1 Salmonella E. Coli Coliform Salmonella E. Coli Coliform
1 + + + - - +
2 - + + - - +
4 - - + - - +
6 - - + - - +
12 - - + - - +
24 - - + - - +
Summer Compost 2 Winter Compost 3
Table 2. A comparison of microbial content between the summer compost and the winter compost.
1Day of the trial samples was collected from the summer and winter composts.
2 Days during the trial when summer compost tested positive or negative for Salmonella, E. coli or coliform.
3 Days during the trial when winter compost tested positive or negative for Salmonella, E. coli or coliform.
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 13
ConCluSIonS
Recently there has been an increased aware-
ness of fruits and vegetables as potential sources of
pathogenic microbes that causes human illness. Ani-
mal manure is an economical fertilizer for crop pro-
ducers. However, animal manure frequently contains
enteric pathogenic microbes (Pell, 1997) and spread-
ing it on the land can result in pathogen entry into
the food chain. Composting daily mortality can be
done effectively and efficiently during the summer or
winter. The temperature profiles that are attained in
the winter compost compares favorably to that of the
summer compost. Results from this study show that
composting bird carcasses can eliminate the patho-
genic microbes that may be present in the birds or
the litter. Using the composted material to fertil-
ize vegetable plots did not result in contamination
of the vegetables by pathogenic microorgansisms.
Also there were no indications of re-emergence of
SE or E. coli in the soil.
Composting as a means of dead bird disposal is
effective no matter what time of the year it is prac-
ticed. It is an economical means of disposal as it uses
everything in the production of chickens, nothing is
wasted and the composted material can be used as
a nutrient rich soil amendment.
ACknowledgeMenT
The authors would like to acknowledge Dr. S. Ra-
jeev for her assistance in conducting the microbial
analyses of the project samples. We also thank young
scholar Tyler Reeves for working on this project.
RefeRenCeS
Beuchat, L. R. 1996. Pathogenic microorganisms as-
sociated with fresh produce. J. Food Prot. 59:204-
216.
Conner, D. E., J. P. Blake, and J. O. Donald. 1991.
Microbiological safety of composted poultry farm
Coliform Coliform ColiformLog10 Log10 Log10
1 - - 1.70±.01 - - 1.22±.01 - - 0±0
3 - - 1.73±.01 - - 1.25±.01 - - 1.52±.01
7 - - 1.78±.02 - - 1.26±.01 - - 0±0
10 - - 3.48±.03 - - 1.30±.01 - - 0±0
Wee
k
Salmonella E. Coli Salmonella E. Coli
Treatment 1 2 Treatment 2 3 Control 4
Salmonella E. Coli
Table 3. Microbial presence on cabbage samples.
1 Week of the trial when cabbage samples were collected.
2 Presence or absence of Salmonella, E. coli or coliform on cabbage plants sampled from treatment 1- compost top-dressed
3 Presence or absence of Salmonella, E. coli or coliform on cabbage plants sampled from treatment 2- compost incorporated.
4 Presence or absence of Salmonella, E. coli or coliform on cabbage plants sampled from the control plot- no compost was added.
5 Log10 coliform numbers isolated on from the cabbage plants sampled from each of the experimental plots.
1
5 5
14 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
mortalities. ASAE/CSAE Meeting Paper No. 91-
4053. St. Joseph, MO: The American Society of
Agricultural Engineers.
Epstein, E. 1997. The science of composting. Tech-
nomic Publishing AG, Lancaster, PA. 487 p.
González, J. L. and M. Sánchez. 2005. Treatment of
poultry mortalities on poultry farms. Compost Sci.
Util. 13:136-140.
Grewal, S. K., S. Rajeev, S. Sreevatsan and F. C. Mi-
chel, Jr. 2005. Persistence of Mycobacterium avium
subsp. paratuberculosis and other zoonotic patho-
gens during simulated composting, manure pack-
ing, and liquid storage of dairy manure. Appl. En-
viron. Microbiol. 72:565-574.
Haug, R. T. 1993. The practical handbook of compost
engineering. CRC Press, Boca Raton, FL. 717 p.
Hess, T. F., I. Grdzelishvili, H. Q. Sheng and C. J.
Hovde. 2004. Heat inactivation of E. coli during
manure composting. Compost Sci. Util. 12:314-
322.
Islam, M., J. Morgan, M. P. Doyle, S. C. Phatak, P.
Millner and X. Jiang. 2004. Fate of Salmonella en-
terica serovar Typhimurium on carrots and radishes
grown in fields treated with contaminated manure
composts or irrigation water. Appl. Environ. Micro-
biol. 70:2497-2502.
Johannessen, G. S., G. B. Bengtsson, B. T. Heier, S.
Bredholt, Y. Wasteson and L. M. Rørvik. 2004. Po-
tential uptake of Escherichia coli O157:H7 from or-
ganic manure in Crisphead lettuce. Appl. Environ.
Microbiol. 71:2221-2225.
Jones, D. L. 1999. Potential health risks associated
with the persistence of Escherichia coli O157 in ag-
ricultural environments. Soil Use Manag. 15:76-83.
Kalbasi, K., S. Mukhtar, S. E. Hawkins, and B. W. Au-
vermann. 2005. Carcass composting for manage-
ment of farm mortalities: A review. Compost Sci.
Util. 13:180-193.
Keener, H. M., W. A. Dick and H. A. J. Hoitink.
2000. Composting and beneficial utilization of
composted by-product materials. In: J. F. Power
and W. A. Dick. Eds. Land application of agricul-
tural, industrial, and municipal by-products. Soil
Science Society of America, Madison, Wisconsin.
p 316-341.
Larney, F. J., L. J. Yanke, J. J. Miller and T. A. McAl-
lister. 2003. Fate of coliform bacteria in composted
beef cattle feedlot manure. J. Environ. Qual.
32:1508-1515.
Malone, G. S. 2006. Mass mortality composting pro-
grams. Pages 26-31 in Proc. Natl. Poult. Waste
Manage. Symp., Springdale, AR.
Millner, P. D., K. E. Powers, N. K. Enkiri and W. D.
Burge. 1987. Microbially mediated growth sup-
pression and death of Salmonella in composted
sewage sludge. Microb. Ecol. 14:255-265.
Mukhtar, S., A. Kalasi and A. Ahmed. 2004.
Composting in carcass disposal: A comprehensive
review. USDA APHIS Cooperative Agreement Proj-
ect, Carcass Disposal Working Group.
Murphy, D. W. and L. E. Carr. 1981. Composting dead
birds. FS-537. University of Maryland, Cooperative
Extension Service, College Park, MD.
Natvig, E. E., S. C. Ingham, B. H. Ingham, L. R.
Cooperband, and T. R. Roper. 2002. Salmonella
enterica serovar Typhimurium and Escherichia coli
contamination of root and leaf vegetables grown
in soils with incorporated bovine manure. Appl.
Environ. Microbiol. 68:2737-2744.
Pell, A. N. 1997. Manure and microbes: public and
animal health problem? J. Dairy Sci. 80:2673-2610.
Ritz, C. W., and J. W. Worley. 2005. Poultry mortal-
ity composting management guide. Bulletin 1266.
The University of Georgia, Cooperative Extension
Service, Athens, GA.
Senne, D. A., B. Panigraphy and R. Morgan. 1994.
Effect of composting poultry carcasses on survival
of exotic avian viruses: HPAI virus and adenovirus
of egg drop syndrome- 76. Avian Dis. 38:733-737.
Turner, C. 2002. The thermal inactivation of E. coli in
straw and pig manure. Bioresour. Technol. 84:57-
61.
Wilkinson, K. G. 2007. The biosecurity of on-farm
mortality composting. J. Appl. Microbiol. 102:609-
618.
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 15
www.afabjournal.comCopyright © 2011
Agriculture, Food and Analytical Bacteriology
ABSTRACT
It has frequently been reported that chitinolytic soil bacteria, in particular biocontrol strains, can lyse
viable fungal hyphae and thereby release potential substrates for bacterial growth. The present work was
carried out with an objective to get a better understanding of the relationship between chitinolytic and
antifungal properties of bacteria that occur naturally in coastal soils, i.e. without artificial selection. Among
the bacterial, strain A3 was identified as Pseudomonas sp. A3 based on morphologic observation and 16S
rRNA analysis. Strain A3 exhibited a maximum chitinase production of 1.44 U/ml in CC broth after 3 days of
cultivation. Besides having chitinolytic activity, the molecular weight of the crude enzyme was estimated to
be 56 kDa by SDS-PAGE and zymogram. In vitro assays revealed that the crude chitinase inhibited activity
of Fusarium oxysporum as identified by dual plate assay and microscopic methods. Hydrolysis products of
the fungal cell wall by the crude enzymes of Pseudomonas sp. A3 were analyzed by high-pressure liquid
chromatography (HPLC) and identified as oligosaccharides, which included monomers (GlcNAc), dimers
(GlcNAc)2, and trimers (GlcNAc)3 using chitin oligomer standards. The crude chitinase isolated from strain
A3 can be directly applied for suppressing growth of viable fungal hyphae.
Keywords: Antifungal activity, Pseudomonas, Fusarium oxysporum, chitinase, fungus, 16S rRNA, Zymo-
gram, HPLC
InTRoduCTIon
Biological control of plant pathogens by soil bac-
teria is a well established phenomenon and chitin-
ase production has been shown to play an important
role in the suppression of various diseases (Chernin
et al., 1995; Hong and Hwang, 2005; Hoster et al.,
2005). Chitin (C8H13O5N)n is an unbranched long
Received: September 3, 2010, Accepted: November 26, 2010. Released Online Advance Publication: March 1, 2011. Correspondence: P. Velusamy, [email protected]: - +91-44-22127331 , Fax: +91-44-22121155
chain polymer of glucose derivatives, composed of
ß-1,4 linked units of the amino sugar N-acetyl-D-
glucosamine (NAGA), which is speculated to play
a vital role in fungal defense against toxic stresses.
The interest in chitin degrading enzymes and their
application in management of fungal pathogens are
significant. Chitinases (EC 3.2.1.14), a group of anti-
fungal proteins, catalyse the hydrolytic cleavage of
the ß-1,4-glycoside bond present in the biopolymers
of N-acetyl-D-glucosamine, mainly in chitin.
Determination of antifungal activity of Pseudomonas sp. A3 against Fusarium oxysporum by high performance liquid chromatography (HPLC)
P. Velusamy1, H. S. Ko2, K. Y. Kim2
1Department of Biotechnology, School of Bioengineering, SRM University, Chennai-603 203, India2Division of Applied Bioscience and Biotechnology, Chonnam National University, Gwangju 500-757, Korea
Agric. Food Anal. Bacteriol. 1: 15-23, 2011
16 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
Antagonistic bacteria are considered as ideal bio-
logical control agents that mediate one or several
mechanism of disease suppression. Among these,
hyper parasitism relies on chitinase for degrada-
tion of the cell walls of fungi (Chet et al., 1990). The
soil-borne Enterobacter agglomerans IC1270 has a
broad spectrum of antifungal activity and secretes
a number of chitinolytic enzymes, including two N-
acetyl-ß-D-glucosaminidases and chitinase. Its bio-
control activity has been demonstrated with Rhizoc-
tonia solani in cotton using Tn5 mutants deficient
in chitinolytic activity (Chernin et al., 1995). Hence,
chitinolytic enzyme might be considered to have an
important role in biological control of fungal patho-
gens.
It has frequently been reported that chitinase
producing microorganisms, in particular biocontrol
strains, can lyse viable fungal hyphae, thereby re-
leasing substantial level of oligomers and other sub-
stances (Cohen-Kupiec and Chet, 1998; Dahiya et al.,
2006; De Boer et al., 2001). In the present work, we
report a new strain Pseudomonas sp. A3 possessing
strong chitinolytic activity, which exhibited an antag-
onism toward F. oxysporum. Moreover, the antiungal
activity of the crude chitinase from strain A3 was also
partially characterized.
MATeRIAlS And MeThodS
Screening of bacteria
Five soil samples were obtained from different
sites of the coastal soils enriched with crab shells
in Buan area, Korea. Soils were serially diluted with
sterile water until a dilution of 106 colony form-
ing units (CFU) g-1 of soils, inoculated on colloidal
chitin (CC) agar medium containing 0.5% colloidal
chitin, 0.2% Na2HPO4, 0.1% KH2PO4, 0.05% NaCl,
0.1% NH4Cl, 0.05% MgSO4 7H2O, 0.05% CaCl2 2H2O,
0.05% yeast extract and 2% agar, and incubated at
30°C for 3 days. Strains exhibiting a clear zone (deg-
radation of chitin) around the colony were picked
and further subjected to antifungal activity against F.
oxysporum f. sp. cucumerinum (KACC 40032, Korean
Agricultural Culture Collection, Suwon, Korea) grown
on potato dextrose agar (PDA) medium containing
0.5% colloidal chitin at 30°C for 7 days.
Bacterial identification
To identify the bacterium, a polymerase chain re-
action (PCR) was performed to amplify the 16S rRNA
gene from the genomic DNA of strain A3 using uni-
versal primers fD1 (5’-GAGTTTGATCCTGGCTCA-3’)
and rP2 (5’-ACGGCTACCTTGTTACGACTT-3’) as de-
scribed earlier (Weisburg et al., 1991). The PCR prod-
uct was cloned in a pGEM-T easy vector (Promega,
Madison, WI, USA). The nucleotide sequence of the
16S rRNA gene was determined by an ABI Prism 377
DNA sequencer (PE Applied Biosystems, Foster City,
CA, U.S.A) and compared with published 16S rRNA
sequences using Blast search at Genbank data base
of NCBI (Bethesda, MD, USA).
Chitinase assay
For determination of chitinase activities, strain A3
was grown in CC broth at 30°C, and samples were
taken at 1, 2, 3, 4, and 5 days. Each sample was cen-
trifuged at 8000 ×g for 5 min and the supernatant
was used for enzyme activities. Chitinase activity was
determined by incubating 1 ml of culture supernatant
with 1 ml of 1% colloidal chitin in a 0.05M phosphate
buffer, pH 7.0 at 37°C for 1 h. After centrifugation of
reaction mixture, the amount of N-acetyl-d-glucos-
amine released in the supernatant was determined
by the standard method (Lingappa and Lockwood,
1962) using N-acetyl-d-glucosamine (GlcNAc) as
a standard. GlcNAc present in 0.5 mL of aliquot of
supernatant was determined by adding 0.1 ml of
K2B4O7 and then boiled for 3 min in a water bath.
The tubes were cooled and 3 ml of p-dimethylami-
nobenzaldehyde was added. Absorbance was read
within 10 min at 585 nm against the blank prepared
with distilled water without the enzyme present. One
unit of chitinase is defined as the amount of enzyme
which releases 1 μM N-acetyl-d-glucosamine per
hour under the conditions of the study.
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 17
Preparation of crude enzyme
Strain A3 was cultured in a 2000 ml Erlenmeyer
flask containing 1000 ml of CC broth at 30°C for 3
days in a shaking incubator (180 rpm). After centrifu-
gation of the broth culture at 8000 ×g for 30 min, am-
monium sulfate was added to the supernatant at 50%
saturation, and the mixture was left overnight at 4°C.
The precipitate was centrifuged at 12 000 ×g for 30
min and the pellet was resuspended in 50 mM Tris-
HCl buffer [pH 8.0], and dialyzed against the same
buffer for overnight. The dialyzate was concentrated
by lyophilization, and the concentration of protein
was determined using bovine serum albumin (Sigma
Chemical Co., St. Louis, MO, USA) as the standard
(Bradford, 1976).
Electrophoresis
The concentrated enzyme sample was subjected
to electrophoresis in 12% SDS-PAGE, according to
the method described previously (Laemmli, 1970).
Subsequently, zymogram was demonstrated by co-
polymerizing 0.01% of glycol chitin (Sigma) in SDS-
PAGE for the detection of chitinase activity (Trudel
and Asselin, 1989).
Antifungal activity
The crude chitinase was assayed for antifungal
activity against F. oxysporum by well diffusion assay
on a PDA plate. A fungal plug (6 mm diameter) was
removed from the 5 day old culture. The plug was
transferred onto the center of the PDA plate, which
had been loaded with chitinase in the right well and
the left well was loaded with the same volume of
buffer. The plate was incubated for 5 days at 30ºC
and was monitored for a zone of inhibition around
the well. However, the antifungal effect was also ob-
served by a light microscope (Nikon, Tokyo, Japan).
Two milliliters of the F. oxysporum suspension with
crude chitinase (final concentrations of 2.5 U/ml) in
50 mM of sodium acetate buffer [pH 6.0] was added
into a 12 well plates (12 mm, Corning, NY, USA). A
mixture of hyphae suspension and buffer was treat-
ed as control. In order to determine the deformation
of hyphae, the experiment was carried out at vary-
ing conditions such as pH, temperatures, incubation
time, and different buffers.
Hydrolysis of fungal hyphae
To determine the hydrolysis of F. oxysporum
by crude chitinase (as described in the section on
microscope analysis) after 24 h, the reaction was
stopped by addition of 200 μl 1 M NaOH. The reac-
tion product was centrifuged at 6000 ×g for 30 min,
and the supernatant was passed through a 0.22 μm
membrane filter (Nalgene, Rochester, NY, U.S.A).
The enzyme hydrolysate was analyzed by high-per-
formance liquid chromatography (HPLC). The HPLC
was performed with acetonitrile:water (70:30, v/v) as
the mobile phase at the flow rate of 1 m/min and
detected at 210 nm with NH2P50-4E column (Sho-
dex, Tokyo, Japan) (Kuk et al., 2005). The retention
times for the peaks obtained in the crude samples
of hydrolytic products were compared with the chitin
oligomer standard.
ReSulTS And dISCuSSIon
Isolation and identification of antago-nistic bacterium
In our pilot scale screening, various microbial
colonies were able to degrade chitin on CC agar
medium. Among these, a bacterial isolate that ex-
hibited the maximum halo zone around the colonies,
was designated to be the strain A3. Subsequent
antimicrobial activity was examined through dual
plate assays using various phytopathogens. Interest-
ingly, strain A3 exhibited a strong antifungal activity
against F. oxysporum (Fig. 1). From the morphologic
observation, strain A3 was found to be a Gram-neg-
ative, rod-shaped and polar-flagella bacterium with
permissive temperature ranging between 20 ºC to
37 ºC with an optimum at 30 ºC. The genomic DNA
of the strain A3 was amplified with universal primers
and 16S rRNA gene sequence was analyzed. Align-
ment of this sequence (1464 bp) through matching
18 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
with reported 16S rRNA gene sequences in the Gen-
bank showed high similarity (99 to 100%) to Pseudo-
monas sp. The phylogenetic tree determined by the
Neighbor-joining method showed that Pseudomo-
nas sp. and P. aeruginosa were most closely related
to strain A3 (Fig. 2). On the basis of these results,
strain A3 was identified as a member of Pseudo-
monas sp, and designated as Pseudomonas sp. A3
(Genbank accession number EU784845).
Members of the genus Pseudomonas are ubiq-
uitous in soil microorganisms. They are believed to
serve as a promising group of biocontrol agents and
have been widely evaluated with the production of
chitinases (Ajit et al., 2006; Choi et al., 2006; Fogliano
et al., 2002; Folders et al., 2001; Neiendam and So-
rensen, 1999).
Determination of chitinolytic activity
Strain A3 was investigated for the production of
extracellular chitinase in CC broth by spectropho-
tometry. At 24 h intervals, aliquots of cell cultures
were taken, and the chitinase activity was deter-
mined by a standard method. The results from cul-
ture filtrate of strain exhibited maximum chitinase
activity of 1.44 U/ml after 3 days of cultivation and
gradually decreased thereafter (Fig. 3).
SDS-PAGE and zymogram
The molecular weight of crude chitinase was de-
termined by gel electrophoresis using a standard
marker (iNtRON Biotech, Inc., Gyeonggi-Do, Korea).
Figure 2. Phylogenetic location of strain A3 based on 16S rRNA sequences by Neighbor-joining method program. Phylogenetic tree based on 16S rRNA sequences displaying the relationship between strain A3 and that of the other species. Reference species with accession numbers were obtained from Genbank databases. Bar indicates 0.10 nucleotide substitutions per site.
Figure 1. Inhibition of the growth of F. oxys-porum by Pseudomonas sp. A3 on potato dex-trose agar (PDA) medium containing 0.5% of colloidal chitin at 30ºC for 7 days.
Pseudomonas otitidis isolate WL15 (EF687744)
Pseudomonas sp. M11 (EU375657)
Pseudomonas aeruginosa strain MML2212 (EU344794)
Pseudomonas aeruginosa strain CMG860 (EU037096)
Pseudomonas sp. G3DM-81 (EU037286)
Strain A2
Pseudomonas sp. AHL 2 (AY379974)
97
97
98
99
0.10
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 19
The crude samples of strain A3 revealed several
bands on 12% SDS-PAGE and the chitinase activ-
ity were identified as 56 kDa by zymogram (Fig. 4).
The molecular weights of bacterial chitinases ranged
from 20,000 to 120,000, with little consistency. Differ-
ent molecular masses have been reported for other
bacterial chitinases as well (Jung et al., 2002; Wang
and Chang, 1997).
Relationship between chitinase produc-tion and F. oxysporum suppression by Pseudomonas sp A3
The inhibitory effect on the growth of hyphae of
F. oxysporum was investigated under different con-
centrations of crude chitinase by a well diffusion
assay on a PDA plate and a concentration of 50 μl
(2.5 U/ml) yielded the maximum inhibition against F.
oxysporum. However, in the control well, the same
volume of sodium acetate buffer did not inhibit the
pathogen (data not shown). Microscopic observation
revealed a morphology of hyphae that appeared as
swelled, fragmented and distorted in the wells treat-
ed with the enzyme, whereas the hyphae from the
control were normal and intact without any distor-
tion (Fig. 5). The results presented here support an-
tibiosis as the mechanism of antagonism against F.
oxysporum by strain A3 mediated through chitinase
production. Several studies have demonstrated that
Figure 3. Determination of chitinolytic activity by Pseudomonas sp. A3 in CC broth medium at 30ºC for 7 days. Mean values were 3 replicates. Bars represent standard error.
Figure 4. SDS-PAGE and zymogram of crude chitinase from culture supernatant of Pseudomo-nas sp. A3 by ammonium sulfate precipitation. Lanes 1 and 2 were obtained from 12% SDS-PAGE of Coomassie brilliant blue R-250 where lane 1 is mo-lecular weight (MW) of standard markers in kilodal-tons, and lane 2 is crude chitinase sample. Lane 3 in-dicates zymogram demonstrated by copolymerizing 0.1% of glycol chitin in 12% SDS-PAGE. The chitinase activity was detected by visualization under UV light.
144
84
63
45
34
26.5
1 2 3LaneMW
(kDa)
56 kDa
Chi
tinas
e A
ctiv
ity(u
nits
/ml)
Period of Incubation (days)
20 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
chitinases of potential biocontrol strains can cause
deformation of viable hyphae and result in inhibi-
tion of hyphae of the test fungi (Fogliano et al., 2002;
Giambattista et al., 2001; Mathivanan et al., 1998).
When the crude chitinase was further assayed for an-
timicrobial activity against various microorganisms.
As listed in the Table 1, the percentage of inhibitory
effect of crude chitinase against R. solani and D. bry-
oniae were recorded to be approximately 50% and
the remaining microorganisms were not significant.
Analysis of antifungal activity
Fusarium wilt is a widespread plant disease caused
by many forms of the soil-inhabiting fungus F. oxyspo-
rum. Several attempts have been made to exploit the
biological control of F. oxysporum by chitinase pro-
ducing bacteria (Chung and Kim, 2007; De la Vega et
al., 2006; Giambattista et al., 2001). To investigate the
antifungal effect of crude chitinase, samples of treat-
ed hyphae of F. oxysporum was analyzed by HPLC. As
shown in Fig. 6, various products of chitin oligosaccha-
rides such as monomers (GlcNAc), dimers (GlcNAc)2,
and trimers (GlcNAc)3 were identified using chitin
oligomer standards. Other peaks that were detected
may have belonged to chitosan or glucan oligomers.
However, there was no detectable number of peaks
found in control. Yet, in the present study, chitinolytic
bacterium strain A3 may have an important role in the
hydrolysis of fungal hyphae and release of substantial
Table 1. Inhibitory effects of crude chitinase against various microorganisms by in vitro dual plate assays
*Antimicrobial effect of crude enzyme 50 μl (2.5 U/ml) was assayed by well diffusion assay on agar medium. The percentage of inhibition of growth was calculated from the mean values as:
% Inhibition = (A-B)/A x 100, where A = microorgan-ism growth in control, and B = microorganism growth in chitinase.
The inhibition was reported as (ND) for any unde-tected inhibition of growth from below 5%, (-) between 5% and 15%, (±) between 15% and 25%, (+) between 25% and 35%, (++) between 35% and 50%. Triplicates were run simultaneously to obtain each value.
Figure 5. Morphological study of the hyphae of F. oxysporum in sodium acetate buffer supple-mented with crude chitinase at a concentration 2.5 U/ml incubated at 40 °C for 24 h. (A) Control: hyphae of F. oxysporum + buffer, (B) Treatment: hyphae of F. oxysporum + crude chitinase
Name of Organisms Inhibition Ratio*
Bacteria
Pectobacterium carotovorum subsp. carotovorum KACC 10057 ND
Xanthomonas oryzae pv. oryzae KACC 10378 -
Fungi
Phytophthora capsici KACC 40483 +
Didymella bryoniae KACC 40900 ++Botrytis cinerea KACC 40573 ±Rhizoctonia solani KACC 40117 ++Colletotrichum gloeosporioides KACC 40689 -
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 21
Figure 6. HPLC chromatograms of hydrolysis products from the hyphae of F. oxysporum and crude chitinase mixture after 24 h of incubation at 40 °C. [A] Chitin oligomer standard (GlcNAc)n, [B] Hydrolytic products of crude sample obtained from hyphae of F. oxysporum + crude chitinase. Peak numbers are referred as 1- monomer (GlcNAc) ; 2- dimer (GlcNAc)2 ; 3- trimer (GlcNAc)3 ; 4- tetramer (GlcNAc)4 ; and 5- pentamer GlcNAc)5.
[A] Standard (GlcNAc)n
(1)
(2)
(3)
(4)(5)
[B]
(1)
(2)
(3)
Hyphae + Crude chitinase
Retention Time (min.)
22 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
levels of chitin oligomers. A previous study reported
that chitinase ChiA71 from Bacillus thuringiensis sub-
sp. pakistani completely hydrolyzed colloidal chitin to
GlcNAc monomers after incubation for 24 h (Thamthi-
ankul et al., 2001). More recently, Van et al. (2008) sug-
gested that chitinases from Trichoderma aureoviride
DY-59 and Rhizopus microsporus VS-9 could release
different oligosaccharides after hydrolysis from the
hyphae of Fusarium solani.
ConCluSIon
From the results presented in this experiment, a
positive correlation can be inferred between the pro-
duction of chitinase and suppression of the growth
of F. oxysporum. However, it is necessary to study
the secretion of other lytic enzymes as well, espe-
cially cellulase, ß-1,3-glucanase, and laminarinase,
as chitinase may combine with other lytic enzymes
to exhibit synergism, and result in high levels of an-
tifungal activity. Therefore, we suggest that Pseudo-
monas sp. A3 may be an optimal candidate for use
as a biocontrol agent of Fusarium wilt in tomato, but
further studies are needed to evaluate more exten-
sively this possibility.
ACknowledgeMenT
This study was supported by the Korean Research
Foundation - second stage of BK21, and National
Research Laboratory (NRL) Program from the Minis-
try of Science and Technology (MOST), Korea.
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24 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
www.afabjournal.comCopyright © 2011
Agriculture, Food and Analytical Bacteriology
ABSTRACT
Kalmi Shak or water spinach (Ipomoea aquatica) is a Bangladeshi indigenous green leafy vegetable
and herbaceous aquatic or semi aquatic perennial plant. A primary study was conducted to elucidate the
multi functionalities of this vegetable. Extract of Kalmi Shak exhibited high antioxidant properties with
hydrophilic-oxygen radical absorbance capacity (H-ORAC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scav-
enging activity being 341.92 ± 1.32 and 37.67 ± 2.63 μmol Trolox equivalent / gram of dry weight (TE/g
DW), respectively. The total polyphenols content was estimated to be 12.56 ± 0.08 mg gallic acid equiva-
lent / gram of dry weight (mg GAE/g DW), and moisture content was found to be 85%. The extract also
showed anti-mutagenic effect on Trp-P2 induced mutagenicity to Salmonella Typhimurium TA98, and anti-
tumor activity to mouse myeloma cell line P388. The extract of this vegetable also exhibited anti-bacterial
activities against several spoilage and pathogenic bacteria. The multi functionalities, economic price and
availability during the entire year have made this indigenous Bangladeshi vegetable important from both
medicinal and industrial aspects.
InTRoduCTIon
Leafy vegetables have been extensively investi-
gated as new sources of natural antioxidants as well
as other bioactive compounds of human health ben-
efits (Lakshmi and Vimala, 2000). Epidemiological
studies have shown that consumption of vegetables
is associated with reduced risk of chronic diseases. It
has been reported that leafy vegetable extracts could
Received: September 7, 2010, Accepted: October 21, 2010. Released Online Advance Publication: March 25, 2011. Correspondence: Hossain Uddin Shekhar, [email protected],Tel: - + 81-298-38-8055, Fax: +81-298-38-7996
be used to reduce blood sugar level (Villansennor
et al., 1998) and as an antibiotic against Escherichia
coli, Pseudomonas aeruginosa, Bacillus subtilis and
other microorganisms (Bhakta et al., 2009). Increased
consumption of vegetables containing high levels of
phytochemicals has been recommended to prevent
chronic diseases related to oxidative stress in the
human body (Chu et al., 2002). Natural antioxidants
increase the antioxidant capacity of the plasma and
reduce the risk of certain diseases such as cancer,
heart diseases and stroke (Prior and Cao, 2000). The
secondary metabolites including phenolics and
Multi Food Functionalities of Kalmi Shak (Ipomoea aquatica) Grown in Bangladesh
H. U. Shekhar1, M. Goto1, J. Watanabe1, I. Konishide-Mikami1, Md. L. Bari2 and Y. Takano-Ishikawa1
1National Food Research Institute, Kannondai 2-1-12, Tsukuba, Ibaraki 305-8642, Japan2Center for Advanced Research in Sciences, University of Dhaka, Dhaka-1000, Bangladesh
Keywords: Kalmi Shak, water spinach, antioxidant, anti-mutagenic activity, anti-tumor activity, anti-bacterial activity
Agric. Food Anal. Bacteriol. 1: 24-32, 2011
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 25
flavonoids from plants have been reported to be
potent free radical scavengers (Chiang et al., 2004).
They are found in all parts of plants such as leaves,
fruits, seeds, roots and bark (Mathew and Abraham,
2006). There are many synthetic antioxidants in use,
however, it is reported that they have several side ef-
fects, such as risk of liver damage and carcinogen-
esis in laboratory animals (Gao et al., 1999; Ito et al.,
1983; Osawa and Namiki, 1981). Therefore, a search
for natural antioxidants from plant may help to find
safer, more potent, less toxic and cost effective an-
tioxidants.
Kalmi Shak, a semi-aquatic plant water spinach (Ip-
omoea aquatica) belongs to Convolvulaceae family,
not only grows wild but is also cultivated throughout
Southeast Asia, and is one of the widely consumed
vegetable in the region (Huang et al., 2002). It is a ten-
der, trailing or floating perennial aquatic plant, found
in most soils along the margins of fresh water, ditch-
es, marshes and wet rice field. It is usually found year
round and treated as a leafy vegetable unlike other
common vegetables in Bangladesh which are mostly
seasonal. Kalmi Shak represents one of the richest
sources of carotenoids and chlorophylls (Chen and
Chen, 1992). The leaves contain adequate quantities
of most of the essential amino acids in accordance with
the WHO recommendation pattern for an ideal dietary
protein (Prasad et al., 2008). Consequently, when com-
pared with conventional food crops such as soybeans
or whole egg, it has potential for utilization as a food
supplement. Ayurveda, a system of traditional medi-
cine native to the Indian subcontinent, has identified
many medicinal properties of Kalmi Shak, and it is
effectively used against nosebleeds and high blood
pressure (Perry, 1980). However, very limited scientific
studies have been conducted on its functional aspects.
Most of the studies have focused on the inhibition of
prostaglandin synthesis (Tseng et al., 1992), effects on
liver diseases (Badruzzaman and Husain, 1992), con-
stipation (Samuelsson et al., 1992) and hypoglycemic
effects (Malalavidhane et al., 2003). There have been
no reports on the systematic study of the indigenous
Kalmi Shak of Bangladesh to evaluate its potentiality
as a functional food or food supplement. The objective
of this study was to investigate the antioxidant activity,
total phenolic content, anti-tumor, anti-mutagenic, and
antimicrobial properties of the extracts of indigenous
fresh green Kalmi Shak.
MATeRIAlS And MeThodS
Materials
RPMI-1640, penicillin-streptomycin solution (Hy-
bri-Max®), Dulbecco’s phosphate buffered saline
(PBS), DPPH, and 0.4% Trypan Blue solution, 6-hy-
droxy-2,5,7,8,-tetramethylchroman-2-carboxylic acid
(Trolox), and fluorescein sodium salt (FL) were pur-
chased from Sigma Chemical Co. (St. Louis, MO,
USA). Fetal calf serum (FCS) and Folin-Ciocalteau
(F-C) reagent were purchased from JRH Biosciences
(Lenexa, KS, USA) and MP Biomedical, LLC (Illkirch,
France), respectively. S9-mix (rat liver homogenate
containing rat liver microsome S9 fraction) was ob-
tained from Kikkoman Co. Ltd. (Tokyo, Japan). Cell
proliferation reagent WST-1 was purchased from
Takara Bio Inc. (Siga, Japan). Methanol, acetone,
gallic acid, 3-amino-1-methyl-5H-pyrido[4,3-b]in-
dole (Trp-P2), 2,2’-azobis(2-amidinopropane) di-
hydrochloride (AAPH) and dimethyl sulfoxide
(DMSO) and all other chemicals were purchased
from Wako Pure Chemical Co. (Osaka, Japan).
Plant material and sample preparation
Fresh Kalmi Shak was collected within 24 hour of
harvest from the Dhaka (Dhaka is the capital of Ban-
gladesh and one of the major cities of south Asia)
new market during the summer period (mid April to
June onwards). One hundred grams of green leaves
and veins were cleaned with water, and finally freeze-
dried and kept at -20°C until use. One gram of the
freeze-dried sample was sequentially extracted with
hexane: dichloromethane (1:1) (v/v) and with metha-
nol: water: acetic acid (MWA) solvent at the ratio of
90:9.5:0.5 (v/v/v) using an automatic accelerated sol-
vent extraction system (ASE 350; Dionex, Sunnyvale,
CA, USA). Lipohilic fraction was collected (3 times) by
hexane: dichloromethane at 70°C, 5 min stand at 1500
psi. Hydrophilic fraction was collected thrice by MWA
26 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
solvent at 80°C, 5 minute stand at 1500 psi. The result-
ing MWA extract of the Kalmi Shak which was used
for subsequent experiments was filled up to 50 ml by
MWA. For cell culture and microbiological analyses,
MWA fraction was dried in vacuo and dissolved in
DMSO.
Determination of hydrophilic-oxygen radical absorbance capacity (H-ORAC)
H-ORAC assay was performed according to the
method described by Cao et al. (1993), and Prior
et al. (2003) with slight modifications. In brief, MWA
extracts or Trolox standard solution diluted with 75
mmol/L potassium phosphate buffer (pH 7.4) were
added to a 96-well microplate (#3072, Becton Dickin-
son, NJ, USA). Following the addition of 115 μl of 111
nmol/ LFL to the wells, the plates were incubated at
37°C for 10 min. After the addition of 50 μl of 31.7
mmol/l AAPH to the wells, fluorescence intensities
were measured every two min. for 90 min. by a mi-
croplate reader (Powerscan HT; DS Pharma Biomedi-
cal, Osaka, Japan) with excitation wavelength of 485
nm and emission wave length of 530 nm. H-ORAC
values were expressed as micromole Trolox equiva-
lent per gram of dry sample weight (μmol TE/g DW).
All measurements were done in triplicate.
Measurement of total polyphenols content
Total polyphenols content was measured by the
Folin-Ciocalteu assay according to Sun et al. (2005)
and Velioglu et al. (1998) with slight modifications.
Briefly, three volumes of F-C reagent was diluted by
five volume of water before use. Reaction mixture
containing 80 μl of samples or gallic acid standard
(diluted with MWA) and 56 μl of diluted F-C reagent
was placed in 96 well-microplate (Sumilon, Sumito-
mobakelite, Tokyo, Japan), and incubated for five
min at room temperature. After the addition of 120
μl of 2% (w/v) sodium carbonate, the plate was al-
lowed to stand for 15 min at room temperature. Ab-
sorbance at 750 nm was measured by a microplate
reader (Powerscan HT; DS Pharma Biomedical). To-
tal polyphenols content was expressed as milligram
gallic acid equivalent per gram of dry sample weight
(mg GAE/g DW). All measurements were conducted
in triplicate.
DPPH radical scavenging activity (DPPH-RSA)
DPPH-RSA of MWA extract was examined ac-
cording to the method of Oki et al. (2001) with slight
modifications. Briefly, the same volume of 10% meth-
anol and MWA extract were mixed, and the mixture
was further diluted with 50% methanol. A 50 μl of
diluted MWA extract and 50 μl of 0.2 M morpho-
linoethanesulfonic acid (MES) buffer (pH 6.0) were
subsequently placed in a 96-well microplate (Sum-
ilon, Sumitomobakelite). The reaction was initiated
by adding 50 μl of 800 μM DPPH in ethanol. After
incubation for 20 min. at room temperature, the ab-
sorbance at 520 nm was measured using a micro-
plate reader (Powerscan HT; DS Pharma Biomedical).
DPPH radical scavenging activity was expressed as
micromole Trolox equivalent per gram of dry sample
weight (μmol TE/g DW). All the determinations were
conducted in triplicate.
Determination of moisture content
The moisture content was determined by dry-
ing the samples in a drying oven at 105°C for 24 h
(AOAC, 1984). The leaf and vein (edible portion) of
fresh Kalmi Shak (5.0 g) were cut by dual razor blades
into small pieces, subsequently placed in aluminum
cups and weighed before and after drying. The per-
centages of moisture content were calculated by
subtracting the two values. At least 10 samples per
experiment were replicated, and mean values for
each replicate were calculated.
Determination of the anti-mutagenic effect on Trp-P2 induced mutagenicity to Salmonella Typhimurium TA98
The assay was carried out according to the modi-
fied Ames test (Ames et al., 1975) with Salmonella
Typhimurium TA98. In brief, TA98 strain was cultured
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 27
aerobically in Nutrient broth no. 2 (Oxoid Ltd., Bas-
ingstoke, UK) at 37°C for 12-14h. Trp-P2 was dis-
solved in DMSO to give a working concentration
of 100 ng/ml. The reaction mixture consisted of 0.7
ml of 0.1 mol/l phosphate buffer (pH 7.0), 50 μl of
sample (40 μg/ml in DMSO), 100 μl of S-9 mix, 50 μl
of Trp-P2 and 100 μl of S. Typhimurium TA98. The
positive control contained the same concentration
of perilla leaves extract in DMSO instead of the
sample. Following incubation at 37°C for 20 min in
water bath shaker, two milliliters of soft agar contain-
ing histidine and biotin was added, and the mixture
was immediately plated on a minimal glucose agar.
After incubation at 37°C for two days, the number
of developed revertants was scored. The experi-
ment was performed in triplicate and the mean val-
ues are presented. Anti-mutagenic activities of the
Kalmi Shak extract were calculated according to the
equation described by Hosoda et al. (1992).
Anti-tumor effects to mouse myeloma P388 cells
Anti-tumor activities were measured by the vi-
abilities of myeloma P388 cells using WST-1 cell pro-
liferation reagent (Shinmoto et al., 2001). In brief,
P388 cells (Japan Health Sciences Foundations,
Osaka, Japan) were seeded in 96-well culture plates
(#353072, Falcon) at a density of 5,000 cells (100 μl)
per well in RPMI-1640 medium supplemented with
10% heat-inactivated fetal calf serum (FCS) and 100
units/ml penicillin and 100 μg/ml streptomycin and
incubated at 37°C in a humidified atmosphere with
5% CO2. DMSO solutions of Kalmi Shak with vari-
ous concentrations were added to each well (final
concentrations of 0 (negative control), 50, 100 and
200 μg/ml). Final concentration of DMSO was 0.4%.
Rosemary (Rosmarinus officinalis) extract at varying
concentrations (50 to 200 μg/ml) in DMSO were used
for positive control. After 48 hours incubation, 10 μl
of premixed WST-1 cell proliferation assay reagent
was added to each well. Two hours after the addition
of WST-1, the degree of cell viability was measured
by the absorbance at 450-650 nm of the cell culture
media using microplate reader (Thermomax, Molecu-
lar Devices Co., Tokyo, Japan). Results were reported
as percentage of the inhibition of cell viability, where
the optical density measured from DMSO-treated
control cells was considered to be 100% of viability.
Percentage of inhibition of cell viability was calcu-
lated as follows:
Test organisms
Fifteen strains/species of frequently reported
food borne pathogens or food spoilage bacteria
were used in the study (Table 2). The stock cultures
of the test organisms in 20% glycerol (Sigma) con-
taining medium in cryogenic vials were maintained
at -84°C. Working cultures were kept at 4°C on Tryp-
to Soy Agar (TSB) slants (Nissui Chemical Co. Ltd,
Tokyo, Japan) and were periodically transferred to
fresh slants.
Anti-microbial sensitivity testing
The anti-microbial activity of the Kalmi Shak ex-
tracts was done according to the method of Bauer
et al. (1966). The 8 mm in diameter discs (Toyo Roshi
Kaisha, Ltd. Tokyo, Japan) were impregnated with 50
μl of different concentration of Kalmi Shak extract
before being placed on the inoculated agar plates.
The inocula of the test organisms were prepared by
transferring a loopful of respective bacterial culture
into 9 ml of sterile TSB medium and incubated at
37°C for 5 to 6 h. The bacterial culture was compared
with McFarland (Jorgensen et al., 1999) turbidity
standard (108 CFU/ml) and streaked evenly in three
planes maintaining a 60° angle onto the surface of
the Mueller Hinton agar plate (5 x 40 mm) with ster-
ile cotton swab. Surplus suspensions were removed
from the swabs by rotation against the side of the
tube before the plate was inoculated. After the inoc-
ula dried, the impregnated discs were placed on the
agar using an ethanol dipped and flamed forceps
and were gently pressed down to ensure contact.
Plates were kept at refrigeration temperature (4°C)
1-Aexp group
Acontrolx 100( )
28 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
for 30 to 60 min for better absorption, during which
microorganisms should not grow but absorption of
extracts should take place. Negative controls were
prepared using the same solvent without the plant
extract. Reference antibiotics (streptomycin, genta-
mycin, and rifampicin) were used as positive control.
The inoculated plates containing the impregnated
discs were incubated in an upright position at 37°C
for overnight and/or 24 to 48 h (depending on the
appearance of colonies). The results were expressed
as positive/negative depending on the zone of inhibition.
Statistical analysis
Statistical analysis was performed using Microsoft
Excel (2007). The data were expressed as means ±
standard deviation (SD) for foods having sample
numbers greater than 2.
ReSulTS And dISCuSSIon
It was observed that Kalmi Shak possessed 341.92
± 1.32 μmol TE/g DW of H-ORAC value (Table 1).
From the moisture content, the H-ORAC value in
fresh weight basis can be calculated as 51.28 μmol
TE/g fresh weight (FW). Wu et al. (2004) reported
that H-ORAC values of common vegetables in USA
were between 0.87 (cucumber) and 145.39 (small
red beans) μmol TE/g FW. The most values were in
a range from 5 to 20 μmol TE/g FW. It is suggested
that H-ORAC value of water spinach is relatively high
when compared with those of common vegetables
and fruits.
Mikami et al. (2009) studied antioxidant activi-
ties of 11 crops from Ibaraki prefecture, Japan, and
found that DPPH-RSA ranged from 0.38 (melon) to
91.0 (ginger) μmol TE/g FW. Pellegrini et al. (2003)
studied 34 vegetables and found that spinach ex-
hibited the highest antioxidant capacity (8.49 μmol
TE/g FW). The DPPH-RSA of water spinach in our
study (Table 1) was nearly equal to that of spinach,
though the methodologies of determination were
slightly different.
It has been reported that the total polyphe-
nols contents of 10 vegetables examined by Cieslik
et al. (2006) were between 0.59 to 2.90 mg GAE/g
FW of samples. Wu et al. (2004) observed that total
polyphenols of 23 vegetables were between 0.24 ±
0.05 (cucumber) and 12.47 (red kidney beans). Water
spinach is available in Bangladesh during the entire
year and our collection period originated from early
onset of summer. It has been reported that leaves
harvested in the spring exhibited much higher levels
of total polyphenols content and ORAC value than
the leaves harvested in the fall (Howard et al., 2002).
Consequently, further study should be undertaken
to see the seasonal variation of antioxidant content
of this leafy vegetables.
MWA extract of Kalmi Shak exhibited anti-muta-
genic effects on Trp-P2 induced mutagenicity to S.
Typhimurium TA98 when tested with perilla as posi-
tive control (Fig. 1). Kanazawa et al. (1995) reported
Table 1. Anti-oxidative activity, total polyphenols content and moisture content of indigenous Kalmi Shak in Bangladesh
acμmol Trolox equivalent (TE)/g DW ± SD
bdμmol Trolox equivalent (TE)/g FW
emg galic acid equivalent (GAE)/g DW ± SD
fmg galic acid equivalent(GAE)/g FW g
Percentage
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 29
that flavonoids were very strong anti-mutagens
against Trp-P2. In our study, anti-mutagenic activi-
ties against Trp-P2 were observed to be 52.62% and
50.79% for perilla and Kalmi Shak, respectively (Fig.
1). However, other established mutagens such as
MNNG, AF-2, AB1 etc. were not tested. Therefore,
it is necessary to check the anti-mutagenic activities
of Kalmi Shak extract on the mutagenicity of these
agents in future studies.
MWA extract of Kalmi Shak yielded detectable
anti-tumor activity in the mouse myeloma P388 cell
line. Rosemary extract was used as a positive control
in this experiment, since rosemary leaves exhibit po-
tent anti-tumor and anti-inflammation effects (Peng
et al., 2007). Dose-dependent increase on the anti-
tumor activity was observed in both Kalmi Shak and
rosemary extract (Fig. 2). At a concentration of 50
μg/ml, the corresponding cell viability of rosemary
and Kalmi Shak was 61.36% and 67.56 %, respective-
ly (Fig. 2). At a concentration of 200 μg/ml, cell viabil-
ity of rosemary was 1.66%. On the other hand, that of
Kalmi Shak was 47.59%. Since Kalmi Shak inhibited
the cell viability by more than 50% cell at this con-
centration, and almost 100% cells were not viable in
the case of rosemary. We reported, here, that Kalmi
Shak extract is capable of working against P388 cell
viability.
MWA extract of Kalmi Shak exhibited anti-microbi-
al activities against several spoilage and food borne
pathogenic bacteria within tested fifteen selected
bacteria. The result is presented in Table 2. The extract
of Kalmi Shak exhibited in vitro anti-microbial activi-
ties against spoilage bacteria P. aeroginosa, P. putida,
and pathogenic bacteria such as E. coli O157:H7 and
C. freundii. The result of this study also suggests that
Kalmi Shak extracts include compounds possessing
anti-microbial properties that may be useful to con-
trol food borne pathogens and spoilage organisms.
Further studies need to be done with other food
borne pathogens and spoilage organisms to see the
anti-microbial activities of Kalmi Shak. It would also
be of interest to apply this extract to actual food to
assess the microbiological condition of the particular
food or food products with an extended shelf life.
Figure 2. Anti-tumor activity on mouse myeloma P388 cells
Figure 1. Anti-mutagenic activity on Trp-P2 induced mutagenicity to Salmonella Typhimurium TA98
Concentration μg/ml
30 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
ConCluSIon
In conclusion, the results from in vitro experiments,
including H-ORAC, DPPH-RSA, total polyphenols con-
tent, anti-mutagenic activity, anti-tumor activity, and
anti-bacterial activity demonstrated that Bangladeshi
water spinach variety possessed potent anti-oxidative
and anti-tumor activities. Hence water spinach can be
used as an easy accessible source of natural antioxi-
dants, as a food supplement or in the pharmaceutical
or medical industries. Further work should be per-
formed to isolate and identify the anti-oxidative, anti-
mutagenic, anti-cancer, and anti-bacterial components
of this indigenous vegetable of Bangladesh.
Test Organisms Origin Anti-microbial activity
Spoilage bacteria
Lactobacillus planterum (ATCC 8014) Mexican style cheese -
Perdicoccus pentosaceus(JCM 5890) Dried American beer yeast -
Lactoccus lactis (IFO 12007) Unknown -
Salmonella Enteritidis (SE1) Chicken feces -
Pseudomonas aeroginosa (PA 01) Unknown +
Enterobacter faecalis (NFRI 010618-8) Unknown -
Klebsilla pneumonia (JCM 1662) Trevisan 1887 -
Bacillus subtilis (IFO 13719) Wound -
Pseudomonas putida (KT 2440) Unknown +
Pathogenic bacteria
Escherichia coli (NFRI 080618-8) Celery +
Escherichia coli O157:H7 (CR 3) Bovine feces +
Escherichia coli O157:H7 (MY 29) Bovine feces +
Citrobacter freundi (JCM 1657) Werkman and Gillen 1932 +
Bacillus cereus (IFO 3457) Unknown -
Alcaligenes faecalis (IFO 12669) Unknown -
Table 2. Test organisms used, their source and antibacterial activity of DMSO suspended MWA extract of Kalmi Shak against selected food borne pathogens and spoilage bacteria
+ , - : indicates positive and no positive activity found in preliminary screening, respectively
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 31
ACknowledgeMenT
This research work was supported by Kirin Holdings
Co., Ltd. (former Kirin Brewery Co., Ltd.) Tokyo, dur-
ing UNU-Kirin fellowship at National Food Research
Institute, Tsukuba, Japan in 2010-11, and its Follow-up
Project in 2011-2013. Authors expressed their sincere
gratitude to the authorities of the NFRI for providing
laboratory facilities and logistic supports to carry out
this investigation.
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Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 33
www.afabjournal.comCopyright © 2011
Agriculture, Food and Analytical Bacteriology
ABSTRACT
Hydrogen—limited continuous culture was used to isolate autotrophic acetogenic bacteria from rumen
contents of cattle on either a high roughage or a high concentrate diet. Twenty bacterial isolates were ob-
tained and were presumptively identified as acetogenic bacteria. They were able to use H2:CO2 and they
produced acetic acid as their sole end—product. Two isolates were selected for further studies based upon
their low hydrogen threshold values. The acetogenic strain H3HH was a strictly anaerobic gram positive
coccus with a hydrogen threshold of 1390 ppm. The acetogenic strain Al0 was a facultatively anaerobic
gram positive coccus with a hydrogen threshold of 209 ppm. The use of H2 limited continuous culture to
isolate low H2 threshold ruminal acetogens suggests that not only do acetogens with these properties exist
in the rumen but this approach could be used in other ecosystems as well.
Keywords: Methane, greenhouse gasses, methanogens, acetogens, ruminants, rumen, hydrogen,
continuous culture, carbon dioxide
InTRoduCTIon
Ruminants are characterized by having a four com-
partment stomach (Russell and Rychlik, 2001). The
largest compartment, the rumen, has a volume of
nearly 80 liters and is located before the gastric com-
partment (Weimer et al., 2009). The rumen ecosys-
tem is essentially isothermal, there is a constant flux
of feed and H2O and the fermentation of substrates
Received: September 12, 2010, Accepted: November 17, 2010. Released Online Advance Publication: April 2011. Correspondence: John Patterson, [email protected]: +1 -765-494-4826 Fax: +1-765-494-9347
results in the production of a large amount of acids
(Weimer et al., 2009). Functionally important rumen
microorganisms representing a varied and mixed
population of bacteria, archaea, protozoa, and fungi
hydrolyze complex and soluble feedstuffs primar-
ily to sugars and other hydrolysis products such as
ammonia (Ricke et al., 1996; Stevenson and Weimer,
2007; Uyeno et al., 2007). Glucose is subsequently
fermented in the rumen by rumen microorganisms
to short chain volatile fatty acids (VFA) with the end
products of fermentation including acetate, H2, CO2,
and reduced fermentation products (lactate, butyr-
Using Hydrogen- Limited Anaerobic Continuous Culture to Isolate Low Hydrogen Threshold Ruminal Acetogenic Bacteria
P. Boccazzi¹,² and J. A. Patterson²
¹ Current address: Massachusetts Institute of Technology, Department of Biology and Health Sciences and Technology, 77 Massachusetts Ave. Room 68-370, Cambridge, MA 02139
² Department of Animal Sciences, Purdue University, 1026 Poultry Building, Room 115, West Lafayette, IN 47907-1026
Agric. Food Anal. Bacteriol. 1: 33-44, 2011
34 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
ate, propionate, ethanol) along with microbial cells
(Stevenson and Weimer, 2007; Weimer et al., 2009).
Hydrogen and formate are produced by many mi-
croorganisms in the rumen; however, methanogens
are also present in the rumen and convert H2, and
CO2 to CH4 (Wright et al., 2006). Methanogenesis
represents the primary H2 consumer in the rumen
and energy captured as methane escapes the rumen
via eructation (Boadi et al., 2004, Martin et al., 2010).
Energy lost as methane represents a 2 to 7% loss in
gross energy intake energy of the animal (Branine
and Johnson, 1990) and a loss of 10 to 15% of the
apparently digestible feed energy to the host animal
(Blaxter and Clapperton, 1965). However, direct inhi-
bition of rumen methane production also results in
energy loss in the form of eructated H2 and reduced
microbial protein (Chalupa, 1980).
Chemo-lithoautotrophic acetogens are bacteria
that utilize CO2 as their sole source of carbon and
reduce it to acetate with H2 as the source of energy
(Drake et al., 2008; Ragsdale, 2008). Acetogens are
known to be present in the rumen but they are less
numerous and considered to be less efficient than
methanogens for utilization of hydrogen as a sub-
strate (Martin et al., 2010). Replacement of metha-
nogenesis with acetogenesis could decrease energy
losses and increase the efficiency of ruminant pro-
duction. Consequently, research on acetogenesis in
ruminant animals has been focused toward two re-
lated areas of interest and application. First of all,
since methane formed as a result of ruminal fermen-
tation is subsequently eructated and is lost to the
animal; thus, it would increase energetic efficiency
of the host animal if this loss of feed energy and car-
bon could be minimized (Boadi et al., 2004; Martin
et al., 2010). Secondly, there is increasing interest in
global warming forced by the production of green-
house gasses such as CO2, CH4, and NO2 (Boadi et
al., 2004; Morrison, 2009). Reductive acetogenesis
is a means for developing alternative H2 sinks away
from methanogens that produce CH4 (Joblin, 1999).
Acetogenesis may provide an important model to
find solutions for limiting CH4 emissions from live-
stock and livestock wastes (Morrison, 2009). Efforts
to enhance in vivo acetogenesis in the rumen have
not been as successful as in vitro studies (Fonty et
al., 2007). Methanogens are thought to outcompete
acetogens because methanogens have a lower hy-
drogen threshold (Martin et al., 2010); however, most
acetogens have been isolated in batch culture in the
presence of high hydrogen concentrations and have
not been selected for low hydrogen thresholds. A
key may be a better understanding of hydrogen use
by acetogens. The objective of this study was to use
H2-limited continuous culture to demonstrate that it
could be used to isolate ruminal acetogenic bacte-
ria able to grow on low threshold concentrations of
H2 utilizing CO2 as their sole carbon source.
MATeRIAlS And MeThodS
Source of Organisms
Acetogenic bacterial strains were isolated ei-
ther from rumen contents collected either from
a ruminally fistulated Angus steer fed a diet of al-
falfa and orchard grass hay at maintenance or of a
ruminally fistulated lactating Holstein Friesian dairy
cow consuming a 60:40 percent hay and corn silage:
corn grain diet at 2.6% of her body weight. Rumen
contents were used to inoculate H2-limiting continu-
ous cultures. Individual strains were isolated after at
least 8 turnovers of the continuous culture.
Media and Growth Conditions
All media were prepared by the anaerobic tech-
niques of (Hungate, 1966) as modified by (Balch and
Wolfe, 1976; Bryant, 1972). The basal semidefined
acetogen medium used for growth and nutritional
studies and the methanogen medium are listed in
Table 1. The medium was boiled under a stream of
oxygen-free CO2, sealed, and autoclaved (120°C, 18
Ib/in², 15 min). The pH of the medium was adjusted
to 6.8 with NaOH before boiling. The cooled me-
dium was transferred into an anaerobic glove box
(Coy Laboratories, Ann Arbor, MI) containing 95%
CO2: 5% H2. For all media, the reducing agents, car-
bonate buffer and vitamins were added separately
to the medium in the anaerobic glove box as sterile
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 35
anaerobic solutions. The medium was subsequently
dispensed into 120 ml serum bottles or into 20 ml se-
rum tubes which were then closed with sterile black
butyul rubber serum stoppers and aluminum crimp
closures (BellCo Inc., Vineland, NJ). Solid medium
for isolation of pure cultures consisted of acetogen
medium with the addition of 2% (w/v) agar (Difco).
Continuous culture medium was the same as the
acetogen medium, but the rumen fluid was previ-
ously incubated at 37°C for 6 days to remove carbo-
hydrates (Greening and Leedle, 1989). For chemo-
lithoautotrophic growth of bacterial cultures, serum
bottles (120 ml) and Erlenmeyer flasks (330 ml) were
flushed for 30 seconds and then pressurized at 2.0
atm with either a H2:CO2 (80:20) or a N2:CO2 (75:25)
gas phase, by insertion of sterile disposable needles
through the black butyl stoppers. In all growth and
nutritional studies cultures were incubated at 39°C.
Isolation Procedures
Hydrogen-limited continuous cultures were uti-
lized in an attempt to isolate acetogenic bacteria
with low H2 thresholds from the bovine rumen. The
isolation medium contained 5 mM 2-bromoeth-
anesulfonic acid (BES) (LeVan et al., 1998) to inhibit
methanogens. The growth vessel (200 ml) was initially
half filled with isolation medium, and BES was added
to give an initial concentration of 40 mM for the full
volume of the growth vessel. The inoculum, 40 ml
of rumen fluid, was collected from either the steer
or the lactating dairy cow prior to morning feeding
and strained through a bilayer of cheesecloth under
a stream of CO2 and was added to the growth vessel.
The medium pump was started immediately after in-
oculation and the medium contained 5 mM BES.
The reservoir and growth vessel of the continu-
ous culture were flushed with a stream of humidified
oxygen-free 100% CO2 gas through a glass diffusion
stone. Although the flow rate was not measured,
humidified oxygen-free 100% H2 gas was bubbled
into the growth vessel at a rate to provide 5 to 10
bubbles/ minute. The dilution rate of the continuous
culture was 0.06 h-1 during isolation of acetogenic
bacteria from the steer and 0.28 h-1 during isolation
of acetogenic bacteria from the lactating dairy cow.
The growth vessels were incubated at 39°C. After
8 fluid volume turnovers, 1 ml of fermentation fluid
from the growth vessel was serially diluted in anaero-
bic dilution solution. Each dilution was plated in trip-
licate on solid acetogen medium containing 5 mM
BES. Plates were subsequently incubated anaero-
bically under 1.5 atm of H2:CO2 (80:20) for 6 days
at 39°C. Ten single colonies from each continuous
culture were selected at random and transferred
a Na2CO3 (8% w/v), Cys·HCl (2.5% w/v) and Na2S·9H2O (2.5% w/v) were added separately as sterile anaerobic solutions, to autoclaved and cooled medium.b Greening and Leedle (1989)c Balch and Wolf (1976)
Table 1. Media Composition
Components Acetogen Methanogen(g/L or ml/L) (g/L or ml/L)
K2HPO4 0.24 0.3KH2PO4 0.24 0.3(NH4)SO4 0.24 0.3NaCl 0.48 0.6MgS04·7H2O 0.1 0.13CaCl2·2H20 0.07 0.008NH4Cl 0.54 1
Na2CO3a 4 5
Cys·HCl a 0.25 0.25
Na2S·9H2O a 0.25 0.25
Yeast Extract 0.5 2Resazurin 0.001 0.001Hemin 0.0001 0.001Trypticase - 2CoM - 0.01FeSO4·7H2O - 0.2
Clarified Rumen Fluid (CRF) 50.0ml 100.0ml
Vitamin Sol.b 10.0ml 10.0mlTrace Min. Sol. 10.0ml 10.0ml
Wolf's Trace Min. Sol. 10.0ml -
KH2PO4 (200nM, pH=7)
- 50.0ml
VFA-Mc - 10.0ml
c
36 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
anaerobically to 10 ml of acetogen medium in dupli-
cate serum bottles. The bottles were pressurized at
2 atm of either H2:CO2 (80:20) or N2:CO2 (75:25) and
incubated on their sides in a rotatory shaker (New
Brunswick Scientific Co., Inc. Model M52, Edison,
NJ) operating at 200 rpm for 3 days at 39°C.
Volatile Fatty Acid (VFA) Assay
After incubation, the supernatant of each culture
was analyzed by gas chromatography using a Varian
3700 (Varian, Inc., Palo Alto, CA) gas chromatograph,
to determine VFA composition. Bacterial isolates
producing at least a 4 fold increase in acetate in
bottles containing H2:CO2 over that produced in
bottles containing N2:CO2 were retained for further
characterization.
H2 Threshold Assay
In order to isolate acetogenic strains with low H2
thresholds, a series of three experiments were per-
formed. The general protocol was to grow cultures
in a complex medium to increase cell number, then
adapt the cells to H2:CO2 flush excess H2:CO2, and
determine H2 thresholds using lower concentrations
of H2. Culture vessels were incubated at 39°C on their
sides in a rotatory shaker operating at 200 rpm.
Experiment 1
Triplicate cultures of each acetogenic isolate were
grown in acetogen medium containing 27.8 mM
glucose for 60 h. Serum bottles were pressurized to
1.5 atm with H2:CO2 (80:20) and the cultures were
incubated an additional 60 h. Cultures were sub-
sequently flushed and pressurized to 1.5 atm with
N2:CO2 (75:25) and incubated for 36 h to lower re-
sidual H2 concentration. Cultures were subsequently
flushed and pressurized to 1.5 atm with H2:CO2:N2
(1:24:75) and incubated for 60 h. Methanogens were
grown on methanogen medium for 120 h on H2:CO2
(80:20) at 1.5 atm, then were flushed with N2:CO2
(75:25) and incubated with H2:CO2:N2 (1:24:75) at
1.5 atm for 60 h.
Experiment 2
The format was similar to experiment 1 in incuba-
tion times and sequence of gas phases. Differences
were duplicate cultures were used and the initial me-
dium contained 0.2% (w/v) Brain Heart Infusion Broth
(BHI) instead of glucose. Cultures were incubated for
130 h under H2:CO2:N2 (1:24:75) after flushing with
N2:CO2 (75:25).
Experiment 3
The format was similar to experiment 2 where
bacterial cultures were initially grown in BHI and
then flushed with N2:CO2 (75:25), except that 10 ml
fresh acetogen medium (without glucose or BHI) was
added prior to pressurizing with H2:CO2:N2 (1:24:75).
After incubation, the head space of each culture
vessel was analyzed by gas chromatography using
a varian 3700 gas chromatograph, to determine H2
concentration. Bacterial isolates with the lowest H2
thresholds were retained for further characteriza-
tion. Selected strains were further purified on solid
acetogen medium under H2:CO2 (80:20) and stored
as broth cultures in glycerol at -4°C as described by
(Teather, 1982).
Characterization studies
Gram stain, flagella stain, optimum pH, and heat
test for spore determination were performed accord-
ing to (Holdeman et al., 1977). Optimum tempera-
ture of growth was determined by growing cultures
in acetogen medium containing 5.6 mM glucose at
the respective temperatures for 48 h with optimum
temperature being defined as that temperature that
yielded the highest OD measured at 660 nm at 48
hours. Oxygen sensitivity was tested by three meth-
ods: a) degree of growth throughout stab cultures
in acetogen medium containing 0.5% (w/v) glucose
and 0.4% (w/v) agar, in which the topmost layer was
allowed to oxidize; b) zone of growth in PYG mol-
ten agar medium (Holdeman et al, 1977); and c)
growth on non-reduced, aerobically prepared solid
acetogen medium or in aerobic acetogen broth con-
taining 0.5% (w/v) glucose. For colony formation,
isolates were plated on solid acetogen medium con-
taining 5.6 mM glucose and incubated aerobically
or anaerobically at 39° C. Nitrate reduction, cata-
lase, oxidase, esculin hydrolysis and utilization, and
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 37
starch-hydrolysis tests were performed according to
(Holdeman et al., 1977). GC-fatty acid methyl ester
(FAME) analysis was performed on strains H3HH and
A10 grown in acetogen medium (Table 1) by Mi-
crobe Inotech Laboratories (St. Louis, MO). Similari-
ty and distance coefficients were evaluated between
strains A10 and H3HH and known bacterial species
using the anaerobe database (Moore, ver. 3.7).
Nutritional Studies
The ability of isolates to utilize organic substrates
as energy sources was determined using acetogen
medium containing 0.5% (w/v) of the substrate tested.
Each organic substrate tested was added to the me-
dium as a sterile anaerobic stock solution. Substrate
utilization was assessed by an increase in OD, 660 nm,
after 36 h of growth at 39°C. Determination of cell dry
mass was performed directly on cells washed in saline
solution (NaCl, 0.1% w/v) and harvested from distilled
water. For molar growth yields, cell net dry weight
of 500 ml cultures were compared with the amount
of substrate consumed. Glucose concentration was
measured enzymatically using glucose oxidase re-
agents from Sigma Chemical Co. (St. Louis, MO).
The requirement of isolates for rumen fluid and
yeast extract was determined using Erlenmeyer flasks
(300 ml, BellCo Inc., Vineland, NJ) modified by addi-
tion of a side arm (130 mm x 16 mm) and a serum bot-
tle (20 mm) closure at the top. The bottles were filled
with 20 ml of acetogen medium and then were pres-
surized to 2 atm with either H2:CO2 (80:20) or N2:CO2
(75:25). The inoculum was 0.2 ml (1%, w/v) of a third
transfer of a culture grown under H2:CO2 (80:20). The
flasks were incubated upright at 39°C and agitated
at 200 rpm on a rotatory shaker. The growth of each
organism was followed by measuring the increase in
optical density at 660 nm with time.
Growth Studies
For the assessment of growth and stoichiometry of
acetic acid production and H2 consumption, serum
bottles (120 ml, BellCo Inc.) were filled with either
10 ml of basal acetogen medium or with acetogen
medium containing 5.6 mM glucose and pressurized
to 2 atm with either H2:CO2 (80:20) or N2:CO2 (75:25).
Cultures from each isolate were transferred three
times in medium containing the test substrate and
then a 0.1 ml of the culture was used to inoculate se-
rum bottles for growth determination. Serum bottles
were incubated on their sides and agitated at 200
rpm on a rotatory shaker (New Brunswick Scientific
Co.). Growth of each isolate was measured as an in-
crease in OD. Hydrogen utilization was determined
by measuring reduction in gas volume with a system
similar to that described by (Balch and Wolfe, 1976).
For each sample time, a 4 ml sample of the culture
liquid from each culture was frozen (-4°C) for subse-
quent VFA analysis.
Analytical Methods
Optical density was measured at 660 nm using a
Spectronic 70 spectrophotometer (Bausch & Lomb,
Rochester, NY). Volatile fatty acid production by iso-
lates was measured by gas-liquid chromatography
(GLC) (Holdeman et al., 1977). The frozen samples
were thawed and centrifuged at 15,000 rpm for 5
min, the supernatant was subsequently acidified by
adding 20% (w/v) of methaphopsphoric acid (25%,
w/v) and analyzed. A 3 ft long column, packed with
SP 1220 (Supelco, Bellefonte, PA, USA), was used in
a Varian 3700 GLC with a flame ionization detector.
Oven temperature was 130° C (isothermal), injector
temperature was 170° C, and detector temperature
was 180°C, with carrier gas (N2) flowing at rate of 30
ml per minute.
For the measurement of H2 uptake and CH4 produc-
tion, gas samples were analyzed using a Varian 3700
gas chromatograph equipped with a thermal con-
ductivity detector and a silica gel column (Supelco).
Temperatures of the injector, oven and detector were
room temperature, 130°C, and 120°C respectively,
with carrier gas (N2) flowing at 30 ml per minute.
Microscopy
Determination of cell morphology and presence
of spores and flagella were assessed by phase con-
38 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
trast microscopy (Carl Zeiss D-7082, Oberkochen,
Germany). Scanning electron micrographs were pre-
pared from cells grown to midlog or early stationary
phase in acetogen medium containing 27.8 mM glu-
cose. For scanning electron microscopy, a poly-D-
lysine coated cover slip was immersed in culture fluid
for one hour. Each coverslip was then fixed for 3 h
with 5% (w/v) glutaraldehyde and 1% (w/v) osmium
tetroxide in 0.1 M phosphate buffer, pH = 6.8. The
material was dehydrated by a series of graded etha-
nol solutions (Lamed et al., 1987). The cells on the
cover slips were critical point dried with liquid CO2.
The samples were sputter coated with gold palladi-
um in a Technics Hummer I and viewed with a JEOL
JSMM840 scanning electron microscope (JEOL Ltd.
Tokyo, Japan).
DNA Base Composition
For determination of mole percent guanine plus
cytosine, chromosomal DNA was extracted from
bacterial cells using the procedure described by
(Marmur, 1961). The mole percentage guanine plus
cytosine was calculated from the inflection point of
the temperature melting profile of isolated DNA with
DNA from Escherichia coli strain K12 as the reference
(Marmur and Doty, 1962). The temperature melting
profiles were analyzed using a Perkin-Elmer Lambda
3A spectrophotometer (Norwalk, CT) equipped with
a thermal cuvette.
ReSulTS And dISCuSSIon
Isolation of Bacteria
Twenty strains of acetogenic bacteria were iso-
lated from rumen contents of either an Angus steer
fed a high forage diet or a lactating dairy cow (Hol-
stein Friesian) fed a 40% concentrate diet. All iso-
lates produced at least five fold more acetate un-
der H2:CO2 than under N2:CO2. Acetate production
ranged from 40 to 75 mM on H2:CO2 and from 2
to 8 mM on N2:CO2. The production of other short
chain VFA was minimal for all strains designated.
Bacterial Characterization
All isolates stained gram positive. Strain A4 and
A9 were short rods while strains A2, A10, H3HH, and
H3HP were oval cocci (Table 3). However, H3HH was
pleomorphic, especially during exponential growth
on a rich carbohydrate medium. No flagella were ob-
On the basis of morphology, growth characteristics,
and H2 threshold values, acetogenic strains H3HH,
H3HP, A10, A2, A4, and A9 were selected for further
characterization. Hydrogen threshold of strain A10
and H3HH were the closest to those of the metha-
nogen strain NI4A (Table 2) and were more com-
pletely characterized. These threshold values are
lower than those reported by (LeVan et al., 1998) for
other ruminal acetogens and are comparable to the
values for non-ruminal acetogens (Cord-Ruwisch et
al., 1998).
Table 2. Hydrogen threshold values of methano-gen strain NI4A and of acetogenic strains A10, A2, A9, A4, and H3HH.
a Acetogenic isolates initially grown in 10 ml acetogen medium containing 27.8 mM glucose then flushed and incubated with 1% H2 for an additional 60 h
b Acetogenic isolates initially grown in 0.2% (w/v) BHI then flushed and incubated with 1% H2 for an ad-ditional 130 h
c Acetogenic isolates initially grown in 0.2% (w/v) BHI with 10 ml fresh medium added prior to incubation with 1% H2 for additional 130 h
d ND=not done
H2 Concentration (ppm)
Culture EXP 1a EXP 2b EXP 3c
Initial 10702 9993 9993NI4A 92 90 NDd
A10 1284 994 208A2 2516 1852 1383A9 5383 66157 1619A4 8007 NDd NDH3HH 1390 ND ND
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 39
served by either electron microscopy (Figures 1 and
2 for A10 and H2HH) or by standard staining proce-
dures using light microscopy. No spores were ob-
served by phase contrast microscopy and no spores
were produced by heating the cultures at 80° C for 30
min (Holdeman et al., 1976).
All strains when grown in H2:CO2 or glucose were
mesophilic. Strains H3HH, A10 and A2 grown in ace-
togen medium plus 0.1 (w/v) glucose, reached higher
OD at 30° C, however all strains grew faster at 39°C
(Table 4). Thus 39°C was considered the optimum tem-
perature of growth. Cells grew within a temperature
range of 17° to 45°C. strains H3HH and A2 reached
higher OD at 30°C and grew poorly or did not grow at
17°C. Strain A10 grew at almost the same rate at 30° C
and 39°C and was uniquely different from strain H3HH
and A2 in that it was able to grow at 17° C. The opti-
mum pH for growth in acetogen medium plus 0.5%
(w/v) glucose was 7.0 for strains A10 and H3HH and
7.5 for strains A2 and H3HP (Table 3). The pH range for
growth was 5.5 to 8.0 (data not shown). While strains
H3HH, H3HP, and A2 were strict anaerobes, strain A10
was considered to be facultative, because it grew in
all the media and conditions used to determine oxy-
gen sensitivity (Table 3). Most acetogens isolated have
been more strict anaerobes although several can tol-
erate low concentrations of O2 after exhibiting a lag
phase in growth (Karnholz et al., 2002).
Table 3. Morphological characteristics of isolates.
Cat. = catalase Sens. = sensitivity pleom. = pleomorphic
Figure 1. Picture A10: Morphology of strain A10 at late log-phase grown in acetogen medium containing 27.8 mM glucose (Scanning Electron Microscopy, x 11, 000).
Figure 2. Picture H3HH: Morphology of strain H3HH at late log-phase grown in acetogen medium containing 27.8 mM glucose (Scanning Electron Microscopy, x 11, 000).
Culture Gram stain Shape Cell size
µmOxygen
Sens. Cat. Spore MotileOptimum
Temp. (°C)
Optimum pH
G+C Mol%
H3HH + cocci-pleom 0.6-0.8 x 1.0-1.2 anaerobe - No No 39 6.8-7.0 ND
H3HP + oval-cocci 0.6-0.8 x 1.0-1.3 anaerobe - No No 39 7.5 ND
A10 + oval-cocci 0.6-0.8 x 1.0-1.4 facultative + No No 39 6.8-7.5 51.5
A2 + oval-cocci 0.6-0.8 x 1.0-1.5 anaerobe - No No 39 6.8-7.5 ND
40 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
After 5 days of autotrophic growth on solid ace-
togen medium under H2:CO2, strain H3HH colonies
were 1 mm in diameter, entire, slightly convex with a
regular margin and the colonies were white in color.
Colonies of strain A10 were approximately 0.7 mm
in diameter, the colonies were entire with a regular
margin and were transparent. When incubated aero-
bically on solid acetogen medium containing 27.8
mM glucose, strain A10 colonies were 1.2 mm in di-
ameter, were convex with a regular margin and were
white in color (data not shown). The FAME compo-
sition of strain A10 and H3HH was not found to be
similar to any of the species existing in the anaerobe
database (Moore, ver. 3.7). Species of interest clos-
est to these isolates that were in the FAME database
included Peptostreptococcus productus, Clostridi-
um thermoaceticum, Clostridium thermoautotrophi-
cum, and Eubacterium limosum. The mol% G+C for
strain A10 was 51.5% but was not determined for the
other isolates.
Nutrition Studies, Growth Studies, and Fermentation
Yeast extract, rumen fluid, or both were required
to support initial growth of strains A10 and H3HH
(data not shown). Both strains reached higher op-
tical density when growing on a basal acetogen
medium plus yeast extract, but initially they grew
faster when both yeast extract and rumen fluid were
added to the basal acetogen medium. All strains
utilized a wide range of carbohydrates as an energy
source (Table 4). Cellobiose, lactose, and sucrose
supported the highest growth. No strain was able
to utilize arabinose. Esculin was utilized poorly even
though all strains were able to hydrolyze it. Pectin
and casein were poorly utilized. Strain A10 was the
only strain that did not utilize glycerol. Simple acids
listed in Table 4 were either poorly or not utilized.
Strains H3HH, H3HP, and A2 were all oxidase and
catalase negative (Table 3). Strain A10 was oxidase
negative, but showed a weakly positive response in
the catalase test. Strains A10, A2, H3HH, and H3HP
were able to hydrolyze esculin and both strains A10
and H3HH were able to reduce nitrate (Table 5).
Strain A10, but not strain H3HH, was able to hydro-
lyze starch.
Strain H3HH differed from the ruminal acetogen
Acetitomaculum ruminis, in cell shape, absence of
flagella, and substrate utilization (Greening and
Leedle, 1989). Strain H3HH also differed from the
ruminal acetogen Eubacterium limosum, in sub-
strate utilization (Genthner et al., 1981; Genthner
and Bryant, 1982, 1987) and the ability to reduce
nitrate to nitrite. The species that most closely re-
sembled strain H3HH was Peptostreptococcus pro-
ductus which had been isolated from the calf rumen
(Bryant et al., 1958). However, strain H3HH was not
Table 4. Growtha of selected acetogenic strains on various substrates.
a Absorbance (660 nm) values represent the increase in OD after 36 hours of incubation at 39°C.
b Growth tests were carried out in acetogen medium plus 0.5% (w/v) of the desired substrate. Substrates were added separately as sterile anaerobic solutions to the autoclaved and cooled medium.
Substrateb A10 H3HP H3HH A2
Arabinose 0.13a 0.09 0.12 0.08Cellobiose 3.1 1.71 2.6 2.39Fructose 1.54 0.89 1.29 1.56Galactose 1.38 0.76 1.93 1.35Glucose 1.5 1.1 2.1 1.7Lactose 2.16 1.28 2.48 2.12Maltose 1.97 1.25 2.13 1.56Sucrose 2.58 1.23 2.14 1.99Casein 0.33 0.34 0.37 0.43Esculin 0.59 0.72 0.45 0.23Glycerol 0.22 1.04 0.69 1.2Mannitol 0.38 0.36 1.19 0.21Pectin 0.23 0.25 0.28 0.2Starch 1.5 1.35 0.12 0.15Glutamic acid 0.11 0.05 0.18 0.09
Formic acid 0.17 0.48 0.17 0.1
Fumaric acid 0.08 0.03 0.03 0.05
Lactic acid 0.13 0.17 0.03 0.13Succinic acid 0.05 0.08 0.09 0.05
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 41
related to P. productus based on GC-FAME analysis.
Strain H3HH differed from Clostridium thermoaceti-
cum and C. thermoautotrophicum in cell shape and
because it did not form spores (Fontaine et al., 1941;
Wiegel et al., 1981). Both strain H3HH and A. kivui
lack flagella and do not produce spores but strain
H3HH differed from A. kivui in cell shape, growth
temperature range and substrate utilized (Klemps et
al., 1987; Leigh et al., 1981; Leigh and Wolfe, 1983).
Strain A10 differed from strain H3HH in substrate
utilization profile (Table 4), and because strain A10
is catalase positive, could also hydrolyze starch, and
is a facultative anaerobe. Strain A10 differed from
the ruminal acetogen Acetitomaculum ruminis, in
cell shape, absence of flagella, substrates utilized,
and DNA base composition (Greening and Leedle,
1989). Strain A10 also differed from the ruminal ace-
togen Eubacterium limosum, in being able to reduce
nitrate and in substrate utilization profile (Genthner
et al., 1981; Genthner and Bryant, 1982, 1987). Strain
A10 was not related to P. productus based on GC-
FAME analysis. Strain A10 differed from Clostridium
thermoaceticum, and C. thermoautotrophicum in
cell shape, lack of flagella and a lower optimum
temperature for growth (Fontaine et al., 1941; Wie-
gel et al., 1981). Strain A10 differed from Acetoge-
nium kivui in cell shape, growth temperature range,
and substrate utilization profile ( Klemps et al., 1987;
Leigh et al., 1981; Leigh and Wolfe, 1983).
Acetate was the major VFA produced when strains
A10 and H3HH were growing in H2:CO2, glucose, or
glucose plus H2:CO2 When strains A10 and H3HH
were grown under H2:CO2 (80:20) strain A10 achieved
a maximum OD of 0.84 and a doubling time of 16 h
(Table 5), strain H3HH achieved a maximum OD of
0.81 and a doubling time of 13 h. These OD values
are lower than those achieved by E. limosum but the
doubling time is shorter (Genthner et al., 1981; Gen-
thner and Bryant, 1982, 1987). The maximum acetate
production of strains A10 and H3HH growing under
H2:CO2 (80:20) was 69 and 35 mM respectively. Both
C. thermoautotrophicum and E. limosum have been
shown to produce slightly more acetate (Genthner
et al., 1981; Genthner and Bryant, 1982, 1987; Wie-
gel et al., 1981). When strains A10 and H3HH were
grown on glucose (5.6 mM) strain A10 achieved a
maximum OD of 0.79 and a doubling time of 1.4 h,
strain H3HH achieved a maximum OD of 0.65 and a
doubling time of 1 h. The maximum acetate produc-
tion of strains A10 and H3HH growing on glucose (5.6
mM) was 14 and 15 mM respectively. When grown on
glucose plus H2:CO2 strain A10 and H3HH achieved
a maximum OD of 1.27 and 1.22 respectively.
When grown on H2:CO2 as an energy source, H2
consumption by strain A10 and H3HH was close to
the theoretical stoichiometry:
4H2 + 2CO2 g 1CH3COOH + 2H2O (Table 6)
Molar growth yields (g dry weight cell/mol sub-
strate consumed) for strain A10 and strain H3HH
were 0.67 and 0.51 g/mole respectively which were
Table 5. Growth and fermentation characteristics of isolates
Doub. = doubling Prod. = production
Culture
O.D. H2:CO2
Doub. Time
H2:CO2
Acetate Prod. (mM)
H2 Threshold
(ppm)
y (H2) (g
DW/mole)
Esculin Hydrol.
Nitrate Reduction
Starch Hydrol.
H3HH 0.81 13h 35 1390 0.51 + + -H3HP 0.73 15h 62 ND ND + ND NDA10 0.84 16h 68.8 209 0.67 + + ND
A2 0.43 24h 30 1383 ND + ND ND
Hydrol. = hydrolysis ND = Not Determined
42 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
lower than the values reported for C. thermoauto-
trophicum and E. limosum (Genthner et al., 1981;
Genthner and Bryant, 1982, 1987; Wiegel et al.,
1981). When grown on glucose as energy source,
glucose consumption by strain A10 and H3HH
was consistent with the theoretical stoichiometry:
C6H12O6 (glucose)g 3CH3COOH (Table 7)
Molar yields (g dry weight cell/mol substrate con-
sumed) were 36.9 for strain A10 and 47.4 for strain
H3HH.
ConCluSIon
Ruminants are one of the many sources of bio-
genic methane, hence the interest in reducing emis-
sions (Boadi et al, 2004; Fonty et al., 2007; Morrison,
2009). Ruminants may provide an important model
to enhance the animal production efficiency while
at the same time reduce global warming effects. In
addition, assuming that the energy lost as methane
by ruminants represents a loss of potential energy
to the animal (Branine and Johnson, 1990), sig-
nificant savings in feed cost to the producer could
Table 6. Growth yields and stoichiometry of fermentation of strains A10 and H3HH, grown on H2 + CO2(80:20)
aCell dry weights were determined from 500 ml cultures grown in acetogen medium under a H2:CO2 (80:20) gas atmosphere.
b Assimilation of acetate into cell material was calculated by the equation: 17C2H3O2 + 11H20 g 8<C4H7O3> + 2HCO3 + 150H; thus, 20.6 μmol acetate was required for 1.0 mg of cell dry matter (Eichler and Schink, 1984).
cH2 present in fermentation products as percentage of H2 consumed
Table 7. Growth yields and stoichiometry of fermentation of strains A10 and H3HH, grown in acetogen medium containing 5.6 mM glucose.
a Cell dry weights were determined from 500 ml cultures grown in acetogen medium under a H2:CO2 (80:20) gas atmosphere.
b Assimilation of acetate into cell material was calculated by the equation: 17C2H3O2 + 11H20 g 8<C4H7O3> + 2HCO3 + 150H; thus, 20.6 μmol acetate was required for 1.0 mg of cell dry matter (Eichler and Schink, 1984).
Culture
H2 uptake mM
Cell Dry
Weighta mg/ml
Acetate
Assimilatedb
mM
Acetate Produced mM
%H2
Recoveryc
Y(H2) g/mole
A10 421.6 0.28 5.83 68.86 71 0.67
H3HH 128.3 0.07 1.36 35 114 0.51
Culture
Initial Glucose Conc. mM
Cell Dry
Weighta mg/ml
Acetate
Assimilatedb
mM
Acetate Produced
mM
%Carbon Recovery
Y(Glc) g/mole
A10 5.6 0.22 4.54 14.37 113 36.9
H3HH 5.6 0.26 5.42 11.18 99.6 47.4
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 43
be realized if the potential energy presently lost as
methane is captured as fermentation products. Con-
sequently, methanogenesis in the rumen could be
inhibited and acetogenesis in the rumen enhanced
sufficiently to act as an electron sink and convert en-
ergy in H2 to acetate, which in turn can be utilized
by the animal (Boadi et al., 2004; Morrison, 2009).
Several limitations remain in the study because un-
fortunately these isolates have now been lost. First,
the taxonomy of the isolates was not resolved since
standard nutritional and physiological methods were
used instead of molecular methods. A next step
would have been to use 16S rRNA gene sequence
analysis of these isolates to provide phylogenetic
identification of the isolates. Such information would
have allowed design of FISH probes or PCR primers
for quantifying these acetogens both in vivo and in
vitro, thus expanding greatly the direction of future
research in this area. Unfortunately, the current iso-
lates were lost due to freezer malfunction and further
phylogenetic characterization is not possible. Despite
these limitations, the current study does demonstrate
that H2 limited continuous culture is a possible ap-
proach for isolating low H2 threshold isolates from the
rumen or other anaerobic ecosystems.
ACknowledgeMenT
We thank Kenneth Maciorowski, Purdue University,
for performing the scanning electron micrographs.
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Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 45
www.afabjournal.comCopyright © 2011
Agriculture, Food and Analytical Bacteriology
ABSTRACT
The objective of this experiment was to determine the effect of plant-based protein meals (soybean and
canola) in poultry feed on colonization and shedding of Salmonella Heidelberg in broiler birds over a 42-
day period. One-day old chicks were randomly assigned to 4 different dietary treatments (n=360 birds per
treatment) with 6 replicates per treatment, 60 birds per replication. Three all plant protein meal diets and
one commercial diet containing animal protein meal (meat and bone) were used in the study. Half of the
birds (n=30) per pen were challenged with nalidixic acid-resistant (NA) S. Heidelberg on day one (called
seeders), and the remaining unchallenged birds were called contacts. Drag swabs were collected from all
pens on days 0 (prior to placement), 14, and 42. Ceca samples were collected from 20 birds per pen (10
seeders and 10 contacts) on day 42. Drag swabs and ceca samples were examined for NA- S. Heidelberg
using enrichment and enumeration/enrichment, respectively. All drag swabs were negative on day 0, but
positive for S. Heidelberg on both days 14 and 42. Within seeder and contact birds, there was no signifi-
cant differences in:1) NA-S. Heidelberg concentration (cfu/g of ceca), and 2) proportions of positive ceca
among the treatment groups. It can be concluded that all plant-based protein meal diets did not signifi-
cantly reduce the environmental contamination with S. Heidelberg nor did it reduce the concentration and
proportion of positive S. Heidelberg in contact and seeder birds compared to commercial diet containing
animal protein meal. Keywords: Salmonella Heidelberg, broiler, canola, soybean, performance, plant-based protein meal, animal protein meal, shedding, colonization
InTRoduCTIon
Foodborne salmonellosis is a major public health
concern in the United States. Poultry continues to be
Received: Sepetember 22, 2010, Accepted: October 19, 2010. Released Online Advance Publication: May 6, 2011. Correspondence: W. Q. Walid , [email protected]: +1 (770) 467-6066 Fax: +1-(770) 229-3216
an important frequent vehicle of Salmonella trans-
mission to humans, mainly via contaminated chicken
meat. Therefore, reducing Salmonella colonization
and shedding in live chickens and consequently
chicken meat contamination can lead to a decline
in the burden of salmonellosis in humans. Poultry
feed is at the beginning of the food safety chain in
Effect of Plant-based Protein Meal Use in Poultry Feed on Colonization and Shedding of Salmonella Heidelberg in Broiler Birds
W. Q. Alali1, C. L. Hofacre2, G. F. Mathis3, A. B. Batal4
1Center for Food Safety, University of Georgia, Griffin, Georgia2Department of Population Health, Poultry Diagnostic and Research Center, University of Georgia, Athens, Georgia
3Southern Poultry Research, Athens, Georgia4Department of Poultry Science, University of Georgia, Athens, Georgia
Agric. Food Anal. Bacteriol. 1: 45-53, 2011
46 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
the farm-to-table continuum. Broiler bird infection,
colonization and subsequent fecal shedding of Sal-
monella is prevalent ( Bailey et al., 2001; Liljebjelke
et al., 2005; Renwick et al., 1992; Rodriguez et al.,
2006). Several studies have examined the effect of
various on-farm management practices (e.g., vac-
cine, use of antibiotics and probiotics, cereal grains)
on colonization and shedding of Salmonella in poul-
try (Barrow et al., 1984; Bjerrum et al., 2005; Engberg
et al., 2004; Hofacre et al., 2007; Van Immerseel et al.,
2005). However, very little is known about how dif-
ferent feed rations/ingredients; specifically, how the
type of protein meals in broiler feed affect the colo-
nization and shedding of Salmonella in broiler birds.
Globally, the feed industry is considering alterna-
tives (e.g., soybean, cottonseed meal, fishmeal, and
legumes) to the rising cost of animal protein meals
(e.g., meat and bone meal and animal by-products)
and the worldwide growth in intensive poultry pro-
duction (FAO, 2004). Furthermore, the high preva-
lence of contaminants (e.g., Salmonella spp.) de-
tected in the animal protein meal ( Hacking et al.,
1978; Hofacre et al., 2001; Isa et al., 1963; Veldman et
al., 1995) is another reason to look for suitable alter-
natives. Soybean and canola meals (i.e., rapeseed)
are the largest protein meals produced worldwide
(USDA, 2010a). These meals are high in protein and
can be used in broiler feed to provide up to 60% of
the crude protein in a typical diet (Newkirk and Clas-
sen, 2002). Canola meal has lower protein content
than soybean meal (34 to 38 % compared to 44 to
49 %) and lower concentration of essential amino
acids. Canola meal is considered a protein source
for animals that do not have high energy demand.
It has been largely fed to cattle and pigs as part of
their diet rations (Brzoska, 2008; Caine et al., 2008).
Additionally, canola meal is used in broiler diet as
a protein source (up to 10%) (Dale, 1996; Montazer-
Sadegh, et al. 2008). The U.S. produced 41 million
tons of soybean meal in 2009 (valued at $12.5 bil-
lion) and 787,000 tons canola meal in the same
year (valued at $196 million) (USDA, 2010b). On the
other hand, the U.S. imported 1.9 million tons of
canola meal, mainly from Canada, in 2009 valued at
$469 million (USDA, 2010b).
This study was conducted to compare the effect
of plant-based protein meals (soybean and canola)
in poultry feed on colonization and shedding of Sal-
monella Heidelberg in broiler birds over a 42-day
period compared to broiler fed animal protein meals
(meat and bone). We also compared the effect of the
diets used in the study on the broiler birds perfor-
mance (feed conversion ratio [FCR] and weight gain)
and mortality percentages.
MATeRIAlS And MeThodS
Study Design
Chicks were assigned randomly to 4 different di-
etary treatments (n=360 birds per treatment) with
6 replicates per treatment, 60 birds per replication.
Treatment 1 was an all plant-based feed containing
diet, 35% soybean protein meal (SBM). Treatment
2 was an all plant-based diet containing 30% SBM
and 10% canola meal (CM). Treatment 3 was an all
plant-based diet containing 10% SBM and 40% CM.
Treatment 4 (control diet) was a commercial diet con-
taining animal protein meal (10% meat and bone)
and plant-based protein meal (17% SBM and 10%
CM). The study consisted of 24 pens starting with 60
male broiler chickens. The birds were Cobb×Cobb
one-day-old male broilers purchased from Cobb-
Vantress hatchery (Cleveland, GA). All birds were
spray vaccinated with Coccivac-B on day one. Sixty
birds were housed in each floor pen (0.77 ft2/bird,
stocking density) that contained litter of approxi-
mately 4 inches of fresh pine shavings. Feed and
water were available ad libitum.
Feed Formulation
All diets were formulated to meet or exceed the
animal nutrient requirements (NRC, 1994). Three diet
phases were used: a starter (0 to 14 days of age),
grower (14 to 28 days), and finisher (28 to 42 days).
Tables 1 through 3 show the composition of the
starter, grower, and finisher diets used in this study.
Broiler diets were fed as crumbles (starter feed) or
as pellets (grower and finisher). All feed contained
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 47
1Vitamin mix provided the following (per kilogram of diet): vitamin A, 2481 IU; vitamin D3, 496 IU; vitamin E, 4.96 IU; vitamin B12, 5.46 μg; riboflavin, 2.0 mg; niacin, 19.8 mg, d-Pantothenic acid, 4.96 mg; choline, 86.1 mg; menadione, 0.33 mg; folic acid, 0.25 mg; thiamine, 0.99 mg, pyridoxine, 0.99 mg; biotin, 0.49 mg; and ethoxyquin, 57.1 mg.
2Trace mineral mix provided the following (per kilogram of diet): calcium, 0.022 mg; iron, 0.02 mg; magnesium, 0.02 mg; maganese, 0.1 mg; zinc, 0.08 mg; copper, 4000 ppm; iodine, 1000 ppm; and selemium, 400 ppm.
3Coban: coccidiostat
4BMD: bacitracin methylene disalycilate
5TBCC: tribasic copper chloride
Table 1. Composition of dietary treatments (as-fed basis) for broiler starter (0-14 days of age)
Table 2. Composition of dietary treatments (as-fed basis) for broiler grower (14-28 days of age)
Ingredient 35% SBM
30% SBM
+10% CM
10% SBM
+40% CM
10% Animal protein meal
Corn, yellow, ground
57.68 54.23 45.2 58.78
Soybean Meal (49) 36.45 28.95 9.95 20.19
Canola _ 10 35.55 10
Poultry meat & bone meal
_ _ _ 7
Fat 2.57 3.61 6.32 1.86
Dicalcium phosphate 1.17 1.12 0.99 0.17
Limestone 0.93 0.87 0.7 0.62
Salt 0.6 0.34 0.32 0.38
Methionine 0.29 0.25 0.29 0.24
Vitamin premix 0.25 0.25 0.25 0.25
Lysine 0.08 0.15 0.16 0.29
Trace mineral premix
0.08 0.08 0.08 0.08
Coban 0.05 0.05 0.05 0.05
BMD 0.05 0.05 0.05 0.05
L-Threonine _ _ 0.04 0.02
TBCC 0.02 0.02 0.02 0.02
Ronozyme 0.02 0.02 0.02 0.02
Ingredient 35% SBM
30% SBM
+10% CM
10% SBM
+40% CM
10% Animal protein meal
Corn, yellow, ground
65.11 61.67 50.83 64.34
Soybean Meal (49) 28.66 21.12 9.8 15.82
Canola _ 10 50.83 9
Poultry meat & bone meal
_ _ _ 6
Fat 2.82 3.87 6.46 2.46
Dicalcium phosphate 1.07 1.02 0.89 0.2
Limestone 0.94 0.87 0.73 0.66
Salt 0.37 0.35 0.32 0.39
Methionine 0.3 0.26 0.15 0.24
Vitamin premix 0.25 0.25 0.25 0.25
Lysine 0.23 0.29 0.31 0.36
Trace mineral premix
0.08 0.08 0.08 0.08
Coban 0.05 0.05 0.05 0.05
BMD 0.05 0.05 0.05 0.05
L-Threonine 0.04 0.06 0.05 0.07
TBCC 0.02 0.02 0.02 0.02
Ronozyme 0.02 0.02 0.02 0.02
1
2
3
4
5
1
2
3
4
5
48 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
bacitracin methylene disalycilate (BMD) antibiotic.
Bird weights (kg) by pen were recorded on days 1
and 42. Feed intake was measured at 21, 35, and 42
days of age. Feed conversion ratios (feed intake/
weight gain) were also calculated.
Salmonella Challenge and Sample Col-lection
Nalidixic-acid (NA) Salmonella Heidelberg isolate
was grown for 6 h in Tryptic Soy Broth (TSB; Difco),
whereafter the number of colony-forming units (cfu)
per ml was determined by plating 10-fold dilutions
of the bacterial suspension on Xylose Lys Tergitol-4
(XLT-4; Difco) medium. Then, a bacterial colony was
diluted in Phosphate Buffered Saline (PBS) solution
to reach the inoculation titer (5×107 cfu/ml).
Chick-box liners (i.e., paper pads) were collected,
and stored in sterile sampling bags for Salmonella
analysis. At day 1, prior to placement, 30 of the 60
chicks per pen (all treatments) were orally adminis-
tered by gavage 0.1 ml of a NA (25 μg/ml) S. Heidel-
berg (5×107 cfu/ml). Each of these 30 chicks chal-
lenged with S. Heidelberg were also wing-banded
and were called seeders, whereas the remaining 30
chicks were called contacts. Contact birds in our
study model the horizontal transmission of Salmo-
nella in broiler houses (i.e., infected broilers with Sal-
monella horizontally spread this organism to other
birds (contact) in the same poultry house).
Drag swab samples were tested for Salmonella
environmental contamination from all pens on days
0 (prior to placement), 14, and 42. Dragging sterile
gauze swabs soaked in double strength skim milk
across the birds bedding material is considered
to be the most sensitive method of environmental
sampling by the National Poultry Improvement Plan
(NPIP) (USDA, 1996). To reduce the possibility of
cross contaminating a sample, gloves were changed
between completing each drag swab. Appropriate
dress in clean coveralls and plastic shoe covers was
used on entry to any pen by the farm workers. Self
contained Solar-Cult sterile drag swabs (Solar Bio-
logicals Inc., Ogdensburg, NY) were used. Samples
were transported on ice within 24 h to Center for
1Vitamin mix provided the following (per kilogram of diet): vitamin A, 2481 IU; vitamin D3, 496 IU; vita-min E, 4.96 IU; vitamin B12, 5.46 μg; riboflavin, 2.0 mg; niacin, 19.8 mg, d-Pantothenic acid, 4.96 mg; choline, 86.1 mg; menadione, 0.33 mg; folic acid, 0.25 mg; thiamine, 0.99 mg, pyridoxine, 0.99 mg; biotin, 0.49 mg; and ethoxyquin, 57.1 mg.
2Trace mineral mix provided the following (per kilo-gram of diet): calcium, 0.022 mg; iron, 0.02 mg; mag-nesium, 0.02 mg; maganese, 0.1 mg; zinc, 0.08 mg; copper, 4000 ppm; iodine, 1000 ppm; and selemium, 400 ppm.
3Coban: coccidiostat
4BMD: bacitracin methylene disalycilate
5TBCC: tribasic copper chloride
Table 3. Composition of dietary treatments (as-fed basis) for broiler grower (28-42 days of age)
Ingredient 35% SBM
30% SBM
+10% CM
10% SBM
+40% CM
10% Animal protein meal
Corn, yellow, ground
69.6 66.85 58.73 69.06
Soybean Meal (49) 25.16 19.14 6.36 16.26
Canola _ 8 26.95 7Poultry meat & bone meal
_ _ _ 2.89
Fat 2.86 3.7 5.88 2.87Dicalcium phosphate 0.43 0.39 28 _
Limestone 0.7 0.65 0.52 0.56
Salt 0.44 0.43 0.4 0.45
Methionine 0.19 0.16 0.08 0.16Vitamin premix 0.25 0.25 0.25 0.25
Lysine 0.15 0.2 0.28 0.25Trace mineral premix
0.08 0.08 0.08 0.08
Coban 0.05 0.05 0.05 0.05
BMD 0.05 0.05 0.05 0.05
L-Threonine _ 0.02 0.04 0.03
TBCC 0.02 0.02 0.02 0.02
Ronozyme 0.02 0.02 0.02 0.02
1
2
3
4
5
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 49
Food Safety Laboratory (Griffin, GA).
On day 42, 20 birds per pen (10 seeders and 10
contacts) were randomly selected, euthanized by
cervical dislocation, weighed, and the ceca were
aseptically removed and then placed into sterile
plastic sampling bags for Salmonella isolation. Sam-
ples were transported on ice within 24 h to Center
for Food Safety Laboratory.
Salmonella Analysis
Chick paper pads were enriched in 400 ml tet-
rathionate broth (TT- Difco™) containing 0.001%
aqueous brilliant green and 2 ml of iodine solution
(25% iodine and 30% potassium iodide), then in-
cubated at 42ºC for 24 h. A loopful (~10 μL) of the
broth was streaked on XLT4 agar without and with
NA supplement (final concentration was 25 parts per
million [ppm]). The XLT4 plates were then incubated
at 37ºC for 24 h.
Drag swab samples were enriched with 100 ml of
TT as above and incubated (42ºC, 24 h). One loop-
ful was streaked onto XLT4 agar supplemented
with NA. The XLT4 plates were then incubated at
37ºC for 24 h, and one black colony per plate was
streaked on blood agar (Remel, Lenexa, KS). One
colony per blood agar plate was tested against Sal-
monella O group B antiserum (Difco) and confirmed
to be S. Heidelberg if agglutination was noted. If
no black colonies were present from a sample, the
enriched drag swab was held at room temperature
for 5 days and a delayed secondary enrichment was
performed as follow: 1:10 dilution into new TT broth
was made and then incubated at 37ºC for 24 h. A
loopful of the mixture was streaked onto XLT4 agar
supplemented with NA. The remaining isolation
and confirmation of S. Heidelberg was carried out
similarly as the primary enrichment.
Fifty milliliters of phosphate buffered saline (PBS-
Fisher) solution was added to the ceca, and then
mechanically homogenized using a stomacher
(Technar Company, Cincinnati, OH) for 20 seconds.
A serial dilution (10 fold) was performed using 0.5
ml portion of the stomacher bag content added to
4.5 ml of saline solution. From each dilution, a 500
μl aliquot was spread plated onto XLT4 plate con-
taining NA (25 ppm). After incubation (37ºC, 24 h),
black colonies were counted. One colony per plate
was randomly selected and struck onto blood agar
(Remel, Lenexa, KS). One colony per blood agar
plate was tested against Salmonella O group B an-
tiserum (Difco) and confirmed to be S. Heidelberg if
agglutination was noted. Five milliliters of TT broth
containing brilliant green and iodine solution was
then added to the ceca and PBS mixture, and in-
cubated at 42ºC for 24 h. One loopful per sample
was streaked on XLT4 containing NA, incubated
(37ºC, 24 h), and black colonies were identified as
described earlier. If no black colonies were pres-
ent from a sample, the enriched ceca were held at
room temperature for an additional 7 days and a
delayed secondary enrichment was tested for Sal-
monella as for the drag swabs.
Statistical Analysis
A sample was considered positive if Salmonella
was recovered from direct plating, primary or sec-
ondary enrichments. A randomized complete block
design was used in the study with 6 blocks and 4
treatments. The experimental unit was the pen. The
study outcomes were: 1) the proportion of Salmo-
nella isolates from drag swabs or ceca, 2) Salmo-
nella counts (cfu/g) from ceca samples, and 3) per-
formance data (FCR, weight gain, and mortality).
First, proportion of Salmonella isolates were com-
pared between treatment groups by contact and
seeders using a Generalized Linear Model (GLM)
with binomial distribution and a logit link (GEN-
MOD procedure, SAS Inst. version 9.1.3, Inc., Cary,
NC). Second, Salmonella counts (concentrations)
were compared between treatment groups by con-
tacts and seeders using MIXED procedure in SAS
software. Third, performance data were compared
between treatment groups using MIXED procedure
in SAS software. Treatment was a fixed effect and
the block was a random effect in the model. Counts
(cfu/g) were logarithmically transformed by use of
log base 10 to approximate normality. Treatment
effects were considered significant if P < 0.05.
50 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
ReSulTS And dISCuSSIon
Data on FCR, average weight gain, and percent-
age of mortality for the 4 treatment groups are
shown in Table 4.
ference in mortality percentages in our study. This
indicates that all plant-based protein meal diets did
not adversely affect the bird mortality compared to
the animal protein diet. The BMD used in the feed
diets in our study would have no or little effect on S.
Heidelberg since it was applied across all the treat-
ments (with the same amount), and it would have no
effect on performance. This antibiotic was used to
prevent necrotic enteritis infections caused by Clos-
tridium perfringens and as a growth promoter; a
common practice by the poultry industry.
Paper pads were Salmonella negative which indi-
cated that chicks were Salmonella-free prior the in-
oculation. Feed was not tested for Salmonella prior
consumption by chicks. However, we used Salmo-
nella inoculum that was nalidixic acid resistant and
the media we used for culture had nalidixic acid in
it. Therefore, even if the feed had a small amount of
Salmonella, the counts reported in this study were
only NA-S. Heidelberg strain that we confirmed with
nalidixic acid supplement XLT4 and Salmonella O
group B antiserum.
All drag swabs collected on day 0 from the 24
pens (i.e., 4 treatment groups) were negative for
NA-S. Heidelberg; whereas swabs collected on both
days 14 and 42 were all positives. This suggests that
none of the treatment groups in our study lowered
the shedding of S. Heidelberg throughout the 42-
day study period to a level below the detection limit
of the enrichment method used in this study. Newly
hatched chicks can be colonized with Salmonella
early in life (i.e., during hatching process and first few
days post hatching) and that could lead to presence
of this organism in their intestinal tract and subse-
quent shedding during the grow out period (Cason
et al., 1994).
There was no significant difference in percentage
of cecal samples positive for S. Heidelberg, by chal-
lenge type (seeder or contact birds) among the treat-
ment groups (Table 5). Furthermore, there was no
significant difference in S. Heidelberg populations
(cfu/g of ceca), by challenge type, among the treat-
ment groups (Table 6). We challenge the birds (i.e.,
seeders) with 5 log cfu of NA-S. Heidelberg, howev-
er, both proportions (percentages) of positive cecal
The FCR and average weight gain for birds in
treatments 3 and 4 were significantly different (P
<0.05). The highest FCR was observed in treatment
group 3 (10% SBM and 40% CM), and lowest FCR
was in group 4 (animal protein diet); whereas aver-
age weight gain was highest in group 4 and lowest
in group 3. This may indicate that higher percent-
age of CM in the diet significantly increased FCR in
broiler birds in our study. However, the higher weight
gain in group 4 was due to an increase in feed con-
sumption compared to other treatment groups (data
not shown). This is in contrast to Newkirk and Clas-
sen (2002) findings that replacement of up to 60% of
SBM with CM in broiler diets has no adverse effect
on growth performance. There was no significant dif-
FCR Mortality(0 to 42 d) (%)
35% SBM 1.754a 2.306a 4.67a
30% SBM +10% CM 1.776a 2.287a 1.67a
10% SBM +40% CM 1.789b 2.119b 2.33a
10% Animal protein
meal21.710c 2.345c 2.33a
TreatmentAverage weight gain
Table 4. Performance results and mortality percentages in broilers following exposure to Salmonella Heidelberg1
a-c Means in a column with no common superscript letters are significantly different (P <0.05)
1 Means represent 6 pens per treatment, with 60 birds per pen.
2 The 10% Animal protein meal treatment included plant-based protein meal (17% SBM and 10% CM).
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 51
samples and the concentrations of NA-S. Heidelberg
were low. Birds with fully functioning T-cell immune
systems will naturally begin to clear Salmonella from
their ceca over time after exposure (i.e., initial inocula-
tion). Furthermore, even though we challenged them
with 5 logs NA-S. Heidelberg, that does not mean all
5 logs colonized. It has been reported in several stud-
ies that different types of feed may influence Salmo-
nella populations in multiple parts of the gastrointes-
tinal tract (GIT) of broiler birds. Bjerrum et al. (2005)
found that broiler birds fed pelleted commercial feed
had higher Salmonella Typhimurium population in
the gizzard and ileum compared to birds fed whole
wheat based diet. Similarly, whole wheat based diet
lowered Lactobacillus, enterococci, and Clostridium
perfringens populations in broiler bird GIT compared
to pelleted commercial feed (Engberg et al., 2004).
In another study, triticale-soybean based diet fed to
broilers reduced Salmonella cecal colonization com-
pared to corn-based diet (Santos et al., 2008). The
authors used a cocktail of 4 Salmonella serotypes (Ty-
phimurium, Newport, Heidelberg, and Kentucky) for
their inoculum. It is difficult to assess whether Salmo-
nella populations in the ceca samples were Kentucky,
Heidelberg, Typhimurium, Newport or a combination
of serotypes. Furthermore, since Salmonella is pres-
ent in the environment, there is no way to distinguish
between the sources of Salmonella (inoculated versus
environmental). In our study, we used NA- S. Heidel-
berg (one serotype) as a surrogate for the following
reasons: 1) the ability to differentiate the source of
Salmonella (inoculated versus environmental) which
could confound the results and 2) inoculation with
one serotype could reduce the possibility of multiple
Table 5. Mean percentages of positive Salmo-nella Heidelberg ceca culture results in broilers following exposure to Salmonella Heidelberg1
1 Means represent 6 pens per treatment, with 60 birds per pen. At day 42, 20 birds per pen (10 seeders and 10 contacts) were selected and the ceca were removed for S. Heidelberg isolation. There were no significant differences detected among treatments within seeder and contact birds. Statistical analysis was conducted using MIXED procedure in SAS software (GENMOD procedure, SAS Inst., Inc., Cary, NC). Treatment was a fixed effect and the block was a random effect in the model.
2Seeders were chicks challenged with S. Heidelberg and wing-banded (n=30 per pen).
3Contacts were chicks unchallenged with S. Heidelberg (i.e., the remaining 30 chicks per pen).
4The 10% Animal protein meal treatment included plant-based protein meal (17% SBM and 10% CM).
All(seeders+contacts)
35% SBM 70 36.67 33.33
30% SBM +10% CM 63.33 31.67 31.67
10% SBM +40% CM 70 35 35
10% Animal protein
meal476.67 40 36.67
Contacts3Treatment Seeders2
Table 6. Mean Salmonella Heidelberg enu-meration results per gram of cecal content in broilers following exposure to Salmonella Heidelberg1
Treatment Seeder2 Contacts3
35% SBM 872.2 63.9
30% SBM +10% CM 409.7 44
10% SBM +40% CM 136.1 111.1
10% Animal
protein meal4291.1 1212.2
52 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
serotypes interaction which could confound the re-
sults (treatment effect versus interaction effect).
To the best of our knowledge, this is the first study
to investigate the effect of canola and soybean in
combination in all plant-based protein diet on the
colonization and shedding of Salmonella in broiler
birds. The results obtained herein suggests that
plant-based protein meal diets did not significantly
reduced the environmental contamination with S.
Heidelberg nor did it reduce the concentration and
proportion of positive S. Heidelberg in contact and
seeder birds compared to commercial diet contain-
ing animal protein meal.
ACknowledgeMenT
This study was supported in part by the University
of Georgia, Center for Food Safety seed grant. We
thank Rebekah Turk, Aparna Petkar, Kim Hortz, and
Christine Lobsinger for the technical assistance in
processing samples and performing microbiological
assays at the laboratory.
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54 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
www.afabjournal.comCopyright © 2011
Agriculture, Food and Analytical Bacteriology
ABSTRACT
Microbial keratinases have become biotechnologically important since they target the hydrolysis of
highly rigid, strongly cross-linked structural polypeptide “keratin” recalcitrant to the commonly known
proteolytic enzymes trypsin, pepsin and papain. Keratinases are produced in a medium containing kerati-
nous substrates such as feathers and hair. This paper reports on the optimization of keratinase production
by Bacillus subtilis NCIM 2724. One factor-at-a-time method was used to investigate the effect of carbon
sources, nitrogen sources and pH on keratinase production. An L8 orthogonal array design was adopted to
select the most important fermentation parameters influencing the yield of keratinase. Response surface
methodology (RSM) was used to develop a mathematical model to identify the optimum concentrations
of the key parameters for higher keratinase production, and confirm its validity experimentally. The effect
of various amino acids on the production of keratinase was also studied. The final optimized medium gave
a maximum yield of 12.32 KU ml-1 of keratinase. Keratinases are commercially important among the prote-
ases that have been studied since they attack the keratin residues and hence find application in developing
cost-effective feather by-products for feeds and fertilizers.
Keywords: Keratinase, Fermentation, Bacillus subtilis, Optimization, Orthogonal Array Design, Response
surface methodology
InTRoduCTIon
Keratin is a fibrous and insoluble structural protein
extensively cross linked with hydrogen, disulphide
and hydrophobic bonds. It forms a major component
of the epidermis and its appendages viz. hair, feath-
ers, nails, horns, hoofs, scales and wool (Anbu et al.,
Received: September 27, 2010, Accepted: October 29, 2010. Released Online Advance Publication: May 6, 2011. Correspondence: Ishwar B. Bajaj, [email protected] Tel: - +91 22 24145616, Fax: +91 22 24145614
2007). Feather keratin exhibits an elevated content of
several amino acids such as glycine, alanine, serine,
cysteine and valine. The intensive cross-linkage in ker-
atins hinders their degradation by commonly known
proteolytic enzymes (Gupta and Ramnani, 2006). Deg-
radation of feathers will not only decrease the envi-
ronmental problem caused due to their accumulation
but could also act as source of some nutritionally im-
portant amino acids.
Currently, some industries have produced feather
Optimization of Fermentative Production of Keratinase From Bacillus Subtilis NCIM 2724
S. M. Harde1, I. B. Bajaj1, R. S. Singhal1
1Food Engineering and Technology Department, Institute of Chemical Technology,
Matunga, Mumbai, Maharashtra, India, 400 019
Agric. Food Anal. Bacteriol. 1: 54-65, 2011
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 55
meal by steam pressure cooking. This technique re-
quires high energy input and may degrade amino
acids. The enzymatic hydrolysis of feather may be a
viable alternative to steam pressure cooking (Grazzi-
otin et al., 2006). The use of crude enzymes from Ba-
cillus species particularly Bacillus licheniformis and
Bacillus subtilis have been extensively studied due
to their effectiveness in terms of feather degradation
(Manczinger et al., 2003).
Keratinases [EC.3.4.99.11] belong to the group of
serine proteases capable of degrading keratin. It is
an extracellular enzyme produced in a medium con-
taining keratinous substrates such as feathers and
hair. Keratinases have applications in traditional in-
dustrial sectors including feed, detergent, medicine,
cosmetics and leather manufacturers (Farag and
Hassan, 2004), they also find application in more re-
cent fields such as prion degradation for treatment
of the dreaded mad cow disease (Langeveld et al.,
2003), biodegradable plastic manufacture and feath-
er meal production and thus can be appropriately
called “modern proteases”. The use of keratinases
to enhance drug delivery in some tissues and hydro-
lysis of prion proteins arise as novel potentially high
impact applications for these enzymes (Brandelli,
2007). Although many applications of keratinases are
still in the stage of infancy, a few have found their
way to commercialization, particularly the use of Bio-
resource International’s (BRI) Versazyme for feather
meal production. The crude enzyme can also serve
as a nutraceutical product, leading to significant im-
provement in broiler performance (Odetallah et al.,
2003). The most promising application of keratinase
is in the production of nutritious, cost effective and
environmentally benign feather meal (Gupta and
Ramnani, 2006). Nutritional enhancement can be
achieved by hydrolysis of feather meal/raw feather
using keratinase which significantly increases the lev-
els of essential amino acids methionine, lysine and
arginine (Williams et al., 1991). The present work focuses on trying to produce
keratinase from nonpathogenic microorganisms
and utilization of chicken feathers as a sole carbon
source. Several bacteria produce keratinase as an
extracellular material. Most of these belong to the
genus Bacillus. These bacteria use keratinous sub-
strates such as chicken feathers as carbon sources
for the production of keratinase. Aspergillus fu-
migatus was previously reported to be able to
use chicken feather flour as carbon and nitrogen
source (Santos et al., 1996). Addition of glucose,
sucrose and lactose resulted in strong inhibition
of keratinase production (Brandelli, 2007). The
production of keratinase is usually most notice-
able when chicken feathers are used as a sole car-
bon source (Williams et al., 1990). Farag and Has-
san (2004) used chicken feathers as a sole carbon,
nitrogen and sulphur sources for keratinase pro-
duction and observed 26.69 U/mg of keratinase
activity. Lin et al. (1992) used chicken feathers as a
sole carbon, nitrogen and energy sources for ker-
atinase production. Suntornsuk and Suntornsuk
(2003) reported that keratinase activity increased
upto 0.9 U/ml by using chicken feathers as a sub-
strate and sole carbon source from Bacillus sp. FK
46. They also varied the feather concentration for
production of keratinase and observed that higher
feather concentrations cause substrate inhibition
or repression of keratinase production, resulting
in a low percentage of feather degradation. El-
Refai et al. (2005) used different substrates for ker-
atinase production from Bacillus pumilus FH9 like
feather, muscle protein and wool. They observed
that wool gave the maximum keratinase activity of
647 U/ml. According to Kim et al. (2001), B. cereus
gave the maximum keratinase activity of 117 U/ml
by using feathers as a carbon source. Anbu et al.
(2007) produced keratinase from Scopulariopsis
brevicaulis by using glucose and feather as car-
bon sources and observed 1% glucose and 1.5%
feather to achieve a maximum keratinase activity
of 6.2 KU/ml.
Besides carbon sources, factors such as nitro-
gen sources (Thyes et al., 2006) and medium pH
(Suntornsuk and Suntornsuk, 2003) can influence
the productivity of keratinase. B. licheniformis
produced keratinase at neutral pH (Wang and
Shih, 1999). Anbu et al. (2007) studied the effect
of several organic and inorganic nitrogen sources
on keratinase production and found maximum
56 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
production in the presence of 1.5 to 2% sodium ni-
trate (6.2 KU/ml) followed by peptone (6 KU/ml) and
potassium nitrate (5.5 KU/ml). Sodium nitrate below
1.0 to 1.5% permitted enzyme synthesis, but was in-
hibitory above 2%. Thyes et al. (2006) studied the ef-
fect of feather meal, soybean, gelatin, casein, yeast
extract, cheese whey and peptone at 10 g/L for pro-
duction of protease from Microbacterium arbores-
cenes. Among the various nitrogen sources studied,
maximum keratinase was produced in feather meal
(96.5 U/ml), followed by soybean protein (73.8 U/ml)
and gelatin (45.8 U/ml).
The initial pH of the medium greatly affects the bac-
terial growth, percentage of feather degradation and
keratinase production (Suntornsuk and Suntornsuk,
2003). It was observed that Bacillus species is most ac-
tive under neutral or basic conditions. The optimum
pH for B. cereus was 7.0 (Kim et al., 2001), while that
for B. pumilus was 8.0 (El-Refai et al., 2005). For B. sub-
tilis, highest enzyme production was obtained over a
broad range of pH 5 to 9.
According to Wang and Shih (1999) maximum
growth rate and keratinase productivity of B. subtilis
occurred at 42°C instead of 37°C, and the fermenta-
tion time could also be shortened. However, the maxi-
mum keratinase activity was observed at 37°C. Elevat-
ed temperature increased cell growth, but not enzyme
production. The temperature differential effect on
growth versus keratinase production was more obvi-
ous in B. licheniformis, where cells grew best at 50°C,
but keratinase production was best at 37°C. High tem-
perature may increase the protein turnover rate. Ac-
cording to El-Refai et al. (2005) the optimal reaction
temperature recorded for B. pumilus FH9 keratinase is
higher than those reported for other B. pumilus strains.
This paper reports on optimization of keratinase
production using a statistical approach. Effects of
pH, carbon source and nitrogen source were inves-
tigated by using one factor at-a-time method. Initial
screening of the medium components was done by
using an L8 orthogonal array design to understand
the significance of their effect on the product forma-
tion, and then a few of the more significant param-
eters were selected for further optimization using
response surface methodology (RSM).
MATeRIAlS And MeThodS
Materials
Chicken feathers were collected from the Devgiri
poultry farm, Wadegavhaon, Pune, India. Chicken
feathers were washed three times with distilled wa-
ter followed by defattening with chloroform: metha-
nol (1:1), dried and ground. All chemicals used were
of the AR grade and were purchased from Hi Media
Limited, Mumbai, India.
Bacterial strain and medium
A bacterial strain of Bacillus subtilis NCIM 2724 was
used in the present study. The medium used for the
growth and maintenance contained (g L-1), ammoni-
um chloride, 0.5; magnesium sulphate, 0.1; yeast ex-
tract, 0.1; sodium chloride, 0.5; dipotassium hydrogen
phosphate, 0.3; potassium hydrogen phosphate, 0.3;
feathers, 10 (pH 7.5 ± 0.2). Bacterial cells in agar slants
were incubated at 37°C for 24 h and stored at 4°C.
The medium was sterilized in an autoclave for 15 min
at 121°C.
For the production of keratinase, a medium re-
ported by El-Refai et al. (2005) was used, which con-
tained (g L-1) Feather, 10; Yeast extract, 0.1; MgSO4,
0.1; NH4Cl, 0.5; K2HPO4, 0.3; KH2PO4, 0.3; NaCl, 0.5.
Initial pH of the medium was adjusted to 7.5 ± 0.2 with
Tris–HCl buffer. The medium was sterilized in an auto-
clave for 15 min at 121°C.
Inoculum and fermentation
One ml cell suspension from a slant was trans-
ferred to 20 ml of the seed medium containing (g
L-1) peptone, 5; yeast extract, 1.5; beef extract, 1.5
and sodium chloride, 5; (pH 7 ± 0.2) and incubated
at 37°C and 200 rpm for 24 h. This was used as the
inoculum. Fermentation was carried out in 250 ml Er-
lenmeyer flasks, each containing 50 ml of the sterile
production medium. The medium was inoculated
with 5% (v/v) of 12 h old B. subtilis culture containing
approximately 2×106 cells/ml. The flasks were inoculat-
ed on a rotary shaker at 37 ± 2 °C and 200 rpm for 48 h.
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 57
All the experiments were carried out at least in triplicate.
Optimization of fermentation medium us-ing one factor-at-a-time method
In order to investigate the effect of initial pH of me-
dium on keratinase production, fermentation runs were
carried out by adjusting initial pH of the medium in the
pH range of 5 to 8, and analyzing the samples for kera-
tinase production after 48 h. To study the effects of dif-
ferent nitrogen sources on keratinase production, yeast
extract in the medium was replaced with different or-
ganic nitrogen sources, such as peptone, malt extract,
and beef extract at 0.1 g L-1, and ammonium chloride
was replaced with different inorganic nitrogen sources,
such as sodium nitrate, potassium nitrate, ammonium
sulphate or ammonium nitrate at 0.5 g L-1 and fermen-
tation was carried out as described in the previous sec-
tion. To check the effect of additional carbon sources
on the production of keratinase, fermentation medium
containing chicken feathers was supplemented with ad-
ditional carbon sources, viz., glycerol, sucrose, soluble
starch, maltose, lactose, fructose, glucose. All carbon
sources were used at 10 g L-1.
Optimization of fermentative production by using Orthogonal Array Design
An L8 orthogonal array method was used for screen-
ing of the most significant fermentation parameters
influencing keratinase production. The design for
the L8 orthogonal array was developed and analyzed
using MINITAB 13.30 software (Pennsylvania State
university, University Park, Pennsylvania). The L8 or-
thogonal array design is shown in Table 1. Seven fac-
tors at two levels were studied viz. chicken feather,
ammonium chloride, beef extract, potassium dihy-
drogen phosphate, potassium hydrogen phosphate,
Table 1. Orthogonal project design for 2 levels of 7 variables used for media optimization for keratinase production.
a Results are mean ± SD of three determinations Values in the parenthesis indicate the real values of variables
1 1 (5) 1 (0.05) 1 (0.05) 1 (0.1) 1 (0.1) 1(0.15) 1 (0.15) 1.33 ± 0.04
2 1 (5) 1 (0.05) 1 (0.05) 2 (0.5) 2 (0.5) 2 (0.75) 2 (0.75) 2.37 ± 0.1
3 1 (5) 2 (0.25) 2 (0.25) 1 (0.1) 1 (0.1) 2 (0.75) 2 (0.75) 1.1 ± 0.23
4 1 (5) 2 (0.25) 2 (0.25) 2 (0.5) 2 (0.5) 1 (0.15) 1 (0.15) 1.05 ± 0.04
5 2 (25) 1 (0.05) 2 (0.25) 1 (0.1) 2 (0.5) 1 (0.15) 2 (0.75) 3.66 ± 0.07
6 2 (25) 1 (0.05) 2 (0.25) 2 (0.5) 1 (0.1) 2 (0.75) 1 (0.15) 1.90 ± 0.25
7 2 (25) 2 (0.25) 1 (0.05) 1 (0.1) 2 (0.5) 2 (0.75) 1 (0.15) 2.88 ± 0.2
8 2 (25) 2 (0.25) 1 (0.05) 2 (0.5) 1 (0.1) 1 (0.15) 2 (0.75) 4.20 ± 0.08
KH2PO4 K2HPO4Keratinasea
(KU ml-1)NaCl NH4ClRun Feathers Beef extract MgSO4 -1
58 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
magnesium sulphate and sodium chloride for their
significance in production of keratinase by B. subtilis
NCIM 2724.
Optimization of concentrations of the selected medium components using Response Surface Methodology (RSM)
Response surface methodology is an empirical
statistical modeling technique employed for mul-
tiple regression analysis using quantitative data
obtained from properly designed experiments to
solve multivariable equations simultaneously (Puri
et al., 2002). RSM was used to determine the opti-
mum nutrient concentrations for the production of
keratinase. A central composite design (CCD) for
four independent variables was used to obtain the
combination of values that optimizes the response
within the region of three dimensional observation
spaces, which allows one to design a minimal num-
ber of experiments. The experiments were designed
using the software, Design Expert Version 6.0.10 trial
version (State Ease, Minneapolis, MN).
The medium components (independent variables)
selected for the optimization were chicken feather,
ammonium chloride, magnesium sulphate, and di-
potassium hydrogen phosphate. Regression analysis
was performed on the data obtained from the de-
sign experiments. Coding of the variables was done
according to the following equation:
where: xi, dimensionless value of an independent
variable; Xi, real value of an independent variable;
Xcp, real value of an independent variable at the
center point; and ∆Xi, step change of real value of
the variable i corresponding to a variation of a unit
for the dimensionless value of the variable i.
The experiments were carried out at least in trip-
licate, which was necessary to estimate the vari-
ability of measurements, i.e. the repeatability of
the phenomenon. Replicates at the center of the
domain in three blocks permit the checking of ab-
sence of bias between several sets of experiments.
The relationship of the independent variables and
the response was calculated by the second order
polynomial equation:
Y is the predicted response; ß0 a constant; ßi the
linear coefficient; ßii the squared coefficient; and ßij
the cross-product coefficient, k is number of factors.
The second order polynomial coefficients were cal-
culated using the software package Design Expert
Version 6.0.10 to estimate the responses of the de-
pendent variable. Response surface plots were also
obtained using Design Expert Version 6.0.10.
Effect of amino acids on keratinase production by B. subtilis NCIM 2724
To study the effect of amino acids on keratinase
production, various amino acids including L-cyste-
ine, L-serine, L-valine, L-alanine, L-methionine, L-glu-
tamic acid, L-threonine, L-histidine, L-arginine and L-
lysine were added individually at 0.05 g L-1, 0.10 g L-1
and 0.50 g L-1 in the RSM optimized medium.
Keratinase assay
Keratinase activity was determined by the
method reported by Yu et al. (1968). Chicken
feathers (20 mg) were suspended in 3.8 ml of 100
mM Tris–HCl buffer (pH 7.8), to which 0.2 ml of
the culture filtrate (enzyme source) was added.
The reaction mixture was incubated at 37°C for 1
h. After incubation, the assay mixture was dipped
into the ice cold water for 10 min and the remain-
ing feathers were filtered out by Whatman filter
paper (Whatman® Schleicher and Schuell, Mum-
bai, India). The absorbance of the clear mixture
was measured at 280 nm. The keratinase activity
was expressed as one unit of the enzyme corre-
sponding to an increase in the absorbance value
0.1 (1KU= 0.100 corrected absorbance).
∆Xi
(Xi -Xcp)xi = i= 1,2,3,... k
i=1 i=1 i<ji<j
kkk k
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 59
ReSulTS And dISCuSSIon
Optimization using one-factor-at-a-time
An initial pH of 8.0 supported maximum produc-
tion of keratinase of 3.3 KU ml-1. pH is a significant
factor that influences the physiology of a microor-
ganism by affecting nutrient solubility and uptake,
enzyme activity, cell membrane morphology, by-
product formation and oxidative-reductive reac-
tions. During production of keratinase, keratin utili-
zation occurs more rapidly and to a great extent at
pH 7.5 (Suntornsuk and Suntornsuk, 2003). Friedrich
and Antranikian (1996) described maximum kerati-
nase production at alkaline pH. Alkaline pH favors
keratin degradation at higher pH, probably by modi-
fying the cystine residues to lanthionine and making
it accessible for keratinase action. The optimum pH
reported for keratinase production by B. cereus is 7.0
(Kim et al., 2001), chryseobacterium sp. is 9.0 (Casa-
rin et al., 2008), while that by B. pumilus FH9 is 8.0
(El-Refai et al., 2005). For B. subtilis, highest enzyme
production has been reported over a range of pH
of 7 to 9. It was observed that maximum keratinase
production occurs at alkaline pH.
It was found that ammonium chloride and beef
extract supported maximum keratinase activity of
4.05 KU ml-1 and 4.15 KU ml-1 respectively. These re-
sults are in accordance with the results obtained by
El-Refai et al. (2005), where ammonium chloride and
yeast extract supported maximum keratinase pro-
duction in B. pumilus FH9. Some researchers have
considered feather meal as a nitrogen source for
keratinase production (Thyes et al., 2006).
B. subtilis NCIM 2724 produced keratinase in pres-
ence of chicken feathers as the sole carbon source
which supported a maximum production of 4.14 KU
ml-1. Addition of simple carbon sources reduced the
production of keratinase. A decrease in the keratin-
ase production due to the addition of conventional
carbon sources is reported in literature. Addition of
fructose and maltose in medium decreased the kera-
tinase production in Trichophyton rubrum (Meevoo-
tisom and Niederpruem, 1979) and B. licheniformis
(Sen and Satyanarayana, 1993), respectively. These
results may be due to the catabolic repression of
keratinase (Anbu et al., 2007; Ignatova et al., 1999;
Yamamura et al., 2002; Santos et al., 1996). It has
been reported that chicken feathers act as the best
carbon source for keratinase production.
Statistical media optimization Optimization of fermentative production
by using Orthogonal Array Design
Once the best carbon and nitrogen sources were
selected, the medium was subjected to screening
of the most significant parameters for keratinase
production using the L8 orthogonal array. The re-
sponses for means (larger is better) and for signal
to noise ratios obtained using the L8 orthogonal ar-
ray are shown in Table 2. The last two rows in the
tables show delta values and ranks for the system.
Rank and delta values help in assessing which factors
have the greatest effect on the response character-
istic of interest. Delta measures the size of the effect
by taking the difference between the highest and
lowest characteristic average for a factor. A higher
delta value indicates a greater effect of that compo-
nent. Rank orders the factors from the greatest effect
(on the basis of the delta values) to the least effect
on the response characteristic. The order in which
the individual components affected the fermenta-
tion process were feather > ammonium chloride >
magnesium sulphate > dipotassium hydrogen phos-
phate > sodium chloride > beef extract > potassium
dihydrogen phosphate suggesting that feathers had
a major effect, while K2HPO4 had the least effect on
keratinase production by B. subtilis NCIM 2724.
Optimization by RSM
Based on the L8 orthogonal array design, feather
(A), ammonium chloride (B), magnesium sulphate (C)
and dipotassium hydrogen phosphate (D) were se-
lected for further optimization by RSM. To examine
the combined effect of these medium components
(independent variables) on keratinase production,
a central composite factorial design of 24 =16 plus
6 center points and (2 × 4 = 8) star points lead-
60 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
ing to a total of 30 experiments were performed.
A CCRD matrix of independent variables along
with responses of each experimental trial is given
in Table 3.
The ANOVA of the quadratic regression model
indicated the model to be significant (P < 0.05) (Ta-
ble 4). The P values were used as a tool to check
the significance of each of the coefficients, which,
in turn, are necessary to understand the pattern of
the mutual interactions among the test variables.
The smaller the magnitude of the P, the more sig-
nificant is the corresponding coefficient (Thys et al.,
2006). Among the test variables used in the study,
A (feather), B (ammonium chloride), C (magnesium
sulphate), D (dipotassium hydrogen phosphate), A2
(feather2), B2 (ammonium chloride2) and D2 (dipotas-
sium hydrogen phosphate2) are significant model
terms. Interactions between B (ammonium chlo-
ride) and C (magnesium sulphate); B (ammonium
chloride) and D (dipotassium hydrogen phosphate);
and C (magnesium sulphate) and D (dipotassium
hydrogen phosphate) are also significant. Other in-
teractions were found to be insignificant.
The corresponding second-order response
model found after analysis for the regression was
keratinase (KU ml-1) = 3.66 + 1.09 * feather + 0.51 *
ammonium chloride + 0.72* magnesium sulphate
+ 1.26 * dipotassium hydrogen phosphate + 1.20
* feather2 + 0.36 * ammonium chloride2 + 0.12 *
magnesium sulphate2 + 0.36 * dipotassium hydro-
gen phosphate2 - 0.084 * feather * ammonium chlo-
ride + 0.15 * feather *magnesium sulphate - 0.23 *
feather * dipotassium hydrogen phosphate - 0.24 *
ammonium chloride * magnesium sulphate + 0.68 *
ammonium chloride * dipotassium hydrogen phos-
phate + 0.43 * magnesium sulphate * dipotassium
hydrogen phosphate.
The fit of the model was also expressed by the
coefficient of regression R2, which was found to
be 0.98, indicating that 98.0% of the variability in
keratinase yield could be explained by the model.
Other parameters of ANOVA for response surface
quadratic model were also studied. The ‘Pred R-
Squared’ of 0.92 is in reasonable agreement with
the ‘Adj R-Squared’ of 0.96. ‘Adeq Precision’ mea-
sures the signal to noise ratio.
The special features of the RSM tool, “contour
plot generation” and “point prediction” were also
studied to find optimum value of the combination
of the four media constituents. It was observed
that medium containing (g L-1), feather, 60.0; am-
monium chloride, 1.0; magnesium sulphate, 0.08;
and dipotassium hydrogen phosphate, 0.2 yielded
maximum (10.6 KU ml-1) keratinase.
Accordingly, three-dimensional graphs were gener-
ated for the pair-wise combination of the four factors,
LevelA
FeathersB
Beef ExtractC
MgSO4
DKH2PO4
EK2HPO4
FNaCl
GNH4CL
S/N Mean S/N Mean S/N Mean S/N Mean S/N Mean S/N Mean S/N Mean
1 2.82 2.82 6.70 2.31 7.71 2.60 5.94 2.24 5.14 2.04 6.48 2.47 4.43 1.79
2 9.42 9.42 5.54 2.21 4.54 1.92 6.31 2.29 7.11 2.49 5.77 2.06 7.81 2.74
Delta 6.60 6.60 1.15 0.09 3.17 0.67 0.37 0.04 1.96 0.44 0.70 0.40 3.38 0.95
Rank 1 6 3 7 4 5 2
Table 2. Response table for means and S/N ratio.
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 61
Sr. no.
Feather (g L-1)
NH4CL(g L-1)
MgSO4
(g L-1)K2HPO4
(g L-1)Keratinase a
(KU ml-1)
1 -1 (30) -1 (0.5) -1 (0.04) -1 (0.1) 2.77 ± 0.14
2 1 (60) -1 (0.5) -1 (0.04) -1 (0.1) 5.5 ± 0.11
3 -1 (30) 1 (1.0) -1 (0.04) -1 (0.1) 3.1 ± 0.20
4 1 (60) 1 (1.0) -1 (0.04) -1 (0.1) 5.13 ± 0.13
5 -1 (30) -1 (0.5) 1 (0.08) -1 (0.1) 3.65 ± 0.10
6 1 (60) -1 (0.5) 1 (0.08) -1 (0.1) 6.81 ± 0.18
7 -1 (30) 1 (1.0) 1 (0.08) -1 (0.1) 3.41 ± 0.13
8 1 (60) 1 (1.0) 1 (0.08) -1 (0.1) 5.75 ± 0.17
9 -1 (30) -1 (0.5) -1 (0.04) 1 (0.2) 3.45 ± 0.16
10 1 (60) -1 (0.5) -1 (0.04) 1 (0.2) 4.59 ± 0.14
11 -1 (30) 1 (1.0) -1 (0.04) 1 (0.2) 6.36 ± 0.17
12 1 (60) 1 (1.0) -1 (0.04) 1 (0.2) 7.75 ± 0.23
13 -1 (30) -1 (0.5) 1 (0.08) 1 (0.2) 6.14 ± 0.20
14 1 (60) -1 (0.5) 1 (0.08) 1 (0.2) 8.24 ± 0.18
15 -1 (30) 1 (1.0) 1 (0.08) 1 (0.2) 7.91 ± 0.24
16 1 (60) 1 (1.0) 1 (0.08) 1 (0.2) 9.93 ± 0.13
17 -2 (15) 0 (0.75) 0 (0.06) 0 (0.15) 6.18 ± 0.21
18 2 (75) 0 (0.75) 0 (0.06) 0 (0.15) 10.84 ± 0.8
19 0 (45) -2 (0.25) 0 (0.06) 0 (0.15) 4.15 ± 0.04
20 0 (45) 2 (1.25) 0 (0.06) 0 (0.15) 6.12 ± 0.03
21 0 (45) 0 (0.75) -2 (0.02) 0 (0.15) 3.14 ± 0.20
22 0 (45) 0 (0.75) 2 (0.1) 0 (0.15) 5.21 ± 0.20
23 0 (45) 0 (0.75) 0 (0.06) -2 (0.05) 2.13 ± 0.05
24 0 (45) 0 (0.75) 0 (0.06) 2 (0.25) 8.14 ± 0.17
25 0 (45) 0 (0.75) 0 (0.06) 0 (0.15) 3.55 ± 0.11
26 0 (45) 0 (0.75) 0 (0.06) 0 (0.15) 2.75 ± 0.25
27 0 (45) 0 (0.75) 0 (0.06) 0 (0.15) 4.12 ± 0.04
28 0 (45) 0 (0.75) 0 (0.06) 0 (0.15) 3.86 ± 0.02
29 0 (45) 0 (0.75) 0 (0.06) 0 (0.15) 3.86 ± 0.17
30 0 (45) 0 (0.75) 0 (0.06) 0 (0.15) 3.8 ± 0.20
Table 3. The CCRD matrix of independent variables in coded form and actual values with their corresponding response in terms of production of keratinase by B. subtilis NCIM 2724.
a Results are mean ± SD of three determinations Values in the parenthesis indicate the real values of variables
62 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
Factor a CoefficientEstimate
Sum of squares
Standard Error DF b F value p
Intercept 3.66 139.52 0.18 1 53.27 < 0.0001
A 1.09 28.67 0.088 1 153.24 < 0.0001
B 0.51 6.13 0.088 1 32.77 < 0.0001
C 0.72 12.51 0.088 1 66.89 < 0.0001
D 1.16 38.18 0.088 1 204.07 < 0.0001
A2 1.20 39.46 0.083 1 210.94 < 0.0001
B2 0.36 3.47 0.083 1 18.55 0.0006
C2 0.12 0.37 0.083 1 1.96 0.1815
D2 0.36 3.47 0.083 1 18.55 0.0006
AB -0.084 0.11 0.11 1 0.61 0.4474
AC 0.15 0.34 0.11 1 1.81 0.1981
AD -0.23 0.81 0.11 1 4.35 0.0544
BC -0.24 0.94 0.11 1 5.0 0.0409
BD 0.68 7.38 0.11 1 39.47 < 0.0001
CD 0.43 3.02 0.11 1 16.14 0.0011
Table 4. Analysis of variance (ANOVA) for the experimental results of the central-composite design (Quadratic Model).
a A = Feathers, B = NH4Cl, C = MgSO4, D =K2HPO4b Degree of freedom
Figure 1. Contour plot for keratinase production (-Effect of MgSO4 and NH4Cl).
Figure 2. Contour plot for keratinase production (Effect of K2HPO4 and NH4Cl).
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 63
a A = Feathers, B = NH4Cl, C = MgSO4, D =K2HPO4b Degree of freedom
while keeping the other two at their center point levels.
Graphs for interactions are given here to highlight the
roles played by these factors (Figure 1 and Figure 2).
From the central point of the contour plot the optimal
process parameters were identified. The keratinase yield
(10.6 KU/ml) in the present study is quite high as com-
pared to the literature reports. The maximum keratinase
production reported till date by using most widely used
strain Bacillus subtilis S1 MTCC 2616 is 4.89 KU/ml and
Scopulariopsis brevicaulis MTCC 2170 is 6.2 KU/ml.
The effect of amino acids on keratinase pro-duction by B. subtilis NCIM 2724
Effect of amino acids on production of keratinase is
shown in Figure 3. All of the amino acids examined sup-
ported keratinase production, but the maximum kerati-
nase activity of 12.32 KU ml-1 was observed with 0.5 g L-1
of L-valine. Further increases in L-valine concentration
did not increase keratinase activity (Data not shown).
Addition of amino acids is of considerable impor-
tance in the protease synthesis in terms of metabolic
driving force. Feather keratin is composed of vari-
ous amino acids including glycine, alanine, serine,
cysteine and valine that are extensively cross linked
with hydrogen, disulphide and hydrophobic bonds.
Degraded feathers may act as source of some nutri-
tionally important amino acids and also serves as an
inducer for keratinase production.
ConCluSIon
Statistical nutrient optimization was done to op-
timize keratinase production from B. subtilis NCIM
2724. Taguchi design (L8 orthogonal array) demon-
strated the effect of feather, ammonium chloride,
K2HPO4 and MnSO4 to be significant. Further optimi-
zation of the most significant factors by RSM revealed
complex nutrient interactions among them, and also
increased the production of keratinase by B. subtilis
NCIM 2724 from 3.0 KU ml-1 to 10.6 KU ml-1. All amino
acids supported keratinase production and the maxi-
mum keratinase activity of 12.32 KU ml-1 was observed
with 0.5 g L-1 of L-valine.
Figure 3. Effect of amino acids on keratinase production by B. subtilis NCIM 2724.
64 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
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www.afabjournal.comCopyright © 2011
Agriculture, Food and Analytical Bacteriology
ABSTRACT
Listeria monocytogenes adapts to diverse stress conditions including cold, osmotic, heat, acid, and alkali
stresses encountered during food processing and preservation which is a serious food safety threat. In this
review, we have presented the major findings on this bacterium’s stress response proteomes to date along
with the different approaches used for its proteomic analysis. The key proteome findings on cold, heat
shock, salt, acid, alkaline and HHP stresses illustrate that the cellular stress responses in this organism are
a culmination of multiple protein expression changes in response to a particular stress stimuli. Moreover,
a number of key proteins may be involved in conferring the cross protective effects against various stress
environments. As an example, ferritin-like protein (designated as Fri or Flp) is induced during cold, heat,
and HHP stresses. Similarly, general stress protein Ctc is induced in cold and osmotic stresses while mo-
lecular chaperones such as GroEL and DnaK are induced in cold and heat stresses. Furthermore, a number
of stress proteins also contribute towards L. monocytogenes virulence and pathogenicity. Future research
may lead to understanding the stress proteomes of this pathogen induced on various food matrices and
processing environments in which it can persist for long periods of time.
Keywords: Listeria monocytogenes, proteome, cold stress, osmotic stress, heat stress, acid stress, alkali stress.
InTRoduCTIon
Listeria monocytogenes is an important food-
borne pathogen with significant public health threats
and economic impacts on the food industry. It causes
Received: November 22, 2010, Accepted: April 9, 2011. Released Online Advance Publication: April 1, 2011. Correspondence: Ramakrishna Nannapaneni,
[email protected]: +1 -662-325-7697 Fax: +1-662-325-8728
“listeriosis” in humans, which is associated with a
variety of symptoms ranging from flu-like illness to
severe life threatening meningitis as well as high
mortality (Lennon et al., 1984). Epidemiological stud-
ies estimate that listeriosis to be responsible for ap-
proximately 19 % of food-related deaths in the Unit-
ed States annually (Scallan et al., 2011). Suspected
L. monocytogenes contamination is also among the
leading causes of food recalls resulting in significant
REVIEWAn Overview of Stress Response Proteomes in Listeria monocytogenes
K. A. Soni1, R. Nannapaneni1*, and T. Tasara2
1Department of Food Science, Nutrition and Health Promotion, Mississippi State University, Mississippi State, MS 39762, USA
2Institute for Food Safety and Hygiene, Vetsuisse Faculty University of Zurich, Zurich, Switzerland
Agric. Food Anal. Bacteriol. 1: 66-85, 2011
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 67
financial losses to the food industry due to the “zero
tolerance” standard adopted for the ready-to-eat
food products in the USA (Kramer et al., 2005; Mars-
den, 2001; Teratanavat and Hooker, 2004).
The prevalence of L. monocytogenes is mainly
due to its wide-spread distribution and its ability to
withstand adverse environmental conditions. This
includes the ability of this pathogen to survive and
grow at low temperatures, and resistance to high
osmolarity, acidic and alkaline environments. Cold
adaptation of this organism is of growing concern
due to the changing life styles over the years that
have increased the consumption of refrigerated
and minimally processed food products. Besides
cold storage, elevated salt concentrations are an al-
ternative means of food preservation, but L. mono-
cytogenes is also highly salt tolerant and has been
documented to grow in the presence of as high
as 10% NaCl (McClure et al., 1991). Jensen et al.
(2007) recently showed that L. monocytogenes cells
can display increased aggregation and biofilm for-
mation when exposed to NaCl stress. Additionally
this bacterium exhibits acid tolerance responses
(ATR), which significantly increases its resistance to
a subsequent lethal acid (pH 3.0-3.5) stress expo-
sure after an initial encounter with the non-lethal
acidic (pH 5.0-5.5) conditions. As an example, 4-log
higher survival was observed in L. monocytogenes
cells exposed to acid stress at pH 3.5 for 6 h after
an initial 90 minute exposure to a mild acidic condi-
tion at pH 5.5 (Koutsoumanis et al., 2004). Similarly,
L. monocytogenes may also acquire an increased
alkaline stress tolerance subsequent to sublethal al-
kaline stress exposure (Mendonca et al., 1994). Dur-
ing food processing and preservation, L. monocyto-
genes cells may become exposed to multiple forms
of sublethal stresses, leading to “stress hardening”.
Consequently, L. monocytogenes exposure to mild
forms of particular stresses may inadvertently in-
duce cross protection against subsequent expo-
sures to lethal levels of other unrelated stresses. For
example, it has been shown that acid (pH 4.5 for 1
h) or cold (10°C for 4 h) stressed L. monocytogenes
LO28 (serotype 1/2c) cells tend to be more resistant
to high hydrostatic pressure (HHP) in comparison
to the non-stress adapted cells (Wemekamp-Kam-
phuis et al., 2002). Lou and Yousef (1997) reported
that the heat stress of L. monocytogenes results
in cell-hardening and subsequent osmoprotection
and higher resistance of these cells to ethanol treat-
ment. Likewise, L. monocytogenes cells were also
found to be more thermotolerant after a combined
acid and heat shock or after osmotic and heat shock
treatments (Skandamis et al., 2008).
Stress adaptation events in L. monocytogenes, as
in other microorganisms, includes coordinated in-
duction of different stress protection systems within
the affected cells. Proteomics and transcriptomics
are both invaluable tools in delineation of the dif-
ferent mechanisms of stress response in microbes.
Transcriptome analysis technologies while impor-
tant in deciphering the global mRNA expression
changes during stress responses, fail to capture all
aspects of these molecular responses since mRNA
transcripts changes may not directly correlate with
protein expression due to the fact that transcripts
produced in abundance may be rapidly degraded,
translated poorly, or influenced through post-trans-
lational modifications. Therefore complementa-
tion of the transcriptome based analysis of stress
responses with the proteome studies is important
to get a clearer picture as proteins are the key func-
tional units involved in physiological stress respons-
es. As a result of new developments in microbial
cell global protein profiling based on the protein
identification approaches and bioinformatics, re-
searchers are now also able to monitor and deter-
mine the importance of stress induced proteins in
L. monocytogenes during its adaptation to diverse
conditions. A number of proteome profiling studies
performed on this organism so far have already pro-
vided extensive preliminary insights into gene and
protein expression changes that are associated with
the environmental stress adaptation in this bacte-
rium. The purpose of this review is to discuss the
significant developments in proteomic analysis of
the stress-adaptation in L. monocytogenes with fo-
cus on cold, heat, osmotic, acid, alkaline, and HHP
adaptation along with cross linking between stress
proteins and virulence.
68 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
PRoTeoMIC TeChnologIeS APPlIed In L. monocytogenes AnAlySIS
The summary of different gel-based and non-gel
based techniques and their assay principles are dis-
cussed in-depth in recent review articles by Haynes
et al. (2007) and Nesatyy and Suter (2007). For L.
monocytogenes, the most commonly applied pro-
tocols to date have used two-dimensional gel elec-
trophoresis (2DE) for protein separation (Folio et al.,
2004; Mujahid et al., 2007; Ramnath et al., 2003; Scha-
umburg et al., 2004). The majority of the L. mono-
cytogenes stress proteome studies utilized soluble
cellular proteins (excluding the extracellular fraction)
that were fractionated using mechanical disruption
alone or protocols combining mechanical disrup-
tion and enzymatic lysis. In the earlier studies most of
the proteins were not identified (; Bayles et al., 1996;
Phan-Thanh and Gormon, 1995), although in later
studies a significant number of the 2DE separated
proteins were identified by mass spectrophotometry
(MS) (Abram et al., 2008b; Dumas et al., 2008; Folio
et al., 2004; Mujahid et al., 2007; 2008; Phan-Thanh
and Jansch, 2006; Schaumburg et al., 2004 ). More re-
cently however, non-gel based approaches that com-
bine liquid chromatography (LC) separation and MS
(LC-MS/MS) are increasingly used. The fractionated
complex bacterial protein mixtures are digested into
peptides, separated by liquid chromatography and
analyzed in MS, taking advantage of the advances in
bioinformatics to identify even larger numbers of the
fractionated proteins (Abram et al., 2008b; Calvo et
al., 2005; Trost et al., 2005).
The majority of studies that have compared pro-
tein expression between normal versus stress ex-
posed L. monocytogenes cells using 2DE gel-based
protein separation with or without subsequent appli-
cation of MS to identify separated proteins (Bayles et
al., 1996; Duche et al., 2002a; Esvan et al., 2000; Phan-
Thanh and Gormon, 1995; 1997; Phan-Thanh and
Mahouin, 1999; Wemekamp-Kamphuis et al., 2004a).
A 2DE reference map covering an estimated 28.8%
of potential gene products was generated from the
soluble subproteome of L. monocytogenes EGDe
serotype 1/2a strain (Folio et al., 2004). Ramnath et
al. (2003) also used this approach and detected two
proteins found in L. monocytogenes EGDe but were
absent in some food isolates. The identification of
these proteins revealed they were involved in glyco-
lytic pathway and metabolism of coenzymes, but the
relevance of their differential expression specifically
in such food isolates remains unknown. The draw-
backs of gel based 2DE proteomics include poor
reproducibility in separation of highly basic or hydro-
phobic proteins, gel-to-gel variations and poor reso-
lution of high molecular weight protein complexes.
Attempts to overcome these drawbacks include the
recent use of 2D-DIGE (Two dimensional-difference
gel electrophoresis) based proteomics analysis. By
using different fluorescent dyes such as Cy2, Cy3
or Cy5 for protein labeling, such approaches allow
protein mixtures of different origins to be analyzed
within the same gel run. Thus these approaches are
more amenable to stress proteome response studies
where protein expression patterns of stress-adapted
cells and control samples can be directly compared
within the same gel run to minimize the influence of
gel-to-gel variations. Folsom and Frank (2007) used
a 2DE-DIGE based proteomics approach to analyze
protein expression changes associated with chlorine
resistance and biofilm formation in a hypochlorous
acid tolerant variant of the L. monocytogenes Scott
A (4b) strain. They found 19 proteins that were dif-
ferentially expressed between planktonic and biofilm
cells of a hypochlorous acid tolerant cultural variant
of this strain (Folsom and Frank, 2007). Six of these
differentially expressed proteins were subsequently
identified by peptide-mass mapping. They included
three ribosomal proteins (L7, L10 and L12), perox-
ide resistance protein (Dpr/Flp/Fri), sugar-binding
protein (Lmo0181), and a putative protein Lmo1888
of yet unknown function. This study also revealed
that peroxide stress resistance proteins Fri that is in-
volved in multitude of other stresses was expressed
2.2-fold times higher in biofilm than in planktonic
cells. At phenotypic level it was observed that L.
monocytogenes cells present in biofilm mass were
more resistant to sanitization treatments compared
to planktonic cells (Pan et al., 2006). Although not yet
widely adapted for L. monocytogenes analysis, LC
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based techniques seem capable of detecting even
higher numbers of proteins compared to tradition-
al 2DE gel-based techniques. As an example when
the same protein fraction of cell free supernatant
(extracellular) of L. monocytogenes EGDe was ana-
lyzed, 105 proteins were identified using LC-MS/MS
compared to 58 detected by 2DE (Trost et al., 2005).
Forty-five of the detected proteins were found to be
common between the two methods. The analysis of
differential protein expression between L. monocyto-
genes 10403S and its σB null mutant strain using the
LC-MS/MS with iTRAQ (isotope tag for relative and
absolute quantification) identified 35 σB regulated
proteins, whereas the 2DE approach only managed
to detect 13 proteins. Four proteins were common
between the two methods (Abram et al., 2008b). A
combination of SDS-PAGE and LC-MS/MS detected
301 membrane associated proteins of L. monocy-
togenes EGDe (Wehmhoner et al., 2005). This was
greater than 79 proteins detected using the 2DE ap-
proach by Mujahid et al. (2007). One possible reason
for increased protein detection with SDS-PAGE/LC-
MS/MS might be increased protein solubilization in
the SDS-PAGE sample buffer in comparison to the
urea based sample buffer applied in the 2DE-MS ap-
proach (Haynes and Roberts, 2007).
In another example LC-LC-MS/MS combination
also called the multidimensional protein identifica-
tion technique (MudPIT), has also been used for pro-
teome analysis of L. monocytogenes cells. Fifteen
proteins that covalently bound the LPXTG motif were
identified in the subproteome fraction of cell wall as-
sociated proteins of L. monocytogenes strain EGDe
(Calvo et al., 2005). The SrtA and SrtB enzymes an-
chor surface proteins to the cell wall. Surface proteins
recognized by these two sortases were also analyzed
using LC-LC-MS/MS in the EGDe strain. A total of
13 and 2 LPXTG-containing proteins were identi-
fied in srtA and srtB null mutant strains (Pucciarelli
et al., 2005). Recently, MudPIT was used to study the
differences that exist between serotype 1/2a (strain
EGD) and 4b (strain F2365) (Donaldson et al., 2009).
In total, 1754 EGD proteins and 1427 F2365 proteins
were detected representing 50-60% of total Liste-
ria proteome coverage. In total 1077 proteins were
common to both serotypes and of these 413 proteins
displayed significantly differential expression level
between the two serotypes.
PRoTeoMe AnAlySIS In STReSS-AdAPTed L. monocytogenes CellS
The ability of L. monocytogenes to sense and re-
spond to a particular stress factor has implications
for both survival and virulence properties of this bac-
terium. Stress exposure elicits various fundamental
changes in this organism’s cellular physiology. These
changes are mediated via multiple and specific
changes in gene and protein expression profiles in
cells. Proteins associated with cold, heat, osmotic,
acid, and high hydrostatic pressure stress adaptation
will be discussed in the following sections.
Cold stress adaptation
The growth of L. monocytogenes on cold pre-
served food products is one of its important food
safety challenges. In addition to decreased meta-
bolic capacity, cold stress exposed microorgan-
isms are faced with a wide range of structural and
functional impediments in membrane structures,
nucleic acids (DNA and RNA), and macromolecular
assemblies such as ribosomes (Schumann, 2009).
The putative integral membrane protein PgpH,
whose deletion leads to impaired cold growth, has
been proposed as a possible cold sensing factor in
L. monocytogenes (Liu et al., 2006). Based on the
proposed model, environmental cold stress sensed
through membrane bound PgpH proteins is con-
veyed intracellularly through homeodomain depen-
dent signaling pathways.
Using 2DE gel-based proteome analysis, initial
studies revealed modulation in expression of be-
tween 10 to 38 proteins in association with cold stress
adaptation of this organism (Bayles et al., 1996; He-
braud and Guzzo, 2000; Phan-Thanh and Gormon,
1995). Of these differentially expressed proteins vi-
sualized, the predominating cold shock protein was
subsequently identified through microsequencing as
ferritin (Fri) (designated as Flp or Fri) (Hebraud and
70 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
Guzzo, 2000). The role of this protein in cold adap-
tation was also later phenotypically confirmed when
Dussurget et al. (2005) created a fri null mutant strain
in L. monocytogenes EGDe, which exhibited a cold
sensitive phenotype. Although physiological and
cold adaptation roles of Fri are not yet well under-
stood, it is hypothesized that it might facilitate alle-
viation of oxidative stress environments developing
in cold stress exposed L. monocytogenes cells (Liu
et al., 2002; Tasara and Stephan, 2006). Wemekamp-
Kamphuis et al. (2002) described four approximately
7 kDa protein that were cold inducible in L. mono-
cytogenes LO28 as determined by using a combi-
nation of 2DE gel electrophoresis and immunoblot-
ting. These proteins designated Csp1-Csp4, were
described as the L. monocytogenes cold shock fam-
ily proteins based on their cross reactivity with anti-B.
subtilis CspB polyclonal antibodies. Although their
identity as such was not confirmed by peptide mass
fingerprinting (PMF) in this work, genomic informa-
tion show that L. monocytogenes harbors three pro-
teins of the cold shock domain protein family (Glaser
et al., 2001). Two of these L. monocytogenes Csp
proteins, CspL (CspA) and CspD have now been
confirmed to be functionally vital for efficient cold
growth in this bacterium (Schmid et al., 2009). CspA
and CspD proteins, based on knowledge from oth-
er microorganisms, are also presumed to facilitate
cold growth possibly through nucleic acid (DNA and
RNA) chaperone-like functions (Horn et al., 2007).
This facilitates DNA replication and gene expression
events that may otherwise be hampered through
secondary structures that tend to form in bacterial
cells at low temperatures.
Meanwhile, a more comprehensive cold adapta-
tion proteome analysis in this bacterium has been
recently described. Cacace et al. (2010) performed
detailed proteome analysis on L. monocytogenes
cells grown for 13 days at 4°C with subsequent MAL-
DI (Matrix-assisted laser desorption/ionization) anal-
ysis. Proteome analysis revealed that 57 proteins in
total were over-expressed and eight were repressed
in cold grown cells compared to cells cultivated at
37°C. Proteome changes detected in this study in-
dicated the increased synthesis of proteins linked to
energy production, oxidative stress resistance, nutri-
ent uptake, lipid synthesis, and protein synthesis and
folding. Cold stress adaptation proteins identified
by this study that are of particular interest include:
OppA, Ctc, GroEL and DnaK. The OppA protein,
which facilitates accumulation of short peptide sub-
strates, is important for efficient cold growth in this
bacterium and at phenotypic level oppA null mutant
of this bacterium was unable to grow at low tem-
perature (5°C) (Borezee et al., 2000). Ctc is a gen-
eral stress protein which has been found to promote
the adaptation of L. monocytogenes cells to high
osmolarity conditions (Gardan et al., 2003b). The
GroEL and DnaK proteins are molecular chaperones
that promote proteins refolding and degradation of
stress damaged proteins that accumulate under dif-
ferent suboptimal conditions including heat stress
(Sokolovic et al., 1990). The cold growth associated
induction of the Ctc, GroEL and DnaK proteins, which
have been previously associated with adaptation to
other stresses (i.e. Ctc for cold and osmotic stress
and GroEL-DnaK for cold and heat stress) conditions
may thus indicate commonality of some stress adap-
tive responses in this bacterium (Cacace et al., 2010;
Duche et al., 2002a,b; Gardan et al., 2003b; Soko-
lovic et al., 1990).
The accumulation of compatible solutes espe-
cially glycine, betaine, and carnitine also promotes
cold growth in various bacteria including L. mono-
cytogenes (Mendum and Smith, 2002; Smith, 1996;
Wemekamp-Kamphuis et al., 2004b). There are no
enzymatic systems for the de novo synthesis of main
cryoprotective compatible solutes glycine, betaine,
and carnitine in L. monocytogenes, but transport
systems (Gbu, BetL and OpuC) that accumulate
them from environmental sources are present, and
deletion of genes coding for these transporters has
confirmed that they facilitate efficient cold growth of
this bacterium (Angelidis et al., 2002; Ko and Smith,
1999; Sleator et al., 1999). Analysis of cold-sensitive
mutants in which Lmo1078 (Chassaing and Auvray,
2007), and LtrC (Chan et al., 2007) proteins are in-
activated also indicates that these proteins func-
tionally contribute to cold adaptation processes in
L. monocytogenes. The Lmo1078 protein is a UDP-
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 71
glucose pyrophosphorylase proposed to promote
cold adaptation through enhanced UDP-glucose
production at low temperatures. UDP glucose is an
essential substrate in lipoteichoic acid production
and might facilitate maintenance of architectural in-
tegrity in cell wall and membrane structures leading
to protection of bacterial cells from cold stress dam-
age (Chassaing and Auvray, 2007).
Heat stress adaptation
The understanding of heat stress adaptation in
food-borne pathogens is an important issue since
heating constitutes one of the major food process-
ing and preservation methods. The heat shock re-
sponse is one of the most universal and extensively
studied physical stress responses in living organisms.
This process involves increased production of vari-
ous cell protective protein systems, which ultimately
promotes general environmental stress resistance
and enhanced thermal tolerance (Gandhi and Chi-
kindas, 2007; Klinkert and Narberhaus, 2009; Muga
and Moro, 2008; van der Veen et al., 2007). Similar
to other bacteria, L. monocytogenes synthesizes
a highly conserved set of proteins, also defined as
heat shock proteins (Hsps), upon exposure to high
temperatures (>45°C). Hsps include highly con-
served molecular chaperones and proteases that
functionally prevent nonproductive protein aggre-
gations under different stress environments. GroEL
and DnaK are major Hsps that promote refolding
and degradation of damaged proteins through ATP-
dependent mechanisms (Kandror et al., 1994; Sher-
man and Goldberg, 1996; van der Veen et al., 2007).
These two proteins are highly conserved among liv-
ing organisms and also constitute as the main Hsp
chaperones observed in L. monocytogenes (Gahan
et al., 2001; Hanawa et al., 2000)
Using proteome analysis, the induction of up to
15 Hsps in response to heat shock (48°C/30 min) was
observed using SDS-PAGE (Sokolovic et al., 1990). Of
these, two Hsps were identified as GroEL and DnaK
in L. monocytogenes CLIP 54149 (serotype 1/2a)
based on immunological detection. In another study
the induction of as many as 32 Hsps was observed
using preparative 2DE gels of L. monocytogenes
EGD in response to a temperature shock of 49°C/15
min (Phan-Thanh and Gormon, 1995). One identified
predominant protein, Fri, with molecular weight 18
kDa and pI of 5.1 showed 50.6-fold inductions due
to heat shock. This very same protein spot was 10.5-
fold induced in response to cold shock (Phan-Thanh
and Gormon, 1995). Similarly, other researchers have
also observed the transcriptional induction of fri
transcripts in response to heat (Hebraud and Guz-
zo, 2000; van der Veen et al., 2007) and cold stress
(Dussurget et al., 2005). Phenotypically fri gene null L.
monocytogenes EGDe cells also failed to reach the
maximal optical density compared to the wild type
strain during growth under heat at 45°C (Dussur-
get et al., 2005). These findings together suggest
that ferritin-like protein is important for high and
low temperature adaptation in L. monocytogenes.
Recently, Agoston et al. (2009) compared the effect
of mild and prolonged heat treatments on L. mono-
cytogenes cells using 2DE analysis. In line with the
reduced metabolic activity at suboptimal tempera-
ture, large numbers of metabolic proteins were sup-
pressed during heat exposure in this study which is
also consistent with the observation from other stud-
ies (Phan-Thanh and Gormon, 1995; Phan-Thanh and
Jansch, 2006). Importantly, L. monocytogenes stress
protein DnaN, a beta subunit of polymerase III, was
highly induced in response to different heat shock
treatments. Observed induced expression of DnaN,
involved in DNA synthesis process, may indicate its
role in increased synthesis of some HSPs.
Osmotic stress adaptation
The osmotolerance of L. monocytogenes is an-
other property crucial to survival and growth of this
pathogen at high salt levels and low water activity
environments encountered in conserved food prod-
ucts. Osmotic stress adaptation in microorganisms
depends on the modulation of both ionic and or-
ganic solute pools so as to sustain cytoplasmic water
and turgor pressure at levels, which are compatible
with cell viability and growth at low water activity
(Booth and Louis, 1999; Wood, 2007).
72 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
L. monocytogenes cells cope with elevated levels
of osmolarity through appropriate changes in pro-
tein expression levels. Significant modulation (ap-
proximately 32 proteins) in protein expression under
hyper osmotic conditions (3.5% to 6.5% NaCl con-
centration) was first visualized in preparative 2DE
gels (Esvan et al., 2000) and some of the salt stress
adaptation proteins were later identified (Duche
et al., 2002a,b). Identified osmotic stress proteins
include those related to general stress (Ctc and
DnaK), transporters (GbuA and AppA), ribosomal
proteins (RpsF, 30S ribosomal protein S6), as well as
proteins involved in general metabolism processes
(Ald, CcpA, CysK, TufA (EF-Tu), Gap, GuaB, PdhA,
PdhD, and Pgm) (Duche et al., 2002a,b). Among the
salt stress induced proteins, the role of Ctc in osmo-
tolerance was further characterized by Gardan et al.
(2003b), who demonstrated that ctc gene is involved
in L. monocytogenes osmotolerance. They found
that growth of the ctc mutant strain was signifi-
cantly impaired compared to its isogenic wild type
L. monocytogenes LO28 strain in minimal medium
with 3.5% NaCl.
Other than the differential expression of salt stress
proteins, increased uptake of glycine betaine and
carnitine osmolytes via betL, gbu, and opuC en-
coded transporter porters is crucial under hyper-os-
motic conditions. Accumulation of these osmolytes
prevents the intracellular water loss by counteracting
external osmolarity and keeping the macromolecu-
lar structure of the cells intact. Indeed, the induced
expression (>2-fold) of GbuA transporter protein un-
der high osmolarity (3.5% NaCl) has been observed
in 2DE analysis of L. monocytogenes LO28 (Duche
et al., 2002a). Meanwhile the induction of compat-
ible solute transporter encoding genes, betL, gbu,
and opuC in response to higher osmolarity has been
reported at the transcriptional level in L. monocyto-
genes cells (Fraser et al., 2003). Interestingly these
transporter systems expressed under hyper-osmotic
stress conditions are the same as the ones expressed
under cold stress (Mendum and Smith, 2002; Smith,
1996; Wemekamp-Kamphuis et al., 2004b), suggest-
ing that some of the mechanisms counteracting os-
motic and cold stress may be common in L. mono-
cytogenes. Moreover, the cold shock protein CspD
also facilitates both osmotic and cold stress adapta-
tion in L. monocytogenes and a mutant strain lack-
ing cspD gene also display a stress sensitive phe-
notype under NaCl salt stress conditions (Schmid et
al., 2009). Other important proteins in L. monocyto-
genes salt stress adaptation are HtrA (Wonderling
et al., 2004) and Lmo 1078 (Chassaing and Auvray,
2007). The HtrA protein is a general stress response
serine protease that contributes to osmotic stress
adaptation functions through its role in degradation
of salt stress damaged proteins. At the phenotypic
level the L. monocytogenes htrA null mutant displays
diminished growth in presence of NaCl stress. The
Lmo1078 promotes both cold and osmotic tolerance
based on its proposed functional contribution to
maintenance of cell wall and membrane architectur-
al integrity in this bacterium. The CstR transcriptional
repressor protein is also involved in modulation of L.
monocytogenes osmotic stress tolerance functions
since a CstR null mutant of this bacterium displays
improved growth under NaCl salt stress conditions
(Nair et al., 2000b).
Acid stress adaptation
The adaptation of microorganisms to acid stress
environments includes significant gene and protein
expression changes associated with, among other
response, the mobilization of cellular mechanisms
that consume acids and generate basic amines
(Foster, 2004; Merrell and Camilli, 2002). L. monocy-
togenes cells face acid stress conditions in low pH
foods and at various stages during human infection.
L. monocytogenes counteracts acidic stress condi-
tions by production of various acid stress response
proteins (ASPs). ASPs were initially designated based
on their location on the preparative 2DE gels (Davis
et al., 1996; O’Driscoll et al., 1997) and some were
later identified by PMF (Phan-Thanh and Mahouin,
1999; Wemekamp-Kamphuis et al., 2004a). Some
of the identified ASPs include: proteins involved in
respiration (enzyme dehydrogenases and reductas-
es), osmolyte transport (GbuA), protein folding and
repair (Chapronin, GroEL, ClpP), general stress re-
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 73
sponse (sigma H homologous of B. subtilis), flagella
synthesis (FlaA), and metabolism (Pfk, GalE) (Phan-
Thanh and Mahouin, 1999; Wemekamp-Kamphuis
et al., 2004a).
The acid tolerance response (ATR) is character-
ized by increased microbial cell resistance to le-
thal acid after an exposure to mild acidic condition
(Koutsoumanis et al., 2004). This phenomenon has
been examined by a number of studies in L. mono-
cytogenes cells. When the synthesis of ASPs in L.
monocytogenes LO28 produced under both mild
(pH 5.5 for 2 h) and lethal (pH 3.5 for 15 min) acidic
conditions were compared to a normal pH (~7.2), a
total of 37 proteins were induced under mild acidic
treatment and 47 under lethal acidic treatment, with
23 of the induced proteins being common under
both conditions (Phan-Thanh and Mahouin, 1999).
The different aspects of acid stress adaptive mecha-
nisms in L. monocytogenes are well elucidated from
acid stress adaptation mechanisms studies in this
bacterium (Abram et al., 2008a; Ferreira et al., 2003;
Phan-Thanh and Jansch, 2006; Ryan et al., 2008b ). In
brief, when exposed to a lower external pH, bacte-
rial cells attempt to maintain their cytoplasmic pH by
decreasing the membrane permeability to protons,
buffering their cytoplasm, and by equilibrating the
external pH through catabolism (Phan-Thanh and
Jansch, 2006). One of the ways that limit the bac-
terial permeability to proton is through changes in
the lipid bilayer of cell membrane. Giotis et al. (2007)
suggested that there was an increased concentration
of straight chain fatty acids and decreased concen-
tration of branched chain fatty acids in L. monocy-
togenes 10403S cells grown under acidic conditions
(pH 5.0 to 6.0) compared to neutral pH. Another im-
portant approach that the bacterial cells use for dis-
pelling the protons outside the cells is to accelerate
electron transferring reactions through enhanced
oxidation reduction potential. The ASPs identified
as dehydrogenases (GuaB, PduQ and lmo0560) and
reductases (YcgT) together with respiratory enzymes
are implicated to play an important role in main-
taining pH homeostasis by active proton transport
(Phan-Thanh and Jansch, 2006).
Organic acid salts such as sodium lactate and
sodium diacetate are extensively used in ready-to-
eat (RTE) meat products as antiListerial food preser-
vatives. Recently Mbandi et al. (2007) used 2DE to
evaluate the protein induction in L. monocytogenes
Scott A by these organic salts. Experiments were
conducted in defined medium with either sodium
lactate (2.5%) or sodium diacetate (0.2%) or in com-
bination. Some of the proteins that showed substan-
tial up or down regulation (>10 fold) were identified
using PMF. Oxidoreductase and lipoproteins were
upregulated whereas DNA-binding proteins, alpha
amylase and SecA were repressed during exposure
to these organic acid salts. Identified enzyme protein
oxidoreductase in L. monocytogenes has been pre-
viously suggested to be involved in dispelling proton
molecules to maintain cell homeostasis (Phan-Thanh
and Jansch, 2006).
The glutamate decarboxylase (GAD) and arginine
deiminase (ADI) are well described major acid adap-
tive mechanisms in L. monocytogenes. L. monocyto-
genes LO28 strain with a mutation in genes of GAD
proteins GadA, GadB and GadC displayed higher
acid stress sensitivity in an acidified reconstituted
skim milk background (Cotter et al., 2001b) and gas-
tric fluid (Cotter et al., 2001a). The L. monocytogenes
ADI system includes proteins ArcA, ArcB and ArcC
and ArcD for the conversion and transfer of arginine
into ornithine and deletion in functional genes of
ADI leads impaired growth in mildly acidic condi-
tions (pH 4.8) and survival in lethal pH conditions (pH
3.5) (Ryan et al., 2009).
Alkaline stress adaptation
L. monocytogenes cells are more resistant to
alkaline stress in comparison to other foodborne
pathogens such as Salmonella Enteritidis and E. coli
O157:H7 (Mendonca et al., 1994). At pH 12, L. mono-
cytogenes F5069 (serotype 4b) cell concentrations
decreased by only 1-log in 10 min compared to 8-log
reductions observed for E. coli and S. Enteritidis
within 15 s. Earlier, 2DE analysis of alkaline stressed
(pH 10.0 for 35 min) L. monocytogenes EGDe cells by
Phan-Thanh and Gormon (1997) showed induction
of 16 proteins, synthesis of 11 novel proteins, and
74 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
repression of nearly half of the total proteins in com-
parison to non-stressed cells. Recently, Giotis et al.
(2008) also reported the repression of a large number
of proteins along with synthesis of 8 novel proteins
in response to alkaline stress of L. monocytogenes
10403S strain. In addition to proteomic analysis, they
also evaluated the alkaline stress adaptive mecha-
nism using microarray transcriptional profiling and
found 390 gene transcripts differentially expressed
(Giotis et al., 2008). Protein identification of four dif-
ferentially expressed proteins by peptide-mass map-
ping revealed induction of heat shock proteins DnaK
and GroEL and repression of DdlA (alanine ligase)
and AtpD (ATP synthase). These identified proteins
spots were also found to be induced or repressed in
microarray analysis. In addition, screening library of
Tn917- lac insertional mutants in L. monocytogenes
LO28 identified 12 mutants sensitive to alkaline con-
ditions, though identification of transposition target
suggest they all carried mutations in only putative
transporter genes (Gardan et al., 2003a).
High hydrostatic pressure (HHP) stress adaptation
L. monocytogenes cells undergo mechanical
stress following HHP treatment. The usual pressure
range employed in HHP is in the range of 200-600
MPa for 5-10 min depending on the food matrices.
Such high pressure damages the cell membrane and
results in leakage of cell content along with disso-
ciation of protein complexes (Gross and Jaenicke,
1994). However, HHP treated L. monocytogenes
cells were found to be sublethally injured with their
metabolic-activity largely maintained and had the
potential for a gradual recovery (Ritz et al., 2006). In
addition although L. monocytogenes cells in HHP
treated cooked ham displayed a lag phase lasting
up to 1.5 months, they subsequently recovered to
grow more than 5-logs over 3 months (Aymerich et
al., 2005).
To characterize the HHP induced proteins en-
abling resistance to mechanical stress, Jofre et al.
(2007) conducted 2DE analysis of L. monocytogenes
CTC1011 (serotype 1/2c) after treatment with 400
MPa for 2 h and observed expression of 23 proteins
being modulated. These high pressure induced pro-
teins were related to ribosomal function (RplJ, RplL,
RpsF, RpsB, IleS, GatA), transcription (GreA), protein
degradation (PepF, PepT), protein folding (GroES),
metabolism (PflB, Pta, Zwf, Ald,), general stress (Fri)
and unknown functions. Of these high pressure in-
duced proteins, chaperone GroES may be necessary
in refolding of dissociated protein complexes follow-
ing HHP treatment, and peptidases (PepF, PepT) may
contribute to degradation of proteins that cannot be
folded by molecular chaperones. Flp has been pre-
viously elucidated to have roles in cold, heat, and
oxidative stress adaptation (Dussurget et al., 2005).
Moreover, L. monocytogenes shows increased resis-
tant to HHP treatment following prior exposure to
cold stress along with induced expression of cold
shock proteins following HHP treatment.
Implications of L. monocytogenes stress adaptation to virulence responses
The stress responses of L. monocytogenes are
not only important in survival of hostile external
and food-associated environments but also dur-
ing host colonization processes. The pathogenicity
of food-borne L. monocytogenes also depends on
their physiological status at infection, which is deter-
mined by, among other factors, the environmental
stress challenges encountered and stress responses
activated prior to interaction with susceptible hosts.
Besides the fact that acid stress adaptation of this
bacterium promotes survival in acidic food environ-
ments, this process has been also shown to modu-
late various aspects of virulence in this pathogen. As
an example, the pathogenic potential of this bacte-
rium can be increased through improved viability in
the gastrointestinal tract, which includes increased
survival of the gastric acid stress challenges. The
increased expression of virulence genes as well as
enhanced cell adhesion and invasion has been re-
ported in association with acid stress adaptation of
L. monocytogenes cells (Conte et al., 2000; Garner et
al., 2006; Olsen et al., 2005; Werbrouck et al., 2009).
Conte et al. (2000) detected enhanced Caco-2 cell
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 75
invasion ability, in addition to improved survival and
proliferation in activated murine macrophages of L.
monocytogenes cells preadapted by mild organic
acid stress exposure. Werbrouck et al. (2009) also
described increased cellular invasiveness and inlA
mRNA levels in their analysis of acid stress adapted
L. monocytogenes cells. In similar fashion there was
an increased transcription of virulence genes such as
prfA, inlA and inlB, as well as enhanced adhesion and
invasion of Caco-2 cells in two L. monocytogenes
strains adapted to prolonged acid stress (Olesen et
al., 2009). Another stress commonly encountered by
L. monocytogenes cells in food associated environ-
ments considered to potentially influence virulence
of this bacterium is NaCl osmotic stress. NaCl stress
exposure is associated with increased expression of
various general stress resistance and virulence genes
in this bacterium suggesting that osmotic stress ad-
aptation events along the food supply chain may
enhance subsequent pathogenicity (Kazmierczak et
al., 2003; Olesen et al., 2009; Sue et al., 2004). Phe-
notypically increased cell adhesion and invasion in
vitro has been observed in NaCl stress adapted L.
monocytogenes cells (Garner et al., 2006; Olesen
et al., 2009). The significance of these phenotypic
observations however remains to be further exam-
ined. One study, which examined the growth of
some food environment persistent strains and clini-
cal isolates under NaCl osmotic stress, was not able
to detect significant influence of this stress exposure
on pathogenicity of these strains using several viru-
lence models (Jensen et al., 2008). Similarly, Wałecka
et al., (2011) did not find increased expression of in-
ternalins with salt stress and suggested that bacterial
growth phase instead of salt stress was direct deter-
minant of L. monocytogenes invasiveness. Hence
the above mentioned reports determining the in-
volvement of salt stress show conflicting findings
and more work in this direction would be required
to understand the factors that result in such differ-
ing view. The expression of prfA controlled virulence
genes and cell invasion capacity of L. monocyto-
genes cells is temperature dependent and pathoge-
nicity in some meat-processing plant derived strains
of this bacterium was reported to decrease during
long term cold storage at 4°C (Duodu et al., 2010;
Johansson et al., 2002; McGann et al., 2007). Simi-
larly, cold stress exposed wild type and mutants lack-
ing csp genes in the L. monocytogenes EGDe strain
were significantly impaired in cell invasion relative
to corresponding controls grown at 37°C (Loepfe et
al., 2010). Temperature dependent virulence gene
expression repression as well as membrane damage
and cell surface modifications in these organisms ex-
posed at low temperatures might lead to phenotypic
virulence defects observed in cold adapted L. mono-
cytogenes organisms.
Van de Velde et al. (2009) compared proteomes
between L. monocytogenes cells grown in human
THP-1 monocytes versus those growing extracel-
lularly in TSB broth using 2D-DIGE. Down regula-
tions of general stress protein Ctc and oxidative
stress protein Sod was detected suggesting that
compared to extra cellular environment the intra-
cellular uptake by host cells may be more favorable
environment for L. monocytogenes survival and ad-
aptations. Shin et al. (2010) observed the increased
σB activity, as measured by ß-galactosidase lacZ pro-
moter assay, to vancomycin antibiotic stress. While
subsequent proteome analysis of L. monocytogenes
σB wild type and null mutant strains using LC-ESI-
MS/MS also revealed among other proteins the in-
creased production of the virulence protein InlD. Fri
protein is another general stress response protein
with virulence promoting functions in L. monocyto-
genes. It has been shown by using both mice chal-
lenge and macrophage cell virulence models that
fri null strains of L. monocytogenes are significantly
impaired (Dussurget et al., 2005; Mohamed et al.,
2006; Olsen et al., 2005). Proteome analysis of the fri
mutant and wild type strain was compared to reveal
repression in Hly (Listeriolysin O) and stress response
proteins CcpA (Catabolite control protein A) and
OsmC (Dussurget et al., 2005).
The stress induced chaperone proteins ClpB,
ClpC, ClpE, ClpP have all been shown to provide
virulence promoting activities in L. monocytogenes
and thus it is possible that their induction in this
bacterium in response to stress in food associated
environments also increases the capacity of stress
76 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
adapted organism to survive hostile host envi-
ronments as well as enhance their pathogenicity
(Chastanet et al., 2004; Gaillot et al., 2000; Nair et
al., 1999; 2000a;). Meanwhile Olesen et al. (2009)
in their recent study showed that acid exposed L.
monocytogenes cells displaying increased Caco-
2 cell virulence also displayed increased expres-
sion of genes encoding the ClpC and ClpP. The
RNA binding regulatory protein Hfq, is another
general stress response modulating protein which
has been shown to protects cells from osmotic and
ethanol stress as well as facilitate enhanced patho-
genicity in L. monocytogenes infected mice (Chris-
tiansen et al., 2004). Stack et al. (2005) found that
the HrtA serine protease, which protects L. mono-
cytogenes from various stresses including expo-
sure to acidic conditions also contributes towards
virulence capablities of this bacterium. The gen-
eral stress response protein σB, which facilitates L.
monocytogenes adaptation to multiple stresses
has also been shown to promote virulence and cell
invasiveness in this bacterium (Garner et al., 2006;
Ivy et al., 2010). Recently it was shown that the im-
portance of σB responses in these aspects might
be lineage specific with its activity being important
in lineage I, II, IIIB strains but not in IIIA (Oliver et
al., 2010).
Role of alternative sigma factor (σB ) in L. monocytogenes stress adaptation
In L. monocytogenes, σB is a major stress re-
sponse regulator and mutant strain lacking σB ac-
tivity shows increased sensitivity to a wide range of
stresses including cold (Becker et al., 2000; Chan
et al., 2007; 2008; Moorhead and Dykes, 2004;
Raimann et al., 2009; Wemekamp-Kamphuis et al.,
2004a; ), heat (Hu et al., 2007a,b; van der Veen et
al., 2007), osmotic (Becker et al., 1998, Fraser et al.,
2003; Okada et al 2008; Raimann et al., 2009), acid
(Cotter et al., 2001a,b; Ryan et al., 2008a; Weme-
kamp-Kamphuis et al., 2004a), and HHP (Weme-
kamp-Kamphuis et al., 2004a). The main role of σB
in L. monocytogenes is to regulate the expression
of various stress response associated genes. As an
example, Flp is a general stress protein involved
in cold, oxidative and heat stress adaptation. The
expression of fri gene encoding Flp protein is par-
tially regulated through σB-dependent pathways in
L. monocytogenes 10403S (Chan et al., 2007).
To identify the proteins that show σB dependent
expression in the acidic conditions, 2DE analysis of
acid adapted (pH 4.5) and non-adapted cells (both
wild type and σB mutant) was performed (Weme-
kamp-Kamphuis et al., 2004a). The expression of 9
proteins was dependent on σB during acid stress
and some of these proteins were identified using
PMF. The identified proteins with σB dependent
expression in response to HHP stress included Pfk,
GalE, ClpP, and Lmo1580. The Pfk (6-phospho-
fructokinase) and GalE are enzymes involved in
glycolysis and sugar metabolism, respectively, and
ClpP is the ATP-dependent chaperone protease
that plays a role in preventing the accumulation of
misfolded proteins. The induction of ClpP protein
expression may be necessary in acidic conditions
to help in resolution of protein aggregations that
are likely to occur due to acid stress induced pro-
tein damage.
Recently, the role of σB regulon on L. monocyto-
genes 10403S cells grown to stationary phase in the
presence or absence of 0.5 M NaCl was evaluated
using both 2DE and iTRAQ (Abram et al., 2008b).
Using a combination of these two approaches a
total of 38 proteins (17 induced and 21 repressed)
were identified whose expression was σB depen-
dent. Among these σB controlled proteins, 10
proteins (7 positively regulated and 3 negatively
regulated by σB) were further classified based on
their potential role in stress related functions. Of
these 7 σB positively regulated proteins, two pro-
teins OpuC and HtrA were previously conferred to
have role in L. monocytogenes stress adaptation
(Fraser et al., 2003; Wonderling et al., 2004). OpuC
is involved in osmolyte transfer needed for os-
motic and cold adaptation (Fraser et al., 2003) and
HtrA serves as a protease whose deletion leads to
growth defects under NaCL stress (Wonderling et
al., 2004). Intracellular accumulation of glycine be-
taine and carnitine osmolytes is necessary in cold
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 77
as well as osmotic stress. Expressions of the os-
molyte transporter proteins, Gbu and Opu, have
been shown to be at least partially dependent
on the σB activity (Cetin et al., 2004; Fraser et al.,
2003;). Also L. monocytogenes 10403S strain with
a null mutation in the σB gene showed substantial
defects in its ability to accumulate glycine betaine
and carnitine osmolytes (Becker et al., 1998; 2000).
Moreover, σB deletion impairs the ability of L.
monocytogenes 10403S cells to withstand against
heat stress (55°C for 30-60 min) and class II heat
shock genes, which also includes the osmolyte
transporter gene opuC, are positively upregulated
following heat shock (48°C for 3 min) in L. mono-
cytogenes EGDe strain (Hu et al., 2007a; van der
Veen et al., 2007). Using transcriptional analysis,
Ryan et al. (2008a) reported the induction of the σB
in response to sublethal levels of detergent stress.
In addition, following HHP treatment of 300 MPa
of 20 min, the parent strain (EGDe) showed 100-
fold higher survival compared to σB mutant strain
(Wemekamp-Kamphuis et al., 2004a).
Apart from σB, other sigma factors σc, σH, and
σL (RpoN) are also known to play important roles
in stress adaptation of L. monocytogenes. L.
monocytogenes strain lacking σB, σc, σH encod-
ing proteins have been shown to have significantly
impaired growth compared to wild type strain at
4°C for 12 days (Chan et al., 2008). Raimann et
al. (2009) reported that L. monocytogenes strain
lacking σL has impaired cold growth due to in
part by the repressed transcript production of oli-
gopeptide-binding OppA protein that facilitates
accumulation of short peptide substrates which
are also important for efficient cold growth in this
bacterium (Borezee et al., 2000). Absence of σc in-
creases the L. monocytogenes sensitivity to ther-
mal treatment, thus highlighting the importance
of this regulatory factor in conferring L. monocy-
togenes adaptation to heat stress (Zhang et al.,
2005). σL aids in L. monocytogenes ability to grow
at high salt concentrations (Okada et al., 2006) as
well as control carbohydrate metabolism through
its influence on expression of phosphotransferase
system genes (Arous et al., 2004).
ConCluSIon And fuTuRe PeRSPeCTIveS
The ability of L. monocytogenes cells to survive
adverse physiological conditions is a serious food
safety and public health concern. The physiological
changes in response of environmental stress stimuli’s
reflect key changes instituted by microbial cells at
gene or protein expression levels. In the future an
improved understanding of fundamental changes
occurring at genes or proteins level in L. monocy-
togenes cells in response to adverse environmental
conditions will provides new insights that can be har-
nessed in developing more effective practical food
preservation approaches (Gandhi and Chikindas,
2007; Tasara and Stephan, 2006).
The physiological changes mounted in response
to particular environmental stress stimuli in L. mono-
cytogenes are a consequence of changes at gene
transcription and/or protein expression levels. The
cold adaptive nature of this organism is probably
one of the most important concerns to food produc-
tion due to the ability of this pathogen to grow and
achieve high concentrations in long shelf life ready-
to-eat products preserved by refrigeration. Vari-
ous cold adaptive mechanisms such as synthesis of
conserved cold shock proteins (Schmid et al., 2009),
increased uptake of cryoprotective osmolytes (An-
gelidis and Smith, 2003), increased membrane per-
miablity (Borezee et al., 2000), increased production
of general stress proteins Fri (Dussurget et al., 2005),
etc have been identified that may directly or indi-
rectly confer this bacterium with an ability to multiply
and/or survive at lower temperatures. However, at
this stage it is unclear if these different mechanisms
work in any coordinated manner or if they work on
separate niches leading overall cold stress resistance
of L. monocytogenes cells. Future experiments are
warranted to understand the complex hierarchy be-
tween these different stress response mechanisms.
One way to do this would be to conduct gene knock
out studies where the related genes/proteins of a
particular stress adaptive mechanism (i.e. deletion of
cold shock proteins) is deleted and use these strains
to understand the modulations in genes/proteins of
other stress mechanisms. The adaptation of this bac-
78 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
terium to osmotic stress also involves complex sets
of cellular responses. Notably some osmotic stress
response mechanisms, such as compatible solute
uptake systems as well as alternative sigma factors
are also involved in cold stress adaptation (Fraser et
al., 2003; Wemekamp-Kamphuis et al., 2004b), which
suggests that some cellular response mechanism to-
wards food related environmental (cold and osmotic)
stresses in this bacterium are common.
The main limitation of current studies is that
large numbers of genes/proteins are tabulated as
being differentially expressed but there is little or
no insight on what the modulations in these gene/
proteins mean. In any event, perturbation in physiol-
ogy of living cells is likely to change the expression
levels of various genes/proteins. Such information is
of limited value without further functional character-
izations of such putative stress adaptation genes or
proteins. While it may not be practical to use such
approach for hundreds of genes/proteins that are
differentially expressed along with each stress, it is
necessary to do follow-up studies on genes/proteins
that exhibit substantially large changes in expression
level. So far only in a few cases of stress proteins has
the follow-up work been done in elucidating their
molecular roles during stress adaptation of L. mono-
cytogenes. Some key examples are: (a) Flp protein,
first identified to be highly induced in cold and heat
stress, and subsequently confirmed through fri mu-
tant strain of L. monocytogenes EGDe, which is im-
paired under both stress conditions (Dussurget et
al., 2005; Hebraud and Guzzo, 2000; Phan-Thanh and
Gormon, 1995); (b) Ctc protein is induced under salt
stress and L. monocytogenes LO28 ctc mutant strain
is found defective in growth under NaCl stress con-
ditions (Duche et al., 2002a; Gardan et al., 2003b);
and (c) GbuA osmolyte transporter protein, induced
under high osmolarity, (at 3.5% NaCl) was confirmed
by gbu mutant strain of L. monocytogenes LTG59
as defective in growth in the absence of osmolyte
uptake activity (Duche et al., 2002a; Mendum and
Smith, 2002). Moreover most of the current stress
adaptation findings are based on laboratory media
and it is crucial that to design new experimental
strategies that detect stress adaption response in L.
monocytogenes cells exposed to different food ma-
trices. The experiments with food substrate may be
designed to see how different food components and
food preservatives modulate the expression of stress
proteins identified using broth media.
ACknowledgeMenT
This research was supported in part by Food Safe-
ty Initiative award to RN by the Mississippi Agricul-
tural and Forestry Experiment Station (MAFES), Mis-
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as that described for unsolicited review papers. Con-
ference papers must be prepared in accordance with
the guidelines for review articles and are subject to
peer review. The conference chair must decide
whether or not they wish to serve as Special Issue
Editor and conduct the editorial review process. If
the conference chair/organizer chooses to serve as
special issue editor, this will involve review of the pa-
pers and, if necessary, returning them to the authors
for revision. The conference organizer then submits
the revised manuscripts to the journal editorial of-
fice for further editorial examination. Final revisions
by the author and recommendations for acceptance
or rejection by the chair must be completed by a
mutually agreed upon date between the editor and
the conference organizer. Manuscripts not meeting
this deadline will not be included in the published
symposium proceedings but if submitted later can
still be considered as unsolicited review papers. Al-
though offprints and costs of pages are the same
as for all other papers, the symposium chair may be
asked to guarantee an agreed upon number of hard
copies to be purchased by conference attendees. If
the decision is not to publish the symposium as a
special issue, the individual authors retain the right
to submit their papers for consideration for the jour-
nal as ordinary unsolicited review manuscripts.
Book Reviews
AFAB publishes reviews of books considered to
be of interest to the readers. The editor-in-chief ordi-
narily solicits reviews. Book reviews shall be prepared
in accordance to the style and form requirements of
the journal, and they are subject to editorial revision.
No page charges will be assessed solicited reviews
while unsolicited book reviews will be assigned the
regular page charge rate.
Opinions and Current Viewpoints
The purpose of this section will be to discuss, cri-
tique, or expand on scientific points made in articles
recently published in AFAB. Drafts must be received
within 6 months of an article’s publication. Opinions
and current perspectives do not have page limits.
They shall have a title followed by the body of the
text and references. Author name(s) and affiliation(s)
shall be placed between the end of the text and list
of references. If this document pertains to a par-
ticular manuscript then the author(s) of the original
paper(s) will be provided a copy of the letter and of-
fered the opportunity to submit for consideration a
reply within 30 days. Responses will have the same
page restrictions and format as the original opinion
and current viewpoint, and the titles shall end with
“Opinions.” They will be published together. Letters
and replies shall follow appropriate AFAB format
and may be edited by the editor-in-chief and a tech-
nical editor. If multiple letters on the same topic are
received, a representative set of opinions concern-
ing a specific article will be published. A disclaimer
will be added by the editorial staff that the opinion
expressed in this viewpoint is the authors alone and
does not necessarily represent the opinion of AFAB
or the editorial board.
CoPyRIghT AgReeMenT
The copyright form is published in AFAB as space
permits and is available online (www.afabjournal.com).
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 89
AFAB grants to the author the right of re-publication
in any book of which he or she is the author or edi-
tor, subject only to giving proper credit to the original
journal publication of the article by AFAB. AFAB re-
tains the copyright to all materials accepted for pub-
lication in the journal. If an author desires to reprint
a table or figure published from a non-AFAB source,
written evidence of copyright permission from an au-
thority representing that source must be obtained by
the author and forwarded to the AFAB editorial office.
PeeR RevIew PRoCeSS
Authors will be requested to provide the names
and complete addresses including emails of five (5) potential reviewers who have expertise in the research
area and no conflict of interest with any of the authors.
Except for manuscripts designated as Rapid Commu-
nication each reviewer has two (2) weeks to review
the manuscript, and submit comments electronically
to the editorial office. Authors have three (3) weeks
to complete the revision, which shall be returned to
the editorial office within six (6) weeks after which the
authors risk having their manuscript removed from
AFAB files if they fail to ask the editorial office for
an extension by email. Deleted manuscripts will be
reconsidered, but they will have to be processed as
new manuscripts with an additional processing fee as-
sessed upon submission. Once reviewed, the author
will be notified of the outcome and advised accord-
ingly. Editors handle all initial correspondence with
authors during the review process. The editor-in chief
will notify the author of the final decision to accept or
reject. Rejected manuscripts can be resubmitted only
with an invitation from the editor or editor-in chief. Re-
vised versions of previously rejected manuscripts are
treated as new submissions.
PRoduCTIon of PRoofS
Accepted manuscripts are forwarded to the edito-
rial office for technical editing and layout. The manu-
script is then formatted, figures are reproduced, and
author proofs are prepared as PDFs. Author proofs
of all manuscripts will be provided to the correspond-
ing author. Author proofs should be read carefully and
checked against the typed manuscript, because the
responsibility for proofreading is with the author(s).
Corrections must be returned by e-mail. Changes
sent by e-mail to the technical editor must indicate
page, column, and line numbers for each correction
to be made on the proof. Corrections can also be
marked using “track changes” in Microsoft Word or
using e-annotation tools for electronic proof correc-
tion in Adobe Acrobat to indicate necessary chang-
es. Author alterations to proofs exceeding 5% of the
original proof content will be charged to the author. All
correspondence of proofs must be agreed to by the
editorial office and the author within 48 hours or proof
will be published as is and AFAB will assume no re-
sponsibility for errors that result in the final publication.
PuBlICATIon ChARgeS
AFAB has two publication charge options: conven-
tional page charges and rapid communication. The
current charge for conventional publication is $25 per printed page in the journal. There is no additional
charge for the publication of pages containing color
images, micrographs or pictures. For authors who
wish to have their papers processed as a rapid com-
munication, authors will pay the rapid communication
fee when proofs are returned to the editorial office
in addition to twice the conventional page charges.
Charges for rapid communications are $1000 per manuscript for guaranteed peer review within one
week and $100 per journal page.
hARd CoPy offPRInTS
If you are wishing to obtain a physical hard copy of
the AFAB journal, offprints are available in any quan-
tity at an additional charge: $100/page for black-white
and $150/page for color prints. You may order your
offprints at any time after publication on our website.
Scientific conference organizers may be expected to
agree to a set number of offprints as a part of their
agreement with AFAB.
90 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
MAnuSCRIPT ConTenT ReQuIReMenTS
Preparing the Manuscript File
Manuscripts must be written in grammatically
correct English. AFAB offers a fee based language
service upon request ([email protected]).
Manuscripts should be typed double-spaced, with
lines and pages numbered consecutively. All docu-
ments must be submitted in Microsoft Word (.doc or
.docx, PC or Mac). All special characters (e.g., Greek,
math, symbols) should be inserted using the sym-
bols palette available in this font. Tables and figures
should be placed in separate sections at the end of
the manuscript (not placed in the text). Failure to fol-
low these instructions will cause delays of the pro-
cessing and review of the manuscript.
Title Page
At the very top of the title page, include a title of
not more than 100 characters. Format the title with
the first letter of each word capitalized. No abbre-
viations should be used. Under the title, the authors
names are listed. Use the author’s initials for both first
and middle names with a period (full-stop) between
initials (e.g., W. A. Afab). Underneath the authors, a
list affiliations must be listed. Please use numerical
superscripts after the author’s names to designate
affiliation. If an authors address has changed since
the research was completed, this new information
must be designated as “Current address:”. The cor-
responding author should be indicated with an aster-
isk e.g., * Corresponding author. The title page shall
include the name and full address of the correspond-
ing author. Telephone and e-mail address must also
be provided for the corresponding author, and email-addresses must be provided for all authors.
Editing
Author-derived abbreviations should be defined
at first use in the abstract and again in the body of
the manuscript. If abbreviations are extensive au-
thors may need to provide a list of abbreviations
at the beginning of the manuscript. In vivo, in vitro
and bacterial names must be italicized (obligatory).
Authors must avoid single sentence paragraphs and
merge such paragraphs appropriately. Authors must
not begin sentences with “Figure or Table shows…”
as these are inanimate objects and cannot “show”
anything. When number are reported in text or in ta-
bles, always put a zero in front of decimal numbers:
“0.10” instead of “.10”.
MAnuSCRIPT SeCTIonS
Abstract
The abstract provides an abridged version of the
manuscript. Please submit your abstract on a sepa-
rate page after the title page. The abstract should
provide a justification of your work, objectives, meth-
ods, results, discussion and implications of study or
review findings . Your abstract must consist of com-
plete sentences without references to other work or
footnotes and must not exceed 250 words. On the
same page as your abstract, please provide at least ten (10) keywords to be used for linking and index-
ing. Ideally, these keywords should include signifi-
cant words from the title.
Introduction
The introduction should clearly present the foun-
dation of the manuscript topic and what makes the
research or the review unique. The introduction
should validate why this topic is important based on
previously published literature, and the relevance of
the current research. Overall goals and project ob-
jectives must be clearly stated in the final sentence
of the last paragraphs of the introduction.
Materials and Methods
Information on equipment and chemicals used
must include the full company name, city, and state
(country if outside the United States or Province if
in Canada) [i.e., (Model 123, ACME Inc., Afab, AR)].
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 91
Variability, Replication, and Statistical Analysis
To properly assess biological systems indepen-
dent replication of experiments and quantification
of variation among replicates is required by AFAB.
Reviewers and/or editors may request additional
statistical analysis depending on the nature of the
data and it will be the responsibility of the authors
to respond appropriately. Statistical methods com-
monly used in the bacteriology do not need to be
described in detail, but an adequate description
and/or appropriate references should be provided.
The statistical model and experimental unit must
be designated when appropriate. The experimen-
tal unit is the smallest unit to which an individual
treatment is imposed. For bacterial growth stud-
ies, the average of replicate tubes per single study
per treatment is the experimental unit; therefore,
individual studies must be replicated. Repeated
time analyses of the same sample usually do not
constitute independent experimental units. Mea-
surements on the same experimental unit over time
are also not independent and must not be consid-
ered as independent experimental units. For analy-
sis of time effects, assess as a rate of change over
time. Standard deviation refers to the variability
in the biological response being measured and is
presented as standard deviation or standard error
according to the definitions described in statistical
references or textbooks.
Results
Results represent the presentation of data in
words and all data should be described in same
fashion. No discussion of literature is included in
the results section.
DiscussionThe discussion section involves comparing the
current data outcomes with previously published
work in this area without repeating the text in the
results section. Critical and in-depth dialogue is
encouraged.
Results and Discussion
Results and discussion can be under combined or
separate headings.
Conclusions
State conclusions (not a summary) briefly in one
paragraph
Acknowledgments
Acknowledgments of individuals should include
institution, city, and state; city and country if not U.S.;
and City or Province if in Canada. Copies being re-
viewed shall have authors’ institutions omitted to re-
tain anonymity.
References
a) Citing References In Text
Authors of cited papers in the text are to be pre-
sented as follows: Adams and Harry (1992) or Smith
and Jones (1990, 1992). If more than two authors of
one article, the first author’s name is followed by the
abbreviation et al. in italics. If the sentence structure
requires that the authors’ names be included in pa-
rentheses, the proper format is (Adams and Harry,
1982; Harry, 1988a,b; Harry et al., 1993). Citations to a
group of references should be listed first alphabeti-
cally then chronologically. Work that has not been
submitted or accepted for publication shall be listed
in the text as: “G.C. Jay (institution, city, and state,
personal communication).” The author’s own un-
published work should be listed in the text as “(J.
Adams, unpublished data).” Personal communica-
tions and unsubmitted unpublished data must not
be included in the References section. Two or more
publications by the same authors in the same year
must be made distinct with lowercase letters after
the year (2010a,b). Likewise when multiple author ci-
tations designated by et al. in the text have the same
first author, then even if the other authors are differ-
ent these references in the text and the references
section must be identified by a letter. For example
92 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
“(James et al., 2010a,b)” in text, refers to “James,
Smith, and Elliot. 2010a” and “James, West, and Ad-
ams. 2010b” in the reference section.
b) Citing References In Reference Section
In the References section, references are listed in
alphabetical order by authors’ last names, and then
chronologically. List only those references cited in the
text. Manuscripts submitted for publication, accepted
for publication or in press can be given in the refer-
ence section followed by the designation: “(submit-
ted)”, “(accepted)’, or “(In Press), respectively. If the
DOI number of unpublished references is available,
you must give the number. The year of publication fol-
lows the authors’ names. All authors’ names must be
included in the citation in the Reference section. Jour-
nals must be abbreviated. First and last page num-
bers must be provided. Sample references are given
below. Consult recent issues of AFAB for examples
not included in the following section.
Journal manuscript:
Examples:
Chase, G. and L. Erlandsen. 1976. Evidence for a
complex life cycle and endospore formation in the
attached, filamentous, segmented bacterium from
murine ileum. J. Bacteriol. 127:572-583.
Jiang, B., A.-M. Henstra, L. Paulo, M. Balk, W. van
Doesburg, and A. J. M. Stams. 2009. A typical
one-carbon metabolism of an acetogenic and
hydrogenogenic Moorella thermioacetica strain.
Arch. Microbiol. 191:123-131.
Book:
Examples:
Hungate, R. E. 1966. The rumen and its microbes.
Academic Press, Inc., New York, NY. 533 p.
Book Chapter:
Examples:
O’Bryan, C. A., P. G. Crandall, and C. Bruhn. 2010.
Assessing consumer concerns and perceptions
of food safety risks and practices: Methodologies
and outcomes. In: S. C. Ricke and F. T. Jones. Eds.
Perspectives on Food Safety Issues of Food Animal
Derived Foods. Univ. Arkansas Press, Fayetteville,
AR. p 273-288.
dissertation and thesis:
Maciorowski, K. G. 2000. Rapid detection of Salmo-
nella spp. and indicators of fecal contamination
in animal feed. Ph.D. Diss. Texas A&M University,
College Station, TX.
Donalson, L. M. 2005. The in vivo and in vitro effect
of a fructooligosacharide prebiotic combined with
alfalfa molt diets on egg production and Salmo-
nella in laying hens. M.S. thesis. Texas A&M Uni-
versity, College Station, TX.
Van Loo, E. 2009. Consumer perception of ready-to-
eat deli foods and organic meat. M.S. thesis. Uni-
versity of Arkansas, Fayetteville, AR. 202 p.
web sites, patents:
Examples:
Davis, C. 2010. Salmonella. Medicinenet.com.
http://www.medicinenet.com/salmonella /article.
htm. Accessed July, 2010.
Afab, F. 2010, Development of a novel process. U.S.
Patent #_____
Author(s). Year. Article title. Journal title [abbreviated].
Volume number:inclusive pages.
Author(s) [or editor(s)]. Year. Title. Edition or volume (if
relevant). Publisher name, Place of publication. Number
of pages.
Author(s) of the chapter. Year. Title of the chapter. In:
author(s) or editor(s). Title of the book. Edition or vol-
ume, if relevant. Publisher name, Place of publication.
Inclusive pages of chapter.
Author. Date of degree. Title. Type of publication, such
as Ph.D. Diss or M.S. thesis. Institution, Place of institu-
tion. Total number of pages.
Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011 93
Abstracts and Symposia Proceedings:
Fischer, J. R. 2007. Building a prosperous future in
which agriculture uses and produces energy effi-
ciently and effectively. NABC report 19, Agricultural
Biofuels: Tech., Sustainability, and Profitability. p.27
Musgrove, M. T., and M. E. Berrang. 2008. Presence
of aerobic microorganisms, Enterobacteriaceae and
Salmonella in the shell egg processing environment.
IAFP 95th Annual Meeting. p. 47 (Abstr. #T6-10)
Vianna, M. E., H. P. Horz, G. Conrads. 2006. Options
and risks by using diagnostic gene chips. Program
and abstracts book , The 8th Biennieal Congress of
the Anaerobe Society of the Americas. p. 86 (Abstr.)
Data Presentation in Tables and Figures
Figures and tables to be published in AFAB must
be constructed in such a fashion that they are able
to “stand alone” in the published manuscript. This
means that the reader should be able to look at
the figure or table independently of the rest of the
manuscript and be able to comprehend the experi-
mental approach sufficiently to interpret the data.
Consequently, all statistical analyses should be very
carefully presented along with variation estimates
and what constitutes an independent replication
and the number of replicates used to calculate the
averages presented in the table or figure.
Each table and figure must be on a separate
page in the submitted paper. If your manuscript
is accepted for publication, you will need to sub-
mit all data for charts, tables and figures in Excel
spreadsheet format.
All figures should be clearly presented with well
defined axis and units of measurement. Symbols,
lines, and bars must be made distinct as “stand
alone” black and white presentations. Stippling,
dashed lines etc. are encouraged for multiple com-
parison but shades of gray are discouraged. Color
images, micrographs, pictures are recommended
and there is no additional fee for their submission.
AFAB Online Edition is Now Available!
www.AFABjournal.com
• Free Access
• Print PDFs
• Flip Through Issues
• Search Article Archives
• Order Reprints
• Submit a Paper
94 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011
www.foodandfunction.net
www.foodandfunction.net
International Scientific Conference on Nutraceuticals and Functional Foods - FF2011
The next International Scientific Conference on Nutraceuticals and Functional Foods, Food and Function 2011, will be held during 25th-27th October 2011 in the university city of Kosice, Slovakia. The conference programme will focus on current advances in the research of nutraceuticals and functional foods and their present and future role in maintaining health and preventing diseases. Nutraceuticals and functional foods are intensively researched for their role in maintaining health and the preventing diseases. The science behind is growing rapidly not only because of the growing number of new substances or type of novel foods, but also while regulatory bodies require more and more evidence on efficacy, mode-of-action and safety. The goal of the conference is to provide a scientific forum for all stakeholders of nutraceuticals, functional foods and enable interactive exchange of state-of-the-art knowledge. The conference will focus on the evidence-based benefits of nutraceuticals and functional foods. Meet those who influenced the past, influence the present and most importantly will enable the future of nutraceuticals and functional foods. At Food and Function 2011, leading scientists will present and discuss current advances in the research of nutraceuticals and functional foods. New scientific evidences that support or question the efficacy of already existing or prospective substances and applications will be conferred. In addition novel compounds, controversial but scientifically solid ideas, approaches and visions will be presented as well. Food and Function 2011 is a networking event. A unique opportunity to meet all the stakeholders of nutraceuticals and funtional foods. Initiation of cross-border cooperations between scientists and institutions will be also facilitated during the conference.
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