SW_PosterSummer Symposium 2015 Final Edit

1
The addi(on of grape leaves to CaCl 2 brined fermenta(ons did not improve the texture of the fermented cucumbers, likely because there was no polygalacturonase in the brine to inhibit. There seems to be a synergis(c effect between the highest concentra(on of grape leaf (36g/gal) and ace(c acid (25mM). These treatments were less likely to contain less spoilage metabolites and held a stable pH throughout fermenta(on. A grape leaf extract has poten(al to be used as a natural preserva(ve in CaCl 2 fermenta(ons. However, the tested concentra(ons of grape leaves contain tannins within the known limit of detec(on, which may contribute an astringent taste. PG and yeast and molds were not associated with the soKening of CaCl 2 fermented cucumbers, and the soKening mechanism in CaCl 2 fermenta(ons is s(ll unknown. Sean Whitfield 1,2 , Dr. Suzanne Johanningsmeier 1,2 , Dr. Ilenys Perez-Diaz 1,2 1 USDA-ARS Food Science Research Unit, Raleigh, NC; 2 Department of Food, Bioprocessing, and Nutrition Sciences During the 1950s and 1960s, research on the inhibi(on of polygalacturonase (PG), an enzyme that degrades cucumber texture, was conducted with a variety plants. Grape leaves of the Muscadine family, specifically the Scuppernong Variety (GL), were indicated as one of the best contenders to inhibit PG in mul(ple experiments. The inhibitory effects of GL were never inves(gated in cucumber fermenta(ons with 1.1% CaCl 2 brine, which has an increased likelihood for texture variability and spoilage compared to tradi(onal NaCl fermenta(ons. HYPOTHESIS STATEMENT Adding GL to fermenta(ons with CaCl 2 brines will improve the texture of the cucumbers by inhibi(ng PG To determine the impact of ace(c acid and grape leaf (GL) in the reten(on of texture quality of CaCl 2 fermented cucumbers. To inves(gate the effect of ace(c acid on the microbiota of the brine of CaCl 2 fermenta(ons Gregory Vineyards, for allowing grape leaf samples to be taken from their muscadine cul(vars (275 Bowling Spring Dr, Angier, NC 27501) Dr. PerezDiaz Lab members for supplies for microbiological analyses. Cucumber fermenta(on in CaCl 2 brines (1.1% CaCl 2 , 6mM potassium sorbate) has undergone commercial trials to reduce salt waste, but can result in variable texture and increased risk of spoilage. Lab scale fermenta(ons were conducted to determine if ace(c acid and/or grape leaves improve firmness of CaCl 2 fermented cucumbers. A full factorial treatment structure in a completely randomized design was applied to the CaCl 2 fermented cucumbers for ace(c acid (0, 25 mM) and grape leaf (0, 18, 36 g/gal). A control group fermented in 6% NaCl, 2 mM potassium sorbate, and 25mM ace(c acid was included. Fermenta(ons were inoculated with 10 6 CFU/ml Lactobacillus Planetarium. Fermenta(on metabolites and pH were analyzed during fermenta(on and storage, and texture analysis was performed on days 28 and 129. The drip plate method was used for microbiological counts of enterobacteriaceae, lac(c acid bacteria , clostridia, yeast and molds on cucumbers, grape leaves, and in fermenta(on brines. It was found that 25mM ace(c acid reduced enterobacteriaceae counts by two orders of magnitude by the second day of fermenta(on, 5.1 ± 0.8 CFU/ml vs 7.8 ± 0.4 CFU/ml for fermenta(ons without ace(c acid (P = 0.0419). No improvement in firmness was observed for cucumbers fermented in CaCl 2 brines and grape leaf (P = 0.6522), but CaCl 2 fermenta(ons with 36 g/gal grape leaf and 25 mM ace(c acid had less spoilage metabolites (P < 0.05) and had a stable pH during storage. SPOILAGE METABOLITES Legend 0 10 20 30 40 50 60 2 3 4 5 6 0 25 50 75 100 125 150 Spoilage Metabolites (mM) Brine pH Time (Day) Spoilage of Cucumbers Fermented in CaCl 2 (100mM) & AceJc (25mM) 0 10 20 30 40 50 60 2 3 4 5 6 0 25 50 75 100 125 150 Spoilage Metabolites (mM) Brine pH Time (Day) Cucumbers Fermented in CaCl 2 (100mM), GL (36g/gal), & AceJc (25mM) 0 10 20 30 40 50 60 2 3 4 5 6 0 25 50 75 100 125 150 Spoilage Metabolites (mM) Brine pH Time (Day) Spoilage of Cucumbers Fermented in CaCl 2 0 10 20 30 40 50 60 2 3 4 5 6 0 25 50 75 100 125 150 Spoilage Metabolites (mM) Brine pH Time (Day) Cucumbers Fermented in CaCl 2 & GL (36g/gal) 0 10 20 30 40 50 60 2 3 4 5 6 0 25 50 75 100 125 150 Spoilage Metabolites (mM) Brine pH Time (Day) NaCl Control Effect of GL and AceJc Acid on LacJc Acid ProducJon 0 20 40 60 80 100 120 140 0 25 50 75 100 125 150 LacJc Acid (mM) Time (Day) NaCl Control CaCl2 (100mM) GL(0g/gal) AceJc (0mM) CaCl2 (100mM) GL (18g/gal) AceJc (0mM) CaCl2 (100mM) GL (36g/gal) AceJc (0mM) Effect of GL on LacJc Acid ProducJon & RetenJon During Storage 0 20 40 60 80 100 120 140 0 25 50 75 100 125 150 LacJc Acid (mM) Time (Day) NaCl Control CaCl2 (100mM) AceJc (0mM) CaCl2 (100mM) AceJc (25mM) Effect of AceJc Acid on LacJc Acid ProducJon & RetenJon During Storage MICROBIOLOGY Legend CaCl2 (100mM) AceJc (0mM) CaCl2 (100mM) AcerJc (25mM) NaCl Control Effect of AceJc Acid on Enterobacteriaceae Counts Effect of AceJc Acid on LacJc Acid Bacteria Counts TEXTURE Effect of AceJc Acid on Cucumber Firmness Effect of Grape Leaf and AceJc Acid on Cucumber Firmness Effect of Grape Leaf on Cucumber Firmness No polygalacturonase ac(vity was detected in any treatments throughout fermenta(on and storage. POLYGALACTURONASE ACTIVITY pH AceJc Acid Propionic Acid Propanol 1, 2Propanediol *Potassium Sorbate was effecJve in inhibiJng yeast and molds during the first 9 days of fermentaJon LACTIC ACID PRODUCTION Full Factorial Treatment Structure with Complete RandomizaJon Two Levels of AceJc Acid (0,25mM) Three Levels of Grape Leaf (0, 18, 36g/gal) CaCl 2 FermentaJon Treatments: (100mM CaCl 2, 6mM Potassium Sorbate) NaCl Control (6% NaCl, 2mM Potassium Sorbate, 25mM AceJc Acid) Two Texture Time Points 28 FermentaJons In Total Day 28 Day 129 Day 28 Day 129 Two Replicates for Each Treatment Two Replicates for Each Treatment Incubated at 30 °C Inoculated with 10 6 CFU/ml Lactobacillus plantarum HPLC: FermentaJon Metabolites Microbiological analysis: Drip Plate Method AsepJc Brine Sampling with Syringe and Septa Diffusion Plate Assay: PG AcJvity Firmness of Fermented Cucumber Mesocarp Tissue: Average Peak Puncture Force Indicators of Secondary FermentaJon 0 2 4 6 8 10 12 14 28 129 Average Peak Force (N) Time (Day) NaCl Control CaCl2 (100mM) AceJc (0mM) CaCl2 (100mM) AceJc Acid (25mM) 0 2 4 6 8 10 12 14 28 129 Average Peak Force (N) Time (Day) NaCl Control CaCl2 (100mM) AceJc (0mM) GL(0g/gal) CaCl2 (100mM) AceJc (0mM) GL(18g/gal) CaCl2 (100mM) AceJc (0mM) GL (36g/gal) 0 2 4 6 8 10 12 14 28 129 Average Peak Force (N) Time (Day) NaCl Control CaCl2 (100mM) AceJc Acid (25mM) GL (0g/gal) CaCl2 (100mM) AceJc Acid (25mM) GL (18g/gal) CaCl2 (100mM) AceJc Acid (25mM) GL(36mM) 1.E+02 1.E+03 1.E+04 1.E+05 1.E+06 1.E+07 1.E+08 1.E+09 0 1 2 3 4 5 6 7 8 9 MRS (CFU/ml) Time (Day) 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 LacJc Acid (mM) Time (Day) NaCl Control CaCl2 (100mM) GL(0g/gal) AceJc (25mM) CaCl2 (100mM) GL (18g/gal) AceJc (25mM) CaCl2 (100mM) GL (36g/gal) Lactobacillus plantarum on Cucumber Tissue 1.E+02 1.E+03 1.E+04 1.E+05 1.E+06 0 1 2 3 4 5 6 7 8 9 VRBG (CFU/ml) Time (Day) Under Limit of DetecJon by day 5

Transcript of SW_PosterSummer Symposium 2015 Final Edit

Page 1: SW_PosterSummer Symposium 2015 Final Edit

•  The  addi(on  of  grape  leaves  to  CaCl2  brined  fermenta(ons  did  not  improve  the  texture  of  the  fermented  cucumbers,  likely  because  there  was  no  polygalacturonase  in  the  brine  to  inhibit.    

•  There  seems  to  be  a  synergis(c  effect  between  the  highest  concentra(on  of  grape  leaf  (36g/gal)  and  ace(c  acid  (25mM).  These  treatments  were  less  likely  to  contain  less  spoilage  metabolites  and  held  a  stable  pH  throughout  fermenta(on.    

•  A  grape  leaf  extract  has  poten(al  to  be  used  as  a  natural  preserva(ve  in  CaCl2  fermenta(ons.  However,  the  tested  concentra(ons  of  grape  leaves  contain  tannins  within  the  known  limit  of  detec(on,  which  may  contribute  an  astringent  taste.      

•  PG  and  yeast  and  molds  were  not  associated  with  the  soKening  of  CaCl2  fermented  cucumbers,  and  the  soKening  mechanism  in  CaCl2  fermenta(ons  is  s(ll  unknown.  

 

Sean Whitfield1,2, Dr. Suzanne Johanningsmeier1,2, Dr. Ilenys Perez-Diaz1,2 1USDA-ARS Food Science Research Unit, Raleigh, NC; 2Department of Food, Bioprocessing, and Nutrition Sciences

During  the  1950s  and  1960s,  research  on  the  inhibi(on  of  polygalacturonase  (PG),  an  enzyme  that  degrades  cucumber  texture,  was  conducted  with  a  variety  plants.  Grape  leaves  of  the  Muscadine  family,  specifically  the  Scuppernong  Variety  (GL),  were  indicated  as  one  of  the  best  contenders  to  inhibit  PG  in  mul(ple  experiments.  The  inhibitory  effects  of  GL  were  never  inves(gated  in  cucumber  fermenta(ons  with  1.1%  CaCl2  brine,  which  has  an  increased  likelihood  for  texture  variability  and  spoilage  compared  to  tradi(onal  NaCl  fermenta(ons.      HYPOTHESIS  STATEMENT    Adding  GL  to  fermenta(ons  with  CaCl2  brines  will  improve  the  texture  of  the  cucumbers  by  inhibi(ng  PG    

• To  determine  the  impact  of  ace(c  acid  and  grape  leaf  (GL)  in  the  reten(on  of  texture  quality  of  CaCl2  fermented  cucumbers.  • To  inves(gate  the  effect  of  ace(c  acid  on  the  microbiota  of  the  brine  of  CaCl2  fermenta(ons  

• Gregory   Vineyards,   for   allowing   grape   leaf   samples   to   be   taken   from   their  muscadine  cul(vars  (275  Bowling  Spring  Dr,  Angier,  NC  27501)  • Dr.  Perez-­‐Diaz  Lab  members  for  supplies  for  microbiological  analyses.  

Cucumber  fermenta(on  in  CaCl2  brines  (1.1%  CaCl2,  6mM  potassium  sorbate)  has  undergone  commercial  trials  to  reduce  salt  waste,  but  can  result  in  variable  texture  and  increased  risk  of  spoilage.    Lab  scale  fermenta(ons  were  conducted  to  determine  if  ace(c  acid  and/or  grape  leaves  improve  firmness  of  CaCl2  fermented  cucumbers.  A  full  factorial  treatment  structure  in  a  completely  randomized  design  was  applied  to  the  CaCl2  fermented  cucumbers  for  ace(c  acid  (0,  25  mM)  and  grape  leaf  (0,  18,  36  g/gal).    A  control  group  fermented  in  6%  NaCl,  2  mM  potassium  sorbate,  and  25mM  ace(c  acid  was  included.  Fermenta(ons  were  inoculated  with  106  CFU/ml  Lactobacillus  Planetarium.  Fermenta(on  metabolites  and  pH  were  analyzed  during  fermenta(on  and  storage,  and  texture  analysis  was  performed  on  days  28  and  129.    The  drip  plate  method  was  used  for  microbiological  counts  of  enterobacteriaceae,  lac(c  acid  bacteria  ,  clostridia,  yeast  and  molds  on  cucumbers,  grape  leaves,  and  in  fermenta(on  brines.  It  was  found  that  25mM  ace(c  acid  reduced  enterobacteriaceae  counts  by  two  orders  of  magnitude  by  the  second  day  of  fermenta(on,    5.1  ±  0.8  CFU/ml  vs  7.8  ±  0.4  CFU/ml  for  fermenta(ons  without  ace(c  acid  (P  =  0.0419).  No  improvement  in  firmness  was  observed  for  cucumbers  fermented  in  CaCl2  brines  and  grape  leaf  (P  =  0.6522),  but  CaCl2  fermenta(ons  with  36  g/gal  grape  leaf  and  25  mM  ace(c  acid  had  less  spoilage  metabolites  (P  <  0.05)  and  had  a  stable  pH  during  storage.  

SPOILAGE METABOLITES

Legend  

0  

10  

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2  

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0   25   50   75   100   125   150  

Spoilage  M

etab

olite

s  (m

M)  

Brine  pH

 

Time  (Day)  

Spoilage  of  Cucumbers  Fermented  in  CaCl2  (100mM)  &  AceJc  (25mM)  

0  

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0   25   50   75   100   125   150  

Spoilage  M

etab

olite

s  (m

M)  

Brine  pH

 

Time  (Day)  

Cucumbers  Fermented  in  CaCl2  (100mM),  GL  (36g/gal),  &  AceJc  (25mM)  

0  

10  

20  

30  

40  

50  

60  

2  

3  

4  

5  

6  

0   25   50   75   100   125   150  

Spoilage  M

etab

olite

s  (m

M)  

Brine  pH

 

Time  (Day)  

Spoilage  of  Cucumbers  Fermented  in  CaCl2  

0  

10  

20  

30  

40  

50  

60  

2  

3  

4  

5  

6  

0   25   50   75   100   125   150  

Spoilage  M

etab

olite

s  (m

M)  

Brine  pH

 

Time  (Day)  

Cucumbers  Fermented  in  CaCl2  &  GL  (36g/gal)    

0  

10  

20  

30  

40  

50  

60  

2  

3  

4  

5  

6  

0   25   50   75   100   125   150  

Spoilage  M

etab

olite

s  (m

M)  

Brine  pH

 

Time  (Day)  

NaCl  Control  

Effect  of  GL  and  AceJc  Acid  on  LacJc  Acid  ProducJon  

0  20  40  60  80  100  120  140  

0   25   50   75   100   125   150  

LacJc  Ac

id  (m

M)  

Time  (Day)  

NaCl  Control  CaCl2  (100mM)  GL(0g/gal)  AceJc  (0mM)  CaCl2  (100mM)  GL  (18g/gal)  AceJc  (0mM)  CaCl2  (100mM)  GL  (36g/gal)  AceJc  (0mM)  

Effect  of  GL  on  LacJc  Acid  ProducJon  &  RetenJon  During  Storage  

0  20  40  60  80  

100  120  140  

0   25   50   75   100   125   150  

LacJc  Ac

id  (m

M)  

Time  (Day)  

NaCl  Control  CaCl2  (100mM)  AceJc  (0mM)  CaCl2  (100mM)  AceJc  (25mM)  

Effect  of  AceJc  Acid  on  LacJc  Acid  ProducJon  &  RetenJon  During  Storage    

MICROBIOLOGY

Legend  CaCl2  (100mM)  AceJc  (0mM)  CaCl2  (100mM)  AcerJc  (25mM)  NaCl  Control  

Effect  of  AceJc  Acid  on  Enterobacteriaceae    Counts  

Effect  of  AceJc  Acid  on  LacJc  Acid  Bacteria  Counts  

TEXTURE

Effect  of  AceJc  Acid  on  Cucumber  Firmness  

Effect  of  Grape  Leaf  and  AceJc  Acid  on  Cucumber  Firmness  

Effect  of  Grape  Leaf  on  Cucumber  Firmness  

• No  polygalacturonase  ac(vity  was  detected  in  any  treatments  throughout  fermenta(on  and  storage.  

POLYGALACTURONASE ACTIVITY

pH  AceJc  Acid  Propionic  Acid  Propanol  1,  2-­‐Propanediol  

*Potassium  Sorbate  was  effecJve  in  

inhibiJng  yeast  and  molds  during  the  first  9  days  of  fermentaJon  

LACTIC ACID PRODUCTION Full  Factorial  Treatment  Structure    with  Complete  RandomizaJon  

Two  Levels  of  AceJc  Acid  (0,25mM)  

Three  Levels  of  Grape  Leaf          

(0,  18,  36g/gal)  

 CaCl2  FermentaJon  Treatments:  (100mM  CaCl2,  6mM  Potassium  

Sorbate)  

NaCl  Control  (6%  NaCl,  2mM  Potassium  Sorbate,  25mM  AceJc  Acid)  

Two  Texture  Time  Points  

28  FermentaJons  In  Total  

Day  28  

Day  129  

Day  28  

Day  129  

Two  Replicates  for  Each  

Treatment  

Two  Replicates  for  Each  

Treatment   Incubated  at  30  °C  

Inoculated  with  106  CFU/ml  Lactobacillus  plantarum  

HPLC:  FermentaJon  Metabolites  

Microbiological  analysis:  Drip  Plate  Method  

AsepJc  Brine  Sampling  with  Syringe  and  Septa  

Diffusion  Plate  Assay:  PG  AcJvity  

Firmness  of  Fermented  Cucumber    Mesocarp    Tissue:  Average  Peak  Puncture  Force  

Indicators  of    

Secondary  

FermentaJon  

0  2  4  6  8  

10  12  14  

28   129  Average  Pe

ak  Force  (N

)  

Time  (Day)  

NaCl  Control  CaCl2  (100mM)  AceJc  (0mM)  CaCl2  (100mM)  AceJc  Acid  (25mM)  

0  2  4  6  8  

10  12  14  

28   129  Average  Pe

ak  Force  (N

)  

Time  (Day)  

NaCl  Control  CaCl2  (100mM)  AceJc  (0mM)  GL(0g/gal)  CaCl2  (100mM)  AceJc  (0mM)  GL(18g/gal)  CaCl2  (100mM)  AceJc  (0mM)  GL  (36g/gal)  

0  2  4  6  8  10  12  14  

28   129  

Average  Pe

ak  Force  (N

)  

Time  (Day)  

NaCl  Control  CaCl2  (100mM)  AceJc  Acid  (25mM)  GL  (0g/gal)  CaCl2  (100mM)  AceJc  Acid  (25mM)  GL  (18g/gal)  CaCl2  (100mM)  AceJc  Acid  (25mM)  GL(36mM)  

1.E+02  

1.E+03  

1.E+04  

1.E+05  

1.E+06  

1.E+07  

1.E+08  

1.E+09  

0   1   2   3   4   5   6   7   8   9  

MRS

 (CFU

/ml)  

Time  (Day)  

0  20  40  60  80  

100  120  140  

0   20   40   60   80   100   120   140  

LacJc  Ac

id  (m

M)  

Time  (Day)  

NaCl  Control  CaCl2  (100mM)  GL(0g/gal)  AceJc  (25mM)  CaCl2  (100mM)  GL  (18g/gal)  AceJc  (25mM)  CaCl2  (100mM)  GL  (36g/gal)  

Lactobacillus  plantarum  on  Cucumber  Tissue  

1.E+02  

1.E+03  

1.E+04  

1.E+05  

1.E+06  

0   1   2   3   4   5   6   7   8   9  

VRBG

 (CFU

/ml)  

Time  (Day)  

Under  Limit  of  DetecJon  by  day  5