Accepted Manuscript Investigation of Shelf life of Potency and Activity of the Lactobacilli Produced...

7/29/2019 Accepted Manuscript Investigation of Shelf life of Potency and Activity of the Lactobacilli Produced Bacteriocinsthro

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*Corresponding author. Tel. +44(0)7413541769.

E-mail address: [email protected]

Investigation of Shelf life of Potency and Activity of theLactobacilli Produced Bacteriocins

through their Exposure to Various Physicochemical Stress factors

M.P.Zacharof* and R.W. Lovittb

aMultidisciplinary Nanotechnology Centre, Swansea University, Swansea, SA2 8PP, UKb

College of Engineering, Multidisciplinary Nanotechnology Centre, Swansea University, Swansea, SA2 8PP, UK

Abstract

ThreeLactobacilli strains,Lactobacillus casei NCIMB 11970, Lactobacillus plantarum NCIMB 8014, Lactobacillus

lactis NCIMB 8586 have been used for the production of bacteriocins. Though, their production phase, their

biochemical nature , their mode of activity even their genetic structure have been widely investigated, there are

hardly any studies investigating their potency and activity in depth of time, in other words their self life under

several physicochemical conditions that may occur during their production in large scale. As such the effect of

several factors influencing the activity and the potency of bacteriocins when produced in large scale were examined

as due to bacteriocins peptide nature degradation or denaturation might occur, under extreme physicochemical

conditions. During scale up process, differences between the output data may occur, such as concerning biomass,

metabolic by products and limiting substrate concentrations. These may affect negatively, the activity and the

potency of the bacteriocins. For investigating these effects and minimising them, numerous studies were conducted,

which were related to the exact phase of the production of these substances, the effect of dilution and temperature

changes. These studies could be used in order to minimise the scaling up effect when decided to produce these

peptides in large scale.

Keywords: Lactic acid bacteria,Lactobacilli, Bacteriocins, Heat Treatment, Potency, Activity, Nisin

Introduction

A great number of Gram positive (+) bacteria and Gram negative (-) bacteria produce during their growth,

substances of protein structure (either proteins or polypeptides) possessing antimicrobial activities, called

bacteriocins[3]. Bacteriocins limit their activity to strains of species related to the producing species and particularly

to strains of the same species. Bacteriocins are ribosomally synthesised and produced during the primary phase of

growth, though antibiotics are usually secondary metabolites [1].

They mostly have low molecular weight (rarely over 10 kDa). They are posttranslational modified and are quickly

degraded by proteolytic enzymes especially by the proteases of the human gastrointestinal tract, that makes them


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safe for human consumption. Bacteriocins, in general, are cationic, amphipathic molecules as they have an excess of

lysyl and arginyl residues [3, 4]. Usually unstructured when incorporated in aqueous solutions, but when exposed to

structure promoting solvents such as triofluroethanol or with anionic phospholipids membranes, they form a helical

scheme. [5]

LAB have gained extensive attention nowadays, because of their ability to produce of bacteriocins [10], as the use

of LAB and of their metabolic products is generally considered as safe (GRAS, Grade One). The application of their

produced antimicrobial compounds as a natural barrier against pathogens and food spoilage caused by bacterial

agents has been proven to be efficient [2].

Numerous preservation methods have been used in order to prevent food poisoning and spoilage. These techniques

include thermal treatment (pasteurization, heating sterilisation), pH and water activity reduction (acidification,

dehydration) and addition of preservatives (antibiotics, organic compounds such as propionate, sorbate, benzoate,

lactate, and acetate). Although these methods have been proven to be highly successful, there is an increasing

demand for natural, microbiologically safe products providing the consumers with high health benefits [20].

Examples of the use of bacteriocins in the food industry include their application on dairy, egg, vegetable and meat

products, and these have been extensively investigated. Among the LAB bacteriocins, nisin A and its natural

variant nisin Z has been proven to be highly effective against microbial agents causing food poisoning and spoilage.

Furthermore nisin is the only bacteriocin that has been officially employed in the food industry and its use has been

approved worldwide [5].

Bacteriocins can be applied on a purified or on a crude form or through the use of a product previously fermented

with a bacteriocin producing strain as an ingredient in food processing or incorporated through a bacteriocin

producing strain (starter culture). The incorporation of a bacteriocin producing strain has the disadvantage of the

lack of compatibility between the bacteriocin producing strain and the other cultures required for fermentation [10],

[9]. However, it has been proven that a bacteriocin alone in a food is not likely to ensure complete safety; especially

in the case of Gram negative (-) bacteria this has been apparent. Then the use of bacteriocins has to be combined

with other technologies that are able to disrupt the cellular membrane so bacteriocins can kill the pathogenic

bacteria [9], [11]. For example the use of non-thermal treatments such as pulsed electric field (PEF) is advantageous

as it does not have any effect on food functionality and nutritional qualities. This technique may not be financially

viable when used alone, but in lower levels and combined with other treatments such as bacteriocins may be highly

effective [6]. Furthermore bacteriocins could be combined with other antimicrobial compounds such as sodium

acetate and sodium lactate resulting in enhanced inactivation of bacteria. Bacteriocins can also be used to improve

food quality and sensory properties, for example increasing the rate of proteolysis or in the prevention of gas

blowing defect in cheese [12].


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[3]

Another application of bacteriocins is bioactive packaging, a process that can protect the food from external

contaminants. For instance the spoilage of refrigerated food commonly begins with microbial growth on the surface

that reinforces the attractive use of bacteriocins being used in conjunction with packaging to improve food safety

and self-life [10]. Bioactive packaging can be prepared by directly immobilising the bacteriocin to the food

packaging or by addition of a sachet containing the bacteriocin into the packaged food, which will be released

during storage of the food product.

The gradual release of bacteriocins from a packaging film on the food surface may have an advantage over dipping

and spraying foods with bacteriocins, because antimicrobial activity may be lost or reduced due to inactivation of

the bacteriocins by food components or dilution below active concentration due to migration into the foods [10], [7].

The only commercially available bacteriocin from LAB is nisin which is produced by strains ofLactobacillus lactis

var. lactis. Several other bacteriocins from LAB have been identified throughout the last decade where research on

their production and purification techniques has been highly intensive, due to the growing need of replacement of

chemical food preservatives. Contemporary purification techniques of bacteriocins include chemical precipitation,

separation through solvents such as used a combination of acid treatment of the culture followed by removal of the

cells and then solvent extraction and precipitation to obtain nisin with high potency and mainly high performance

liquid chromatography or reverse phase chromatography [8],[14,15,16,17],[19],[22].Most methods rely on

ammonium sulphate precipitation of the bacteriocins from cell-free cultured broth. These methods have been used to

obtain bacteriocins from LAB such asLactobacillus spp.,Leuconostoc spp., Pediococcus spp. and Lactococcus spp.

Nevertheless, it has been agreed that low yields of bacteriocins are achieved with these methods. This is due, to

many other proteins from the medium can also be precipitated. For further purification of precipitated bacteriocins

and for the determination of the amino acid composition and sequence, several researchers [19], [8], [14, 15, 16,

17], [22] have implemented have used various column chromatography techniques and sodium dodecyl sulfate-

polyacrylamide gel electrophoresis (SDS-PAGE). Commercial nisin preparations are available in highly purified

food-grade form. However, the methods used commercially are not known [29]. Other researchers [24], [23], [25]

have tried several different methods based on propanol- sodium chloride or butanol-acetic acid extraction from

culture supernatant and breaking of cells and extraction with acid. Although the nisin preparations had high potency,

the methods were laborious and total yields were low.

As such, the need for investigating the production of bacteriocins in large scale emerged. One step towards thatdirection, was to examine the effect of several factors influencing the activity and the potency of bacteriocins. These

substances, if and when produced in large scale due to their peptide nature, degradation or denaturation might occur.

For investigating these effects and minimising them, numerous studies were conducted. These were related to the

exact phase of the production of these substances, the effect of dilution and temperature changes. These studies

could be used in order to minimise the scaling up effect when decided to produce these peptides in large scale.


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Materials and Methods

Materials

The yeast extract, peptone, glucose, sodium acetate, trisodium citrate, NaOH were bought from Sigma-Aldrich

Chemicals, UK.

Inoculum source

All the Lactobacilli, Lactobacillus casei NCIMB 11970, Lactobacillus plantarum NCIMB 8014, Lactobacillus

lactis NCIMB 8586and the target strain Lactobacillus delbruckii subsp. lactis NCIMB 8117were provided in a

lyophilised form by National Collection of Food and Marine bacteria(NCIMB) , Aberdeen , Scotland.

Culturing Conditions

All the three bacteriocin producing strains bacteria were cultured in modified optimised liquid medium containing

2% w/v glucose, yeast extract (Y.E) 2% w/v, sodium acetate 1% w/v, tri-sodium citrate 1% w/v, potassium

hydrogen phosphate. This medium was used to perform anaerobic, temperature and pH controlled batch

fermentation in a 2.5 L stirring tank pyrex glass reactor. The temperature maintained was 36C while the stirring

speed was 200 rpm allowing sufficient mixing and agitation.

Membrane Filtration

A bench membrane apparatus (stirred cell unit reactor, Amicon 8200) was used for the filtration of the media. The

reactor system was composed of an ultrafiltration stirred cell unit of 200 ml maximum process volume, a magnetic

stirrer and an effective area of 28.7 cm. (Millipore Co., UK). The stirrer speed was set at 50 rpm through the series

of experiments concerning the bacteriocin concentration, which was achived through filration by a serie of

membranes. The molecular weight cut-off (MWCO) of ultrafiltration polysulphone membranes in use was 30 kDa,

4 kDa and 1kDa. The filters were provided from Millipore Co., UK, (30 kDa) from Microdyn-Nadir Co., Germany

(4 kDa),and from General Electric-Osmonics Co. USA (1kDa). The cell unit was pressurizes by constant

compressed nitrogen at 200 kPa. The operating temperature was controlled at 25C constantly by connecting via

rubber tubes the cell unit water jacket with a water bath (Grant Water bath, UK). The stirred cell unit was operated

in a batch dead-end mode. After each experiment, the components of the unit cell were soaked into an ethanol

solution (50%) for 24h. The membranes were rinsed with distilled water and sterilised with 25% v/v ethanol

solution.


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Measurement of cellular growth and biomass

The cellular growth was measured by placing the pressure tubes into a spectrophotometer fitted with a test tube

holder (PU 8625 UV/VIS Philips, France) at 600 nm. The tube had a 1.8 cm. light path. The doubling time and the

specific growth rate of the strain in the presence of bacteriocin were evaluated according to the formula:

(1h )=

DTdt

xd

dt

dx

x

2ln)(ln1== where DT (h) (Equation 1)

DT (h) =n

tt )( 12 (O.D. at 600nm hourly basis) (Equation 2)

Determination of Nisin and Bacteriocin Activity and Potency

The activity and the potency of nisin and the produced bacteriocins was tested according to a simple turbidometric

assay[26].This assay was based on the effect of several different concentration of commercial nisin against a target

strain , in terms of growth rate. Into 25 ml of 0.02 M of HCl 25mg of nisin are dispersed. This solution equals to

1000 IU/ml of nisin. According to this formula the necessary quantities of solid nisin were calculated to fabricate

standard solution at the following concentrations: 0, 25, 50, 75, 85, 100, 110, 125, 150, 175, 200, 250, 500, 750,

1000, 1250, 1500, 1750, 2000 IU/ml. The solutions are preserved stable (up to 30 days) into 4C [26].

Lactobacillus delbruckii subsp.lactis 8117was selected as the target strain. The inoculum was consistent in growth

phase,as it was frozen when the growth reached 1.5 g/L. The target strain was grown on a liquid medium containing

2% w/v glucose, 2% w/v Y.E., 1% w/v sodium acetate, 1% w/v tri-sodium citrate, 0.5% w/v, magnesium sulphate

0.05% w/v ,manganese sulphate 0.005% w/v. This medium was also used when testing the effect of bacteriocins

and nisin

Into glass tubes containing 8 ml of optimised medium including metals ,so to ensure that any effect on growth of the

tested microorganism results from the bacteriocin produced and not due to any other factors such as nutrient

exhaustion of optimum anaerobic medium for the growth of the tested strain L.delbruckii. 1 ml of the frozen

inoculum ofL.delbruckii and 1 ml of the supernatant resulting from pH control fermentations of differential

concentration is added [26].The samples are gently mixed, and incubated statically at 36C. The biomass was

recorded on an hourly basis by measuring the turbidity is a photometer(PU 8625 UV/VIS Philips, France) at 600

nm.


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The amount of the bacteriocin produced by each under investigation strain was primarily defined on the samples

taken at the end of pH and temperature controlled fermentations. The selected samples (pH fermentation at 6.5)

were transferred into 10 ml conical plastic tubes (Fisherbrand, UK) and centrifuged (10.000 rpm for 15 min.)

(Biofuge Stratos Sorall, Kendro Products, Germany) in order to remove completely the biomass. The clarified liquid

was filtrated through a 0.2 m pore size filter for sterilisation. The sterilised liquids pH was adjusted at 6.0 to

eliminate the antimicrobial effect of lactic acid and then it was diluted with fresh medium [25].

Testing the Activity and the Potency of the Commercially Available Nisin and the Produced Bacteriocins

through Dilution

The bacteriocins were harvested by separating the cells through centrifugation (4000 rpm for 10 min.) (Biofuge

Stratos Sorall, Kendro Products, Germany) and collecting the remaining supernatants into 50 ml plastic conical

sterile tubes (Fisherbrand, UK).The collected supernatants, namely crude extracts were diluted with distilled water

in a factor of 4. The remaining supernatants were filtered through a 0.2 m pore size filter (Whatman qualitative

filters, UK) and were neutralised to avoid any interference with lactic acid with 1 M solution of NaOH and were

serially diluted with distilled sterilised water in a factor of 4. The samples were tested for potency against the

indicator strainL.delbruckii and their activity was calculated in international units per millilitre (IU/ml). In the case

of the commercially available nisin a positive and a negative control were firstly tested .The negative control is

consisted of by 8 ml of nutrient medium, 1 ml of distilled water and 1ml of tested indicator strain though the

positive control is consisted of 8ml of nutrient medium 1ml of nisin solution 1000 IU/ml and 1ml of indicator strain.

The tubes were gently mixed and incubated at 36C (Thermo Scientific Series 6000 Incubator, USA) for 10 h. (O.D

at 600nm hourly basis). The crude extracts were also filtered through 4 kDa (Microdyn-Nadir Co., Germany) and 1

kDa MWCO membrane filters, so to be concentrated. (General Electric- Osmonics Co. USA). The concentrated

bacteriocins were then serially diluted in a factor of 4 with distilled sterilised water so to test their potency and

activity.


with Heat Treatment

Initially a stock solution of 1000 IU/ml of nisin was made up. The solution was equally dispersed in a glass serum

vials which were incubated at temperatures of 40, 60, 80 and 100C for 15, 30, 45, and 60 minutes time interval in

each temperature. After the end of each set of incubation experiment the solutions were tested again using the

indicator strain. Microfiltration of the 1000 IU/ml Nisin solution of was filtered through a 30 kDa membrane filter.

The resulting supernatant was tested for activity against the indicator strain. The same process was followed for the

bacteriocins which were produced and harvested as stated previously.


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Through Storage effect on low temperatures

The stability of the bacteriocins in low temperatures had to be tested. Initially a 1000 IU/ml nisin solution was

fabricated. The solutions were heat treated for 80C and 15 minutes and then kept in 4C for 12, 48, 60, 72, 84 and

96h. After each set of storage the solutions were tested against the indicator strain. The same procedure was kept

also in the case of microfiltration of nisin and of the bacteriocins produced.

Numerical Analysis of the Experimental Data

All the experimental data that gathered were processed through Microsoft Excel software Version 2003. Each

differential parameter was triplicated to obtain the average data (statistical data variation


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(Figure 2) but is roughly linear with respect to the log of the concentration of the preparation. There are small

numerical differences in terms of growth parameters between the effect of all the bacteriocin on the target strain

although it can clearly be seen that L.plantarum and L.casei bacteriocin have the strongest potency even under

extreme dilution conditions.

Studies were conducted on the concentrated retentates derived from each medium using 4kD and 1 kDa MWCO

membrane filters. Using these retentate preparations in a dilution assay, the potency was then estimated.

The extracted broth treated so as to avoid any interference from other antimicrobial agents existing in the nutrient

broth was filtered through a 4 kDa MWCO membrane and diluted with sterilised distilled water in a factor of 1:4.

The resulting retentates were tested against the target strain L.delbruckii, in parallel with undiluted concentrated

retentate. Under the influence ofL.casei concentrated and diluted permeateL.delbruckii has a maximum growth rate

of 0.171h (DT: 4.05 h) (Figure 3) was observed equivalent to 90 IU/ml bacteriocin equivalent to nisin. While the

concentrated undiluted sample a maximum growth rate of 0.141h (DT: 4.92 h) with 105 IU/ml bacteriocin

equivalent to nisin. As for L.plantarum (Figure 4) the concentrated and diluted permeates ofL.delbruckii had a

maximum growth rate of 0.161h and a DT: 4.31 h was observed, with 95 IU/ml bacteriocin produced. The

concentrated undiluted sample had a maximum growth rate of 0.131h (DT: 6.30 h) with 110 IU/ml bacteriocin,

equivalent to nisin.

Tested against the concentrated undiluted and diluted permeate ofL.lactis though L.delbruckii has a maximum

growth rate of 0.161h and a doubling time of 4.31 h were achieved, (95 IU/ml bacteriocin) though for the

concentrated undiluted sample a maximum growth rate of 0.131

h and a doubling time of 6.30 h were achieved,

(110 IU/ml bacteriocin) (Figure 5). L.delbruckii normal growth on the optimized unfiltered medium was used as

control.L.delbruckii has a maximum growth rate of 0.311h and a doubling time of 2.22 h.

The same process was also repeated concentrating the crude extracts with 1kDa MWCO. For L.casei concentrated

and diluted retentate L.delbruckii has a maximum growth rate of 0.161h and a doubling time of 4.31 h are

achieved, (95 IU/ml bacteriocin) (Figure 6) though for the concentrated undiluted sample a maximum growth rate

of 0.121h and a doubling time of 6.75 h are achieved (115 IU/ml bacteriocin). The concentrated and diluted

retentate ofL.plantarum (Figure 7) onL.delbruckii has a maximum growth rate of 0.171h and a doubling time of

4.05 h are achieved (90 IU/ml bacteriocin). Though for the concentrated undiluted sample a maximum growth rate

of 0.141h and a doubling time of 4.92 h are achieved (105 IU/ml bacteriocin). When tested against the

concentrated and washed retentates ofL.lactis (Figure 8) though L.delbruckii has a maximum growth rate of

0.171h and a doubling time of 4.05 h are achieved, (90 IU/ml bacteriocin) though for the concentrated undiluted

sample a maximum growth rate of 0.131h and a doubling time of 6.30 h are achieved (110 IU/ml bacteriocin).


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L.delbruckii normal growth on the optimized unfiltered medium was used as control; L.delbruckii has a maximum

growth rate of 0.231h and a doubling time of 3.0 h.

Dilution was chosen to study the potency of the produced substances. It had an effect on all the crude extracts of

bacteriocins produced on the optimised media. The preparations were more potent when grown on unfiltered.

Interestingly, the effect of dilution gave a correlation that was a function of the concentration of the bacteriocin

preparation. This implies that the specific potency of the materials inhibition per unit amount of bacteriocin is more

effective at low concentration that at high concentrations. The mode of action may be that the first interaction of

bacteriocin with the membrane is very powerful and after this initial stage the effects are reduced.

Stability of Potency and Activity of Nisin and Bacteriocins against the target strainL.delbruckii

As the selected bacteriocins were successfully extracted and concentrated from the nutrient broths, their stability of

the activity, (the duration of their antimicrobial activity against the target strain) was determined. Primarily the

stability of commercially available nisin in a concentration of 1000 IU/ml was tested against the target strain

L.delbruckii in a time length of 36 h. The testing included untreated nisin solution and nisin retentates samples as a

1000 IU/ml was filtered through a 4 kDa and a 1 kDa MWCO membrane filters. This experiment was used as a

guideline in order to test the stability of the crude extracts of bacteriocins as well as the stability of the retentates of

the substances collected after filtration through a 4 kDa and1 kDa MWCO membrane filters. Under the solutions of

nisin both concentrated and untreated no growth occurs within 36 h. A slight difference in biomass in the case of

filtered nisin in second 24 h is due to the change of scaling. The positive control, the normal growth ofL.delbruckii,

has reached the stationary phase during the second 24 h., having a growth rate 0.321h and a doubling time of 2.15

h. during the first 24h. The stability of bacteriocins produced from the selected Lactobacilli was tested against the

target strainL.delbruckii. The stability was tested in a 72 h time length and as samples the crude extracts from the

cultured broths were used. The crude extracts produced from the selectedLactobacilli, when grown on an unfiltered

optimised medium, have a clear bacteriostatic effect that deactivates the growth of the target strain. The positive

control ofL.delbruckii, has reached the stationary phase during the second and third 24 h., having a growth rate 0.28

1h and a doubling time of 2.45 h. during the first 24h.

The life span following the same experimental process, of the highly concentrated bacteriocins through a 1 kDa

membrane (concentration factor of 1:10) and, produced from the selected Lactobacilli on all the 3 media categories,

was tested against the target strainL.delbruckii. The stability was tested in a 36 h time length as under the influence

of the concentrated bacteriocin the target strain was entering death phase soon after the 1st

24h.

The numerical values are tabulated in Table 1; though in the case of the treated samples in the 48h the target strain

has already started entering the death phase.


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The concentrated bacteriocins can indeed have a stronger bacteriostatic effect when compared with the crude

extracts as indicated by the rapid decline in the stationary culture. The target strain gets in the death phase in the

second 24h declaring a much stronger effect of the bacteriocin strain although the positive control is still in the

stationary phase.

Testing the Activity and Potency of the Heat treated solutions of Nisin

Substances of protein nature easily deteriorate and become inactive when exposed into high temperatures. The

potency of nisin and of the produced bacteriocins was tested through heat treatment. Nisin has been reported to be a

thermostable molecule though this fact had to be confirmed. Also the produced bacteriocins had to be tested for heat

stability. It has also been reported that nisin is sensitive to proteases produced during growth which may degrade the

bacteriocins quickly. In an effort to remove any such protease existing in the solutions the commercially available

nisin and the produced bacteriocins were filtered through a 30 kDa membrane filter (Millipore Co., UK) which was

previously soaked overnight in sterile distilled water, to enhance their porosity, and then tested against the indicator

strain.

According to several researchers nisin is a thermostable molecule that can maintain its bacteriostatic activity against

a wide range of bacteria without denaturising [27, 28]. To confirm this and to further investigate the potency of

bacteriocins under several physicochemical conditions nisin and the bacteriocins produced from the selected

Lactobacilli on all the 3 media categories were treated with heat .The temperatures selected were 40C, 60C, 80C

and 100 C and the duration of treatment with heat varied between 15, 30 45 and 60 minutes. Nisin activity against

the target strain remains relatively unaffected from heat indicating that the molecule is indeed quite thermostable.

The activity of nisin though is slightly degraded after certain amount of time in temperatures above 40C implyingthat the molecule gets denaturised under high temperature conditions. The numerical values are tabulated below

(Table 2). The previous results serve as a guideline in order to further test the effect of heat on the several

substances produced on the three different media categories.

Testing Shelf life of Potency and Activity of the treated solutions of Nisin and selected Bacteriocins

through Storage on Low Temperature

In order to test the shelf life- in a depth of time- of the activity and potency of the commercially available nisin and

bacteriocin solutions produced by the selected Lactobacilli all the solutions were stored in 4 C up to 96 h. As

methods of treatment, in order to test the shelf life, microfiltration and heat were chosen. These methods were used

so as to avoid any interference factors such as protease enzymes in the case of nisin or other antimicrobial factors

such as lactic acid or degrading enzymes in the case of bacteriocins.

In the case ofL.casei microfiltration is proven to be the most effective method of treatment of the substance as heat

diminishes the substances activity against the target strain (Figure 9). The substance remains strongly activity up to


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48h of storage. When L.plantarum crude extracts, untreated and treated with heat, microfiltration and stored up to

96h in 4C, were tested for potency and activity against the target strain, the results were similar in all the cases,

(Figure 10) implying that the bacteriocins are not losing their activity under extreme physical conditions and can

withstand storage up to 72h. In the case ofL.lactis crude extracts, which are treated with heat are losing partially

their potency when compared with the untreated or the micro filtered samples and stored up to 96h in 4C (Figure

11). The bacteriocin activity is remains strong up to 48h storage. In the case of nisin 1000 IU/ml its antimicrobial

activity remains unaffected even up to 96h of storage in low temperature (4 C) (data not shown).

It can be said that bacteriocins are thermostable molecules maintaining their potency up top 96h when stored in low

temperature. These studies imply that bacteriocins can be extracted from the nutrient broths even up to two days

after the end of fermentation provided the samples are stored in low temperatures.

As the potency was tested through dilution, the next phase was to establish the duration, the stability or the shelf life

of the antimicrobial activity of the produced bacteriocins against the target strain. Firstly, the duration of the

antimicrobial activity of the only commercially available bacteriocin, nisin was tested in order to model its effect and

then implement these results in the experimental processes developed for the testing of the shelf life of the produced

bacteriocins. The produced bacteriocins on all the three media categories were concentrated in a factor of 1:10

through a 4kDa membrane. Then their potency was tested against the target strain for a period of 72h. The

bacteriostatic effect was strong up to the first 48h though the target strain started getting in the death phase. As the

concentrated samples were highly active, further concentration was done with a 1kDa membrane filter.

The concentrated samples deriving from each media category were tested against the target strain. The target strain in

this case is entering in the death phase already from the 24h. Indeed the death phase of the organism seems to be

stimulated by the bacteriocin. This is consistent with the idea that an energy -nutrient limitation at the end of growth

is affected such that resistance this stress is reduced, i.e. the maintenance of cell viability is reduced in the presence in

the bacteriocin and this expressed as reduced growth rate and survival. As the duration of bacteriocin activity against

the target strain was established, further studies were carry out concerning the potency of the substance under several

physical conditions. The crude extracts were treated with heat and with filtration through a 30 kDa membrane filter.

A comparative study was made between the treated and the untreated crude extracts in order to establish the effect of

these conditions on the bacteriocin activity. The most sensitive bacteriocin is proven to be the one deriving from

L.lactis, as it is partially loses its potency when heated in 80C. Interesting are the results of nisin where when heat

treated, loses partially its activity against the target strain though when micro filtered its potency and activity remains

unaffected. This could be justified due to the encapsulation of nisin in casein micelles that they may be more sensitive

to heat treatment.


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Conclusions

The above studies indicate the existence of an antimicrobial peptide substances developing during growth in the

nutrient broth of the selected Lactobacilli. The mode of action and potency of the preparation was concentration

dependent and this deserved further investigation. These substances are proven to be sensitive when treated with in

very high temperatures but are relatively stable. They maintain though their stability and potency even up to 72 h of

storage facilitating their extraction and purification processes. These results are encouraging as they indicate that

these can be used when upscaling the bacteriocin production and purification.

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[12] Board R. G., A Modern Introduction to Food Microbiology. 1st ed., Blackwell Scientific Publications: 1983,p 1-50.

[13] Todorov S.D., Dicks L. M. T., Screening for bacteriocin -producing lactic acid bacteria from boza, a

traditional cereal beverage from Bulgaria. Comparison of bacteriocins. Process Biochemistry Journal 2006, 41, 11-

19.

[14] Todorov, S. D., Van Reenen, C., Dicks, L.M., Optimization of bacteriocin production byLactobacillus

plantarum ST13BR, a strain isolated from barley beer. Journal of General Applied Microbiology 2004, 50, 149-157.


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[15] Todorov, S. D., Vaz-Velho, M., Gibbs, D. , Comparison of two methods for purification of Plantaricin

ST31, a bacteriocin produced byLactobacillus plantarum ST31 Brazilian Journal of Microbiology 2004, 35, 157-

160.

[16] Todorov, S. D., Dicks L.M.T.,Lactobacillus plantarum isolated from molasses produces bacteriocins

active against Gram-negative bacteria. Enzyme and Microbial Technology Journal 2005, 36, 318-326.[17] Todorov S. D., Dicks L. M. T., Influence of Growth conditions on the production of a bacteriocin by

Lactococcus lactis subp. lactis ST 34BR, a strain isolated from barley beer. Journal of Basic Microbiology 2004, 44,

305-316.

[18] Todorov S. D. D., Dicks L.M.T., Effect on Growth medium on bacteriocin production by Lactobacillus

plantarum ST194BZ, a strain isolated from boza. Journal of Food Technology and Biotechnology 2005, 43, 165-

173.

[19] Uteng M. et al., Rapid two-step procedure for large-scale purification of pediocin-like bacteriocins and

other cationic antimicrobial peptides from complex culture medium Applied and Environmental Microbiology

Journal 2002, 5, 952-956.

[20] Carr J. G., Cutting, C. V., Whiting, G. C., Lactic Acid Bacteria in Beverage and Food. 1st ed., Academic

Press LTD.: 1975, p 17-28, 233-266.[21] Daw M.A, Falkiner F. R., Bacteriocins: nature, function and structure Micron Journal 1996, 27, 467-479.

[22] Deraz S., Karlsson E., Hedstorm M., Andersoon M., Mattiason B., Purification and characterisation of

acidocin D20079, a bacteriocin produced byLactobacillus acidophilus DSM 20079. Journal of Biotechnology 2005,

117, 343-354.

[23] Cheeseman GC, Berridge NJ., Observations on the molecular weight and chemical composition of nisin A.

Biochem J. 1959, 185194.

[24] Berridge NJ., Preparation of the antibiotic nisin. Biochem J. 1949,486493.

[25] White, H. R., Hurst A., The location of nisin in the producer organism Streptococcus lactis. J. Gen.

Microbiol. 1968, 3, 171-179.

[26] M.P.Zacharof and R.W. Lovitt, "Development of an Optimised Growth Strategy for Intensive Propagation,

Lactic Acid and Bacteriocin Production of Selected Strains ofLactobacilli Genus," International Journal of

Chemical Engineering and Applications vol. 1, no. 1, pp. 55-62, 2010.

[27] Delgrado A., Brito D., Feveiro P., Tenreiro R., Peres C., Bioactivity quantification of crude bacteriocin

solution Journal of Microbiological Methods 2005, 62, 121-124.

[28] Aymerich M. T., Garriga, M., Monfort J.M., Nes I., Hugas M., Bacteriocin-producingLactobacilli in

Spanish-style fermented sausages: characterisation of bacteriocins. Journal of Food Microbiology 2000, 17, 33-45.

[29] Yang R, Johnson MC, Ray B. Novel method to extract large amounts of bacteriocins from lactic acid

bacteria. Appl Environ Microbiol. 1992, 33553359.


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Lactobacilli

Bacteriocins

Bacteriocins Concentrated through a 4kDa MWCO Filter Bacterioc

24 48 72 24

Growth

rate

(, h-1)

Doublin

g time

(Td, h)

Potenc

y

(IU/ml)

Growt

h rate

(, h-

1)

Doublin

g time

(Td, h)

Potenc

y

(IU/ml)

Growt

h rate

(, h-

1)

Doublin

g time

(Td, h)

Potenc

y

(IU/ml)

Growt

h rate

(, h-

1)

D

g

(

L.casei 0.15 4.60 100 0.001 Stationa

ry phase

1000.0

No

growth

100 0.07 9

L.plantarum 0.16 4.31 95 0.007 Stationa

ry phase

950.0

No

growth

95 0.06 1

L.lactis 0.14 4.92 103 0.006 Stationary phase

1030.0

Nogrowth

103 0.06 1

Table 1 Growth of the target strainL.delbruckii under the influence of produced bacteriocin retentates (4 k

optimised medium


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[15]

Nisin 1000 IU/ml

15 min 30 min 45 min

TemperatureGrowth

rate

(, h-1)

Doubling

time

(Td, h)

Growth

rate

(, h-1)

Doubling

time

(Td, h)

Growth

rate

(, h-1)

Doubling

time

(Td, h)

40C No

growth

No

growth

No

growth

No

growth

No

growth

No

growth

60C No

growth

No

growth

No

growth

No

growth

No

growth

No

growth

80C No

growth

No

growth

No

growth

No

growth

0.02 34.5

100C 0.03 23 0.03 23 0.04 17.25

Table 2 Testing the effect of heat treated nisin solutions against the growth of the target strainL.delbruckii


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Figure 1 Effect of relative dilution on the potency of nisin solution 1000 IU/ml


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[17]

Figure 2 Potency ofL.casei () L.plantarum () &L.lactis () produced bacteriocin on optimised

media and relativedilution


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[18]

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 2 4 6 8 10

Time (h)

Biomass(g/L)

Figure 3 Effect of dilution on the concentrated retentate (4 kDa) ofL.casei bacteriocin () in parallel to undiluted concentrated retentate (4kDa)

()L.casei bacteriocin () and normal growth of the target strainL.delbruckii ()


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[19]

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 2 4 6 8 10

Time (h)

Biomass(g/L)

Figure 4 Effect of dilution on the concentrated retentate (4 kDa) ofL. plantarum bacteriocin () in parallel to undiluted concentrated retentate

(4kDa) ()L.plantarum bacteriocin () and normal growth of the target strainL.delbruckii ()


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[20]

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 2 4 6 8 10Time (h)

Biomass(g/L)

Figure 5 Effect of dilution on the concentrated retentate (4 kDa) ofL.lactis bacteriocin () in parallel to undiluted concentrated retentate (4kDa)

()L.lactis bacteriocin () and normal growth of the target strainL.delbruckii ()


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[21]

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 2 4 6 8 10Time (h)

Biomass(g/L)

Figure 6 Effect of dilution on the concentrated retentate (1 kDa) ofL.casei bacteriocin () in parallel to undiluted concentrated retentate (1 kDa)

()and normalgrowth of the target strainL.delbruckii ()


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[22]

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 2 4 6 8 10Time (h)

Biomass(g/L)

Figure 7 Effect of dilution on the concentrated retentate (1 kDa) ofL.casei bacteriocin () in parallel to undiluted concentrated retentate (1 kDa)

()and normalgrowth of the target strainL.delbruckii ()


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[23]

0

20

40

60

80

100

120

140

0 20 40 60 80 100 120

Time (h)

Bacteriocinamount(IU/ml)

Figure 9 Stability of potency ofL.casei bacteriocin up to 96h treated with heat (80 C) () treated with microfiltration (

) &without any

treatment ()


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[24]

0

20

40

60

80

100

120

140

0 20 40 60 80 100 120

Time (h)


Figure 10 Stability of potency of L.plantarum bacteriocin up to 96h treated with heat (80 C) () treated with microfiltration ()&without any treatment ()


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[25]

0

20

40

60

80

100

120

140

0 20 40 60 80 100 120

Time (h)


Figure 11 Stability of potency ofL.lactis bacteriocin up to 96h treated with heat (80 C) () treated with microfiltration () &without

any treatment ()

Accepted Manuscript Investigation of Shelf life of Potency and Activity of the Lactobacilli Produced...

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Transcript of Accepted Manuscript Investigation of Shelf life of Potency and Activity of the Lactobacilli Produced...