Accepted Manuscript Low molecular weight liquid media development for Lactobacilli producing bacteriocins Journal of Chemical Technology and Biotechnology

7/29/2019 Accepted Manuscript Low molecular weight liquid media development for Lactobacilli producing bacteriocins Journa

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Low Molecular Weight Liquid Media Development

For Lactobacilli Producing Bacteriocins

Journal: Journal of Chemical Technology & Biotechnology

Manuscript ID: JCTB-12-0436.R1

Wiley - Manuscript type: Original Article

Date Submitted by the Author: n/a

Complete List of Authors: Zacharof, Myrto; Swansea University, College of EngineeringLovitt, Robert; Swansea University, College of Engineering

Key Words:Process Optimisation, Industrial Microbiology, Filtration, Fermentation,Biotechnology, Biomass

http://mc.manuscriptcentral.com/jctb-wiley

Journal of Chemical Technology & Biotechnology


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Correspondence concerning this article shold be addressed to M.P.Zacharof at [email protected]

_____________________________________________________________________________

Low Molecular Weight Liquid Media Development For Lactobacilli Producing Bacteriocins

Myrto-Panagiota Zacharof

Multidisciplinary Nanotechnology Centre, Swansea University, Swansea, SA2 8PP, UK

Robert W. Lovitt

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

___________________________________________________________________________________________

Background: Contemporary purification techniques of Lactobacilli bacteriocins include chemical precipitation and

separation through solvents to obtain highly potent semi purified bacteriocins. These methods are laborious and

bacteriocinsyields are low. To address this problem a set of new, efficient, cost effective media, was created,

containing low molecular weight nutrient sources (LMWM). Using these media future separation and concentration

of the desired metabolic products, using ultra- and nanofiltration from the cultured broth was possible.

Results: The LMWM were made through serial filtration (filters varying in pore size 30kDa, 4 kDa and 1 kDa

MWCO) of a modified optimum liquid medium for Lactobacilli growth. The developed media were tested for

bacteriocin production and biomass growth, using three known bacteriocin producing Lactobacilli strains,

Lactobacillus casei NCIMB 11970, Lactobacillus plantarum NCIMB 8014, Lactobacillus lactis NCIMB 8586. All

were successfully grown ( max 0.16 to 0.181

h ) on the LMWM and produced a significant amount of

bacteriocins in a range of 110 to 130 IU/ml.

Conclusions: LMWM do support Lactobacilli growth and bacteriocin production, establishing an alternative to the

current production nutrient media. The uptake of the nutrient sources is facilitated as nitrogen sources which were

primarily responsible for growth were supported in less complex forms.

Keywords: Lactobacilli, bacteriocins, low molecular weight medium, yield, filtration

Introduction

Since the industrialisation of food production, food safety has been an issue of great importance. Naturally

occurring, food deterioration and spoilage due to microbial agents has been the main source of hardship in todays

food industry. Numerous preservation methods, have been used to prevent food poisoning and contamination. These

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,

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lactate, and acetate). Regardless their proven success and effectiveness, there is an increasing demand for naturally

accruing , non artificial, biologically safe products providing the consumers with high health benefits1,5

.

Currently lactic acid bacteria and especially Lactobacilli have attracted great attention, due to the production of

antimicrobial peptide compounds namely bacteriocins2. Lactobacilli are widely applied in the food industry as

natural acidifiers. Their potential use as bacteriocidal agents would constitute a great commercial benefit. The use

of Lactobacilli produced bacteriocins, is generally considered as safe (GRAS, Grade One). Most of Lactobacilli

bacteriocins are small (


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Although the bacteriocin preparations had high potency, the methods were laborious and total recovery yields were

low17-20

. This is due to many other proteins from the medium can also be precipitated, since for the culturing of

Lactobacilli complex media are used such as Man de Rogosa (MRS) broth21

.

However, for the successful development of cellular biomass and bacteriocin productivity the use of suitable

nutrient media is of crucial importance, as growth media assimilate and define the nutritional conditions

determining the growth yield and the metabolites productivity of the selected bacteria. Lactobacilli have complex

nutritional needs, with several researchers34-39, 42-44

highlighting their growth dependence over minerals, such as

manganese and magnesium, vitamins of the B complex, amino acids such as serine and adenine and organic

compounds. Commercially available media for Lactobacilli propagation include Man De Rogosa medium, (MRS)

which is the most commonly used, Elliker broth, Lactobacillus -Streptococcus Differential Agar (LS agar) and Al

Purpose Tween agar (APT). Although these media, often used for research purposes, do ensure bacterial growth,

they do not support fastidious growth, or high biomass yields due to the plethora of nitrogen sources they contain39-

41. Especially in the case of MRS extensive use of beef or poultry extract (peptone) does causes environmental

(undischarged waste) and health(potential CJD- prion disease or H1N1 virus) hazards, while the complexity of

nutrients leads to highly expensive media fabrication, unsuitable for economically viable mass production

process22-24

.

MRS, though is a well established growth medium specifically designed to support the growth of Lactobacilli. It

contains rich nutrient sources suitable to support the high auxotrophic needs of these organisms. It can be easily

prepared and it was highly selective, its rich content of nitrogen sources and minerals ensure the bacterial growth

but do not support fastidious growth and high biomass yields. In addition, its cost of fabrication due to the materials

needed remains relatively high.

To address these hurdles a serie of new, efficient, cost effective media, capable of further improvements was

created, containing low molecular weight nutrient sources (LMWM). The development of the LMWM was

proposed mainly to facilitate the future separation and concentration of the desired metabolic products, using ultra-

and nanofiltration from the cultured broth. Additionally, the uptake of the nutrient sources would be and was indeed

facilitated as nitrogen sources which were primarily responsible for growth were supported in less complex forms.

The LMWM were made through serial filtration (filters varying in pore size 30kDa, 4 kDa and 1 kDa MWCO) of an

modified liquid medium which had been already established as the most suitable of the selected Lactobacilli. The

developed media were tested for bacteriocin production and biomass growth, using three known bacteriocin

producing Lactobacilli strains, Lactobacillus casei NCIMB 11970, Lactobacillus plantarum NCIMB 8014,

Lactobacillus lactis NCIMB 8586.

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Materials and Methods

Bacterial strains

Lactobacillus plantarum NCIMB 8014, Lactobacillus lactis NCIMB 8586, Lactobacillus casei NCIMB 11970 andthe target strain Lactobacillus delbruckii subsp. lactis NCIMB 8117 were provided in a lyophilised form by

National Collection of Industrial Food and Marine bacteria(NCIMB) , Aberdeen , Scotland, United Kingdom.

Culturing Conditions

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

20 g L-1

glucose, yeast extract (Y.E) 20 g L-1

, sodium acetate 10 g L-1

, tri-sodium citrate 10 g L-1

, potassium

hydrogen phosphate 5 g L-1

. In all the experimental procedures the media are dispersed in the 100ml capacity serum

vials, under anaerobic conditions (nitrogen flow), and sealed with butyl rubber stoppers (Fischer Scientific, UK) and

alumina seals (Wheaton Industries, USA). They are autoclaved (120C for 15 min) (Priorclave: Tactrol 2, RSC/E,

UK) and left to cool down, for 12h. The inoculum size was is 10% v/v. The tubes were incubated for 12 h at 36C.

Measurement of cellular growth and biomass

Determination of cell growth was monitored as an increase of turbidity in terms of optical density (O.D.) at 660 nm

wavelength into a spectrophotometer (PU 8625 UV/VIS Philips, France). The light path of the tube was 1.8 cm.

Measuring the O.D. was carried out on an hourly basis until it reached the late stationary phase. The growth curves

were obtained by plotting the O.D. against time. The maximum and specific growth rates ( max ,1

h and , 1h )

of bacteria were calculated from the logarithmic plots of the O.D. versus time during the exponential growth phase,

according to the formula:

(1

h )=DT

2ln

dt

)x(lnd

dt

dx

x

1== (1)

where DT (h) =x

)1t2t( (O.D. at 660nm hourly basis) (2)

Nutrient media Membrane Filtration

The modified optimised liquid medium containing 20 g L-1

glucose, yeast extract (Y.E) 20 g L-1

, sodium acetate 10 g

L-1

, tri-sodium citrate 10 g L-1

, potassium hydrogen phosphate 5 g L-1

was used for fabrication low molecular

weight media (LMWM). A bench membrane apparatus (stirred cell unit reactor, Amicon 8200 ,Millipore Co., UK)

was used for the filtration of the nutrient media, operated batchwise (Figure 1). The reactor system was composed

of a stirred cell unit of 200 ml maximum process volume, a magnetic stirrer and a filtration effective area of 28.7

cm. The stirrer speed was set at 150 rpm. Filtration of media was achieved through series of ultrafiltration and

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nanofiltration membranes The molecular weight cut-off (MWCO) of ultrafiltration polysulphone membranes in use

was 30 kDa (cellulose acetate, Microdyn-Nadir Co., Germany) and 4 kDa (polysulfone, Microdyn-Nadir Co.,

Germany) while nanofiltration 1kDa (polysulfone, General Electric-Osmonics Co. USA). 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% v/v) for 24h. The

membranes were rinsed with distilled water and sterilised with 25% v/v ethanol solution.

Determination of permeate flux, membrane resistance and cake resistance were resulting from the standard

equations25

used for evaluating membrane performance, the flux was defined as

=

mA

fQJ

(3)

for the determination of transmembrane pressure (P) was defined as

permeateoutinl P

2

PPTMPP

+==

(4)

The membrane resistance was defined by Darcys law as

=*J

PR m (5)

Each membrane was characterised under different pressure conditions varying between 0 to 400 kPa with the

following solutions, sterilised distilled water, 10mM phosphate buffer )( 42POKH buffer (Sigma-Aldrich, UK) and

sterilised basal medium. For each experimental run 150ml of the selected solution was inserted in the reactor.

Determination of Protein Sources in Low Molecular Weight Media by Gravimetry

In order to measure the content of proteins in the resulting solutions the gravimetric method was used26

. 2 ml of

each medium category were placed in glass plates of 10 mm diameter equipped with membrane filters (Whatman

0.2 m qualitative filters, UK) and weighted in a high precision electronic scale (0.1 mg Ohaus, V12140 Voyager,

Switzerland). The samples were placed in 100C furnace (Heraus Furnace, UK) for 24 h. After that, the samples

were weighted again in the same scale and the estimated difference was the content of solids in the medium.

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Determination of Protein Sources in Low Molecular Weight Media through High Precision Particle Sizer (HPPS)

The principle of function of High Precision Particle Sizer is based onDynamic Light Scattering (DLS also known as

PCS - Photon Correlation Spectroscopy, or QELS - Quasi-Elastic Light Scattering), which measures Brownian

motion of the particles in a solution and relates this to the size of the particles 27. This is done by illuminating the

particles with a laser beam and analysing the intensity fluctuations of the scattered light..The relationship between

the size of a particle and its speed due to Brownian motions is defined as the Stokes-Einstein equation

d3

kTDr

= (8)

where k (1.3807*10 J/K) is the Boltzmann constant, T is the absolute temperature in Kelvin (K), and is the

vicosity (8.937*10 kg/ms) of the medium in which the particles of diameter d (meters, m) are suspended. The

HPPS system measures the rate of the intensity fluctuation and then uses this to calculate the size of the particles.

The size of the particles is graphical represented into curves where the highest peak represents the majority of

molecules in the specific size given by the peak28

.In order to measure the size of the molecules 4 ml of each

medium, both autoclaved and non autoclaved (unfiltered, 4 kDa LMWM &1 kDa LMWM) were placed in plastic

cuvettes and put in the apparatus. The apparatus was connected with a personal computer equipped with the special

software programme (Malvern Instruments LDT. DTS 4.20, 2002) and all the measurements were done

automatically.

Determination of Protein Sources in Low Molecular Weight Media through High Performance Liquid Chromatography

In order to further purify and also to confirm the fact that bacteriocins were indeed produced by the selected strains,

purification techniques had to be used. All the analysis of the commercially available nisin and bacteriocins was

done using a High Performance Liquid Chromatography (HPLC) method. The HPLC system was connected with a

UV/Vis detector (Dionex, UK) and fitted with a C18 reverse phase column (Vydac 238 TP54, HPLC Columns,

UK.) which is used to detect small polypeptides less than 4,000-5,000 MW, enzymatic digest fragments, natural and

synthetic peptides and complex carbohydrates. The solvent (mobile phase) delivery system was constructed by 2

pumps (pumps A and B) (Varian Co.Canada.) with a pressure operation range between 1500 and 1900 mbar.

Temperature control of the solvents was maintained with a hotplate (Millipore Co., UK) at 25C.

The mobile phase was represented by two solutions, solvent A consisted of 99% pure acetonitrile (ACN) 10% v/v in

distilled water and 1% v/v of standard buffer solution and solvent B of 99% pure ACN75% in distilled water and

1% v/v of standard buffer solution. The standard buffer solution consisted of 7.5% trifluroacetic acid (TFA) 5 % v/v

triethylamine (TEA) and 65% of 99% pure ACN in distilled water. The solutions were delivered to the pumps via

plastic tubes and valves. The mobile phase was organised as gradient, consisting of 65% of solvent A and 35% of

solvent B. The flow rate of the samples and of the mobile phase was set at 1.5 ml/min for 15 minutes, and the

wavelength used was 220nm.The operation of the system was controlled automatically using Prostar Workstation

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Data analysis software package (Varian Co., Canada). Each run lasted for 17 min. All the samples were injected in

the system via a sterile HPLC plastic syringe (1 ml sterile syringe, Fischerbrand, UK) at a 20 l injection loop

connected with the HPLC system.

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

assay29

.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 .

Lactobacillus delbruckii subsp.lactis 8117 was 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

20 g L-1

glucose, 20 g L-1

Y.E., 10 g L-1

sodium acetate, 10 g L-1

tri-sodium citrate, 5 g L-1

di-hydrogen

orthophosphate, magnesium sulphate 0.5 g L-1

,manganese sulphate 0.05 g L-1

. This medium was also used when

testing the effect of bacteriocins and nisin.

Into glass tubes containing 8 ml of optimised medium including metals , 1 ml of the frozen inoculum of L.delbruckii

and 1 ml of the supernatant resulting from pH control fermentations of differential concentration is added29

. 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.

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) for complete biomass removal. 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 medium29

.

Separation of Produced Bacteriocins on Low Molecular Weight Media Using Filtration Technology

A bench membrane apparatus (stirred cell unit reactor, Amicon 8200, Millipore Co., UK) was used for the filtration

of the cultured LMWM and unfiltered optimised media cell free (via centrifugation) supernatants, operatedbatchwise. The cell unit was constantly pressurized by compressed nitrogen at 200 kPa. The reactor system was

composed of a stirred cell unit of 200 ml maximum process volume, a magnetic stirrer and a filtration effective area

of 28.7 cm. The cultured cell free supernatant were filtered through a nanofiltration membrane of 1 kDa weight cut-

off (polysulfone, General Electric-Osmonics Co. USA).

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Numerical Analysis of the Experimental Data

Each differential parameter was triplicated to obtain the average data. The data were statistically analysed for

accuracy and precision calculating standard deviation, standard error, experimental error, regression factor and

reading error (Microsoft Excel software Version 2003). All the numerical data proven to be highly accurate and

reproducible having standard deviation of mean below 5% and experimental error below 5% offering highly

significant results.

Results and Discussion

Membrane Characterisation and Filtrability of the Nutrient Medium

In order to determine the membrane resistance and the influence of pressure during the operation of the equipment,

membrane characterization studies were carried out.

The permeability of distilled water, optimized nutrient medium and phosphate buffer (10mM) solution through the

membranes of different MWCO was measured in order to analyze the behavior of the reactor system. The

permeability of distilled water, phosphate buffer solution and optimum nutrient medium through the membrane was

measured to analyze the membraness behavior (30 kDa, 4 kDa and 1 kDa MWCO) when incorporated in the unit.

The flux values linearly increased with increasing pressure. In the case of 30 kDa MWCO membrane, for pure water

the flux increased from 7.90 to 28.00 m/m/h with an increase in pressure from 50 to 400 kPa. For phosphate

buffer solution the flux increased from 1.95 to 9.05 m/m/h with an increase in pressure from 50 to 400 kPa. While

operating with optimized nutrient medium the flux was lower, from 0.79 to 2.80 m/m/h, with an increase in

pressure from 50 to 400 kPa respectively. For the 4kDa MWCO membrane, the flux values from for all solutions

linearly increased with increasing pressure. Pure water the flux increased from 0.23 to 1.20 m/m/h, with an

increase in pressure from 50 to 400 kPa. For phosphate buffer solution the flux increased from 0.14 to 1.11 m/m/h,

with an increase in pressure from 50 to 400 kPa. While operating with optimized nutrient medium the flux was

lower from 0.09 to 0.56 m/m/h with an increase in pressure from 50 to 400 kPa respectively. Lastly, for 1 kDa

MWCO membrane, For pure water the flux increased from 0.04 to 0.16 m/m/h, with an increase in pressure from

50 to 400 kPa. For phosphate buffer solution the flux increased from 0.02 to 0.13 m/m/h with an increase in

pressure from 50 to 400 kPa. While operating with optimized nutrient medium the flux was lower, from 0.008 to

0.08 m/m/h with an increase in pressure from 50 to 400 kPa respectively. The membrane resistance values were

rising during the filtration of the solutions at different pressure; in the case of 4 kDa and 1 kDa MWCO membranes,

there were smaller when compared with the values of the 30 kDa membrane although the operating conditions were

the same. This was probably due to the difference of the fabrication material of the membrane itself as well as due

to the pore size and the general porosity of the filter.

During the filtration of the nutrient medium, flux decline during the course of time was noticed due to the deposition

of organic macromolecules on the surface of the selected membranes, suggesting successful retention of larger

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molecules by the membranes. This is also assumed by the membrane resistance numerical values ( Table 1), due to

the cake layer formed on the membrane surfaces (Figure 2). Having proven the filterability of the developed

medium through the chosen membranes, the next step was to test the efficacy and the efficiency of the filtration

method for the formation of low molecular weight nutrient media.

Determination of the Low molecular weight nutrient sources in the Developed nutrient media

Gravimetry was used, so to measure the remaining nutrient sources in the autoclaved nutrient media after each

filtration process (Table 2). The nutrient sources were partially retained from the membrane filter during the

filtration process, allowing low molecular weight nutrient sources to pass through the membrane filters, resulting in

the production of the desired nutrient medium. All gravimetric analyses depend on final determination of weight as

a means of quantifying an analyte. Weight can be measured with greater accuracy than any other fundamental

property, gravimetric analysis is possibly one of the most accurate and commonly used methods of analytical

chemistry available. In this case though only the suspended solids can be defined, suggesting that there is successfulremoval of solids by the membrane filters. So to define the size and the volume of the remaining nitrogen sources in

the filtered media were measured through the Dynamic Light Scattering (DLS). This method provided higher

accuracy and credibility of the results as solely the protein sources deriving from yeast extract were measured in the

medium, due to the methods high sensitivity(


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membrane filter. When the medium was filtered through this filter the protein sources were sizing up to 1 nm

suggesting that only oligopeptides were present in the solutions.

High performance liquid chromatography (HPLC) was selected to further characterise the nutrient sources in the

developed media. This method was highly suitable for quantifying and analyzing mixtures of chemical compounds

due to its high sensitivity and specificity, especially in peptides and oligopeptides, and has been used by numerous

researchers30-33

in an effort to investigate protein substances in complex solutions. The protein sources were

successfully detected (Figure 5) suggesting sufficient presence of protein sources in the resulting media ( Table 3),

certifying as well the removal of larger protein molecules.

Testing the low molecular weight nutrient media for Lactobacilli growth and bacteriocin production

As LMWM were successfully developed, next step was to investigate whether they could sufficiently support

Lactobacilli growth providing high biomass yields and amounts of bacteriocin. A comparative study was made

between the standard nutrient media used for this study, and the developed LMWM of 4 kDa and 1 kDa molecular

weight sources. The LMWM can support the growth of the selected Lactobacilli, although the maximum growth

rates achieved were small, when compared to the optimised unfiltered medium. Further investigation in order to

achieve higher growth yields, was made by incorporating the metal ions of manganese (0.5 g L-1

) and magnesium

(0.05 g L-1

) salts in the media of 4kDa and 1 kDa sources, as there was a strong possibility the ions to be retained by

the membranes, potentially due to their aggregation with higher molecular weight nutrient sources. The selected

Lactobacilli were growing better proving as well the dependence of growth of the selected bacteria from the metal

ions (Table 4).

Successfully grown on LMWM, Lactobacilli strains had to be tested for bacteriocin productivity. The pre-treated

supernatants of each selected Lactobacilli, grown on optimised modified media and LMWM, were tested for

bacteriocin activity against the selected target strain L.delbruckii subsp.lactis. All the three media categories can

equally support bacteriocin production and even in higher amounts when the bacteria were grown in LMWM (Table

5). The comparative studies conducted served also in investigating whether there was a qualitative difference in the

activity of bacteriocins against the target strain due to the growth of their producers on different media categories. It

can be though seen that the bacteriocins deriving from the Lactobacilli grown on LMW medium of 1 kDa had the

weaker potency.

Separation of Produced Bacteriocins on Low Molecular Weight Media Using Filtration Technology

Filtration was the selected extraction and concentration method that could also enhance the potency of the

produced bacteriocins. Cultured broths solutions produced on all the media categories, were filtered through a 4

kDa and 1kDa MWCO membrane filter. The resulting retentates were tested for bacteriocin activity (Table 6). The

resulting retentates containing produced bacteriocins had a stronger antimicrobial activity, with the bacteriocins

becoming more potent. In the case though of the bacteriocins developed on the optimised unfiltered medium , the

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bacteriocin yield is only slightly enhanced. On the contrary, in the case of LMWM bacteriocins the potency is

significantly reinforced, resulting in a successfull separation. Filtration is proven to be a highly successful method

for separation of the substances from the nutrient broths, being relatively inexpensive and quite easy to implement.

Conclusions

The above studies indicate the ability of the developed media of 4kDa and 1 kDa LMWM , to support the

production of antimicrobial peptide substances during growth of the selected Lactobacilli. These substances are

proven to be equally effective towards the target strain, being highly potent, regardless the fact that Lactobacilli

were grown on different media categories. These results are encouraging as they indicate that these media can be

used when upscaling the bacteriocin production and purification using filtration as separation method, having solved

the problem of excess of proteins.

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[13]

26. Demicri A, Pomento AL, Lee B, Hinz P. Media evaluation of lactic acid repeated-batch fermentation with

Lactobacillus plantarum and Lactobacillus casei subsp.rhamnosus. J Agricul Food Chem 1998; 46:4771-

4774.

27. Callister WDJ. Fundamentals of Materials Science and Engineering: An Integrated Approach(2nd edition).

New York :John Wiley and Sons, Inc.,2004.

28. Moachon N et al. Influence of the charge of low molecular weight proteins on their efficacy of filtration

and/or adsorption on dialysis membranes with different intrinsic properties. J Biomaterials 2001; 23: 651-

658.

29. Zacharof MP, Lovitt RW. (2012) Investigation of Shelf Life of Potency and Activity of the Lactobacilli

Produced Bacteriocins Through Their Exposure to Various Physicochemical Stress Factors. Probiot &

Antimicrob Prot DOI 10.1007/s12602-012-9102-2

30. Van Reenen ML, Dicks LMT, Chikindas ML. Isolation, purification and partial characterisation of

plantaricin 423 a bacteriocin produced by Lactobacillus plantarum. J Appl Microbiol 1998; 84: 1131-1137.

31. Todorov SD, Vaz-Velho M, Gibbs D. Comparison of two methods for purification of Plantaricin ST31, a

bacteriocin produced by Lactobacillus plantarum ST31 JBraz Microbiol 2004; 35:157-160

32. Mierau I, Lei JP. Industrial scale production and purification of an heterogenous protein in L.lactis using

the Nisin-controlled gene expression system NICE: The case of lysostaphin. Microb Cell Fact 2005; 4: 1-9.

33. Zendo T, Nakayama J, Fujita. K, Sonomoto K. Bacteriocin detection by liquid chromatography /mass

spectrometry for rapid identification J Applied Microbiol 2008; 104: 449-507.

34. Dembczynski R., Jankowski, T. Growth characteristics and acidifying activity of Lactobacillus rhamnosus

in alginate/ starch liquid core capsules. Enz Microb Technol 2002; J 31: 111-115.

35. Desjardins P., Meghrous J., Lacroix C. Effect of aeration and dilution rate of nisin Z production during

continuous fermentation with free and immobilized Lactococcus lactis UL719 in supplemented whey

permeate Int Dairy J 2001;11:943-951.

36. Hoefnagel M. H. N.(2002) Metabolic engineering of lactic acid bacteria, the combined approach: kinetic

modelling , metabolic control and experimental analysis. J Microbiol 2002;148: 1003-1013.

37. Bober J. A., Demicri A. Nisin Fermentation by Lactococcus Lactis subsp.lactis using plastic composite

supports in biofilm reactors. Agr Eng Int: the CIGR J Scien Resear Devel 2004; 6: 1-15.

38. Deegan L.H., Cotter P.D., Colin H., Ross P. Bacteriocins: biological tools for bio-preservation and shelf-

life extension J Int Dairy 2006; 16:1058-1071.

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[14]

39. Konings W.N., Kok J., Kulipers O., Poolman B. Lactic Acid Bacteria : The bugs of the new millennium.

Cur Opin Microbiol 2000; 3: 276-282.

40. Liew S.L., Ariff A. B., Racha A.R., Ho Y.W. Optimization of medium composition for the production of a

probiotic microorganism Lactobacillus rhamnosus using response surface methodology. Int J Food

Microbiol 2005; 102:137-142.

41. Ostlie H.M., Helland M. H., Narvhus J.A., Growth and metabolism of selected strains of probiotic bacteria

in milk Int J Food Microb 2003; 87: 17-27.

42. Todorov S.D., Dicks M.T. Growth parameters influencing the production of Lactobacillus rhamnosus

bacteriocins STR461BZ and ST462BZ Annals Microb 2005;55:283-289

43. Mollendorff von J.W., Todorov S.D., Dicks M.T Optimization of growth medium for production of

bacterocin produced by Lactobacillus plantarum JW3BZ and JW6BZ and Lactobacillus fermentum

JW11BZ and JW15BZ isolated from boza Trakia J Sci 2009:7, 22-33

44. A Aktypis A., Tychowski M., George Kalantzopoulos G., Aggelis G. Studies on bacteriocin (thermophilin

T) production by Streptococcus thermophilus ACA-DC 0040 in batch and fed-batch fermentation modes

Ant van Leeuw 2007; 92:207-220

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Figure 1. : Schematic diagram of the stirred cell (Amicon cell 8200 Manual, Sterlitech USA) (1) cap, (2) pressure relief valve, (3) pre

ring, provides seal to maintain pressure in the unit, (5) magnetic impeller , provides cross flow conditions , (6) main body of the s

seal to maintain pressure in the unit and prevent loss of sample, (8) base with permeate outlet, (9) screw in bottom to secure bas

and (11) retaining stand prevents displacement of cap when pressure is used in the unit.

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Figure 2: Deposition of solids forming a cake on the outer layer of the ultrafiltration (a,30 kDa) (b, 4 kDa) and nanofiltration (c, 1 k




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Figure 3: Size distribution of media particles in a non autoclaved media , unfiltered (a) and filtered through (b,30 kDa) (c,

membranes

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Figure 4: Size distribution of media particles in autoclaved media, unfiltered (a) and filtered through (b,30 kDa) (c, 4 kDa) and nan




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Figure 5: HPLC Analysis of autoclaved media, unfiltered (a) and filtered through (b,30 kDa) (c, 4 kDa) and nanofiltration (d, 1 kDa)

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Nutrient Media Permeate Flux (J, m3 s-1) Membrane Resistance (Rm,m

30 kDa filtered medium 1.86*10-

1.17*10

LMWM (4 kDa) 9.43*10-

2.24*10

LMWM (1 kDa) 1.05*10-

2.14*10

Table 1: Flux and membrane resistance of the serial filtration of the developed media

Table 2: The effect of filtration on the dry weight content of the media

Nutrient medium Solids (g L-

)

Unfiltered medium 0.09

30 kDa filtered medium 0.08

LMWM (4 kDa) 0.03

LMWM (1 kDa) 0.02




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Table 3:Chromatographic analysis of the developed media

Nutrient Media Retention Time (min) Width Area (m

Optimised Unfiltered Medium 1.063 6.061.225 19.281.534 19.361.831 32.81

30 kDa filtered nutrient medium 1.087 6.281.209 14.531.297 6.281.575 1.022

LMWM (4 kDa) 1.104 6.341.214 16.53

LMWM (1 kDa) 1.112 5.721.237 15.63

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Selected

Strains

Unfiltered MediumLMWM

(4kDa)

LMWM

(4kDa incl. Metal ions)

LM

(1

Maximum growth

rate( 1h )

Final Biomass(g/L) Maximum

growth

rate( 1h )

Final

Biomass(g/L)

Maximum

growth

rate( 1h )

Final

Biomass(g/L)

M

ra

L.casei0.24 2.43 0.18 1.65 0.19 1.60 0.

L.plantarum0.30 2.63 0.16 1.33 0.22 2.03 0.

L.lactis

0.22 1.81 0.16 1.65 0.21 1.70 0.

Table 4: Growth of the selected Lactobacilli on the developed media




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Table 5: Bacteriocin Production on the three different media categories

Unfiltered Medium LMWM


LMWM


Lactobacilli Maximum

growth

rate( 1h )

Indicator

strain

Amount of

Bacteriocin

Produced

(IU/ml).

Maximum

growth

rate( 1h )

Indicator

strain

Amount of

Bacteriocin

Produced

(IU/ml)

Maximum growth

rate( 1h )

Indicator strain

L.casei 0.13 110 0.12 115 0.12

L.plantarum 0.13 110 0.09 130 0.12

L.lactis 0.10 125 0.10 125 0.11

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Table 6: Activity of Extracted Bacteriocins of the three different media categories

Unfiltered Medium LMWM


LMWM


Lactobacilli Maximum

growth

rate(1h )

Indicator

strain

Amount of

Bacteriocin

Produced

(IU/ml).

Maximum

growth

rate(1h )

Indicator

strain

Amount of

Bacteriocin

Produced

(IU/ml)

Maximum growth

rate(1h )

Indicator strain

L.casei 0.12 115 0.005 165 0.004

L.plantarum 0.11 120 0.003 180 0.002

L.lactis 0.09 130 0.002 185 0.007




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Literature Cited

1. Carr JG, Cutting CV, Whiting GC, Lactic Acid Bacteria in Beverage and Food. (1stedition)New York: Academic Press LTD., 1975.

2. Ross RP., Desmond C, Fitzgerald GF, Stantch C. Overcoming the technological hurdlesin the development of probiotic foods. J Appl Microbiol 2005; 98: 1410-1417.

3. Rodriguez E., Martinez MI, Horn N, Dodd HM, Heterologous production of bacteriocinsby Lactic Acid Bacteria. Int J Food Microbiol 2003;80: 101-116.

4. Rodriguez EGB, Gaya P, Nanez M, Medina M. Diversity of bacteriocins produced byLactic Acid Bacteria isolated from raw milk. Int Dairy J 2000; 10:7-15.

5. Chen H, Hoover DG. Bacteriocins and their food applications. Compr Reviews Food ScFood Saf 2003; 2: 83-97.

6. Moll GN., Konings WN, Driessen, AJM., Bacteriocins: mechanism of membraneinsertion and pore formation Anton van Leeuw 1999;3: 185-195.

7. Daw MA, Falkiner FR. Bacteriocins: nature, function and structure Micron J 1996;27:467-479.

8. Jack RW, Tagg, JR, Ray B. Bacteriocins of Gram-positive bacteria. Microbiol Reviews1995; 3:171-200.

9. Mierau I. Optimization of the Lactococcus lactis nisin-controlled gene expression systemNICE for industrial applications. Microb Cell Fact 2005;4: 16-28.

10. Ross RP., Desmond C, Fitzgerald GF, Stantch C. Overcoming the technological hurdlesin the development of probiotic foods. J Appl Microbioly 2005; 98:1410-1417.

11. Cleeveland J, Montville TJ., Nes IF, Chikindas ML, Bacteriocins : safe, naturalantimicrobial for food preservation Int J Food Microbiol 2001; 71: 1-20.

12. Board RG. A Modern Introduction to Food Microbiology. (1st edition), New York:Blackwell Scientific Publications, 1983.

13. Paul Ross R Morgan S, Hill S, Preservation and Fermentation : past , present and future.Int J Food Microbiol 2002; 79: 3-16.

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14. Berridge N J. Preparation of the antibiotic nisin Biochem 1949; 45: 486-492.15. Cheeseman, GC, Berridge NJ. Observation on molecular weight and chemical

composition of nisin A. Biochemistry J 1968; 71:185-195.

16. White HR, Hurst A. The location of nisin in the producer organism Streptococcus lactis.J. Gen Microbiol 1968; 3, 171-179.

17. Maldonado A, Barda-Ruiz J, Jimenez-Diez R, Purification and Genetic characterizationof plantaricin NC8, a novel culture-inducible two-peptide bacteriocin from Lactobacillus

plantarum NC8. J Appl and Environm Microbiol 2003; 69: 383-389.

18. Todorov SD, Van Reenen C, Dicks LM, Optimization of bacteriocin production byLactobacillus plantarum ST13BR, a strain isolated from barley beer. J Gen Appl

Microbiol 2004; 50: 149-157.

19. Uteng M. et al. Rapid two-step procedure for large-scale purification of pediocin-likebacteriocins and other cationic antimicrobial peptides from complex culture medium J

App and Environ Microbiol 2002; 5: 952-956.

20. Deraz S, Karlsson E, Hedstorm M, Andersoon M, Mattiason B. Purification andcharacterisation of acidocin D20079, a bacteriocin produced by Lactobacillus acidophilus

DSM 20079. J Biotech 2005; 117: 343-354.

21. Bujalance C, Jimenez-Valera M, Moreno E, Ruiz-Bravo A. A selective differentialmedium for Lactobacillus plantarum. J Microbiol Meth 2006; 66: 572-575.

22. Prusiner S, Scott MR, Stephen J, DeArmond, SJ, Cohen FE. Prion Protein BiologyCell,1998; 93: 337348

23. Johnson RT, Gibbs CJ. CreutzfeldtJakob Disease and Related TransmissibleSpongiform Encephalopathys Review Article New England J Medicine 1998; 339:

1994-2004.

24. Foster PR. Prions and blood products Annals Medicine, 2000; 32:1365-2060.

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25. Coulson JM, Richardson, JF. Chemical Engineering, Chemical and BiochemicalReactors and Process control. (3rd edition), Oxford, Pergamon Press,1994.

26. Demicri A, Pomento AL, Lee B, Hinz P. Media evaluation of lactic acid repeated-batchfermentation with Lactobacillus plantarum and Lactobacillus casei subsp.rhamnosus. J

Agricul Food Chem 1998; 46:4771-4774.

27. Callister WDJ. Fundamentals of Materials Science and Engineering: An IntegratedApproach(2nd edition). New York :John Wiley and Sons, Inc.,2004.

28. Moachon N et al. Influence of the charge of low molecular weight proteins on theirefficacy of filtration and/or adsorption on dialysis membranes with different intrinsic

properties. J Biomaterials 2001; 23: 651-658.

29. Zacharof MP, Lovitt RW. (2012) Investigation of Shelf Life of Potency and Activity ofthe Lactobacilli Produced Bacteriocins Through Their Exposure to Various

Physicochemical Stress Factors. Probiot & Antimicrob Prot DOI 10.1007/s12602-012-

9102-2

30. Van Reenen ML, Dicks LMT, Chikindas ML. Isolation, purification and partialcharacterisation of plantaricin 423 a bacteriocin produced by Lactobacillus plantarum. J

Appl Microbiol 1998; 84: 1131-1137.

31. Todorov SD, Vaz-Velho M, Gibbs D. Comparison of two methods for purification ofPlantaricin ST31, a bacteriocin produced by Lactobacillus plantarum ST31 J Braz

Microbiol 2004; 35:157-160

32. Mierau I, Lei JP. Industrial scale production and purification of an heterogenous proteinin L.lactis using the Nisin-controlled gene expression system NICE: The case of

lysostaphin. Microb Cell Fact 2005; 4: 1-9.

33. Zendo T, Nakayama J, Fujita. K, Sonomoto K. Bacteriocin detection by liquidchromatography /mass spectrometry for rapid identification J Applied Microbiol 2008;

104: 449-507.

ge 27 of 29




29/30

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[4]

34. Dembczynski R., Jankowski, T. Growth characteristics and acidifying activity ofLactobacillus rhamnosus in alginate/ starch liquid core capsules. Enz Microb Technol

2002; J 31: 111-115.

35. Desjardins P., Meghrous J., Lacroix C. Effect of aeration and dilution rate of nisin Zproduction during continuous fermentation with free and immobilized Lactococcus lactis

UL719 in supplemented whey permeate Int Dairy J 2001;11:943-951.

36. Hoefnagel M. H. N.(2002) Metabolic engineering of lactic acid bacteria, the combinedapproach: kinetic modelling , metabolic control and experimental analysis. J Microbiol

2002;148: 1003-1013.

37. Bober J. A., Demicri A. Nisin Fermentation by Lactococcus Lactis subsp.lactis usingplastic composite supports in biofilm reactors. Agr Eng Int: the CIGR J Scien Resear

Devel 2004; 6: 1-15.

38. Deegan L.H., Cotter P.D., Colin H., Ross P. Bacteriocins: biological tools for bio-preservation and shelf-life extension J Int Dairy 2006; 16:1058-1071.

39. Konings W.N., Kok J., Kulipers O., Poolman B. Lactic Acid Bacteria : The bugs of thenew millennium. Cur Opin Microbiol 2000; 3: 276-282.

40. Liew S.L., Ariff A. B., Racha A.R., Ho Y.W. Optimization of medium composition forthe production of a probiotic microorganism Lactobacillus rhamnosus using response

surface methodology. Int J Food Microbiol 2005; 102:137-142.

41. Ostlie H.M., Helland M. H., Narvhus J.A., Growth and metabolism of selected strains ofprobiotic bacteria in milk Int J Food Microb 2003, 87, 17-27.

42. Todorov S.D., Dicks M.T. Growth parameters influencing the production ofLactobacillus rhamnosus bacteriocins STR461BZ and ST462BZ Annals Microb2005;55:283-289

43. Mollendorff von J.W., Todorov S.D., Dicks M.T Optimization of growth medium forproduction of bacterocin produced by Lactobacillus plantarum JW3BZ and JW6BZ and

Page 28




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[5]

Lactobacillus fermentum JW11BZ and JW15BZ isolated from boza Trakia J Sci 2009:7,

22-33

44. A Aktypis A., Tychowski M., George Kalantzopoulos G., Aggelis G. Studies onbacteriocin (thermophilin T) production by Streptococcus thermophilus ACA-DC 0040

in batch and fed-batch fermentation modes Ant van Leeuw 2007; 92:207-220

ge 29 of 29 Journal of Chemical Technology & Biotechnology

Documents

Accepted Manuscript Low molecular weight liquid media development for Lactobacilli producing bacteriocins Journal of Chemical Technology and Biotechnology