Accepted Manuscript Low Molecular Weight Liquid Media Development For Lactobacilli Producing Bacteriocins Journal of Chemical Technology and Biotechnology

Journal of Chemical Technology & Biotechnology
Low Molecular Weight Liquid Media Development For Lactobacilli Producing Bacteriocins
Journal: Manuscript ID: Wiley - Manuscript type:
Journal of Chemical Technology & Biotechnology JCTB-12-0436.R1 Original Article n/a
Date Submitted by the Author:
Complete List of Authors:
Key Words:
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Zacharof, Myrto; Swansea University, College of Engineering Lovitt, Robert; Swansea University, College of Engineering Process Optimisation, Industrial Microbiology, Filtration, Fermentation, Biotechnology, Biomass
http://mc.manuscriptcentral.com/jctb-wiley
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_____________________________________________________________________________ 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 0.18 h 1 ) 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
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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,
Correspondence concerning this article shold be addressed to M.P.Zacharof at myrtozacharof1981@yahoo.com
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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 (<10 kDa) cationic, heat-stable, amphiphilic and membrane permeabilizing peptides. Many of these bacteriocins appear to exhibit relatively little adsorption specificity and have greater antibacterial activity at lower pH values (below 5) by means that their adsorption to the cell surface of Gram positive (+) bacteria including the producing cells is pH dependent5. Lactobacilli bacteriocins have been proven to be a highly effective natural barrier against microbial agents causing food poisoning and spoilage5,6. Antimicrobial activity of bacteriocins is directed principally against other Gram positive (+) bacteria. The majority of Lactobacilli bacteriocins has been shown to be effective when used in sufficient amounts, towards a wide spectrum of Gram positive (+) bacteria, including Listeria and other species of Lactobacilli. However, a bacteriocin alone induced in a food product 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 bacteria7,8. Several other bacteriocins from Lactobacilli 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 10-13.
Regardless the wide variety of bacteriocins being produced by Lactobacilli, only nisin produced by Lactococcus lactis var. lactis previously known as Lactobacillus lactis var lactis is the commercially produced by Dupont (Nisaplin) and Sigma Aldrich (Nisin 2.5% purified). Nisin is utilised worldwide as a food additive, under the number E234 (ECCU 1983 EEC Commission Directive 8314631EEC) 9. The methods used for the commercially nisin available nisin are not known8 .
Contemporary purification techniques of bacteriocins include chemical precipitation, separation through solvents used in a combination with acid treatment
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extraction and precipitation, and high performance liquid chromatography or reverse phase chromatography. Currently, most methods rely on ammonium sulfate precipitation of the bacteriocins from cell-free cultured broth. These methods have been used to obtain bacteriocins from Lactobacillus spp., Leuconostoc spp., Pediococcus spp. and Lactococcus spp.
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of the culture followed by removal of the cells and then solvent
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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 contain3941
. 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 ultraand 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 and the 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 , h 1 and , h 1 ) of bacteria were calculated from the logarithmic plots of the O.D. versus time during the exponential growth phase, according to the formula:
where DT (h) =
Nutrient media Membrane Filtration
The modified optimised liquid medium containing 20 g L-1glucose, 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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( h 1 )=
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( t 2 t1 ) x
1 dx d (ln x ) ln 2 = = x dt dt DT
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(O.D. at 660nm hourly basis)
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(1) (2)
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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 equations 25 used for evaluating membrane performance, the flux was defined as
Qf J= A m
for the determination of transmembrane pressure (P) was defined as
The membrane resistance was defined by Darcys law as

Rm = P J *
Each membrane was characterised under different pressure conditions varying between 0 to 400 kPa with the following solutions, sterilised distilled water, 10mM phosphate buffer ( KH 2 PO4 ) 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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(3)
P +P P = TMP = inl out Ppermeate 2
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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 on Dynamic 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 particles27. 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
Dr = kT 3d
(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
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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 36 C. 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, operated batchwise. 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 cutoff (polysulfone, General Electric-Osmonics Co. USA).
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[8]
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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[9]
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 successful removal 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(<nm).
The nutrient sources contained in the non autoclaved medium filtered through a 30 kDa MWCO membrane filter were found to range in size between 1 nm to 30 nm. The nutrient medium was therefore thought to contain mostly polypeptides and needed further treatment. When filtered through the 4 kDa MWCO filter the nutrient medium contains protein sources sizing between 1 nm to 15 nm as it can be clearly stated from the nutrient medium contains mostly oligopeptides. Further filtration was performed through a 1 kDa MWCO membrane filter, where the protein sources were sizing between 1 nm to 5 nm suggesting that only oligopeptides were present in the solutions (Figure 3).
To use these LMWM for growth of Lactobacilli and bacteriocin production, sterilization is necessary. The LMWM were autoclaved and analysed again (Figure 4). During autoclaving, due to the high temperature and pressure applied, often reactions, such as caramelization of glucose, agglomeration, deterioration or inactivation of protein sources, as substances of protein nature easily deteriorate and become inactive when exposed into high temperatures of detrimental for the media quality, occur. When filtered through a 30 kDa MWCO membrane filter the nutrient sources contained in the autoclaved medium range in size between 1 nm to 30 nm, but with a higher percentage of proteins ranging in size between 1 nm to 10nm when compared with the non autoclaved media. The nutrient medium contains mostly polypeptides and needs further filtration. When filtered through the 4 kDa MWCO filter the nutrient medium contains particles between 1 nm to 20 nm. Most of the proteins range in size between 1 nm to 3 nm. The nutrient medium contained mostly oligopeptides. Further filtration was performed through a 1 kDa MWCO
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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
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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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ew
83-97. 6.
Moll GN., Konings WN, Driessen, AJM., Bacteriocins: mechanism of membrane insertion and pore formation Anton van Leeuw 1999;3: 185-195.
7. 8. 9.
Daw MA, Falkiner FR. Bacteriocins: nature, function and structure Micron J 1996;27: 467-479. Jack RW, Tagg, JR, Ray B. Bacteriocins of Gram-positive bacteria. Microbiol Reviews 1995; 3:171-200. Mierau I. Optimization of the Lactococcus lactis nisin-controlled gene expression system NICE for industrial applications. Microb Cell Fact 2005;4: 16-28.
10. Ross RP., Desmond C, Fitzgerald GF, Stantch C. Overcoming the technological hurdles in the development of probiotic foods. J Appl Microbioly 2005; 98:1410-1417.
Page 12 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
[12]
11. Cleeveland J, Montville TJ., Nes IF, Chikindas ML, Bacteriocins : safe, natural antimicrobial 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. 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 characterization of 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 by Lactobacillus 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-like bacteriocins 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 and characterisation 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 differential medium for Lactobacillus plantarum. J Microbiol Meth 2006; 66: 572-575. 22. Prusiner S, Scott MR, Stephen J, DeArmond, SJ, Cohen FE. Prion Protein Biology Cell, 1998; 93: 337348 23. Johnson RT, Gibbs CJ. CreutzfeldtJakob Disease and Related Transmissible Spongiform
Encephalopathys Review Article New England J Medicine 1998; 339: 1994-2004. 24. Foster PR. Prions and blood products Annals Medicine, 2000; 32:1365-2060. 25. Coulson JM, Richardson, JF. Chemical Engineering, Chemical and Biochemical Reactors and Process control. (3rd edition), Oxford, Pergamon Press,1994.
r Fo
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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; 46:47714774. 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: 651658. 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 J Braz 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 shelflife extension J Int Dairy 2006; 16:1058-1071.
r Fo
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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
r Fo
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er Re vi ew
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Fo
rP
ee
rR
Figure 1. : Schematic diagram of the stirred cell (Amicon cell 8200 Manual, Sterlitech USA) (1) cap, (2) pressure relief valve, (3) pressure tube fitting assembly, (4) top oring, provides seal to maintain pressure in the unit, (5) magnetic impeller , provides cross flow conditions , (6) main body of the stirred cell , (7) bottom o-ring provides seal to maintain pressure in the unit and prevent loss of sample, (8) base with permeate outlet, (9) screw in bottom to secure base in the main body, (10) permeate line and (11) retaining stand prevents displacement of cap when pressure is used in the unit.
ev
iew
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Fo
rP
ee
rR
ev
iew
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 kDa) membranes
Page 17 of 29
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Fo
rP
ee
rR
ev
iew
Figure 3: Size distribution of media particles in a non autoclaved media , unfiltered (a) and filtered through (b,30 kDa) (c, 4 kDa) and nanofiltration (d, 1 kDa) membranes
Page 18 of 29
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Fo
rP
ee
rR
ev
iew
Figure 4: Size distribution of media particles in autoclaved media, unfiltered (a) and filtered through (b,30 kDa) (c, 4 kDa) and nanofiltration (d, 1 kDa) membranes
Page 19 of 29
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Fo
rP
ee
rR
ev
iew
Figure 5: HPLC Analysis of autoclaved media, unfiltered (a) and filtered through (b,30 kDa) (c, 4 kDa) and nanofiltration (d, 1 kDa) membranes
Page 20 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
Nutrient Media 30 kDa filtered medium LMWM (4 kDa) LMWM (1 kDa)
Permeate Flux (J, m3 s-1)
Membrane Resistance (Rm,m-1)

1.17*1013 2.24*1014 2.14*1015
Fo
1.86*10-1 9.43*10-3
Table 1: Flux and membrane resistance of the serial filtration of the developed media
rP
1.05*10-4
Nutrient medium
ee
Unfiltered medium
30 kDa filtered medium LMWM (4 kDa) LMWM (1 kDa)
rR
Solids (g L-1)
ev
0.08 0.03 0.02
0.09
Table 2: The effect of filtration on the dry weight content of the media
iew
Page 21 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
Nutrient Media Optimised Unfiltered Medium
Retention Time (min)
Width Area (mVolts)
30 kDa filtered nutrient medium
LMWM (4 kDa) LMWM (1 kDa)
Fo
Table 3:Chromatographic analysis of the developed media
rP
ee
1.063 1.225 1.534 1.831 1.087 1.209 1.297 1.575 1.104 1.214 1.112 1.237
6.06 19.28 19.36 32.81 6.28 14.53 6.28 1.022 6.34 16.53 5.72 15.63
rR
ev
iew
Page 22 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
Unfiltered Medium
LMWM (4kDa)
LMWM (4kDa incl. Metal ions) Final Biomass(g/L) Maximum growth rate( h 1 ) Final Biomass(g/L)
LMWM (1kDa incl. Metal ions) Maximum rate( h 1 ) growth Final Biomass(g/L)
Selected Strains
Maximum rate( h 1 )
growth
Final Biomass(g/L)
Maximum growth rate( h 1 )
Fo
2.43 2.63 1.81
L.casei L.plantarum
0.24 0.30
rP
0.18 0.16 0.16
1.65
0.19 0.22
1.60 2.03
0.18 0.16
1.64 1.65
L.lactis
0.22
ee
1.33
rR
1.65
0.21
1.70
0.16
1.60
Table 4: Growth of the selected Lactobacilli on the developed media
ev
iew
Page 23 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
Unfiltered Medium
LMWM (4kDa incl. Metal ions)
LMWM (1kDa incl. Metal ions) of Maximum rate( h 1 ) Indicator strain growth Amount Bacteriocin Produced (IU/ml) of
Lactobacilli
Maximum growth
rate( h 1 ) Indicator strain L.casei L.plantarum L.lactis
Fo
Amount Bacteriocin
of
Maximum growth rate( h 1 ) Indicator
Amount Bacteriocin Produced (IU/ml)
rP
Produced (IU/ml).
0.13 0.13 0.10
110 110 125
ee
0.12 0.09 0.10
strain
rR
115 130 125
0.12 0.12 0.11
115 115 120
Table 5: Bacteriocin Production on the three different media categories
ev
iew
Page 24 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
Unfiltered Medium
LMWM (4kDa incl. Metal ions)
LMWM (1kDa incl. Metal ions) of Maximum rate( h 1 ) Indicator strain growth Amount Bacteriocin Produced (IU/ml) of
Lactobacilli
Maximum growth
rate( h 1
Indicator strain L.casei L.plantarum L.lactis
Fo
)
Amount Bacteriocin Produced
of
Maximum growth rate( h 1 Indicator
Amount Bacteriocin
0.12 0.11 0.09
rP
(IU/ml).
Produced (IU/ml)
115 120
ee
strain
0.005 0.003
165 180
0.004 0.002 0.007
170 185 155
130
0.002
rR
185
Table 6: Activity of Extracted Bacteriocins of the three different media categories
ev
iew
Page 25 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Literature Cited
1.
Carr JG, Cutting CV, Whiting GC, Lactic Acid Bacteria in Beverage and Food. (1st edition)New York: Academic Press LTD., 1975.
2.
Ross RP., Desmond C, Fitzgerald GF, Stantch C. Overcoming the technological hurdles in the development of probiotic foods. J Appl Microbiol 2005; 98: 1410-1417.
3.
Rodriguez E., Martinez MI, Horn N, Dodd HM, Heterologous production of bacteriocins by Lactic Acid Bacteria. Int J Food Microbiol 2003;80: 101-116.
4.
Rodriguez EGB, Gaya P, Nanez M, Medina M. Diversity of bacteriocins produced by Lactic 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 Sc Food Saf 2003; 2: 83-97.
6.
Moll GN., Konings WN, Driessen, AJM., Bacteriocins: mechanism of membrane insertion and pore formation Anton van Leeuw 1999;3: 185-195.
7.
Daw MA, Falkiner FR. Bacteriocins: nature, function and structure Micron J 1996;27:
r Fo
Pe
er
467-479.
8.
Jack RW, Tagg, JR, Ray B. Bacteriocins of Gram-positive bacteria. Microbiol Reviews 1995; 3:171-200.
9.
Mierau I. Optimization of the Lactococcus lactis nisin-controlled gene expression system NICE for industrial applications. Microb Cell Fact 2005;4: 16-28.
10.
Ross RP., Desmond C, Fitzgerald GF, Stantch C. Overcoming the technological hurdles in the development of probiotic foods. J Appl Microbioly 2005; 98:1410-1417.
11.
Cleeveland J, Montville TJ., Nes IF, Chikindas ML, Bacteriocins : safe, natural antimicrobial 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.
Re
vi
ew
Page 26 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
[2]
14. 15.
Berridge N J. Preparation of the antibiotic nisin Biochem 1949; 45: 486-492. 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 characterization of 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 by Lactobacillus 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-like bacteriocins 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 and characterisation 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 differential medium for Lactobacillus plantarum. J Microbiol Meth 2006; 66: 572-575.
22.
Prusiner S, Scott MR, Stephen J, DeArmond, SJ, Cohen FE. Prion Protein Biology Cell, 1998; 93: 337348
23.
Johnson RT, Gibbs CJ. CreutzfeldtJakob Disease and Related Transmissible Spongiform Encephalopathys Review Article New England J Medicine 1998; 339: 1994-2004.
24.
Foster PR. Prions and blood products Annals Medicine, 2000; 32:1365-2060.
r Fo
Pe
er
Re
vi
ew
Page 27 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
[3]
25.
Coulson JM, Richardson, JF. Chemical Engineering, Chemical and Biochemical Reactors and Process control. (3rd edition), Oxford, Pergamon Press,1994.
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-
r Fo
Pe
er
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 J Braz 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.
Re
vi
ew
Page 28 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
[4]
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 biopreservation 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 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
r Fo
Pe
er
Re
vi
ew
Page 29 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
[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 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
r Fo
Pe er Re vi ew

Accepted Manuscript Low Molecular Weight Liquid Media Development For Lactobacilli Producing Bacteriocins Journal of Chemical Technology and Biotechnology

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Accepted Manuscript Low Molecular Weight Liquid Media Development For Lactobacilli Producing Bacteriocins Journal of Chemical Technology and Biotechnology

Transféré par

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Journal: Manuscript ID: Wiley - Manuscript type:

Journal of Chemical Technology & Biotechnology JCTB-12-0436.R1 Original Article n/a

Date Submitted by the Author:

Complete List of Authors:

Journal of Chemical Technology & Biotechnology

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

Correspondence concerning this article shold be addressed to M.P.Zacharof at myrtozacharof1981@yahoo.com

Journal of Chemical Technology & Biotechnology

of the culture followed by removal of the cells and then solvent

Journal of Chemical Technology & Biotechnology

Journal of Chemical Technology & Biotechnology

Materials and Methods

(O.D. at 660nm hourly basis)

Journal of Chemical Technology & Biotechnology

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

The membrane resistance was defined by Darcys law as

P +P P = TMP = inl out Ppermeate 2

Journal of Chemical Technology & Biotechnology

Journal of Chemical Technology & Biotechnology

Journal of Chemical Technology & Biotechnology

Results and Discussion

Journal of Chemical Technology & Biotechnology

Journal of Chemical Technology & Biotechnology

ions (Table 4).

Journal of Chemical Technology & Biotechnology

Academic Press LTD., 1975. 2.

Journal of Chemical Technology & Biotechnology

Journal of Chemical Technology & Biotechnology

Journal of Chemical Technology & Biotechnology

Journal of Chemical Technology & Biotechnology

Journal of Chemical Technology & Biotechnology

Journal of Chemical Technology & Biotechnology

Journal of Chemical Technology & Biotechnology

Journal of Chemical Technology & Biotechnology

Journal of Chemical Technology & Biotechnology

Nutrient Media 30 kDa filtered medium LMWM (4 kDa) LMWM (1 kDa)

Permeate Flux (J, m3 s-1)

Membrane Resistance (Rm,m-1)

30 kDa filtered medium LMWM (4 kDa) LMWM (1 kDa)

Journal of Chemical Technology & Biotechnology

Nutrient Media Optimised Unfiltered Medium

Retention Time (min)

Width Area (mVolts)

30 kDa filtered nutrient medium

LMWM (4 kDa) LMWM (1 kDa)

Table 3:Chromatographic analysis of the developed media

Journal of Chemical Technology & Biotechnology

Maximum growth rate( h 1 )

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

Journal of Chemical Technology & Biotechnology

LMWM (4kDa incl. Metal ions)

rate( h 1 ) Indicator strain L.casei L.plantarum L.lactis

Maximum growth rate( h 1 ) Indicator

Amount Bacteriocin Produced (IU/ml)

0.13 0.13 0.10

110 110 125

115 130 125

0.12 0.12 0.11

115 115 120

Table 5: Bacteriocin Production on the three different media categories

Journal of Chemical Technology & Biotechnology

LMWM (4kDa incl. Metal ions)