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Research Article

Received: 22 May 2012 Revised: 10 June 2012 Accepted: 11 June 2012 Published online in Wiley Online Library: 20 July 2012

(wileyonlinelibrary.com) DOI 10.1002/jctb.3892

Low molecular weight liquid media development for Lactobacilli producing bacteriocins
Myrto-Panagiota Zacharof and Robert W. Lovitt
Abstract
BACKGROUND: Contemporary purication techniques of Lactobacilli bacteriocins include chemical precipitation and separation through solvents to obtain highly potent semi-puried bacteriocins. These methods are laborious and bacteriocin yields are low. To address this problem a set of new, efcient, 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 nano-ltration from the cultured broth was possible. RESULTS: The LMWM were made through serial ltration (lters varying in pore size 30 kDa, 4 kDa and 1 kDa MWCO) of a modied 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 h1 ) on the LMWM and produced a signicant amount of bacteriocins in the range 110 to 130 IU mL1 . 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. c 2012 Society of Chemical Industry Keywords: Lactobacilli; bacteriocins; low molecular weight medium; yield; ltration

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 (acidication, dehydration) and addition of preservatives (antibiotics, organic compounds such as propionate, sorbate, benzoate, lactate, and acetate). Regardless of their proven success and effectiveness, there is an increasing demand for naturally developed, non-articial, biologically safe products providing the consumers with high health benets.1,5 Currently lactic acid bacteria and especially Lactobacilli have attracted great attention, due to the production of antimicrobial peptide compounds namely bacteriocins.2 Lactobacilli are widely applied in the food industry as natural acidiers. Their potential use as bacteriocidal agents would constitute a great commercial benet. The use of Lactobacilli-produced bacteriocins, is generally considered safe (GRAS, Grade One). Most Lactobacilli bacteriocins are small (<10 kDa) cationic, heat-stable, amphiphilic and membrane permeabilizing peptides. Many of these bacteriocins appear to exhibit relatively little adsorption specicity and have greater antibacterial activity at lower pH values (below 5). By means that their adsorption to the cell surface of Gram positive (+) bacte-

ria, eitherto the producing species or to the target strains, is pH dependent. Lactobacilli bacteriocins have been proven to be a highly effective natural barrier against microbial agents causing food poisoning and spoilage.5,6 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 sufcient 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; 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 that bacteriocins can kill the pathogenic bacteria.7,8 Several other bacteriocins from Lactobacilli have been identied throughout the last decade where research on their production and purication techniques has been highly intensive, due to the growing need for replacement of chemical food preservatives.10 13

Correspondence to: Myrto-Panagiota Zacharof, College of Engineering, Multidisciplinary Nanotechnology Centre, Swansea University, Swansea, SA2 8PP, UK. E-mail: myrtozacharof1981@yahoo.com College of Engineering, Multidisciplinary Nanotechnology Centre, Swansea University, Swansea, SA2 8PP, UK

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Liquid media development for Lactobacilli producing bacteriocins Regardless of 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 commercially produced by Dupont (Nisaplin) and Sigma Aldrich (Nisin 2.5% puried). Nisin is utilised worldwide as a food additive, under the number E234 (ECCU 1983 EEC Commission Directive 8 314 631EEC).9 The production methods used for the commercially available nisin are not known.8 Contemporary purication techniques for bacteriocins include chemical precipitation, separation through solvents used in combination with acid treatment14 16 of the culture followed by removal of the cells and then solvent 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. Although the bacteriocin preparations had high potency, the methods were laborious and total recovery yields were low.17 20 This is because 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) broth.21 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 dene 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 on 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 most commonly used, Elliker broth, LactobacillusStreptococcus differential agar (LS agar) and all 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 contain.39 41 Especially in the case of MRS, extensive use of beef or poultry extract (peptone) 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 an economically viable mass production process.22 24 MRS, though is a well established growth medium specically 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 is highly selective, its rich content of nitrogen sources and minerals ensure 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 issues a series of new, efcient, 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 desired metabolic products, using ultra- and nano-ltration, 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 ltration (lters varying in pore size 30 kDa, 4 kDa

www.soci.org and 1 kDa MWCO) of a modied liquid medium which had already been established as the most suitable for the selected Lactobacilli. The developed media were tested for bacteriocin production and biomass growth, using three known bacteriocin-producing Lactobacilli strains, Lactobacillus casei NCIMB 11 970, Lactobacillus plantarum NCIMB 8014, Lactobacillus lactis NCIMB 8586.

MATERIALS AND METHODS


Bacterial strains Lactobacillus plantarum NCIMB 8014, Lactobacillus lactis NCIMB 8586, Lactobacillus casei NCIMB 11 970 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, UK. Culturing conditions All three bacteriocin-producing strains bacteria were cultured in modied optimised liquid medium containing 20 g L1 glucose, yeast extract (YE) 20 g L1 , sodium acetate 10 g L1 , tri-sodium citrate 10 g L1 , potassium hydrogen phosphate 5 g L1 . In all the experimental procedures the media are dispersed in the 100 mL capacity serum vials, under anaerobic conditions (nitrogen ow), and sealed with butyl rubber stoppers (Fischer Scientic, UK) and alumina seals (Wheaton Industries, USA). They were autoclaved (120 C 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 36 C. Measurement of cellular growth and biomass Determination of cell growth was monitored as an increase of turbidity in terms of optical density (OD) at 660 nm wavelength using a spectrophotometer (PU 8625 UV/VIS Philips, France). The light path of the tube was 1.8 cm. Measuring OD was carried out on an hourly basis until the late stationary phase. The growth curves were obtained by plotting OD against time. The maximum and specic growth rates (max , h1 and , h1 ) of bacteria were calculated from logarithmic plots of the OD versus time during the exponential growth phase, according to the formula: (h1 ) = where DT (h) = (t2 t1 ) x (OD at 660 nm, hourly basis) (2) d( ln x) ln 2 1 dx = = x dt dt DT (1)

Nutrient media membrane ltration The modied optimised liquid medium containing 20 g L1 glucose, YE 20 g L1 , sodium acetate 10 g L1 , tri-sodium citrate 10 g L1 , potassium hydrogen phosphate 5 g L1 was used for fabrication of low molecular weight media (LMWM). A bench membrane apparatus (stirred cell unit reactor, Amicon 8200, Millipore Co., UK) was used for ltration of the nutrient media, operated batchwise (Fig. 1). The reactor system was composed of a stirred cell unit of 200 mL maximum process volume, a magnetic stirrer and ltration effective area of 28.7 cm2 . The stirrer speed was set at 150 rpm. Filtration of media was achieved through a series of ultraltration and nanoltration membranes The molecular weight cut-off (MWCO) of ultraltration polysulphone membranes in use was 30 kDa (cellulose acetate, Microdyn-Nadir Co., Germany)

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M-P Zacharof, RW Lovitt 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 used.26 2 mL of each medium category were placed in glass plates of 10 mm diameter equipped with membrane lters (Whatman 0.2 m qualitative lters, UK) and weighted in a high precision electronic scale (0.1 mg Ohaus, V12 140 Voyager, Switzerland). The samples were placed in 100 C furnace (Heraus Furnace, UK) for 24 h. After that, the samples were weighed again using the same scales and the difference was the content of solids in the medium. Determination of protein sources in low molecular weight media through high precision particle sizer (HPPS) The principle of the 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 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 uctuations of the scattered light. The relationship between the size of a particle and its speed due to Brownian motion is dened as the StokesEinstein equation D = k T/3 d (8)

Figure 1. Schematic diagram of the stirred cell (Amicon cell 8200 Manual, Sterlitech USA) (1) cap, (2) pressure relief valve, (3) pressure tube tting assembly, (4) top o-ring, provides seal to maintain pressure in the unit, (5) magnetic impeller, provides cross ow 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.

and 4 kDa (polysulfone, Microdyn-Nadir Co., Germany) while nanoltration 1 kDa (polysulfone, General Electric-Osmonics Co. USA). The cell unit was pressurized by constant compressed nitrogen at 200 kPa. The operating temperature was controlled to 25 C using a water jacket with water bath (Grant Water bath, UK). The stirred cell unit was operated in batch dead-end mode. After each experiment, the components of the unit cell were soaked in an ethanol solution (50% v/v) for 24 h. The membranes were rinsed with distilled water and sterilised with 25% v/v ethanol solution. Determination of permeate ux, membrane resistance and cake resistance were obtained from the standard equations25 used for evaluating membrane performance; the ux was dened as J= Qf Am (3)

where K (1.3807 1023 J K1 ) is the Boltzmann constant, T is the absolute temperature in Kelvin (K), and is the vicosity (8.937 104 kg m1 s1 ) of the medium in which the particles of diameter d (meters, m) are suspended. The HPPS system measures the rate of the intensity uctuation and then uses this to calculate the size of the particles. The size of the particles is graphically represented in curves where the highest peak represents the majority of molecules in the specic size given by the peak.28 In order to measure the size of the molecules 4 mL of each medium, both autoclaved and non autoclaved (unltered, 4 kDa LMWM and 1 kDa LMWM) were placed in plastic cuvettes and put in the apparatus. The apparatus was connected to a personal computer equipped with 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 conrm the fact that bacteriocins were indeed produced by the selected strains, purication techniques had to be used. All the analysis of the commercially available nisin and bacteriocins was done using a high performance liquid chromatograph (HPLC) method. The HPLC system was connected to a UV/Vis detector (Dionex, UK) and tted with a C18 reverse phase column (Vydac 238 TP54, HPLC Columns, UK) which is used to detect small polypeptides less than 40005000 MW, enzymatic digest fragments, natural and synthetic peptides and complex carbohydrates. The solvent (mobile phase) delivery system was formed of two pumps (pumps A and B) (Varian Co. Canada.) with a pressure operating range between 1500 and 1900 mbar. Temperature control of the solvents was maintained with a hotplate (Millipore Co., UK) at 25 C. 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

the transmembrane pressure ( P) was dened as P = TMP = Pinl + Pout 2 Ppermeate (4)

The membrane resistance was dened by Darcys law as Rm = P J (5)

Each membrane was characterised under different pressure conditions varying between 0 and 400 kPa with the following solutions, sterilised distilled water, 10 mmol L1 phosphate buffer (KH2 PO4 ) buffer (Sigma-Aldrich, UK) and sterilised basal medium. For each experimental run 150 mL of the selected solution was inserted in the reactor.

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Liquid media development for Lactobacilli producing bacteriocins ACN75% in distilled water and 1% v/v of standard buffer solution. The standard buffer solution consisted of 7.5% triuroacetic 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 a gradient, consisting of 65% of solvent A and 35% of solvent B. The ow rate of the samples and of the mobile phase was set at 1.5 mL min1 for 15 min, and the wavelength used was 220 nm. The operation of the system was controlled automatically using Prostar Workstation Data analysis software package (Varian Co., Canada). Each run lasted 17 min. All samples were injected into the system by sterile HPLC plastic syringe (1 mL sterile syringe, Fischerbrand, UK) at a 20 L injection loop connected to the HPLC system. Determination of nisin and bacteriocin activity and potency The activity and potency of nisin and the bacteriocins produced were tested according to a simple turbidometric assay.29 This assay was based on the effect of several different concentrations of commercial nisin against a target strain, in terms of growth rate. Into 25 mL of 0.02 mol L1 of HCl, 25 mg of nisin were dispersed. This solution equals 1000 IU mL1 of nisin. According to this formula the necessary quantities of solid nisin were calculated to fabricate standard solutions at the following concentrations: 0, 25, 50, 75, 85, 100, 110, 125, 150, 175, 200, 250, 500, 750, 1000, 1250, 1500, 1750, 2000 IU mL1 . The solutions were preserved (up to 30 days) at 4 C. Lactobacillus delbruckii subsp. lactis 8117 was selected as the target strain. The inoculum was consistent in the growth phase, as it was frozen when the growth reached 1.5 g L1 . The target strain was grown on a liquid medium containing 20 g L1 glucose, 20 g L1 YE, 10 g L1 sodium acetate, 10 g L1 tri-sodium citrate, 5 g L1 di-hydrogen orthophosphate, magnesium sulphate 0.5 g L1 , manganese sulphate 0.05 g L1 . This medium was also used when testing the effect of bacteriocins and nisin. Into glass tubes containing 8 mL of nutrient, 1 mL of the frozen inoculum of L. delbruckii and 1 mL of the supernatant resulting from pH control fermentations of differential concentration was added.29 The samples were gently mixed, and incubated statically at 36 C. The biomass was recorded on an hourly basis by measuring the turbidity using a spectrophotometer (PU 8625 UV/VIS Philips, France) at 600 nm. The amount of bacteriocin produced by each tested strain was dened primarily on 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 claried liquid was ltered through a 0.2 m pore size lter for sterilisation. The sterilised liquid pH was adjusted to 6.0 to eliminate the antimicrobial effect of lactic acid and then it was diluted with fresh medium.29 Separation of bacteriocins produced on low molecular weight media using ltration technology A bench membrane apparatus (stirred cell unit reactor, Amicon 8200, Millipore Co., UK) was used for ltration of the cultured LMWM and unltered 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

www.soci.org process volume, a magnetic stirrer and a ltration effective area of 28.7 cm2 . The cultured cell free supernatant was ltered through a nanoltration membrane of 1 kDa weight cut-off (polysulfone, General Electric-Osmonics Co. USA). 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 proved to be highly accurate and reproducible having mean standard deviation below 5% and experimental error below 5%, offering highly signicant results.

RESULTS AND DISCUSSION


Membrane characterisation and ltrability of the nutrient medium In order to determine the membrane resistance and the inuence of pressure during operation of the equipment, membrane characterisation studies were carried out. The permeability of distilled water, optimised nutrient medium, and phosphate buffer (10 mmol L1 ) solution through membranes of different MWCO was measured in order to analyse the behaviour of the reactor system. The permeability of distilled water, phosphate buffer solution and optimum nutrient medium through the membrane was measured to analyse the membraness behaviour (30 kDa, 4 kDa and 1 kDa MWCO) when incorporated in the unit. The ux values linearly increased with increasing pressure. In the case of 30 kDa MWCO membrane, for pure water the ux increased from 7.90 to 28.00 m3 m2 h1 with an increase in pressure from 50 to 400 kPa. For phosphate buffer solution the ux increased from 1.95 to 9.05 m3 m2 h1 with an increase in pressure from 50 to 400 kPa. While operating with optimised nutrient medium the ux was lower, from 0.79 to 2.80 m3 m2 h1 , with an increase in pressure from 50 to 400 kPa, respectively. For the 4 kDa MWCO membrane, the ux values from for all solutions linearly increased with increasing pressure. Pure water the ux increased from 0.23 to 1.20 m3 m2 h1 , with an increase in pressure from 50 to 400 kPa. For phosphate buffer solution the ux increased from 0.14 to 1.11 m3 m2 h1 , with an increase in pressure from 50 to 400 kPa. While operating with optimized nutrient medium the ux was lower from 0.09 to 0.56 m3 m2 h1 with an increase in pressure from 50 to 400 kPa, respectively. Lastly, for 1 kDa MWCO membrane, For pure water the ux increased from 0.04 to 0.16 m3 m2 h1 , with an increase in pressure from 50 to 400 kPa. For phosphate buffer solution the ux increased from 0.02 to 0.13 m3 m2 h1 with an increase in pressure from 50 to 400 kPa. While operating with optimized nutrient medium the ux was lower, from 0.008 to 0.08 m3 m2 h1 with an increase in pressure from 50 to 400 kPa, respectively. The membrane resistance values were rising during ltration of the solutions at different pressures; in the case of 4 kDa and 1 kDa MWCO membranes, these 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 in the fabrication material of the membrane itself as well as due to the pore size and the general porosity of the lter. During ltration of the nutrient medium, ux decline over time was noticed due to the deposition of organic macromolecules on the surface of the selected membranes, suggesting successful

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Table 1. Flux and membrane resistance of the serial ltration of the developed media Nutrient media 30 kDa ltered medium LMWM (4 kDa) LMWM (1 kDa) Permeate ux (J m3 s1 ) 1.86 101 9.43 103 1.05 104 Membrane resistance (Rm , m1 ) 1.17 1013 2.24 1014 2.14 1015

Table 2.

The effect of ltration on the dry weight media content Solids (g L1 ) 0.09 0.08 0.03 0.02

Nutrient Media Unltered medium 30 kDa ltered medium LMWM (4 kDa) LMWM (1 kDa)

retention of larger 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 (Fig. 2). Having proven the ltrability of the developed medium through the chosen membranes, the next step was to test the efcacy and the efciency of the ltration 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 to measure the remaining nutrient sources in the autoclaved nutrient media after each ltration process (Table 2). The nutrient sources were partially retained from the membrane lter during the ltration process, allowing low molecular weight nutrient sources to pass through the membrane lters, resulting in the production of the desired nutrient medium. All gravimetric analyses depend on nal 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 dened, suggesting that there is successful removal of solids by the membrane lters. So to dene the size and the volume of the remaining nitrogen sources in the ltered media were measured by dynamic light scattering

(DLS). This method provided higher accuracy and credibility of the results as only the protein sources derived from yeast extract were measured in the medium, due to the methods high sensitivity (<nm). The nutrient sources contained in the non autoclaved medium ltered through a 30 kDa MWCO membrane lter were found to range in size between 1 and 30 nm. The nutrient medium was therefore thought to contain mostly polypeptides and needed further treatment. When ltered through the 4 kDa MWCO lter the nutrient medium contains protein sources sized between 1 and 15 nm as the nutrient medium contains mostly oligopeptides. Further ltration was performed through a 1 kDa MWCO membrane lter, where the protein sources were sized between 1 and 5 nm suggesting that only oligopeptides were present in the solutions (Fig. 3). To use these LMWM for growth of Lactobacilli and bacteriocin production, sterilisation is necessary. The LMWM were autoclaved and analysed again (Fig. 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 occur, as proteins easily deteriorate and become inactive when exposed to high temperatures. When ltered through a 30 kDa MWCO membrane lter the nutrient sources contained in the autoclaved medium range in size between 1 and 30 nm, but with a higher percentage of proteins of size between 1 and 10 nm when compared with the non-autoclaved

(a)

(b)

(c)

Figure 2. Deposition of solids forming a cake on the outer layer of the ultraltration (a, 30 kDa) (b, 4 kDa) and nanoltration (c, 1 kDa) membranes.

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Figure 3. Size distribution of media particles in a non-autoclaved media: unltered (a) and ltered through ultraltration (b, 30 kDa) (c, 4 kDa) and nanoltration (d, 1 kDa) membranes.

Figure 4. Size distribution of media particles in autoclaved media: unltered (a) and ltered through ultraltration (b, 30 kDa) (c, 4 kDa) and nanoltration (d, 1 kDa) membranes.

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Figure 5. HPLC analysis of autoclaved media: unltered (a) and ltered through ultraltration (b, 30 kDa) (c, 4 kDa) and nanoltration (d, 1 kDa) membranes.

media. The nutrient medium contains mostly polypeptides and needs further ltration. When ltered through the 4 kDa MWCO lter the nutrient medium contains particles between 1 and 20 nm, with most of the proteins between 1 and 3 nm. The nutrient medium contained mostly oligopeptides. Further ltration was performed through a 1 kDa MWCO membrane lter, resulting in protein sources sized 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 is highly suitable for quantifying and analysing mixtures of chemical compounds due to its high sensitivity and specicity, especially in peptides and oligopeptides, and has been used by numerous researchers30 33 to investigate protein substances in complex solutions. The protein sources were successfully detected (Fig. 5) suggesting sufcient presence of protein sources in the resulting media (Table 3), certifying also the removal of larger protein molecules. Testing the low molecular weight nutrient media for lactobacilli growth and bacteriocin production As LMWM were successfully developed, the next step was to investigate whether they could sufciently support Lactobacilli growth providing high biomass yields and amounts of bacteriocin.

Table 3.

Chromatographic analysis of the developed media Retention time (min) 1.063 1.225 1.534 1.831 1.087 1.209 1.297 1.575 1.104 1.214 1.112 1.237 Width area (mV) 6.06 19.28 19.36 32.81 6.28 14.53 6.28 1.022 6.34 16.53 5.72 15.63

Nutrient media Optimised unltered medium

30 kDa ltered nutrient medium

LMWM (4 kDa) LMWM (1 kDa)

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

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Table 4.

Growth of the selected Lactobacilli on the developed media Unltered medium LMWM (4 kDa) Maximum growth rate (h1 ) 0.18 0.16 0.16 Final biomass (g L1 ) 1.65 1.33 1.65 LMWM (4 kDa incl. metal ions) Maximum growth rate (h1 ) 0.19 0.22 0.21 Final biomass (g L1 ) 1.60 2.03 1.70 LMWM (1 kDa incl. metal ions) Maximum growth rate (h1 ) 0.18 0.16 0.16 Final biomass (g L1 ) 1.64 1.65 1.60

Selected strains L. casei L. plantarum L. lactis

Maximum growth rate (h1 ) 0.24 0.30 0.22

Final biomass (g L1 ) 2.43 2.63 1.81

Table 5.

Bacteriocin production on the three different media categories Unltered Medium Maximum growth rate (h1 ) Indicator strain 0.13 0.13 0.10 Amount of Bacteriocin Produced (IU mL1 ). 110 110 125 LMWM (4 kDa incl. metal ions) Maximum growth rate (h1 ) Indicator strain 0.12 0.09 0.10 Amount of Bacteriocin Produced (IU mL1 ) 115 130 125 LMWM (1 kDa incl. metal ions) Maximum growth rate (h1 ) Indicator strain 0.12 0.12 0.11 Amount of bacteriocin produced (IU mL1 ) 115 115 120

Lactobacilli L. casei L. plantarum L. lactis

growth rates achieved were small, when compared with the optimised unltered medium. Further investigation to achieve higher growth yields was made by incorporating metal ions of manganese (0.5 g L1 ) and magnesium (0.05 g L1 ) salts in the 4 kDa and 1 kDa media, as there was a strong possibility that the ions were retained by the membranes, potentially due to their aggregation with higher molecular weight nutrient sources. The selected Lactobacilli grew better, proving the dependence of growth of the selected bacteria on 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 modied 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 to investigate 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 seen that the bacteriocins derived from Lactobacilli grown on LMW medium of 1 kDa had the weaker potency.

Separation of bacteriocins produced on low molecular weight media using ltration technology Filtration was the selected extraction and concentration method that could also enhance the potency of the bacteriocins produced. Cultured broth solutions produced on all the media categories, were ltered through 4 kDa and 1 kDa MWCO membrane lters. The resulting retentates were tested for bacteriocin activity (Table 6). The resulting retentates containing bacteriocins had stronger antimicrobial activity, with the bacteriocins becoming more potent. In the case of bacteriocins developed on the optimised unltered medium, the bacteriocin yield was only slightly enhanced. In contrast, in the case of LMWM bacteriocins the potency was signicantly reinforced, resulting in successful 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 4 kDa and 1 kDa LMWM media to support the production of antimicrobial peptide substances during growth of the selected Lactobacilli. These substances were proven to be equally effective towards

Table 6.

Activity of extracted bacteriocins of the three different media categories Unltered medium LMWM (4 kDa incl. metal ions) LMWM (1 kDa incl. metal ions)

Lactobacilli L. casei L. plantarum L. lactis

Maximum growth Maximum growth Maximum growth Amount of bacteriocin Amount of bacteriocin Amount of bacteriocin rate (h1 ) rate (h1 ) rate (h1 ) Indicator strain produced (IU mL1 ) Indicator strain produced (IU mL1 ) Indicator strain produced (IU mL1 ) 0.12 0.11 0.09 115 120 130 0.005 0.003 0.002 165 180 185 0.004 0.002 0.007 170 185 155

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www.soci.org the target strain, being highly potent, regardless of the fact that Lactobacilli were grown on different media. These results are encouraging as they indicate that these media can be used when upscaling bacteriocin production and purication using ltration as the separation method, having solved the problem of excess proteins.

M-P Zacharof, RW Lovitt


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