low molecular weight liquid media development for lactobacilli producing bacteriocins

Research ArticleReceived: 22 May 2012 Revised: 10 June 2012 Accepted: 11 June 2012 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jctb.3892

Low molecular weight liquid mediadevelopment for Lactobacilli producingbacteriocinsMyrto-Panagiota Zacharof∗ and Robert W. Lovitt

Abstract

BACKGROUND: Contemporary purification techniques of Lactobacilli bacteriocins include chemical precipitation and separationthrough solvents to obtain highly potent semi-purified bacteriocins. These methods are laborious and bacteriocin yields arelow. To address this problem a set of new, efficient, cost effective media, was created, containing low molecular weight nutrientsources (LMWM). Using these media future separation and concentration of the desired metabolic products, using ultra- andnano-filtration from the cultured broth was possible.

RESULTS: The LMWM were made through serial filtration (filters varying in pore size 30 kDa, 4 kDa and 1 kDa MWCO) of amodified optimum liquid medium for Lactobacilli growth. The developed media were tested for bacteriocin production andbiomass growth, using three known bacteriocin-producing Lactobacilli strains, Lactobacillus casei NCIMB 11970, Lactobacillusplantarum NCIMB 8014, Lactobacillus lactis NCIMB 8586. All were successfully grown (µmax 0.16 to 0.18 h−1) on the LMWM andproduced a significant amount of bacteriocins in the range 110 to 130 IU mL−1.

CONCLUSIONS: LMWM do support Lactobacilli growth and bacteriocin production, establishing an alternative to the currentproduction nutrient media. The uptake of the nutrient sources is facilitated as nitrogen sources, which were primarily responsiblefor growth, were supported in less complex forms.c© 2012 Society of Chemical Industry

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

INTRODUCTIONSince the industrialisation of food production, food safetyhas been an issue of great importance. Naturally occurringfood deterioration and spoilage due to microbial agents hasbeen the main source of hardship in today’s food industry.Numerous preservation methods have been used to prevent foodpoisoning and contamination. These include thermal treatment(pasteurization, heating sterilisation), pH and water activity re-duction (acidification, dehydration) and addition of preservatives(antibiotics, organic compounds such as propionate, sorbate,benzoate, lactate, and acetate). Regardless of their proven successand effectiveness, there is an increasing demand for naturallydeveloped, non-artificial, biologically safe products providing theconsumers with high health benefits.1,5

Currently lactic acid bacteria and especially Lactobacilli haveattracted great attention, due to the production of antimicrobialpeptide compounds namely bacteriocins.2 Lactobacilli are widelyapplied in the food industry as natural acidifiers. Their potentialuse as bacteriocidal agents would constitute a great commercialbenefit. The use of Lactobacilli-produced bacteriocins, is generallyconsidered safe (GRAS, Grade One). Most Lactobacilli bacteriocinsare small (<10 kDa) cationic, heat-stable, amphiphilic and mem-brane permeabilizing peptides. Many of these bacteriocins appearto exhibit relatively little adsorption specificity and have greaterantibacterial activity at lower pH values (below 5). By means thattheir adsorption to the cell surface of Gram positive (+) bacte-

ria, eitherto the producing species or to the target strains, is pHdependent. Lactobacilli bacteriocins have been proven to be ahighly effective natural barrier against microbial agents causingfood poisoning and spoilage.5,6

Antimicrobial activity of bacteriocins is directed principallyagainst other Gram positive (+) bacteria. The majority ofLactobacilli bacteriocins has been shown to be effective when usedin 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 notlikely to ensure complete safety; in the case of Gram negative(−) bacteria this has been apparent. Then the use of bacteriocinshas to be combined with other technologies that are able todisrupt the cellular membrane so that bacteriocins can kill thepathogenic bacteria.7,8 Several other bacteriocins from Lactobacillihave been identified throughout the last decade where researchon their production and purification techniques has been highlyintensive, due to the growing need for replacement of chemicalfood preservatives.10 – 13

∗ Correspondence to: Myrto-Panagiota Zacharof, College of Engineering, Multi-disciplinary Nanotechnology Centre, Swansea University, Swansea, SA2 8PP,UK. E-mail: [email protected]

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

J Chem Technol Biotechnol (2012) www.soci.org c© 2012 Society of Chemical Industry

www.soci.org M-P Zacharof, RW Lovitt

Regardless of the wide variety of bacteriocins being producedby Lactobacilli, only nisin produced by Lactococcus lactis var.lactis, previously known as Lactobacillus lactis var lactis, iscommercially produced by Dupont (Nisaplin) and Sigma Aldrich(Nisin 2.5% purified). Nisin is utilised worldwide as a foodadditive, under the number E234 (ECCU 1983 EEC CommissionDirective 8 314 631EEC).9 The production methods used for thecommercially available nisin are not known.8

Contemporary purification techniques for bacteriocins includechemical precipitation, separation through solvents used incombination with acid treatment14 – 16 of the culture followed byremoval of the cells and then solvent extraction and precipitation,and high performance liquid chromatography or reverse phasechromatography. Currently, most methods rely on ammoniumsulfate precipitation of the bacteriocins from cell-free culturedbroth. These methods have been used to obtain bacteriocinsfrom Lactobacillus spp., Leuconostoc spp., Pediococcus spp. andLactococcus spp.

Although the bacteriocin preparations had high potency, themethods were laborious and total recovery yields were low.17 – 20

This is because many other proteins from the medium can also beprecipitated, since for the culturing of Lactobacilli, complex mediaare used such as Man de Rogosa (MRS) broth.21

However, for the successful development of cellular biomassand bacteriocin productivity the use of suitable nutrient mediais of crucial importance, as growth media assimilate and definethe nutritional conditions determining the growth yield and themetabolites’ productivity of the selected bacteria. Lactobacilli havecomplex nutritional needs, with several researchers34 – 39,42 – 44

highlighting their growth dependence on minerals, such asmanganese and magnesium, vitamins of the B complex, aminoacids such as serine and adenine and organic compounds.Commercially available media for Lactobacilli propagation includeMan De Rogosa medium (MRS), which is most commonly used,Elliker broth, Lactobacillus–Streptococcus differential agar (LS agar)and all purpose Tween agar (APT). Although these media, oftenused for research purposes, do ensure bacterial growth, they donot support fastidious growth, or high biomass yields due to theplethora of nitrogen sources they contain.39 – 41 Especially in thecase of MRS, extensive use of beef or poultry extract (peptone)causes environmental (undischarged waste) and health (potentialCJD-prion disease or H1N1 virus) hazards, while the complexity ofnutrients leads to highly expensive media fabrication, unsuitablefor an economically viable mass production process.22 – 24

MRS, though is a well established growth medium specificallydesigned to support the growth of Lactobacilli. It contains richnutrient sources suitable to support the high auxotrophic needs ofthese organisms. It can be easily prepared and it is highly selective,its rich content of nitrogen sources and minerals ensure bacterialgrowth but do not support fastidious growth and high biomassyields. In addition, its cost of fabrication due to the materialsneeded remains relatively high.

To address these issues a series of new, efficient, cost effectivemedia, capable of further improvements was created, containinglow molecular weight nutrient sources (LMWM). The developmentof the LMWM was proposed mainly to facilitate the futureseparation and concentration of desired metabolic products, usingultra- and nano-filtration, from the cultured broth. Additionally, theuptake of the nutrient sources would be and was indeed facilitatedas nitrogen sources, which were primarily responsible for growth,were supported in less complex forms. The LMWM were madethrough serial filtration (filters varying in pore size 30 kDa, 4 kDa

and 1 kDa MWCO) of a modified liquid medium which had alreadybeen established as the most suitable for the selected Lactobacilli.The developed media were tested for bacteriocin productionand biomass growth, using three known bacteriocin-producingLactobacilli strains, Lactobacillus casei NCIMB 11 970, Lactobacillusplantarum NCIMB 8014, Lactobacillus lactis NCIMB 8586.

MATERIALS AND METHODSBacterial strainsLactobacillus plantarum NCIMB 8014, Lactobacillus lactis NCIMB8586, Lactobacillus casei NCIMB 11 970 and the target strainLactobacillus delbruckii subsp. lactis NCIMB 8117 were provided ina lyophilised form by National Collection of Industrial Food andMarine bacteria (NCIMB), Aberdeen, Scotland, UK.

Culturing conditionsAll three bacteriocin-producing strains bacteria were cultured inmodified optimised liquid medium containing 20 g L−1 glucose,yeast extract (YE) 20 g L−1, sodium acetate 10 g L−1, tri-sodiumcitrate 10 g L−1, potassium hydrogen phosphate 5 g L−1. In all theexperimental procedures the media are dispersed in the 100 mLcapacity serum vials, under anaerobic conditions (nitrogen flow),and sealed with butyl rubber stoppers (Fischer Scientific, UK) andalumina seals (Wheaton Industries, USA). They were autoclaved(120 ◦C for 15 min) (Priorclave: Tactrol 2, RSC/E, UK) and left to cooldown, for 12h. The inoculum size was is 10% v/v. The tubes wereincubated for 12 h at 36 ◦C.

Measurement of cellular growth and biomassDetermination of cell growth was monitored as an increase ofturbidity in terms of optical density (OD) at 660 nm wavelengthusing a spectrophotometer (PU 8625 UV/VIS Philips, France). Thelight path of the tube was 1.8 cm. Measuring OD was carried outon an hourly basis until the late stationary phase. The growthcurves were obtained by plotting OD against time. The maximumand specific growth rates (µmax, h−1 and µ, h−1) of bacteria werecalculated from logarithmic plots of the OD versus time during theexponential growth phase, according to the formula:

µ(h−1) = 1

x

dx

dt= d( ln x)

dt= ln 2

DT(1)

where

DT (h) = (t2 − t1)

x(OD at 660 nm, hourly basis) (2)

Nutrient media membrane filtrationThe modified optimised liquid medium containing 20 g L−1

glucose, YE 20 g L−1, sodium acetate 10 g L−1, tri-sodium citrate10 g L−1, potassium hydrogen phosphate 5 g L−1 was used forfabrication of low molecular weight media (LMWM). A benchmembrane apparatus (stirred cell unit reactor, Amicon 8200,Millipore Co., UK) was used for filtration of the nutrient media,operated batchwise (Fig. 1). The reactor system was composed ofa stirred cell unit of 200 mL maximum process volume, a magneticstirrer and filtration effective area of 28.7 cm2. The stirrer speed wasset at 150 rpm. Filtration of media was achieved through a seriesof ultrafiltration and nanofiltration membranes The molecularweight cut-off (MWCO) of ultrafiltration polysulphone membranesin use was 30 kDa (cellulose acetate, Microdyn-Nadir Co., Germany)

wileyonlinelibrary.com/jctb c© 2012 Society of Chemical Industry J Chem Technol Biotechnol (2012)

Liquid media development for Lactobacilli producing bacteriocins www.soci.org

Figure 1. Schematic diagram of the stirred cell (Amicon cell 8200 Manual,Sterlitech USA) (1) cap, (2) pressure relief valve, (3) pressure tube fittingassembly, (4) top o-ring, provides seal to maintain pressure in the unit,(5) magnetic impeller, provides cross flow conditions, (6) main body of thestirred cell, (7) bottom o-ring provides seal to maintain pressure in theunit and prevent loss of sample, (8) base with permeate outlet, (9) screwin bottom to secure base in the main body, (10) permeate line and (11)retaining stand prevents displacement of cap when pressure is used in theunit.

and 4 kDa (polysulfone, Microdyn-Nadir Co., Germany) whilenanofiltration 1 kDa (polysulfone, General Electric-Osmonics Co.USA). The cell unit was pressurized by constant compressednitrogen at 200 kPa.

The operating temperature was controlled to 25 ◦C using a waterjacket with water bath (Grant Water bath, UK). The stirred cell unitwas operated in batch dead-end mode. After each experiment, thecomponents of the unit cell were soaked in an ethanol solution(50% v/v) for 24 h. The membranes were rinsed with distilled waterand sterilised with 25% v/v ethanol solution.

Determination of permeate flux, membrane resistance and cakeresistance were obtained from the standard equations25 used forevaluating membrane performance; the flux was defined as

J =(

Qf

Am

)(3)

the transmembrane pressure (�P) was defined as

�P = TMP =(

Pinl + Pout

2

)− Ppermeate (4)

The membrane resistance was defined by Darcy’s law as

Rm = �P

J × µ(5)

Each membrane was characterised under different pressureconditions varying between 0 and 400 kPa with the followingsolutions, sterilised distilled water, 10 mmol L−1 phosphate buffer(KH2PO4) buffer (Sigma-Aldrich, UK) and sterilised basal medium.For each experimental run 150 mL of the selected solution wasinserted in the reactor.

Determination of protein sources in low molecular weightmedia by gravimetryIn order to measure the content of proteins in the resultingsolutions the gravimetric method was used.26 2 mL of eachmedium category were placed in glass plates of 10 mm diameterequipped with membrane filters (Whatman 0.2 µm qualitativefilters, UK) and weighted in a high precision electronic scale(0.1 mg Ohaus, V12 140 Voyager, Switzerland). The samples wereplaced in 100 ◦C furnace (Heraus Furnace, UK) for 24 h. After that,the samples were weighed again using the same scales and thedifference was the content of solids in the medium.

Determination of protein sources in low molecular weightmedia through high precision particle sizer (HPPS)The principle of the high precision particle sizer is basedon dynamic light scattering (DLS also known as PCS – photoncorrelation spectroscopy, or QELS – quasi-elastic light scattering),which measures Brownian motion of particles in a solution andrelates this to the size of the particles.27 This is done by illuminatingthe particles with a laser beam and analysing the intensityfluctuations of the scattered light. The relationship between thesize of a particle and its speed due to Brownian motion is definedas the Stokes–Einstein equation

D = kβT/3πηd (8)

where Kβ (1.3807 × 10−23 J K−1) is the Boltzmann constant, Tis the absolute temperature in Kelvin (K), and η is the vicosity(8.937 × 10−4 kg m−1 s−1) of the medium in which the particles ofdiameter d (meters, m) are suspended. The HPPS system measuresthe rate of the intensity fluctuation and then uses this to calculatethe size of the particles. The size of the particles is graphicallyrepresented in curves where the highest peak represents themajority of molecules in the specific size given by the peak.28

In order to measure the size of the molecules 4 mL of eachmedium, both autoclaved and non autoclaved (unfiltered, 4 kDaLMWM and 1 kDa LMWM) were placed in plastic cuvettes andput in the apparatus. The apparatus was connected to a personalcomputer equipped with special software programme (MalvernInstruments LDT. DTS 4.20, 2002) and all the measurements weredone automatically.

Determination of protein sources in low molecular weightmedia through high performance liquid chromatographyIn order to further purify and also to confirm the fact thatbacteriocins were indeed produced by the selected strains,purification techniques had to be used. All the analysis of thecommercially available nisin and bacteriocins was done usinga high performance liquid chromatograph (HPLC) method. TheHPLC system was connected to a UV/Vis detector (Dionex, UK) andfitted with a C18 reverse phase column (Vydac 238 TP54, HPLCColumns, UK) which is used to detect small polypeptides less than4000–5000 MW, enzymatic digest fragments, natural and syntheticpeptides and complex carbohydrates. The solvent (mobile phase)delivery system was formed of two pumps (pumps A and B) (VarianCo. Canada.) with a pressure operating range between 1500 and1900 mbar. Temperature control of the solvents was maintainedwith a hotplate (Millipore Co., UK) at 25 ◦C.

The mobile phase was represented by two solutions; solvent Aconsisted of 99% pure acetonitrile (ACN) 10% v/v in distilled waterand 1% v/v of standard buffer solution, and solvent B of 99% pure

J Chem Technol Biotechnol (2012) c© 2012 Society of Chemical Industry wileyonlinelibrary.com/jctb


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 indistilled water. The solutions were delivered to the pumps viaplastic tubes and valves. The mobile phase was organised as agradient, consisting of 65% of solvent A and 35% of solvent B.The flow rate of the samples and of the mobile phase was set at1.5 mL min−1 for 15 min, and the wavelength used was 220 nm.The operation of the system was controlled automatically usingProstar Workstation Data analysis software package (Varian Co.,Canada). Each run lasted 17 min. All samples were injected intothe system by sterile HPLC plastic syringe (1 mL sterile syringe,Fischerbrand, UK) at a 20 µL injection loop connected to the HPLCsystem.

Determination of nisin and bacteriocin activity and potencyThe activity and potency of nisin and the bacteriocins producedwere tested according to a simple turbidometric assay.29 Thisassay was based on the effect of several different concentrationsof commercial nisin against a target strain, in terms of growth rate.Into 25 mL of 0.02 mol L−1 of HCl, 25 mg of nisin were dispersed.This solution equals 1000 IU mL−1 of nisin. According to thisformula the necessary quantities of solid nisin were calculated tofabricate 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 mL−1. The solutions were preserved (up to30 days) at 4 ◦C.

Lactobacillus delbruckii subsp. lactis 8117 was selected asthe target strain. The inoculum was consistent in the growthphase, as it was frozen when the growth reached 1.5 g L−1. Thetarget strain was grown on a liquid medium containing 20 g L−1

glucose, 20 g L−1 YE, 10 g L−1 sodium acetate, 10 g L−1 tri-sodiumcitrate, 5 g L−1 di-hydrogen orthophosphate, magnesium sulphate0.5 g L−1, manganese sulphate 0.05 g L−1. This medium was alsoused when testing the effect of bacteriocins and nisin.

Into glass tubes containing 8 mL of nutrient, 1 mL of the frozeninoculum of L. delbruckii and 1 mL of the supernatant resultingfrom pH control fermentations of differential concentration wasadded.29 The samples were gently mixed, and incubated staticallyat 36 ◦C. The biomass was recorded on an hourly basis bymeasuring the turbidity using a spectrophotometer (PU 8625UV/VIS Philips, France) at 600 nm.

The amount of bacteriocin produced by each tested strainwas defined primarily on samples taken at the end of pH andtemperature controlled fermentations. The selected samples (pHfermentation at 6.5) were transferred into 10 mL conical plastictubes (Fisherbrand, UK) and centrifuged (10 000 rpm for 15 min)(Biofuge Stratos Sorall, Kendro Products, Germany) for completebiomass removal. The clarified liquid was filtered through a 0.2 µmpore size filter for sterilisation. The sterilised liquid pH was adjustedto 6.0 to eliminate the antimicrobial effect of lactic acid and thenit was diluted with fresh medium.29

Separation of bacteriocins produced on low molecular weightmedia using filtration technologyA bench membrane apparatus (stirred cell unit reactor, Amicon8200, Millipore Co., UK) was used for filtration of the culturedLMWM and unfiltered optimised media cell free (via centrifugation)supernatants, operated batchwise. The cell unit was constantlypressurized by compressed nitrogen at 200 kPa. The reactorsystem was composed of a stirred cell unit of 200 mL maximum

process volume, a magnetic stirrer and a filtration effective area of28.7 cm2. The cultured cell free supernatant was filtered througha nanofiltration membrane of 1 kDa weight cut-off (polysulfone,General Electric-Osmonics Co. USA).

Numerical analysis of the experimental dataEach differential parameter was triplicated to obtain the averagedata. The data were statistically analysed for accuracy and precisioncalculating standard deviation, standard error, experimental error,regression factor and reading error (Microsoft Excel softwareVersion 2003). All the numerical data proved to be highly accurateand reproducible having mean standard deviation below 5% andexperimental error below 5%, offering highly significant results.

RESULTS AND DISCUSSIONMembrane characterisation and filtrability of the nutrientmediumIn order to determine the membrane resistance and the influenceof pressure during operation of the equipment, membranecharacterisation studies were carried out.

The permeability of distilled water, optimised nutrient medium,and phosphate buffer (10 mmol L−1) solution through membranesof different MWCO was measured in order to analyse thebehaviour of the reactor system. The permeability of distilledwater, phosphate buffer solution and optimum nutrient mediumthrough the membrane was measured to analyse the membranes’sbehaviour (30 kDa, 4 kDa and 1 kDa MWCO) when incorporatedin the unit. The flux values linearly increased with increasingpressure. In the case of 30 kDa MWCO membrane, for pure waterthe flux increased from 7.90 to 28.00 m3 m−2 h−1 with an increasein pressure from 50 to 400 kPa. For phosphate buffer solution theflux increased from 1.95 to 9.05 m3 m−2 h−1 with an increasein pressure from 50 to 400 kPa. While operating with optimisednutrient medium the flux was lower, from 0.79 to 2.80 m3 m−2

h−1, with an increase in pressure from 50 to 400 kPa, respectively.For the 4 kDa MWCO membrane, the flux values from for allsolutions linearly increased with increasing pressure. Pure waterthe flux increased from 0.23 to 1.20 m3 m−2 h−1, with an increasein pressure from 50 to 400 kPa. For phosphate buffer solution theflux increased from 0.14 to 1.11 m3 m−2 h−1, with an increasein pressure from 50 to 400 kPa. While operating with optimizednutrient medium the flux was lower from 0.09 to 0.56 m3 m−2 h−1

with an increase in pressure from 50 to 400 kPa, respectively.Lastly, for 1 kDa MWCO membrane, For pure water the fluxincreased from 0.04 to 0.16 m3 m−2 h−1, with an increase inpressure from 50 to 400 kPa. For phosphate buffer solution theflux increased from 0.02 to 0.13 m3 m−2 h−1 with an increasein pressure from 50 to 400 kPa. While operating with optimizednutrient medium the flux was lower, from 0.008 to 0.08 m3 m−2

h−1 with an increase in pressure from 50 to 400 kPa, respectively.The membrane resistance values were rising during filtration ofthe solutions at different pressures; in the case of 4 kDa and1 kDa MWCO membranes, these were smaller when comparedwith the values of the 30 kDa membrane although the operatingconditions were the same. This was probably due to the differencein the fabrication material of the membrane itself as well as due tothe pore size and the general porosity of the filter.

During filtration of the nutrient medium, flux decline over timewas noticed due to the deposition of organic macromoleculeson the surface of the selected membranes, suggesting successful



Table 1. Flux and membrane resistance of the serial filtration of thedeveloped media

Nutrient mediaPermeate flux

(J m3 s−1)Membrane resistance

(Rm, m−1)

30 kDa filtered medium 1.86 × 10−1 1.17 × 1013

LMWM (4 kDa) 9.43 × 10−3 2.24 × 1014

LMWM (1 kDa) 1.05 × 10−4 2.14 × 1015

retention of larger molecules by the membranes. This is alsoassumed by the membrane resistance numerical values (Table 1),due to the cake layer formed on the membrane surfaces (Fig. 2).Having proven the filtrability of the developed medium throughthe chosen membranes, the next step was to test the efficacy andthe efficiency of the filtration method for the formation of lowmolecular weight nutrient media.

Determination of the low molecular weight nutrient sourcesin the developed nutrient mediaGravimetry was used to measure the remaining nutrient sourcesin the autoclaved nutrient media after each filtration process(Table 2). The nutrient sources were partially retained fromthe membrane filter during the filtration process, allowing lowmolecular weight nutrient sources to pass through the membranefilters, resulting in the production of the desired nutrient medium.All gravimetric analyses depend on final determination of weightas a means of quantifying an analyte. Weight can be measured withgreater accuracy than any other fundamental property, gravimetricanalysis is possibly one of the most accurate and commonly usedmethods of analytical chemistry available. In this case though onlythe suspended solids can be defined, suggesting that there issuccessful removal of solids by the membrane filters. So to definethe size and the volume of the remaining nitrogen sources inthe filtered media were measured by dynamic light scattering

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

Nutrient Media Solids (g L−1)

Unfiltered medium 0.09

30 kDa filtered medium 0.08

LMWM (4 kDa) 0.03

LMWM (1 kDa) 0.02

(DLS). This method provided higher accuracy and credibility of theresults as only the protein sources derived from yeast extract weremeasured in the medium, due to the method’s high sensitivity(<nm).

The nutrient sources contained in the non autoclaved mediumfiltered through a 30 kDa MWCO membrane filter were foundto range in size between 1 and 30 nm. The nutrient mediumwas therefore thought to contain mostly polypeptides andneeded further treatment. When filtered through the 4 kDaMWCO filter the nutrient medium contains protein sources sizedbetween 1 and 15 nm as the nutrient medium contains mostlyoligopeptides. Further filtration was performed through a 1 kDaMWCO membrane filter, where the protein sources were sizedbetween 1 and 5 nm suggesting that only oligopeptides werepresent in the solutions (Fig. 3).

To use these LMWM for growth of Lactobacilli and bacteriocinproduction, sterilisation is necessary. The LMWM were autoclavedand analysed again (Fig. 4). During autoclaving, due to thehigh temperature and pressure applied, often reactions, suchas caramelization of glucose, agglomeration, deterioration orinactivation of protein sources occur, as proteins easily deteriorateand become inactive when exposed to high temperatures. Whenfiltered through a 30 kDa MWCO membrane filter the nutrientsources contained in the autoclaved medium range in size between1 and 30 nm, but with a higher percentage of proteins of sizebetween 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 ultrafiltration (a, 30 kDa) (b, 4 kDa) and nanofiltration (c, 1 kDa) membranes.



Figure 3. Size distribution of media particles in a non-autoclaved media: unfiltered (a) and filtered through ultrafiltration (b, 30 kDa) (c, 4 kDa) andnanofiltration (d, 1 kDa) membranes.

Figure 4. Size distribution of media particles in autoclaved media: unfiltered (a) and filtered through ultrafiltration (b, 30 kDa) (c, 4 kDa) and nanofiltration(d, 1 kDa) membranes.



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

media. The nutrient medium contains mostly polypeptides andneeds further filtration. When filtered through the 4 kDa MWCOfilter the nutrient medium contains particles between 1 and 20 nm,with most of the proteins between 1 and 3 nm. The nutrientmedium contained mostly oligopeptides. Further filtration wasperformed through a 1 kDa MWCO membrane filter, resultingin protein sources sized up to 1 nm, suggesting that onlyoligopeptides were present in the solutions.

High performance liquid chromatography (HPLC) was selectedto further characterise the nutrient sources in the developedmedia. This method is highly suitable for quantifying and analysingmixtures of chemical compounds due to its high sensitivityand specificity, especially in peptides and oligopeptides, andhas been used by numerous researchers30 – 33 to investigateprotein substances in complex solutions. The protein sourceswere successfully detected (Fig. 5) suggesting sufficient presenceof protein sources in the resulting media (Table 3), certifying alsothe removal of larger protein molecules.

Testing the low molecular weight nutrient mediafor lactobacilli growth and bacteriocin productionAs LMWM were successfully developed, the next step was toinvestigate whether they could sufficiently support Lactobacilligrowth providing high biomass yields and amounts of bacteriocin.

Table 3. Chromatographic analysis of the developed media

Nutrient mediaRetentiontime (min)

Widtharea (mV)

Optimised unfiltered medium 1.063 6.06

1.225 19.28

1.534 19.36

1.831 32.81

30 kDa filtered nutrient medium 1.087 6.28

1.209 14.53

1.297 6.28

1.575 1.022

LMWM (4 kDa) 1.104 6.34

1.214 16.53

LMWM (1 kDa) 1.112 5.72

1.237 15.63

A comparative study was made between the standard nutrientmedia used for this study, and the developed LMWM of 4 kDaand 1 kDa molecular weight sources. The LMWM can supportthe growth of the selected Lactobacilli, although the maximum



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

Unfiltered mediumLMWM(4 kDa)

LMWM(4 kDa incl. metal ions)

LMWM(1 kDa incl. metal ions)

Selectedstrains

Maximumgrowth

rate (h−1)

Finalbiomass(g L−1)

Maximumgrowth

rate (h−1)


Maximumgrowth

rate (h−1)


Maximumgrowth

rate (h−1)


L. casei 0.24 2.43 0.18 1.65 0.19 1.60 0.18 1.64

L. plantarum 0.30 2.63 0.16 1.33 0.22 2.03 0.16 1.65

L. lactis 0.22 1.81 0.16 1.65 0.21 1.70 0.16 1.60

Table 5. Bacteriocin production on the three different media categories

Unfiltered MediumLMWM

(4 kDa incl. metal ions)LMWM

(1 kDa incl. metal ions)

Lactobacilli

Maximum growthrate (h−1)

Indicator strain

Amount ofBacteriocin

Produced (IU mL−1).


Indicator strain

Amount ofBacteriocin

Produced (IU mL−1)


Indicator strain

Amount ofbacteriocin

produced (IU mL−1)

L. casei 0.13 110 0.12 115 0.12 115

L. plantarum 0.13 110 0.09 130 0.12 115

L. lactis 0.10 125 0.10 125 0.11 120

growth rates achieved were small, when compared with theoptimised unfiltered medium. Further investigation to achievehigher growth yields was made by incorporating metal ionsof manganese (0.5 g L−1) and magnesium (0.05 g L−1) salts inthe 4 kDa and 1 kDa media, as there was a strong possibilitythat the ions were retained by the membranes, potentially dueto their aggregation with higher molecular weight nutrientsources. The selected Lactobacilli grew better, proving thedependence of growth of the selected bacteria on the metalions (Table 4).

Successfully grown on LMWM, Lactobacilli strains had to betested for bacteriocin productivity. The pre-treated supernatantsof each selected Lactobacilli, grown on optimised modifiedmedia and LMWM, were tested for bacteriocin activity againstthe selected target strain L. delbruckii subsp.lactis. All the threemedia categories can equally support bacteriocin productionand even in higher amounts when the bacteria were grown inLMWM (Table 5). The comparative studies conducted served alsoto investigate whether there was a qualitative difference in theactivity of bacteriocins against the target strain due to the growthof their producers on different media categories. It can be seenthat the bacteriocins derived from Lactobacilli grown on LMWmedium of 1 kDa had the weaker potency.

Separation of bacteriocins produced on low molecular weightmedia using filtration technologyFiltration was the selected extraction and concentration methodthat could also enhance the potency of the bacteriocins produced.Cultured broth solutions produced on all the media categories,were filtered through 4 kDa and 1 kDa MWCO membrane filters.The resulting retentates were tested for bacteriocin activity(Table 6). The resulting retentates containing bacteriocins hadstronger antimicrobial activity, with the bacteriocins becomingmore potent. In the case of bacteriocins developed on theoptimised unfiltered medium, the bacteriocin yield was onlyslightly enhanced. In contrast, in the case of LMWM bacteriocinsthe potency was significantly reinforced, resulting in successfulseparation. Filtration is proven to be a highly successful methodfor separation of the substances from the nutrient broths, beingrelatively inexpensive and quite easy to implement.

CONCLUSIONSThe above studies indicate the ability of the developed 4 kDa and1 kDa LMWM media to support the production of antimicrobialpeptide 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

Unfiltered mediumLMWM

(4 kDa incl. metal ions)LMWM

(1 kDa incl. metal ions)

Lactobacilli


Indicator strainAmount of bacteriocin








L. casei 0.12 115 0.005 165 0.004 170

L. plantarum 0.11 120 0.003 180 0.002 185

L. lactis 0.09 130 0.002 185 0.007 155



the target strain, being highly potent, regardless of the fact thatLactobacilli were grown on different media. These results areencouraging as they indicate that these media can be used whenupscaling bacteriocin production and purification using filtrationas the separation method, having solved the problem of excessproteins.

REFERENCES1 Carr JG, Cutting CV and Whiting GC, Lactic Acid Bacteria in Beverage

and Food, 1st edn. Academic Press, New York (1975).2 Ross RP, Desmond C, Fitzgerald GF and Stantch C, Overcoming the

technological hurdles in the development of probiotic foods. J ApplMicrobiol 98:1410–1417 (2005).

3 Rodriguez E, Martinez MI, Horn N and Dodd HM, Heterologousproduction of bacteriocins by lactic acid bacteria. Int J Food Microbiol80:101–116 (2003).

4 Rodriguez EGB, Gaya P, Nanez M and Medina M, Diversity ofbacteriocins produced by lactic acid bacteria isolated from rawmilk. Int Dairy J 10:7–15 (2000).

5 Chen H and Hoover DG, Bacteriocins and their food applications.Compr Reviews Food Sci Food Safety 2:83–97 (2003).

6 Moll GN, Konings WN and Driessen AJM, Bacteriocins: mechanismof membrane insertion and pore formation. Anton van Leeuw3:185–195 (1999).

7 Daw MA and Falkiner FR, Bacteriocins: nature, function and structure.Micron J 27:467–479 (1996).

8 Jack RW, Tagg JR and Ray B, Bacteriocins of gram-positive bacteria.Microbiol Rev 3:171–200 (1995).

9 Mierau I, Optimization of the Lactococcus lactis nisin-controlled geneexpression system NICE for industrial applications. Microbiol CellFact 4:16–28 (2005).

10 Ross RP, Desmond C, Fitzgerald GF and Stantch C, Overcoming thetechnological hurdles in the development of probiotic foods. J ApplMicrobiol 98:1410–1417 (2005).

11 Cleeveland J, Montville TJ, Nes IF and Chikindas ML, Bacteriocins: safe,natural antimicrobial for food preservation. Int J Food Microbiol71:1–20 (2001).

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

13 Paul Ross R, Morgan S and Hill S, Preservation and fermentation: past,present and future. Int J Food Microbiol 79:3–16 (2002).

14 Berridge NJ, Preparation of the antibiotic nisin. Biochemistry45:486–492 (1949).

15 Cheeseman GC and Berridge NJ, Observation on molecular weightand chemical composition of nisin A. Biochem J 71:185–195 (1968).

16 White HR and Hurst A, The location of nisin in the producer organismStreptococcus lactis. J Gen Microbiol 3:171–179 (1968).

17 Maldonado A, Barda-Ruiz J and Jimenez-Diez R, Purification andgenetic characterization of plantaricin NC8, a novel culture-inducible two-peptide bacteriocin from Lactobacillus plantarumNC8. J Appl Environ Microbiol 69:383–389 (2003).

18 Todorov SD, Van Reenen C and Dicks LM, Optimization of bacteriocinproduction by Lactobacillus plantarum ST13BR, a strain isolatedfrom barley beer. J Gen Appl Microbiol 50:149–157 (2004).

19 Uteng M, Hauge HH, Brondz I, Nissen-Meyer J and Fimland G, Rapidtwo-step procedure for large-scale purification of pediocin-likebacteriocins and other cationic antimicrobial peptides fromcomplex culture medium. J Appl Environ Microbiol 5:952–956 (2002).

20 Deraz S, Karlsson E, Hedstorm M, Andersoon M and Mattiason B,Purification and characterisation of acidocin D20079, a bacteriocinproduced by Lactobacillus acidophilus DSM 20079. J Biotechnol117:343–354 (2005).

21 Bujalance C, Jimenez-Valera M, Moreno E and Ruiz-Bravo A, A selectivedifferential medium for Lactobacillus plantarum. J Microbiol Meth66:572–575 (2006).

22 Prusiner S, Scott MR, Stephen J, DeArmond SJ and Cohen FE, Prionprotein biology cell. 93:337–348 (1998).

23 Johnson RT and Gibbs CJ, Creutzfeldt–Jakob disease and relatedtransmissible spongiform encephalopathy’s review article. NewEngland J Med 339:1994–2004 (1998).

24 Foster PR, Prions and blood products. Annals Med 32:1365–2060(2000).

25 Coulson JM and Richardson JF, Chemical Engineering, Chemical andBiochemical Reactors and Process Control, 3rd edn. Pergamon Press,Oxford (1994).

26 Demicri A, Pomento AL, Lee B and Hinz P, Media evaluation of lacticacid repeated-batch fermentation with Lactobacillus plantarumand Lactobacillus casei subsp.rhamnosus. J Agricul Food Chem46:4771–4774 (1998).

27 Callister WDJ, Fundamentals of Materials Science and Engineering: anIntegrated Approach, 2nd edn. John Wiley and Sons, Inc, New York(2004).

28 Moachon N, Boullanger C, Fraud S, Vial E, Thomas M and Quash G,Influence of the charge of low molecular weight proteins on theirefficacy of filtration and/or adsorption on dialysis membranes withdifferent intrinsic properties. J Biomater 23:651–658 (2001).

29 Zacharof MP and Lovitt RW, Investigation of shelf life of potencyand activity of the lactobacilli produced bacteriocins throughtheir exposure to various physicochemical stress factors.Probiot Antimicrobiol Prot (2012). DOI 10.1007/s12602-012-9102-2.http://www.springerlink.com/content/g4451u146008736g/

30 Van Reenen ML, Dicks LMT and Chikindas ML, Isolation, purificationand partial characterisation of plantaricin 423 a bacteriocinproduced by Lactobacillus plantarum. J Appl Microbiol84:1131–1137 (1998).

31 Todorov SD, Vaz-Velho M and Gibbs D, Comparison of two methodsfor purification of Plantaricin ST31, a bacteriocin produced byLactobacillus plantarum ST31. J Braz Microbiol 35:157–160 (2004).

32 Mierau I and Lei JP, Industrial scale production and purification of anheterogenous protein in L. lactis using the Nisin-controlled geneexpression system NICE: the case of lysostaphin. Microbiol Cell Fact4:1–9 (2005).

33 Zendo T, Nakayama J, Fujita K and Sonomoto K, Bacteriocindetection by liquid chromatography/mass spectrometry for rapididentification. J Appl Microbiol 104:449–507 (2008).

34 Dembczynski R and Jankowski T, Growth characteristics and acidifyingactivity of Lactobacillus rhamnosus in alginate/starch liquid corecapsules. Enzyme Microbiol Technol J 31:111–115 (2002).

35 Desjardins P, Meghrous J and Lacroix C, Effect of aeration and dilutionrate of nisin Z production during continuous fermentation with freeand immobilized Lactococcus lactis UL719 in supplemented wheypermeate. Int Dairy J 11:943–951 (2001).

36 Hoefnagel MHN, Metabolic engineering of lactic acid bacteria, thecombined approach: kinetic modelling, metabolic control andexperimental analysis. J Microbiol 148:1003–1013 (2002).

37 Bober JA and Demicri A, Nisin fermentation by Lactococcus Lactissubsp.lactis using plastic composite supports in biofilm reactors.Agric Eng Int: the CIGR J Sci Res Devel 6:1–15 (2004).

38 Deegan LH, Cotter PD, Colin H and Ross P, Bacteriocins: biologicaltools for bio-preservation and shelf-life extension. Int Dairy J16:1058–1071 (2006).

39 Konings WN, Kok J, Kulipers O and Poolman B, Lactic acid bacteria: thebugs of the new millennium. Curr Opin Microbiol 3:276–282 (2000).

40 Liew SL, Ariff AB, Racha A and Ho YW, Optimization of mediumcomposition for the production of a probiotic microorganismLactobacillus rhamnosus using response surface methodology. IntJ Food Microbiol 102:137–142 (2005).

41 Ostlie HM, Helland MH and Narvhus JA, Growth and metabolism ofselected strains of probiotic bacteria in milk. Int J Food Microbiol87:17–27 (2003).

42 Todorov SD and Dicks MT, Growth parameters influencing theproduction of Lactobacillus rhamnosus bacteriocins STR461BZ andST462BZ. Annals Microbiol 55:283–289 (2005).

43 Mollendorff von JW, Todorov SD and Dicks MT, Optimization of growthmedium for production of bacterocin produced by Lactobacillusplantarum JW3BZ and JW6BZ and Lactobacillus fermentum JW11BZand JW15BZ isolated from boza Trakia. J Sci 7:22–33 (2009).

44 Aktypis A, Tychowski M, George Kalantzopoulos G and AggelisG, Studies on bacteriocin (thermophilin T) production byStreptococcus thermophilus ACA-DC 0040 in batch and fed-batchfermentation modes. Antony van Leeuwen 92:207–220 (2007).


low molecular weight liquid media development for lactobacilli producing bacteriocins

Documents