Journal of Education Chemistry

7/28/2019 Journal of Education Chemistry

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Research Article

Recei ved: 22 May 201 2 Revi sed: 10 Jun e 201 2 Accepted: 11 Ju ne 2012 Pu blish ed on lin e i n Wil ey Onl ine Lib rar y: 20 July 20 12

(wileyonlinelibrary.com) DOI 10.1002/jctb.3892

Low molecular weight liquid media

development for Lactobacilli producingbacteriocinsMyrto-Panagiota Zacharof ∗ and Robert W. Lovitt

Abstract

BACKGROUND: Contemporarypurification techniquesof 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 molecularweight 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, Lactobacillus

plantarum NCIMB 8014, Lactobacillus lactis NCIMB 8586. All were successfully grown (µ max 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 currentproductionnutrient media. The uptake of thenutrientsourcesis facilitated as nitrogensources,whichwere primarilyresponsiblefor 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 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 today’s 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 re-

duction (acidification, 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-artificial, biologically safe products providing theconsumers with high health benefits.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 acidifiers. Their potential

use as bacteriocidal agents would constitute a great commercial

benefit. 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 mem-

branepermeabilizing 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 (+) 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

Lactobacillibacteriocinshas beenshownto beeffectivewhen 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; 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

pathogenicbacteria.7,8 Severalother 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 for replacement of chemical

food 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, Swansea

University, Swansea, SA2 8PP, UK

J Chem Technol Biotechnol 2013; 88: 72–80 www.soci.org c 2012 Society of Chemical Industry



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

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% purified). 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 purification 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, sincefor the culturingof Lactobacilli, complex mediaare 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 define

the nutritional conditions determining the growth yield and the

metabolites’productivity ofthe selectedbacteria.Lactobacillihave

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 availablemedia for Lactobacilli propagation include

Man 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 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 specifically

designed to support the growth of Lactobacilli. It contains rich

nutrientsourcessuitableto support the highauxotrophic needs of

these organisms. It canbe easily prepared andit is highlyselective,

its rich content of nitrogen sources and minerals ensure bacterialgrowth 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, efficient, cost effective

media, capable of further improvements was created, containing

low molecularweight nutrientsources (LMWM). The development

of the LMWM was proposed mainly to facilitate the future

separation andconcentration of desiredmetabolic products,using

ultra-andnano-filtration,fromtheculturedbroth.Additionally,the

uptake of the nutrientsourceswould 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 30 kDa, 4 kDa

and1 kDa MWCO) of a modified liquidmedium which had already

been established as themost suitable forthe selected Lactobacilli.

The developed media were tested for bacteriocin production

and biomass growth, using three known bacteriocin-producing

Lactobacilli strains, Lactobacilluscasei NCIMB 11 970, Lactobacillus

plantarum NCIMB 8014, Lactobacillus lactis NCIMB 8586.

MATERIALS AND METHODSBacterial 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

modified optimised liquid medium containing 20 g L−1 glucose,

yeast extract (YE) 20 g L−1, sodium acetate 10 g L−1, tri-sodium

citrate 10 g L−1, potassium hydrogen phosphate 5 g L−1 . Inall the

experimental 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) and

alumina seals (Wheaton Industries, USA). They were autoclaved

(120 ◦C for15 min)(Priorclave: Tactrol 2, RSC/E,UK) andleft 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 growthcurves were obtained by plotting OD against time. The maximum

and specific growth rates (µmax, h−1 and µ, h−1) of bacteria were

calculated from logarithmic plots of theOD versustime duringthe

exponential growth phase, according to the formula:

µ(h−1) =1

x

dx

dt=

d(lnx)

dt=

ln 2

DT(1)

where

DT (h) =(t2 − t1)

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

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

glucose, YE 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 of low molecular weight media (LMWM). A bench

membrane 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 of

a stirred cell unit of 200 mL maximum process volume, a magnetic

stirrerand filtrationeffectivearea of 28.7 cm2.Thestirrerspeedwas

set at 150 rpm. Filtration of media was achieved through a series

of ultrafiltration and nanofiltration membranes The molecular

weight cut-off (MWCO) of ultrafiltrationpolysulphonemembranes

in usewas 30 kDa (cellulose acetate,Microdyn-Nadir Co.,Germany)

J Chem Technol Biotechnol 2013; 88: 72–80 c 2012 Society of Chemical Industry wileyonlinelibrary.com/jctb



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

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) magneticimpeller,provides cross flowconditions, (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)retainingstand prevents displacement of cap when pressure is usedin theunit.

and 4 kDa (polysulfone, Microdyn-Nadir Co., Germany) while

nanofiltration 1 kDa (polysulfone, General Electric-Osmonics Co.

USA). The cell unit was pressurized by constant compressed

nitrogen at 200 kPa.

The operatingtemperaturewas controlledto 25 ◦Cusingawater

jacket with water bath (Grant Water bath, UK). The stirred cell unitwas operatedin batch dead-endmode. After eachexperiment,the

components of the unit cell were soaked in an ethanol solution

(50% v/v) for24 h. Themembraneswererinsed with distilledwater

and sterilised with 25% v/v ethanol solution.

Determinationof permeateflux, membraneresistance and cake

resistance were obtained from the standard equations25 used for

evaluating 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 pressure

conditions varying between 0 and 400 kPa with the following

solutions, sterilised distilled water, 10 mmol L−1 phosphate buffer

(KH 2PO4) buffer (Sigma-Aldrich, UK) and sterilised basal medium.

For each experimental run 150 mL of the selected solution was

inserted in the reactor.

Determination of protein sources in low molecular weightmedia 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 filters (Whatman 0.2 µm qualitative

filters, 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 weightmedia 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 tothe size ofthe particles.27 This is done by illuminating

the particles with a laser beam and analysing the intensity

fluctuations of the scattered light. The relationship between the

size of a particle and its speed due to Brownian motion is definedas the Stokes–Einstein equation

D = k β T/3πηd (8)

where K β (1.3807 × 10−23 J K −1) is the Boltzmann constant, T

is the absolute temperature in Kelvin (K), and η is the vicosity

(8.937× 10−4 kg m−1 s−1) of the mediumin which the particles of

diameterd (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 graphically

represented in curves where the highest peak represents the

majority 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 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 weightmedia 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 usinga high performance liquid chromatograph (HPLC) method. The

HPLC systemwas connectedto 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

4000–5000MW,enzymaticdigestfragments,naturalandsynthetic

peptides and complex carbohydrates. The solvent (mobile phase)

delivery systemwasformedof twopumps (pumpsA andB) (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

wileyonlinelibrary.com/jctb c 2012 Society of Chemical Industry J Chem TechnolBiotechnol 2013; 88: 72–80




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 a

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−1 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 20µL 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 L−1 of HCl, 25 mg of nisin were dispersed.

This solution equals 1000 IU mL−1 of nisin. According to this

formula the necessary quantities of solid nisin were calculated to

fabricatestandard 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 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 L−1. The

target 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-sodium

citrate,5 g L−1 di-hydrogenorthophosphate,magnesiumsulphate

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 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 defined 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 clarifiedliquid was filteredthrougha 0.2 µmporesize filter forsterilisation.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 bacteriocinsproducedon low molecular weightmedia using filtration technology

A bench membrane apparatus (stirred cell unit reactor, Amicon

8200, Millipore Co., UK) was used for filtration of the cultured

LMWMand unfilteredoptimisedmediacellfree (viacentrifugation)

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 cm2. The cultured cell free supernatant was filtered through

a nanofiltration 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.Thedatawerestatisticallyanalysedforaccuracyandprecisioncalculating standard deviation, standard error, experimental error,

regression factor and reading error (Microsoft Excel software

Version 2003). All the numerical data provedto be highly accurate

and reproducible having mean standard deviation below 5% and

experimental error below 5%, offering highly significant results.

RESULTS AND DISCUSSIONMembrane characterisation and filtrability of the nutrientmedium

In order to determine the membrane resistance and the influence

of pressure during operation of the equipment, membrane

characterisation studies were carried out. The permeability of distilled water, optimised nutrient medium,

and phosphatebuffer(10 mmol L−1) solution throughmembranes

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

throughthemembranewasmeasuredtoanalysethemembranes’s

behaviour (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

theflux increased from 7.90 to 28.00 m3 m−2 h−1 with an increase

in pressure from 50 to 400 kPa. For phosphate buffer solution the

flux increased from 1.95 to 9.05 m3 m−2 h−1 with an increase

in pressure from 50 to 400 kPa. While operating with optimised

nutrient 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 all

solutions linearly increased with increasing pressure. Pure water

the flux increased from 0.23 to 1.20 m3 m−2 h−1, with an increase

in pressure from 50 to 400 kPa. For phosphate buffer solution the

flux increased from 0.14 to 1.11 m3 m−2 h−1, 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 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 flux

increased from 0.04 to 0.16 m3 m−2 h−1, with an increase in

pressure from 50 to 400 kPa. For phosphate buffer solution the

flux increased from 0.02 to 0.13 m3

m−2

h−1

with an increasein pressure from 50 to 400 kPa. While operating with optimized

nutrient 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 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 thesame. This wasprobably dueto thedifference

in thefabrication material of themembrane itself as well as dueto

the pore size and the general porosity of the filter.

During filtration of the nutrient medium, flux decline over time

was noticed due to the deposition of organic macromolecules

on 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 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 filtrability 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 sourcesin the developed nutrient media

Gravimetry was used to measure the remaining nutrient sourcesin 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

molecularweight nutrient sources to pass through the membrane

filters, resulting in theproduction of thedesired nutrient medium.

All gravimetric analyses depend on final determination of weight

as a means of quantifying an analyte.Weightcan be measuredwith

greateraccuracythananyotherfundamentalproperty,gravimetric

analysis is possibly one of the most accurate and commonly used

methods of analytical chemistryavailable. 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 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 the

results as only theproteinsources derived from yeast extract were

measured in the medium, due to the method’s 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 and 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 sized

between 1 and 15 nm as the nutrient medium contains mostly

oligopeptides. Further filtration was performed through a 1 kDaMWCO membrane filter, 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

filtered through a 30 kDa MWCO membrane filter the nutrient

sourcescontainedintheautoclavedmediumrangeinsizebetween

1 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 distributionof media particlesin autoclavedmedia:unfiltered (a) andfiltered 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 and

needs further filtration. When filtered through the 4 kDa MWCO

filter thenutrient medium containsparticles between1 and20 nm,

with most of the proteins between 1 and 3 nm. The nutrient

medium contained mostly oligopeptides. Further filtration was

performed through a 1 kDa MWCO membrane filter, 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. Thismethodis highly suitablefor quantifying andanalysing

mixtures of chemical compounds due to its high sensitivity

and specificity, 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 sufficient presence

of protein sources in the resulting media (Table 3), certifying also

the removal of larger protein molecules.

Testing the low molecular weight nutrient mediafor lactobacilli growth and bacteriocin production

As LMWM were successfully developed, the next step was to

investigate whether they could sufficiently support Lactobacilli

growth providing highbiomass 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 6.281.575 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 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





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 1.64L. 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 of Bacteriocin

Produced (IU mL−1).


Indicator strain

Amount of Bacteriocin

Produced (IU mL−1)


Indicator strain

Amount of bacteriocin

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 115L. lactis 0.10 125 0.10 125 0.11 120

growth rates achieved were small, when compared with the

optimised unfiltered medium. Further investigation to achieve

higher growth yields was made by incorporating metal ions

of manganese (0.5 g L−1) and magnesium (0.05 g L−1) 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 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

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 bacteriocinsproduced on low molecular weightmedia using filtration 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 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 had

stronger antimicrobial activity, with the bacteriocins becoming

more potent. In the case of bacteriocins developed on theoptimised unfiltered medium, the bacteriocin yield was only

slightly enhanced. In contrast, in the case of LMWM bacteriocins

the potency was significantly 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

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 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 purification using filtration

as the separation method, having solved the problem of excess

proteins.

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