______________________________________________________________

Separation of lactobacilli Bacteriocins from Fermented Broths Using Membranes

Myrto-Panagiota Zacharof *a,b,c, Gregory M. Coss a ,Stephen J. Mandale a,c, and Robert
W. Lovitta,b,c
a

Centre for Complex Fluid Processing (CCFP), College of Engineering, Swansea University, Talbot building,

Swansea, SA2 8PP, UK

b

Multidisciplinary Nanotechnology Centre (MNC), College of Engineering, Swansea University, Swansea, Talbot

building, SA2 8PP, UK

c

Centre for Water Advanced Technologies and Environmental Research (CWATER), College of Engineering,

Talbot building, Swansea University, Swansea, SA2 8PP, UK

______________________________________________________________________________

Highlights

UF and NF separated bacteriocins of cell debris and medium components successfully.

Improved recovery yields between 53 % to 57% were achieved employing an UF 4 kDa MWCO
membrane.

A total activity yield between 44% to 53% was achieved with a UF 4 kDa MWCO filter.

Further improved yields between 64 % to 68% were attained using a NF 1kDa MWCO membrane.

Further enhancement of activity up to 36% was accomplished through NF 1 kDa MWCO filter.

*Corresponding author. Tel. +44(0)7413541769.

E-mail address: myrtozacharof1981@yahoo.com (Myrto-Panagiota Zacharof)

[2]

Abstract
Current separation, isolation and purification techniques to obtain highly potent purified lactobacilli and lactococcoi
bacteriocins include chemical precipitation, separation employing solvents and chromatographic techniques. These
methods are arduous, costly, with limited scalability, offering low bacteriocin yields (<20%). To address these
challenges, the alternatives of ultrafiltration and nanofiltration, as separation methods were tested. Three promising
bacteriocin producing strains, Lactobacillus casei NCIMB 11970, Lactobacillus plantarum NCIMB 8014 and
Lactococcus lactis NCIMB 8586 were selected to investigate the applicability and feasibility of the method.
To facilitate separation, the microorganisms were grown on specially developed low molecular weight medium
(LMWM) mainly containing nutritive sources up to 4 kDa molecular weight. Bacterial cells were removed by
centrifugation. The clarified broths were filtered using 4 and 1 kDa MWCO. Bacteriocin activity was determined by
an antimicrobial activity test using nisin, which has an inhibitory effect on the growth of susceptible
microorganisms.

Recovery yields using filtration were found to range between 53 to 68%, a high recovery

performance.
The bacteriocin activity of crude extracts of all the three lactobacilli were between 95-105 IU ml-1. When the
substances were separated using ultrafiltration membrane (4kDa MWCO) their activity was enhanced to 145-150 IU
ml-1, achieving a total potency yield of 44% to 53%. Further enhancement of yields up to 36% was attained
employing nanofiltration (1 kDa MWCO) membranes with an activity increased up to 200 IU ml-1.
Bacteriocin isolation from crude extracts using filtration was found to be effective, offering high recovery yields,
optimising their activity as well as presenting a realistic option towards the formulation of these as commercially
available antibacterial agents.

Keywords: Bacteriocins; lactobacilli; Downstream processing; Separation; Ultrafiltration, Nanofiltration

[3]

1. Introduction

Numerous Lactic Acid Bacteria (LAB) including lactobacilli [1], produce bacteriocins as primary metabolites [2].
These are substances of peptide structure (either peptides or polypeptides). Bacteriocins are of low molecular weight
(usually below 10 kDa) possessing strong antimicrobial activities towards a wide sprectrum of bacterial agents [3].
Lactobacilli bacteriocins have strong potential for application in the food and feed industry [4], as preservative and
anticontaminant agents, as the use of LAB and of their metabolic products is generally considered as safe (GRAS,
Grade One) [5].

The application of bacteriocins has been considered to be compatible with other antimicrobial compounds (acetic,
lactic acid) resulting in the inhibition of growth of pathogenic microorganisms, simultaneously improving food
quality and sensory properties [6]. In addition, bacteriocins are easily degradable during mammalian digestion by
proteases of the gastrointestinal tract, a phenomenon that constitutes them safe for human consumption [7].

Bacteriocins can be applied in a food product in multiple ways. For instance either as an ingredient of purified or
crude form, or added through a bacteriocin producing strain (starter culture) [8, 9]. Their application as a barrier
against food spoiling microbial agents and bacterial pathogens has been proven to be efficient [10].

Their employment in a highly purified form is possibly preferable. If induced through a bacteriocin producing
strain, different to the strain or strains used to ferment the product, might result in minimizing the antimicrobial
activity of the bacteriocins towards the food spoiling agents or producing an negative effect on the fermentative
culture, an aspect that can potentially be avoided if the compound is introduced timely independently to a starter
culture. Furthermore, if available in purified form , bacteriocins can be easily applied in meat and fish products,
products of vegetable origin including fruit juice, beverages and fresh vegetables [65] as food additives, as well as
integrated in food packaging [63,64,70]. Other applications of purified bacteriocins could be their inclusion in

[4]

pharmaceutical and cosmetic products since nisin has been successfully included in dental-care products,
pharmaceutical products such as stomach ulcers and colon infection treatment products and potential birth control
[69].

Regardless of the high number of identified bacteriocins having vigorous antibacterial action, only nisin A produced
by Lactococcus lactis subsp. lactis has been approved as a purified food additive for use in the industry worldwide
(food additive, under the number E234 ECCU 1983 EEC Commission Directive 8314631EEC) [8-10].

This is possibly due to the difficulty of mass production and effective recovery of these substances,

as

contemporary separation and purification techniques rely on solvent extraction [11,12], chemical precipitation
[13,14], ion exchange chromatography [14-16] and liquid chromatography techniques [17-19,29], that are complex
to carry out in large scale, requiring multiple processing steps leading to poor yields.

Even for the commercially available nisin, industrial isolation and purification methods used have not been
extensively described in the literature [66]. However it has been referred that nisin is produced biotechnologically,
and its isolation follows a pattern of foam or filtration concentration, followed by salt precipitation, centrifugation,
spray drying and pin milling [71].

Nevertheless, highly potent bacteriocins are achieved at low yields (<20%) [20, 21] with these methods. Yields are
possibly low due to the presence and precipitation of numerous other peptides resulting from the complex culturing
media used.

These peptides have been found to mask the activity of bacteriocins, resulting in reduction of their effectiveness
against the target strains [21]. The development of low molecular weight nutrient media (LMWM) has been
proposed [39] and proven highly effective to address this hurdle [39]. However, the need to extract the produced

[5]

antimicrobial substances in a highly purified form, available for further processing towards the fabrication of a
commercial products necessary.

Several researchers [22, 23] have used methods based on propanol- sodium chloride or butanol-acetic acid
extraction from culture supernatants, [24] various column chromatography techniques (ion exchange, reverse phase)
and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [25,26]. These methods are laborious,
demanding extensive sample preparation. They are complicated due to the multi-step procedures required,
protracted processing time required, and expensive due to the high resin cost of equipment resins, solvents and
chemicals [21]. Their industrial scale implementation to produce a highly potent purified bacteriocin [21-26] has
therefore been considered challenging.

Contrary to these methods, membrane filtration [27, 29, 30], is an efficient, functional, economical and easily
scalable process [31, 32]. Nowadays is a well established process in the food and pharma industries, with a
complete range of membrane filtration technologies being commercially available. These include microfiltration
(MF), ultrafiltration (UF), nanofiltration (NF) and reverse osmosis (RO) successfully employed to clarify,
concentrate, desalt, fractionate and recover components from complex effluent streams. Examples include lactic
acid [33], low molecular weight substances such as hormones and endocrine disrupters [58-60] and antibiotics from
complex sources [61] such as cheese whey [34] and fermented broths [35].

Evident benefits of this approach do include the use of a solely based upon physical function processes, an
advantage that can lead to the reduction or avoidance of chemical agents usage such as emulsifiers, acidifiers or
coagulation agents [33, 36]. The separation of aqueous systems is based on selectivity by size (sieving), polarity or
electrical properties rather than phase change means that they have a low energy process [37, 38]. Most if not all, of
lactobacilli bacteriocins are hydrophobic molecules [14, 21, 73-75]. Taking advantage of this ability, hydrophilic
UF - and NF membranes were used to separate, concentrate and partially purify these small size extracellular

[6]

metabolites [39,40,57] and expected to achieve an enhanced retention by the filters due to their hydrophobicity
[41,36]. Using these membranes, additionally to the development of

bacteriocin-producing strains on low

molecular weight media (LMWM) it would be expected to facilitate the recovery of bacteriocins.

Isolation from crude extracts could possibly enhance their potency and activity [21, 42] as well as offer a viable
option towards the formulation of biological preservatives. Three promising bacteriocin producing candidates,
namely Lactobacillus casei NCIMB 11970, Lactobacillus plantarum NCIMB 8014 and Lactobacillus lactis
NCIMB 8586 [43, 44, 47], were selected to investigate the applicability of the membrane recovery of bacteriocins
from LMWM.

Consequently, this paper reports on the investigation of the production of bacteriocins on LMWM and the
subsequent isolation of production materials using ultrafiltration and nanofiltration. The paper also reports isolation
of nisin from model solutions. The concentrated materials were characterised by several methods, including reverse
phase chromatography, peptide content and trypnination.

2. Materials and Methods

2.1. Bacterial strains
Lactobacillus plantarum NCIMB 8014, Lactobacillus lactis NCIMB 8586, Lactobacillus casei NCIMB 11970 [43,
44, 47] and the target strain Lactobacillus delbruckii subsp. lactis NCIMB 8117 were provided in a lyophilised
form by the National Collection of Industrial Food and Marine bacteria (NCIMB) , Aberdeen , Scotland, United
Kingdom.

2.2. Culturing conditions

The bacteriocin producing strains L. casei, L. plantarum, and L. lactis, were cultured in a 2L capacity continuously
stirred tank reactor (CSTR) (Fig.1) operated batchwise.

[7]

The 2L round glass fermenter was set to operate at a constant temperature of 36C and provide pH control (6.5). pH
was controlled through the use of a pump supplying alkali/acid to the culture, while the provision of gaseous
nitrogen to the headspace ensured anaerobic conditions were maintained at all times. The fermenter was equipped
with an autoclavable pH probe (Fisher Scientific, UK) connected to an automated pH controller (Electrolab FerMac
260, UK). This worked a pump to supply alkali/acid feed to the culture when the level of pH was detected to be
below or above the set point. Agitation was provided by a magnetic stirrer coupled bar for agitation (350 rpm). The
fermenter had several ports for control and sampling including a sampling and inoculation port and was sealed with
silicone rubber. Anaerobic conditions were maintained through the use of a glass air lock, while gas in and gas out
ports were fitted with filters (Polyvent filter, 0.2m, Whatman Filters, United Kingdom) to prevent contamination.
The temperature of the fermentation media was monitored with the use of a glass thermometer and was controlled
via the use of stainless steel coils connected to a thermostatically controlled water bath.
A specially designed and developed LMWM was used, as previously described [39]. This had been proven to
achieve high biomass yields [39, 45-47] simultaneously with elevated yields of bacteriocins.

The medium

comprised of 20 g L-1 glucose, 20 g L-1 yeast extract, 10 g L-1 sodium acetate, 10 g L-1 tri-sodium citrate, and 5 g L-1
potassium hydrogen phosphate. The medium was filtered initially through a microfiltration membrane (0.2m
Whatman filters), using a bench scale unit (Amicon 8200) followed by filtration through two ultrafiltration
membranes 30 kDa (Millipore Co. United Kingdom) and then a 4 kDa (Microdyn-Nadir, Germany) molecular
weight cut-off (MWCO) respectively. Into the final collected volume magnesium sulphate and manganese sulphate
were added into trace quantities of 0.05 g L-1 and 0.005 g L-1 respectively [39].
During the fermentation, samples were taken aseptically on an hourly basis from the sampling port and transferred
into 10 ml conical plastic tubes (Fisherbrand, UK) and centrifuged (Biofuge Stratos Sorall, Kendro Products,
Germany) (4C, 4000 g, 15 min) for complete biomass removal. When it was determined that growth was at early
death phase, the fermentation was stopped. Collected samples which had been clarified by centrifugation were then
filtered (0.2 m pore size filter) and the pH adjusted to 6.0 to eliminate the antimicrobial effect of lactic acid and

[8]

then they were diluted with fresh medium [48, 49]. These crude extracts samples were then tested for antimicrobial
activity.

2.3. Measurement of cellular growth and biomass

Determination of cell growth was measured as a change in turbidity by optical density (O.D.) at 660 nm wavelength
using a spectrophotometer (PU 8625 UV/VIS Philips, France). Cellular sedimentation was minimised by gently
mixing each tube prior to measurement. A blank of nutrient media was used as a control to prevent interference of
colour. The maximum and specific growth rates (max, h-1 and , h-1) of bacteria were calculated from the
logarithmic plots of the O.D. versus time during the exponential growth phase. To convert the O.D. measurements
into dry weight units (g L-1) of the bacteria dry weight determination assays [50, 51] were performed.
The following equations Y = 0.5471x + 0.014639, Y = 0.7850 x + 0.1709, Y = 0.5755 x + 0.02066 and Y = 0.4266
x + 0.3814 [35] were obtained for L. casei, L. plantarum, L. lactis and L .delbruckii subsp. lactis respectively. In
these linear functions, the Y value represented the intercept slope and the X value was the optical density units.

2.4. Membrane filtration of nisin solutions of known concentrations and crude extracts of lactobacilli
A bench membrane apparatus (stirred cell unit module, Amicon 8200, Millipore Co., UK) was used for the filtration
of the solutions operated batchwise (Fig 2.). The system was composed of a stirred cell unit of 200 ml maximum
process volume, a magnetic stirrer and a filter with an effective area of 28.7 cm. The stirrer speed was set at 100
rpm. The cell unit was pressurized by constant compressed nitrogen to 200 kPa. The operating temperature was
controlled at a constant 25C using a water jacket and water bath (Grant Water bath, UK). The stirred cell unit was
operated in a batch dead-end mode. At the end of each experimental process, the components of the unit cell system
were placed into an ethanol solution (50% v/v) for 24 hours to achieve effective cleaning and sterilisation. The
molecular weight cut-off (MWCO) of the ultrafiltration membrane used was 4 kDa and nanofiltration was 1kDa
(Table 1).

[9]

Prior to each experiment, the membranes were rinsed with distilled water and sterilised with 25% v/v ethanol
solution. Each membrane was characterised under different pressure conditions varying between 0 to 400 kPa using,
sterilised distilled water (Millipore RiOs) and 10mM phosphate buffer (KH2PO4) buffer (Sigma-Aldrich, UK).
During the progression of the solutions flux, in dependence to the transmembrane pressure, membrane resistance
and hydraulic permeability were determined. The hydraulic membrane permeability (Lp) of each membrane was
defined by the slope of the linear functions using the plots of the flux over the TMP and is characteristic to each
unfouled membrane [55]. For each experimental run 150 ml of the selected solution was inserted in the unit.
Commercially available nisin (2.5% Sigma Aldrich, Dorset, United Kingdom) has a molecular weight range
between 3kDa to 4 kDa. This was used as a substitute for bacteriocins to test the viability of ultra- and nanofiltration
membranes to selectively separate these substances by their molecular size range from cell free crude extracts.
Solutions of 1000 IU ml-1 of 2.5% nisin were formed and incorporated in the liquid medium in order to test the
feasibility of membrane filtration as a separation method. The activity of the model solutions was calculated
according the commercial nisin reference (Nisin, Sigma Aldrich) where one gram is equivalent to 1*106 IU ml-1.
Cultured fermented broth samples were collected in conical 50 ml tubes (Fisherbrand, UK). These tubes were
centrifuged (4C, 4000g, 15 min) for complete biomass removal. Filtration of the resulting supernatants was
achieved through a series of ultrafiltration and nanofiltration membranes (concentration factor 1:10).

These

solutions were filtered under constant pressure of 150 kPa and a stirring speed of 100 rpm.

2.5. Determination of bacteriocin concentration effect of filtration through High Precision Particle Sizer
(HPPS)
In order to measure the size of the filtered bacteriocin samples, 4 ml of retentate and permeate sample were placed
in plastic cuvettes and analysed using the (HPPS Malvern Instruments, USA) instrument. The HPPS was connected
to a personal computer equipped with proprietary control and logging software (Malvern Instruments LDT. DTS
4.20, 2002) and all the measurements were done automatically.

[10]

2.6. Characterisation of the filtered crude extracts

2.6.1. Peptide content
Filtered samples of the cell free crude extracts were analysed for peptide content using the Lowry method [56]. The
method is sensitive to low peptide concentrations ranging from 0.005 to 0.10 mg of peptide per ml. Based on the
peptide content analysed in all the samples (crude extracts, retentates, permeates of 4 kDa and 1 kDa MWCO
membrane filters) the recovery yields were calculated using the following equation [35]
Recovery %= [1- (CP/CR)]*100
where CP (g ml-1) and CR (g ml-1) are the concentrations of permeate and retentate fractions respectively.
2.6.2. Trypsinisation
Crude extracts containing bacteriocins were collected between the 20th - 24th hour of the fermentation and
centrifuged (4C, 4000 g 15min, Biofuge Stratos Sorall, Kendro Products, Germany) for complete biomass removal.
The bacteriocins contained in these crude extracts were further concentrated using a 1kDa polyamide membrane
filter in a 1:10 factor. The resulting retentates containing concentrated bacteriocins were dispersed in pressure tubes
(1ml) and 40 l of trypsin (Sigma Aldrich, UK) of 10000 BAEE IU per mg-1 of protein were added. The tubes were
carefully mixed, sealed and then incubated for 18 hours at 37C. After the incubation period each fraction was
tested for antimicrobial activity against the indicator strain.
2.6.3. Reverse phase chromatography
High Performance Liquid Chromatography (HPLC) was used to further characterise the collected retentates. The
HPLC system, controlled automatically (Prostar Workstation Data, Varian Co., Canada). was connected to a
UV/Vis detector (Dionex, UK) and fitted with a C18 reverse phase column (Vydac 238 TP54, HPLC Columns,
UK). Temperature control of the solvents was maintained using a column incubator (Millipore Co., UK) at 25C.

[11]

The mobile phase was represented by two solutions, solvent A consisted of 99% pure acetonitrile (ACN) 10% v/v in
distilled water and 1% v/v of standard buffer solution and solvent B of 99% pure ACN75% in distilled water and
1% v/v of standard buffer solution. The standard buffer solution consisted of 7.5% trifluroacetic acid (TFA) 5 % v/v
triethylamine (TEA) and 65% of 99% pure ACN in distilled water. The mobile phase was organised as gradient,
consisting of 65% of solvent A and 35% of solvent B. The flow rate of the samples and of the mobile phase was set
at 1.5 ml min-1 for 15 minutes, at a wavelength of 220nm. Each run lasted for 17 min. All the samples were injected
into the system via a sterile HPLC plastic syringe (1 ml sterile syringe, Fischer brand, UK) at a 20 l injection loop
connected with the HPLC system [39].

2.8. Determination of nisin and bacteriocin activity and potency

The activity and the potency of nisin and the produced bacteriocins were tested according to a simple turbidometric
assay. This assay was based on the effect of several different concentrations of commercial nisin against a target
strain, in terms of growth rate, by developing a calibration curve of different nisin concentration (0-2000 IU ml-1)
effect on the growth of the indicator organism [52-54].
Based on the commercial reference of nisin (Nisin, Sigma Aldrich 2.5%), 1 gram equals to 1*106 IU , 25 mg of
nisin were used and dissolve into 25 ml of HCl (0.02 M). Thus, this solution had a concentration of 1000 IU ml-1 of
nisin [53]. The following quantities of nisin were calculated to do the standard solutions: 0, 25, 50, 75, 85, 100, 110,
125, 150, 175, 200, 250, 500, 750, 1000, 1250, 1500, 1750, 2000 IU ml-1, which were needed for nisin curve
pattern.
Lactobacillus delbruckii subsp. lactis NCIMB 8117 was selected as the target strain, as lactobacilli bacteriocins
often restrict their activity to strains of species related to the producing species and particularly to strains of the
same species [1, 10, 21, 44].The inoculum of the target strain was consistent in the growth phase, as it was deep
frozen when the growth reached 1.5 g L-1 [39, 46, 47]. The target strain was grown on an optimised liquid medium
containing 20 g L-1 glucose, 20 g L-1 yeast extract, 10 g L-1 sodium acetate, 10 g L-1 tri-sodium citrate, 5 g L-1 di-

[12]

hydrogen orthophosphate, magnesium sulphate 0.5 g L-1 and manganese sulphate 0.05 g L-1. This medium was also
used when testing the effect of bacteriocins and nisin. 8 ml of optimised medium was placed into glass tubes. To
these, 1 ml of the frozen inoculum of L. delbruckii subsp. lactis and 1 ml of the supernatant resulting from pH
control fermentations of differential concentration were added. The samples were gently mixed, and incubated
statically at 36C. The biomass concentration was recorded on an hourly basis by measuring the optical density in a
spectrophotometer (PU 8625 UV/VIS Philips, France) at 600 nm. The developed assay had a maximum detection
limit of 200 IU ml-1 equivalent to nisin.
The amount of the bacteriocin produced by each strain was primarily defined on the samples taken at the end of pH
and temperature controlled fermentations. The selected samples (pH fermentation at 6.5) were transferred into 10 ml
conical plastic tubes (Fisherbrand, UK) and centrifuged (10.000 g, 15 min.) (Biofuge Stratos Sorall, Kendro
Products, Germany) for complete biomass removal. The clarified liquid was filtered through a 0.2 m pore size
syringe filter (Whatman Filters, United Kingdom) for sterilisation. The pH of the sterilised liquid was adjusted to
6.0 to eliminate the antimicrobial effect of lactic acid and then it was diluted with fresh medium.

2.9. Statistical analysis of the experimental data

Standard statistical analysis (Microsoft Excel software Version 2003) was applied to the data to test accuracy,
reproducibility and precision. All the numerical data proved to be highly accurate and reproducible having a
standard deviation of mean below 6% and experimental error below 4% offering highly significant results. Each
differential parameter was triplicated to obtain the average data.

3. Results and Discussion

3.1. Lactobacilli growth on a pH controlled batch reactor and bacteriocin productivity
Previous studies have shown that the production of bacteriocins also is related to cellular growth curve [4, 14, 19,
42]. The optimum bacteriocin production phase is -in general- a phenomenon occurring during the late exponential
or early stationary phase of bacterial growth so the bacteriocins can be considered to be primary metabolites rather

[13]

than secondary [18, 48,49]. In order to investigate whether there is indeed a correlation between the growth phase
and the cellular yields and the potency of the produced metabolites several batch cultures in a pH controlled CSTR
were carried out. LMWM was used as the means of growth and bacteriocins production. This approach has multiple
benefits, for example reusing the waste nutrient sources occurring from the LMWM development, minimising the
cost of the production and at the same time enhancing the recovery of the bacteriocins of the crude extracts.
th

In L.

th

casei (Fig.3a) bacteriocin the strongest antimicrobial effect is noted in late exponential phase, 20 to 24 hour of
growth (95 IU ml-1 of bacteriocin). L. plantarum (Fig.3b) bacteriocin also had strong antimicrobial effect that was
highest in early stationary of growth with a slight difference of potency being between the 22nd to 24th hours of
growth curve (95 IU ml-1 of bacteriocin). Finally for the bacteriocin produced by L. lactis (Fig.3c) its antimicrobial
effect was strongest between the 21st to 24th hours of growth (105 IU ml-1 of bacteriocin). These conclusions are
further substantiated by the growth rates (max 0.22 h-1 to 0.30 h-1).
This is possibly an important finding; it offers the prospect of continuous production of bacteriocin which,
theoretically, is a more productive process technology than batch culture. For all further studies on the produced
antimicrobial compounds, the samples were taken on the optimization phase of production determined in this study.

3.2. Determination of membrane permeability using standard solutions

Prior to use the membranes selected to separate, concentrate and partially purify the produced bacteriocins from the
crude extracts, were tested for membrane permeability using distilled water and phosphate buffer (10mM, pH 6.5)
solution, in an effort to analyze the behavior of the membranes when incorporated in the stirred cell unit. For both
membranes, flux values increased linearly with increasing pressure and the membrane resistance values were found
to rise during the filtration of the solutions at different pressures.
In the case of the 4kDa MWCO membrane pure water flux increased from 13.73 to 55.31 L/m h, respectively with
a pressure increase from 50 to 400 kPa. When employing phosphate buffer solution the flux increased from 7.55 to
46.09 L/m h, with an increase in pressure from 50 to 400 kPa. In the case of 1 kDa MWCO for pure water the flux
increased from 8.15 to 42.55 1 L/mh, simultaneously to an increase in pressure from 50 to 400 kPa, while for
phosphate buffer solution the flux increased from 5.02 to 39.36 L/mh with an equal increase in pressure (Fig. 4).
Coarse particulates are absent from the model solutions, so flux is a linear correlation to the TMP, depending solely

[14]

on the membrane resistance. Membrane resistance (Rm) values rise concurrently with the rise of TMP. Differences
between the numerical results of membrane resistance in the case of 4 kDa (deionised water mean Rm 1.06 *10-11 m1

, phosphate buffer mean Rm 1.67 *10-11 m-1) and 1 kDa (deionised water mean Rm 1.09 *10-11 m-1, phosphate buffer

mean Rm 2.05 *10-11 m-1) are due to the difference of the fabrication material of the membrane itself and to the pore
size and permeability of the filter. These are typical, descriptive results of the behavior of these membranes.

3.3. Bacteriocins crude extracts separation and concentration using membrane filtration
The potential separation efficiency, permeability and ability to concentrate bacteriocins from crude extracts using
membrane technologies was initially tested using synthetic solutions i.e. nutrient media containing known
concentration of nisin of 1000 IU ml-1. The resulting permeates and retentate solutions were tested for antimicrobial
activity against the selected target strain L. delbruckii, whose positive control had a max of 0.28 h-1. The resulting
permeate solution had an activity equivalent to 90 IU ml-1 units of nisin (L. delbruckii max 0.17 h-1) while under the
presence of the retentate (concentration factor 1:10) no growth occurred.
Bacteriocin activity was also tested on the crude extracts drawn from CSTR fermentations, with L. lactis extracts
having the highest activity (105 IU ml-1) and L. casei and L. plantarum had an activity of 95 IU ml-1. Having
identified the strains as bacteriocin producers, membrane filtration was selected as a separation and recovery
method of the desired substances.
Cultured broths of the selected lactobacilli were filtered using an ultrafiltration membrane (4kDa MWCO). The
permeates and the retentates were tested for antimicrobial activity against the target strain. In this series of
experiments the normal growth of L. delbruckii was used as a positive control had a maximum growth rate of 0.28
h-1. L. casei clarified retentate had a potency of approximately 145 IU ml-1 bacteriocin equivalent to nisin, while the
permeate had a 25 IU ml-1 potency. For L. plantarum clarified retentate, 140 IU ml-1 of bacteriocin equivalent to
nisin was measured in the permeate 25 IU ml-1 are identified. Lastly the influence of bacteriocin of L. lactis was
studied. It was found to have an activity of 150 IU ml-1 in the retentate and 30 IU ml-1 activity in the permeate.

[15]

The bacteriocins were concentrated over the control using a 4 kDa membrane (Table 2), enhancing their activity up
to 53%. However, permeates still had an antimicrobial effect suggesting that some of the antimicrobial substances
were partially passing through the membrane. Filtration has been shown to be a successful extraction method
facilitating the separation of bacteriocins from the nutrient broth. Further improvement of bacteriocins activity was
attempted using a 1 kDa MWCO membrane.
The 4 kDa retentate solutions produced were filtered through a 1kDa MWCO membrane filter and the resulting
retentates of the solutions were tested for antimicrobial activity against the target strain. L. casei clarified retentate
had a potency of approximately 185 IU ml-1 bacteriocin equivalent to nisin, L. plantarum, clarified retentate had an
activity 190 IU ml-1 of bacteriocin and L. lactis retentate was found having an activity of 200 IU ml-1. Permeates
resulting from the 1 kDa filtration did not have any antimicrobial effect on the indicator strain (L. delbruckii max
0.31 h-1). The resulting retentates of the produced bacteriocins have an even inhibitory stronger effect against the
target strain increasing their effect up to 36% (Table 3).
Having confirmed the antimicrobial activity due to the existence of bacteriocins in the crude extracts and the
enhancement of their activity through separation using membrane filtration the substances were further analyzed by
measuring their peptide nature [57] and calculating the recovery yield. Recovery yields between 53 % to 57%
(Table 4) were achieved when using the 4 kDa MWCO filter. High peptide content was found in the retentates and a
small amount of peptide was present in the permeates. This finding correlates with the limited antimicrobial activity
of the permeates towards the target strain. Further optimisation of yields between 64% to 68% was attained by
employing nanofiltration (1 kDa MWCO) membrane (Table 4).

The use of membrane technology in the case of bacteriocins recovery can offer a viable option towards large scale
production of these compounds. In situ separation of the substances is an achievable process, as commercially
available modules of ultrafiltration and nanofiltration membranes can be attached to reactors treating sequentially
(MF
UF
NF) the crude extracts [67,68,72]. Bacteriocins are extracellular metabolites, facilitating their

[16]

separation from the fermented broths, rich in cellular biomass, by using a microfiltration membrane. Further
separation of the substances from the remaining elements in the broth can be achieved by ultrafiltration,

while

nanofiltration membranes can be used to concentrate the substances. Using the LMWM growth approach can
probably help minimise membrane fouling as most if not all of the nutritive sources in the medium would be used,
leaving a relatively small amount of substances in excess that can form an insoluble cake upon the membranes
[69,70].Consequently, membrane filtration as a fractionation and separation method does have great potential
because of its ease of scalability, due to numerous commercially available robust modules, its economic feasibility
and practicality [27,28,36-39,55,58].

3.4. Demonstration of filtration separation and concentration effect using High Precision Particle Sizer
(HPPS)
The crude extracts and associated supernatants and retentates (4 kDa, 1 kDa MWCO), as well as the model
solutions were sized using dynamic light scattering to define the exact size of the peptides. These measurements
allowed the evaluation of the proper function of the filtration method concerning the removal of the nutritive
sources of yeast extract avoiding much of the interference from high molecular weight peptides. The bacteriocins
were successfully separated and further concentrated by the selected filters. However, in the case of nisin, (Table 5)
it seems that the filters could not successfully separate the substance from the other solids existing in the clarified
solution. This is possibly due to the entrapment of nisin by casein micelles [71]. Casein and its hydrolysate is the
basic peptide existing in the nutrient media used for nisin production while powdered nisin is a mixture of
phosphate, casein, nisin and other solids [71].

3.5. Characterization of content of the filtered crude extracts

3.5.1. Trypsinisation

[17]

To confirm that bacteriocins are peptide nature substances, possessing antimicrobial activities, it was decided to
deactivate any peptide substances existing in the concentrated extracts using trypsin, and then test these samples for
antimicrobial activity. Trypsin is a protease with a broad specificity and can hydrolyze a wide range of peptides. If
treated samples were found to have antimicrobial activity against the target, it could be concluded that the
antimicrobial activity of the extracts was due to substances other than bacteriocins. After the treatment of the
concentrated samples with trypsin their antimicrobial activity was tested against the target strain. It was found that
there was no longer any antimicrobial activity in the concentrated samples. This confirmed the peptide nature of the
bactericidal activity, which can therefore be attributed to bacteriocins. Trypsin was found not to affect the growth of
L. delbruckii (max 0.28 h-1).
3.5.2. Reverse phase Chromatography
Research regarding bacteriocin characterization and purification [15, 16] was performed on bacteriocins using high
performance liquid chromatography. The resulting filtrates, permeates and retentates were tested (Fig.5 [a, b, c]).
The peaks occurring at the start of the chromatogram were the supernatants of the filtration of the samples through
the 1 kDa MWCO membranes; these are peptide substances associated with the growth medium (yeast extract). In
the case of the tested retentates the selected method of separation and extraction of the substance produced the
desired results, with retentates resulting from 1 kDa MWCO filtration demonstrating the best results. For L.
plantarum bacteriocin two peaks are shown in the chromatogram, possibly indicating with the fact that the detected
bacteriocin composed by a two-peptide system (Table 6). Similar results have been obtained by other researchers
[15, 18, 54] however the indentified planaticin [72] by L. plantarum ATCC 8014 is of a 122 kDa molecular weight.
In this study though a different target strain has been used suggesting that a different bacteriocin has been detected.

4. Conclusions
Using membrane filtration technology, an extraction and concentration technique has been developed and
demonstrated for the recovery of bacteriocins from crude fermentation extracts. The use of ultra- and nanofiltration

[18]

membranes was proven to be efficient, achieving a 68% recovery of the antimicrobial compounds up. Membrane
filtration is easy to implement on a large scale and a cost effective separation and recovery process of the desired
products. Membrane filtration employed here did not cause significant loss of bacteriocin activity and minimised
denaturation, foaming and precipitation, all of which can potentially occur during processing due to the peptide
nature of bacteriocins. It was found that bacterocin activity (IU ml-1) against the target strain was enhanced in
process retentates by 56%. Peptide content measurements, sizing and further purification using liquid
chromatography confirmed the peptide nature of these substances. The use of digesting enzymes, which deactivated
the antimicrobial activity of the concentrated bacteriocins, confirmed their peptide nature.

Acknowledgements
The authors would like to thank Dr. Paul M. Williams, Centre for Complex Fluids Processing (CCFP), Centre for
Water Advanced Technologies and Environmental Research (CWATER), Swansea University for his valuable help
in the experimental part of the research summarised here.

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Figure 1: Schematic representation of the CSTR used for bacteriocins production studies from the selected lactobacilli

*Corresponding author. Tel. +44(0)7413541769.

E-mail address: myrtozacharof1981@yahoo.com (Myrto-Panagiota Zacharof)

[25]

Figure 2: Schematic representation of membrane-integrated system for separation of synthetic solutions and bacteriocins crude extracts

[26]

Figure 3[a, b,c]: Bacteriocin Production studies (

) and biomass growth (
) of [a] L.casei [b] L.plantarum and [c] L.lactis

[27]

Figure 4 [a,b]: The effect of pressure on flux during ultrafiltration [a] and nanofiltration [b] of water () and phosphate buffer (
)in
the unit at constant temperature 25C

Figure 5 [a,b,c]: HPLC separation results (1kDa MWCO retentates) of (a) L.casei (b) L.plantarum (c) L.lactis extracts

*Corresponding author. Tel. +44(0)7413541769.

E-mail address: myrtozacharof1981@yahoo.com (Myrto-Panagiota Zacharof)

[29]

Characteristics

Membranes

Model

UH004

YMGESP475

Manufacturer

Nadir (Germany)

GE-Osmonics (USA)

Manufacturer

MICRODYN-NADIR GmbH

Sterlitech Corporation

Address

Kalle Albert Industriepark

22027 70th Avenue S.

Rheingaustrasse 190-196

Kent, WA 98032-1911

D-65203 Wiesbaden

USA

http://www.microdyn-

http://www.sterlitech.com/

nadir.de/cms/index.php?lang=en
Geometry

Flat sheet

Flat sheet

4000 Da

1000 Da

Material

Organic (Polyethersulfone, PESH)

Organic (Composite Polyamide)

Effective Membrane area (cm2)

28.70

28.70

pH range

0-14

2-11

Water affinity

Hydrophilic

Hydrophilic

Rejection size

4000 Da (Polyethylene glycol)

1000 Da (Polyethylene glycol)

Water permeability coefficient

20

18

Molecular

weight

cut-off

(MWCO)

-1

-1

(L m h bar, 20C at 3 bar)

Table1: Membranes characteristics provided by the manufacturers

[30]

lactobacilli

Crude Extracts

Bacteriocins

Ultrafiltration
Membrane (4 kDa

Bacteriocins Activity

Bacteriocins Activity

Optimisation

-1

(IU ml )
Percentage (%)

MWCO)

(IU ml-1)
95

L.casei

L.plantarum

L.lactis

95

105

Retentate

145

Permeate

25

Retentate

140

Permeate

25

Retentate

150

Permeate

30

52.6

47.3

42.8

Table 2: Effect of filtration on the bacteriocins activity (crude extracts, 4 kDa filtrates)

[31]

lactobacilli
Bacteriocins

Ultrafiltration
Membrane (4 kDa

Bacteriocins

Nanofiltration
-1

Bacteriocins

Optimisation
-1

Activity (IU ml )

(1 kDa MWCO)

Activity (IU ml )

Percentage (%)

Retentate

145

Retentate

185

27.6

Permeate

25

Permeate

Retentate

140

Retentate

190

Permeate

25

Permeate

Retentate

150

Retentate

200

Permeate

30

Permeate

MWCO)
L.casei

L.plantarum

L.lactis

35.7

33.3

Table 3: Effect of filtration on the bacteriocins activity (4 kDa filtrates, 1 kDa filtrates)

[32]

lactobacilli

Crude

Ultrafiltration Membrane

Recovery

Ultrafiltration Membrane (1

Recovery

Extracts

(4 kDa MWCO)

Percentage

kDa MWCO)

Percentage

Protein

Protein content (g ml-1)

(%)

Protein content (g ml-1)

(%)

Retentate

55.32

56.92

Retentate

57.05

Permeate

23.83

Permeate

17.88

Retentate

55.36

Retentate

55.09

Permeate

25.34

Permeate

19.63

Retentate

55.11

Retentate

57.89

Permeate

25.43

Permeate

18.45

content
(g ml-1)
L.casei

L.plantarum

L.lactis

75.98

43.02

72.08

54.22

53.85

Table 4: Recovery of proteins using membrane filtration (4 kDa, 1 kDa filters)

68.65

64.36

68.12

[33]

Crude

Ultrafiltration

Ultrafiltration

Extracts

Membrane

Membrane

(nm)

(4 kDa MWCO)

(1 kDa MWCO)

Protein content (nm)

Protein

content

(nm)
901

L.casei
lactobacilli
Bacteriocins

L.plantarum

L.lactis

Nisin (2.5%)

896

882

985

Retentate

459

Retentate

129.6

Permeate

242

Permeate

459

Retentate

531

Retentate

270

Permeate

255

Permeate

396

Retentate

532

Retentate

171

Permeate

296

Permeate

342

Retentate

868

Retentate

615

Permeate

95

Permeate

190

Table 5: Size distribution of bacteriocins and nisin