Antimicrobial microbes-bacteriocin producing lactic acid bacteria · Antimicrobial...

Antimicrobial microbes-bacteriocin producing lactic acid bacteria

Lihua Fan and Jun Song

Atlantic Food and Horticulture Research Centre, Agriculture and Agri-Food Canada, Kentville, Nova Scotia, B4N 1J5, Canada

The contribution of lactic acid bacteria (LAB) to the improvement of food safety and stability of fermented foods has long been known. LAB overgrow other microorganisms of the same ecological niche in food products mainly due to lowering the pH, competing for nutrients, producing various antimicrobial substances such as organic acids, hydrogen peroxide, antibiotics and bacteriocins. Among these antimicrobials, bacteriocins have received great attention in recent years. Bacteriocins are ribosomally synthesized antimicrobial peptides that are inhibitory to other microorganisms, either within the same species (narrow spectrum) or across genera (medium to broad spectrum).

This book chapter focus on bacteriocins producing LAB and covers the following topics:

1. Introduction of LAB

2. Methods of screening bacteriocins producing LAB

3. Antimicrobial spectrum of bacteriocins

4. Optimization of growth conditions for LAB and bacteriocin production

5. Purification and identification

6. Potential application of LAB and bacteriocins in the food industry.

Keywords lactic acid bacteria, bacteriocins, antimicrobials, food safety

1. Introduction

The lactic acid bacteria (LAB) comprise a clade of Gram-positive, low-GC, acid-tolerant, generally non-sporulating, and non-respiring rod-shaped bacilli or cocci that are associated by their common metabolic and physiological characteristics. LAB naturally occur in a large variety of natural environments where soluble carbohydrates, protein breakdown products and low oxygen tension are found. Use of a combination of traditional and molecular techniques has divided bacteria belonging to the LAB family into the following genera: Lactobacillus, Streptococcus, Lactococcus, Enterococcus, Melissococcus, Tetragenococcus, Vagococcus, Trichococcus, Carnobacterium, Desemzia, Leuconostoc, Weisella and Pediococcus [1]. LAB have been historically linked with food fermentations, as acidification inhibits the growth of spoilage microorganisms. The antimicrobial activities of the LAB have been known to play important roles in food fermentations, food preservation and intestinal ecology [2]. In addition, lactic acid and other metabolic products produced by LAB contribute to the organoleptic and textural profile as well as shelf life of the foods [3]. The industrial importance of the LAB is further evinced by their generally recognized as safe (GRAS) status. The basis of LAB’s protection of foods is mainly due to their production of organic acids, carbon dioxide, ethanol, hydrogen peroxide, diacetyl, antifungal compounds such as free fatty acids or phenyllactic acid, antibiotics such as reutericyclin and bacteriocins [4]. Bacteriocins are ribosomally synthesized, extracellularly released low molecular mass peptides which have a bactericidal or bacteriostatic effect on bacteria either from the same species or across genera [2, 5-6]. Furthermore, it is also recognized that LAB have antifungal properties and may be used to eliminate fungal spoilage and/or formation of mycotoxins [7-8]. Proteinaceous bacteriocins are produced by several LAB strains and provide an additional hurdle for controlling spoilage and pathogenic microorganisms. Investigations on bacteriocins have focused on bacteriocin expression; production and mode of action; characterization of plasmids encoding bacteriocin production and immunity and their use as vehicles for gene transfer; identification and description of novel bacteriocins produced by bacteria not previously recognized as bacteriocinogenic; and taxonomic characterization of bacteria based on bacteriocin production or susceptibility [2]. The LAB bacteriocins represent a heterogeneous group of molecules whose classification is periodically revised [5, 9] because much has been learned about the biosynthesis, structure, and mode of action of LAB bacteriocins over the last decade. Generally speaking, LAB bacteriocins are divided into three major classes [10]. Classes I and II are the main classes of bacteriocins due to their abundance and potential use in commercial applications. Class I bacteriocins are also called lantibiotics, they are small (<5 kDa) peptides. Lantibiotics are divided into two groups on the basis of their structural and functional features. Type A lantibiotics are elongated and cationic peptides up to 34 residues in length (for example, nisin). These peptides primarily act by disrupting the membrane integrity of target organisms. Type B lantibiotics are globular, up to 19 residues in length and act through disruption of enzyme function. Class II

Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)

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bacteriocins are also small (<10 kDa) and generally consist of heat-stable, non-lanthionine-containing, and membrane-active peptides. Class II bacteriocins are further divided into three subclasses. Class IIa is the largest group with activity against Listeria (for example Pediocin PA-1). These bacteriocins are promising for industrial applications because of their strong antilisterial activity. They do not have a broad inhibitory spectrum and thus do not kill starter cultures. Class IIb includes bacteriocins with two peptides (requires two different peptides for activity). Other Class II bacteriocins are grouped together as Class IIc (circular bacteriocins). These bacteriocins have a wide range of effects on membrane permeability, cell wall formation and pheromone actions of target cells. Class III bacteriocins are defined as large heat-labile proteins (>30 kDa) such as Acidophilucin A, Lactacin A, and B produced from members of the genus Lactobacillus [11-12]. A fourth class of bacteriocins with a complex structure has also been suggested, and it contains bacteriocins with lipid or carbohydrate moieties which appear to be necessary for activity.

2. Methods of isolation, morphological characterization, screening of antimicrobial activity and identification of LAB isolates

2.1. Isolation and morphological characterization of LAB

LAB can be isolated from a variety of food products of industrial and natural origins including meat products, dairy products, fresh or fresh-cut fruits and vegetables. In our studies, a 25 g sample of fruit, vegetable or dairy product was mixed with 225 ml of sterile peptone water (0.1% w/v peptone, Fisher Scientific, Nepean, ON, Canada) in stomacher bags with filters (Fisherbrand, Filtra-bag, Fisher Scientific) and processed using a stomacher (Seaward, 400 C stomacher circulator, Seaward, England) at 280 rpm for 3 min [13-14]. 100 µL of appropriate serial dilutions were spread linearly on LAB elective de Man, Rogosa and Sharp (MRS, Oxoid, Fisher Scientific) agar and LAB selective Nitrite Actidione Polymyxin (NAP) agar (antibiotically modified all-purpose tween agar) using a Whitley automatic spiral plater (WASP2, Don Whitley Scientific Limited, West Yorkshire, England). Morphologically distinct colonies were isolated from the MRS and NAP plates after incubation at 37 °C for 48 h and streaked on MRS until pure cultures were obtained. Long-term storage of the pure isolates was kept in MRS broth with 30% (v/v) sterile glycerol at -80 ˚C. All potential LAB isolates were Gram stained and catalase tested using 3% (v/v) hydrogen peroxide. MRS cultures of Gram positive and catalase negative isolates were negatively stained using nigrosin to determine cell size and morphology under light microscopy (1000 x magnification, Eclipse E400, Nikon, Tokyo, Japan).

2.2. Screening of antibacterial activity of LAB isolates using the agar diffusion bioassay

The agar diffusion bioassay is the most commonly used by researchers to study the antibacterial effect of LAB isolates. In our studies, the agar diffusion bioassay was performed following the method of Herreros et al. [15] with minor modifications. Listeria innocua subsp. innocua (ATCC 33090TM) and Lactobacillus sakei subsp. sakei (ATCC 15521 TM) were used as indicator organisms. Li. innocua was grown aerobically overnight in Brain Heart Infusion (BHI) broth at 37 ˚C while Lact. sakei was incubated anaerobically (using anaerobic jars with the Gas Pak Plus System from BBL, Fisher Scientific) overnight in MRS broth at 37 ˚C. One mL of the cultured indicator bacteria (5x105 cfu mL-1) was mixed with 15 mL of semi-solid BHI (BHI broth plus 0.7% bacteriological agar, used for Li. innocua) or MRS agar (MRS broth plus 0.75% bacteriological agar, used for Lact. sakei) tempered to 45 ˚C and poured into petri dishes. After solidification of the agar, three wells (5 mm in diameter) were cut into the agar using a sterile cork borer. 35 µL of untreated or treated cell-free supernatant (CFS) obtained from each LAB isolates was added to cut wells as indicated below. The CFS was prepared as follows: LAB isolates from frozen stock were grown overnight and 500 µL was then sub-cultured aerobically overnight in 25 mL MRS broth at 37 °C. Cells were removed by centrifugation at 14,000 x g for 5 min (Sorvall RC6 PLUS, Thermo-electron Corporation, Asheville, NC, USA). The supernatant was filtered through sterile 0.22 µm syringe filters (Chromspec 0.2 µm, 13 mm, PVDF syringe filters, Chromatographic Specialties Inc., Brockville, ON, Canada) to obtain the untreated CFS, which was added to the first well. The pH of the remaining CFS was adjusted from 4.3-4.55 to 6.0 with sterilized 1 N NaOH to rule out antimicrobial inhibition resulting from the production of organic acids, then filtered through sterile 0.22 µm syringe filters and added to the second well. The neutralized CFS was then treated with 1 mg mL-1 of catalase at 25 ˚C for 30 min, filtered through a sterile 0.22 µm syringe filter and added to the third well to eliminate the possible inhibitory action due to hydrogen peroxide and organic acids produced by the LAB isolates (picture 1). The BHI plates were incubated under aerobic conditions at 37 °C for 24 h, and the MRS plates were incubated anaerobically at 37 ˚C for 48 h for the Li. innocua and Lact. sakei indicator bacteria, respectively. Following incubation, inhibition zones were measured using an electronic calliper with digital display (Master-craft MD, Miami, FL, USA).


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Fig. 1 Agar diffusion bioassay showing inhibition zones produced by Ent. faecium (13.2) in a lawn of Lact. sakei grown in MRS medium.

2.3. Enzymatic test for presence of bacteriocins in LAB CFS

The possible presence of proteinaceous compounds in the CFS from LAB isolates, which showed antibacterial activity after acid neutralization and H2O2 elimination, was further analysed in our studies by incubating the treated CFS with 1 mg mL-1 proteolytic enzymes (α-chymotrypsin [C4129], protease [P4630] or trypsin [T8802], all from Sigma-Aldrich Corporation, St. Louis, Missouri, USA) at 37 ˚C for 2 h. The treated LAB CFS (pH adjusted, and H2O2 eliminated) without addition of proteolytic enzymes served as the control. The agar diffusion assay was carried out as described in section 2.2. to determine if the inhibition zones were disappeared. If the pH neutralized and catalase- treated LAB CFSs lose their antimicrobial activity following treatment with proteolytic enzymes, they can be confirmed as bacteriocins producing LAB because of the proteinaceous nature of the bacteriocins compounds. A method for rapid screening for LAB antimicrobial activity was reported by Budde and Rasch [16]. Indicator organisms were exposed to bacteriocins after staining with carboxyfluorescein diacetate. Fluorescence was measured by flow cytometry and the effect of bacteriocin was found as a decrease of fluorescence when the fluorescent compound was leaked out from cells. Ouwehand and Vesterlund [10] reported that using bioluminescent indicator strains, luciferase genes were transformed into indicator strains, and indicator strains started to produce light in reaction. When bioluminescence is closely linked into the energy metabolism of bacteria, changes in light production are rapid. This method increases the sensitivity of the assay and allows for real-time assessment of antimicrobial activity of bacteriocins.

2.4. Identification of bacteriocins producing LAB isolates

Sequencing of 16S ribosomal RNA genes was used to identify the unknown bacteriocins producing LAB isolates in our studies. Each isolate was inoculated into 5 mL of MRS broth and incubated anaerobically at 37 ˚C for 24 h. Genomic DNA was extracted from isolates using the UltraCleanTM Microbial DNA Isolation kit (MO BIO laboratories, Inc., Carlsbad, CA, USA) according to the manufacturer’s instructions. 16S rRNA genes from the DNA of each isolate were amplified by PCR using the universal gene primers F44 (5'-RGT TYG ATY MTG GCT CAG-3') and R1543 (5'-GGN TAC CTT KTT ACG ACT T-3') as described by Abnous et al. [17]. The PCR reaction consisted of a 25 µL master mixture containing 0.5 µL IDTaqTM DNA Polymerase (5U µL-1) (ID Labs Inc., London, ON, Canada), 0.2 mM deoxynucleoside triphosphates (dNTPs), 0.4 mM of each primer, 1x reaction buffer (2 mM MgCl2), and 2.0 µL of genomic DNA. PCR was performed in a Thermocycler (Biometra, Montreal Biotech Inc., Kirkland, QC, Canada) with initial denaturation at 94 ˚C for 2 min, followed by 35 cycles of denaturation at 94 ˚C for 30 s, annealing at 52 ˚C for 30 s, and elongation at 72 ˚C for 30 s. A final elongation step was performed at 72 ˚C for 5 min. PCR amplification was confirmed by agarose gel electrophoresis (1% w/v) and staining with ethidium bromide to visualize products. The PCR products were cloned using the StrataClone PCR Cloning kit (Stratagene, La Jolla, CA, USA) and transformed into E. coli according to the manufacturer’s instructions. Plasmids were purified from selected E. coli clones using the UltraCleanTM mini plasmid prep kit (MO BIO Laboratories) following the manufacturer’s recommendations, and used for sequencing in the laboratory of the Bureau of Nutritional Sciences, Health Canada (Ottawa, ON, Canada). DNA sequencing of each gene was carried out as described by Abnous et al. [17]. An aligned sequence file generated by the ARB program [18] was used to identify sequences which were within 97% similarity as they would be considered to belong to the same genus and species. Analysis of sequence homology with previously reported cultivated bacterial species was carried out using the SEQMATCH function of the Ribosomal Database Program (RDP) [19]. Also, carbohydrate fermentation patterns for LAB identification can be obtained using


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API 50 CH strips according to the manufacturer’s instructions (BioMérieux, Lyon, France). Results of reaction can be input into the apiwebTM database at http://apiweb.biomerieux.com

3. Antimicrobial spectrum of bacteriocins producing LAB isolates

3.1. In vitro antibacterial effects of bacteriocins producing LAB isolates

In our studies, the agar diffusion bioassay was used to further study the antibacterial spectrum of the bacteriocins producing LAB isolates which were tested against six common food/plant bacterial pathogens and spoilage organisms at 5 and 20 ˚C, respectively [13, 20]. These six indicator organisms and their respective media used were as follows: Li. innocua (ATCC 33090) (BHI), E. coli K-12 (ATCC 10798TM) (tryptic soy broth), Bacillus cereus (ATCC 14579TM) (nutrient broth), Pseudomonas fluorescens (A7B) (nutrient broth), Erwinia carotovora (ATCC 15713TM) (nutrient broth) and Leuconostoc mesenteroides subsp. mesenteroides (ATCC 8293TM) (MRS). All tested indicator organisms were revived from frozen stock, cultured in their respective broth and plated in the corresponding semi-solid media containing 0.7% agar, except semi-solid MRS medium was prepared using 0.75% agar. Indicator organisms were incubated aerobically for 24 h at their optimal growth temperatures with 26 °C for Erw. carotovora, Ps. fluorescens and Leuc. mesenteroides, 30 °C for B. cereus and 37 °C for E. coli and Li. innocua according to the ATCC instructions. One mL of the indicator bacteria (5x105 cfu mL-1) was mixed with 15 mL of semi-solid agar tempered to 45 °C, poured into sterile petri dishes and allowed to solidify before the cutting of wells as described above. The appropriately treated CFSs from each LAB isolate under investigation were added to wells as described previously. All plates were incubated aerobically and examined for inhibition zones after 8 days at 5 °C or after 24 h at 20 ˚C. Using the same method, the antibacterial spectrum of LAB isolates would be tested against any other target bacteria.

3.2. In vitro antifungal effects of bacteriocins producing LAB isolates

Microdilution tests were performed in our studies to determine the antifungal activity of the LAB isolates against three common food spoilage fungi of Penicillium expansum (Pex 03-10.1), Botrytis cinerea (B94-b), and Monilinia fructicola (Mof 03-25) at 5 and 20 ˚C. The microdilution method was slightly modified from report by Lavermicocca et al. [21]. Fungi were obtained from the culture collection at the Atlantic Food and Horticultural Research Center, AAFC (Nova Scotia, Canada). The stock cultures were stored as spore or mycelial suspensions in 15% glycerol (v/v) at -80 ˚C. To produce fungal conidia spores, P. expansum was cultured at 25 ˚C for 4 days in a para filmed petri dish on potato dextrose agar (PDA), Bo. cinerea at 22 ˚C for 10 days under a 12 h light/dark cycle in a petri dish on Pseudomonas Agar F medium (BDH; with 10 g L-1 glucose instead of glycerol) and M. fructicola at 25 ˚C for 10 days in a para filmed petri dish on modified V-8TM medium. The conidia spores were collected from the plates using sterile water and a sterile cell spreader. The suspension was filtered through sterilized cheese cloth into a sterile 50 mL beaker to remove mycelia. The spore concentration was diluted to 6.5x104 spores mL-1. Conidial suspension density was calculated using a Haemocytometer (Hausser Scientific, PA, USA) and the viability of spores was calculated by randomly counting the number of germinating spores out of 200 spores under a light microscopy. The CFS from the LAB isolates was prepared and treated as previously described in section 2.2. Microdilution tests were performed in sterile, disposable 96-well microdilution plates. 185 µL of the LAB CFSs (CFS, pH adjusted CFS or pH adjusted and catalase treated CFS) and 15 µL of the appropriate conidial spore suspension were dispensed into the wells (picture 2). Microdilution plates were then incubated in a humid environment at 5 °C or 20 ˚C with measurements of the optical density (OD580 nm) values after 48, 72, 96 and 120 h at 5 °C and 24, 40 and 48 h at 20 ˚C using a microdilution plate reader/spectrophotometer (Spectra MAX 190, Molecular Devices, CA, USA). In each experiment, a negative control for each fungus composed of 185 µL MRS broth and 15 µL autoclaved conidia (dead spores), and a positive control containing 185 µL MRS broth and 15 µL of conidia suspension were prepared. Using the same method, the antifungal spectrum of LAB isolates would be tested against any other target fungi. Bioscreen C (OY Growth Curves AB, Finland) can also be used for antimicrobial spectrum tests. Bioscreen C measures the growth of microorganisms and the effect of chemicals, antimicrobials, pH, temperature, water activity, salt concentration and other parameters on the growth of microorganisms. It measures optical density vs. time to generate growth curves. Each well in the 100 well micro-plates can generate a growth curve allowing us to vary the organism, growth media and experimental parameters to obtain valid and reproducible results.


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Fig. 2 Microdilution method showing inhibition of fungal spore germination by Ent. faecium (13.2). Inhibition of fungi as a result of organic acids (rows 4, 7, and 10), H2O2 (rows 5, 8, and 11), and bacteriocins (rows 6, 9, and 12).

4. Optimization of growth condition for LAB and bacteriocin production

In order to effectively use bacteriocinogenic LAB strains and bacteriocins to enhance the safety and shelf life of many foods, a better understanding of the factors affecting LAB growth and bacteriocin production is required. Bacteriocins are usually produced in complex growth media such as de Man, Rogosa and Sharpe (MRS), tryptone glucose extract (TGE), all- purpose with tween (APT), brain heart infusion (BHI), trypticase soy broth (TSB) and trypticase soy broth yeast extract (TSBYE) [22]. Studies on complex media and on some wastes from the food industry have demonstrated that bacteriocin production was depended on the medium composition [23-25]. Guerra et al. [22] studied nutritional factors affecting the production of two bacteriocins produced by Lactococcus lactis and Pediococcus acidilactici strains and the kinetic behaviour of bacteriocin production on whey. They also studied the influence of pH, temperature and proteolytic enzymes on the stability of the bacteriocin extracts produced by the two LAB strains and found that although the whey supported the growth and bacteriocin production, both biomass and bacteriocin productions were lower than those obtained on MRS broth. Both bacteriocins showed the highest heat stability at acidic pH and short incubation times. In another study, Guerra and Pastrana [26] reported that enhanced pediocin and nisin productions on mussel-processing waste were achieved in a medium supplemented with yeast extract or Bacto casitone. Gänzle et al. [27] reported that bacteriocin production was influenced by medium composition, incubation atmosphere, pH, temperature, and microorganism growth phase. Several studies have also shown that bacteriocin production was dependent on the biomass of LAB [28-32]. However, optimal cell growth does not always result in high bacteriocin production levels. Mataragas et al. [29] founded that the highest bacteriocin levels were obtained at temperatures and pH values lower than the optimal condition for growth. In our studies, four selected bacteriocinogenic LAB strains of Ent. faecium (JFR-1), Strep. thermophilus (TSB-8), Lact. casei (JFR-5) and Lact. sakei subsp. sakei (Arla-10) were cultured at different culture media with various of initial medium pH and temperature [20]. The optimal conditions for LAB growth and bacteriocin production as well as the correlation between LAB growth kinetics and bacteriocins production were determined as follows.

4.1. Inoculums preparation and growth conditions for LAB

One mL of frozen LAB strains was cultured in 20 mL MRS or M17 broth (for Strep. thermophilus) at 37 °C for 24 h and then 1 mL of culture was sub-cultured in 20 mL MRS broth at 37 °C for 16 h. Cells were collected by centrifuging at 14,000 g for 5 min. The pellets were re-suspended using sterile water to a final OD600 nm of 0.4. The cell suspensions were used for the following growth curve experiments. Two culture media of MRS and BHI broth were used for the four bacteriocinogenic LAB strains to grow at 20, 37 or 44 °C. The initial pH of MRS and BHI broth was adjusted to 4.5, 5.5, 6.2, 7.4 and 8.5 using 0.5 N HCl or NaOH. The growth curves were generated using a Bioscreen C®, which was able to monitor turbidity of samples kinetically. 15 µL of LAB cell suspensions (OD600 nm = 0.4) was inoculated into 100-well plates filled with 285 µL MRS or BHI broth (5%, v/v) of each growth condition descripted above. The Bioscreen C multi-well plates were incubated under aerobic conditions at 37 or 44 °C for 48 h or at 20 °C for 72 h. OD values were recorded under a brown filter setting (wavelength of 600nm) at 20 min interval. Logistic model was used in our study to fit the growth curves generated by Bioscreen C and the lag stage, log stage, slopes and maximum OD values of the growth curves were statistically analysed and compared.


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4.2. Determination of OD, pH, bacteriocin activity and viability of LAB

Based on growth kinetics, the combination of growth condition with MRS pH 6.2, MRS pH 7.4, BHI pH 5.5, and BHI pH 6.2 at 37 and 44 °C were selected for further investigation of the relationship between growth kinetics and bacteriocins production (Fig. 1, table 1). 0.5 mL of LAB cell suspensions (OD600 = 0.4) was inoculated to 9.5 mL of each condition in test tubes and incubated at 37 or 44 °C, respectively. OD, pH, bacteriocin activity and viability of LAB were determined following 6, 8, 10, 13, 16 and 24 h at 37 °C and 5, 7, 9, 11, 13 and 18 h at 44 °C. The OD values were measured at 600nm using an UV/visible spectrophotometer (Ultrospec 3100 Pro, Biochrom Ltd. England). The pH of LAB samples was determined using a pH meter (Accumet AR 20, Fisher Scientific, Ottawa, Canada) that was calibrated with standard pH buffers of pH 4.0, 7.0 and 10.0. For viability of LAB, 100 µL of appropriate serial dilutions were spirally plated on MRS or M17 agar using a Whitley automatic spiral plater. The petri dishes were incubated at 37 °C for 48 h. The counts of LAB were conducted using an aCOLyte automated colony counter (Synbiosis, Cambridge, England). The agar diffusion bioassay was again used to test bacteriocin activity with Li. innocua as an indicator. Li. innocua was grown overnight in BHI broth at 37 °C, the cells were then collected and diluted to a final concentration at 5×105 cfu mL-1. At each sampling time the LAB cell free supernatants (CFS), and pH adjusted and catalase treated CFS (treated CFS) were prepared and filtered through sterile 0.22 µm syringe filters. 35 µL of CFS or treated CFS was added to the wells (5 mm in diameter) in BHI agar plates (0.7 % agar) containing Li. innocua. The plates were incubated at 37 °C for 24 h in order to measure the inhibitory zones. To quantify the bacteriocin activity, CFS or treated CFS was serially diluted two-fold with sterile deionized water and 35 µL of each dilution was added into the wells described above. The titer was defined as 2n, where n is the reciprocal of the highest dilution that resulted in inhibition of the indicator bacteria. Thus, the arbitrary unit (AU) of antibacterial activity of LAB bacteriocins was defined as 2n×1000 µL 35 µL-1 [33].

Fig. 3 Changes of OD values (A), viability (B) and bacteriocin activity (C, D) of Ent. faecium (JFR-1).


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Table 1 The correlation of optical density, pH, bacteriocin activity of CFS and treated CFS, and viability of four LAB strains.

O.D600nm pH CFS Treated CFS Viable counts

(AU mL-1) (AU mL-1) (log CFU mL-1)

Arla-10

O.D600nm 1

pH -0.505 1

CFS (AU mL-1) 0.750 -0.426 1

Treated CFS (AU mL-1) 0.781 -0.291 0.892 1

Viable counts (log CFU mL-1) 0.959 -0.531 0.815 0.812 1

JFR-1

O.D600nm 1

pH -0.503 1

CFS (AU mL-1) 0.775 -0.401 1

Treated CFS (AU mL-1) 0.737 -0.345 0.822 1


JFR-5

O.D600nm 1

pH -0.533 1

CFS (AU mL-1) 0.709 -0.433 1

Treated CFS (AU mL-1) 0.754 -0.477 0.938 1


TSB-8

O.D600nm 1

pH -0.465 1

CFS (AU mL-1) 0.817 -0.306 1

Treated CFS (AU mL-1) 0.847 -0.346 0.946 1


Our results indicated that four selected bacteriocinogenic LAB strains of Ent. faecium (JFR-1), Lact. casei (JFR-5) and Lact. sakei subsp. sakei (Arla-10) grew well on both culture media at 20, 37 and 43 °C while Strep. thermophilus (TSB-8) was only able to grow at 37 and 43 °C. The OD values were significantly affected by the initial pH of media (p < 0.001). The optimal pH for LAB growth was found between 6.2 and 7.4 and no growth was found at initial pH 4.5. In addition, bacteriocin production was significantly affected by the culture media (p <0.001). The optimal condition for LAB strains to produce bacteriocins was in MRS broth with initial pH 6.2 incubated at 37 °C [20]. These research findings provide useful information on application of LAB and bacteriocins efficiently for the food industry.

5. Purification and identification of bacteriocins

5.1. Purification and isolation of bacteriocin

Purification and isolation of bacteriocins are important steps for their identification and for in-depth studying their mechanisms of action. In addition to the traditional biochemical methods of protein purification, ammonium sulfate precipitation and various forms of chromatography, alternative precipitation methods have also been studies. A technique based on the pH dependent adsorption-desorption principle was successfully used in extracting different cell-associated bacteriocins in large quantities and relatively pure forms from the cultures [34]. Bacteriocins were adsorbed on the cells of producing LAB strains and some other gram-positive bacteria. pH was a crucial factor in determining the degree of adsorption of these peptides onto cell surfaces. In general, between 93 and 100% of the bacteriocin molecules were adsorbed at pHs near 6.0, and the lowest (<5%) adsorption took place at pH 1.5 to 2.0. Based on this finding, an isolation method was developed for bacteriocins produced from four genera of LAB with a higher yield of pediocin AcH, nisin, sakacin A, and leuconocin Lcm1 [34] compared with other isolation procedures which rely on precipitation of the bacteriocins from the cell-free culture liquor. This method descripted below is simple and would be used to produce large quantities of bacteriocins from LAB to be used as food biopreservatives. For extraction of pediocin AcH, nisin, leuconocin Lcml, and sakacin A, each LAB strain was grown to its early stationary phase (about 5x108 cfu ml-1) in 1 L of the appropriate medium. The culture broths were adjusted to pH 6.5 for


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pediocin AcH, nisin, and sakacin A and pH 5.5 for leuconocin Lcml. Each culture broth was heated to 70 °C for 25 min to kill the cells, and the cells were harvested by centrifugation at 15,000 x g for 15 min. Supernatant was assayed to calculate the amount of bacteriocin not adsorbed onto the cells. After the cells had been washed with 5 mM sodium phosphate (pH 6.5), they were re-suspended in 50 mL of 100 mM NaCl at pH 2.0 (adjusted with 5% phosphoric acid) for pediocin AcH, leuconocin Lcml and sakacin A, and pH 2.5 for nisin and mixed with a magnetic stirrer at 4 °C for 1 h. Cell suspensions were then centrifuged at 29,000 x g for 20 min, the cells were re-suspended in 5 mM sodium phosphate (pH 6.5) and assayed to calculate the amount of bacteriocin lost with the cells. The supernatants were dialyzed in 1,000-molecular-weight-cutoff dialysis bags against dH2O at 4 °C for 24 h and then freeze-dried. The total dry matter, protein concentration, and bacteriocin activity of each product were determined. Freeze- dried extractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A 10-cm layer of 16.5% acrylamide resolving gel and a 2-cm layer of 10% acrylamide spacer gel were prepared. The electrophoresis was run at 15 °C and 20 mA for the first 2 h and then at 30 mA for another 12 h. Sample preparation, application of samples, and gel division after electrophoresis were conducted following the descriptions by Bhunia et al. [35]. One-half of each gel was stained with Coomassie brilliant blue, and the other half was used for identification of the bacteriocin band by using growth inhibition of Ent. faecalis MB1. For growth inhibition, the gel half was washed in deionized water for 2 h at 25 °C, placed on a TGE agar pre-poured plate, and overlaid with soft agar seeded with Ent. faecalis MB1. The plate was incubated at 30 °C for 24 h, and examined for the location of the zone of growth inhibition. The stained gel half was destained and photographed. Based on the bands in the stained gel, the gel half showing inhibitory zones and the bands in the standard marker, one can identify the activity bands and their molecular weights. In the study by Deba et al. [36], a bacteriocin-cell extraction procedure without SDS combined with reverse-phase high performance liquid chromatography (HPLC) was used to isolate the bacteriocin from Ped. acidiiactici UL5. The amino acid sequence of the peptide was determined from the pure bacteriocin extract. The molecular weight of HPLC-purified bacteriocin in 10-30% acetic acid was determined using a triple quadruppole mass spectrometer (MS) with an electrospray interface (API III system, SCIEX, Thornhill, ON, Canada) as described by Feng and Konishi [37]. In our studies, protein concentration was determined according to Bradford [38]. Bio-Rad Protein Assay Kit II (Bio-Rad Laboratories, Inc. USA) was used with bovine serum albumin as the standard. For the determination of the molecular weight of bacteriocin, tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis (Tricine-SDS- PAGE) was performed by using a 16.5% acrylamide slab running gel on a Mini-Protean Tetra Cell for Ready Gel Precast Gels (Bio-Rad Laboratories, USA) according to Schägger [39]. Ultra-low range molecular weight marker M 3546 (Sigma-Aldrich, USA) ranging from 1.06 KDa to 26.6 KDa was used as protein standard. After electrophoresis at 30 V for 3 h and followed at 140 V for 7 h, the gel was fixed for 30 min in 20% (v/v) isopropanol and 10% (v/v) acetic acid in water [40], then washed three times (5 min each time) using dH2O. The gels were then divided vertically into two pieces. One half of gel contained the samples and markers was stained with 0.25% Coomassie blue R 250 in 10% (v/v) acetic acid for 1 h, and the other half was used for identification of the bacteriocin bands by using growth inhibition of Li. innocua (5×105 cfu mL-1) and incubated at 37 °C for 24 h. Based on the location of the bands, molecule weights of bacteriocins were determined using a gel analysis software (Quantity One, Version 4.6.3. Bio-Rad Laboratories).

5.2. Identification of bacteriocins

In recent years, liquid chromatograph/mass spectrometry (LC/MS) has been employed for rapid detection and identification of bacteriocins. MS analysis allows the researchers not only to detect known bacteriocins but also to identify novel bacteriocins by accurate mass determination. In our studies, selected bacteriocin bands were excised from SDS-PAGE gels with a spot cutter under the exposure of UV (Bio-Rad Laboratories). Bands were reduced with DTT to break disulfide linkages, alkylated with iodoacetamide, and then digested with trypsin (Promega, USA). The resultant peptides were extracted in washes of ammonium bicarbonate solution, acetonitrile and 1.0% formic acid. Extraction solvent was removed under vacuum and the peptides were re-suspended in 30 μL of 5% MeOH with 0.5% formic acid. HPLC was performed on a nano-flow system LC system (Ultimate 3000, Dionex, Sunnyvale, CA, USA). 6.4 μL of sample was injected directly into a reversed phase capillary column of 75 µm x 150 mm RP - C18 (Acclaim PepMap100, Dionex, Sunnyvale, CA). The flow rate was 200 nL min-1 and the sample was sprayed through a distal coated fused silica emitter tip (New Objective, Woburn, MA, USA). MS was performed on a hybrid triple quadrupole linear ion trap (QTRAP4000, LC/MS/MS, Applied Biosystems, Foster City, CA, USA) equipped with a nano-spray ion source. Spectra were acquired as follows using Information Dependent Acquisition mode in the Analyst 1.4.1. software. For each MS scan, 370–1400 Da, four most intense ions were selected for an Enhanced Resolution (ER) scan in order to determine their charge states. Enhanced Product Ion scans, 100 to 1700, were initiated by the presence of doubly and triply charged ions with intensities exceeding 1x105 counts. Former target ions were excluded for 30 s after four occurrences. The ion spray voltage was 2400 V, the curtain gas was set to 20 (arbitrary units) and the de-clustering potential was 60 V. MS/MS peak lists were generated for database searching using MASCOT version 2.4 (Matrix Science, London, UK) or using ProID (Applied Biosystems) software. Product ion scans for precursors within a 1.0 Da range were summed. All spectra were centroided and de-isotoped.


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The raw MS/MS data were searched against NCBI entries of bacteria (Eu Bacteria) with 11368323 sequences and 3877152002 residues (NIH, Bethesda, MD, USA) using the MASCOT algorithm (Matrix Science). The MS and MS/MS mass tolerance was 1.2 and 0.5 Da, respectively, and one missed cleavage was allowed. Carboxamidomethyl cysteins and oxidized methionine were set as fixed and variable modifications, respectively. Proteins with significant peptide matches were selected for error tolerant searching. The data was also searched against the SwissProt database, 234112 sequences (Sprot Version 57.15) to ensure no peptides from trypsin or keratin were present. Peptide ion scores greater than 47 indicate identity or extensive homology (p < 0.05) and were referred to as significant hits. Peptides below the significance threshold were only reported where other significant hits to the same protein were present. As comparison, the raw MS/MS data were also searched against a bacteriocins database, BACTIBASE (www.bactibase.pfba-lab-tun.org) with 181 sequences, 10781 residues. Search parameters were the same as those used with MASCOT. In order to detect and identify bacteriocins without purification, various methods have been developed and examined. For example, detection methods for nisin included antimicrobial activity assay and immunoassay [41-42]. Genetic methods such as colony hybridization were also investigated [43]. Nisin-specific two-component regulatory system has been developed with high sensitivity and specificity [44-45]. A new system to detect and identify bacteriocins in the early stage of screening for novel bacteriocins was also established, the methods for pre-treatments to improve bacteriocin detection and LC/MS analysis were reported [46]. The methods achieved not only concentration of the bacteriocins but also easy removal of impurities which often inhibit the ionization of target molecules. These pre-treatment methods can also be applied to make bacteriocin preparations, as easy partial purification technique. In addition, the uses of matrix-assisted laser desorption/ionization time- of- flight mass spectrometry were reported for rapid detection and identification of bacteriocins from culture supernatants [47] and bacterial cells [48].

6. Potential application of LAB and bacteriocins in the food industry

Many LAB used in the food industry produce bacteriocins and thus are a rich source of these natural inhibitors. These bacteriocins have potential applications in preservation for improving the safety and quality of foods. For example, nisin produced by several strains of Lactococcus lactis is the best known, most well characterized and the only lantibiotic to have realized widespread commercial use. Lactobacillus plantarum produces serial bacteriocins like plantaricin 35d, bacteriocins ST28MS and ST26MS that are active against the pathogens and spoilage microorganisms in foods [49-50]. Another bacteriocin has potential to be used in the food industry is pediocin which is an antilisterial bacteriocin produced by several Pediococcus strains. Up to now, different strategies have been used to incorporate bacteriocins into foods. They can be produced by live cultures directly in fermented food by using bateriocin producing starter cultures or they can be applied to the food as a protective culture by spaying them onto the surface of cheese [51]. Bacteriocins can be added directly to the food as an additive, such as the enriched nisin product, Nisaplin®. They may also be included in packaging materials such as nisin coatings on low density polyethylene film or barrier films with methyl cellulose [52]. Nisin is commercially available as a powder called Nisaplin® and NovasinTM (Danisco), which contains the active ingredient of 2.5% nisin, along with 77.5% NaCl and non-fat dry milk (12% protein and 6% carbohydrate). Nisin has a broad spectrum of antimicrobial activity against gram-positive bacteria and has been shown to be bactericidal to Staphylococcus aureus, Listeria monocytogenes, vegetative cells of Bacillus spp. and Clostridium spp. as well as preventing outgrowth of spores in Bacillus and Clostridium species [12]. In canned foods, there are two commonly associated spoilage organisms, B. stearothermophilus and C. thermosaccharolyticum. Addition of nisin in the range of 100-200 IU g-1 controlled these pathogens and positively contributed to overall nutritional and organoleptic properties of the canned product [53]. Nisin was also used in high acid canned foods such as in canned tomato and fruit juices to control Clostridium pasteurianum and Bacillus spp. [54]. Combinations of nisin with other preservative regimes were also the most effective method of preservation and extension of shelf life for foods. For example, treatment of caviar with nisin (500 IU mL-1) reduced the L. monocytogenes numbers by 2.5 logs, while when used in combination with heat at 60 °C for 3 min the pathogen was no longer detected after storage of 28 days at 4 °C [55]. Ukuku et al. [56] reported that melons were washed in a solution containing nisin (25µg mL-1), hydrogen peroxide (1%), sodium lactate (1%) and citric acid (0.5%) and population of E. coli O157:H7 or L. monocytogenes was reduced form 5.27 and 4.07 log cfu cm-2 to less than 1 log cfu cm-2. This treatment reduced transfer of bacterial pathogens from whole melon surfaces to fresh-cut pieces. As biopreservation of fresh and/or fresh-cut products using bacteriocinogenic LAB isolated from vegetables also presents an innovative approach to improve the safety and quality of the products. We applied Ent. faecium (13.2) and Lact. lactis (7.17) onto fresh-cut salads to investigate their effect on the growth of the natural microflora and Li. innocua inoculated on salads. Results showed that the addition of the LAB significantly reduced the loads of natural occurring Listeria spp. (p= 0.033), yeasts (p = 0.011), Pseudomonas spp. (p = 0.010) and coliforms (p = 0.011). Ent. faecium and Lact. lactis also significantly reduced growth of Li. innocua inoculated on fresh-cut salads (p = 0.005) during a 10 days storage at 5 ˚C. Scanning electron microscopy (SEM) revealed a significantly reduced presence of Li. innocua on the surface of fresh-cut salads after addition of the bacteriocinogenic LAB [13]. The bacteriocin producing


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LAB have the potential to be used as protective cultures in fresh-cut produce to control spoilage and pathogenic microorganisms thereby improving the product shelf-life and safety. Although knowledge about bacteriocins has increased greatly, there are still many questions regarding immunity (self-protection) and the molecular basis of target-cell specificity. Use of genetic methods for the construction and improvement of bacteriocin-producing LAB provides great promise for the food fermentation and preservation industries. Genetic strategies are forthcoming that will direct genes for bacteriocin production and immunity into the desired industrial strains [2]. Further characterization of the bacteriocins and their mechanism of action may result in a better understanding of their implications in food systems. However, applications of bacteriocins for food protection should not substitute good manufacturing practice.

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