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Bioresource Technology 99 (2008) 5384–5390
Pediocin SA-1, an antimicrobial peptide from Pediococcus acidilactici NRRL B5627: Production conditions, purification and characterization Sofia Anastasiadou, Maria Papagianni *, George Filiousis, Ioannis Ambrosiadis, Pavlos Koidis Department of Hygiene and Technology of Food of Animal Origin, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki 54006, Greece Received 3 October 2007; received in revised form 6 November 2007; accepted 7 November 2007 Available online 21 February 2008
Abstract Fermentation broths of Pediococcus acidilactici NRRL B5627 exhibited a certain antimicrobial activity due to a bacteriocin produced during early growth and until the stationary phase of growth was reached (at optimum of 60% dissolved oxygen saturation). Its size was determined by electrospray ionization mass spectrometric analysis as 3.660 kDa. N-terminal sequencing showed that the bacteriocin had 19 amino acid residues in the order KYYGXNGVXTXGKHSXVDX. The purified bacteriocin is similar to pediocins isolated by various Pediococci and therefore, it belongs to the class IIa of bacteriocins and is thus designated pediocin SA-1. Sensitivity of the purified pediocin to various storage temperatures and enzyme treatments was examined. Purified pediocin SA-1 is heat stable for up to 60 min at 121 °C. Pediocin SA-1 is inhibitory to several food-borne pathogens and food spoilage bacteria. It appears to be significantly more effective against Listeria spp. compared to pediocin PD-1 produced by P. damnosus. The mode of action of the purified bacteriocin appears to be bactericidal. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Pediococcus acidilactici; Pediocin; Bacteriocin; Biopreservative
1. Introduction Lactic acid bacteria (LAB) are well known for their production of peptides and proteins with antimicrobial properties, known as bacteriocins. The potential applications of bacteriocins from LAB in the food and health care sectors have attracted the strong interest of academia and the industry resulting in an impressive amount of published research on their production, purification, genetics and applications (Papagianni, 2003). So far, only nisin produced by L. lactis, is a commercial product and an approved food additive in most major food producing countries. Another bacteriocin that attracts research interest and will likely be the next to be used in the food industry is pediocin (Ray, 1992; Turcotte et al., 2004), which is *
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[email protected] (M. Papagianni).
0960-8524/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.11.015
an antilisterial bacteriocin (Guyonnet et al., 2000; Simon et al., 2002) produced by several Pediococcus strains. However, unlike nisin and L. lactis, studies on the physiology and genetics of pediocin producing Pediococcus, are still rather limited. Most commonly, strains of Pediococcus acidilactici and P. pentosaceus have been reported to produce bacteriocins (Gonzales and Kunka, 1987; Biswas et al., 1991; Kim et al., 1992; Elegado et al., 1997; Gurira and Buys, 2005). Some of these are designated as pediocin AcH by P. acidilactici H, E, F, and M (Bhunia et al., 1987; Ray et al., 1989; Kim et al., 1992), pediocin PA-1 by P. acidilactici PAC 1.0 (Gonzales and Kunka, 1987), pediocin JD by P. acidilactici SJ-1 (Schved et al., 1993), pediocin 5 by P. acidilactici UL5 (Huang et al., 1996), pediocin A by P. pentosaceus FBB-61 (Etchells et al., 1964; Flemming et al., 1975), pediocin N5p by P. pentosaceus (Stra¨sser de Saad et al., 1995), and pediocin ST18 by P. pentosaceus (Todorov and Dicks,
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2005). P. damnosus has also been reported to produce a pediocin, designated as pediocin PD-1 (Green et al., 1997). It is now known for most studied pediocins that they are plasmid encoded (Ray, 1995; Le Marrec et al., 2000) and posttranslationally modified hydrophobic molecules (Green et al., 1997; Yin et al., 2003; Wu et al., 2004), which also share a similar N-terminal sequence (Henderson et al., 1992; Wu et al., 2004). Pediocins form a group of bacteriocins belonging to the class IIa of bacteriocins, characterized as ‘‘antilisterial” (Papagianni, 2003). They inhibit several gram-positive spoilage and pathogenic bacteria. The spectra of antimicrobial activity of pediocins produced by strains of P. acidilactici and P. pentosaceus have been found to be similar and this has been attributed to the phylogenetically close relation of the producer organisms (Collins et al., 1991). Pediocin PD-1 by P. damnosus however, has been found to be inactive against other pediococci, a characteristic that makes it different from the known pediocins produced by P. acidilactici and P. pentosaceus strains (Green et al., 1997). We have undertaken studies on the physiology of the meat isolated, bacteriocin producer, P. acidilactici NRRL B5627, grown in stirred tank bioreactor cultures. In this work, we report information on production, isolation, purification, antimicrobial activity and mechanism of activity of the bacteriocin produced by this microorganism. 2. Methods 2.1. Bacterial strains and growth conditions The microorganism used in this study was Pediococcus acidilactici NRRL B5627. This was maintained in MRS agar from which was transferred in 250 ml Erlenmeyer flasks, containing 100 ml of MRS broth. Following growth until the mid-logarithmic phase (OD600 = 1.4), the culture was transferred (2% v/v) in a stirred tank bioreactor – BIOFLO 110, New Brunswick Scientific – with a working volume of 2 l. The reactor was equipped with baffles. The agitation system consisted of two 6-bladed Rushton-type impellers (52 mm), operating at the stirrer speed of 150 rpm. Process temperature was maintained at 30 °C. The culture pH was left uncontrolled (pH at inoculation time was around 6.0). The reactor was equipped with a polarographic oxygen sensor (Mettler Toledo, Urdorf, Switzerland). The oxygen electrode was calibrated by sparging the medium with air (dissolved oxygen tension, DOT, 100%) and N2 (DOT, 0%); the 100% saturation value was based on air. Experiments were carried out under semiaerobic conditions, with the DOT kept at 60% by sparging the reactor with a mixture of N2 and atmospheric air, adjusted by using two mass flow controllers, and the DOT was kept constant by feedback regulation of the ratio. Samples were taken every two hours for biomass, lactic acid, and bacteriocin activity determination. All runs were carried out in triplicate and repeated if experimental variation exceeded 10%.
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2.2. Determination of biomass, lactate, and glucose concentrations Biomass concentration was monitored spectrophotometrically by measuring the optical density at 600 nm and correlating the optical density with cell dry weight measurements. One unit of optical density at 600 nm was shown to be equivalent to 0.25 g (dry weight) of cells per liter. Cell dry weight was determined by filtering 10 ml of broth through nitrocellulose filters (pore size, 0.45 lm). The filters were tared after having been dried in a microwave oven at 150 W for 15 min. The biomass collected on the filter was washed twice with 10 ml distilled water before being dried and tared as indicated above. Lactic acid concentration was determined with the EnzyPlus D/L Lactic Acid kit by Diffchamb AB (Diffchamb, Sweden). Glucose was determined using the glucose oxidase/peroxidase method of Kunst et al. (1986). 2.3. Determination of antimicrobial activity Antimicrobial activity was assessed by the agar diffusion assay (Tramer and Fowler, 1964). For each indicator organism the appropriate solid medium was used. Solid media were prepared by adding 1.5% wt/vol technical agar to the broth media. Anaerobic conditions for plates were generated in a GasPak anaerobic jar. Anaerobic and microaerophilic growth for liquid cultures was ensured by growing the cultures in an incubator with controlled CO2 partial pressure. An aliquot of 10 ll of cell-free culture supernatant fluid (pH adjusted at 6.0) was spotted on the appropriate solid media (1.5% w/v agar) seeded with a fresh culture of test cells (106 cells/ml). Plates were incubated at the optimum conditions for each test microorganism and examined for the presence of 2 mm or larger clear zones of inhibition. 2.4. Determination of bacteriocin activity Production of bacteriocin in P. acidilactici cultures was examined in cell-free samples and the arbitrary units (AU) of activity (reciprocal of the highest dilution at which activity was still obtained) were determined (Wu et al., 2004). To eliminate the antimicrobial effect of lactic acid, the pH of the samples was adjusted to 6.0 with 1 N NaOH. In order to find a suitable indicator microorganism to be used in bacteriocin activity assays, ten microorganisms were examined for their sensitivity levels and linearity of response to bacteriocin according to Papagianni et al. (2006). Based on these criteria, Micrococcus luteus CECT 241 was chosen among the tested P. acidilactici ATCC 25740, P. pentosaceus ATCC 33316, Lactococcus lactis 11454, Lactobacillus curvatus ATCC 51436, Lb. sakei CECT 906 T, M. luteus CECT 241, Lb. plantarum NCAIM B 01133, Lb. plantarum NCAIM B 1074, Lb. plantarum CECT 220, and Lb. plantarum ATH.
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2.5. Detection, isolation and purification of the bacteriocin Cell-free filtrates from fermentation broths were subjected to tricine–SDS–polyacrylamide gel electrophoresis according to the method of Scha¨gger and Von Jagow (1987) for the separation of proteins in the range of 1 to 100 kDa. One half of the gel was stained with Coomassie brilliant blue R250 according to Sambrook and Russell (2001). The Rainbow protein molecular weight marker (2.35–46 kDa) of Amersham International (Amersham, UK) was used. Comparisons of bands from filtrates obtained at various points during fermentation and bacteriocin activity determination with the agar diffusion assay at the same time, showed that bands positioned between 3 and 4 kDa should be further analyzed. The position of the pediocin was determined by overlaying the other half of the gel (prewashed, as described by Van Belcrum et al. (1991)) with cells of M. luteus (106/ml), embedded in nutrient agar. Gel excising and destaining was done according to Cohen and Chait (1997) from 10% polyacrylamide SDS–PAGE gels containing 0.1% bromophenol blue. This method of extraction elutes protein directly into an aqueous solution of formic acid/water/2-propanol (1:3:2 v/v/v). 2.6. Molecular mass determination and N-terminal sequencing of the bacteriocin Molecular mass determination of the purified bacteriocin was done by electrospray ionization mass spectrometric analysis (ESI-MS). Following elution of the protein into the aqueous solution as described above, the sample was further processed according to Cohen and Chait (1997) and the protein was finally eluted into the electrospray source with a solution consisting of 70% acetonitrile/2.5% acetic acid at 6 ml/min. ESI-MS was performed on a TSQ-700 triple-quadrupole mass spectrometer (Finnigan MAT Corp., San Jose, CA) using the standard conditions for protein analysis by ESI-MS according to Mirza et al. (1995). N-terminal sequencing was determined by Edman degradation sequencing of the protein using an Applied Biosystems Procise Sequenser. 2.7. Effect of temperature on bacteriocin activity Purified bacteriocin (pH 6.0, 130 AU/ml) was exposed to heat treatments (with a heat block) of 40, 60, 80, and 100 °C for 10, 30, and 60 min and 121 °C for 10 and 20 min. The samples were then tested for antimicrobial activity. Purified bacteriocin was also stored for 4-weeks at 80, 20, 4, and 30 °C and the bacteriocin activity was assayed at 1-week intervals. 2.8. Effect of pH on bacteriocin activity Purified bacteriocin was dissolved in distilled water at a concentration of 130 AU/ml. The pH of the samples was adjusted to the range of pH values of 2.0–14.0, using 1 N
HCl or 1 N NaOH solutions. Following incubation at 37 °C for 30 min, the samples were neutralized to pH 5.5, and tested for antimicrobial activity. 2.9. Sensitivity of the bacteriocin to proteases Resistance to proteolytic enzymes was determined by incubating samples of the purified bacteriocin in the presence of proteinase K, trypsin, a-chymotrypsin (0.05 M sodium phosphate buffer, pH 7.0), pepsin and papain (0.2 M citric acid buffer, pH 2.0), at 37 °C for 1 h. All enzymes were from Sigma and used at a final concentration of 2 mg/ml. Purified bacteriocin in buffers without enzyme, enzyme-buffer solutions, and buffers were used as controls. Following incubation, enzymes were inactivated by heating for 3 min at 100 °C, and samples were examined on SDS– PAGE on 10% gels (w/v) and also for antimicrobial activity. 2.10. Mode of action of the bacteriocin Addition of purified bacteriocin, at a final concentration of 130 AU/ml, was done to M. luteus CECT 241 cultures at mid-logarithmic phase of growth. Changes in turbidity were recorded at 600 nm and the number of colony-forming units (cfu) was determined for samples and controls (bacteriocin-free cultures of M. luteus). All samples in which bacteriocin was added, were then centrifuged and the supernatant was examined for the presence of proteins and DNA by measuring the OD at 280 and 260 nm, respectively. 3. Results and discussion Tricine–SDS–PAGE analysis of cell-free filtrates of P. acilactici culture broths confirmed a bactericidal peptide band of 3–4 kDa (Fig. 1). Further analysis of the purified peptide by ESI-MS gave the exact molecular mass of 3.660 kDa, while N-terminal sequencing showed that the bacteriocin had 19 amino acid residues in the order KYYGXNGVXTXGKHSXVDX (X, are the unidentified residues at positions 5, 9, 11, 16, and 19 from the N-terminal). In this respect, the purified bacteriocin is similar to pediocins isolated by various Pediococci (Wu et al., 2004) and therefore, it belongs to the class IIa of bacteriocins and is thus designated pediocin SA-1. The isolated pediocin SA-1 is a larger molecule compared to the 2.7 kDa molecule of pediocin AcH/PA-1 and a smaller compared to the 4 kDa of pediocin SJ-1, both produced by P. acidilactici strains (Bhunia et al., 1987; Marugg et al., 1992; Schved et al., 1993). The M.W. of another pediocin produced by P. damnosus (Green et al., 1997) was found to be 3.5 kDa. Growth and metabolism of P. acidilactici were studied under different aeration conditions by controlling the inlet gas composition in the bioreactor, as described in the previous section. Figs. 2–4, show the fermentation plots under aerobic, anaerobic and semi-aerobic conditions.
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25
25
20 Biomass (g/L) Lactic acid (g/L)
15
15
Glucose (g/L) pH
10
10
5
5
0
0 0
10
20
30
40
50
60
Time (hours) Fig. 3. Cultivation of Pediococcus acidilactici NRRL B5627 in a stirred tank bioreactor at 150 rpm under anaerobic conditions: Time-courses of biomass, lactic acid, residual glucose, pediocin concentrations and pH of the culture broth.
20
20 Biomass (g/L) Lactic acid (g/L) Glucose (g/L) pH Pediocin (AU/ml)
15 10
15 10
5
5
0
0 0
10
20 30 40 Time (hours)
50
60
Fig. 2. Cultivation of Pediococcus acidilactici NRRL B5627 in a stirred tank bioreactor at 150 rpm under aerobic conditions: Time-courses of biomass, lactic acid, residual glucose, pediocin concentrations and pH of the culture broth.
Biomass, Glucose, Lactic acid (g/l)
25
Pediocin (AU/ml)
Biomass, Glucose, Lactic acid (g/L)
25 25
Pediocin (AU/ml)
20
180 160
20
140 120
Biomass (g/l) Lactic acid (g/l) Glucose (g/l) pH Pediocin (AU/ml)
15
10
100 80 60
Pediocin (AU/ml)
Anaerobic, compared to other, conditions led to higher biomass and lactate production levels, while the levels of the produced bacteriocin were very low (Fig. 3). Fully aerobic conditions were again not favorable for bacteriocin production (Fig. 3). Studies on aeration levels revealed that the optimum conditions for pediocin production were the semi-aerobic, with the DOT maintained at 60% of saturation. Fig. 4 shows the time-courses for the production of biomass, pediocin, and lactic acid, assimilation of glucose and the broth pH, in a P. acidilactici NRRL B5627 fermentation in the stirred tank reactor in semi-aerobic fermentation and under the conditions described in the methods section. Biomass levels reached 5.2 g/l, while lactic acid production approximated 7 g/l in 52 h of fermentation. The initial culture pH of 6.0, declined steadily during fermentation to the final value of 3.7. Because of the increased productivity of bacteriocin under semi-aerobic conditions, these were applied throughout this study. At 60% DOT, Pediococcus growth commenced with inoculation and the maximum growth rate
Biomass, Glucose, Lactic acid (g/L)
Fig. 1. Separation of pediocin SA-1 by tricine–SDS–PAGE. The arrows denote the active pediocin bands.
40
5
20 0
0 0
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20 30 40 Time (hours)
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Fig. 4. Cultivation of Pediococcus acidilactici NRRL B5627 in a stirred tank bioreactor at 150 rpm under semi-aerobic conditions: Time-courses of biomass, lactic acid, residual glucose, pediocin concentrations and pH of the culture broth.
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0.40
specific pediocin production rate (h-1)
16
0.35
specific growth rate (h-1)
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12
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8
0.15
6 4
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2
0.05
0
-1
Specific production rate of pediocin (h -1)
18
Specific growth rate (h )
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0.00 0
20 40 Time (hours)
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Fig. 5. Cultivation of Pediococcus acidilactici NRRL B5627 in a stirred tank bioreactor at 150 rpm under semi-aerobic conditions: Time-courses of specific growth rate of Pediococcus acidilactici NRRL B5627 and specific pediocin production rate.
was detected within 2 h from inoculation (0.62 g/l/h), while the maximum pediocin production rate was obtained at the same time (27 AU/ml/h). Maximum pediocin levels were detected at 14 h (160 AU/ml) and remained stable until 28 h to decrease steadily beyond that point (127 AU/ml at 52 h). Production of the pediocin followed the trend of biomass production. The relationship between the two becomes obvious in Fig. 5, which shows the plot of the specific rates of growth and pediocin production (h 1) vs. time. Production of the pediocin ceased once the stationary phase of growth was reached. From a metabolic point of view, this trend is characteristic of a primary metabolite production, like e.g. nisin, the bacteriocin produced by Lactococcus lactis (Cabo et al., 2001; Papagianni et al., 2007). Aeration studies revealed that an oxygen-enriched atmosphere enhanced pediocin production significantly. The effects of aeration on pediocin production have not been studied previously. However, as the oxygen tolerance of lactic acid bacteria is associated to different metabolic pathways which give rise to different yields of various products, one can expect that aeration should affect pediocin production levels. Although production of many bacterio-
cins produced by LAB has been studied under anaerobic conditions, there are certain cases like e.g. nisin (Cabo et al., 2001) and amilovorin (De Vuyst et al., 1996) in which an oxygen-enriched atmosphere enhanced production considerably. The drop in pediocin production levels observed under fully aerobic conditions in the present study suggests that an optimum DOT exists. The same optimum DOT level of 60%, was found to be the case for nisin Z production by L. lactis (Amiali et al., 1998). The results of the present study point to a direct effect of dissolved oxygen on pediocin production with no correlative increase of biomass. This suggests that such a production is associated with an oxidative metabolic pathway. A similar mode for bacteriocin production was observed with pediocin SJ-1 (Schved et al., 1993), plantaricin C (Gonzales et al., 1994), and reutericin 6 (Toba et al., 1991). As shown in Figs. 4 and 5, large amounts of pediocin are excreted in the fermentation broth which is of a significantly lower pH since the onset of fermentation and contains the produced in the meantime lactate. The results suggest that prepediocin is produced as a primary metabolite during growth but it is processed posttranslationally and excreted at low pH, even when the cells have stopped growing. Posttranslational modification of prepediocin AcH was studied by Johnson et al. (1992) and described as an enzymatic process that requires high lactate and low pH. That process includes removal of a leader segment of 18 amino acids form the NH2-terminal to produce a 44 amino acid molecule that is biologically active (Ray, 1995). Studies by other researchers (Henderson et al., 1992; Lozano et al., 1992) have also shown that pediocin PA-1 undergoes a similar prosttranslational processing. Amino acid sequencing of prepediocin and its processed active form and possible existence of disulfide rings will elucidate the processing details of pediocin SA-1 of the present study. Pediocin SA-1 is inhibitory to several food spoilage bacteria and food-borne pathogens. It is not active against the Gram-negative Salmonella spp., while it shows antilisterial activity, which is characteristic for this type of bacteriocins (Papagianni, 2003). However, compared with the data given by Green et al. (1997) for pediocin PD-1 by
Table 1 Pediocin SA-1: heat, pH and proteolytic enzyme treatments Treatment* Pediocin
Temperature 100 °C/60 min
121 °C/60 min
2–10
4–7
Pepsin
Papain
Trypsin
a-Chymotrypsin
SA-1 PD-1 SJ-1 N5p AcH PA-1
+ + + + + +
+ + + + + ±
+ + +
+ + + + + +
+ + ND ND ND
+ +
+ +
+ +
ND
ND
ND
ND
ND
ND
ND
*
pH
+ +
Proteolytic enzymes
+: Activity; : absence of any activity; ND: not determined. Properties of other pediocins are given for comparisons.
Proteinase K
References This study Green et al. (1997) Schved et al. (1993) Stra¨sser de Saad et al. (1995) Bhunia et al. (1987) Gonzales and Kunka (1987)
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P. damnosus, pediocin SA-1 appeared to be significantly more effective against Listeria spp. Pediocin SA-1 exhibits intermediate inhibitory activity against other P. acidilactici strains and the phylogenetically close P. pentosaceus, unlike pediocin PD-1 by P. damnosus which showed no activity against these species, while small inhibitory activity against known bacteriocin producers Lb. sakei CECT906T, Lb. plantarum CECT220, Lc. lactis ATTC 11454. Pediocin SA-1 appears to be very effective against the anaerobic Clostridium sporogenes and C. thiaminolyticum. Literature reports on the spectra of antimicrobial activities of various pediocins and the present case reveal that although these molecules are classified under the same class of bacteriocins their effectiveness against other LAB or various common food spoilage bacteria and food pathogens may vary widely. Purified pediocin SA-1 is heat stable for up to 60 min at 121 °C. Storage for 4-weeks at 80, 20, 4 and 30 °C did not affect bacteriocin activity. Full activity was retained even following incubation at 30 °C for 1-week at pH values ranging from 3.0 to 12.0. No antimicrobial activity was detected after 30 min of incubation in the buffers of pH 2.0, 13.0, and 14.0. Purified pediocin SA-1 is resistant to treatment with trypsin, a-chymotrypsin, pepsin and papain, but not to proteinase K. The various biochemical properties examined for pediocin SA-1 are listed in Table 1 in which the properties of other described pediocins are given for comparisons. The obvious differences found may result in a different way of action and explain the differences found regarding the inhibitory spectra of various pediocins. The cell numbers of M. luteus CECT 241 declined from 109 cfu to zero within 2 h following addition of pediocin SA-1 to the cells, in the way described in the previous section. The decrease in cfu, along with absence of differences in turbidity measurements, which indicates that no lysis took place, of M. luteus cell suspensions treated with the pediocin, are indicative of a bactericidal mode of action. Cell suspensions of pediocin treated cells were examined for the presence of DNA and the increase in protein levels, which in both cases were negative. These characteristics relate to the described pediocin by P. damnosus by Green et al. (1997). 4. Conclusions A new bacteriocin has been isolated from P. acidilactici NRRL B5627 culture broths. Molecular mass determination and N-terminal sequencing showed that it resembles known pediocins and belongs therefore to the class IIa of bacteriocins. The new bacteriocin, designated pediocin SA-1, is inhibitory to several food spoilage and food-borne pathogens and shows a remarkable stability to heat treatment. Pediocin SA-1 appears to be a potential biopreservative. Further studies are required regarding suitable bioprocessing strategies for an efficient bacteriocin production process.
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Acknowledgements We thank the ARS Patent Culture Collection, Peoria, Illinois and the curator Mr. J.L. Swezey, for kindly providing us with the Pediococcus acidilactici strain. References Amiali, M.N., Lacroix, C., Simard, R.E., 1998. High nisin Z production by Lactococcus lactis UL719 in whey permeate with aeration. World J. Microbiol. Biotechnol. 14, 887–894. Bhunia, A.K., Johnson, M.C., Ray, B., 1987. Direct detection of an antimicrobial peptide of Pediococcus acidilacti by SDS–PAGE. J. Ind. Microbiol. 2, 312–319. Biswas, S.R., Ray, P., Johnson, M.C., Ray, B., 1991. Influence of growth conditions on the production of a bacteriocin pediocin AcH, by Pediococcus acidilactici H. Appl. Environ. Microbiol. 57, 1265– 1267. Cabo, M.L., Murado, M.A., Gonzales, M.P., Pastoriza, L., 2001. Effects of aeration and pH gradient on nisin production. A mathematical model. Enzyme Microb. Technol. 29, 264–273. Cohen, S.L., Chait, B.T., 1997. Mass-spectrometry of whole proteins eluted from sodium dodecyl sulphate-polyacrylamade gel electrophoresis gels. Anal. Biochem. 247, 257–267. Collins, M.D., Rodriguess, U.M., Ash, C., Aguirre, M., Farrow, J.A.E., Martinez-Murcia, A., 1991. Phylogenetic analysis of the genus Lactobacillus and related lactic acid bacteria determined by reverse transcriptase sequencing of 16S rRNA. FEMS Microbiol. Lett. 77, 5– 12. De Vuyst, L., Callewaert, R., Crabbe, K., 1996. Primary metabolic kinetics of bacteriocin biosynthesis by Lactobacillus amylovorus and evidence for stimulation of bacteriocin production under unfavorable growth conditions. Microbiology 142, 817–827. Elegado, F.B., Kim, W.J., Kwon, D.Y., 1997. Rapid purification, partial characterization, and antimicrobial spectrum of the bacteriocin, pediocin AcM, from Pediococcus acidilactici M. Int. J. Food Microbiol. 37, 1–11. Etchells, J.L., Costilow, R.N., Anderson, T.E., Bell, T.A., 1964. Pure culture fermentation of brined cucumbers. Appl. Environ. Microbiol. 12, 523–535. Flemming, H.P., Etchells, J.L., Costilow, R.N., 1975. Microbial inhibition by an isolate of Pediococcus from cucumber brines. Appl. Microbiol. 30, 1040–1042. Green, G., Dicks, L.M.T., Bruggeman, G., Vandamme, E.J., Chikindas, M.L., 1997. Pediocin PD-1, a bactericidal antimicrobial peptide from Pediococcus damnosus NCFB 1832. J. Appl. Microbiol. 83, 127–132. Gonzales, B., Arca, P., Mayo, B., Suarez, J.E., 1994. Detection, purification and partial characterization of plantaricic C, a bacteriocin produced by a Lactobacillus plantarum strain of dairy origin. Appl. Environ. Microbiol. 60, 2158–2163. Gonzales, C.F., Kunka, B.S., 1987. Plasmid-associated bacteriocin production and sucrose fermentation in Pediococcus acidilactici. Appl. Environ. Microbiol. 53, 2534–2538. Gurira, O.Z., Buys, E.M., 2005. Characterization and antimicrobial activity of Pediococcus species isolated from South African farm-style cheese. Food Microbiol. 22, 159–168. Guyonnet, D., Fremaux, C., Cenatiempo, Y., Berjeaud, J.M., 2000. Method for rapid purification of class IIa bacteriocins and comparison of their activities. Appl. Environ. Microbiol. 66, 1744–1748. Henderson, J.T., Chopco, A.L., van Wassenaar, P.D., 1992. Purification and primary structure of pediocin PA-1 produced by Pediococcus acidilactici PAC1 0. Arch. Biochem. Biophys. 295, 5–12. Huang, J., Lacroix, C., Daba, H., Simard, R.E., 1996. Pediocin 5 production and plasmid stability during continuous free and immobilized cell cultures of Pediococcus acidilactici UL5. J. Appl. Bacteriol. 80, 635–644.
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