Use of the gfp gene in monitoring bacteriocin-producing Lactobacillus plantarum N014, a potential starter culture in nham fermentation

Lactobacillus plantarum N014 is a bacteriocin-producing lactic acid bacteria originally isolated from nham, a traditional Thai fermented sausage, and in the process of development to be used as a starter culture for nham fermentation. During the fermentation process, there is a need to identify the starter culture among several naturally occurring bacteria. In this study, a new plasmid carrying the gfp (green fluorescent protein) gene was constructed based on pGKV210, an Escherichia coli/ Lactococcus shuttle vector containing an erythromycin resistance marker. The gfp gene derived from pGFPuv was placed under the control of an L-lactate dehydrogenase promoter and then inserted at the EcoRI site of pGKV210, leading to pN014-GFP. The novel plasmid was used to transform L. plantarum N014, which is a bacteriocin-producing lactic acid bacteria isolated from nham. The resulting transformant, L. plantarum N014-GFP+, was brightly fluorescent and harbored the expected plasmid. A plasmid stability test revealed that pN014-GFP was stable after 100 generations of growth under nonselective pressure. L. plantarum N014-GFP+ and its parent strain were shown to be very similar in growth rate, bacteriocin production, and lactate production. L. plantarum N014-GFP+ was able to survive in a nham model. The survival clones were still fluorescent and harbored pN014-GFP.     

Detection and characterization of a novel antibacterial substance produced by Lactobacillus plantarum ST 31 isolated from sourdough.

Lactobacillus plantarum ST31 isolated from sourdough produced an antimicrobial substance inhibiting other strains of the genera Lactobacillus, Leuconostoc, Pediococcus, Streptococcus, Bacillus and some foodborne pathogens including Staphylococcus aureus. This antimicrobial substance was inactivated by proteolytic enzymes. Consequently, it was characterized as a bacteriocin and was designated plantaricin ST31. This bacteriocin was stable in the pH range 3-8 and it was not affected by amylolytic enzymes. Production of plantaricin was pH and temperature dependent, and maximum yields were obtained in MRS broth cultures maintained at pH 6 and incubated at 30 degrees C in the exponential phase to the early stationary growth phase of the producer organism. This bacteriocin was purified by using consecutive ammonium sulfate and reversed-phase chromatography. It is a peptide of 20 amino acid residues with a mass of 2755+/-0.3 Da, as determined by electrospray mass spectrometry. The sequence of Plantaricin ST31 showed no similarity to those of other bacteriocins. Plantaricin ST31 production appeared to be chromosomally encoded.     

Detection and preliminary characterization of a bacteriocin (plantaricin 35d) produced by a Lactobacillus plantarum strain

Lactic acid bacteria (134) from Italian sausages were tested for the production of antimicrobial substances (bacteriocins). Six percent of these showed antibacterial activity against one or several closely related microorganisms used as indicators. Lactobacillus plantarum 35d in particular produced a bacteriocin of high activity (320 AU ml(-1)) and a wide range of antimicrobial activity including S. aureus, L. monocytogenes, and A. hydrophila. The bacteriocin withstood heating at 80 degrees C for 120 min and storage at 4 degrees C for 6 months. The mode of action was identified as bactericidal. The apparent molecular weight of the bacteriocin extracted with n-butanol was estimated to be 4.5 kDa.     

Growth inhibition of Listeria monocytogenes by a bacteriocin of Pediococcus acidilactici M during fermentation of kimchi

Four lactic acid bacteria known to produce bacteriocins were evaluated for the inhibitory effect against Listeria monocytogenes strain Scott A. Lactococcus lactis subsp. lactis biovar diacetylactis 26-2 was the least antilisterial, whereas Pediococcus acidilactici M resulted in the largest zone of inhibition on an agar medium. Leuconostoc paramesenteroides OX and Lactobacillus sake Lb 706 were intermediate. Aqueous solutions of crude bacteriocin powders derived from supernatant fluids of L. sake Lb 706 and P. acidilactici M. cultures inhibited the growth of Lactobacillus plantarum UGA8 in MRS agar. The addition of 10 mg of crude bacteriocin powder from L. sake to 150 g of kimchi fermented at 14 ± 1°C for 16 days had no effect on the growth of L. monocytogenes. The same amount of crude bacteriocin derived from P. acidilactici, however, had an initial lethal effect on L. monocytogenes and controlled growth of the pathogen throughout the 16-day fermentation. The successful application of bacteriocins to control the growth of L. monocytogenes in lightly fermented foods such as kimchi would appear to be achievable.     

Bacteriocin activity by Lactobacillus curvatus CWBI-B28 to inactivate Listeria monocytogenes in cold-smoked salmon during 4 degrees C storage

The inhibition effectiveness of a bacteriocin produced by Lactobacillus curvatus CWBI-B28 against Listeria monocytogenes was investigated in cold-smoked salmon during storage at 4 degrees C. Three bacteriocin-based strategies for the control of L. monocytogenes in foods (i.e., producing bacteriocin in situ, spraying with partially purified bacteriocin, and packaging in bacteriocin-coated plastic film), plus a newly developed method that uses cell-adsorbed bacteriocin (i.e., a suspension of producer cells on which maximum bacteriocin has been immobilized by pH adjustments), were assessed. Although all the approaches inactivated L. monocytogenes in cold-smoked salmon, various efficacy levels were observed. The behavior of L. monocytogenes was similar in samples treated with either partially purified bacteriocin or in situ bacteriocin production. In both of these cases, the counts of the pathogen declined to below the detectable limit of 0.7 log CFU/cm2 within the first week, but a approximately 0.95- and 1.3-log increase, respectively, occurred after day 14. The bioactive packaging film resulted in a slower inactivation of the pathogen but prevented any subsequent increase in the CFU throughout 22 days of storage at 4 degrees C. Application of the cell-adsorbed bacteriocin was shown to be the most effective means, as it resulted in a complete inactivation of the pathogen within 3 days, and no increase in Listeria counts occurred up to 22 days.     

Use of bacteriocin-producing starter cultures of Lactobacillus plantarum and Lactobacillus curvatus in production of ostrich meat salami

Ostrich meat salami was produced by using Lactobacillus plantarum strain 423 and Lactobacillus curvatus strain DF126. The strains produce the bacteriocins plantaricin 423 and curvacin DF126, respectively. The specific activity of plantaricin 423 in MRS broth at 30?°C increased as the pH decreased from 6.5 to 3.5, but activity subsequently decreased. The activity of curvacin DF126 increased under the same conditions, but remained stable for the duration of the growth cycle. Maximum curvacin DF126 and plantaricin 423 activity levels were recorded at a culture pH of around 4. The spectra of antimicrobial activity recorded for plantaricin 423 and curvacin DF126 were similar. Neither of the two bacteriocins inhibited the growth of Micrococcus sp. MC50 and did not have any inhibitory effect on either of the producer strains. Curvacin DF126 and plantaricin 423 inhibited the growth of L. monocytogenes in salami meat. However, after 15 h of fermentation the viable count of L. monocytogenes LM1 increased, probably due to a decrease in activity of the bacteriocins and/or the development of resistant bacterial cells. This is the first report on the inhibition of L. monocytogenes in ostrich meat salami by using bacteriocinogenic starter cultures.     

Coculture-inducible bacteriocin activity of Lactobacillus plantarum strain J23 isolated from grape must

Detection and characterization of bacteriocin production by Lactobacillus plantarum strain J23, recovered from a grape must sample in Spain, have been carried out. Bacteriocin activity was degraded by proteolytic enzymes (trypsin, alfa-chymotrypsin, papaine, protease, proteinase K and acid proteases), and it was stable at high temperatures (121 degrees C, 20min), in a wide range of pH (1-12), and after treatment with organic solvents. L. plantarum J23 showed antimicrobial activity against Oenococcus oeni, and a range of Lactobacillus and Pediococcus species. Bacteriocin production was detected in liquid media only when J23 was cocultivated with some inducing bacteria, and induction took place when intact cells or 55 degrees C heated cells of the inducer were cocultivated with J23, but not with their autoclaved cells. Bacteriocin activity of J23 was not induced by high initial J23 inocula, and it was detected in cocultures during the exponential phase. The presence of ethanol or acidic pH in the media reduced bacteriocin production in the cocultures of J23 with the inducing bacteria. The presence of plantaricin-related plnEF and plnJ genes was detected by PCR and sequencing. Nevertheless, negative results were obtained for plnA, plnK, plNC8, plS and plW genes.     

源自泡菜的植物乳杆菌产新型广谱抑菌细菌素的特性分析

筛选对食源性致病菌和腐败菌有特异性抑制作用的产细菌素乳酸菌菌株,并研究细菌素的特性,为其在食品防腐中的应用提供参考。从分离自泡菜的112 株乳酸菌中反复筛选得到1 株产细菌素（BacH32）菌株（Hlh32）,经鉴定菌株Hlh32为植物乳杆菌（Lactobacillus plantarum）,其在35 ℃ MRS培养基中发酵至稳定期即18～24 h达到最大BacH32产量（对金黄色葡萄球菌的抑菌活性达到1 850 AU/mL）。通过硫酸铵盐析、DEAE Sepharose Fast Flow离子交换层析、Sephadex G-15凝胶过滤层析、制备型高效液相色谱等方法进行纯化。Trinice十二烷基硫酸钠-聚丙烯酰氨凝胶电泳结果显示BacH32分子质量为6.7 kDa,与文献报道的植物乳杆菌细菌素分子质量有显著差异。细菌素BacH32对蛋白酶敏感,而脂肪酶、α-淀粉酶和β-淀粉酶对其无影响,表明BacH32为蛋白质。BacH32在pH 2～9的环境下孵育4 h,在37、60、80、100 ℃保持30 min或121 ℃保持15 min仍能保持较好抑菌活性。细菌素BacH32对革兰氏阳性菌和革兰氏阴性菌具有广谱抑菌活性,对真菌也有一定的抑菌活性。向培养至对数初期的金黄色葡萄球菌中添加BacH32,菌体生长完全受到抑制。以上特性表明植物乳杆菌Hlh32合成的细菌素BacH32有潜力作为天然食品防腐剂在食品中使用。     

Identification and partial characterization of lacticin BH5, a bacteriocin produced by Lactococcus lactis BH5 isolated from Kimchi

Strain BH5 was isolated from naturally fermented Kimchi and identified as a bacteriocin producer that has bactericidal activity against Micrococcus flavus ATCC 10240. Strain BH5 was identified tentatively as Lactococcus lactis by API test. Lactococcus lactis BH5 showed a broad spectrum of activity against most of the nonpathogenic and pathogenic microorganisms tested by the modified deferred method. The activity of lacticin BH5, named tentatively as the bacteriocin produced by L. lactis BH5, was detected at the mid-log growth phase, reached its maximum during the early stationary phase, and decreased after the late stationary phase. Lacticin BH5 also showed a relatively broad spectrum of activity against nonpathogenic and pathogenic microorganisms as tested by the spot-on-lawn method. Its antimicrobial activity on sensitive indicator cells was completely destroyed by protease XIV. The inhibitory activities of lacticin BH5 were detected during treatments up to 100 degrees C for 30 min. Lacticin BH5 was very stable over a pH range of 2.0 to 9.0 and was stable with all the organic solvents examined. It demonstrated a typical bactericidal mode of inhibition against M. flavus ATCC 10240. The apparent molecular mass of lacticin BH5 was estimated to be in the region of 3 to 3.5 kDa, by the direct detection of bactericidal activity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

一株后生元菌株的抑菌特性研究及其细菌素基因簇的挖掘

本文旨在筛选一株可拮抗中国大鲵源嗜水气单胞菌的后生元菌株,并对其进行抑菌特性的研究和细菌素基因簇的挖掘。以中国大鲵病原性嗜水气单胞菌（Aeromonas hydrophila, Ah2）为指示菌,筛选一株产细菌素乳酸菌株并评估其药敏特性;采用有机溶剂萃取法初步纯化菌株产细菌素;通过pH、温度和消化酶耐受性、贮藏稳定性、抑菌谱及最小抑菌（MIC）和杀菌浓度（MBC）共六类指标评价细菌素的抑菌特性;溶血反应与细胞毒性实验测试细菌素对Ah2的抑菌效果,经扫描电子显微镜初步探究细菌素的抑菌机制;经由紫外全波长扫描定性细菌素以及Tricine-SDS-PAGE电泳测定细菌素的分子量范围;最后根据菌株的全基因组序列挖掘其潜在的细菌素基因簇（RiPPs）。结果显示,从青岛市售腐乳中筛出一株产细菌素植物乳植物杆菌M4L1（Lactiplantibacillus plantarum M4L1）。初步纯化M4L1产细菌素并命名为LP01。细菌素LP01具备良好的消化酶耐受性,且在pH2~10、?20~121 ℃和9个月贮藏期内均表现出稳定的抑菌活性,并对单增李斯特菌、弗氏柠檬酸杆菌等14株革兰氏阳性和阴性菌均有不同程度的抑制作用;另外,LP01对Ah2的MIC和MBC分别为12.94和25.88 μg/mL。经MBC浓度的LP01处理后的Ah2,溶血活性和细胞毒性均得到明显缓解;SEM观察其通过破坏Ah2的细胞壁具有抑制或杀伤作用。LP01在波长200~220 nm处的肽类特征吸收峰显著,电泳后条带显示其分子量在3.3~4.0 kDa,LP01为小分子肽类细菌素。此外,基因注释到M4L1有2个细菌素基因簇（RiPPs）,对应产物分别为Plantaricin K和Plantaricin E,均属于植物乳杆菌II类细菌素。菌株M4L1不仅含有多种抗菌物质相关基因簇,而且其细菌素LP01具备了优良的抑菌性能,能有效抑制多种水产病原菌、食物腐败菌及食源性致病菌等。产细菌素植物乳植物杆菌M4L1作为一株潜在的后生元菌株具有较好的应用前景。     

Partial characterization of lacticin NK24, a newly identified bacteriocin of Lactococcus lactis NK24 isolated from Jeot-gal

Strain NK24 was isolated from Jeot-gal and identified as a bacteriocin producer, which has bactericidal activity against Leuconostoc mesenteroides KCCM 11324. Strain NK24 was identified tentatively as Lactococcus lactis by the API test. The activity of lacticin NK24, named tentatively as the bacteriocin produced by L. lactis NK24, was detected during the mid-log growth phase, reached a maximum during the early stationary phase, and decreased after the late stationary phase. Lacticin NK24 showed a relatively broad spectrum of activity against non-pathogenic and pathogenic micro-organisms as assessed using the spot-on-lawn method. Its antimicrobial activity on sensitive indicator cells was completely destroyed by protease IX or protease XIV. The inhibitory activities of lacticin NK24 were detected during treatments up to 100°C for 30 min. Lacticin NK24 was very stable over a pH range of 2·0–9·0 and to all organic solvents examined. It demonstrated a typical bactericidal mode of inhibition against L. mesenteroides KCCM 11324. The apparent molecular mass of lacticin NK24 was estimated to be in the region of 3–3·5 kDa, which was determined by the direct detection of bactericidal activity after SDS-PAGE.     

Survival of Listeria monocytogenes on sliced cooked sausage after treatment with pediocin AcH

A preparation with pediocin AcH bound to its heat-killed producer cells Lactobacillus plantarum WHE 92 (starter culture ALC01, Wisby, Denmark) by adjusting the pH of the preparation to 6.0 was studied for its effects against Listeria monocytogenes ATCC 7644 and (spoilage) lactic acid bacteria on sliced cooked sausage. The pediocin AcH preparation or 0.9% (w/w) NaCl dilution (as a control) were randomly distributed dropwise on the surface of the slices. Treated slices were vacuum-packed and stored at 6 degrees C. Microbiological analysis and determination of pH values were performed after 3, 6, 9, 14 and 21 days of storage. Flavour of the sausages was evaluated after 7 and 11 days of storage. The pediocin preparation had effect (p > 0.05) neither on the growth of lactic acid bacteria, on the pH value nor on the flavour of vacuum-packed sliced sausage during 21 days of storage compared to control. However, during 6 days of storage, the number of L. monocytogenes decreased from the initial level of 2.7 log cfu/g sausage to < 2 log cfu/g, while on the control sausages the number of L. monocytogenes remained at the inoculated level. The numbers of L. monocytogenes remained at those levels to the end of storage period (21 days). However, the treated samples were determined to be Listeria positive, which indicates that the pediocin preparation was not efficient enough to kill all L. monocytogenes.     

Inhibition of Listeria monocytogenes by enterocin EJ97 produced by Enterococcus faecalis EJ97

Enterocin EJ97 from Enterococcus faecalis EJ97 showed a concentration-dependent antimicrobial activity against Listeria monocytogenes CECT 4032. Activity of enterocin EJ97 against L. monocytogenes CECT 4032 increased slightly at 4 degrees C, and cold-adapted cells did not show any increased resistance. Sensitivity of L. monocytogenes CECT 4032 to enterocin EJ97 was not modified by the addition of sodium benzoate, sodium acetate, NaCl or sodium tripolyphosphate. Anti-listeria activity was enhanced by potassium nitrate, and especially by sodium nitrite at concentrations of 50 microg/ml or above. E. faecalis EJ97 produced bacteriocin activity during cocultivation with L. monocytogenes CECT 4032 at 37 degrees C and also at 15 degrees C, but not at 4 degrees C. Growth of L. monocytogenes CECT 4032 was inhibited by bacteriocin produced during cocultivation at 37 and 15 degrees C, and the degree of inhibition was influenced by the incubation temperature and the initial concentrations of enterococci and listeria. E. faecalis EJ97 also produced bacteriocin during cocultivation in half-skimmed milk, although its capacity to control L. monocytogenes was limited to populations of 10(3) CFU/ml or lower.     

Inhibition of Listeria monocytogenes by Lactobacillus sake strains of meat origin

The ability of two Lactobacillus sake strains of meat origin to inhibit the growth of Listeria monocytogenes at 4, 8, 15, 24 and 32°C in a conventional liquid media was investigated. Growth of L. monocytogenes was affected by Lac. sake strains at all temperatures. The inhibition was higher at 15, 24 and 32°C than at refrigeration temperatures. The inhibitory activity of both lactobacilli was similar perhaps due to the fact that Lac. sake 148 produces a bacteriocin inhibitory to L. monocytogenes, while Lac. sake 23 is a strong lactic acid producer. The antagonism exhibited by the lactobacilli on the L. monocytogenes strains seems to display a bacteriostatic rather than a bacteriocidal effect.     

植物乳杆菌CGMCC.5297产细菌素条件的优化及应用

用响应面法对植物乳杆菌CGMCC.5297生产细菌素的培养基进行了优化。通过Plackett-Burman设计和中心组合试验设计,植物乳杆菌CGMCC.5297代谢产细菌素的最佳培养条件为酵母粉5.09g/L,牛肉膏10.85g/L,葡萄糖55.34g/L。此时的细菌素上清与指示菌单核细胞增多性李斯特菌CVCC1595共培养4h后,指示菌OD600nm为0.001 73,效价为4499 IU/mL,提高了1.4倍。在最优发酵条件下获得的试验结果与模型预测值吻合,说明所建立的模型是切实可行的。将优化后的植物乳杆菌上清加入到自制酸奶中,发现此细菌素对酸奶具有良好的保鲜效果。     

Lactobacillus plantarum inhibits growth of Listeria monocytogenes in an in vitro continuous flow gut model, but promotes invasion of L. monocytogenes in the gut of gnotobiotic rats

The ability of the pediocin AcH producing Lactobacillus plantarum DDEN 11007 and its non-producing plasmid-cured isogenic variant, DDEN 12305 to prevent the persistence and growth of Listeria monocytogenes EP2 in two gastrointestinal (GI) tract models was examined. In vitro studies conducted in a two-stage continuous flow system showed that L. plantarum DDEN 11007 inhibited L. monocytogenes EP2 under these conditions, while less effect was seen of the non-bacteriocin producing variant. The inhibitory effect was more pronounced at pH 5 than at pH 7. No effect on persistence of L. monocytogenes in the GI tract was seen in gnotobiotic rats colonized with either the pediocin AcH producing or the non-bacteriocin producing variant of L. plantarum when compared to rats inoculated with L. monocytogenes EP2 alone. Surprisingly, inoculation of the gnotobiotic animals with either of the L. plantarum strains prior to inoculation with L. monocytogenes EP2 resulted in increased occurrence of L. monocytogenes in liver and spleen when compared to the animals inoculated with L. monocytogenes EP2 alone. Our results indicate that the presence of L. plantarum in the gut of gnotobiotes facilitates L. monocytogenes invasion by an unknown mechanism. This observation is however not necessarily specifically related to L. plantarum, and should not be interpreted as the expected effect in animals carrying a conventional intestinal microflora.     

Purification, characterization and amino acid sequencing of divergicin M35: a novel class IIa bacteriocin produced by Carnobacterium divergens M35

Carnobacterium divergens M35, isolated from a commercial sample of frozen smoked mussels, produces a new bacteriocin, divergicin M35, a class IIa bacteriocin. Divergicin M35 is sensitive to pronase-E, alpha-chymotrypsin and proteinase K, but not to trypsin and withstands thermal treatments up to 121 degrees C for 30 min. Divergicin M35 was extracted from the culture supernatant of C. divergens M35 using an SP-Sepharose cation-exchange column, desalted and purified on a C18 Sep-Pack column and further purified by reverse phase-high pressure liquid chromatography. This procedure allowed the recovery of 10% of the bacteriocin present in the culture supernatant with purity higher than 99%. Divergicin M35 had a molecular mass of 4518.75 Da as determined by mass spectrometry, a pI value of 8.3 and positive net charge (+3). The amino acid sequence of divergicin M35 was found to consist of 43 amino acid with four cysteine residues (Cys10, 15, 25, 43) and showed 80.5% homology with divercin V41 (80.5%) and 80.0% with bavaricin MN. Divergicin M35 showed powerful antilisterial activity, especially against Listeria monocytogenes and was also active against carnobacteria but not against strains of Lactococcus, Lactobacillus, Enterococcus, Bifidobacteria and Escherichia. Divergicin M35 production began in late exponential phase and reached a maximum activity of 65,000 AU/ml in early stationary phase. Initial broth pH, Tween 80 and acetate did not affect C. divergens M35 growth or divergicin production. This bacteriocin may be a potential tool for inhibiting L. monocytogenes in seafood products that do not usually undergo an adequate heat treatment.     

An α-amylase sensitive bacteriocin of Leuconostoc carnosum

Leuconostoc carnosum LA54A isolated from processed meat produces an antimicrobial substance that inhibits the growth of strains of lactic acid bacteria, Listeria monocytogenes and Enterococcus faecalis. The antibacterial substance was sensitive to proteases suggesting that Lc. carnosum is producing a bacteriocin. The bacteriocin was in addition sensitive against the activity of different α-amylases indicating that the molecule consists of a protein and a carbohydrate moiety. The bacteriocin was active over a wide pH range with an optimum between pH 3 and 5. It was stable after heat treatment at 100°C for 15 min. Optimum production of the bacteriocin by Lc. carnosum was during the late logarithmic phase. The purified bacteriocin showed a molecular weight of about 4000 Da when analyzed by SDS-polyacrylamide gel electrophoresis.     

Development of a lacticin 3147-enriched whey powder with inhibitory activity against foodborne pathogens.

The broad-spectrum bacteriocin lacticin 3147, produced by Lactococcus lactis DPC3147, is inhibitory to a wide range of gram-positive food spoilage and pathogenic organisms. A 10% solution of demineralized whey powder was fermented with DPC3147 at a constant pH of 6.5. The fermentate was spray dried, and the resulting powder exhibited inhibitory activity. The ability of the lacticin 3147-enriched powder to inhibit Listeria monocytogenes Scott A and Staphylococcus aureus 10 was assessed in buffer at both acidic (pH 5) and neutral (pH 7) pH. In addition, the ability of the powder to inhibit L. monocytogenes Scott A in an infant milk formulation was assessed. Resuspension of approximately 10(8) midexponential phase L. monocytogenes Scott A cells in a 10% solution of the lacticin 3147-enriched powder resulted in a 1,000-fold reduction in viable cells at pH 5 and pH 7 after 3 h at 30 degrees C. In the case of S. aureus 10, resuspension of 2.5 x 10(7) midexponential phase cells in a 15% solution of the lacticin 3147-enriched powder at pH 5 resulted in only a 10-fold reduction in viable cell counts, compared with a 1,000-fold reduction at pH 7, following incubation for 3 h at 30 degrees C. The use of the lacticin 3147 powder in an infant milk formulation resulted in greater than a 99% kill of L. monocytogenes within 3 h at 30 degrees C. These results suggest that this bioactive lacticin 3147 food ingredient may find applications in many different foods, including those with pH close to neutrality.     

Behavior of Listeria monocytogenes and Staphylococcus aureus in yogurt fermented with a bacteriocin-producing thermophilic starter

Streptococcus salivarius subsp. thermophilus B producing a bacteriocin active against Listeria monocytogenes ATCC 7644 and Staphylococcus aureus SAD 30 was isolated from bakery yeast. The bacteriocin was partially purified by an adsorption/desorption technique, and its spectrum of action was compared to that of a neutralized cell-free supernatant (CFS). Although the CFS inhibited a number of gram-positive and -negative bacteria of health and spoilage significance, the spectrum of action of the partially purified bacteriocin was limited to gram-positive bacteria. L. monocytogenes was the most sensitive to both preparations. The bacteriocin-producing streptococcal strain was used in combination with a Bac- Lactobacillus delbrueckii subsp. bulgaricus CY strain isolated from commercial yogurt to assess the effectiveness of the resulting thermophilic starter in controlling L. monocytogenes and S. aureus in yogurt during fermentation and storage at refrigeration (ca. 7 degrees C) or abuse (ca. 22 degrees C) temperature. Yogurt samples were contaminated with L. monocytogenes or S. aureus to the approximate levels of 10(3) and 10(6) CFU/ml of milk, respectively. The results showed that in situ bacteriocin production was more active against L. monocytogenes than against S. aureus in vitro and in contaminated samples. While L. monocytogenes leveled off below the detectable limit in a 1-ml sample of yogurt within 24 h of processing, S. aureus survived in Bac+ and Bac- samples during 10 days of storage at room temperature (ca. 22 degrees C). Use of a Bac+ starter resulted in a 5-day extension of the shelf life.