Production,mode of action and sequencing of the corresponding gene of a bacteriocin produced by Lactococcus lactis 19.3

Lactococcus lactis 19.3 produces a bacteriocin with a wide inhibitory spectrum, including the food pathogen Listeria monocytogenes. The producing strain was able to grow and produce similar amounts of bacteriocin in MRS medium, cow's milk and soya milk, respectively. The mode of action of this bacteriocin was further investigated using two indicator strains, Lactobacillus delbrueckii subsp. bulgaricus LMG 6901^T and Listeria monocytogenes ATCC 1911‐1. The bacteriocin displayed a bactericidal effect, causing a rapid decrease of the cell viability. Bacteriocin treatment of the sensitive strains resulted in major morphological changes, including pore formation, as shown by scanning electron microscopy. Finally, the bacteriocin‐encoding gene was identified by sequencing. The presence of nisin gene was confirmed. Based on all our data gathered so far, L. lactis 19.3 is a good candidate for a starter or protective culture in the manufacturing of both fermented dairy and vegetarian food products.     

Biopreservation by Lactobacillus paracasei in coculture with Streptococcus thermophilus in potentially probiotic and synbiotic fresh cream cheeses

The viability of Lactobacillus paracasei and its effect on growth of the microbiota in potentially probiotic and synbiotic fresh cheeses during storage at 4 +/- 1 degree C was investigated. Three cheese-making trials (T1, T2, and T3) were prepared in quadruplicate, all supplemented with a Streptococcus thermophilus culture. L. paracasei subsp. paracasei was added to cheeses in T1 and T2, and inulin was added to cheeses in T2. Counts of L. paracasei, S. thermophilus, coliforms, Escherichia coli, Staphylococcus spp., DNase-positive Staphylococcus, and yeasts and molds were monitored during storage for up to 21 days. Viable counts of L. paracasei in probiotic (T1) and synbiotic (T2) cheeses remained above 7 log CFU/g during the entire storage period, whereas counts of S. thermophilus remained above 9.5 log CFU/g for cheeses from TI, T2, and T3. Populations of coliforms, Staphylococcus spp., and DNase-positive Staphylococcus were higher in T3 cheese and differed significantly from those in cheeses from T1 and T2 (P < 0.05). Inhibition of contaminants prevailed when both L. paracasei and S. thermophilus were present in fresh cream cheese and probably was due to acid production by both strains; bacteriocin production was not found. Addition of inulin in T2 did not impact microbial viability (P > 0.05). L. paracasei subsp. paracasei in coculture with S. thermophilus was inhibitory against microbial contaminants in fresh cream cheese with or without the addition of inulin, indicating the potential use of this combination in a probiotic and synbiotic product.     

Characterisation of the Lactobacillus community in traditional Karst ewe's cheese

Ewe's cheese, produced in the Slovenian Karst region, was analysed for its lactobacilli population. Biochemical fingerprinting with PhenePlate™ LB grouped lactobacilli in 16 distinct PhP types. One representative from each PhP type was further analysed. Members of Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus curvatus, and Lactobacillus brevis species were determined phenotypically. The genus-specific DNA band was amplified with all PhP representatives, whereas species-specific polymerase chain reaction efficiently determined only L. paracasei and L. brevis. Lactobacilli were highly to moderate sensitive to six antibiotics, and resistant to vancomycin. From halo formation in the agar spot method, indicators exerted a promising level of sensitivity for antimicrobials, produced by test microorganisms. Lactobacilli from Karst ewe's cheese offer a remarkable reservoir of 'natural' microbes.     

Incorporation and Survival of Probiotic Bacteria in Whey Cheese Matrices

ABSTRACT: The viabilities of probiotic strains of Lactobacillus acidophilus, Lactobacillus paracasei, Bifidobacterium animalis , and Lactobacillus brevis were studied following incorporation in a whey cheese matrix. Experimental production of plain, as well as sugar-added or salt-added whey cheeses, was based on the traditional manufacture protocol of Requeijäo, a Portuguese whey cheese that essentially results from protein denaturation via heating of whey at about 85°C. After inoculation, the experimental whey cheeses were incubated at 7°C for 28 d. Our results have shown that all strains considered were able to maintain (or even increase) their initial viable numbers; L. paracasei ssp. paracasei strain LCS-1 and L. acidophilus strain Ki exhibited the highest cell viability in plain Requeijão by the end of the storage period—an increase of ca. 2 log cycles in their viable numbers was actually recorded. Among the parameters studied, bacterial species and matrix nature had the most important effect upon viable counts, whereas time of storage was the least important.     

Characterization of certain bacterial strains for potential use as starter or probiotic cultures in dairy products

The present work was aimed at characterizing 12 strains of lactic acid bacteria (LAB) to obtain improved potential starter or probiotic cultures that could be used for making dairy products from ewe's milk and cow's milk. Eight strains with antimicrobial properties, isolated from ewe's milk and from cheese made from ewe's and/or cow's milk, were studied. They were identified as Enterococcus faecalis (five strains), Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides, and Lactobacillus paracasei subsp. paracasei (one strain of each species). Additionally, four strains were obtained from the American Type Culture Collection: Lactobacillus casei 393 (isolated from cheese), L. lactis subsp. lactis 11454 (origin nonspecified and a producer of nisin), and two strains isolated from human feces (L. paracasei subsp. paracasei 27092 and Lactobacillus rhamnosus 53103, antibacterial agent producer). All E. faecalis strains showed at least one virulence factor (either hemolysin or gelatinase), which emphasizes the importance of these studies in this species. Both L. lactis strains and most Lactobacillus spp. were good acidifiers in ewe's milk and cow's milk at 30°C. High β-galactosidase activity, as well as aminopeptidase activities that favor the development of desirable flavors in cheese, were detected in all Lactobacillus spp. strains. Furthermore, L. rhamnosus ATCC 53103 showed α-fucosidase activity (thought to help colonization of the intestine) and lack of α-glucosidase activity (a trait considered positive for diabetic and obese humans). This last enzymatic activity was also lacking in L. lactis ATCC 11454. L. mesenteroides was the only strain D(2)-lactic acid producer. The selection of any particular strain for probiotic or dairy cultures should be performed according to the technological and/or functional abilities needed.     

西藏地区发酵牛乳中产细菌素乳酸菌的筛选及鉴定

从西藏地区藏族传统发酵乳中分离乳酸菌,采用生理生化特性和16S基因序列同源性分析对其进行鉴定,通过双层琼脂平板扩散法筛选具有抑菌活性的菌株。结果表明,共分离37株乳酸菌,其中,乳杆菌属(Lactobacillus)35株、明串珠菌属(Leuconostoc)2株;35株乳酸杆菌为Lactobacillus casei 16株、Lactobacillus paracasei 7株、Lactobacillus plantarum 4株、Lactobacillus fermentum 2株、L.delbrueckii subsp.bulgaricus 2株、Lactobacillus helveti-cus 1株、Lactobacillus diolivorans 3株;7株L.casei和L.paracasei的发酵上清液对3株细菌指示菌表现出明显抑制作用,所有菌株对真菌无抑菌活性;在排除有机酸、H2O2等的干扰和经蛋白酶K处理后,初步确定7株乳酸菌发酵上清液中的抑菌物质为细菌素。     

1株产细菌素乳酸菌的鉴定及所产细菌素的诱导合成现象

从发酵大蒜中筛选到一株具有抑菌作用的菌株Br26,通过用不同蛋白酶处理判断该菌株是否为细菌素产生菌。随后,通过分析自身发酵上清液与其他乳酸菌对细菌素合成的影响,探讨该细菌素的诱导合成现象。结果表明：菌株Br26的发酵上清液经胃蛋白酶和木瓜蛋白酶处理后抑菌活性降低,确定该菌为细菌素产生菌,经16S rDNA鉴定为Weissella sp. Br26。Weissella sp. Br26在42 ℃、1/4 MRS培养时,细菌素抑菌活性消失,而当添加不同生长时期的发酵上清液时,抑菌活性显著增加,表明该细菌素可进行自我诱导。另一方面,Weissella sp. Br26与卷曲乳杆菌、瑞士乳杆菌、副干酪乳杆菌和肠膜明串珠菌的活菌共同培养后,发酵液pH值未有明显变化,但细菌素抑菌活性显著增加,表明细菌素的合成亦可受其他乳酸菌的诱导。综上,Weissella sp. Br26细菌素的合成是受自身发酵液及其他菌诱导调控的。     

Characterization of lactic acid bacteria isolated from Bukuljac, a homemade goat's milk cheese

The Bukuljac cheese is traditionally homemade cheese, produced from heat-treated goat's milk without the addition of any bacterial starter culture. The presence of lactic acid bacteria (LAB) in Bukuljac cheese has been analyzed by using a polyphasic approach including microbiological and molecular methods such as rep-PCR with (GTG)5 primer. Lactobacillus paracasei subsp. paracasei represents a dominant strain in the microflora of analyzed cheese. Out of 55 Gram-positive and catalase-negative isolates, 48 belonged to L. paracasei subsp. paracasei species. Besides lactobacilli, five Lactococcus lactis subsp. lactis and two Enterococcus faecalis were found. Results of PCR-denaturing gradient gel electrophoresis (DGGE) of DNA extracted directly from the fresh cheese revealed the presence of Leuconostoc mesenteroides. Only lactobacilli showed a high proteolytic activity and hydrolyzed alpha(s1)- and beta-caseins. They are also producers of diacetyl. In addition, 34 out of 55 isolates, all determined as lactobacilli, showed the ability of auto-aggregation. Among 55 isolates, 50 also exhibited antimicrobial activity.     

Manufacture of Fior di Latte cheese by incorporation of probiotic lactobacilli

This work aimed to select heat-resistant probiotic lactobacilli to be added to Fior di Latte (high-moisture cow milk Mozzarella) cheese. First, 18 probiotic strains belonging to Lactobacillus casei, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus reuteri were screened. Resistance to heating (65 or 55°C for 10 min) varied markedly between strains. Adaptation at 42°C for 10 min increased the heat resistance at 55°C for 10 min of all probiotic lactobacilli. Heat-adapted L. delbrueckii ssp. bulgaricus SP5 (decimal reduction time at 55°C of 227.4 min) and L. paracasei BGP1 (decimal reduction time at 55°C of 40.8 min) showed the highest survival under heat conditions that mimicked the stretching of the curd and were used for the manufacture of Fior di Latte cheese. Two technology options were chosen: chemical (addition of lactic acid to milk) or biological (Streptococcus thermophilus as starter culture) acidification with or without addition of probiotics. As determined by random amplified polymorphic DNA-PCR and 16S rRNA gene analyses, the cell density of L. delbrueckii ssp. bulgaricus SP5 and L. paracasei BGP1 in chemically or biologically acidified Fior di Latte cheese was approximately 8.0 log(10)cfu/g. Microbiological, compositional, biochemical, and sensory analyses (panel test by 30 untrained judges) showed that the use of L. delbrueckii ssp. bulgaricus SP5 and L. paracasei BGP1 enhanced flavor formation and shelf-life of Fior di Latte cheeses.     

Effect of lactobacillus adjunct cultures on the microbiological and physicochemical characteristics of Roncal-type ewes'-milk cheese

The effect of two different experimental adjunct cultures composed of native facultatively heterofermentative lactobacilli (FHL) on the development of various groups of micro-organisms in Roncal-type ewes' milk cheese was studied. Four cheese batches were manufactured from raw milk (C), pasteurized milk (P), pasteurized milk and an adjunct culture of Lactobacillus paracasei (PP); and pasteurized milk and adjunct culture of Lactobacillus paracasei plus Lactobacillus plantarum (PPP). Retention of the two adjunct cultures in the cheeses was good, and population levels remained constant at around 10(7) cfu g(-1) of cheese throughout ripening. Levels of Enterobacteriaceae and enterococci fell off more abruptly in the batches made with the Lactobacillus adjunct cultures, suggesting competition between the added lactobacilli and those groups of micro-organisms. The inhibitory effect was greater for the adjunct culture composed of L. paracasei plus L. plantarum. Lactococcal levels were higher in the batches made with added FHL, which may be indicative of a synergistic effect between these two groups.     

Characterization and antimicrobial activity of bacteriocin 217 produced by natural isolate Lactobacillus paracasei subsp. paracasei BGBUK2-16

The strain Lactobacillus paracasei subsp. paracasei BGBUK2-16. which was isolated from traditionally homemade white-pickled cheese, produces bacteriocin 217 (Bac217; approximately 7 kDa). The onset of Bac217 biosynthesis was observed in the logarithmic phase of growth, and the production plateau was reached after 9 or 12 h of incubation at 37 and 30 degrees C, respectively, when culture entered the early stationary phase. Biochemical characterization showed that Bac217 retained antimicrobial activity within the range of pH 3 to 12 or after treatment at 100 degrees C for 15 min. Bac217 antimicrobial activity also remained unchanged after storage at 4 degrees C for 6 months or -20 degrees C for up to 12 months. However, Bac217 activity was completely lost after treatment with different proteolytic enzymes. BGBUK2-16 contains only one plasmid about 80 kb in size. Plasmid curing indicated that genes coding for Bac217 synthesis and immunity seem to be located on this plasmid. Bac217 exhibited antimicrobial activity against some pathogenic bacteria, such as Staphylococcus aureus and Bacillus cereus. Interestingly, Bac217 showed activity against Salmonella sp. and Pseudomonas aeruginosa ATCC27853. The inhibitory effect of BGBUK2-16 on the growth of S. aureus in mixed culture was observed. S. aureus treatment with Bac217 led to a considerable decrease (CFU/ml) within a short period of time. The mode of Bac217 action on S. aureus was identified as bactericidal. It should be noted that the strain BGBUK2-16 was shown to be resistant to bacteriocin nisin, which is otherwise widely used as a food additive for fermented dairy products.     

Isolation and partial biochemical characterization of a proteinaceous anti-bacteria and anti-yeast compound produced by Lactobacillus paracasei subsp. paracasei strain M3

New proteinaceous active substance produced by Lactobacillus paracasei subsp. paracasei strain M3 used as a starter for Bulgarian yellow cheese was identified and studied. It displayed bactericidal and fungistatic activities. Its activity was checked against over 60 bacterial and yeast strains. It was efficient against Bacillus subtilis ATCC 6633, several L. delbrueckii species, Helicobacter pylori NCIPD 230 and some yeast species, for example Candida albicans, C. pseudointermedia NBIMCC 1532, C. blankii NBIMCC 85 and Saccharomyces cerevisiae NBIMCC 1812. The synthesis of the substance by producing strain was detected in the late logarithmic growth phase during batch fermentation. Anion exchange chromatography, reversed phase chromatography (RPC) on C4 column and HPLC on C18 column were used for partial purification of this antimicrobial compound. The gene responsible for the synthesis of the active substance is located on the bacterial chromosome.     

Effect of probiotic lactobacilli on faecal enzyme and genotoxic activity in human faecal water in the presence of the carcinogen PhIP in vitro

The average β‐glucuronidase activity for children was 0.48 ± 0.04 U/mg, for adults 0.75 ± 0.27 U/mg and for elderly 1.55 ± 0.06 U/mg. For β‐glucosidase, it was 0.19 ± 0.02 U/mg for children, 0.77 ± 0.26 U/mg for adults and 1.18 ± 0.27 U/mg for elderly. In the presence of probiotics, the highest decrease in genotoxicity was observed for Lactobacillus casei 0908 (to 7.99 ± 1.32) and Lactobacillus paracasei 0919 (to 6.19 ± 1.44) for all children. In adults, lower mean genotoxicity was regarded after incubation of PhIP with L. casei 0908 (it was 5.27 ± 1.13) and L. paracasei 0919 (it was o 6.01 ± 1.00). For elderly, statistically significant decrease was maintained after incubation of PhIP with L. casei 0900 (to 6.72 ± 2.67).     

Lactobacillus strains isolated from Danbo cheese as adjunct cultures in a cheese model system

Isolates of Non-Starter Lactic Acid Bacteria (NSLAB) from six ripened Danbo cheeses of different ages and of different brands were examined. Special emphasis was on the genus Lactobacillus with the aim of investigating their role in cheese maturation. Thirty-three isolates were typed by the PCR-based method, Randomly Amplified Polymorphic DNA (RAPD). Ten RAPD types were found and 70% of the isolates were of RAPD types found in more than one cheese. The different RAPD types were identified to species level by Temporal Temperature Gradient Gel Electrophoresis (TTGE). Most of the isolates were identified as Lactobacillus paracasei (76%), but also Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus rhamnosus and some taxa originating from the starter culture were detected. In one cheese, no lactobacilli were found.One strain of the most frequent Lactobacillus RAPD type from each of the five cheeses with a Lactobacillus flora was used as adjunct cultures in a cheese model system. Four of the five adjuncts were re-isolated during ripening. Two adjunct containing model cheeses received higher flavour scores than the control while two other were associated with off-flavours. The two model cheeses with off-flavour had a similar microflora and both were after 13 weeks of ripening dominated by a strain identified as L. plantarum.     

产细菌素乳酸菌的筛选及对农家干酪保质期的影响

从实验室保存的7 株乳酸菌中,经分离纯化,以金黄色葡萄球菌为指示菌,筛选获得1 株高产细菌素的菌株KLDS 1.0338,排除有机酸和过氧化氢的干扰以及蛋白酶敏感性实验,确定抑菌作用是由细菌素产生的。通过菌株形态学分析,生理生化及16S rDNA分析鉴定菌株KLDS 1.0338为干酪乳杆菌ATCC 334。进一步利用该菌株制作农家干酪,考察4 ℃贮藏期间干酪的变化,添加KLDS 1.0338干酪乳杆菌的实验组抑菌活性显著大于未添加KLDS 1.0338干酪乳杆菌组（P＜0.01）,实验组的pH值下降平缓,可以有效延长农家干酪的货架期,并保持产品原有的色泽、风味、质构等感官特性。     

Identification and first characterization of lactic acid bacteria isolated from the artisanal ovine cheese Pecorino del Poro

A total of 225 lactic acid bacteria, isolated from the artisanal raw ovine milk cheese Pecorino del Poro, were identified and given an initial characterization; the best 18 strains were tested for their antagonistic activity against autochthonous Escherichia coli . An intense antagonistic activity against E. coli growth, induced by strains of Lactobacillus paracasei ssp. paracasei or L. curvatus , was observed; the E. coli inhibition can be ascribed to the production of bacteriocin-like compounds. The lactobacilli seem able to improve the hygienic quality of other artisanal raw milk cheeses.     

The design of a three strain starter system for Cheddar cheese manufacture exploiting bacteriocin-induced starter lysis

A three strain starter system was developed for increasing the level of starter cell lysis during cheese manufacture and ripening. The composition of this starter combination includes a bacteriocin (lactococcin A, B and M) producer which causes the lysis of a second strain (sensitive to bacteriocin activity) during cheese manufacture, and a third strain resistant to bacteriocin activity. The latter strain plays an essential role in ensuring acid production during cheese manufacture. Cheeses manufactured at pilot-scale (450 L vats), with the three strain starter combination were assessed for levels of the intracellular enzyme, lactate dehydrogenase, released into the cheese matrix during ripening. Experimental cheeses, manufactured with the bacteriocin-producing adjunct, exhibited higher levels of free amino acids and greater release of intracellular LDH than control cheeses manufactured in its absence. Cheese was subject to sensory analysis which revealed that experimental cheese showed a decrease in bitterness over cheeses manufactured without the bacteriocin-producing adjunct. Thus, this three strain system offers manufacturers a reliable starter system exhibiting increased lysis with concomitant improvements in cheese flavour.     

Fate of Listeria monocytogenes in Gouda microcheese: No growth,and substantial inactivation after extended ripening times

This challenge study demonstrates that Listeria monocytogenes does not grow in Gouda cheese: during the first 8 weeks of ripening no growth was observed and between 8 and 52 weeks viable numbers declined significantly in a well-established Gouda microcheese system. Cheese milk was artificially contaminated just prior to addition of the starter culture. Three individual L. monocytogenes strains were used, including strains originating from cheese, a cheese plant environment and a reference strain. During curd formation, viable numbers of L. monocytogenes increased by 0.5 log cfu g⁻¹, resulting from entrapment in the curd. No growth was observed during the first 8 weeks of ripening. A significant decline in the viable numbers of L. monocytogenes was observed in Gouda cheese that was ripened for longer than 8 weeks. Two factors that could possibly control the fate of L. monocytogenes in Gouda cheese were lactic acid and water activity.     

Elevated enzyme release from lactococcal starter cultures on exposure to the lantibiotic lacticin 481, produced by Lactococcus lactis DPC5552

A Lactococcus lactis subsp. lactis strain (DPC5552), which causes the lysis of other lactococcal cultures, was isolated during a screening of raw milk samples for bacteriocin producers. Purification of the bacteriocin produced revealed that production of the lantibiotic, lacticin 481, was associated with the bacteriolytic capability of the strain. However, unlike bacteriocin-induced lysis observed with bacteriocins such as lacticin 3147 and lactococcins A, B, and M (where the target strain is killed), the DPC5552 supernatant gave rise to a situation whereby the target strain continued to grow (albeit at a lower rate) with simultaneous release of the intracellular enzymes lactate dehydrogenase (LDH) and post-proline dipeptidyl aminopeptidase (Pep X). In parallel experiments, 32 AU/ml of the inhibitory activity from L. lactis DPC5552 resulted in a 10- and 6-fold-higher LDH release after 5 h than that with 32 AU/ml of either lacticin 3147 or lactococcin A, B, and M. Laboratory-scale Cheddar cheese-making trials also demonstrated that lacticin 481-producing cultures induced the release of elevated levels of LDH from the starter L. lactis HP, without severely compromising its acid-producing capabilities. These results indicate that lacticin 481-producing strains may provide improved adjuncts for delivering lactococcal intracellular enzymes into the cheese matrix and, thus, improve cheese quality and flavor.     

An anti-listerial Lactococcus lactis strain isolated from Azorean Pico cheese produces lacticin 481

A bacteriocin produced by a strain of Lactococcus lactis (L3A21M1), isolated from an Azorean cheese, was purified, characterised and identified. This bacteriocin, of molecular mass 2900.23 Da, similar to that of lacticin 481, was heat stable, active across a wide pH range and exhibited a broad spectrum of activity. It revealed a bacteriostatic mode of action against Listeria monocytogenes both in culture media and in model cheeses. Also, the strain presented good auto-aggregation, as well as co-aggregation ability with L. monocytogenes. The strain cell surface was relatively hydrophobic and basic, presented low tolerance to acidic conditions, but good resistance to the gut environment. The strain was able to reduce the adhesion of L. monocytogenes to Caco-2 and HT-29 cells in competition, displacement, inhibition and invasion experiments. The Lc. lactis L3A21M1 strain therefore has significant anti-listerial activity and so may find application for both food protection and decreased risk of infection.