Incidence of nisin Z production in Lactococcus lactis subsp. lactis TFF 221 isolated from Thai fermented foods

Lactic acid bacteria isolated from various Thai fermented foods were screened for the presence of nisin gene by using PCR with primers specific to nisin A structural gene. Only one strain, Lactococcus lactis subsp. lactis TFF 221, isolated from kung jom, a traditional shrimp paste, was found to carry a nisin gene. The TFF 221 nisin had antimicrobial activity against not only closely related lactic acid bacteria but also some foodborne pathogens. It was heat stable and inactivated by alpha-chymotrypsin and proteinase K. Some characteristics of TFF 221 nisin were found to be very similar to those of nisin A produced by Lactococcus lactis subsp. lactis NCDO 2111. Both of them had the same antimicrobial spectrum and MICs against all indicator bacteria. However, when assayed with indicator organisms, in all cases the TFF 221 nisin produced larger zones of inhibition in agar diffusion assays than the nisin A did. Sequencing of the TFF 221 nisin gene showed that it was the natural nisin variant, nisin Z, as indicated by the substitution of asparagine residue instead of histidine at position 27. The nisin determinant in strain TFF 221 was found to be located on a conjugative transposon residing in the chromosome. The ability of the nisin produced by L. lactis subsp. lactis TFF 221 to inhibit a wide range of foodborne pathogens may be useful in improving the food safety of the fermented product, especially in the Thai environment, which suffers from perennial problems of poor food hygiene.     

Inhibition of Clostridium tyrobutyricum in Vidiago cheese by Lactococcus lactis ssp. lactis IPLA 729, a nisin Z producer

Lactococcus lactis ssp. lactis IPLA 729 is a nisin Z producer isolated from raw milk cheese able to grow and produce nisin Z in milk. The ability of this strain to inhibit the growth of Clostridium tyrobutyricum CECT 4011, a late blowing agent, in Vidiago cheese, a semi-hard farmhouse variety, manufactured in Asturias, Northern Spain, was investigated. For control purposes, cheeses were manufactured with the mesophilic mixed starter IPLA-001. In experimental cheeses, the nisin-producing strain L. lactis IPLA 729 was combined with this starter. Nisin Z activity reached a concentration of 1600 AU/ml in 1-day cheeses and this level was maintained until 15 days of ripening. Furthermore, to compare the inhibitory activity of the nisin-producing strain to nitrate, cheeses were also manufactured with a commercial starter culture and potassium nitrate as anti-blowing agent was added in accordance with Vidiago's cheesemakers. The control, experimental and commercial cheeses were contaminated with C. tyrobutyricum CECT 4011. The composition of the three different cheeses showed only slight differences with respect to total solids, protein and fat, although control and experimental cheeses showed a richer flavour-compound profile than commercial cheeses. The level of the spoilage strain C. tyrobutyricum CECT 4011 decreased from 1.2x10(6) to 1.3x10(3) cfu/g during ripening in presence of the nisin Z producer, while it increased to 1.99x10(9) cfu/g in control cheeses and to 3.5x10(7) cfu/g in commercial cheeses.     

Lactococcin MMT24, a novel two-peptide bacteriocin produced by Lactococcus lactis isolated from rigouta cheese

Lactococcin MMT24 is a novel bacteriocin produced by Lactococcus lactis MMT24, a strain isolated from a Tunisian traditional cheese. The bacteriocin shows a narrow antimicrobial activity against closely related lactic acid bacteria. Lactococcin MMT24 is heat resistant, remains active after incubation at pH 3 to 10, lyophilization, long-term storage at -20 degrees C and is sensitive to treatment with proteolytic enzymes. The mode of action of lactococcin MMT24 was identified as bactericidal. Purification of the active compound showed that lactococcin MMT24 consists of two distinct peptides, named pepalpha and pepbeta, whose complementary action is necessary for full antibacterial activity. Optimal antibacterial activity was obtained when the complementary peptides pepalpha and pepbetawere present in equal amounts. Mass spectrometry analysis showed masses of 3765.33 Da and 3255.26 Da for pepalpha and pepbeta, respectively. These molecular masses do not correspond to those of so far described bacteriocins. Addition of 50 nmol l(-1) of lactococcin MMT24 to cells of L. lactis ssp. cremoris ATCC11603 induced increase in the concentration of K+ in supernatant indicating a massive leakage of this ion from the cells. This release was most likely caused by pores formation by the pepalphaand pepbeta peptides in the target bacterial membrane.     

Optimisation of bacteriocin production of Lactococcus lactis subsp. lactis MA23, a strain isolated from Boza

Lactococcus lactis subsp. lactis MA23 produces a bacteriocin (6400 AU/mL) that inhibits the growth of many Gram‐positive bacteria but is not active against Gram‐negative bacteria. This bacteriocin inhibits growth of lactococcal strains that are producing nisin, lacticin or lactococcin suggesting it to be different from these bacteriocins. The nutritional requirements and optimal growth conditions for MA23 bacteriocin production were studied with fed‐batch fermentations. The optimal pH, carbon source and nitrogen source for bacteriocin production were pH 6.5, sucrose (0.5%) and yeast extract (1%), respectively.     

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.     

Phenotypic and genetic characterization of a selected set of Lactococcus lactis strains isolated from a starter-free farmhouse cheese

Eleven strains of lactococci isolated from a farmhouse starter-free cheese manufactured from raw cow's milk were analysed in detail for some technologically-related properties. Large phenotypic differences were encountered between the isolates, some of which could be of practical relevance. Several strains produced lactic acid at a rate and at a final concentration suitable for large-scale cheesemaking. The enzymatic capabilities assayed with the API ZYM system showed that all strains possess similar profiles with weak proteinase and moderate leucine-arylamidase and esterase-lipase activities. Interestingly, two related strains presented a strong β -galactosidase activity. Plasmid and chromosomal analyses indicated a high degree of diversity among wild strains and showed low homology with some well-known Lactococcus lactis strains. Under highly stringent conditions, only one plasmid from a single strain gave a clear positive hybridization signal with lactococcal-derived probes for the β -phospho- galactosidase and the proteinase genes. In general, wild strains produced more odorous compounds and in higher amounts than the reference strains.     

Preventing phage lysis of Lactococcus lactis in cheese production using a neutralizing heavy-chain antibody fragment from llama

Bacteriophage infection is still a persistent problem in large dairy processes despite extensive studies over the last decades. Consequently, new methods are constantly sought to prevent phage infection. In this paper, we show that phage neutralizing heavy-chain antibody fragments, obtained from Camelidae and produced at a large scale in the generally regarded as safe microorganism Saccharomyces cerevisiae, can effectively be used to impede phage induced lysis during a cheese process. The growth inhibition of the cheese starter culture by 10(5) pfu/ml cheese-milk of the small isometric-headed 936-type phage p2 was prevented by the addition of only 0.1 microg/ml (7 nM) of the neutralizing antibody fragment. The use of such antibody fragments in cheese manufacturing are a realistic and interesting option because of the small amount of antibody fragments that are needed. Moreover the antibodies are produced in a food grade microorganism and can easily be isolated from the fermentation liquid in a pure and DNA free form.     

Inhibition of a methicillin-resistant Staphylococcus aureus strain in Afuega'l Pitu cheese by the nisin Z-producing strain Lactococcus lactis subsp. lactis IPLA 729

Methicillin-resistant Staphylococcus aureus strains are a potential threat for food safety because foodborne illness caused by methicillin-resistant Staphylococcus aureus has been reported even though these strains were only associated with nosocomial infections until recently. This article focuses on the inhibitory effect of the nisin Z-producing strain Lactococcus lactis subsp. lactis IPLA 729 on the growth of Staphylococcus aureus CECT 4013, a methicillin-resistant strain. S. aureus was inhibited by the presence of the nisin producer IPLA 729 in buffered Trypticase soy broth, milk, and Afuega'l Pitu cheese, an acid-coagulated cheese manufactured in Asturias, Northern Spain. A reduction of 3.66 log units was observed in Trypticase soy broth at the end of the incubation period. In milk, viable counts of S. aureus were undetectable or were reduced by 2.16 log units in 24 h depending on the initial inoculum (1.8 x 10(4) and 7.2 x 10(6) CFU/ml). The staphylococcal strain was also undetected in test cheeses in which the nisin Z producer was present whereas 2 log units were detected in control cheeses at the end of ripening.     

Effects of mixed starter composition on nisin Z production by lactococcus lactis subsp. lactis biovar. diacetylactis UL 719 during production and ripening of Gouda cheese

A starter culture system that produced both acid and nisin at acceptable rates in milk for manufacture of Gouda cheese was developed using nisin Z-producing L. lactis subsp. lactis biovar. diacetylactis UL 719 (UL 719) and a commercial Flora Danica (FD) starter culture. Different compositions of mixed cultures (0, 0.2, 0.4, 0.6 or 0.8% UL 719 with 1.4% FD) were tested for acidification and nisin Z production in milk after 12 h incubation at 30 degrees C. The 0.6/1.4% combination, selected as the optimal mixture of starter cultures, acidified milk to a suitable pH and produced nisin Z at a high concentration of 512 IU/ml. With this optimal combination, FD numbers of citrate-fermenting and non-fermenting bacteria did not change compared with the control (1.4% FD). However, with 0.8% of L. lactis strain UL 719 and 1.4% of the FD starter culture, the numbers of citrate-fermenting and non-fermenting bacteria in fermented milk decreased compared with those obtained when milk was inoculated with 0.2, 0.4 or 0.6% of UL 719 added to 1.4% FD or control cultures (1.4% FD). Mixed starter culture ratios 0.6/1.4%, 0.4/1.4% and 0.5/1.4% (UL 719/FD) were used to manufacture nisin Z containing Gouda cheese which was ripened up to 45 weeks. The composition of control cheeses made with 1.4% FD, and nisin Z-containing Gouda cheeses were similar with respect to percent moisture, fat, salt and protein. During the ripening period, the cell counts observed were approximately two logs higher in cheese made with the 0.6/1.4% mixed starter culture than in control cheese. In experimental cheese produced with 0.6/1.4% (UL 719/FD) mixed starter culture, nisin activity increased from 256 IU/g at the end of manufacture to a maximum of 512 IU/g after 6 weeks of ripening; the levels then decreased to 128 and 32 IU/g after 27 and 45 weeks of ripening, respectively. In contrast, nisin Z was not detected in experimental cheeses made with 0.4/1.4% or 0.5/1.4% (UL 719/FD) mixed starters. Using an affinity purified anti-nisin polyclonal antibody, anti-rabbit gold-conjugate and transmission electron microscopy, nisin Z was found to be localized in the cheese matrix, in fat globules, in the casein phase and concentrated at the fat-casein interface. After 27 weeks of ripening, nisin Z was detected preferentially in the fat globules of the experimental cheese.     

Screening for natural defence mechanisms of Lactococcus lactis strains isolated from traditional starter cultures

Three different bacterial defence mechanisms were identified in the seventeen Lactococcus lactis isolates from starter cultures in three Slovenian dairy plants. Isolates MB18, KR7, PT4, PT13 and PT19 inhibited phage adsorption by means of exopolysaccharides production. The most extensive polysaccharides production was detected in PT19 isolate, which was susceptible only to phage ΦPT19. Eight isolates exhibited nuclease activity, and seven of them were susceptible up to four phages out of thirteen from our collection. Eight isolates possessed the abiB gene, fourteen isolates abiH, two isolates abiJ and one isolate abiQ. Isolates PT27 and PT28 possessed AbiB, AbiH and AbiJ mechanisms as well as inhibition of phage adsorption. Isolate MB18, which was susceptible to one phage only, possessed the abiQ gene, nuclease activity and ability to prevent adsorption of most phages. Isolates PT67 and PT70, possessing only AbiH mechanism, were susceptible to only two phages.     

Inhibition of toxicogenic Bacillus licheniformis 553/1 in Nigerian Wara soft cheese by nisin-producing Lactococcus lactis LAC309

Wara soft cheese is a traditionally produced cheese in Nigeria. The production of this cheese includes a heating step for killing vegetative cells. Therefore, mainly spore-forming bacteria surviving the process may function as the first spoilers resulting in lowered shelf-life and safety of the product. In this study, we investigated if the addition of a nisin-producing Lactococcus lactis LAC309 starter after the high temperature treatment could result in nisin production and inhibition of the toxicogenic Bacillus licheniformis 553/1 strain spiked into the cheese. The results showed that L. lactis LAC309 could produce nisin in Wara cheese and that the strain inhibited (3 log reduction) B. licheniformis 553/1 in Wara cheese. Food technologists developing industrialised Wara cheese should therefore consider including a nisin-producing starter strain in the cheese-making process.     

Fate of enterotoxigenic Staphylococcus aureus in the presence of nisin-producing Lactococcus lactis strain during manufacture of Jben,a Moroccan traditional fresh cheese

The inhibitory effect of nisin-producing Lactococcus lactis subsp. lactis UL730 on the growth of enterotoxigenic Staphylococcus aureus J10 during manufacture of Jben, a Moroccan traditional fresh cheese prepared from recombined milk, was investigated.With an inoculum level of 10³ cfu mL⁻¹, S. aureus was absent in Jben four days after inoculation when the nisin-producing lactococcus was used as lactic starter. In contrast, it survived after that period, when the starter was non-nisin-producing. No staphylococcal thermonuclease was detected in all Jben samples made from milk inoculated with S. aureus at the level of 10³ cfu mL⁻¹.With a higher inoculum of 10⁵ cfu mL⁻¹, S. aureus was still present in Jben after manufacture and persisted during the storage of the product for 3 days in the laboratory, even when the starter used was nisin-producing. Staphylococcal thermonuclease and type C enterotoxin were detected in all Jben samples made from milk inoculated with 10⁵ cfu mL⁻¹. Thermonuclease and enterotoxin were already produced in the coagulum, at 24 h after milk inoculation with S. aureus.     

Genotypic and phenotypic diversity of Lactococcus lactis isolates from Batzos, a Greek PDO raw goat milk cheese

The genotypic and phenotypic variability of 40 Lactococcus lactis isolates obtained from three cheese-making trials of Batzos cheese made one in each, winter, spring and summer was investigated. RAPD-PCR, plasmid profiling and PFGE were used to study the genetic variability and distinguish closely related isolates. Results showed a high degree of heterogeneity among strains. According to PFGE data, all strains except one were clustered together (at a similarity level of approximately 50%) with the L. lactis subsp. lactis reference strain and eleven groups of isolates consisting of 2-8 strains each were distinguished. Plasmid profiling results revealed that there were eight isolates lacking plasmids and nine having unique plasmids. Twenty-three isolates were allocated into six groups. There was an interesting similarity between the plasmid profiling groups and those formed according to PFGE. Clustering of strains according to RAPD-PCR was in agreement with results obtained by both plasmid profiling and PFGE for the majority of the strains. In addition, results obtained by molecular methods indicate a grouping of most of the strains according to the season of cheese production. All strains inhibited the growth of Escherichia coli O157:H7. Their ability to affect the growth of Yersinia enterocolitica, Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes and Enterococcus faecalis was strain dependent. In 42.5% of the isolates high acidifying ability in milk after 24 h was recorded and these were isolates, mainly, from fresh cheese. The 75% of the isolates from winter cheese exhibited higher Lys- than Leu-aminopeptidase activity while the approximately 67% of the isolates from summer cheese showed higher Leu- than Lys-aminopeptidase activity. Their caseinolytic activity after growth in milk for 24 h was significant with preference for alpha(s)-casein degradation. The majority (90%) of the strains formed methanethiol from methionine and this ability was strain dependent. These results suggest that among the wild lactococcal population from Batzos cheese there are interesting strains appropriate to be used as starters for the dairy industry.     

Isolation of nisin-producing Lactococcus lactis WNC 20 strain from nham,a traditional Thai fermented sausage

A total of 14,020 lactic acid bacteria (LAB) were isolated from nham and screened for bacteriocin production. One Lactococcus lactis strain WNC 20 produced a bacteriocin that not only inhibited closely related LAB, but also some food-borne pathogens including Listeria monocytogenes, Clostridium perfringens, Bacillus cereus and Staphylococcus aureus. Biochemical studies revealed that the bacteriocin was heat-stable even at autoclaving temperature (121 degrees C for 15 min) and was active over a wide pH range (2-10). The bacteriocin was inactivated by alpha-chymotrypsin and proteinase K but not other proteases. The antimicrobial spectrum and some characteristics of this bacteriocin were nearly identical to that of nisin. The gene encoding this bacteriocin was amplified by polymerase chain reaction (PCR) with nisin gene-specific primer. Sequencing of this gene showed identical sequences to nisin Z as indicated by the substitution of asparagine residue instead of histidine at position 27. The ability of the bacteriocin produced by Lc. lactis WNC 20 may be useful in improving the food safety of the fermented product.     

Selection of starters and a starter-mediated novel procedure for production of wara, a West African soft cheese

Summary Wara serves as an important protein source for people in West Africa. The ten most frequently isolated lactic acid bacteria from abomasum-soured milk samples were screened singly or in combinations as starter organisms for the production of wara . The screening was based on the bacteria's ability to produce lactic acid, diacetyl, β-galactosidase and an average weighted firm curd at 30 °C (6 h, 72 h). With lactic acid concentration of 10.0 g l⁻¹, β-galactosidase of 25.2 U mL⁻¹ and a firm curd weight of 16.0 g l⁻¹ milk at 30 °C, pH 5.1 after 6 h, a single-organism starter culture of Lactococcus lactis was chosen as the best suited. Compared with the commercial market samples, wara produced by this starter culture showed a 54% increase in percentage protein, 17.9% increase in ash, 23.6% increase in iron and a 150% increase in vitamin A content. Statistical analysis of sensory evaluation results showed a preference for the market sample only in terms of appearance and texture, while the starter-produced wara matched the market sample in terms of flavour and palatability. Treatment with 3% brine reduced microbial counts although dipping in 5% NaCl-in-whey gave better results in terms of the total viable count of mesophiles, psychrotrophs and coliforms compared with the untreated sample.     

Characterization of four new Lactococcus lactis bacteriophages isolated from dahi whey

Four lytic phages of Lactococcus lactis ssp. diacetylactis isolated from indigenous dahi whey were examined for their stability, growth characteristics and morphology. All these phages were partially inactivated by CHCl₃ , remained stable at 40°C and were partially inactivated at pH 3. There was a marked difference among these phages with respect to latent period, rise period and burst size. All phages belonged to Bradley's group B.     

Phenotypic,genetic and technological characterization of Lactococcus garvieae strains isolated from a raw milk cheese

A series of Lactococcus garvieae strains isolated as the majority population of a Spanish traditional, starter-free cheese made from raw milk were phenotypically and genotypically characterised to address their biochemical potential, safety requirements, and technological properties. As expected, all L. garvieae cheese strains fermented lactose but grew slowly in UHT-treated milk. Enzymatic activities of L. garvieae were similar to those of Lactococcus lactis, although higher esterase and lipase activities were recorded for L. garvieae strains. Profiles of the volatile compounds produced from milk by L. garvieae and L. lactis strains were also comparable. L. garvieae strains did not produce haemolysin, gelatinase and the biogenic amines tyramine and histamine. Five L. garvieae stains showed tetracycline resistance encoded by a tet(M) gene. The use of L. garvieae strains as starter or adjunct cultures might be recommended for certain cheese types, provided that the safety of the strains has been demonstrated.