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.     

Characterization of bacteriocins from two Lactococcus lactis subsp. lactis isolates

In this study, bacteriocins from two Lactococcus lactis subsp. lactis isolates from raw milk samples in Turkey designated OC1 and OC2, respectively, were characterized and identified. The activity spectra of the bacteriocins were determined by using different indicator bacteria including Listeria, Bacillus and Staphylococcus spp. Bacteriocins were tested for their sensitivity to different enzymes, heat treatments and pH values. Loss of bacteriocin activities after alpha-amylase treatment suggested that they form aggregates with carbohydrates. Molecular masses of the purified bacteriocins were determined by SDS-PAGE. PCR amplification was carried out with specific primers for the detection of their structural genes. As a result of these studies, the two bacteriocins were characterized as nisin and lacticin 481, respectively. Examination of plasmid contents of the isolates and the results of plasmid curing and conjugation experiments showed that in L. lactis subsp. lactis OC1 strain the 39.7-kb plasmid is responsible for nisin production, lactose fermentation and proteolytic activity, whereas the 16.0-kb plasmid is responsible for lacticin 481 production and lactose fermentation in L. lactis subsp. lactis OC2 strain.     

Characterization of microflora in homemade semi-hard white Zlatar cheese

The Zlatar cheese belongs to the group of traditionally homemade cheeses, which are produced from nonpasteurized cow's milk, without adding of any bacterial starter culture. Changes were followed in lactic acid bacteria population and chemical composition during the ripening period of cheese up to 60 days. Results showed that the percentage of lactic acid cocci was higher in raw milk and one day old cheese and their percentage was gradually decreasing, whereas the number of lactobacilli was increasing. After 30 days of cheese ripening the number of cocci increased again, reaching the number of lactobacilli. The results of API 50 CH system and rep-PCR analysis showed that Lactobacillus paracasei subsp. paracasei, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Enterococcuus faecium and Enterococcus faecalis were the main groups present during the ripening of Zlatar cheese. Results revealed that in older cheeses (45 and 60 days old) enterococci were the main group present. It was also demonstrated that 57 isolates showed antimicrobial activity. The number of bacteria showing antimicrobial activity slowly decreased during the ripening period and in samples of 60 days old cheese producers of antimicrobial activities were not detected.     

Potential of bacteriocinogenic Lactococcus lactis subsp. lactis inhabiting low pH vegetables to produce nisin variants

Antimicrobial behavior of lactic acid bacteria (LAB) has been explored since many years to assess their ability to produce bacteriocin, a natural preservative, to increase the shelf life of food. This study aims to characterize bacteriocin producing strains of lactic acid bacteria isolated from acidic to slightly acidic raw vegetables including tomato, bell pepper and green chili and to investigate their potential to inhibit food related bacteria. Among twenty nine LAB screened for antimicrobial activity, three exhibited antagonism against closely related bacterial isolates which was influenced by varying temperature and pH. They were identified up to strain level as Lactococcus lactis subsp. lactis TI-4, L. lactis subsp. lactis CE-2 and L. lactis subsp. lactis PI-2 based on 16S rRNA gene sequence. Their spectrum of inhibition was observed against food associated strains of Bacillus subtilis and Staphylococcus aureus. Moreover, L. lactis subsp. lactis PI-2 selected on the basis of higher antimicrobial activity was further evaluated for bacteriocin production which was detected as nisin A and nisin Z. These findings suggest the possible use of L. lactis strains of vegetable origin as protective cultures in slightly acidic as well as slightly alkaline food by the bio-preservative action of bacteriocins.     

Characterization of the lactic acid bacteria in ewe's milk and cheese from northwest Argentina

Indigenous lactic acid bacteria in ewe's milk and artisanal cheese were studied in four samples of fresh raw milk and four 1-month-old cheeses from the provinces of northwest Argentina. Mean growth counts on M17, MRS, and MSE agar media did not show significant differences (P < 0.05) in raw milk and cheeses. Isolates of lactic acid bacteria from milk were identified as Enterococcus (48%), lactococci (14%), leuconostocs (8%), and lactobacilli (30%). All lactococci were identified as Lactococcus lactis (subsp. lactis and subsp. cremoris). Lactobacilli were identified as Lactobacillus plantarum (92%) and Lactobacillus acidophilus (8%). Enterococci (59%) and lactobacilli (41%) were isolated from cheeses. L. plantarum (93%), L. acidophilus (5%), and Lactobacillus casei (2%) were most frequently isolated. L. lactis subsp. lactis biovar diacetylactis strains were considered as fast acid producers. L. lactis subsp. cremoris strains were slow acid producers. L. plantarum and L. casei strains identified from the cheeses showed slow acid production. The majority of the lactobacilli and Lactococcus lactis strains utilized citrate and produced diacetyl and acetoin in milk. Enzyme activities (API-ZYM tests) of lactococci were low, but activities of L. plantarum strains were considerably higher. The predominance of L. plantarum in artisanal cheese is probably important in the ripening of these cheeses due to their physiological and biochemical characteristics.     

Diversity of bacteriocins produced by lactic acid bacteria isolated from raw milk

Bacteriocin-producing lactic acid bacteria were isolated from 298 samples of raw ewes', goats’ or cows’ milk. Eighty-two bacteriocin producers were phenotypically and genotypically identified as L. lactis subsp. lactis (59 isolates), L. lactis subsp. cremoris (2 isolates), L. lactis subsp. lactis biovar diacetylactis (6 isolates), E. faecalis (7 isolates), E. faecium (1 isolate), L. paracasei subsp. paracasei (4 isolates), L. plantarum (1 isolate) and Leuconostoc spp. (2 isolates). By means of PCR-techniques, nisin was characterized in 39 of the 67 bacteriocin-producing lactococci and lacticin 481 in 23 isolates, some of which presented antilisterial activity. Enterocin AS-48 was produced by four enterococcal isolates. Four non-identified bacteriocins produced by 16 isolates showed a broad inhibitory spectrum. Nisin-producing lactic acid bacteria were the most abundant, but lacticin 481-producing lactococci and AS-48-producing enterococci were found at relatively high rates.     

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.     

Impedance measurement of growth of lactic acid bacteria in the presence of nisin and lysozyme

Effect of nisin (0.500 g/l), lysozyme (0.035 g/l) and the mix of nisin (0.500 g/l) and lysozyme (0.035 g/l) against two nisin-producing lactococci strains, three non-nisin-producing lactococci strains and four lactobacilli strains with antimicrobial activity was determined by the impedimetric method. None of the tested lactococci and lactobacilli strains were inhibited by lysozyme at concentration of 0.035 g/l. Only the nisin-producing lactococci strains were resistant to nisin at 0.500 g/l, the other lactococci and lactobacilli were inhibited by nisin at 0.500 g/l intensively. The mix of lysozyme and nisin displayed a comparable effect to the nisin addition against the used lactococci strains and was very variable depending on the strain of tested lactobacilli. The impedimetric method was shown to be convenient for screening the growth abilities of lactococci and lactobacilli in the presence of antimicrobial components.     

Lactic Microflora Present in Liqvan Ewes' Milk Cheese

This study aimed to isolate and characterize Lactic Acid Bacteria (LAB) in Liqvan Ewes' milk cheese. A total of 117 Lactic Acid Bacteria were isolated and identified phenotypically. They belonged to 4 genera and 17 species. The dominant LAB found in Liqvan cheese were from the genus Lactobacillus (75.21%) consisted of 70.08% facultatively heterofermentative and 5.12% obligately heterofermentative lactobacillus species. Other isolates were classified as Pediococci (5.12%), Enterococci (5.98%), and Leuconostocs (13.67%). Lb. paracasei subsp. paracasei was the predominant species accounted for 36.75%. Likewise, predominant species of each genus were Lb. paracasei subsp. paracasei, P. pentosaceus, E. faecalis, and Leu. lactis. The preponderance of isolates (86.32%) was referred to be as members of Non Starter Lactic Acid Bacteria (NSLAB).     

Microbiological characterization of artisanal Raschera PDO cheese: analysis of its indigenous lactic acid bacteria

The aim of this research was to study the bacterial populations involved in the production of artisanal Raschera PDO cheese (Italian Maritime Alps, northwest Italy) in order to collect preliminary knowledge on indigenous lactic acid bacteria (LAB). A total of 21 samples of Raschera PDO cheese, collected from six dairy farms located in the production area, were submitted to microbiological analysis. LAB were randomly isolated from M17 agar, MRS agar and KAA plates and identified by combining PCR 16S-23S rRNA gene spacer analysis, species-specific primers and 16S rRNA gene sequencing. Biodiversity of Lactococcus lactis subsp. lactis isolates was investigated by RAPD-PCR. LAB microflora showed the highest count values among all microbial groups targeted. They reached counts of 10(9) colony forming unit (cfu)/g in cheese samples after 3 days of salting and 15 days of ripening. Yeast population also showed considerable count values, while enterococci and coagulase-negative cocci (CNC) did not overcome 10(7)cfu/g. L. lactis subsp. lactis was the species most frequently isolated from Raschera PDO samples at all different production stages while in aged cheeses Lactobacillus paracasei was frequently isolated. RAPD-PCR highlighted that isolates of L. lactis subsp. lactis isolated from Raschera PDO were highly homogeneous.     

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.     

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.     

Proteolysis and formation of volatile compounds in cheese manufactured with a bacteriocin-producing adjunct culture

Hispánico cheese, a semi-hard Spanish variety, was manufactured from a mixture of pasteurized cows' and ewes' milks (4:1) using a commercial mesophilic LD-type starter comprising Lactococcus lactis subsp. cremoris, Lc. lactis subsp. lactis, Lc. lactis subsp. lactis var diacetylactis and Leuconostoc mesenteroides subsp. cremoris. Varying amounts (0-1.0 g/kg) of an Enterococcus faecalis INIA 4 culture in milk were added as a bacteriocin-producing adjunct. Differences in pH between cheeses manufactured with and without the bacteriocin producer did not exceed 0.11 pH units. Starter lactococci lost viability more rapidly in cheeses made with the bacteriocin producer, which reached counts of up to 6 x 10(7) cfu/g during ripening. Aminopeptidase activity in 1-d-old cheese made from milk inoculated with 1.0 g bacteriocin-producing culture/kg was twice that in control cheese. Degrees of overall proteolysis and levels of total free amino acids in 45-d-old cheese made with 1.0 g bacteriocin-producing culture/kg were 1.80-fold and 2.17-fold those in control cheese of the same age. Inoculating milk with 1.0 g/kg bacteriocin-producing culture reduced the level of hydrophobic peptides in the resultant cheese, increased the concentrations of 3-methyl-1-butanal, diacetyl and acetoin, and resulted in the highest scores for flavour quality and flavour intensity throughout ripening.     

Purification and characterization of a bacteriocin produced by Lactococcus lactis subsp. lactis H-559 isolated from kimchi

Lactic acid bacteria were isolated from kimchi and screened for bacteriocin production. Strain H-559, identified as Lactococcus lactis subsp. lactis, exhibited the strongest antibacterial activity among them and was active against pathogenic bacteria such as Listeria monocytogenes and Staphylococcus aureus as well as many lactic acid bacteria. The antimicrobial substance produced by L. lactis subsp. lactis H-559 was inactivated by alpha-chymotrypsin, and protease type IX and XIV and was confirmed to be a bacteriocin. The bacteriocin activity was stable from pH 2.0-11.0 and up to 10 min heating at 100 degrees C. The bacteriocin was sequentially purified by ammonium sulfate precipitation, ion-exchange chromatography, and reversed-phase high-performance liquid chromatography (HPLC). Its molecular weight was determined to be 3343.7 Da by MALDI-mass spectrometry. Isoleucine was detected as the first N-terminal amino acid residue but the remaining amino acid sequence could not be determined by the Edman degradation method. It was different from other bacteriocins in terms of pH stability, molecular weight, amino acid composition, and the partial amino acid sequences of peptides obtained by acid hydrolysis.     

Antibiotic Resistance in Lactic Acid Bacteria and Micrococcaceae/Staphylococcaceae Isolates from Artisanal Raw Milk Cheeses, and Potential Implications on Cheese Making

ABSTRACT:  Antibiotic susceptibility against 19 antimicrobial agents was evaluated in isolates of the genera  Lactococcus  (46 isolates),  Leuconostoc  (22),  Lactobacillus  (19),  Staphylococcus  (8),  Enterococcus  (7), and  Microccoccus/Kocuria  (5) obtained from the predominant microflora of nonrecent and recent types of artisanal raw cow's milk cheeses. Beta-lactams showed broad activity against all genera, although leuconostocs and lactobacilli were highly resistant to oxacillin (80% to 95.5%). Resistance to aminoglycosides was frequent for lactococci and enterococci (particularly for streptomycin), whereas lower rates of resistance were detected for lactobacilli and leuconostocs. Technologically interesting traits for the food industry were distributed among isolates that showed different degrees of resistance to common antibiotics. However, isolates showing resistance to less than 2 antibiotics were mainly those with properties of greatest technological interest (acidifying activity, proteolytic/lipolytic activities, or diacetyl production).     

Bioluminescence-based identification of nisin producers - A rapid and simple screening method for nisinogenic bacteria in food samples

We present a simple and rapid method for screening nisin producers that directly identifies nisinogenic bacteria by induction of bioluminescence within the Lactococcus lactis NZ9800lux biosensor strain (Immonen and Karp, 2007, Biosensors and Bioelectronics 22, 1982-7). An overlay of putative nisinogenic colonies with the biosensor strain gives identification results within 1h. Functionality and specificity of the method were verified by screening nisin producers among 144 raw milk colonies and a panel of 91 lactococcal strains. Studies performed on strains and colonies that did not induce bioluminescence but inhibited growth of the biosensor demonstrated that only nisinogenic bacteria can cause induction. Bacteria known to produce bacteriocins other than nisin failed to induce bioluminescence, further verifying the specificity of the assay. We discovered a non-inducing but inhibitory lactococcal strain harboring a modified nisin Z gene, and demonstrated that the source of the inhibitory action is not a non-inducing variant of nisin, but a bacteriocin of lower molecular weight. The concentration of nisin producers in a raw milk sample was 1.3×10(2)CFU/ml. We identified from raw milk a total of seven nisin Z producing L. lactis subsp. lactis colonies, which were shown by genetic fingerprinting to belong to three different groups. Among the panel of 91 lactococci, four strains were nisin A producers, and one strain harbored the modified nisin Z gene. The method presented here is robust, cost-effective and simple to perform, and avoids the pitfalls of traditional screening methods by directly specifying the identity of the inhibitory substance.     

Antagonism of Helicobacter pylori by bacteriocins of lactic acid bacteria

Antimicrobial activity of seven bacteriocins produced by lactic acid bacteria against Helicobacter pylori strains (ATCC 43504, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH [DSM] 4867, DSM 9691, and DSM 10242) was investigated in vitro using a broth microdilution assay. The bacteriocins chosen for the study were nisin A; lacticins A164, BH5, JW3, and NK24; pediocin PO2; and leucocin K. Antimicrobial activity of the bacteriocins varied among the H. pylori strains tested, of which strain ATCC 43504 was the most tolerant. Among the bacteriocins tested, lacticins A164 and BH5 produced by Lactococcus lactis subsp. lactis A164 and L. lactis BH5, respectively, showed the strongest antibacterial activity against H. pylori strains. MICs of the lacticins against H. pylori strains, when assessed by the critical dilution micromethod, ranged from 0.097 to 0.390 mg/liter (DSM strains) or from 12.5 to 25 mg/liter (ATCC 43504), supporting the strain-dependent sensitivity of the pathogen. Pediocin PO2 was less active than the lacticins against four strains of H. pylori, and leucocin K was the least active peptide, with no inhibition toward H. pylori ATCC 43504. Anti-Helicobacter activity of lacticin A164 was dependent on initial inoculum size as well as concentration of the bacteriocin added.     

Homofermentative lactic acid bacteria of a traditional cheese, Comlek peyniri from Cappadocia region

Comlek peyniri is a typical artisanal cheese in Central Anatolia. This type of cheese was made by using the indigenous lactic acid bacteria (LAB) flora of cow or ewes' milk. Majority of the samples were taken from fresh cheese because the aim was to isolate homofermentative LAB. Initially 661 microbial isolates were obtained from 17 cheese samples. Only 107 were found to be homofermentative LAB. These isolates were selected and identified by using both phenotypic and molecular methods. Phenotypic identification included curd formation from skim milk, catalase test, Gram staining and light microscopy, growth at different temperatures and salt concentrations, arginine hydrolysis, gas production from glucose, and carbohydrate fermentation. Molecular identification was based on the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the 16S rRNA gene-ITS (internally transcribed spacer) region. By combining the phenotypic and molecular identification results, isolates belonging to each of the following genera were determined at species or subspecies level: 54 Lactococcus lactis subsp. lactis, 21 Enterococcus faecium, 3 Ec. faecalis, 2 Ec. durans, 10 Ec. sp., 15 Lactobacillus paracasei subsp. paracasei, and 2 Lb. casei strains. Technological characterisation was also performed by culturing each of the strains in UHT skim milk, and by monitoring pH change and lactic acid production at certain time intervals through the 24 h incubation. Results of the technological characterisation indicated that 33% of the isolates (35 strains) were capable of lowering the pH of UHT milk below 5.3 after 6 h incubation at 30 degrees C. Thirty four of these strains were Lc. lactis subsp. lactis, and only one was an Ec. faecium strain.     

INHIBITION OF STAPHYLOCOCCUS AUREUS IN BUFFER, CULTURE MEDIA AND FOODS BY LACIDIN A, A BACTERIOCIN PRODUCED BY LACTOBACILLUS ACIDOPHILUS OSU133

Thirty seven strains of lactobacilli and leuconostocs were screened for bacteriocin production. A bacteriocin-like substance (designated lacidin A) was detected in culture filtrate of Lactobacillus acidophilus OSU133. Lacidin A inhibited Enterococcus faecalis, L. acidophilus, Lactobacillus delbrueckii subsp. lactis, and Staphylococcus aureus. The bacteriocinogenic nature of lacidin A was confirmed by eliminating the inhibitory effects of acid, hydrogen peroxide and phage and by loss of activity upon hydrolysis with proteolytic enzymes. The bacteriocin is relatively heat-stable and has considerable bactericidal action against S. aureus in phosphate buffer. Lacidin A also demonstrated bacteriostatic action against S. aureus in brain heart infusion and heat-treated milk and liquid whole egg.     

Streptococcus thermophilus 580 produces a bacteriocin potentially suitable for inhibition of Clostridium tyrobutyricum in hard cheese

A strain of Streptococcus thermophilus that inhibits Clostridium tyrobutyricum has been isolated from raw milk. The active compound produced disappears after a treatment with protease. However, unlike most bacteriocins, it is not thermoresistant, and the activity is completely lost after 1 h at 60 degrees C. Its inhibitory spectrum is limited to other thermophilic streptococci, Brochothrix, and sporulated gram-positive rods. So this bacteriocin could be different from those already described. This bacteriocin-producing strain could be used in thermophilic starter for hard cheese making because the bacteriocin is not active against thermophilic lactobacilli. It is produced in M17 medium during the decreasing temperature phase of the hard cheese-making process temperature cycle and is also produced in milk. Moreover, when Streptococcus thermophilus was cocultured with a Lactobacillus delbrueckii subsp. lactis starter strain, it seems to enhance the bacteriocin production. However the level of activity always decreases drastically during the stationary phase. But inhibition of Clostridium tyrobutyricum spores can be obtained in small-scale curds.