Lactococcal Strains Research Articles

Cloning and Expression of Listeriolysin O in Lactobacillus plantarum

Background: The protein listeriolysin O (LLO) encoded by hly gene, is one of the most important virulence factors of Listeria monocytogenes, responsible for phagosomal membrane disruption and bacterial escape to the cytoplasm, stimulation of CD8+ T cells and Th1 response. Recently pathobiotechnological vaccination using probiotic bacteria have been proposed. One of these strategies is expression of LLO in non-pathogenic bacteria such as lactic acid bacteria as delivery strains. Objective: In the current study, we aimed to clone hly gene in a Lactobacillus species via pNZ8110, an inducible expression vector which is specific for Lactococcus species. Materials and Methods: Hly gene was amplified by polymerase chain reaction (PCR) and inserted into pNZ8110 by restriction enzymes cutting and ligation method. After transformation and propagation in Escherichia coli MC1061 intermediate host, it was successfully electrotransformed into Lactobacillus plantarum. Results: Gel electrophoresis of colony PCR, extracted plasmids and restriction analysis along with sequencing confirmed the transformation. After induction with supernatant of nisin producer, strain Lactococcus lactis NZ9700, expression of LLO was confirmed by SDS-PAGE and western blot. Conclusion: Here, we employed a nonpathogenic probiotic strain, L. plantarum for the first time to express hly gene of L. monocytogenes in order to propose a new vaccine candidate.

Another Brick in the Wall: a Rhamnan Polysaccharide Trapped inside Peptidoglycan of Lactococcus lactis.

ABSTRACTPolysaccharides are ubiquitous components of the Gram-positive bacterial cell wall. In Lactococcus lactis, a polysaccharide pellicle (PSP) forms a layer at the cell surface. The PSP structure varies among lactococcal strains; in L. lactis MG1363, the PSP is composed of repeating hexasaccharide phosphate units. Here, we report the presence of an additional neutral polysaccharide in L. lactis MG1363 that is a rhamnan composed of α-l-Rha trisaccharide repeating units. This rhamnan is still present in mutants devoid of the PSP, indicating that its synthesis can occur independently of PSP synthesis. High-resolution magic-angle spinning nuclear magnetic resonance (HR-MAS NMR) analysis of whole bacterial cells identified a PSP at the surface of wild-type cells. In contrast, rhamnan was detected only at the surface of PSP-negative mutant cells, indicating that rhamnan is located underneath the surface-exposed PSP and is trapped inside peptidoglycan. The genetic determinants of rhamnan biosynthesis appear to be within the same genetic locus that encodes the PSP biosynthetic machinery, except the gene tagO encoding the initiating glycosyltransferase. We present a model of rhamnan biosynthesis based on an ABC transporter-dependent pathway. Conditional mutants producing reduced amounts of rhamnan exhibit strong morphological defects and impaired division, indicating that rhamnan is essential for normal growth and division. Finally, a mutation leading to reduced expression of lcpA, encoding a protein of the LytR-CpsA-Psr (LCP) family, was shown to severely affect cell wall structure. In lcpA mutant cells, in contrast to wild-type cells, rhamnan was detected by HR-MAS NMR, suggesting that LcpA participates in the attachment of rhamnan to peptidoglycan.

Phytoestrogen metabolism by lactic acid bacteria: Enterolignan production by Lactobacillus salivarius and Lactobacillus gasseri strains

Phytoestrogens are plant polyphenols similar to human estrogens. Isoflavones, ellagitanins, and lignans are metabolized by intestinal bacteria into equol, urolithins, and enterolignans, respectively, which are more bioavailable and bioactive, having beneficial effects on health. In this paper, we analysed the production of equol, 5-hydroxy-equol, enterodiol and enterolactone by 70 strains of Lactobacillus, Lactococcus and Enterococcus. Enterodiol and enterolactone production was found in Lactobacillus salivarius INIA P183, Lb. salivarius INIA P448 and Lactobacillus gasseri INIA P508, in levels around 46µM and 6µM respectively. However, we did not find neither equol nor 5-hydroxy-equol producing strains. Furthermore, analysis of the ellagic acid metabolism was extended to a total of 138 lactic acid bacteria strains, although none of them produced urolithins. This is the first time that simultaneous production of enterolactone and enterodiol is described in bacteria with probiotic potential, open their possible application in functional foods.

Discrimination of lactic acid bacteria Enterococcus and Lactococcus by infrared spectroscopy and multivariate techniques

Raw milk is often described as a major source of lactic acid bacteria for indigenous lactic starter. These indigenous starters contribute to the sensorial quality of cheese. Raw milk, rich in Lactoccocus lactis may therefore be very interesting for the cheese making. Currently, the most commonly used methods to differentiate lactic acid bacteria, and particularly the closely related phenotypes Lactococcus and Enterococcus, are based on DNA sequencing, but the cost and time required for these analytical methods hinder their use for rapid screening of raw material. The present study therefore proposes a simple alternative method to identify and discriminate against Lactococcus and Enterococcus, at the genus, but also at the species level, that is based on collecting near infrared spectra directly from bacterial colonies in Petri dishes. The infrared spectra of 280 strains of Lactococcus and Enterococcus cultured on solid media were collected by using a spectrometer with a wavelength range of 908 to 1684 nm and a remote probe. The best Classification And Regression Trees models for genus and species discrimination gave an excellent classification rate of 87% on an external validation set (30 strains). Loading line plots, with prominent bands at 900–960 and 1270–1390 nm, confirmed that the source of variation was due to changes in the polysaccharides.

Rapid detection of Lactococcuslactis isolates producing the lantibiotics nisin, lacticin 481 and lacticin 3147 using MALDI-TOF MS

The aim of the study was to evaluate the potential use of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) for fast and reliable detection of strains producing the lantibiotics nisin, lacticin 481 and lacticin 3147 in a large collection of lactococci. A total of one hundred lactococcal isolates from traditional ewe's and goat's raw milk cheeses were identified to the species level as Lactococcuslactis by MALDI-TOF MS based on comparison with lactococcal entries in the BioTyper database. Mass spectra in the range 2000–4000Da of the identified isolates were compared to reference spectra of three lactococcal strains producing lacticin 481 (IFPL 330), lacticin 3147 (IFPL 105) and nisin (IFPL 503). Only eight isolates had mass spectra with peaks that could be unequivocally identified as lacticin 481 (2900.47Da) or nisin (3330.31Da). None of the assayed isolates matched the mass spectra corresponding to the two-peptide lacticin 3147 (2847.97 and 3306.29Da). The results obtained by MALDI-TOF MS were genetically validated by amplification of the corresponding structural gene coding for lacticin 481, nisin and lacticin 3147. MALDI-TOF MS can be used as a fast and reliable technique to screen a large number of lactococcal isolates for the ability to produce the lantibiotics nisin, lacticin 481 and lacticin 3147.

Effects of rare sugar D-allulose on acid production and probiotic activities of dairy lactic acid bacteria

It has recently been reported that the rare sugar d-allulose has beneficial effects, including the suppression of postprandial blood glucose elevation in humans, and can be substituted for sucrose as a low-calorie food ingredient. To examine the applications of d-allulose in the dairy industry, we investigated the effects of d-allulose on the acid production of 8 strains of yogurt starter (Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus) and 4 strains of lactococci, including potential probiotic candidates derived from dairy products. Acid production by 2 L. delbrueckii ssp. bulgaricus yogurt starter strains in milk was suppressed by d-allulose, but this phenomenon was also observed in some strains with another sugar (xylose), a sugar alcohol (sorbitol), or both. In contrast, among the dairy probiotic candidates, Lactococcus lactis H61, which has beneficial effects for human skin when drunk as part of fermented milk, was the only strain that showed suppression of acid production in the presence of d-allulose. Strain H61 did not metabolize d-allulose. We did not observe suppression of acid production by strain H61 with the addition of xylose or sorbitol, and xylose and sorbitol were not metabolized by strain H61. The acid production of strain H61 after culture in a constituted medium (tryptone-yeast extract-glucose broth) was also suppressed with the addition of d-allulose, but growth efficiency and sugar fermentation style were not altered. Probiotic activities-such as the angiotensin-converting enzyme inhibitory activity of H61-fermented milk and the superoxide dismutase activity of H61 cells grown in tryptone-yeast extract-glucose broth-were not affected by d-allulose. d-Allulose may suppress acid production in certain lactic acid bacteria without altering their probiotic activity. It may be useful for developing new probiotic dairy products from probiotic strains such as Lactococcus lactis H61.

Influence of temperature and pH on the production of biosurfactant, bacteriocin and lactic acid by Lactococcus lactis CECT-4434

ABSTRACTOperational conditions such as temperature and pH are well known to influence the production of biosurfactants, bacteriocins, and lactic acid by lactic acid bacteria. The bacterial strain Lactococcus lactis subsp. lactis CECT-4434 was used to investigate the effects of temperature (30°C, 37°C and 40°C) and pH control on the production of these biomolecules, in order to establish whether they are able to preferentially address its metabolism towards one product or another. It was observed that the pH control within the range 5.0–5.3 favored the production of lactic acid, which was 38% higher than that obtained without control. The bacteriocin production was higher in the temperature range between 30°C and 37°C, evidenced by the formation of inhibition zones against Lactobacillus sakei and Staphylococcus aureus, 5–22% and 4–14% higher, respectively, compared with those achieved at 40°C. Finally, the best results for biosurfactant synthesis occurred at 37°C without pH control, with a surface tension reduction between 22.5 and 24.7 mN/m after 6–24 h of culture, respectively.

Efficient production of acetoin by fermentation using the newly isolated mutant strain Lactococcus lactis subsp. lactis CML B4

Abstract With the aim of applying biotechnology to produce acetoin, a chemical that can be used as an aroma and as a building block for other compounds, several putative mutants with reduced lactic acid synthesis were obtained from a wild-type homolactic strain of L. lactis subjected to chemical mutagenesis. Among these mutants, a strain was isolated, CML B4, that showed reduced lactate dehydrogenase (LDH) and increased NADH oxidase (NOX) activities. Shaken flask cultures of this mutant strain mainly produced acetoin, increasing the levels produced compared to the wild-type strain by 15-fold. A point mutation detected in the ldh gene encoding LDH was probably the genetic defect responsible for this phenotype. In pH-controlled aerobic batch fermentation, the CML B4 strain produced more than 40 gL −1 acetoin, which was increased by up to 59 gL −1 in fed-batch fermentations, with yields close to 88 and 74%, respectively, and productivities exceeding 2 gL −1 h −1 . These results indicate that this strain could be used industrially as a cell factory for the production of acetoin from bioresources.

Coproduction of colicin V and lactic acid bacteria bacteriocins in lactococci and enterococci strains of biotechnological interest.

The aim of this study was the coproduction in a single strain of the Gram-negative bacteriocin colicin V with other bacteriocins from lactic acid bacteria (LAB). Colicin V was expressed in Lactococcus and Enterococcus strains by replacing the colicin V leader peptide by the leader peptide and promoter of d-alanyl-d-alanine carboxypeptidase from Lactobacillus reuteri CECT925 in pNZ8048 (pNZ:LR-colV). The antimicrobial activity of colicin V against the indicator organism Escherichia coli DH5α in transformed strains was checked by agar diffusion assay and SDS-PAGE analysis. Lactococcus and Enterococcus transformed with pNZ:LR-colV were able to coproduce colicin V at high levels together with other LAB bacteriocins such as nisin A, nisin Z, lacticin 481 or enterocins A and B, obtaining broad-spectrum activity strains with large potential applications. The construction showed in this work could be used for the heterologous expression of other bacteriocins active against Gram-negative bacteria or wide-spectrum bacteriocins from LAB.

Отримання казеїнових фосфопептидів за дії протеолітичних систем лактококів

Протеїни молока, зокрема протеїни казеїнового комплексу, є попередниками біологічно активних пептидів. Ці пептиди утворюються в процесі розщеплення казеїнів травними протеазами у шлунково-кишковому тракті. Також вони можуть звільнятися під час технологічних процесів виробництва молочних продуктів за дії молокозгортальних препаратів та ензимів протеолітичних систем молочнокислих бактерій. Одними із найважливіших біологічно активних пептидів, які утворюються з казеїнів, є фосфопептиди. Вони позитивно впливають на засвоєння кальцію та інших двовалентних іонів металів. Для отримання фосфопептидів із казеїну використовують активні протеолітичні препарати тваринного, рослинного і мікробіологічного походження. Не з’ясованим залишається питання можливості утворення фосфопептидів за дії протеаз молочнокислих бактерій, зокрема лактококів, які поширені в молоці та молочних продуктах. Метою нашої роботи було встановити можливість утворення казеїнових фосфопептидів за дії ензимів протеїназа-позитивних протеолітично активних лактококів. Для досліджень була використана протеолітична система, яка дозволила збільшити активність протеаз лактококів. Були відібрані три штами протеїназа-позитивних протеолітично активних лактококів різних підвидів. Як субстрати були виділені загальний кислотний казеїн, нативний міцелярний казеїн, суміш фракцій αS1-CN і αS2-CN, β-CN. У всіх випадках вихід фосфопептидів не перевищував 3%. Такий вихід є занадто низьким для виробництва фосфопептидів, але може мати значення при їх утворенні у ферментованих молочних продуктах.

Isolation and Characterization of Bacteriocinogenic Enterococcal and Lactococcal Strains from South of Morocco Dairy Product

Isolation and Characterization of Bacteriocinogenic Enterococcal and Lactococcal Strains from South of Morocco Dairy Product

Anti-Staphylococcal Enterotoxinogenesis of Lactococcus lactis in Algerian Raw Milk Cheese.

Staphylococcus aureus is a potential pathogen contaminating raw milk and dairy products, where it is able to produce thermostable enterotoxins that can cause staphylococcal food poisoning. This study was undertaken to investigate the inhibitory activity of a Lactococcus lactis strain (isolated from milk) on S. aureus growth and staphylococcal enterotoxin A (SEA) production. In the presence of L. lactis, the number of the pathogen decreased significantly (p<0.05) after 6 h of incubation in a laboratory medium and milk (3 log CFU/mL reduction compared to pure cultures). SEA concentration was reduced by 79% in the co-cultures. S. aureus was unable to reach population levels permitting SEA production in the cheese inoculated with L. lactis during 32 days of storage. In contrast, during the same period, it attained 7 log CFU/g in the cheese manufactured without the lactococcal strain, a level which permitted SEA detection in the cheese extracts. However, this enterotoxin was never detected in the cheese harbouring L. lactis. These results demonstrate the anti-staphylococcal enterotoxinogenesis potential of the L. lactis strain and its usefulness in raw milk cheese biopreservation.

Technological Properties and Biogenic Amines Production by Bacteriocinogenic Lactococci and Enterococci Strains Isolated from Raw Goat's Milk

Technological properties and biogenic amine production were analyzed in 56 bacteriocinogenic lactococci and enterococci strains isolated from raw goat's milk. Fifteen lactococci strains were able to reduce milk pH to 5.3 or lower after 6 h, while enterococci strains were initially slow in producing acids. Lactococcus lactis subsp. lactis GLc06 and three strains of Enterococcus faecalis (GEn20, GEn22, and GEn23) presented high proteolytic activity. L. lactis subsp. lactis GLc06 and E. faecalis GEn22 also showed a high percentage of autolysis after only 4 h, reaching 71.11 and 97.67%, respectively, after 24 h. No strain was able to secrete exopolysaccharides, and L. lactis subsp. lactis GLc22 and 25 of the Enterococcus strains were able to produce diacetyl. L. lactis subsp. lactis GLc05 and 23 of the Enterococcus strains presented a high tolerance to NaCl at 10% (wt/vol). Regarding biogenic amine production, 12 strains (5 lactococci and 7 enterococci) were capable of forming tyramine and 4 strains (1 lactococcus and 3 enterococci) were capable of forming 2-phenylethylamine, but in very low amounts. GLc06 presented great acidifying, proteolytic, and autolytic activities, and GLc05 was capable of growing at high NaCl concentrations (10%, wt/vol), possessing medium autolytic and proteolytic activities. Some enterococci strains produced diacetyl and high autolytic and extracellular proteolytic activities and also presented resistance to high NaCl concentrations. The interesting technological properties presented by some bacteriocinogenic strains can justify their use by the dairy industry, with the aim of ensuring both safety due to bacteriocin production and technological transformations in fermented products.

Natural Microflora of Raw Cow Milk and their Enzymatic Spoilage Potential

Abstract The aim of this work was to identify the main microbiota in raw cow milk from dairy farm of Slovakia and to describe the selected microorganisms responsible for thermostable protease and lipase production which can affected the quality of dairy products. The main bacterial classes identifying by MALDI-TOF MS were Gammaproteobacteria (62 %), Actinobacteria (19 %) and Bacilli (12 %). The dominant microbial genus of raw cow milk was Pseudomonas. From milk bacteria, the strain Lactococcus lactis and from the family Enterobacteriaceae, namely Enterococcus faecalis, Hafnia alvei, Citrobacter braakii and Raoultella ornithinolytica were observed in raw milk. The spoilage of milk products is caused by thermostable enzymes with lipolytic and proteolytic activity. Qualitative proteolytic and lipolytic activities were performed on skin milk agar and olive oil, respectively. From 16 identified microorganisms, only 8 strains (P. fragii, P. gessardii, P. lundesis, H. alvei, C. braakii, R. ornithinolytica, Kocuria rhizophila and Candida inconspicua) showed protease activity. Quantitative protease and lipase activities were determined by casein and olive oil, respectively. The highest both activities were measured for the genus Pseudomonas. While lipases produced by all isolated microbial species lose enzymatic activity at 77 °C for 30 – 40 min, almost proteases showed comparable activities during whole pasteurization experiment at selected experimental conditions (70 °C, 40 min).

Lactococcus species for conventional Karish cheese conservation

Karish cheese is one of the most popular, oldest cheese varieties in Egypt and Arab countries. As a low fat cheese; it is a promising food in particularly for old people and whom suffering from obesity related diseases. The objective of this work was to isolate, identify and select Lactococcus lactic acid bacteria (LAB) from traditional Karish cheese to conserve and enhance the characteristics of conventional Karish cheese made of pasteurized milk to meet the new regulations for dairy production. Two Lactococcus strains were selected out of 11 Lactococcus LAB isolates from 15 Karish cheese samples, phenotypically and technologically characterized then identified using genotypic 16S rRNA approach. The two unique strains; L. lactis subsp cremoris (KM746) and the exopolysaccharide (EPS) producing strain L. lactis subsp lactis (KM721) were applied as single starter cultures in comparison with commercial starter culture used in Karish cheese making. Selected LAB showed decent growth throughout processing and storage at 4 °C for 10 days preventing growth of coliform and staphylococci. Chemical composition of tested cheese samples was similar to the conventional Karish cheese, whilst rheological and sensorial properties were enhanced which encourage recommending these strains as starter cultures conserving and enhancing this type of traditional cheese.

Genome Sequences of Eight Prophages Isolated from Lactococcus lactis Dairy Strains.

P335 group phages represent the most divergent phage group infecting dairy Lactococcus lactis strains and have significant implications for the dairy processing industry. Here, we report the complete genome sequences of eight lactococcal prophages chemically induced from industrial lactococcal strains that propagate lytically on one of two laboratory strains.

Antimicrobial Activities of Five Strains Of Lactococcus Isolated from Beef Against Indicator Organisms of Public Health Significance

Five strains of Lactococcus, including L. garviae K2, L. piscium SU4, L. lactis subsp. cremoris E22, L. plantarum L7 and L. lactis subsp. hordinae E91 were screened for production of antimicrobial agents. The strains were also analysed for antimicrobial activities against spoilage and pathogenic organisms, including Staphylococcus aureus, Salmonella Typhimurium, Escherichia coli, Yersinia enterocolitica, Pseudomonas aeruginosa, Klebsiella pneumoniae, Listeria monocytogenes, Bacillus cereus and Pseudomonas fluorescens. Result revealed that L. piscium SU4, L. plantarum L7 and L. lactis subsp. cremoris E22 had lactic acid production (g/107 colony forming units, CFU) of 7.23, 7.20 and 7.19. The value of 6.83 recorded as lactic acid produced by L. garviae K2 was significantly different from those obtained for others. The highest acetic acid production (3.55 g/107 CFU) was recorded for L. garviae K2 while L. piscium SU4 had the lowest (2.99 g/107 CFU). L. lactis subsp. hordinae E91 had diacetyl production of 71.99 (µg/107 CFU), which was higher than those recorded for other Lactococcus strains. Test for antimicrobial activities showed that Escherichia coli NCTC 86, Yersinia enterocolitica NCTC 10460, Pseudomonas aeruginosa NCIMB 10848, Bacillus cereus NCTC 21113 and Pseudomonas flourescens recorded higher susceptibilities to the antimicrobial action of the Lactococcus strains than others; zones of inhibition of 5 mm and above were recorded for the indicator organisms compared to lower values (

Characterization of prophages containing "evolved" Dit/Tal modules in the genome of Lactobacillus casei BL23.

Lactic acid bacteria (LAB) have many applications in food and industrial fermentations. Prophage induction and generation of new virulent phages is a risk for the dairy industry. We identified three complete prophages (PLE1, PLE2, and PLE3) in the genome of the well-studied probiotic strain Lactobacillus casei BL23. All of them have mosaic architectures with homologous sequences to Streptococcus, Lactococcus, Lactobacillus, and Listeria phages or strains. Using a combination of quantitative real-time PCR, genomics, and proteomics, we showed that PLE2 and PLE3 can be induced-but with different kinetics-in the presence of mitomycin C, although PLE1 remains as a prophage. A structural analysis of the distal tail (Dit) and tail associated lysin (Tal) baseplate proteins of these prophages and other L. casei/paracasei phages and prophages provides evidence that carbohydrate-binding modules (CBM) located within these "evolved" proteins may replace receptor binding proteins (RBPs) present in other well-studied LAB phages. The detailed study of prophage induction in this prototype strain in combination with characterization of the proteins involved in host recognition will facilitate the design of new strategies for avoiding phage propagation in the dairy industry.

Investigation of genes involved in nisin production in Enterococcus spp. strains isolated from raw goat milk.

Different strains of Lactococcus lactis are capable of producing the bacteriocin nisin. However, genetic transfer mechanisms allow the natural occurrence of genes involved in nisin production in members of other bacterial genera, such as Enterococcus spp. In a previous study, nisA was identified in eight enterococci capable of producing antimicrobial substances. The aim of this study was to verify the presence of genes involved in nisin production in Enterococcus spp. strains, as well as nisin expression. The nisA genes from eight Enterococcus spp. strains were sequenced and the translated amino acid sequences were compared to nisin amino-acid sequences previously described in databases. Although containing nisin structural and maturation related genes, the enterococci strains tested in the present study did not present the immunity related genes (nisFEG and nisI). The translated sequences of nisA showed some point mutations, identical to those presented by Lactococcus strains isolated from goat milk. All enterococci were inhibited by nisin, indicating the absence of immunity and thus that nisin cannot be expressed. This study demonstrated for the first time the natural occurrence of nisin structural genes in Enterococcus strains and highlights the importance of providing evidence of a link between the presence of bacteriocin genes and their expression.

Cultivation and characterization of symbiotic bacteria from the gut of Reticulitermes chinensis

The complex symbiotic relationship between wood-feeding termites and microorganisms inhabiting their intestinal tracts is a fascinating phenomenon in nature. To understand the physiological functions of symbiotic micro-organisms, bacteria were isolated from the gut homogenate of Reticulitermes chinensis with different media and culture conditions. Under aerobic conditions, 105 bacterial strains were isolated with 1/5 LB medium, 1/3 TSB medium and a modified basal mineral medium, MM-4. Most dominant isolates were bacteria in the genera Bacillus (27.6%) and Lactococcus (21.9%). Under anaerobic conditions, 60 bacterial strains were isolated with 1/5 LB medium, 1/3 TSB medium and a modified Peptone-Yeast medium. The predominant isolates were bacteria in the genus Enterobacter (41.7%) and Citrobacter (33.3%). Many of these bacterial isolates shared high sequence similarity (&gt;98%) in 16S rRNA genes to bacterial clones obtained from the same termite and the other wood-feeding termites or cockroaches. Several bacterial species such as Deinococcus and Gryllotalpicola were isolated from termite gut for the first time. Characterization of these isolates showed that (i) most of Enterobacteriaceae and Lactococcus strains were able to hydrolyze uric acid; and (ii) many of the Bacillus and Streptomyces strains presented endo-β-1,4-glucanase activity. The preliminary results of this work gave us hints of possible functions of symbiotic bacteria in nitrogen recycling and cellulose degradation in the gut of wood-feeding termites