Lactococcus Lactis Strains Research Articles

Complete Genome Sequence of Lactococcus lactis Strain AI06, an Endophyte of the Amazonian Açaí Palm.

We report the genome, in a single chromosome, of Lactococcus lactis strain AI06, isolated from the mesocarp of the açaí fruit (Euterpe oleracea) in eastern Amazonia, Brazil. This strain is an endophyte of the açaí palm and also a component of the microbiota of the edible food product.

Texture Properties of Miniature Chihuahua‐Type Cheese Manufactured with Different Strains of Lactococcus Lactis Isolated from Plants and Raw Milk Cheese

AbstractThere is a growing interest to explore new wild strains of Lactococcus lactis because of their ability to generate certain flavor compounds due to differences in their metabolic activities. However, it is believed that plant‐derived strains of lactococci have a limited ability to ferment lactose and hydrolyze the milk proteins. It is therefore hypothesized that cheese manufactured with plant‐derived strains of L. lactis might develop a harder texture than cheese manufactured with commercial strains or strains isolated from raw milk products. The aim of this work was to describe the texture properties of cheese manufactured with different strains of L. lactis isolated from raw milk cheese and vegetables. The miniature cheese manufactured with diverse strains of L. lactis showed a wide range of textures. The source of isolation of L. lactis did not predispose the proteolytic activities of the strains or the cheese texture properties. The plant‐derived strains did not produce the hardest cheese.Practical ApplicationsNowadays, cheese makers from Chihuahua are encouraged to produce cheese using pasteurized milk. However, these pasteurized milk cheese have shown differences in flavor and texture in comparison with artisanal raw milk cheese. One of the most promising options to emulate the flavor and texture of artisanal cheese is the utilization of native lactic acid bacteria isolated from raw milk cheese and vegetables. Some of these bacteria are able to produce flavor similar to that of artisanal cheese but their effect on cheese texture is still unclear. From the results of this work, it was observed that some of these wild strains of lactic acid bacteria are able to produce cheese with texture similar to that observed in artisanal Chihuahua cheese. Therefore, these bacteria could be used as a starter culture in the manufacture of Chihuahua cheese with pasteurized milk.

Oral immunization with recombinant hepatitis E virus antigen displayed on the Lactococcus lactis surface enhances ORF2-specific mucosal and systemic immune responses in mice.

Hepatitis E virus (HEV) as a recognized zoonotic pathogen has posed global burden on public health, which is exacerbated by lack of efficient vaccine. In this study, we constructed a recombinant (inaQ-ORF2 gene fusion) Lactococcus lactis (L. lactis) strain NZ3900 that expresses and displays the hepatitis E virus antigen ORF2 utilizing an ice uncleation protein-based anchor system. After oral vaccination of BALB/c mice, significantly higher levels of ORF2-specific mucosal IgA and serum IgG were detected and cellular immunity was also induced. These findings further support that L. lactis-based HEV antigen vaccines could be used for human and animal protection against infection.

Classification of Lactococcus lactis cell envelope proteinase based on gene sequencing, peptides formed after hydrolysis of milk, and computer modeling

Lactococcus lactis strains depend on a proteolytic system for growth in milk to release essential AA from casein. The cleavage specificities of the cell envelope proteinase (CEP) can vary between strains and environments and whether the enzyme is released or bound to the cell wall. Thirty-eight Lc. lactis strains were grouped according to their CEP AA sequences and according to identified peptides after hydrolysis of milk. Finally, AA positions in the substrate binding region were suggested by the use of a new CEP template based on Streptococcus C5a CEP. Aligning the CEP AA sequences of 38 strains of Lc. lactis showed that 21 strains, which were previously classified as group d, could be subdivided into 3 groups. Independently, similar subgroupings were found based on comparison of the Lc. lactis CEP AA sequences and based on normalized quantity of identified peptides released from αS1-casein and β-casein. A model structure of Lc. lactis CEP based on the crystal structure of Streptococcus C5a CEP was used to investigate the AA positions in the substrate-binding region. New AA positions were suggested, which could be relevant for the cleavage specificity of CEP; however, these could only explain 2 out of 3 found subgroups. The third subgroup could be explained by 1 to 5 AA positions located opposite the substrate binding region.

Isolation of Enterococcus faecium NM113, Enterococcus faecium NM213 and Lactobacillus casei NM512 as novel probiotics with immunomodulatory properties.

Probiotics, defined as living bacteria that are beneficial for human health, mainly function through their immunomodulatory abilities. Hence, these microorganisms have proven successful for treating diseases resulting from immune deregulation. The aim of this study was to find novel candidates to improve on and complement current probiotic treatment strategies. Of 60 lactic acid bacterial strains that were isolated from fecal samples of healthy, full-term, breast-fed infants, three were chosen because of their ability to activate human immune cells. These candidates were then tested with regard to immunomodulatory properties, antimicrobial effects on pathogens, required pharmacological properties and their safety profiles. To identify the immunomodulatory structures of the selected isolates, activation of specific innate immune receptors was studied. The three candidates for probiotic treatment were assigned Enterococcus faecium NM113, Enterococcus faecium NM213 and Lactobacillus casei NM512. Compared with the established allergy-protective strain Lactococcus lactis G121, these isolates induced release of similar amounts of IL-12, a potent inducer of T helper 1 cells. In addition, all three neonatal isolates had antimicrobial activity against pathogens. Analysis of pharmacological suitability showed high tolerance of low pH, bile salts and pancreatic enzymes. In terms of safe application in humans, the isolates were sensitive to three antibiotics (chloramphenicol, tetracycline and erythromycin). In addition, the Enterococcus isolates were free from the four major virulence genes (cylA, agg, efaAfs and ccf). Moreover, the isolates strongly activated Toll-like receptor 2, which suggests lipopeptides as their active immunomodulatory structure. Thus, three novel bacterial strains with great potential as probiotic candidates and promising immunomodulatory properties have here been identified and characterized.

Correlation between fibronectin binding protein A expression level at the surface of recombinant lactococcus lactis and plasmid transfer in vitro and in vivo.

BackgroundFibronectin Binding Protein A (FnBPA) is an invasin from Staphylococcus aureus that allows this pathogen to internalize into eukaryote cells. It was previously demonstrated that recombinant Lactococcus lactis expressing FnBPA were invasive and able to transfer a plasmid to eukaryotic cells in vitro and in vivo. In this study, the invasivity of recombinant strains of Lactococcus lactis that express FnBPA under the control of its constitutive promoter or driven by the strong nisin inducible expression system (NICE) were studied.ResultsIt was demonstrated that the nisA promoter allows an increase of FnBPA expression on the surface of Lactococcus lactis surface, as shown by flow cytometry, which subsequently enhanced internalization and plasmid transfer properties in vitro in Caco2 cells and Bone Marrow Dendritic Cells. In vivo, the use of nisA promoter increase the plasmid transfer in cells of both the small and large intestine of mice.ConclusionFnBPA expression at the surface of recombinant L. lactis is positively correlated to internalization and DNA transfer properties. The recombinant strains of L. lactis that expresses FnBPA under the control of the nisin inducible expression system could thus be considered as an improved tool in the field of DNA transfer.

Inhibitory Effect of Isolated Lactic Acid Bacteria from Scomberomorus commerson Intestines and their Bacteriocin on Listeria innocua

ABSTRACT- Lactic acid bacteria are commonly found in the gastrointestinal tract of various endothermic animals and humans, in milk, dairy and seafood products and on some plant surfaces. The aim of this study was to investigate the inhibitory effect of isolated lactic acid bacteria from intestines of narrow-barred Spanish mackerel (Scomberomorus commerson) on Listeria innocua growth. Five strains of lactic acid bacteria (LAB) were isolated. Lactobacillus buchneri, Lactococcus lactis, Lactobacillus acidophilus, Lactobacillus fermentum and Sterptococcus salivarius strains were presumptively identified by biochemical and physiological tests. Anti-listerial activities of these isolates and their cell free supernatants (CFS) were evaluated using agar spot tests, agar well diffusion assays and BHI broth. The results revealed that all cultures and their cell free supernatants were able to inhibit L. innocua growth. Furthermore, the study showed that wastes of this kind of warm water sea fish, have antagonistic activities against L. innocua and can be potentially used as a free source of LAB.

Secretion of biologically active human interleukin 22 (IL-22) by Lactococcus lactis.

Interleukin-22 (IL-22) participates in the modulation of innate immunity and inflammation. This cytokine has important therapeutic potential, such as with ulcerative colitis, liver and lung injury, and infection, in different animal models. We generated a Lactococcus lactis strain that secretes human IL-22 under the regulation of the nisin-inducible promoter. Identification and secretion of this cytokine was demonstrated using western blots of culture supernatants from IL-22-expressing bacteria. The recombinant IL-22 protein produced by L. lactis was biologically active as determined by its ability to induce IL-10 secretion when co-cultured with a colon epithelial cell line in vitro. We consider this novel strain a promising live vaccine for various therapeutic applications.

Efficient production of secreted staphylococcal antigens in a non-lysing and proteolytically reduced Lactococcus lactis strain.

Cell surface-exposed and secreted proteins are attractive targets for vaccination against pathogenic gram-positive bacteria. To obtain sufficient amounts of such antigens, efficient protein production platforms are needed. In this study, a pipeline for the production and purification of surface-exposed and secreted antigens of the gram-positive bacterial pathogen Staphylococcus aureus is presented. Cytoplasmic or extracellular production of S. aureus antigens was achieved using the Lactococcus lactis strain PA1001, which lacks the major extracellular protease HtrA and the autolysin AcmA to minimize proteolysis and cell lysis, respectively. For most tested S. aureus antigens, secretory production directed by the signal peptide of the major secreted protein Usp45 of L. lactis resulted in higher yields than intracellular production without a signal peptide. Additionally, secretory production of His-tagged antigens allowed their facile one-step purification from the growth medium by metal affinity chromatography. For three of the purified antigens, biological activity was confirmed through enzyme activity assays. We, furthermore, show that the present pipeline can be used to produce staphylococcal antigens with an N-terminal AVI-tag for site-specific labeling with biotin or a C-terminal cell wall-binding domain for cell surface display. We conclude that our L. lactis-based pipeline allows the efficient production of S. aureus antigens and their subsequent purification in one step.

The C-terminus of nisin is important for the ABC transporter NisFEG to confer immunity in Lactococcus lactis.

The lantibiotic nisin is a small 3.4 kDa antimicrobial peptide, which acts against Gram-positive bacteria in the nmol/L range. Nisin is produced and secreted by several Lactococcus lactis strains to ensure advantages against other bacteria in their habitat. Nisin contains five specific lanthionine rings of which the first two are important for Lipid II binding and the last two are crucial for the pore formation in the membrane. To gain immunity against nisin, the producing strain is expressing an ABC transporter called NisFEG, which expels nisin from the membrane. As a result six to eightfold more nisin is needed to affect the cells. The hydrolysis of ATP by NisFEG is required for this immunity as shown by a mutant, where the ATP hydrolysis is disrupted (NisFH181AEG). Furthermore, NisFEG recognizes the C-terminus of nisin, since deletion of the last six amino acids as well as of the last ring lowered the fold of immunity displayed by NisFEG.

Lactococcus lactis carrying the pValac DNA expression vector coding for IL-10 reduces inflammation in a murine model of experimental colitis.

BackgroundInflammatory bowel diseases (IBD) are intestinal disorders characterized by inflammation in the gastrointestinal tract. Interleukin-10 is one of the most important anti-inflammatory cytokines involved in the intestinal immune system and because of its role in downregulating inflammatory cascades, its potential for IBD therapy is under study. We previously presented the development of an invasive strain of Lactococcus lactis (L. lactis) producing Fibronectin Binding Protein A (FnBPA) which was capable of delivering, directly to host cells, a eukaryotic DNA expression vector coding for IL-10 of Mus musculus (pValac:il-10) and diminish inflammation in a trinitrobenzene sulfonic acid (TNBS)-induced mouse model of intestinal inflammation. As a new therapeutic strategy against IBD, the aim of this work was to evaluate the therapeutic effect of two L. lactis strains (the same invasive strain evaluated previously and the wild-type strain) carrying the therapeutic pValac:il-10 plasmid in the prevention of inflammation in a dextran sodium sulphate (DSS)-induced mouse model.ResultsResults obtained showed that not only delivery of the pValac:il-10 plasmid by the invasive strain L. lactis MG1363 FnBPA+, but also by the wild-type strain L. lactis MG1363, was effective at diminishing intestinal inflammation (lower inflammation scores and higher IL-10 levels in the intestinal tissues, accompanied by decrease of IL-6) in the DSS-induced IBD mouse model.ConclusionsAdministration of both L. lactis strains carrying the pValac:il-10 plasmid was effective at diminishing inflammation in this murine model of experimental colitis, showing their potential for therapeutic intervention of IBD.

Cell wall-anchored nuclease of Streptococcus sanguinis contributes to escape from neutrophil extracellular trap-mediated bacteriocidal activity.

Streptococcus sanguinis, a member of the commensal mitis group of streptococci, is a primary colonizer of the tooth surface, and has been implicated in infectious complications including bacteremia and infective endocarditis. During disease progression, S. sanguinis may utilize various cell surface molecules to evade the host immune system to survive in blood. In the present study, we discovered a novel cell surface nuclease with a cell-wall anchor domain, termed SWAN (streptococcal wall-anchored nuclease), and investigated its contribution to bacterial resistance against the bacteriocidal activity of neutrophil extracellular traps (NETs). Recombinant SWAN protein (rSWAN) digested multiple forms of DNA including NET DNA and human RNA, which required both Mg2+ and Ca2+ for optimum activity. Furthermore, DNase activity of S. sanguinis was detected around growing colonies on agar plates containing DNA. In-frame deletion of the swan gene mostly reduced that activity. These findings indicated that SWAN is a major nuclease displayed on the surface, which was further confirmed by immuno-detection of SWAN in the cell wall fraction. The sensitivity of S. sanguinis to NET killing was reduced by swan gene deletion. Moreover, heterologous expression of the swan gene rendered a Lactococcus lactis strain more resistant to NET killing. Our results suggest that the SWAN nuclease on the bacterial surface contributes to survival in the potential situation of S. sanguinis encountering NETs during the course of disease progression.

Lantibiotic immunity: inhibition of nisin mediated pore formation by NisI.

Nisin, a 3.4 kDa antimicrobial peptide produced by some Lactococcus lactis strains is the most prominent member of the lantibiotic family. Nisin can inhibit cell growth and penetrates the target Gram-positive bacterial membrane by binding to Lipid II, an essential cell wall synthesis precursor. The assembled nisin-Lipid II complex forms pores in the target membrane. To gain immunity against its own-produced nisin, Lactococcus lactis is expressing two immunity protein systems, NisI and NisFEG. Here, we show that the NisI expressing strain displays an IC50 of 73±10 nM, an 8–10-fold increase when compared to the non-expressing sensitive strain. When the nisin concentration is raised above 70 nM, the cells expressing full-length NisI stop growing rather than being killed. NisI is inhibiting nisin mediated pore formation, even at nisin concentrations up to 1 µM. This effect is induced by the C-terminus of NisI that protects Lipid II. Its deletion showed pore formation again. The expression of NisI in combination with externally added nisin mediates an elongation of the chain length of the Lactococcus lactis cocci. While the sensitive strain cell-chains consist mainly of two cells, the NisI expressing cells display a length of up to 20 cells. Both results shed light on the immunity of lantibiotic producer strains, and their survival in high levels of their own lantibiotic in the habitat.

Effects of ingesting milk fermented by Lactococcus lactis H61 on skin health in young women: A randomized double-blind study

We conducted a randomized double-blind trial to evaluate the effects of fermented milk produced using only Lactococcus lactis strain H61 as a starter bacterium (H61-fermented milk) on the general health and various skin properties of young women. Healthy female volunteers (n=23; age=19–21r) received H61-fermented milk (1010 cfu of strain H61/d) or conventional yogurt (1010 cfu of both Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus per day), as a reference food, daily for 4 wk. Before and at the end of 4 wk, blood samples were taken, and skin hydration (inner forearms and cheek) and melanin content, elasticity, and sebum content (cheek only) were measured. Skin hydration at the inner forearm was higher at wk 4 than at wk 0 in both groups. Sebum content in cheek rose significantly after intervention in the H61-fermented milk group, but not the conventional yogurt group. Other skin parameters did not differ in either group. Serum analysis showed that total protein concentration and platelet count were elevated and reactive oxygen species decreased in both groups after the intervention. Although H61-fermented milk and conventional yogurt had similar effects on skin status and some blood characteristics of participants, an increase of sebum content in cheek is preferable to H61-fermented milk. As skin lipids contribute to maintaining the skin barrier, H61-fermented milk would provide beneficial effects on skin for young women.

Altered Superoxide Dismutase Activity by Carbohydrate Utilization in a Lactococcus lactis Strain

Reactive oxygen species, such as superoxide, can damage cellular components, such as proteins, lipids, and DNA. Superoxide dismutase (SOD) enzymes catalyze the conversion of superoxide anions to hydrogen peroxide and dioxygen. SOD is present in most lactococcal bacteria, which are commonly used as starters for manufacturing fermented dairy products and may have health benefits when taken orally. We assessed the effects of carbohydrate use on SOD activity in lactococci. In Lactococcus lactis ssp. lactis G50, the SOD activity of cells grown on lactose and galactose was higher than that on glucose; in Lactococcus lactis ssp. cremoris H61, SOD activity was independent of the type of carbohydrate used. We also investigated the activity of NADH oxidase, which is related to the production of superoxide in strains G50 and H61. Activity was highest in G50 cells grown on lactose, lower on galactose, and lowest on glucose, whereas activity in H61 cells did not differ with the carbohydrate source used. The SOD and NADH oxidase activities of strain G50 in three carbohydrates were linked. Strain G50 fermented lactose and galactose to lactate, acetate, formate, and ethanol (mixed-acid fermentation) and fermented glucose to mainly lactate (homolactic fermentation). Strain H61 fermented glucose, lactose, and galactose to mainly lactate (homolactic fermentation). In strain G50, when growth efficiency was reduced by adding a metabolic inhibitor to the growth medium, SOD activity was higher than in the control; however, the metabolism was homofermentative. Aerobic conditions, but not glucose-limited conditions, increased SOD activity, and mixed-acid fermentation occurred. We conclude that the effect of carbohydrate on SOD activity in lactococci is strain dependent and that the activity of commercial lactococci can be enhanced through carbohydrate selection for mixed-acid fermentation or by changing the energy distribution, thus enhancing the value of the starter and the resulting dairy products.

Comparison of volatile compounds produced by wild Lactococcus lactis in miniature Chihuahua-type cheeses

Strains of Lactococcus lactis used for a long time as starter cultures in the production of cheese have not only acquired special features like fast utilization of lactose, but it is also believed that they have lost certain metabolic capabilities. Certain wild strains of L. lactis isolated from vegetables or raw milk products are able to generate flavors different from those produced by industrial strains. The aim of this work was to assess the production of volatile compounds in miniature Chihuahua-type cheeses manufactured with different strains of L. lactis isolated from vegetables, raw milk products, and industrial cultures. There was variation among volatile profiles in the miniature cheeses manufactured with different strains of L. lactis. However, some compounds were seen in most of the cheeses such as acetic, lactic, butyric, and caproic acids, acetoin, 3-methyl-1-butanol, and 2,3-butanediol. The source of isolation of the strains (plants, raw milk products, and industrial cultures) did not have a clear influence on the production of volatile compounds in miniature cheeses. According to principal component analysis, 19 out of 21 strains of L. lactis produced volatile profiles similar to the three pasteurized Chihuahua cheeses analyzed, but only 2 strains generated profiles similar to the commercial raw milk cheese. However, further research is required to understand the metabolic and genetic differences of these strains.

The P170 expression system enhances hyaluronan molecular weight and production in metabolically-engineered Lactococcus lactis

Recombinant Lactococcus lactis strains based on the P170 expression system were developed for hyaluronan (HA) production, by incorporating genes from the has operon of Streptococcus zooepidemicus and compared with nisin-inducible recombinant L. lactis strains containing the hasABC and hasABD constructs. It was found across all batch and fed-batch experimental studies that HA concentration and molecular weight (MW) were higher for the P170 expression systems than the corresponding NICE-based strains. The highest hyaluronan MW was obtained for all constructs in batch studies at 60g/L initial glucose concentration, the highest being 2.94MDa for the P170 strains with hasABC construct (L. lactis APJ3). In fed-batch studies with constant feed rate, the L. lactis APJ3 gave better HA yield (0.03g/g) than the NICE-based strain. A higher hyaluronan MW was obtained for all strains in pulse fed-batch compared to constant feed experiments, the highest being 2.52MDa for L. lactis APJ3.

Sensitivity to the two‐peptide bacteriocin lactococcin G is dependent on UppP, an enzyme involved in cell‐wall synthesis

Most bacterially produced antimicrobial peptides (bacteriocins) are thought to kill target cells by a receptor-mediated mechanism. However, for most bacteriocins the receptor is unknown. For instance, no target receptor has been identified for the two-peptide bacteriocins (class IIb), whose activity requires the combined action of two individual peptides. To identify the receptor for the class IIb bacteriocin lactococcin G, which targets strains of Lactococcus lactis, we generated 12 lactococcin G-resistant mutants and performed whole-genome sequencing to identify mutations causing the resistant phenotype. Remarkably, all had a mutation in or near the gene uppP (bacA), encoding an undecaprenyl pyrophosphate phosphatase; a membrane protein involved in peptidoglycan synthesis. Nine mutants had stop codons or frameshifts in the uppP gene, two had point mutations in putative regulatory regions and one caused an amino acid substitution in UppP. To verify the receptor function of UppP, it was shown that growth of non-sensitive Streptococcus pneumoniae could be inhibited by lactococcin G when L. lactis uppP was expressed in this bacterium. Furthermore, we show that the related class IIb bacteriocin enterocin 1071 also uses UppP as receptor. The approach used here should be broadly applicable to identify receptors for other bacteriocins as well.

Cloning and optimization of a nisin biosynthesis pathway for bacteriocin harvest.

Nisin is an important antimicrobial peptide that has enormous applications in biotechnology. Despite many encouraging efforts, its overproduction has been a long-standing challenge due to the complexity of the underlying pathway and the difficulty in genetic modification of lactic acid bacteria. Here, we cloned an entire nisin biosynthesis pathway from a nisin-producing strain (Lactococcus lactis K29) into a plasmid and transplanted the plasmid into a nisin deficient strain Lactococcus lactis MG1363, resulting in successful heterologous expression of bioactive recombinant nisin. To increase nisin harvest, we also overexpressed nisA, a gene responsible for nisin precursor production, with a set of constitutive promoters. To further optimize nisin yield, we minimized the metabolic cost of the engineered strains by integrating nisA overexpression cassettes and the recombinant pathway into a single circuit. With our rational construction and optimization, our engineered optimized strain is able to produce bioactive nisin with a yield of 1098 IU/mL, which is more than six times higher than that of the original strain.

Effects of strains of Lactococcus lactis on the production of nitric oxide and cytokines in murine macrophages.

Nitric oxide (NO) is a multifunctional mediator that is involved in a variety of pathologic and physiologic processes. Few studies have addressed the effect of lactic acid bacteria (LAB), especially Lactococcus lactis strains used in dairy products, on inducible nitric oxide synthase (iNOS) induction as a component of the host's gastrointestinal immune response. We investigated the ability of L. lactis strains to induce NO synthesis in the murine macrophage-like cell line J774.1 and in peritoneal macrophages from mice. The degree of NO induction was specific to the L. lactis strain used. Compared with the no-treatment control, heat treatment of L. lactis cells decreased NO and TNF-α levels but further stimulated interleukin (IL)-12 production. Adding L. lactis cells to peritoneal macrophages dose-dependently increased the production of NO and IL-10 but decreased that of IL-12p70. Adding L. lactis cells to interferon-γ-stimulated J774.1 cells enhanced cell death and the production of NO and IL-12p40, whereas addition of 1400W, a specific inhibitor of iNOS, decreased NO production and cell death. Conversely, adding 1400W to J774.1 cells further enhanced IL-12p40 production, suggesting that IL-12 production is perturbed by excess endogenous NO. IL-12 production is thought to be a marker of improved immunostimulation. Our results suggest that IL-12 production could be increased by limiting endogenous NO production.