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Heavy metals (HMs) contaminated fish is a threat to humans when consumed. Dietary probiotics have evolved as a successful HMs removal approach. In this study, probiotics Enterococcus (EC) sp. and Lactococcus (LC) sp. were evaluated for toxicity alleviation and gut microbiota maintenance in Cyprinus carpio (single and combined approach) on Cr, Cd, and Cu mixture (0.8 mg/L and 1.6 mg/L) exposure (28 days). HMs removal, oxidative stress, cytokines response, histology, and gut microbiota were investigated. LC alone showed remarkable HMs removal for Cr (62.28%–87.57%), Cd (89%–90.42%), and Cu (72%–88%) than LC + EC. Probiotics up-regulated superoxide dismutase and total protein levels, while decreased the activity of malondialdehyde than the control. Pro-inflammatory cytokine (TNF-α) and chemokine (IL-8) expressions were higher at 1.6 mg/L concentration, whereas anti-inflammatory cytokine (IL-10) was higher in the 0.8 mg/L group. LC mitigated the histological alterations of gills, kidneys, and intestines, particularly at the lower concentration. Sequencing results revealed that Proteobacteria (44%–61%) was the most dominant phylum in all groups, followed by Fusobacteria (34%–36%) at 0.8 mg/L and Firmicutes (19%–34%) at 1.6 mg/L. The current study presented LC and EC potential separately and in combination to countermeasure HMs mixture induced toxicity and gut microbial dysbiosis, in which the conjoint group was less effective.

Lactococcus lactis ssp. lactis isolated from raw milk was used for titanium dioxide (TiO2) nanoparticles biosynthesis. Biosynthesized TiO2 nanoparticles were characterized using UV-vis spectroscopy, Atomic Force Microscopy (AFM) (1.97 nm), X-ray diffraction (XRD) appa-ratus, Field Emission Scanning Electron Microscopy (FE-SEM), Energy dispersive X-ray anal-ysis (EDX) spectra and Fourier Transform Infrared Spectroscopy (FTIR). Result was 408.21 cm-1 that belong to anatase Titania. L. lactis ssp. Lactis isolates had the ability to synthesize TiO2 nanoparticles, the characterization results presented that the biosynthesized nanoparti-cles were at wavelength (344-347) nm; approving the formation of anatase phase of TiO2 NPs; spherical crystals, with particles, average diameter of 47.22 nm.

Lanthipeptides are ribosomally synthesized and post-translationally modified peptides characterized by the presence of lanthionine rings that provide stability and functionality. Genome mining techniques have shown their huge diversity and potential for the discovery of novel active molecules. However, in many cases, they are not easily produced under laboratory conditions. The heterologous expression of these molecules using well-characterized lanthipeptide biosynthetic enzymes is rising as an alternative system for the design and production of new lanthipeptides with biotechnological or clinical properties. Nevertheless, the substrate-enzyme specificity limits the complete modification of the desired peptides and hence, their full stability and/or biological activity. New low substrate-selective biosynthetic enzymes are therefore necessary for the heterologous production of new-to-nature peptides. Here, we have identified, cloned, and heterologously expressed in Lactococcus lactis the most promiscuous lanthipeptide synthetase described to date, i.e., SyncM from the marine cyanobacteria Synechococcus MITS9509. We have characterized the functionality of SyncM by the successful expression of 15 out of 18 different SyncA substrates, subsequently determining the dehydration and cyclization processes in six representatives of them. This characterization highlights the very relaxed substrate specificity of SyncM toward its precursors and the ability to catalyze the formation of exceptionally large rings in a variety of topologies. Our results suggest that SyncM could be an attractive enzyme to design and produce a wide variety of new-to-nature lanthipeptides with a broad range of ring topologies.

Aflatoxins (AFs) are known to be oncogenic mycotoxins. This study investigated the mitigation effects of lactic acid bacteria (LAB) isolated from four types of vegetable, cucumber, Chinese cabbage, Japanese radish and eggplant, which are used to make Japanese traditional fermented pickles, on AFs. Using aflatoxin M1 (AFM1) binding assay for screening, four representative strains were selected (one from each vegetable) from total 94 LAB strains, based on the highest binding ratio. The ranges of the binding ratio of these representative strains to aflatoxin B1 (AFB1), aflatoxin B2, aflatoxin G1, aflatoxin G2 and AFM1 were 57.5%-87.9% for the LAB strain derived from cucumber, 18.9%-43.9% for the LAB strain derived from Chinese cabbage, 26.4%-41.7% for the LAB strain derived from Japanese radish, and 15.0%-42.6% for the LAB strain derived from eggplant. The strains isolated from cucumber, Chinese cabbage, Japanese radish and eggplant were identified as Lactococcus lactis subsp. lactis, Weissella cibaria, Leuconostoc mesenteroides and Leu. mesenteroides, respectively. An in vitro binding assay of the four strains under acidic conditions showed that the number of living bacteria decreased, while the binding ratio increased in some strains, suggesting that the LAB maintained their capacity to bind aflatoxins even in an environment that imitated the stomach. An in vivo experiment using L. lactis subsp. lactis derived from cucumber revealed that the bacteria significantly inhibited the absorption of AFB1 into blood. These results showed that the LAB used for Japanese vegetable pickles was an effective binding agent of AFs and suggested that they might play a role in mitigating AF absorption.

Viral diseases are extremely widespread infections that change constantly through mutations. To produce vaccines against viral diseases, transient expression systems are employed, and Nicotiana benthamiana (tobacco) plants are a rapidly expanding platform. In this study, we developed a recombinant protein vaccine targeting the major capsid protein (MCP) of iridovirus fused with the lysine motif (LysM) and coiled-coil domain of coronin 1 (ccCor1) for surface display using Lactococcus lactis. The protein was abundantly produced in N. benthamiana in its N-glycosylated form. Total soluble proteins isolated from infiltrated N. benthamiana leaves were treated sequentially with increasing ammonium sulfate solution, and recombinant MCP mainly precipitated at 40–60%. Additionally, affinity chromatography using Ni–NTA resin was applied for further purification. Native structure analysis using size exclusion chromatography showed that recombinant MCP existed in a large oligomeric form. A minimum OD600 value of 0.4 trichloroacetic acid (TCA)-treated L. lactis was required for efficient recombinant MCP display. Immunogenicity of recombinant MCP was assessed in a mouse model through enzyme-linked immunosorbent assay (ELISA) with serum-injected recombinant MCP-displaying L. lactis. In summary, we developed a plant-based recombinant vaccine production system combined with surface display on L. lactis.

The current research was performed to evaluate the potential protective effect of Lactobacillus paracasei ssp. paracasei, Pediococcus acidilactici, Lactococcus lactis ssp. lactis, and silymarin in the alleviation of health (hepatic and renal) complications caused by carbon tetrachloride (CCl4) in rats. Healthy sixty albino rats were divided into six groups, the first group was control (negative), the second group (control positive) was injected CCl4 (1 ml/kg, 1:1 v/v paraffin oil mixture, i.p. every third day for 8 weeks), the third group (CCl4 + silymarin group) receiving both CCl4 and daily silymarin therapy (50 mg/kg, oral), and the fourth group: CCl4 + Lactobacillus paracasei (1 ml orally). The fifth group (CCl4 + Pediococcus acidilactici 1 ml orally) and the sixth group (CCl4 + Lactococcus lactis 1 ml orally) for 8 weeks per day. Biochemical markers were tested for blood, liver, and kidney tissue. Histopathological examination of the liver and kidney tissues was performed. The findings obtained have shown that Lactobacillus paracasei ssp. paracasei, Pediococcus acidilactici, and Lactococcus lactis ssp. lactis improved the disrupted biochemical parameters caused by CCl4 therapy. Besides, the findings of the histopathology are in consistent with biochemical parameters and the protective ability of lactic acid bacteria suggesting that the best lactic acid bacteria were Pediococcus acidilactici that helped strengthen liver fibrosis caused by CCl4 therapy, while the best bacterium for improving renal damage was Lactococcus lactis.

ComX can improve bacterial competence by modulating global gene expression. Although competence induction may also be a protective mechanism under stress, this has not been investigated in detail. Here, we demonstrated that ComX improved the acid tolerance and nisin yield of Lactococcus lactis, which is an important gram-positive bacterium increasingly used in modern biotechnological applications. We found that overexpression of comX could improve the survival rate up to 36.5% at pH 4.0, compared with only 5.4% and 1.1% with the wild-type and comX knockout strains, respectively. Moreover, quantitative real-time PCR results indicated that comX overexpression stimulated the expression of late competence genes synergistically with exposure to acid stress. Finally, electrophoretic mobility shift assay demonstrated the binding of purified ComX to the cin-box in the promoters of these genes. Taken together, our results reveal a regulation mechanism by which ComX and acid stress can synergistically modulate the expression of late competence genes to enhance cells' acid tolerance and nisin yield.

Ionizing radiation (IR) is widely used in the diagnosis and treatment of various cancers. However, IR can cause damage to human health by producing reactive oxygen species. Lactococcus lactis is a type of microorganism that is beneficial to human health and has a strong antioxidant capacity. In this study, the protective effect of normal and IR-induced L. lactis IL1403 cell-free extracts (CFE and IR-CFE, respectively) against oxidative damage in vitro and the radioprotective effect of IR-CFE in vivo was evaluated using 60Coγ-induced oxidative damage model in mice. Results showed that IR-CFE exhibited a stronger oxidative damage-protective effect than CFE for L. lactis IL1403 under H2O2 in vitro. Moreover, IR-CFE also showed strong radioprotective effect on hepatocyte cells (AML-12) under radiation condition, and the effect was better than that of CFE. Animal experiment indicated that IR-CFE could reduce the IR-induced damage to the hematopoietic system by increasing the number of white blood cells and red blood cells in peripheral blood of irradiated mice. It was also observed that IR-CFE could markedly alleviate the 60Coγ-induced oxidative stress via increasing the activities of superoxide dismutase and glutathione peroxidase, enhancing the levels of glutathione, and decreasing the contents of malondialdehyde in serum, liver, and spleen. In addition, IR-CFE also could reduce the activities of alanine transaminase and aspartate aminotransferase in serum, thereby reducing radiation damage to the liver. These results suggested that IR-CFE could be considered as potential candidates for natural radioprotective agents. This study provides a theoretical basis for improving the application of lactic acid bacteria.

Background:: The microbiome is now known for its important role in whole-body homeostasis. A dysbiosis of the normal microbiota is correlated with metabolic disorders. In this sense, the search for compounds able to modulate the microbiome is needed. Resveratrol, a natural compound found in grapes seems to be a promising candidate. Objective:: In this study, our motivation was to evaluate the effects of the association between Resveratrol and Lactococcus lactis, a probiotic, on the composition of the gastrointestinal microbiota and body weight of mice. Methods:: Twenty female mice were divided into 4 groups: (1) standard diet, (2) standard diet plus Lactococcus lactis, (3) standard diet plus resveratrol, and (4) standard diet plus Lactococcus lactis and resveratrol. At the end of the treatment period, samples of blood, mucus, stomach, and small and large intestines were collected for analysis. Total levels of Immunoglobulin A and Immunoglobulin E, Lac+ and Lac- bacteria and Lactobacillus were measured. Results:: The main results indicate that the association between resveratrol and probiotics was able to decrease mice body weight, as compared to the other groups, in addition to decrease the number of Lac- bacteria and increasing the number of Lac+ bacteria. The levels of secretory IgA were also decreased, compared to the animals treated with only probiotics or resveratrol. Conclusion:: We observed potential synergism between Resveratrol and Lactococcus lactis mainly in modulating the stomach and intestinal microbiota.

An attempt was made, to characterize natural antibiotics or lantibiotics from unconventional sources and its antibacterial spectrum against food borne pathogens and drug resistant bacteria. Six different traditional fermented foods i.e., fermented fish, fermented soybeans, Soibum (fermented bamboo shoots), milk, idly and dosa batter were used for the isolation of bacteriocin producing Lactic acid bacteria (LAB). Among all bacterial cultures isolated from the various sources, 129 cultures have found to produce antimicrobial compounds. Nisin specific reporter bacteria was utilized as biosensor to identify the Nisin like bacteriocin, where 10 cultures found to be positive Nisin producer. Identified Nisin like bacteriocin was partially concentrated by using ammonium sulphate followed by butanol extraction. Minimum inhibitory concentration (MIC) was analyzed against food borne pathogen and drug resistant bacteria. MIC of partially purified Nisin (pp-Nisin) of all the LAB isolates against food-borne pathogens are ranged between 0.5 and 92 µg/ml respected to various Gram-positive bacteria. Similarly, the drug resistant bacteria were also inhibited by pp-Nisin (MIC ranged between 15 and 175 µg/ml). All samples of ppnisin exhibited auto induction ability. Taxonomic identification of the nisin producers was done by whole genome sequencing which reveals that cultures belongs to Lactococcus lactis ssp. lactis. Also it was found that Lactococcus lactis ssp. lactis C2d and Lactococcus lactis ssp. lactis SP2C4 harbor nisA gene and Lactococcus lactis ssp. lactis FS2 (L. lactis FS2) harbor nisQ gene. The finding of this study highlights the first case of L. lactis FS2 isolated from fermented fish harbor nisQ gene. Antibacterial activity of pp-Nisin against drug resistant LAB is also reported.

Imaging flow cytometry (IFC) combines microscopy-based capturing of cell images with conventional flow cytometry-based high-throughput analysis. Thus, IFC approach may limit false-positive results and provide more accurate and unambiguous data. This may be particularly important in the case of the single-cell analysis of relatively small biological objects such as bacteria. The microbial quality control of milk samples is of a special interest during milk processing and several methods have been previously developed to address the presence of bacterial species in milk and other economically important dairy products. However, the vast majority of screening assays is time-consuming and cost-ineffective to be used as a routine test. In the present study, we propose combined acridine orange (AO) staining and IFC-based profiling of microbial species as a novel alternative method for the microbial quality control of milk samples. Eight bacterial profiles (Lactococcus lactis, Lactobacillus rhamnosus GG, Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Staphylococcus aureus, Salmonella typhimurium and Sarcina lutea) were characterized using a number of IFC parameters and IFC approach was also validated using commercially available milk samples and probiotic supplement. The obtained results suggest that IFC-based microbial profiling may be considered as a potentially useful method for rapid screening for selected bacterial species in milk samples.

High values of agitation and temperature lead to stressful conditions in the fermentations of Lactococcus lactis due to its aero-tolerant and mesophilic nature. Here, the adaptive laboratory evolution (ALE) technique was applied to increase biomass and nisin production yields by enhancing L. lactis subsp. lactis robustness at higher growth temperature and aeration rates. In two separate ALE experiments, after 162 serial transfers, optimum agitation and growth temperature of L. lactis were shifted from 40 rpm and 30 °C to 200 rpm and 37 °C, respectively. Oxidative and acid resistance were enhanced in the evolved strain. Whole-genome sequencing revealed the emergence of five single-nucleotide polymorphisms in the genome of the evolved strain in jag, DnaB, ArgR, cation transporter genes, and one putative protein. The evolved strain of L. lactis in this study has more industrial desirable features and improved nisin production capability and can act more efficiently in nisin production in stressful conditions.

Acanthamoeba spp. are free-living amoebas with worldwide distribution and play an important role as disease-causing agents in humans. Drug inability to completely eradicate these parasites along with their toxic effects suggest urgent need for new antimicrobials. Nisin is a natural antimicrobial peptide produced by Lactococcus lactis. Nisin is also the only bacteriocin approved for use in food preservation. In this work, we analyzed the effect of nisin on the growth of Acanthamoeba castellanii trophozoites. A total of 8 × 104 trophozoites were exposed to increasing concentrations of nisin to determine its activity. Changes in cell membrane and cellular cycle of trophozoites were investigated by flow cytometry, and nisin cytotoxicity in mammalian cells was evaluated in L929 cells by MTT method. After 24 h exposure to increasing nisin concentrations, an IC50 of 4493.2 IU mL−1 was obtained for A. castellanii trophozoites. However, after 72 h a recovery in amoebic growth was observed, and it was no longer possible to determine IC50. Flow cytometry analysis showed that nisin has no effect on the membrane integrity. Treatment with nisin induced cell-cycle arrest during G1 and S phases in A. castellanii trophozoites, which recovered their growth after 72 h. This is one of the first studies showing the effect of internationally approved nisin against A. castellanii trophozoites. Nisin caused cell-cycle arrest in trophozoites, momentarily interfering with the DNA replication process. The data highlight the amoebostatic activity of nisin, and suggest its use as an adjuvant for the treatment of infections caused by Acanthamoeba spp.

The goal of the present study was oriented to extraction of a bioactive compound 2,4-di-tertiary-butyl phenol (2,4-DTBP) from Lactococcus lactis subsp. lactis CAU: 3138-GM2 of goat milk origin (accession no. MF354327.1). The 2,4-DTBP was extracted by methanol: chloroform. Radioiodination of 2,4-DTBP using Na125I and chloramine -T was carried out. Effect of radiolabeled tracer and non-radiolabeled of 2,4-DTBP on the inhibition of growth myeloma cells were evaluated. The bioactivity of 2,4-DTBP was tested against Staphylococcus aureus MRSA ATCC 43300, Listeria monocytogenes ATCC 19116, Pseudomonas aeruginosa MTCC 1934, and Escherichia coli MTCC 1610, Clostridium bifermentans MTCC 11273 and Candida albicans MTCC 183.

International audience; Wheat is a worldwide staple food, yet some people suffer from strong immunological reactions after ingesting wheat-based products. Lactic acid bacteria (LAB) constitute a promising approach to reduce wheat allergenicity because of their proteolytic system. In this study, 172 LAB strains were screened for their proteolytic activity on gluten proteins and alpha-amylase inhibitors (ATIs) by SDS-PAGE and RP-HPLC. Gliadins, glutenins, and ATI antigenicity and allergenicity were assessed by Western blot/Dot blot and by degranulation assay using RBL-SX38 cells. The screening resulted in selecting 9 high gluten proteolytic strains belonging to two species: Enterococcus faecalis and Lactococcus lactis. Proteomic analysis showed that one of selected strains, Lc. lactis LLGKC18, caused degradation of the main gluten allergenic proteins. A significant decrease of the gliadins, glutenins, and ATI antigenicity was observed after fermentation of gluten by Lc. lactis LLGKC18, regardless the antibody used in the tests. Also, the allergenicity as measured by the RBL-SX38 cell degranulation test was significantly reduced. These results indicate that Lc. lactis LLGKC18 gluten fermentation can be deeply explored for its capability to hydrolyze the epitopes responsible for wheat allergy.

The purpose of this study was to evaluate the impacts of two Lactic Acid Bacteria strains (LAB) Pediococcus acidilactici (P. acidilactici) and Lactococcus Lactis (L. Lactis)) isolated from fermented dairy products. In this study, 24 female rats were divided into three groups (8 rats per each group): (1) untreated group, (2) P. acidilactici group: rats fed orally with 108 CFU /ml skim milk, (3) L. Lactis group: rats fed orally with 108 CFU /ml skim milk for 6 weeks. Bodyweight gain, daily feed intake, relative weight organs, hematological parameters, ALT, AST, protein, albumin, globulin, urea, creatinine, and histopathological examinations of liver and kidney were determined at the end of the experiment. Results: In this analysis, statistical significance was found for certain parameters e.g. organ weights, hematology, and clinical chemistry; none of these improvements were expected due to the treatment since the changes remained close to controls, also, histological studies of the liver and kidney observed no and mild morphology changes in the liver or kidney. The current study indicates that administration of P. acidilactici and L. Lactis strains of LAB didn't harm laboratory rats and safe for animal or human consumption.

The sensory properties and flavor of sour cream are important factors that influence consumer acceptability. The present study aimed to isolate lactic acid bacteria with excellent diacetyl production ability and to optimize the fermentation conditions for sour cream manufacture. Lactococcus lactis ssp. cremoris was isolated as a lactic acid bacterium derived from raw milk. This strain showed the greatest diacetyl production among other strains and was named LRCC5306. Various culture conditions were optimized to improve the diacetyl production of LRCC5306. The highest diacetyl production was found to be at 105.04±2.06 mg/L, when 0.2% citric acid and 0.001% Fe2+ were added and cultured at 20°C for 15 h. Based on the optimal cultivation conditions, sour cream was manufactured using LRCC5306, with a viable count of 1.04×108 CFU/g and a diacetyl concentration of 106.56±1.53 mg/g. The electronic tongue system was used to compare the sensory properties of the sour cream; the fermented product exhibited sweetness and saltiness which was similar to that of an imported commercial product, but with slightly reduced bitterness and a significantly greater degree of sour taste. Therefore, our study shows that if cream is fermented using the LRCC5306, it is possible to produce sour cream with greatly improved sensory attractiveness, resulting in increased acceptance by consumers. Since this sour cream has a higher viable count of lactic acid bacteria, it is also anticipated that it will have a better probiotic effect.

Gamma-aminobutyric acid (GABA), an important bioactive compound, is synthesized through the decarboxylation of L-glutamate (L-Glu) by glutamate decarboxylase (GAD). The use of lactic acid bacteria (LAB) as catalysts opens interesting avenues for the biosynthesis of food-grade GABA. However, a key obstacle involved in the improvement of GABA production is how to resolve the discrepancy of optimal pH between the intracellular GAD activity and cell growth. In this work, a potential GAD candidate (LpGadB) from Lactobacillus plantarum was heterologously expressed in Escherichia coli. Recombinant LpGadB existed as a homodimer under the native conditions with a molecular mass of 109.6 kDa and exhibited maximal activity at 40°C and pH 5.0. The Km value and catalytic efficiency (kcat/Km) of LpGadB for L-Glu was 21.33 mM and 1.19 mM-1s-1, respectively, with the specific activity of 26.67 μM/min/mg protein. Subsequently, four C-terminally truncated LpGadB mutants (GadBΔC10, GadBΔC11, GadBΔC12, GadBΔC13) were constructed based on homology modeling. Among them, the mutant GadBΔC11 with highest catalytic activity at near-neutral pH values was selected. In further, the GadBΔC11 and Glu/GABA antiporter (GadC) of Lactococcus lactis were co-overexpressed in the host L. lactis NZ3900. Finally, after 48 h of batch fermentation, the engineered strain L. lactis NZ3900/pNZ8149-gadBΔC11C yielded GABA concentration up to 33.52 g/L by applying a two-stage pH control strategy. Remarkably, this is the highest yield obtained to date for GABA from fermentation with L. lactis as a microbial cell factory.Key points• The GadB from L. plantarum was heterologously expressed in E. coli and biochemically characterized.• Deletion of the C-plug in GadB shifted its pH-dependent activity toward a higher pH.• Reconstructing the GAD system of L. lactis is an effective approach for improving its GABA production.

Background Endometritis is the most common disease of dairy cows and traditionally treated with antibiotics. Lactic acid bacteria can inhibit the growth of pathogens and also have potential for treatment of endometritis. Using PacBio single-molecule real-time sequencing technology, we sequenced the full-length l6S rRNA of the microbiota in uterine mucus samples from 31 cows with endometritis, treated with lactic acid bacteria (experimental [E] group) and antibiotics (control [C] group) separately. Microbiota profiles taken before and after treatment were compared. Results After both treatments, bacterial species richness was significantly higher than before, but there was no significant difference in bacterial diversity. Abundance of some bacteria increased after both lactic acid bacteria and antibiotic treatment: Lactobacillus helveticus, Lactococcus lactis, Lactococcus raffinolactis, Pseudomonas alcaligenes and Pseudomonas veronii. The bacterial species that significantly decreased in abundance varied depending on whether the cows had been treated with lactic acid bacteria or antibiotics. Abundance of Staphylococcus equorum and Treponema brennaborense increased after lactic acid bacteria treatment but decreased after antibiotic treatment. According to COG-based functional metagenomic predictions, 384 functional proteins were significantly differently expressed after treatment. E and C group protein expression pathways were significantly higher than before treatment (p Conclusions In this study, we found that lactic acid bacteria could cure endometritis and restore a normal physiological state, while avoiding the disadvantages of antibiotic treatment, such as the reductions in abundance of beneficial microbiota. This suggests that lactic acid bacteria treatment has potential as an alternative to antibiotics in the treatment of endometritis in cattle. How to cite: Yang L, Huang W, Yang C, et al. Using PacBio sequencing to investigate the effects of treatment with lactic acid bacteria or antibiotics on cow endometritis. Electron J Biotechnol 2021:51. https://doi.org/10.1016/j.ejbt.2021.02.004

Heat-killed lactic acid bacteria perform immunomodulatory functions and are advantageous as probiotics, considering their long product shelf-life, easy storage, and convenient transportation. In this study, we aimed to develop appropriate heat treatments for industrial preparation of probiotics with antioxidant activity. Among 75 heat-killed strains, Lactococcus lactis MG5125 revealed the highest nitric oxide inhibition (86.2%), followed by Lactobacillus acidophilus MG4559 (86.0%), Lactobacillus plantarum MG5270 (85.7%), Lactobacillus fermentum MG4510 (85.3%), L. plantarum MG5239 (83.9%), L. plantarum MG5289 (83.2%), and L. plantarum MG5203 (81.8%). Moreover, the heat-killed selected strains markedly inhibited lipopolysaccharide-induced nitric oxide synthase and cyclooxygenase-2 expression. The use of heat-killed bacteria with intact bio-functionality can elongate the shelf-life and simplify the food processing steps of probiotic foods, given their high stability. The antioxidant and immune-modulatory activities of the heat-killed strains selected in this study indicate a strong potential for their utilization probiotic products manufacturing.

Nisin is commonly used as a biopreservative in foods. For industrial production, nisin-producing Lactococcus lactis strains are usually grown to high cell densities to achieve the highest possible nisin titer. However, accumulation of lactic acid eventually halts production, even in pH-controlled fermentations. Here, we describe a nisin-producing L. lactis strain Ge001, which was obtained after transferring the nisin gene cluster from L. lactis ATCC 11454, by conjugation, into the natural mutant L. lactis RD1M5, with low lactate dehydrogenase activity. The ability of Ge001 to produce nisin was tested using dairy waste as the fermentation substrate. To accommodate redox cofactor regeneration, respiration conditions were used, and to alleviate oxidative stress and to reduce adsorption of nisin onto the producing cells, we found it to be beneficial to add 1 mM Mn2+ and 100 mM Ca2+, respectively. A high titer of 12 084 IU/mL nisin could be reached, which is comparable to the highest titers reported using expensive, rich media. Summing up, we here present a 100% natural, robust, and sustainable approach for producing food-grade nisin and acetoin from readily available dairy waste.

During industrial handling, Lactococcus lactis needs to adapt to different culture conditions by regulating its metabolic pathways. Modifying culture conditions may be an important way to control the biomass and functional metabolites of lactic acid bacteria. In this study, we identified the differentially expressed genes and proteins of L. lactis under different culture conditions by integrating transcriptomics and proteomics. We also analyzed the data using a bioinformatic approach to reveal the regulatory mechanisms affected by culture conditions. The transcriptome and proteome studies indicated that different culture conditions (fructose, calcium ion, palmitic acid, low pH) affected gene and protein expressions. The levels of differentially expressed proteins did not significantly correlate with the expression levels of their corresponding genes. Our results highlight the importance of comparative transcriptomics and proteomics analyses. In this study, fructose and pH significantly affected sugar metabolism of L. lactis. When lactose was replaced by fructose, fructokinase expression was promoted, and fructose metabolism was accelerated, whereas starch and sucrose metabolism and galactose metabolism system were inhibited. Low pH may be beneficial to homofermentation of L. lactis, which may also metabolize galactose through the tagatose pathway and the Leloir pathway. Fatty acid metabolism and fatty acid biosynthesis were significantly downregulated under calcium ion and palmitic acid. The purine metabolism was upregulated under fructose treatment and downregulated under palmitic acid treatment.

Nisin is a bacteriocin that is globally employed as a biopreservative in food systems to control gram-positive, and some gram-negative, bacteria. Here we tested the bioactivity of nisin A-producing Lactococcus lactis NZ9700 and producers of bioengineered variants thereof against representatives of the gram-negative genus Thermus, which has been associated with the pink discoloration defect in cheese. Starting with a total of 73 nisin variant-producing Lactococcus lactis, bioactivity against Thermus was assessed via agar diffusion assays, and 22 variants were found to have bioactivity greater than or equal to that of the nisin A-producing control. To determine to what extent this enhanced bioactivity was attributable to an increase in specific activity, minimum inhibitory concentrations were determined using the corresponding purified form of these 22 nisin A derivatives. From these experiments, nisin M17Q and M21F were identified as peptides with enhanced antimicrobial activity against the majority of Thermus target strains tested. In addition, several other peptide variants were found to exhibit enhanced specific activity against a subset of strains.

Comparative genomic analysis was performed on eight species of lactic acid bacteria (LAB)-Lactococcus (L.) lactis, Lactobacillus (Lb.) plantarum, Lb. casei, Lb. brevis, Leuconostoc (Leu.) mesenteroides, Lb. fermentum, Lb. buchneri, and Lb. curvatus-to assess their glutamic acid production pathways. Glutamic acid is important for umami taste in foods. The only genes for glutamic acid production identified in the eight LAB were for conversion from glutamine in L. lactis and Leu. mesenteroides, and from glucose via citrate in L. lactis. Thus, L. lactis was considered to be potentially the best of the species for glutamic acid production. By biochemical analyses, L. lactis HY7803 was selected for glutamic acid production from among 17 L. lactis strains. Strain HY7803 produced 83.16 pmol/μl glutamic acid from glucose, and exogenous supplementation of citrate increased this to 108.42 pmol/μl. Including glutamic acid, strain HY7803 produced more of 10 free amino acids than L. lactis reference strains IL1403 and ATCC 7962 in the presence of exogenous citrate. The differences in the amino acid profiles of the strains were illuminated by principal component analysis. Our results indicate that L. lactis HY7803 may be a good starter strain for glutamic acid production.

Objective Topical sinonasal rinse therapies may alter the local microbiome and improve disease control in chronic rhinosinusitis (CRS). The objective of this study was to examine microbiome changes in post-surgical CRS patients when rinsing with commercially available products containing xylitol or Lactococcus lactis. Methods A crossover-type protocol with a washout period was designed. Swab samples from anterior ethmoid cavities of CRS patients were collected prospectively at baseline. Subjects were provided packets containing either L. lactis W136 or xylitol in non-blinded fashion and instructed to add it to their rinse bottles daily for 28 days, after which another swab was taken. A saline wash-out period was completed and a third swab taken. A final 28-day regimen of the opposite product was followed by a final swab. DNA extraction and sequencing of the 16S rRNA gene allowed for global microbiome analysis. Results We enrolled 25 subjects with CRS and 10 controls resulting in 70 adequate samples. Increased detection of Lactococcus was observed after use of L. lactis. No significant trends in alpha or beta diversity as a result of treatment were observed. SNOT-22 score did not change significantly following treatment with xylitol, L. lactis, or saline. Conclusion We did not detect any major clinical or microbiome-level effect due to treatment with two topical rinse products. Further research is needed to elucidate their clinical utility and possible probiotic effect. Level of evidence 3.

L-5-methyltetrahydrofolate (5-MTHF) is one of the biological active forms of folate, which is widely used as a nutraceutical. However, low yield and serious pollution associated with the chemical synthesis of 5-MTHF hampers its sustainable supply. In this study, 5-MTHF production was improved by engineering the 5-MTHF biosynthesis pathway and NADPH supply in Lactococcus lactis for developing a green and sustainable biosynthesis approach. Specifically, overexpressing the key rate-limiting enzyme methylenetetrahydrofolate reductase led to intracellular 5-MTHF accumulation, reaching 18 μg/l. Next, 5-MTHF synthesis was further enhanced by combinatorial overexpression of 5-MTHF synthesis pathway enzymes with methylenetetrahydrofolate reductase, resulting in 1.7-fold enhancement. The folate supply pathway was strengthened by expressing folE encoding GTP cyclohydrolase I, which increased 5-MTHF production 2.4-fold to 72 μg/l. Furthermore, glucose-6-phosphate dehydrogenase was overexpressed to improve the redox cofactor NADPH supply for 5-MTHF biosynthesis, which led to a 60% increase in intracellular NADPH and a 35% increase in 5-MTHF production (97 μg/l). To reduce formation of the by-product 5-formyltetrahydrofolate, overexpression of 5-formyltetrahydrofolate cyclo-ligase converted 5-formyltetrahydrofolate to 5,10-methyltetrahydrofolate, which enhanced the 5-MTHF titer to 132 μg/l. Finally, combinatorial addition of folate precursors to the fermentation medium boosted 5-MTHF production, reaching 300 μg/l. To the best of our knowledge, this titer is the highest achieved by L. lactis. This study lays the foundation for further engineering of L. lactis for efficient 5-MTHF biosynthesis.

The antifungal cyclo-depeptide and the fatty acid were isolated and purified from an indigenous strain of Lactococcus lactis subsp. cremoris. Maximal activity was observed at pH 5.5 and 6.5, and at 30 °C under stationary conditions, which was detected in the culture supernatant 8 h post-inoculation in MRS broth until 22 h. The activity of antifungal compounds in the culture supernatant was sensitive to pH and temperature; and was protease-resistant. The antifungal compounds were concentrated by freeze-drying and ultrafiltration with activity retained in 1 kDa filtrates indicating low molecular weight metabolites. The compounds were further extracted by using different solvents amongst which, ethyl acetate provided the highest recovery. Antifungal compounds were separated on a silica gel column into two active fractions that were revealed to be tetradecanoic acid and cyclo-(Leu-Pro), a cyclic dipeptide, by GC–MS. Herein, we describe and attribute the biocontrol potential of L. lactis subsp. cremoris to the low molecular weight antifungal compounds isolated, which is the first report of their isolation from this strain. The broad antifungal spectrum of this candidate advocates further exploration of its biocontrol potential in managing fungal infections in different food and feed systems. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12088-020-00917-z.

The ability of nosocomial opportunistic microorganism, K. pneumoniae, to produce biofilms on food and hospital surfaces is crucial to the development of infection which has adverse effects on patient outcome and survival. The present study aimed to evaluate the impact of probiotic Nisin producing L. lactis and TiO2 NPs on the biofilm formation of K. pneumoniae. L. lactis isolates were commercially obtained and TiO2 NPs were produced using a sol–gel method. 62 K. pneumoniae strains were isolated in three hospitals in Tehran, and food samples. The antimicrobial activity of L. lactis and TiO2 NPs against antibiotic-resistant biofilm producing strains was determined via well-diffusion method. Definitive identification of isolates was performed through conventional biochemical tests and 16SrRNA PCR. The biofilm formation ability among the isolates obtained from sputum was higher than other isolates (p < 0.001). The L. lactis inhibited biofilm production in 4 isolates, but failed to prevent the growth of the 13 isolates. Additionally, TiO2 NPs inhibited 14 isolates to form a biofilm at 256 μg/mL. Comparison of LuxS gene expression in K. pneumoniae untreated and treated with TiO2 NPs alone and TiO2 + L. lactis showed –2.04 and –3.85 fold changes (CI = 95%, p = 0.006 and p = 0.002, respectively). The synthesized TiO2 nanoparticles are effective against these biofilms forming multidrug resistant K. pneumoniae strains and can be considered as reliable and operative inorganic antimicrobial agents. Antimicrobial and antibiofilm effects of TiO2 nanoparticles can be synergically elevated in combination with probiotic Nisin producing L. lactis.

In as much as spontaneous fermentation of cowpea leaves enhances product diversification, the process is rather slow with poor product quality. Limited work has been undertaken to provide input toward standardization of the process and enhancing of product quality. The current study sought to evaluate the in‐process fermentative bacteria profile changes due to the effect of optimization of fermentation process of cowpea leaves. Lactic acid bacteria (LAB) isolates from spontaneous and optimized fermentation were characterized using biochemical tests, whereby optimization was done using the Response Surface Methodology model of the central composite design in the Design Expert Software. The RSM models accounted for 89% and 60% variability in the response variables of pH and titratable acidity, respectively (p, Evaluation of acidity was a good indicator of fermentation process. Addition of sugar and salt improved the fermentation process of the cowpea leaves. Carbohydrate fermentation trends showed dominance of thirteen different LAB isolates.

In this study, we have investigated the cheese starter culture as a microbial community through a question: can the metabolic behaviour of a co‐culture be explained by the characterized individual organism that constituted the co‐culture? To address this question, the dairy‐origin lactic acid bacteria Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, Streptococcus thermophilus and Leuconostoc mesenteroides, commonly used in cheese starter cultures, were grown in pure and four different co‐cultures. We used a dynamic metabolic modelling approach based on the integration of the genome‐scale metabolic networks of the involved organisms to simulate the co‐cultures. The strain‐specific kinetic parameters of dynamic models were estimated using the pure culture experiments and they were subsequently applied to co‐culture models. Biomass, carbon source, lactic acid and most of the amino acid concentration profiles simulated by the co‐culture models fit closely to the experimental results and the co‐culture models explained the mechanisms behind the dynamic microbial abundance. We then applied the co‐culture models to estimate further information on the co‐cultures that could not be obtained by the experimental method used. This includes estimation of the profile of various metabolites in the co‐culture medium such as flavour compounds produced and the individual organism level metabolic exchange flux profiles, which revealed the potential metabolic interactions between organisms in the co‐cultures., Co‐cultures of lactic acid bacteria were investigated using dynamic genome‐scale metabolic modelling techniques. Özcan and co‐workers estimated the metabolic capacity, microbial abundance, potential of flavour compounds production and metabolic interactions of the co‐cultures using the features of individual organisms that constituted the co‐cultures.

11 p.-5 fig.-3 tab., Background: Isobutanol is a candidate to replace gasoline from fossil resources. This higher alcohol can be produced from sugars using genetically modified microorganisms. Shimwellia blattae (p424IbPSO) is a robust strain resistant to high concentration of isobutanol that can achieve a high production rate of this alcohol. Nevertheless, this strain, like most strains developed for isobutanol production, has some limitations in its metabolic pathway. Isobutanol production under anaerobic conditions leads to a depletion of NADPH, which is necessary for two enzymes in the metabolic pathway. In this work, two independent approaches have been studied to mitigate the co-substrates imbalance: (i) using a NADH-dependent alcohol dehydrogenase to reduce the NADPH dependence of the pathway and (ii) using a transhydrogenase to increase NADPH level., Results: The addition of the NADH-dependent alcohol dehydrogenase from Lactococcus lactis (AdhA) to S. blattae (p424IbPSO) resulted in a 19.3% higher isobutanol production. The recombinant strain S. blattae (p424IbPSO, pIZpntAB) harboring the PntAB transhydrogenase produced 39.0% more isobutanol than the original strain, reaching 5.98 g L−1 of isobutanol. In both strains, we observed a significant decrease in the yields of by-products such as lactic acid or ethanol., Conclusions: The isobutanol biosynthesis pathway in S. blattae (p424IbPSO) uses the endogenous NADPH-dependent alcohol dehydrogenase YqhD to complete the pathway. The addition of NADH-dependent AdhA leads to a reduction in the consumption of NADPH that is a bottleneck of the pathway. The higher consumption of NADH by AdhA reduces the availability of NADH required for the transformation of pyruvate into lactic acid and ethanol. On the other hand, the expression of PntAB from E. coli increases the availability of NADPH for IlvC and YqhD and at the same time reduces the availability of NADH and thus, the production of lactic acid and ethanol. In this work it is shown how the expression of AdhA and PntAB enzymes in Shimwellia blattae increases yield from 11.9% to 14.4% and 16.4%, respectively., This work was supported by grants from Biopolis S.L., Spanish Ministry of Science and Innovation BIOSOS CEN20091040 (CENIT-CDTI), RTI2018-095584-B-C44 and MINECO CTQ2017- 84963-C2-1-R and the Community of Madrid and the Structural Funds from European Union (Ref: S2018/BAA-4532 (ALGATEC-CM)). The grant for one of the authors (MGA) with reference BES-2014-068344 is gratefully recognized.

Background In bioengineering, growth of microorganisms is limited because of environmental and industrial stresses during fermentation. This study aimed to construct a nisin-producing chassis Lactococcus lactis strain with genome-streamlined, low metabolic burden, and multi-stress tolerance characteristics. Results The Cre-loxP recombination system was applied to reduce the genome and obtain the target chassis strain. A prophage-related fragment (PRF; 19,739 bp) in the L. lactis N8 genome was deleted, and the mutant strain L. lactis N8-1 was chosen for multi-stress tolerance studies. Nisin immunity of L. lactis N8-1 was increased to 6500 IU/mL, which was 44.44% higher than that of the wild-type L. lactis N8 (4500 IU/mL). The survival rates of L. lactis N8-1 treated with lysozyme for 2 h and lactic acid for 1 h were 1000- and 10,000-fold higher than that of the wild-type strain, respectively. At 39 ℃, the L. lactis N8-1 could still maintain its growth, whereas the growth of the wild-type strain dramatically dropped. Scanning electron microscopy showed that the cell wall integrity of L. lactis N8-1 was well maintained after lysozyme treatment. Tandem mass tags labeled quantitative proteomics revealed that 33 and 9 proteins were significantly upregulated and downregulated, respectively, in L. lactis N8-1. These differential proteins were involved in carbohydrate and energy transport/metabolism, biosynthesis of cell wall and cell surface proteins. Conclusions PRF deletion was proven to be an efficient strategy to achieve multi-stress tolerance and nisin immunity in L. lactis, thereby providing a new perspective for industrially obtaining engineered strains with multi-stress tolerance and expanding the application of lactic acid bacteria in biotechnology and synthetic biology. Besides, the importance of PRF, which can confer vital phenotypes to bacteria, was established.

Shark can be a potential source for host-associated probiotics destined to be used in aquaculture. In this study, intestinal tissues of Black tip shark (Carcharhinus limbatus) was used as the source sample for inhibition screening based on spot-on-lawn and agar well diffusion methods. Out of the 80 isolates, only four showed antagonistic activities against selected indicator strains. All of the 4 isolates FA1, FA2, FA3 and FA4 were found to be Gram positive coccus, non-spore former, oxidase and catalase negatives, as well as lactose fermenters. The isolate FA1, FA2, FA3 and FA4 were found to demonstrate broad range of inhibitory spectrum on gram-positive indicator bacterium (Staphylococcus aureus and Bacillus cereus) and the gram-negative bacterium (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Vibrio alginolyticus and Vibrio parahaemolyticus); with the highest inhibition zone at 20.0±0.1 mm, recorded on V. parahaemolyticus. The genotype of the isolates was characterized using 16S rRNA sequencing. Each sequence with a given GenBank (NCBI) accession number (MN975529 for FA1; MN982712 for FA2; MN982711 for FA3; and MN982710 for FA4) showed at least 99% similarity with Lactococcus lactis subspecies. The crude cell free supernatant (CFS) of the isolates potentially contained a putative bacteriocin displaying nisin-like properties. The pH and catalase treatments showed that neither organic acid nor hydrogen peroxide (H2O2) was the inhibitory component. An almost 95% inactivation of the CFS’s antimicrobial activity was observed following treatment with trypsin suggested a presence of proteinaceous agent. Growth and inhibition studies on all isolated strains demonstrated that the inhibition properties were growth associated, with maximum inhibition achieved at ~20.0 ± 0.1 mm (or ~168 ± 17 AU/ml) at 17 hours by FA2 strain against V. parahaemolyticus. A study on the mode of inhibition demonstrated a bactericidal killing against V. parahaemolyticus. The isolation of Lactococcus lactis strain from shark is rather unique since it was commonly reported to be isolated from human and animals. These strains show bacteriocinogenic properties, broad spectrum inhibition, and more importantly, they are able to antagonise some pathogens implicated in fish or shrimp diseases. Therefore, these strains have potential for use as probiotics in aquaculture.

Biosynthesis of zinc oxide nanoparticles was carried out by two bacterial strains namely Lactococcus lactis NCDO1281(T) and Bacillus sp PTCC 1538. The produced particles were characterized using X-...

International audience; Nisin is a small peptide produced byLactococcus lactisssplactisthat is currently industrially produced. This preservative is often used for growth prevention of pathogenic bacteria contaminating the food products. However, the use of nisin as a food preservative is limited by its low production during fermentation. This low production is mainly attributed to the multitude of parameters influencing the fermentation progress such as bacterial cells activity, growth medium composition (namely carbon and nitrogen sources), pH, ionic strength, temperature, and aeration. This review article focuses on the main parameters that affect nisin production byLactococcus lactisbacteria. Moreover, nisin applications as a food preservative and the main strategies generally used are also discussed.

1,2,4-Butanetriol (BT) is used as a precursor for the synthesis of various pharmaceuticals and the energetic plasticizer 1,2,4-butanetriol trinitrate. In Saccharomyces cerevisiae, BT is biosynthesized from xylose via heterologous four enzymatic reactions catalyzed by xylose dehydrogenase, xylonate dehydratase, 2-ketoacid decarboxylase, and alcohol dehydrogenase. We here aimed to improve the BT yield in S. cerevisiae by genetic engineering. First, the amount of the key intermediate 2-keto-3-deoxy-xylonate as described previously was successfully reduced in 41% by multiple integrations of Lactococcus lactis 2-ketoacid decarboxylase gene kdcA into the yeast genome. Since the heterologous BT synthetic pathway is independent of yeast native metabolism, this manipulation has led to NADH/NADPH imbalance and deficiency during BT production. Overexpression of the NADH kinase POS5Δ17 lacking the mitochondrial targeting sequence to relieve NADH/NADPH imbalance resulted in the BT titer of 2.2 g/L (31% molar yield). Feeding low concentrations of glucose and xylose to support the supply of NADH resulted in BT titer of 6.6 g/L with (57% molar yield). Collectively, improving the NADH/NADPH ratio and supply from glucose are essential for the construction of a xylose pathway, such as the BT synthetic pathway, independent of native yeast metabolism.

Bacteriocins produced by various lactic acid bacteria (LAB) have found enormous use in the food industries as biopreservatives. This study evaluated the effect of varied culture conditions (temperature, pH, and NaCl concentration) on bacteriocin production by Lactococcus lactis MT186647 isolated from fermenting African oil bean seeds (Pentaclethra macrophylla Benth) using response surface methodology. A three-factor central composite design (CCD) was adopted with the interest of estimating the optimal conditions for its production using the response surface regression model, which evaluated the linear, squared, and interactive relationship between the response variables. The analysis of variance (ANOVA) using Minitab statistical software version 14.13 showed an R2 = 0.869 variations in the response variable for culture conditions. It was accounted for by the predictors suggesting that the model was adequate. The optimal culture condition for bacteriocin production by L. lactis was estimated at 30.5C, pH 5.9, 1.94% NaCl concentration at Y = 12.31 mm where Y represents the response (zone of inhibition) against Staphylococcus aureus ATCC 19095 using the agar well diffusion assay method. Validation of optimal values according to the regression model, produced an inhibition zone at Y = 13.33 ± 0.29 mm. There was a 19.33% increase in bacteriocin activity compared to an OFAT optimized medium from a previous study.

Lactococcus lactis has been reported unable to directly incorporate mononucleotides but instead requires their external dephosphorylation by nucleotidases to the corresponding nucleosides prior to ...

The development of sustainable methods with robust strains and renewable substrates for the production of high-value chemicals has gained much attention recently. This study investigates the effect of ten different microbial strains for lactic acid production from food waste through simultaneous saccharification and fermentation process at optimal conditions of pH 5.5–6.5 at 30 °C. The highest lactic acid concentration of 18.69 g L-1 was obtained from Lacobacillus manihotivorans DSM 13343, followed by 17.03 g L-1 from Lactobacillus plantarum DSM 20174 and 15.88 g L-1 from mixed culture during fermentation of food waste, whereas the strains produced only 12.72 g L-1, 17.75 g L-1, and 9.38 g L-1 respectively from MRS broth, therefore showing that the food waste was a superior substrate compared to the MRS broth for lactic acid fermentation. The maximum lactic acid yield was 0.73 g g-1, 0.71 g g-1, and 0.69 g g-1 with Lacobacillus manihotivorans DSM 13343, Lactococcus lactis subsp. lactis DSM 20481, and Lactobacillus plantarum DSM 20174 respectively with high selectivity for lactic acid up to 84%. Furthermore, this study has achieved significant lactic acid production with increased substrate utilization in lower processing time and reactor volume.

Lactobacillus paracasei SMN-LBK (serial number: CCTCC M 2017429) is an ethanol-resistant lactic acid bacteria (LAB) in kumiss. However, the anti-ethanol stress mechanism of L. paracasei SMN-LBK remains unclear. Hence, we performed a transcriptome analysis between L. paracasei SX10 (L. paracasei SMN-LBK under 10% ethanol stress strain, abbreviated as SX10) and L. paracasei SMN-LBK (abbreviated as S10) by RNA sequencing. We performed real-time quantitative PCR (RT-qPCR) to verify the accuracy of the transcription data. The transcriptome data revealed that 315 genes exhibited upregulated expression, and 332 genes were downregulated in the SX10 compared with the S10 group. The PFK, LDH, GPDH, and GK genes were upregulated, with a log2-fold change of 1.10, 0.30, 0.56, and 1.512, respectively. A gene ontology enrichment analysis revealed significant enrichment of ribosomes, ribonucleoprotein complex, non-membrane-bounded organelles, and intracellular non-membrane-bound organelles. Analysis using the Kyoto Encyclopedia of Genes and Genomes database revealed differential genes associated with ribosome function, pyruvate metabolism, biosynthesis of amino acids, fatty acid biosynthesis, fatty acid metabolism, ATP-binding cassette (ABC) transporter, glycolysis, and glycerophospholipid metabolism. The RT-qPCR results were consistent with the transcriptome results. Lactococcus lactis NZ9000 is a typical host bacterium. We performed PFK and GK overexpression to verify the function of the L. paracaseiSX10 resistance gene in Lactococcus lactis NZ9000. Using sodium dodecyl sulfate (SDS)-PAGE electrophoresis, these resistance genes were successfully expressed in Lactococcus lactis NZ9000. The survival rate and key enzyme activity of the recombinant strains were determined under ethanol stress. The survival rate of Lactococcus lactis NZ9000-pNZ8148-PFK and Lactococcus lactis NZ9000-pNZ8148-GK under 10% ethanol stress were 3.43- and 3.80-fold higher compared with the Lactococcus lactis NZ9000-pNZ8148 control, respectively. These results indicate that PFK and GK are important for the ethanol tolerance of LAB and can increase the ethanol tolerance of Lactococcus lactis NZ9000. Hence, PFK and GK were identified as key genes of L. paracasei SX10 with a high ethanol tolerance. Our results provide novel insight for further studies to perform a systematic analysis of the differentially expressed genes and to determine their potential functions in the ethanol tolerance mechanism of LAB.

Food preservation is a big challenge for the industries. The mechanisms of preservation involved microorganisms such as lactic acid bacteria. Modeling the micro-organisms growth is a very useful information to well-understand their behavior and to propose the best conditions for food preservation. In this way, this study was to propose a mathematical model of the dynamical growth of Lactococcus lactis CWBI-B1410 which play a main role for the bio-conservation of fish. This study is conducted by varying the concentration of glucose and nitrogenized matter. The results showed that the mathematical model predicted the growth of L. lactis CWBI-B1410. The curves that are predicted are exactly those that can be observed experimentally. The results showed that the production of biomass by L. lactis CWBI-B1410 is obtained on the MRS medium composed of 15 g of glucose per liter and 40 g of nitrogenous material per liter. From the obtained results, it could be clearly advocated that for two tests with the same amount of nitrogenous matter, it is possible to achieve economies of scale in glucose while maintaining the same biomass performance. Key words: Mathematical models, lactic bacteria, Lactococcus lactis CWBI-B1410, glucose, nitrogenous matter.

Glycolate is an important α-hydroxy acid with a wide range of industrial applications. The current industrial production of glycolate mainly depends on chemical synthesis, but biochemical production from renewable resources using engineered microorganisms is increasingly viewed as an attractive alternative. Crude glycerol is an abundant byproduct of biodiesel production and a widely investigated potential sustainable feedstock. Here, we constructed a novel biosynthetic pathway for the production of glycolate from glycerol in Escherichia coli. The pathway starts from the oxidation of glycerol to d-glycerate by alditol oxidase, followed by sequential enzymatic dehydrogenation and decarboxylation as well as reduction reactions. We screened and characterized the catalytic activity of candidate enzymes, and a variant of alditol oxidase from Streptomyces coelicolor A3(2), 2-hydroxyglutarate-pyruvate transhydrogenase from Saccharomyces cerevisiae, α-ketoisovalerate decarboxylase from Lactococcus lactis, and aldehyde dehydrogenase from Escherichia coli were selected and assembled to create an artificial operon for the biosynthetic production of glycolate from glycerol. We also characterized the native strong constitutive promoter Plpp from E. coli and compared it with the PT7 promoter, which was employed to express the artificial operon on the plasmid pSC105-ADKA. To redirect glycerol flux toward glycolate synthesis, we deleted key genes of the native glycerol assimilation pathways and other branches of native E. coli metabolism, and we introduced a second plasmid expressing Dld3 to reduce the accumulation of the intermediate d-glycerate. Finally, the engineered strain TZ-108 harboring pSC105-ADKA and pACYC184-Plpp-Dld3 produced 0.64 g/L glycolate in shake flasks, which was increased to 4.74 g/L in fed-batch fermentation. This study provides an alternative pathway for glycolate synthesis and demonstrates the potential for producing other commodity chemicals by redesigning glycerol metabolism.

Temperate bacteriophages can enter one of two life cycles following infection of a sensitive host: the lysogenic or the lytic life cycle. The choice between the two alternative life cycles is dependent upon a tight regulation of promoters and their cognate regulatory proteins within the phage genome. We investigated the genetic switch of TP901-1, a bacteriophage of Lactococcus lactis, controlled by the CI repressor and the modulator of repression (MOR) antirepressor and their interactions with DNA. We determined the solution structure of MOR, and we solved the crystal structure of MOR in complex with the N-terminal domain of CI, revealing the structural basis of MOR inhibition of CI binding to the DNA operator sites. 15N NMR Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion and rotating frame R1ρ measurements demonstrate that MOR displays molecular recognition dynamics on two different time scales involving a repacking of aromatic residues at the interface with CI. Mutations in the CI:MOR binding interface impair complex formation in vitro, and when introduced in vivo, the bacteriophage switch is unable to choose the lytic life cycle showing that the CI:MOR complex is essential for proper functioning of the genetic switch. On the basis of sequence alignments, we show that the structural features of the MOR:CI complex are likely conserved among a larger family of bacteriophages from human pathogens implicated in transfer of antibiotic resistance.

Aim of the study Effect of internalized phthalyl starch nanoparticles (PSNs) on the antimicrobial ability of Lactococcus lactis (LL) KCTC 2013. Methods and results Phthalyl starch nanoparticles were prepared by self-assembly of phthalyl starch and the amount of the hydrophobic phthalic moieties were characterized by nuclear magnetic resonance: PSN1 (DS: 14·3 mol.%), PSN2 (DS: 17·8 mol.%) and PSN3 (DS: 30·4 mol.%). The sizes of PSN1, PSN2 and PSN3 measured by dynamic light scattering were 364·7, 248·4 and 213·4 nm, respectively, and the surface charges of PSNs measured by electrophoretic light scattering were negative charges and PSNs were spherical in shape according to scanning electron microscope. It was found that when PSNs were treated with LL, the PSNs were internalized into LL through nanoparticle size-, energy- and glucose transporter-dependent mechanisms. The internalization was confirmed by confocal laser scanning microscopy and fluorescence-activated cell sorting. Nisin was isolated and identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Also, more nisin was produced from PSNs-treated LL than untreated- or starch-treated LL. Co-culture assay and agar diffusion test were performed to test the antimicrobial ability. Antimicrobial ability against Gram-negative Escherichia coli k88, Salmonella gallinarum and Gram-positive Listeria monocytogenes of LL treated with PSNs was higher than that of untreated or starch-treated group. Finally, it was found that the expression level of stress response genes dnaK, dnaJ and groES was significantly higher in PSNs-treated groups compared with starch-treated group or LL alone. Conclusion The internalization of PSNs into LL enhanced the production of nisin through mild intracellular stimulation, resulting in enhanced antimicrobial ability. Significance and impact of the study This study shows the promising potential of PSNs as new prebiotics for increasing the production of nisin, thus demonstrating a new method for the biological production of such antimicrobial peptides.

In this study, a fermented whey-based formulate with enhanced levels of gamma aminobutyric acid (GABA) was produced by using a selected lactic acid bacteria isolated from traditional cabbage pickle. 16S rRNA gene sequencing resulted in identification of high GABA-producing isolate as Lactobacillus plantarum which was used as single or co-culture with Lactococcus lactis subsp. lactis (a proteolytic strain from the microbial bank). The isolated L. plantarum showed higher proteolytic, acidification and GABA-producing activity in cheese whey compared to Lc. lactis Subsp. lactis. However, the highest concentration of GABA (365.6 mg/100 mL) was found in the samples fermented using co-cultures of these strains. Moreover, addition of soy protein hydrolysate (SPH) to whey resulted in a significant enhancement in GABA concentration in the final product upon fermentation. Moreover, in the co-cultured sample containing higher amount of SPH, the lower fermentation time was observed. The results of this study showed that L. plantarum isolated from cabbage pickle is a good candidate to be used as p starter culture to manufacture a novel functional whey formulate enriched with GABA.

Lactococcus lactis is a Gram-positive bacterium that is widely used as a cell factory for the expression of heterologous proteins that are relevant in the pharmaceutical and nutraceutical fields. The signal peptide of the major secreted protein of L. lactis, Usp45, has been employed extensively in engineering strategies to secrete proteins of interest. However, the biological function of Usp45 has remained obscure despite more than 25 years of research. Studies on Usp45 homologs in other Gram-positive bacteria suggest that Usp45 may play a role in cell wall turnover processes. Here, we show the effect of inactivation and overexpression of the usp45 gene on L. lactis growth, phenotype, and cell division. Our results are in agreement with those obtained in streptococci and demonstrate that the L. lactis Usp45 protein is essential for proper cell division. We also show that the usp45 promoter is highly activated by galactose. Overall, our results indicate that Usp45 mediates cell separation, probably by acting as a peptidoglycan hydrolase. IMPORTANCE The cell wall, composed mainly of peptidoglycan, is key to maintaining the cell shape and protecting the cell from bursting. Peptidoglycan degradation by peptidoglycan hydrolysis and autolysins occurs during growth and cell division. Since peptidoglycan hydrolases are important for virulence, envelope integrity, and regulation of cell division, it is valuable to investigate their function and regulation. Notably, PcsB-like proteins such as Usp45 have been proposed as new targets for antimicrobial drugs and could also be target for the development of food-grade suicide systems. In addition, although various other expression and secretion systems have been developed for use in Lactococcus lactis, the most-used signal peptide for protein secretion in this bacterium is that of the Usp45 protein. Thus, elucidating the biological function of Usp45 and determining the factors affecting its expression would contribute to optimize several applications.

The objective of this study was to investigate the effects of cellulolytic lactic acid bacteria on the lignocellulose degradation, sugar profile and lactic acid fermentation of high-moisture alfalfa during anaerobic bioprocessing in low-temperature seasons. Last cut and low dry matter (DM) alfalfa was ensiled with five additives, commercial cellulase (EN), a combination of Lactobacillus plantarum and commercial cellulase (LPEN), a combination of engineered Lactococcus lactis strains (HT2) containing cellulase gene, Enterococcus faecium Y83 (EF83), or Enterococcus faecalis JF85 (EF85), and without additives, as the control, with four replicates each. After anaerobic preservation in a silo from late fall through winter (3 to 20 °C) for 140 days, the ensiled alfalfa was sampled and analysed. Compared with the control, EF85 increased the relative contents of neutral detergent fibre (NDF), acid detergent fibre (ADF), and cellulose and decreased contents of water-soluble carbohydrates (WSCs) and lactic acid, and the conversion of WSCs into lactic acid. Consequently, lactic acid fermentation was decreased in EF85. Compared with the control, supplementation with EN, EF83, HT2, and LPEN decreased contents of NDF, ADF, cellulose, and hemicellulose and increased the contents of WSCs, disaccharide, xylose, and arabinose; interestingly, compared with EN, supplementation with EF83, HT2 and LPEN increased the lactic acid content and the ratio of lactic acid to acetic acid as indicated by strong homo-lactic acid fermentation. Furthermore, HT2 increased the lactic acid concentration to a maximum of 72.2 g/kg DM and had a higher conversion of WSCs into lactic acid than EF83 (36.3%) and LPEN (17.9%), indicating that HT2 had a greater ability to ferment lignocellulose of alfalfa into lactic acid. Therefore, HT2 could enhance the lactic acid production of alfalfa during ensiling in low-temperature seasons.

This work aimed to study the potential of defatted rice bran (DRB) and defatted soybean meal (DSM) as carbon and nitrogen sources for Lactococcus lactis growth and nisin production. First, a maximum nisin yield of 3630 IU/mL was achieved using 40% DRB hydrolysates and 30% DSM hydrolysates, which was 1.13 times greater than that found in commercial media. Second, to simplify the operation and shorten the length of the entire process, the processes of combined hydrolysis of DRB-DSM followed by fermentation, and simultaneous hydrolysis and fermentation of DRB-DSM were developed. Neutral proteinase enhanced the saccharification of DRB by cellulase and α-amylase. Furthermore, the strategy of NADH oxidase expression and hemin addition was innovatively proposed to overcome the oxygen stress in a simultaneous hydrolysis and fermentation process, which could alleviate the lag period following inoculation of L. lactis and result in a 77.3% increase in nisin titer.

Lactic acid bacteria (LAB) has been documented to promoting growth, enhancing immunity and disease resistance. In this study, we aimed to evaluate the single or conjoint effects of Lactococcus lactis L19 (Genbank: MT102745.1) and Enterococcus faecalis W24 (Genbank: MT102746.1) isolated from the intestine of Channa argus (C. argus) on growth performance, immune response and disease resistance of C. argus. A total of 720 apparently healthy C. argus (9.50 ± 0.03 g) were randomly divided into four equal groups. Fish were fed with a basal diet (CK) supplemented with L. lactis (L19), E. faecalis (W24), and L. lactis L19 + E. faecalis W24 (L + W) at 1.0 × 108 cfu/g basal diet for 56 days. After feeding, the final body weight (FBW), weight gain (WG), feed efficiency ratio (FER), specific growth rate (SGR) and protein efficiency ratio (PER) had significantly increased (p

Chemical and organoleptic properties of dairy products largely depend on the action of microorganisms that tend to be selected in cheese during ripening in response to the availability of specific substrates. The aim of this work was to evaluate the effects of a diet enriched with hemp seeds on the microbiota composition of fresh and ripened cheese produced from milk of lactating ewes. Thirty-two half-bred ewes were involved in the study, in which half (control group) received a standard diet, and the other half (experimental group) took a diet enriched with 5% hemp seeds (on a DM basis) for 35 d. The dietary supplementation significantly increased the lactose in milk, but no variations in total fat, proteins, caseins, and urea were observed. Likewise, no changes in total fat, proteins, or ash were detected in the derived cheeses. The metagenomic approach was used to characterize the microbiota of raw milk and cheese. The phyla Proteobacteria and Firmicutes were in equally high abundance in both control and experimental raw milk samples, whereas Bacteroidetes was less abundant. The scenario changed when considering the dairy products. In all cheese samples, Firmicutes was clearly predominant, with Streptococcaceae being the most abundant family in the experimental group. The reduction of taxa observed during ripening was in accordance with the increment (relative abundance) of the starter culture Lactococcus lactis and Streptococcus thermophilus, which together dominate the microbial community. The analysis of the volatile profile in ripened cheeses led to the identification of 3 major classes of compounds: free fatty acids, ketones, and aldehydes, which indicate a prevalence of lipolysis compared with the other biochemical mechanisms that characterize the cheese ripening.