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Tetrahydrofolate is a cofactor involved in C1 metabolism including biosynthesis pathways for adenine and serine. In the classical tetrahydrofolate biosynthesis pathway, the steps removing three phosphate groups from the precursor 7,8-dihydroneopterin triphosphate (DHNTP) remain unclear in many bacteria. DHNTP pyrophosphohydrolase hydrolyzes pyrophosphate from DHNTP and produces 7,8-dihydroneopterin monophosphate. Although two structurally distinct DHNTP pyrophosphohydrolases have been identified in the intestinal bacteria Lactococcus lactis and Escherichia coli, the distribution of their homologs is limited. Here, we aimed to identify a third DHNTP pyrophosphohydrolase gene in the intestinal lactic acid bacterium Limosilactobacillus reuteri. In a gene operon including genes involved in dihydrofolate biosynthesis, we focused on the lreu_1276 gene, annotated as Ham1 family protein or XTP/dITP diphosphohydrolase, as a candidate encoding DHNTP pyrophosphohydrolase. The Lreu_1276 recombinant protein was prepared using E. coli and purified. Biochemical analyses of the reaction product revealed that the Lreu_1276 protein displays significant pyrophosphohydrolase activity toward DHNTP. The optimal reaction temperature and pH were 35°C and around 7, respectively. Substrate specificity was relatively strict among 17 tested compounds. Although previously characterized DHNTP pyrophosphohydrolases prefer Mg2+, the Lreu_1276 protein exhibited maximum activity in the presence of Mn2+, with a specific activity of 28.2 ± 2.0 µmol min-1 mg-1 in the presence of 1 mM Mn2+. The three DHNTP pyrophosphohydrolases do not share structural similarity to one another, and the distribution of their homologs does not overlap, implying that the Lreu_1276 protein represents a third structurally novel DHNTP pyrophosphohydrolase in bacteria. [ABSTRACT FROM AUTHOR]

We previously showed through clinical trials that one plant-derived lactic acid bacteria (LAB) can improve constipation. We preliminarily found that the plant-derived LAB Lactococcus lactis BM32-1 can grow in a mixture of sericin and fibroin, which are extracted from silk and have been reported to help promote health. Thus, in the present study, we evaluated the favorable effect of a sericin/fibroin mixture (S/F-M), which was extracted from silk prepared from cocoons reared in an aseptic rearing system using an artificial diet, fermented with the BM32-1 strain through a clinical trial. The trial was conducted at Hiroshima University from June to October 2022 as a double-blind, placebo-controlled, randomized parallel-group comparative study with 50 eligible subjects (aged 23-71) who had an average defecation frequency of less than 5 times per week. The subjects were instructed to drink 100 mL of fermented S/F-M or placebo every day. After the 12 weeks of the clinical trial period, the average defecation frequency increased significantly--1.4 times higher than that at baseline in the test group-- as compared with the placebo group. Furthermore, the fecal microbiota was also compared before and after treatment, revealing that intake of the fermented S/F-M significantly multiplied the relative abundance of the genera Enterococcus and Clostridium, which have been reported to contribute to the amelioration of constipation by improving the gut microbiota and producing butyric acid, respectively. In conclusion, the S/F-M fermented using the BM32-1 strain improves defecation frequency through alteration of the gut microbiota. [ABSTRACT FROM AUTHOR]

This study aimed to unleash the potential of indigenous lactic acid bacteria (LAB) originating from traditionally made Serbian goat cheese. Following the isolation and identification of the LAB, the safety aspects of the isolates were evaluated through tests for hemolytic activity and antibiotic sensitivity. The selected isolates were then tested for various technological properties, including growth in methylene blue, proteolytic activity, acidification, curd formation ability in both pure and enriched goat milk, diacetyl production, antagonistic potential against other LAB, and biofilm formation ability. The results indicated that Lactococcus spp., Lacticaseibacillus spp., and Lactiplantibacillus spp. did not exhibit α or β hemolysis, while enterococci displayed α hemolysis. A higher number of isolates demonstrated sensitivity to ampicillin, tetracycline, and streptomycin, while sensitivity to gentamicin and vancomycin was strain-dependent. Based on the evaluation of technological properties, Lacticaseibacillus paracasei M-1 and Lactiplantibacillus plantarum C7-7, C7-8, and C14-5 showed promising characteristics. Additionally, Lactococcus lactis subsp. lactis strains C0-14 and C21-8 emerged as promising candidates with notable technological properties. Notably, certain indigenous strains LAB exhibit promising technological properties and safety profiles. These characteristics make them suitable candidates for use as starter or adjunct cultures in goat's milk cheese production, potentially enhancing the quality and safety of the cheese as well as hygiene practices among small-scale dairy producers. [ABSTRACT FROM AUTHOR]

During malolactic fermentation (MLF) of vinification, the harsh L–malic acid undergoes transformation into the milder L–lactic acid, and via decarboxylation reactions it is catalyzed by malolactic enzymes in LAB. The use of bacterial malolactic starter cultures, which usually present challenges in the industry as the suboptimal conditions after alcoholic fermentation (AF), including nutrient limitations, low temperatures, acidic pH levels, elevated alcohol, and sulfur dioxide concentrations after AF, lead to "stuck" or "sluggish" MLF and spoilage of wines. Saccharomyces uvarum has interesting oenological properties and provides a stronger aromatic intensity than Saccharomyces cerevisiae in AF. In the study, the biological pathways of deacidification were constructed in S. uvarum, which made the S. uvarum carry out the AF and MLF simultaneously, as different genes encoding malolactic enzyme (mleS or mleA), malic enzyme (MAE2), and malate permease (melP or MAE1) from Schizosaccharomyces pombe, Lactococcus lactis, Oenococcus oeni, and Lactobacillus plantarum were heterologously expressed. For further inquiry, the effect of L–malic acid metabolism on the flavor balance in wine, the related flavor substances, higher alcohols, and esters production, were detected. Of all the recombinants, the strains WYm1SN with coexpression of malate permease gene MAE1 from S. pombe and malolactic enzyme gene mleS from L. lactis and WYm1m2 with coexpression of gene MAE1 and malate permease gene MAE2 from S. pombe could reduce the L–malic acid contents to about 1 g/L, and in which the mutant WYm1SN exhibited the best effect on the flavor quality improvement. [ABSTRACT FROM AUTHOR]

In recent years, the use of natural preservatives in food products has gained significant attention due to their potential health benefits and effectiveness. A standardized microbiological analysis was conducted on Slovak farm-produced lump goat cheese samples to determine the antibacterial activity of dry herbs and essential oils added to vacuum-packed goat cheese. We employed five dried herbs and five essential oils derived from the same plants. The microbiological quality of 145 fresh and vacuum-packed goat cheese samples was assessed. The number of coliform bacteria, total viable count, lactic acid bacteria, and microscopic filamentous fungi were examined in raw cheese samples stored for 12 days at 4 °C. All cheese samples were vacuum-packed (control samples were packed without vacuum). This study evaluated the potential benefits of using essential oils and dried herbs from thyme (Thymus serpyllum L.), black pepper (Piper nigrum L.), clove (Eugenia caryophyllus Thunb.), mint (Mentha × piperita L.), and basil (Ocimum basilicum L.) as preservatives. The essential oils were obtained from Hanus Ltd., Nitra, Slovakia, and were applied at a concentration of 2%. The dried herbs were obtained from Popradský čaj (Poprad, Slovakia) and Mäspoma Ltd. (Zvolen, Slovakia). The results showed that all microorganism groups were significantly reduced in cheese samples following the application of essential oils throughout the entire storage period. During the preservation of cheese samples in polyethylene bags used for vacuum packing food, Lactococcus garvieae, L. lactis, Enterobacter cloacae, and Serratia liquefaciens were the most frequently isolated microbiota. Essential oils and dried herbs demonstrated antimicrobial potential during the storage of vacuum-packed goat cheese. [ABSTRACT FROM AUTHOR]

In the global food industry, fermented dairy products are valued for their unique flavors and nutrients. Lactococcus lactis is crucial in developing these flavors during fermentation. Meeting diverse consumer flavor preferences requires the careful selection of fermentation agents. Traditional assessment methods are slow, costly, and subjective. Although electronic-nose and -tongue technologies provide objective assessments, they are mostly limited to laboratory environments. Therefore, this study developed a model to predict the electronic sensory characteristics of fermented milk. This model is based on the genomic data of Lactococcus lactis, using the DBO (Dung Beetle Optimizer) optimization algorithm combined with 10 different machine learning methods. The research results show that the combination of the DBO optimization algorithm and multi-round feature selection with a ridge regression model significantly improved the performance of the model. In the 10-fold cross-validation, the R2 values of all the electronic sensory phenotypes exceeded 0.895, indicating an excellent performance. In addition, a deep analysis of the electronic sensory data revealed an important phenomenon: the correlation between the electronic sensory phenotypes is positively related to the number of features jointly selected. Generally, a higher correlation among the electronic sensory phenotypes corresponds to a greater number of features being jointly selected. Specifically, phenotypes with high correlations exhibit from 2 to 60 times more jointly selected features than those with low correlations. This suggests that our feature selection strategy effectively identifies the key features impacting multiple phenotypes, likely originating from their regulation by similar biological pathways or metabolic processes. Overall, this study proposes a more efficient and cost-effective method for predicting the electronic sensory characteristics of milk fermented by Lactococcus lactis. It helps to screen and optimize fermenting agents with desirable flavor characteristics, thereby driving innovation and development in the dairy industry and enhancing the product quality and market competitiveness. [ABSTRACT FROM AUTHOR]

This study aimed to assess the molecular screening and safety of 14 lactic acid bacteria isolates for their capacity to produce antimicrobial peptides from milk proteins. The isolates were molecularly identified as Enterococcus faecium, Enterococcus durans, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. hordniae, Lactiplantibacillus plantarum, and Weissella confusa. The tested lactococci isolates produced bioactive peptides with a wild‐type antimicrobial spectrum against Salmonella typhimurium, Pseudomonas aeruginosa, Bacillus cereus, and Listeria monocytogenes. The majority of isolates were susceptible to chloramphenicol, tetracycline, and ampicillin but resistant to nalidixic acid. Lactococcus lactis FFNL‐2034 and Weissella confusa FFNL‐1850 presented antibiotic multiresistance. Antibiotic resistance genes bla, ermB, tetK, cat, vanA, and vanC were absent in most of the tested isolates. The ermC, strA, strB, and tetM genes were positive in some isolates. These results provide safe LAB, which could act as a potent biopreservative agent for functional dairy products. [ABSTRACT FROM AUTHOR]

Introduction: This study aimed to explore the anti-oxidative and antiinflammatory properties of Lactococcus lactis subsp. lactis HFY14 (LLSLHFY14) and investigate its effects on the intestinal barrier, cranial nerve, and motor function in mice treated with antibiotics. Methods: Mice were administered an antibiotic mixture (neomycin 5 mg/mL, vancomycin 25 mg/mL, amphotericin B 0.1 mg/mL, ampicillin 10 mg/mL, metronidazole file 5 mg/mL, and lipopolysaccharide 1.5 mg/mL) intraperitoneally, and oxidative stress and inflammatory markers in the serum and brain tissues, and liver index were measured. H&E staining was performed to detect pathological alterations in brain tissues. The expression of intestinalbarrier- related genes and that of genes involved in inflammatory pathways in the brain were detected using polymerase chain reaction (PCR). Results: LLSLHFY14 administration extended the weight-loaded swimming and running times of mice and decreased the liver index. Moreover, the levels of malondialdehyde (MDA), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-a) in the serum and brain tissue were reduced, whereas those of superoxide dismutase (SOD), glutathione (GSH), and interleukin-10 (IL-10) were elevated. Elevated brain expression of the protein kinase B (AKT)/cAMPresponse element binding protein (CREB)/brain-derived neurotrophic factor (BDNF)/extracellular signal-regulated kinase 1 (ERK1) pathway, decreased brain expression of the IL-6 gene, and elevated cecum expression of zonula occludens-1 (ZO-1), occludin-1, and claudin-1 genes were noted. LLSLHFY14 supplementation significantly increased Bacteroidetes expression but decreased Firmicutes expression, thus increasing the Bacteroidetes/Firmicutes ratio. Discussion: Overall, LLSLHFY14 supplementation ameliorated antibioticinduced oxidative stress and inflammation in the mouse central nervous system, intestinal barrier dysfunction, and increased motor function, thus confirming its potential application as probiotics. [ABSTRACT FROM AUTHOR]

The application of transition metal complexes for antimicrobial photodynamic therapy (PDT) has emerged as an attractive alternative in mitigating a broad range of bacterial pathogens, including multidrug‐resistant pathogens. In view of their photostability, long excited‐state lifetimes, and tunable emission properties, transition metal complexes also contribute as bioimaging agents. In the present work, we designed mono and trinuclear cyclometalated iridium (III) complexes to explore their imaging application and antibacterial potential. For this, we used Methicillin‐resistant S. aureus (MRSA), the most prevalent of community‐associated (CA) multidrug‐resistant (MDR) bacteria (CA MDR) and Lactococcus lactis (L. lactis) as Gram‐positive while Campylobacter jejuni (C. jejuni) and E. coli as Gram‐negative bacteria. In addition to differential bioimaging of these bacteria, we assessed the antibacterial effects of both mono and trinuclear Ir(III) complexes under exposure to 427 nm LED light. The data presented herein strongly suggest better efficacy of trinuclear Ir(III) complex over the mononuclear complex in imparting photoinduced cell death of MRSA. Based on the safety profile of these complexes, we propose that trinuclear cyclometalated iridium(III) complex holds great promise for selective recognition and targeting MDR bacteria with minimal off‐target effect. [ABSTRACT FROM AUTHOR]

Introduction: Kefir beverage has beneficial microorganisms that have healthgiving properties; therefore, they have a good potential to be probiotic. This study evaluated the probiotic potential, technological, and safety characteristics of Enterococcus faecalis, Lactococcus lactis, and Pichia fermentans isolated from traditional kefir beverages. Method: First, isolates were evaluated in terms of resistance to acid, alkali, bile salts, trypsin, and pepsin of the gastrointestinal tract. The auto-aggregation and co-aggregation ability of isolates were measured using spectrophotometry. Antimicrobial activities were assayed against important food-borne pathogens using the agar well diffusion method. Moreover, gamma-aminobutyric acid (GABA) production was investigated by thin-layer chromatography (TLC). Result: Among the isolates, P. fermentans had an 85% total survival rate, but its amount reached below 6 log CFU/ml which is considered non-resistant, and it showed the highest auto-aggregation (74.67%). Moreover, only L. lactis showed antimicrobial activity and had the highest co-aggregation with E. coli PTCC 1338 (54.33%) and L. monocytogenes ATCC 7644 (78%). Finally, an evaluation of the technological and safety characteristics of the strains showed that the strains produced GABA and were safe. Discussion: Although the isolates were not resistant to the gastrointestinal tract, their supernatant contained valuable natural compounds, including antioxidants, GABA, and antimicrobials, which can be used to produce functional foods and medicines. In addition, other approaches, such as increasing the initial number of strains, using foods as carriers of isolates, and encapsulating the isolates, can effectively increase the survivability of isolates in the gastrointestinal tract. [ABSTRACT FROM AUTHOR]

[Display omitted] • The biological CO production in the compost pile was carried out by bacteria. • Bacteria isolated from compost produced CO with concentration > 1000 ppm. • The most common genus in compost was Bacillus with four representatives. • B. paralicheniformis and B. licheniformis produced the highest concentrations of CO. • High CO concentrations were accompanied by low O 2 (<6%) and high CO 2 levels (>8%) Carbon monoxide (CO) formation has been observed during composting of various fractions of organic waste. It was reported that this production can be biotic, associated with the activity of microorganisms. However, there are no sources considering the microbial communities producing CO production in compost. This preliminary research aimed to isolate and identify microorganisms potentially responsible for the CO production in compost collected from two areas of the biowaste pile: with low (118 ppm) and high CO concentration (785 ppm). Study proved that all isolates were bacterial strains with the majority of rod-shaped Gram-positive bacteria. Both places can be inhabited by the same bacterial strains, e.g. Bacillus licheniformis and Paenibacillus lactis. The most common were Bacillus (B. licheniformis, B. haynesii, B. paralicheniformis, and B. thermolactis). After incubation of isolates in sealed bioreactors for 4 days, the highest CO levels in the headspace were recorded for B. paralicheniformis (>1000 ppm), B. licheniformis (>800 ppm), and G. thermodenitrificans (∼600 ppm). High CO concentrations were accompanied by low O 2 (<6%) and high CO 2 levels (>8%). It is recommended to analyze the expression of the gene encoding CODH to confirm or exclude the ability of the identified strains to convert CO 2 to CO. [ABSTRACT FROM AUTHOR]

Raw milk cheeses harbor complex microbial communities. Some of these microorganisms are technologically essential, but undesirable microorganisms can also be present. While most of the microbial dynamics and cross-talking studies involving interaction between food-derived bacteria have been carried out on agar plates in laboratory-controlled conditions, the present study evaluated the modulation of the resident microbiota and the changes of metabolite production directly in ripening raw milk cheese inoculated with Listeria innocua strains. Using a proxy of the pathogenic Listeria monocytogenes, we aimed to establish the key microbiota players and chemical signals that characterize Latteria raw milk cheese over 60 days of ripening time. The microbiota of both the control and Listeria-inoculated cheeses was analyzed using 16S rRNA targeted amplicon sequencing, while direct analysis in real time mass spectrometry (DART-HRMS) was applied to investigate the differences in the metabolic profiles of the cheeses. The diversity analysis showed the same microbial diversity trend in both the control cheese and the inoculated cheese, while the taxonomic analysis highlighted the most representative genera of bacteria in both the control and inoculated cheese: Lactobacillus and Streptococcus. On the other hand, the metabolic fingerprints revealed that the complex interactions between resident microbiota and L. innocua were governed by continuously changing chemical signals. Changes in the amounts of small organic acids, hydroxyl fatty acids, and antimicrobial compounds, including pyroglutamic acid, hydroxy-isocaproic acid, malic acid, phenyllactic acid, and lactic acid, were observed over time in the L. innocua-inoculated cheese. In cheese that was inoculated with L. innocua, Streptococcus was significantly correlated with the volatile compounds carboxylbenzaldheyde and cyclohexanecarboxylic acid, while Lactobacillus was positively correlated with some volatile and flavor compounds (cyclohexanecarboxylic acid, pyroxidal acid, aminobenzoic acid, and vanillic acid). Therefore, we determined the metabolic markers that characterize a raw milk cheese inoculated with L. innocua, the changes in these markers with the ripening time, and the positive correlation of flavor and volatile compounds with the resident microbiota. This multi-omics approach could suggest innovative food safety strategies based on the enhanced management of undesirable microorganisms by means of strain selection in raw matrices and the addition of specific antimicrobial metabolites to prevent the growth of undesirable microorganisms. [ABSTRACT FROM AUTHOR]

Background: Intramammary infection is the result of invasion and multiplication of microorganisms in the mammary gland and commonly leads to mastitis in dairy animals. Although much has been done to improve cows' udder health, mastitis remains a significant and costly health issue for dairy farmers, especially if subclinical. In this study, quarter milk samples from clinically healthy cows were harvested to detect pathogens via quantitative PCR (qPCR) and evaluate changes in individual milk traits according to the number of quarters infected and the type of microorganism(s). A commercial qPCR kit was used for detection of Mycoplasma bovis, Mycoplasma spp., Staphylococcus aureus, coagulase-negative staphylococci (CNS), Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Prototheca spp., Escherichia coli, Klebsiella spp., Enterococcus spp. and Lactococcus lactis ssp. lactis. Quarter and pooled milk information of 383 Holstein, 132 Simmental, 129 Rendena, and 112 Jersey cows in 9 Italian single-breed herds was available. Results: Among the cows with pathogen(s) present in at least 1 quarter, CNS was the most commonly detected DNA, followed by Streptococcus uberis, Mycoplasma bovis, and Streptococcus agalactiae. Cows negative to qPCR were 206 and had the lowest milk somatic cell count. Viceversa, cows with DNA isolated in ≥ 3 quarters were those with the highest somatic cell count. Moreover, when major pathogens were isolated in ≥ 3 quarters, milk had the lowest casein index and lactose content. In animals with pathogen(s) DNA isolated, the extent with whom milk yield and major solids were impaired did not significantly differ between major and minor pathogens. Conclusions: The effect of the number of affected quarters on the pool milk quality traits was investigated in clinically healthy cows using a commercial kit. Results remark the important negative effect of subclinical udder inflammations on milk yield and quality, but more efforts should be made to investigate the presence of untargeted microorganisms, as they may be potentially dangerous for cows. For a smarter use of antimicrobials, analysis of milk via qPCR is advisable – especially in cows at dry off - to identify quarters at high risk of inflammation and thus apply a targeted/tailored treatment. [ABSTRACT FROM AUTHOR]

Background: Emerging evidence emphasized the role of oral microbiome in oral lichen planus (OLP). To date, no dominant pathogenic bacteria have been identified consistently. It is noteworthy that a decreased abundance of Streptococcus, a member of lactic acid bacteria (LAB) in OLP patients has been commonly reported, indicating its possible effect on OLP. This study aims to investigate the composition of LAB genera in OLP patients by high-throughput sequencing, and to explore the possible relationship between them. Methods: We collected saliva samples from patients with OLP (n = 21) and healthy controls (n = 22) and performed 16 S rRNA gene high-throughput sequencing. In addition, the abundance of LAB genera was comprehensively analyzed and compared between OLP and HC group. To verify the expression of Lactococcus lactis, real time PCR was conducted in buccal mucosa swab from another 14 patients with OLP and 10 HC. Furthermore, the correlation was conducted between clinical severity of OLP and LAB. Results: OLP and HC groups showed similar community richness and diversity. The members of LAB, Lactococcus and Lactococcus lactis significantly decreased in saliva of OLP cases and negatively associated with OLP severity. In addition, Lactococcus and Lactococcus lactis showed negative relationship with Fusobacterium and Aggregatibacter, which were considered as potential pathogens of OLP. Similarly, compared with healthy controls, the amount of Lactococcus lactis in mucosa lesion of OLP patients was significantly decreased. Conclusions: A lower amount of Lactococcus at genus level, Lactococcus lactis at species level was observed in OLP cases and associated with disease severity. Further studies to verify the relationship between LAB and OLP, as well as to explore the precise mechanism is needed. [ABSTRACT FROM AUTHOR]

γ-Aminobutyric acid (GABA) is an inhibitory neurotransmitter of the central nervous system that impacts physical and mental health. Low GABA levels have been documented in several diseases, including multiple sclerosis and depression, and studies suggest that GABA could improve disease outcomes in those conditions. Probiotic bacteria naturally produce GABA and have been engineered to enhance its synthesis. Strains engineered thus far use inducible expression systems that require the addition of exogenous molecules, which complicates their development as therapeutics. This study aimed to overcome this challenge by engineering Lactococcus lactis with a constitutive GABA synthesis gene cassette. GABA synthesizing and transport genes (gadB and gadC) were cloned onto plasmids downstream of constitutive L. lactis promoters [P2, P5, shortened P8 (P8s)] of different strengths and transformed into L. lactis. Fold increase in gadCB expression conferred by these promoters (P2, P5, and P8s) was 322, 422, and 627, respectively, compared to the unmodified strain (P  = 0.0325, P8s). GABA synthesis in the highest gadCB expressing strain, L. lactis -P8s-glutamic acid decarboxylase (GAD), was dependent on media supplementation with glutamic acid and significantly higher than the unmodified strain (P  < 0.0001, 125 mM, 200 mM glutamic acid). Lactococcus lactis -P8s-GAD is poised for therapeutic testing in animal models of low-GABA-associated disease. [ABSTRACT FROM AUTHOR]

The objectives of this paper were to investigate the existence of Listeria monocytogenes (L. monocytogenes) in different types of cheese (Mozzarella, Thalaga, and Romy cheeses, 40 samples each) sold in Assiut dairy shops and to evalute antibacterial properties of chitosan and its nanoparticles against isolated strains. L. monocytogenes was isolated in higher percentage from Mozzarella cheese (25%) followed by Thalaga and Romy cheese samples. L. monocytogenes isolates were validated by PCR utilising the 16S rRNA gene. Zeta-sizer and TEM were used to characterise nano-chitosan. The minimum inhibitory concentrations of chitosan and nano-chitosan on L. monocytogenes were detected by MIC test. Nano-chitosan (3.125%) could prevent organism growth in cheese as early as the third week. The findings revealed that 3.125% nano-chitosan had a sufficient effect on L. monocytogenes while also providing good consumer satisfaction; therefore, nano-chitosan should be utilised as a natural food additive in Egyptian dairy products. [ABSTRACT FROM AUTHOR]

Ice cream manufacture commonly results in the accumulation of wasted product that contains valuable food-grade quality components, including fat, carbohydrates, and protein. Methods have been developed for recovering the fat from this waste stream, but this results in the generation of a co-product rich in fermentable carbohydrates. This study aimed to investigate the potential for using this co-product as a fermentation substrate for production of antimicrobial peptides, called bacteriocins, by dairy starter cultures. Results showed that Streptococcus thermophilus B59671 and Lactococcus lactis 11454 produced the broad-spectrum bacteriocins thermophilin 110 and nisin, respectively, when the fermentation substrate was melted ice cream, or a co-product generated by a modified butter churning technique. Bacteriocin production varied depending on the brand and variety of vanilla ice cream used in this study. When an alternate enzyme-assisted fat extraction technique was used, S. thermophilus metabolism was impaired within the resulting co-product, and thermophilin 110 production was not observed. Lactococcus lactis was still able to grow in this co-product, but antimicrobial activity was not observed. Results from this study suggest the co-product generated when using the churning technique is a better choice to use as a base medium for future studies to optimize bacteriocin production. [ABSTRACT FROM AUTHOR]

Summary: This research was the first to investigate the effects of Haematococcus pluvialis microalgal biomass and colour pigment astaxanthin for the enhancement of the functional characteristics of water kefir. Kefir was evaluated for physicochemical, biochemical, and lactic acid bacteria (LAB) content after fermentation using biomass (0.25%; 0.50% with and without sugar) and astaxanthin (0.50%, no sugar) for 30 h. The addition of 0.25% biomass (W1) and astaxanthin (W4) to water kefir increased the C16:0 content almost three times and the protein content increased by 58% in W2 sample. Nanopore sequencing provided thorough insights into LAB content, revealing that in kefir containing 0.50% biomass replacing sugar (W3), Liquorilactobacillus nagelii proliferated from 8.05% to 46.57% and Lactobacillus hordei increased 15 times, while 0.25% biomass addition (W2) increased Lactococcus lactis abundance 13 times. H. pluvialis biomass was found to be an alternative prebiotic source, and it increased LAB content of water kefir. [ABSTRACT FROM AUTHOR]

In vitro ruminal culture was carried out to assess the effect of the concentration of an antibiotic resistant transgenic bacterium (Lactococcus lactis, L. lactis) along with different dietary lactose/starch ratios on first order kinetic disappearance of nutrients using an exponential model. Dietary treatments were designed to include lactose (L), using whey powder which was substituted of corn grain, as zero (L0.0), 36 (L36) and 72 (L72) g/kg dry matter (DM), with different L. lactis count (CFU/mL) as 0.0 (Bact0.0), 1.25x108 (Bact1.25) and 2.5x108 (Bact2.50) in a 3x3 factorial arrangement. The DM and CP disappearance was significantly influenced by the dietary inclusion of L. lactis. Constant rate of crude protein (CP) disappearance significantly decreased in both L36 and L72 using bacteria as Bact2.50. Bact2.50 enhanced the constant rate of neutral detergent fiber (NDF) disappearance of the diets. DM disappearance was significantly increased in lactose containing diets, while crude protein disappearance was significantly enhanced in L36 only. NDF disappearance decreased significantly in L36 and L72 treatments, and the indigestible fraction of NDF was significantly higher for L72 compared with L0.0 or L36. NFC digestible fraction was significantly improved in L36 and L72.There was significant dietx bacteria interaction in most of the studied parameters. Ruminal L. lactis abundance measured using RT-PCR altered in all diets, and the highest values were recorded in L72 diet. Regarding the dietary inclusion of lactose, some alterations were detected, while the effect of the bacteria on nutrient disappearance was consider and related to the dietary lactose/starch ratios. [ABSTRACT FROM AUTHOR]

There is a growing demand from consumers on functional food and beverages. Potential probiotics such as L. lactis could be incorporated into food and beverages as a value-added ingredient. However, sufficient viable cell counts are required to be present in the food or beverages products to confer health benefits to hosts. Therefore, this study aimed to encapsulate food-grade recombinant L. lactis NZ3900 in 1:1, 2:1, and 3:1 ratio of sodium alginate-resistant starch matrix using co-extrusion technique and evaluate its storage stability in coconut water. All the microbeads were spherical with size ranged from 528.33 to 560.67 μm. The microbeads with 2:1 ratio sodium alginate-resistant starch had the highest microencapsulation efficiency (91.1%) and highest survivability (78.4%) after 6 h of simulated sequential digestion. After 4 weeks of storage, L. lactis microbeads had higher survivability in coconut water with 11.4 log10 CFU/g and lesser reduction in total soluble solids than free cells. There was no significant difference (p > 0.05) between the coconut water with free cells and microbeads in terms of pH, titratable acidity, and viscosity after 4 weeks of storage. At the end of the storage, the coconut water with microbeads had pH 3.9, 0.32% malic acid, 5.4 °Brix, 1.4 cP of viscosity, and total colour changes of < 1.26. This study shows that co-extrusion technique with sodium alginate-resistant starch matrix are suitable for the formation of L. lactis microbeads. Moreover, the stability of the L. lactis microbeads in coconut water showed its potential to be marketed as a functional isotonic beverage. [ABSTRACT FROM AUTHOR]

A Lactococcus (L.) lactis strain producing antimicrobial and anti-inflammatory biomolecules (mainly 1,4-Diaza-2,5-dioxobicyclo[4.3.0]nonanes and pyrazine-derivatives) was tested for its capacity to cure clinical endometritis in buffaloes compared to conventional antibiotic-based treatment. Clinical endometritis-diagnosed buffaloes (n = 16/group) were infused intrauterine with four doses of 109 CFU-free (FLC group) or nanoencapsulated L. lactis (NLC group) and compared to those that received three doses of saline + a single dose of 500 mg cephapirin benzathin (AB group) or four doses of saline (control, C group) every other day. Endometrium samples were analyzed for cytological (polymorphonuclear cells, PMN), bacteriological, and proinflammatory mRNA expression. Uterine wash and blood samples were collected to determine proinflammatory cytokine concentrations and metabolites in the blood samples. The reproductive performance of buffaloes was assessed. Compared to the C group, the AB and NLC groups had the lowest percentage of PMN, followed by those in the FLC group (p < 0.05). All treated buffaloes had significantly lower numbers of pathogens than the control buffaloes. Compared to control, all treatments significantly down-regulated endometrial proinflammatory encoding mRNA expression. The concentrations of IL1B, TNFAIP7, and leukocyte esterase activity in the uterine washings were significantly decreased in the AB and NLC groups compared to the C and FLC groups. All treatments significantly decreased concentrations of serum proinflammatory cytokines compared to control. Both the AB and NLC groups had significantly lower concentrations of serum NEFA than the C and FLC groups. The percentage of control buffaloes having an echogenic uterus and PVD score > 2 was significantly higher than those in the treated buffaloes with higher numbers of corpora lutea, higher conception rates, and shorter days open than control buffaloes (p < 0.05). In conclusion, L. lactis-producing antimicrobial and anti-inflammatory metabolites reduce uterine inflammatory responses and improve fertility in buffaloes. [ABSTRACT FROM AUTHOR]

Featured Application: In this work, cheese whey, which is a by-product of the cheese industry, was used to obtain lactic acid by biotechnological processes (using strains isolated from the same cheese whey). Lactic acid has several uses in the cosmetic, food, medical, textile, and manufacturing industries. Therefore, while lactic acid can have multiple uses, we want to use it as a raw material for biopolymers for the manufacturing of bioplastics in future research. Cheese whey is a byproduct of the cheese industry that causes high levels of pollution in the environment, but its high lactose content means that it can be used as a source to obtain lactic acid. In this study, two strains, one belonging to a yeast and the other one to a bacteria (Kluyveromyces lactis and Enterococcus faecalis), were isolated from cheese whey and molecularly characterized, and the optimal growth conditions were determined. Then, using proteinized and deproteinized cheese whey, batch fermentation was carried out with the strains arranged in suspension and immobilized. The consumption of lactose and the production of lactic acid were measured through Brix degrees and acidity analysis. Afterwards, the lactic acid was purified, and its yield and physical and chemical characteristics were determined. It was proven that there were differences between each of the strains; arranged in free or encapsulated cells, the proteinized and deproteinized cheese wheys, under the same purification conditions, achieved different yields, colors, and densities of lactic acid. Immobilized Enterococcus faecalis had the highest yield (50.61 ± 34.94 g/L) using the deproteinized cheese whey compared to the immobilized Kluyveromyces lactis (35.70 ± 0.15 g/L) using the proteinized cheese whey. [ABSTRACT FROM AUTHOR]

Nisin is a bacteriocin produced by Lactococcus lactis and it is generally recognised as safe by the U.S. Food and Drug Administration. The study aimed to determine the presence of nisin-resistant Staphylococcus , Enterococcus, and Listeria spp. in 157 raw milk and 125 cheese samples. As a result of the isolation procedure, a total of 282 bacterial strains were isolated. 83/282 strains showed resistance to 300 ppm nisin concentration and they were identified by 16S rRNA sequencing. The two most common species were Enterococcus faecalis and Staphylococcus aureus. In both raw milk and cheese samples, nisin-resistant Listeria could not be detected. Generally, enterococci (59/65) showed higher biofilm formation ability than staphylococci (13/18). 38% (25/65) of enterococci and 88% (16/18) of staphylococci were determined to have protease activity in at least one of Skim Milk Agar, Casein Agar, or Milk Agar. In addition, the most common antibiotic resistance in nisin-resistant enterococci was against chloramphenicol. This study revealed that nisin-resistant staphylococci and enterococci are present in raw milk and cheese. In addition, the presence of some virulence factors such as biofilm production, protease activity, and antibiotic resistance in resistant isolates needs to be drawn attention to. Consumption of nisin-resistant microorganisms with virulence factors through food is a food safety risk. [ABSTRACT FROM AUTHOR]

Lactic acid bacteria (LAB) are the microorganisms most commonly used as probiotics. Since probiotic benefits are strain-dependent, there is a continuous need for research into new cultures with probiotic properties. Fruits such as juçara (Euterpe edulis Martius), a palm species from the Atlantic Forest threatened with extinction, are rich niches for microorganisms, including LAB. This study investigated the probiotic properties of Lactococcus lactis J7 and Leuconostoc pseudomesenteroides JF17 strains isolated from juçara fruits native to the Atlantic Forest. Probiotic characteristics, such as tolerance to simulated gastrointestinal fluids or juices, hydrophobicity, autoaggregation, coaggregation properties, inhibition of pathogenic microorganisms, and technological properties were evaluated. The survival rate of L. lactis J7 and L. pseudomesenteroides JF17 decreased after exposure to simulated gastrointestinal conditions; however, L. lactis J7 was more resistant, maintaining viability at the end of the enteric phase of 6.07 ± 0.16 log CFU mL-1. The J7 strain also exhibited the highest values in hydrophobicity (12.55 ± 0.52%), autoaggregation (25.63 ± 0.75%), and coaggregation capacity with E. coli ATCC 25922 (21.52 ± 0.98%) and S. Enteritidis ATCC 13076 (22.68 ± 1.01%). Both J7 and JF17 strains demonstrated antimicrobial activity, confirmed by the multilayer agar plate system. Additionally, the growth of the strains was temperature-dependent, and they were able to thrive in high concentrations of sodium chloride (6%). Thus, L. lactis J7 is a promising probiotic candidate for the development of functional products. Nevertheless, further studies using animal models are necessary to explore the properties of these probiotic bacteria. Novel strains isolated from fruits should be studied to broaden the application of probiotic microorganisms in the development of foods and medicines.

In this study, the pyrF screening marker and the genomic DNA fragments were used to construct the expression vectors in food-grade Lactococcus lactis (L. lactis). Such expression system could potentially be used to express and produce food-grade and medicinal polypeptides. Firstly, the NZ3900 ΔpyrF auxotrophic strain was created from the homologous recombination mutant cassette. Secondly, the repA and repC genes were used as the replication elements, the pyrF gene as the screening marker, the P32 and P8 elements from L. lactis as the promoters, and the Tusp45 and TpepN from L. lactis as the terminators, all of which were constructed in the expression plasmid pLD. Finally, the reporter gene ZsGreen (a fluorescent protein) was used to verify the expression of recombinant protein in the NZ3900 ΔpyrF mutant strain and the genetic stability of pLD-ZsG plasmid. The result showed that the prototrophic ZsGreen positive transformants could grow normally in common Elliker culture medium, and the green fluorescent signal was observed under a fluorescence microscope. In addition, ZsGreen protein could be highly expressed in NZ3900 ΔpyrF and the expression plasmid could be stably transmitted through at least 30 generations, according to the results of the PCR and Western blotting, indicating that the recombinant protein was expressed in L. lactis in a stable manner. Based on the above results, the approach for creating an L. lactis expression vector (without antibiotic resistance gene) based on the pyrF auxotrophic marker is feasible and offers a basis for further investigation into the use of L. lactis to manufacture food- and pharmaceutical-grade polypeptides.

Abstract Lactococcus lactis is widely applied by the dairy industry for the fermentation of milk into products such as cheese. Adaptation of L. lactis to the dairy environment often depends on functions encoded by mobile genetic elements (MGEs) such as plasmids. Other L. lactis MGEs that contribute to industrially relevant traits like antimicrobial production and carbohydrate utilization capacities belong to the integrative conjugative elements (ICE). Here we investigate the prevalence of ICEs in L. lactis using an automated search engine that detects colocalized, ICE-associated core-functions (involved in conjugation or mobilization) in lactococcal genomes. This approach enabled the detection of 36 candidate-ICEs in 69 L. lactis genomes. By phylogenetic analysis of conserved protein functions encoded in all lactococcal ICEs, these 36 ICEs could be classified in three main ICE-families that encompass 7 distinguishable ICE-integrases and are characterized by apparent modular-exchangeability and plasticity. Finally, we demonstrate that phylogenetic analysis of the conjugation-associated VirB4 ATPase function differentiates ICE- and plasmid-derived conjugation systems, indicating that conjugal transfer of lactococcal ICEs and plasmids involves genetically distinct machineries. Our genomic analysis and sequence-based classification of lactococcal ICEs creates a comprehensive overview of the conserved functional repertoires encoded by this family of MGEs in L. lactis, which can facilitate the future exploitation of the functional traits they encode by ICE mobilization to appropriate starter culture strains.

In this study, foodborne microorganisms capable of fermenting and decomposing soy proteins were screened, and molecular weight analysis was performed for the peptides produced during decomposition. Subsequently, oligopeptides were obtained via isolation and purification, and their antioxidant activities were studied. The experiment results showed that a PZ1 strain was isolated from homemade kimchi and identified as Lactococcus lactis based on morphology and 16S rDNA sequence analysis. Whole genome analysis showed that the PZ1 strain contained a variety of peptidases and protease genes that had the potential to decompose proteins. Soybean proteins were then fermented by PZ1, and the polypeptides produced during fermentation were analyzed via gel permeation chromatography, revealing that 85% of polypeptides had a molecular weight below 1000 Da. The oligopeptides with molecular weight 300～1000 Da were obtained via ultrafiltration purification, and their antioxidant activity was studied. The oligopeptides demonstrated a good scavenging effect on DPPH, hydroxyl (·OH), and superoxide anion (O2−·) radicals, at an oligopeptide concentration of 2 mg/mL, the clearance rates were 79.31%, 78.27%, and 84.62%, respectively. Therefore, L. lactis PZ1 could degrade soybean protein efficiently and could be used as a probiotic for developing functional soybean products.

Abstract Amino acid transporters are promising targets for engineering acid‐tolerant strains of Lactococcus lactis. However, the simple overexpression of transporters alone is insufficient to achieve a highly acid‐resistant phenotype. This study investigated the effects of amino acid transporters on the acid‐stress tolerance of L. lactis. Here, we first verified the contribution of amino acid transporters to acid tolerance by overexpressing the ctrA, glnP, and glnQ genes in L. lactis. Transcriptome analysis revealed that most genes associated with specific amino acid transport, pyrimidine metabolism, and functional proteins, were upregulated in the overexpression strains. Among them, arginine biosynthesis (argG and argH), amino acid transport (yjeM and azlC), and pyrimidine metabolism were considered to be the most important regulatory mechanisms. Importantly, metabolite profiling revealed that the overexpression strains had higher intracellular levels of amino acids, particularly aspartate, glutamate, and arginine at low pH, as well as higher intracellular ATP levels, which was consistent with the corresponding gene‐expression levels. Finally, the simultaneous overexpression of glnP and glnQ led to a further improvement of acid tolerance in L. lactis. This study reveals the regulatory mechanisms of amino acid transporters, and provides a novel strategy for achieving higher acid tolerance via positive tandem expression approaches.

Lactococcus lactis and Streptococcus thermophilus are considered as ideal chassis of engineered probiotics, while food-grade genetic tools are limited in those strains. Here, a Zn2+-controlled gene expression (ZICE) system was identified in the genome of S. thermophilus CGMCC7.179, including a transcriptional regulator sczAst and a promoter region of cation transporter czcD (PczcDst). Specific binding of the SczAst to the palindromic sequences in PczcDst was demonstrated by EMSA analysis, suggesting the regulation role of SczAst on PczcDst. To evaluate their possibility to control gene expression in vivo, the sczAst-PczcDst was employed to drive the expression of green fluorescence protein (GFP) gene in L. lactis NZ9000 and S. thermophilus CGMCC7.179, respectively. Both of the transformants could express GFP under Zn2+ induction, while no fluorescence without Zn2+ addition. For optimal conditions, Zn2+ was used at a final concentration of 0.8 mM in L. lactis and 0.16 mM in S. thermophilus at OD600 close to 0.4, and omitting yeast extract powder in the medium unexpectedly improved GFP expression level by 2.2-fold. With the help of the ZICE system, engineered L. lactis and S. thermophilus strains were constructed to secret cytokine interleukin-10 (IL-10) with immunogenicity, and the IL-10 content in the supernatant of the engineered L. lactis was 59.37 % of that under the nisin controlled expression system. This study provided a tightly controlled expression system by the food-grade inducer Zn2+, having potential in development of engineered probiotics.

This document discusses the manufacture of fermented dairy products, which are produced globally using various fermentation processes. Different species of mammals, such as cows, sheep, goats, and camels, provide milk for these products, each with its own microbiota and chemical composition. The diversity of manufacturing practices contributes to the wide variety of fermented milk products available. The document also highlights recent research on dairy fermentation, including studies on the use of autochthonous LAB cultures in cheese production, the addition of adjunct cultures to improve the quality of traditional cheese, the health benefits of probiotics and prebiotics in yogurt, the evaluation of LAB strains for potential probiotic use, and the application of protective cultures in cheese production. Additionally, the challenges and potential of plant-based and hybrid cheese products are discussed. The document emphasizes the importance of further research on the characterization and application of autochthonous LAB cultures in the production of novel dairy, non-dairy, and hybrid products. [Extracted from the article]

Encapsulation in alginate hydrogel microspheres is an effective method for protecting and improving the survival of lactic acid bacteria in different environments. This research aims to expand the knowledge about the structure/property relationship of calcium alginate microspheres loaded with a mixture of autochthonous probiotic bacteria (Lactococcus lactis and Lactiplantibacillus plantarum). A novel hydrogel formulation (FORMLAB) was prepared by ionic gelation and the molecular interactions between the FORMLAB constituents, surface morphology, structure, swelling degree, and release profile were characterized. The simultaneous encapsulation of two bacterial cultures in the same compartment does not diminish their viability. The binding of calcium ions to bacterial cells creates favorable conditions for the propagation of the encapsulated bacteria. The molecular interactions between the FORMLAB constituents are complex, involving mainly hydrogen bonds and electrostatic interactions. With a very high degree of swelling followed by low crosslinking, the surface of the microspheres covered with bacterial cells and diffusion through the hydrogel matrix allow for the delivery of probiotics at the right time. The findings suggest that bacterial cells are efficiently delivered from calcium alginate microspheres, offering promising applications in the development of functional foods, especially in cheese production.

The lack of sustainable strategies for combating Xylella fastidiosa (Xf) highlights the pressing need for novel practical antibacterial tools. In this study, Lactococcus lactis subsp. lactis strain ATCC 11454 (L. lactis), known for its production of nisin A, was in vitro tested against Xf subsp. pauca. Preliminary investigations showed that nisin A was involved in a strong antagonistic activity exhibited by L. lactis against Xf. Thus, the efficacy of nisin A was comprehensively assessed through a combination of in vitro and in planta experiments. In vitro investigations employing viable-quantitative PCR, spot assay, turbidity reduction assay, fluorescence microscopy, and transmission electron microscopy demonstrated nisin's robust bactericidal effect on Xf at a minimal lethal concentration of 0.6 mg/mL. Moreover, results from fluorescence and transmission electron microscopies indicated that nisin directly and rapidly interacts with the membranes of Xf cells, leading to the destruction of bacterial cells in few minutes. In in planta tests, nisin also demonstrated the ability to tackle Xf infections within Nicotiana benthamiana plants that remained asymptomatic 74 days post inoculation. Furthermore, RPLC-ESI-MS/MS analyses showed that nisin translocated to all parts of the plants and remains intact for up to 9 days. For the first time, this study underscores the nisin-based strategy as a realistic and eco-friendly approach to be further investigated against Xf infections in the field. [ABSTRACT FROM AUTHOR]

Studies have determined that nonredox enzymes that are cofactored with Fe(II) are the most oxidant-sensitive targets inside Escherichia coli. These enzymes use Fe(II) cofactors to bind and activate substrates. Because of their solvent exposure, the metal can be accessed and oxidized by reactive oxygen species, thereby inactivating the enzyme. Because these enzymes participate in key physiological processes, the consequences of stress can be severe. Accordingly, when E. coli senses elevated levels of H2O2, it induces both a miniferritin and a manganese importer, enabling the replacement of the iron atom in these enzymes with manganese. Manganese does not react with H2O2 and thereby preserves enzyme activity. In this study, we examined several diverse microbes to identify the metal that they customarily integrate into ribulose-5-phosphate 3-epimerase, a representative of this enzyme family. The anaerobe Bacteroides thetaiotaomicron, like E. coli, uses iron. In contrast, Bacillus subtilis and Lactococcus lactis use manganese, and Saccharomyces cerevisiae uses zinc. The latter organisms are therefore well suited to the oxidizing environments in which they dwell. Similar results were obtained with peptide deformylase, another essential enzyme of the mononuclear class. Strikingly, heterologous expression experiments show that it is the metal pool within the organism, rather than features of the protein itself, that determine which metal is incorporated. Further, regardless of the source organism, each enzyme exhibits highest turnover with iron and lowest turnover with zinc. We infer that the intrinsic catalytic properties of the metal cannot easily be retuned by evolution of the polypeptide. [ABSTRACT FROM AUTHOR]

The rapid emergence of invasive infections caused by azole-resistant Candida tropicalis has become a public health concern, and there is an urgent need for alternative treatment strategies. Studies have demonstrated the antibacterial effects of nisin, a well-known peptide naturally produced by Lactococcus lactis subsp. lactis. However, there is scant information about the antifungal effect of nisin against C. tropicalis. The present study aims to investigate the in vitro antifungal activity of nisin against clinical isolates of azole-resistant C. tropicalis strains, as well as its inhibitory effect on biofilm formation. A total of 35 C. tropicalis strains isolated from patients with invasive fungal infections were divided into the azole-resistant group and the azole-sensitive group, containing 21 and 14 strains, respectively. The relative expression levels of the ERG11 and UPC2 genes in the azole-resistant group were higher than those in the azole-sensitive group (p < 0.0001), while no significant differences were observed in the expression levels of the MDR1 and CDR1 genes. The minimum inhibitory concentration of nisin against C. tropicalis ranged from 2 to 8 μg/ mL. Nisin treatment inhibited the growth of azole-resistant C. tropicalis, with over a four-fold reduction in OD600 nm values observed at the 8-h time point, while it promoted the transition of C. tropicalis from the spore phase to the hyphal phase, as observed on cryo-scanning electron microscopy. The results of biofilm quantification using crystal violet staining indicated a significant decrease in OD570 nm values in the nisin-treated group compared to the controls (p < 0.0001). Among the 21 azole-resistant C. tropicalis strains, the biofilm formation was inhibited in 17 strains (17/21, 81%), and more than 85% inhibition of biofilm formation was observed in the representative strains. With regard to the molecular mechanisms, the expression of the BCR1 and UPC2 genes in the azole-resistant strains was down-regulated on nisin treatment (p < 0.05). In conclusion, we demonstrated, for the first time, that nisin has antifungal activity and significant anti-biofilm activity against clinical isolates of azole-resistant C. tropicalis strains. Based on the findings, nisin could be a promising alternative antifungal agent for combating azole-resistant C. tropicalis infections. [ABSTRACT FROM AUTHOR]

Microbial nitrogen sources are promising, and soy protein as a plant-based nitrogen source has absolute advantages in creating microbial culture medium in terms of renewability, eco-friendliness, and greater safety. Soy protein is rich in variety due to different extraction technologies and significantly different in the cell growth and metabolism of microorganisms as nitrogen source. Therefore, different soy proteins (soy meal powder, SMP; soy peptone, SP; soy protein concentrate, SPC; soy protein isolate, SPI; and soy protein hydrolysate, SPH) were used as nitrogen sources to culture Bacillus subtilis, Streptococcus lactis, and Streptomyces clavuligerus to evaluate the suitable soy nitrogen sources of the above strains. The results showed that B. subtilis had the highest bacteria density in SMP medium; S. lactis had the highest bacteria density in SPI medium; and S. clavuligerus had the highest PMV in SPI medium. Nattokinase activity was the highest in SP medium; the bacteriostatic effect of nisin was the best in SPI medium; and the clavulanic acid concentration was the highest in SMP medium. Based on analyzing the correlation between the nutritional composition and growth metabolism of the strains, the results indicated that the protein content and amino acid composition were the key factors influencing the cell growth and metabolism of the strains. These findings present a new, high-value application opportunity for soybean protein. [ABSTRACT FROM AUTHOR]

Widespread manganese-sensing transcriptional riboswitches effect the dependable gene regulation needed for bacterial manganese homeostasis in changing environments. Riboswitches – like most structured RNAs – are believed to fold co-transcriptionally, subject to both ligand binding and transcription events; yet how these processes are orchestrated for robust regulation is poorly understood. Through a combination of single-molecule and bulk approaches, we discover how a single Mn2+ ion and the transcribing RNA polymerase (RNAP), paused immediately downstream by a DNA template sequence, are coordinated by the bridging switch helix P1.1 in the representative Lactococcus lactis riboswitch. This coordination achieves a heretofore-overlooked semi-docked global conformation of the nascent RNA, P1.1 base pair stabilization, transcription factor NusA ejection, and RNAP pause extension, thereby enforcing transcription readthrough. Our work demonstrates how a central, adaptable RNA helix functions analogous to a molecular fulcrum of a first-class lever system to integrate disparate signals for finely balanced gene expression control. Here the authors unveil an intermediate state during the folding of the manganese riboswitch from L. lactis. This transient state allows the integration of multiple cellular signals including RNA polymerase pausing and transcription factor NusA. [ABSTRACT FROM AUTHOR]

The alteration of structure and function of gut microbiota (dysbiosis) appears to be a major factor associated with metabolic disorders such as dyslipidemia and subsequent development of cardiovascular diseases. However, the consumption of fermented milks is a promising strategy to enhance health and restore the function of gut microbiota; specifically, in individuals with intestinal dysbiosis and hypercholesterolemia. Therefore, the aim of the present study was to evaluate the potential association between gut microbiota and the hypocholesterolemic effect of fermented milks with Lactococcus lactis NRRL B-571 (FM-571), NRRL B-572 (FM-572) and NRRL B-600 (FM-600) in Sprague–Dawley rats. Fermented milks were administered to hypercholesterolemic Sprague–Dawley rats during seven weeks. At the end of the experimental period, fecal and colonic microbiota were characterized using 16S RNA gene sequencing. Also, the short chain fatty acids (SCFAs) content was quantified in feces. Results showed that a high-cholesterol diet (HCD) altered the bacterial community in both fecal and mucosal samples. The consumption of fermented milks, specifically FM-572 promoted changes in the structure (beta diversity) in fecal, but not in mucosal microbiota. The levels of SCFAs in feces were improved after fermented milks consumption. From all SCFAs, butyrate was negatively correlated with total cholesterol, LDL-C (p < 0.05) and positively correlated with HDL-C (p < 0.05). Furthermore, Ruminococcaceae, Lactobacillaceae, Lachnospiraceae and Oscillospiraceae families, were negatively associated with total cholesterol, LDL-C (p < 0.05) and positively associated with HDL-C (p < 0.05). The abundance of these families was increased in groups treated with fermented milks, particularly with FM-572 (p < 0.05). Thus, the in vivo hypocholesterolemic effect after the consumption of milks fermented with Lactococcus lactis strains may be related with the modulation of fecal microbiota associated with the increase of butyrate-producing bacteria. Furthermore, these associations may suggest that butyrate may influence the cholesterol metabolism, resulting in the decreasing cholesterol levels. [ABSTRACT FROM AUTHOR]

The integration of aqueous clove extract (ACE) and spray-dried probiotics into an edible film presents numerous health advantages, such as enhanced probiotic stability and antibacterial efficacy against harmful bacteria like Klebsiella pneumoniae and Pseudomonas aeruginosa. This study aimed to formulate a probiotic edible film with antibacterial properties by incorporating milk powder containing ACE and probiotic strains, namely Streptococcus thermophilus ATCC 19258, Lactobacillus bulgaricus ATCC 11842, and Lactococcus lactis MG1363. The milk powders, containing 7.5% (w/v) ACE with various probiotic strains, were combined with 2% (w/v) pectin biopolymer and 60% (w/w) glycerol plasticizer to produce the probiotic edible film with ACE. Antibacterial effects were investigated using disc diffusion, while probiotic film viability was assessed through the pour plate method. The study revealed higher viability in the probiotic strains L. lactis and S. thermophilus within the edible film, compared to L. bulgaricus. The specific strains exhibited increased antibacterial activity against K. pneumoniae and P. aeruginosa, underscoring the critical role of probiotic strain selection in determining functional properties. Notably, the edible film containing L. lactis and S. thermophilus demonstrated substantial antibacterial activity against both pathogens. However, further research is imperative to optimize industrial-scale production methods, assess efficacy in diverse food items, evaluate sensory properties, gauge barrier attributes, and investigate the impact of storage conditions on stability and performance. [ABSTRACT FROM AUTHOR]

Bacteria utilize various strategies to prevent internal dehydration during hypertonic stress. A common approach to countering the effects of the stress is to import compatible solutes such as glycine betaine, leading to simultaneous passive water fluxes following the osmotic gradient. OpuA from Lactococcus lactis is a type I ABC-importer that uses two substrate-binding domains (SBDs) to capture extracellular glycine betaine and deliver the substrate to the transmembrane domains for subsequent transport. OpuA senses osmotic stress via changes in the internal ionic strength and is furthermore regulated by the 2nd messenger cyclic-di-AMP. We now show, by means of solution-based single-molecule FRET and analysis with multi-parameter photon-by-photon hidden Markov modeling, that the SBDs transiently interact in an ionic strength-dependent manner. The smFRET data are in accordance with the apparent cooperativity in transport and supported by new cryo-EM data of OpuA. We propose that the physical interactions between SBDs and cooperativity in substrate delivery are part of the transport mechanism. [ABSTRACT FROM AUTHOR]

This research investigated the physicochemical, microbiological, and bacterial diversity of Jben cheese, a popular artisanal variety in Morocco. The bacterial diversity was explored using culture-independent methods, including temporal temperature gel electrophoresis (TTGE), denaturing gradient gel electrophoresis (DGGE), and high-throughput sequencing (HTS). Significant intra-sample differences were observed for most physicochemical parameters within each milk type, while inter-sample differences occurred between cow and goat cheeses for dry matter and ash. Jben cheese exhibited distinct characteristics, with low pH values of 3.96, 4.16, and 4.18 for cow, goat, and mixed cheeses, respectively. Goat cheeses had higher fat (49.23 g/100 g), ash (1.91 g/100 g), and dry matter (36.39 g/100 g) than cow cheeses. All cheeses displayed high microbial counts, with a notable prevalence of the lactic acid bacteria (LAB) group, averaging 8.80 ± 0.92 log CFU/g. Jben cheese also displayed high contamination levels with total coliforms, faecal coliforms, yeast, and molds. Fatty acid profiling revealed fraudulent practices in Jben cheese marketing, with cow or mixed cheeses sold as goat cheese, as proven by low capric acid concentration. HTS analysis of Jben cheese identified ten genera and twenty-four species, highlighting Lactococcus lactis as predominant. TTGE and DGGE confirmed the presence of L. lactis but failed to provide the detailed profile achieved through HTS analysis. HTS has been demonstrated to be more reliable, whereas TTGE/DGGE methods, though informative, were more time-consuming and less reliable. Despite limitations, the combined use of TTGE, DGGE, and HTS provided a comprehensive view of indigenous bacterial communities in Jben cheese, identifying L. lactis as the main species. [ABSTRACT FROM AUTHOR]

Cheese taste and flavour properties result from complex metabolic processes occurring in microbial communities. A deeper understanding of such mechanisms makes it possible to improve both industrial production processes and end-product quality through the design of microbial consortia. In this work, we caracterise the metabolism of a three-species community consisting of Lactococcus lactis , Lactobacillus plantarum and Propionibacterium freudenreichii during a seven-week cheese production process. Using genome-scale metabolic models and omics data integration, we modeled and calibrated individual dynamics using monoculture experiments, and coupled these models to capture the metabolism of the community. This model accurately predicts the dynamics of the community, enlightening the contribution of each microbial species to organoleptic compound production. Further metabolic exploration revealed additional possible interactions between the bacterial species. This work provides a methodological framework for the prediction of community-wide metabolism and highlights the added value of dynamic metabolic modeling for the comprehension of fermented food processes. [ABSTRACT FROM AUTHOR]

Lactococcus lactis, a lactic acid bacterium used in food fermentations and commonly found in the human gut, is known to possess a fermentative metabolism. L. lactis, however, has been demonstrated to transfer metabolically generated electrons to external electron acceptors, a process termed extracellular electron transfer (EET). Here, we investigated an L. lactis mutant with an unusually high capacity for EET that was obtained in an adaptive laboratory evolution (ALE) experiment. First, we investigated how global gene expression had changed, and found that amino acid metabolism and nucleotide metabolism had been affected significantly. One of the most significantly upregulated genes encoded the NADH dehydrogenase NoxB. We found that this upregulation was due to a mutation in the promoter region of NoxB, which abolished carbon catabolite repression. A unique role of NoxB in EET could be attributed and it was directly verified, for the first time, that NoxB could support respiration in L. lactis. NoxB, was shown to be a novel type-II NADH dehydrogenase that is widely distributed among gut microorganisms. This work expands our understanding of EET in Gram-positive electroactive microorganisms and the special significance of a novel type-II NADH dehydrogenase in EET. [ABSTRACT FROM AUTHOR]

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Probiotic microorganisms are used in a variety of food supplements and medical formulations to promote human health. In periodontal therapy, probiotics are mainly used in the form of gels, tablets or rinses that often tend to leak from the periodontal pocket, resulting in a strongly reduced therapeutic effect. In this pilot in vitro study, we present biodegradable alginate-based particles as an alternative, highly efficient system for a periodontal delivery of probiotic bacteria to the inflammation site. For this purpose, Lactococcus (L.) lactis was encapsulated using a standardized pump-controlled extrusion-dripping method. Time-dependent bacterial release in artificial saliva was investigated over 9 days. The effect of freeze drying was explored to ensure long-term storage of L. lactis-loaded particles. Additionally, the particles were bound to dentin surface using approved bioadhesives and subjected to shear stress in a hydrodynamic flow chamber that mimics the oral cavity in vitro. Thus, round particles within the range of 0.80–1.75 mm in radius could be produced, whereby the diameter of the dripping tip had the most significant impact on the size. Although both small and large particles demonstrated a similar release trend of L. lactis, the release rate was significantly higher in the former. Following lyophilization, particles could restore their original shape within 4 h in artificial saliva; thereby, the bacterial viability was not affected. The attachment strength to dentin intensified by an adhesive could resist forces between 10 and 25 N/m2. Full degradation of the particles was observed after 20 days in artificial saliva. Therefore, alginate particles display a valuable probiotic carrier for periodontal applications that have several crucial advantages over existing preparations: a highly stable form, prolonged continuous release of therapeutic bacteria, precise manufacturing according to required dimensions at the application site, strong attachment to the tooth with low risk of dislocation, high biocompatibility and biodegradability. [ABSTRACT FROM AUTHOR]