Efficacy of Lactococcus lactis subsp. lactis LY-66 and Lactobacillus plantarum PL-02 in Enhancing Explosive Strength and Endurance: A Randomized, Double-Blinded Clinical Trial.

Probiotics are posited to enhance exercise performance by influencing muscle protein synthesis, augmenting glycogen storage, and reducing inflammation. This double-blind study randomized 88 participants to receive a six-week intervention with either a placebo, Lactococcus lactis subsp. lactis LY-66, Lactobacillus plantarum PL-02, or a combination of both strains, combined with a structured exercise training program. We assessed changes in maximal oxygen consumption (VO2max), exercise performance, and gut microbiota composition before and after the intervention. Further analyses were conducted to evaluate the impact of probiotics on exercise-induced muscle damage (EIMD), muscle integrity, and inflammatory markers in the blood, 24 and 48 h post-intervention. The results demonstrated that all probiotic groups exhibited significant enhancements in exercise performance and attenuation of muscle strength decline post-exercise exhaustion (p < 0.05). Notably, PL-02 intake significantly increased muscle mass, whereas LY-66 and the combination therapy significantly reduced body fat percentage (p < 0.05). Analysis of intestinal microbiota revealed an increase in beneficial bacteria, especially a significant rise in Akkermansia muciniphila following supplementation with PL-02 and LY-66 (p < 0.05). Overall, the combination of exercise training and supplementation with PL-02, LY-66, and their combination improved muscle strength, explosiveness, and endurance performance, and had beneficial effects on body composition and gastrointestinal health, as evidenced by data obtained from non-athlete participants.

Investigating lactic acid bacteria genus Lactococcus lactis properties: Antioxidant activity, antibiotic resistance, and antibacterial activity against multidrug-resistant bacteria Staphylococcus aureus.

Background: Lactic acid bacteria (LAB) are utilized as a starter culture in the manufacturing of fermented dairy items, as a preservative for various food products, and as a probiotic. In our country, some research has been carried out, even if LAB plays a principal role in food preservation and improves the texture and taste of fermented foods, that is why we tried to evaluate their probiotic effect. The objective of this research was to determine the antibacterial activity of Lactococcus lactis (L. lactis) against Staphylococcus aureus (S. aureus) ATCC 29213, investigate their antioxidant activity, and characterize their sensitivity against 18 antibiotics. Methods: A total of 23 LAB (L. lactis subsp. cremoris, L. lactis subsp. Lactis diacetylactis, L. lactis subsp. lactis) were isolated from cow's raw milk. The antibacterial activity was performed using two techniques, competition for nutrients and a technique utilizing components nature, using the disk diffusion method. The sensitivity of the studied LAB to different antibiotics was tested on Man rogosa sharp (MRS) agar using commercial antibiotic disks. All strains of LAB were examined for their antioxidant activity. The antioxidant activity of L. lactis was tested by 2,2-diphenyl-1 picrylhydrazyl (DPPH). Results: The results showed that the MRS medium was more adapted than Muller Hinton Agar (MHA) to investigate the antibacterial activity of L. lactis against S. aureus ATCC 29213. Also, L. lactis exhibited a notable degree of antibacterial activity against S. aureus ATCC 29213. L. Lactis subsp. Lactis displayed higher antibacterial activities, followed by L. lactis ssp. lactis biovar. diacetylactis, and lastly, L. lactis ssp. cremoris against S. aureus ATCC 29213. Lc 26 among all strains of L. lactis showed a high potential antibacterial activity reaching 40 ± 3 mm against S. aureus ATCC 29213. All strains of L. lactis showed a slightly moderate antioxidant activity (10.56 ± 1.28%-26.29 ± 0.05 %). The results of the antibiotic resistance test indicate that all strains of L. lactis were resistant to cefotaxime, sulfamethoxazole-trimethoprim, and streptomycin and were sensitive to Ampicillin, Amoxicillin, Penicillin G, Teicoplanin, Vancomycin, Gentamicin 500, Tetracycline, and Chloramphenicol. These test results indicate that this strain falls within the criteria of not posing any harmful effects on human health. The important antibacterial properties recorded for all L. Lactis strains were derived from the production of antibacterial active metabolites, such as protein, diacetyl, hydrogen peroxide, and lactic acid, together with the fight for nutrients. Conclusion: This study suggests that the strains of L. lactis could be added as an antibacterial agent against S. aureus ATCC 29213 and can provide an important nutritional property for their antioxidant potential.

Potential role of salivary lactic acid bacteria in pathogenesis of oral lichen planus.

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.

Heat-Killed Lactococcus Lactis KC24 Ameliorates Scopolamine-Induced Memory Impairment in ICR Mice.

Heat-killed Lactococcus lactis KC24 (H-KC24) has been examined for its neuroprotective effects in SH-SY5Y cells. We hypothesized that H-KC24 could alleviate memory impairment through the gut-brain axis. Scopolamine (1 mg/kg/day) was administered to ICR mice to induce memory impairment. Low- and high-dose H-KC24 cells (1 × 109 and 2 × 109 CFU/day, respectively) or donepezil (DO, 2 mg/kg) were administered for 14 days. H-KC24 treatment alleviated the deleterious scopolamine-induced memory effects on the recognition index and object recognition ability in the novel object recognition test and the Y-maze test. Changes in neurotransmitters and synaptic plasticity were confirmed by measuring acetylcholine, acetylcholinesterase, choline acetyltransferase, brain-derived neurotrophic factor, cyclic AMP response element-binding protein, and phosphorylated cyclic AMP response element-binding protein expression in brain tissues. In the H-KC24 and DO groups, ß-secretase levels increased, whereas amyloid ß levels decreased, demonstrating that H-KC24 can improve memory impairment caused by oxidative stress. This study demonstrated the positive effects of H-KC24 in a scopolamine-induced memory impairment mouse model.

Dietary Supplementation of Lactococcus lactis subsp. lactis BIONCL17752 on Growth Performance, and Gut Microbiota of Broiler Chickens.

The rapid rise of antimicrobial resistance (AMR) is a global concern, being triggered by the overuse or misuse of antibiotics in poultry farming sector. We evaluated Lactococcus lactis subsp. lactis BIONCL17752 strain, and characterized its probiotic potential to endure hostile gastrointestinal conditions. Genome sequencing analysis revealed probiotics traits, and gene clusters involved in bacteriocins, lactococcin A, and sactipeptides production. The absence of genes for antibiotic resistance, virulence, and biogenic amine production indicates the potential of probiotic strain. The BIONCL17752 strain was explored for antibiotic-free feed supplement for growth promotor in broiler chicken. The feed supplemented with 4 × 109 CFU/kg of probiotic strain, in combination with various concentrations of fructooligosaccharides (FOS) 1.0, 2.5, and 5.0 kg/tonne in starter, grower, and finisher diets, respectively. A significant improvement of body weight 152 to 171 g/bird (p < 0.05), and a low feed conversion ratio (FCR) of 1.62, was achieved without using synthetic antibiotics for growth promotion. The results of biochemical, hematological, and histological examinations showed normal features, indicating that the treatment had no harmful effects on the bird's health. Reduced levels of cholesterol, triglycerides, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) in serum are an indication of the health benefits for the treated birds. Microbial community analysis of fecal samples of poultry birds exhibited a higher abundance of Bacteroidetes, Firmicutes, Proteobacteria, Actinobacteria, and Fusobacteria. Probiotic treatment resulted in reduced Firmicutes and increased Bacteroidetes (F/B ratio) in the broiler's gut which highlights the benefits of probiotic dietary supplements. Importantly, the probiotic-fed group exhibited a high abundance of carbohydrate-active enzymes (CAZyme) such as glycoside hydrolases (GH), glycoside transferases (GT), and carbohydrate-binding module (CBM) hydrolases which are essential for the degradation of complex sugar molecules. The probiotic potential of the BIONCL17752 strain contributes to broilers' health by positively affecting intestinal microbiota, achieving optimal growth, and lowering mortality, demonstrating the economic benefits of probiotic treatment in organic poultry farming.

AcrR1, a novel TetR/AcrR family repressor, mediates acid and antibiotic resistance and nisin biosynthesis in Lactococcus lactis F44.

Lactococcus lactis, widely used in the manufacture of dairy products, encounters various environmental stresses both in natural habitats and during industrial processes. It has evolved intricate machinery of stress sensing and defense to survive harsh stress conditions. Here, we identified a novel TetR/AcrR family transcription regulator, designated AcrR1, to be a repressor for acid and antibiotic tolerance that was derepressed in the presence of vancomycin or under acid stress. The survival rates of acrR1 deletion strain ΔAcrR1 under acid and vancomycin stresses were about 28.7-fold (pH 3.0, HCl), 8.57-fold (pH 4.0, lactic acid) and 2.73-fold (300 ng/mL vancomycin) as that of original strain F44. We also demonstrated that ΔAcrR1 was better able to maintain intracellular pH homeostasis and had a lower affinity to vancomycin. No evident effects of AcrR1 deletion on the growth and morphology of strain F44 were observed. Subsequently, we characterized that the transcription level of genes associated with amino acids biosynthesis, carbohydrate transport and metabolism, multiple drug resistance and DNA repair proteins significantly upregulated in ΔAcrR1 using transcriptome analysis and quantitative reverse transcription-PCR (qRT-PCR) assays. Additionally, AcrR1 could repress the transcription of nisin post-translational modification gene, nisC, leading to a 16.3% increase in nisin yield after AcrR1 deletion. Our results not only refined the knowledge of the regulatory mechanism of TetR/AcrR family regulator in L. lactis, but presented a potential strategy to enhance industrial production of nisin.

Genetically engineered Lactococcus lactis strain constitutively expresses GABA-producing genes and produces high levels of GABA.

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.

Heterologous expression of P9 from Akkermansia muciniphila increases the GLP-1 secretion of intestinal L cells.

Glucagon-like peptide-1(GLP-1) is an incretin hormone secreted primarily from the intestinal L-cells in response to meals. GLP-1 is a key regulator of energy metabolism and food intake. It has been proven that P9 protein from A. muciniphila could increase GLP-1 release and improve glucose homeostasis in HFD-induced mice. To obtain an engineered Lactococcus lactis which produced P9 protein, mature polypeptide chain of P9 was codon-optimized, fused with N-terminal signal peptide Usp45, and expressed in L. lactis NZ9000. Heterologous secretion of P9 by recombinant L. lactis NZP9 were successfully detected by SDS-PAGE and western blotting. Notably, the supernatant of L. lactis NZP9 stimulated GLP-1 production of NCI-H716 cells. The relative expression level of GLP-1 biosynthesis gene GCG and PCSK1 were upregulated by 1.63 and 1.53 folds, respectively. To our knowledge, this is the first report on the secretory expression of carboxyl-terminal processing protease P9 from A. muciniphila in L. lactis. Our results suggest that genetically engineered L. lactis which expressed P9 may have therapeutic potential for the treatment of diabetes, obesity and other metabolic disorders.

Incorporation of Lactococcus lactis and Chia Mucilage for Improving the Physical and Biological Properties of Gelatin-Based Coating: Application for Strawberry Preservation.

In this work, a gelatin/chia mucilage (GN/CM) composite coating material doped with Lactococcus lactis (LS) was developed for strawberry preservation applications. The results of the scanning electron microscope and Fourier transform infrared spectroscopy stated that the enhanced molecular interaction between the CM and GN matrix strengthened the density and compactness of the GN film. Antifungal results indicated that the addition of LS significantly (p < 0.05) improved the ability of the GN coating to inhibit the growth of Botrytis cinerea (inhibition percentage = 62.0 ± 4.6%). Adding CM significantly (p < 0.05) decreased the water vapour permeability and oxygen permeability of the GN coating by 32.7 ± 4.0% and 15.76 ± 1.89%, respectively. In addition, the incorporated CM also significantly (p < 0.05) improved the LS viability and elongation at break of the film by 13.11 ± 2.05% and 42.58 ± 1.21%, respectively. The GN/CM/LS composite coating material also exhibited an excellent washability. The results of this study indicated that the developed GN/CM/LS coating could be used as a novel active material for strawberry preservation.

Recombinant lactococcal-based oral vaccine for protection against Streptococcus agalactiae infections in tilapia (Oreochromis niloticus).

Streptococcosis outbreaks caused by Streptococcus agalactiae infection in tilapia aquaculture have been consistently reported and associated with high mortality and morbidity leading to significant economic losses. Existing vaccine candidates against Streptococcus spp. are designed for intraperitoneal injections that are not practical and labor-intensive which have prompted farmers to protect aquatic animals with antibiotics, thus encouraging the emergence of multidrug resistant bacteria. In this study, a live recombinant L. lactis vaccine expressing a 1403 bp surface immunogenic protein (SIP) and a 1100 bp truncated SIP (tSIP) gene was developed and evaluated against S. agalactiae infection in tilapia. Both SIP and tSIP sequences were cloned and transformed into L. lactis. The recombinant L.lactis vaccine was orally administered to juvenile tilapia for a month. Detection of SIP-specific serum IgM in vaccinated groups compared to control groups indicated that recombinant proteins expressed from L. lactis could elicit immunogenic reactions in tilapia. Fish immunized with the tSIP vaccine also showed the highest level of protection compared to other test groups, and the mortality rate was significantly reduced compared to both control groups. The relative percentage of survival (RPS) against S. agalactiae for both SIP and tSIP-vaccinated groups was 50 % and 89 %, respectively, at 14 days post-challenge. Significant up-regulation of IgM, IL-1ß, IL-10, TNF-α and IFN-γ were observed at day 34 between the vaccinated and control groups. These results indicated that the recombinant lactococcal tSIP vaccine can elicit both cell-mediated and humoral responses and is recommended as a potential oral vaccine against S. agalactiae infection. Future work will include further in vivo challenge assessments of this vaccine candidate fused with adjuvants to boost immunogenicity levels in tilapia.

Detection and classification of the integrative conjugative elements of Lactococcus lactis.

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.

Scrutinizing a Lactococcus lactis mutant with enhanced capacity for extracellular electron transfer reveals a unique role for a novel type-II NADH dehydrogenase.

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.IMPORTANCEElectroactive microorganisms with extracellular electron transfer (EET) ability play important roles in biotechnology and ecosystems. To date, there have been many investigations aiming at elucidating the mechanisms behind EET, and determining the relevance of EET for microorganisms in different niches. However, how EET can be enhanced and harnessed for biotechnological applications has been less explored. Here, we compare the transcriptomes of an EET-enhanced L. lactis mutant with its parent and elucidate the underlying reason for its superior performance. We find that one of the most significantly upregulated genes is the gene encoding the NADH dehydrogenase NoxB, and that upregulation is due to a mutation in the catabolite-responsive element that abolishes carbon catabolite repression. We demonstrate that NoxB has a special role in EET, and furthermore show that it supports respiration to oxygen, which has never been done previously. In addition, a search reveals that this novel NoxB-type NADH dehydrogenase is widely distributed among gut microorganisms.

A Point Mutation in Cassette Relieves the Repression Regulation of CcpA Resulting in an Increase in the Degradation of 2,3-Butanediol in Lactococcus lactis.

In lactic acid bacteria, the global transcriptional regulator CcpA regulates carbon metabolism by repressing and activating the central carbon metabolism pathway, thus decreasing or increasing the yield of certain metabolites to maximize carbon flow. However, there are no reports on the deregulation of the inhibitory effects of CcpA on the metabolism of secondary metabolites. In this study, we identified a single-base mutant strain of Lactococcus lactis N8-2 that is capable of metabolizing 2,3-butanediol. It has been established that CcpA dissociates from the catabolite responsive element (cre) site due to a mutation, leading to the activation of derepression and expression of the 2,3-butanediol dehydrogenase gene cluster (butB and butA). Transcriptome analysis and quantitative polymerase chain reaction (Q-PCR) results showed significant upregulation of transcription of butB and butA compared to the unmutated strain. Furthermore, micro-scale thermophoresis experiments confirmed that CcpA did not bind to the mutated cre. Furthermore, in a bacterial two-plasmid fluorescent hybridization system, it was similarly confirmed that the dissociation of CcpA from cre eliminated the repressive effect of CcpA on downstream genes. Finally, we investigated the differing catalytic capacities of the 2,3-butanediol dehydrogenase gene cluster in L. lactis N8-1 and L. lactis N8-2 for 2,3-butanediol. This led to increased expression of butB and butA, which were deregulated by CcpA repression. This is the first report on the elimination of the deterrent effect of CcpA in lactic acid bacteria, which changes the direction of enzymatic catalysis and alters the direction of carbon metabolism. This provides new perspectives and strategies for metabolizing 2,3-butanediol using bacteria in synthetic biology.

Iron-tannic acid nano-coating: A promising treatment approach for enhancing Lactococcus lactis antibiotic resistance.

The objective of this study was to explore a novel methodology for the synthesis of nanocoated probiotics following their collection and cultivation under optimized conditions, in light of their significant contribution to human health. Probiotics are instrumental in sustaining immune health by modulating the gastrointestinal microbiota and facilitating digestion. However, the equilibrium they maintain can be adversely affected by antibiotic treatments. It is critical to investigate the vulnerability of probiotics to antibiotics, considering the potential implications. This research aimed to assess whether nanoparticle coating could augment the probiotics' resistance to antibiotic influence. A strain of Lactococcus lactis (L. lactis) was isolated, cultured, and comprehensively characterized utilizing state-of-the-art methodologies, including the VITEK® 2 compact system, VITEK® MS, and 16S rRNA gene sequencing. The nanoparticle coating was performed using iron (III) chloride hexahydrate and tannic acid, followed by an evaluation of the probiotics' resistance to a range of antibiotics. The analysis through scanning electron microscopy (SEM) and atomic force microscopy (AFM) demonstrated a partial nanoparticle coating of the probiotics, which was further supported by UV/Vis spectroscopy findings, suggesting enhanced resistance to standard antibiotics. The results revealed that this strain possesses a unique protein profile and is genetically similar to strains identified in various other countries. Moreover, nano-encapsulation notably increased the strain's resistance to a spectrum of standard antibiotics, including Benzylpenicillin, Teicoplanin, Oxacillin, Vancomycin, Tetracycline, Rifampicin, Erythromycin, and Clindamycin. These findings imply that nanoparticle-coated probiotics may effectively counteract the detrimental effects of extended antibiotic therapy, thus preserving their viability and beneficial influence on gastrointestinal health.

The impact of Lactococcus lactis KUST48 on the transcription profile of Aeromonas hydrophila-infected zebrafish spleen.

Aeromonas hydrophila, an aquatic pathogenic bacterium, has been found to infect many fish species and cause huge aquaculture losses. Antibiotics are the most common drugs used to treat these infections. However, antibiotic abuse can lead to the development of antibiotic resistance. Probiotics have the potential to replace antibiotics for preventing infections. Zebrafish (Danio rerio) is a model organism used to study the innate immune system and host-pathogen interactions. Currently, there is little information on how the fish immune system responds to A. hydrophila and probiotic treatment. To increase the understanding of the molecular mechanisms behind the zebrafish defense against A. hydrophila and provide evidence that antibiotics can be replaced by probiotics, a transcriptome analysis of the zebrafish spleen was conducted 48 hours after infection by A. hydrophila, as well as after treatment using Lactococcus lactis KUST48 4 hours after infection. A total of 36,499 genes were obtained. There were 3,337 genes found to have significant differential expression between treatment and control groups. According to further annotation and enrichment analysis, differentially expressed genes (DEGs) were involved in signal transduction, endocrine system cancer, and the immune system. Insulin resistance disappeared in the zebrafish after treatment. Quantitative real-time PCR was performed to confirm the significant regulation of immune defense DEGs, the results of which were consistent with the RNA-sequencing data. These results could serve as a basis for future studies on the immune response to A. hydrophila and provide suggestions for probiotic alternatives to antibiotics, which will be of great significance to aquaculture and environmental protection.IMPORTANCEIn recent years, the unreasonable use of antibiotics has led to the emergence of drug-resistant pathogenic bacteria, antibiotic residues, cross infection, toxic side effects, and so on, which has caused a serious threat to human food safety and life health. In recent years, many studies have demonstrated the potential of probiotics as a substitute for antibiotics, but there is still a lack of understanding of the molecular mechanisms underlying probiotic therapy. We conduct a research on the impact of Lactococcus lactis KUST48 on the transcription profile of Aeromonas hydrophila-infected zebrafish spleen. Mortality of zebrafish infected with A. hydrophila was significantly reduced after treatment with L. lactis KUST48. Our results can help to strengthen our understanding of the pathogenic mechanisms of zebrafish and provide a valuable reference for the molecular mechanisms of probiotic therapy.

The strain-dependent cytostatic activity of Lactococcus lactis on CRC cell lines is mediated through the release of arginine deiminase.

BACKGROUND: Colorectal cancer (CRC) is one of the most commonly diagnosed cancers, posing a serious public health challenge that necessitates the development of new therapeutics, therapies, and prevention methods. Among the various therapeutic approaches, interventions involving lactic acid bacteria (LAB) as probiotics and postbiotics have emerged as promising candidates for treating and preventing CRC. While human-isolated LAB strains are considered highly favorable, those sourced from environmental reservoirs such as dairy and fermented foods are also being recognized as potential sources for future therapeutics. RESULTS: In this study, we present a novel and therapeutically promising strain, Lactococcus lactis ssp. lactis Lc4, isolated from dairy sources. Lc4 demonstrated the ability to release the cytostatic agent - arginine deiminase (ADI) - into the post-cultivation supernatant when cultured under conditions mimicking the human gut environment. Released arginine deiminase was able to significantly reduce the growth of HT-29 and HCT116 cells due to the depletion of arginine, which led to decreased levels of c-Myc, reduced phosphorylation of p70-S6 kinase, and cell cycle arrest. The ADI release and cytostatic properties were strain-dependent, as was evident from comparison to other L. lactis ssp. lactis strains. CONCLUSION: For the first time, we unveil the anti-proliferative properties of the L. lactis cell-free supernatant (CFS), which are independent of bacteriocins or other small molecules. We demonstrate that ADI, derived from a dairy-Generally Recognized As Safe (GRAS) strain of L. lactis, exhibits anti-proliferative activity on cell lines with different levels of argininosuccinate synthetase 1 (ASS1) expression. A unique feature of the Lc4 strain is also its capability to release ADI into the extracellular space. Taken together, we showcase L. lactis ADI and the Lc4 strain as promising, potential therapeutic agents with broad applicability.

The Acetic Acid Produced by Lactobacillus Species Regulates Immune Function to Alleviate PEDV Infection in Piglets.

Porcine epidemic diarrhea virus (PEDV) infection results in significant mortality among newborn piglets, leading to substantial economic setbacks in the pig industry. Short-chain fatty acids (SCFA), the metabolites of intestinal probiotics, play pivotal roles in modulating intestinal function, enhancing the intestinal barrier, and bolstering immune responses through diverse mechanisms. The protective potential of Lactobacillus delbrueckii, Lactobacillus johnsonii, and Lactococcus lactis was first noted when administered to PEDV-infected piglets. Histological evaluations, combined with immunofluorescence studies, indicated that piglets receiving L. lactis displayed less intestinal damage, with diminished epithelial cell necrosis and milder injury levels. Differences in immunofluorescence intensity revealed a significant disparity in antigen content between the L. lactis and PEDV groups, suggesting that L. lactis might suppress PEDV replication, the intestine. We then assessed short-chain fatty acid content through targeted metabolomics, finding that acetate levels markedly varied from other groups. This protective impact was confirmed by administering acetate to PEDV-infected piglets. Data suggested that piglets receiving acetate exhibited resistance to PEDV. Flow cytometry analyses were conducted to evaluate the expression of innate and adaptive immune cells in piglets. Sodium acetate appeared to bolster innate immune defenses against PEDV, marked by elevated NK cell and macrophage counts in mesenteric lymph nodes, along with increased NK cells in the spleen and macrophages in the bloodstream. Acetic acid was also found to enhance the populations of CD8+ IFN-γ T cells in the blood, spleen, and mesenteric lymph, CD4+ IFN-γ T cells in mesenteric lymph nodes and spleen, and CD4+ IL-4+T cells in the bloodstream. Transcriptome analyses were carried out on the jejunal mucosa from piglets with PEDV-induced intestinal damage and from healthy counterparts with intact barriers. Through bioinformatics analysis, we pinpointed 189 significantly upregulated genes and 333 downregulated ones, with the PI3K-AKT, ECM-receptor interaction, and pancreatic secretion pathways being notably enriched. This transcriptomic evidence was further corroborated by western blot and qPCR. Short-chain fatty acids (SCFA) were found to modulate G protein-coupled receptor 41 (GPR41) and 43 (GPR43) in porcine intestinal epithelial cells (IPEC-J2). Post-acetic acid exposure, there was a notable upsurge in the ZO-1 barrier protein expression in IPEC-J2 compared to the unexposed control group (WT), while GPR43 knockdown inversely affected ZO-1 expression. Acetic acid amplified the concentrations of phosphorylated PI3K and AKT pivotal components of the PI3K/AKT pathway. Concurrently, the co-administration of AKT agonist SC79 and PI3K inhibitor LY294002 revealed acetic acid's role in augmenting ZO-1 expression via the P13K/AKT signaling pathway. This study demonstrates that acetic acid produced by Lactobacillus strains regulates intestinal barrier and immune functions to alleviate PEDV infection. These findings provide valuable insights for mitigating the impact of PEDV in the pig industry.

Probiotic Properties of Lactococcus lactis Strains Isolated from Natural Whey Starter Cultures.

Lactococcus lactis is a lactic acid bacterium (LAB), generally recognized as safe, and has been widely used in the food industry, especially in fermented dairy products. Numerous studies have evaluated the technological and probiotic properties of lactococci; however, few studies have reported the probiotic characteristics of L. lactis strains isolated from dairy products. In this work, probiotic potential, including survival in simulated gastric juice, tolerance to bile salts, hydrophobicity, and auto- and co-aggregation, was evaluated in L. lactis strains from natural whey starter cultures. The results highlighted the potential probiotic properties of some strains under study, which showed high values of hydrophobicity and auto-aggregation and low values of co-aggregation with the tested pathogenic strains. In addition, studies of safety parameters, such as antibiotic susceptibility and haemolytic activity, confirmed the safety status of all strains under study. Finally, the four most promising strains were investigated for their ability to inhibit the enteroinvasive Escherichia coli (EIEC) and Salmonella Typhimurium adhesion to epithelial cells, using a model of co-cultured epithelial cells. The results demonstrated that L. lactis strains A3-A5-I4-I7 showed the ability to compete with pathogens as well as the ability to exert a protective effect on cells previously infected with E. coli or S. Typhimurium. The identification of new probiotic LAB strains from dairy products aims to produce novel functional foods.

Human Soluble TRAIL Secreted by Modified Lactococcus lactis Bacteria Promotes Tumor Growth in the Orthotopic Mouse Model of Colorectal Cancer.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis of sensitive cancer cells, including colorectal cancer (CRC). Due to its short biological half-life after intravenous administration and related clinical ineffectiveness, novel formulations of TRAIL need to be developed. Here we propose Lactococcus lactis bacteria as a vehicle for local delivery of human soluble TRAIL (hsTRAIL) in CRC. The use of common probiotics targeting guts as carriers for TRAIL could ensure its sustained release at the tumor site and extend the duration of its activity. We have already engineered hsTRAIL-secreting L.lactis bacteria and showed their effectiveness in elimination of human CRC cells in vitro and in vivo in a mouse subcutaneous model. Here, L.lactis(hsTRAIL+) were administered by gastric gavage to SCID mice with orthotopically developed HCT116 tumor in cecum, in monotherapy or in combination with metformin (MetF), already shown to enhance the hsTRAIL anti-tumor activity in subcutaneous CRC model. Oral administration of L.lactis(hsTRAIL+) resulted in significant progression of HCT116 tumors and shortening of the colon crypts. Secretion of hsTRAIL in the colon was accompanied by infiltration of the primary tumor with M2-macrophages, while MetF promoted transient colonization of the gut by L.lactis. Our study indicates that L.lactis bacteria after oral administration enable delivery of biologically active hsTRAIL to colon, however its potential therapeutic effect in CRC treatment is abolished by its pro-tumorigenic signalling, leading to the recruitment of M2-macrophages and tumor growth promotion.

Enhancing nutritional and sensory properties of plant-based beverages: a study on chickpea and Kamut® flours fermentation using Lactococcus lactis.

The study aimed to set up a protocol for the production of a clean-label plant-based beverage (PBB), obtained by mixing chickpeas and Kamut® flours and using a commercial Lactococcus lactis (LL) as fermentation starter, and to characterize it, from nutritional, microbiological, textural, shelf-life, and sensory points of view. The effect of using the starter was evaluated comparing the LL-PBB with a spontaneously fermented beverage (CTRL-PBB). Both PBBs were high in proteins (3.89/100 g) and could be considered as sources of fiber (2.06/100 g). Notably, L. lactis fermentation enhanced the phosphorus (478 vs. 331 mg/kg) and calcium (165 vs. 117 mg/kg) concentrations while lowering the raffinose content (5.51 vs. 5.08 g/100 g) compared to spontaneous fermentation. Cell density of lactic acid bacteria increased by ca. two log cycle during fermentation of LL-PBB, whereas undesirable microbial groups were not detected. Furthermore, L. lactis significantly improved the beverage's viscosity (0.473 vs. 0.231 Pa s), at least for 10 days, and lightness. To assess market potential, we conducted a consumer test, presenting the LL-PBB in "plain" and "sweet" (chocolate paste-added) variants. The "sweet" LL-PBB demonstrated a higher acceptability score than its "plain" counterpart, with 88 and 78% of participants expressing acceptability and a strong purchase intent, respectively. This positive consumer response positions the sweet LL-PBB as a valuable, appealing alternative to traditional flavored yogurts, highlighting its potential in the growing plant-based food market.