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This study describes the discovery and characterization of raffinocyclicin, a novel plasmid-encoded circular bacteriocin, produced by the raw milk isolate Lactococcus raffinolactis APC 3967. This bacteriocin has a molecular mass of 6,092 Da and contains 61 amino acids with a three-amino acid leader peptide. It shows the highest identity to the circular bacteriocins bacicyclicin XIN-1 (42.62%), aureocyclicin 4185 (42.62%), and garvicin ML (41.53%). A broad inhibitory spectrum includes strains from Staphylococcus , Enterococcus , Streptococcus , Micrococcus , Lactobacillus , Leuconostoc, and Listeria , in addition to a pronounced inhibitory effect against Lactococcus and Clostridium . It displays low sensitivity to trypsin, most likely as a result of its circular nature. The raffinocyclicin gene cluster is composed of 10 genes: 6 core genes, genes encoding an accessory three-component ABC transporter ( rafCDE ), and a putative transcriptional regulator related to the MutR family. A lack of inhibitory activity in the cell-free supernatant combined with the pronounced activity of cell extracts suggests that the majority of raffinocyclicin is associated with the cell rather than being released to the extracellular environment. This is the first report of a bacteriocin produced by the L. raffinolactis species.IMPORTANCEThe present study aimed to characterize raffinocyclicin, a novel circular bacteriocin produced by the lactic acid bacteria Lactococcus raffinolactis APC 3967. Bacteriocins are generally cationic and hydrophobic peptides with antimicrobial activity, which present diverse biotechnological properties of interest for the food industry. Raffinocyclicin inhibits a wide range of bacteria, including foodborne pathogens, and is stable against different treatments which suggest its potential as a natural biopreservative. Whole-genome sequencing and the genetic analysis of the raffinocyclicin gene cluster showed that it is encoded by plasmid that could be used in the future to transfer the ability to produce the bacteriocin to other lactic acid bacteria for industrial applications. These results together highlight the potential of this novel antimicrobial as a biopreservative to be used by the food industry., Competing Interests: The authors declare no conflict of interest.

Lactococcus (Lc.) paracarnosus and the phylogenetically closely related Lc. carnosus species are common members of the microbiota in meat stored under modified atmosphere and at low temperature. The effect of these strains on meat spoilage is controversially discussed. While some strains are known to cause spoilage, others are being studied for their potential to suppress the growth of spoilage and pathogenic bacteria. In this study, Lc. paracarnosus DSM 111017 T was selected based on a previous study for its ability to suppress the growth of meat spoilers, including Brochothrix thermosphacta. The mechanism by which this bioprotective strain inhibits competing bacteria and how it contributes to spoilage are not yet known. To answer these two questions, we investigated the effect of four different headspace gas mixtures (simulated air (21 % O 2 /79 % N 2 ); HiOx-MAP (70 % O 2 /30 % CO 2 ); nonOx-MAP (70 % N 2 / 30 % CO 2 ); simulated vacuum (100 % N 2 ) and the presence of Brochothrix (B.) thermosphacta TMW 2.2101 on the growth and transcriptional response of Lc. paracarnosus DSM 111017 T when cultured on a meat simulation agar surface at 4 °C. Analysis of genes specifically upregulated by the gas mixtures used revealed metabolic pathways that may lead to different levels of spoilage metabolites production. We propose that under elevated oxygen levels, Lc. paracarnosus preferentially converts pyruvate from glucose and glycerol to uncharged acetoin/diacetyl instead of lactate to counteract acid stress. Due to the potential production of a buttery off-flavour, the strain may not be suitable as a protective culture in meat packaged under high‑oxygen conditions. 70 % N 2 / 30 % CO 2 , simulated vacuum- and the presence of Lc. paracarnosus inhibited the growth of B. thermosphacta TMW 2.2101. However, B. thermosphacta did not affect gene regulation of metabolic pathways in Lc. paracarnosus, and genes previously predicted to be involved in B. thermosphacta growth suppression were not regulated at the transcriptional level. In conclusion, the study indicates that the gas mixture used in packaging significantly affects the metabolism and spoilage potential of Lc. paracarnosus and its ability to inhibit B. thermosphacta growth., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Victoria Werum reports financial support was provided by German Federal Ministry of Food and Agriculture. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

In this study, the feasibility of promoting the lactic acid (LA) fermentation of food waste (FW) with iron tailings (ITs) addition was explored. The best LA yield was 0.91 g LA/g total sugar when 1 % ITs were added into the system. The mechanisms for promoting LA production were acidification alleviation effects and reduction equivalent supply of ITs. Furthermore, the addition of ITs promoted carbohydrate hydrolysis, and the carbohydrates digestibility reached 88.85 % in the 1 % ITs group. The ITs also affected the microbial communities, Lactococcus gradually replaced Streptococcus as the dominant genus, and results suggested that Lactococcus had a positive correlation with LA production and carbohydrate digestibility. Finally, the complex LAB in FW had significant effects on heavy metal removal from ITs, and the removal efficiency Cr, As, Pb, Cd, and Hg can reach 50.84 %, 26.72 %, 59.65 %, 49.75 % and 78.87 % in the 1 % ITs group, respectively., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Lactococcus garvieae (L. garvieae) is a pathogenic bacterium that is Gram-positive and catalase-negative (GPCN), and it is capable of growing in a wide range of environmental conditions. This bacterium is associated with significant mortality and losses in fisheries, and there are concerns regarding its potential as a zoonotic pathogen, given its presence in cattle and dairy products. While we have identified and characterized virulent strains of L. garvieae through phenotyping and molecular typing studies, their impact on mammary tissue remains unknown. This study aims to investigate the pathogenicity of strong and weak virulent strains of L. garvieae using in vivo mouse models. We aim to establish MAC-T cell model to examine potential injury caused by the strong virulent strain LG41 through the TLR2/NLRP3/NF-kB pathway. Furthermore, we assess the involvement of NLRP3 inflammasome-mediated pyroptosis in dairy mastitis by silencing NLRP3. The outcomes of this study will yield crucial theoretical insights into the potential mechanisms involved in mastitis in cows caused by the L. garvieae-induced inflammatory response in MAC-T cells., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The wild-type Lactococcus lactis strain LAC460 produces two bacteriocin-like phage lysins, LysL and LysP. This study aimed to produce and secrete LysL in various heterologous hosts and an in vitro cell-free expression system for further functional studies. Initially, the lysL gene from L. lactis LAC460 was cloned into Lactococcus cremoris NZ9000 and L. lactis N8 strains, with and without the usp45 signal sequence (SSusp45), under a nisin-inducible promoter. Active LysL was primarily produced intracellularly in recombinant L. lactis N8, with some secretion into the supernatant. Recombinant L. cremoris NZ9000 lysed upon nisin induction, indicating successful lysL expression. However, fusion with Usp45 signal peptide (SPUsp45-LysL) weakened LysL activity, likely due to incomplete signal peptide cleavage during secretion. Active LysL was also produced in vitro, and analysed in SDS-PAGE, giving a 42-kDa band. However, the yield of LysL protein was still low when produced from recombinant lactococci or by in vitro expression system. Therefore, His-tagged LysL was produced in Escherichia coli BL21(DE3). Western blot confirmed the intracellular production of about 44-kDa His-tagged LysL in E. coli. His-tagged active LysL was then purified by Ni-NTA affinity chromatography yielding sufficient 4.34 mg of protein to be used in future functional studies., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

In Gram-positive bacteria, cell wall polysaccharides (CWPS) play critical roles in bacterial cell wall homeostasis and bacterial interactions with their immediate surroundings. In lactococci, CWPS consist of two components: a conserved rhamnan embedded in the peptidoglycan layer and a surface-exposed polysaccharide pellicle (PSP), which are linked together to form a large rhamnose-rich CWPS (Rha-CWPS). PSP, whose structure varies from strain to strain, is a receptor for many bacteriophages infecting lactococci. Here, we examined the first two steps of PSP biosynthesis, using in vitro enzymatic tests with lipid acceptor substrates combined with LC-MS analysis, AlfaFold2 modeling of protein 3D-structure, complementation experiments, and phage assays. We show that the PSP repeat unit is assembled on an undecaprenyl-monophosphate (C 55 P) lipid intermediate. Synthesis is initiated by the WpsA/WpsB complex with GlcNAc-P-C 55 synthase activity and the PSP precursor GlcNAc-P-C 55 is then elongated by specific glycosyltransferases that vary among lactococcal strains, resulting in PSPs with diverse structures. Also, we engineered the PSP biosynthesis pathway in lactococci to obtain a chimeric PSP structure, confirming the predicted glycosyltransferase specificities. This enabled us to highlight the importance of a single sugar residue of the PSP repeat unit in phage recognition. In conclusion, our results support a novel pathway for PSP biosynthesis on a lipid-monophosphate intermediate as an extracellular modification of rhamnan, unveiling an assembly machinery for complex Rha-CWPS with structural diversity in lactococci., Competing Interests: Conflicts of interest C. C. is an employee of Alphagraphix (cambillau.alphagraphix@gmail.com). Authors and Alphagraphix declare that they have no competing interests. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Paucilactobacillus wasatchensis can use gluconate (GLCN) as well as galactose as an energy source and because sodium GLCN can be added during salting of Cheddar cheese to reduce calcium lactate crystal formation, our primary objective was to determine if the presence of GLCN in cheese is another risk factor for unwanted gas production leading to slits in cheese. A secondary objective was to calculate the amount of CO 2 produced during storage and to relate this to the amount of gas-forming substrate that was utilized. Ribose was added to promote growth of Pa. wasatchensis WDC04 (P.waWDC04) to high numbers during storage. Cheddar cheese was made with lactococcal starter culture with addition of P.waWDC04 on 3 separate occasions. After milling, the curd was divided into six 10-kg portions. To the curd was added (A) salt, or salt plus (B) 0.5% galactose + 0.5% ribose (similar to previous studies), (C) 1% sodium GLCN, (D) 1% sodium GLCN + 0.5% ribose, (E) 2% sodium GLCN, (F) 2% sodium GLCN + 0.5% ribose. A vat of cheese without added P.waWDC04 was made using the same milk and a block of cheese used as an additional control. Cheeses were cut into 900-g pieces, vacuum packaged and stored at 12°C for 16 wk. Each month the bags were examined for gas production and cheese sampled and tested for lactose, galactose and GLCN content, and microbial numbers. In the control cheese, P.waWDC04 remained undetected (i.e., <10 4 cfu/g), whereas in cheeses A, C, and E it increased to 10 7 cfu/g, and when ribose was included with salting (cheeses B, D, and F) increased to 10 8 cfu/g. The amount of gas (measured as headspace height or calculated as mmoles of CO 2 ) during 16 wk storage was increased by adding P.waWDC04 into the milk, and by adding galactose or GLCN to the curd. Galactose levels in cheese B were depleted by 12 wk while no other cheeses had residual galactose. Except for cheese D, the other cheeses with GLCN added (C, E and F) showed little decline in GLCN levels until wk 12, even though gas was being produced starting at wk 4. Based on calculations of CO 2 in headspace plus CO 2 dissolved in cheese, galactose and GLCN added to cheese curd only accounted for about half of total gas production. It is proposed that CO 2 was also produced by decarboxylation of amino acids. Although P.waWDC04 does not have all the genes for complete conversion and decarboxylation of the amino acids in cheese, this can be achieved in conjunction with starter culture lactococcal. Adding GLCN to curd can now be considered another confirmed risk factor for unwanted gas production during storage of Cheddar cheese that can lead to slits and cracks in cheese. Putative risk factors now include having a community of bacteria in cheese leading to decarboxylation of amino acids and release of CO 2 as well autolysis of the starter culture that would provide a supply of ribose that can promote growth of Pa. wasatchensis., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Lactococcus garvieae causes infectious diseases in animals and is considered an emerging zoonotic pathogen involved in human clinical conditions. In silico analysis of plasmid pLG50 of L. garvieae Lg-Granada, an isolate from a patient with endocarditis, revealed the presence of two gene clusters ( orf 46-47 and orf 48-49), each one encoding a novel putative bacteriocin, i.e., garvicin AG1 (GarAG1; orf 46) and garvicin AG2 (GarAG2; orf 48), and their corresponding immunity proteins ( orf 47 and orf 49). The chemically synthesised bacteriocins GarAG1 and GarAG2 presented inhibitory activity against pathogenic L. garvieae strains, with AG2 also being active against Listeria monocytogenes , Listeria ivanovii and Enterococcus faecalis . Genetic organisation, amino acid sequences and antimicrobial activities of GarAG1 and GarAG2 indicate that they belong to linear non-pediocin-like one-peptide class IId bacteriocins. Gram-positive bacteria that were sensitive to GarAG2 were also able to ferment mannose, suggesting that this bacteriocin could use the mannose phosphotransferase transport system (Man-PTS) involved in mannose uptake as a receptor in sensitive strains. Intriguingly, GarAG1 and GarAG2 were highly active against their own host, L. garvieae Lg-Granada, which could be envisaged as a new strategy to combat pathogens via their own weapons.

Human gut-originated lactic acid bacteria were cultivated, and high γ-aminobutyric acid (GABA)-producing Lactococcus garvieae MJF010 was identified. To date, despite the importance of GABA, no studies have investigated GABA-producing Lactococcus species, except for Lc. lactis. A recombinant glutamate decarboxylase of the strain MJF010 (rLgGad) was successfully expressed in Escherichia coli BL21(DE3) with a size of 53.9 kDa. rLgGad could produce GABA, which was verified using the silylation-derivative fragment ions of GABA. The purified rLgGad showed the highest GABA-producing activity at 35 °C and pH 5. rLgGad showed a melting temperature of 43.84 °C. At 30 °C, more than 80% of the activity was maintained even after 7 h; however, it rapidly decreased at 50 °C. The kinetic parameters, K m , V max , and k cat , of rLgGad were 2.94 mM, 0.023 mM/min, and 12.3 min - 1 , respectively. The metal reagents of CaCl 2 , MgCl 2 , and ZnCl 2 significantly had positive effects on rLgGad activity. However, most coenzymes including pyridoxal 5'-phosphate showed no significant effects on enzyme activity. In conclusion, this is the first report of Gad from Lc. garvieae species and provides important enzymatic information related to GABA biosynthesis in the Lactococcus genus., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

A multiparameter study was performed to evaluate the effect of fondaco, a traditional ripening cellar without any artificial temperature and relative humidity control, on the chemical, microbiological, and sensory characteristics of Protected Geographical Indication Canestrato di Moliterno cheese. Ripening in such a nonconventional environment was associated with lower counts of lactococci, lactobacilli, and total viable bacteria, and higher presence of enterococci, in comparison with ripening in a controlled maturation room. Moreover, fondaco cheese underwent accelerated maturation, as demonstrated by faster casein degradation, greater accumulation of free AA, and higher formation of volatile organic compounds. Secondary proteolysis, as assessed by liquid chromatography-mass spectrometry of free AA and low molecular weight peptides, did not show any qualitative difference among cheeses, but fondaco samples evidenced an advanced level of peptidolysis. On the other hand, significant qualitative differences were observed in the free fatty acid profiles and in the sensory characteristics. Principal component analysis showed a clear separation of the fondaco and control cheeses, indicating that ripening in the natural room conferred unique sensory features to the product., (© 2022, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

In this study, we expressed rAvBD1-2-6-13 protein through Lactococcus lactis NZ3900, and the effects of the recombinant L. lactis NZ3900 as an immune enhancer and immune adjuvant were verified using in vivo and in vitro tests. In vitro tests revealed that recombinant L. lactis NZ3900 significantly activated the NF-κB signaling pathway and IRF signaling pathway in J774-Dual™ report cells and significantly increased the transcript levels of IL-10, IL-12p70, CD80, and CD86 in chicken PBMCs and chicken HD11 cells. In vivo experiments revealed that the immunized group supplemented with recombinant L. lactis NZ3900 as an adjuvant had significantly higher serum antibody titers and higher proliferative activity of PBMCs in the blood of the chickens immunized with NDV live and inactivated vaccines. Our study shows that the recombinant L. lactis NZ3900 has strong immunomodulatory activity both in vivo and in vitro and is a potential immune enhancer. Our work lays the foundation for the research and development of new animal immune enhancers for application in the poultry industry., (© 2021. The Author(s).)

Strains of Lactococcus lactis subsp. cremoris are used to produce yogurt containing exopolysaccharides with a sticky texture. When strain G3-2 producing exopolysaccharides was grown at elevated temperatures, a spontaneous mutant EPSC, which had lost exopolysaccharides biosynthesis, was isolated. Genomes of the two strains were determined to be composed of a 2.4-Mb chromosome and up to eleven plasmids, and it was revealed that one of the plasmids encoding the gene cluster for exopolysaccharides biosynthesis was lost selectively in EPSC.

Listeria monocytogenes is an important food-borne pathogen and a serious concern to food industries. Bacteriocins are antimicrobial peptides produced naturally by a wide range of bacteria mostly belonging to the group of lactic acid bacteria (LAB), which also comprises many strains used as starter cultures or probiotic supplements. Consequently, multifunctional strains that produce bacteriocins are an attractive approach to combine a green-label approach for food preservation with an important probiotic trait. Here, a collection of bacterial isolates from raw cow's milk was typed by 16S rRNA gene sequencing and MALDI-Biotyping and supernatants were screened for the production of antimicrobial compounds. Screening was performed with live Listeria monocytogenes biosensors using a growth-dependent assay and pHluorin, a pH-dependent protein reporting membrane damage. Purification by cation exchange chromatography and further investigation of the active compounds in supernatants of two isolates belonging to the species Pediococcus acidilactici and Lactococcus garvieae suggest that their antimicrobial activity is related to heat-stable proteins/peptides that presumably belong to the class IIa bacteriocins. In conclusion, we present a pipeline of methods for high-throughput screening of strain libraries for potential starter cultures and probiotics producing antimicrobial compounds and their identification and analysis.

The effect of different types of sugar (sucrose, demerara, brown, fructose, coconut sugar, and honey) on sheep milk kefir was evaluated. Microbial counts (Lactobacillus, Lactococcus, Leuconostoc, yeast), antagonistic activity against foodborne pathogens, microstructure (scanning electron microscopy), and antiproliferative activity of cancer cells were evaluated. Furthermore, the antioxidant activity (DPPH), inhibitory activity of angiotensin-converting enzyme (ACE), α-amylase, and α-glucosidase, lactose content, lactic and acetic acids and ethanol, fatty acid profile and volatile organic compounds were determined. The addition of sugars increased the Lactobacillus population (up to 2.24 log CFU/mL), metabolites concentration, antagonistic activity against pathogens, antioxidant activity (11.1 to 24.1%), ACE inhibitory activity (27.5 to 37.6%), α-amylase inhibition (18 to 37.4%), and anti-proliferative activity. Furthermore, it improved the fatty acid profile and volatile compounds. The results suggest that the replacement of sucrose with different types of sugar constitute an interesting option to be used in sheep milk kefir formulations., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

A mechanistic, spatio-temporal model to predict early stage semi-solid food ripening, exemplary for semi-solid casein matrices, was created using software based on the finite element method (FEM). The model was refined and validated by experimental data obtained during 8 wk of ripening of a casein matrix that was inoculated by one single central injection of starter culture. The resulting spatio-temporal distributions of lactococci strains, lactose, lactic acid/lactate and pH allowed us to optimize a number of parameters of the predictive model. Using the optimized model, the agreement between simulation and experiment was found to be satisfactory, with the pH matching best. The predictive model unveiled that effective diffusion of substrate and metabolites were crucial for an eventual homogeneous distribution of the measured substances. Hence, while using the optimized parameters from the single injection model, an injection technology for starter culture to inoculate and ferment casein matrices homogeneously was developed by means of solving another optimization problem with respect to injection positions. The casein matrix inoculated by the proposed injection pattern (21 injections, distance = 19 mm) showed sufficient homogeneity (bacterial activity and pH distribution) after the early stages of ripening, demonstrating the potential of application of the injection technology for fermentation of casein-based foods e.g. cheese., (Copyright © 2021. Published by Elsevier B.V.)

Microbiological spoilage of meat is considered as a process which involves mainly bacterial metabolism leading to degradation of meat sensory qualities. Studying spoilage requires the collection of different types of experimental data encompassing microbiological, physicochemical and sensorial measurements. Within this framework, the objective herein was to carry out a multiblock path modelling workflow to decipher causality relationships between different types of spoilage-related responses: composition of microbiota, volatilome and off-odour profiles. Analyses were performed with the Path-ComDim approach on a large-scale dataset collected on fresh turkey sausages. This approach enabled to quantify the importance of causality relationships determined a priori between each type of responses as well as to identify important responses involved in spoilage, then to validate causality assumptions. Results were very promising: the data integration confirmed and quantified the causality between data blocks, exhibiting the dynamical nature of spoilage, mainly characterized by the evolution of off-odour profiles caused by the production of volatile organic compounds such as ethanol or ethyl acetate. This production was possibly associated with several bacterial species like Lactococcus piscium, Leuconostoc gelidum, Psychrobacter sp. or Latilactobacillus fuchuensis. Likewise, the production of acetoin and diacetyl in meat spoilage was highlighted. The Path-ComDim approach illustrated here with meat spoilage can be applied to other large-scale and heterogeneous datasets associated with pathway scenarios and represents a promising key tool for deciphering causality in complex biological phenomena., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

A bioactive peptide of 8595 Da was purified from the cell free supernatant of Lactococcus garvieae subsp. bovis BSN307 T . MALDI MS/MS peptide mapping and the data base search displayed no significant similarity to any reported antimicrobial peptide of LAB. This peptide at a dose concentration of 200 µg ml -1 inhibited the growth of both Gram-positive and Gram-negative bacteria by 58-89% and a dose of 500 µg ml -1 scavenged 50% of DPPH-free radicals generated. Interestingly, cytotoxicity assay demonstrated that 17 µg ml -1 of peptide selectively inhibited 50% proliferation of mammalian cancer cell lines HeLa and MCF-7 whereas normal H9c2 cells remained unaffected. Fluorescent microscopic analysis after DAPI nuclear staining of HeLa cells showed characteristics of apoptosis and activation of caspase-3 was ascertained by caspase-3 fluorescence assay., (© 2021 The Society for Applied Microbiology.)

Efficient genome editing methods are essential for biotechnology and fundamental research. Homologous recombination (HR) is the most versatile method of genome editing, but techniques that rely on host RecA-mediated pathways are inefficient and laborious. Phage-encoded single-stranded DNA annealing proteins (SSAPs) improve HR 1,000-fold above endogenous levels. However, they are not broadly functional. Using Escherichia coli, Lactococcus lactis, Mycobacterium smegmatis, Lactobacillus rhamnosus and Caulobacter crescentus, we investigated the limited portability of SSAPs. We find that these proteins specifically recognize the C-terminal tail of the host's single-stranded DNA-binding protein (SSB) and are portable between species only if compatibility with this host domain is maintained. Furthermore, we find that co-expressing SSAPs with SSBs can significantly improve genome editing efficiency, in some species enabling SSAP functionality even without host compatibility. Finally, we find that high-efficiency HR far surpasses the mutational capacity of commonly used random mutagenesis methods, generating exceptional phenotypes that are inaccessible through sequential nucleotide conversions.

We report the supramolecular assembly of artificial metalloenzymes (ArMs), based on the Lactococcal multidrug resistance regulator (LmrR) and an exogeneous copper(II)-phenanthroline complex, in the cytoplasm of E. coli cells. A combination of catalysis, cell-fractionation, and inhibitor experiments, supplemented with in-cell solid-state NMR spectroscopy, confirmed the in-cell assembly. The ArM-containing whole cells were active in the catalysis of the enantioselective Friedel-Crafts alkylation of indoles and the Diels-Alder reaction of azachalcone with cyclopentadiene. Directed evolution resulted in two different improved mutants for both reactions, LmrR&#95;A92E&#95;M8D and LmrR&#95;A92E&#95;V15A, respectively. The whole-cell ArM system required no engineering of the microbial host, the protein scaffold, or the cofactor to achieve ArM assembly and catalysis. We consider this a key step towards integrating abiological catalysis with biosynthesis to generate a hybrid metabolism., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Today, cheese is valued because of its high nutritional value and unique characteristics. Improving the texture and flavor of cheese by selecting suitable starter cultures is an important way to promote the development of cheese industry. The effect of starter cultures on the physicochemical and textural properties and volatile compounds during the ripening of semihard goat cheese were investigated in this work. Different starter cultures-mesophilic (M) and thermophilic starters (T), Lactobacillus plantarum ssp. plantarum ATCC 14917 (Lp), a mix of the M and T starters (M1), and mix of the M, T, and Lp starters (M2)-were used in the production of the goat cheeses. Volatile compounds were determined by a solid-phase microextraction/gas chromatography-mass spectrometric (SPME/GC-MS) method. The results showed that the moisture content of cheeses produced with the 5 kinds of starter cultures decreased after maturation, whereas ash content increased. The pH values of goat cheeses decreased first and then increased during maturity, and the pH value of M2 cheese was the lowest among the cheeses. The hardness and chewiness of the cheeses increased with increasing maturity, whereas cohesiveness, springiness, and resilience showed the opposite tendency. The 60-d-old cheese made with Lp had the highest chewiness, cohesiveness, springiness, and resilience, whereas the 60-d-old cheese made with M2 had the highest hardness. A total of 53 volatile components were identified by SPME/GC-MS, and carboxylic acids, alcohols, ketones, and esters were the 4 major contributors to the characteristic flavors of the cheeses. Volatile components and their contents differed greatly among the produced cheeses. The M2 cheese contained the highest relative content of the main volatile compounds (90.10%), especially butanoic acid and acetoin. Through a comprehensive comparison of the results, we concluded that M2 cheese had a dense texture and milky flavor, and M2 is a potential starter culture candidate for the production of goat cheese., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Fermented camel milk possesses a weak (liquid-like) gel structure. We aimed to 1) investigate the characteristics, bioactivities and rheological properties of the exopolysaccharide (EPS) produced by Lactococcus garvieae-C47 (exopolysaccharide-C47 product), a potential probiotic bacterium, on milk extracted from camels and 2) examine the rheological properties of the fermented camel milk produced by L. garvieae-C47. Exopolysaccharide-C47 product (molecular weight: 7.3 × 10 6 Da) was composed of the following monosaccharides: glucose (82.51%), arabinose (5.32%) and xylose (12.17%). The antioxidant, antitumor and α-amylase inhibitory activities of exopolysaccharide-C47 product reached up to 67.52, 59.35 and 91.0%, respectively. The apparent viscosity of exopolysaccharide-C47 product decreased with the increase in shear rate and declined by increasing the temperature up to 50 °C. The rheological properties of exopolysaccharide-C47 product are influenced by the salt type and pH value. The exopolysaccharide product produced by L. garvieae C47 possesses valuable health benefits and has the ability to improve the weak structure of fermented camel milk., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Bacteria can gain resistance to antimicrobials by acquiring and expressing genetic elements that encode resistance determinants such as efflux pumps and drug-modifying enzymes, thus hampering treatment of infection. Previously we showed that acquisition of spectinomycin resistance in a lactococcal strain was correlated with a reversible genomic inversion, but the precise location and the genes affected were unknown. Here we use long-read whole-genome sequencing to precisely define the genomic inversion and we use quantitative PCR to identify associated changes in gene expression levels. The boundaries of the inversion fall within two identical copies of a prophage-like sequence, located on the left and right replichores; this suggests possible mechanisms for inversion through homologous recombination or prophage activity. The inversion is asymmetrical in respect of the axis between the origin and terminus of the replication and modulates the expression of a SAM-dependent methyltransferase, whose heterologous expression confers resistance to spectinomycin in lactococci and that is up-regulated on exposure to spectinomycin. This study provides one of the first examples of phase variation via large-scale chromosomal inversions that confers a switch in antimicrobial resistance in bacteria and the first outside of Staphylococcus aureus., (Crown Copyright © 2020. Published by Elsevier GmbH. All rights reserved.)

Obesity, which has become a major public health problem, can arise from complex dyslipidemia, insulin resistance, and immune responses, among other mechanisms. Some Lactobacillus strains effectively ameliorate obesity; however, the beneficial effects of Lactococcus spp., which are often used as dairy starters, remain unclear. In the present study, we evaluated the efficacy of Lactococcus chungangensis CAU 28 using the 3T3-L1 cell line and obese mice fed a high-fat diet. Overall, administration of Lc. chungangensis CAU 28 effectively resolved obesity associated with weight gain and lipid accumulation. In differentiated 3T3-L1 cells, Lc. chungangensis CAU 28 treatment significantly diminished the total lipid quantity, inhibited triglyceride formation, and prevented the proliferation of adipogenic transcription factors (fatty acid synthase, adiponectin, peroxisome proliferator-activated receptor-gamma, and CCAAT-enhancer-binding protein-α) associated with lipid accumulation. In the obesity mouse model, wherein the intake of Lc. chungangensis CAU 28 effectively reduced body weight gain, along with fat differentiation and accumulation (white fat; abdominal and subcutaneous). Furthermore, Lc. chungangensis CAU 28 increased serum adiponectin levels, decreased serum leptin levels, and effectively regulated adipokine secretion. It also increased the high-density lipoprotein:cholesterol ratio, reduced total cholesterol and triglyceride levels, reduced the low-density lipoprotein:cholesterol ratio, and affected obesity-regulated inflammatory cytokines IL-6, tumor necrosis factor-α, IFN-γ, and IL-1β. Additionally, Lc. chungangensis CAU 28 was associated with an increase in the CD3 + CD4 + CD8 - phenotype among obese mice. Thus, the administration of Lc. chungangensis CAU 28 induced antiobesity effects, suggesting potential applications of this species as a supplement for obesity mitigation., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The biosynthetic machinery for cell wall polysaccharide (CWPS) production in lactococci is encoded by a large gene cluster, designated cwps. This locus displays considerable variation among lactococcal genomes, previously prompting a classification into three distinct genotypes (A-C). In the present study, the cwps loci of 107 lactococcal strains were compared, revealing the presence of a fourth cwps genotype (type D). Lactococcal CWPSs are comprised of two saccharidic structures: a peptidoglycan-embedded rhamnan backbone polymer to which a surface-exposed, poly/oligosaccharidic side-chain is covalently linked. Chemical structures of the side-chain of seven lactococcal strains were elucidated, highlighting their diverse and strain-specific nature. Furthermore, a link between cwps genotype and chemical structure was derived based on the number of glycosyltransferase-encoding genes in the cwps cluster and the presence of conserved genes encoding the presumed priming glycosyltransferase. This facilitates predictions of several structural features of lactococcal CWPSs including (a) whether the CWPS possesses short oligo/polysaccharide side-chains, (b) the number of component monosaccharides in a given CWPS structure, (c) the order of monosaccharide incorporation into the repeating units of the side-chain (for C-type strains), (d) the presence of Galf and phosphodiester bonds in the side-chain, and (e) the presence of glycerol phosphate substituents in the side-chain., (© 2020 John Wiley & Sons Ltd.)

Lactic acid bacteria (LAB) play an important role in dairy fermentations, notably as cheese starter cultures. During the cheese production and ripening period, various enzymes from milk, rennet, starter cultures, and non-starter LABs are involved in flavor formation pathways, including glycolysis, proteolysis, and lipolysis. Among these three pathways, starter LABs are particularly related to amino acid degradation, presumably as the origins of major flavor compounds. Therefore, we used several enzymes as major criteria for the selection of starter bacteria with flavor-forming ability. Lactococcus lactis subsp. lactis LDTM6802 and Lactococcus lactis subsp. cremoris LDTM6803, isolated from Korean raw milk and cucumber kimchi, were confirmed by using multiplex PCR and characterized as starter bacteria. The combinations of starter bacteria were validated in a miniature Gouda-type cheese model. The flavor compounds of the tested miniature cheeses were analyzed and profiled by using an electronic nose. Compared to commercial industrial cheese starters, selected starter bacteria showed lower pH, and more variety in their flavor profile. These results demonstrated that LDTM6802 and LDTM6803 as starter bacteria have potent starter properties with a characteristic flavor-forming ability in cheese.

This study explores the ability of lactic acid bacteria (LAB) to ferment soy juice. The ability of 276 LAB strains from 25 species to ferment the principal soy carbohydrates, sucrose, raffinose or stachyose was tested in synthetic media and a soy juice. Fermented soy juices (FSJs) were characterized for their odor. Selected FSJs were characterized by targeted metabolomics. All Streptococcus, 83% of Leuconostoc and Lactobacillus and 41% of Lactococcus strains were sucrose-positive, while only 36% of all the LAB strains tested were raffinose-positive and 6% stachyose-positive. Nearly all (97%) the sucrose-positive strains fermented soy juice, indicating that an ability to use sucrose is a good criterion to select strains for soy juice fermentation. Among the most efficient acidifying strains, 46 FSJs had an odor deemed to be acceptable. FSJ composition was dependent on both species and strains: 17/46 strains deglycosylated soy juice isoflavones, the 27 S. thermophilus strains converted a mean 4.4 ± 0.1 g/L of sucrose into 3.0 ± 0.1 g/L of lactic acid versus 5.2 ± 0.1 g/L into 2.2 ± 0.1 g/L for the 18 Lactobacillus and one Lactococcus strains. This study highlights the diversity of the metabolic profiles of LAB strains in soy juice fermentation., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Lactic acid bacteria (LAB) can be used to increase the folate in foods by in situ fortification. Seventy LAB were screened for their ability to produce folate during growth in de Man, Rogosa and Sharpe/M17 broth. Lactobacillus casei, Lactobacillus plantarum, Lactobacillus paracasei subsp. paracasei, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactococcus lactis subsp. lactis, Enterococcus faecium and Enterococcus lactis were able to synthetize folates in the medium, even if to a different extent. The 47 folate-producing strains were further analyzed by microbiological assay, for total, extra and intracellular folate. Enterococcus faecium VC223 and E. lactis BT161 were able to produce in cultural medium 123,625.74 ± 8.00 ng/ml and 384.22 ± 5.00 ng/ml of folate, respectively. Five strains were further examined for their ability to synthesize folate in cheese. The folate content increased with ripening up to by 54% after 30 d when L. casei VC199 was used and up to 108% and 113% after 60 d, with L. paracasei SE160 and E. lactis BT161 respectively exceeding 100 ng/100g. Results encourage the use of specific LAB to obtain natural folate bio-enriched dairy products improving folate intake., (© FEMS 2020.)

Lactococcus lactis subsp. cremoris MG1363 is a model for the lactic acid bacteria (LAB) used in the dairy industry. The proteolytic system, consisting of a proteinase, several peptide and amino acid uptake systems, and a host of intracellular peptidases, plays a vital role in nitrogen metabolism and is of eminent importance for flavor formation in dairy products. The dipeptidase PepV functions in the last stages of proteolysis. A link between nitrogen metabolism and peptidoglycan (PG) biosynthesis was underlined by the finding that deletion of the dipeptidase gene pepV (creating strain MGΔ pepV ) resulted in a prolonged lag phase when the mutant strain was grown with a high concentration of glycine. In addition, most MGΔ pepV cells lyse and have serious defects in their shape. This phenotype is due to a shortage of alanine, since adding alanine can rescue the growth and shape defects. Strain MGΔ pepV is more resistant to vancomycin, an antibiotic targeting peptidoglycan d-Ala-d-Ala ends, which confirmed that MGΔ pepV has an abnormal PG composition. A mutant of MGΔ pepV was obtained in which growth inhibition and cell shape defects were alleviated. Genome sequencing showed that this mutant has a single point mutation in the codY gene, resulting in an arginine residue at position 218 in the DNA-binding motif of CodY being replaced by a cysteine residue. Thus, this strain was named MGΔ pepVcodY R218C Transcriptome sequencing (RNA-seq) data revealed a dramatic derepression in peptide uptake and amino acid utilization in MGΔ pepVcodY R218C A model of the connections among PepV activity, CodY regulation, and PG synthesis of L. lactis is proposed. IMPORTANCE Precise control of peptidoglycan synthesis is essential in Gram-positive bacteria for maintaining cell shape and integrity as well as resisting stresses. Although neither the dipeptidase PepV nor alanine is essential for L. lactis MG1363, adequate availability of either ensures proper cell wall synthesis. We broaden the knowledge about the dipeptidase PepV, which acts as a linker between nitrogen metabolism and cell wall synthesis in L. lactis ., (Copyright © 2020 Huang et al.)

Paocai is a widely consumed Chinese traditional fermented vegetable product. To understand the effect of temperature on paocai fermentation flora, the bacterial community structure of paocai fermented at 10 °C, 15 °C, 25 °C and 35 °C was analyzed by culture-dependent and culture-independent methods. The results showed that increasing the fermentation temperature in a certain range is beneficial for rapid paocai acid production and shortening of the maturity period. Illumina Miseq sequencing was performed on 56 samples at different fermentation process temperatures using a culture-independent method. A total of 1,964,231 high-quality reads of 16S rRNA V3-V4 regions were obtained, and they were divided into 405 operational taxonomic units (OTUs) and identified as 213 bacterial genera. The bacterial diversity decreased with the progression of fermentation, and some spoiled samples had an increased diversity. The culture-independent method found that at 10 °C, Lactococcus appeared at the start of fermentation, Leuconostoc and Weissella appeared in the middle of fermentation, and Lactobacillus and Leuconostoc dominated fermentation in the late stage. At 15 °C, Lactococcus started fermentation, Leuconostoc appeared in the middle stage, and Lactobacillus was dominant in the late stage. At 25 °C, Lactococcus started fermentation, Weissella and Lactobacillus appeared in the middle stage, and Lactobacillus dominated fermentation in the late stage. Finally, at 35 °C, Lactococcus, Weissella, and Lactobacillus started fermentation, Weissella and Lactobacillus appeared in the middle stage, and Lactobacillus dominated fermentation in the late stage. A total of 647 strains of bacteria were isolated by culture-dependent methods and were divided into 12 genera and 19 species by randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) and 16S ribosomal RNA gene (rDNA) sequencing technology. More types of bacteria were isolated in the early stage of fermentation. At 10 °C, Lactococcus lactis began fermentation, and Lactobacillus brevis and Leuconostoc mesenteroides dominated acid production in the middle and late stages of paocai fermentation. At 15 °C, L. lactis initiates fermentation, while Lactobacillus plantarum dominates the acid fermentation of paocai. At 25 °C and 35 °C, there were a large number of Enterobacteriaceae bacteria in the start-up fermentation stage, and L. plantarum was dominant after 1-2 days of fermentation. Redundancy analysis (RDA) found that the lower the temperature, the more bacterial species that are produced, and the higher the temperature and the longer the time, the more obvious are the effects of L. plantarum on paocai. The results of dominant bacteria studied by culture-dependent and culture-independent methods are similar. The results indicate that most of the dominant microorganisms in the paocai fermentation system are culturable. This discovery can provide data and physical support for modernization and regulation of different types of paocai production., Competing Interests: Declaration of competing interest The authors state no conflict of interest., (Copyright © 2019. Published by Elsevier B.V.)

Exopolysaccharides (EPS) synthesized by lactic acid bacteria during fermentation significantly affect the rheology of set-style acid milk gels and that of acid gel suspensions, produced from the gels by stirring. In this study, shear and uniaxial extensional flow of acid gel suspensions made with seven single strains of Streptococcus thermophilus or Lactococcus lactis was investigated. Six strains produced ropy EPS, and maximum filament length determined by using a continuous stretching method was up to four times higher than filament length of an EPS-negative control strain. The systems containing ropy EPS showed a different response to shear and extensional deformation. In shear rheology, higher apparent viscosities and an enhanced structural breakdown was observed for acid gel suspensions with more pronounced ropiness. Breakup time and extensional viscosity, determined by using a Capillary Breakup Extensional Rheometer (CaBER™), significantly increased with increasing ropiness. The increase of extensional viscosity with increasing ropiness was, however, much higher than the effects of ropiness on shear viscosity. As relaxation times also depended on ropiness, it is concluded that ropiness is caused by EPS-EPS interactions that can be better discriminated in extensional rheology. PRACTICAL APPLICATIONS: To improve the texture of fermented milk, lactic acid bacteria that are able to produce ropy exopolysaccharides (EPS) are increasingly used in the dairy industry. The EPS exhibit a significant influence on processing properties and sensory characteristics of the resulting products, which can be estimated by means of shear and extensional rheology. The current work provides information on these respective properties of acid gel suspensions, which facilitate product design by supporting the selection of appropriate starter cultures., (© 2019 Wiley Periodicals, Inc.)

Preparation of curd vary worldwide due to which its taste, texture and impact on human health also differ. In Assam, curd prepared from raw milk (RMC) is preferred over curd prepared from boiled milk (BMC), a tradition believed to have originated from the Mongoloid customs. Microbial diversity of raw milk (RM), boiled milk (BM), RMC and BMC collected from three farms were investigated by culture dependent and independent techniques. Additionally, metabolite profiles of RMC and BMC were studied by gas chromatography and mass spectroscopy. A total of 59 bacterial isolates were identified from the four different dairy products. In RM, lactic acid bacteria such as Lactococcus, Enterococcus, Lactobacillus and Leuconostoc were obtained along with the environmental bacteria like Bacillus, Staphylococcus, Acetobacter, Chryseobacterium, Streptococcus, Acinetobacter, Kocuria, Klebsiella and Macrococcus. Additionally, Prevotella, Oscillospira, Phascolarctobacterium and Akkermansia were also detected in BM by culture independent technique. In RMC and BMC, Lactococcus, Leuconostoc and Lactobacillus were prevalent. RM and RMC shared Enterococcus, Lactococcus, Streptococcus and Acinetobacter as common bacterial genera. However, no bacterial genus was common in BM and BMC. The correlation analysis revealed that Lactobacillus was negatively correlated to other bacterial genera. Oligotyping analysis revealed that Lactobacillus brevis and L.fermentum were abundant in RMC and BMC, respectively. In metabolomic study, ascorbic acid, dodecanoic acid and hexadecanoic acid were found to be significantly higher in RMC. Presence of different types of probiotics in these curds samples opens a new avenue to understand their effects on human health.

Dengue virus (DENV), a mosquito‑borne flavivirus, causes an acute febrile illness that is a major public health problem in the tropics and subtropics globally. However, methods to prevent or treat DENV infection have not been well established. It was previously demonstrated that Lactococcus lactis strain plasma (LC‑plasma) has the ability to stimulate plasmacytoid dendritic cells (pDCs). As pDCs are key immune cells that control viral infection by producing large amounts of type I interferons (IFN), the present study evaluated the effect of LC‑plasma on DENV infection using a mouse infectious DENV strain. Mice were divided into two groups and the test group was orally administered LC‑plasma for two weeks. Two weeks following administration, the mice were infected with DENV and the relative viral titers and the expression of the inflammatory genes in DENV‑infected tissue were measured using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). The relative viral titers were notably lower in the DENV‑infected tissues compared with the control group when LC‑plasma was orally administered prior to DENV infection. Furthermore, the expression of the inflammatory genes associated with DENV infection was also reduced by LC‑plasma administration. To investigate how LC‑plasma administration controls DENV infection, the present study examined anti‑viral gene expression, which is critical for the viral clearance induced by type I IFN. Two weeks subsequent to the administration of LC‑plasma, the expression of anti‑viral gene was measured using RT‑qPCR. Oral intake of LC‑plasma enhanced anti‑viral gene expression in DENV‑infected spleen tissue. To clarify the detailed mechanism, in vitro co‑culture studies using bone‑marrow derived DC (BMDC) were performed. BMDC were stimulated with LC‑plasma in combination with anti‑IFN‑α/β antibody and the expression of anti‑viral genes was measured. In vitro studies revealed that the effect of LC‑plasma on anti‑viral genes was dependent on type I IFN. Based on these results, LC‑plasma may be effective against DENV infection by stimulating pDCs, which results in the increased production of anti‑viral factors.

Background: Staphylococcus aureus is an important foodborne pathogen. Lactococcus garvieae is a lactic acid bacterium found in dairy products; some of its strains are able to inhibit S. aureus growth by producing H 2 O 2 . Three strains of L. garvieae from different origins were tested for their ability to inhibit S. aureus SA15 growth. Two conditions were tested, one in which H 2 O 2 was produced (high aeration) and another one in which it was not detected (low aeration). Several S. aureus genes related to stress, H 2 O 2 -response and virulence were examined in order to compare their level of expression depending on the inoculated L. garvieae strain. Simultaneous L. garvieae H 2 O 2 metabolism gene expression was followed., Results: The results showed that under high aeration condition, L. garvieae strains producing H 2 O 2 (N201 and CL-1183) inhibited S. aureus SA15 growth and impaired its ability to deal with hydrogen peroxide by repressing H 2 O 2 -degrading genes. L. garvieae strains induced overexpression of S. aureus stress-response genes while cell division genes and virulence genes were repressed. A catalase treatment partially or completely restored the SA15 growth. In addition, the H 2 O 2 non-producing L. garvieae strain (Lg2) did not cause any growth inhibition. The SA15 stress-response genes were down-regulated and cell division genes expression was not affected. Under low aeration condition, while none of the strains tested exhibited H 2 O 2 -production, the 3 L. garvieae strains inhibited S. aureus SA15 growth, but to a lesser extent than under high aeration condition., Conclusion: Taken together, these results suggest a L. garvieae strain-specific anti-staphylococcal mechanism and an H 2 O 2 involvement in at least two of the tested L. garvieae strains.

Psychrotrophic lactic acid bacteria (LAB) are the prevailing spoilage organisms in packaged cold-stored meat products. Species composition and metabolic activities of such LAB spoilage communities are determined by the nature of the meat product, storage conditions, and interspecies interactions. Our knowledge of system level responses of LAB during such interactions is very limited. To expand it, we studied interactions between three common psychrotrophic spoilage LAB ( Leuconostoc gelidum , Lactococcus piscium , and Lactobacillus oligofermentans ) by comparing their time course transcriptome profiles obtained during their growth in individual, pairwise, and triple cultures. The study revealed how these LAB employed different strategies to cope with the consequences of interspecies competition. The fastest-growing bacterium, Le. gelidum , attempted to enhance its nutrient-scavenging and growth capabilities in the presence of other LAB through upregulation of carbohydrate catabolic pathways, pyruvate fermentation enzymes, and ribosomal proteins, whereas the slower-growing Lc. piscium and Lb. oligofermentans downregulated these functions. These findings may explain the competitive success and predominance of Le. gelidum in a variety of spoiled foods. Peculiarly, interspecies interactions induced overexpression of prophage genes and restriction modification systems (mechanisms of DNA exchange and protection against it) in Lc. piscium and Lb. oligofermentans but not in Le. gelidum Cocultivation induced also overexpression of the numerous putative adhesins in Lb. oligofermentans These adhesins might contribute to the survival of this slowly growing bacterium in actively growing meat spoilage communities. IMPORTANCE Despite the apparent relevance of LAB for biotechnology and human health, interactions between members of LAB communities are not well known. Knowledge of such interactions is crucial for understanding how these communities function and, consequently, whether there is any possibility to develop new strategies to interfere with their growth and to postpone spoilage of packaged and refrigerated foods. With the help of controlled experiments, detailed regulation events can be observed. This study gives an insight into the system level interactions and the different competition-induced survival strategies related to enhanced uptake and catabolism of carbon sources, overexpression of adhesins and putative bacteriocins, and the induction of exchange of genetic material. Even though this experiment dealt with only three LAB strains in vitro , these findings agreed well with the relative abundance patterns typically reported for these species in natural food microbial communities., (Copyright © 2018 American Society for Microbiology.)

The aim of this study was to evaluate whether Lactococcus hircilactis and Lactococcus laudensis can be used as starter cultures. To this end, the two lactococci were characterized for traits of technological and functional interest. Tests in milk included growth at 20, 25, 30, and 37 °C, flavor production, antioxidant (AO) activity, folate and exopolysaccharide (EPS) production. At 30 °C, which resulted the best growth temperature for both strains, Lc. hircilactis and Lc. laudensis lowered the pH of the milk to 4.8 and 5.5, respectively, after 24 h of incubation. Sugar and organic acid composition indicated a higher lactose utilization, coupled with a higher lactate accumulation, by Lc. hircilactis, while galactose was completely consumed by both species. Both strains showed a Cit - phenotype after growth in a selective medium containing citrate as the sole carbon source. Nevertheless, a small amount of citrate was used by both lactococci when grown in milk. The two strains were characterized by a different flavor production, showed high AO activity, and produced small amounts of EPS (~30 mg/L). Lactococcus laudensis showed a weak proteolytic activity while Lc. hircilactis was able to accumulate folate at levels four times higher than uninoculated milk. When the two lactococci were tested as starter cultures in small-scale cheesemaking trials, cheeses resulted of satisfying quality and contained amounts of ethanol, acetic acid, diacetyl and acetoin higher than controls, obtained using a commercial culture. The application of Lc. hircilactis and Lc. laudensis as aromatic cultures in cheesemaking is proposed., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Previous studies have shown that fermentations can enhance the bioactivity and absorption rate of soybean products. Fermented soybean products can alleviate hyperlipidemia and decrease risks of atherosclerosis and cardiovascular diseases. This study aimed to investigate the effects and mechanisms of soymilk fermented by Lactococcus acidophilus on blood lipids and antioxidant enzyme activities of rats fed with a high fat diet. Sixty rats were randomly assigned to six groups: normal control group (NC), high-fat control group (HFC), positive control group (cholestyramine, PC), Lactococcus acidophilus group (LA), soymilk group (SM), and fermented soymilk group (FSM), respectively. The NC group was fed with a basic diet, while the other groups were fed with a high-fat diet. After the experimental period (6 W), rats were sacrificed by decapitation. Blood and liver were collected to measure the concentrations of lipids and antioxidant enzyme activities. Results demonstrated that fermented soymilk could regulate lipid levels, restore HDL-c and TG to normal levels, and lower the concentrations of LDL-c than hypolipidemic drugs in hyperlipidemia rats. More importantly, fermented soymilk caused significant reduction in arteriosclerosis index and coronary risk index. Fermented soymilk also improved antioxidant capacities of hyperlipidemia rats. The increase of aglycone isoflavones in fermented soymilk could explain the above phenomena. In conclusion, soymilk fermented by Lactococcus acidophilus reduced risks of arteriosclerosis and coronary heart disease by regulating lipid levels and improving the antioxidant capacities of hyperlipidemia rats.

It has recently been reported that the rare sugar d-allulose has beneficial effects, including the suppression of postprandial blood glucose elevation in humans, and can be substituted for sucrose as a low-calorie food ingredient. To examine the applications of d-allulose in the dairy industry, we investigated the effects of d-allulose on the acid production of 8 strains of yogurt starter (Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus) and 4 strains of lactococci, including potential probiotic candidates derived from dairy products. Acid production by 2 L. delbrueckii ssp. bulgaricus yogurt starter strains in milk was suppressed by d-allulose, but this phenomenon was also observed in some strains with another sugar (xylose), a sugar alcohol (sorbitol), or both. In contrast, among the dairy probiotic candidates, Lactococcus lactis H61, which has beneficial effects for human skin when drunk as part of fermented milk, was the only strain that showed suppression of acid production in the presence of d-allulose. Strain H61 did not metabolize d-allulose. We did not observe suppression of acid production by strain H61 with the addition of xylose or sorbitol, and xylose and sorbitol were not metabolized by strain H61. The acid production of strain H61 after culture in a constituted medium (tryptone-yeast extract-glucose broth) was also suppressed with the addition of d-allulose, but growth efficiency and sugar fermentation style were not altered. Probiotic activities-such as the angiotensin-converting enzyme inhibitory activity of H61-fermented milk and the superoxide dismutase activity of H61 cells grown in tryptone-yeast extract-glucose broth-were not affected by d-allulose. d-Allulose may suppress acid production in certain lactic acid bacteria without altering their probiotic activity. It may be useful for developing new probiotic dairy products from probiotic strains such as Lactococcus lactis H61., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Aims: The aim of this study was the coproduction in a single strain of the Gram-negative bacteriocin colicin V with other bacteriocins from lactic acid bacteria (LAB)., Methods and Results: Colicin V was expressed in Lactococcus and Enterococcus strains by replacing the colicin V leader peptide by the leader peptide and promoter of d-alanyl-d-alanine carboxypeptidase from Lactobacillus reuteri CECT925 in pNZ8048 (pNZ:LR-colV). The antimicrobial activity of colicin V against the indicator organism Escherichia coli DH5α in transformed strains was checked by agar diffusion assay and SDS-PAGE analysis., Conclusions: Lactococcus and Enterococcus transformed with pNZ:LR-colV were able to coproduce colicin V at high levels together with other LAB bacteriocins such as nisin A, nisin Z, lacticin 481 or enterocins A and B, obtaining broad-spectrum activity strains with large potential applications., Significance and Impact of the Study: The construction showed in this work could be used for the heterologous expression of other bacteriocins active against Gram-negative bacteria or wide-spectrum bacteriocins from LAB., (© 2017 The Society for Applied Microbiology.)

The study of microbial ecosystems in artisanal foodstuffs is important to complete in order to unveil its diversity. The number of studies performed on dairy products has increased during the last decade, particularly those performed on milk and cheese derivative products. In this work, we investigated the bacterial content of "Tomme d'Orchies" cheese, an artisanal pressed and uncooked French cheese. To this end, a metagenetic analysis, using Illumina technology, was utilized on samples taken from the surface and core of the cheese at 0, 1, 3, 14 and 21days of ripening process. In addition to the classical microbiota found in cheese, various strains likely from environmental origin were identified. A large difference between the surface and the core content was observed within samples withdrawn during the ripening process. The main species encountered in the core of the cheese were Lactococcus spp. and Streptococcus spp., with an inversion of this ratio during the ripening process. Less than 2.5% of the whole population was composed of strains issued from environmental origin, as Lactobacillales, Corynebacterium and Brevibacterium. In the core, about 85% of the microbiota was attributed to the starters used for the cheese making. In turn, the microbiota of the surface contained less than 30% of these starters and interestingly displayed more diversity. The predominant genus was Corynebacterium sp., likely originating from the environment. The less abundant microbiota of the surface was composed of Bifidobacteria, Brevibacterium and Micrococcales. To summarize, the "Tomme d'Orchies" cheese displayed a high diversity of bacterial species, especially on the surface, and this diversity is assumed to arise from the production environment and subsequent ripening process., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The aim of this study was to characterise the gut microbiota composition of piglets fed bovine colostrum (BC), milk replacer (MR) or sow milk (SM) in the post-weaning period. Piglets (n 36), 23-d old, were randomly allocated to the three diets. Faecal samples were collected at 23, 25, 27 and 30 d of age. Digesta from the stomach, ileum, caecum and mid-colon was collected at 30 d of age. Bacterial DNA from all samples was subjected to amplicon sequencing of the 16S rRNA gene. Bacterial enumerations by culture and SCFA analysis were conducted as well. BC-piglets had the highest abundance of Lactococcus in the stomach (P<0·0001) and ileal (P<0·0001) digesta, whereas SM-piglets had the highest abundance of Lactobacillus in the stomach digesta (P<0·0001). MR-piglets had a high abundance of Enterobacteriaceae in the ileal digesta (P<0·0001) and a higher number of haemolytic bacteria in ileal (P=0·0002) and mid-colon (P=0·001) digesta than SM-piglets. BC-piglets showed the highest colonic concentration of iso-butyric and iso-valeric acid (P=0·02). Sequencing and culture showed that MR-piglets were colonised by a higher number of Enterobacteriaceae, whereas the gut microbiota of BC-piglets was characterised by a change in lactic acid bacteria genera when compared with SM-piglets. We conclude that especially the ileal microbiota of BC-piglets had a closer resemblance to that of SM-piglets in regard to the abundance of potential enteric pathogens than did MR-piglets. The results indicate that BC may be a useful substitute for regular milk replacers, and as a feeding supplement in the immediate post-weaning period.

Branched chain aldehyde, 3-methylbutanal is associated as a key flavor compound with many hard and semi-hard cheese varieties. The presence and impact of this flavor compound in bread, meat, and certain beverages has been recently documented, however its presence and consequences regarding cheese flavor were not clearly reported. This paper gives an overview of the role of 3-methylbutanal in cheese, along with the major metabolic pathways and key enzymes leading to its formation. Moreover, different strategies are highlighted for the control of this particular flavor compound in specific cheese types.

Lactococcus garvieae is a relevant worldwide fish pathogen affecting various farmed and wild marine and freshwater species. It has also been isolated from other animals, such as ruminants with subclinical mastitis and pigs with pneumonia. From the early 90s, L. garvieae has been associated with different human infections, mainly endocarditis. During the last five years, human infections by this bacterium appear to be increasing, likely due to the improvement in microbiological methods for bacterial identification and the alertness of this bacterium by physicians. Human L. garvieae infections have been associated with the consumption or the handling of contaminated raw fish or seafood, and recently, a genetic study showed that meat, raw milk and dairy products may also be food sources of human L. garvieae infections. However, the status of L. garvieae as a potential zoonotic bacterium is still controversial to date. In this work, we describe four new human infections by L. garvieae in elderly and inmunocompromised patients, and we show an overview on L. garvieae microbiology, epidemiology, virulence factors and relationship with its presence in foods., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Extracytoplasmic thiol-disulfide oxidoreductases (TDORs) catalyze the oxidation, reduction, and isomerization of protein disulfide bonds. Although these processes have been characterized in Gram-negative bacteria, the majority of Gram-positive TDORs have only recently been discovered. Results from recent studies have revealed distinct trends in the types of TDOR used by different groups of Gram-positive bacteria, and in their biological functions. Actinobacteria TDORs can be essential for viability, while Firmicute TDORs influence various physiological processes, including protein stability, oxidative stress resistance, bacteriocin production, and virulence. In this review we discuss the diverse extracytoplasmic TDORs used by Gram-positive bacteria, with a focus on Gram-positive Firmicutes., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: This study aimed to characterize microorganisms responsible for accumulation of ethyl carbamate precursor citrulline in fermented grains during Luzhou-flavor spirits fermentation process, to provide theoretical basis for clarifying mechanisms of ethyl carbamate formation., Methods: High-throughput strain screening approach was used to isolate strains with high arginine utilization and high citrulline accumulation capability. Essential genes that comprise the arginine deiminase pathway of these isolates were verified and a modified Chinese liquor fermentation process was conducted by adding these strains., Results: A total of 20 strains with high arginine utilizing ability were obtained. Among them, Lactococcus garvieae LD3, Bacillus amyloliquefaciens BG5, Pediococcus acidilactici PH7 and Staphylococcus pasteuri SH11 exhibited high efficacy to produce citrulline from arginine. These strains could also increase citrulline in fermented grains., Conclusion: The accumulation of citrulline in fermented grains was confirmed to be corresponding to arginine utilization by Lactococcus garvieae, Bacillus amyloliquefaciens, Pediococcus acidilactici and Staphylococcus pasteuri through the arginine deiminase pathway.

Unlabelled: From raw milk we found 10 Lactococcus garvieae isolates that produce a new broad-spectrum bacteriocin. Though the isolates were obtained from different farms, they turned out to possess identical inhibitory spectra, fermentation profiles of sugars, and repetitive sequence-based PCR (rep-PCR) DNA patterns, indicating that they produce the same bacteriocin. One of the isolates (L. garvieae KS1546) was chosen for further assessment. Purification and peptide sequencing combined with genome sequencing revealed that the antimicrobial activity was due to a bacteriocin unit composed of three similar peptides of 32 to 34 amino acids. The three peptides are produced without leader sequences, and their genes are located next to each other in an operon-like structure, adjacent to the genes normally involved in bacteriocin transport (ABC transporter) and self-immunity. The bacteriocin, termed garvicin KS (GarKS), showed sequence homology to four multipeptide bacteriocins in databases: the known staphylococcal aureocin A70, consisting of four peptides, and three unannotated putative multipeptide bacteriocins produced by Bacillus cereus All these multipeptide bacteriocin loci show conserved genetic organization, including being located adjacent to conserved genetic determinants (Cro/cI and integrase) which are normally associated with mobile genetic elements or genome rearrangements. The antimicrobial activity of all multipeptide bacteriocins was confirmed with synthetic peptides, and all were shown to have broad antimicrobial spectra, with GarKS being the most active of them. The inhibitory spectrum of GarKS includes important pathogens belonging to the genera Staphylococcus, Bacillus, Listeria, and Enterococcus, Importance: Bacterial resistance to antibiotics is a very serious global problem. There are no new antibiotics with novel antimicrobial mechanisms in clinical trials. Bacteriocins use antimicrobial mechanisms different from those of antibiotics and can kill antibiotic-resistant bacteria, but the number of bacteriocins with very broad antimicrobial spectra is very small. In this study, we have found and purified a novel three-peptide bacteriocin, garvicin KS. By homology search, we were able to find one known and three novel sequence-related bacteriocins consisting of 3 or 4 peptides. None of the peptides has modified amino acids in its sequence. Thus, the activity of all bacteriocins was confirmed with chemically synthesized peptides. All of them, especially garvicin KS, have very broad antibacterial spectra, thus representing a great potential in antimicrobial applications in the food industry and medicine., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Multispecies solid-state fermentation (MSSF), a natural fermentation process driven by reproducible microbiota, is an important technique to produce traditional fermented foods. Flavours, skeleton of fermented foods, was mostly produced by microbiota in food ecosystem. However, the association between microbiota and flavours and flavour-producing core microbiota are still poorly understood. Here, acetic acid fermentation (AAF) of Zhenjiang aromatic vinegar was taken as a typical case of MSSF. The structural and functional dynamics of microbiota during AAF process was determined by metagenomics and favour analyses. The dominant bacteria and fungi were identified as Acetobacter, Lactobacillus, Aspergillus, and Alternaria, respectively. Total 88 flavours including 2 sugars, 9 organic acids, 18 amino acids, and 59 volatile flavours were detected during AAF process. O2PLS-based correlation analysis between microbiota succession and flavours dynamics showed bacteria made more contribution to flavour formation than fungi. Seven genera including Acetobacter, Lactobacillus, Enhydrobacter, Lactococcus, Gluconacetobacer, Bacillus and Staphylococcus were determined as functional core microbiota for production of flavours in Zhenjiang aromatic vinegar, based on their dominance and functionality in microbial community. This study provides a perspective for bridging the gap between the phenotype and genotype of ecological system, and advances our understanding of MSSF mechanisms in Zhenjiang aromatic vinegar.

The identification of DNA coding sequences contained in the genome of many organisms coupled to the use of high throughput approaches has fueled the field of recombinant protein production. Apart from basic research interests, the growing relevance of this field is highlighted by the global sales of the top ten biopharmaceuticals on the market, which exceeds the trillion USD in a steady increasing tendency. Therefore, the demand of biological compounds seems to have a long run on the market. One of the most popular expression systems is based on Escherichia coli cells which apart from being cost-effective counts with a large selection of resources. However, a significant percentage of the genes of interest are not efficiently expressed in this system, or the expressed proteins are accumulated within aggregates, degraded or lacking the desired biological activity, being finally discarded. In some instances, expressing the gene in a homologous expression system might alleviate those drawbacks but then the process usually increases in complexity and is not as cost-effective as the prokaryotic systems. An increasing toolbox is available to approach the production and purification of those difficult-to-express proteins, including different expression systems, promoters with different strengths, cultivation media and conditions, solubilization tags and chaperone coexpression, among others. However, in most cases, the process follows a non-integrative trial and error strategy with discrete success. This review is focused on the design of the whole process by using an integrative approach, taken into account the accumulated knowledge of the pivotal factors that affect any of the key processes, in an attempt to rationalize the efforts made in this appealing field.

Lactococcus garvieae is the causative agent of lactococcosis and has been isolated from a wide variety of animals. In the present study, 34 strains of L. garvieae isolated from fish from different sources and locations were tested for the presence or absence of the following putative virulence genes: a capsule gene cluster (CGC), hemolysins 1, 2, and 3 (hly1, -2, -3), NADH oxidase, superoxide dismutase (sod), phosphoglucomutase (pgm), adhesin Pav (adhPav), adhesin PsaA (adhPsaA), enolase (eno), LPxTG-containing surface proteins 1, 2, 3, and 4 (LPxTG-1, LPxTG-2, LPxTG-3, LPxTG-4; where LPxTG means Leu-Pro-any-Thr-Gly), adhesin clusters 1 and 2 (adhCI, adhCII), and adhesin (adh). To determine the presence of the CGC, we developed a multiplex PCR. All strains of L. garvieae had the hly1, -2, -3, NADH oxidase, pgm, adhPav, LPxTG-2, LPxTG-3, sod, eno, adhPsaA, adhCII, and adhCII genes, while only the Lg2 strain contained the CGC. The virulent Lg2 strain contained all 17 virulent genes. All Turkish, Spanish, Italian, and French strains did not contain the CGC. The multiplex PCR assay was useful for the detection of the CGC genes. In conclusion, the CGC is not the only virulent factor in L. garvieae because strains that lack the CGC are virulent to rainbow trout. Single genes also might not be responsible for the virulence of L. garvieae.

This study assessed the inhibitory effects of the essential oil from Origanum vulgare L. (OVEO) on Staphylococcus aureus, Listeria monocytogenes, and a mesophilic starter coculture composed of lactic acid bacteria (Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris) in Brazilian coalho cheese systems. The MIC of OVEO was 2.5 μl/ml against both S. aureus and L. monocytogenes and 0.6 μl/ml against the tested starter coculture. In cheese broth containing OVEO at 0.6 μl/ml, no decrease in viable cell counts (VCC) of both pathogenic bacteria was observed, whereas the initial VCC of the starter coculture decreased approximately 1.0 log CFU/ml after 24 h of exposure at 10°C. OVEO at 1.25 and 2.5 μl/ml caused reductions of up to 2.0 and 2.5 log CFU/ml in S. aureus and L. monocytogenes, respectively, after 24 h of exposure in cheese broth. At these same concentrations, OVEO caused a greater decrease of initial VCC of the starter coculture following 4 h of exposure. Higher concentrations of OVEO were required to decrease the VCC of all target bacteria in semisolid coalho cheese slurry compared with cheese broth. The VCC of Lactococcus spp. in coalho cheese slurry containing OVEO were always lower than those of pathogenic bacteria under the same conditions. These results suggest that the concentrations of OVEO used to control pathogenic bacteria in semihard cheese should be carefully evaluated because of its inhibitory effects on the growth of starter lactic acid cultures used during the production of the product.

Listeria monocytogenes is a pathogenic Gram positive bacterium and the etiologic agent of listeriosis, a severe food-borne disease. Lactococcus piscium CNCM I-4031 has the capacity to prevent the growth of L. monocytogenes in contaminated peeled and cooked shrimp. To investigate the inhibititory mechanism, a chemically defined medium (MSMA) based on shrimp composition and reproducing the inhibition observed in shrimp was developed. In co-culture at 26 °C, L. monocytogenes was reduced by 3-4 log CFU g(-1) after 24 h. We have demonstrated that the inhibition was not due to secretion of extracellular antimicrobial compounds as bacteriocins, organic acids and hydrogen peroxide. Global metabolomic fingerprints of these strains in pure culture were assessed by liquid chromatography coupled with high resolution mass spectrometry. Consumption of glucose, amino-acids, vitamins, nitrogen bases, iron and magnesium was measured and competition for some molecules could be hypothesized. However, after 24 h of co-culture, when inhibition of L. monocytogenes occurred, supplementation of the medium with these compounds did not restore its growth. The inhibition was observed in co-culture but not in diffusion chamber when species were separated by a filter membrane. Taken together, these data indicate that the inhibition mechanism of L. monocytogenes by L. piscium is cell-to-cell contact-dependent., (Copyright © 2015 Elsevier Ltd. All rights reserved.)