Showing papers on "Lactococcus lactis published in 2019"
TL;DR: The results are consistent with the hypothesis that dairy isolates evolved from plant isolates and suggest that genomes of cremoris phenotype strains are so eroded that they are restricted to a dairy environment.
Abstract: Lactococcus lactis is one of the most important micro-organisms in the dairy industry for the fermentation of cheese and buttermilk. Besides the conversion of lactose to lactate it is responsible for product properties such as flavor and texture, which are determined by volatile metabolites, proteolytic activity and exopolysaccharide production. While the species Lactococcus lactis consists of the two subspecies lactis and cremoris their taxonomic position is confused by a group of strains that, despite of a cremoris genotype, display a lactis phenotype. Here we compared and analyzed the (draft) genomes of 43 L. lactis strains, of which 19 are of dairy and 24 are of non-dairy origin. Machine-learning algorithms facilitated the identification of orthologous groups of protein sequences (OGs) that are predictors for either the taxonomic position or the source of isolation. This allowed the unambiguous categorization of the genotype/phenotype disparity of ssp. lactis and ssp. cremoris strains. A detailed analysis of phenotypic properties including plasmid-encoded genes indicates evolutionary changes during niche adaptations. The results are consistent with the hypothesis that dairy isolates evolved from plant isolates. The analysis further suggests that genomes of cremoris phenotype strains are so eroded that they are restricted to a dairy environment. Overall the genome comparison of a diverse set of strains allowed the identification of niche and subspecies specific genes. This explains evolutionary relationships and will aid the identification and selection of industrial starter cultures.
130 citations
TL;DR: LAB inoculants and a relatively low environmental temperature could be effective to improve the quality of MOL silage.
78 citations
TL;DR: 3 L. lactis strains screened from common carp were effective in improving growth, innate immunity and disease resistance, and might be used as potential probiotics in aquaculture.
61 citations
TL;DR: Data suggest that L. lactis ML2018 could have therapeutic potential for treating IBDs and could inhibit DSS-induced intestinal inflammation by preventing the overproduction of proinflammatory factors, suppressing the infiltration of macrophages, controlling the fibrosis, improving the integrity of the intestinal epithelial barrier and upregulating the concentrations of short-chain fatty acids.
Abstract: Multiple articles have confirmed that an imbalance of the intestinal microbiota is closely related to aberrant immune responses of the intestines and to the pathogenesis of inflammatory bowel diseases (IBDs). Probiotic strains have been identified for the treatment and prevention of IBDs. The aim of this study was to screen a new probiotic strain with anti-inflammatory activity and investigate the potential mechanisms underlying its activity. We identified a new probiotic strain, L. lactis ML2018, that has anti-inflammatory properties and was isolated from traditional fermented food. In an in vitro experiment, L. lactis ML2018 prevented the release of nitric oxide (NO) and the production of inflammatory factors induced by lipopolysaccharides (LPS) in RAW264.7 cells. The in vivo anti-inflammatory effects of L. lactis ML2018 were evaluated using a dextran sulfate sodium (DSS)-induced animal model of colitis. Oral administration of L. lactis ML2018 significantly ameliorated colitis induced by DSS, which included preventing a decrease in body weight, shortening of the colon length and apoptosis of epithelial cells. L. lactis ML2018 could inhibit DSS-induced intestinal inflammation by preventing the overproduction of proinflammatory factors, suppressing the infiltration of macrophages, controlling the fibrosis, improving the integrity of the intestinal epithelial barrier and upregulating the concentrations of short-chain fatty acids (SCFAs). Moreover, L. lactis ML2018 could prevent inflammation by inhibiting the activation of the NF-κB and MAPK signaling pathways. These data suggest that L. lactis ML2018 could have therapeutic potential for treating IBDs.
58 citations
TL;DR: The work provided a rapid, versatile and precise tool for L. lactis genomic engineering by combination of ssDNA recombineering with improved CRISPR/Cas9 counterselection, which will simplify the production of isogenic strains for assessment of gene function or construction of biosynthetic host.
Abstract: Lactococcus lactis is one of the most extensively characterized lactic acid bacteria, from physiological traits to industrial exploitation. Since last decade, L. lactis has been developed into cell factories for the production of bioactive compounds such as enzymes, vaccine antigens and natural products. However, its precise and efficient genome editing tools is still required to make L. lactis more suitable candidate for engineered functionality. A high active recombinase, RecT of Enterococcus faecalis ATCC14506, was selected from six candidates and mediated homologous recombination between single-stranded DNA (ssDNA) and the L. lactis chromosomal rpoB locus with an efficiency of 100% after rifampin selection. To screen mutants without an externally selectable phenotype, the CRISPR/Cas9 system was used for counterselection, yielding an upp mutant with an efficiency of 46%. By optimization of the copy number of plasmid carrying the CRISPR/Cas9 system and the length of spacer sequence, the off-target efficiency of the recA, galK, hemN and noxD genes were eliminated. The ability of this optimized tool to perform sequential point mutation was demonstrated using the upp and galK gene loci as targets with improved efficiencies > 75%. Moreover, seamless genomic DNA deletions (50/100 bp) or insertion (a loxP site, 34 bp) was efficiently accomplished within 72 h. The work provided a rapid, versatile and precise tool for L. lactis genomic engineering by combination of ssDNA recombineering with improved CRISPR/Cas9 counterselection. This tool will simplify the production of isogenic strains for assessment of gene function or construction of biosynthetic host.
58 citations
TL;DR: Results showed the potential of LAB obtained from the indigenous microbiota of wild marine fishes for use as probiotics in aquaculture.
Abstract: With the increase of antimicrobial resistances due to the widespread use of antibiotics, the search of new probiotics to control aquaculture diseases has a growing public interest. The aim of this study was to isolate bacteria with antimicrobial effect from the gut of marine healthy fishes and select lactic acid bacteria (LAB) as potential probiotics, being strains considered as generally regarded as safe (GRAS) by the European Food Safety Agency (EFSA). Of a total of 45 Gram-positive strains with antimicrobial activity found in a screening of the gut microbiota of 13 marine fishes, nine were identified as LAB by 16S rRNA gene sequencing. LAB strains (five Lactococcus lactis subsp. lactis, two Enterococcus spp., one Lactobacillus plantarum, and one Leuconostoc mesenteroides subsp. mesenteroides) also showed a broad-spectrum antibacterial activity against aquaculture pathogens such as Vibrio harveyi, V. splendidus, and Photobacterium damselae and survived in experimental gastrointestinal conditions when grown in culture media modified with different values of pH and bile salts. These results showed the potential of LAB obtained from the indigenous microbiota of wild marine fishes for use as probiotics in aquaculture.
58 citations
TL;DR: The results indicate that Csm6 ribonuclease activity rather than Csm1-mediated DNase activity effects anti-plasmid immunity by the three Type III-A systems investigated.
Abstract: CRISPR-Cas systems provide prokaryotes with RNA-based adaptive immunity against viruses and plasmids. A unique feature of Type III CRISPR-Cas systems is that they selectively target transcriptionally-active invader DNA, and can cleave both the expressed RNA transcripts and source DNA. The Type III-A effector crRNP (CRISPR RNA-Cas protein complex), which contains Cas proteins Csm1-5, recognizes and degrades invader RNA and DNA in a crRNA-guided, manner. Interestingly, Type III-A systems also employ Csm6, an HEPN family ribonuclease that does not stably associate with the Type III-A effector crRNP, but nevertheless contributes to defense via mechanistic details that are still being determined. Here, we investigated the mechanism of action of Csm6 in Type III-A CRISPR-Cas systems from Lactococcus lactis, Staphylococcus epidermidis, and Streptococcus thermophilus expressed in Escherichia coli. We found that L. lactis and S. epidermidis Csm6 cleave RNA specifically after purines in vitro, similar to the activity reported for S. thermophilus Csm6. Moreover, L. lactis Csm6 functions as a divalent metal-independent, single strand-specific endoribonuclease that depends on the conserved HEPN domain. In vivo, we show that deletion of csm6 or expression of an RNase-defective form of Csm6 disrupts crRNA-dependent loss of plasmid DNA in all three systems expressed in E. coli. Mutations in the Csm1 palm domain, which are known to deactivate Csm6 ribonuclease activity, also prevent plasmid loss in the three systems. The results indicate that Csm6 ribonuclease activity rather than Csm1-mediated DNase activity effects anti-plasmid immunity by the three Type III-A systems investigated.
57 citations
TL;DR: Biochemical and probiotic properties of L. lactis strains varied depending on the strain and some of these strains could be used as functional cultures depending on their properties, however, these strains did not possess all of the properties required to meet the definition of a probiotic.
53 citations
TL;DR: The mentioned findings indicated that EPS could be utilized as a natural additive in pharmaceutical, food, and cosmetic industries.
48 citations
TL;DR: It is demonstrated that the KupA and KupB proteins of L. lactis IL1403 are high-affinity potassium transporters and that their transport activity is inhibited by the second messenger c-di-AMP.
Abstract: Cyclic di-AMP (c-di-AMP) is a second messenger involved in diverse metabolic processes, including osmolyte uptake, cell wall homeostasis, and antibiotic and heat resistance. In Lactococcus lactis, a lactic acid bacterium which is used in the dairy industry and as a cell factory in biotechnological processes, the only reported interaction partners of c-di-AMP are the pyruvate carboxylase and BusR, the transcription regulator of the busAB operon for glycine betaine uptake. However, recent studies uncovered a major role of c-di-AMP in the control of potassium homeostasis, and potassium is the signal that triggers c-di-AMP synthesis. In this study, we have identified KupA and KupB, which belong to the Kup/HAK/KT family, as novel c-di-AMP binding proteins. Both proteins are high-affinity potassium transporters, and their transport activities are inhibited by binding of c-di-AMP. Thus, in addition to the well-studied Ktr/Trk potassium channels, KupA and KupB represent a second class of potassium transporters that are subject to inhibition by c-di-AMP.IMPORTANCE Potassium is an essential ion in every living cell. Even though potassium is the most abundant cation in cells, its accumulation can be toxic. Therefore, the level of potassium has to be tightly controlled. In many Gram-positive bacteria, the second messenger cyclic di-AMP plays a key role in the control of potassium homeostasis by binding to potassium transporters and regulatory proteins and RNA molecules. In the lactic acid bacterium Lactococcus lactis, none of these conserved c-di-AMP-responsive molecules are present. In this study, we demonstrate that the KupA and KupB proteins of L. lactis IL1403 are high-affinity potassium transporters and that their transport activity is inhibited by the second messenger c-di-AMP.
43 citations
TL;DR: Electrospinning represents a promising method for the incorporation of lactic acid bacteria into solid delivery systems, while drying the bacterial dispersion at the same time.
Abstract: Lactic acid bacteria can have beneficial health effects and be used for the treatment of various diseases. However, there remains the challenge of encapsulating probiotics into delivery systems with a high viability and encapsulation efficacy. The electrospinning of bacteria is a novel and little-studied method, and further investigation of its promising potential is needed. Here, the morphology, zeta potential, hydrophobicity, average cell mass, and growth characteristics of nine different species of Lactobacillus and one of Lactococcus are characterized. The electrospinning of polymer solutions containing ~10 log colony forming units (CFU)/mL lactic acid bacteria enabled the successful incorporation of all bacterial species tested, from the smallest (0.74 µm; Lactococcus lactis) to the largest (10.82 µm; Lactobacillus delbrueckii ssp. bulgaricus), into poly(ethylene oxide) nanofibers with an average diameter of ~100 nm. All of these lactobacilli were viable after incorporation into nanofibers, with 0 to 3 log CFU/mg loss in viability, depending on the species. Viability correlated with the hydrophobicity and extreme length of lactic acid bacteria, whereas a horizonal or vertical electrospinning set-up did not have any role. Therefore, electrospinning represents a promising method for the incorporation of lactic acid bacteria into solid delivery systems, while drying the bacterial dispersion at the same time.
TL;DR: In this paper, an autochthonous Lactococcus lactis subsp. R7 (L. lactis R7) microencapsulated with whey and inulin by spray drying was evaluated.
Abstract: This study was conducted to characterize autochthonous Lactococcus lactis subsp. lactis R7 (L. lactis R7) microencapsulated with whey and inulin by spray drying and evaluate the microencapsulation's ability to protect against adverse conditions. Symbiotic microcapsules produced by spray drying showed high viability (13.0 log CFU.g−1) and high encapsulation yield (94.61%). The viable cell counts of the microcapsules remained stable for 6 months of storage (>8.0 log CFU.g−1), at the different temperatures analyzed. Microencapsulation showed protection of L. lactis R7 through a simulated gastrointestinal tract passage, as well as at the temperatures applied in the thermal resistance test. The thermal analyses evidenced the protection conferred by the encapsulating material because no endothermic peak of free cells was observed in the thermogram of the microcapsules. The study demonstrated that the combination of inulin and whey as encapsulating materials was adequate and effective in the microencapsulation process by spray drying, with potential application in foods.
TL;DR: The results indicate that incorporation of Lactococcus lactis changed the physical properties of edible films, and was highest in films composed of sodium alginate and methylcellulose.
TL;DR: In this study, Lactococcus lactis Gh1 was encapsulated via spray-drying with gum Arabic together with Synsepalum dulcificum or commonly known as miracle fruit and retained a higher viability, when incorporated into yogurt compared to non-microencapsulated cells ~105 CFU/mL.
Abstract: There has been an explosion of probiotic incorporated based product. However, many reports indicated that most of the probiotics have failed to survive in high quantity, which has limited their effectiveness in most functional foods. Thus, to overcome this problem, microencapsulation is considered to be a promising process. In this study, Lactococcus lactis Gh1 was encapsulated via spray-drying with gum Arabic together with Synsepalum dulcificum or commonly known as miracle fruit. It was observed that after spray-drying, high viability (~109 CFU/mL) powders containing L. lactis in combination with S. dulcificum were developed, which was then formulated into yogurt. The tolerance of encapsulated bacterial cells in simulated gastric juice at pH 1.5 was tested in an in-vitro model and the result showed that after 2 h, cell viability remained high at 1.11 × 106 CFU/mL. Incubation of encapsulated cells in the presence of 0.6% (w/v) bile salts showed it was able to survive (~104 CFU/mL) after 2 h. Microencapsulated L. lactis retained a higher viability, at ~107 CFU/mL, when incorporated into yogurt compared to non-microencapsulated cells ~105 CFU/mL. The fortification of microencapsulated and non-microencapsulated L. lactis in yogurts influenced the viable cell counts of yogurt starter cultures, Lactobacillus delbrueckii subs. bulgaricus and Streptococcus thermophilus.
TL;DR: Data indicated that a high concentration of the probiotic strain JCM5805 upregulated the expression of IFN&agr; via the TLR7/TLR9‐Myd88 pathway and enhanced disease resistance of larvae.
TL;DR: It is found that overexpression of the ABC transporters RbsB and MsmK increased intracellular ATP concentrations to protect cells against acidic damage in the initial stage of acid stress.
Abstract: Microbial cell factories are widely used in the production of acidic products such as organic acids and amino acids. However, the metabolic activity of microbial cells and their production efficiency are severely inhibited with the accumulation of intracellular acidic metabolites. Therefore, it remains a key issue to enhance the acid tolerance of microbial cells. In this study, we investigated the effects of four ATP-binding cassette (ABC) transporters on acid stress tolerance in Lactococcus lactis. Overexpressing the rbsA, rbsB, msmK, and dppA genes exhibited 5.8-, 12.2-, 213.7-, and 5.2-fold higher survival rates than the control strain, respectively, after acid shock for 3 h at pH 4.0. Subsequently, transcriptional profile alterations in recombinant strains were analyzed during acid stress. The differentially expressed genes associated with cold-shock proteins (csp), fatty acid biosynthesis (fabH), and coenzyme A biosynthesis (coaD) were up-regulated in the four recombinant strains during acid stress. Additionally, some genes were differentially expressed in specific recombinant strains. For example, in L. lactis (RbsB), genes involved in the pyrimidine biosynthetic pathway (pyrCBDEK) and glycine or betaine transport process (busAA and busAB) were up-regulated during acid stress, and the argG genes showed up-regulations in L. lactis (MsmK). Finally, we found that overexpression of the ABC transporters RbsB and MsmK increased intracellular ATP concentrations to protect cells against acidic damage in the initial stage of acid stress. Furthermore, L. lactis (MsmK) consistently maintained elevated ATP concentrations under acid stress. This study elucidates the common and specific mechanisms underlying improved acid tolerance by manipulating ABC transporters and provides a further understanding of the role of ABC transporters in acid-stress tolerance.
TL;DR: The positive salutary effects of a beneficial bacterium, namely, L. lactis subsp.
TL;DR: Biosynthesized SeNPs by L. lactis NZ9000 are a promising selenium supplement with antioxidant and anti-inflammatory activities and maintain the intestinal epithelial barrier integrity through eco-friendly and economic biotechnology methods.
Abstract: Lactococcus lactis (L lactis) NZ9000, which has been genetically modified, is the most commonly used host strain for nisin regulated gene expression Selenium (Se) is an essential trace element in the diet of humans and animals important for the maintenance of health and growth Biosynthesized Se nanoparticles (SeNPs) that use microorganisms as a vehicle are uniquely advantages in terms of low costs, low toxicity and high bioavailability This study was aimed at preparing novel functionalized SeNPs by L lactis NZ9000 through eco-friendly and economic biotechnology methods Moreover, its physicochemical characteristics, antioxidant and anti-inflammatory activities were investigated L lactis NZ9000 synthesized elemental red SeNPs when co-cultivated with sodium selenite under anaerobic conditions Biosynthesized SeNPs by L lactis NZ9000 were mainly capped with polysaccharides and significantly alleviated the increase of malondialdehyde (MDA) concentration, the decrease of glutathione peroxidase (GPx) and total superoxide dismutase (T-SOD) activity in porcine intestinal epithelial cells (IPEC-J2) challenged by hydrogen peroxide (H2O2) SeNPs also prevented the H2O2-caused reduction of transepithelial electrical resistance (TEER) and the increase of FITC-Dextran fluxes across IPEC-J2 Moreover, SeNPs attenuated the increase of reactive oxygen species (ROS), the reduction of adenosine triphosphate (ATP) and the mitochondrial membrane potential (MMP) and maintained intestinal epithelial permeability in IPEC-J2 cells exposed to H2O2 In addition, SeNPs pretreatment alleviated the cytotoxicity of Enterotoxigenic Escherichia coli (ETEC) K88 on IPEC-J2 cells and maintained the intestinal epithelial barrier integrity by up-regulating the expression of Occludin and Claudin-1 and modulating inflammatory cytokines Biosynthesized SeNPs by L lactis NZ9000 are a promising selenium supplement with antioxidant and anti-inflammatory activities
TL;DR: The strains selected in the present study showed indispensable characteristics for their inclusion in a probiotic formulation to be used at dry-off period for the prevention of bovine mastitis.
Abstract: Bovine mastitis causes economic losses on dairy farms worldwide. Lactic acid bacteria (LAB) in animal health are an alternative tool to avoid antibiotic therapy on the prevention of bovine mastitis. In previous studies, 12 LAB isolated from bovine milk were selected taking into account some of the following characteristics: hydrophobicity, auto aggregative capability, inhibition of indicator pathogens, hydrogen peroxide, and capsular polysaccharide production. These LAB were considered because of their beneficial properties. In this work, we also analyzed the antimicrobial activity and the co-aggregation against mastitis causing bacteria, auto-inhibition, adhesion to bovine teat canal epithelial cells (BTCEC), and growth kinetic curves for the 12 LAB. Two of them, Lactococcus lactis subsp. lactis CRL 1655 and Lactobacillus perolens CRL 1724, were selected because they had an interesting pattern of adhesion to BTEC, the inhibition of pathogens and the co-aggregation with the 100% of the assayed pathogens. They showed a predictable difference in the PFGE genomic pattern bands. The kinetic growth of these two strains was similar between them and with the rest of the assayed LAB. The strains selected in the present study showed indispensable characteristics for their inclusion in a probiotic formulation to be used at dry-off period for the prevention of bovine mastitis.
TL;DR: Replacement of the NSC with the CSC controlled growth of dairy enterococci in favor of mesophilic nonstarter lactobacilli during ripening, and safety concerns associated with the inefficiency of NSCs to prevent outgrowth of indigenous Enterococci suggest that CSCs should be preferred by traditional Greek Graviera cheese processors.
TL;DR: A review of the different levels at which Gram-positive bacteria Lactococcus lactis and Bacillus subtilis can be engineered and their various application possibilities.
TL;DR: Seven strains belonging to the species that showed a more broad-spectrum activity, L. paracasei and L. plantarum, were tested for their ability to inhibit the growth of fungi and all of them showed, in different degree, activity against Fusarium oxysporum.
Abstract: Lactic acid bacteria isolated from wine fermentations, particularly from the malolactic fermentation, and belonging to Lactobacillus plantarum, Lactobacillus hilgardii, Lactobacillus paracasei and Lactococcus lactis species were tested for their effectiveness in inhibiting the development of different microorganisms. The different strains showed, to varying degrees, an antagonistic effect against bacteria of the genera Bacillus and Staphylococcus. The specificity of the species L. hilgardii that inhibits only strains of the genus Bacillus is remarkable, on the other hand, L. plantarum was more effective against the strains of the genus Staphylococcus. The greatest effectiveness, considering both the degree of inhibition and the number of inhibited species, was presented by strains of L. lactis and L. paracasei. Seven strains belonging to the species that showed a more broad-spectrum activity, L. paracasei and L. plantarum, were also tested for their ability to inhibit the growth of fungi. All of them showed, in different degree (55–76%), activity against Fusarium oxysporum. Finally, the ability of the L. paracasei LPAUV12 and L. plantarum LPLUV10 strains was evaluated to protect Lycopersicon esculentum plants against the fungus F. oxysporum and promote its growth. Strain LPLUV10, showed capacity to significantly inhibit the harmful effect of F. oxysporum in tomato plants as well as to significantly stimulate their growth.
TL;DR: This study investigates the effect of four selected autochthonous co-cultures on the free amino acid profile, with special emphasis on GABA and ornithine, and on the biogenic amine content of pasteurized sheep milk cheese during ripening to find a good approach to the development of functional sheep milk Cheese.
TL;DR: Either alone, in mixtures, or in combination with industrial starter or adjunct cultures, these strains might be useful in the development of health-oriented dairy products.
Abstract: γ-Aminobutyric acid (GABA), an amino acid not used in protein synthesis, intervenes in several physiological functions and has both diuretic and calming effects in humans. Lactic acid bacteria (LAB) strains that produce GABA could be exploited for the manufacture of health-promoting GABA-enriched dairy products. In this study, 262 LAB strains isolated from traditional dairy products made from raw milk without starter cultures were screened for GABA production in culture media supplemented with 1% monosodium glutamate (MSG) using an enzymatic (GABase) method. About half of the strains (123) were found to be GABA producers. Of these, 24, among which were 16 Lactococcus lactis subsp. lactis and three Streptococcus thermophilus strains, produced >1 mM of GABA (range 1.01-2.81 mM) and were selected for further characterisation. GABA production was confirmed in most strains by culturing in 5 mM MSG followed by HPLC quantification. A majority of the strains were confirmed to be GABA producers by this method, although lower production levels were recorded. Using species-specific primers, the gene encoding glutamate decarboxylase (GAD) was PCR-amplified in all but one of the GABA producers analysed. Amplicons sequences were compared to one another and to those held in databases. Except for one Lactobacillus brevis strain, none of the 24 GABA producers investigated produced toxic biogenic amines, such as tyramine, histamine or cadaverine. They were therefore considered safe. Either alone, in mixtures, or in combination with industrial starter or adjunct cultures, these strains might be useful in the development of health-oriented dairy products.
TL;DR: In this paper, the composition of protective blends for manufacture of L. lactis probiotic powders was optimized using a statistical experimental design, and the powders, generated by either spray- or freeze-drying techniques, were subsequently subjected to storage testing, and in vitro digestion in simulated stomach and small intestine.
TL;DR: In this study, the BC/HA (hyaluronic acid) nanocomposites in the pellicle form were directly produced through co-culturing Gluconacetobacter hansenii ATCC 23769 and Lactococcus lactis APJ3 in a novel two-vessel circulating system.
TL;DR: A synthetic metabolic pathway enabling direct biosynthesis of 1,3-propanediol (PDO) from glucose via the Krebs cycle intermediate malate and the simultaneous overexpression of the genes encoding these enzymes together with the endogenous ydhD-encoded aldehyde reductase enabled PDO biosynthesis from (L)-DHB.
Abstract: In this work, we describe the construction of a synthetic metabolic pathway enabling direct biosynthesis of 1,3-propanediol (PDO) from glucose via the Krebs cycle intermediate malate. This non-natural pathway extends a previously published synthetic pathway for the synthesis of (L)-2,4-dihydroxybutyrate (L-DHB) from malate by three additional reaction steps catalyzed respectively, by a DHB dehydrogenase, a 2-keto-4-hydroxybutyrate (OHB) dehydrogenase and a PDO oxidoreductase. Screening and structure-guided protein engineering provided a (L)-DHB dehydrogenase from the membrane-associated (L)-lactate dehydrogenase of E. coli and OHB decarboxylase variants derived from the branched-chain keto-acid decarboxylase encoded by kdcA from Lactococcus lactis or pyruvate decarboxylase from Zymomonas mobilis. The simultaneous overexpression of the genes encoding these enzymes together with the endogenous ydhD-encoded aldehyde reductase enabled PDO biosynthesis from (L)-DHB. While the simultaneous expression of the six enzymatic activities in a single engineered E. coli strain resulted in a low production of 0.1 mM PDO from 110 mM glucose, a 40-fold increased PDO titer was obtained by co-cultivation of an E. coli strain expressing the malate-DHB pathway with another strain harboring the DHB-to-PDO pathway.
TL;DR: Evidence is provided for the use of Lactococcus lactis carrying an anti-TNFα scFv expression plasmid on a DSS-induced colitis model in mice, paving the way for a novel low-cost and site-specific biotechnological route for the treatment of inflammatory disorders.
Abstract: Anti-Tumor Necrosis Factor-alpha therapy has become clinically important for treating inflammatory bowel disease. However, the use of conventional immunotherapy requires a systemic exposure of patients and collateral side effects. Lactic acid bacteria have been shown to be effective as mucosal delivering system for cytokine and single domain antibodies, and it is amenable to clinical purposes. Therefore, lactic acid bacteria may function as vehicles for delivery of therapeutic antibodies molecules to the gastrointestinal tract restricting the pharmacological effect towards the gut. Here, we use the mucosal delivery of Lactococcus lactis carrying an anti-TNFα scFv expression plasmid on a DSS-induced colitis model in mice. Experimental colitis was induced with DSS administered in drinking water. L. lactis carrying the scFv expression vector was introduced by gavage. After four days of treatment, animals showed a significant improvement in histological score and disease activity index compared to those of untreated animals. Moreover, treated mice display IL-6, IL17A, IL1β, IL10 and FOXP3 mRNA levels similar to health control mice. Therefore, morphological and molecular markers suggest amelioration of the experimentally induced colitis. These results provide evidence for the use of this alternative system for delivering therapeutic biopharmaceuticals in loco for treating inflammatory bowel disease, paving the way for a novel low-cost and site-specific biotechnological route for the treatment of inflammatory disorders.
TL;DR: It is demonstrated that certain LAB strains can be employed for efficient production of long-chain vitamin K2, providing a basis for biotechnological production of vitamin K1 and in situ fortification of this vitamin in food products.
Abstract: Vitamin K2 (menaquinone, MK-n) is a lipid-soluble vitamin that functions as a carboxylase co-factor for maturation of proteins involved in many vital physiological processes in humans. Notably, long-chain vitamin K2 is produced by bacteria, including some species and strains belonging to the group of lactic acid bacteria (LAB) that play important roles in food fermentation processes. This study was performed to gain insights into the natural long-chain vitamin K2 production capacity of LAB and the factors influencing vitamin K2 production during cultivation, providing a basis for biotechnological production of vitamin K2 and in situ fortification of this vitamin in food products. We observed that six selected Lactococcus lactis strains produced MK-5 to MK-10, with MK-8 and MK-9 as the major MK variant. Significant diversities between strains were observed in terms of specific concentrations and titres of vitamin K2. L. lactis ssp. cremoris MG1363 was selected for more detailed studies of the impact of selected carbon sources tested under different growth conditions [i.e. static fermentation (oxygen absent, heme absent); aerobic fermentation (oxygen present, heme absent) and aerobic respiration (oxygen present, heme present)] on vitamin K2 production in M17 media. Aerobic fermentation with fructose as a carbon source resulted in the highest specific concentration of vitamin K2: 3.7-fold increase compared to static fermentation with glucose, whereas aerobic respiration with trehalose resulted in the highest titre: 5.2-fold increase compared to static fermentation with glucose. When the same strain was applied to quark fermentation, we consistently observed that altered carbon source (fructose) and aerobic cultivation of the pre-culture resulted in efficient vitamin K2 fortification in the quark product. With this study we demonstrate that certain LAB strains can be employed for efficient production of long-chain vitamin K2. Strain selection and optimisation of growth conditions offer a viable strategy towards natural vitamin K2 enrichment of fermented foods, and to improved biotechnological vitamin K2 production processes.
TL;DR: Back-slopping fermentation offers greater abundance and diversity compared to spontaneous fermentation in dadih, and was showed to be one log cycle higher than spontaneous fermented dadih.
Abstract: Aim Dadih samples from two different origins (Kamang and Gadut in West Sumatra) manufactured with different methods (back-slopping or spontaneous fermentation) were evaluated for the diversity of lactic acid bacteria (LAB). Materials and methods Four dadih samples manufactured with two different fermentation methods were obtained from Kamang and Gadut regions. Both genotypic and phenotypic characteristic (16S rRNA partial gene sequence analysis and carbohydrate fermentation profile) were used to analyze the diversity of dadih LAB population. Results This study showed that LAB count in back-slopping fermented dadih was one log cycle higher than spontaneous fermented dadih. LAB isolates from the two regions were divided into three genera, namely Lactococcus, Lactobacillus, and Pediococcus. Sequencing results showed that 41.6% (five isolates) were identified as Lactococcus lactis ssp. lactis, 25% (three isolates) were identified as Lactobacillus plantarum ssp. plantarum, 16.6% (two isolates) were identified as L. lactis ssp. cremoris, and 8.3% (one isolate each) were identified as Pediococcus pentosaceus and Lactobacillus pentosus. Conclusion Five species were determined in back-slopping fermented dadih, i.e., L. lactis ssp. lactis, L. lactis ssp. cremoris, L. plantarum ssp. plantarum, L. pentosus, and P. pentosaceus. On the other hand, spontaneous fermented dadih only contained three different species, namely L. lactis ssp. lactis, L. lactis ssp. cremoris, and L. plantarum ssp. plantarum. This research showed that back-slopping fermentation offers greater abundance and diversity compared to spontaneous fermentation in dadih.