Search

The bacteriocins produced by lactic acid bacteria (LAB) are attracting attention due to their promising applications in food and pharmaceuticals fields. Hence, a LAB strain, GCNRC_GA15, was isolated from Egyptian goat cheese, and molecularly identified as Lactiplantibacillus plantarum. This strain showed a wide antimicrobial spectrum, which was found to be of proteineous nature, suggesting that L. plantarum GCNRC_GA15 is a bacteriocin-producer. This bacteriocin (bacteriocin GA15) was partially purified using cation exchange, and hydrophobic interaction chromatography. Tricine SDS-PAGE analysis for the fraction showing bacteriocin activity has estimated the molecular mass to be 4369 Da. Furthermore, amino acid sequencing of this peptide has detected 34 amino acids, and comparing its amino acid sequence with those of some pediocin-like bacteriocins revealed that bacteriocin GA15 has the conserved sequence (YYGNGV/L) in its N-terminal region which identified bacteriocin GA15 as a pediocin-like bacteriocin. Bacteriocin GA15 showed good heat and pH stabilities, and its activity was enhanced after treatment with Tween 80 or Triton X-100. Bacteriocin production medium was statistically optimized using the Plackett-Burman and Central Composite designs. As a result, bacteriocin production increased from 800 to 12,800 AU/ml using the optimized medium in comparison with result recorded for the un-optimized medium.

The multiple bacteriocins produced by Lactiplantibacillus plantarum PUK6 isolated from misozuke-tofu (tofu pickled in miso) were identified as plantaricins A, EF, and NC8. The pln locus (21,847 bp) containing the three plantaricin structural genes and another newly found putative bacteriocin structural genes (orf1 and orf2) were determined, and a biosynthesis mechanism was proposed. Reverse transcription-PCR analysis revealed that orf1 and orf2, the putative two-peptide bacteriocin structural genes, were expressed after 8 h (logarithmic growth phase) and 20 h (stationary growth phase) of cultivation of the PUK6 strain. Additionally, the growth inhibition profile obtained using the chemically synthesized mature peptides of Orf1 and Orf2 (referred to as mOrf1 and mOrf2) showed that the equimolar mixture of mOrf1 and mOrf2 exhibited bactericidal effect against the indicator strain Latilactobacillus sakei subsp. sakei JCM 1157

Enterococcus faecium NKR-5-3 produces multiple-bacteriocins, enterocins NKR-5-3A, B, C, D, and Z (Ent53A, Ent53B, Ent53C, Ent53D, and Ent53Z). However, the biosynthetic mechanisms on how their productions are regulated are yet to be fully understood. In silico analysis revealed putative promoters and terminators in the enterocin NKR-5-3ACDZ gene cluster, and the putative direct repeats (5'-ATTTTAGGATA-3') were conserved upstream of each promoter. Transcriptional analysis by quantitative real-time polymerase chain reaction (PCR) of the biosynthetic genes for the enterocins NKR-5-3 suggested that an inducing peptide (Ent53D) regulates the transcription of the structure genes and corresponding biosynthetic genes of enterocins NKR-5-3, except for Ent53B (a circular bacteriocin), thus consequently regulating their production. Moreover, transcriptional analysis of some knock-out mutants showed that the production of Ent53A, C, D and Z is controlled by a three-component regulatory system (TCS) consisting of Ent53D, EnkR (response regulator), and EnkK (histidine kinase). The production of the circular bacteriocin Ent53B appeared to be independent from this TCS. Nevertheless, disrupting the TCS by deletion of a single component (enkD, enkR and enkK) resulted in a slight increase of enkB transcription and consequently the production of Ent53B, presumably, as an indirect consequence of the increase of available energy to the strain NKR-5-3. Here, we demonstrate the regulatory control of the multiple bacteriocin production of strain NKR-5-3 likely through the TCS consisting of Ent53D, EnkR, and EnkK. The information of the sharing of the regulatory machinery between bacteriocins in strain NKR-5-3 can be useful in its future application such as designing strategies to effectively dispense its multiple bacteriocin arsenal.

Bacteriocin production in lactic acid bacteria (LAB) has always been considered as a highly desirable trait as it enhances the strain's utility in different industrial applications. Bacteriocin producing LAB strains are considered to have higher bacterial fitness as they are able to easily establish themselves into target microbial niche and hence are more effective starter cultures in food fermentation and/or probiotic strains. The rapid advancement in genomic research revealed the true bacteriocin producing capacity of some select novel LAB strains capable of producing multiple bacteriocins which further improves their utility in different application systems. What is common to these novel strains is the remarkable sharing of some elements in the biosynthetic process enabling them to accomplish the extraordinary feat of producing multiple bacteriocins without exhausting its energy. Contrary to the common understanding that biosynthetic enzymes are specific to their cognate bacteriocins, multiple bacteriocin producing strains employ shared biosynthetic elements between their multiple bacteriocins. The quorum-sensing three-component regulatory system, bacteriocin maturation and transport mechanisms are shared among multiple bacteriocins in these strains. Nevertheless, although these novel strains possess enormous application potential, their safety with regards to their potential virulence and pathogenicity needs to be confirmed through comprehensive genotypic characterization. Here, we compile the occurrence of multiple bacteriocin production in some novel LAB strains and highlight specific examples of the unique sharing mechanism of its biosynthetic machinery because a good understanding how these novel strains synthesize their multiple bacteriocins can aid in maximizing their application potential.

Bacteriocins from lactic acid bacteria (LAB) are industrially important compounds that have been utilized as a safe food preservative replacing the potentially harmful synthetic chemical preservatives, Enterococcus faecium NKR- 5-3 is a novel LAB strain that produces five different bacteriocins‘ The critical fermentation factors, such as specific medium components and optimum incubation temperature that support the maximum production of multiple bacteriocins of strain NKR-5-3, were identified. Sucrose and yeast extract were found to be the preferred carbon and nitrogen sources for bacteriocin production of this strain, respectively. The highest bacteriocin production was observed when strain NKR-5-3 was incubated at 25°C. At incubation temperatures beyond 30°C, bacteriocin production was significantly reduced and completely ceased when further raised to 40°C, These findings possess remarkable practical implications as they can be vital in the future design of a cost-effective production system for these bacteriocins. Such a system would address the issue of the high production cost, which has remained the major barrier to the development of the large-scale industrial utilization of these important compounds.

In this study, a non-sterile (open) continuous fermentation (OCF) process with no-carbon loss was developed to improve lactic acid (LA) productivity and operational stability from the co-utilization of lignocellulose-derived sugars by thermophilic Enterococcus faecium QU 50. The effects of different sugar mixtures on LA production were firstly investigated in conventional OCF at 50°C, pH 6.5 and a dilution rate of 0.20 hr-1 . The xylose consumption ratio was greatly lower than that of glucose in fermentations with glucose/xylose mixtures, indicating apparent carbon catabolite repression (CCR). However, CCR could be efficiently eliminated by feeding solutions containing the cellobiose/xylose mixture. In OCF at a dilution rate ca. 0.10 hr-1 , strain QU 50 produced 42.6 g L-1 of l-LA with a yield of 0.912 g g-1 -consumed sugars, LA yield of 0.655 g g-1 based on mixed sugar-loaded, and a productivity of 4.31 g L-1 hr-1 from simulated energy cane hydrolyzate. In OCF with high cell density by cell recycling, simultaneous and complete co-utilization of sugars was achieved with stable LA production at 60.1 ± 3.25 g L-1 with LA yield of 0.944 g g-1 -consumed sugar and LA productivity of 6.49 ± 0.357 g L-1 hr-1 . Besides this, a dramatic increase in LA yield of 0.927 g g-1 based on mixed sugar-loaded with prolonged operational stability for at least 500 hr (>20 days) was established. This robust system demonstrates an initial green step with a no-carbon loss under energy-saving toward the feasibility of sustainable LA production from lignocellulosic sugars.

The fraction of administered antibiotics that reach the cecum and colon causes dysbiosis of the gut microbiome, resulting in various diseases. Protection of the gut microbiome from antibiotics using antibiotic adsorbents in the cecum and colon is a promising method to overcome this issue. Previously, activated charcoal (AC) has been reported to protect the gut microbiome of host animals. AC is an adsorbent that is widely used to capture toxic compounds and overdosed drugs in the gastrointestinal tract. The specificity of adsorbents for antibiotics is critical to avoid the risk of unexpected side effects caused by nonspecific adsorption of biological compounds in the intestinal fluid, such as bile acids and essential micronutrients. Here, we have developed specific adsorbents for vancomycin (VCM), which is known to cause gut dysbiosis. The adsorbents were composed of polyethyleneglycol-based microparticles (MPs) in which a specific ligand for VCM, D-Ala-D-Ala-OH, was attached via dendrons of D-lysine to raise the content of the ligand in the MPs. The MPs successfully protected Staphylococcus lentus from VCM in vitro because of the adsorption of VCM in the culture media. Pre-administration of MPs to mice reduced the amount of free VCM in the feces to an undetectable level. This treatment minimized the effect of VCM on gut microbiota and provided protection against Clostridioides difficile infection after oral challenge with spores.

In the present study, we investigated the glucose-decreasing action of lactic acid bacteria (LAB). The finding of this study could be helpful for people in controlling their blood sugar levels. The LAB candidate was isolated from a Japanese fermented food and identified as Pediococcus pentosaceus by an analysis of its genome sequence. Postprandial blood glucose elevation was investigated using oral starch tolerance tests in mice. Normal mice were fed starch and lyophilized cells of P. pentosaceus QU 19 at the same time. Even without pre-administration of P. pentosaceus QU 19, elevation of the blood glucose level was significantly suppressed by the intake of P. pentosaceus QU 19 at the same time as oral administration of starch. According to the results for its survival in simulated digestive juice and the reduction of blood glucose level in mice, P. pentosaceus QU 19 has potential hypoglycemic activity. In vitro measurements revealed that the glucose-decreasing action of P. pentosaceus QU 19 is probably caused by the glucose assimilation of the strain, not the inhibition of carbohydrate-splitting enzymes which has been reported for other LABs previously. These findings indicate that specific strains of LAB, especially P. pentosaceus QU 19, and foods fermented by LAB may be beneficial for people who must manage glucose ingestion.

Enterocin F4-9 belongs to the glycocin family having post-translational modifications by two molecules of N-acetylglucosamine β-O-linked to Ser37 and Thr46. In this study, the biosynthetic gene cluster of enterocin F4-9 was cloned and expressed in Enterococcus faecalis JH2-2. Production of glycocin by the JH2-2 expression strain was confirmed by expression of the five genes. The molecular weight was greater than glycocin secreted by the wild strain, E. faecalis F4-9, because eight amino acids from the N-terminal leader sequence remained attached. This N-terminal extension was eliminated after treatment with the culture supernatant of strain F4-9, implying an extracellular protease from E. faecalis F4-9 cleaves the N-terminal sequence. Thus, leader sequences cleavage requires two steps: the first via the EnfT protease domain and the second via extracellular proteases. Interestingly, the long peptide, with N-terminal extension, demonstrated advanced antimicrobial activity against Gram-positive and Gram-negative bacteria. Furthermore, enfC was responsible for glycosylation, a necessary step prior to secretion and cleavage of the leader peptide. In addition, enfI was found to grant self-immunity to producer cells against enterocin F4-9. This report demonstrates specifications of the minimal gene set responsible for production of enterocin F4-9, as well as a new biosynthetic mechanism of glycocins.

Bioactive peptides were successfully produced from fish (Gadidae) and beef skeletal muscles after being hydrolyzed for 8 h with pepsin. Subsequently, they were purified using a Sep-Pak C18 cartridge and reversed-phase high-performance liquid chromatography (RP-HPLC). The molecular weights of pure fish and beef peptides were determined to be 2364.4 and 3771.8, respectively. According to Edman degradation, the fish peptide was composed of 21 amino acid residues (F21), while the beef peptide was composed of 34 amino acid residues (B34). F21 and B34 displayed angiotensin-converting enzyme inhibitory activity with a half maximal inhibitory concentration (IC50) values of 7.3 µg/mL and 5.8 µg/mL, respectively. F21 exhibited antioxidant activity with an IC50 value of 389.9 µg/mL, whereas B34 exhibited no antioxidant activity. Moreover, F21 and B34 displayed antimicrobial effects against a wide spectrum of food-borne pathogens and spoilage bacteria. Bioactive peptides derived from muscle proteins are a promising strategy for the production of functional food materials and safe food preservatives.

The spontaneous microbiota of wheat sourdough, often comprising one yeast species and several lactic acid bacteria (LAB) species, evolves over repeated fermentation cycles, which bakers call backslopping. The final product quality largely depends on the microbiota functions, but these fluctuate sometimes during the initial months of fermentation cycles due to microbiota evolution in which three phases of LAB relay occur. In this study, the understanding of yeast-LAB interactions in the start of the evolution of the microbiota was deepened by exploring the timing and trigger interactions when sourdough yeast entered a preestablished LAB-relaying community. Monitoring of 32 cycles of evolution of 6 batches of spontaneous microbiota in wheat sourdoughs revealed that sourdough yeasts affected the LAB community when the 2nd- or 3rd-relaying types of LAB genera emerged. In in vitro pairwise cocultures, all 12 LAB strains containing the 3 LAB-relaying types arrested the growth of a Saccharomyces cerevisiae strain, a frequently found species in sourdoughs, to various extents by sugar-related interactions. These findings suggest competition due to different affinities of each LAB and a S. cerevisiae strain for each sugar. In particular, maltose was the driver of S. cerevisiae growth in all pairwise cocultures. The functional prediction of sugar metabolism in sourdough LAB communities showed a positive correlation between maltose degradation and the yeast population. Our results suggest that maltose-related interactions are key factors that enable yeasts to enter and then settle in the LAB-relaying community during the initial part of evolution of spontaneous sourdough microbiota. IMPORTANCE Unpredictable evolution of spontaneous sourdough microbiota sometimes prevents bakers from making special-quality products because the unstable microbiota causes the product quality to fluctuate. Elucidation of the evolutionary mechanisms of the sourdough community, comprising yeast and lactic acid bacteria (LAB), is fundamental to control fermentation performance. This study investigated the mechanisms by which sourdough yeasts entered and settled in a bacterial community in which a three-phase relay of LAB occurred. Our results showed that all three layers of LAB restricted the cohabiting yeast population by competing for the sugar sources, particularly maltose. During the initial evolution of spontaneous sourdough microbiota, yeasts tended to grow synchronously with the progression of the lactic acid bacterial relay, which was predictably associated with changes in the maltose degradation functions in the bacterial community. Further study of ≥3 species’ interactions while considering yeast diversity can uncover additional interaction mechanisms driving the initial evolution of sourdough microbiota.

The probiotic Lactobacillus brevis KB290 is a natural producer of cell-bound exopolysaccharide (EPS), and the plasmid-encoded glycosyltransferase genes are responsible for this EPS production. KB290 forms unique rugose colonies inside an agar medium; this characteristic is useful for detecting and enumerating KB290 in the gut or feces. However, the genetic elements associated with this morphology remain unclear. Here, we aimed to investigate the relation between the plasmid eps genes and rugose colony morphology in KB290. The plasmid-cured mutants formed smooth colonies, and the rugose colony morphology was restored after complementation with the eps genes. The eps genes were successfully cloned and expressed in other L. brevis and L. plantarum strains. In these transformant strains, the presence of the EPS, consisting of glucose and N-acetylglucosamine, correlated with rugose colonies, indicating that EPS is responsible for rugose colony formation. To the best of our knowledge, this is the first report identifying the genetic factors influencing rugose colonies in Lactobacillus strains. This rugose colony formation may serve as a useful selective marker for KB290 in routine laboratory and research settings and can be used to detect the spontaneous loss of plasmids in this strain.

We demonstrate here that exopolysaccharide (EPS) production, cell aggregation, and bile resistance in Lactobacillus brevis KB290 are conferred by three eps genes (gtf27, gtf28, and orf29) located on the 42.4-kb plasmid pKB290-1. The predicted products of gtf27 and gtf28 belong to the membrane-bound glycosyltransferase family whereas the orf29 gene product showed homology with the ABC transporter. On in silico analysis, these genes were found to be widely distributed among lactobacilli from publicly available genomes and metagenomes, and their function is not yet elucidated. RT-PCR analysis showed that the eps genes were organised in an operon and their expression was markedly lower in arabinose- and xylose-containing media than in a glucose-containing medium. The three eps genes were cloned and expressed in homologous and heterologous strains. Considerably less EPS was produced by the plasmid-cured KB1802 strain than by the parental KB290 strain, whereas a similar amount was produced by the KB1802 strain expressing the three eps genes. The KB1802 strain expressing gtf27 and gtf28 but not orf29 did not produce EPS. Cell aggregation and bile resistance were also decreased in KB1802 strains but were complemented by eps genes. Moreover, the three eps genes conferred these phenotypes to a Lactobacillus plantarum strain. In conclusion, the three eps genes in pKB290-1 were sufficient for EPS biosynthesis with glucose and N-acetylglucosamine, and were responsible for cell aggregation and bile resistance. We consider these phenotypes to be at least partly responsible for KB290-specific properties.

Wheat sourdough is a common traditional fermented food that is produced worldwide. However, product quality of spontaneous sourdough is not easy to control because it depends on natural fermentation and backslopping, about which little is known, notably after ten backslopping steps. To this end, we tracked the spontaneous fermentation of three sourdoughs made from wheat flours during 32 backslopping steps for 60 days. At 24 time points, the microbial community was analyzed by both culture-dependent and culture-independent methods and its chemical constituents were assessed. Dynamic changes were observed in the microbial community, which showed a common succession pattern among the three sourdoughs at the bacterial family level and differences at the species level. The bacterial communities evolved through three phases that were driven by different groups of lactic acid bacteria (LAB) species. The dynamism among the metabolites also differed, depending on the species composition of the LAB and yeast communities. In one sourdough, the growth of Saccharomyces cerevisiae was detected along with a concentration of increased ethanol, while in the other two sourdoughs, Wickerhamomyces anomalus was detected without ethanol production. Regarding the LAB communities, two sourdoughs were eventually co-dominated by Lactobacillus plantarum and L. brevis, while the other sourdough was eventually dominated solely by the heterolactic fermentative bacterium Lactobacillus fermentum, and ethanol was produced at the same level as lactic acid. Further research is needed to determine the bacterial and yeast species involved in the fermentation of sourdough, to help improve the design and quality control of the final product.

Phosphoketolase (PK) is responsible for heterolactic fermentation; however, the PK gene of Enterococcus mundtii QU 25, xfpA, is transcribed constitutively, even under homolactic fermentation conditions. In order to deduce the regulatory mechanisms of PK activity in QU 25, XfpA levels in QU 25 cells under hetero- and homolactic fermentation conditions were tested using western blotting. The results showed that the XfpA protein expression was similar under both conditions and that the expression products formed complexes, most likely homodimers, indicating that the regulation of PK activity is downstream of translation.

Lactic acid bacteria (LAB) are a diverse group of microorganisms that can be found in natural habitats and are used in a wide range of industries, especially in the food sector. They are regarded "Generally Recognized as Safe" and are commonly used as a starter culture, probiotics, and biopreservative. However, some LAB genera are excluded from the "Qualified Presumption of Safety" (QPS) list due to the possibility that they have virulence and antibiotic resistance genetic determinants. The objective of this study was to investigate the beneficial, virulence, and antibiotic resistance-related genes in 10 well-characterized bacteriocinogenic LAB strains. The auto-aggregation, co-aggregation, cell surface hydrophobicity, and bile salt tolerance were also evaluated to examine their potential as bacteriocinogenic probiotics and/or starter culture. Results showed that all tested strains showed abilities for auto-aggregation at 4oC and 37oC, co-aggregation with S. aureus JCM8704, S. typhimurium BIOTECH1826, and E. coli DH5α, and significant cell-surface hydrophobicity. However, only a few strains were able to withstand the media treated with 0.3% bile salt. Among the tested bacteriocinogenic LAB strains, L. lactis IO-1 and C. divergens V41 had the maximum values for auto-aggregation at 4oC and 37oC, respectively. C. divergens V41 also exhibited the highest percentage for cell surface hydrophobicity. E. faecium NKR-5-3 showed the highest co-aggregation percentages with all indicator strains. Our findings also showed that the tested isolates presented distinct combinations of virulence-related genes. Only two of ten bacteriocinogenic LAB strains exhibited presence of multiple virulence genes. Lactococcus sp. QU12 was found to have a high frequency of beneficial and virulence genes, with 2 out of 7 genes present encoding beneficial factor and 11 out of 13 genes encoding virulence factor. Lactococcus sp. QU12 and L. lactis IO-1 were also positive for tetracycline resistance gene tetM and aminoglycoside resistance gene aphA-2, respectively, and transposon genes. Moreover, only a few LAB isolates tested positive for 2 out of 8 antibiotic resistance classes. Although few, the substantial danger of these genes being transferred and acquired cannot be overlooked as this could potentially cause serious health concerns in the future. These results suggest that despite the promising properties of bacteriocinogenic LAB, careful safety evaluation of these strains should be a prerequisite before using these in food systems.

Sourdough is a naturally fermented dough that is used worldwide to produce a variety of baked foods. Various lactic acid bacteria (LAB), which can determine the quality of sourdough baked foods by producing metabolites, have been found in the sourdough ecosystem. However, spontaneous fermentation of sourdough leads to unpredictable growth of various micro-organisms, which result in unstable product quality. From an ecological perspective, many researchers have recently studied sourdough LAB diversity, particularly the elucidation of LAB community interactions and the dynamic mechanisms during the fermentation process, in response to requests for the control and design of a desired sourdough microbial community. This article reviews recent advances in the study of sourdough LAB diversity and its dynamics in association with unique characteristics of the fermentation system; it also discusses future perspectives for better understanding of the complex sourdough microbial ecosystem, which can be attained efficiently by both in vitro and in situ experimental approaches.

EnkT is an ATP-binding cassette (ABC) transporter produced by Enterococcus faecium NKR-5-3, which is responsible for the secretion of multiple bacteriocins; enterocins NKR-5-3A, C, D, and Z (Ent53A, C, D, and Z). EnkT has been shown to possess a tolerant recognition mechanism that enables it to secrete the mature Ent53C from a chimeric precursor peptide containing the leader peptide moieties that are derived from different heterologous bacteriocins. In this study, to further characterize EnkT, we aimed to investigate the capacity of EnkT to recognize, process, and secrete non-cognate bacteriocins, which belong to different subclasses of class II. For this, the non-cognate bacteriocin precursor peptides, including enterocin A, pediocin PA-1, lactococcin Q, lactococcin A, and lacticin Q were co-expressed with EnkT, and thereafter, the production of the mature forms of these non-cognate bacteriocins was assessed. Our results revealed that EnkT could potentially recognize, process, and secrete the non-cognate bacteriocins with an exception of the leaderless bacteriocin, lacticin Q. Moreover, the processing and secretion efficiencies of these heterologous non-cognate bacteriocins by EnkT were further enhanced when the leader peptide moiety was replaced with the Ent53C leader peptide (derived from a native NKR-5-3 bacteriocin). The findings of this study describe the wide substrate tolerance of this ABC transporter, EnkT, that can be exploited in the future in establishing effective bacteriocin production systems adaptive to complex fermentation conditions common in many food systems.

Enterococcus mundtii QU25, a non-dairy lactic acid bacterium of the phylum Firmicutes, is capable of simultaneously fermenting cellobiose and xylose, and is described as a promising strain for the industrial production of optically pure l-lactic acid (≥ 99.9%) via homo-fermentation of lignocellulosic hydrolysates. Generally, Firmicutes bacteria show preferential consumption of sugar (usually glucose), termed carbon catabolite repression (CCR), while hampering the catabolism of other sugars. In our previous study, QU25 exhibited apparent CCR in a glucose-xylose mixture phenotypically, and transcriptional repression of the xylose operon encoding initial xylose metabolism genes, likely occurred in a CcpA-dependent manner. QU25 did not exhibit CCR phenotypically in a cellobiose-xylose mixture. The aim of the current study is to elucidate the transcriptional change associated with the simultaneous utilization of cellobiose and xylose. To this end, we performed RNA-seq analysis in the exponential growth phase of E. mundtii QU25 cells grown in glucose, cellobiose, and/or xylose as either sole or co-carbon sources. Our transcriptomic data showed that the xylose operon was weakly repressed in cells grown in a cellobiose-xylose mixture compared with that in cells grown in a glucose-xylose mixture. Furthermore, the gene expression of talC, the sole gene encoding transaldolase, is expected to be repressed by CcpA-mediated CCR. QU25 metabolized xylose without using transaldolase, which is necessary for homolactic fermentation from pentoses using the pentose-phosphate pathway. Hence, the metabolism of xylose in the presence of cellobiose by QU25 may have been due to 1) sufficient amounts of proteins encoded by the xylose operon genes for xylose metabolism despite of the slight repression of the operon, and 2) bypassing of the pentose-phosphate pathway without the TalC activity. Accordingly, we have determined the targets of genetic modification in QU25 to metabolize cellobiose, xylose and glucose simultaneously for application of the lactic fermentation from lignocellulosic hydrolysates.

Lactic acid (LA) fermentation requires a neutralizer for a physiologically acceptable range. However, a neutralizer generates a large amount of gypsum, an environmental pollutant. Furthermore, the downstream processing is complicated and expensive, comprising 50–70% of the total cost. We previously developed a Lactobacillus delbrueckii FM1, which can produce undissociated LA without neutralizer. Here, we improved FM1 by adaptive evolution at pH 4.5, which generated Adp FM1 showing an ~ 1.80-fold increase in LA production compared to FM1. The LA production via fed-batch fermentation yielded 36.2 g/L of LA, with a productivity of 0.500 g/L/h. However, cell viability was reduced due to the acidic pH and/or end-product inhibition. Therefore, an in situ LA recovery process using an extractive solvent was employed to maintain cell viability. Adp FM1 produced 49.2 g/L of LA via in situ LA-extractive fed-batch fermentation, which was ~ 1.4-fold higher than that without LA extraction. Adp FM1 provided a total LA productivity of 0.512 g/L/h in 96 h. Among the tested strains, Adp FM1 exhibited the highest H+-ATPase activity and a 415-fold increase in H+-ATPase gene expression compared to the parent strain. These results suggest that the in situ LA extractive fermentation process will ease downstream processing and prove to be a more economical and environmentally friendly option compared to the present fermentation. To our knowledge, this is the first report on the production of undissociated L-LA by Lactobacillus using an in situ recovery process, with high LA production levels and productivity.

This study aimed to develop an antimicrobial biodegradable food packaging for control of pathogens in pangasius fish fillets, one of the world's most consumed freshwater fish. Bacteriocin 7293 (Bac7293), a novel bacteriocin from Weissella hellenica BCC 7293, was diffusion coated onto poly (lactic acid)/sawdust particle biocomposite film (PLA/SP) with the maximum amount of 19.54 ± 2.87 μg/cm2. According to the JIS Z 2801:2000 standard testing method, the produced PLA/SP film impregnated with Bac7293 (PLA/SP + Bac7293) effectively inhibited both Gram-positive (Listeria monocytogenes and Staphylococcus aureus) and Gram-negative bacteria (Pseudomonas aeruginosa, Aeromonas hydrophila, Escherichia coli and Salmonella Typhimurium) which have been considered as a reason for the rejection of pangasius fish fillets. This antimicrobial film inhibited the growth of target microorganisms by about 2–5 log CFU/cm2, depended on bacterial strains, compared with control, both in vitro and in pangasius fish fillets by means of challenge test. The overall migration of PLA/SP + Bac7293 film to food simulants was maximized in isooctane (3.67 ± 0.47 mg/dm2) which was much lower than the overall migration limit regulated by the commission of the European communities. This suggested high potential application of PLA/SP + Bac7293 as a good antimicrobial packaging for pangasius fish fillets.

Bacteriocins are gene-encoded antimicrobial peptides that are traditionally appreciated as safe food preservative in the food industry and as possible alternative to conventional antibiotics in the pharmaceutical industry. Enterocin NKR-5-3B is a well characterized circular bacteriocin that possess exceptional stability due to the circular nature of its structure. In this paper, a mutant library of this bacteriocin was constructed through NNK-scanning whereby the presumed critical residues were targeted hoping to obtain bacteriocin derivatives with enhanced bioactivity. Thirteen (13) mutant phenotypes exhibited bioactivity enhancement relative to the native bacteriocin. The most notable bioactivity increases were observed from the phenotypes expressing V32C, V32A, and L40G that exhibited 233, 217, and 200% relative bioactivity, respectively. In-silico analyses of the resulting bacteriocin derivatives showed significant changes in the physico-chemical properties of the bacteriocin derivatives, particularly its hydrophobicity index, as a consequence of the introduced mutation. The V32C bacteriocin derivative which exhibited the strongest bioactivity enhancement was found to exhibit a reduction of the molecular surface hydrophobicity and isoelectric point. These changes may have contributed in the enhancement of its bioactivity. The identification of these critical mutations is highly valuable as basis for future studies on the rational design of bioengineered bacteriocins with enhanced bioactivity.

The present study revealed the effect of nitrogen sources on lactic acid production and stimulation of d- and l-lactate dehydrogenases (LDH) of parent Lactobacillus lactis NCIM 2368 and its mutant RM2-24 generated after UV mutagenesis. Both the parent and mutant strains were evaluated for d-lactic acid production in control and modified media. The modified media did not show remarkable effect on lactic acid production in case of parent whereas mutant exhibited significant enhancement in d-lactic acid production along with the appearance of l-lactic acid in the broth. Both LDH activities and specific activities were found to be higher in mutant than the parent strain. These results suggested that the diammonium hydrogen phosphate in modified media triggered the expression of LDH genes leading to enhanced lactic acid production. This observation has been proved by studying the expression levels of d- and l-LDH genes of parent and mutant in control and modified media using quantitative RT-PCR technique. In case of mutant, the transcriptional levels of d-LDH and l-LDH increased ∼17 fold and ∼1.38 fold respectively in modified medium compared to the values obtained with control medium. In case of parent, no significant change in transcriptional levels of d- and l-LDH was found when the cells were grown in either control medium or modified medium. This study suggested that the mutant, RM2-24 has l-LDH gene which is expressed in presence of (NH4)2HPO4 resulting in l-lactic acid production. Co-production of l-lactic acid in d-lactic acid fermentation may be detrimental in the PLA production.

Bacteriocins are antimicrobial peptides that possess a number of desirable properties that can be utilized for application in various industries including the food and pharmaceutical industries. Enterocin NKR-5-3B (Ent53B) and lacticin Q (LnqQ) are well characterized bacteriocins that exhibit exceptional stability and potent antimicrobial activity. Here we report the establishment of a dual-plasmid expression system of these bacteriocins by exploiting the quorum sensing production system of nisin known as the nisin inducible controlled expression (NICETM) system. Using this dual-plasmid expression system, the production of Ent53B and LnqQ can be controlled by the addition of exogenous nisin at sub-lethal concentration, either in pure or crude form, to induce the transcription of the genes responsible for the production of these bacteriocins. Using this system, cheap cultivation media instead of the commonly used complex and expensive cultivation media, can be utilized in the production of these bacteriocins thereby significantly lowering the production cost – one of the major limiting factors for its large-scale application. Additionally, this system provides a robust control of bacteriocin production thereby creating an effective bacteriocin application delivery system.

Screening for bacteriocin-producing lactic acid bacteria (LAB) showing probiotic properties is of high significance due to their applications in food, and health fields. Hence, the aim of the current study was to isolate, identify, and study the in vitro probiotic properties, antimicrobial and antioxidant activities of LAB isolates obtained from Egyptian sources. Out of 79 isolated bacteria, 53 isolates were selected as potential LAB and their antimicrobial activities were investigated. Seven out of them (isolates 33, 34, and 35 from feces of Nile Lechwe, and isolate 42 from feces of Grant's zebra) exhibited wide antimicrobial spectra, which were caused by a proteineous compound that were suggested to be bacteriocin. These isolates are nominated as potential probiotic strains due to their high pH tolerance and good thermal stability. Moreover, they exhibited high in vitro antioxidant activities by recording 84.47 ± 1.83, 86.84 ± 0.93, 82.37 ± 1.11, and 83.16 ± 0.84% DPPH scavenging activity, respectively. Furthermore, isolates 33, 34, and 35 showed high in vitro hydrophobic properties, which reached 93.93 ± 0.83, 87.75 ± 1.75, and 80.20 ± 1.40%, respectively. Isolates were identified using 16S rRNA sequencing as Enterococcus faecium except for isolate 10, which was classified to Enterococcus hirae. These results nominated Egyptian sources as a promising repository for potent LAB. This is the first study describing bacterial isolates from Egyptian zoo samples as potential bacteriocin producers, possible probiotic strains, and antioxidant agents,. Further studies are encouraged to evaluate the in vivo potency of these isolates for future application as antioxidant dietary supplements for feeding and treatment of human and animals.

Enterocin NKR-5-3B (Ent53B) is a 64-residue novel circular bacteriocin synthesized from an 87-residue prepeptide. Albeit through a still unknown mechanism, the EnkB1234 biosynthetic enzyme complex processes the prepeptide to yield its mature active, circular form. To gain insights into the key region/residue that plays a role in Ent53 maturation, several mutations near the cleavage site on the precursor peptide were generated. The interaction of the precursor peptide and EnkB1234 appeared to be hydrophobic in nature. At the Leu1 position, only mutations with helix structure-promoting hydrophobic residues (Ala, Ile, Val or Phe) were able to yield the mature Ent53B derivative. In this study, we also highlight the possible conformation-stabilizing role of the Ent53B leader peptide on the precursor peptide for its interaction with its biosynthetic enzyme complex. Any truncations of the leader peptide moiety interfered in the processing of the prepeptide. However, when propeptides of other circular bacteriocins (circularin A, leucocyclicin Q or lactocyclicin Q) were cloned at the C-terminus of the leader peptide, EnkB1234 could not process them to yield a mature bacteriocin. Taken together, these findings offer new perspectives in our understanding of the possible molecular mechanism of the biosynthesis of this circular bacteriocin. These new perspectives will help advance our current understanding to eventually elucidate circular bacteriocin biosynthesis. Understanding the biosynthetic mechanism of circular bacteriocins will materialize their application potential.

First-generation cephalosporins such as cefazolin (CEZ) have been widely used for mastitis treatment in dairy cattle. However, the use of antibiotics results in the presence of antibiotic residues in milk, which is used for human consumption. Nisin A, a bacteriocin produced by Lactococcus lactis, has been used as a broad-spectrum food preservative for over 50 years. Therefore, a combination of CEZ and nisin A might provide an extended activity spectrum against mastitis pathogens and reduce the antibiotic dose for mastitis treatment. This study aimed to evaluate the combined effect of CEZ and nisin A against mastitis pathogens using the checkerboard and time-kill assays. In the checkerboard assay, the CEZ-nisin A combination exhibited a synergistic effect against Staphylococcus aureus (n=20/20) and Enterococcus faecalis (n=13/18), and meanwhile exhibited a mostly additive effect against Staphylococcus intermedius (n=12/20), Streptococcus agalactiae (n=10/10), Streptococcus dysgalactiae (n=18/18), and Escherichia coli (n=14/18). There were no indifferent or antagonistic effects between CEZ and nisin A. In the time-kill assay, the CEZ-nisin A combination at 0.5 × or 1 × minimum inhibitory concentration exhibited synergistic reduction of bacterial growth by over 3 log10 colony forming units per ml relative to that observed with either antimicrobial substance alone. These results suggest that the CEZ-nisin A combination can be used for developing an intramammary infusion for mastitis treatment, with lower antibiotic concentrations than normal.

Enterococcus faecium QU 50, a novel thermophilic L-lactic acid (LA) producing strain, was used in this study to ferment sugar mixtures into LA. Under the optimal fermentation conditions (50 °C, pH 6.5), strain QU 50 could ferment both mixed glucose/xylose sugars with relaxed CCR and mixed cellobiose/xylose sugars simultaneously without CCR to produce homo L-LA. The activity of enzymes related to xylose metabolism was also investigated. In the cells grown in a medium containing cellobiose/xylose, the activity of xylose isomerase and xylulose kinase, was 3.22 and 1.91 times higher, respectively, as compared to that of cells grown in a glucose/xylose medium. Strain QU 50 produced 70.8 g L−1 of L-LA with a yield of 1.04 g g−1 and a productivity of 2.95 g L−1 h−1 from simulated energy cane hydrolysate in batch fermentation. Immobilisation of strain QU 50 improved the operational stability of open repeated fermentation (three cycles), resulting in 61.1–64.3 g L−1 of L-LA with a yield of 1.01–1.02 g g−1 and a productivity of 3.22–3.82 g L−1 h−1. Thus, an efficient and cost-effective fermentation system was successfully established for L-LA production from sugar mixtures.

In the present study, we investigated the glucose-decreasing action of lactic acid bacteria (LAB). The finding of this study could be helpful for people in controlling their blood sugar levels. The LAB candidate was isolated from a Japanese fermented food and identified as Pediococcus pentosaceus by an analysis of its genome sequence. Postprandial blood glucose elevation was investigated using oral starch tolerance tests in mice. Normal mice were fed starch and lyophilized cells of P. pentosaceus QU 19 at the same time. Even without pre-administration of P. pentosaceus QU 19, elevation of the blood glucose level was significantly suppressed by the intake of P. pentosaceus QU 19 at the same time as oral administration of starch. According to the results for its survival in simulated digestive juice and the reduction of blood glucose level in mice, P. pentosaceus QU 19 has potential hypoglycemic activity. In vitro measurements revealed that the glucose-decreasing action of P. pentosaceus QU 19 is probably caused by the glucose assimilation of the strain, not the inhibition of carbohydrate-splitting enzymes which has been reported for other LABs previously. These findings indicate that specific strains of LAB, especially P. pentosaceus QU 19, and foods fermented by LAB may be beneficial for people who must manage glucose ingestion.

Herein, we report the complete genome sequence of Enterococcus faecium QU50, isolated from Egyptian soil and exhibiting intermediate susceptibility to vancomycin. The genome contains a 2,535,796-bp circular chromosome and two plasmids of 196,595 bp and 17,267 bp., Herein, we report the complete genome sequence of Enterococcus faecium QU50, isolated from Egyptian soil and exhibiting intermediate susceptibility to vancomycin. The genome contains a 2,535,796-bp circular chromosome and two plasmids of 196,595 bp and 17,267 bp. IS1062-like sequences were not found.

Phosphoketolase (PK) is responsible for heterolactic fermentation; however, the PK gene of Enterococcus mundtii QU 25, xfpA, is transcribed constitutively, even under homolactic fermentation conditions. In order to deduce the regulatory mechanisms of PK activity in QU 25, XfpA levels in QU 25 cells under hetero- and homolactic fermentation conditions were tested using western blotting. The results showed that the XfpA protein expression was similar under both conditions and that the expression products formed complexes, most likely homodimers, indicating that the regulation of PK activity is downstream of translation.

Two-component systems (TCSs) are regulatory systems in bacteria that play important roles in sensing and adapting to the environment. In this study, we systematically evaluated the roles of TCSs in the susceptibility of the group A Streptococcus (GAS; Streptococcus pyogenes ) SF370 strain to several types of lantibiotics. Using individual TCS deletion mutants, we found that the deletion of srtRK (spy_1081–spy_1082) in SF370 increased the susceptibility to nisin A, which is produced by Lactococcus lactis ATCC 11454, but susceptibility to other types of lantibiotics (nukacin ISK-1, produced by Staphylococcus warneri , and staphylococcin C55, produced by Staphylococcus aureus ) was not altered in the TCS mutants tested. The expression of srtFEG (spy_1085 to spy_1087), which is located downstream of srtRK and is homologous to ABC transporters, was increased in response to nisin A. However, srtEFG expression was not induced by nisin A in the srtRK mutant. The inactivation of srtFEG increased the susceptibility to nisin A. These results suggest that SrtRK controls SrtFEG expression to alter the susceptibility to nisin A. Further experiments showed that SrtRK is required for coexistence with L. lactis ATCC 11454, which produces nisin A. Our results elucidate the important roles of S. pyogenes TCSs in the interactions between different bacterial species, including bacteriocin-producing bacteria. IMPORTANCE In this study, we focused on the association of TCSs with susceptibility to bacteriocins in S. pyogenes SF370, which has no ability to produce bacteriocins, and reported two major new findings. We demonstrated that the SrtRK TCS is related to susceptibility to nisin A by controlling the ABC transporter SrtFEG. We also showed that S. pyogenes SrtRK is important for survival when the bacteria are cocultured with nisin A-producing Lactococcus lactis . This report highlights the roles of TCSs in the colocalization of bacteriocin-producing bacteria and non-bacteriocin-producing bacteria. Our findings provide new insights into the function of TCSs in S. pyogenes .