Reducing Immunoreactivity of Gluten Peptides by Probiotic Lactic Acid Bacteria for Dietary Management of Gluten-Related Diseases.

Immunoreactive gluten peptides that are not digested by peptidases produced by humans can trigger celiac disease, allergy and non-celiac gluten hypersensitivity. The aim of this study was to evaluate the ability of selected probiotic strains to hydrolyze immunoreactive gliadin peptides and to identify peptidase-encoding genes in the genomes of the most efficient strains. Residual gliadin immunoreactivity was measured after one- or two-step hydrolysis using commercial enzymes and bacterial peptidase preparations by G12 and R5 immunoenzymatic assays. Peptidase preparations from Lacticaseibacillus casei LC130, Lacticaseibacillus paracasei LPC100 and Streptococcus thermophilus ST250 strains significantly reduced the immunoreactivity of gliadin peptides, including 33-mer, and this effect was markedly higher when a mixture of these strains was used. In silico genome analyses of L. casei LC130 and L. paracasei LPC100 revealed the presence of genes encoding peptidases with the potential to hydrolyze bonds in proline-rich peptides. This suggests that L. casei LC130, L. paracasei LPC100 and S. thermophilus ST250, especially when used as a mixture, have the ability to hydrolyze immunoreactive gliadin peptides and could be administered to patients on a restricted gluten-free diet to help treat gluten-related diseases.

Reducing transmission of high-risk antibiotic resistance genes in whole-crop corn silage through lactic acid bacteria inoculation and increasing ensiling temperature.

The microbial hosts of antibiotic resistance genes (ARGs) found epiphytically on plant materials could grow and flourish during silage fermentation. This study employed metagenomic analysis and elucidated the occurrence and transmission mechanisms of ARGs and their microbial hosts in whole-crop corn silage inoculated with homofermentative strain Lactiplantibacillus plantarum or heterofermentative strain Lentilactobacillus buchneri ensiled under different temperature (20 and 30 °C). The results revealed that the corn silage was dominated by Lactobacillus, Leuconostoc, Lentilactobacillus, and Latilactobacillus. Both the ensiling temperature and inoculation had greatly modified the silage microbiota. However, regardless of the ensiling temperature, L. buchneri had significantly higher ARGs, while it only exhibited significantly higher mobile genetic elements (MGEs) in low temperature treatments. The microbial community of the corn silage hosted highly diverse form of ARGs, which were primarily MacB, RanA, bcrA, msbA, TetA (58), and TetT and mainly corresponded to macrolides and tetracyclines drug classes. Plasmids were identified as the most abundant MGEs with significant correlation with some high-risk ARGs (tetM, TolC, mdtH, and NorA), and their abundances have been reduced by ensiling process. Furthermore, higher temperature and L. buchneri reduced abundances of high-risk ARGs by modifying their hosts and reduced their transmission in the silage. Therefore, ensiling, L. buchneri inoculation and higher storage temperature could improve the biosafety of corn silage.

The oral cavity and intestinal microbiome in children with functional constipation.

Constipation is a widespread problem in paediatric practice, affecting almost 30% of children. One of the key causal factors of constipation may be disturbances in the homeostasis of the gastrointestinal microbiome. The aim of the study was to determine whether the oral and fecal microbiomes differ between children with and without constipation. A total of 91 children over three years of age were included in the study. Of these, 57 were qualified to a group with constipation, and 34 to a group without. The saliva and stool microbiomes were evaluated using 16S rRNA gene amplicon sequencing. Functional constipation was associated with characteristic bacterial taxa in the fecal microbiota. Statistically significant differences were found at the family level: Burkholderiaceae (q = 0.047), Christensenellaceae (q = 0.047), Chlostridiaceae (q = 0.047) were significantly less abundant in the constipation group, while the Tannerellaceae (q = 0.007) were more abundant. At the genus level, the significant differences were observed for rare genera, including Christensenellaceae r-7 (q = 2.88 × 10-2), Fusicatenibacter (q = 2.88 × 10-2), Parabacteroides (q = 1.63 × 10-2), Romboutsia (q = 3.19 × 10-2) and Subdoligranulum (q = 1.17 × 10-2). All of them were less abundant in children with constipation. With the exception of significant taxonomic changes affecting only feces, no differences were found in the alpha and beta diversity of feces and saliva. Children with functional constipation demonstrated significant differences in the abundance of specific bacteria in the stool microbiome compared to healthy children. It is possible that the rare genera identified in our study which were less abundant in the constipated patients (Christensellaceae r-7, Fusicatenibacter, Parabacteroides, Romboutsia and Subdoligranulum) may play a role in protection against constipation. No significant differences were observed between the two groups with regard to the saliva microbiome.

Lactic acid bacteria reduce bacterial diarrhea in rabbits via enhancing immune function and restoring intestinal microbiota homeostasis.

BACKGROUND: Numerous previous reports have demonstrated the efficacy of Lactic acid bacteria (LAB) in promoting growth and preventing disease in animals. In this study, Enterococcus faecium ZJUIDS-R1 and Ligilactobaciiius animalis ZJUIDS-R2 were isolated from the feces of healthy rabbits, and both strains showed good probiotic properties in vitro. Two strains (108CFU/ml/kg/day) were fed to weaned rabbits for 21 days, after which specific bacterial infection was induced to investigate the effects of the strains on bacterial diarrhea in the rabbits. RESULTS: Our data showed that Enterococcus faecium ZJUIDS-R1 and Ligilactobaciiius animalis ZJUIDS-R2 interventions reduced the incidence of diarrhea and systemic inflammatory response, alleviated intestinal damage and increased antibody levels in animals. In addition, Enterococcus faecium ZJUIDS-R1 restored the flora abundance of Ruminococcaceae1. Ligilactobaciiius animalis ZJUIDS-R2 up-regulated the flora abundance of Adlercreutzia and Candidatus Saccharimonas. Both down-regulated the flora abundance of Shuttleworthia and Barnesiella to restore intestinal flora balance, thereby increasing intestinal short-chain fatty acid content. CONCLUSIONS: These findings suggest that Enterococcus faecium ZJUIDS-R1 and Ligilactobaciiius animalis ZJUIDS-R2 were able to improve intestinal immunity, produce organic acids and regulate the balance of intestinal flora to enhance disease resistance and alleviate diarrhea-related diseases in weanling rabbits.

Brazilian indigenous nonstarter lactic acid bacteria enhance the diversification of volatile compounds in short-aged cheese.

There is growing interest in using autochthonous lactic acid bacteria (LAB) that provide unique sensory characteristics to dairy products without affecting their safety and quality. This work studied the capacity of three Brazilian indigenous nonstarter LABs (NSLAB) to produce biogenic amines (BAs) and evaluated their effect on the volatile organic compounds (VOCs), microbial LAB communities, and physicochemical profile of short-aged cheese. Initially, the strain's potential for biosynthesis of BAs was assessed by PCR and in vitro assays. Then, a pilot-scale cheese was produced, including the NSLAB, and the microbial and VOC profiles were analyzed after 25 and 45 days of ripening. As a results, the strains did not present genes related to relevant BAs and did not produce them in vitro. During cheese ripening, the Lactococci counts were reduced, probably in the production of alcohols and acid compounds by the NSLAB. Each strain produces a unique VOC profile that changes over the ripening time without the main VOCs related to rancid or old cheese. Particularly, the use of the strain Lacticaseibacillus. paracasei ItalPN16 resulted in production of ester compounds with fruity notes. Thus, indigenous NSLAB could be a valuable tool for the enhancement and diversification of flavor in short-aged cheese.

New Genetic Determinants for qPCR Identification and the Enumeration of Selected Lactic Acid Bacteria in Raw-Milk Cheese.

Lactic acid bacteria (LAB) play an important role in the ripening of cheeses and contribute to the development of the desired profile of aroma and flavor compounds. Therefore, it is very important to monitor the dynamics of bacterial proliferation in order to obtain an accurate and reliable number of their cells at each stage of cheese ripening. This work aimed to identify and conduct a quantitative assessment of the selected species of autochthonous lactic acid bacteria from raw cow's milk cheese by the development of primers and probe pairs based on the uniqueness of the genetic determinants with which the target microorganisms can be identified. For that purpose, we applied real-time quantitative PCR (qPCR) protocols to quantify Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and Lactococcus lactis subsp. cremoris cells in cheese directly after production and over three-month and six-month ripening periods. While L. lactis subsp. cremoris shows good acidification ability and the ability to produce antimicrobial compounds, L. delbrueckii subsp. bulgaricus has good proteolytic ability and produces exo-polysaccharides, and S. thermophilus takes part in the formation of the diacetyl flavor compound by metabolizing citrate to develop aroma, they all play an important role in the cheese ripening. The proposed qPCR protocols are very sensitive and reliable methods for a precise enumeration of L. delbrueckii subsp. bulgaricus, S. thermophilus, and L. lactis subsp. cremoris in cheese samples.

Bumble bee microbiota shows temporal succession and increase of lactic acid bacteria when exposed to outdoor environments.

Question: The large earth bumble bee (Bombus terrestris) maintains a social core gut-microbiota, similar as known from the honey bee, which plays an important role for host health and resistance. Experiments under laboratory conditions with commercial hives are limited to vertically transmitted microbes and neglect influences of environmental factors or external acquisition of microbes. Various environmental and landscape-level factors may have an impact on the gut-microbiota of pollinating insects, with consequences for pollinator health and fitness in agroecosystems. Still, it is not fully clear whether access to different flower diversities will have a significant influence on the bumble bee microbiota. Here, we tested in a semi-field experiment if the bumble bee microbiota changes over time when exposed to different flower diversities within outdoor flight cages. We used commercial hives to distinguish between vertically and horizontally transmitted bacteria, respectively from the nest environment or the exposed outside environment. Result: The sequential sampling of foraging workers over a period of 35 days indicated a temporal progression of the bumble bee microbiota when placed outside. The microbiota increased in diversity and changed in composition and variability over time. We observed a major increase in relative abundance of the families Lactobacillaceae, Bifidobacteriaceae and Weeksellaceae. In contrast, major core-taxa like Snodgrassella and Gilliamella declined in their relative abundance over time. The genus Lactobacillus showed a high diversity and strain specific turnover, so that only specific ASVs showed an increase over time, while others had a more erratic occurrence pattern. Exposure to different flower diversities had no significant influence on the progression of the bumble bee microbiota. Conclusion: The bumble bee microbiota showed a dynamic temporal succession with distinct compositional changes and diversification over time when placed outdoor. The exposure of bumble bees to environmental conditions, or environmental microbes, increases dissimilarity and changes the gut-community composition. This shows the importance of environmental influences on the temporal dynamic and progression of the bumble bee microbiota.

Biocontrol activities of yeasts or lactic acid bacteria isolated from Robusta coffee against Aspergillus carbonarius growth and ochratoxin A production in vitro.

Biocontrol Agents (BCAs) can be an eco-friendly alternative to fungicides to reduce the contamination with mycotoxigenic fungi on coffee. In the present study, different strains of bacteria and yeasts were isolated from Ivorian Robusta coffee. Their ability to reduce fungal growth and Ochratoxin A (OTA) production during their confrontation against Aspergillus carbonarius was screened on solid media. Some strains were able to reduce growth and OTA production by 85 % and 90 % and were molecularly identified as two yeasts, Rhodosporidiobolus ruineniae and Meyerozyma caribbica. Subsequent tests on liquid media with A. carbonarius or solely with OTA revealed adhesion of R. ruineniae to the mycelium of A. carbonarius through Scanning Electron Microscopy, and an OTA adsorption efficiency of 50 %. For M. caribbica potential degradation of OTA after 24 h incubation was observed. Both yeasts could be potential BCAs good candidates for Ivorian Robusta coffee protection against A. carbonarius and OTA contamination.

In Vitro Lactic Acid Bacteria Anti-Hepatitis B Virus (HBV) Effect and Modulation of the Intestinal Microbiota in Fecal Cultures from HBV-Associated Hepatocellular Carcinoma Patients.

Hepatocellular carcinoma (HCC), being ranked as the top fifth most prevalent cancer globally, poses a significant health challenge, with a considerable mortality rate. Hepatitis B virus (HBV) infection stands as the primary factor contributing to HCC, presenting substantial challenges in its treatment. This study aimed to identify lactic acid bacteria (LAB) with anti-HBV properties and evaluate their impact on the intestinal flora in HBV-associated HCC. Initially, two LAB strains, Levilactobacillus brevis SR52-2 (L. brevis SR52-2) and LeviLactobacillus delbrueckii subsp. bulgaicus Q80 (L. delbrueckii Q80), exhibiting anti-HBV effects, were screened in vitro from a pool of 498 LAB strains through cell experiments, with extracellular expression levels of 0.58 ± 0.05 and 0.65 ± 0.03, respectively. These strains exhibited the capability of inhibiting the expression of HBeAg and HBsAg. Subsequent in vitro fermentation, conducted under simulated anaerobic conditions mimicking the colon environment, revealed a decrease in pH levels in both the health control (HC) and HCC groups influenced by LAB, with a more pronounced effect observed in the HC group. Additionally, the density of total short-chain fatty acids (SCFAs) significantly increased (p < 0.05) in the HCC group. Analysis of 16S rRNA highlighted differences in the gut microbiota (GM) community structure in cultures treated with L. brevis SR52-2 and L. delbrueckii Q80. Fecal microflora in normal samples exhibited greater diversity compared to HBV-HCC samples. The HCC group treated with LAB showed a significant increase in the abundance of the phyla Firmicutes, Bacteroidetes and Actinobacteria, while Proteobacteria significantly decreased compared to the untreated HCC group after 48 h. In conclusion, the findings indicate that LAB, specifically L. brevis SR52-2 and L. delbrueckii Q80, possessing antiviral properties, contribute to an improvement in gastrointestinal health.

Effect of Fermented Milk Supplemented with Nisin or Plantaricin Q7 on Inflammatory Factors and Gut Microbiota in Mice.

Lactic-acid-bacteria-derived bacteriocins are used as food biological preservatives widely. Little information is available on the impact of bacteriocin intake with food on gut microbiota in vivo. In this study, the effects of fermented milk supplemented with nisin (FM-nisin) or plantaricin Q7 (FM-Q7) from Lactiplantibacillus plantarum Q7 on inflammatory factors and the gut microbiota of mice were investigated. The results showed that FM-nisin or FM-Q7 up-regulated IFN-γ and down-regulated IL-17 and IL-12 in serum significantly. FM-nisin down-regulated TNF-α and IL-10 while FM-Q7 up-regulated them. The results of 16S rRNA gene sequence analysis suggested that the gut microbiome in mice was changed by FM-nisin or FM-Q7. The Firmicutes/Bacteroides ratio was reduced significantly in both groups. It was observed that the volume of Akkermansia_Muciniphila was significantly reduced whereas those of Lachnospiraceae and Ruminococcaceae were increased. The total number of short-chain fatty acids (SCFAs) in the mouse feces of the FM-nisin group and FM-Q7 group was increased. The content of acetic acid was increased while the butyric acid content was decreased significantly. These findings indicated that FM-nisin or FM-Q7 could stimulate the inflammation response and alter gut microbiota and metabolic components in mice. Further in-depth study is needed to determine the impact of FM-nisin or FM-Q7 on the host's health.

Genomic characterization and probiotic potential of lactic acid bacteria isolated from feces of guinea pig (Cavia porcellus).

Background: Presently, there exists a growing interest in mitigating the utilization of antibiotics in response to the challenges emanating from their usage in livestock. A viable alternative strategy encompasses the introduction of live microorganisms recognized as probiotics, exerting advantageous impacts on the immune system and nutritional aspects of the host animals. Native lactic acid bacteria, inherently possessing specific properties and adaptive capabilities tailored to each animal, are deemed optimal contenders for probiotic advancement. Aim: In the current investigation, microorganisms exhibiting probiotic potential were isolated, characterized, and identified from the fecal samples of guinea pigs (Cavia porcellus) belonging to the Peruvian breed. Methods: The lactic acid bacteria isolated on Man, Rogosa, and Sharpe agar underwent Gram staining, catalase testing, proteolytic, amylolytic, and cellulolytic activity assays, low pH tolerance assessment, hemolytic evaluation, antagonism against Salmonella sp., determination of autoaggregation and coaggregation capacity, and genotypic characterization through sequencing of the 16S rRNA gene. Results: A total of 33 lactic acid bacteria were isolated from the feces of 30 guinea pigs, also 10 isolates were selected based on Gram staining and catalase testing. All strains exhibited proteolytic activity, while only one demonstrated amylolytic capability, and none displayed cellulase activity. These bacteria showed higher tolerance to pH 5.0 and, to a lesser extent, to pH 4.0. Furthermore, they exhibited antagonistic activity against Salmonella sp. Only two bacteria demonstrated hemolytic activity, and were subsequently excluded from further evaluations. Subsequent assessments revealed autoaggregation capacities ranging from 4.55% to 23.19%, with a lesser degree of coaggregation with Salmonella sp. ranging from 3.53% to 8.94% for the remaining eight bacterial isolates. Based on these comprehensive tests, five bacteria with notable probiotic potential were identified by molecular assays as Leuconostoc citreum, Enterococcus gallinarum, Exiguobacterium sp., and Lactococcus lactis. Conclusion: The identified bacteria stand out as promising probiotic candidates, deserving further assessment in Peruvian breed guinea pigs. This exploration aims to enhance production outcomes while mitigating the adverse effects induced by pathogenic microorganisms.

Resuscitation of baijiu pit mud bacteria based on Rpf protein of Umezawaea beigongshangensis.

The pit mud in the Baijiu fermentation cellar is an abundant microbial resource that is closely related to the quality of baijiu. However, many naturally existing species might be in a viable but nonculturable (VBNC) state, posing challenges to the isolation and application of functional species. Herein, a previously isolated strain from baijiu mash, Umezawaea beigongshangensis, was found to contain the rpf gene that encodes resuscitation promotion factor (Rpf). Therefore, the gene was cloned and heterologously expressed, and the recombinant protein (Ub-Rpf 2) was purified. Ub-Rpf 2 was found to significantly promote the growth of resuscitated VBNC state Corynebacterium beijingensis and Sphingomonas beigongshangensis. To determine the resuscitation effect of Ub-Rpf 2 on real ecological samples, the protein was supplemented in pit mud for enrichment culture. Results revealed that specific families and genera were enriched in abundance upon Ub-Rpf 2 incubation, including a new family of Symbiobacteraceae and culturable Symbiobacterium genus. Furthermore, 14 species belonging to 12 genera were obtained in Ub-Rpf 2 treated samples, including a suspected novel species. This study lays a foundation for applying Rpf from U. beigongshangensis to exploit microbial resources in baijiu pit mud.

Characterization of the broad-spectrum antibacterial activity of bacteriocin-like inhibitory substance-producing probiotics isolated from fermented foods.

Antimicrobial peptides, such as bacteriocin, produced by probiotics have become a promising novel class of therapeutic agents for treating infectious diseases. Selected lactic acid bacteria (LAB) isolated from fermented foods with probiotic potential were evaluated for various tests, including exopolysaccharide production, antibiotic susceptibility, acid and bile tolerance, antibacterial activity, and cell adhesion and cytotoxicity to gastric cell lines. Six selected LAB strains maintained their high viability under gastrointestinal conditions, produced high exopolysaccharides, showed no or less cytotoxicity, and adhered successfully to gastric cells. Furthermore, three strains, Weissella confusa CYLB30, Lactiplantibacillus plantarum CYLB47, and Limosilactobacillus fermentum CYLB55, demonstrated a strong antibacterial effect against drug-resistant Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enterica serovar Choleraesuis, Enterococcus faecium, and Staphylococcus aureus. Whole genome sequencing was performed on these three strains using the Nanopore platform; then, the results showed that all three strains did not harbor genes related to toxins, superantigens, and acquired antimicrobial resistance, in their genome. The bacteriocin gene cluster was found in CYLB47 genome, but not in CYLB30 and CYLB55 genomes. In SDS-PAGE, the extract of CYLB30 and CYLB47 bacteriocin-like inhibitory substance (BLIS) yielded a single band with a size of less than 10 kDa. These BLIS inhibited the growth and biofilm formation of drug-resistant P. aeruginosa and methicillin-resistant S. aureus (MRSA), causing membrane disruption and inhibiting adhesion ability to human skin HaCaT cells. Moreover, CYLB30 and CYLB47 BLIS rescued the larvae after being infected with P. aeruginosa and MRSA infections. In conclusion, CYLB30 and CYLB47 BLIS may be potential alternative treatment for multidrug-resistant bacteria infections.

Comparative genomics of four lactic acid bacteria identified with Vitek MS (MALDI-TOF) and whole-genome sequencing.

Lactic acid bacteria (LAB) can be used as a probiotic or starter culture in dairy, meat, and vegetable fermentation. Therefore, their isolation and identification are essential. Recent advances in omics technologies and high-throughput sequencing have made the identification and characterization of bacteria. This study firstly aimed to demonstrate the sensitivity of the Vitek MS (MALDI-TOF) system in the identification of lactic acid bacteria and, secondly, to characterize bacteria using various bioinformatics approaches. Probiotic potency-related genes and secondary metabolite biosynthesis gene clusters were examined. The Vitek MS (MALDI-TOF) system was able to identify all of the bacteria at the genus level. According to whole genome sequencing, the bacteria were confirmed to be Lentilactobacillus buchneri, Levilactobacillus brevis, Lactiplantibacillus plantarum, Levilactobacillus namurensis. Bacteria had most of the probiotic potency-related genes, and different toxin-antitoxin systems such as PemIK/MazEF, Hig A/B, YdcE/YdcD, YefM/YoeB. Also, some of the secondary metabolite biosynthesis gene clusters, some toxic metabolite-related genes, and antibiotic resistance-related genes were detected. In addition, Lentilactobacillus buchneri Egmn17 had a type II-A CRISPR/Cas system. Lactiplantibacillus plantarum Gmze16 had a bacteriocin, plantaricin E/F.

Co-culture fermentation by Saccharomycopsis fibuligera and lactic acid bacteria improves bioactivity and aroma profile of wheat bran and the bran-containing Chinese steamed bread.

Co-culture fermentation with yeast and lactic acid bacteria (LAB) exhibits advantages in improving the bioactivity and flavor of wheat bran compared to single-culture fermentation, showing application potentials in bran-containing Chinese steamed bread (CSB). To explore the effects of combination of yeast and different LAB on the bioactivity and flavor of fermented wheat bran, this study analyzed the physicochemical properties, phytate degradation capacity, antioxidant activities, and aroma profile of wheat bran treated with co-culture fermentation by Saccharomycopsis fibuligera and eight different species of LAB. Further, the phenolic acid composition, antioxidant activities, texture properties, aroma profile, and sensory quality of CSB containing fermented wheat bran were evaluated. The results revealed that co-culture fermentation brought about three types of volatile characteristics for wheat bran, including ester-feature, alcohol and acid-feature, and phenol-feature, and the representative strain combinations for these characteristics were S. fibuligera with Limosilactobacillus fermentum, Pediococcus pentosaceus, and Latilactobacillus curvatus, respectively. Co-culture fermentation by S. fibuligera and L. fermentum for 36 h promoted acidification with a phytate degradation rate reaching 51.70 %, and improved the production of volatile ethyl esters with a relative content of 58.47 % in wheat bran. Wheat bran treated with co-culture fermentation by S. fibuligera and L. curvatus for 36 h had high relative content of 4-ethylguaiacol at 52.81 %, and exhibited strong antioxidant activities, with ABTS•+ and DPPH• scavenging rates at 65.87 % and 69.41 %, respectively, and ferric reducing antioxidant power (FRAP) at 37.91 µmol/g. In addition, CSB containing wheat bran treated with co-culture fermentation by S. fibuligera and L. fermentum showed a large specific volume, soft texture, and pleasant aroma, and received high sensory scores. CSB containing wheat bran treated with co-culture fermentation by S. fibuligera and L. curvatus, with high contents of 4-ethylguaiacol, 4-vinylguaiacol, ferulic acid, vanillin, syringaldehyde, and protocatechualdehyde, demonstrated strong antioxidant activities. This study is beneficial to the comprehensive utilization of wheat bran resources and provides novel insights into the enhancement of functions and quality for CSB.

Sugar Utilization-Associated Food-Grade Selection Markers in Lactic Acid Bacteria and Yeast.

This comprehensive review explores the development of food-grade selection markers in lactic acid bacteria and yeast; some of their strains are precisely defined as safe microorganisms and are crucial in the food industry. Lactic acid bacteria, known for their ability to ferment carbohydrates into lactic acid, provide essential nutrients and contribute to immune responses. With its strong fermentation capabilities and rich nutritional profile, yeast finds use in various food products. Genetic engineering in these microorganisms has grown rapidly, enabling the expression of enzymes and secondary products for food production. However, the focus is on ensuring safety, necessitating food-grade selection markers. Traditional antibiotic and heavy metal resistance selection markers pose environmental and health risks, prompting the search for safer alternatives. Complementary selection markers, such as sugar utilization markers, offer a promising solution. These markers use carbohydrates as carbon sources for growth and are associated with the natural metabolism of lactic acid bacteria and yeast. This review discusses the use of specific sugars, such as lactose, melibiose, sucrose, D-xylose, glucosamine, and N-acetylglucosamine, as selection markers, highlighting their advantages and limitations. In summary, this review underscores the importance of food-grade selection markers in genetic engineering and offers insights into their applications, benefits, and challenges, providing valuable information for researchers in the field of food microbiology and biotechnology.

Automated workflow for characterization of bacteriocin production in natural producers Lactococcus lactis and Latilactobacillus sakei.

BACKGROUND: Lactic acid bacteria are commonly used as protective starter cultures in food products. Among their beneficial effects is the production of ribosomally synthesized peptides termed bacteriocins that kill or inhibit food-spoiling bacteria and pathogens, e.g., members of the Listeria species. As new bacteriocins and producer strains are being discovered rapidly, modern automated methods for strain evaluation and bioprocess development are required to accelerate screening and development processes. RESULTS: In this study, we developed an automated workflow for screening and bioprocess optimization for bacteriocin producing lactic acid bacteria, consisting of microcultivation, sample processing and automated antimicrobial activity assay. We implemented sample processing workflows to minimize bacteriocin adsorption to producer cells via addition of Tween 80 and divalent cations to the cultivation media as well as acidification of culture broth prior to cell separation. Moreover, we demonstrated the applicability of the automated workflow to analyze influence of media components such as MES buffer or yeast extract for bacteriocin producers Lactococcus lactis B1629 and Latilactobacillus sakei A1608. CONCLUSIONS: Our automated workflow provides advanced possibilities to accelerate screening and bioprocess optimization for natural bacteriocin producers. Based on its modular concept, adaptations for other strains, bacteriocin products and applications are easily carried out and a unique tool to support bacteriocin research and bioprocess development is provided.

Genomics-based analysis of four porcine-derived lactic acid bacteria strains and their evaluation as potential probiotics.

The search for probiotics and exploration of their functions are crucial for livestock farming. Recently, porcine-derived lactic acid bacteria (LAB) have shown great potential as probiotics. However, research on the evaluation of porcine-derived LAB as potential probiotics through genomics-based analysis is relatively limited. The present study analyzed four porcine-derived LAB strains (Lactobacillus johnsonii L16, Latilactobacillus curvatus ZHA1, Ligilactobacillus salivarius ZSA5 and Ligilactobacillus animalis ZSB1) using genomic techniques and combined with in vitro tests to evaluate their potential as probiotics. The genome sizes of the four strains ranged from 1,897,301 bp to 2,318,470 bp with the GC contents from 33.03 to 41.97%. Pan-genomic analysis and collinearity analysis indicated differences among the genomes of four strains. Carbohydrate active enzymes analysis revealed that L. johnsonii L16 encoded more carbohydrate active enzymes than other strains. KEGG pathway analysis and in vitro tests confirmed that L. johnsonii L16 could utilize a wide range of carbohydrates and had good utilization capacity for each carbohydrate. The four strains had genes related to acid tolerance and were tolerant to low pH, with L. johnsonii L16 showing the greatest tolerance. The four strains contained genes related to bile salt tolerance and were able to tolerate 0.1% bile salt. Four strains had antioxidant related genes and exhibited antioxidant activity in in vitro tests. They contained the genes linked with organic acid biosynthesis and exhibited antibacterial activity against enterotoxigenic Escherichia coli K88 (ETEC K88) and Salmonella 6,7:c:1,5, wherein, L. johnsonii L16 and L. salivarius ZSA5 had gene clusters encoding bacteriocin. Results suggest that genome analysis combined with in vitro tests is an effective approach for evaluating different strains as probiotics. The findings of this study indicate that L. johnsonii L16 has the potential as a probiotic strain among the four strains and provide theoretical basis for the development of probiotics in swine production.

Metabolism mechanism of glycosaminoglycans by the gut microbiota: Bacteroides and lactic acid bacteria: A review.

Glycosaminoglycans (GAGs), as a class of biopolymers, play pivotal roles in various biological metabolisms such as cell signaling, tissue development, cell apoptosis, immune modulation, and growth factor activity. They are mainly present in the colon in free forms, which are essential for maintaining the host's health by regulating the colonization and proliferation of gut microbiota. Therefore, it is important to explain the specific members of the gut microbiota for GAGs' degradation and their enzymatic machinery in vivo. This review provides an outline of GAGs-utilizing entities in the Bacteroides, highlighting their polysaccharide utilization loci (PULs) and the enzymatic machinery involved in chondroitin sulfate (CS) and heparin (Hep)/heparan sulfate (HS). While there are some variations in GAGs' degradation among different genera, we analyze the reputed GAGs' utilization clusters in lactic acid bacteria (LAB), based on recent studies on GAGs' degradation. The enzymatic machinery involved in Hep/HS and CS metabolism within LAB is also discussed. Thus, to elucidate the precise mechanisms utilizing GAGs by diverse gut microbiota will augment our understanding of their effects on human health and contribute to potential therapeutic strategies for diseases.

A Review of N-Heterocycles: Mousy Off-Flavor in Sour Beer.

Beer has over 600 flavor compounds and creates a positive tasting experience with acceptable sensory properties, which are essential for the best consumer experience. Spontaneous and mixed-culture fermentation beers, generally classified as sour beers, are gaining popularity compared to typical lager or ale styles, which have dominated in the USA for the last few decades. Unique and acceptable flavor compounds characterize sour beers, but some unfavorable aspects appear in conjunction. One such unfavorable flavor is called "mousy". This description is usually labeled as an unpleasant odor, identifying spoilage of fermented food and beverages. It is related as having the odor of mouse urine, cereal, corn tortilla chips, or freshly baked sour bread. The main compounds responsible for it are N-heterocyclic compounds: 2-acetyltetrahydropyridine, 2-acetyl-1-pyrroline, and 2-ethyltetrahydropyridine. The most common beverages associated with mousy off-flavor are identified in wines, sour beers, other grain-based beverages, and kombucha, which may contain heterofermentative lactic acid bacteria, acetic acid bacteria, and/or yeast/fungus cultures. In particular, the fungal species Brettanomyces bruxellensis are associated with mousy-off flavor occurrence in fermented beverages matrices. However, many factors for N-heterocycle formation are not well-understood. Currently, the research and development of mixed-cultured beer and non/low alcohol beverages (NABLAB) has increased to obtain the highest quality, sensory, functionality, and most notably safety standards, and also to meet consumers' demand for a balanced sourness in these beverages. This paper introduces mousy off-flavor expression in beers and beverages, which occurs in spontaneous or mixed-culture fermentations, with a focus on sour beers due to common inconsistency aspects in fermentation. We discuss and suggest possible pathways of mousy off-flavor development in the beer matrix, which also apply to other fermented beverages, including non/low alcohol drinks, e.g., kombucha and low/nonalcohol beers. Some precautions and modifications may prevent the occurrence of these off-flavor compounds in the beverage matrix: improving raw material quality, adjusting brewing processes, and using specific strains of yeast and bacteria that are less likely to produce the off-flavor. Conceivably, it is clear that spontaneous and mixed culture fermentation is gaining popularity in industrial, craft, and home brewing. The review discusses important elements to identify and understand metabolic pathways, following the prevention of spoilage targeted to off-flavor compounds development in beers and NABLABs.