Synbiotics, the combination of probiotics and prebiotics, are thought to be a pragmatic approach for the treatment of various diseases, including inflammatory bowel disease (IBD). The synergistic therapeutic effects of probiotics and prebiotics remain underexplored. Clostridium tyrobutyricum, a short-chain fatty acid (SCFA) producer, has been recognized as a promising probiotic candidate that can offer health benefits. In this study, the treatment effects of synbiotics containing C. tyrobutyricum and chitooligosaccharides (COSs) on IBD were evaluated. The results indicated that the synbiotic supplement effectively relieved inflammation and restored intestinal barrier function. Additionally, the synbiotic supplement could contribute to the elimination of reactive oxygen species (ROS) and improve the production of SCFAs through the SCFAs-producer of C. tyrobutyricum. Furthermore, such the synbiotic could also regulate the composition of gut microbiota. These findings underscore the potential of C. tyrobutyricum and COSs as valuable living biotherapeutics for the treatment of intestinal-related diseases.

Ethyl hexanoate and ethyl butyrate are indispensable flavor metabolites in strong-flavor Baijiu (SFB), but batch production instability in fermenting grains can reduce the quality of distilled Baijiu. Biofortification of the fermentation process by designing a targeted microbial collaboration pattern is an effective method to stabilize the quality of Baijiu. In this study, we explored the metabolism under co-culture liquid fermentation with Clostridium tyrobutyricum DB041 and Saccharomyces cerevisiae YS219 and investigated the effects of inoculation with two functional microorganisms on physicochemical factors, flavor metabolites, and microbial communities in solid-state simulated fermentation of SFB for the first time. The headspace solid-phase microextraction-gas chromatography-mass spectrometry results showed that ethyl butyrate and ethyl hexanoate significantly increased in fermented grain. High-throughput sequencing analysis showed that Pediococcus, Lactobacillus, Weissella, Clostridium_sensu_stricto_12, and Saccharomyces emerged as the dominant microorganisms at the end of fermentation. Co-occurrence analysis showed that ethyl hexanoate and ethyl butyrate were significantly correlated (|r| > 0.5, P < 0.05) with a cluster of interactions dominated by lactic acid bacteria (Pediococcus, Lactobacillus, Weissella, and Lactococcus), which was driven by the functional C. tyrobutyricum and S. cerevisiae. Mantel test showed that moisture and reducing sugars were the main physicochemical factor affecting microbial collaboration (|r| > 0.7, P < 0.05). Taken together, the collaborative microbial pattern of inoculation with C. tyrobutyricum and S. cerevisiae showed positive results in enhancing typical flavor metabolites and the synergistic effects of microorganisms in SFB.

Enumeration of endospores of butyric acid-forming clostridia in cheese milk is an essential part of milk quality monitoring for cheese producers to avoid late blowing, severe spoilage caused by clostridia during ripening. However, due to the lack of an internationally standardized method, different methods are used and it is important to consider how the choice of method affects the results. This is particularly relevant when clostridial spore counts in milk are considered for quality payments. The aim of this study was to evaluate the specificity of the AMP-6000 method for the enumeration of endospores of cheese spoiling clostridia in milk. First, to assess the prevalence of Clostridium diversity and to determine potential non-target species, we identified isolates from positive reactions of the AMP-6000 method used to quantify clostridial endospores in raw milk and teat skin samples by MALDI-TOF MS. Based on these results, a strain library was designed to evaluate method inclusivity and exclusivity using pure cultures of target and non-target strains according to ISO 16140-2:2016. Most target Clostridium tyrobutyricum strains, as well as all tested C. butyricum and C. sporogenes strains were inclusive. However, C. beijerinckii may be underestimated as only some strains gave positive results. All non-target strains of bacilli and lysinibacilli, but not all paenibacilli, were confirmed to be exclusive. This study provides performance data to better understand the results of microbiological enumeration of butyric acid-forming clostridia in milk and serves as a basis for future methodological considerations and improvements.

Microbial conversion of agri-food waste to valuable compounds offers a sustainable route to develop the bioeconomy and contribute to sustainable biorefinery. Clostridium tyrobutyricum displays a series of native traits suitable for high productivity conversion of agri-food waste, which make it a promising host for the production of various compounds, such as the short-chain fatty acids and their derivative esters products. In this study, a butanol synthetic pathway was constructed in C. tyrobutyricum, and then efficient butyl butyrate production through in situ esterification was achieved by the supplementation of lipase into the fermentation. The butyryl-CoA/acyl-CoA transferase (cat1) was overexpressed to balance the ratio between precursors butyrate and butanol. Then, a suitable fermentation medium for butyl butyrate production was obtained with xylose as the sole carbon source and shrimp shell waste as the sole nitrogen source. Ultimately, 5.9 g/L of butyl butyrate with a selectivity of 100%, and a productivity of 0.03 g/L·h was achieved under xylose and shrimp shell waste with batch fermentation in a 5 L bioreactor. Transcriptome analyses exhibited an increase in the expression of genes related to the xylose metabolism, nitrogen metabolism, and amino acid metabolism and transport, which reveal the mechanism for the synergistic utilization of xylose and shrimp shell waste. This study presents a novel approach for utilizing xylose and shrimp shell waste to produce butyl butyrate by using an anaerobic fermentative platform based on C. tyrobutyricum. This innovative fermentation medium could save the cost of nitrogen sources (~97%) and open up possibilities for converting agri-food waste into other high-value products.

Clostridium tyrobutyricum is an anaerobe known for its ability to produce short-chain fatty acids, alcohols, and esters. We aimed to develop inducible promoters for fine-tuning gene expression in C. tyrobutyricum. Synthetic inducible promoters were created by employing an Escherichia coli lac operator to regulate the thiolase promoter (PCathl) from Clostridium acetobutylicum, with the best one (LacI-Pto4s) showing a 5.86-fold dynamic range with isopropyl β- d-thiogalactoside (IPTG) induction. A LT-Pt7 system with a dynamic range of 11.6-fold was then created by combining LacI-Pto4s with a T7 expression system composing of RNA polymerase (T7RNAP) and Pt7lac promoter. Furthermore, two inducible expression systems BgaR-PbgaLA and BgaR-PbgaLB with a dynamic range of ~40-fold were developed by optimizing a lactose-inducible expression system from Clostridium perfringens with modified 5\' untranslated region (5\' UTR) and ribosome-binding site (RBS). BgaR-PbgaLB was then used to regulate the expressions of a bifunctional aldehyde/alcohol dehydrogenase encoded by adhE2 and butyryl-CoA/acetate Co-A transferase encoded by cat1 in C. tyrobutyricum wild type and Δcat1::adhE2, respectively, demonstrating its efficient inducible gene regulation. The regulated cat1 expression also confirmed that the Cat1-catalyzed reaction was responsible for acetate assimilation in C. tyrobutyricum. The inducible promoters offer new tools for tuning gene expression in C. tyrobutyricum for industrial applications.

The genus Clostridium is a large and diverse group of species that can cause food spoilage, including late blowing defect (LBD) in cheese. In this study, we investigated the taxonomic status of strain FAM25158 isolated from Emmental cheese with LBD using a polyphasic taxonomic and comparative genomic approach. A 16S rRNA gene sequence phylogeny suggested affiliation to the Clostridium sensu stricto cluster, with Clostridium tyrobutyricum DSM 2637T being the closest related type strain (99.16% sequence similarity). Average Nucleotide Identity (ANI) analysis revealed that strain FAM25158 is at the species threshold with C. tyrobutyricum, with ANI values ranging from 94.70 to 95.26%, while the digital DNA-DNA hybridization values were below the recommended threshold, suggesting that FAM25158 is significantly different from C. tyrobutyricum at the genomic level. Moreover, comparative genomic analysis between FAM25158 and its four closest C. tyrobutyricum relatives revealed a diversity of metabolic pathways, with FAM25158 differing from other C. tyrobutyricum strains by the presence of genes such as scrA, srcB, and scrK, responsible for sucrose utilization, and the absence of many important functional genes associated with cold and osmolality adaptation, which was further supported by phenotypic analyses. Surprisingly, strain FAM25158 exhibited unique physiologic traits, such as an optimal growth temperature of 30°C, in contrast to its closest relatives, C. tyrobutyricum species with an optimal growth temperature of 37°C. Additionally, the growth of FAM25158 was inhibited at NaCl concentrations higher than 0.5%, a remarkable observation considering its origin from cheese. While the results of this study provide novel information on the genetic content of strain FAM25158, the relationship between its genetic content and the observed phenotype remains a topic requiring further investigation.

Obesity and its complications constitute a main threat to global human health. The purpose of this investigation was to explore the influences of Clostridium tyrobutyricum (Ct) on lipid metabolism, intestinal barrier function, and intestinal microbiome in obese mice induced by a high-fat diet (HFD). After establishing the obesity model, 107 CFU/mL and 108 CFU/mL C. tyrobutyricum were used to intervene in HFD-fed mice by gavage for six weeks, and indexes related to obesity were measured. In the liver of HFD-fed mice, the results revealed that C. tyrobutyricum reduced liver weight and the levels of triglyceride (TG), total cholesterol (TC), and nonesterified fatty acid (NEFA), along with decreasing red lipid droplets and fat vacuoles. After C. tyrobutyricum intervention, the mRNA expression of peroxisome proliferator-activated receptor-γ (PPARγ) was downregulated, and AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor-α (PPARα), adipose triglyceride lipase (ATGL), and hormone-sensitive lipase (HSL) were upregulated in the liver. Additionally, C. tyrobutyricum alleviated intestinal morphology injury caused by HFD, decreased the expression of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), and IL-1β in the colon, and upregulated tight junction protein expression. In addition, 16S rRNA sequencing revealed that C. tyrobutyricum increases the diversity of intestinal microbiota. Overall, C. tyrobutyricum improved HFD-induced lipid metabolism disorders, preserved the intestinal barrier\'s integrity, and modulated the structure of the intestinal microbiome. These findings provide a novel insight into the role of C. tyrobutyricum as a probiotic in regulating lipid metabolism.

Phenolic compounds (PCs) generated during pretreatment of lignocellulosic biomass severely hinder the biorefinery by Clostridia. As a hyperbutyrate-producing strain, Clostridium tyrobutyricum has excellent tolerance to PCs, but its tolerance mechanism is poorly understood. In this study, a comprehensive transcriptome analysis was applied to elucidate the response of C. tyrobutyricum to four typical PCs. The findings revealed that the expression levels of genes associated with PC reduction, HSPs, and membrane transport were significantly altered under PC stress. Due to PCs being reduced to low-toxicity alcohols/acids by C. tyrobutyricum, enhancing the reduction of PCs by overexpressing reductase genes could enhance the strain\'s tolerance to PCs. Under 1.0 g/L p-coumaric acid stress, compared with the wild-type strain, ATCC 25755/sdr1 exhibited a 31.2 % increase in butyrate production and a 38.5 % increase in productivity. These insights contribute to the construction of PC-tolerant Clostridia, which holds promise for improving biofuel and chemical production from lignocellulosic biomass.

Clostridium tyrobutyricum strain NRRL B-67062 was previously isolated from an ethanol production facility and shown to produce high yields of butyric acid. In addition, the cell-free supernatant of the fermentation broth from NRRL B-67062 contained antibacterial activity against certain Gram-positive bacteria. To determine the source of this antibacterial activity, we report the genome and genome mining of this strain. The complete genome of NRRL B-67062 showed one circular chromosome of 3,242,608 nucleotides, 3114 predicted coding sequences, 79 RNA genes, and a G+C content of 31.0%. Analyses of the genome data for genes potentially associated with antimicrobial features were sought after by using BAGEL-4 and anti-SMASH databases. Among the leads, a polypeptide of 66 amino acids (PEG 446) contains the DUF4177 domain, which is an uncharacterized highly conserved domain (pfam13783). The cloning and expression of the peg446 gene in Escherichia coli and Bacillus subtilis confirmed the antibacterial property against Lactococcus lactis LM 0230, Limosilactobacillus fermentum 0315-25, and Listeria innocua NRRL B-33088 by gel overlay and well diffusion assays. Molecular modeling suggested that PEG 446 contains one alpha-helix and three anti-parallel short beta-sheets. These results will aid further functional studies and facilitate simultaneously fermentative production of both butyric acid and a putative bacteriocin from agricultural waste and lignocellulosic biomass materials.

The qualified presumption of safety (QPS) process was developed to provide a safety assessment approach for microorganisms intended for use in food or feed chains. The QPS approach is based on an assessment of published data for each taxonomic unit (TU), with respect to its taxonomic identity, the body of relevant knowledge and safety concerns. Safety concerns identified for a TU are, where possible, confirmed at the species/strain or product level and reflected by \'qualifications\'. In the period covered by this Statement, no new information was found that would change the status of previously recommended QPS TUs. Of 71 microorganisms notified to EFSA between April and September 2023 (30 as feed additives, 22 as food enzymes or additives, 7 as novel foods and 12 from plant protection products [PPP]), 61 were not evaluated because: 26 were filamentous fungi, 1 was Enterococcus faecium, 5 were Escherichia coli, 1 was a bacteriophage (all excluded from the QPS evaluation) and 28 were TUs that already have a QPS status. The other 10 notifications belonged to 9 TUs which were evaluated for a possible QPS status: Ensifer adhaerens and Heyndrickxia faecalis did not get the QPS recommendation due to the limited body of knowledge about their occurrence in the food and/or feed chains and Burkholderia ubonensis also due to its ability to generate biologically active compounds with antimicrobial activity; Klebsiella pneumoniae, Serratia marcescens and Pseudomonas putida due to safety concerns. K. pneumoniae is excluded from future QPS evaluations. Chlamydomonas reinhardtii is recommended for QPS status with the qualification \'for production purposes only\'; Clostridium tyrobutyricum is recommended for QPS status with the qualification \'absence of genetic determinants for toxigenic activity\'; Candida oleophila has been added as a synonym of Yarrowia lipolytica. The Panel clarifies the extension of the QPS status for genetically modified strains.