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Abstract Chronic endometritis is associated with the imbalance of female reproductive tract microbiota and pathogenic microbial infection. This study aimed to identify the specific changes in the endometrial microbiome in patients with endometritis and to explore how Clostridium tyrobutyricum (C.t) influences the progression of endometritis in mice for further elucidating endometritis pathogenesis. For this purpose, endometrial tissues from 100 participants were collected and divided into positive, weakly positive, and negative groups based on CD138 levels, while endometrial microbiome differences were detected and analyzed using 16S rRNA gene sequencing. Staphylococcus aureus (S. aureus)-induced endometritis mouse model was established, followed by treatment with C.t, and inflammatory response, epithelial barrier, and TLR4/NF-κB pathway were evaluated. Results showed that α- and β-diversity was significantly lower in the positive group compared with the weakly positive or negative groups, where the negative group had more unique operational taxonomic units. The abundance of Proteobacteria was found to be increased, while that of Actinobacteria, Firmicutes, and Bacteroidetes was found to be reduced in the positive group, while the area under the curve value was found to be 0.664. Furthermore, C.t treatment resulted in the alleviation of S. aureus-induced inflammatory response, epithelial barrier damage, and activation of the TLR4/NF-κB pathway in mice. Clinical samples analysis revealed that the diversity and abundance of microbiota were altered in patients with endometritis having positive CD138 levels, while mechanistic investigations revealed C.t alleviated S. aureus-induced endometritis by inactivating TLR4/NF-κB pathway. The findings of this study are envisaged to provide a diagnostic and therapeutic potential of microbiota in endometritis.

Chronic endometritis is associated with the imbalance of female reproductive tract microbiota and pathogenic microbial infection. This study aimed to identify the specific changes in the endometrial microbiome in patients with endometritis and to explore how Clostridium tyrobutyricum (C.t) influences the progression of endometritis in mice for further elucidating endometritis pathogenesis. For this purpose, endometrial tissues from 100 participants were collected and divided into positive, weakly positive, and negative groups based on CD138 levels, while endometrial microbiome differences were detected and analyzed using 16S rRNA gene sequencing. Staphylococcus aureus (S. aureus)-induced endometritis mouse model was established, followed by treatment with C.t, and inflammatory response, epithelial barrier, and TLR4/NF-κB pathway were evaluated. Results showed that α- and β-diversity was significantly lower in the positive group compared with the weakly positive or negative groups, where the negative group had more unique operational taxonomic units. The abundance of Proteobacteria was found to be increased, while that of Actinobacteria, Firmicutes, and Bacteroidetes was found to be reduced in the positive group, while the area under the curve value was found to be 0.664. Furthermore, C.t treatment resulted in the alleviation of S. aureus-induced inflammatory response, epithelial barrier damage, and activation of the TLR4/NF-κB pathway in mice. Clinical samples analysis revealed that the diversity and abundance of microbiota were altered in patients with endometritis having positive CD138 levels, while mechanistic investigations revealed C.t alleviated S. aureus-induced endometritis by inactivating TLR4/NF-κB pathway. The findings of this study are envisaged to provide a diagnostic and therapeutic potential of microbiota in endometritis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Butyric acid, a four-carbon fatty acid, is an important industrial chemical and feedstock. To produce this chemical, a control fermentation was run with a 126.5 g.L−1 glucose concentration in the feed medium. In this medium, the strain produced 44.8 g.L−1 total acid with a productivity of 0.23 g.L−1h−1 and a yield of 0.41 g.g−1. The strain (Clostridium tyrobutyricum ATCC 25755) was also able to utilize glucose and xylose simultaneously with similar fermentation performance. The culture was also used to produce butyric acid from wheat straw hydrolysate (WSH) employing a hot water pretreatment. In a batch system, the strain resulted in a productivity and yield of 0.27 g.L−1h−1 and 0.44 g.g−1, respectively, which was an improvement over the use of glucose or xylose alone or mixtures of both. To improve reactor productivity, a membrane cell recycle bioreactor was used which resulted in a productivity of 1.89 g.L−1h−1. This productivity was 822% of that achieved in the glucose or xylose batch fermentation. Furthermore, a butyric acid recovery method was developed using XAD-4 adsorbent resin. In this system, up to 206.1 g.L−1 of butyric acid was used in the feed and, as a result of the quick adsorption, the residual butyric acid concentration was 29.5 g.L−1. In this experiment, the rate of acid removal of 1059.4 g.L−1h−1 was achieved. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

Abstract 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.

ABSTRACT Necrotizing enterocolitis (NEC) is a challenging gastrointestinal disease that disproportionately affects premature neonates, with high incidence and case-fatality rates. Despite extensive research efforts, the pathogenesis and mechanisms of NEC remain unclear, making it difficult to effectively eradicate. However, it has been established that dysbiosis of gut microbes occurs before the onset of NEC, providing compelling evidence for the potential use of probiotic therapy. As such, we have focused our attention on two probiotics in particular: Clostridium butyricum and Clostridium tyrobutyricum, especially in light of recent breakthroughs that have linked several Clostridia species with NEC. To determine whether C. butyricum and C. tyrobutyricum are pathogenic or probiotic, we conducted a comparison of the phenotypic traits of NEC mice treated with each bacterium. Our results confirm that treatment with C. tyrobutyricum restores intestinal barrier integrity and alleviates inflammatory immune responses associated with NEC. In contrast, treatment with C. butyricum exacerbates intestinal barrier damage and promotes immune disorder, including increased numbers of macrophages, monocytes, and neutrophils in the intestinal lamina propria. Further analysis of the gut microbiome suggests that the positive effects of C. tyrobutyricum treatment are associated with an increase in Akkermansia muciniphila, while C. butyricum treatment decreases the level of A. muciniphila, which accounts for its negative effect on NEC. This study sheds light on the fact that treatment with C. tyrobutyricum, but not C. butyricum, has the potential to protect against NEC development. The opposite effects of these two probiotics on NEC may result from their different modulation of the level of A. muciniphila, a gut microbe that is closely associated with intestinal homeostasis. In summary, by improving the abundance of A. muciniphila to alleviate intestinal inflammation and enhance intestinal barrier integrity, supplementation with C. tyrobutyricum may become a promising therapy for NEC.IMPORTANCEThis study sheds light on that treatment with Clostridium tyrobutyricum but not Clostridium butyricum is entitled to protect against necrotizing enterocolitis (NEC) development potentially. The mechanisms behind the opposite effect on NEC may result in different modulation on the level of Akkermansia muciniphila, which is deeply associated with intestinal homoeostasis. Briefly, through improving the abundance of A. muciniphila to alleviate intestinal inflammation and enhance intestinal barrier integrity, C. tyrobutyricum supplement may become a promising therapy for NEC.

This study compared the differences in the growth and metabolism of Clostridium celerecrescens JSJ-1, a strain able to produce caproic acid, and Clostridium tyrobutyricum D-1, a stain able to produce butyric acid, when cultured separately or co-cultured in different systems (solid medium, and liquid medium with or without use of dialysis bag). Furthermore, this study investigated the effect and mechanism of co-culture with C. tyrobutyricum on caproic acid metabolism in caproic acid-producing bacteria by culturing JSJ-1 alone in the presence of butyric acid or sodium butyrate at different concentrations. The results showed that when co-cultured on a composite solid medium containing glucose in anaerobic conditions at 34 ℃, D-1 grew better than JSJ-1, and could preferentially utilize glucose for growth. The generation time of caproic acid was three days earlier when D-1 and JSJ-1 were co-cultured in a liquid medium than when JSJ-1 was cultured alone under the same conditions, and the concentration of caproic acid was 9.93 and 9.79 g/L on the 12th day in the two cases, respectively. Glucose had a strong inhibitory effect on the production of caproic acid by JSJ-1, which could be relieved by preferential utilization of by D-1 upon co-culture. The co-culture in the liquid medium with and without use of dialysis bag were compared, revealing that whether there was direct contact between JSJ-1 and D-1 had no effect on the yield of caproic acid. By purely culturing JSJ-1 in the presence of butyric acid or sodium butyrate, it was found that butyric acid generated by D-1 at pH 6–6.5 could promote the synthesis of caproic acid by JSJ-1. During co-culture, the preferential utilization of glucose by D-1 could relieve the inhibitory effect of glucose on the production of caproic acid, and butyric acid produced by D-1 could promote the synthesis of caproic acid.

Milk allergy is one of the most common food allergies, affecting 6 % of young children, and β-lactoglobulin (β-LG) is the main milk allergen. Clostridium tyrobutyricum Z816 was selected for the degradation of β-LG, which was successfully reduced by about 90 % using permeabilized bacteria under the optimized conditions. The hydrolyzed peptides were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and analyzed by molecular modeling, which indicated that C. tyrobutyricum Z816 could effectively degrade the antigenic epitopes of β-LG. Finally, the concentration and digestibility of β-LG in actual samples was quantified using enzyme-linked immunosorbent assay (ELISA) and gastrointestinal digestion simulation experiments. The results showed more than 92 % of β-LG in actual samples was hydrolyzed, and the gastric and total digestibility of whey protein isolate (WPI) was improved by 85.96 % and 64.51 %, respectively. Therefore, C. tyrobutyricum Z816 offers an effective method to degrade β-LG and reduce the occurrence of milk allergies, which has great significance for the development of hypoallergenic dairy products.

Abstract As a next‐generation probiotic, Clostridium tyrobutyricum, which is considered obligate anaerobe, has been extensively considered a promising candidate for future use to promote health benefits. The industrial applications were limited by its weak tolerance to oxygen stress during fermentation. Here, we enhanced the oxidative stress tolerance of C. tyrobutyricum L319 by deleting the peroxide response regulator gene perR using an endogenous CRISPR‐Cas system. The mutant strain exhibited greatly improved growth when exposed to oxygen, which also showed a shortened lag phase compared with the control strain. Consistently, a concomitant increase in butyric acid productivity from 0.08 to 0.13 g/L/h in the presence of oxygen was observed. Decreased intracellular ROS level sand NAD+/NADH ratios were also observed in the mutant strain. Furthermore, transcriptome and binding target analysis revealed that genes encoding transcriptional regulators and proteins related to redox balance were upregulated. Changes in carbohydrate, amino acid and purine metabolisms upon oxygen exposure were also observed. Importantly, phylogenomic analysis indicated that PerR may be a universal engineering target in diverse Clostridium species. These findings will contribute to the development of robust C. tyrobutyricum strains for efficient butyric acid production in the food industry.

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.

Butyric acid, a four-carbon fatty acid, is an important industrial chemical and feedstock. To produce this chemical, a control fermentation was run with a 126.5 g.L−1 glucose concentration in the feed medium. In this medium, the strain produced 44.8 g.L−1 total acid with a productivity of 0.23 g.L−1h−1 and a yield of 0.41 g.g−1. The strain (Clostridium tyrobutyricum ATCC 25755) was also able to utilize glucose and xylose simultaneously with similar fermentation performance. The culture was also used to produce butyric acid from wheat straw hydrolysate (WSH) employing a hot water pretreatment. In a batch system, the strain resulted in a productivity and yield of 0.27 g.L−1h−1 and 0.44 g.g−1, respectively, which was an improvement over the use of glucose or xylose alone or mixtures of both. To improve reactor productivity, a membrane cell recycle bioreactor was used which resulted in a productivity of 1.89 g.L−1h−1. This productivity was 822% of that achieved in the glucose or xylose batch fermentation. Furthermore, a butyric acid recovery method was developed using XAD-4 adsorbent resin. In this system, up to 206.1 g.L−1 of butyric acid was used in the feed and, as a result of the quick adsorption, the residual butyric acid concentration was 29.5 g.L−1. In this experiment, the rate of acid removal of 1059.4 g.L−1h−1 was achieved.

Molasses, a sugar industry waste product with low cost and high sugar content, can be used as a high-quality carbon source for microbial fermentation. In this study, molasses was pretreated by means of acid–base treatment combined with 0.6% mass fraction of activated carbon in order for its sugar concentration (78.42 g/L) and composition to meet the needs of Clostridium tyrobutyricum fermentation. C. tyrobutyricum was able to produce butyric acid from the sugars of treated molasses by microbial adaptive laboratory evolution. After 120 h of shake flask fermentation, high-performance liquid chromatography and gas chromatography showed that 46.39% of fructose and 32.56% of glucose in molasses after treatment were converted to 31.52 g/L butyric acid by C. tyrobutyricum. The results showed that the two-step process, combined with microbial adaptive laboratory evolution technology used to hydrolyze and reuse sugar, is an adapted method for the utilization of molasses, a by-product of the sugarcane industry. [ABSTRACT FROM AUTHOR]

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Butyl butyrate has broad applications in foods, cosmetics, solvents, and biofuels. Microbial synthesis of bio-based butyl butyrate has been regarded as a promising approach recently. Herein, we engineered Clostridium tyrobutyricum ATCC 25755 to achieve de novo biosynthesis of butyl butyrate from fermentable sugars. Through introducing the butanol synthetic pathway (enzyme AdhE2), screening alcohol acyltransferases (AATs), adjusting transcription of VAAT and adhE2 (i.e., optimizing promoter), and efficient supplying butyryl-CoA, an excellent engineered strain, named MUV3, was obtained with ability to produce 4.58 g/L butyl butyrate at 25 °C with glucose in serum bottles. More NADH is needed for butyl butyrate synthesis, thus mannitol (the more reduced substrate) was employed to produce butyl butyrate. Ultimately, 62.59 g/L butyl butyrate with a selectivity of 95.97%, and a yield of 0.21 mol/mol was obtained under mannitol with fed-batch fermentation in a 5 L bioreactor, which is the highest butyl butyrate titer reported so far. Altogether, this study presents an anaerobic fermentative platform for de novo biosynthesis of butyl butyrate in one step, which lays the foundation for butyl butyrate biosynthesis from renewable biomass feedstocks. • Development of an upp /5-FU counter-selection system for efficient iterative genome-editing in C. tyrobutyricum. • Establishment of the de novo biosynthetic pathway of butyl butyrate from fermentable sugars in C. tyrobutyricum. • Mannitol as substrate for efficiency butyl butyrate production at a low temperature of 25°C. • Performed fermentation strategy with fed-batch fermentation in 5 L bioreacter for high level of butyl butyrate production. [ABSTRACT FROM AUTHOR]

Introduction: Poor quality silage can derive from the presence of deleterious microorganisms such as clostridia. Their dissemination along the food chain, especially in milk, causes issues such as the cheese late-blowing defect, particularly triggered by Clostridium tyrobutyricum. The scope of our study was to determine the C. tyrobutyricum occurrence in three different farms across four time periods in relation to the animal diets, specifically the Total Mixed Ration (TMR), by using real-time PCR. Methods: For this purpose, molecular-derived data were exploited to optimize a predictive model that simulated the farm conditions favoring the growth of butyric acid bacteria such as C. tyrobutyricum. Results: Our results showed that the originally utilized predictive model strongly underestimated the growth of C. tyrobutyricum in comparison to the molecular data. At the same time, our findings uncovered an additional source of contamination in the TMR related to silage and dietary residues that represent a reservoir of microbial contamination during successive TMR preparation. Based on these findings, the optimization of the model parameters such as growth rate range and the inclusion of the residues in the model, allowed a more accurate prediction of the contamination levels. Therefore, this study revealed that proper hygiene practices such as the removal of silage and TMR residues within the farm environment is essential to control the contamination by C. tyrobutyricum and avoid food waste and economic losses. [ABSTRACT FROM AUTHOR]

Abstract Background Butyric acid, an essential C4 platform chemical, is widely used in food, pharmaceutical, and animal feed industries. Clostridium tyrobutyricum is the most promising microorganism for industrial bio-butyrate production. However, the metabolic driving mechanism for butyrate synthesis was still not profoundly studied. Results This study reports a first-generation genome-scale model (GEM) for C. tyrobutyricum, which provides a comprehensive and systematic analysis for the butyrate synthesis driving mechanisms. Based on the analysis in silico, an energy conversion system, which couples the proton efflux with butyryl-CoA transformation by two redox loops of ferredoxin, could be the main driving force for butyrate synthesis. For verifying the driving mechanism, a hydrogenase (HydA) expression was perturbed by inducible regulation and knockout. The results showed that HydA deficiency significantly improved the intracellular NADH/NAD+ rate, decreased acetate accumulation (63.6% in serum bottle and 58.1% in bioreactor), and improved the yield of butyrate (26.3% in serum bottle and 34.5% in bioreactor). It was in line with the expectation based on the energy conversion coupling driving mechanism. Conclusions This work show that the first-generation GEM and coupling metabolic analysis effectively promoted in-depth understanding of the metabolic driving mechanism in C. tyrobutyricum and provided a new insight for tuning metabolic flux direction in Clostridium chassis cells.

IntroductionPoor quality silage can derive from the presence of deleterious microorganisms such as clostridia. Their dissemination along the food chain, especially in milk, causes issues such as the cheese late-blowing defect, particularly triggered by Clostridium tyrobutyricum. The scope of our study was to determine the C. tyrobutyricum occurrence in three different farms across four time periods in relation to the animal diets, specifically the Total Mixed Ration (TMR), by using real-time PCR.MethodsFor this purpose, molecular-derived data were exploited to optimize a predictive model that simulated the farm conditions favoring the growth of butyric acid bacteria such as C. tyrobutyricum.ResultsOur results showed that the originally utilized predictive model strongly underestimated the growth of C. tyrobutyricum in comparison to the molecular data. At the same time, our findings uncovered an additional source of contamination in the TMR related to silage and dietary residues that represent a reservoir of microbial contamination during successive TMR preparation. Based on these findings, the optimization of the model parameters such as growth rate range and the inclusion of the residues in the model, allowed a more accurate prediction of the contamination levels. Therefore, this study revealed that proper hygiene practices such as the removal of silage and TMR residues within the farm environment is essential to control the contamination by C. tyrobutyricum and avoid food waste and economic losses.

Introduction: Clostridium tyrobutyricum has considerable prospect in the production of organic acids. Globally, refinery final molasses is rich in sugar and reported to have high levels of accumulation and high emission costs, recognized as an excellent substrate for C. tyrobutyricum fermentation, but there is no suitable method available at present. Methods: In this study, an acid-base treatment combined with a new green membrane treatment technology – a dynamic ion-exchange membrane -was used to pretreat refinery final molasses, so that it could be used for C. tyrobutyricum to produce butyric acid. A high-performance liquid chromatography method was established to determine the conversion of a large amount of sucrose into fermentable sugars (71.88 g/L glucose and 38.06 g/L fructose) in the treated refinery final molasses. The process of sequential filtration with 3, 1, and 0.45 µm-pore diameter dynamic ion-exchange membranes could remove impurities, pigments, and harmful substances from the refinery final molasses, and retain the fermentable sugar. Results and discussion: This means that refinery final molasses from the sugar industry could be utilized as a high-value by-product and used for the growth of C. tyrobutyricum, with industrial feasibility and economic competitiveness. Using the treated refinery final molasses as a carbon source, C. tyrobutyricum was screened by the method of adaptive evolution. The strain with butyric acid yielded 52.54 g/L, and the yield of the six carbon sugar was increased from 0.240 to 0.478 g/g. The results showed that combination of C. tyrobutyricum and ionic membrane technology broke through the bottleneck of its utilization of refinery final molasses. This study provided an innovative idea for the C. tyrobutyricum fermentation to produce butyric acid. [ABSTRACT FROM AUTHOR]

Clostridium tyrobutyricum has great potential for bio-based chemicals and biofuel production from mannitol; however, the mannitol metabolic pathway and its metabolic regulatory mechanism have not been elucidated. To this end, the RNA-seq analysis on the mid-log growth phase of C. tyrobutyricum grown on mannitol or xylose was performed. Comparative transcriptome analysis and co-transcription experiment indicated that mtlARFD, which encodes the mannitol-specific IIA component, transcription activator, mannitol-specific IIBC components, and mannitol-1-phosphate 5-dehydrogenase, respectively, formed a polycistronic operon and could be responsible for mannitol uptake and metabolism. In addition, comparative genomic analysis of the mtlARFD organization and the MtlR protein structural domain among various Firmicutes strains identified the putative cre (catabolite-responsive element) sites and conserved phosphorylation sites, but whether the expression of mannitol operon was affected by CcpA- and MtlR-mediated metabolic regulation during mixed substrate fermentation needs to be further verified experimentally. Based on the gene knockout and complementation results, the predicted mannitol operon mtlARFD was confirmed to be responsible for mannitol utilization in C. tyrobutyricum. The results of this study could be used to enhance the mannitol metabolic pathway and explore the potential metabolic regulation mechanism of mannitol during mixed substrate fermentation. [ABSTRACT FROM AUTHOR]

Lignocellulosic biomass is considered the most abundant and renewable feedstock for biobased butyric acid production. However, the furan derivatives (FAs, mainly furfural and 5-hydroxymethylfurfural) generated from the pretreatment of lignocellulose severely inhibit the growth of Clostridium tyrobutyricum, which is the best strain for producing butyric acid. The tolerance mechanism of C. tyrobutyricum to FAs has not been investigated thus far. Here, the response of C. tyrobutyricum ATCC 25755 to FA challenge was first evaluated by using comprehensive transcriptional analysis. The results indicated that the genes related to membrane transport, heat shock proteins, and transcriptional regulation were upregulated under FA stress. However, the expression of almost all genes encoding reductases was not changed, and only the ad gene CTK_RS02625 and the bud gene CTK_RS07810 showed a significant increase of ~ 1.05-fold. Then, the enzyme activity assays indicated that BUD could catalyze the reduction of FAs with relatively low activity and that AD could not participate in the conversion of FAs, indicating that the inability to rapidly convert FAs to their low-toxicity alcohols may be the main reason for the low FA tolerance of C. tyrobutyricum. This research provides insights into the development of FA-tolerant strains, thereby enhancing the bioconversion of lignocellulosic biomass to butyric acid. Key points: • The response of C. tyrobutyricum to FAs was evaluated for the first time. • Genes encoding membrane transporters and heat shock proteins were triggered by FAs. • A lack of effective FA reductases leads to low FA tolerance in C. tyrobutyricum. [ABSTRACT FROM AUTHOR]

Clostridium tyrobutyricum is a promising microbial host for the anaerobic production of bio-based chemicals, especially butyric acid. However, the limited genetic traits of C. tyrobutyricum resulted in its constrained applicability in the food industry due to the absence of publicly available data. We performed a comparative genomic analysis of 13 C. tyrobutyricum strains isolated from different habitats to investigate the adaptation mechanisms and metabolic potentials of the C. tyrobutyricum strains isolated from fermentation environments (FE). The results showed that the genetic diversity of FE-associated C. tyrobutyricum strains was higher than that of strains isolated from non-fermentation environments (NFE). Based on evaluating safety-related genes, FE-associated strains were likely non-pathogenic to humans or animals. Many environmental adaptation-related genes involved in energy production and conversion, phosphotransferase system (PTS), etc., were significantly enriched in FE-associated strains, which was conducive to their survival in the corresponding habitats. The analysis of carbohydrate-active enzymes revealed that the CAZyme categories of glycoside hydrolases (GHs) and carbohydrate esterases (CEs) were found to be significantly enriched in FE-associated strains, which was beneficial for carbohydrate utilization (including starch, cellulose, etc.). Fermentation experiments revealed that strains isolated from the Chinese liquor fermentation environment were capable of decomposing carbohydrates and producing organic acids. Collectively, this study provided insights into the genomic features of C. tyrobutyricum and the theoretical basis for further use, research, and development of these strains. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] • H 2 fermentation process of C. tyrobutyricum was optimized systematically. • The effect of endogenous hydrogenases overexpression on H 2 production was studied. • Introducing sucrose metabolic pathway realized H 2 production from sugarcane molasses. • The highest H 2 production achieved 1098.4 mL with a yield of 248.2 mL/g sugar. Clostridia is the dominant microorganism in dark-fermentative biohydrogen production, but there have been relatively few studies on hydrogen production by Clostridium tyrobutyricum using cheap biomass. In this study, the important process parameters for hydrogen fermentation, such as substrate concentration, pH and temperature, were optimized systematically. Then, the effect of endogenous hydrogenases overexpression on hydrogen production was studied. To achieve economical hydrogen production from cheap substrate sugarcane molasses, the sucrose metabolic pathway was further introduced into C. tyrobutyricum. As a result, the engineered C. tyrobutyricum produced 1098.4 mL hydrogen with a yield of 248.2 mL/g sugar and productivity of 199.6 mL/L/h under the optimum fermentation conditions, which is the highest hydrogen production (910.37 mmol/L) reported so far using molasses. This study demonstrates that C. tyrobutyricum is a promising cell factory for hydrogen production from cheap and renewable biomass resources. [ABSTRACT FROM AUTHOR]

As a pleiotropic regulator, Antibiotic resistant protein B (AbrB) was reported to play important roles in various cellular processes in Bacilli and some Clostridia strains. In Clostridium tyrobutyricum, abrB (CTK_C 00640) was identified to encode AbrB by amino acid sequence alignment and functional domain prediction. The results of abrB deletion or overexpression in C. tyrobutyricum showed that AbrB not only exhibited the reported characteristics such as the negative regulation on sporulation, positive effects on biofilm formation and stress resistance but also exhibited new functions, especially the negative regulation of carbon metabolism. AbrB knockout strain (Ct/ΔabrB) could alleviate glucose-mediated carbon catabolite repression (CCR) and enhance the utilization of xylose compared with the parental strain, resulting in a higher butyrate titer (14.79 g/L vs. 7.91 g/L) and xylose utilization rate (0.19 g/L·h vs. 0.02 g/L·h) from the glucose and xylose mixture. This study confirmed the pleiotropic regulatory function of AbrB in C. tyrobutyricum, suggesting that Ct/ΔabrB was the potential candidate for butyrate production from abundant, renewable lignocellulosic biomass mainly composed of glucose and xylose. [ABSTRACT FROM AUTHOR]

Feedback inhibition is one of the main challenges of fermentative hydrogen production. In this study, the effects of butyrate and acetate on hydrogen production of Clostridium tyrobutyricum were investigated. Substrate consumption and hydrogen production were accelerated when acetate ≤15 g/L was fed. Exogenous acetate induced acetate assimilation and increased the metabolic flux of butyrate synthesis. Exogenous butyrate significantly decreased biomass formation, and slowed substrate consumption and hydrogen production. Metabolic and gene expression analyses showed that butyrate impaired glycolysis and acetate production pathway. The increased butyrate/acetate molar ratio was deemed as a strategy for cells to alleviate pH decrease and reduce the inhibition of undissociated butyric acid. Inhibition model analyses indicated butyrate was the main inhibitor in butyrate-type hydrogen production. This study demonstrates the different feedback effects of acetate and butyrate on hydrogen production of C. tyrobutyricum and provides strategies to relieve the feedback inhibition for efficient hydrogen production. [Display omitted] • Acetate (≤15 g/L) benefited substrate consumption and H 2 productivity. • Exogenous acetate induced acetate assimilation and lactate production. • Butyrate inhibited cell growth, glucose consumption and H 2 productivity. • The molar butyrate/acetate ratio increased in response to exogenous butyrate. • Removing butyrate would avoid feedback inhibition for C. tyrobutyricum. [ABSTRACT FROM AUTHOR]

Rice husk, as a cellulose-rich by-product in agriculture, has been considered as a low-cost substrate for the production of bioethanol and chemicals. In this study, rice husk was pretreated with an alkali, followed by cellulose and β-glucosidase hydrolysis optimized by an orthogonal experiment and response surface methodology (RSM), respectively. Under the optimal treatment conditions, a hydrolysate containing a high reducing sugar yield (77.85%) was obtained from the rice husk. Then, the hydrolysate was used as a carbon substrate for butyric acid production through Clostridium tyrobutyricum Δpta fermentation. Compared to free-cell fermentation, higher concentrations of butyric acid (50.01 g/L vs. 40.8 g/L and 49.03 g/L vs. 27.49 g/L) were observed in immobilized-cell fermentation for the carbon source of glucose and hydrolysate, respectively. A final butyric acid concentration of 16.91 g/L, a yield of 0.31 g/g, and an overall productivity of 0.35 g/L/h from rice husk hydrolysate were obtained in the repeated-fed-batch mode. Taken together, rice husk hydrolysate can be effectively utilized for the bioproduction of butyrate with immobilized-cell fermentation. [ABSTRACT FROM AUTHOR]

In this study, the ability of a novel strain of Clostridium tyrobutyricum NRRL 67062 to produce butyric acid during glucose fermentation was evaluated. The strain was evaluated for substrate and product inhibition in batch experiments using anaerobic tubes. To characterize glucose inhibition, initial glucose concentrations ranging from 60 to 250 g L−1 were used, and it was demonstrated that a glucose concentration of 250 g L−1 exerted strong inhibition on cell growth and fermentation. To evaluate butyric acid inhibition, the culture was challenged with 5–50 g L−1 of butyric acid at an initial pH of 6.5. These experiments were performed without pH control. When challenged with a butyric acid concentration of 50 g L−1, cell growth was slow; however, it produced 8.25 g L−1 of butyric acid. This suggested that the butyric acid tolerance of the culture was 58 g L−1. In a scaled-up batch experiment, which was performed in a 2.5 L fermentor with an initial glucose concentration of 100 g L−1, the pH was controlled at 6.5. In this experiment, the strain produced 57.86 g L−1 of butyric acid and 12.88 g L−1 of acetic acid, thus producing 70.74 g L−1 of total acids with a productivity of 0.69 g·L−1·h−1. A concentration of 70.74 g L−1 of acids equates to a yield of 0.71 g of acid per g consumed glucose. The maximum cell concentration was 3.80 g L−1, which may have been the reason for high productivity in the batch culture. Finally, corn steep liquor (CSL; a commercial nutrient solution) provided greater growth and acid production than the refined medium. [ABSTRACT FROM AUTHOR]

Probiotics contribute a lot to human health and the occurrence of diseases. Correspondingly, probiotics' safety evaluation and probiotic properties have received increasing attention in the food industry and disease treatment. Clostridium tyrobutyricum L319 is a short-chain fatty acid (SCFA)-producing strain isolated from Grana Padano cheese with a blowing defect. Our previous study has shown its safety at the genomic level. This study focused more on the safety evaluation and probiotic properties in vitro. According to the results, this strain has no potential virulence factors or the possibility of antibiotic resistance genes propagation. It also fulfilled several criteria to be used as a probiotic, including significant hydrophobicity under an acidic condition (pH 5.0) and resistance to simulate gastric juice and intestinal juice. Additionally, this strain was found to be tolerant to the harsh conditions of the external environment, including resistance to low (20°C) and high (50°C) temperatures, high salts (3% NaCl), and low pH (pH 5.0). Finally, we found that this strain could ferment prebiotics, such as chito-oligosaccharides, to produce SCFAs. It exhibited excellent growth performance whether using chito-oligosaccharide as a sole carbon source or combining glucose as the mixed carbon source. Furthermore, chito-oligosaccharide and glucose (1:1) mixed carbon sources were the optimal strategy for the production of SCFAs. Our findings demonstrated that this strain might be considered a promising candidate for future use as a probiotic to promote health benefits. [ABSTRACT FROM AUTHOR]

Clostridium tyrobutyricum has been recognized as the main cause of late blowing defects (LBD) in cheese leading to considerable economic losses for the dairy industry. Although differences in spoilage ability among strains of this species have been acknowledged, potential links to the genetic diversity and functional traits remain unknown. In the present study, we aimed to investigate and characterize genomic variation, pan-genomic diversity and key traits of C. tyrobutyricum by comparing the genomes of 28 strains. A comparative genomics analysis revealed an "open" pangenome comprising 9,748 genes and a core genome of 1,179 genes shared by all test strains. Among those core genes, the majority of genes encode proteins related to translation, ribosomal structure and biogenesis, energy production and conversion, and amino acid metabolism. A large part of the accessory genome is composed of sets of unique, strain-specific genes ranging from about 5 to more than 980 genes. Furthermore, functional analysis revealed several strain-specific genes related to replication, recombination and repair, cell wall, membrane and envelope biogenesis, and defense mechanisms that might facilitate survival under stressful environmental conditions. Phylogenomic analysis divided strains into two clades: clade I contained human, mud, and silage isolates, whereas clade II comprised cheese and milk isolates. Notably, these two groups of isolates showed differences in certain hypothetical proteins, transcriptional regulators and ABC transporters involved in resistance to oxidative stress. To the best of our knowledge, this is the first study to provide comparative genomics of C. tyrobutyricum strains related to LBD. Importantly, the findings presented in this study highlight the broad genetic diversity of C. tyrobutyricum , which might help us understand the diversity in spoilage potential of C. tyrobutyricum in cheese and provide some clues for further exploring the gene modules responsible for the spoilage ability of this species. [ABSTRACT FROM AUTHOR]

Clostridium tyrobutyricum has been recognized as the main cause of late blowing defects (LBD) in cheese leading to considerable economic losses for the dairy industry. Although differences in spoilage ability among strains of this species have been acknowledged, potential links to the genetic diversity and functional traits remain unknown. In the present study, we aimed to investigate and characterize genomic variation, pan-genomic diversity and key traits of C. tyrobutyricum by comparing the genomes of 28 strains. A comparative genomics analysis revealed an “open” pangenome comprising 9,748 genes and a core genome of 1,179 genes shared by all test strains. Among those core genes, the majority of genes encode proteins related to translation, ribosomal structure and biogenesis, energy production and conversion, and amino acid metabolism. A large part of the accessory genome is composed of sets of unique, strain-specific genes ranging from about 5 to more than 980 genes. Furthermore, functional analysis revealed several strain-specific genes related to replication, recombination and repair, cell wall, membrane and envelope biogenesis, and defense mechanisms that might facilitate survival under stressful environmental conditions. Phylogenomic analysis divided strains into two clades: clade I contained human, mud, and silage isolates, whereas clade II comprised cheese and milk isolates. Notably, these two groups of isolates showed differences in certain hypothetical proteins, transcriptional regulators and ABC transporters involved in resistance to oxidative stress. To the best of our knowledge, this is the first study to provide comparative genomics of C. tyrobutyricum strains related to LBD. Importantly, the findings presented in this study highlight the broad genetic diversity of C. tyrobutyricum, which might help us understand the diversity in spoilage potential of C. tyrobutyricum in cheese and provide some clues for further exploring the gene modules responsible for the spoilage ability of this species.

Probiotics contribute a lot to human health and the occurrence of diseases. Correspondingly, probiotics’ safety evaluation and probiotic properties have received increasing attention in the food industry and disease treatment. Clostridium tyrobutyricum L319 is a short-chain fatty acid (SCFA)-producing strain isolated from Grana Padano cheese with a blowing defect. Our previous study has shown its safety at the genomic level. This study focused more on the safety evaluation and probiotic properties in vitro. According to the results, this strain has no potential virulence factors or the possibility of antibiotic resistance genes propagation. It also fulfilled several criteria to be used as a probiotic, including significant hydrophobicity under an acidic condition (pH 5.0) and resistance to simulate gastric juice and intestinal juice. Additionally, this strain was found to be tolerant to the harsh conditions of the external environment, including resistance to low (20°C) and high (50°C) temperatures, high salts (3% NaCl), and low pH (pH 5.0). Finally, we found that this strain could ferment prebiotics, such as chito-oligosaccharides, to produce SCFAs. It exhibited excellent growth performance whether using chito-oligosaccharide as a sole carbon source or combining glucose as the mixed carbon source. Furthermore, chito-oligosaccharide and glucose (1:1) mixed carbon sources were the optimal strategy for the production of SCFAs. Our findings demonstrated that this strain might be considered a promising candidate for future use as a probiotic to promote health benefits.

[Display omitted] • Application of cellar mud-sourced acid-producing bacteria and acid-tolerant esterifying yeast. • Co-culture solid-state fermentation of functional microbial to improve key flavor ethyl esters. • Ethyl hexanoate and ethyl butyrate were correlated with a cluster of interactions dominated by lactic acid bacteria. • Positive synergistic relationship between Clostridium tyrobutyricum and Saccharomyces cerevisiae. • Conversion of acids to esters by microorganisms in a high-acid fermentation system by stress reactions. 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. [ABSTRACT FROM AUTHOR]

Aims: The adsorption of lead ions from aqueous solution by macroporous Ca‐alginate‐lignin (MCAL) beads immobilized with Clostridium tyrobutyricum and free strains was evaluated. Methods and Results: The effects of different factors including pH, adsorption time, adsorbent dosage and initial concentration of lead ions were explored. Different characterization methods were used to evaluate the adsorption process of lead ions. Meanwhile, the adsorption kinetics models and adsorption isotherm models were applied. The fitting results showed that the adsorption behaviour of C. tyrobutyricum immobilized in MCAL beads and free strains was better described by the pseudo‐second‐order kinetic model and the adsorption process followed the Langmuir isotherm model. The maximum biosorption of lead ions by C. tyrobutyricum immobilized in MCAL beads and free strains was 144.9 and 106.4 mg/g respectively. Conclusions: The C. tyrobutyricum immobilized in MCAL beads proved to be practicable and had better adsorption effects on lead ions compared with the free strains. Significance and Impact of the Study: The paper demonstrated a new insight and strategy for the effective treatment of lead ions from aqueous solutions by the novel function of C. tyrobutyricum. [ABSTRACT FROM AUTHOR]

The generally accepted concept of the necessity of producing safe foods has indirectly influenced the decision to replace chemical preservatives with natural ones. Bacteriocins, and in particular those synthesized by lactic acid bacteria (LAB) in the food industry, are considered to be their effective replacement. In controlling the growth of microbial pathogens and/or the occurrence of pathogenic bacteria in food, with the permitted nisin and pediocin, a significant antibacterial effect has been shown for most LAB bacteriocins. However, the use of purified bacteriocins as bio preservatives in cheese production is limited. To inhibit the growth of bacteria L. monocytogenes, S. aureus and C. tyrobutyricum in cheese, bacteriocinogenic LAB strains contained in primary, adjunct or protective culture are much more acceptable in cheese production.

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.

Purpose: To optimize the process parameters for the encapsulation of Clostridium tyrobutyricum (Ct) and to determine its in vitro characteristics. Methods: The process parameters, including the concentration of the wall and hardening material, Ct to gelatin ratio and hardening time, were studied by single factor analysis, while optimization was performed by orthogonal experimental design for the encapsulation rate of Ct. Results: Optimal conditions exhibited by orthogonal experimental design at a 92.17 % encapsulation rate with a viable count of 9.61 ± 0.06 lgCFU/g were: 6 % modified starch, 3 % sodium alginate, and 2 % CaCl2 at a Ct to gelatin ratio of 1:1 with a hardening time of 30 min. The survival rates of encapsulated Ct were higher than free Ct in simulated gastric (6.22 %) and intestinal juices (15.55 %). Reduction in viable counts of Ct at 90 °C were higher for free cells (44.76 %) than encapsulated cells (28.09 %) after 30 min of heat treatment. Correspondingly, encapsulation boosted the capacity of Ct to withstand the strong acidic conditions of the stomach and improved the storage properties of Ct. Conclusion: The results suggested that extrusion is a good technique for the encapsulation of Ct, as it enhances the viability of Ct during their transit through the gastrointestinal tract. Furthermore, encapsulation is favorable for Ct if planned for use in formulations where high temperature treatment is required. [ABSTRACT FROM AUTHOR]

As a pleiotropic regulator, Antibiotic resistant protein B (AbrB) was reported to play important roles in various cellular processes in Bacilli and some Clostridia strains. In Clostridium tyrobutyricum, abrB (CTK_C 00640) was identified to encode AbrB by amino acid sequence alignment and functional domain prediction. The results of abrB deletion or overexpression in C. tyrobutyricum showed that AbrB not only exhibited the reported characteristics such as the negative regulation on sporulation, positive effects on biofilm formation and stress resistance but also exhibited new functions, especially the negative regulation of carbon metabolism. AbrB knockout strain (Ct/ΔabrB) could alleviate glucose-mediated carbon catabolite repression (CCR) and enhance the utilization of xylose compared with the parental strain, resulting in a higher butyrate titer (14.79 g/L vs. 7.91 g/L) and xylose utilization rate (0.19 g/L·h vs. 0.02 g/L·h) from the glucose and xylose mixture. This study confirmed the pleiotropic regulatory function of AbrB in C. tyrobutyricum, suggesting that Ct/ΔabrB was the potential candidate for butyrate production from abundant, renewable lignocellulosic biomass mainly composed of glucose and xylose.

Rice husk, as a cellulose-rich by-product in agriculture, has been considered as a low-cost substrate for the production of bioethanol and chemicals. In this study, rice husk was pretreated with an alkali, followed by cellulose and β-glucosidase hydrolysis optimized by an orthogonal experiment and response surface methodology (RSM), respectively. Under the optimal treatment conditions, a hydrolysate containing a high reducing sugar yield (77.85%) was obtained from the rice husk. Then, the hydrolysate was used as a carbon substrate for butyric acid production through Clostridium tyrobutyricum Δpta fermentation. Compared to free-cell fermentation, higher concentrations of butyric acid (50.01 g/L vs. 40.8 g/L and 49.03 g/L vs. 27.49 g/L) were observed in immobilized-cell fermentation for the carbon source of glucose and hydrolysate, respectively. A final butyric acid concentration of 16.91 g/L, a yield of 0.31 g/g, and an overall productivity of 0.35 g/L/h from rice husk hydrolysate were obtained in the repeated-fed-batch mode. Taken together, rice husk hydrolysate can be effectively utilized for the bioproduction of butyrate with immobilized-cell fermentation.