Tyrobutyricum Strains Research Articles

Comparative genomics of Clostridium tyrobutyricum reveals signatures of environment-specific adaptation and metabolic potentials

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.

Specificity of the AMP-6000 Method for Enumerating Clostridium Endospores in Milk.

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.

Clostridium strain FAM25158, a unique endospore-forming bacterium related to Clostridium tyrobutyricum and isolated from Emmental cheese shows low tolerance to salt.

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.

Deciphering the contribution of PerR to oxidative stress defense system in Clostridium tyrobutyricum

AbstractAs 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.

Comparative Genomics Provides Insights Into Genetic Diversity of Clostridium tyrobutyricum and Potential Implications for Late Blowing Defects in Cheese.

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.

Draft Genome Sequences of 12 Clostridium tyrobutyricum Strains Isolated from Raw Milk and Cheese.

ABSTRACTClostridium tyrobutyricum is recognized as the main causative agent of late blowing defect—severe spoilage of hard and semihard cheeses. In this work, we present the draft genome sequences of 12 C. tyrobutyricum strains isolated from raw milk and cheese.

Strain-Dependent Cheese Spoilage Potential of Clostridium tyrobutyricum.

Clostridium tyrobutyricum, a Gram-positive, anaerobic, spore-forming bacterium, is considered as one of the main causative agents for spoilage of hard and semihard cheeses. Growth of C. tyrobutyricum in cheese is critically influenced by ripening temperature and time, pH, salt and lactic acid concentration, moisture and fat content, and the presence of other microorganisms. Previous studies revealed high intraspecies diversity of C. tyrobutyricum strains and variable tolerance toward pH, temperatures, and salt concentrations. These findings indicate that strain-dependent characteristics may be relevant to assess the risk for cheese spoilage if clostridial contamination occurs. In this study, we aimed to compare the phenotypes of 12 C. tyrobutyricum strains which were selected from 157 strains on the basis of genotypic and proteotypic variability. The phenotypic analysis comprised the assessment of gas production and organic acid concentrations in an experimental cheese broth incubated at different temperatures (37, 20, and 14 °C). For all tested strains, delayed gas production at lower incubation temperatures and a strong correlation between gas production and the change in organic acid concentrations were observed. However, considering the time until gas production was visible at different incubation temperatures, a high degree of heterogeneity was found among the tested strains. In addition, variation among replicates of the same strain and differences due to different inoculum levels became evident. This study shows, that, among other factors, strain-specific germination and growth characteristics should be considered to evaluate the risk of cheese spoilage by C. tyrobutyricum.

Recent advances in n-butanol and butyrate production using engineered Clostridium tyrobutyricum.

Acidogenic clostridia naturally producing acetic and butyric acids has attracted high interest as a novel host for butyrate and n-butanol production. Among them, Clostridium tyrobutyricum is a hyper butyrate-producing bacterium, which re-assimilates acetate for butyrate biosynthesis by butyryl-CoA/acetate CoA transferase (CoAT), rather than the phosphotransbutyrylase-butyrate kinase (PTB-BK) pathway widely found in clostridia and other microbial species. To date, C. tyrobutyricum has been engineered to overexpress a heterologous alcohol/aldehyde dehydrogenase, which converts butyryl-CoA to n-butanol. Compared to conventional solventogenic clostridia, which produce acetone, ethanol, and butanol in a biphasic fermentation process, the engineered C. tyrobutyricum with a high metabolic flux toward butyryl-CoA produced n-butanol at a high yield of > 0.30g/g and titer of > 20g/L in glucose fermentation. With no acetone production and a high C4/C2 ratio, butanol was the only major fermentation product by the recombinant C. tyrobutyricum, allowing simplified downstream processing for product purification. In this review, novel metabolic engineering strategies to improve n-butanol and butyrate production by C. tyrobutyricum from various substrates, including glucose, xylose, galactose, sucrose, and cellulosic hydrolysates containing the mixture of glucose and xylose, are discussed. Compared to other recombinant hosts such as Clostridium acetobutylicum and Escherichia coli, the engineered C. tyrobutyricum strains with higher butyrate and butanol titers, yields and productivities are the most promising hosts for potential industrial applications.

Characterization of Clostridium tyrobutyricum Strains Using Three Different Typing Techniques

Clostridium tyrobutyricum is well known as one of the main causative agents of severe cheese spoilage. The metabolism of this anaerobic bacterium during ripening leads to textural and sensory defects in cheese and consequential loss of product value. The potential to induce cheese spoilage, however, may vary among different strains of the same species. Therefore, a better understanding of the intra-species diversity of C. tyrobutyricum may be of practical relevance for the dairy industry. In the present study, we compared the ability of three typing techniques to differentiate 95 C. tyrobutyricum strains on the subspecies level: (1) repetitive element palindromic PCR (rep-PCR) fingerprinting combined with conventional agarose gel electrophoresis, (2) hexaplex-PCR followed by an automated capillary electrophoresis and (3) matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) typing. MALDI-TOF MS fingerprinting provided only moderate reproducibility and low discriminatory power. Both PCR-based methods were highly reproducible and discriminative, with hexaplex-PCR fingerprinting being slightly more discriminative than rep-PCR typing. Overall, a high intra-species diversity was observed among the tested strains, indicating that further investigations on the strain level may be of interest.

Production of butyric acid at constant pH by a solventogenic strain of Clostridium beijerinckii

A solventogenic strain of Clostridium beijerinckii, NRRL B-598, was cultured for the production of butyric acid as the main fermentation product. However, unlike typical acetone-butanol-ethanol (ABE) fermentations, where pH is not regulated, in this study the pH was kept constant during fermentation. From the five pH values tested, 6.0, 6.5, 7.0, 7.5 and 8.0, pH 6.5 and 7.0 resulted in the highest concentrations of butyric acid, at 9.69 ± 0.09 g L–1 and 11.5 ± 0.39 g L–1, respectively. However, a low concentration of solvents, 1.8 ± 0.22 g L–1, was only reached at pH 7.0. These results are comparable with those from typical butyric acid producers, i.e. Clostridium butyricum and Clostridium tyrobutyricum strains. At pH 7.0, we succeeded in suppressing sporulation and prolonging the population viability, which was confirmed by flow cytometry combined with double fluorescence staining.

Application of high pressure processing for controlling Clostridium tyrobutyricum and late blowing defect on semi-hard cheese

In this study we evaluated the application of different high pressure (HP) treatments (200–500 MPa at 14 °C for 10 min) to industrial sized semi-hard cheeses on day 7, with the aim of controlling two Clostridium tyrobutyricum strains causing butyric acid fermentation and cheese late blowing defect (LBD). Clostridium metabolism and LBD appearance in cheeses were monitored by sensory (cheese swelling, cracks/splits, off-odours) and instrumental analyses (organic acids by HPLC and volatile compounds by SPME/GC–MS) after 60 days. Cheeses with clostridial spores HP-untreated and HP-treated at 200 MPa showed visible LBD symptoms, lower concentrations of lactic, citric and acetic acids, and higher levels of pyruvic, propionic and butyric acids and of 1-butanol, ethyl and methyl butanoate, and ethyl pentanoate than cheeses without spores. However, cheeses with clostridial spores and HP-treated at ≥ 300 MPa did not show LBD symptoms and their organic acids and volatile compounds profiles were comparable to those of their respective HP-treated control cheeses, despite HP treatments caused a low spore reduction. A decrease in C. tyrobutyricum spore counts was observed after curd pressing, which seems to indicate an early spore germination, suggesting that HP treatments ≥300 MPa were able to inactivate the emerged C. tyrobutyricum vegetative cells and, thereby, prevent LBD.

Genomic approach to studying nutritional requirements of Clostridium tyrobutyricum and other Clostridia causing late blowing defects

Clostridium tyrobutyricum is the main microorganism responsible for the late blowing defect in hard and semi-hard cheeses, causing considerable economic losses to the cheese industry. Deeper knowledge of the metabolic requirements of this microorganism can lead to the development of more effective control approaches. In this work, the amino acids and B vitamins essential for sustaining the growth of C. tyrobutyricum were investigated using a genomic approach. As the first step, the genomes of four C. tyrobutyricum strains were analyzed for the presence of genes putatively involved in the biosynthesis of amino acids and B vitamins. Metabolic pathways could be reconstructed for all amino acids and B vitamins with the exception of biotin (vitamin B7) and folate (vitamin B9). The biotin pathway was missing the enzyme amino-7-oxononanoate synthase that catalyzes the condensation of pimeloyl-ACP and l-alanine to 8-amino-7-oxononanoate. In the folate pathway, the missing genes were those coding for para-aminobenzoate synthase and aminodeoxychorismate lyase enzymes. These enzymes are responsible for the conversion of chorismate into para-aminobenzoate (PABA). Two C. tyrobutyircum strains whose genome was analyzed in silico as well as other 10 strains isolated from cheese were tested in liquid media to confirm these observations. 11 strains showed growth in a defined liquid medium containing biotin and PABA after 6–8 days of incubation. No strain showed growth when only one or none of these compounds were added, confirming the observations obtained in silico. Furthermore, the genome analysis was extended to genomes of single strains of other Clostridium species potentially causing late blowing, namely Clostridium beijerinckii, Clostridium sporogenes and Clostridium butyricum. Only the biotin biosynthesis pathway was incomplete for C. butyricum and C. beijerincki. In contrast, C. sporogenes showed missing enzymes in biosynthesis pathways of several amino acids as well as biotin, folate, and cobalamin (vitamin B12). These observations agree with the results of growth experiments of these species in liquid media reported in the literature. The results of this study suggest that biotin and folate are potential targets for reducing late blowing in cheese and highlight the usefulness of genomic analysis for identifying essential nutrients in bacteria.

Draft Genome Sequences of Clostridium tyrobutyricum Strains FAM22552 and FAM22553, Isolated from Swiss Semihard Red-Smear Cheese.

Clostridium tyrobutyricum is the main microorganism responsible for late blowing defect in cheeses. Here, we present the draft genome sequences of two C. tyrobutyricum strains isolated from a Swiss semihard red-smear cheese. The two draft genomes comprise 3.05 and 3.08 Mbp and contain 3,030 and 3,089 putative coding sequences, respectively.

Multilocus variable-number of tandem repeat analysis (MLVA) for Clostridium tyrobutyricum strains isolated from cheese production environment

Clostridium tyrobutyricum is a gram-positive spore-forming anaerobe that is considered as the main causative agent for late blowing in cheese due to butyric acid fermentation. In this study, multilocus variable-number of tandem repeat (VNTR) analysis (MLVA) for C. tyrobutyricum was developed to identify the source of contamination by C. tyrobutyricum spores in the cheese production environment. For each contig constructed from the results of a whole genome draft sequence of C. tyrobutyricum JCM11008T based on next-generation sequencing, VNTR loci that were effective for typing were searched using the Tandem Repeat Finder program. Five VNTR loci were amplified by polymerase chain reaction (PCR) to determine their number of repeats by sequencing, and MLVA was conducted. 25 strains of C. tyrobutyricum isolated from the environment, raw milk, and silage were classified into 18 MLVA types (DI=0.963). Of the C. tyrobutyricum strains isolated from raw milk, natural cheese, and blown processed cheese, strains with identical MLVA type were detected, which suggested that these strains might have shifted from natural cheese to blown processed cheese. MLVA could be an effective tool for monitoring contamination of natural cheese with C. tyrobutyricum in the processed cheese production environment because of its high discriminability, thereby allowing the analyst to trace the source of contamination.

Clostridium tyrobutyricum strains show wide variation in growth at different NaCl, pH, and temperature conditions.

Outgrowth from Clostridium tyrobutyricum spores in milk can lead to butyric acid fermentation in cheeses, causing spoilage and economical loss to the dairy industry. The aim of this study was to investigate the growth of 10 C. tyrobutyricum strains at different NaCl, pH, and temperature conditions. Up to 7.5-fold differences among the maximum growth rates of different strains in the presence of 2.0% NaCl were observed. Five of 10 strains were able to grow in the presence of 3.0% NaCl, while a NaCl concentration of 3.5% was completely inhibitory to all strains. Seven of 10 strains were able to grow at pH 5.0, and up to 4- and 12.5-fold differences were observed among the maximum growth rates of different strains at pH 5.5 and 7.5, respectively. The maximum growth temperatures varied from 40.2 to 43.3°C. The temperature of 10°C inhibited the growth of all strains, while 8 of 10 strains grew at 12 and 15°C. Despite showing no growth, all strains were able to survive at 10°C. In conclusion, wide variation was observed among different C. tyrobutyricum strains in their ability to grow at different stressful conditions. Understanding the physiological diversity among the strains is important when designing food control measures and predictive models for the growth of spoilage organisms in cheese.

Effects of copper on germination, growth and sporulation of Clostridium tyrobutyricum

The effects of copper (Cu 2+) on spore germination, vegetative growth and sporulation of Clostridium tyrobutyricum, which is capable to causing texture and flavour defects in Emmental cheese, were studied. Spore suspensions of three different strains were used as starting material for two experimental set-ups. The first studied the effects of supplemented (0–30 ppm) copper in RCM medium during spore germination and vegetative growth of C. tyrobutyricum measured by plating. The second set-up studied the effects of copper (0–30 ppm) in RCM medium during growth and sporulation of C. tyrobutyricum as measured by optical density at 550 nm and by platings after heat treatment of the samples respectively. Inhibition of germination, vegetative growth and sporulation processes by copper was strain-dependent. Both sporulation and germination were more sensitive than vegetative growth of C. tyrobutyricum to the inhibitory effects of copper. Copper, at the concentrations investigated in this study, inhibits spore germination of C. tyrobutyricum strains. Consequently copper may reduce the risk of late blowing spoilage from in the germination of C. tyrobutyricum spores during the ripening period of Emmental cheese.

Influence of milk centrifugation, brining and ripening conditions in preventing gas formation by Clostridium spp. in Gouda cheese

This study examined milk centrifugation, increased salt concentration, and low ripening temperature as potential strategies to prevent late blowing caused by gas-forming Clostridium spp. in Gouda cheese. The survival of clostridia spores in cheese brine and their ability to enter Gouda cheese during brining was also evaluated. Centrifugation (3000× g for 30 s) of contaminated milk resulted in >60% spore reduction, with increased spore reduction at greater centrifugal forces. Low levels of C. tyrobutyricum and C. sporogenes spores survived in saturated (23%, w/v) brine with 2% (v/v) added whey at 15°C for 63 days, while C. beijerinckii and C. butyricum spores were not detectable on days 4 and 35, respectively. Spores of C. tyrobutyricum in brine infiltrated Gouda cheese during 2 h of brining at 13°C resulted in production of small gas holes during ripening. In Gouda cheese slurry stored at 13°C, three C. tyrobutyricum strains plus one of three C. sporogenes strains germinated in the slurry with no added salt. Of three C. tyrobutyricum strains stored at 13°C in slurries with higher water-phase salt concentrations of 2.4 and 3.6%, two strains and one strain germinated, respectively. No germination of spores was detected in any cheese slurry stored at 5 or 8°C. Milk centrifugation, increased percent water-phase salt, absence of spores in brine, and decreased ripening temperature are all potentially important measures against gas production by Clostridium spp. in Gouda cheese.

Differentiation of lactate-fermenting, gas-producing Clostridium spp. isolated from milk

Endospores of Clostridium spp. capable of producing gas in a lactate-containing medium were enumerated from 14 pasteurized milk samples from Wisconsin cheese plants. Concentrations of endospores of lactate-fermenting, gas-producing Clostridium spp. were between 5.0×10 −2 and 1.7×10 0 MPN ml −1. Concentrations of presumptive C. tyrobutyricum endospores (defined by subterminal endospore position and lactate dehydrogenase activity) were lower, not exceeding 2.0×10 −2 MPN ml −1. Based on subterminal endospore position, lactate dehydrogenase activity, and a carbohydrate fermentation profile identical to C. tyrobutyricum strain ATCC 25755, five isolates (Ct) were initially characterized as C. tyrobutyricum, a known cause of late-blowing in high-pH cheeses. Twenty-eight other isolates (Cx) produced gas from lactate, but differed from ATCC 25755 in either endospore position, lactate dehydrogenase activity or carbohydrate fermentation profile. When inoculated at high concentrations in Gouda cheese, strain ATCC 25755, two Ct isolates and 18 Cx isolates tested produced gas during ripening. Among the five Ct isolates obtained and two reference strains confirmed as C. tyrobutyricum, there were four qualitatively different volatile organic acid byproduct profiles. Each of the two confirmed C. tyrobutyricum reference strains and five Ct isolates had distinct quantitative cell membrane fatty acid (CMFA) profiles. The Cx isolates represented 14 different volatile organic acid byproduct profiles and each isolate had a unique CMFA profile. Pulsed field gel electrophoresis (PFGE) of DNA from the two confirmed reference C. tyrobutyricum strains, four Ct and three Cx isolates, showed a low degree of relatedness. The results of this study suggest that a heterogeneous group of lactate-fermenting, gas-producing Clostridium spp. may be found in milk. Gas chromatographic analysis of volatile organic acid byproducts or CMFA, and PFGE of DNA are highly discriminating methods for differentiating Clostridium spp. that may cause late blowing in high-pH cheeses.

Identification of Clostridium tyrobutyricum as the causative agent of late blowing in cheese by species-specific PCR amplification.

Butyric acid fermentation, the late-blowing defect in cheese, caused by the outgrowth of clostridial spores present in raw milk, can create considerable loss of product, especially in the production of semihard cheeses like Gouda cheese, but also in grana and Gruyère cheeses. To demonstrate the causative relationship between Clostridium tyrobutyricum and late blowing in cheese, many cheesemaking experiments were performed to provoke this defect by using spores from several strains of the major dairy-related clostridia. A method of PCR amplification of a part of the 16S rRNA gene in combination with hybridization with species-specific DNA probes was developed to allow the specific detection of clostridial sequences in DNAs extracted from cheeses. The sensitivity was increased by using nested PCR. Late blowing was provoked in experimental cheeses with 28 of the 32 C. tyrobutyricum strains tested, whereas experimental cheeses made with spores from C. beijerinckii, C. butyricum, and C. sporogenes showed no signs of butyric acid fermentation. In all experimental and commercial cheeses with obvious signs of late blowing, DNA from C. tyrobutyricum was detected; in some cheeses, signals for C. beijerinckii were also found. It was concluded that only C. tyrobutyricum strains are able to cause butyric acid fermentation in cheese.

Occurrence of Clostridium tyrobutyricum in cattle slurry and fresh forage grasses

A comparative, numerical, taxonomic analysis was carried out on 24 selected strains of Clostridium spp. isolated from cattle slurries and fresh forage grasses fertilized with slurries, as well as on 10 culture-collection Clostridium tyrobutyricum strains for reference. The results showed an unexpectedly low occurrence of the Clostridium tyrobutyricum, with only one of the 24 strains isolated from slurry belonging to this species. The majority of Clostridium strains isolated from grasses were identified as C. malenominatum. Although the predominant anaerobic, spore-forming colonizers of the cattle slurry belonged taxonomically to the genus Clostridium they could not be firmly identified, but appeared to show some resemblance to C. propionicum.