Clostridium tyrobutyricum strains exhibit high genetic diversity and differ in their ability to cause Late Blowing Defect in washed-curd cheese | Late Blowing Defect in Washed-Curd Cheese

Supplementary Figure 1: Phylogenetic tree of C. tyrobutyricum strains based on 16S rRNA gene sequences. The phylogenetic tree was constructed using the maximum-likelihood method and Kimura 2-parameter model. The percentage numbers at the nodes indicate the level of bootstrap support based on 500 replicates. Supplementary Figure 2: Phylogenetic tree based on a 657-core-SNP matrix genome-wide SNPs among the six C. tyrobutyricum strains. A Maximum-Likelihood tree was constructed using kSNP 4.0. “LBD+” and “LBD-“ indicate strains that did or did not show the ability to cause a Late Blowing Defect in cheese at 15°C, respectively. Bar indicates 0.2 substitutions per nucleotide position. Supplementary Table 1: Growth and gas production of six strains of C. tyrobutyricum in liquid Reinforced Clostridial Medium (RCM) and Enriched Milk Medium (EM) at temperatures between 8 ⁰C and 37 ⁰C. Supplementary Table 2: Growth and gas production of six strains of C. tyrobutyricum in liquid Reinforced Clostridial Medium (RCM) at temperature 37⁰C and pH values between 6.9 and 4.1. Supplementary Table 3: Microbiological and physicochemical characteristics of washed-curd cheeses individually inoculated with six different strains of C. tyrobutyricum and corresponding uninoculated control cheeses. Supplementary Table 4: General and specific genomic features of six C. tyrobutyricum strains. Supplementary Table 5: The ANIb values (%) between six experimental C. tyrobutyricum strains and C. tyrobutyricum ATCC 25755T strain calculated using the standard BLAST algorithm. Supplementary Table 6: Gene presence-absence in C. tyrobutyricum pangenome. “LBD+” and “LBD-“ indicate strains that did or did not show the ability to cause a Late Blowing Defect in cheese at 15 °C, respectively. White cell with number “1” represents the presence of a specific gene; empty grey cell represents the absence of a specific gene; orange cell represents the presence of a specific gene in all three “LBD+” strains and absence in all three “LBD-” strains; blue cell represents the absence of a specific gene in all three “LBD+” strains and presence in all three “LBD-” strains.

Clostridium tyrobutyricum is a spore-forming bacterium and is considered to be the main causative agent of Late Blowing Defect (LBD) of hard and semi-hard cheeses. However, the spoilage potential of C. tyrobutyricum appears to be strain-dependent. Since previous studies have been mostly limited to laboratory liquid media or experimental food models imitating cheese matrix, we (i) characterized six strains of C. tyrobutyricum for their ability to cause LBD in washed-curd cheese aged at 8 and 15 °C, and (ii) probed the whole genome data for the genetic markers linked to LBD. Washed-curd cheeses were made with pasteurized cow milk individually inoculated with different C. tyrobutyricum strains. During cheese aging at 8 °C, none of the six evaluated strains caused changes in cheese consistent with LBD (i.e., accumulation of gas, formation of cracks, and production of butyric acid). Three of the six tested strains were, however, able to cause LBD at 15 °C; the remaining three C. tyrobutyricum strains showed little or no gas and butyric acid production during 160 d aging at 15 °C. All six strains were additionally evaluated in three separate experiments performed in two different liquid media (Reinforced Clostridial Medium and Enriched Milk Medium) to determine germination, growth, and gas formation at temperatures between 8 and 37 °C, and pH values between 6.9 and 4.1. All six evaluated strains were able to grow and form gas in liquid media in at least one replicate at temperatures and pH values as low as 12.5 °C and 5.1, respectively. Comparative genomics showed that the six strains exhibit high genetic diversity, without specific genetic features that would explain the observed differences in the ability to cause LBD in washed-curd cheese. Future studies would need to compare a larger set of C. tyrobutyricum strains with demonstrated ability or inability to cause LBD in cheese. This ability to cause LBD will most likely have to be determined in actual cheese experiments since our results show that growth and gas formation in liquid media models are not always representative of growth and gas formation in cheese.

This work is part of the project “Control of Clostridium tyrobutyricum, a remerging concern in hard cheese production” (Project No.: C012388) funded by the New York Dairy Promotion Order (Albany, NY). The authors wish to thank the New York State Dairy Promotion Advisory Board for its continued support of our research and extension work. The authors would like to thank Larissa Bischof from the University of Natural Resources and Life Sciences, Vienna, Austria for her assistance during the experiments in EM broth. The authors would also like to thank Gavin Lavi Sacks from the Department of Food Science, Cornell University for supplying the equipment, material, and expertise to quantify butyric acid in cheese. The authors acknowledge that N. H. Martin is a section editor for the Journal of Dairy Science. All coauthors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. No human or animal subjects were used, so this analysis did not require approval by an Institutional Animal Care and Use Committee or Institutional Review Board.