Microbiota assessments for the identification and confirmation of slit defect-causing bacteria in milk and cheddar cheese

Authors: XUE, ZHENGYAO - University Of California, Davis; BROOKS, JASON - University Of California, Davis; QUART, ZACHARY - University Of California, Davis; STEVENS, ERIC - University Of California, Davis; Kable, Mary; HEIDENRIECH, JESSIE - Hilmar Cheese Company; MCLEOD, JEREMY - Hilmar Cheese Company; MARCO, MARIA - University Of California, Davis

Submitted to: mSystems
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/16/2021
Publication Date: 2/9/2021
Citation: Xue, Z., Brooks, J.T., Quart, Z., Stevens, E.T., Kable, M.E., Heidenriech, J., McLeod, J., Marco, M.L. 2021. Microbiota assessments for the identification and confirmation of slit defect-causing bacteria in milk and cheddar cheese. mSystems. 6(1). Article e01114-20. https://doi.org/10.1128/mSystems.01114-20.
DOI: https://doi.org/10.1128/mSystems.01114-20

Interpretive Summary: Bacteria present in milk vary in their susceptibility to pasteurization and in their impact on the development, or spoilage, of milk products like cheese. Therefore, we characterized the live bacteria present in milk before and after high temperature short time (HTST) pasteurization within a single pasteurizer over time and in the cheddar cheese produced from this pasteurized milk during aging. Pasteurization reduced the number and types of bacteria present in milk. However, the bacterial genera Thermus was found to thrive post-pasturization. Despite this observation, the comparison of normal and defective cheeses (with slits) showed that lactic acid bacteria present in much lower abundance (Lactobacillus fermentum, Leuconostoc mesenteroides, and Leuconostoc lactis) were associated with slit formation in cheddar cheese. This association was confirmed by the development of slits in aged cheddar from cheese curds inoculated with these lactic acid bacteria.

Technical Abstract: Background: Milk and dairy foods contain a variety of microorganisms that can either be necessary for the production of those products (e.g. cheese) or cause spoilage and defects. To identify bacterial taxa and consortia in milk responsible for slit defects in cheese, we measured the bacterial composition in milk pre- and post-pasteurization for a single pasteurizer over time on multiple days, and in Cheddar cheese during aging. For this purpose, we employed propidium monoazide treatment combined with 16S rRNA gene amplicon DNA sequencing for viable cell detection, quantitative PCR for bacterial enumeration, laboratory culture, isolate identification, and pilot-scale cheese production to verify the importance of certain bacteria and consortia in causing slit defects. Results: Milk was sampled over 10 h periods on 10 dates immediately before and after the final High Temperature Short Time (HTST) pasteurization prior to the initiation of Cheddar cheese fermentations. Overall, HTST reduced the diversity of the viable bacteria in milk, reduced the proportions of most bacterial taxa such as Acinetobacter, Enterobacteriaceae and Pseudomonas, and increased the proportions of Clostridiales, Streptococcus, Thermus, and Turicibacter. These changes were also dependent on the time since pasteurizer cleaning. There was a 3-fold increase in viable, bacterial cell numbers in milk post-HTST within 10 h after equipment cleaning. This increase was associated with a 68-fold rise in Thermus cell numbers (from 22 cells/mL to 1500 cells/mL 9 h post-CIP estimated by qPCR). Comparisons of cheese blocks with and without slits showed that slit defects were associated with several low-abundance bacterial taxa, including thermoduric heterofermentative Lactobacillus, which were also enriched in the pasteurized milk used to make that cheese. Pilot-scale cheeses inoculated with bacterial consortia from milk or milk-derived isolates of Lactobacillus fermentum, Leuconostoc mesenteroides, and Leuconostoc lactis developed slits, thereby confirming that low abundance taxa in milk are important agents of cheese defects. Conclusions: We identified and verified that certain low-abundance, bacterial taxa in milk are responsible for causing slit defects in Cheddar cheese. The likelihood for microorganisms in milk to cause defects could be predicted based on comparisons of the bacteria present in the pre- and post-HTST milk used for cheesemaking.