Graduate Thesis Or Dissertation
 

Contribution of Farm Level Milk Sourcing on Non-Starter Lactic Acid Bacteria (NSLAB) in Cheddar, and the Assessment of High Resolution Melt Analysis used for Species Identification

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/hx11xk21j

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  • Non-starter lactic acid bacteria (NSLAB) are found at low levels in fresh raw milk and are important to the dairy industry because of their potential impact on the flavor and texture of yogurt, sour cream, and cheese. The purpose of this study was to evaluate three methods for the identification of NSLAB and investigate NSLAB contribution from raw milk sourced from different farms on the microbiological profile of Cheddar cheese during aging. Three methods were evaluated: P1V1/P2V1 PCR with HRM analysis, 16s rDNA sequencing using MicroSEQ® 500, and API 50 CHL. HRM analysis and API 50 CHL were evaluated using NSLAB reference strains which included Lb. paracasei, Lb. casei, Lb. plantarum, Lb. rhamnosus, and L. lactis subsp. lactis. In addition, 16s rDNA sequencing and HRM analysis of the P1V1/P2V1 amplicon were used for the identification of unknown isolates from raw milk and 6.5 mo aged Cheddar cheese. Five out of seven reference strains were correctly identified using API 50 CHL. HRM analysis was in agreement with 16s rDNA sequencing 75% and 66% of the time for L. lactis and Lb. paracasei, respectively. Melt curves of isolates identified as Lb. paracasei and L. lactis subps. lactis by 16s rDNA sequencing showed a significant amount of overlap. Farm level contribution of NSLAB was investigated by making Cheddar cheeses using a standardized recipe with raw milk sourced from dairies on the Oregon Coast and in the Willamette Valley and aged. Isolates were selected for preliminary speciation using HRM analysis and further subtyped using a second HRM repetitive sequence-based polymerase chain reaction (rep-PCR). Lb. paracasei/L. lactis were identified in raw milk and cheeses sourced from the Oregon Coast and from the Willamette Valley. Lb. curvatus was only identified in raw milk samples. Species diversity decreased throughout aging in all cheeses with the exception of cheese made from milk sourced from the southern Oregon Coast. After 6.5 mo of aging, the predominant species across all cheeses was Lb. paracasei/L. lactis (70.79% of isolates). Strain diversity was highest in milk sourced from the northern Oregon Coast. 16S rDNA sequencing identified additional species not reported using HRM analysis which included Lb. brevis, L. lactis cremoris, E. saccharolyticus, E. faecalis, S. bovis, S. chromogenes, Leuc. lactis, and W. paramesenteroides. Based on comparison to 16s rDNA identification, HRM analysis of the P1V1/P2V1 amplicon is not suitable for unknown NSLAB identification. For NSLAB contribution, evidence suggests that milk sourcing at the farm level contributes to the strain diversity of NSLAB present in raw milk and Cheddar cheese. This information can help larger producers understand the contribution their farmers have on their product and steps they may need to take to mitigate it or potentially capitalize on the differences in a small batch capacity.
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