Microbiological analysis through aging: 1 Day, 1, 3 & 6.5 months Regional milk sourcing impact on non-starter lactic acid bacteria (NSLAB) in raw milk and Cheddar cheese during aging Christopher Baird*, Julia Wilson, Nicolas Hergert, Lisbeth Meunier-Goddik, and Joy Waite-Cusic Department of Food Science and Technology ABSTRACT INTRODUCTION OBJECTIVES Non-starter lactic acid bacteria (NSLAB), which include lactobacilli, are found at low levels in fresh raw milk. Lactobacilli are important to the dairy industry because of their potential impact on the flavor and texture of yogurt, sour cream, and cheese. The objective of this study was to investigate the contribution of lactobacilli from raw milk on the microbiological profile of Cheddar cheese during aging (0-6 mos). Using a standardized recipe, Cheddar cheeses were made with raw milk sourced from dairies on the Oregon Coast (n =4) and in the Willamette Valley (n = 2) and aged up to 6.5 mos at 53°C. Lactic acid bacteria counts (LAB) were determined in raw milk and cheese samples using standard serial dilution and spread plating techniques on Lactobacilli MRS Agar with anaerobic incubation at 30°C for 48 hrs. Isolates (n=5-10/sample) were selected for preliminary speciation using High Resolution Melt Analysis (HRM) PCR assay targeting the V1 region of the 16S rDNA. Strains were further subtyped using a second HRM repetitive sequence-based PCR (rep-PCR). Lactobacillus curvatus and L. paracasei were identified in raw milk sourced from the Oregon Coast. Lactobacillus paracasei were also identified in the Willamette Valley cheeses. Species diversity decreased throughout aging in all cheeses with the exception of cheese made from milk sourced from a single dairy on the southern Oregon Coast. After 6.5 months of aging, the predominant species across all cheeses was L. paracasei (40.8% of the identified isolates). Strain diversity was highest in milk sourced from the northern Oregon Coast with six unique strains of L. paracasei. Evidence suggest that milk sourcing impacts the strain diversity of NSLAB present in raw milk and Cheddar cheese. Further investigation is needed to evaluate HRM analysis of the P1V1/P2V1 amplicon as an effective tool for identifying L. lactis and Lb. paracasei which were found to have similar Tm. Cheddar cheese flavor from different regions within the United States have been found to be distinct in flavor with the major factor identified as differences in production facility (Drake et al., 2008). With production being limited to a single facility Cheddar cheese flavor was found to be distinct based on the region milk was sourced from (Turbes et. al, 2016). Farm environment and pasture are likely sources for NSLAB diversity in raw milk. The purpose of this study was to determine the impact of regional milk sourcing on NSLAB in raw milk and Cheddar cheese while dairy herds were on pasture by: •Investigate the differences in NSLAB between the Oregon Coast and the Willamette Valley •Investigate the differences in NSLAB at the farm level METHODS Transport to OSU Creamery Sample Locations of individual dairies providing milk for Cheddar cheese production. Microbiological analysis through aging: 1 Day, 1, 3 & 6.5 months 11 g Cheese Dilute HRM Analysis MRS Blend Fingerprint rep-PCR Isolate Plate Incubate 30C, 48 hr (anaerobic) MRS 10-1 dilution Sodium Citrate Blender jar

Department of Food Science and Technology Regional milk sourcing impact on non-starter lactic acid bacteria (NSLAB) in raw milk and Cheddar cheese during aging Christopher Baird*, Julia Wilson, Nicolas Hergert, Lisbeth Goddik, and Joy Waite-Cusic Department of Food Science and Technology RESULTS DISCUSSION ` Levels of NSLAB in raw milk were initially low then quickly increased in Cheddar cheese samples (Figure 1). NSLAB bacteria diversity was greatest in raw milk samples. Differences in NSLAB species were observed between milk sourced from farm NCA on different days. Within the North Coast NSLAB were different between farms with the most shared NSLAB species between NCA and NCC (Figure 2). Based on HRM analysis of the P1V1/P2V1 amplicon species diversity between farms in the North Coast were unique compared to the species observed in the Willamette Valley farms (Figure 3). Lb. paracasei was the major NSLAB species (40.8%) identified in aged cheese samples through HRM analysis. Lb. paracasei also made up 25 of the 56 strains observed between all species (Figure 4). A fingerprint related to a single starter strain used in cheese making, strain S (Figure 4), was identified in all species suggesting that HRM analysis of the P1V1/P2V1 amplicon may not be sensitive enough to distinguish L. lactis and Lb. paracasei. Additionally, specific fingerprints for L. lactis subsp. lactis and Lb. paracasei were found over a range of melt temperatures between 80-83C. Further investigation is needed to determine the sensitivity of this method. Oregon Coast Willamette Valley Figure 1: NSLAB density (log CFU/ml) in raw milk to aged Cheddar cheese. Figure 3: NSLAB species variability in raw milk between the Oregon Coast and Willamette Valley REFERENCES Drake, M. A., Yates, M. D., & Gerard, P. D. 2008. Determination of regional flavor differences in U.S. Cheddar cheeses aged for 6 mo or longer. J. Food Sci, 73(5), 199-208. Turbes, G., Linscott, T. D., Tomasino, E., Waite-Cusic, J., Lim, J., & Meunier-Goddik, L. 2016. Evidence of terroir in milk sourcing and its influence on Cheddar cheese. J. Dairy Sci. 1–11. ACKNOWLEDGMENTS Figure 2. NSLAB species in raw milk from the North Oregon Coast; (●) NCA, (●) NCB, (●) NCC, (●) NCA and NCC, (●) NCA and NCB Thank authors would like to thank the Eckelman Endowment for funding the research. The authors would also like to thank Eva Kuhn for assistance with lab work. Figure 4: Lb. paracasei isolate fingerprints determined through rep-PCR.