Graduate Thesis Or Dissertation
 

Characterization of Spoilage Events on Flavored Milk Products by Paenibacillus odorifer

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  • Flavored milk is a growing category for dairy products; however, it is a perishable product that is subject to microbial spoilage which can lead to a negative consumer experience and lead to lost sales. Flavored milk processors communicated unique spoilage events related to their products, including color loss of strawberry milk products and off-odor development in chocolate milk products. While there is a body of knowledge in the scientific literature related to milk spoilage, there is an absence of information available about spoilage defects in flavored milk. Paenibacillus spp. are a particularly problematic group of psychrotrophic Grampositive sporeforming bacteria that are commonly associated with refrigerated milk products. Therefore, there was interest in exploring the behavior of Paenibacillus strains in flavored milk products to evaluate their potential contribution to spoilage defects described by commercial processors. The color of strawberry milk is commonly due to the addition of FD&C Red No. 40 which is typically considered to be a very stable dye in food products. Commercial processors described color loss occurring in their products before the end of shelflife (21 days). Without more information and the cause of the problem or potential solution, processors decided to reduce their shelflife to 14 days; however, this creates other logistical problems. Retail strawberry milk products were purchased, and color loss was monitored, and potential causative organisms were isolated from products experiencing color loss. Several strains representing the Paenibacillus genus were isolated from defective products. Additional Paenibacillus strains, particularly P. odorifer, were tested to determine the capabilities of dye degradation at the species and genus level. Individual strains were inoculated into UHT strawberry milk in triplicate and monitored to measure growth rates and confirm dye degradation ability. Inoculated strawberry milk was stored at 7°C and sampled every 2-3 days to determine P. odorifer cell density by spread plating on Tryptic Soy Agar + Yeast Extract (TSAYE) with incubation at 25°C for 48 hours. Growth rates of individual P. odorifer strains ranged between 0.71-1.14 CFU/ml/day. Color loss in strawberry milk occurred within 4 days of incubation for P. odorifer strains JWC-L2, JWC-L3 and JWC-2503 which corresponded to average cell densities of 5.74, 4.07 and 4.24 log CFU/ml respectively. By Day 6, all strains (JWC-L2, JWC-L3, JWCU1, JWC-363, JWC-150 and JWC-2503) had degraded FD&C Red No. 3 and had achieved cell densities between 5.78-7.57 log CFU/ml. Additional inoculation studies were performed to compare growth rates of these strains in strawberry and unflavored milk at 4C and 7C. In regular fluid milk at 7°C, growth rates ranged between 0.54- 0.74 log CFU/ml/day. As expected, growth rates were slower at 4°C (0.26-0.46 log CFU/ml/day). The additional sucrose in strawberry milk did not impact the growth rate of P. odorifer strains at these temperatures. Regarding the comparison between strawberry milk and regular fluid, we found that Paenibacillus odorifer doesn’t have significant different growth in both products at the same temperatures. Added ingredients in strawberry milk, specifically sucrose, may be the causative factor for this difference in growth of Paenibacillus odorifer between the two products, although our results did not indicate that this is true when compared to regular fluid milk at both 7°C and 4°C. “Band-Aid” odor is a defect that is commonly reported in chocolate milk and it is typically linked to concentration of 4-ethyl phenol in various food systems. Our approach was to determine if Paenibacillus odorifer could be responsible for this problem. Paenibacillus odorifer strains previously isolated from chocolate milk, regular fluid milk and strawberry milk were used in this study. Chocolate milk inoculated with individual strains and stored at 7ºC for up to 8 days. Inoculated chocolate milk was sampled every 2 days for changes in microbial cell density and odor changes were noted. Results showed that final cell densities of P. odorifer in chocolate milk after 8 days of storage at 7°C ranged from 6.96 to 7.38 log CFU/ml. Average growth rates for each of the strains ranged from 0.71 to 0.97 log CFU/ml/day. Chemical analyses using head-space solid phase microextraction gas chromatography mass spectrometry (HS-SPME-GCMS) was used to measure the concentration of 4-ethyl phenol present on chocolate milk. Using the GCMS we also quantified the volatile phenols extracted from the chocolate milk using standard isotope dilution assay. 4 ethyl phenol concentration did not change on four out of the six strains, but a higher concentration was noted in samples inoculated with strains FSL E2-0150 and FSL A6-0363. At day 0 FSL E2-0150 had 7.30 ug/L, but on day 6 and day 8 it had 24.78 and 21.56 ug/L respectively. FSL A6-0363 followed the same pattern where the day 6 concentration (35.16 ug/L) was higher than the day 0 (4.27 ug/L) and the day 8 (16.26 ug/L). Odors associated with inoculated chocolate milk were not considered to be characteristic of “Band-Aid or “rubber”, but instead, smelled “fermented” or “alcoholic”. Quantitative analysis of the GCMS data supported this observation as the growth of most strains were associated with an increase in 2-pentanol. Concentration levels of this compound were low on day 0 but higher on day 6. On day 8 the levels were lower than in day 6, possibly meaning that production of 2-pentanol is happening at its highest rate when microbial cells are in log phase or that the 2-pentanol is further metabolized over time. Overall results from the two studies in strawberry milk and chocolate milk show that Paenibacillus odorifer is responsible for spoilage events relevant to the dairy industry. This work provides insight to guide mitigation strategies to better control spoilage events in real world settings.
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  • Pending Publication
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  • 2022-12-26 to 2023-07-27

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