Control of Spoilage Bacteria in Lamb Meat by Using Bacteriophage

Abstract

Lamb meat, known for its distinctive taste and high nutritional value, is a significant part of the human diet worldwide. However, it is susceptible to bacterial contamination, which can compromise its quality and safety. This thesis explores the bacterial community dynamics of fresh and chilled backstrap lamb meat and investigates the feasibility of using bacteriophages (viruses that infect and kill bacteria), as a novel method to control bacterial growth, including spoilage and pathogenic bacteria, in lamb meat. The research aimed to assess changes in bacterial communities, particularly specific spoilage organisms (SSO), in Modified Atmosphere Packaged (MAP) lamb meat stored at 4°C over 35 days. Methods used included 16S rRNA-gene-based sequencing, MALDI-TOF MS, and sequencing for bacterial identification. Culture-based analysis using Brain Heart Infusion (BHI) media was used to monitor changes in the bacterial population of Modified atmosphere packaging (MAP) lamb meat stored at 4°C. This comprehensive approach allowed for detailed bacterial community profiling and an understanding of bacterial stability and spoilage trajectories in stored meat products. The investigation revealed a diverse culturable bacterial community in lamb backstrap meat packaged under modified atmosphere conditions over time in chilled storage. MALDI- TOF profiling identified spoilage-associated taxa such as Pseudomonas and Acinetobacter, which thrive in refrigerated, MAP meats. Quantitative assessments of viable counts depicted an increase over time in aerobic bacterial loads and a variable yet overall increasing anaerobic population. These trends were most pronounced post-day 14, indicating significant increases in bacterial numbers present on meat. 1 Principal Coordinate Analysis (PCoA) based on Bray-Curtis, Jaccard, unweighted emperor, and weighted emperor dissimilarities was used to chart successional changes in bacterial community structure and composition based on molecular analysis of total bacterial communities. Changes in bacterial diversity was assessed using Chao1 and Shannon indices, revealing a decrease in both richness and evenness, suggesting a simplification of the communities over time. This trend was mirrored in phylogenetic diversity measures, indicating a potential loss of less dominant taxa. The findings underscore significant shifts in bacterial community structure as typified by the loss and or appearance of new species during meat storage and highlight the importance of bacterial diversity in maintaining meat quality and shelf life. High-throughput DNA sequencing data elucidated predominant and minor bacterial taxa, highlighting the dominance of bacterial groups belonging to the class Gammaproteobacteria and Firmicutes at the end of 35-day incubation period. The data also showed that the community composition changed over time as exemplified by the appearance or disappearance of new species. Additionally, the study also explored isolating bacteriophages from lamb meat as biocontrol agents. Fresh lamb backstrap meat samples were collected, packed under MAP and non-MAP conditions, and used to attempt to isolate bacteriophages active against Pseudomonas fragi, Brochothrix thermosphacta, and Carnobacterium divergens. These taxa were selected for bacteriophage assay because they were the prevalent bacterial groups at the later stages of MAP and non-MAP incubated meat samples. Optimal growth conditions for these isolates were determined with growth curve studies of bacterial cultures incubated at 25°C. The results showed optimal OD600 values of 1.0 for Pseudomonas fragi after 6 hours, 2

0.65 for Brochothrix thermosphacta after 4 hours, and 0.44 for Carnobacterium divergens after 14 hours. Isolation of bacteriophages was performed on Double layer agar plates (DAL) using two methods: Direct isolation method and Isolation of phage using a phage amplification method. The direct isolation method detected plaques only from Pseudomonas fragi DAL plates incubated at 25°C and Carnobacterium divergens DAL plates incubated at 4°C. No plaques were seen on Brochothrix thermosphacta DAL plates. The isolation of phage using the amplification method, applied to samples from days 0 to 35, detected plaques only from Brochothrix thermosphacta DAL plates incubated at 25°C. Unfortunately, none of the plaques could be re-propagated despite many attempts, indicating a need to optimize propagation methodologies. To investigate the potential use and efficacy of phage to reduce the numbers of spoilage bacteria present on meat, a commercially available phage (Pseudomonas phage vB_pfrM-S117) from a culture collection was used to investigate phage treatment of Pseudomonas fragi populations in irradiated meat samples at two time points (Day 2 and Day 7) using Tryptic Soy Agar (TSA) and Cetrimide Fucidin Nalidixic Acid (CFN) agar. On Day 2, bacteriophage treatment reduced bacterial counts by approximately 25% on TSA and 50% on CFN agar compared to samples in which phage were absent. (p < 0.05). By Day 7, the reduction increased to 70% on TSA and 57% on CFN agar. Control samples remained sterile, confirming the effectiveness of the irradiation process. These findings underscore the potential of bacteriophage therapy as a promising biocontrol strategy to managing and reduce Pseudomonas fragi in meat products. Bacteriophage-treated samples showed significant reductions in bacterial counts, highlighting the efficacy of phages in lysing bacterial cells and reducing bacterial loads. This is particularly 3

important for food safety, where controlling spoilage bacteria like Pseudomonas fragi is crucial for extending shelf life and ensuring meat quality. Additionally, bacteriophages preserve the organoleptic properties of food, unlike traditional preservation methods, by naturally controlling bacterial groups responsible for off-flavors and odors. These findings align with previous studies demonstrating the prolonged efficacy of bacteriophages in reducing bacterial populations in various food matrices, highlighting the potential of bacteriophages as effective biocontrol agents. Specifically, the key spoilage bacterial genera on lamb meat after chilled storage were identified. Subsequent assays designed to reduce bacterial population numbers using bacteriophages was carried out and for one of them, P. fragi, the application of bacteriophage (P.phage vB pfrM-S117) successfully resulted in reductions in P. fragi numbers on packaged meat. Overall, this study provides valuable insights into the potential application of bacteriophages for controlling Pseudomonas fragi in meat products. Given the significant reduction in bacterial count, the findings support the potential integration of bacteriophages into existing food safety protocols, offering a natural, targeted, and effective method for enhancing food microbiological safety. Future studies should refine bacteriophage application strategies, address potential resistance issues, and explore long-term stability and effectiveness of phage treatments in various food matrices.