Effect of chilled storage, packaging, and antimicrobial treatment upon the lamb meat microbiota
Creators: name: ALADHADH, Mohammed Abdullah M
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 310703
subject: Microbial ecology

description: Lamb meat is an excellent source of high-value protein, micronutrients, and vitamins important for the normal functioning of the human body. This makes lamb meat a key component of human diets. Lamb meat has a unique flavour, it is culturally important and is widely consumed in many countries in Africa and the Middle East. Despite this, there is limited scientific information (compared to beef) on the microbiology of lamb meat and how this affects its quality, safety, and nutritional characteristics. This information is important to ensure that lamb meat is safely processed and packaged for human consumption. Therefore, this thesis aimed to assess the bacterial composition of fresh and chilled lamb meat cuts from different sources and the impact of Modified Atmosphere and Vacuum Packaging with and without the presence on nisin on the key meat taxa (including spoilage bacteria) using culture-dependent and independent approaches.

In this study, lamb meat (leg, shoulder, and loin cuts) obtained from three butchers were stored at 5 °C for 6 days. Analysis of the day 0 bacterial OTU numbers after amplicon-based next-generation sequencing indicated no significant differences between the meat cuts. However, substantial differences in bacterial composition (OTU) were observed between samples from different butchers based on nMDS plots. These differences were reflected in a significant ANOSIM R-value of 0.509 at P=0.1 and dissimilarity percentages of 51-75% (SIMPER) when day 0 samples from the three butchers were compared. This indicated that the source (possibly the hygiene and meat processing conditions) of lamb meat could be an important determinant of the meat's bacterial community. Over 6 days, bacterial OTU numbers, Shannon diversity values and Chao1 indices decreased significantly (P &lt; 0.05). Overall, the day 6 lamb meat bacterial community was substantially different in samples from different butchers (SIMPER dissimilarity percentages of 40-57% and an overall R-value of 0.51 at P = 0.1) as a result of chilled storage. Twenty-four different phyla were identified in this study in lamb meat with the most abundant phyla based their abundance order being Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria in days 0 and 6 samples. Lamb meat taxa analysis showed that the most abundant bacterial genus at day 0 was Acinetobacter, followed by Pseudomonas, Psychrobacter, Brochothrix and Photobacterium. Cold storage for 6 days resulted in changes in community composition, with Pseudomonas, Psychrobacter, Acinetobacter, Photobacterium and Brochothrix species as the most abundant taxa. Heatmap analysis showed differences in the bacterial composition between meat cuts and samples from different butchers.

Subsequently, the effects of packaging and cold storage on culturable bacteria within the lamb meat micrbiota were assessed in meat samples subject to modified atmosphere packaging (MAP) (20% CO2 and 80% O2) and vacuum packaging (VP) and with and without nisin (1.8 mg ml-1) treatment with storage at 5 °C for 22 days. Overall, a general increase in the total viable bacterial count was observed in all samples with or without nisin over 22 days. However, the addition of nisin to MAP meat generally reduced the total bacterial viable count at all time-points when compared to non-nisin containing MAP samples. Similar but less pronounced effects were observed in VP lamb meat. The meat packaging method had some influence on the key cultured bacterial groups as assessed with MALDI-TOF MS analysis. At day 22, in MAP-treated meat, Brochothrix, Pseudomonas, Serratia and Carnobacterium species were identified. In chilled MAP + nisin treated-meat, Brochothrix was replaced by Pseudomonas, Carnobacterium and Serratia species as the key genera by day 22. In chilled day 22 VP samples, Carnobacterium, Serratia along with Staphylococcus, Yersinia and Hafnia species were detected. In chilled day 22 VP + nisin meat samples, Carnobacterium and Lactobacillus (in high proportions) and Serratia (in low proportions) were identified. 

The effects of MAP and VP with and without nisin addition were also assessed in the total bacterial community present in chilled lamb meat stored for 22 days using a next-generation sequencing-based approach. The number of bacterial OTUs generally increased in all meat samples over 22 days. The bacterial diversity (Shannon diversity values) was, however, lower at day 22 when compared to day 0 in MAP and VP samples. While nisin application was associated with reduced bacterial diversity (compared to controls) in chilled VP lamb meat, no similar trend was observed in chilled MAP meat. Bacterial diversity was higher in some MAP samples compared to nisin + MAP lamb meat. This suggested that nisin use was ineffective in reducing bacterial diversity in MAP meat. By day 22, bacterial evenness had decreased by up to 3 fold while Chao1 indices had increased in VP and MAP meat with and without nisin. Furthermore, in day 22 MAP-treated meat samples, the dominant phylum was the Actinobacteria (relative abundance of 91%) with Proteobacteria present at a much lower relative abundance (8.7%). In contrast, in nisin-containing MAP samples, the Proteobacteria (86.6%) and the Firmicutes (13,4%) were the key phyla showing nisin use was associated with a shift in bacterial community composition at the phylum level. A similar trend was observed at the genus level. At day 22, Brochothrix species were dominant in MAP treated samples without nisin (75%) followed by Lactobacillus and Lactococcus species while Pseudomonas species (~70%) was the dominant group in MAP + nisin samples. The relative abundance of Brochothrix was higher MAP-treated samples (75 %) when compared to MAP + nisin -treated samples (~12%) suggesting that nisin could be used to control the population of Brochothrix species (meat spoilage bacteria). In chilled VP meat, Proteobacteria (~54%) was the most dominant phylum at day 22 followed by the Firmicutes (~13%) and this trend was unaffected by nisin addition. A similar trend was observed at the genus level.

Overall, this research has shown that the source of lamb meat can be a key determinant of the bacterial composition of lamb meat.  Cold storage significantly reduced bacterial diversity in air-packed, MAP and VP lamb meat.  MALDI-TOF MS, a cheaper and faster method (compared to NGS) was successfully used to identify meat bacteria indicating that it can potentially be used as an accurate adjunct or optional or alternative method to existing NGS-based identification approaches. The packaging method used (VP or MAP) determined the dominant bacterial groups at the end of the storage period. Unlike in VP meat, nisin had no reductive effect on bacterial diversity in MAP samples but substantial reductions in Brochothrix abundance and significant shifts in bacterial community composition were observed.