Analysis of Microbial Diversity and Dynamics During Bacon Storage Inoculated With Potential Spoilage Bacteria by High-Throughput Sequencing

Abstract

Staphylococcus xylosus, Leuconostoc mesenteroides, Carnobacterium maltaromaticum, Leuconostoc gelidum, and Serratia liquefaciens were investigated for their roles in in the spoilage of sterilized smoked bacon. These five strains, individually and in combination, were applied as starters on sliced bacon at 4-5 log₁₀ CFU/g using a hand-operated spraying bottle and stored for 45 days at 0-4°C. Dynamics, diversity, and succession of microbial community during storage of samples were studied by high-throughput sequencing (HTS) of the V3-V4 region of the 16S rRNA gene. A total of 367 bacterial genera belonging to 21 phyla were identified. Bacterial counts in all the inoculated specimens increased significantly within the first 15 days while the microbiota developed into more similar communities with increasing storage time. At the end of the storage time, the highest abundance of Serratia (96.46%) was found in samples inoculated with S. liquefaciens. Similarly, for samples inoculated with C. maltaromaticum and L. mesenteroides, a sharp increase in Carnobacterium and Leuconostoc abundance was observed as they reached a maximum relative abundance of 97.95 and 81.6%, respectively. Hence, these species were not only the predominant ones but could also have been the more competitive ones, potentially inhibiting the growth of other microorganisms. By analyzing the bacterial load of meat products using the SSO model, the relationships between the microbial communities involved in spoilage can be understood to assist further research.

Keywords: bacon; high-throughput sequencing; microbial diversity; spoilage bacteria; storage.

FIGURE 1

pH values and total volatile basic nitrogen (TVB-N) of bacon inoculated with potential spoilage bacteria, during refrigerated storage. The error bars were derived from the standard deviation between replicates (n = 3). CK, Control; P2, Staphylococcus xylosus; P6, Leuconostoc mesenteroides; P9, Carnobacterium maltaromaticum; P16, Leuconostoc gelidum; P20, Serratia liquefaciens; Pm, The five strains in combination.

FIGURE 2

Dynamics in relative abundance (%) of bacterial taxa at the phylum level in bacon inoculated with potential spoilage bacteria. Each bar represents the relative abundance of each group, below the top-30 abundances (at genus level) data were merged. CK, Control; P2, Staphylococcus xylosus; P6, Leuconostoc mesenteroides; P9, Carnobacterium maltaromaticum; P16, Leuconostoc gelidum; P20, Serratia liquefaciens; Pm, The five strains in combination.

FIGURE 3

Dynamics in relative abundance (%) of bacterial taxa at the genus level in bacon inoculated with potential spoilage bacteria. Each bar represents the relative abundance of each group, below the top 30 abundance (at genus level) data were merged. CK, Control; P2, Staphylococcus xylosus; P6, Leuconostoc mesenteroides; P9, Carnobacterium maltaromaticum; P16, Leuconostoc gelidum; P20, Serratia liquefaciens; Pm, The five strains in combination.

FIGURE 4

Heatmap showing the changes in the microbial communities of bacon inoculated with potential spoilage bacteria. The group heatmaps represent a homogenization of numerical values of corresponding points. CK, Control; P2, Staphylococcus xylosus; P6, Leuconostoc mesenteroides; P9, Carnobacterium maltaromaticum; P16, Leuconostoc gelidum; P20, Serratia liquefaciens; Pm, The five strains in combination.

FIGURE 5

Flower figure showing the unique and shared OTUs of the bacterial communities across different stages. Only with more than five groups can the flower figure be shown. The plotting was carried out after homogenization of all samples. CK, Control; P2, Staphylococcus xylosus; P6, Leuconostoc mesenteroides; P9, Carnobacterium maltaromaticum; P16, Leuconostoc gelidum; P20, Serratia liquefaciens; Pm, The five strains in combination.

FIGURE 6

Non-metric multi-dimensional scaling analysis of the bacterial community structure of bacon inoculated with potential spoilage bacteria. The analysis was based on the relative abundances of all genera of microbiota detected by high-throughput sequencing. CK, Control; P2, Staphylococcus xylosus; P6, Leuconostoc mesenteroides; P9, Carnobacterium maltaromaticum; P16, Leuconostoc gelidum; P20, Serratia liquefaciens; Pm, The five strains in combination.