Microbial Successions Are Associated with Changes in Chemical Profiles of a Model Refrigerated Fresh Pork Sausage during an 80-Day Shelf Life Study

Rarefaction and Shannon diversity estimates of 16S rRNA gene sequences from untreated pork sausage stored at 4°C. Phylogeny-based analysis was used to develop OTU from pyrosequencing data of the day 0 and day 80 samples of the untreated product using the cmALIGN and complete linkage clustering at 97% identity (RDP Pyrosequencing Pipeline). The data were rarefied using the RDP pipeline rarefaction application (y axis on left), while the Shannon diversity was estimated using the standard equation Σprop1 × ln prop1, where prop1 is the proportion of each individual taxon of the total sequences from a sample (y axis on the right).

Dynamic changes in relative abundances of taxa over time in untreated pork sausage during refrigeration at 4°C. The relative abundances of dominant taxa identified by pyrosequencing of 16S rRNA gene amplicons are plotted from samples of the untreated pork sausage after thawing and storage at refrigeration over 80 days. Graphs were plotted with each of the 15-day time points over the 80-day storage period. Taxa are colored by phyla, with Firmicutes in shades of blue-purple, Proteobacteria in shades of green, Bacteroidetes in yellow, and Actinobacteria in red.

High-resolution snapshot of microbial population dynamics in untreated pork sausage. (A) Total mesophilic APCs, psychrotrophic APCs, and LAB, Enterobacteriaceae, coliforms, and generic E. coli were enumerated from untreated pork sausage after thawing and storage at refrigeration temperature for 80 days. The log-transformed data from each treatment group is plotted from each of the 15-day time points taken over the 80-day refrigeration period, and the means and standard deviations of the log-transformed data are plotted at each of the 15-day time points. The pH of the samples is plotted on the second y axis on the right. The light red arrow indicates onset of sour odors, and the red arrow at day 45 marks the onset of a sour, putrid, or spoiled odor that would yield the product objectionable. (B) Relative abundances of taxa of interest from the pyrosequencing data derived from paired untreated pork sausage after thawing and storage at refrigeration temperatures for the same 80-day period.

High-resolution snapshots of microbial population dynamics in untreated and treated pork sausage. (A) The total mesophilic APCs, psychrotrophic APCs, and lactic acid bacteria, and Enterobacteriaceae were enumerated from pork sausage containing 0, 3, 4, 5, and 6% LD. The log-transformed data from each treatment group are plotted from each of the 15-day time points taken over the 80-day refrigeration period, along with the standard deviations of the log-transformed data (whiskers). (B) The relative abundances of dominant taxa identified by pyrosequencing of 16S rRNA gene amplicons are plotted from paired samples taken at 15-day time points over the course of the 80-day shelf life (x axis) for the 3, 4, 5, and 6% LD treatments.

Dynamic behavior of different Lactobacillus species in untreated and LD-treated pork sausage. (A) The total mesophilic APCs, psychrotrophic APCs, and LAB, and Enterobacteriaceae were enumerated from pork sausage containing 0, 3, 4, 5, and 6% LD. The log-transformed data from each treatment group are plotted from each of the 15-day time points taken over the 80-day refrigeration period. (B) The relative abundances of the six different species of Lactobacillus that were detectable in the pyrosequencing data are plotted over time across the untreated and treated samples.

Correlation analysis of detectable taxa in the microbiota of the pork sausage. Correlations were calculated between all pairwise combinations of taxa for each time point in treated and untreated samples. The correlation matrix was sorted to group samples together with similar behavior. The scale for the R values is shown at the bottom left of the figure, with dark blue corresponding to positive R values and bright red corresponding to negative R values. Groups A to E show taxa that share similar dynamics.

Dynamic behavior of correlation group E taxa in untreated and treated pork sausage. (A) Total mesophilic APCs, psychrotrophic APCs, and LAB, and Enterobacteriaceae were enumerated from pork sausage containing 0, 3, 4, 5, and 6% LD. The log-transformed data from each treatment group are plotted from each of the 15-day time points taken over the 80-day refrigeration period. (B) Relative abundances of group E taxa from the pyrosequencing data are at the 15-day sampling intervals during the 80-day storage at refrigeration. The pH of the samples is plotted in royal blue.

Box-and-whisker plots of responses from individual eNose sensors showing statistically significant responses. Each plot shows the responses of a single sensor from the eNose array for each individual sausage sample. Boxes represent the 95% confidence intervals, and whiskers depict maximum and minimum values for sensor responses over the 60-s measurements. The time points (days) are indicated above each individual sample, and boxes are color-coded as depicted at the bottom. Individual sensors detect aromatics (W1C), ammonia (W3C), aromatic-aliphatics (W5C), broad-range alcohols (W2S), and broad range with sensitivity to nitrogen oxides (W5S). The values for days 30 to 80 were significant (P < 0.05) as determined by ANOVA for each of the sensors shown, with the exception of the W5S sensor, where only the day 60 and day 80 untreated samples were significant.

Association of eNose sensor responses and relative of individual taxa. (A) Correlation matrix (R values) of the relative abundances of microbial taxa measured by pyrosequencing and the responses of individual eNose sensors. The color scale on the left and the positions of the eNose rows and rows of taxa are indicated on the right. (B) Dot plots of the relative abundances of specific taxa and responses of individual eNose sensors in untreated sausage samples. The correlation coefficients (R² values) are highlighted by color-coded boxes that correspond to regression lines and data points from each sensor.

Source-tracking taxa from the pork sausage and its spice blend ingredient. The distribution of taxa is illustrated from the day 0 sample of the untreated pork sausage and from a sample of the spice blend mix that was used to develop the model pork sausage. The dendrogram was developed from representative sequences derived from each of the 82 different genera that were detected in the samples. Representative sequences were developed from each of the genus-level Multi-CLASSIFIER taxonomic bins by first collapsing to 97% identity using cmALIGN and then aligning it to a phylogenetic framework established from 16S reference sequences. The individual pie charts depict species-level OTU for each of the 82 genera and the relative abundance of each OTU in the spice blend (dark blue) or the meat-spice blend (light blue) is illustrated by the proportion in each pie.