Microbial Profiling of Biltong Processing Using Culture-Dependent and Culture-Independent Microbiome Analysis

Abstract

Biltong is a South African air-dried beef product that does not have a heat lethality step, but rather relies on marinade chemistry (low pH from vinegar, ~2% salt, spices/pepper) in combination with drying at ambient temperature and low humidity to achieve microbial reduction during processing. Culture-dependent and culture-independent microbiome methodologies were used to determine the changes in the microbial community at each step during biltong processing through 8 days of drying. Culture-dependent analysis was conducted using agar-based methods to recover viable bacteria from each step in the biltong process that were identified with 16S rRNA PCR, sequencing, and BLAST searching of the NCBI nucleotide database. DNA was extracted from samples taken from the laboratory meat processing environment, biltong marinade, and beef samples at three stages of processing (post-marinade, day 4, and day 8). In all, 87 samples collected from two biltong trials with beef obtained from each of three separate meat processors (n = six trials) were amplified, sequenced with Illumina HiSeq, and evaluated with bioinformatic analysis for a culture-independent approach. Both culture-dependent and independent methodologies show a more diverse population of bacteria present on the vacuum-packaged chilled raw beef that reduces in diversity during biltong processing. The main genera present after processing were identified as Latilactobacillus sp., Lactococcus sp., and Carnobacterium sp. The high prevalence of these organisms is consistent with extended cold-storage of vacuum-packaged beef (from packers, to wholesalers, to end users), growth of psychrotrophs at refrigeration temperatures (Latilactobacillus sp., Carnobacterium sp.), and survival during biltong processing (Latilactobacillus sakei). The presence of these organisms on raw beef and their growth during conditions of beef storage appears to ‘front-load’ the raw beef with non-pathogenic organisms that are present at high levels leading into biltong processing. As shown in our prior study on the use of surrogate organisms, L. sakei is resistant to the biltong process (i.e., 2-log reduction), whereas Carnobacterium sp. demonstrated a 5-log reduction in the process; the recovery of either psychrotroph after biltong processing may be dependent on which was more prevalent on the raw beef. This phenomenon of psychrotrophic bloom during refrigerated storage of raw beef may result in a natural microbial suppression of mesophilic foodborne pathogens that are further reduced during biltong processing and contributes to the safety of this type of air-dried beef.