Isolation and Characterization of Causative Organisms of “Blown-pack” Phenomenon in Ground Beef Chubs

ABSTRACT

“Blown-pack” spoilage of ground beef chubs is characterized by the accumulation of copious amounts of gas in the package, which leads to pack distention and spoilage and consequently results in substantial economic losses in the beef industry. The objective of this study was to enumerate and identify the microflora associated with blown appearance of spoiled ground beef chubs obtained from commercial beef processing plants in the United States. Ground beef samples in blown-pack packaging were analyzed for the following microflora using selective and non-selective media: total mesophilic bacteria, total psychrotrophic bacteria, lactic acid bacteria, Enterobacteraceae, Pseudomonas, and Brochothrix. In addition, samples were enriched in gas assay media containing Durham tubes at 7°C and 25°C until visual sings of gas productions were observed. Gas producing organisms were isolated and identified using phenotypic characterization and 16s rRNA sequencing. Total mesophilic plat counts ranged from 3 to 5 log CFU/g while psychrotrophic bacterial counts ranged from 5 to 7 log CFU / g, 3 log CFU /g and less than 2 log CFU /g, respectively. Leuconostoc gasicomitatum, L. gelidum, L. mesenteroides, Hafnia alvei and Clostridium spp. were identified as the most common organisms from the samples. This study presents the bacterial populations in the “blow-pack” spoilage of ground beef chubs and identified potential gas-producing spoilage organisms.

OBJECTIVES

  1. Evaluate the overall microbiological profile of gassed “Blown Pack” ground beef chubs
  2. Identify potential gas-producing spoilage organisms associated with commercial gassed ground beef chubs

INTRODUCTION

“Blown Pack”, a quality defect characterized by excessive amount of gas production and swelling, has been a significant problem associated with ground beef chubs. “Blown Pack” spoilage affects the beef industry dramatically by financial losses for returned product and customer dissatisfaction. Previous studies suggested that in samples which were subjected to temperature abuse the causative organisms of the “blown pack” spoilage could be species of the Enterobacteriaceae family, specifically Serratia liquefaciens, Enterobacter aerogenes, and Hafnia alvei. However, in the absence of temperature abuse the causative agents of “Blown Pack” spoilage samples could be lactic acid bacteria and/or psychrotrophic Clostridium spp. The psychrotrophic Clostridium spp. are generally associated with gas production in chilled vacuum-packed whole muscle meats and no literature addresses whether these species also affect ground beef product quality. In the present study, nine spoiled ground beef chubs showing gas distention obtained from three commercial beef processing plants were tested for the potential gas-producing spoilage organisms.

METHODS

Ground Beef Chubs 

Ground beef chubs (n=9) exhibiting visual signs of gas production were obtained from 2 commercial beef processing plants in the United States (establishments A and B).

Enumeration of bacteria 

Ground beef samples (50g) were analyzed for microbial populations including: total plate counts (TPC), anaerobic total plate counts (An-TPC), psychrotrophic total plate counts (Psy-TPC), psychrotrophic anaerobic total plate counts (Psy-An-TPC), Enterobacteriaceae counts (EBC), total coliform counts (TCC), E. coli counts (ECC), lactic acid bacteria (LAB) counts, Pseudomonas spp. counts, and Brochothrix thermosphacta counts. The following media and incubation conditions were used: 3M™ Petrifilm™ Aerobic Count Plates (3M™, St. Paul, MN) for TPC and An-TPC, incubated aerobically and anaerobically respectively at 35°C for 24-48 h; 3M™ Petrifilm™ Aerobic Count Plates (3M™, St. Paul, MN) for Psy- TPC and Psy-An-TPC, incubated aerobically and anaerobically respectively at 35°C for 24-48 h; 3M™ Petrifilm™ Enterobacteriaceae count Plates (3M™, St. Paul, MN) for EBC, incubated at 35°C for 24-48 h; 3M™ Petrifilm™ E.coli and coliform count Plates (3M™, St. Paul, MN) for ECC and TCC, incubated at 35°C for 24-48 h; Pseudomonas isolation agar (PIA, Oxoid) for Pseudomonas spp. count, incubated at 25°C for 48h, de Man, Rogosa and Sharpe agar (MRS agar, Oxoid) for LAB counts, incubated at 25°C for 72-96 h; streptomycin thallous acetate agar (STA agar, Oxoid) with STA selective supplement (Oxoid) for Brochothrix spp. counts, incubated at 25°C for 48h.

Isolation and identification of Gas Producing Microflora 

MRS agar. Lactic acid bacteria (3-5 isolates/sample) were randomly taken from MRS agar plates and cultured on MRS agars again. Each pure culture was identified using phenotypic characterization and 16s rRNA sequencing.

Cooked meat medium. Portions (10 g) of ground beef samples were enriched in tubes containing cooked meat medium (CM, Oxoid) equipped with Durham vials. The tubes were incubated anaerobically at 7°C and 25°C for 14 and 3 days respectively until visual signs of gas production were observed. Once gas production was observed, the enrichment was streaked onto pre-reduced Columbia Blood agar and incubated anaerobically at 7°C and 25°C for 14 and 3 days respectively. Plates exhibiting colony growth were streaked for purity onto tryptic soy agar containing 5% sheep blood (SBA; Becton Dickinson and Co.) and characterized via phenotypic identification and 16S rRNA sequencing.

PCR detection of psychrophilic Clostridium spp. Ground beef samples were enriched in tubes containing cooked meat medium and incubated anaerobically at 7°C and 25°C for 14 and 3 days respectively. Following enrichment, all samples were screened using PCR reactions (Molecular Epidemiology, Inc., Seattle, WA) designed to detect the psychrotrophic Clostridia including Cl. algidixylanolyticum, Cl. psychrophilum, Cl. frigidicarnis, Cl. vincentii, Cl. gasigenes, Cl. estertheticum, and Cl. algidicarnis/Cl. putrefaciens.

RESULTS

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Establishment A (3 samples)

  • The low counts of Enterobacteriaceae detected in the 3 samples suggest that this group of organisms was not the causative agent of the “blown pack” spoilage.
  • The low numbers of total plate counts, anaerobic total plate count and Enterobacteriaceae indicate that spoilage had occurred at non-abusive storage temperature.
  • Three kinds of lactic acid bacteria including Leuconostoc gasicomitatum, L. gelidum, L. mesenteroides were isolated. These bacteria have been previously reported as causative agents in “blown pack” vacuum beef samples by other authors, therefore they were probably the causative organisms of the 3 “blown pack” ground beef chubs from the establishment A.

Establishment B (6 samples)

  • The Enterobacteriaceae coutns detected in the 6 samples from the establishment B were higher than those of the 3 samples from the establishment A, but they were not the dominant flora and therefore they were not the sole causative organisms of “blown pack” spoilage.
  • The fact that the psychrotrophic anaerobic total plate counts were higher than the LAB counts suggest that the psychrotrophic anaerobic group may represent Clostridium spp. in the spoilage flora.
  • Lactic acid bacteria identified by sequencing and Clostridia spp. detected by PCR in the 6 “blown pack” samples in the establishment B have been previously reported as causative agents in “blown pack” vacuum beef samples by other authors. It is, therefore, possible that “blown pack” spoilage of the 6 samples were a synergistic effect of several groups of bacteria.