Hilde Nissen

The storage life of beef and pork packaged in an atmosphere with low carbon monoxide and high carbon dioxide

Ground beef, beef loin steaks and pork chops were packaged in modified atmospheres of 0.4% CO/60% CO(2)/40% N(2) and 70% O(2)/30% CO(2). In addition ground beef was packaged in clipped chub packs, beef loin steaks were vacuum packaged, and pork chops were packaged in an atmosphere of 60% CO(2)/40% N(2) with each pack containing an O(2) absorber. The packs were stored in the dark at 4 or 8°C for up to 21 days. Meat in 0.4% CO/60% CO(2)/40% N(2) had a stable bright red colour that lasted beyond the time of spoilage. The storage lives in this gas mixture at 4°C, as limited by off-odours, were 11, 14 and 21 days for ground beef, beef loin steaks and pork chops, respectively. The 70% O(2)/30% CO(2) atmosphere resulted in an initially bright red to red colour of the meat, but the colour was unstable and off-odours developed rapidly. The off-odours probably were caused by Brochothrix thermosphacta, which grew in all meat types, or by pseudomonads in ground beef. Meat stored in chub packs, vacuum packs or 60% CO(2)/40% N(2) with an O(2) absorber developed off-odours and microflora similar to those of meat in 0.4% CO/60% CO(2)/40% N(2), but with less acceptable appearances. These results show that a low CO/high CO(2) atmosphere is effective for preserving retail-ready meat. ©

Addition of 2.5% lactate and 0.25% acetate controls growth of Listeria monocytogenes in vacuum-packed, sensory-acceptable servelat sausage and cooked ham stored at 4°C

A study of the inhibitory effects of propylparaben and of a combination of lactate and acetate against growth of Listeria monocytogenes in inoculated liquid medium, sliced servelat sausage and cooked ham, were performed using rifampicin resistant Listeria strains in inoculation experiments. A consumer acceptance test of products produced with and without the compounds was also performed. Propylparaben was found to be effective in a model liquid non-fat medium, but was without effect in the actual products. This illustrates the potential pitfalls in translating results from studies in liquid media to fat-containing food products. The combined inhibitory and sensory results showed that a mixture of 2.5% lactate and 0.25% acetate (w/w, calculated on the water phase), could be used to increase the margins of safety for sliced and spreadable vacuum-packed ready-to-eat cooked meat products stored for 4-6 weeks. In addition, strict control of temperature during production and storage is very important.

High levels of background flora inhibits growth of Escherichia coli O157:H7 in ground beef.

The influence of natural background flora under aerobic and anaerobic incubation on the growth of Escherichia coli O157:H7 in ground beef was investigated. The background flora from eight different commercial ground beef were added to ground beef spiked with E. coli O157:H7 and stored either aerobically or anaerobically at 12 degrees C. The results showed that the presence of a large number of background bacteria in the ground meat inhibited the growth of E. coli O157:H7 both aerobically and anaerobically. Inhibition was more pronounced under anaerobic conditions. The background floras consisted mainly of lactic acid bacteria of which approximately 80% were Lactobacillus sakei. These results show the importance of the natural background flora in meat for inhibition of growth of E. coli O157:H7.

Development and Evaluation of a 16S Ribosomal DNA Array-Based Approach for Describing Complex Microbial Communities in Ready-To-Eat Vegetable Salads Packed in a Modified Atmosphere

ABSTRACT There is a clear need for new approaches in the field of microbial community analyses, since the methods used can be severely biased. We have developed a DNA array-based method that targets16S ribosomal DNA (rDNA), enabling the direct detection and quantification of microorganisms from complex communities without cultivation. The approach is based on the construction of specific probes from the 16S rDNA sequence data retrieved directly from the communities. The specificity of the assay is obtained through a combination of DNA array hybridization and enzymatic labeling of the constructed probes. Cultivation-dependent assays (enrichment and plating) and cultivation-independent assays (direct fluorescence microscopy and scanning electron microscopy) were used as reference methods in the development and evaluation of the method. The description of microbial communities in ready-to-eat vegetable salads in a modified atmosphere was used as the experimental model. Comparisons were made with respect to the effect of storage at different temperatures for up to 12 days and with respect to the geographic origin of the crisphead lettuce (Spanish or Norwegian), the main salad component. The conclusion drawn from the method comparison was that the DNA array-based method gave an accurate description of the microbial communities. Pseudomonas spp. dominated both of the salad batches, containing either Norwegian or Spanish lettuce, before storage and after storage at 4°C. The Pseudomonas population also dominated the batch containing Norwegian lettuce after storage at 10°C. On the contrary, Enterobacteriaceae and lactic acid bacteria dominated the microbial community of the batch containing Spanish lettuce after storage at 10°C. In that batch, the Enterobacteriaceae also were abundant after storage at 4°C as well as before storage. The practical implications of these results are that microbial communities in ready-to-eat vegetable salads can be diverse and that microbial composition is dependent both on the origin of the raw material and on the storage conditions.

Explorative Multivariate Analyses of 16S rRNA Gene Data from Microbial Communities in Modified-Atmosphere-Packed Salmon and Coalfish

ABSTRACT Modified-atmosphere packaging (MAP) of foods in combination with low-temperature storage extends product shelf life by limiting microbial growth. We investigated the microbial biodiversity of MAP salmon and coalfish by using an explorative approach and analyzing both the total amounts of bacteria and the microbial group composition (both aerobic and anaerobic bacteria). Real-time PCR analyses revealed a surprisingly large difference in the microbial loads for the different fish samples. The microbial composition was determined by examining partial 16S rRNA gene sequences from 180 bacterial isolates, as well as by performing terminal restriction fragment length polymorphism analysis and cloning 92 sequences from PCR products of DNA directly retrieved from the fish matrix. Twenty different bacterial groups were identified. Partial least-squares (PLS) regression was used to relate the major groups of bacteria identified to the fish matrix and storage time. A strong association of coalfish with Photobacterium phosphoreum was observed. Brochothrix spp. and Carnobacterium spp., on the other hand, were associated with salmon. These bacteria dominated the fish matrixes after a storage period. Twelve Carnobacterium isolates were identified as either Carnobacterium piscicola (five isolates) or Carnobacterium divergens (seven isolates), while the eight Brochothrix isolates were identified as Brochothrix thermosphacta by full-length 16S rRNA gene sequencing. Principal-component analyses and PLS analysis of the growth characteristics (with 49 different substrates) showed that C. piscicola had distinct substrate requirements, while the requirements of B. thermosphacta and C. piscicola were quite divergent. In conclusion, our explorative multivariate approach gave a picture of the total microbial biodiversity in MAP fish that was more comprehensive than the picture that could be obtained previously. Such information is crucial in controlled food production when, for example, the hazard analysis of critical control points principle is used.

Survival and growth of Escherichia coli O157:H7, Yersinia enterocolitica and Salmonella enteritidis on decontaminated and untreated meat

Decontamination of meat or carcasses may have an effect in reducing the number of pathogens. Recontamination with other pathogens during cutting or packaging may, however, result in higher growth on decontaminated than on untreated meat due to the lack of competing non-pathogenic microorganisms. In this study we compared the growth of pathogens during storage at 10°C (worst case condition) on untreated meat and meat that had been decontaminated by steam vacuuming combined with spraying with 0.2 M lactic acid. Salmonella enteritidis inoculated on chicken multiplied quickly and reached log 7 cfu per cm(2) after 4 days of aerobic storage at 10°C, but growth was not significantly higher on decontaminated than on untreated chicken. The number of Yersinia enterocolitica inoculated on decontaminated pork skin reached log 9 cfu per cm(2) after 5 days of aerobic storage at 10°C. Overall, growth on vacuum-packed decontaminated and untreated pork under the same conditions was not significantly different, although there tended to be less growth on the untreated samples. The number of Escherichia coli O157:H7 on decontaminated beef increased by nearly 3 log cycles after 5 days of aerobic storage at 10°C compared to only a 1 log cycle increase on untreated beef. For the vacuum-packed beef, growth of E. coli O157:H7 on the fresh meat was very slow, while there was about a 3 log increase on the decontaminated beef. A higher average growth on the decontaminated beef was also found in an experiment with a very low inoculum (27 cfu per cm(2)). During storage of vacuum-packed samples there was multiplication of E. coli O157:H7 on the decontaminated beef, but virtually none on the untreated beef. This study shows that multiplication of S. enteritidis on chicken and Y. enterocolitica on pork skin was not significantly higher on decontaminated compared to untreated meat. The increased multiplication of E. coli O157:H7 on decontaminated beef, especially when vacuum-packed, gives cause for concern. Preventive measures might be a strict HACCP approach to the handling of the decontaminated meat before packaging or use of a protective culture of lactic acid bacteria.

Development and application of new nucleic acid-based technologies for microbial community analyses in foods.

Several challenges still persist in the analysis of microorganisms in foods, particularly in studies of complex communities. Nucleic acid-based methods are promising tools in addressing new questions concerning microbial communities. We have developed several new methods in the field of nucleic acid-based microbial community analyses. These methods cover both sample preparation and detection approaches. The sample preparation method involves simplified DNA purification using paramagnetic beads. As an extension of this method, the same paramagnetic beads are used for both cell separation and DNA purification. This enables full automation. The separate detection of viable and dead bacteria is a major issue in nucleic acid-based diagnostics. We have applied a living/dead dye that binds covalently to DNA and inhibits the PCR from dead cells. In addition, a DNA array-based detection assay has been developed. The assay combines the specificity obtained by enzymatic labeling of DNA probes with the possibility of detecting several targets simultaneously by DNA array hybridization. In combination with 16S rDNA amplification, this is a promising tool for community analyses. Also, we have developed a novel approach for multiplex quantitative PCR. The multiplex PCR has been combined with our DNA array-based detection method. Finally, we are now in the process of adapting a system for monitoring microbial growth and death in real-time through the tagging of bacteria with green fluorescent protein (GFP) combined with fluorescence detection using a high-resolution confocal laser scanner.

Comparison between the growth of Yersinia enterocolitica, listeria monocytogenes, escherichia coli O157:H7 and salmonella spp. in ground beef packed by three commercially used packaging techniques.

Growth of the pathogens Yersinia enterocolitica, Listeria monocytogenes, Escherichia coli O157:H7 and strains of Salmonella were compared in ground beef packed in modified atmospheres of 60% CO2/40% N2/0.4% CO (high CO2/low CO mixture), 70% O2/30% CO2 (high O2 mixture) and in chub packs (stuffed in plastic casings). The ground beef was inoculated with rifampicin-resistant or nalidixic acid/streptomycin-resistant strains of the pathogens (final concentration 10(2) - 10(3) bacteria/g) and stored at 4 and 10 degrees C for up to 14 days. At 4 degrees C the shelf life, based on colour stability and background flora development, was prolonged for the high CO2/low CO mixture compared to the two other packaging methods, but at 10 degrees C the shelf life was < 8 days for all the packaging methods. Growth of Y. enterocolitica was nearly totally inhibited both at 4 and 10 degrees C in the high CO2/low CO mixture, while the bacterial numbers in the samples packed in the high O2 mixture increased from about 5 x 10(2) bacteria/g at day 0 to about 10(4) at day 5 at 4 degrees C and to 10(5) at 10 degrees C. Growth in the chub packs was even higher. L. monocytogenes showed very little growth at 4 degrees C in all treatments. At 10 degrees C there was slow growth from about 5 x 10(3) bacteria/g to about 10(4) at day 5 in the high CO2/low CO mixture, while the numbers in the high O2 mixture and the chub packs were about 10 times higher. Growth of E. coli O157:H7 at 10 degrees C in the ground beef was nearly totally inhibited in both the high CO2/low CO mixture and the high O2 mixture. Growth in the chub packs was higher, as the number of bacteria increased 3 log in 5 days. The Salmonella strains (S. typhimurium, S. dublin, S. enteritidis and S. enterica 61:k:1,5,(7)) in the ground beef stored at 10 degrees C for 5 and 7 days grew to a higher number in the high CO2/low CO mixture than in the high O2 mixture. This study shows that the growth of Y. enterocolitica and L. mononcytogenes in ground beef stored in the high CO2 /low CO mixture was not increased as a result of prolonging the shelf life. However, the observed growth of strains of Salmonella at 10 degrees C in this mixture and in chub packs does emphasise the importance of temperature control during storage.

Comparison of the use of rRNA probes and conventional methods in identifying strains of Lactobacillus sake and L. curvatus isolated from meat

Published 23S rRNA directed probes for the species Lactobacillus sake and L. curvatus were used for rapid identification of lactic acid bacteria (LAB) isolated from meat. Of 96 strains of LAB from pork loin, 23 hybridised with the probe for L. sake and 16 with the probe for L. curvatus. Of 20 LAB strains from bologna, 19 strains hybridised with L. sake. By comparison with conventional biochemical tests, the probes provided a reliable identification.

Identification of Carnobacterium spp. and Leuconostoc spp. in meat by genus‐specific 16S rRNA probes

Oligonucleotide probes specific for Carnobacterium and Leuconostoc species were constructed from the variable regions of 16S rRNA obtained from the literature and sequence data bases. The probes were hybridized with crude nucleic acid extract from 32 type strains of lactic acid bacteria (LAB) commonly found on meat. Two of the probes hybridized only to the four Carnobacterium species whereas the other two hybridized only to five of the six Leuconostoc species tested. The probes were also hybridized with nucleic acids from unknown strains of LAB. The identification was consistent with the results of biochemical tests used to characterize the two genera.