Elisabeth Borch

Bacterial spoilage of meat and cured meat products

The influence of environmental factors (product composition and storage conditions) on the selection, growth rate and metabolic activity of the bacterial flora is presented for meat (pork and beef) and cooked, cured meat products. The predominant bacteria associated with spoilage of refrigerated beef and pork, are Brochothrix thermosphacta, Carnobacterium spp., Enterobacteriaceae, Lactobacillus spp., Leuconostoc spp., Pseudomonas spp. and Shewanella putrefaciens. The main defects in meat are off-odours and off-flavours, but discolouration and gas production also occur. Bacteria associated with the spoilage of refrigerated meat products, causing defects such as sour off-flavours, discolouration, gas production, slime production and decrease in pH, consist of B. thermosphacta, Carnobacterium spp. Luctobacillus spp. Leuconostoc spp. and Weissella spp. Analysis of spoilage as measured by bacterial and chemical indicators is discussed. It is concluded that a multivariate approach based on spectra of chemical compounds, may be helpful in order to analyse spoilage, at least for spoilage caused by lactic acid bacteria. The consequences of bacteria bacteria interactions should be evaluated more.

Hazard identification in swine slaughter with respect to foodborne bacteria

Swine slaughter is an open process with many opportunities for the contamination of the pork carcass with potentially pathogenic bacteria; however, it does not contain any point where hazards are completely eliminated. Data on the prevalence of various pathogenic bacteria (Aeromonas hydrophila, Campylobacter coli/jejuni, Listeria monocytogenes, Salmonella spp., Staphylococcus aureus and Yersinia enterocolitica) in pigs, their growth and survival characteristics and ability to become established on the slaughter line are presented. The presentation covers the processing steps from lairage to chilling and is based on swine slaughter practices in Denmark, Norway and Sweden. The major contamination points during swine slaughter are pig-related, such as faecal and pharyngeal, and environmental. HACCP (Hazard Analysis Critical Control Point) and GMP (Good Manufacturing Practice) in swine slaughter must be focused on limiting this spread. The pathogenic bacteria show differences in their general mechanism of distribution. The major contamination source of Campylobacter spp., Salmonella spp. and Y. enterocolitica is the pig, and the contamination of carcasses with these bacteria may be limited, provided that only strict slaughtering procedures are used. Other organisms such as Aeromonas spp., L. moncytogenes/Listeria spp. and S. aureus can be endemic in the processing environment. Since endemic bacteria can be controlled by proper cleaning and disinfection, these organisms are useful as indicators for the success of GMP rules. The following affiliation to CPs or CCPs made for specific steps during slaughter and dressing may serve as a guidance: (i) lairage (CP), (ii) killing (CP), (iii) scalding (CP), (iv) dehairing (CP), (v) singeing/flaming (CP), (vi) polishing (CP), (vii) circumanal incision and removal of the intestines (CCP), (viii) excision of the tongue, pharynx, and in particular the tonsils (CCP), (ix) splitting (CP), (x) post mortem inspection procedures (CCP) and (xi) deboning of the head (CCP).

Lipolysis, proteolysis and formation of volatile components during ripening of a fermented sausage with Pediococcus pentosaceus and Staphylococcus xylosus as starter cultures

Bacterial growth, formation of acids, lipolysis, proteolysis, fat oxidation, formation of volatile compounds and flavour characteristics were followed during ripening and storage of a fermented sausage. The starter culture used was composed of Pediococcus pentosaceus and Staphylococcus xylosus. The number of Pediococcus sp. increased by 1.5 log cfu/g during the first day of processing and remained constant at this level for 3 weeks. The corresponding initial increase in the numbers of Staphylococcus sp., 0.4 log cfu/g, was followed by a rapid decrease in the viable numbers. Lactic acid, mainly d-lactic acid, and acetic acid were formed during ripening. The triglycerides were hydrolysed to 1,2-diglycerides and free fatty acids at the beginning of ripening, followed by the formation of 1,3-diglycerides and monoglycerides, indicating lipolytic activity. Moreover, the nonprotein nitrogen increased during ripening as a result of the proteolytic activity. Most of the changes with respect to pH, formation of d-lactic acid, acetic acid, peroxides and flavour development occurred during the initial 3 days of ripening, when growth of Pediococcus sp. and Staphylococcus sp. occurred. Lipolysis as well as proteolysis continued after this initial period. The volatile compounds identified belonged to several chemical families, viz. aliphatic and aromatic hydrocarbons, aldehydes, ketones, alcohols, phenols, carboxylic acids, esters, nitrogen compounds, sulphur compounds, chloride compounds, terpenes and furans. Many of the volatile compounds probably originated from smoke and seasoning (onion/garlic and pepper), while others were a result of the activities of muscle enzymes and bacteria.

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.

Bacteriological safety issues in red meat and ready-to-eat meat products, as well as control measures

The importance of Eschericha coli O157, Listeria monocytogenes and Salmonella typhimurium DT104 as meat-borne pathogens is well established. Pathogenic bacteria such as Aeromonas spp., Arcobacter spp., psychrotrophic Bacillus cereus, Campylobacter spp., Clostridium botulinum and non-invasive Listeria monocytogenes can be regarded as rookies, but not yet firmly associated with todays production of red meat and meat products. The development of PCR and other DNA-based techniques will shed new light on so called emerging pathogens. Important safety issues in meat production, such as insufficient cleaning and disinfection (including the stable/lairage, processing environment), carcass decontamination and chilling, and cross contamination are discussed. Furthermore, probability modelling of survival and growth is identified as an important way to achieve a better understanding of how to deal with the complexity of further processing, including heat treatment and storage.

Comparison of shelf life of vacuum-packed pork and beef.

The relevance of the intrinsic factors of meat to the sensorial shelf life of vacuum-packed, cold-stored minced pork and beef was investigated. The intrinsic factors studied were the pH and the concentrations of glycogen, glucose, glucose-6-phosphate, l-lactate and fat. The initial bacterial loading was the same on all the meat. High correlations were found between the initial values of pH, fat and l-lactate, respectively, and the rate of spoilage. Using partial least square regression, it was shown that changes in the pH and the concentrations of l-lactate and glucose-6-phosphate during storage were able to explain 68% of the variation observed in the rate of spoilage. No relationship was found between spoilage and the origin of the meat (pork or beef).

Detection of pathogenic Yersinia enterocolitica in enrichment media and pork by a multiplex PCR: a study of sample preparation and PCR-inhibitory components

A multiplex PCR assay including sample preparation was developed to detect viable pathogenic strains of Yersinia enterocolitica in PCR-inhibitory samples, such as pork and enrichment media. The method developed was used to simultaneously detect the plasmid-borne virulence gene yadA and a Yersinia-specific region of the 16S rRNA gene. According to an auto-agglutination test for virulence-plasmid-bearing strains of Y. enterocolitica, all potential pathogenic strains tested were detected by the assay. A DNA extraction procedure, an aqueous two-phase system composed of polyethylene glycol 4000 and dextran 40 and a buoyant density centrifugation method, based on Percoll, were compared with regard to their efficiency in separating Yersinia enterocolitica from PCR inhibitors originating from enrichment media and pork. Using the density gradient centrifugation method resulted in a detection level of 4.0 x 10(2) CFU Y. enterocolitica per ml enrichment media. To ensure detection of viable bacteria a short enrichment step was included in the sample preparation together with the density gradient centrifugation. When this sample treatment method was evaluated with a selective enrichment medium together with a background flora inoculated with approximately 1.0 x 10(1) CFU per ml of Y. enterocolitica and incubated at 25 degrees C, a positive PCR result was obtained after 6 to 8 h. Our results indicate that selective enrichment followed by buoyant density gradient centrifugation provides a convenient and user-friendly sample preparation method prior to PCR.

Reduction of Yersinia enterocolitica and Listeria spp. on pig carcasses by enclosure of the rectum during slaughter

By sealing off the rectum with a plastic bag immediately after it had been freed, the spread of Y. enterocolitica O:3/biovar 4 to pig carcasses could be considerably reduced. The organism was recovered from only 0.8% of carcasses when the plastic bag technique was employed. Y. enterocolitica O:3/biovar 4 was recovered from 10% of pig carcasses when eviscerating procedures did not include the use of the plastic bag technique. There was thus an obvious risk of the bacteria further contaminating meat cuts and other meat products. The plastic bag technique was effective both in connection with manual excision of the rectum/low throughput (90 per h), and mechanical freeing of the rectum/high slaughter rate (240 per h). L. monocytogenes was not detected in any of the samples taken from 120 pig carcasses in Norway or from 120 pig carcasses in Sweden. The plastic bag technique was used on half of these pigs. L. innocua was tested for in 120 pigs slaughtered in Sweden. The bacterium was recovered from 33% of the carcasses eviscerated without using a plastic bag, and from 10% of the carcasses in which this technique was employed. The results suggested that there were other, non-faecal, sources of contamination. Other measures in addition to the plastic bag technique are therefore required to limit the spread of Listeria spp. By incorporating the plastic bag technique into the slaughtering procedures, the meat industry would contribute to preventing the dissemination of Y. enterocolitica and other pathogens which spread via the faeces.

Using an electronic nose for determining the spoilage of vacuum-packaged beef

The use of an electronic nose in the quantitative determination of the degree of spoilage of vacuum-packaged beef was evaluated. Beef from four different slaughterhouses was sliced, vacuum-packaged and stored at 4 degrees C for 8 weeks. Samples were withdrawn for bacterial (aerobic bacteria, lactic acid bacteria, Brochothrix thermosphacta, Pseudomonas and Enterobacteriaceae) and sensorial analyses and analysis of the volatile compounds during the storage period. A trained panel was used for the sensorial evaluations. The volatile compounds were analysed using an electronic nose containing a sensory array composed of 10 metal oxide semiconductor field-effect transistors, four Tagushi type sensors and one CO2-sensitive sensor. Four of the 15 sensors were excluded due to lack of response or overloading. Partial least-squares regression was used to define the mathematical relationships between the degree of spoilage of vacuum-packaged beef, as determined by the sensory panel, and the signal magnitudes of the sensors of the electronic nose. The mathematical models were validated after 6 months using a new set of samples. The stability of the sensors during this period was examined and it was shown that the sensitivity of five of the 11 sensors used had changed. Using the six remaining sensors, the signal patterns obtained from the meat from the different slaughterhouses did not change over a period of 6 months. It was shown that the degree of spoilage, as calculated using a model based on two Tagushi sensors, correlated well with the degree of spoilage determined by the sensory panel (r2 = 0.94).

Development of a combined selection and enrichment PCR procedure for Clostridium botulinum types B, E, and F and its use to determine prevalence in fecal samples from slaughtered pigs

ABSTRACT A specific and sensitive combined selection and enrichment PCR procedure was developed for the detection of Clostridium botulinum types B, E, and F in fecal samples from slaughtered pigs. Two enrichment PCR assays, using the DNA polymeraserTth, were constructed. One assay was specific for the type B neurotoxin gene, and the other assay was specific for the type E and F neurotoxin genes. Based on examination of 29 strains of C. botulinum, 16 strains of other Clostridium spp., and 48 non-Clostridium strains, it was concluded that the two PCR assays detect C. botulinum types B, E, and F specifically. Sample preparation prior to the PCR was based on heat treatment of feces homogenate at 70°C for 10 min, enrichment in tryptone-peptone-glucose-yeast extract broth at 30°C for 18 h, and DNA extraction. The detection limits after sample preparation were established as being 10 spores per g of fecal sample for nonproteolytic type B, and 3.0 × 103 spores per g of fecal sample for type E and nonproteolytic type F with a detection probability of 95%. Seventy-eight pig fecal samples collected from slaughter houses were analyzed according to the combined selection and enrichment PCR procedure, and 62% were found to be PCR positive with respect to the type B neurotoxin gene. No samples were positive regarding the type E and F neurotoxin genes, indicating a prevalence of less than 1.3%. Thirty-four (71%) of the positive fecal samples had a spore load of less than 4 spores per g. Statistical analysis showed that both rearing conditions (outdoors and indoors) and seasonal variation (summer and winter) had significant effects on the prevalence of C. botulinum type B, whereas the effects of geographical location (southern and central Sweden) were less significant.