Isolation of Yersinia enterocolitica-resembling Organisms and Alteromonas putrefaciens from Vacuum-Packed Chilled Beef Cuts

Yersinia enterocolitica -resembling organisms were found at levels of 10⁷/g on a high pH (pH ≧ 6·0) vacuum-packaged beef striploin held for 6 weeks at 0·2°C, but did not exceed 10⁵/g on normal pH (pH < 6·0) striploins held for 10 weeks. Gram negative bacteria that produced H₂S on peptone iron agar were isolated from high pH vacuum packed striploins. These organisms were identified as Alteromonas putrefaciens . They attained levels of about 10⁷/g in 6 weeks at 0–2°C, at which time greening of the fat surface and 'drip'had occurred. On meat of normal pH, counts of A. putrefaciens were less than 10⁴/g after 6 weeks and no greening was evident.     

Microbial Spoilage of Fresh Refrigerated Beef Liver

The types and numbers of micro-organisms involved in the spoilage of refrigerated beef liver were studied together with pH, hydration and organoleptic changes of the material. Fresh liver harboured a mixed population ( c . 1 × 10⁵ organisms/g) of Gram positive cocci, chromogens and non-chromogens, sporeformers, presumptive coliforms and Gram negative rods. When samples were rejected organoleptically, after 7–10 days at 5°, the contamination attained levels of c . 7–8 × 10⁷ organisms/g. Spoilage was due to souring; the pH fell from 6·3 (fresh liver) to c . 5·9. Lactic acid bacteria were predominant and Gram negative bacteria did not exceed 1·0 × 10⁶ organisms/g. This type of spoilage is explained by the carbohydrate content of c . 5% in liver. The value of pH appears to be a reliable indicator of liver freshness, with a pH of 6·1 indicating incipient spoilage.     

Changes in the Microbiology of Vacuum-packaged Beef

The development of the microbial flora on meat stored in vacuum-bags at 0–2° for up to 9 weeks was studied. Although the proportion of lactic acid bacteria increased relative to the aerobic spoilage organisms, the numbers of the latter continued to increase throughout storage. The initial contamination of the meat before vacuum-packaging was important; meat with a very low initial number had lower numbers of bacteria throughout storage for up to 9 weeks and steaks cut from such meat which had been stored always had 1–2 days' additional aerobic shelf life at 4°. Spoilage of these steaks was due either to slime formation and off-odour associated with high counts of presumptive Pseudomonas spp., or by discoloration and souring (lactic acid bacteria). Extract release volume and pH measurements performed on the vacuum-packaged primal joints were only of value in determining the onset of aerobic spoilage when large numbers of Gram negative organisms were present, whereas the titrimetric method of spoilage evaluation of the vacuum-packaged meat showed a correlation with spoilage due to lactic organisms.     

Factors affecting the production of hydrogen sulphide by Lactobacillus sake L13 growing on vacuum-packaged beef

Lactobacillus sake L13 produced hydrogen sulphide during growth at 0°C on vacuum-packaged beef of normal pH (5·6–5·8) when the packaging films used had oxygen permeabilities as high as 200 ml/m²/24 h/atm (measured at 25°C and 98% relative humidity. No hydrogen sulphide was detected when the film permeability was 300 ml/m²/24 h/atm. Sulphmyoglobin was formed whenever hydrogen sulphide was present except when the film permeability was very low (1 ml of oxygen/m²/24 h/atm). Lactobacillus sake L13 also produced hydrogen sulphide when grown on beef under anaerobic conditions at 5°C. When meat pH was high (6·4–6·6) hydrogen sulphide was first detected after incubation for 9 d. When 250 μg of glucose was added to each g of high pH meat, or when meat pH was normal (5·6–5·8), hydrogen sulphide was first detected after incubation for 18 d. The spoilage of beef by hydrogen sulphide-producing lactobacilli is more rapid when the pH of the meat is high because high-pH meat contains less glucose. Sulphmyoglobin formation and greening can be prevented by the use of packaging films of very low oxygen permeability.     

The effect of variation of thermal processing on the microbial spoilage of chub-packed luncheon meat

Process pasteurization values for reference temperature 70°C (P₇₀) were calculated from the temperature profiles of 250 g luncheon meat chubs cooked under experimental conditions. A simple equation relating Process P₇₀-value and the time and temperature of cooking was derived. With minimal cooking (P₇₀= 40) the surviving microflora (10³/g) was dominated by species of Lactobacillus, Brochothrix and Micrococcus. These organisms were destroyed by more intensive cooking (P₇₀= 105), leaving a flora (10²/g) composed of Bacillus and Micrococcus species. The spoilage that developed after 14 d storage at 25°C reflected the severity of the heat treatment received by each chub: with P₇₀ between 40 and 90, a Streptococcus spoilage sequence occurred; with P₇₀ between 105 and 120, a Bacillus/Streptococcus spoilage sequence occurred; with P₇₀ of 135 and above, a Bacillus spoilage sequence occurred. Cooking to a P₇₀= 75 was adequate to reduce the surviving microflora to the 10²/g level associated with current good manufacturing practice.     

Effects of gaseous environment and temperature on the storage behaviour of Listeria monocytogenes on chicken breast meat

Portions of skinless chicken breast meat (pH 5·8) were inoculated with a strain of Listeria monocytogenes and stored at 1, 6 or 15°C in (1) aerobic conditions; (2) 30% CO₂+ air; (3) 30% CO₂+ N₂; and (4) 100% CO₂. When samples were held at 1°C the organism failed to grow under any of the test conditions, despite marked differences between treatments in spoilage rate and ultimate microflora. At 6°C counts of L. monocytogenes increased ca 10-fold in aerobic conditions before spoilage of the meat, but only when the inoculum culture was incubated at 1°C rather than 37°C. In CO₂ atmospheres growth of L. monocytogenes was inhibited on meat held at 6°C, especially under 100% CO₂. By contrast, storage at 15°C led to spoilage of the meat within 2 d, in all gaseous environments, and listeria levels increased up to 100-fold. Differences in the behaviour of L. monocytogenes on poultry and red meats are discussed.     

OBSERVATIONS ON THE MICROFLORA OF MINCED CHICKEN MEAT IRRADIATED WITH 4 MeV CATHODE RAYS

SUMMARY: Experiments are described in which minced chicken meat, packed anaerobically, was irradiated at room temperature and in the frozen state with a wide range of doses of 4 MeV cathode rays. Sterility was achieved in 14 out of 15 samples which had received 2 × 10⁶ rads or more. Doses of 0·5 and 1·0 × 10⁶ rads allowed survival of a few bacteria/g, usually spore formers. Bacterial counts indicated an approximately logarithmic decrease in numbers at lower doses, while freezing reduced the bactericidal effect.
The storage life at 5° was prolonged only slightly by doses of 5 × 10⁴ and 10 × 10⁴ rads, and highly variable results were obtained with 17·5 × 10⁴ rads. A dose of 25 × 10⁴ rads, however, increased the storage life very considerably. The types of bacteria present initially, and after irradiation with low doses and storage at 5°, were studied. After storage for 12 days or more various types of nonsporing Gram-positive rods were predominant in almost all samples, both control and irradiated. Streptococci were also important where irradiation with 17·5 × 10⁴ and 25 × 10⁴ rads was followed by long storage.     

Amines in fresh beef of normal pH and the role of bacteria in changes in concentration observed during storage in vacuum packs at chill temperatures

E dwards , R.A., D ainty , R.H., H ibbard , C.M. & R amantanis , S.V. 1987. Amines in fresh beef of normal pH and the role of bacteria in changes in concentration observed during storage in vacuum packs at chill temperatures.
The amine content of fresh and vacuum-packaged beef of normal pH stored at 1°C was evaluated by high performance liquid chromatography of dansyl derivatives. Fresh samples contained five amines, viz. putrescine, cadaverine, histamine, sperm-ine and spermidine. Development of a natural spoilage flora during storage led to increases in concentration of putrescine and cadaverine and the production of a sixth amine, tyramine. Pure culture meat inoculation experiments showed tyramine formation to be restricted to lactobacilli and to strains of Lactobacillus divergens and Lact. carnis in particular; strains of leuconostocs, Enterobacteriaceae, Pseudomonas spp. and Brochothrix thermosphacta were negative. Production of tyramine at cell densities >log₁₀ 6/cm² indicated its potential as an objective measure of acceptability/spoilage.     

The Limulus Lysate Endotoxin Assay as a Test of Microbial Quality of Ground Beef

The Limulus lysate test (LLT) for endotoxin assay has been found to be an excellent, simple and rapid test of microbial quality of refrigerated ground beef. In fresh ground beef held at 5°C for 7–12 d, LLT titres increased from 10²–10⁵ and correlated very highly with extract-release volume (ERV) data and total viable Gram negative counts at both 5° and 30°C. The LLT was negative for fresh beef containing low numbers of bacteria and on aged beef in the absence of increasing numbers of Gram negative bacteria. Of 14 Gram negative meat isolates, all gave a positive LLT while none of eight miscellaneous Gram positive bacteria did. The use of this test provides objective information on the microbial quality of fresh refrigerated ground meats in 1 h. Based upon this study, it is suggested that a 0·1 ml inoculum from a 10³ dilution of good quality ground beef should produce a negative lysate test and thus serve as an additional rapid screening test of meat microbial quality.     

Fate of low temperature and acid-adapted Yersinia enterocolitica and Listeria monocytogenes that contaminate lactic acid decontaminated meat during chill storage

Pathogens found in the environment of abattoirs may become adapted to lactic acid used to decontaminate meat. Such organisms are more acid tolerant than non-adapted parents and can contaminate meat after lactic acid decontamination (LAD). The fate of acid-adapted Yersinia enterocolitica and Listeria monocytogenes, inoculated on skin surface of pork bellies 2 h after LAD, was examined during chilled storage. LAD included dipping in 1%, 2% or 5% lactic acid solutions at 55°C for 120 s. LAD brought about sharp reductions in meat surface pH, but these recovered with time after LAD at ≈1–1·5 pH units below that of water-treated controls. Growth permitting pH at 4·8–5·2 was reached after 1% LAD in less than 0·5 d (pH 4·8–5·0), 2% LAD within 1·5 d (pH 4·9–5·1) and after 5% LAD (pH 5·0–5·2) within 4 d. During the lag on 2% LAD meat Y. enterocolitica counts decreased by 0·9 log₁₀ cfu per cm² and on 5% LAD the reduction was more than 1·4 log₁₀ cfu per cm². The reductions in L. monocytogenes were about a third of those in Y. enterocolitica . On 1% LAD the counts of both pathogens did not decrease significantly. The generation times of Y. enterocolitica and L. monocytogenes on 2–5% LAD meats were by up to twofold longer than on water-treated controls and on 1% LAD-treated meat they were similar to those on water-treated controls. Low temperature and acid-adapted L. monocytogenes and Y. enterocolitica that contaminate skin surface after hot 2–5% LAD did not cause an increased health hazard, although the number of Gram-negative spoilage organisms were drastically reduced by hot 2–5% LAD and intrinsic (lactic acid content, pH) conditions were created that may benefit the survival and the growth of acid-adapted organisms.     

The use of the bacteriocin, nisin, as a preservative in ricotta-type cheeses to control the food-borne pathogen Listeria monocytogenes

The efficacy of nisin to control the food-borne pathogen Listeria monocytogenes in ricotta-type cheeses over long storage (70 d) at 6–8°C was determined. Cheeses were prepared from unpasteurized milk by direct acidification with acetic acid (final pH 5·9) and/or calcium chloride addition during heat treatment. Nisin was added in the commercial form of Nisaplin^® pre-production to the milk. Each batch of cheese was inoculated with 10²–10³ cfu g⁻¹ of a five-strain cocktail of L. monocytogenes before storage. Shelf-life analysis demonstrated that incorporation of nisin at a level of 2·5 mg l⁻¹ could effectively inhibit the growth of L. monocytogenes for a period of 8 weeks or more (dependent on cheese type). Cheese made without the addition of nisin contained unsafe levels of the organism within 1–2 weeks of incubation. Measurement of initial and residual nisin indicated a high level of retention over the 10-week incubation period at 6–8°C, with only 10–32% nisin loss.     

Factors affecting the growth of Listeria monocytogenes on minimally processed fresh endive

The influence of various factors on the fate of Listeria monocytogenes on cut leaves of broad-leaved endive has been studied. Factors considered were temperature, characteristics of the leaves (age, quantity and quality of the epiphytic microflora) and characteristics of the L. monocytogenes inoculum (concentration, strain). The increases in numbers of L. monocytogenes were lower than those of the aerobic mesophilic microflora at 3°, 6°, 10° and 20°C. Doubling times of the populations of L. monocytogenes were in the same order of magnitude as those of aerobic bacteria at 10° and 20°C, but longer at 3° and 6°C. There were positive significant correlations between growth of L. monocytogenes and populations of aerobic bacteria, and between growth of L. monocytogenes and extent of spoilage on the leaves.
Of 225 bacteria isolated from the leaves, 84% were identified as fluorescent pseudomonads; there was no difference in the species isolated from leaves that showed a low growth of L. monocytogenes and leaves that showed a high growth of L. monocytogenes. Populations of L. monocytogenes increased faster during the first 2 and 4 d of storage at 10°C on leaves inoculated with 10–10³ cfu g^-1 than on leaves inoculated with about 10⁵ cfu g^-1, but the population reached after 7 d was lower. The behaviour of L. monocytogenes was similar among the three strains tested.     

Survival of Escherichia coli O157 : H7 in yoghurt during preparation and storage at 4°C

Cow's milk was inoculated with ca 10³ and 10⁷ cfu ml⁻¹ Escherichia coli O157 : H7. After fermentation at 42°C for 0–5 h, the yoghurt was stored at 4°C. Two kinds of yoghurt were used : traditional yoghurt (TY), made with Streptococcus thermophilus and Lactobacillus bulgaricus starter cultures, and 'bifido' yoghurt (BY), made with the two starter cultures plus Bifidobacterium bifidum . After 7 d E. coli O157 : H7 decreased from 3·52 to 2·72 log₁₀ cfu ml⁻¹ and from 7·08 to 5·32 log₁₀ cfu ml⁻¹ in TY, and from 3·49 to 2·73 log₁₀ cfu ml⁻¹ and from 7·38 to 5·41 log₁₀ cfu ml⁻¹ in BY. The pH values of yoghurt dropped from 6·6 to 4·5 and 4·4 in TY (for low and high pathogen inocula, respectively), and from 6·6 to 4·6 and 4·5 in BY (for low and high pathogen inocula, respectively).     

Poikilothermic traits and thermoregulation in the Afrotropical social subterranean Mashona mole-rat (Cryptomys hottentotus darlingi) (Rodentia: Bathyergidae)

When acclaimated for two months at 26 C the social Mashona mole-rat Cryptomys hottentotus darlingi (±S.D.) resting metabolic rate (RMR) of 0·98±0.·14cm²O₂g _-1 h_-1 ( n =21), within a thermal neutral zone (TNZ) of 28 31·5 C ambient temperature (Ta). The body temperature (Tb) of the mole-rat is very low. 33·3±0·5 C, and remained stable between 25 31·5 C ( n =28). Above 33 C. Tb increased to a mean of 34·±0· C (n=28) (Ta range 33 39 C). Below Ta 25 C. Tb showed strong poikilothermic tendencies, with Tb dropping to a mean of 26·8±1·16 C. whereas above Ta25 C. Tb varied in a typically endothermic pattern. The conductance is high 0·19±0·03 cm² O_2g¹ C ¹ (n=28) at the lower limit of thermoneutrality. The mean RMR at 18 C (the lowest Ta tested) was 2·63 ± 0·55 cm³ O₂g ¹ h ¹ (n=7) which is 2·6 times that of the resting metabolic rate in the TNZ.     

Effect of Iysozyme concentration, heating at 90°C, and then incubation at chilled temperatures on growth from spores of non-proteolytic Clostridium botulinum

The heat treatment necessary to inactivate spores of non-proteolytic Clostridium botulinum in refrigerated, processed foods may be influenced by the occurrence of lysozyme in these foods. Spores of six strains of non-proteolytic Cl. botulinum were inoculated into tubes of an anaerobic meat medium, to give 10⁶ spores per tube. Hen egg white lysozyme (0–50 μg ml^-1) was added, and the tubes were given a heat treatment equivalent to 19·8 min at 90°C, cooled, and incubated at 8°, 12°, 16° and 25°C for up to 93 d. In the absence of added lysozyme, neither growth nor toxin formation were observed. A 6–D inactivation was therefore achieved. In tubes to which lysozyme (5–50 μg ml^-1) had been added prior to heating, growth and toxin formation were observed. With lysozyme added at 50 μg ml^-1, growth was first observed after 68 d at 8°C, 31 d at 12°C, 24 d at 16°C, and 9 d at 25°C. Thus, in these circumstances, a heat treatment equivalent to 19·8 min at 90°C was not sufficient, on its own, to give a 6–D inactivation. A combination of the heat treatment, maintenance at less than 12°C, and a shelf-life not more than 4 weeks reduced the risk of growth of non-proteolytic Cl. botulinum by a factor of 10⁶.     

Effects of essential oil from mint (Mentha piperita) on Salmonella enteritidis and Listeria monocytogenes in model food systems at 4° and 10°C

The effect of mint ( Mentha piperita ) essential oil (0·5, 1·0, 1·5 and 2·0%, v/w) on Salmonella enteritidis and Listeria monocytogenes in a culture medium and three model foods; tzatziki (pH 4·5), taramosalata (pH 5·0) and pâté (pH 6·8), inoculated at 10⁷ cfu g^-1, at 4° and 10°C for ca 1 week was studied. In the culture medium supplemented with the essential oil, no growth was observed over 2 d at 30°C determined by a conductance method with a Malthus 2000 growth analyser. Salmonella enteritidis died in tzatziki in all treatments and declined in the other foods except for pâté at 10°C as judged with viable counts. Listeria monocytogenes populations showed a declining trend towards the end of the storage period but was increased in pâté. Mint essential oil antibacterial action depended mainly on its concentration, food pH, composition, storage temperature and the nature of the micro-organism.     

The Effect of Film Permeability on the Storage Life and Microbiology of Vacuum-packed meat

Joints of beef were stored in packaging films with oxygen permeabilities ranging from 0–920 ml/m²24 h/atm at 25 °C and 100% r.h. The storage life of the 'vacuum-packaged' meat, as assessed by discolouration and the development of putrefactive odours, was inversely related to film permeability; the best results were obtained with meat which received 'zero oxygen' treatment. The growth rates and final counts of Pseudomonas spp. increased with increasing film permeability; the storage life of the meat corresponded with the time taken for the counts of the organism to reach ca. 10⁶/cm² for putrefactive odours to be produced. Although their growth rate was unaffected, the final counts of Brochothrix thermosphacta also increased with increasing film permeability. These results are discussed in terms of the influence of film permeability on the inhibitory effects of Lactobacillus spp., whose numbers were unaffected by the permeability of the film used, and the substrates in the meat available to the bacteria.     

Production of amylase by the intestinal microflora in cultured freshwater fish

The amylase-producing ability of the intestinal microflora in cultured specimens of ayu, carp, channel catfish, Japanese eel and tilapia was determined. Mean viable counts of aerobes and anaerobes ranged from 1·1×10⁶ to 3·7×10⁸ cfu g⁻¹ and from 1·3×10³ to 1·6×10⁸ cfu g⁻¹, respectively. Aeromonas spp. and Bacteroidaceae were predominant in four to five fish species. Of 206 strains examined, 65 (31·6%) produced ≥0·01 U amylase ml⁻¹. The percentage of producers differed among families and genera of bacteria and fish species. While 56% of the anaerobes produced amylase, only 20% of the aerobes did. More than 50% of Aeromonas , Bacteroidaceae and Clostridium strains produced amylase efficiently while Acinetobacter , coryneforms, Enterobacteriaceae, Moraxella , Plesiomonas and Streptococcus strains did not. High amylase production (≥0·05 U ml⁻¹) was found in 12 strains, 11 from Aeromonas and one Pseudomonas . The percentage of high amylase producers in Japanese eel was lower than the other four fish (2–30%). These results strongly suggest that the amylase produced by the intestinal microflora play an important role in the digestion of starch in freshwater fish to some extent.     

Detection and characterization of a bacteriocin produced by Lactococcus lactis subsp. cremoris R isolated from radish

Bacteria isolated from radish were identified as Lactococcus lactis subsp. cremoris R and their bacteriocin was designated lactococcin R. Lactococcin R was sensitive to some proteolytic enzymes (proteinase-K, pronase-E, proteases, pepsin, α-chymotrypsin) but was resistant to trypsin, papain, catalase, lysozyme and lipase, organic solvents, or heating at 90 °C for 15, 30 and 60 min, or 121 °C for 15 min. Lactococcin R remained active after storage at −20 and −70 °C for 3 months and after exposure to a pH of 2–9. The molecular weight of lactococcin R was about 2·5 kDa. Lactococcin R was active against many food-borne pathogenic and food spoilage bacteria such as Clostridium, Staphylococcus, Listeria, Bacillus, Micrococcus, Enterococcus, Lactobacillus, Leuconostoc, Streptococcus and Pediococcus spp., but was not active against any Gram-negative bacteria. Lactococcin R was produced during log phase and reached a maximum activity (1600 AU ml⁻¹) at early stationary phase. The highest lactococcin R production was obtained in MRS broth with 0·5% glucose, at 6·5–7·0 initial pH values, 30 °C temperature and 18–24-h incubation times. Lactococcin R adsorbed maximally to its heat-killed producing cells at pH 6–7 (95%). Crude lactococcin R at 1280 AU ml⁻¹ was bactericidal, reducing colony counts of Listeria monocytogenes by 99·98% in 3 h. Lactococcin R should be useful as a biopreservative to prevent growth of food-borne pathogenic and food spoilage bacteria in ready-to-eat, dairy, meat, poultry and other food products. Lactococcin R differs from nisin in having a lower molecular weight, 2·5 kDa vs 3·4 kDa, and in being sensitive to pepsin and α-chymotrypsin to which nisin is resistant.     

The effect of citric acid on growth of proteolytic strains of Clostridium botulinum

In strictly anaerobic conditions in a culture medium adjusted to pH 5·2 with HCl and incubated at 30°C, inocula containing < 10 vegetative bacteria of Clostridium botulinum ZK3 (type A) multiplied to give > 10⁸ bacteria per ml in 3 d. Growth from an inoculum of between 10 and 100 spores occurred after a delay of 10–20 weeks. Citric acid concentrations of 10–50 mmol/l at pH 5·2 inhibited growth from both vegetative bacteria and spore inocula, a concentration of 50 mmol/l increasing the number of vegetative bacteria or of spores required to produce growth by a factor of approximately 10⁶. The citric acid also reduced the concentration of free Ca²⁺ in the medium. The inhibitory effect of citric acid on vegetative bacteria at pH 5·2 could be prevented by the addition of Ca²⁺ or Mg²⁺ and greatly reduced by Fe²⁺ and Mn²⁺. The addition of Ca²⁺, but not of the remaining divalent metal ions, restored the concentration of free Ca²⁺ in the medium to that in the citrate-free medium. The inhibitory effect of citric acid on growth from a spore inoculum was only partially prevented by Ca²⁺. Citric acid (50 mmol/l) did not inhibit growth of strain ZK3 at pH 6 despite the greater chelating activity of citrate at pH 6 than at pH 5·2. The effect of citric acid and Ca²⁺ at pH 5·2 on vegetative bacteria of strains VL1 (type A) and 2346 and B6 (proteolytic type B) was similar to that on strain ZK3.