The effect of temperature abuse on Staphylococcus aureus and Salmonellae in raw beef and chicken substrates during frozen storage

Raw beef and chicken substrates inoculated with Staphylococcus aureus and two serotypes of Salmonella were subjected to severe temperature abuse at intervals during six months frozen storage at ?18°C. A method was developed for enumeration of freeze-damaged Salmonellae in a mixed population. After 24 h S. hadar in chicken at c 20 or 27°C, increased by 2·87 and 5·40 log cycles respectively. In beef, S. typhimurium increased by 1·80 and 2·93 log cycles. Refreezing thawed samples reduced the Salmonellae by up to 99%. Growth of St. aureus during thawing was generally slight in comparison but there was little change in viable counts on refreezing. Substantial growth of St. aureus occurred in chicken at 27°C. Some samples were assayed for staphylococcal enterotoxin A but this was not detected in samples containing St. aureus at c 10⁷ cfu.g^?1. Growth of natural saprophytes had caused obvious spoilage by 24 h of thawing.     

Enterotoxin formation by Clostridium perfringens during sporulation and vegetative growth

Growth, sporulation and enterotoxin production have been followed for three different strains of Clostridium perfringens on a vegetative (FTG) medium and a sporulation (DS) medium. Enterotoxin production was followed by use of enzyme-linked immunosorbent assay (ELISA). All strains produced enterotoxin under both vegetative growth and sporulation in spite of the fact that one strain is known to be enterotoxin negative. The presumed negative strain produced about the same amount of enterotoxin on both media (1 ng/ml). The two other strains produced about 1000–2000 times more enterotoxin during sporulation than during vegetative growth.     

Molecular methods to evaluate biodiversity in Bacillus cereus and Bacillus thuringiensis strains from different origins

The spore-forming genus Bacillus includes species of industrial, clinical and environmental significance. The possibility of differentiating between Bacillus cereus and Bacillus thuringiensis, toxin producers associated with illness, is a real need in monitoring potentially contaminated foods to understand the real distribution of B. cereus/B. thuringiensis in different outbreak cases. As the use of DNA comparison obtains clearer results than classical microbiological methods in distinguishing B. cereus from B. thuringiensis in this work PCR-TTGE (Temporal Temperature Gradient gel Electrophoresis), rep-PCR and RAPD-PCR methods have been compared to assess the intra- and inter-specific variability of B. cereus and B. thuringiensis. 80 strains of B. cereus and B. thuringiensis isolated from food, patients and pesticides were analyzed using a gyrB gene DNA sequence in TTGE; primer M13 in the RAPD-PCR and primers REP1DT and REP2DT in the rep-PCR methods. A widespread distribution of the electrophoretic profiles was obtained either for B. cereus or for B. thuringiensis using TTGE. rep-PCR and RAPD-PCR were not always able to group strains from the same origin or belonging to the same species. The fingerprints obtained with the rep- and RAPD-PCR methods confirm the high intraspecific variability present in B. cereus and B. thuringiensis indicating the difficulty to discriminate between these two species in outbreak cases.     

Isolation and characterization of a novel analyte from Bacillus subtilis SC-8 antagonistic to Bacillus cereus

In this study, an effective substance was isolated from Bacillus subtilis SC-8, which was obtained from traditionally fermented soybean paste, cheonggukjang. The substance was purified by HPLC, and its properties were analyzed. It had an adequate antagonistic effect on Bacilluscereus, and its spectrum of activity was narrow. When tested on several gram-negative and gram-positive foodborne pathogenic bacteria such as Salmonella enterica, Salmonella enteritidis, Staphylococcus aureus, and Listeria monocytogenes, no antagonistic effect was observed. Applying the derivative from B. subtilis SC-8 within the same genus did not inhibit the growth of major soybean-fermenting bacteria such as Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloquefaciens. The range of pH stability of the purified antagonistic substance was wide (from 4.0 to >10.0), and the substance was thermally stable up to 60 °C. In the various enzyme treatments, the antagonistic activity of the purified substance was reduced with proteinase K, protease, and lipase; its activity was partially destroyed with esterase. Spores of B. cereus did not grow at all in the presence of 5 μg/mL of the purified antagonistic substance. The isolated antagonistic substance was thought to be an antibiotic-like lipopeptidal compound and was tentatively named BSAP-254 because it absorbed to UV radiation at 254 nm.     

Characterisation and profiling of Bacillus subtilis,Bacillus cereus and Bacillus licheniformis by MALDI-TOF mass fingerprinting

The Bacillus genus includes species such as Bacillus cereus, Bacillus licheniformis and Bacillus subtilis, some of which may be pathogenic or causative agents in the spoilage of food products. The main goal of this work was to apply matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass fingerprinting to the classification of these Bacillus species. Genetic analyses were also compared to phyloproteomic analyses. A collection of 57 Bacillus strains isolated from fresh and processed food and from culture collections were studied and their mass spectra compiled. The resulting mass fingerprints were compared and characteristic peaks at the strain and species levels were assigned. The results showed that MALDI-TOF was a good complementary approach to 16S rRNA sequencing and even a more powerful tool in the accurate classification of Bacillus species, especially for differentiating B. subtilis and B. cereus from Bacillus amyloliquefaciens and Bacillus thuringiensis, respectively. MALDI-TOF was also found to provide valuable information at both intra- and interspecies levels in the Bacillus species studied.     

A simple procedure for the detection of small numbers of enterotoxigenic Clostridium perfringens in frozen meat and cod paste

By use of commercially available pouches and dehydrated modified Handford medium, a simple method for the selective detection of small numbers of Clostridium perfringens spores in frozen meat and cod paste has been developed. By a direct method, 1 × 10²/g or more spores were detectable and after enrichment culture, the sensitivity of the direct method was increased by 10 to 100 times. One hundred and thirty of 319 samples (40.8%) of frozen meat and cod fish paste proved positive for presumptive C. perfringens by the enrichment method. The incidences of presumptive C. perfringens in frozen mutton (50.0%), rabbit meat (65.0%), cod paste (50.0%) and horse meat (42.4%) were relatively higher than those in frozen beef (25.0%) and pork (23.8%). By the direct method, the incidence decreased to 3.1% (10/319). Two enterotoxigenic strains were found among 100 verified C. perfringens isolates from 58 samples.     

The fracture toughness of pea testa in relation to temperature abuse during frozen storage

Obtaining information on the textural quality of food materials in SI units is desirable for objective quality control. Adapting standard materials testing methods to biological specimens has the potential of providing such objective measurements. Based on the approach of linear elastic fracture mechanics, a tear test was used to measure the fracture toughness of the testa of frozen peas. The mean fracture toughness obtained for an extension rate of 0.13 mm s⁻¹ was 212 J m⁻². The effect of extension rate was investigated and showed that fracture toughness increased with increasing extension rate. Measurement of fracture toughness was applied to identify the effect of “temperature abuse” in the pea testa. Following three thaw-freeze cycles, the specimen became more fragile and fracture toughness of pea testa decreased by about 18%. Brightfield microscopy and low temperature scanning electron microscopy were used to observe tissue changes resulting from temperature abuse. Cell rupture in the osteosclereids and parenchyma layers was associated with temperature-abused tissue. Slow freezing during the thaw-freeze cycles caused formation of large ice crystals which disrupted the pea testa tissue.     

Effect of packaging and frozen storage temperature on beef pigments

The influence of final freezing and storage temperature on pigment modifications of beef samples wrapped in polyethylene and EVA/SARAN/EVA was analysed. Met-, oxy- and myoglobin relative surface concentrations were determined using reflectance spectrophotometry. Pigment behaviour during storage of beef packaged in both films and oxygenation of vacuum-packaged samples was described by different reaction schemes and modelled to obtain kinetic and equilibrium constants. Comparative studies performed with beef samples (a) refrigerated, (b) frozen to –5°C and stored at this temperature (partial freezing), and (c) frozen to -25°C and stored at -5 and -20°C, indicated that for aerobic storage periods of 90 days, metmyoglobin levels of partially frozen samples upon thawing were comparable to those of frozen samples stored at –20°C. For vacuum-packaged beef, pigment concentrations remained practically constant during frozen storage; partial freezing increased oxygenation capacity of the tissue compared with chilled and frozen conditions.     

Formation of pyrazines and 2-acetyl-1-pyrroline by Bacillus cereus

The production of alkylpyrazines and 2-acetyl-1-pyrroline by different Bacillus cereus strains, which has been previously reported, was studied in detail. B. cereus ATCC 27522 produced the highest amounts of flavour compounds when grown as surface cultures on plate count agar. Pyrazine, methylpyrazine, 2,5-dimethylpyrazine, trimethylpyrazine and 3-ethyl-2,5-dimethylpyrazine were produced in low amounts. Since they were also detected in control flasks, an enzymatic formation was concluded to be unlikely. Only the production of 3-ethyl-2,5-dimethylpyrazine was in all cases significantly different from the control. Detailed precursor studies revealed that the production of 2-acetyl-1-pyrroline by B. cereus ATCC 27522 proceeds via acetylation of 1-pyrroline, a metabolic degradation product of proline and ornithine. Comparison of results obtained from dynamic headspace and simultaneous steam distillation – solvent extraction showed that the use of a non-thermal extraction method is essential to obtain reliable results on the biological formation of these Maillard flavour compounds.     

Isolation and partial purification of a proteolytic enzyme preparation from Clostridium perfringens

Although two strains of Clostridium perfringens (ATCC 12915 and 13124) exhibited excellent growth on amino acid and peptone media, only one (ATCC 13124) produced measurable proteolytic enzyme activity. Thus, subsequent purification steps concentrated on isolation of a proteolytic enzyme preparation produced by this strain. Purification and concentration were carried out by precipitating the crude enzyme fraction from the culture filtrate with ZnCl₂, extracting with saturated disodium phosphate and reprecipitating by 60% saturation with (NH₄)₂SO₄. The precipitate was then redissolved in borate buffered saline solution and further purified by successively passing it through a Bio-Gel P-100 column, a DEAE-cellulose column and a Bio-Gel P-200 column. The final step resulted in a 159-fold purification with 12% recovery and a final specific activity of 79 azocoll units/milligramme protein. Although each successive purification step eliminated some of the impurities, the final fraction still showed considerable heterogeneity upon disc-gel electrophoresis.     

Wine industry residues extracts as natural antioxidants in raw and cooked chicken meat during frozen storage

The effect of Isabel (IGE) and Niagara (NGE) grape seed and peel extracts on lipid oxidation, instrumental colour, pH and sensory properties of raw and cooked processed chicken meat stored at -18°C for nine months was evaluated. The pH of raw and cooked samples was not affected by the addition of grape extracts. IGE and NGE were effective in inhibiting the lipid oxidation of raw and cooked chicken meat, with results comparable to synthetic antioxidants. The extracts caused alterations in colour, as evidenced by the instrumental (darkening and lower intensity of red and yellow colour) and sensory results of cooked samples. In the sensory evaluation of odour and flavour, IGE produced satisfactory results, which did not differ from synthetic antioxidants. These findings suggest that the IGE and NGE are effective in retarding lipid oxidation of raw and cooked chicken meat during frozen storage.     

Modelling the influence of palmitic, palmitoleic, stearic and oleic acids on apparent heat resistance of spores of Bacillus cereus NTCC 11145 and Clostridium sporogenes Pasteur 79.3

Heat resistance of spores is affected by many factors such as temperature, pH, water activity (aw) and others. Previous studies have reported that free fatty acids can affect the germination and growth of bacterial spores. In this study, we investigated the influence of free fatty acids in heating medium or in recovery medium on the heat resistance of spores of Bacillus cereus NTCC 11145 and Clostridium sporogenes Pasteur 79.3. Four free fatty acids were studied: palmitic, palmitoleic, stearic and oleic acids. During thermal treatments, the impact of these FFA in heating media was generally low, but the presence of free fatty acids in the recovery medium highly decreases bacterial spore apparent heat resistance, particularly with unsaturated fatty acids. A mathematical model was developed to describe and quantify the influence of free fatty acids in recovery media on the D-values. The z′_FFA parameter values which quantify the impact of free fatty acids were determined. The variation of this parameter value according to the free fatty acid type was compared with MIC value variation given in the literature. The model enables the decrease in D-values in the presence of free fatty acids to be estimated. The high concentrations of free fatty acids in liver or canned duck may explain the microbial stability with low sterilization values applied.     

The effects of colorifico on lipid oxidation,colour and vitamin E in raw and grilled chicken patties during frozen storage

Colorifico is a spice consisting essentially of a mixture of annatto (Bixa orellana) and corn flour. The effects of colorifico addition (0.4 g/100 g) to chicken meat on the development of lipid oxidation, colour stability, and degradation of bixin and vitamin E was investigated in raw and grilled patties during storage at −18 °C for 120 days. Colorifico was able to provide a more stable and intense red and yellow colour in both raw and grilled chicken patties when compared to the meat without spice. Lipid oxidation was delayed by colorifico in the grilled patties until 30 days of storage; however, no effect was observed in the raw patties. Vitamin E content was significantly higher in raw meat with colorifico and heat treatment resulted in relatively less loss when compared to the meat without spice; however, during storage both presented the same degradation pattern. Bixin content was stable during storage but not after grilling. The observed antioxidant mechanism suggests that vitamin E, probably the tocotrienols, is acting along with bixin to protect the unsaturated lipids from oxidation.     

Response of Bacillus cereus spores to high hydrostatic pressure and moderate heat

The effects of high pressure (400-600 MPa) and moderate heat (60-80 °C) treatments at various process times (10-20 min) on the reduction of Bacillus cereus As 1.1846 spores, suspended in milk buffer were investigated. In the present work, response surface methodology (RSM) was employed, and a quadratic equation of high hydrostatic pressure inactivation was built with RSM. By analyzing response surface plots and corresponding contour plots and by solving the quadratic equation, experimental values were shown to be significantly in agreement with predicted values, since the adjusted determination coefficient was 0.9752 and the level of significance was P < 0.0001. Optimum process parameters for a six-log cycle reduction of B. cereus spores were obtained: pressure, 540.0 MPa; temperature, 71 °C; and pressure-holding time, 16.8 min. The adequacy of the model equation in predicting optimum response values was verified effectively using experimental test data that was not used in the development of the model.     

Changes in extensibility of raw beef muscle during storage

The mechanical properties of raw meat under tension were studied to characterise the factors responsible for tenderisation during storage. Extensibility and irreversible lengthening in the direction of muscle fibres was determined continuously from 2 h to 10 days after stunning by cycling the muscle between two applied forces. In post-rigor meat, extensibility increased slightly and the muscle lengthened by 40%. Lengthening occurred earlier at higher post-rigor temperatures and was more sensitive to changes in temperature than the changes in texture of cooked meat. Increasing the maximum applied stress also caused lengthening to occur earlier in post-rigor meat. No lengthening was detected pre-rigor. The relationship between the maximum applied stress and the rate of lengthening showed that lengthening would not have occurred with a stress of 0·07 Ncm(-2) or less. These findings suggest that weakening which occurs during conditioning commenced at rigor and that the component responsible for tenderising was about 30 times weaker than indicated using previous techniques. Histological sections showed regular cracks spaced at about 17 μm apart. This was believed to be indicative of the involvement of extracellular components in the texture of conditioned meat.     

冷冻保藏对鸡肉保鲜效果的研究

主要研究鲜鸡肉在-18℃下冷冻保鲜过程中的肉品质的变化。通过对肉样色差值、菌落总数、理化指标(pH值、TVB-N)的测定和比较分析,探讨鲜鸡肉在冻藏条件下肉品质的变化情况。结果表明:随着贮藏时间的增加,各项理化指标和细菌总数均呈现先下降后上升的趋势;随着储藏期的延长,色差L*值呈下降趋势,而a*值呈上升趋势;经过90d冻藏,鸡肉还是二级鲜度。     

The effect of dietary oregano essential oil on lipid oxidation in raw and cooked chicken during refrigerated storage

The antioxidative effect of dietary supplementation with oregano essential oil on susceptibility of raw and cooked breast and thigh muscle meat of chickens to lipid oxidation during refrigerated storage for 9 days was investigated. Day-old chickens (n=80) were randomly divided into four groups and fed a basal diet containing 30 mg α-tocopheryl acetate kg(-1) feed as control, or basal diet plus 200 mg α-tocopheryl acetate kg(-1) feed, or basal diet plus 50 or 100 mg oregano essential oil kg(-1) for 38 days prior to slaughter. Lipid oxidation was assessed by monitoring malondialdehyde (MDA) formation in raw and cooked meat during 0, 3, 6 and 9 days of refrigerated storage, using the thiobarbituric acid (TBA) assay and third-order derivative spectrophotometry. Results showed that dietary oregano essential oil supplementation exerted antioxidative effects, the supplementation being most effective in retarding lipid oxidation in stored raw and cooked meat at the 100 mg oregano essential oil kg(-1) feed. However, dietary α-tocopheryl acetate supplementation at 200 mg kg(-1) feed displayed greater antioxidant activity than oregano treatments. Thigh muscle was more susceptible to oxidation compared to breast muscle in all treatments, although the former tissues contained α-tocopherol at markedly higher levels.     

Intensity of lipid oxidation and formation of cholesterol oxidation products during frozen storage of raw and cooked chicken

Raw and cooked chicken breasts were stored at ?18 °C for 3 months under aerobic and vacuum conditions, and the intensity of lipid oxidation and the formation of COP (cholesterol oxidation products) were studied. Raw samples showed low COP levels (4.60–7.40 µg g^?1 fat), TBARS (thiobarbituric acid reactive substances) levels (0.01–0.03 mg kg^?1) and peroxide values (not detected) under both aerobic and vacuum conditions. Cooked samples (grilled and roasted) showed TBARS levels of 0.36–0.99 mg kg^?1 in aerobic conditions and 0.21–0.70 mg kg^?1 in vacuum conditions, whilst peroxide levels reached 38–40 and 19–23 meq O₂ kg^?1 in samples stored under aerobic and vacuum conditions respectively. Total COP levels in grilled and roasted samples were 28.91 and 39.34 µg g^?1 fat in aerobic packaging and 4.90 and 20.24 µ g g^?1 fat in vacuum packaging respectively. Significant correlations were found between the lipid oxidation parameters and cholesterol oxidation indices. In general, TBARS were better correlated with total COP than with only 7‐ketocholesterol. Vacuum packaging was particularly efficient in slowing down the oxidation process during frozen storage of cooked samples. Copyright © 2004 Society of Chemical Industry