Changes in the Spoilage-Related Microbiota of Beef during Refrigerated Storage under Different Packaging Conditions

The microbial spoilage of beef was monitored during storage at 5°C under three different conditions of modified-atmosphere packaging (MAP): (i) air (MAP1), (ii) 60% O₂ and 40% CO₂ (MAP2), and (iii) 20% O₂ and 40% CO₂ (MAP3). Pseudomonas, Enterobacteriaceae, Brochothrix thermosphacta, and lactic acid bacteria were monitored by viable counts and PCR-denaturing gradient gel electrophoresis (DGGE) analysis during 14 days of storage. Moreover, headspace gas composition, weight loss, and beef color change were also determined at each sampling time. Overall, MAP2 was shown to have the best protective effect, keeping the microbial loads and color change to acceptable levels in the first 7 days of refrigerated storage. The microbial colonies from the plate counts of each microbial group were identified by PCR-DGGE of the variable V6-V8 region of the 16S rRNA gene. Thirteen different genera and at least 17 different species were identified after sequencing of DGGE fragments that showed a wide diversity of spoilage-related bacteria taking turns during beef storage in the function of the packaging conditions. The countable species for each spoilage-related microbial group were different according to packaging conditions and times of storage. In fact, the DGGE profiles displayed significant changes during time and depending on the initial atmosphere used. The spoilage occurred between 7 and 14 days of storage, and the microbial species found in the spoiled meat varied according to the packaging conditions. Rahnella aquatilis, Rahnella spp., Pseudomonas spp., and Carnobacterium divergens were identified as acting during beef storage in air (MAP1). Pseudomonas spp. and Lactobacillus sakei were found in beef stored under MAP conditions with high oxygen content (MAP2), while Rahnella spp. and L. sakei were the main species found during storage using MAP3. The identification of the spoilage-related microbiota by molecular methods can help in the effective establishment of storage conditions for fresh meat.     

Multivariate study of different beef quality traits from local Spanish cattle breeds

Different raw beef quality traits from four local Spanish cattle breeds were studied using correlation, factorial, discriminant and multiple regression analysis. The following variables were studied after 0, 5, 10 and 15 days of storage under 60% O₂, 30% CO₂ and 10% N₂ modified atmosphere packaging (MAP): colour physical variables, meat pigments, sensory degradation of odour and colour, microbial counts, thiobarbituric acid (TBA), pH, drip loss, lipid composition and volatile compounds. The degradation of raw beef quality was related to the increase in 2,3,3-trimethylpentane, 2,2,5-trimethylhexane, 3-methyl-2-heptene, 2-octene, 3-octene, 2-propanone, Enterobacteriaceae and aerobial plate counts (APC), metmyoglobin (MMb), lightness (L*), yellowness (b*), drip loss and TBA. Among these variables, TBA, b* and MMb may be useful in evaluating raw beef quality. No variables related to fat, except for TBA, including pH were limiting factors of the colour and odour shelf-life of raw beef under MAP. Each breed had some characteristics that were unique and these differences may influence the stability of meat to oxidation depending on myoglobin concentration and the polyunsaturated fatty acid (PUFA)/saturated fatty acid (SFA) ratio.     

Development of a Microbial Model for the Combined Effect of Temperature and pH on Spoilage of Ground Meat, and Validation of the Model under Dynamic Temperature Conditions

The changes in microbial flora and sensory characteristics of fresh ground meat (beef and pork) with pH values ranging from 5.34 to 6.13 were monitored at different isothermal storage temperatures (0 to 20°C) under aerobic conditions. At all conditions tested, pseudomonads were the predominant bacteria, followed by Brochothrix thermosphacta, while the other members of the microbial association (e.g., lactic acid bacteria and Enterobacteriaceae) remained at lower levels. The results from microbiological and sensory analysis showed that changes in pseudomonad populations followed closely sensory changes during storage and could be used as a good index for spoilage of aerobically stored ground meat. The kinetic parameters (maximum specific growth rate [μ_max] and the duration of lag phase [λ]) of the spoilage bacteria were modeled by using a modified Arrhenius equation for the combined effect of temperature and pH. Meat pH affected growth of all spoilage bacteria except that of lactic acid bacteria. The “adaptation work,” characterized by the product of μ_max and λ(μ_max × λ) was found to be unaffected by temperature for all tested bacteria but was affected by pH for pseudomonads and B. thermosphacta. For the latter bacteria, a negative linear correlation between ln(μ_max × λ) and meat pH was observed. The developed models were further validated under dynamic temperature conditions using different fluctuating temperatures. Graphical comparison between predicted and observed growth and the examination of the relative errors of predictions showed that the model predicted satisfactorily growth under dynamic conditions. Predicted shelf life based on pseudomonads growth was slightly shorter than shelf life observed by sensory analysis with a mean difference of 13.1%. The present study provides a “ready-to-use,” well-validated model for predicting spoilage of aerobically stored ground meat. The use of the model by the meat industry can lead to effective management systems for the optimization of meat quality.     

Effect of Lactobacillus-protective cultures with bacteriocin-like inhibitory substances' producing ability on microbiological, chemical and sensory changes during storage of refrigerated vacuum-packaged sliced beef

AIMS: To investigate the effect of applying two different Lactobacillus-protective cultures, with bacteriocin-like inhibitory substances' (BLIS) producing ability, individually or in combination, on microbiological, chemical and sensory changes during storage of refrigerated vacuum-packaged sliced beef meat. METHODS AND RESULTS: Lactobacillus sakei CECT 4808 and Lactobacillus curvatus CECT 904(T), which were shown to be producers of BLIS, were inoculated individually or in combination on slices of beef M. semitendinosus. The samples were vacuum packaged and stored at 4 +/- 1 degrees C and were assessed during a 28-day storage period for microbiological [Enterobacteriaceae, Pseudomonas spp., lactic acid bacteria (LAB), Brochothrix thermosphacta and yeasts and moulds], chemical (pH, protein hydrolysis degree, lipid oxidation), sensory (abnormal odour) parameters and instrumental colour. Samples inoculated with the Lact. sakei strain and samples inoculated with the combination of the two strains had significantly (P < 0.05) lower spoilage microbial counts than those inoculated with the Lact. curvatus strain alone or the controls, while both chemical parameters (including lipid oxidation) and abnormal odour scores were also significantly (P < 0.05) improved by the former. Moreover, Lact. sakei alone showed a better preserving effect (P < 0.05) than the combination of both strains in the majority of the parameters tested. Instrumental colour measurements changed with storage time, but no treatment effects (P >or= 0.05) were observed during the whole 28-day storage period. CONCLUSIONS: The BLIS producer Lact. sakei CECT 4808 strain may be used for improving preservation of vacuum-packaged beef slices, as regards spoilage microbial counts and the chemical parameters tested in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: Inoculation with the BLIS producer Lact. sakei CECT 4808 strain would provide an additional hurdle to improve storage life of refrigerated vacuum-packaged sliced beef. Furthermore, this strain demonstrated limited antioxidative ability, which could make a contribution to the prevention of lipid oxidation in meat and meat products.     

Spoilage-related activity of Carnobacterium maltaromaticum strains in air-stored and vacuum-packed meat

One hundred three isolates of Carnobacterium spp. from raw meat were analyzed by random amplification of polymorphic DNA (RAPD) and PCR and were identified by 16S rRNA gene sequencing. Forty-five strains of Carnobacterium maltaromaticum were characterized for their growth capabilities at different temperatures, NaCl concentrations, and pH values and for in vitro lipolytic and proteolytic activities. Moreover, their spoilage potential in meat was investigated by analyzing the release of volatile organic compounds (VOCs) in meat stored in air or vacuum packs. Almost all the strains were able to grow at 4, 10, and 20°C, at pH values of 6 to 9, and in the presence of 2.5% NaCl. The release of VOCs by each strain in beef stored at 4°C in air and vacuum packs was evaluated by headspace solid-phase microextraction (HS-SPME)-gas chromatography-mass spectrometry (GC-MS) analysis. All the meat samples inoculated and stored in air showed higher numbers of VOCs than the vacuum-packed meat samples. Acetoin, 1-octen-3-ol, and butanoic acid were the compounds most frequently found under both storage conditions. The contaminated meat samples were evaluated by a sensory panel; the results indicated that for all sensory odors, no effect of strain was significant (P > 0.05). The storage conditions significantly affected (P < 0.05) the perception of dairy, spoiled-meat, and mozzarella cheese odors, which were more intense in meat stored in air than in vacuum packs but were never very intense. In conclusion, different strains of C. maltaromaticum can grow efficiently in meat stored at low temperatures both in air and in vacuum packs, producing volatile molecules with low sensory impacts, with a negligible contribution to meat spoilage overall.     

Estimation of bacteriological spoilage of pork cutlets by electronic nose

The utility of chemosensor array (EN) signals of head-space volatiles of aerobically stored pork cutlets as a non-invasive technique for monitoring their microbiological load was studied during storage at 4, 8 and 12 degrees C, respectively. The bacteriological quality of the meat samples was determined by standard total aerobic plate counts (TAPC) and colony count of selectively estimated Pseudomonas (PS) spp., the predominant aerobic spoilage bacteria. Statistical analysis of the electronic nose measurements were principal component analysis (PCA), and canonical discriminant analysis (CDA). Partial least squares (PLS) regression was used to model correlation between microbial loads and EN signal responses, the degree of bacteriological spoilage, independently of the temperature of the refrigerated storage. Sensor selection techniques were applied to reduce the dimensionality and more robust calibration models were computed by determining few individual sensors having the smallest cross correlations and highest correlations with the reference data. Correlations between the predicted and "real" values were given on cross-validated data from both data reduced models and for full calibrations using the 23 sensor elements. At the same time, sensorial quality of the raw cutlets was noted subjectively on faultiness of the odour and colour, and drip formation of the samples. These preliminary studies indicated that the electronic nose technique has a potential to detect bacteriological spoilage earlier or at the same time as olfactory quality deterioration.     

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.     

Changes in the spoilage-related microbiota of beef during refrigerated storage under different packaging conditions

The microbial spoilage of beef was monitored during storage at 5 degrees C under three different conditions of modified-atmosphere packaging (MAP): (i) air (MAP1), (ii) 60% O2 and 40% CO2 (MAP2), and (iii) 20% O2 and 40% CO2 (MAP3). Pseudomonas, Enterobacteriaceae, Brochothrix thermosphacta, and lactic acid bacteria were monitored by viable counts and PCR-denaturing gradient gel electrophoresis (DGGE) analysis during 14 days of storage. Moreover, headspace gas composition, weight loss, and beef color change were also determined at each sampling time. Overall, MAP2 was shown to have the best protective effect, keeping the microbial loads and color change to acceptable levels in the first 7 days of refrigerated storage. The microbial colonies from the plate counts of each microbial group were identified by PCR-DGGE of the variable V6-V8 region of the 16S rRNA gene. Thirteen different genera and at least 17 different species were identified after sequencing of DGGE fragments that showed a wide diversity of spoilage-related bacteria taking turns during beef storage in the function of the packaging conditions. The countable species for each spoilage-related microbial group were different according to packaging conditions and times of storage. In fact, the DGGE profiles displayed significant changes during time and depending on the initial atmosphere used. The spoilage occurred between 7 and 14 days of storage, and the microbial species found in the spoiled meat varied according to the packaging conditions. Rahnella aquatilis, Rahnella spp., Pseudomonas spp., and Carnobacterium divergens were identified as acting during beef storage in air (MAP1). Pseudomonas spp. and Lactobacillus sakei were found in beef stored under MAP conditions with high oxygen content (MAP2), while Rahnella spp. and L. sakei were the main species found during storage using MAP3. The identification of the spoilage-related microbiota by molecular methods can help in the effective establishment of storage conditions for fresh meat.     

Exploring the Sources of Bacterial Spoilers in Beefsteaks by Culture-Independent High-Throughput Sequencing

Microbial growth on meat to unacceptable levels contributes significantly to change meat structure, color and flavor and to cause meat spoilage. The types of microorganisms initially present in meat depend on several factors and multiple sources of contamination can be identified. The aims of this study were to evaluate the microbial diversity in beefsteaks before and after aerobic storage at 4°C and to investigate the sources of microbial contamination by examining the microbiota of carcasses wherefrom the steaks originated and of the processing environment where the beef was handled. Carcass, environmental (processing plant) and meat samples were analyzed by culture-independent high-throughput sequencing of 16S rRNA gene amplicons. The microbiota of carcass swabs was very complex, including more than 600 operational taxonomic units (OTUs) belonging to 15 different phyla. A significant association was found between beef microbiota and specific beef cuts (P<0.01) indicating that different cuts of the same carcass can influence the microbial contamination of beef. Despite the initially high complexity of the carcass microbiota, the steaks after aerobic storage at 4°C showed a dramatic decrease in microbial complexity. Pseudomonas sp. and Brochothrix thermosphacta were the main contaminants, and Acinetobacter, Psychrobacter and Enterobacteriaceae were also found. Comparing the relative abundance of OTUs in the different samples it was shown that abundant OTUs in beefsteaks after storage occurred in the corresponding carcass. However, the abundance of these same OTUs clearly increased in environmental samples taken in the processing plant suggesting that spoilage-associated microbial species originate from carcasses, they are carried to the processing environment where the meat is handled and there they become a resident microbiota. Such microbiota is then further spread on meat when it is handled and it represents the starting microbial association wherefrom the most efficiently growing microbial species take over during storage and can cause spoilage.     

Bacterial sources of putrescine and cadaverine in chill stored vacuum-packaged beef

Of the meat strains of streptobacteria, leuconostocs, Enterobacteriaceae and Brochothrix thermosphacta tested, only Hafnia alvei and Serratia liquefaciens showed diamine-producing potential during growth in pure culture on beef stored in vacuum packs at 1°C. Both organisms produced cadaverine at concentrations similar to those reported previously in naturally contaminated beef stored under the same conditions. Putrescine concentrations produced by the two organisms, however, were an order of magnitude lower. During the growth on beef of either H. alvei or S. liquefaciens in mixed culture with arginine-utilizing strains of streptobacteria, putrescine as well as cadaverine concentrations were similar to those detected in naturally contaminated samples.     

Putrescine and cadaverine formation in vacuum packed beef

Bacterial numbers, putrescine and cadaverine concentrations and pH were measured at regular intervals during the chill storage of vacuum packed beef. Odours on opening the packs were also assessed. Cadaverine concentration increased more rapidly than that of putrescine and measurable increases were evident before maximum bacterial numbers were attained and before any permanent off-odours were detected. Diamine concentrations correlated better with total viable count (TVC) than with counts of Gram negative organisms.     

Viability of lyophilized microorganisms after storage

Bacteria, yeasts and fungi lyophilized in a dextran-sodium glutaminate solution were stored under vacuum in the dark at room temperature. After 6 years the survival was tested and viability counts were made. Of the 48 genera or groups tested 31 survived well and 9 moderately (respectively, 80–100% and 50–80% of the strains were still alive). Many strains of the generaAgrobacterium; Azotobacter; Beijerinckia; Erwinia; Pseudomonas; Lipomyces andMicrocyclus failed to survive. Lyophilized mushroom mycelium did not grow after 6 years storage. The results of this experiment are compared with those obtained with the same strains preserved on sealed agar slants (Antheunisse, 1972). The results of both methods in relation to some genera are discussed.     

Study of changes in physicochemical and microbiological characteristics of shrimps (Melicertus kerathurus) stored under modified atmosphere packaging

Fresh minimally processed shrimps were stored under modified atmosphere packaging (60% CO₂:40% N₂ for MAP A and 92.9% N₂:5.1% CO₂:2% O₂ for MAP B) for 5 days at 3 °C. Total mesophiles, H₂S forming bacteria, Pseudomonas spp., Brochothrix thermosphacta, firmness, color and sensory parameters were investigated throughout the whole time of the experiment. During storage period samples stored under MAP B managed to retain firmness values close to the initial values. All microbial populations growth was suppressed by the presence of MAP A. Samples stored under MAP B managed to maintain their firmness values close to the initial ones while MAP A samples were significantly less firm (p < 0.05).     

Investigation on the potential application of biological agents in the extension of shelf life of fresh beef in Nigeria

The objective of this study was to evaluate the ability of lactic acid bacteria (LAB) cultures to preserve fresh beef at room temperature, with a view to promoting safety and availability of the product in Nigeria. Two LAB strains, Pediococcus pentosaceus LIV 01 and P. acidilactici FLE 01, were applied as starters (10⁶ cfu/g) on sliced fresh beef samples, and were stored for 7 days at 30°C. Analyses of microbiological, thiobarbituric acid (TBA) and free fatty acids (FFA) were carried out during storage. Results indicated reduction in the Enterobacteriaceae, Staphylococcus and coliforms in starter inoculated samples. TBA and FFA were lower in starter culture inoculated samples compared to controls during storage. In a challenge experiment against the LAB cultures during a 7-day storage, two sets of meat were inoculated separately with 10⁶ cfu/g each of pathogenic organisms Listeria monocytogenes and Salmonella Typhimurium. There was about 1 log reduction in the L. monocytogenes on day 1 while counts were below detection limit (<2 log) on day 2 in meat samples inoculated with P. pentosaceus alone and in combination with P. acidilactici. Counts of S. Typhimurium showed about 2 log reduction in starter inoculated samples during storage while an increase by about 3 log was observed in control samples. The protective ability of the LAB strains could be exploited in shelf life extension and control of foodborne pathogens in fresh beef; their use as biological preservatives may help in promoting public health, safety and availability of the product in Nigeria.     

The use of Alcalase 2·5L in the acridine orange direct count technique for the rapid enumeration of bacteria in beef mince

The acridine orange direct count (AODC) technique was used to count bacterial numbers in beef mince. The ability of Alcalase 2·5L to degrade beef mince was compared with the previously used Alcalase 0·6L. Both methods were evaluated against the standard plate count. The results showed that Alcalase 2·5L can be used in the AODC technique for the rapid counting of bacterial numbers in beef mince. Prediction equations obtained for the relationship between the AODC and standard plate counts were validated with commercial beef mince samples.     

Manganese Carbonate Precipitation Induced by Calcite-Forming Bacteria

Abstract

Several dissimilatory metal-reducing bacteria and a halophilic bacterium are able to induce manganese carbonate (rhodochrosite) precipitation. In this study, it was revealed that Ensifer adhaerens JCM 21105^T, Microbacterium testaceum JCM 1353^T, Pseudomonas protegens DSM 19095^T, and Rheinheimera texasensis DSM 17496^T, which are calcite-forming bacteria, were able to aerobically induce the precipitation of manganese carbonate crystals on an agar medium. In the case of all four strains, the principal morphology of the precipitated manganese carbonate crystals was that of micro-sized spheres, when they were aerobically cultivated over the entire surface of the agar medium at 28?°C for 7?days.     

Effect of medium and temperature of storage on viability of lactic acid bacteria immobilized in κ-carrageenan-locust bean gum gel beads

Summary The influence of various storage solutions and temperature (4°C and 25°C) on viability ofStreptococcus salivarius subsp.thermophilus andLactobacillusdelbrueckii subsp.bulgaricus entrapped in κ-carrageenan-locust bean gum mixed gel beads was studied. The immobilized strains could be stored at 4°C in all storage solutions studied for at least 14 and 11 days respectively before counts decreased to 10⁵c.f.u./mL, which was considered to be the practical limit for their use as inoculum in a fermentation process. The most effective storage solutions for preserving cell viability at 4°C were NaCl, glycerol and sorbitol solutions forS. thermophilus, and PO₄ buffer and sorbitol solutions forL. bulgaricus. At 25°C,S. thermophilus could be stored for over 14 days in all solutions except glycerol, andL. bulgaricus for 4 days in 10% sorbitol.     

The aerobic growth and product formation of Lactobacillus,Leuconostoc, Brochothrix,and Carnobacterium in batch cultures

Summary The aerobic growth and metabolism of eleven homofermentative and three heterofermentative Lactobacillus strains, three Leuconostoc strains, two Brochothrix thermosphacta strains and two Carnobacterium strains were studied in batch cultures at pH 6.0 and 25°C on a complex substrate containing 10.0 g glucose per litre. All strains, except Carnobacterium divergens 69, grew well aerobically. An oxygen consumption was registered for 18 of the strains—the exceptions being Lactobacillus alimentarius DSM 20249^T, Lactobacillus farciminis DSM 20284^T and Lactobacillus sharpeae DSM 20505^T. The homofermentative lactobacilli showed a maximal oxygen consumption during the stationary growth phase and this was coupled with a low final viable count. Leuconostoc strains, heterofermentative lactobacilli, Brochothrix thermosphacta and Carnobacterium strains showed a maximal oxygen consumption during the exponential growth phase together with a high final viable count. The maximum specific growth rate varied from 0.19 to 0.54 h^-1 while the growth yield varied from 19 to 86 g dry weight per mol glucose consumed. In general, homofermentative lactobacilli produced dl-lactic acid, acetic acid and acetoin. The three heterofermentative lactobacilli produced dl-lactic acid and acetic acid, two strains also produced ethanol Leuconostoc spp. formed d-lactic acid, acetic acid, and ethanol. B. thermosphacta produced acetoin, acetic acid, formic acid, isobutyric acid and isovaleric acid but no lactic acid. Carnobacterium produced l-lactic acid, acetic acid and acetoin. All strains accumulated hydrogen peroxide except L. alimentarius DSM 20249^T, Carnobacterium piscicola 3 and B. thermosphacta.née Blickstad     

Survival of Campylobacter Jejuni/Coli in Ground Refrigerated and in Ground Frozen Beef Liver and in Frozen Broiler Carcasses

Survival of 5 strains of Campylobacter jejuni/coli in ground beef liver stored at 4° C and at –20° C was studied. After 6 days of storage at 4° C the beef liver was spoiled, which was indicated by APG log 7.25 and lactobacilli count log 7.0. During this storage Campylobacter counts decreased only slightly. After 12 weeks of storage at –20° C Campylobacter counts decreased by 2–3 logs in frozen ground beef liver. Survival of 4 strains of C. jejuni/coli on frozen broiler carcasses was also studied. Two inoculation levels, 103–104/g and 104–105/g were used. On frozen broiler carcasses Campylobacter counts decreased by 0.5–2.0 logs during 12 weeks at –20° C.