Microbial interaction in cooked cured meat products under vacuum or modified atmosphere at 4 degrees C

AIMS: To investigate the antagonistic activity of two lactic acid strains against the spoilage microflora in cooked cured meat products, vacuum or modified atmosphere packed at 4 degrees C and to determine the inhibitory capacity of their bacteriocins. METHODS AND RESULTS: Frankfurter-type sausages and sliced cooked cured pork shoulder were inoculated with Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442 or with their bacteriocins. The microbial, physico-chemical (pH, L- and D-lactate, acetate and ammonia) and colour changes were studied. Results under vacuum packaging showed that in the uninoculated samples of the pork product the spoilage microflora grew but in the inoculated ones the spoilage microorganisms (e.g. Brochothrix thermosphacta and enterococci) reduced during the storage. This observation was more pronounced in the samples with the addition of bacteriocins. In the frankfurter-type sausages the spoilage microflora did not grow in the uninoculated and inoculated samples. In the modified atmosphere enriched in CO2 the population of spoilage microflora remained at low levels in both products, indicating that CO2 has an effect on the spoilage microorganisms' growth. In the pork product the concentrations of acetate and d-lactate increased while L-lactate decreased, but in the frankfurter-type sausages increase of acetate and D-lactate was not observed. CONCLUSIONS: Lactic acid strains had an effect on the spoilage microflora growth but did not affect, negatively, the organoleptic properties of the products. These strains may be used as biopreservative cultures or their bacteriocins could be an important contribution to microbiological quality of meat products. SIGNIFICANCE AND IMPACT OF STUDY: Establishment of biopreservation as a method for extension of shelf life of meat products.     

Microbial spoilage and formation of biogenic amines in fresh and thawed modified atmosphere-packed salmon (Salmo salar) at 2 degrees C

AIMS: To evaluate the microbial spoilage, formation of biogenic amines and shelf life of chilled fresh and frozen/thawed salmon packed in a modified atmosphere and stored at 2 degrees C. METHODS AND RESULTS: The dominating microflora, formation of biogenic amines and shelf life were studied in two series of storage trials with naturally contaminated fresh and thawed modified atmosphere-packed (MAP) salmon at 2 degrees C. Photobacterium phosphoreum dominated the spoilage microflora of fresh MAP salmon at more than 10(6) cfu g(-1) and the activity of this specific spoilage organism (SSO) limited the shelf life of the product to ca 14 and 21 d in the two experiments. Despite the high levels of P. phosphoreum, less than 20 mg kg(-1) histamine was observed in fresh MAP salmon prior to sensory spoilage. Freezing eliminated P. phosphoreum and extended the shelf life of MAP salmon at 2 degrees C by 1-2 weeks. Carnobacterium piscicola dominated the spoilage microflora of thawed MAP salmon and probably produced the ca 40 mg kg(-1) tyramine detected in this product at the end of its shelf life. CONCLUSIONS: Photobacterium phosphoreum dominated the spoilage microflora of fresh MAP salmon but produced only small amounts of biogenic amines in this product. The elimination of P. phosphoreum by freezing allowed this bacteria to be identified as the SSO in fresh MAP salmon. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of P. phosphoreum as the SSO in fresh MAP salmon facilitates the development of methods to determine and predict the shelf life of this product, as previously shown with fresh MAP cod.     

Nitrite loss and spoilage microflora development in chub-packed luncheon meat

Residual nitrite was lost from chub-packed luncheon meat during storage through both chemical breakdown and microbial consumption. The relative importance of these mechanisms in this pasteurized product was determined by the speed of development of the spoilage microflora, which is influenced by storage conditions. The nitrite half-life due to chemical loss was 13 d at 25°C and 36 d at 10°C. When microbial growth occurred these half-lives were reduced to 2.6 d and 21 d, respectively. Qualitative differences in the microflora that developed at these two temperatures (denitrifying Bacillus spp. at 25°C and non-denitrifying Streptococcus spp. at 10°C) account for the large temperature effect. Growth of Streptococcus spp. increased the rate of chemical nitrite loss in chubs by reducing the pH value. Nitrite did not inhibit the aerobic growth of either Bacillus or Streptococcus species associated with spoilage but did inhibit the anaerobic growth of Bacillus spp. This bacteriostatic effect of residual nitrite in anaerobic conditions will decrease during storage as nitrite level falls and oxygen penetrates the chub pack. Nitrite-mediated bacteriostasis does not obviate the need for refrigerated storage but does afford a real, if ephemeral, safeguard against spoilage occurring during short periods of temperature abuse.     

Heat tolerance of Salmonella typhimurium DT104 isolates attached to muscle tissue

Aerobic and anaerobic plate counts were compared for routine monitoring of the microflora, dominated by lactic acid bacteria, developing on vacuum- and carbon dioxide-packaged raw meat during chilled storage. No statistical differences were observed between aerobic and anaerobic enumerations, made on plate count and blood agar plates, of the microflora developing on beef striploins packaged under vacuum or carbon dioxide during 14 weeks' storage at 0°C. With both techniques the spoilage microflora development differed between the two packaging regimes. The results indicate that there is no necessity for aerobic plate counts to be replaced by anaerobic plate counts in the routine microbiological examination of the spoilage microflora developing on chilled meats packaged under anoxic modified atmospheres.     

Successive Microbial Populations in Calimyrna Figs

Smyrna-type (Calimyrna) figs have essentially sterile internal tissue until visited by the pollinating fig wasp, Blastophaga psenes, which introduces a specific microflora consisting of Candida guilliermondii var. carpophila and Serratia plymuthica. This flora persists and develops in numbers throughout the ripening period until maturity of the fruit. These organisms do not cause spoilage. The presence of C. guilliermondii var. carpophila appears to increase the attractiveness of the fruit to drosophilae. Drosophila (mainly D. melanogaster) carry spoilage yeasts and bacteria on their exterior body parts, and introduce these organisms during ovipositing in the fruit cavity. The spoilage yeasts consist almost entirely of apiculate yeasts (Hanseniaspora valbyensis, H. uvarum, and Kloeckera apiculata) and of Torulopsis stellata, which cause active fermentative spoilage. Spoilage bacteria (primarily Acetobacter melanogenus) are also introduced with the yeasts. Organic acids are produced by these yeasts as well as by the Acetobacter. A number of minor spoilage yeasts were also identified.     

Lactic acid bacteria associated with a heat-processed pork product and sources of variation affecting chemical indices of spoilage and sensory characteristics

Aims:  To evaluate the potential for developing a quality index for a Danish modified atmosphere packaged (MAP) heat-processed and naturally contaminated pork meat product stored at 5°C.
Methods and Results:  The composition of the predominating microflora and changes in contents of tyramine, arginine, organic acids and sensory characteristics were analysed. The microflora was predominated by Lactobacillus sakei , Leuconostoc carnosum and Carnobacterium divergens . The presence of each species varied between products and batches resulting in limited usefulness of the concentrations of these bacteria or their metabolites as indices of quality. Furthermore, the three species differed in their metabolic activities as shown by use of a model meat extract. However, when MAP storage of the processed pork product was followed by aerobic storage then acetic acid showed some potential as a chemical indicator of sensory quality.
Conclusion:  Variation in processing parameters and spoilage microbiota limited the usefulness of concentrations of micro-organisms and their metabolites as indices of spoilage for the studied processed MAP pork product.
Significance and Impact of the Study:  The present study contributes to an understanding of the difficulties experienced in developing quality indices to be used in the control of microbial spoilage of processed MAP meat products.     

Incidence and Spoilage Potential of Isolates from Vacuum-packaged Meat of High pH Value

Meat of high pH value (6·6) showing dark-cutting characteristics was vacuum-packaged and stored for up to 8 weeks at 0–2°C. 'Off'-odours were detected on opening the packages after 6 weeks of storage. Total counts at this stage were ca. 10⁷/cm² of which lactobacilli were the major component, with ca. 10⁶/cm² Gram negative organisms. Psychrotrophic Enterobacteriaceae represented a major proportion of the microflora only after the full 8 weeks of storage and were not detected previously. Aerobic storage of steaks cut from the vacuum packaged meat stored for 8 weeks resulted in a predominantly Gram negative spoilage flora.
Inoculation studies on meat of normal pH value (5·4) and appearance using representative isolates from the vacuum-packaged meat microflora indicated that most of the test organisms were capable of causing spoilage under aerobic conditions but few under vacuum-packaging when incubated at 4°C. On meat of higher pH value (6·15) many of the Gram negative isolates did not grow as well, whereas the Gram positive isolates grew better than on meat of normal pH value when held under aerobic conditions. Under vacuum-packaging all but one isolate grew as well or better on meat of high pH value than on normal meat at 4°C and objectionable odours were more marked.     

A note on microbial spoilage of sheep meat at ambient temperature

Shelf-life and microbial spoilage of sheep carcass meat at ambient temperature under commercial conditions were studied. The initial bacterial count of carcasses ranged 5.6-5.8 log/cm2. Staphylococcus spp. (48%) predominated in the initial microflora of carcasses followed by Micrococcus spp. (19%) and Escherichia spp. (12%). Microbial spoilage of carcasses occurred around 20 h when the bacterial count reached 8.0-9.0 log/cm2. Thus the shelf life of carcasses at ambient temperature was 19 h. The predominant micro-organisms at the time of spoilage were Escherichia and 'Acinetobacter-like' organisms. It was also observed that Enterobacter, Pseudomonas and Staphylococcus spp. could form a major part of the final flora. The presence of Escherichia and Staphylococcus spp. in higher percentages on the surface of carcasses at the time of spoilage presents the scope for health hazards.     

Effect of Oxygen Tension on the Spoilage Microflora of Irradiated and Non-irradiated Haddock (Melanogrammus aeglefinus) Fillets

S ummary . The spoilage microflora of irradiated and non-irradiated air packed or vacuum packed haddock fillets was identified after storage of the fillets at 35°F. With the non-irradiated fillets, both air packed and vacuum packed, the spoilage flora was predominantly pseudomonads. With the irradiated fillets, the spoilage flora of the air packed samples was chiefly achromobacter; that of the vacuum packed samples was chiefly lactobacilli (or possibly Microbacterium thermosphactum ).     

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.     

Review: the human cutaneous microflora and factors controlling colonisation

The human skin is an unusual habitat for microorganisms in that it is open to contamination from the environment and yet is largely unsuitable for microbial colonisation, unlike mucosal surfaces. The normal microflora of human skin consists of resident colonising species capable of maintaining a viable, reproducing population on the skin and transient contaminating species that cannot sustain growth in the cutaneous environment. The structure of the skin and physiological factors such as hydration, pH, O₂ and growth substrates determine the density and diversity of colonisation. Ecological stability is maintained by interactions between the host and the microflora, and between microbial species, and the relative importance of these factors varies between individuals at equivalent sites. The distribution of skin appendages at different sites on the body determines the prevailing environmental conditions, which in turn affects the density and diversity of the microflora. Microbial colonisation is not only restricted to the surface of the skin and there are substantial populations associated with the skin appendages, in particular sebaceous follicles. The aim of this article is to review the factors which determine the composition of the skin microflora under normal conditions and assess their relative importance.     

Diversity of culturable psychrophilic and psychrotrophic anaerobic bacteria isolated from beef abattoirs and their environments

This study identified 431 psychrophilic or psychrotrophic isolates from commercial Irish beef abattoir environments and "blown packs" of vacuum-packed beef, using PCR and 16S rRNA sequencing, and estimated their intraspecies genetic diversity using restriction fragment length polymorphism (RFLP) analysis and spacer region PCR (SR-PCR). Twenty-five species were identified in the 431 isolates, with the most frequently recovered species being Clostridium gasigenes (n=315), Clostridium estertheticum (n=17), and a potentially novel species designated strain TC1 (n=52). These species were previously found to be associated with a particular type of spoilage known as blown-pack spoilage (BPS), which occurs in chilled-stored (i.e., -1.5°C to 4°C) vacuum-packaged meat within 2 to 4 weeks and involves the production of large volumes of gas. Overall, the study demonstrates the considerable and not previously reported diversity of the anaerobic microflora in abattoirs and the presence of a wide range of organisms capable of causing BPS at chilled temperatures.     

The effect of biogenic amine production by single bacterial cultures and metabiosis on cold-smoked salmon

AIM: Biogenic amines are important indicators of spoilage in vacuum-packed cold-smoked salmon. It is the aim of this study to identify bacteria responsible for biogenic amine production in cold-smoked salmon. METHODS AND RESULTS: The present study identified spoilage microflora from cold-smoked salmon and determined biogenic amine production of single and co-cultures growing in cold-smoked salmon. Photobacterium phosphoreum was the only species that produced histamine when inoculated on sterile cold-smoked salmon. Production of putrescine was enhanced 10-15 times when cultures of Serratia liquefaciens or Hafnia alvei were grown with Carnobacterium divergens or Lactobacillus sakei subsp. carnosus. This phenomenon was explained by interspecies microbial metabolism of arginine, i.e., metabiosis. CONCLUSIONS: The amounts of biogenic amines produced by single and co-cultures corresponded to those observed during spoilage of naturally-contaminated cold-smoked salmon. Photobacterium phosphoreum and Lact. curvatus were identified as the specific spoilage organisms in cold-smoked salmon. SIGNIFICANCE AND IMPACT OF THE STUDY: Determination of the specific spoilage organism is needed before a model can be developed for shelf-life predictions of cold-smoked salmon.     

The effect of temperature on the bacterial load and microbial composition in Norway lobster (Nephrops norvegicus) tail meat during storage

Aims: The aim of this study was to update and extend our knowledge of the bacterial load and microbial composition in Norway lobster (Nephrops norvegicus) under commercially relevant storage conditions to optimize handling procedures. Methods and Results: Total viable counts were performed at different storage temperatures (0, 4, 8, 10, 12 or 16°C) and after different storage times (1–7 days). Storage at 16°C was found to be most detrimental, and storage at 0°C was found to be optimal. 16S‐rRNA sequencing was utilized to determine the composition of the bacteria within the microflora. In this way, Photobacterium isolates, especially Photobacterium phosphoreum, were identified as the main specific spoilage organisms. The abilities to reduce trimethylamineoxide (TMAO) and to produce H₂S were analysed in a selection of bacterial isolates. The higher the incubation temperature during storage, the more isolates were found to reduce TMAO and produce H₂S. Conclusions: Nephrops norvegicus possesses an unusually high initial microbial load when fresh. Storage temperature is the most crucial factor affecting microbial growth, microbial activity and spoilage potential in N. norvegicus produce. Spoilage can be attributed mainly to P. phosphoreum. Significance and Impact of the Study: This study presents significant new findings with regard to the progression and causative agents of spoilage in N. norvegicus. Based on the results, we can recommend that N. norvegicus tails should be stored in a 0°C environment immediately after catch. Stored this way, the growth and spoilage activity of the microflora may be reduced significantly and an extension of shelf life might be attained.     

Light butter: natural microbial population and potential growth of Listeria monocytogenes and Yersinia enterocolitica

Yarrowia lipolytica, Bacillus polymyxa and Enterococcus faecium were the most frequent yeast and bacterial spoilage species associated with commercial light butter. Following inoculation and incubation at 4°C, strains of Y. enterocolitica and L. monocytogenes exhibited growth rates higher than those of the naturally occurring microflora. Listeria monocytogenes displayed a higher aptitude to proliferate in such food while the cell increment of Y. enterocolitica was limited.     

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 degrees C in (1) aerobic conditions; (2) 30% CO2 + air; (3) 30% CO2 + N2; and (4) 100% CO2. When samples were held at 1 degree 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 degrees 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 degree C rather than 37 degrees C. In CO2 atmospheres growth of L. monocytogenes was inhibited on meat held at 6 degrees C, especially under 100% CO2. By contrast, storage at 15 degrees 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.     

Changes in the Microflora of Haddock Fillets and Shucked Soft-Shelled Clams After Irradiation with Co60 Gamma Rays and Storage at 0 C and 6 C

Frequency distribution patterns of aerobic-facultative microflora, obtained by random selection of macrocolonies from samples of haddock fillets and shucked soft-shelled clams before and after treatment with doses of from 50,000 to 800,000 rad of Co⁶⁰ gamma rays, are presented, with analyses and interpretation. The data showed that a decided change occurred in the constitution of the microbial populations of both products: from a mixed gram-negative—gram-positive flora to a predominantly gram-positive flora immediately after irradiation. The great majority of these surviving microorganisms were micrococci, sporeforming bacilli, and certain yeasts, molds, and actinomyces. During storage at refrigeration temperatures above freezing, the microflora changed from the descendants of the more radioresistant gram-positive species to the more prolific gram-negative psychrophilic species that flourish at these low temperatures. Micrococci and gram-positive rods declined somewhat during the rise of the actively proteolytic-lipolytic pseudomonads and related species, but still remained at high enough levels to contribute significantly to the spoilage observed at different times in storage. The eventual spoilage of haddock fillets was characterized by discoloration of the cream-white tissue with water-soluble yellow, green, and red bacterial pigments; degradation of the tissue, by proteolytic and other microbial enzymes, to a watery, flaccid mass; and formation of volatile compounds that smelled putrid, rancid, and generally foul and pungent. Shucked soft-shelled clams displayed a different spoilage pattern, changing to a variegated brown-gray and forming a matted or loose gelatinous mass from which arose stale, acrid, putrid, sulfurous odors. These differences are believed to be attributable to the varied biochemical nature of the tissues involved, the Eh potential within the tissues, the time sequence of microfloral change, and the species and types of microorganisms associated with each product. Staphylococci were present in small numbers in some of the samples tested, but did not appear to be species of public health significance. Gram-negative enteric rods were also encountered, but were considered to be of doubtful public health importance. More detailed investigations, designed to study the effects of Co⁶⁰ gamma radiations on such microbes in sea foods, would be useful in assessing the problem further.     

Evaluation of the extent and type of bacterial contamination at different stages of processing of cooked ham

In an attempt to determine the composition and origin of the spoilage flora of refrigerated vacuum-packed cooked ham, the changes in microbial numbers and types were followed along the processing line. Results revealed Lactobacillus sake and Leuconostoc mesenteroides ssp. mesenteroides as the major causative agents of spoilage of sliced ham stored at 4 °C and 12 °C, due to recontamination in the cutting room. On the contrary, the progressive deterioration of whole ham under the same storage conditions was associated with a non-identifiable group of leuconostoc-like bacteria. Except for lactic acid bacteria, no other organism grew in vacuum packs of either sliced or whole ham. Although atypical leuconostocs could not be detected among isolates recovered from freshly produced whole ham, they appeared to survive cooking and proliferate during storage. Neither these organisms however, nor Lact. sake and Leuc. mesenteroides were important in curing and tumbling as carnobacteria, mainly Carnobacterium divergens, and Brochothrix thermosphacta dominated at this stage. A progressive inversion of the ham microflora from mostly Gram-negative at the beginning of processing to highly Gram-positive prior to cooking was noted. Listeria monocytogenes cross-contaminated ham during tumbling. However, the pathogen was always absent from the vacuum-packed product provided that heating to a core temperature of 70 °C occurred and recontamination during slicing and packing was prevented. The percentage distribution of different species of lactic acid bacteria as well as the uncommon phenotypic characteristics of some strains were discussed.     

Cellulolytic Bacteria Associated with Sloughing Spoilage of California Ripe Olives

Sloughing spoilage of California ripe olives during processing is characterized by severe softening, skin rupture, and flesh sloughing. It was assumed that cellulolytic activity was responsible for skin rupture and sloughing of flesh, and so a deliberate search was made for cellulolytic bacteria from olives undergoing sloughing spoilage. A bacterium identified as Cellulomonas flavigena was highly cellulolytic, attacking filter paper, carboxymethyl cellulose (CMC) gel, and olive tissue. Other bacteria attacking CMC, but not filter paper, enhanced the activity of the Cellulomonas strain when grown in mixed culture, although they did not, in pure culture, have any effect on filter paper. These latter cultures (all degraded olive tissue) represented the genera Xanthomonas, Aerobacter, and Escherichia. Other noncellulolytic bacteria belonging to the genera Alcaligenes, Kurthia, and Micrococcus also were used for study of mixed culture fermentation of cellulose by C. flavigena. Cellobiose accumulation at levels of 1.0% (w/v) and above suppressed growth of C. flavigena.     

Microbial profiles of country-cured hams aged in stockinettes, barrier bags, and paraffin wax.

No significant differences were found in surface microflora of county-cured hams covered with stockinettes, barrier bags, or a coating of paraffin wax during aging, except for a reduction in mold growth on waxed hams. The incidence of Clostridium spp. was lost in all treatments. Micrococcus spp. and Streptococcus spp. were the most common contaminants, but caused no apparent spoilage problem in any treatment.