Residual effect of storage in an elevated carbon dioxide atmosphere on the microbial flora of rock cod (Sebastes spp.)

A residual inhibitory effect on microbial growth due to modified-atmosphere (MA) storage (MA, 80% CO2-20% air) was demonstrated for rock cod fillets stored in MA and transferred to air at 4 degrees C. Results of measurements of CO2 concentrations of the fillets suggested that the residual effect after transfer from MA to air was not due to retention of CO2 at the surface of the fillets but was probably due to the microbial ecology of the system. Lactobacillus spp. and tan Alteromonas spp. (TAN) predominated after 7 and 14 days of storage in MA. During storage in MA, Pseudomonas spp. were inhibited or killed. Following transfer from MA to air, the percentage of the total flora represented by Lactobacillus spp. and TAN bacteria decreased, and 6 days after transfer Pseudomonas spp. were again dominant.     

Residual effect of storage in an elevated carbon dioxide atmosphere on the microbial flora of rock cod (Sebastes spp.).

A residual inhibitory effect on microbial growth due to modified-atmosphere (MA) storage (MA, 80% CO2-20% air) was demonstrated for rock cod fillets stored in MA and transferred to air at 4 degrees C. Results of measurements of CO2 concentrations of the fillets suggested that the residual effect after transfer from MA to air was not due to retention of CO2 at the surface of the fillets but was probably due to the microbial ecology of the system. Lactobacillus spp. and tan Alteromonas spp. (TAN) predominated after 7 and 14 days of storage in MA. During storage in MA, Pseudomonas spp. were inhibited or killed. Following transfer from MA to air, the percentage of the total flora represented by Lactobacillus spp. and TAN bacteria decreased, and 6 days after transfer Pseudomonas spp. were again dominant.     

The microbial flora of herring fillets after storage in carbon dioxide, nitrogen or air at 2°C

Herring fillets from the Baltic Sea were stored in glass vessels in air, nitrogen or carbon dioxide (CO₂) at 2°C and the microbial development was studied. The microbiological shelf-life of the herring (the time to reach 10⁷ organisms/g) was prolonged by a factor of 3.5 in CO₂ as compared to air. The corresponding factor in nitrogen was 1.5. The microflora of fresh and spoiled herring was classified. The initial microflora was dominated by coryneforms, Flavobacterium spp., Moraxella -like organisms and Pseudomonas spp. The spoilage-flora in air (after 9 d) was dominated by Pseudomonas spp. and Moraxella -like organisms, and in nitrogen (14 d) Enterobacteriaceae, Vibrionaceae and Lactobacillus spp. were dominant. Homofermentative Lactobacillus spp. were the only organisms isolated from fillets stored in CO₂ (28 d). It was concluded that storing fresh fish in pure CO₂ at low refrigeration temperatures is a method with industrial potential. The method (1) improves the microbiological stability and (2) reduces the microbiological health hazards of the fish.     

Effect of Hyperbaric Carbon Dioxide Pressure on the Microbial Flora of Pork Stored at 4 or 14°C

Changes in the microbial flora of pork stored at 4 or 14°C were studied in 5 atm CO₂, 1 atm CO₂ or 1 atm air. The time needed for the total aerobic count at 4°C to reach 5 × 10⁶ organisms/cm² was about three times longer in 5 atm CO₂ than in 1 atm CO₂, and about 15 times longer in 5 atm CO₂ than in air. At 14°C there was no difference in growth rate between 5 atm CO₂ and 1 atm CO₂. No off-odour was detected after storage in 5 atm CO₂ for 14 d, but the pork in 1 atm CO₂ (6 d) was organoleptically unacceptable.
The predominant organisms on the pork from the processing line were: Flavobacterium spp., Acinetobacter calcoaceticus, Pseudomonas spp., Micrococcus spp. and Moraxella spp. After aerobic storage at 4°C (8 d) or 14°C (3 d) more than 90% of the flora consisted of Pseudomonas spp. At 4°C all Pseudomonas spp. were of the non-fluorescent type, whilst at 14°C 32% were Ps. putida and Ps. fluorescens. After storage in 1 atm CO₂ Lactobacillus spp. represented 66% of the flora at 14°C (6 d) and 100% at 4°C (40 d), with L. xylosus dominating. After storage in 5 atm CO₂ Lactobacillus spp. constituted the total flora at both temperatures with L. lactis (14°C) and L. xylosus (4°C) dominating.
It was concluded that high partial pressures of CO₂ have a considerable shelf-life prolonging effect by (i) selecting the microflora towards Lactobacillus spp. and (ii) reducing the growth rate of these Lactobacillus spp. The controlling and growth inhibitory effect of CO₂ was promoted by reduced temperatures.     

Glucose, the key substrate in the microbiological changes occurring in meat and certain meat products

The literature dealing with the role of glucose in the microbiological changes of meat and certain meat products is reviewed. Discussion is centered on two aspects. First, glucose plays a part in the selection of the dominant spoilage organisms, Pseudomonas fragi, Ps. lundensis, and Ps. fluorescens, on red meat stored aerobically under chill (2-7 degrees C) conditions. It is concluded that the pseudomonads flourish because they convert glucose to the less commonly used substrate, gluconate. The latter serves as an extracellular energy store. With its depletion, the pseudomonads utilize amino acids, thereby producing the characteristic off-odors of spoiled meat. Storage of meat in a modified atmosphere (viz., 20% CO2:80% O2) selects Gram-positive flora (lactobacilli and Brochothrix thermosphacta) which impart a "cheesy odor" through acid production from glucose and volatile fatty acids from amino acids. The first mentioned organisms produce the same off-odors in "acid" meat (pH 5.5) from which oxygen is excluded. So too does the less acid-tolerant Br. thermosphacta in less acid meat (pH greater than 5.8), especially if trace amounts of O2 are present. Such meat may be colonized by Shewanella putrefaciens also, with green discoloration resulting from the release of H2S from amino acids. The addition of glucose and NO2- to, and the exclusion of oxygen from, comminuted meat selects a flora dominated by Lactobacillus spp. and staphylococci such as Staphylococcus carnosus. Second, sulfite, the preservative of British-style sausages, has a sparing action on glucose. As a consequence of its curtailed breakdown there is only a meager acid drift with storage even though a fermentative flora of lactobacilli and Br. thermosphacta is selected. Yeasts also contribute to the microbial association in sausages; members of four of the six commonly occurring genera bind sulfite through acetaldehyde production. Glucose appears to be essential for acetaldehyde synthesis. The role of glucose in spoilage and the conditions which select particular groups of spoilage organisms are considered in the context of chemical probes and/or instrumental methods for routine assessment of the "freshness" of meat and meat products.     

A Numerical Taxonomic Study of Pseudomonas-like Bacteria Isolated from Fish in Southeastern Queensland and their Association with Spoilage

Pseudomonas -like bacteria isolated from fresh and spoiling fish in southeastern Queensland were subjected to a wide range of physiological and nutritional tests. The results of these tests, together with those of 20 named strains, were analysed numerically, resulting in the formation of 11 groups. Most of the isolates clustered into group 1 and group 2 which also contained the bulk of the strains able to produce spoilage odours when grown in a tryptic digest of fish muscle at 2°C. Almost all of the group 1 organisms produced sulphydryl type odours, had only 50 mol % G + C and were identified as strains of Alteromonas putrefaciens which were deficient in the ability to produce H₂S detectable in Peptone Iron Agar. Certain of the group 2 strains produced fruity and sulphydryl type odours, but these organisms were not distinguishable from other strains in this group not producing odours. Group 2 strains were highly related to Pseudomonas fragi and were intermediate in properties between Ps. fluorescens and Ps. putida. The remaining nine minor groups contained few organisms able to produce spoilage odours.     

Microbiological analysis of rock cod (Sebastes spp.) stored under elevated carbon dioxide atmospheres

The numbers and types of microorganisms on fresh rock cod fillets and fillets stored in air or in a modified atmosphere (MA; 80% CO(2), 20% air) at 4 degrees C were compared. Samples were analyzed after 0, 7, 14, and 21 days of storage. The isolation plates were incubated aerobically, anaerobically, or under MA at 4, 20, or 35 degrees C. After 7 days of storage in air, the fillets were obviously spoiled and had a 3- to 4-log cycle increase in microbial counts. Plate counts increased more slowly on MA-stored fillets. After 21 days, the counts on the latter had increased only 2 log cycles, and the fillets did not seem spoiled. The microbial flora changed greatly during MA storage. Only Lactobacillus spp. (70%) and an Aeromonas sp.-like isolate (30%) were found on plates incubated aerobically at 4 and 20 degrees C, and only Lactobacillus spp. was found on plates incubated aerobically and anaerobically at 35 and at 20 degrees C under MA. Isolation plates incubated at 20 degrees C in air gave the highest counts in the shortest incubation time and the greatest diversity of bacterial types recovered. No Vibrio parahaemolyticus, Staphylococcus aureus, or Clostridium botulinum type E were isolated from the fresh or MA-stored fillets.     

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.     

Ecophysiological attributes of a Lactobacillus sp. and a Pseudomonas sp. on sterile beef fillets in relation to storage temperature and film permeability

AIMS: To determine the combined effect of packaging film and temperature on the rate and type of end-products caused by the growth of two main contrasting prevailing organisms in air and 100% CO2, Pseudomonas sp. and Lactobacillus sp., respectively. METHODS AND RESULTS: Pseudomonas sp. and Lactobacillus sp. were inoculated individually on sterile meat fillets. The samples were packed in air or 100% CO2, using a high and a low permeable film, and stored at 0 and 10 degrees C. Pseudomonas sp. grew aerobically and in 100% CO2 using high permeable film at both storage temperatures, while film permeability significantly affected the growth of Lactobacillus sp. only at 10 degrees C. Enzymatic kits and HPLC and GC analysis were used to determine the chemical changes of the samples throughout storage. Pseudomonas sp. presented a greater rate of consumption of glucose and lactate than Lactobacillus sp. in samples stored aerobically or with high permeable film. Propanol-1 and two unidentified organic acids were present only in samples inoculated with Pseudomonas sp., while acetaldehyde, ethanol, diacetyl and acetoin were detected in samples inoculated with Lactobacillus sp. CONCLUSION: Since different microbial species and introduction of new packaging methods affect spoilage reactions of meat either qualitatively or quantitatively, a combination of several chemical indicators should be thoroughly investigated. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study provides information on how and when such potential indicators can be exploited for the benefit of the industry and consumer.     

The microbial association of Greek taverna sausage stored at 4 and 10 degrees C in air, vacuum or 100% carbon dioxide, and its spoilage potential

Strains of the Lactobacillus sakei/curvatus group, mainly non-slime-producing Lact. sakei, dominated the microbial flora of industrially manufactured taverna sausage, a traditional Greek cooked meat, stored at 4 degrees C and 10 degrees C in air, vacuum and 100% CO2. Atypical, arginine-positive and melibiose-negative strains of this group were isolated. The isolation frequency of Lact. sakei/curvatus from sausages stored anaerobically was as high as 92-96%, while other meat spoilage organisms were practically absent. Conversely, in air-stored sausages, leuconostocs, mainly Leuconostoc mesenteroides ssp. mesenteroides, had a considerable presence (14-21%), whereas Brochothrix thermosphacta, pseudomonads and Micrococcaceae grew, but failed to increase above 10(5) cfu g(-1) in all samples during storage. Only yeasts were able to compete against LAB and reached almost 10(7) cfu g(-1) after 30 d of aerobic storage at 10 degrees C. The great dominance (> 10(8) cfu g(-1)) of LAB caused a progressive decrease of pH and an increase of the concentration of L-lactate, D-lactate and acetate in all sausage packs. The growth of LAB and its associated chemical changes were more pronounced at 10 degrees C than 4 degrees C. At both storage temperatures, L-lactate and acetate increased more rapidly and to a higher concentration aerobically, unlike D-lactate, which formed in higher amounts anaerobically. Storage in air was the worst packaging method, resulting in greening and unpleasant off-odours associated with the high acetate content of the sausages. Carbon dioxide had no significant effect on extending shelf-life. The factors affecting the natural selection of Lact. sakei/curvatus in taverna sausage are discussed. Moreover, it was attempted to correlate the metabolic activity of this group with the physicochemical changes and the spoilage phenomena occurring in taverna sausage under the different storage conditions.     

Respective effects of sodium and chloride ions on filament formation and growth and ethanol production in Zymomonas mobilis fermentations

AIMS: To evaluate the antimicrobial effect of nine essential oils (EO) on P. phosphoreum and determine the effect of oregano oil on the shelf-life of modified atmosphere-packed (MAP) cod fillets. METHODS AND RESULTS: The antimicrobial effect of EO was studied in a liquid medium and in product storage trials. Oils of oregano and cinnamon had strongest antimicrobial activity, followed by lemongrass, thyme, clove, bay, marjoram, sage and basil oils. Oregano oil (0.05%, v/w) reduced growth of P. phosphoreum in naturally contaminated MAP cod fillets and extended shelf-life from 11-12 d to 21-26 d at 2 degrees C. CONCLUSIONS: Oregano oil reduced the growth of P. phosphoreum and extended the shelf-life of MAP cod fillets. SIGNIFICANCE AND IMPACT OF THE STUDY: Mild and natural preservation using EO can extend the shelf-life of MAP seafood through inhibiting the specific spoilage organism P. phosphoreum.     

Changes in the Microflora of Irradiated Petrale Sole (Eopsetta jordani) Fillets Stored Aerobically at 0.5 C

The microfloral changes on irradiated petrale sole fillets during aerobic (packaged with oxygen-permeable film), refrigerated storage were determined by the identification of bacterial and yeast isolates to the generic level. The samples were irradiated at 0.0, 0.1, 0.15, 0.2, 0.3, and 0.4 Mrad by use of a cobalt-60 gamma source, were stored at 0.5 C, and were examined periodically for spoilage, total microbial population, and composition. The preirradiation flora of the fresh fillets consisted of coryneforms, Achromobacter, Micrococcus, Flavobacterium, Pseudomonas, and Lactobacillus. Immediately after irradiation, Micrococcus, Achromobacter, coryneforms, and Bacillus were predominant. The flora of the nonirradiated fillets at the time of spoilage consisted of Pseudomonas and Achromobacter. The flora of the irradiated fillets at the time of spoilage consisted of Achromobacter and Trichosporon.     

Classification of the spoilage flora of fish, with special reference to Shewanella putrefaciens

One hundred and fifty-nine Gram-negative strains isolated from refrigerated fish, taken from the Baltic Sea or Swedish inland waters, together with 32 reference strains of Shewanella, Pseudomonas, Aeromonas and Alcaligenes, were phenotypically classified using 124 unit characters. Data were processed by the Simple Matching (SSM) and Jaccard (SJ) coefficients, and unweighted pair group algorithm with arithmetic averages. Fourteen clusters were defined at the 75% SJ similarity level which correspond to the SSM level of 86%. SJ-based clusters containing field strains were designated Pseudomonas fragi (cluster 1; 31% of the field strains), Ps. lundensis (cluster 2; 2% of the field strains), Ps. fluorescens biovar III (cluster 4; 4% of the field strains), Ps. putida biovar A (cluster 5; 3% of the field strains), Ps. fluorescens/putida (clusters 3 and 6; 6% of the field strains), Psychrobacter (clusters 8 and 9; 3% of the field strains), Shewanella putrefaciens (clusters 10, 11, 12 and 13; 44% of the field strains) and Aer. sobria (cluster 14; 6% of the field strains, all isolated from fresh water fish). Each field strain represented the spoilage flora of refrigerated fish at a total aerobic count of about 10(8) cfu/g. Phenotypic characteristics of major clusters are given. The four S. putrefaciens clusters may be separated by key characteristics. Shewanella putrefaciens ATCC 8071T and reference strains from sources other than fish, did not group in any of the clusters. The mol % guanine + cytosine content was on average 47.6 for cluster 10, and 45.3 for cluster 13.     

Effect of Packaging under Carbon Dioxide, Nitrogen or Air on the Microbial Flora of Pork Stored at 4°C

Changes in the microbial flora of pork packed in laminated plastic bags and stored at 4 °C were studied in an initial atmosphere of carbon dioxide, nitrogen or air. The time needed for the total aerobic count at 28 °C to reach 5 × 10⁶ organisms/cm² was about 7 times longer in carbon dioxide than in air, whilst in nitrogen it was about twice as long.
The predominant organisms on fresh pork taken directly from the processing line were: Acinetobacter calcoaceticus , non-fluorescent Pseudomonas spp. and Flavobacterium spp. After storage in air for 7 d, more than 90% of the flora consisted of non-fluorescent Pseudomonas spp. After storage in nitrogen for 10 d, 70% of the flora consisted of non-fluorescent Pseudomonas spp. with lower levels of fluorescent Pseudomonas spp., Kurthia zopfii, Aeromonas hydrophila and Lactobacillus plantarum. The non-fluorescent Pseudomonas spp. could be divided into three different groups, on proteolytic and lipolytic ability; the distribution of the groups was markedly different between pork loins stored in air and nitrogen.
On pork stored in carbon dioxide for 21 d the flora consisted of L. plantarum together with lower levels of heterofermentative lactic acid bacteria. When the storage time in carbon doxide was prolonged to 35 d, the proportion of heterofermentative lactic acid bacteria increased to about 50% of the flora.     

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 ).     

Classification of the spoilage flora offish, with special reference to Shewanella putrefaciens

One hundred and fifty-nine Gram-negative strains isolated from refrigerated fish, taken from the Baltic Sea or Swedish inland waters, together with 32 reference strains of Shewanella, Pseudomonas, Aeromonas and Alcaligenes , were phenotypically classified using 124 unit characters. Data were processed by the Simple Matching (S_SM) and Jaccard (S_J) coefficients, and unweighted pair group algorithm with arithmetic averages. Fourteen clusters were defined at the 75% S_J similarity level which correspond to the S_SM level of 86%. S_J-based clusters containing field strains were designated Pseudomonas fragi (cluster 1; 31% of the field strains), Ps. lundensis (cluster 2; 2% of the field strains), Ps. fluorescens biovar III (cluster 4; 4% of the field strains), Ps. putida biovar A (cluster 5; 3% of the field strains), Ps. fluorescens/putida (clusters 3 and 6; 6% of the field strains), Psychrobacter (clusters 8 and 9; 3% of the field strains), Shewanella putrefaciens (clusters 10, 11, 12 and 13; 44% of the field strains) and Aer. sobria (cluster 14; 6% of the field strains, all isolated from fresh water fish). Each field strain represented the spoilage flora of refrigerated fish at a total aerobic count of about 10⁸ cfu/g.
Phenotypic characteristics of major clusters are given. The four S. putrefaciens clusters may be separated by key characteristics. Shewanella putrefaciens ATCC 8071^T and reference strains from sources other than fish, did not group in any of the clusters. The mol % guanine + cytosine content was on average 47.6 for cluster 10, and 45.3 for cluster 13.     

Pseudomonads and achromobacters in the spoilage of irradiated haddock of different preirradiation quality

The effect of initial quality of fish on postirradiation (100 krad) changes in the bacterial flora of haddock fillets during aerobic storage at 3 C has been investigated, with emphasis on the Pseudomonas and Achromobacter groups. The quality was related to the length of time the eviscerated fish had been stored in ice prior to filleting. Increased numbers of organisms, in particular Pseudomonas putrefaciens, were found initially on fillets cut from older fish. Pseudomonads were reduced by 2 to 3 log orders by irradiation, and achromobacters and gram-positive isolates predominated in the immediate postirradiation flora. Little difference could be detected in either types or relative proportions of organisms occurring during storage of unirradiated fish of different quality. Pseudomonads outgrew achromobacters and dominated the spoilage flora in all cases. After spoilage, however, the growth rate of pseudomonads declined markedly. In irradiated fish, achromobacters predominated throughout storage. In fish of better initial quality, bacterial numbers were 1 to 2 log orders higher at spoilage than in their unirradiated counterparts and in the poorer quality of irradiated samples. The increased number of organisms was accompanied by a radical change in the character of the predominant achromobacters. Pseudomonads were found to increase in numbers during storage of irradiated fish, in particular in poorer quality fish on which they were initially present in higher numbers. Detection of pseudomonads, even when present in high numbers, was found to be limited by the identification techniques normally used.     

Production and specificity of poly- and monoclonal antibodies raised against Shewanella putrefaciens

B. FONNESBECH, H. FRØKIAER, L. GRAM AND C. MOSBY JESPERSEN. 1993. Polyclonal antibodies were raised in rabbits and mice against Shewanella putrefaciens. Murine monoclonal antibodies were produced against the type strain (ATCC 8071) as well as wild type strains isolated from fish products. The specificities of four polyclonal and 12 monoclonal antibodies were tested by dot-blotting, an indirect and a competitive ELISA against 16 Gram-negative strains; including six strains of S. putrefaciens and one strain of Pseudomonas rubescens (NC 10695). All polyclonal antibodies reacted strongly with S. putrefaciens and with Ps. rubescens and cross-reacted with the nine other bacteria ( Pseudomonas spp., Aeromonas spp. and Vibrio anguillarum ). The monoclonal antibodies could be divided into three groups with different patterns of specificity. The largest group (8 monoclonal antibodies) reacted strongly with S. putrefaciens and with Ps. rubescens and showed only weak reactions with the other strains. The results confirm that Ps. rubescens should be classified as S. putrefaciens.     

Spoilage of vacuum-packaged dark, firm, dry meat at chill temperatures.

The flora of vacuum-packaged dark, firm, dry meat included thred organisms not usually found on vacuum-packaged meat, Yersinia enterocolitica, Enterobacter liquefaciens, and Alteromonas putrefaciens. Y. enterocolitica did not affect the meat quality. Production of spoilage odors by E. liquefaciens could be prevented by addition of glucose or citrate to the meat. Greening of meat could be prevented by addition of glucose or citrate to the meat. Greening of meat by A. putrefaciens was not prevented by addition of glucose, as the organism degraded cysteine with the release of H2S even when glucose was present. To prevent greening, growth of A. putrefaciens must be inhibited by reducing the meat pH to less than 6.0.     

Spoilage of vacuum-packaged dark, firm, dry meat at chill temperatures.

The flora of vacuum-packaged dark, firm, dry meat included thred organisms not usually found on vacuum-packaged meat, Yersinia enterocolitica, Enterobacter liquefaciens, and Alteromonas putrefaciens. Y. enterocolitica did not affect the meat quality. Production of spoilage odors by E. liquefaciens could be prevented by addition of glucose or citrate to the meat. Greening of meat could be prevented by addition of glucose or citrate to the meat. Greening of meat by A. putrefaciens was not prevented by addition of glucose, as the organism degraded cysteine with the release of H2S even when glucose was present. To prevent greening, growth of A. putrefaciens must be inhibited by reducing the meat pH to less than 6.0.