Detection and Incidence of Specific Species of Spoilage Bacteria on Fish: I. Methodology

The ability of Pseudomonas putrefaciens to form H(2)S was found to serve as a singularly useful criterion of identity for this species and was used to directly enumerate the organism from haddock fillets by the use of pour plates of Peptone-Iron Agar. Subsurface colonies appear intensely black, whereas surface colonies are black or gray. A highly sensitive soft-agar-gelatin overlay technique has been found useful for directly determining the numbers of weakly and strongly proteolytic organisms from fish tissue.     

Extracellular Nuclease Activity of Fish Spoilage Bacteria, Fish Pathogens, and Related Species

The production of extracellular deoxyribonuclease and ribonuclease by 23 marine and 3 dairy strains of Pseudomonas putrefaciens, 15 strains of fish-pathogenic fluorescent pseudomonads, 38 strains of fluorescent pseudomonads isolated from haddock, and 34 related organisms was determined by an agar plate method. All strains of P. putrefaciens produced both deoxyribonuclease and ribonuclease. Of the other 87 organisms examined, 26.5% produced ribonuclease and 14.5% produced deoxyribonuclease. All organisms which produced deoxyribonuclease also produced ribonuclease. Deoxyribonuclease production by P. putrefaciens is suggested as a useful criterion of identity for members of this intense fish spoilage species.     

Proteolytic Activity of Microorganisms Isolated from Freshwater Fish

A raw fish-juice was prepared and sterilized through the use of (60)Co gamma-irradiation. It was evaluated for suitability in an agar medium for testing the proteolytic activity of bacteria isolated from fish. Microorganism proteolytic activity was also detected by conventional methods with skim milk-agar. We tested 1,145 isolates from fresh and spoiling irradiated (0.0, 0.3, and 0.6 Mrad) yellow perch fillets for proteolytic activity, by the use of both media. Most isolates that showed proteolytic activity exhibited this activity in both media. A few isolates showed proteolytic activity only in one medium or the other. Proteolysis was found mainly among bacteria isolated from nonirradiated perch fillets. Nonproteolytic organisms were slightly more abundant than were proteolytic ones throughout refrigerated storage (6 days); the latter constituted 48% of the total organisms. Irradiation eliminated essentially all proteolytic bacteria when the fillets were stored at 1 C. However, some proteolytic bacteria survived for a few days after irradiation when the fillets were stored at 5 C.     

Effects of Substerilization Doses of Co60 Gamma Radiation on the Cold-Storage Life Extension of Shucked Soft-Shelled Clams and Haddock Fillets

Total aerobic-facultative and anaerobic (clostridia) macrocolony count data are presented, with analysis and interpretation, for both haddock fillets and shucked soft-shelled clams which received doses of from 50,000 to 800,000 rad of Co⁶⁰ gamma rays. These data indicated that haddock fillets may be maintained in good condition at refrigeration temperatures above freezing for about 1 week at 6 C, and approximately 2 weeks at 0 C, when treated with from 50,000 to 150,000 rad of ionizing radiation. In the dose range from 200,000 to 350,000 rad, the storage life may be extended up to some 2 weeks at 6 C, and 3 weeks at 0 C. Treatments in the dose range from 400,000 to 500,000 rad may defer spoilage for about 1 month, and doses of 550,000 to 650,000 rad afford protection against bacterial spoilage up to approximately 1.5 months. At the high substerilization doses of 700,000 to 800,000 rad, haddock fillets may be held for from 2 to 3 months in refrigerated storage before becoming unfit for marketing and consumption. Shucked soft-shelled clams can be held for about 2.5 weeks at 0 C and close to 12 days at 6 C, when given low substerilization doses of from 50,000 to 150,000 rad of ionizing radiation. At doses of from 200,000 to 350,000 rad, the clams may be preserved effectively for periods up to 3 weeks at 0 or 6 C, and some 6 weeks at these temperatures with doses of about 450,000 rad. With treatments of 500,000 to 600,000 rad, the storage life may be extended for some 2 months, and at doses of 650,000 to 800,000 rad the shucked clams remain in a good state of preservation for up to 3 months at temperatures of 0 to 6 C. Thus, it would appear that shucked soft-shelled clams may be maintained for significantly longer periods in refrigerated storage than haddock fillets when the same radiation treatments are applied to each product. Clostridia levels in both products were relatively low initially, and were reduced significantly by the gamma rays at the doses studied. Moreover, those clostridia that survived the radiation treatments were found to remain at safe, low levels during the various periods in refrigerated storage employed for these products, a very encouraging result from the public health, as well as commercial, standpoint.     

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.     

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 ).     

Identification of Major High-Boiling Volatile Compounds Produced During Refrigerated Storage of Haddock Fillets

The two major high-boiling volatile compounds produced during refrigerated storage of haddock fillets were found by gas chromatography and mass spectroscopy to be phenethyl alcohol and phenol.     

Effect of γ Irradiation on the Microflora of Freshwater Fish: III. Spoilage Patterns and Extension of Refrigerated Storage Life of Yellow Perch Fillets Irradiated to 0.1 and 0.2 Megarad1

Maximal shelf life was determined and microbial flora were compared for irradiated (0.1 and 0.2 Mrad) and nonirradiated yellow perch fillets stored at 1 C. Shelf life was estimated by organoleptic determinations. Microbiological studies included determination of the effects of irradiation on the total aerobic microbial population, lag phase, and rate of growth. Genera of organisms isolated from fillets through the course of microbial spoilage were identified, and the proteolytic activity of the organisms was determined. Plate counts for fish prior to irradiation showed the presence of approximately 10(6) organisms per g of sample. Irradiation to 0.1 and 0.2 Mrad produced 1.4 and 3 logarithm reductions of the initial count, respectively. Irradiation to 0.1 and 0.2 Mrad approximately doubled the product's shelf life. Organisms initially isolated from the nonirradiated fillets, in order of decreasing number, consisted of Flavobacterium, Micrococcus-Sarcina, Achromobacter-Alcaligenes-Mima, Pseudomonas, Microbacterium, Vibrio, Bacillus, Corynebacterium, Lactobacillus, Brevibacterium, and Aeromonas. By the 6th and 9th days of fillet storage, Pseudomonas and the Achromobacter group were the predominant organisms. All members of the genus Flavobacterium, but not all members of the genus Pseudomonas, were proteolytically active on raw fish juice-agar and skim milk-agar media. The Achromobacter group was found to be nonproteolytic on both media. Residual flora of fillets irradiated to 0.1 and 0.2 Mrad consisted of the Achromobacter group, Lactobacillus, Micrococcus-Sarcina, and Bacillus. Their sequence in predominance, however, varied with dose level. Not all proteolytic bacteria in the fillets were eliminated by 0.1 and 0.2 Mrad; proteolytic Micrococcus-Sarcina survived these treatments.     

Performance of Pseudomonas CFC-selective medium in the fish storage ecosystems

Pseudomonas agar base supplemented with cephaloridine, fucidin, and cetrimide (CFC) was used to count Pseudomonas populations on fish. Both Enterobacteriaceae and Shewanella putrefaciens were able to grow on the CFC medium. Evaluation of the performance of CFC-selective for pseudomonads medium, on fish samples stored aerobically and under a modified atmosphere at 0, 10 and 20 degrees C was tested.The selectivity of the medium was affected by storage temperatures and the type of packaging of the fish samples. The selectivity of the medium diminished as the population increased and for samples stored at high temperature (20 degrees C) or under modified atmospheres.When designing adequate selectivity of a medium, interfering organisms should be taken into account, especially when the background flora tends to be more robust than the organisms to be counted or detected.     

Effect of γ-Irradiation on the Microflora of Freshwater Fish: I. Microbial Load,Lag Period,and Rate of Growth on Yellow Perch (Perca flavescens) Fillets

Microbial flora were compared in irradiated and nonirradiated yellow perch fillets. These studies included effects of irradiation on the total microbial population, the lag phase, and rate of growth in this freshwater fishery product. The work was conducted concurrently with sensory and chemical evaluation, and constituted part of an investigation designed to evaluate the effect of substerilization doses (0.3 and 0.6 Mrad) of Co⁶⁰ γ rays on the storage life of yellow perch fillets at 1.0 or 6.0 C. In five storage tests, total plate counts prior to irradiation did not exceed 8.7 × 10⁵ per gram of sample; this count was reduced nearly 100% by irradiation with either 0.3 or 0.6 Mrad. Progressively lower maximal bacterial populations and lengthened lag phases were obtained as more radiation was used. The growth rate of the population did not appear to decrease significantly. Microbial data obtained in these studies confirmed the sensory and chemical studies, by indicating that irradiation can significantly extend the refrigerated shelf life of freshwater fish.     

Effect of γ-Irradiation on the Microflora of Freshwater Fish: I. Microbial Load, Lag Period, and Rate of Growth on Yellow Perch (Perca flavescens) Fillets

Microbial flora were compared in irradiated and nonirradiated yellow perch fillets. These studies included effects of irradiation on the total microbial population, the lag phase, and rate of growth in this freshwater fishery product. The work was conducted concurrently with sensory and chemical evaluation, and constituted part of an investigation designed to evaluate the effect of substerilization doses (0.3 and 0.6 Mrad) of Co⁶⁰ γ rays on the storage life of yellow perch fillets at 1.0 or 6.0 C. In five storage tests, total plate counts prior to irradiation did not exceed 8.7 × 10⁵ per gram of sample; this count was reduced nearly 100% by irradiation with either 0.3 or 0.6 Mrad. Progressively lower maximal bacterial populations and lengthened lag phases were obtained as more radiation was used. The growth rate of the population did not appear to decrease significantly. Microbial data obtained in these studies confirmed the sensory and chemical studies, by indicating that irradiation can significantly extend the refrigerated shelf life of freshwater fish.     

Effect of temperature on the microbial flora of herring fillets stored in air or carbon dioxide

The microbial development on fillets of herring from the Baltic Sea was studied at temperatures from 0-15 C in air or 100% carbon dioxide (96-100% CO2). The shelf-life of the fillets , defined as the time for the 'total aerobic count' to reach 1 X 10(7) c.f.u./g increased with decreased temperature from 1 d at 15 degrees C to 7 d at 0 degrees C (air). The corresponding values in CO2 were 3 d and 33 d, respectively. The initial flora of the herring fillets was dominated by Alteromonas putrefaciens and Pseudomonas spp. and so was the spoilage flora after storage in air (together 62-95% of the flora: all temperatures). Alteromonas putrefaciens predominated slightly at 2 degrees C to 15 degrees C, while Pseudomonas spp. dominated at 0 degrees C. The Pseudomonas flora was mainly split between Ps. fragi , Ps. fluorescens and a heterogenous group of unidentified Pseudomonas spp. The proportions were not influenced by temperature. In 100% CO2 at the time of spoilage the flora consisted of a significant number of Lactobacillus spp. Below 4 C the domination was almost complete while at 10 degrees C and 15 degrees C. Enterobacteriaceae, Vibrionaceae and Alt. putrefaciens was also found. It was concluded that the microbiological shelf-life of herring fillets is improved by refrigeration storage in 100% CO2 but for good results the temperature should not exceed 2 degrees C.     

Hydrogen Sulfide Production by Pseudomonas putrefaciens in Shrimp Experimentally Packed in Nitrogen

Shrimp refrigerated in a nitrogen atmosphere develop off-odors not typical of normal spoilage. Investigations of this phenomenon showed that hydrogen sulfide developed in the headspace gas, and a large percentage of the microbial population present on the shrimp stored in nitrogen was capable of hydrogen sulfide production, in contrast to the flora on shrimp stored in air. The predominant hydrogen sulfide-producing organism, Pseudomonas putrefaciens, was present in low numbers on fresh shrimp but usually reached high numbers by day 8 of nitrogen storage. Further studies revealed that cysteine and cystine were the probable substrates in shrimp utilized by this organism for hydrogen sulfide production. When shrimp sterilized by irradiation were inoculated with P. putrefaciens and incubated in an atmosphere of nitrogen, hydrogen sulfide and the characteristic off-odors developed.     

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.     

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.     

New Selective Media for Enumeration and Recovery of Fluorescent Pseudomonads from Various Habitats

New media (S1 and S2) were formulated that provide a high degree of selectivity and detection of fluorescent pseudomonads on initial plating. The selectivity of the S-type media was based on a detergent, sodium lauroyl sarcosine, and an antibiotic, trimethoprim. A total of five soils from different geographical locations and one sewage sludge sample were examined. On S1 medium, isolates from two soils with low fluorescent pseudomonad populations exhibited a high frequency of arginine dihydrolase (78%) and oxidase-positive (95%) phenotypes, but no fermentative isolates were recovered. Medium S2 was more defined and selective than S1, but lower numbers of fluorescent pseudomonads were recovered on S2. In soils in which fluorescent pseudomonads represent a small proportion of the total population, S1 medium consistently recovered high percentages of fluorescent phenotypes (82.5%).     

Numerical taxonomy of proteolytic psychrotrophs from Queensland raw milks

Eighty-seven proteolytic psychrotrophic micro-organisms were isolated from 11 bulk milk supplies of two Queensland factories from different climatic regions, before and after storage at 4°C for 7 d. These isolates together with 15 reference strains formed the basis of a numerical taxonomic study involving 81 attributes. All but six isolates were pseudomonads. The strains clustered into nine groups, of which one group consisted of four yeasts. One group, containing 39 isolates, was designated as Pseudomonas fluorescens biovar 1; three groups, containing 27 isolates, as Ps. fluorescens biovar 5; and one group, containing 10 isolates, as Ps. putida biovar A. This study showed that the proteolytic psychrotrophic microflora of the 11 milks supplying the two factories was substantially different and that the proteolytic flora of 7 d refrigerated milk could not be estimated by examining the flora before storage.     

Numerical taxonomy of proteolytic psychrotrophs from Queensland raw milks

Eighty-seven proteolytic psychrotrophic micro-organisms were isolated from 11 bulk milk supplies of two Queensland factories from different climatic regions, before and after storage at 4 degrees C for 7 d. These isolates together with 15 reference strains formed the basis of a numerical taxonomic study involving 81 attributes. All but six isolates were pseudomonads. The strains clustered into nine groups, of which one group consisted of four yeasts. One group, containing 39 isolates, was designated as Pseudomonas fluorescens biovar 1; three groups, containing 27 isolates, as Ps. fluorescens biovar 5; and one group, containing 10 isolates, as Ps. putida biovar A. This study showed that the proteolytic psychrotrophic microflora of the 11 milks supplying the two factories was substantially different and that the proteolytic flora of 7 d refrigerated milk could not be estimated by examining the flora before storage.     

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.     

The influence of packaging methodology on the microbiological and fatty acid profiles of refrigerated African catfish fillets

AIMS: The shelf-life of refrigerated catfish fillets was determined at 2 degrees C, to simulate retail conditions, using two types of packaging materials, vacuum packing (VP) and oxygen permeable packaging (OPP). METHODS AND RESULTS: Representative samples (n=5) from both types of packaging methods were drawn at random every 2 d until a microbiological count of 106 cfu g-1 was reached. Samples were pooled and screened microbiologically using standard methods. Fatty acid analyses of total lipids, neutral lipid, glycolipid and phospholipid fractions were also conducted, to determine at which point the fish was regarded as spoiled and which packaging method provided a longer shelf-life. OPP limits storage to a maximum of 4 d (aerobic plate count of 8.2 x 105 cfu g-1), whereas VP extends the shelf-life of the fillets to between 6 and 8 d (aerobic plate count of 9.2 x 104 cfu g-1 and 1.66 x 106 cfu g-1, respectively). Similarly, coliform counts increased with time; however, packaging material had no statistical influence thereon. CONCLUSION: Until d 13, when the experiment was terminated, no deterioration in lipid composition of the various fractions was noted. SIGNIFICANCE AND IMPACT OF THE STUDY: An extended shelf-life microbiologically-speaking, for potential processors, could thus be obtained by using VP instead of OPP.