Bacteriology of Spoilage of Fish Muscle: III. Characterization of Spoilers

A total of 807 bacterial isolates from fresh and spoiling fillets of English sole (Parophrys vetulus) stored at 5 C were classified as to genus and tested for various biochemical activities, including the ability to spoil sterile muscle press juice at 5 C. Production of off-odor, volatile reducing substances, and trimethylamine was used to estimate spoilage. It was found that (i) spoilers could be distinguished from nonspoilers on the basis of the juice spoilage test, (ii) differentiation between spoilers and nonspoilers could not be achieved by means of the usual biochemical tests, (iii) no micrococci, flavobacteria, and “coryneforms” were spoilers, (iv) certain specific subgroups of the genus Pseudomonas consisted exclusively of spoilers whereas others were inactive, (v) the genus Achromobacter likewise consisted of spoilers and nonspoilers, and (vi) “coliforms” could produce spoilage. It was concluded that a method is now available to determine directly and unequivocally the role played in spoilage by various bacterial groups and that it is no longer necessary to rely on indirect evidence.     

Bacteriology of Spoilage of Fish Muscle: IV. Role of Protein

Clarified muscle press juice from English sole (Parophrys vetulus) was fractionated by gel filtration into a protein and a protein-free fraction. Upon inoculation with spoilage bacteria, the protein fraction failed to show any signs of spoilage, but the protein-free fraction spoiled according to the usual organoleptic and chemical criteria. Despite its spoilage-resistant qualities, the protein fraction accelerated spoilage of the protein-free fraction when the two were combined. Protein breadkown due to bacterial action was greatest in the unfractionated juice and was least in the protein fraction. No significant proteolysis occurred until after spoilage became evident.     

Bacteriology of Spoilage of Fish Muscle: I. Sterile Press Juice as a Suitable Experimental Medium

A sterile raw fish muscle press juice, diluted 1:4 with saline, has been prepared. This dilution greatly facilitated Seitz filtration and affected the spoilage properties of the medium only negligibly. At 5.5 C, the spoilage pattern of naturally contaminated diluted juice was almost identical to that of naturally contaminated fillets. This was shown by comparing the quantitative and qualitative aspects of the bacterial flora on the two substrates and by measuring the production of volatile reducing substances (VRS) and of trimethylamine (TMA). With the sterile raw muscle press juice, some preliminary data showed that individual members of the genera Achromobacter and Pseudomonas differ markedly in their spoilage capabilities: some grew but did not produce spoilage detectable either organoleptically or chemically; others gave rise to strong off odors and to high levels of VRS and TMA.     

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.     

Detection and Incidence of Specific Species of Spoilage Bacteria on Fish: II. Relative Incidence of Pseudomonas putrefaciens and Fluorescent Pseudomonads on Haddock Fillets

Pseudomonas putrefaciens has been found to constitute one of the major species of spoilage bacteria on haddock fillets. The initial population of this organism on fillets of high bacterial quality is uniformly below 4% and most frequently no greater than 1%. During refrigerated storage, the organism increases at a more rapid rate than the total psychrophilic population, comprising 50 to 90% of the total population when the total count exceeds 10(6)/g of tissue. Fluorescent pseudomonads were shown to constitute a second group of predominant pseudomonads constituting up to 19.3% of the total population after 8 days of refrigerated storage. Of a total of 45 fluorescent pseudomonads isolated from haddock fillets, 14 (31.1%) were found to be potent fish spoilers. The use of a soft-agar-gelatin plating technique showed a parallel increase of proteolytic organisms with total count indicating that proteolytic organisms other than P. putrefaciens and fluorescent pseudomonads increase at a slower rate than these two groups.     

Muscle Dynamics in Fish During Steady Swimming

SYNOPSIS. Recent research in fish locomotion has been dominatedby an interest in the dynamic mechanical properties of the swimmingmusculature. Prior observations have indicated that waves ofmuscle activation travel along the body of an undulating fishfaster than the resulting waves of muscular contraction, suggestingthat the phase relation between the muscle strain cycle andits activation must vary along the body. Since this phase relationis critical in determining how the muscle performs in cycliccontractions, the possibility has emerged that dynamic musclefunction may change with axial position in swimming fish. Quantificationof muscle contractile properties in cyclic contractions relieson in vitro experiments using strain and activation data collectedin vivo. In this paper we discuss the relation between theseparameters and body kinematics. Using videoradiographic datafrom swimming mackerel we demonstrate that red muscle straincan be accurately predicted from midline curvature but not fromlateral displacement. Electromyographic recordings show neuronalactivation patterns that are consistent with red muscle performingnet positive work at all axial positions. The relatively constantcross-section of red muscle along much of the body suggeststhat positive power for swimming is generated fairly uniformlyalong the length of the fish.     

Anaerobic Fish Spoilage by Bacteria II. Kinetics of Bacterial Growth and Substrate Conversions in Herring Extracts

The time course of the conversions of chemical components in herring extracts during anaerobic growth of Proteus sp., str. NTHC 153, Aeromonas sp., str. NTHC 154, and Enterobacter sp., str. NTHC 151 (Strøm & Larsen 1979) has been studied. When the Proteus sp. or the Aeromonas sp. were inoculated into the herring extracts and incubated at 15°C under anaerobic conditions, the sugar components (i.e. mainly ribose, free and bound) were the first substrates utilized. These compounds were converted to acetate and CO₂ by the use of trimethylamine oxide (TMAO) as an external hydrogen acceptor. Growth of bacteria ceased when all TMAO was reduced to trimethylamine (TMA). By adding an extra amount of TMAO to the herring extracts an increased growth of the Proteus sp. and the Aeromonas sp. ensued. The increased growth occurred concomitantly with a further conversion of TMAO to TMA and of lactate to acetate and CO₂. The Enterobacter sp., which did not utilize lactate, did not give an increased growth in herring extracts enriched with TMAO.     

Anaerobic Fish Spoilage by Bacteria I. Biochemical Changes in Herring Extracts

Water extracts of herring fillets were used as laboratory model substrates for the study of anaerobic bacterial spoilage of fish stored in bulk. The organisms studied ( Enterobacter sp., str. NTHC 151, Proteus sp., str. NTHC 153, and Aeromonas sp., str. NTHC 154) were selected for by an enrichment method designed to isolate the bacteria having the highest anaerobic growth yields in these herring extracts. The extracts were then inoculated with pure cultures of the bacteria and incubated anaerobically at 15°C. Chemical analyses of the extracts were carried out at the start and at the end of the incubation period. The compounds determined accounted for 88% of total carbon and 93% of total nitrogen of the sterile extracts. Ribose (free and bound), free hexoses and lactate were the main substrates for the growth of the bacteria. These compounds were converted to acetate and CO₂; hydrogen liberated by these processes was used for the quantitative reduction of trimethylamine oxide to trimethylamine. Fermentation balances support the contention that the biochemical changes observed represent the quantitative dominating chemical events in the herring extracts as a result of the development of the spoilage bacteria.     

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.     

Spoilage Bacteria in Canned Foods: II. Sulfide Spoilage Bacteria in Canned Mushrooms and a Versatile Medium for the Enumeration of Clostridium nigrificans

Clostridium nigrificans was found to be a spoilage organism of canned mushrooms in Taiwan. A modified beef extract tryptone iron medium, both in broth and agar form, was designed for the detection and recovery of the organisms. A procedure of simple plate counting method of C. nigrificans was established.     

The Bacterial Flora of some Queensland Fish and its Ability to cause Spoilage

The bacterial flora were determined qualitatively and quantitatively on samples taken at various stages of handling several species of fish of commercial importance in Queensland. There was an overall increase in the number of bacteria during handling and processing; both the composition and quantity of the bacterial flora of individual samples taken at each stage of handling varied widely. Members of the genus Micrococcus formed the major proportion of the flora of freshly caught fish. Pseudomonas and Moraxella spp. were predominant amongst the bacterial flora able to grow at 2° and constituted the bulk of the population in samples with high bacterial counts. This psychrophilic population was markedly reduced at the filleting stage. A medium prepared by the action of trypsin on a fish muscle homogenate was used to test bacterial isolates for their ability to produce odours. Forty-three per cent of the pseudomonad isolates produced sulphydryl odours at 5°. Only small proportions of the other groups produced detectable odours. Members of the genus Pseudomonas were considered the most important fish spoilage bacteria under the conditions found in Queensland.     

High-Resolution 1H NMR Spectroscopy of Fish Muscle,Eggs and Small Whole Fish via Hadamard-Encoded Intermolecular Multiple-Quantum Coherence

Background and Purpose

Nuclear magnetic resonance (NMR) spectroscopy has become an important technique for tissue studies. Since tissues are in semisolid-state, their high-resolution (HR) spectra cannot be obtained by conventional NMR spectroscopy. Because of this restriction, extraction and high-resolution magic angle spinning (HR MAS) are widely applied for HR NMR spectra of tissues. However, both of the methods are subject to limitations. In this study, the feasibility of HR ¹H NMR spectroscopy based on intermolecular multiple-quantum coherence (iMQC) technique is explored using fish muscle, fish eggs, and a whole fish as examples.

Materials and Methods

Intact salmon muscle tissues, intact eggs from shishamo smelt and a whole fish (Siamese algae eater) are studied by using conventional 1D one-pulse sequence, Hadamard-encoded iMQC sequence, and HR MAS.

Results

When we use the conventional 1D one-pulse sequence, hardly any useful spectral information can be obtained due to the severe field inhomogeneity. By contrast, HR NMR spectra can be obtained in a short period of time by using the Hadamard-encoded iMQC method without shimming. Most signals from fatty acids and small metabolites can be observed. Compared to HR MAS, the iMQC method is non-invasive, but the resolution and the sensitivity of resulting spectra are not as high as those of HR MAS spectra.

Conclusion

Due to the immunity to field inhomogeneity, the iMQC technique can be a proper supplement to HR MAS, and it provides an alternative for the investigation in cases with field distortions and with samples unsuitable for spinning. The acquisition time of the proposed method is greatly reduced by introduction of the Hadamard-encoded technique, in comparison with that of conventional iMQC method.     

Pattern of Skeletal Muscle Differentiation in Fish: Molecular Biological Approaches

The initial stages of myogenesis going in myoblasts include the stages of induction, determination, and differentiation. The induction and determination of cells in the myotomes are controlled by morphogenetic signals from neighboring tissues of the notochord and neural tube manifested as expression of genes of Shh and Wnt families, respectively. In fish (at the example of danio), this signal is passed to somite cells neighboring the notochord; later the cells migrate to the embryo surface and differentiate into slow muscle fibers. Synthesis of the main contractile proteins, primarily the components of myosin molecule—heavy chain (MHC) and individual isoforms of light chains (MLC1, MLC2, and MLC3)—are encoded by different genes during different ontogenetic stages. The peptide maps obtained after -chymotrypsin digestion of MHCs from larvae, fast and slow skeletal muscle of loach are different, which points to differences in their primary structure. In addition, considerable differences were revealed in the structure of MLC isoforms at different ontogenetic stages. The definitive fast muscle contained three light chain types, MLC1, MLC2, and MLC3; slow muscle, MLC1 and MLC3; while the larval muscle fibers included a specific larval MLCL in addition to MLC3.     

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.     

Bacteriology of Manganese Nodules: II. Manganese Oxidation by Cell-free Extract from a Manganese Nodule Bacterium

A cell-free extract from Arthrobacter 37, isolated from a manganese nodule from the Atlantic Ocean, exhibited enzymatic activity which accelerated manganese accretion to synthetic Mn-Fe oxide as well as to crushed manganese nodule. The reaction required oxygen and was inhibited by HgCl₂ and p-chloromercuribenzoate but not by Atebrine dihydrochloride. The rate of enzymatic action depended on the concentration of cell-free extract used. The enzymatic activity had a temperature optimum around 17.5 C and was destroyed by heating at 100 C. The amount of heat required for inactivation depended on the amount of nucleic acid in the preparation. In the cell-free extract, unlike the whole-cell preparation, peptone could not substitute for NaHCO₃ in the reaction mixture. An enzyme-containing protein fraction and a nucleic acid fraction could be separated from cell extract by gel filtration, when prepared in 3% NaCl but not in seawater. The nucleic acid fraction was not required for enzymatic activity.     

THE ULTRASTRUCTURE OF THE CAT MYOCARDIUM : II. Atrial Muscle

The ultrastructure of the cells specialized for contraction in the atrium and ventricle of young adult cats are compared. The cells specialized for conduction are not included. In addition to possessing distinctive atrial granules, the cells of the atrium are smaller in diameter (5–6 µ) than ventricular cells (10–12 µ) and have strikingly fewer T tubules. These latter differences are discussed in terms of their possible significance for the rate of conduction of the action potential. It is suggested that the very small number of T tubules in atrial cells may compensate for the small cell diameter, and thus permit rapid conduction of the action potential across the surface of the atrium. Coated dense vesicles found in association with the sarcoplasmic reticulum at the level of the Z line in ventricular muscle are more evident in atrial cells. In the virtual absence of T tubules in atrial cells, the sub-sarcolemmal cisternae of the sarcoplasmic reticulum are almost exclusively at the cell periphery. The ends of the cells and their processes in ventricular muscle are rectilinear with the interdigitated portions of the intercalated discs oriented transversely, whereas those of the atrium are often oblique to the myofilament axis. This difference may be related to the lower mechanical tension on atrial cells.