The Development of Aerobic Spoilage Flora on Meat Stored at Chill Temperatures

In meat juice medium, aerobic spoilage bacteria utilized the following substrates in the order shown: Pseudomonos , glucose, amino acids, lactic acid; Acinetobacter , amino acids, lactic acid: Enterobacter , glucose, glucose-6-phosphate, amino acids; Microbacterium thermosphactum , glucose, glutamate. All the bacteria grew at their maximum rate utilizing the first and second substrates, but the growth rates declined when these were exhausted. The growth rate of Acinetobacter was reduced at pH 5·7 and below. All other species grew at their maximum rate within the pH range 5·5–7·0. On meat pseudomonads grew faster than the other species at all temperatures between 2° and 15°C. Interactions between any two species were observed only when one organism had attained its maximum cell density. Substrate exhaustion at the meat surface did not limit bacterial growth and it is suggested that the maximum cell density of aerobic spoilage cultures is determined by oxygen limitation of growth.     

Production and utilization of extracellular slime by Pseudomonas solanacearum and its role on survival at different relative humidities

Pseudomonas solanacearum produced maximum slime with glucose and least with glycerol. With increasing concentration of glucose, slime production increased. The maximum slime production was observed in the medium containing 1% glucose. The slime contained sugars, sugar acids and amino acids. The bacterium utilized the slime as a sole source of carbon. The growth on slime was more or less comparable to the growth on 0.1% glucose. The viability of the bacterium in slime increased as the relative humidity (RH) decreased. At higher RH the slime absorbed moisture and the viability was reduced. The bacterium could survive only for short period at higher RH under sunlight not in shade. The sunlight seemed to accentuate the harmful effect of RH on the bacterium.     

Metabolic footprinting of the anaerobic bacterium Fusobacterium varium using 1H NMR spectroscopy

Metabolic footprinting of the anaerobic bacterium Fusobacterium varium demonstrated the accumulation of six carboxylic acids as metabolic end-products and revealed specific growth requirements and utilization capabilities towards amino acids. Guided by (1)H NMR determinations of residual amino acids in spent medium, a modified chemically defined minimal medium (CDMM*) was developed by minimizing the amino acid composition while satisfying nutritional requirements to support abundant growth of F. varium. Quantitative determinations of carboxylate salts and residual substrates were readily performed by (1)H NMR analysis of lyophilized residues from CDMM* cultures without interference from initial medium components. Only small concentrations of alanine, arginine, glycine, isoleucine, leucine, methionine, proline and valine were required to support growth of F. varium, whereas larger quantities of aspartate, asparagine, cysteine, glutamine, glutamate, histidine, lysine, serine and threonine were utilized, most likely as energy sources. Both bacterial growth and the distribution of carboxylate end-products depended on the composition of the chemically defined medium. In cultures provided with glucose as the primary energy source, the accumulation of butyrate and lactate correlated with growth, consistent with the regeneration of reduced coenzyme formed by the oxidative steps of glucose catabolism.     

Growth of Ferrobacillus ferrooxidans on Organic Matter

Following a brief adaptation period to glucose, Ferrobacillus ferrooxidans was grown on glucose, mannitol, several other sugars, and a few amino acids in the absence of an oxidizable iron source. Prolonged growth on an organic substrate free from iron rendered the organism obligately organotrophic. The growth rate of the bacterium was greater in heterotrophic culture; the doubling time was approximately 4.5 hr on glucose. The bacterium retained its acidophilic properties during adaptation and growth on glucose and would not grow in neutral or slightly alkaline media. Addition of p-aminobenzoic acid was necessary for abundant growth of the cells on glucose. Of the eight strains of Fe(++)-oxidizing bacteria studied, only two strains grew on glucose in a nondialyzed system. The results of manometric studies are discussed with regard to metabolic efficiency of organic matter in this organism.     

Formation and distribution of free amino acids in Cunninghamella elegans mycelium during growth on media with glucose and dodecane]

The growth, assimilation of organic nitrogen compounds, and production and distribution of free amino acids were studied during the growth of Cunninghamella elegans (--) 1204 on the defined medium 12 containing glucose and dodecane and without an additional carbon source (control). The culture utilized all leucine, glycine, and asparagine after three days of the growth, irrespective of the source of carbon in the medium. Glutamic acid was assimilated only in the presence of glucose. Some 15 free amino acids and 2 amides were detected in the cells, and also some correlations in the composition of the pool in the presence of different sources of carbon in the medium. The substitution of dodecane for glucose decelerates metabolism of free intracellular amino acids and stimulates their accumulation in the cells, especially if there is a deficiency of carbon sources: the overall content of amino acids and amides on the medium with glucose is from 2.2 to 4.1 mg/g; with dodecane, from 9.8 to 16.5 mg/g; in the control, from 16.4 to 25.8 mg/g.     

Material balance studies on animal cell metabolism using a stoichiometrically based reaction network

A detailed reaction network of mammalian cell metabolism contains hundreds of enzymatic reactions. By grouping serial reactions into single overall reactions and separating overlapped pathways into independent reactions, the total number of reactions of the network is significantly reduced. This strategy of manipulating the reaction network avoids the manipulations of a large number of reactions otherwise needed to determine the reaction extents. A stoichiometric material balance model is developed based on the stoichiometry of the simplified reaction network. Closures of material balances on glucose and each of the 20 amino acids are achieved using experimental data from three controlled fed-batch and one-batch hybridoma cultures. Results show that the critical role of essential amino acids, except glutamine, is to provide precursors for protein synthesis. The catabolism of some of the essential amino acids, particularly isoleucine and leucine, is observed when an excess amount of these amino acids is available in the culture medium. It was found that the reduction of glutamine utilization (for reducing ammonia production) is accompanied by an increase in the uptake of nonessential amino acids (NAAs) from the culture medium. This suggests that NAAs are necessary even though they are not essential for cell growth. A glutamine balance shows that less than 20% of the glutamine nitrogen is utilized for essential roles, such as protein and nucleotide syntheses. A relatively constant percentage (about 45%) of the glutamine nitrogen is utilized for NAA biosynthesis, despite the fact that the absolute amount varies among the four experiments. As to the carbon skeleton of glutamine, a significant portion enters the tricarboxylic acid (TCA) cycle. A material balance on glucose shows that most of the glucose (81%) is converted into lactate when glucose is in excess. On the other hand, when glucose is limited, lactate production is considerably reduced, while a major portion of glucose (48%) enters the TCA cycle. The fraction of glucose used for the synthesis of cellular components ranges from 9 to 28%. (c) 1996 John Wiley & Sons, Inc.     

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.     

Amino acid fermentation byBacteroides melaninogenicus

Each of four strains ofBacteroides melaninogenicus grew well in a trypticaseyeast extract medium, without carbohydrate. Addition of glucose did not increase growth, and the sugar was fermented to only a limited extent. However, growth decreased when the trypticase concentration of the medium was reduced. These observations suggest that amino acid fermentation is of major importance in the energy metabolism ofB. melaninogenicus. Acetic, butyric and isovaleric acids were produced by all four strains. Two of the strains also formed propionic and isobutyric acids. Experiments using media containing either labeled glucose or labeled protein indicated that these acids are primarily derived from the proteinaceous substrates in the medium, rather than from glucose, indicating that amino acid fermentation byB. melaninogenicus is not subject to glucose repression. Resting-cell suspensions ofB. melaninogenicus possessed a limited ability to ferment free acids, as judged by the liberation of ammonia. However the organisms readily fermented amino acids when present as peptides, suggesting that peptides are more readily transported into the cell. Three of the 4 strains studied grew well when incubated under an atmosphere containing 2 to 4 % oxygen in cultures possessing a high surface to volume ratio. Hence not all strains ofB. melaninogenicus can be considered strict anaerobes.This investigation was supported in part by grant DE-02847 from the National Institute of Dental Research, and in part by a grant from the Colgate-Palmolive Co.     

The Development of the Anaerobic Spoilage Flora of Meat Stored at Chill Temperatures

In meat juice medium under anaerobic conditions, spoilage bacteria utilized the following substrates: Microbacterium thermosphactum , glucose; Enterobacter , glucose and glucose-6-phosphate; Lactobacillus , glucose and arginine. On meat stored anaerobically. Lactobacillus grew faster than the other species at all temperatures between 2° and 15 °C. Enterobacter had a greater affinity for glucose than M. thermosphactum. As a result, high numbers of Enterobacter inhibited growth of M. thermosphactum under the glucose limiting conditions at the meat surface. High numbers of Lactobacillus inhibited growth of both competing species, apparently by producing an inhibitory substance.     

Catabolism of leucine to branched-chain fatty acids in Staphylococcus xylosus

AIMS: Staphylococcus xylosus is an important starter culture in the production of flavours from the branched-chain amino acids leucine, valine and isoleucine in fermented meat products. The sensorially most important flavour compounds are the branched-chain aldehydes and acids derived from the corresponding amino acids and this paper intends to perspectivate these flavour compounds in the context of leucine metabolism. METHODS AND RESULTS: GC and GC/MS analysis combined with stable isotope labelling was used to study leucine catabolism. This amino acid together with valine and isoleucine was used as precursors for the production of branched-chain fatty acids for cell membrane biosynthesis during growth. A 83.3% of the cellular fatty acids were branched. The dominating fatty acid was anteiso-C(15:0) that constituted 55% of the fatty acids. A pyridoxal 5'-phosphate and alpha-ketoacid dependent reaction catalysed the deamination of leucine, valine and isoleucine into their corresponding alpha-ketoacids. As alpha-amino group acceptor alpha-keto-beta-methylvaleric acid and alpha-ketoisovaleric acid was much more efficient than alpha-ketoglutarate. The sensorially and metabolic key intermediate on the pathway to the branched-chain fatty acids, 3-methylbutanoic acid was produced from leucine at the onset of the stationary growth phase and then, when the growth medium became scarce in leucine, from the oxidation of glucose via pyruvate. CONCLUSIONS: This paper demonstrates that the sensorially important branched-chain aldehydes and acids are important intermediates on the metabolic route leading to branched-chain fatty acids for cell membrane biosynthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: The metabolic information obtained is extremely important in connection with a future biotechnological design of starter cultures for production of fermented meat.     

The basis of the alkalophilic property of a species of bacillus.

An alkalophilic bacterium belonging to the genus Bacillus was isolated from an indigo ball. The bacterium exhibited a maximum growth rate at pH 10-0 TO 10-5. The incorporation of 14C-labelled amino acids or [14C]uracil, uptake of 14C-labelled alpha-amino isobutyric acid into the bacterium and oxygen consumption of the bacterium with amino acids as substrates were all maximum at pH 9-0 to 10-5. The uptake of [U-14C]glucose into the organism and oxygen consumption with carbohydrates, on the other hand, showed little variation of rate in the pH 8 to 10 region. The oxygen consumption of intact bacteria or protoplasts in culture medium was maximum at pH 10. The membrane of the bacterium oxidized NADH maximally at pH 7-5, and ATPase bound to the membrane exhibited maximum activity at pH 7.L-Lactate, L-alanine and malate dehydrogenases in the soluble fraction exhibited maximum activities at pH 7-4 to 8-4. The alkalophilic property of the bacterium may be due to the behaviour of the membrane towards charged substances admitted into the organisms.     

Control of Fatty Acid Metabolism I. Induction of the Enzymes of Fatty Acid Oxidation in Escherichia coli

Escherichia coli grows on long-chain fatty acids after a distinct lag phase. Cells, preadapted to palmitate, grow immediately on fatty acids, indicating that fatty acid oxidation in this bacterium is an inducible system. This hypothesis is supported by the fact that cells grown on palmitate oxidize fatty acids at rates 7 times faster than cells grown on amino acids and 60 times faster than cells grown on a combined medium of glucose and amino acids. The inhibitory effect of glucose may be explained in terms of catabolite repression. The activities of the five key enzymes of beta-oxidation [palmityl-coenzyme A (CoA) synthetase, acyl-CoA dehydrogenase, enoyl-CoA hydrase, beta-hydroxyacyl-CoA dehydrogenase, and thiolase] all vary coordinately over a wide range of activity, indicating that they are all under unit control. The ability of a fatty acid to induce the enzymes of beta-oxidation and support-growth is a function of its chain length. Fatty acids of carbon chain lengths of C(14) and longer induce the enzymes of fatty acid oxidation and readily support growth, whereas decanoate and laurate do not induce the enzymes of fatty acid oxidation and only support limited growth of palmitate-induced cells. Two mutants, D-1 and D-3, which grow on decanoate and laurate were isolated and were found to contain constitutive levels of the beta-oxidation enzymes. Short-chain fatty acids (相似文献   

Enterococcus faecalis Grows on Ascorbic Acid

We show that Enterococcus faecalis can utilize ascorbate for fermentative growth. In chemically defined media, growth yield was limited by the supply of amino acids, and the cells showed a much higher demand for amino acids than when they were grown on glucose.     

Metabolism of L-amino acids in a marine bacterium isolated from mackerel intestines in relation to eicosapentaenoic acid biosynthesis.

Metabolism of glucose and L-amino acids in an obligately aerobic marine bacterium isolated from Pacific mackerel intestines was investigated for the mechanism and pathway of eicosapentaenoic acid (EPA) biosynthesis. This bacterium could not uptake glucose but the cell-free extract of this bacterium had the enzymatic activities of L-alanine oxidase (EC 1.4.3.2), L-alanine dehydrogenase (EC 1.4.1.1). L-serine dehydratase (EC 4.2.1.13), and malate dehydrogenase (EC 1.1.1.40), and of seven enzymes involved in the TCA cycle of the usual aerobes. On the other hand, the carbon-13 concentration in cellular fatty acids of the bacterium, especially that in their methyl carbon atoms in contrast to their carbonyl carbons, increased drastically when the bacterium was grown in the presence of 13CH3COONa. These results indicate that: (i) the TCA cycle works in this bacterium, (ii) glucose is not utilized and pyruvic acid is in vivo synthesized from L-alanine, L-serine, and malic acid, and (iii) EPA and other cellular fatty acids are in vivo synthesized from acetyl coenzyme A by the usual de novo synthesis route.     

Nutritive effects of d-amino acids on the silkworm, Bombyx mori

Nutritive effects of d-amino acids on the silkworm, Bombyx mori, were investigated by growth experiments using defined diets and also by analysis of free amino acids in the larval haemolymph. None of the d-forms of the usual ten essential amino acids could be utilized effectively, although d-methionine was utilized in lieu of the l-form only to a limited extent and d-histidine gave a positive but smaller effect than d-methionine. d-Proline, its l-form being semi-essential for the silkworm, was not utilized. d-Leucine, and to a lesser extent d-alanine and d-serine, were found to be somewhat toxic. Comparison of free amino acid patterns in the haemolymph of the fifth-instar larvae, which fed on diets either lacking l-forms of histidine, methionine and leucine singly or including the d-forms singly in place of these l-forms, supported the results of the growth experiments.     

The simultaneous biosynthesis and uptake of amino acids by Lactococcus lactis studied by (13)C-labeling experiments

Uniformly (13)C labeled glucose was fed to a lactic acid bacterium growing on a defined medium supplemented with all proteinogenic amino acids except glutamate. Aspartate stemming from the protein pool and from the extracellular medium was enriched with (13)C disclosing a substantial de novo biosynthesis of this amino acid simultaneous to its uptake from the growth medium and a rapid exchange flux of aspartate over the cellular membrane. Phenylalanine, alanine, and threonine were also synthesized de novo in spite of their presence in the growth medium.     

Role of sodium in the growth of a ruminal selenomonad.

The ruminal selenomonad strain H18 grew rapidly (mu = 0.50 h-1) in a defined medium containing glucose, ammonia, purified amino acids, and sodium (95 mM); little if any ammonia was utilized as a nitrogen source. When the sodium salts were replaced by potassium salts (0.13 mM sodium), there was a small reduction in growth rate (mu = 0.34 h-1), and under these conditions greater than 95% of the cell nitrogen was derived from ammonia. No growth was observed when the medium lacked sodium (less than 0.35 mM) and amino acids were the only nitrogen source. At least six amino acid transport systems (aspartate, glutamine, lysine, phenylalanine, serine, and valine) were sodium dependent, and these systems could be driven by an electrical potential (delta psi) or a chemical gradient of sodium. H18 utilized lactate as an energy source for growth, but only when sodium and aspartate were added to the medium. Malate or fumarate was able to replace aspartate, and when these acids were added, sodium was no longer required. Glucose-grown cells accumulated large amounts of polysaccharide (64% of dry weight), and when the exogenous glucose was depleted, this material was converted to acetate and propionate as long as sodium was present. When the cells were incubated in buffers lacking sodium, succinate accumulated and exogenous succinate could not be decarboxylated. Because sodium had little effect on the transmembrane pH gradient at pH 6.7 to 4.5, it did not appear that sodium was required for intracellular pH regulation.     

Growth of Paracoccus denitrificans on [2,3-13C]succinate and [1,4-13C]succinate. III. Biosynthetic pathways

The biosynthesis in vivo of a number of amino acids, sugars, and purines in Paracoccus denitrificans grown on either [2,3-13C]succinate or [1,4-13C]succinate was investigated by using gas chromatography-mass spectrometry. The distribution of label in the TCA-cycle-related amino acids indicated that carbon intermediates of energy metabolism were utilized as precursors for the biosynthesis of these amino acids in vivo. The biosynthesis of glycine, serine, phenylalanine and glycerol from labelled succinate in vivo were consistent with phosphoenol pyruvate as an intermediate. A mechanism for the formation of C4, C5 and C6 sugars without the use of fructose-1,6-bisphosphate aldolase (which has not been detected in P. denitrificans) is proposed. The 13C-enrichments of ribose in the bacterium indicate that there are at least three routes of ribose biosynthesis operating during growth on labelled succinate. The probability distribution of labelled purine molecules was successfully predicted for adenine, guanine and adenosine, thus confirming their generally accepted route of biosynthesis in vivo.     

Role of sodium in the growth of a ruminal selenomonad

The ruminal selenomonad strain H18 grew rapidly (mu = 0.50 h-1) in a defined medium containing glucose, ammonia, purified amino acids, and sodium (95 mM); little if any ammonia was utilized as a nitrogen source. When the sodium salts were replaced by potassium salts (0.13 mM sodium), there was a small reduction in growth rate (mu = 0.34 h-1), and under these conditions greater than 95% of the cell nitrogen was derived from ammonia. No growth was observed when the medium lacked sodium (less than 0.35 mM) and amino acids were the only nitrogen source. At least six amino acid transport systems (aspartate, glutamine, lysine, phenylalanine, serine, and valine) were sodium dependent, and these systems could be driven by an electrical potential (delta psi) or a chemical gradient of sodium. H18 utilized lactate as an energy source for growth, but only when sodium and aspartate were added to the medium. Malate or fumarate was able to replace aspartate, and when these acids were added, sodium was no longer required. Glucose-grown cells accumulated large amounts of polysaccharide (64% of dry weight), and when the exogenous glucose was depleted, this material was converted to acetate and propionate as long as sodium was present. When the cells were incubated in buffers lacking sodium, succinate accumulated and exogenous succinate could not be decarboxylated. Because sodium had little effect on the transmembrane pH gradient at pH 6.7 to 4.5, it did not appear that sodium was required for intracellular pH regulation.     

Metabolic activities of pseudomonads in batch cultures in extract of minced lamb

E.H. DROSINOS AND R.G. BOARD. 1994. Pseudomonas fragi, Ps. lundensis and Ps. fluorescens were studied in axenic batch cultures growing in a lamb juice (pH 6.0) aerobically or in an atmosphere ( Ps. fragi only) enriched with carbon dioxide at 4C. With all but a glucose dehydrogenase-deficient strain of Ps. fluorescens there was a sequential catabolism of glucose and lactate. Diauxic growth was observed in a nutrient-deficient meat juice supplemented with glucose and lactate. A transient peak in the concentration of gluconate and pyruvate was associated with the catabolism of glucose and lactate respectively. With Ps. fluorescens deficient in glucose dehydrogenase there was simultaneous catabolism of glucose and lactate. The stereoisomers of lactate were catabolized simultaneously in a laboratory medium. Glucose-6-phosphate was oxidized to 6-phosphogluconate by Ps. fragi and Ps. lundensis under aerobic conditions only. Creatine and creatinine were catabolized by Ps. fragi under aerobic conditions only. There was a slight decrease in the concentration of total amino acids (ninhydrin-reactive material) during the exponential phase of growth. The results suggest that the dominance of Ps. fragi in the climax populations in meat is due to catabolism of amino acid related substrates, creatine and creatinine.