A Combined Model for Growth and Subsequent Thermal Inactivation of Brochothrix thermosphacta

A mathematical technique for integrating growth and thermal inactivation models of microorganisms into a smooth combined model that can be applied to circumstances under which the temperature gradually rises from growth to inactivation regions is described. For the death part of the model, a correction term is introduced to allow for additional resistance of the cells gained during slow heating. The model was validated with Brochothrix thermosphacta heated in broth at rising temperatures.     

Morphology of Brochothrix thermosphacta phages

Twenty-one Brochothrix thermosphacta phages were examined by electron microscopy. They had isometric heads and contractile or long and noncontractile tails, and were grouped into three species belonging to the Myoviridae (species A19) or Siphoviridae (species NF5 and BL3) families of tailed phages. Species A19 has highly characteristic morphological features and resembles well-known phages of Bacillus, Lactobacillus, Staphylococcus and Streptococcus, indicating phylogenetic relationships between these phages as well as their hosts.     

Inhibition of the Anaerobic Growth of Brochothrix thermosphacta by Lactic Acid

Brochothrix thermosphacta can grow aerobically in the presence of 210 mM l-lactate and anaerobically in its absence at pH values down to at least 5.5. Anaerobic growth is, however, inhibited by l-lactate, the concentration of undissociated lactic acid being the governing factor. Postrigor meat usually contains sufficient lactic acid to select against the anaerobic growth of B. thermosphacta. At least some Lactobacillaceae strains are more resistant to lactic acid and so their growth is favored on vacuum-packaged meat.     

Inhibition of growth of Brochothrix thermosphacta by palmitic acid

Palmitic acid was inhibitory to the growth of Brochothrix thermosphacta in liquid culture at 0.5 mmol/l. Uptake of [1-¹⁴C]-palmitic acid by the organism has been demonstrated, and was reduced at acid pH. These findings are discussed in relation to the known effects of fatty acids on bacteria.     

Lipolytic activity of Brochothrix thermosphacta on natural triglycerides

Four natural glycerides were used to study the lipolytic characteristics of Brochothrix thermosphacta lipase. Hydrolysis of triglycerides from linseed oil and pork fat adipose tissue with a high percentage of unsaturated fatty acids was two to three times greater than in saturated triglycerides from lamb or beef fat adipose tissues. Hydrolysis was not specific as the percentage of the released fatty acids was similar to that of the starting triglycerides.     

Membrane potential-dependent glycine uptake into vesicles of Brochothrix thermosphacta

Glycine uptake in right-side-out membrane vesicles of Brochothrix thermosphacta was investigated. NADH and D-lactate-based potential (-50 mV) supported glycine uptake up to a fivefold accumulation ratio. The uptake was sensitive to the addition of carbonyl cyanide m -chloro-phenyl hydrazone (CCCP), an ionophore.     

Purification and kinetic properties of pyruvate kinase from Brochothrix thermosphacta.

Pyravate kinase (ATP: pyruvate 2-0 phosphotransferase E.C.2.7.1.40) was purified from Brochothrix thermosphacta. The enzyme is a homotetramer of monomer Mr 58,000. Fructose-1,6-bisphosphate stimulates activity and promotes hyperbolic kinetics although it is not essential for enzyme activity. The positive effect of fructose-1,6-bisphosphate on activity is repressed by inorganic phosphate which enhances cooperative kinetics. Unlike pyruvate kinases from other sources, the Brochothrix enzyme is uncompetitively inhibited by glucose-6-phosphate, although at high concentration. ATP is a strong inhibitor of pyruvate kinase and shifts the residual activity/pH profile towards more alkaline values.     

16S rRNA analysis of Listeria monocytogenes and Brochothrix thermosphacta

Abstract Listeria monocytogenes and Brochothrix thermosphacta were investigated by the 16S rRNA cataloguing approach in order to determine their phylogenetic relationship. Both species are specifically, although moderately related, forming one of several sublines within the Bacillus-Lactobacillus-Streptococcus cluster of the ' Clostridium ' subbranch of Gram-positive eubacteria.     

Cell location and partial characterization of Brochothrix thermosphacta and Lactobacillus curvatus lipases

The lipases of Brochothrix thermosphacta and Lactobacillus curvatus were in the soluble fraction of the cell and not membrane- or wall-bound. Their optimal activities were around pH 6mD5 and at 37°C. Enzymes were stable between 4 and 30°C and at freezing temperatures (— 20°C and —60°C). The lipase of Lact. curvatus was stable in the pH range of 4mD0 to 11mD0, whereas that of B. thermosphacta was denatured at pH lower than 5mD0 and greater than 9mD0. Among triglycerides tested, maximal activity was found with tributyrin and hydrolysis of other triglycerides decreased as the length of fatty acid increased.     

Effect of modified atmosphere composition on the metabolism of glucose by Brochothrix thermosphacta

The influence of atmosphere composition on the metabolism of Brochothrix thermosphacta was studied by analyzing the consumption of glucose and the production of ethanol, acetic and lactic acids, acetaldehyde, and diacetyl-acetoin under atmospheres containing different combinations of carbon dioxide and oxygen. When glucose was metabolized under oxygen-free atmospheres, lactic acid was one of the main end products, while under atmospheres rich in oxygen mainly acetoin-diacetyl was produced. The proportions of the total consumed glucose used for the production of acetoin (aerobic metabolism) and lactic acid (anaerobic metabolism) were used to decide whether aerobic or anaerobic metabolism predominated at a given atmosphere composition. The boundary conditions between dominantly anaerobic and aerobic metabolisms were determined by logistic regression. The metabolism of glucose by B. thermosphacta was influenced not only by the oxygen content of the atmosphere but also by the carbon dioxide content. At high CO(2) percentages, glucose metabolism remained anaerobic under greater oxygen contents.     

Precursors of the major end products of aerobic metabolism of Brochothrix thermosphacta

To further our understanding of off-odour production by Brochothrix thermosphacta , the nature and origins of its end products have been compared during aerobic growth in complex and in minimal, defined medium. In complex medium glucose is the major precursor of acetoin and acetic acid but alanine may be an additional minor source. Iso butyric, iso valeric (3-methylbutyric) and 2-methylbutyric acids are derived exclusively from valine, leucine and iso leucine, respectively. In minimal defined medium although the same end products are produced they are all derived from glucose.     

Specific antibody-mediated detection of Brochothrix thermosphacta in situ in British fresh sausage

S.C. STRINGER, B.J. CHAFFEY, C.E.R. DODD, M.R.A. MORGAN AND W.M. WAITES. 1995. A rabbit polyclonal antibody-linked probe was developed which detected 76% of 800 food isolates of the spoilage bacterium Brochothrix thermosphacta when cells were bound to nitrocellulose. In slide cross-reaction tests all six environmental isolates tested were stained but the type strain was not. The antibody did not cross-react with Listeria grayi, L. monocytogenes, Lactobacillus plantarum, Lactococcus lactis, Streptococcus mutans, Bacillus cereus or B. subtilis.
The antibody-linked probe detected Br. thermosphacta in thin sections of British fresh sausage when the viable count was greater than 10⁶ g⁻¹ Cells were detected mainly within 1 or 2 mm of the surface on the loose starchy material. They were not detected within muscle blocks or in the centre of the sausage. Such results suggest that growth of this organism occurs close to the surface of the sausage.     

Pathways of pyruvate metabolism and energetics of growth of Brochothrix thermosphacta

Brochothrix thermosphacta, a psychrophilic, facultative anaerobe, exhibited homolactic fermentation under anaerobic conditions in the presence of excess glucose. In glucose-limited chemostat culture (on synthetic medium), ethanol, acetate, formate and lactate were formed. Formation of ethanol and acetate was accounted for by the formate concentrations in culture filtrates. Acetate, formate and ethanol formation was enhanced at low growth rates in chemostat culture. O₂-limited chemostat studies indicated that formate formation was inhibited by oxygen (<0.2 M) and studies with a variant, strain 301, which lacked pyruvate dehydrogenase activity, showed that cell culture in basal medium did not occur at O₂ tensions greater than that preventing formate production in the wild-type strain. The data are consistent with stimulation of pyruvate formate lyase activity by glucose limitation, possibly because of decreased concentrations of glycolytic intermediates.S.P. Singh was and A. Garrett and P.J. Rogers are with the Division of Science and Technology, Griffith University, Brisbane 4111, Australia. J. McAvoy and A.F. Egan are with the CSIRO Meat Research Laboratory, Cannon Hills, Brisbane 4170, Australia. S.P. Singh is now with the Department of Microbiology, C.B.S. & H., G.B. Pant University of Agriculture & Technology, Pantnagar-263145, India.     

Effect of Modified Atmosphere Composition on the Metabolism of Glucose by Brochothrix thermosphacta

The influence of atmosphere composition on the metabolism of Brochothrix thermosphacta was studied by analyzing the consumption of glucose and the production of ethanol, acetic and lactic acids, acetaldehyde, and diacetyl-acetoin under atmospheres containing different combinations of carbon dioxide and oxygen. When glucose was metabolized under oxygen-free atmospheres, lactic acid was one of the main end products, while under atmospheres rich in oxygen mainly acetoin-diacetyl was produced. The proportions of the total consumed glucose used for the production of acetoin (aerobic metabolism) and lactic acid (anaerobic metabolism) were used to decide whether aerobic or anaerobic metabolism predominated at a given atmosphere composition. The boundary conditions between dominantly anaerobic and aerobic metabolisms were determined by logistic regression. The metabolism of glucose by B. thermosphacta was influenced not only by the oxygen content of the atmosphere but also by the carbon dioxide content. At high CO₂ percentages, glucose metabolism remained anaerobic under greater oxygen contents.     

Regulation of the glucose phosphotransferase system in Brochothrix thermosphacta by membrane energization.

Uptake of 2-deoxyglucose, alpha-methylglucopyranoside, and glucose into intact cells of Brochothrix thermosphacta (formerly Microbacterium thermosphactum, ATCC 11509) was stimulated by KCN or CCCP. The glucose analogs were recovered almost totally as the sugar phosphates. Membrane vesicles were isolated from protoplasts and shown to be right side out by freeze fracturing and by using ATPase as a marker for the cytoplasmic membrane surface. Uptake of glucose into vesicles was dependent on the presence of phosphoenolpyruvate. NADH oxidation, K+ -diffusion gradients, and externally directed lactate gradients (pH greater than 7 initially) were used to generate transmembrane potentials across membrane vesicles. Above a threshold value of about -50 mV, uptake of glucose into membrane vesicles was reduced. Likewise, the maximum uptake of glucose and its two analogs into cells occurred when the protonmotive force was less than about -50 mV.     

Volatile Compounds Associated with Spoilage of Vacuum-Packaged Sliced Luncheon Meat by Brochothrix thermosphacta

By using gas chromatography-mass spectrometry, seven volatile compounds were identified in vacuum-packaged sliced corned beef spoiled by Brochothrix thermosphacta under aerobic conditions. Acetoin and diacetyl appeared to be of major sensory significance.     

The influence of glucose concentration and culture incubation time on end-product formation during aerobic growth of Brochothrix thermosphacta

Fifteen strains of Brochothrix thermosphacta , isolated from various meat sources, produced the same end-products during aerobic growth in a tryptone-based medium containing 0.2% (w/v) glucose. Growth of one strain at different glucose concentrations showed that 2-methylpropanol and 2,3-butanediol were also produced. Formation of the branched chain acids was favoured by low glucose concentrations, that of the alcohols and the diol by higher concentrations. The traces of the diol, but not those of the alcohols, produced during the early stages of growth at low, limiting concentrations of glucose, gradually disappeared following glucose depletion. These findings provide adequate explanations for the observed differences in end-product formation during growth of Broc. thermosphacta on non-processed and corned beef.     

Growth and end product formation of two psychrotrophic Lactobacillus spp. and Brochothrix thermosphacta ATCC 11509T at different pH values and temperatures

Lactobacillus viridescens, Lactobacillus sp. strain 173 (homofermentative), and Brochothrix thermosphacta ATCC 11509T were studied at different pH values and temperatures in aerobic and anaerobic batch cultures. The growth rates were higher in aerobic than in anaerobic cultures. L. viridescens grew faster at pH 5.8 than at pH 6.3, whereas the opposite was true for B. thermosphacta. Lactobacillus sp. strain 173 was inhibited in air or at 8 degrees C in anaerobic culture. B. thermosphacta did not grow in anaerobic culture at pH 5.3. The following variations in growth yields were found in the different environments studied: Lactobacillus sp. strain 173, 23 to 25 g (dry weight) per mol of glucose consumed; L. viridescens, 11 to 23 g/mol; B. thermosphacta, 16 to 38 g/mol. In air, L. viridescens produced D-lactic acid, ethanol, and acetic acid, whereas no acetic acid was produced anaerobically. Acetic acid and ethanol together constituted 41 to 48% of the total product yield irrespective of pH and temperature. Lactobacillus sp. strain 173 produced a racemic mixture of D- and L-lactic acid at pH 6.3, whereas the proportion of L-lactic acid was higher than that of D-lactic acid at pH 5.3. In air, product formation of B. thermosphacta varied from a domination of L-lactic acid to increasing yields of acetoin, acetic acid, 2,3-butanediol and isovaleric acid. The effect of pH and temperature on product formation was difficult to separate from the effect of O2 availability in aerobic cultures. However, it was indicated that more 2,3-butanediol and less acetoin were produced with a decreasing temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction of Brochothrix thermosphacta on beef surfaces following immobilization of nisin in calcium alginate gels

Lean and adipose beef carcass tissues inoculated with Brochothrix thermosphacta (BT) (approx. 4.50 log₁₀ cfu cm⁻²) were left untreated (U) or treated with 100 μg ml⁻¹ nisin (N), calcium alginate (A) or 100 μg ml⁻¹ nisin immobilized in a calcium alginate gel (AN). Tissue samples were refrigerated after treatments and bacterial populations and nisin activity were determined at 0, 1, 2 and 7 d. U, A and N treatments of lean and adipose tissues did not suppress bacterial growth (>6 log₁₀ cfu cm⁻² by day 7) while treatments of lean and adipose tissues with AN suppressed bacteria (>2.42 log₁₀ cfu cm⁻² by day 7). Bacteriocin titres from both tissues were higher in AN vs N samples after the 7 d incubation. This study demonstrates that immobilization of nisin in a gel may be a more effective delivery system of a bacteriocin to the carcass surface than direct application.     

Betaine as a Growth Factor for Pediococcus soyae

Besides P-factor, Pediococcus soyae requires betaine (glycine-betaine) as a specific growth promotant. The maximal growth is obtained with the supplementation of both betaine and P-factor to the synthetic medium, while betaine only gives the half-maximal growth. When the organism is cultured in 18% salted media, the addition of both betaine and P-factor is essential for the occurrence of growth. Thus, betaine is requisite for Ped. soyae as a growth promoting factor and also as a factor bestowing the osmotelerant ability.