Effect of supplementation of micronutrients and phytochemicals to fructooligosaccharides on growth response of probiotics and E. coli.

Probiotics and prebiotics, which can change the colonic microenvironment, are the areas of current interest. Unutilizable fractions of the foods and fortificants, which reach the colon can affect the profile of probiotics. Effects of eight such factors viz. zinc sulphate, zinc carbonate, ferrous sulphate, ferric citrate, quercetin, gallic acid, phytic acid, and oxalic acid were, therefore, investigated on 24 H growth of Lactobacillus acidophilus (L1) and Lactobacillus plantarum (L2), two isolates of bifidobacteria (longum (L3) and bifidum (L4)) and a marketed consortium (L5) of eight probiotic cultures. MRS medium with marketed fructooligosaccharide as the only source of carbon was used for study of dose response curves. Quercetin and zinc sulphate showed significant positive effect for L1 and L5 (P < 0.01), whereas there was slight positive effect or no effect on growth of other probiotics. Phytic acid showed a significant inhibitory effect for L2 and a slight inhibitory effect on L3 and L4 whereas L5 were able to tolerate phytic acid. Oxalic acid had slight positive effect for L1 (P < 0.05) and L5 and no effect on growth of other probiotics (P > 0.05). Further, zinc sulphate, ferrous sulphate, quercetin, and oxalic acid significantly inhibited growth of E. coli (P < 0.05)

In vitro effects of selected synbiotics on the human faecal microbiota composition.

Synbiotics are recognized means of modulating gut microbiota composition and activities. However, whether synbiotics are superior to prebiotics and probiotics alone in moderating the gut microbiota towards a purportedly healthy composition has not been determined. Eight selected synbiotics (short-chain fructooligosaccharides or fructooligosaccharides, each combined with one of four probiotics, Lactobacillus fermentum ME-3, Lactobacillus plantarum WCFS1, Lactobacillus paracasei 8700:2 or Bifidobacterium longum 46) were added to 24-h pH-controlled anaerobic faecal batch cultures. The prebiotic and probiotic components were also tested alone to determine their respective role within the synbiotic for modulation of the faecal microbiota. Effects upon major groups of the microbiota were evaluated using FISH. Rifampicin variant probiotic strains were used to assess probiotic levels. Synbiotic and prebiotics increased bifidobacteria and the Eubacterium rectale-Clostridium coccoides group. Lower levels of Escherichia coli were retrieved with these combinations after 5 and 10 h of fermentation. Probiotics alone had little effect upon the groups, however. Multivariate analysis revealed that the effect of synbiotics differed from the prebiotics as higher levels of Lactobacillus-Enterococcus were observed when the probiotic was stimulated by the prebiotic component. Here, the synbiotic approach was more effective than prebiotic or probiotic alone to modulate the gut microbiota.

Proteiniborus ethanoligenes gen. nov., sp. nov., an anaerobic protein-utilizing bacterium.

A novel anaerobic, mesophilic, protein-utilizing bacterial strain, GW(T), was isolated from the mesophilic hydrogen-producing granular sludge used to treat food industry wastewater. The strain was a Gram-positive, non-spore-forming and non-motile rod. Growth of the strain was observed at 20-48 degrees C and at pH 6.4-10.0. The strain used yeast extract and peptone as carbon and energy sources. Weak growth was also observed with tryptone and Casamino acids as carbon and energy sources. The strain used none of the tested carbohydrates, alcohols or fatty acids. The fermentation products in peptone-yeast broth included ethanol, acetic acid, hydrogen and carbon dioxide. Gelatin was not hydrolysed. Nitrate was reduced. Indole was produced. NH(3) and H(2)S were not produced. The DNA G+C content of strain GW(T) was 38.0 mol%. The predominant cellular fatty acids were the saturated fatty acids C(14:0) (15.58%), C(16:0) (25.40%) and C(18:0) (12.03%). Phylogenetic analysis based on 16S rRNA gene sequence similarity revealed that strain GW(T) represented a new branch within cluster XII of the Clostridium subphylum, with <89.6% 16S rRNA gene sequence similarities to all described species. On the basis of polyphasic evidence from this study, strain GW(T) represents a new genus and novel species, for which the name Proteiniborus ethanoligenes gen. nov., sp. nov. is proposed. The type strain is GW(T) (=CGMCC 1.5055(T)=JCM 14574(T)).

Effect of low temperature and culture media on the growth and freeze-thawing tolerance of Exiguobacterium strains.

Bacteria of the genus Exiguobacterium have been repeatedly isolated from ancient permafrost sediments of the Kolyma lowland of Northeast Eurasia. Here we report that the Siberian permafrost isolates Exiguobacterium sibiricum 255-15, E. sibiricum 7-3, Exiguobacterium undae 190-11 and E. sp. 5138, as well as Exiguobacterium antarcticum DSM 14480, isolated from a microbial mat sample of Lake Fryxell (McMurdo Dry Valleys, Antarctica), were able to grow at temperatures ranging from -6 to 40 degrees C. In comparison to cells grown at 24 degrees C, the cold-grown cells of these strains tended to be longer and wider. We also investigated the effect of growth conditions (broth or surface growth, and temperature) on cryotolerance of the Exiguobacterium strains. Bacteria grown in broth at 4 degrees C showed markedly greater survival following freeze-thawing treatments (20 repeated cycles) than bacteria grown in broth at 24 degrees C. Surprisingly, significant protection to repeated freeze-thawing was also observed when bacteria were grown on agar at either 4 or 24 degrees C.

Tindallia texcoconensis sp. nov., a new haloalkaliphilic bacterium isolated from lake Texcoco, Mexico.

A new alkaliphilic and moderately halophilic, strictly anaerobic, fermentative bacterium (strain IMP-300(T)) was isolated from a groundwater sample in the zone of the former soda lake Texcoco in Mexico. Strain IMP-300(T) was Gram-positive, non-sporulated, motile and rod-shaped. It grew within a pH range from 7.5 to 10.5, and an optimum at 9.5. The organism was obligately dependent on the presence of sodium salts. Growth showed an optimum at 35 degrees C with absence of growth above 45 degrees C. It fermented peptone and a few amino acids, preferentially arginine and ornithine, with production of acetate, propionate, and ammonium. Its fatty acid pattern was mainly composed of straight chain saturated, unsaturated, and cyclopropane fatty acids. The G + C content of genomic DNA was 40.0 mol%. Analysis of the 16S rRNA gene sequence indicated that the new isolate belongs to the genus Tindallia, in the low G + C Gram-positive phylum. Phylogenetically, strain IMP-300(T) has Tindallia californiensis, as closest relative with a 97.5% similarity level between their 16S rDNA gene sequences, but the DNA-DNA re-association value between the two DNAs was only 42.2%. On the basis of differences in genotypic, phenotypic, and phylogenetic characteristics, strain IMP-300(T) is proposed as a new species of the genus Tindallia, T. texcoconensis sp. nov. (type strain IMP-300(T ) = DSM 18041(T) = JCM 13990(T)).

Novel chemolithotrophic, thermophilic, anaerobic bacteria Thermolithobacter ferrireducens gen. nov., sp. nov. and Thermolithobacter carboxydivorans sp. nov.

Three thermophilic strains of chemolithoautotrophic Fe(III)-reducers were isolated from mixed sediment and water samples (JW/KA-1 and JW/KA-2(T): Calcite Spring, Yellowstone N.P., WY, USA; JW/JH-Fiji-2: Savusavu, Vanu Levu, Fiji). All were Gram stain positive rods (approximately 0.5 x 1.8 microm). Cells occurred singly or in V-shaped pairs, and they formed long chains in complex media. All utilized H(2) to reduce amorphous iron (III) oxide/hydroxide to magnetite at temperatures from 50 to 75 degrees C (opt. approximately 73 degrees C). Growth occurred within the pH(60C) range of 6.5-8.5 (opt. pH(60C) 7.1-7.3). Magnetite production by resting cells occurred at pH(60C) 5.5-10.3 (opt. 7.3). The iron (III) reduction rate was 1.3 mumol Fe(II) produced x h(-1) x ml(-1) in a culture with 3 x 10(7) cells, one of the highest rates reported. In the presence or absence of H(2), JW/KA-2(T) did not utilize CO. The G + C content of the genomic DNA of the type strain is 52.7 +/- 0.3 mol%. Strains JW/KA-1 and JW/KA-2(T) each contain two different 16S rRNA gene sequences. The 16S rRNA gene sequences from JW/KA-1, JW/KA-2(T), or JW/JH-Fiji-2 possessed >99% similarity to each other but also 99% similarity to the 16S rRNA gene sequence from the anaerobic, thermophilic, hydrogenogenic CO-oxidizing bacterium 'Carboxydothermus restrictus' R1. DNA-DNA hybridization between strain JW/KA-2(T) and strain R1(T) yielded 35% similarity. Physiological characteristics and the 16S rRNA gene sequence analysis indicated that the strains represent two novel species and are placed into the novel genus Thermolithobacter within the phylum 'Firmicutes'. In addition, the levels of 16S rRNA gene sequence similarity between the lineage containing the Thermolithobacter and well-established members of the three existing classes of the 'Firmicutes' is less than 85%. Therefore, Thermolithobacter is proposed to constitute the first genus within a novel class of the 'Firmicutes', Thermolithobacteria. The Fe(III)-reducing Thermolithobacter ferrireducens gen. nov., sp. nov. is designated as the type species with strain JW/KA-2(T) (ATCC 700985(T), DSM 13639(T)) as its type strain. Strain R1(T) is the type strain for the hydrogenogenic, CO-oxidizing Thermolithobacter carboxydivorans sp. nov. (DSM 7242(T), VKM 2359(T)).

Carnobacterium pleistocenium sp. nov., a novel psychrotolerant, facultative anaerobe isolated from permafrost of the Fox Tunnel in Alaska.

A novel, psychrotolerant, facultative anaerobe, strain FTR1T, was isolated from Pleistocene ice from the permafrost tunnel in Fox, Alaska. Gram-positive, motile, rod-shaped cells were observed with sizes 0.6-0.7 x 0.9-1.5 microm. Growth occurred within the pH range 6.5-9.5 with optimum growth at pH 7.3-7.5. The temperature range for growth of the novel isolate was 0-28 degrees C and optimum growth occurred at 24 degrees C. The novel isolate does not require NaCl; growth was observed between 0 and 5 % NaCl with optimum growth at 0.5 % (w/v). The novel isolate was a catalase-negative chemoorganoheterotroph that used as substrates sugars and some products of proteolysis. The metabolic end products were acetate, ethanol and CO2. Strain FTR1T was sensitive to ampicillin, tetracycline, chloramphenicol, rifampicin, kanamycin and gentamicin. 16S rRNA gene sequence analysis showed 99.8 % similarity between strain FTR1T and Carnobacterium alterfunditum, but DNA-DNA hybridization between them demonstrated 39+/-1.5 % relatedness. On the basis of genotypic and phenotypic characteristics, it is proposed that strain FTR1T (=ATCC BAA-754T=JCM 12174T=CIP 108033T) be assigned to the novel species Carnobacterium pleistocenium sp. nov.

Effects of environmental factors on leucine catabolism by Carnobacterium piscicola.

The metabolites from leucine degradation are involved in dry fermented sausage aroma. The catabolism of leucine by a strain of Carnobacterium piscicola was studied directly in the growth medium with 3H-labelled leucine to investigate the effect of five parameters: phase of growth, pH, oxygen, glucose and alpha-ketoisocaproic acid. Resting cells (RC) were also incubated with 3H-labelled leucine. The radioactive metabolites from leucine catabolism were analysed by high performance liquid chromatography (HPLC). At pH 5.4 and 7.2, the main metabolites detected were 3-methyl butanal, 3-methyl butanol and alpha-ketoisocaproic acid. At pH 6.5, the leucine catabolism was maximum and was characterised by a high production of 3-methyl butanoic acid. Leucine catabolism was most important during the exponential phase of growth. The addition of alpha-ketoisocaproic acid at 1%, glucose at levels of 0.5% to 2% and shaking of the growth medium increased leucine catabolism.

Antimicrobial effect of natural preservatives in a cooked and acidified chicken meat model.

The inhibitory effect of Microgard 100, Microgard 300, nisin, Alta 2002, Perlac 1902, sodium lactate and essential oil of mustard on microorganisms experimentally inoculated was screened in an acidified chicken meat model (pH = 5.0) and stored for 2 weeks at a none restrictive growth temperature of 22 degrees C. All antimicrobials tested were used at the highest concentration recommended by their manufacturer. Sausage batter made with mechanically deboned chicken was inoculated with a mixed culture of Escherichia coli ATCC 25922, Brochothrix thermosphacta CRDAV452, and a protective culture Lactobacillus alimentarius BJ33 (FloraCan L-2). A final cell concentration of 3-4 log CFU g (-1) was targeted after cooking at a core temperature of 55 degrees C for each microorganism in order to assess cell count variation effectively. Composition, water activity (a(w)), pH and redox potential of the sausage model was also evaluated. The E. coli population decreased steadily during storage and was close or below detection level (< 1 log CFU g (-1)) for all treatments, including the control, after 14 days. Sodium lactate was most effective against B. thermosphacta; population was 4 log lower than the control after 14 days of storage. When essential oil of mustard was used, aerobic mesophilic bacteria and lactic acid bacteria were significantly lower than the control after 2 days of storage (P < or = 0.05). The other antimicrobial agents tested had no significant effect on the aerobic mesophilic bacteria, E. coli, B. thermosphacta and lactic acid bacteria counts, when compared to the control.

Microbial growth-promotion activity of 3-hydroxymonoazine- and N-hydroxydiazine-type heterocycles.

Three 3-hydroxymonoazine- and three N-hydroxydiazine-type heterocycles were tested whether they act as artificial siderophores toward Aureobacterium flavescens JG-9 (ATCC No. 25091). Among them, 1-hydroxy-3,5,6-trimethyl-2(1H)-pyrazinone (3) showed the highest growth-promotion activity comparable to desferrioxamine B (DFB), a natural trihydroxamate siderophore, at 48.5 microM or above, followed by 1-hydroxy-5,6-dimethyl-2(1H)-pyrazinone (2), 1-hydroxy-4,6-dimethyl-2(1H)-pyrimidinone (1), and 3-hydroxy-2-methyl-1-phenyl-4(1H)-pyridinone (6), while 3-hydroxy-1,2-dimethyl-4(1H)-pyridinone (5) did not show the bioactivity. These results are the first examples of N-hydroxydiazine-type heterocycles acting as artificial siderophores for A. flavescens JG-9.

Factors affecting production of an antilisterial bacteriocin by Carnobacterium piscicola strain A9b in laboratory media and model fish systems.

AIMS: To investigate factors influencing bacteriocin production and bacteriocin stability of the bioprotective culture Carnobacterium piscicola strain A9b. METHODS AND RESULTS: Maximum activity was obtained in MRS7 broth (MRS adjusted to pH 7.2), with or without glucose. No bacteriocin was produced in APT broth when a low inoculum level (0.001%) was used. In contrast, inoculum level did not influence bacteriocin production in BHI and MRS7 without glucose. Bacteriocin production in APT was induced by the presence of an extracellular compound present in the sterile, filtered, cell-free supernatant fluid of a stationary-phase culture. Increasing concentrations of NaCl (2-7%) reduced bacteriocin production and maximum cell density of C. piscicola A9b when grown in cooked fish juice at 4 degrees C. CONCLUSION: Media composition, inoculum level and sodium chloride concentration affected production. SIGNIFICANCE AND IMPACT OF THE STUDY: The influence of NaCl on bacteriocin production may negate the inhibitory effect of C. piscicola A9b against Listeria monocytogenes in salty foods.

Improved antimicrobial activity of nisin-incorporated polymer films by formulation change and addition of food grade chelator.

AIMS: The following polymers were developed: polyethylene (PE), a PE and polyethylene oxide (70% PE and 30% PEO; PE + PEO) blend, PE and nisin (PE + nisin), PE, nisin, and EDTA (PE + nisin + EDTA), and PE + PEO with nisin (PE + PEO + nisin). METHODS AND RESULTS: Of the polymers tested, PE and PE + PEO did not exhibit any antimicrobial activity against Brochothrix thermosphacta (BT); however, PE + nisin, PE + nisin + EDTA, and PE + PEO + nisin did. Beef surfaces were experimentally inoculated with 3.50 log10 cfu/cm2 of BT, vacuum packaged with each of the five polymers, and held at 4 degrees C for 21 d. After 3 d at 4 degrees C, BT was reduced > 1.70 log(10) by PE + nisin and > 3.50 log(10) with PE + nisin + EDTA or PE + PEO + nisin. By 21 d at 4 degrees C, BT was reduced to 0.30 log(10) cfu/cm(2) when treated with PE + PEO + nisin. CONCLUSION: It appears that PE + PEO + nisin or PE + nisin + EDTA were more effective for reducing BT, as compared to polymers composed of PE + nisin. SIGNIFICANCE AND IMPACT OF THE STUDY: Nisin-incorporated polymers may control the growth of undesirable bacteria, thereby extending the shelf life and possibly enhancing the microbial safety of meats.

Symbiobacterium thermophilum gen. nov., sp. nov., a symbiotic thermophile that depends on co-culture with a Bacillus strain for growth.

A Gram-negative and tryptophanase-positive thermophile, whose growth is dependent on co-culture with an associating Bacillus strain, had been reported and tentatively named Symbiobacterium thermophilum strain T(T). Axenic culture of strain T(T) was recently established by dialysing cultures with the supporting bacterial strains or adding their culture broth. Phylogenetic analysis of strain T(T), based on the 16S rDNA sequence, was conducted for the validation of S. thermophilum. The sequence of strain T(T) was located at the outermost position in the high-G+C Gram-positive group distinctly isolated from any other branches hitherto known. Ten sequences identical to that of strain T(T), and one sequence closely related to it, were identified for the first time from soil and compost samples. The outer membrane of strain T(T) had a three-layered structure, outside the cytoplasmic membrane, which is similar to the S-layer in the cells of members of the Bacillaceae. Chemical analysis of the cells revealed that menaquinone-6 is a major component of the quinone system. According to these results, along with several previous observations (i.e. a G+C DNA content of 65 mol% and the identification of iso-C15:0 and iso-C17:0 acids as major cellular fatty acids), the new taxon Symbiobacterium thermophilum gen. nov., sp. nov. is proposed. The type strain is S. thermophilum strain T(T) (= IAM 14863T).

Papillibacter cinnamivorans gen. nov., sp. nov., a cinnamate-transforming bacterium from a shea cake digester.

A new, strictly anaerobic, Gram-positive, non-sporulating, mesophilic bacterium, designated strain CIN1T (T=type strain) was isolated from an anaerobic digester fed with shea cake rich in tannins and aromatic compounds. Cells of strain CIN1T were rod-shaped, had characteristically pointed ends (1.3-3.0 x 0.5-0.6 microm) and occurred singly, in pairs and sometimes in chains of up to six. The pH range for growth was 6.9-8.5 and the temperature growth range was 15-40 degrees C. Optimum growth occurred with yeast extract and cinnamate at 37 degrees C and a pH of 7.5. The isolate transformed cinnamate by degrading the aliphatic side chain to produce acetate and benzoate rather than by aromatic ring cleavage or demethoxylation. The position of the methoxyl group appears to be important in the degradation of the aliphatic side chain of cinnamate; consequently, 3-methoxycinnamate and 4-methoxycinnamate, but not 2-methoxycinnamate, are transformed to produce acetate and methoxybenzoates, namely 3-methoxybenzoate and 4-methoxybenzoate, respectively. Crotonate is degraded to acetate and butyrate. The G+C content of the DNA is 56 mol%. Phylogenetic analysis of the 16S rRNA gene of strain CIN1T indicated that it was a member of the low-G+C-containing Gram-positive branch with a specific relationship to Sporobacter termitidis (sequence identity of 88%). The phylogenetic results concur with the phenotypic data which reveals that the isolate is a novel bacterium and, based on these findings, strain CIN1T (= DSM 12816T = ATCC 700879T) has been designated Papillibacter cinnamivorans gen. nov., sp. nov.

Growth kinetics of Acetobacterium sp. on methanol-formate in continuous culture.

The fermentative metabolism of Acetobacterium sp. grown on methanol-formate in continuous culture is described. The reaction stoichiometry of methanol-formate, including cells, were as follows: CH3OH + 1.13HCOOH --> 0.87CH3COOH + 0.47 cell C. Formate enhanced growth yields by approximately 60% compared with methanol-CO2-grown cultures. Comparison of yields on methanol-formate allowed calculation of an energy yield of 1.3 mol ATP per mol acetate formed during homoacetate fermentation. The magnitudes of YEG,the theoretical maximum yield of YE, and m, the maintenance coefficient, were determined by growing the organism in methanol-formate and resulted in 16.5 g cell (mol methanol catabolized)-1 and 0.674 mmol methanol catabolized (g cell)-1 h-1, respectively. It is concluded that formate might replace CO2 as a source of carboxyl donor.

Visualization and modelling of the thermal inactivation of bacteria in a model food.

A large number of incidents of food poisoning have been linked to undercooked meat products. The use of mathematical modelling to describe heat transfer within foods, combined with data describing bacterial thermal inactivation, may prove useful in developing safer food products while minimizing thermal overprocessing. To examine this approach, cylindrical agar blocks containing immobilized bacteria (Salmonella typhimurium and Brochothrix thermosphacta) were used as a model system in this study. The agar cylinders were subjected to external conduction heating by immersion in a water bath. They were then incubated, sliced open, and examined by image analysis techniques for regions of no bacterial growth. A finite-difference scheme was used to model thermal conduction and the consequent bacterial inactivation. Bacterial inactivation rates were modelled with values for the time required to reduce bacterial number by 90% (D) and the temperature increase required to reduce D by 90% taken from the literature. Model simulation results agreed well with experimental results for both bacteria, demonstrating the utility of the technique.

Mannan-degrading enzymes from Cellulomonas fimi.

The genes man26a and man2A from Cellulomonas fimi encode mannanase 26A (Man26A) and beta-mannosidase 2A (Man2A), respectively. Mature Man26A is a secreted, modular protein of 951 amino acids, comprising a catalytic module in family 26 of glycosyl hydrolases, an S-layer homology module, and two modules of unknown function. Exposure of Man26A produced by Escherichia coli to C. fimi protease generates active fragments of the enzyme that correspond to polypeptides with mannanase activity produced by C. fimi during growth on mannans, indicating that it may be the only mannanase produced by the organism. A significant fraction of the Man26A produced by C. fimi remains cell associated. Man2A is an intracellular enzyme comprising a catalytic module in a subfamily of family 2 of the glycosyl hydrolases that at present contains only mammalian beta-mannosidases.

Using an electronic nose for determining the spoilage of vacuum-packaged beef.

The use of an electronic nose in the quantitative determination of the degree of spoilage of vacuum-packaged beef was evaluated. Beef from four different slaughterhouses was sliced, vacuum-packaged and stored at 4 degrees C for 8 weeks. Samples were withdrawn for bacterial (aerobic bacteria, lactic acid bacteria, Brochothrix thermosphacta, Pseudomonas and Enterobacteriaceae) and sensorial analyses and analysis of the volatile compounds during the storage period. A trained panel was used for the sensorial evaluations. The volatile compounds were analysed using an electronic nose containing a sensory array composed of 10 metal oxide semiconductor field-effect transistors, four Tagushi type sensors and one CO2-sensitive sensor. Four of the 15 sensors were excluded due to lack of response or overloading. Partial least-squares regression was used to define the mathematical relationships between the degree of spoilage of vacuum-packaged beef, as determined by the sensory panel, and the signal magnitudes of the sensors of the electronic nose. The mathematical models were validated after 6 months using a new set of samples. The stability of the sensors during this period was examined and it was shown that the sensitivity of five of the 11 sensors used had changed. Using the six remaining sensors, the signal patterns obtained from the meat from the different slaughterhouses did not change over a period of 6 months. It was shown that the degree of spoilage, as calculated using a model based on two Tagushi sensors, correlated well with the degree of spoilage determined by the sensory panel (r2 = 0.94).

Incorporation of nisin into a meat binding system to inhibit bacteria on beef surfaces.

In two separate experiments, the bacteriocin, nisin, was incorporated into a commercially available meat binding system (Fibrimex) and applied to meat surfaces as a way of inhibiting the meat spoilage organism, Brochothrix thermosphacta during extended refrigerated storage. In experiment 1, pre-rigor lean beef carcass tissue (BCT) was inoculated with B. thermosphacta, left untreated (U), treated with 10 micrograms ml-1 nisin (N), Fibrimex (F) or Fibrimex containing 10 micrograms ml-1 nisin (FN), held aerobically at 4 degrees C for up to 7 d, and populations of B. thermosphacta and nisin activity determined. Experiment 2 determined the effects of the same treatments but on post-rigor, frozen and thawed lean BCT that was inoculated, vacuum-packaged, and stored at 4 degrees C for up to 14 d. In both experiments, N- and FN-treated tissues exhibited significantly lower populations of B. thermosphacta compared to U- and F-treated tissues, for the duration of refrigerated storage. Nisin activity was detected up to 7 d in N- and FN-treated samples from experiment 1. However, activity was detected only to days 0 and 2 in FN- and N-treated samples, respectively, from experiment 2. These studies indicate that the addition of a bacteriocin to a meat binding system and application to meat surfaces may be useful in reducing undesirable bacteria in restructured meat products.

Tissierella creatinophila sp. nov., a gram-positive, anaerobic, non-spore-forming, creatinine-fermenting organism.

A strictly anaerobic, Gram-positive, non-spore-forming bacterium was isolated from sewage sludge which grew on creatinine as sole source of carbon and energy. This new isolate, designated strain KRE 4T, totally degraded creatinine via creatine, sarcosine and glycine to the products acetate, monomethylamine, ammonia and carbon dioxide. Growth on creatinine or creatine was selenium-dependent and stimulated by formate, indicating the involvement of a creatine reductase, sarcosine reductase and/or glycine reductase. This was substantiated by the fact that creatine, sarcosine and glycine were reduced by cell-free extracts. Growth on creatinine or creatine was also possible in the absence of formate, but with an increase in doubling time. The new bacterium occurred as rod-shaped cells, which exhibited an angular form (2-6 microns long and 0.7-1.1 microns wide) and showed motility by means of peritrichous flagella. The G+C content of the DNA was 30 mol %. Comparative 16S rRNA sequence analysis demonstrated that strain KRE 4T represents a new subline within the genus Tissierella. Due to its very restricted substrate spectrum and the inability of whole cells to utilize sarcosine and glycine as intermediates of creatine breakdown, this organism can be readily separated from currently described species of Tissierella. Therefore, based on the phenotypic and phylogenetic distinctiveness of the new isolate, it si proposed that the bacterium be classified as a new species of the genus Tissierella, Tissierella creatinophila sp. nov. The type strain is KRE 4 (= DSM 6911T).