Characteristics of rumen microbiota and Prevotella isolates found in high propionate and low methane-producing dairy cows.

Ruminal methane production is the main sink for metabolic hydrogen generated during rumen fermentation, and is a major contributor to greenhouse gas (GHG) emission. Individual ruminants exhibit varying methane production efficiency; therefore, understanding the microbial characteristics of low-methane-emitting animals could offer opportunities for mitigating enteric methane. Here, we investigated the association between rumen fermentation and rumen microbiota, focusing on methane production, and elucidated the physiological characteristics of bacteria found in low methane-producing cows. Thirteen Holstein cows in the late lactation stage were fed a corn silage-based total mixed ration (TMR), and feed digestion, milk production, rumen fermentation products, methane production, and rumen microbial composition were examined. Cows were classified into two ruminal fermentation groups using Principal component analysis: low and high methane-producing cows (36.9 vs. 43.2 L/DMI digested) with different ruminal short chain fatty acid ratio [(C2+C4)/C3] (3.54 vs. 5.03) and dry matter (DM) digestibility (67.7% vs. 65.3%). However, there were no significant differences in dry matter intake (DMI) and milk production between both groups. Additionally, there were differences in the abundance of OTUs assigned to uncultured Prevotella sp., Succinivibrio, and other 12 bacterial phylotypes between both groups. Specifically, a previously uncultured novel Prevotella sp. with lactate-producing phenotype was detected, with higher abundance in low methane-producing cows. These findings provide evidence that Prevotella may be associated with low methane and high propionate production. However, further research is required to improve the understanding of microbial relationships and metabolic processes involved in the mitigation of enteric methane.

Comparison of taste characteristics between koji mold-ripened cheese and Camembert cheese using an electronic tongue system.

Koji mold, classified in the genus Aspergillus, is used to produce traditional Japanese fermented foods such as miso, soy sauce, and sake. In recent years, the application of koji mold to cheese ripening has attracted attention, and cheese surface-ripened with koji mold (koji cheese) has been studied. In this study, to evaluate the taste characteristics of koji cheese, an electronic tongue system was employed to measure the taste values of cheese samples ripened using 5 strains of koji mold in comparison with commercial Camembert cheese. All koji cheese samples exhibited lower sourness and greater bitterness, astringency, saltiness, and umami richness than the Camembert cheese samples. The intensity of each taste characteristic differed depending on the koji mold strain. These results indicate that koji cheese has a different taste value than conventional mold-ripened cheese. Furthermore, the results also indicate that various taste characteristics can be achieved by selecting different koji molds.

Prevotella lacticifex sp. nov., isolated from the rumen of cows.

The genus Prevotella plays an important role in polysaccharide degradation and fermentation in the rumen. To further understand the function of the phylogenetically diverse genus Prevotella, it is necessary to explore the individual characteristics at the species level. In this study, Gram-negative anaerobic bacterial strains isolated from the rumen of Holstein cows were identified. Strain R5019T was classified within the genus Prevotella based on 16S rRNA gene sequence-based phylogenetic analysis. The values of 16S rRNA gene sequence similarity, average nucleotide identity and digital DNA-DNA hybridization between strain R5019T and its phylogenetically nearest species Prevotella multisaccharivorax PPPA20T were 89.8, 82.6, and 29.3 %, respectively. The genome size of R5019T was estimated to be ca. 4.19 Mb with a genomic G+C content of 49.5 mol%. The major cellular fatty acids and menaquinones were C15 : 0 anteiso and C17 : 0 anteiso and MK-11 and MK-12, respectively. Succinate, lactate, malate, acetate and formate were produced as the fermentation end products using glucose. Based on phylogenetic, physiological, biochemical and genomic differences between 11 strains and other phylogenetically related Prevotella species, a novel species, Prevotella lacticifex sp. nov., is proposed within the genus Prevotella. The type strain is R5019T (=JCM 34664T=DSM 112675T).

Characterization of unique metabolites in γ-aminobutyric acid-rich cheese by metabolome analysis using liquid chromatography-mass spectrometry.

Fermented dairy products comprise many functional components. Our previous study using fermented milk showed that the γ-aminobutyric acid (GABA)-producing Lactococcus lactis 01-7 strain can produce unique metabolites such as antihypertensive peptides, whereas this study was designed to find the unique metabolites in GABA-rich cheese using the 01-7 strain. Metabolites between cheese ripening with the non-GABA-producing L. lactis 01-1 strain (control) and GABA-rich cheese ripening with a mixture of 01-1 and 01-7 strains were compared. GABA and ornithine were detected in GABA-rich cheese using an amino acid analyzer and citrate was detected in the control cheese using HPLC. Metabolome analysis using LC-MS showed that peptides with unknown function and those with antihypertensive activity were higher in the GABA-rich cheese than in the control cheese. Further analysis of the amount of the YLGY derivatives showed that the amount of YL in the GABA-rich cheese was lower than that in the control. PRACTICAL APPLICATIONS: Clarification of metabolites in cheese contributes to the improvement of cheese ripening, thereby providing consumers with unique cheese with good nutritional and functional characteristics. The use of the 01-7 strain as a cheese starter might provide a functional cheese with antihypertensive-, antioxidative-, and anxiolytic-like activities.

Relative contribution of sensory characteristics for different types of pork loin, assessed by temporal dominance of sensations.

BACKGROUND: Sensory perception is a dynamic process occurring sequentially. The dynamic analysis of the perception of sensory characteristics in meat is important to understand complex meat sensory characteristics. Temporal dominance of sensations (TDS) is a dynamic sensory method that captures the 'dominance of sensations' throughout food consumption, and it is possible to calculate the relative contribution of various sensory characteristics by 'dominance rate' over time. The objective of this study was to assess the major sensory characteristics of the dynamic sensory perception of pork loin. Using the TDS, we evaluated pork loins derived from pigs with different genetic and feeding backgrounds. Moreover, we checked the integrity of the TDS by comparing the TDS results with a descriptive sensory analysis and a physico-chemical analysis. RESULTS: Seven types of pork loin were evaluated by TDS, and 'tender and/or soft,' 'tough and/or hard,' 'juicy,' 'dry,' 'fat melting,' 'smoothness,' 'sourness,' 'umami,' and 'beast odors' became dominant in at least one type of pork loin. The dynamics of the perception of pork sensory characteristics was also depicted by TDS. In addition, TDS and descriptive analysis demonstrated similarity in principal component analysis. CONCLUSION: TDS demonstrated the major sensory characteristics on the dynamic sensory perception of pork loin. © 2019 Society of Chemical Industry.

Diauxic growth of Fibrobacter succinogenes S85 on cellobiose and lactose.

Fibrobacter succinogenes rapidly colonizes the preruminant calf rumen and becomes a dominant cellulolytic bacterium in the rumen after weaning. Although F. succinogenes actively degrades cellulose in the rumen, it seems that there is no or little of its substrate, cellulose, in the rumen of preweaned calves. We thus evaluated the ability of F. succinogenes to utilize lactose, a main sugar of milk, with or without the presence of cellobiose. We grew F. succinogenes S85 on media containing 2.5% lactose combined with 0%-0.2% cellobiose or a medium with 0.2% cellobiose but without lactose. The generation times on the 0.2% cellobiose medium and the 2.5% lactose medium were 1.9 and 16.2 h, respectively. The bacterium showed rapid growth on cellobiose and diauxic growth on the lactose media containing 0.05%-0.2% cellobiose. Moreover, the production of ß-galactosidase was low in the presence of 0.1%-0.2% cellobiose. Since the ß-galactosidase contained a signal peptide and a Por secretion system C-terminal sorting domain, we speculate that the ß-galactosidase would be secreted from the bacterial cells by the Por secretion system. Our data indicate the possibility that F. succinogenes could colonize preruminant calf rumen, consuming the lactose present in cow milk.

Differences in rumen fermentation characteristics between low-yield and high-yield dairy cows in early lactation.

Relationship between rumen fermentation parameters, blood biochemical profiles and milk production traits in different yielding dairy cows during early lactation was investigated. Twelve dairy cows were divided into two groups based on their milk yield, that is low-yield (LY) and high-yield (HY) groups. Rumen fluid and blood were collected at 3 weeks prepartum and 4, 8 and 12 weeks postpartum. Results showed that proportions of acetate, propionate to total short chain fatty acids and acetate : propionate ratio were changed (P < 0.05) in both groups during the peripartum period, whereas butyrate and acetate : butyrate ratio were only altered in the HY group. Blood cholesterol, beta-hydroxybutyric acid (BHBA) and glutamic oxaloacetic transaminase in the HY group were higher (P < 0.01) than those in the LY group. Principal component analysis revealed that milk yield and milk compositions were differently clustered between groups. These parameters showed similar direction with dry matter intake in the HY group and adverse direction in the LY group. Linear regression analysis indicated that butyrate was positively correlated with BHBA (P < 0.05) in the HY group. This study suggests that cows in the HY group seem to accommodate appropriately to negative energy balance in early lactation through rumen fermentation.

Lactobacillus mixtipabuli sp. nov. isolated from total mixed ration silage.

Using a polyphasic taxonomic approach, we investigated three bacterial strains - IWT30T, IWT8 and IWT75 - isolated from total mixed ration silage prepared in Hachimantai, Iwate, Japan. The isolates comprised Gram-stain positive, non-motile, non-spore-forming, catalase-negative, rod-shaped bacteria. Good growth occurred at 15-45 °C and at pH 4.0-7.5. Their major cellular fatty acids were C18:1ω9c and C19:1 cyclo 9,10.The G+C content of genomic DNA of strain IWT30T was 44.6 mol%. Comparative 16S rRNA gene sequence analysis showed that these novel strains belonged to the genus Lactobacillus. These strains shared 100 % 16S rRNA gene sequence similarity and were most closely related to the type strains of Lactobacillus silagei, Lactobacillus odoratitofui, Lactobacillus similis, Lactobacillus collinoides, Lactobacillus paracollinoides and Lactobacillus kimchicus, with sequence similarity values of 99.5, 98.8, 98.7, 97.8, 97.8 and 96.8 %, respectively. The level of DNA-DNA relatedness between these strains and their closest phylogenetic neighbours was less than 30 %. On the basis of additional phylogenetic analysis of pheS and rpoA gene sequences and phenotypic and chemotaxonomic characteristics, we conclude that these three strains represent a novel species of the genus Lactobacillus, for which we propose the name Lactobacillus mixtipabuli sp. nov. The type strain is IWT30T ( = JCM 19805T = DSM 28580T).

Extracellular secretion of noncatalytic plant cell wall-binding proteins by the cellulolytic thermophile Caldicellulosiruptor bescii.

Caldicellulosiruptor bescii efficiently degrades cellulose, xylan, and native grasses at high temperatures above 70°C under anaerobic conditions. C. bescii extracellularly secretes multidomain glycoside hydrolases along with proteins of unknown function. In this study, we analyzed the C. bescii proteins that bind to the cell walls of timothy grass by using mass spectrometry, and we identified four noncatalytic plant cell wall-binding proteins (PWBPs) with high pI values (9.2 to 9.6). A search of a conserved domain database showed that these proteins possess a common domain related to solute-binding proteins. In addition, 12 genes encoding PWBP-like proteins were detected in the C. bescii genomic sequence. To analyze the binding properties of PWBPs, recombinant PWBP57 and PWBP65, expressed in Escherichia coli, were prepared. The PWBPs displayed a wide range of binding specificities: they bound to cellulose, lichenan, xylan, arabinoxylan, glucuronoxylan, mannan, glucomannan, pectin, oligosaccharides, and the cell walls of timothy grass. The proteins showed the highest binding affinity for the plant cell wall, with association constant (Ka) values of 5.2 × 10(6) to 44 × 10(6) M(-1) among the insoluble polysaccharides tested, as measured using depletion binding isotherms. Affinity gel electrophoresis demonstrated that the proteins bound to the acidic polymer pectin most strongly among the soluble polysaccharides tested. Fluorescence microscopic analysis showed that the proteins bound preferentially to the cell wall in a section of grass leaf. Binding of noncatalytic PWBPs with high pI values might be necessary for efficient utilization of polysaccharides by C. bescii at high temperatures.

Segmented filamentous bacteria are a major group in terminal ileum of piglets.

Metabolically active microbiota of the porcine terminal ileum mucosa was analyzed by RT-PCR of 16S rRNAs. The majority of 1951 sequences retrieved (24.8%) displayed the closest similarity to segmented filamentous bacteria (SFB). Phylogenetic analysis inferred the host-specific clusters of SFB sequences suggesting the host-specific selection of this group of bacteria.

An improved dry ash procedure for the detection of titanium dioxide in cattle feces.

We improved the dry ash procedure for detecting titanium dioxide (TiO2 ) in cattle feces containing chromium oxide [corrected] (Cr(2)O(3) ). First, the effect of amount of sodium sulfate (Na2 SO4 ) on the recovery of TiO2 from cattle feces that contained Cr2 O3 was evaluated. Average recovery of TiO2 at the 2.5 g Na2 SO4 level was significantly higher (P < 0.0001) than that at 0.75 g Na2 SO4 . Second, the effect of Cr2 O3 concentration on the recovery of TiO2 of cattle feces by using two levels of Na2 SO4 addition was examined. The recovery of TiO2 decreased with the increase in the amount of Cr2 O3 at the 0.75 g Na2 SO4 level but was consistently high at 2.5 g Na2 SO4 . Third, the recovery of Cr2 O3 from cattle feces was checked. The recoveries of TiO2 and Cr2 O3 were high enough at the 2.5 g Na2 SO4 level. Fourth, the improved dry ash procedure (5 mL of concentrated H2 SO4 and 2.5 g of Na2 SO4 were used for sample digestion) was compared to the wet ash procedure. Average recovery of TiO2 by the improved dry ash procedure was significantly higher (P = 0.0077) than that by the wet ash procedure. Thus, the improved dry ash procedure can be used for TiO2 analysis in cattle feces containing Cr2 O3 .

Degradation of microcrystalline cellulose and non-pretreated plant biomass by a cell-free extracellular cellulase/hemicellulase system from the extreme thermophilic bacterium Caldicellulosiruptor bescii.

Caldicellulosiruptor bescii is a cellulolytic/hemicellulolytic anaerobe, which extracellularly secretes various proteins, including multidomain cellulases with two-catalytic domains, for plant biomass degradation. Degradation by C. bescii cells has been well characterized, but degradation by the cell-free extracellular cellulase/hemicellulase system (CEC) of C. bescii has not been as well studied. In the present study, C. bescii CEC was prepared from cell-free culture supernatant, and the degradation properties for defined substrates and non-pretreated plant biomass were characterized. Four multidomain cellulases (Cbes_1857, Cbes_1859, Cbes_1865, and Cbes_1867), composed of the glycoside hydrolase families 5, 9, 10, 44, and 48, were the major enzymes identified in the CEC by mass spectrometry. The CEC degraded xylan, mannose-based substrates, ß-1,4-linked glucans, including microcrystalline cellulose (Avicel), and non-pretreated timothy grass and rice straw. However, degradation of chitin, pectin, dextran, and wheat starch was not observed. The optimum temperatures for degradation activities were 75°C for timothy grass and Avicel, 85°C for carboxylmethyl cellulose, and >85°C for xylan. The optimum pH for these substrates was 5-6. The degradation activities were compared with a CEC derived from the fungus Trichoderma reesei, the most common enzyme used for plant biomass saccharification. The amounts of degraded Avicel, timothy grass, and rice straw by C. bescii CEC were 2.2-2.4-fold larger than those of T. reesei CEC. The high hydrolytic activity of C. bescii CEC might be attributed to the two-catalytic domain architecture of the cellulases.

Life cycle assessment of animal feeds prepared from liquid food residues: a case study of rice-washing water.

Life cycle assessment (LCA) was used to compare the greenhouse gas (GHG) emissions and energy consumption of three methods used to produce animal feed from concentrated rice-washing water (CRW) and disposing of the rice-washing water through wastewater treatment. Four scenarios were compared using LCA: (i) producing concentrated liquid feed by centrifugation (CC) of CRW with wastewater treatment and discharge of the supernatant, (ii) producing concentrated liquid feed by heating evaporation (HC) of CRW, (iii) producing dehydrated feed by dehydration (DH) of CRW, and (iv) wastewater treatment and discharge of nonconcentrated rice-washing water (WT). The functional unit (FU) was defined as 1 metric ton of rice washed for cooking or processing. Our results suggested that the energy consumptions of CC, HC, DH, and WT were 108, 322, 739, and 242 MJ per FU, respectively, and the amounts of GHG emissions from CC, HC, DH, and WT were 6.4, 15.8, 45.5, and 22.5 kg of CO equivalents per FU, respectively. When the produced feed prepared from CRW was assumed to be transported 200 km to farms, CC and HC still emitted smaller GHGs than the other scenarios, and CC consumed the smallest amount of energy among the scenarios. The present study indicates that liquid feed production from CRW by centrifugation has a remarkably reduced environmental impact compared with the wastewater treatment and discharge of rice-washing water.

Impressions and purchasing intentions of Japanese consumers regarding pork produced by 'Ecofeed,' a trademark of food-waste or food co-product animal feed certified by the Japanese government.

Impressions and purchasing intentions of Japanese consumers regarding pork produced by 'Ecofeed', a trademark of food-waste or co-product animal feeds certified by the Japanese government, were investigated by a questionnaire on the Internet. 'Ecofeed' did not elicit specific impressions as compared to domestic, imported, Kurobuta (in Japan), and specific pathogen-free (SPF) pork. Purchasing intent for 'Ecofeed' pork was the second lowest of the five pork products. Knowledge and purchasing experience regarding 'Ecofeed' pork was the lowest of the five pork products. Respondents were classified into four categories according to their impressions of 'Ecofeed' pork. The largest category of respondents did not have any specific impression of 'Ecofeed' pork and had little knowledge of pork farming. A category that had a positive impression for 'Ecofeed' pork had high knowledge of the pork farming system. In order to establish 'Ecofeed' pork in Japan, our results suggest that information disclosure and education about 'Ecofeed', its certification system, environmental benefits and the current self-efficiency ratio of animal feed, are needed.

Fermented liquid feed enhances bacterial diversity in piglet intestine.

Because of limitations imposed on the antibiotic use in animal industry, there is a need for alternatives to maintain the efficiency of production. One of them may be the use of fermented liquid feed (FLF) but how it affects gut ecology is poorly understood. We investigated the effect of three diets, standard dry feed (control), dry feed supplemented with antibiotics, and fermented liquid feed (FLF, fermented with Lactobacillus plantarum), on gut bacterial diversity in piglets. The structure of the ileal and caecal communities was estimated by sequencing the SSU rRNA gene libraries. Antibiotic-supplemented feed slightly increased bacterial diversity in the ileum but reduced it in the caecum while in FLF-fed animals bacterial diversity was elevated. The majority of bacterial sequences in the ileum of all three groups belonged to lactobacilli (92-98%). In the caecum the lactobacilli were still dominant in control and antibiotic-fed animals (59% and 64% of total bacterial sequences, respectively) but in FLF-fed animals they fell to 31% with the concomitant increase in the Firmicutes diversity represented by the Dorea, Coprococcus, Roseburia and Faecalibacterium genera. Thus FLF affects the gut ecology in a different way than antibiotics and contributes to the enhanced bacterial diversity in the gastrointestinal tract.

Continuous hydrogen production from glucose by using extreme thermophilic anaerobic microflora.

Continuous hydrogen production from glucose at 75 degrees C was examined at HRTs of 0.67, 1, 2, and 3 d by using extreme thermophilic microflora, which consisted of several subspecies of Caldanaerobacter subterraneus. The maximum hydrogen yield of 3.32 mol-H2/mol-glucose degraded was obtained at the highest HRT, 3 d. Both the maximum volumetric and specific rates of hydrogen production (1.22 L-H2/L/d and 4.15 L-H2/g-VSS/d, respectively) were obtained at the lowest HRT, 0.67 d. A 16S rDNA analysis showed that the community structure of the microflora did not change at any of the HRTs.

Brewer's yeast efficiently degrades phytate phosphorus in a corn-soybean meal diet during soaking treatment.

Microbes such as yeast and Aspergillus are known to produce phytase, and Aspergillus phytase has been used as a feed additive for improving phytate-phosphorus bioavailability in monogastric animals. We measured phytase activity in some by-products from fermented food and beverage productions by yeast and Aspergillus. The phytase activity was as high as 3577 and 2225 PU/kg DM in raw and dried brewer's yeasts, respectively. On the other hand, the phytase activity was approximately 400 PU/kg DM in white-wine yeast and red-wine yeast. The phytase activity was further low in natto (fermented soybean) residue, soy sauce cake, rice brewer's grain and the activity was not detected in dried corn-barley distiller's grain with soluble and sweet-potato distiller's residue. The stability of phytase against pepsin was much lower in the brewer's yeast than in an Aspergillus phytase preparation. On the other hand, the addition of raw brewer's yeast effectively degraded phytate phosphorus in a corn-soybean meal diet during soaking. These results suggest that phytase in the examined by-products is not suitable for the phytase source of conventional diets, but that the soaking treatment with a raw brewer's yeast is an alternative method for improving phytate-phosphorus bioavailability in corn-soybean meal diets for pigs.

Reduction of chlortetracycline-resistant Escherichia coli in weaned piglets fed fermented liquid feed.

We investigated the change in chlortetracycline resistance in 360 Escherichia coli strains separated from the feces of piglets fed fermented liquid feed (FLF) in comparison with those fed dry feed (control). The total amount of lactic acid bacteria in feces was 8.77 x 10(8) CFU/g DM at weaning, which increased to 1.23 x 10(12) CFU/g DM (FLF) at 28 days after weaning (P<0.001). As a result of the antibiotic susceptibility test, almost all isolates were chlortetracycline-resistant (CTCR) until 14 days after weaning both in the FLF and control groups. At 28 days, the CTCR E. coli decreased to 22.2% in the FLF group, while the proportion of resistant bacteria was 88.9% in the control group. In addition, as a result of the gene analysis, it was clarified that there is a relation between the decrease in the minimum inhibitory concentration values and the possession rate of the tetracycline-resistance gene tet(A), tet(B) and tet(D). These results show that FLF caused an increase in the number of lactic acid bacteria in the intestines, and suggested that the feeding of FLF can possibly reduce antibiotic-resistance bacteria.

Community analysis of hydrogen-producing extreme thermophilic anaerobic microflora enriched from cow manure with five substrates.

The present study analyzed the community structures of anaerobic microflora producing hydrogen under extreme thermophilic conditions by two culture-independent methods: denaturing gradient gel electrophoresis (DGGE) and clone library analyses. Extreme thermophilic microflora (ETM) was enriched from cow manure by repeated batch cultures at 75 degrees C, using a substrate of xylose, glucose, lactose, cellobiose, or soluble starch, and produced hydrogen at yields of 0.56, 2.65, 2.17, 2.68, and 1.73 mol/mol-monosaccharide degraded, respectively. The results from the DGGE and clone library analyses were consistent and demonstrated that the community structures of ETM enriched with the four hexose-based substrates (glucose, lactose, cellobiose, and soluble starch) consisted of a single species, closely related to a hydrogen-producing extreme thermophile, Caldoanaerobacter subterraneus, with diversity at subspecies levels. The ETM enriched with xylose was more diverse than those enriched with the other substrates, and contained the bacterium related to C. subterraneus and an unclassified bacterium, distantly related to a xylan-degrading and hydrogen-producing extreme thermophile, Caloramator fervidus.

Hydrogen fermentation properties of undiluted cow dung.

Anaerobic treatment of undiluted cow dung (15% total solids), so-called dry fermentation, produced hydrogen (743 ml-H(2)/kg-cow dung) at an optimum temperature of 60 degrees C, with butyrate and acetate formation. The hydrogen production was inhibited by the addition of NH(4)(+) in a dose-dependent manner. A bacterium with similarity to Clostridium cellulosi was detected in the fermented dung by a 16S rDNA analysis.