Evaluation of in vitro ruminal fermentation of ensiled fruit byproducts and their potential for feed use.

OBJECTIVE: Ensiling of tannin-rich fruit byproducts (FB) involves quantitative and qualitative changes in the tannins, which would consequently change the rumen fermentation characteristics. This study aimed to evaluate whether ensiled FBs are effective in mitigating methane emission from ruminants by conducting in vitro assessments. METHODS: Fruit byproducts (grape pomace, wild grape pomace, and persimmon skin) were collected and subjected to four-week ensiling by Lactobacillus buchneri inoculant. A defined feed component with or without FB samples (both fresh and ensiled material) were subjected to in vitro anaerobic culturing using rumen fluid sampled from beef cattle, and the fermentation parameters and microbial populations were monitored. RESULTS: Reduced methane production and a proportional change in total volatile fatty acids (especially enhanced propionate proportion) was noted in bottles containing the FBs compared with that in the control (without FB). In addition, we found lower gene copy number of archaeal 16S rRNA and considerably higher levels of one of the major fibrolytic bacteria (Fibrobacter succinogenes) in the bottles containing FBs than in the control, particularly, when it was included in a forage-based feed. However, in the following cultivation experiment, we observed that FBs failed to exhibit a significant difference in methane production with or without polyethylene glycol, implying that tannins in the FBs may not be responsible for the mitigation of methane generation. CONCLUSION: The results of the in vitro cultivation experiments indicated that not only the composition but also ensiling of FBs affected rumen fermentation patterns and the degree of methane generation. This is primarily because of the compositional changes in the fibrous fraction during ensiling as well as the presence of readily fermented substrates, whereas tannins in these FBs seemed to have little effect on the ruminal fermentation kinetics.

Relationship of Enhanced Butyrate Production by Colonic Butyrate-Producing Bacteria to Immunomodulatory Effects in Normal Mice Fed an Insoluble Fraction of Brassica rapa L.

This study was performed to determine the effects of feeding a fiber-rich fraction of Brassica vegetables on the immune response through changes in enteric bacteria and short-chain fatty acid (SCFA) production in normal mice. The boiled-water-insoluble fraction of Brassica rapa L. (nozawana), which consists mainly of dietary fiber, was chosen as a test material. A total of 31 male C57BL/6J mice were divided into two groups and housed in a specific-pathogen-free facility. The animals were fed either a control diet or the control diet plus the insoluble B. rapa L. fraction for 2 weeks and sacrificed to determine microbiological and SCFA profiles in lower-gut samples and immunological molecules. rRNA-based quantification indicated that the relative population of Bacteroidetes was markedly lower in the colon samples of the insoluble B. rapa L. fraction-fed group than that in the controls. Populations of the Eubacterium rectale group and Faecalibacterium prausnitzii, both of which are representative butyrate-producing bacteria, doubled after 2 weeks of fraction intake, accompanying a marginal increase in the proportion of colonic butyrate. In addition, feeding with the fraction significantly increased levels of the anti-inflammatory cytokine interleukin-10 (IL-10) and tended to increase splenic regulatory T cell numbers but significantly reduced the population of cells expressing activation markers. We demonstrated that inclusion of the boiled-water-insoluble fraction of B. rapa L. can alter the composition of the gut microbiota to decrease the numbers of Bacteroidetes and to increase the numbers of butyrate-producing bacteria, either of which may be involved in the observed shift in the production of splenic IL-10.

Feeding Astragalus membranaceus Root Improves the Rumen Fermentation Rate in Housed Goats through the Alteration of the Rumen Community Composition.

Although Astragalus membranaceus root (AMR) has been noted as an ingredient in ruminant feed, the impacts of AMR feeding on rumen fermentation and the microbial community structure within the rumen are yet to be evaluated. This study investigated the effects of AMR supplementation on rumen fermentation characteristics and microbial community structures in goats. In two sets of feeding experiments, four Japanese native goats were fed AMR (10 g/kg DM/day/head) for three weeks per experiment. The rumen fluid samples were analyzed using high-performance liquid chromatography for fermentation products and next-generation sequencing for microbial analysis. The rumen fluid samples in the second experiment were also subject to an in vitro anaerobic fermentation test. The results indicated a significant modification, with a higher volatile fatty acid (VFA) content in the rumen fluid of goats in the feeding period than before feeding (p < 0.01). The microbial analysis revealed a significant increase in community diversity (p < 0.05) following AMR feeding, and the rumen bacterial community increased in two families belonging to the order Oscillospirales in Firmicutes (p < 0.05). The phylum Verrucomicrobiota was observed to be significantly less abundant after AMR feeding than during the control period (p < 0.05). Notably, the linear discriminant analysis revealed that the families with largely unknown functions in the rumen (Oscillospiraceae, Rikenellaceae, Muribaculaceae, and vadinBB97) were the determinants of the community split between control and AMR feeding. Increased fermentation rate by AMR feeding was also supported by an in vitro culture experiment, which resulted in faster VFA production without affecting methane production in total gas production. The study demonstrated that AMR can significantly facilitate change in the bacterial community structure in the goat rumen involving a shift of the favoring fibrolytic bacteria towards VFA production. The long-term effects of AMR supplementation and its applicability across different ruminant species, with potential benefits for animal health and productivity, should be addressed.

Effects of pellet starch levels in automatic milking systems on rumen fermentation, plasma metabolites, and milk production in mid-lactation cows.

The aim of this study was to evaluate whether the starch levels in pellets fed to cows in automatic milking systems (AMS) affect subacute ruminal acidosis (SARA) occurrence and metabolite parameters. Twenty-four lactating cows (124.4 ± 49.9 days in milk) were studied in a crossover design with two periods of 21 days each and two treatment groups-a control group fed AMS pellets containing 30.0% of starch dry matter (DM) and an experimental group fed AMS pellets containing 23.5% of starch DM. All cows received the same partial mixed ration (PMR). The 1-hr mean ruminal pH in both groups decreased over 4 hr after feeding on PMR but recovered by the next morning. The ruminal pH was unaffected by either treatment, and both groups developed SARA. The groups had no significant differences in the concentrations of ruminal volatile fatty acids, lipopolysaccharides, plasma acute-phase proteins, other metabolites, and hormones. The milk yield and composition were not different in both groups. Feeding low-starch pellets in the AMS did not contribute to the risk of SARA occurrence in cows and had no additive effects on rumen fermentation, plasma metabolites, or milk production.

Biofilm Bacterial Dynamics and Changes in Inorganic Nitrogen Density Due to the Presence of Freshwater Pearl Mussels.

The freshwater pearl mussel (genus Margaritifera) has shown severe declines, while the mussels play important roles in the translocation of nutrients and materials in river water ecosystems. We hypothesized that the biofilm bacterial composition and nutrient flow may reflect the differences in the existence of mussels. We analyzed water from 14 rivers from in multiple regions of Japan, including eight rivers, where the two species of freshwater pearl mussels (Margaritifera laevis and Margaritifera togakushiensis) are predominantly found, to analyze the microbial and nutritional nature of the biofilm artificially formed in the river. Field-produced biofilms, including the bacterial community structure, were examined, using next-generation sequencing of bacterial 16S rRNA gene amplicons followed by analyzing the genomic DNA extracted from the samples, inorganic nitrogen compounds, and chlorophyll a concentration. Compared to those in the control river without freshwater pearl mussels, biofilms of the existing river contained less inorganic nitrogen (ammonia and nitrate), suggesting the involvement of mussels in regulating the river water nutrient flow. Distinct changes were found in biofilms, depending on mussel existence, particularly in biofilms containing fewer photosynthetic bacterial groups, such as Betaproteobacteria and Cyanobacteria. Conversely, bacteria belonging to Bacteroidales in Bacteroidetes and Clostridiales in Firmicutes were predominantly found in biofilm samples where the mussels existed. Mussels alleviated strict nitrogen limitation in streams and possibly caused a concomitant change in the bacterial communities, where populations of bacterial groups exchanging inorganic nitrogen were low. We demonstrate the profound influence of freshwater mussel species on ecosystem processes and community dynamics across rivers. IMPORTANCE The abundance of freshwater unioid mussels exhibited more diverse patterns of inorganic nitrogen flow and bacterial communities than the areas without mussels. This study demonstrates the effect of mussels on different freshwater ecosystem processes with variable organismal densities and biogeochemical factors. Freshwater unionid mussels significantly affect the ecosystem and community dynamics by modulating the relationships, altering nutrient availability, and indirectly manipulating the downstream ecological members, eventually expanding their role in the river ecosystems.

Dietary Supplementation with Fermented Brassica rapa L. Stimulates Defecation Accompanying Change in Colonic Bacterial Community Structure.

Our previous studies have elucidated that oral administration of Brassica rapa L. extract, known as Nozawana in Japan, alters immune responses and gut microbiota composition, increasing the numbers of butyrate-producing bacteria. Therefore, further investigation would help elucidate the mechanism attributable for the changes and health-promoting effects observed after B rapa L. extract ingestion. To reveal the modulation effects of fermented B. rapa L. on immune function and intestinal bacterial community structure, we conducted an intervention study with healthy volunteers followed by a mouse feeding study. The pilot intervention study was conducted for healthy volunteers aged 40-64 years under the hypothesis that the number of subjects exhibiting any change in gut microbiota in response to fermented B. rapa L. consumption may be limited. In total, 20 volunteers consumed 30 g of fermented B. rapa L. per day for 4 weeks. The fecal bacterial community composition of the volunteers was characterized using terminal-restriction fragment length polymorphism patterning followed by clustering analysis. To evaluate the detailed changes in the immune responses and the gut bacterial composition, assessed by high-throughput sequencing, we fed healthy mice with freeze-dried, fermented B. rapa L. for 2 weeks. The fecal bacterial community composition of the volunteers before the intervention was divided into three clades. Regardless of the clade, the defecation frequency significantly increased during the intervention weeks compared with that before the intervention. However, this clustering detected a specific increase of Prevotella in one cluster (low to zero Prevotella and high occupation of Clostridium at clusters IV and XIVa) post-ingestion. The cytokine production of spleen cells significantly increased due to feeding fermented B. rapa L. to the mice. This supplementary in vivo trial provided comparable results to the volunteer study regarding the effects of ingestion of the material given the compositional change complying with that of dietary fiber, particularly in the increase of genera Prevotella, Lachnospira, and genera in the Ruminococcaceae family, and the increase in daily defecation amount during 2 weeks of administration. We conclude that feeding fermented B. rapa L. may be responsible for the observed modulation in gut microbiota to increase fiber-degrading bacteria and butyrate-producing bacteria which may be relevant to the improvement in bowel function such as defecation frequency.

A Soybean Resistant Protein-Containing Diet Increased the Production of Reg3γ Through the Regulation of the Gut Microbiota and Enhanced the Intestinal Barrier Function in Mice.

The maintenance of intestinal homeostasis is necessary for a good quality of life, and strengthening of the intestinal barrier function is thus an important issue. Therefore, we focused on soybean resistant protein (SRP) derived from kori-tofu (freeze-dried tofu), which is a traditional Japanese food, as a functional food component. In this study, to investigate the effect of SRP on the intestinal barrier function and intestinal microbiota, we conducted an SRP free intake experiment in mice. Results showed that ingestion of SRP decreased the serum level of lipopolysaccharide-binding protein and induced the expression of Reg3γ, thereby improving the intestinal barrier function. In addition, SRP intake induced changes in the cecal microbiota, as observed by changes in ß-diversity. In particular, in the microbiota, the up-regulation of functional gene pathways related to the bacterial invasion of epithelial cells (ko05100) was observed, suggesting that Reg3γ expression was induced by the direct stimulation of epithelial cells. The results of this study suggest that SRP is a functional food component that may contribute to the maintenance of intestinal homeostasis.

An rRNA-based analysis for evaluating the effect of heat stress on the rumen microbial composition of Holstein heifers.

We performed a set of heifer feeding trials to investigate the effect of heat and humidity stresses on the rumen bacterial molecular diversity of Holstein heifers (Tajima K, Nonaka I, Higuchi K, Takusari N, Kurihara M, Takenaka A, et al. Anaerobe 2007;13:57-64). To further characterize the response of the microbial community to the physiological changes caused by the stresses, we evaluated changes in the ruminal bacterial community composition in the same trials by applying an RNA-based method (sequence-specific small-subunit (SSU) rRNA cleavage method), which was optimized for a comprehensive description of the predominant bacterial groups inhabiting the rumen. Four Holstein heifers were kept at three temperatures (20 degrees C, 28 degrees C, 33 degrees C) in a climatic chamber for two weeks each, and rumen fluid samples were obtained on the last day of each temperature experiment. For quantitative detection, we applied a set of 15 oligonucleotide probes, including those targeting taxa comprised of uncultured rumen bacteria (URB) belonging to phylum Firmicutes, to the RNAs extracted from the fluid samples. The relative populations of the Clostridium coccoides-Eubacterium rectale group, and the genus Streptococcus increased, and that of the genus Fibrobacter decreased in response to increasing temperature both in the first (nine months old, 80% relative humidity) and second (15 months old, 60% relative humidity) experiments. In addition, the population of a defined URB group was higher at 33 degrees C than at 20 degrees C in the second trial, whereas one of the other URB groups showed a decreasing trend with the temperature rise. These results indicate that the exposure to heat affects the population levels of specific bacterial groups in the ruminal microbial community.

Fermentative Quality and Animal Acceptability of Ensiled Persimmon Skin with Absorbents for Practical Use in Ruminant Feed.

Persimmon skin (PS), while representing an attractive feed source, requires an appropriate preservation procedure to increase its shelf life. We assessed the fermentation quality, in vitro ruminal incubation, and intake of persimmon skin silage ensiled with different dry absorbents. We prepared the silage on a table scale (Experiment 1) and evaluated five different mixtures: PS without an additive, PS plus Lactobacillus buchneri inoculum (LB), and PS plus LB plus each of the absorbents kraft pulp, wheat bran, or beet pulp. We opened the laboratory bags, kept at 25 °C, at 0, 14, 28, and 60 days for fermentation quality and chemical analysis (n = 3 for each measurement). Further, with an in vitro rumen simulated cultivation study (Experiment 2), we evaluated the fermentation pattern of PS with a mixture of two absorbents (kraft pulp and wheat bran) either raw (no fermentation) or ensiled (n = 4 for each treatment). Finally, we conducted an in vivo experiment using six dry ewes assigned based on their body weight to two experimental groups in a crossover design of two periods (Experiment 3). We fed a control group a 100% basal diet (tall fescue hay and concentrate mixture) and ensiled PS (PSS) group, a 20% dry matter substitution of tall fescue with PS silage mixed with kraft pulp as the sole absorbent. The results of Experiment 1 show, regardless of the absorbents used, the effluent volume of the lab bags was lower in absorbent-treated groups (p < 0.001). In Experiment 2, the condition of the PS with absorbents (raw or ensiled) did not affect the total gas production (p > 0.05), but we observed an increased propionate proportion in PSS with absorbents compared to basal diet (p = 0.019). The proportion of methane to the total gas in PSS group was considerably reduced compared with that in the other groups (p < 0.001). As we did this incubation study with a single run, a more detailed evaluation in the future would verify these observations. In the animal trial (Experiment 3), dry matter intake was similar between groups (p > 0.05), but ewes spent a shorter time eating in the PSS-fed group (p = 0.011). Here we present the practical use of PSS as part of ruminant feed in which dry absorbents prevented dry matter loss.

Provision of beta-glucan prebiotics (cellooligosaccharides and kraft pulp) to calves from pre- to post-weaning period on pasture.

We conducted two feeding experiments to evaluate the effects of supplementation with either cellooligosaccharide or kraft pulp on growth performance in grazing beef calves (Japanese Black) from 4 weeks pre-weaning to 12 to 16 weeks post-weaning. In Experiment 1 (20-week duration), nine calves (2.9-month-old females) were assigned to either a control group (CON) or an experimental group (CEL) fed cellooligosaccharide at a rate of 10 g/day mixed with concentrate. Average daily weight gain tended to be greater in CEL than in CON, especially after 1 month of weaning. In Experiment 2 (16-week duration), 10 calves (2.0-month-old females) were assigned to either a control group or an experimental group (KRA) fed kraft pulp at a rate of 10% replacement of total digestible nutrients with concentrate. The proportion of fibrolytic bacteria increased and that of methanogenic Archaea decreased in the rumen microbial community composition of KRA calves in Experiment 2, whereas the decrease in Fibrobacter and Archaea was observed in CEL calves at first 4 weeks in Experiment 1. We conclude that beta-glucan prebiotic supplementation to grazing calves at pre-weaning would affect rumen microbial composition and modified rumen fermentation characteristics, leading to a better rumen environment via different means.

Metagenomic profiles of the rumen microbiota during the transition period in low-yield and high-yield dairy cows.

We investigated potential relationships between rumen microbiota and milk production in dairy cows during the transition period. Twelve dairy cows were divided into a low-yield (LY) or high-yield (HY) group based on their milk yield. Rumen samples were taken from dairy cows at 3 weeks before parturition, and at 4, 8, and 12 weeks after parturition. 16S rDNA-based metagenomic analysis showed that diversities of rumen microbiota in both groups were similar and the number of operational taxonomic units (OTUs) was lower in the postpartum than prepartum period in both groups. The abundance of Bacteroidetes and ratio of Bacteroidetes:Firmicutes was higher in the HY than the LY group. OTUs assigned to Prevotella bryantii, Fibrobacter succinogenes, Ruminococcus albus, Butyrivibrio fibrisolvens, and Succinivibrio sp. were abundant in the HY group. These OTUs were significantly related to the propionate molar proportion of rumen fluids in the HY group. OTUs assigned to Lachnospiraceae, Bifidobacterium sp. and Saccharofermentans were dominant in the LY group. Predictive functional profiling revealed that abundance of gene families involved in amino acid and vitamin metabolism was higher in the HY than the LY group. These results suggest that the community structure and fermentation products of rumen microbiota could be associated with milk production of dairy cows.

Impact of consumption of probiotic lactobacilli-containing yogurt on microbial composition in human feces.

An in vivo study was carried out to determine the effect of consuming probiotic lactobacilli-containing yogurt on the composition of microbiota in the human gut. Fifteen healthy adults ingested a daily serving of one of three commercial yogurts (two of the products contained a probiotic lactobacilli strain) for 20 days. Fecal samples at defined time points before, during, and after the period of yogurt ingestion were collected and analyzed. The fecal population of lactobacilli was determined by a culture-based method and subsequent colony PCR for the identification of species. Six predominant bacterial groups in the fecal samples were quantitatively determined based on a sequence-specific SSU rRNA cleavage method coupled with a suite of oligonucleotide probes, which was optimized for the target-specific detection of bacterial groups inhabiting human feces. In the ingestion period, one probiotic strain was detected in the feces of all five subjects who consumed the yogurt containing the strain, while the other strain was detected in three of another five subjects. The population levels of the two major groups (Bacteroides and Prevotella, and the Clostridium coccoides-Eubacterium rectale group) in the fecal samples tended to change in response to the ingestion but the change did not seem to be dependent on the product-specific property of each yogurt. These results suggest that the human fecal bacterial community could be altered by ingesting yogurt, although whether probiotic lactobacilli are present or absent in the yogurt does not seem to be a factor in this change.

Changes in Gut Microbial Ecology and Immunological Responses of Mice Fed the Insoluble Fraction of Brassica rapa L. that was Fermented or Not.

We aimed to investigate the effects of feeding fermented Brassica rapa L. on ecological and immunological changes in the mouse gut using in vitro cultivation tests and in vivo experiments in normal mice. In the preliminary in vitro study, two B. rapa L. products from different fermentation periods (one d [SF] or six months [LF]) were evaluated along with non-fermented vegetables (NF). Among the components of each product, the insoluble fraction resulted in the most prominent change such as a relative increase in butyrate production during a cultivation inoculated with mouse cecum contents. Based on this result, the boiled water-insoluble fractions of B. rapa L. (SF, LF, and NF samples) were selected as test materials for the subsequent in vivo experiment. Male C57BL/6J mice were divided into four groups and fed either a control diet (CON) or control diet plus one of the insoluble fractions for two weeks. The NF and LF groups had higher relative populations of Faecalibacterium prausnitzii than the CON group. Therefore, colonic butyrate concentrations were higher in the NF and LF groups than in the CON group. The oral administration of B. rapa L. extract induced immune regulatory effects, even when mice were fed NF and SF, but not LF, as assessed by an increase in regulatory T cell numbers. Our results indicate that feeding a purified insoluble fraction from B. rapa L. affects enteric short-chain fatty acid production and immunological responses in the mouse gut in a similar manner, regardless of the fermentation status.

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.

Effects of supplementing an active dry yeast product on rumen microbial community composition and on subsequent rumen fermentation of lactating cows in the mid-to-late lactation period.

The effects of supplementing feed of cows in mid-to-late lactation with an active yeast product (Actisaf Sc 47) were evaluated using 15 Holstein cows in a replicated 3 × 3 Latin square design. The animals were fed a mixed ration with 33% neutral detergent fiber, consisting of timothy hay (29.8%), a commercial concentrate (70.0%) and commercial calcium triphosphate (0.2%), twice daily to meet 105% of their energy requirement. Yeast supplement was set at 0, 5 and 10 g per day over 21-day periods, each of which consisted of 14 days for adaptation followed by 7 days of data collection. Milking performance, plasma metabolite parameters, rumen volatile fatty acids, lipopolysaccharide and microbial properties were measured. Although there were no significant differences in feeding and milking performance or blood parameters associated with supplementation, the acetate to propionate ratio in the rumen fluid tended to decrease (P = 0.08). The population of Bacteroidetes tended to be less prominent (P = 0.07) and the fibrolytic bacterium Fibrobacter significantly increased (P < 0.05) in the rumen fluid of the yeast 10 g group compared with that of the control. These data suggest that effects of supplementing live yeast to cows in mid-to-late lactation may be limited to microbial composition and fermentation characteristics in the rumen.

Effects of supplementation with cellooligosaccharides on growth performance of weaned calves on pasture.

We conducted two feeding experiments to evaluate the effects of cellooligosaccharide (CE) supplementation on growth performance in grazing beef calves. Calf sex and age and duration of the experimental period differed between the experiments. Experiment 1 (10 weeks) used 5.6-month-old castrated males; Experiment 2 (13 weeks) used 3.9-month-old females. Eight Japanese Black calves were assigned to either a control group (CON) or an experimental group (CE) fed CE at a rate of 10 g/day mixed with concentrate. Calves were stocked in fields in which Kentucky bluegrass was dominant. In both experiments, average daily gain tended to be greater in CE than in CON, especially in late stages of experiments, but no significant differences in body weight changes were observed. In Experiment 2, final heart girth was larger in CE than in CON calves. We monitored rumen microbial community composition in Experiment 2 and found increases in fibrolytic bacteria and methanogenic archaea in CE calves, but the overall microbial composition did not differ between the groups. Our results suggest that supplementation with CE may positively affect growth performance in weaned calves on pasture, but longer-term CE supplementation may be required to exhibit the effect.

Effect of Probiotics/Prebiotics on Cattle Health and Productivity.

Probiotics/prebiotics have the ability to modulate the balance and activities of the gastrointestinal (GI) microbiota, and are, thus, considered beneficial to the host animal and have been used as functional foods. Numerous factors, such as dietary and management constraints, have been shown to markedly affect the structure and activities of gut microbial communities in livestock animals. Previous studies reported the potential of probiotics and prebiotics in animal nutrition; however, their efficacies often vary and are inconsistent, possibly, in part, because the dynamics of the GI community have not been taken into consideration. Under stressed conditions, direct-fed microbials may be used to reduce the risk or severity of scours caused by disruption of the normal intestinal environment. The observable benefits of prebiotics may also be minimal in generally healthy calves, in which the microbial community is relatively stable. However, probiotic yeast strains have been administered with the aim of improving rumen fermentation efficiency by modulating microbial fermentation pathways. This review mainly focused on the benefits of probiotics/prebiotics on the GI microbial ecosystem in ruminants, which is deeply involved in nutrition and health for the animal.

Generation of dipeptidyl peptidase-IV-inhibiting peptides from ß-lactoglobulin secreted by Lactococcus lactis.

Previous studies showed that hydrolysates of ß-lactoglobulin (BLG) prepared using gastrointestinal proteases strongly inhibit dipeptidyl peptidase-IV (DPP-IV) activity in vitro. In this study, we developed a BLG-secreting Lactococcus lactis strain as a delivery vehicle and in situ expression system. Interestingly, trypsin-digested recombinant BLG from L. lactis inhibited DPP-IV activity, suggesting that BLG-secreting L. lactis may be useful in the treatment of type 2 diabetes mellitus.

Desulfovibrio marinisediminis sp. nov., a novel sulfate-reducing bacterium isolated from coastal marine sediment via enrichment with Casamino acids.

To obtain amino acid-utilizing sulfate reducers, enrichment culture was carried out with a medium containing Casamino acids and sulfate and inoculated with coastal marine sediment from the eutrophic Tokyo Bay, Japan. A sulfate reducer, designated strain C/L2(T), was isolated from the sulfide-producing enrichment culture after further enrichment with lactate and sulfate by means of the agar shake dilution method. Cells of strain C/L2(T) were vibrio-shaped, Gram-negative, motile rods (0.7-1.0 mum wide and 1.0-3.5 mum long) with single polar flagella. The optimum temperature for its growth was 37 degrees C, the optimum pH was around 7.5 and the optimum NaCl concentration was 20-25 g l(-1). Hydrogen, formate, lactate, pyruvate, fumarate, malate, succinate, ethanol, propanol, glycerol, glycine, alanine, serine, aspartate, Casamino acids, peptone and yeast extract were used as electron donors. Sulfate, sulfite and thiosulfate each served as an electron acceptor, but elemental sulfur, nitrate, fumarate, acrylate and 2,4,6-tribromophenol did not. Disproportionation of thiosulfate was not observed. Desulfoviridin, c-type cytochromes and catalase were present. The major respiratory quinone was MK-6(H(2)). The G+C content of the genomic DNA was 46.2 mol%. Comparisons based on 16S rRNA gene sequences and on dissimilatory sulfite reductase gene sequences clearly showed that strain C/L2(T) belonged to the genus Desulfovibrio: its closest relatives were the uncharacterized Desulfovibrio sp. strain TBP-1 (16S rRNA gene sequence similarity of 99.4 %) and Desulfovibrio acrylicus DSM 10141(T) (16S rRNA gene sequence similarity of 98.7 %). The level of DNA-DNA hybridization with Desulfovibrio acrylicus DSM 10141(T) was 10.3 %. On the basis of the data from this study and the physiological and phylogenetic differences that exist between the isolate and Desulfovibrio acrylicus, strain C/L2(T) represents a novel species of the genus Desulfovibrio, for which the name Desulfovibrio marinisediminis sp. nov. is proposed. The type strain is C/L2(T) (=NBRC [corrected] 101113(T)=JCM 14577(T)=DSM 17456(T)).

Sequence-specific cleavage of small-subunit (SSU) rRNA with oligonucleotides and RNase H: a rapid and simple approach to SSU rRNA-based quantitative detection of microorganisms.

A rapid and simple approach to the small-subunit (SSU) rRNA-based quantitative detection of a specific group of microorganisms in complex ecosystems has been developed. The method employs sequence-specific cleavage of rRNA molecules with oligonucleotides and RNase H. Defined mixtures of SSU rRNAs were mixed with an oligonucleotide (referred to as a "scissor probe") that was specifically designed to hybridize with a particular site of targeted rRNA and were subsequently digested with RNase H to proceed to sequence-dependent rRNA scission at the hybridization site. Under appropriate reaction conditions, the targeted rRNAs were correctly cut into two fragments, whereas nontargeted rRNAs remained intact under the same conditions. The specificity of the cleavage could be properly adjusted by controlling the hybridization stringency between the rRNA and the oligonucleotides, i.e., by controlling either the temperature of the reaction or the formamide concentration in the hybridization-digestion buffer used for the reaction. This enabled the reliable discrimination of completely matched rRNA sequences from single-base mismatched sequences. For the detection of targeted rRNAs, the resulting RNA fragment patterns were analyzed by gel electrophoresis with nucleotide-staining fluorescent dyes in order to separate cleaved and intact rRNA molecules. The relative abundance of the targeted SSU rRNA fragments in the total SSU rRNA could easily be calculated without the use of an external standard by determining the signal intensity of individual SSU rRNA bands in the electropherogram. This approach provides a fast and easy means of identification, detection, and quantification of a particular group of microbes in clinical and environmental specimens based on rRNA.