In vitro Effects of Cellulose Acetate on Fermentation Profiles, the Microbiome, and Gamma-aminobutyric Acid Production in Human Stool Cultures.

Gamma-aminobutyric acid (GABA) is considered as a potential candidate substance that mediates the effects of intestinal bacteria on human mental health. In the present study, we evaluated the effect of water-soluble cellulose acetate (WSCA), a type of cellulose ester, on fermentation and microbial profiles, and GABA production in human stool cultures prepared from fresh feces from volunteers. In addition, the GABA-producing ability of Bacteroides uniformis, which can utilize WSCA, was evaluated in a pure-culture study. All incubations were conducted anaerobically. WSCA supplementation increased (P < 0.05) acetate and propionate production and decreased (P < 0.05) the pH in human fecal cultures. WSCA significantly altered the microbiota, selectively increasing the relative abundance of B. uniformis (P < 0.05). Pure-culture study results revealed that B. uniformis produces GABA, possibly via a glutamate-dependent acid resistance system under low pH conditions. In conclusion, WSCA could be a potential prebiotic material that is fermented by intestinal bacteria and increases short-chain fatty acid and GABA production in the human gut. Bacteroides uniformis might play an important role in both WSCA degradation and GABA production in the intestine.

Cashew nut shell liquid potentially mitigates methane emission from the feces of Thai native ruminant livestock by modifying fecal microbiota.

The methane-mitigating potency of cashew nutshell liquid (CNSL) was evaluated by investigating gas production from batch cultures using feces from Thai native ruminants that had been incubated for different periods. Feces was obtained from four Thai native cattle and four swamp buffaloes reared under practical feeding conditions at the Kasetsart University farm, Thailand. Fecal slurry from the same farm was also included in the analysis. CNSL addition successfully suppressed the methane production potential of feces from both ruminants by shifting short chain fatty acid profiles towards propionate production. Methane mitigation continued for almost 150 days, although the degree of mitigation was more apparent from Day 0 to Day 30. Bacterial and archaeal community shifts with CNSL addition were observed in feces from both ruminants; specifically, Bacteroides increased, whereas Lachnospiraceae and Ruminococcaceae decreased in feces to which CNSL was added. Fecal slurry did not show marked changes in gas production with CNSL addition. The findings showed that the addition of CNSL to the feces of ruminants native to the Southeast Asian region can suppress methane emission. Because CNSL can be easily obtained as a byproduct of the local cashew industry in this region, its on-site application might be ideal.

Effect of Bacillus subtilis C-3102 supplementation in milk replacer on growth and rumen microbiota in preweaned calves.

We aimed to assess the effect of feeding Bacillus subtilis C-3102 on the growth and rumen microbiota in the preweaned calves. Twelve newborn Japanese Black calves were randomly allocated to either the control (n = 6) or the treatment (n = 6) groups in the present study. Calves in the treatment group were offered B. subtilis C-3102 supplemented milk replacer throughout the preweaning period. Rumen fermentation during the first 21 days of life seemed to be slightly suppressed by feeding B. subtilis C-3102. This fermentation shift was probably attributed to the lower abundance of the core members of rumen microbiota until 21 days of age in the calves fed B. subtilis C-3102. However, feeding B. subtilis C-3102 did not influence the abundance of the core members of rumen microbiota at 90 days of age. Distribution of Sharpea spp. and Megasphaera spp., which potentially contribute to low methane production and are regarded as beneficial rumen bacteria, was higher in the rumen of calves fed B. subtilis C-3102 at 90 days of age. These results suggest that B. subtilis C-3102 supplementation in milk replacer could potentially contribute to the improvement of feed efficiency after weaning via the establishment of beneficial rumen bacteria.

Effect of trehalose supplementation in milk replacer on the incidence of diarrhea and fecal microbiota in preweaned calves.

Trehalose, a nonreducing disaccharide consisting of d-glucose with α,α-1,1 linkage, was evaluated as a functional material to improve the gut environment in preweaned calves. In experiment 1, 173 calves were divided into two groups; the trehalose group was fed trehalose at 30 g/animal/d with milk replacer during the suckling period, and the control group was fed nonsupplemented milk replacer. Medication frequency was lower in the trehalose group (P < 0.05). In experiment 2, calves (n = 20) were divided into two groups (control group [n = 10] and trehalose group [n = 10]) based on their body weight and reared under the same feeding regimens as in experiment 1. Fresh feces were collected from individual animals at the beginning of the trial (average age 11 d), 3 wk after trehalose feeding (experimental day 22), and 1 d before weaning, and the fecal score was recorded daily. Fecal samples were analyzed for fermentation parameters and microbiota. The fecal score was significantly lower in the trehalose group than in the control group in the early stage (at an age of 14 to 18 d; P < 0.05) of the suckling period. Calves fed trehalose tended to have a higher proportion of fecal butyrate on day 22 than calves in the control group (P = 0.08). Population sizes of Clostridium spp. were significantly lower (P = 0.036), whereas those of Dialister spp. and Eubacterium spp. tended to be higher in the feces of calves in the trehalose group on day 22 (P = 0.060 and P = 0.083). These observations indicate that trehalose feeding modulated the gut environment and partially contributed to the reduction in medication frequency observed in experiment 1.

Microbial community structure of the bovine rumen as affected by feeding cashew nut shell liquid, a methane-inhibiting and propionate-enhancing agent.

The effect of cashew nut shell liquid (CNSL) feeding on bacterial and archaeal community of the bovine rumen was investigated by analyzing clone libraries targeting 16S rRNA genes, methyl-coenzyme reductase A-encoding genes (mcrA), and their respective transcripts. Rumen samples were collected from three non-lactating cows fed on a hay and concentrate diet with or without CNSL supplementation. DNA and complementary DNA (cDNA) libraries were generated for investigating rumen microbial communities. MiSeq analysis also was performed to understand more comprehensively the changes in the microbial community structures. Following CNSL supplementation, the number of operational taxonomical unit (OTU) and diversity indices of bacterial and archaeal community were decreased. Bacterial OTUs belonging to Proteobacteria, including Succinivibrio, occurred at a higher frequency with CNSL feeding, especially in cDNA libraries. The methanogenic archaeal community became dominated by Methanomicrobium. A bacterial community shift also was observed in the MiSeq data, indicating that CNSL increased the proportion of Succinivibrio and other genera known to be involved in propionate production. Methanogenic archaeal community shifts to increase Methanoplanus and to decrease Methanobrevibacter also were observed. Together, these results imply the occurrence of significant changes in rumen communities, not only for bacteria but also for methanogens, following CNSL feeding.

The bio-surfactant mannosylerythritol lipid acts as a selective antibacterial agent to modulate rumen fermentation.

Methyl-mannosylerythritol lipid (MEL), a new sugar esterified lipid synthesized by Pseudozyma aphidis, was assessed for its functionality in modulating rumen fermentation and microbiota toward more propionate and less methane production. A pure culture study using rumen representatives showed that MEL selectively inhibited the growth of most Gram-positive bacteria including Streptococcus bovis, ruminococci, and Fibrobacter succinogenes, but not Gram-negative bacteria such as Megasphaera elsdenii, Succinivibrio dextrinosolvens, and Selenomonas ruminantium. A batch culture study revealed that MEL significantly decreased methane production in a dose-dependent manner with accumulation of hydrogen, while propionate production was enhanced. A continuous culture (Rusitec) study confirmed all of these changes. A feeding study revealed that sheep fed a MEL diet showed an increased proportion of propionate, while proportions of acetate and butyrate were decreased without affecting total VFA level. These changes disappeared after cessation of MEL feeding. Based on these results, dietary application of MEL can favorably modify rumen fermentation in terms of the efficiency of dietary energy utilization.

Selection of plant oil as a supplemental energy source by monitoring rumen profiles and its dietary application in Thai crossbred beef cattle.

Objective: The present study was conducted to select a plant oil without inhibitory effects on rumen fermentation and microbes, and to determine the optimal supplementation level of the selected oil in a series of in vitro studies for dietary application. Then, the selected oil was evaluated in a feeding study using Thai crossbred beef cattle by monitoring growth, carcass, blood and rumen characteristics. Methods: Rumen fluid was incubated with substrates containing one of three different types of plant oil (coconut oil, palm oil and soybean oil) widely available in Thailand. The effects of each oil on rumen fermentation and microbes were monitored and the oil without a negative influence on rumen parameters was selected. Then, the dose-response of rumen parameters to various levels of the selected palm oil was monitored to determine a suitable supplementation level. Finally, an 8-month feeding experiment with the diet supplemented with palm oil was carried out using 12 Thai crossbred beef cattle to monitor growth, carcass, rumen and blood profiles. Results: Batch culture studies revealed that coconut and soybean oils inhibited the most potent rumen cellulolytic bacterium Fibrobacter succinogenes, while palm oil had no such negative effect on this and on rumen fermentation products at 5% or higher supplementation level. Cattle fed the diet supplemented with 2.5% palm oil showed improved feed conversion ratio (FCR) without any adverse effects on rumen fermentation. Palm oil-supplemented diet increased blood cholesterol levels, suggesting a higher energy status of the experimental cattle. Conclusion: Palm oil had no negative effects on rumen fermentation and microbes when supplemented at levels up to 5% in vitro. Thai crossbred cattle fed the palm oil-supplemented diet showed improved FCR without apparent changes of rumen and carcass characteristics, but with elevated blood cholesterol levels. Therefore, palm oil can be used as a beneficial energy source.

Rumen responses to dietary supplementation with cashew nut shell liquid and its cessation in sheep.

Rumen responses to cashew nut shell liquid (CNSL) were evaluated in a feeding study. Four wethers were fed a hay and concentrate diet for 4 weeks (pre-CNSL period), and then fed the same diet supplemented with low and high levels of CNSL for 2 weeks each (L-CNSL and H-CNSL periods respectively). The diet was then reverted to the unsupplemented control diet for another 2 weeks (post-CNSL period). Rumen parameters were monitored in each feeding period. CNSL, regardless of the two levels tested, did not show any adverse effects on total short chain fatty acid concentration and dry matter digestibility in the rumen. Propionate proportion increased in the H-CNSL period, while methane production potential, acetate and butyrate proportions, viscosity, foam formation and its stability, and ammonia concentration decreased. Values of these parameters returned to those in the unsupplemented control period after cessation of CNSL supplementation. Clone library analysis of 16S rRNA genes revealed the following shifts in the H-CNSL period. For bacteria, Firmicutes was frequently detected, while Bacteroidetes and Spirochetes were not. For archaea, Methanobrevibacter wolinii was predominant. These results indicate that CNSL could be a methane inhibitor and propionate enhancer by altering the rumen microbial community.

Effect of ginkgo extract supplementation on in vitro rumen fermentation and bacterial profiles under different dietary conditions.

Ginkgo extract was applied to a batch culture study and evaluated for its potential as a feed additive for ruminant animals under different forage-to-concentrate (F:C) ratios (1:9, 3:7, 5:5, 7:3 and 9:1). Rumen fluid was mixed with respective diet and incubated at 39°C for 24 h with and without ginkgo extract (1.6% fruit equivalent in culture). Methane production was significantly decreased by ginkgo extract, with the greatest reductions found in the 5:5 (41.9%) followed by the 7:3 ratios (36.7%). Total short chain fatty acid and ammonia levels were not affected by ginkgo extract supplementation in any of the five different diets. However, ginkgo extract increased propionate proportion and decreased acetate proportion in all dietary conditions tested. The levels of total bacteria, Ruminococcus flavefaciens, Ruminococcus albus and Fibrobacter succinogenes were decreased by ginkgo extract. The levels of Selenomonas ruminantium, Anaerovibrio lipolytica, Ruminobacter amylophilus, Succinivibrio dextrinosolvens and Megasphaera elsdenii were increased by ginkgo extract supplementation, possibly contributing to the higher propionate production. These results suggest that rumen modulation by ginkgo extract can be achieved at a wide range of F:C ratios with no adverse impact on feed digestion. Moreover, F:C ratios of 5:5 and 7:3 may be optimal when methane mitigation is expected.

Effect of soybean husk supplementation on the fecal fermentation metabolites and microbiota of dogs.

In vitro fermentation and in vivo feeding experiments were conducted to characterize the effects of soybean (Glycine max) husk on the fecal fermentation metabolites and microbiota of dogs. An in vitro fermentation study using feces from three Toy Poodle dogs (6.5 ± 3.5 months in age and 2.9 ± 0.4 kg in body weight) revealed that the fecal inoculum was able to ferment soybean husk (supplemented at 0.01 g/mL culture) and increased levels of short chain fatty acids (SCFA) and Bifidobacterium, irrespective of pre-digestion of the husk by pepsin and pancreatin. In a feeding experiment, four Shiba dogs (7-48 months in age and 7.5 ± 1.7 kg in body weight) fed a commercial diet supplemented with 5.6% soybean husk showed an increase in SCFA, such as acetate and butyrate, and lactate, and a decrease in indole and skatole in the feces compared to those fed a 5.6% cellulose diet. Real-time PCR assay showed that soybean husk supplementation stimulated the growth of lactobacilli, Clostridium cluster IV including Faecalibacterium prausnitzii, Clostridium cluster XIVa, Bacteroides-Prevotella-Porphyromonas group but inhibited the growth of Clostridium cluster XI. Both in vitro and in vivo experiments indicated that soybean husk supplementation improves gastrointestinal health through optimization of beneficial organic acid production and increase of beneficial bacteria. Therefore, soybean husk is suggested to be applicable as a functional fiber in the formulation of canine diets.

Effect of chickpea husk dietary supplementation on blood and cecal parameters in rats.

Chickpea husk was functionally evaluated for antioxidant status, blood parameters, cecal fermentation and microbial profiles in rats. Fifteen male rats (5 weeks of age) were divided into three groups; they were individually housed and fed one of the following diets for 3 weeks: purified diet containing 5% cellulose (Cellulose), an identical diet in which cellulose was replaced by corn starch (Starch) or by chickpea husk (Chick). Rats were sacrificed to obtain blood and cecal digesta samples. Chickpea husk contained high polyphenolic content and significant superoxide dismutase and 2,2-diphenyl-picrylhydrazyl scavenging activities. In a feeding experiment, Chick showed lowered cholesterol levels and improved antioxidant activity represented by reduced thiobarbituric acid reactive substances in blood. Chick showed increased cecal levels of total short chain fatty acids and butyrate, leading to a lower pH. Chick presented with lowered cecal indole and skatole concentrations, as did Cellulose. Cecal bacterial changes were notable in Chick, evidenced by differences in denaturing gradient gel electrophoresis banding patterns. However, representative bacteria quantified by real-time PCR assay did not support this bacterial change. These results indicate that chickpea husk feeding can improve the antioxidative status of rats through its polyphenolic components and modulate the hindgut environment by its fibrous components.

Use of Asian selected agricultural byproducts to modulate rumen microbes and fermentation.

In the last five decades, attempts have been made to improve rumen fermentation and host animal nutrition through modulation of rumen microbiota. The goals have been decreasing methane production, partially inhibiting protein degradation to avoid excess release of ammonia, and activation of fiber digestion. The main approach has been the use of dietary supplements. Since growth-promoting antibiotics were banned in European countries in 2006, safer alternatives including plant-derived materials have been explored. Plant oils, their component fatty acids, plant secondary metabolites and other compounds have been studied, and many originate or are abundantly available in Asia as agricultural byproducts. In this review, the potency of selected byproducts in inhibition of methane production and protein degradation, and in stimulation of fiber degradation was described in relation to their modes of action. In particular, cashew and ginkgo byproducts containing alkylphenols to mitigate methane emission and bean husks as a source of functional fiber to boost the number of fiber-degrading bacteria were highlighted. Other byproducts influencing rumen microbiota and fermentation profile were also described. Future application of these feed and additive candidates is very dependent on a sufficient, cost-effective supply and optimal usage in feeding practice.

Biochemical mechanism on GABA accumulation during fruit development in tomato.

A large amount of gamma-aminobutyric acid (GABA) was found to accumulate in tomato (Solanum lycopersicum) fruits before the breaker stage. Shortly thereafter, GABA was rapidly catabolized after the breaker stage. We screened the GABA-rich tomato cultivar 'DG03-9' which did not show rapid GABA catabolism after the breaker stage. Although GABA hyperaccumulation and rapid catabolism in fruits is well known, the mechanisms are not clearly understood. In order to clarify these mechanisms, we performed comparative studies of 'Micro-Tom' and 'DG03-9' fruits for the analysis of gene expression levels, protein levels and enzymatic activity levels of GABA biosynthesis- and catabolism-related enzymes. During GABA accumulation, we found positive correlations among GABA contents and expression levels of SlGAD2 and SlGAD3. Both of these genes encode glutamate decarboxylase (GAD) which is a key enzyme of GABA biosynthesis. During GABA catabolism, we found a strong correlation between GABA contents and enzyme activity of alpha-ketoglutarate-dependent GABA transaminase (GABA-TK). The contents of glutamate and aspartate, which are synthesized from GABA and glutamate, respectively, increased with elevation of GABA-TK enzymatic activity. GABA-TK is the major GABA transaminase form in animals and appears to be a minor form in plants. In 'DG03-9' fruits, GAD enzymatic activity was prolonged until the ripening stage, and GABA-TK activity was significantly low. Taken together, our results suggest that GAD and GABA-TK play crucial roles in GABA accumulation and catabolism, respectively, in tomato fruits.