Mycotoxins are secondary metabolites produced by fungi. These toxins pose serious health concerns to animals as well as human beings. Biodegradation of these mycotoxins has been considered as one of the best strategies to decontaminate food and feedstuffs. Biodegradation employs the application of microbes or enzymes to contaminated food and feedstuffs. Ruminants are considered to be resistant to the adverse effects of mycotoxins presumably due to the biodegrading ability of rumen microbes compared to mono-gastric animals. Therefore, rumen microbial source or microbial enzyme could be a great asset in biological detoxification of mycotoxins. Isolation and characterization of pure culture of rumen microorganisms or isolation and cloning of genes encoding mycotoxin-degrading potential would prove to have overall beneficial impact in the food and feed industry.
Influences of plant phenolic acids and their possible metabolites(non-phenolic aromatic acids involved) in the rumen on the cellulolytic activity of mixed rumen populations were examined by a simple in vitro culture technique. Initial concentrations of aromatic acids were 1, 5, 10 and 20 mM/l. All the tested aromatic acids reduced microbial cellulose digestion especially at the higher initial concentration. P-Coumaric acid, ferulic acid and cinnamic acid, those having unhydrogenated propenoic side chain were more inhibitory than were 3-phenylpropinic acid and phloretic acid, those having hydrogenated propanoic side chain. Lag-time for cellulose digestion was prolonged by former three acids by 16 h. Apparent reduction in p-coumaric acid concentration was observed at 24 h when cellulose digestion began. Volatile fatty acid productions from cellulose fermentation were shifted by former three aromatic acids to produce more acetate and less propionate. This suggests that the selection of celluloytic organisms was induced by these aromatic acids.
Effect of a surfactant Tween 80 on the bacterial growth in the rumen was examined on the in vitro pure cultures of Streptococcus bovis, Selenomonas ruminantium, Butyrivibrio fibrisolvens, Prevotella ruminicola, Megasphaera elsidenni, Fibrobacta succinogenes, Ruminanococcus albus and Ruminococcus flavefaciens. Dry matter degradability (DMD), concentrations and compositions of volatile fatty acids (VFA), and the most probable number (MPN) of cellulolytic bacteria and total number of bacteria in the presence of Tween 80 were also examined on the in vitro rumen mixed culture either with barley grain or orchardgrass hay. The growth of S. bovis, S. ruminantium, B. fibrisolvens, P. ruminicola, M. elsidenni and F. succinogenes were significantly higher (p<0.05) at over 0.05% concentrations of Tween 80 than those of the control cultures, while was not changed with R. albus and R. flavefaciens. With rumen mixed culture the DMD of barley grain and orchardgrass hay was significantly higher (p<0.05) at a 0.2% concentration of Tween 80 than the control, being reflected in the significantly higher (p<0.05) VFA production (mmol $g^{-1}$DDM) with orchardgrass hay. The higher (p<0.05) ratio of propionate to acetate at a 0.2% concentration of Tween 80 was also observed with orchardgrass hay, showing a similar trend with barley grain. No changes in the total bacterial number and MPN of cellulolytic bacteria were observed.
An in vitro experiment was conducted to evaluate the effects of Aspergillus oryzae culture (AOC) and 2-hydroxy-4-(methylthio)-butanoic acid (HMB) on rumen fermentation and microbial populations between different roughage sources. Two roughage sources (Chinese wild rye [CWR] vs corn silage [CS]) were assigned in a $2{\times}3$ factorial arrangement with HMB (0 or 15 mg) and AOC (0, 3, or 6 mg). Gas production (GP), microbial protein (MCP) and total volatile fatty acid (VFA) were increased in response to addition of HMB and AOC (p<0.01) for the two roughages. The HMB and AOC showed inconsistent effects on ammonia-N with different substrates. For CWR, neither HMB nor AOC had significant effect on molar proportion of individual VFA. For CS, acetate was increased (p = 0.02) and butyrate was decreased (p<0.01) by adding HMB and AOC. Increase of propionate was only occurred with AOC (p<0.01). Populations of protozoa ($p{\leq}0.03$) and fungi ($p{\leq}0.02$) of CWR were differently influenced by HMB and AOC. Percentages of F. succinogenes, R. albus, and R. flavefaciens (p<0.01) increased when AOC was added to CWR. For CS, HMB decreased the protozoa population (p = 0.01) and increased the populations of F. succinogenes and R. albus ($p{\leq}0.03$). Populations of fungi, F. succinogenes (p = 0.02) and R. flavefacien (p = 0.03) were increased by adding AOC. The HMB${\times}$AOC interactions were noted in MCP, fungi and R. flavefacien for CWR and GP, ammonia-N, MCP, total VFA, propionate, acetate/propionate (A/P) and R. albus for CS. It is inferred that addition of HMB and AOC could influence rumen fermentation of forages by increasing the number of rumen microbes.
An in vitro study was conducted to examine the effect of monensin or fish oil addition on bio-hydrogenation of $C_{18^-} unsaturated fatty acids and CLA production by mixed ruminal bacteria when incubated with safflower oil. Commercially manufactured concentrate (1%, w/v) with safflower oil (0.2%, w/v) were added to mixed solution (600 ml) of strained rumen fluid and McDougalls artificial saliva (control). Monensin $Rumensin^{(R)}$, 10 ppm, w/v, MO), mixed fish oil (0.02%, w/v, absorbed to 0.2 g alfalfa hay, FO) or similar amounts of monensin and fish oil (MO+FO) to MO and FO was also added into the control solution. All the culture solutions prepared were incubated in the culture jar anaerobically at $39^{\circ}C$ up to 12 h. Higher pH (p<0.047) and ammonia concentration (p<0.042) were observed from the culture solution containing MO at 12 h incubation than those from the culture solutions of control or FO. The MO supplementation increased (p<0.0001-0.007) propionate proportion of culture solution but reduced butyrate proportion at 6 h (p<0.018) and 12 h (p<0.001) of incubations. Supplementation of MO or MO+FO increased (p<0.001) the proportions of $C_{18:2}$. The MO alone reduced (p<0.022-0.025) the proportion of c9,t11-CLA compared to FO in all incubation times. The FO supplementation increased the proportion of c9,t11-CLA. An additive effect of MO to FO in the production of c9,t11-CLA was observed at 6 h incubation. In vitro supplementation of monensin reduced hydrogenation of $C_{18^-}$UFAs while fish oil supplementation increased the production of CLA.
Nitrate can serve as a terminal electron acceptor in place of carbon dioxide and inhibit methane emission in the rumen and nitrate reducing bacteria might help enhance the reduction of nitrate/nitrite, which depends on the type of feed offered to animals. In this study the effects of three levels of sodium nitrate (0, 5, 10 mM) on fermentation of three diets varying in their wheat straw to concentrate ratio (700:300, low concentrate, LC; 500:500, medium concentrate, MC and 300:700, high concentrate, HC diet) were investigated in vitro using buffalo rumen liquor as inoculum. Nitrate reducing bacteria, isolated from the rumen of buffalo were tested as a probiotic to study if it could help in enhancing methane inhibition in vitro. Inclusion of sodium nitrate at 5 or 10 mM reduced (p<0.01) methane production (9.56, 7.93 vs. 21.76 ml/g DM; 12.20, 10.42 vs. 25.76 ml/g DM; 15.49, 12.33 vs. 26.86 ml/g DM) in LC, MC and HC diets, respectively. Inclusion of nitrate at both 5 and 10 mM also reduced (p<0.01) gas production in all the diets, but in vitro true digestibility (IVTD) of feed reduced (p<0.05) only in LC and MC diets. In the medium at 10 mM sodium nitrate level, there was 0.76 to 1.18 mM of residual nitrate and nitrite (p<0.01) also accumulated. In an attempt to eliminate residual nitrate and nitrite in the medium, the nitrate reducing bacteria were isolated from buffalo adapted to nitrate feeding and introduced individually (3 ml containing 1.2 to $2.3{\times}10^6$ cfu/ml) into in vitro incubations containing the MC diet with 10 mM sodium nitrate. Addition of live culture of NRBB 57 resulted in complete removal of nitrate and nitrite from the medium with a further reduction in methane and no effect on IVTD compared to the control treatments containing nitrate with autoclaved cultures or nitrate without any culture. The data revealed that nitrate reducing bacteria can be used as probiotic to prevent the accumulation of nitrite when sodium nitrate is used to reduce in vitro methane emissions.
Ethanol was utilized by mixed rumen microbes, but addition of pentachlorophenol (25 mg/l), a methanogen inhibitor, suppressed the utilization of ethanol. Carbon monoxide (50% of the gas phase), a hydrogenase inhibitor, more strongly suppressed the utilization of ethanol, propanol, and butanol. These results suggest that the major ethanol utilizers are $H_2$ producers. Ethanol utilization was depressed at low pH (below 6.0). Since methanogens were shown to be relatively resistant to low pH, it appears that ethanol utilizers are particularly sensitive to low pH. Ruminococcus albus and R. flavefaciens in mono-culture produced ethanol from carbohydrate (glucose and cellobiose), even when a high level (170 mM) of ethanol was present. Ethanol was not utilized even in the absence of carbohydrate, but the co-culture of these bacteria with methanogens resulted in the utilization of ethanol, i.e., when $H_2$ was rapidly converted to $CH_4$, R. albus and R. flavefaciens utilized ethanol. These results suggest that ethanol is utilized when the electrons liberated by the oxidation of ethanol are rapidly removed, and ready electron disposal in ethanol-utilizing, $H_2$-producing bacteria is accomplished by the interspecies transfer of $H_2$.
The effect of different feeding ratios of concentrate to roughage on ruminal lipid synthesis in vitro was examined. Three sheep fitted with a rumen fistula were fed three different ratios (8:2, 4:6 and 0:10) of concentrate and roughage, and their rumen liquor were used for incubation. $^{13}C$-labeled glucose or sodium acetate as substrate was added to cultures of rumen liquor, and they were incubated for 6 h. The total lipid in the culture of the rumen liquor was extracted, and the percentage of $^{13}C$ excess was analyzed. The percentage of $^{13}C$ excess recovered when incubated with glucose increased with increased ratio of concentrate in the diet. The values of cultures incubated with glucose were higher than those incubated with sodium acetate except the roughage-only feeding. In the roughage-only diet, the percentage of $^{13}C$ excess when incubated with sodium acetate was highest of all diets. The recovery percentage of $^{13}C$ from glucose increased with increased ratio of concentrate. The recovery percentage of $^{13}C$ from sodium acetate addition in only roughage feeding was highest among the three diets. The recovery percentage of $^{13}C$ from glucose was markedly higher than that of sodium acetate addition in all feedings. The results indicate that high concentrate feeding facilitates lipid synthesis by rumen microorganisms, and that glucose may be the precursor for lipid synthesis rather than acetic acid.
A series of experiments was carried out to determine the possibility for the non-ionic surfactant (NIS) as a feed additive for ruminant animals. The effect of the NIS on (1) the enzyme distribution in the rumen fluids of Hereford bulls, (2) the growth of pure culture of rumen bacteria and (3) rumen anaerobic fungi, (4) the ruminal fermentation characteristics of Korean native cattle (Hanwoo), and (5) the performances of Holstein dairy cows were investigated. When NIS was added to rumen fluid at the level of 0.05 and 0.1% (v/v), the total and specific activities of cell-free enzymes were significantly (p<0.01) increased, but those of cell-bound enzymes were slightly decreased, but not statistically significant. The growth rates of ruminal noncellulolytic species (Ruminobacter amylophilus, Megasphaera elsdenii, Prevotella ruminicola and Selenomonas ruminantium) were significantly (p<0.01) increased by the addition of NIS at both concentrations tested. However, the growth rate of ruminal cellulolytic bacteria (Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens and Butyrivibrio fibrisolvens) were slightly increased or not affected by the NIS. In general, NIS appears to effect Gram-negative bacteria more than Gram-positive bacteria; and non-cellulolytic bacteria more than cellulolytic bacteria. The growth rates of ruminal monocentric fungi (Neocallimastix patriciarum and Piromyces communis) and polycentric fungi (Orpinomyces joyonii and Anaeromyces mucronatus) were also significantly (p<0.01) increased by the addition of NIS at all concentrations tested. When NIS was administrated to the rumen of Hanwoo, Total VFA and ammonia-N concentrations, the microbial cell growth rate, CMCase and xylanase activities in the rumen increased with statistical difference (p<0.01), but NIS administration did not affect at the time of 0 and 9 h post-feeding. Addition of NIS to TMR resulted in increased TMR intake and increased milk production by Holstein cows and decreased body condition scores. The NEFA and corticoid concentrations in the blood were lowered by the addition of NIS. These results indicated that the addition of NIS may greatly stimulate the release of some kinds of enzymes from microbial cells, and stimulate the growth rates of a range of anaerobic ruminal microorganisms, and also stimulate the rumen fermentation characteristics and animal performances. Our data indicates potential uses of the NIS as a feed additive for ruminant animals.
The present study was conducted to investigate effective starter culture to improve biological activity of Asarum sieboldii. Antibacterial activity, antioxidant activity and reduction of enteric rumen methane production were used as criterions for biological activity. Ground A. sieboldii was added in MRS broth at 10% (w/v) and fermented by different starter cultures. Weissella confusa NJ28, Weissella cibaria NJ33, Lactobacillus curvatus NJ40, Lactobacillus brevis NJ42, Lactobacillus plantarum NJ45 and Lactobacillus sakei NJ48 were used for starter culture strains. Each starter culture was inoculated with 1% (v/v) ratio and fermentation was performed at $30^{\circ}C$ with agitation (150 rpm) for 48 h. MRS broth for the control was employed without starter culture. Then the fermentation growth was dried and extracted using ethyl alcohol. The growth of starter culture was detected at NJ40, NJ42, NJ45 and NJ48. And the highest cell growth was found in NJ40. Antibacterial activity against to Staphylococcus aureus, Listeria monocytogens, Mannheimia haemolytica and Salmonella gallinarum were observed in the extract fermented by NJ40 and NJ45. All treatments showed antioxidant activities, however, there were no significant differences (p>0.05). In in vitro rumen fermentation, negative control (NC) and positive control (PC) were assigned to without extract and with non-fermented A. sieboldii extract. Significant suppression of gas productions were detected in positive control and treatments compared to negative control (p<0.05). However, total volatile fatty acid production was not suppressed. Significant methane reduction per total volatile fatty acid productions were found in positive control and NJ45 treatment (p<0.05). The present study suggested a fermentation of A. sieboldii using NJ45 strain could improve its biological activity and make possible for its use in bio additive for enteric rumen methane mitigation without suppression of animal productivity.
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