• 미생물학회지
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• 제43권2호
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• pp.111-115
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• 2007

반추위에서 분리한 곰팡이(IS-13)의 conjugated linoleic acid 생산 과정을 조사하고conjugated linoleic acid를 생산하는 곰팡이를 동정하기 위하여 본 연구를 실시하였다. IS-13 곰팡이가 함유된 배양액에 linoleic acid를 첨가한 결과 linoleic acid는 배양 12시간 이내에 conjugated linoleic acid와 trans-11 vaccenic acid로 biohydrogenation되었다. IS-13 곰팡이의 동정은 internal transcribed spacer 1 영역(ITS1)을 sequence하여 GenBank 유래의 관련 23속(종)의 반추위 곰팡이와 비교하였다. IS-13 곰팡이의 ITS1의 length는 218 bp인 것으로 확인되었으며, 염기서열 분석 결과 Orpinomyces species와 98% 일치하여 반추위내 Orpinomyces species는 conjugated linoleic acid 생산에 깊이 관여하는 것으로 확인되었다. 연구결과 본 균주는 Orpinomyces 속에 속하는 균으로 동정되었다.

• Asian-Australasian Journal of Animal Sciences
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• 제20권11호
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• pp.1694-1698
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• 2007

The objective of this study was to identify biohydrogenation pathways for linoleic, linolenic, oleic and stearic acids by Orpinomyces species of rumen fungus during in vitro culture. Biohydrogenation of linoleic acid produced conjugated linoleic acid (cis-9, trans-11 C18:2), which was then converted to vaccenic acid (trans-11 C18:1) as the end product of biohydrogenation. Biohydrogenation of linolenic acid produced cis-9, trans-11, cis-15 C18:3 and trans-11, cis-15 C18:2 as intermediates and vaccenic acid as the end product of biohydrogenation. Oleic acid and stearic acid were not converted to any other fatty acid. It is concluded that pathways for biohydrogenation of linoleic and linolenic acids by Orpinomyces are the same as those for group A rumen bacteria.

• Asian-Australasian Journal of Animal Sciences
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• 제14권8호
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• pp.1110-1117
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• 2001

Responses of the rumen fungus, Neocallimastix frontalis RE1, to long chain fatty acid (LCFA) were evaluated by measuring gas production, filter paper (FP) cellulose digestion and polysaccharidase enzyme activities. LCFA (stearic acid, $C_{18:0}$; oleic acid, $C_{18:1}$; linoleic acid, $C_{18:2}$ and linolenic acid, $C_{18:3}$) were emulsitied by ultrasonication under anaerobic condition, and added to the medium. When N frontalis RE1 was grown in culture with stearic, oleic and linoleic acid, the cumulative gas production, gas pool size, FP cellulose digestion and enzymes activities significantly (p<0.05) increased at some incubation times(especially, exponential phases of fungal growth, 48~120 h of incubation) relative to that for control cultures. However, the addition of linolenic acid strongly inhibited all of the investigated parameters up to 120 h incubation, but not after 168 and 216 h of incubation. These results indicated that stearic, oleic and linoleic acids tended to have great stimulatory effects on fungal cellulolysis, whereas linolenic acid caused a significant (p<0.05) inhibitory effects on the cellulolysis by the rumen fungus. These results are the first report of the effect of LCFAs on the ruminal fungi. Further research is needed to identify the mode of action of LCFAs on fungal strains and to verify whether or not ruminal fungi have ability to hydrate unsaturated LCFAs to saturated FAs. There was high correlation between cumulative in vitro gas production and fungal growth (94.78%), FP cellulose degradation (96.34%), CMCase activity(90.86%) or xylanase activity (87.67%). Thus measuring of cumulative gas production could be a useful tool for evaluating fungal growth and/or enzyme production by ruminal fungi.

• Asian-Australasian Journal of Animal Sciences
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• 제14권7호
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• pp.941-946
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• 2001

The effects of various concentrations of saturated fatty acids (SFA; caprylic, capric and stearic acids) on the growth of the anaerobic fungus, Neocallimastix frontalis C5-1 isolated from the rumen of a Korean native goat were investigated. At higher concentrations of fatty acids (0.1%, w/v), the addition of SFA strongly decreased filter paper (FP) cellulose digestion and polysaccharide-degrading enzyme activity. The sensitivity of the rumen anaerobic fungus to the added fatty acids increased in the following order: caprylic ($C_{8:0}$)>capric($C_{10:0}$)>stearic($C_{18:0}$) acid, although stearic acid had no significant (p<0.05) inhibitory effects at any of the concentrations tested. However, the addition of SFA at lower concentrations (0.01 and 0.001% levels), did not inhibit FP cellulose degradation and enzyme activity. Furthermore, although these parameters were slightly stimulated by the addition of SFA, they were not statistically different from control values. This is the first report examining the effects of fatty acids on anaerobic gut fungi. We found that the lower levels of fatty acids used in this experiment were able to stimulate the growth and specific enzyme activities of rumen anaerobic fungi, whereas the higher levels of fatty acids were inhibitory with respect to fungal cellulolysis.

• Asian-Australasian Journal of Animal Sciences
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• 제12권5호
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• pp.752-757
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• 1999

Studies were made of digestion of timothy (Pheleum pretense) hay, tall fescue (Festuca elatior) hay, and rice (Oryza sativa) straw in pure cultures of rumen anaerobic fungus, Neocallimastix frontails MCH3. The fungus was inoculated on ground forages (1%, w/v) in an anaerobic medium and incubated at $39^{\circ}C$. Incubation was continued for 24, 48, 72 and 96 h. The losses of dry matter, xylose and glucose of forage during incubation were determined at the end of these incubation periods. Xylose and glucose were considered to be released from xylan and cellulose, respectively. The digested xylan to digested cellulose (X/C) ratios of the substrate were calculated. Xylanase and carboxymethyl cellulose (CMCase) of culture supernatant and residual substrate was measured at the same time. The X/C ratios in the cultures on timothy hay and rice straw were greater than 0.5 in the first 24-h incubation period. The values were smaller than 0.3 in tall fesque. The ratio of xylanase activity to that of CMCase in the first 24-h incubation period correlated well with the traits in X/C ratio. However xylanase activity was still superior to CMCase in the following incubation period (48 to 96 h), although the glucose (designated as cellulose) was more intensively digested than xylose (designated as xylan). The production of these polysaccharidases appeared to correlate with substrate cell-wall sugar composition, xylose to glucose ratios, at the beginning of fast growing period.

• Asian-Australasian Journal of Animal Sciences
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• 제22권11호
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• pp.1555-1565
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• 2009

Anaerobic rumen fungi have been regarded as good genetic resources for enzyme production which might be useful for feed supplements, bio-energy production, bio-remediation and other industrial purposes. In this study, an expressed sequence tag (EST) library of the rumen anaerobic fungus Neocallimastix frontalis was constructed and functional genes from the EST library were analyzed to elucidate carbohydrate metabolism of anaerobic fungi. From 10,080 acquired clones, 9,569 clones with average size of 628 bp were selected for analysis. After the assembling process, 1,410 contigs were assembled and 1,369 sequences remained as singletons. 1,192 sequences were matched with proteins in the public data base with known function and 693 of them were matched with proteins isolated from fungi. One hundred and fifty four sequences were classified as genes related with biological process and 328 sequences were classified as genes related with cellular components. Most of the enzymes in the pathway of glucose metabolism were successfully isolated via construction of 10,080 ESTs. Four kinds of hemi-cellulase were isolated such as mannanase, xylose isomerase, xylan esterase, and xylanase. Five $\beta$-glucosidases with at least three different conserved domain structures were isolated. Ten cellulases with at least five different conserved domain structures were isolated. This is the first solid data supporting the expression of a multiple enzyme system in the fungus N. frontalis for polysaccharide hydrolysis.

• Asian-Australasian Journal of Animal Sciences
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• 제29권11호
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• pp.1576-1584
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• 2016

Acetyl xylan esterase (AXE), which hydrolyzes the ester linkages of the naturally acetylated xylan and thus known to have an important role for hemicellulose degradation, was isolated from the anaerobic rumen fungus Neocallimastix frontatlis PMA02, heterologously expressed in Escherichi coli (E.coli) and characterized. The full-length cDNA encoding NfAXE1 was 1,494 bp, of which 978 bp constituted an open reading frame. The estimated molecular weight of NfAXE1 was 36.5 kDa with 326 amino acid residues, and the calculated isoelectric point was 4.54. The secondary protein structure was predicted to consist of nine ${\alpha}$-helixes and 12 ${\beta}$-strands. The enzyme expressed in E.coli had the highest activity at $40^{\circ}C$ and pH 8. The purified recombinant NfAXE1 had a specific activity of 100.1 U/mg when p-nitrophenyl acetate (p-NA) was used as a substrate at $40^{\circ}C$, optimum temperature. The amount of liberated acetic acids were the highest and the lowest when p-NA and acetylated birchwood xylan were used as substrates, respectively. The amount of xylose released from acetylated birchwod xylan was increased by 1.4 fold when NfAXE1 was mixed with xylanase in a reaction cocktail, implying a synergistic effect of NfAXE1 with xylanase on hemicellulose degradation.

• Asian-Australasian Journal of Animal Sciences
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• 제24권3호
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• pp.379-385
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• 2011

Ferulic acid esterase (FAE) and acetyl esterase (AE) cleave feruloyl groups substituted at the 5'-OH group of arabinosyl residues and acetyl groups substituted at O-2/O-3 of the xylan backbone, respectively, of arabinoxylans in the cell wall of grasses. In this study, the enzyme profiles of FAE, AE and polysaccharide hydrolases of the anaerobic rumen fungus Neocallimastix sp. YQ1 grown on Chinese wildrye grass hay (CW) or alfalfa hay (AH) were investigated by two $2{\times}4$ factorial experiments, each in 10-day pure cultures. The treatments consisted of two glucose levels ($G^+$: glucose at 1.0 g/L, $G^-$: no glucose) and four N sources (N1: 1.0 g/L yeast extract, 1.0 g/L tryptone and 0.5 g/L $(NH_4)_2SO_4$; N2: 2.8 g/L yeast extract and 0.5 g/L $(NH_4)_2SO_4$; N3: 1.6 g/L tryptone and 0.5 g/L $(NH_4)_2SO_4$; N4: 1.4 g/L tryptone and 1.7 g/L yeast extract) in defined media. The optimal combinations of glucose level and N source for the fungus on CW, instead of AH, were $G^-N4$ and $G^-N3$ for maximum production of FAE and AE, respectively. Xylanase activity peaked on day 4 and day 6 for the fungus grown on CW and AH, respectively. The activities of esterases were positively correlated with those of xylanase and carboxymethyl cellulase. The fungus grown on CW exhibited a greater volatile fatty acid production than on AH with a greater release of ferulic acid from plant cell wall.

• Animal Bioscience
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• 제37권4호
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• pp.655-667
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• 2024

Objective: This study aimed to assess the impact of a hydroethanolic extract of walnut green husks (WGH) on rumen fermentation and the diversity of bacteria, methanogenic archaea, and fungi in sheep fed a high-concentrate diet. Methods: Five healthy small-tailed Han ewes with permanent rumen fistula were selected and housed in individual pens. This study adopted a self-controlled and crossover design with a control period and an experimental period. During the control period, the animals were fed a basal diet (with a ratio of concentrate to roughage of 65:35), while during the treatment period, the animals were fed the basal diet supplemented with 0.5% hydroethanolic extract of WGH. Fermentation parameters, digestive enzyme activities, and microbial diversity in rumen fluid were analyzed. Results: Supplementation of hydroethanolic extract of WGH had no significant effect on feed intake, concentrations of total volatile fatty acids, isovalerate, ammonia nitrogen, and microbial protein (p>0.05). However, the ruminal pH, concentrations of acetate, butyrate and isobutyrate, the ratio of acetate to propionate, protozoa count, and the activities of filter paper cellulase and cellobiase were significantly increased (p<0.05), while concentrations of propionate and valerate were significantly decreased (p<0.05). Moreover, 16S rRNA gene sequencing revealed that the relative abundance of rumen bacteria Christensenellaceae R7 group, Saccharofermentans, and Ruminococcaceae NK4A214 group were significantly increased, while Ruminococcus gauvreauii group, Prevotella 7 were significantly decreased (p<0.05). The relative abundance of the fungus Pseudomonas significantly increased, while Basidiomycota, Fusarium, and Alternaria significantly decreased (p<0.05). However, there was no significant change in the community structure of methanogenic archaea. Conclusion: Supplementation of hydroethanolic extract of WGH to a high-concentrate diet improved the ruminal fermentation, altered the structure of ruminal bacterial and fungal communities, and exhibited beneficial effects in alleviating subacute rumen acidosis of sheep.

• Asian-Australasian Journal of Animal Sciences
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• 제9권3호
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• pp.303-307
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• 1996

The degree of autolysis and presence of cell-wall degrading enzymes in an anaerobic ruminal fungus, Piromyces communis OTSI, grown in liquid medium, was monitored to evaluate the effect of self-digestion on fungal biomass. After a 30 days incubation period fungal dry weight decreased by 45% and the cell wall component chitin decreased by 22%. Chitinase activity detected in the supernatant was mainly of the endotype and peaked at day 6 of the incubation. ${\beta}-1$, 3-glucanase was detected from day 4 and increased throughout the incubation period. Autolysis was a slow process, and under natural conditions it is unlikely that it plays a significant role in the degradation of the spent fungal vegetative stage in the rumen.