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This experiment was designed to investigate the effects of different concentrations (0.00, 0.10, 0.15, 0.20, 0.25, and 0.30 g/L) of dried Cordyceps militaris mushroom on in vitro anaerobic ruminal microbe fermentation and methane production using soluble starch as a substrate. Ruminal fluids were collected from Korean native cattle, mixed with phosphate buffer (1:2), and incubated anaerobically at 38°C for 3, 6, 9, 12, 24, 36, 48, and 72 h. The addition of C. militaris significantly increased total volatile fatty acid and total gas production. The molar proportion of acetate was decreased and that of propionate was increased, with a corresponding decrease in the acetate:propionate ratio. As the concentration of C. militaris increased from 0.10 to 0.30 g/L, methane and hydrogen production decreased. The decrease in methane accumulation relative to the control was 14.1, 22.0, 24.9, 39.7, and 40.9% for the 0.10, 0.15, 0.20, 0.25, and 0.30 g/L treatments, respectively. Ammonia-N concentration and numbers of live protozoa decreased linearly with increasing concentrations of C. militaris. The pH of the medium significantly decreased at the highest level of C. militaris compared with the control. In conclusion, C. militaris stimulated mixed ruminal microorganism fermentation and inhibited methane production in vitro. Therefore, C. militaris could be developed as a novel compound for antimethanogenesis.
Methane (CH4) produced as a result of digestible structural carbohydrate fermentation in the rumen represents 7 to 10% feed energy loss to the host animal (
Improving ruminant production and reducing methane emissions from ruminants by strategic supplementation. EPA/400/1-91/004. US Environmental Protection Agency,
Washington, DC1991
). Several compounds have the potential to reduce methane production from ruminants although their long-term effects have not been well established. Some compounds are toxic or may not be economically feasible (
Cordyceps militaris, a traditional Chinese medicinal mushroom, is an entomogenous fungus belonging to the Ascomycotina. The mushroom is traditionally called “DongChung HaCho” in Korea meaning “summer-plant and winter-worm.” During the past several decades, many kinds of bioactive constituents from Cordyceps spp. have been isolated and characterized. These include cordycepic acid (d-mannitol), cordycepin, ophicordin, polysaccharides, amino acids, galactosaminoglycan, nucleic acids, steroids, and l-tryptophan (
Rapid and specific detection of hydroxyl radical using an ultraweak chemiluminescence analyzer and a low-level chemiluminescence emitter: Application to hydroxyl radical-scavenging ability of aqueous extracts of food constituents.
) effects. Cordyceps militaris mycelia have been shown to alter in vitro rumen microbial fermentation with increased production of gas and VFA, cellulose digestion, and cellulolytic enzyme activities (
). But no information exists with respect to C. militaris modulating methane production in the rumen. Therefore, the present study was conducted to observe the effects of C. militaris on ruminal microorganism fermentation with particular reference to methane production in vitro.
Materials and Methods
Sample Preparation
Because Cordyceps are very difficult to collect due to their very small size and restricted area of growth, mass production of these fungi has been established through artificial cultivation. Dried C. militaris was cultured on floral medium composed of gluten, soybean protein, beer yeast, and corn steep liquor (culturing method and medium composition were patented in Korea, patent registration No.1006442430000;
Lee, H. G. 2006. Composition for cultivating Cordyceps and cultivating process using thereof. AJU International Law & Patent Group, assignee. Korea Pat. No. 1006442430000.
) obtained from EuGene Bio Farm (Hwaseong City, Gyeonggi Province, Korea). The manufacturer reported that C. militaris mycelia used in the present study contained about 2.3 times more cordycepin (1.6 mg/g of DM) than C. militaris traditionally cultured on faunal pupae (0.7 mg/g of DM). It contained 8.6% moisture, 76.2% CP, 12.2% crude fiber, 1.0% ether extract, 3.2% crude ash, and 7.4% nitrogen-free extract.
) except that 200 mg of soluble potato starch (S2004; Sigma-Aldrich Korea, Yongin City, Gyeonggi-do, Korea) was used as a carbon source.
Dried C. militaris was added gravimetrically to achieve final concentrations of 0.00, 0.10, 0.15, 0.20, 0.25, and 0.30 g/L. The bottles (3 replicates per treatment) were closed with butyl rubber stoppers under the Hungate anaerobic gassing system hooked to a source of oxygen-free gas, sealed with aluminum caps, and placed in an incubator at 38°C for 3, 6, 9, 12, 24, 36, 48, and 72 h without shaking. The experimental design was a complete randomized design with 3 replications per treatment.
Total, Hydrogen, and Methane Gas Production
At the end of each incubation time, a needle attached to a glass syringe was inserted through the butyl rubber stopper, and the volume of gas exceeding 1 atm was measured through displacement of the syringe plunger using the technique of
). A 0.5-mL subsample of gas was analyzed for hydrogen and methane content by GC (model CP-3800, Varian Inc., Palo Alto, CA) using a molecular sieve 13×, 45- to 60-mesh column (2.0 mm × 3.2 mm × 2.0 mm, stainless steel) and a thermal conductivity detector (oven temperature = 60°C, injector and thermal conductivity detector temperature = 120°C, flame-ionization detector temperature = 200°C). The carrier gas (N2) flow rate was 50 mL/min.
pH, NH3-N, and VFA
After determination of gas production, the bottles were uncapped, and pH of the culture fluid was determined using a pH meter (MP 230, Mettler-Toledo, Greifensee, Switzerland). For analysis of ammonia-N and VFA, 1 mL of 25% meta-phosphoric acid was added to 5 mL of fermentation fluid and centrifuged (10,000 × g for 10 min at 4°C); supernatants were stored at −30°C until analysis. Volatile fatty acids were analyzed by GC (model GC-14B, Shimadzu Co. Ltd., Tokyo, Japan) using a Thermon-3000 5% Shincarbon A column (1.6m × 3.2 mm i.d., 60 to 80 mesh, Shinwakako, Kyoto, Japan) and flame-ionization detector (column temperature = 130°C, injector and detector temperature = 200°C). The carrier gas (N2) flow rate was 50 mL/min. The micro-diffusion method was used to determine NH3-N (
Bacterial and protozoal interactions with ruminal fungi.
in: Akin D.E. Ljungdahl L.G. Wilson J.R. Harris P.J. Microbial and Plant Opportunities to Improve Lignocellulose Utilization by Ruminants. Elsevier,
New York, NY1981: 311-324
using anaerobic roll tubes. Samples were fixed in methylgreen-formalin-saline (MFS) solution consisting of 900 mL of distilled water, 100 mL of 35% formaldehyde solution, 0.6 g of methylgreen, and 8.0 g of NaCl before enumeration of rumen protozoa by the method of
. Protozoa fixed in MFS were diluted in the same solution and counted under a microscope with a plankton-counting glass (cat. no. 900, Hausser Scientific, Blue Bell, PA).
Relative Quantification of Specific Ruminal Microbes
Total nucleic acid was extracted from the incubated rumen samples using the modified bead-beating protocol (
) with the QIAamp DNA mini kit (Qiagen, Valencia, CA). This was accomplished by taking a 1.0-mL aliquot from the culture medium using a wide-bore pipette to ensure collection of a homogeneous sample. Nucleic acid concentrations were measured using a NanoDrop Spectrophotometer (Thermo Scientific, Wilmington, DE).
Quantitative (q)PCR assays for enumeration of methanogenic archaea, ciliate protozoa, and cellulolytic bacterial species (Fibrobacter succinogenes, Ruminococcus flavefaciens, Ruminococcus albus) were performed according to the methods described by
Development and use of competitive PCR assays for the rumen cellulolytic bacteria: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens..
on a real-time PCR machine (CFX96 Real-Time system, Bio-Rad, Hercules, CA) using the SYBR Green Supermix (QPK-201, Toyobo Co. Ltd., Tokyo, Japan). The PCR primer sets used are shown in Table 1. They included group-specific primers for total bacteria as reference genes and species-specific primers for F. succinogenes, R. flavefaciens, R. albus, methanogenic archaea, and ciliate protozoa. All microbial data were analyzed for calculating relative expressions to total bacteria (
). The values of cycle threshold (Ct) after real-time PCR were used to determine the fold change of different microbial populations relative to control (
). Abundance of these microbes was expressed by the equation relative quantification = 2−[ΔCt (Target) – ΔCt (Control)], where Ct represents threshold cycle. All qPCR reaction mixtures (final volume of 25 µL) contained forward and reverse primers (10 pmol each), the iQ SYBR Green Supermix (Toyobo Co. Ltd.), and DNA template ranging from 10 to 100 ng. A negative control without template DNA was used in every qPCR assay for each primer. The PCR amplification of the target DNA was conducted following the references in Table 1.
Table 1Primers (F = forward; R = reverse) for real-time PCR assay
Target species
Primer sequence (5′→3′)
Size (bp)
Reference
Total bacteria
F: CGG CAA CGA GCG CAA CCC R: CCA TTG TAG CAC GTG TGT AGC C
Development and use of competitive PCR assays for the rumen cellulolytic bacteria: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens..
To give a more precise estimate of gas production throughout fermentation, the following calculation was used to analyze the kinetic data, as described by
where GP is gas production (mL/0.1 g DM of substrate) at time t; a, b, and c are the scaling factor for Y-axis intercept (mL/0.1 g of DM), potential gas production (mL/0.1 g of DM), and the rate constant for gas production per hour (h−1), respectively. Gas production rate was fitted to the model by using the nonlinear (NLIN) procedure (
) using Marquardt’s algorithm while varying a, b, and c. Effective gas production (EGP: substrate availability) from the culture was estimated as EGP = a + b[cd/(cd + cp)], where cd is a gas production rate constant, and cp is a passage rate constant assumed to be 0.05/h (
) software package and differences were tested by Duncan’s multiple range test. Significance was declared at P < 0.05.
Results
Table 2 shows the effects of C. militaris on cumulative gas production and its parameters at different incubation times. Gas production was linearly increased by the addition of C. militaris at all incubation times. The potential gas production (a + b) was significantly higher for C. militaris treatments than for the control treatment. In all treatments, cumulative gas production by mixed rumen microorganisms rapidly increased from 3 to 12 h of incubation. The addition of C. militaris significantly increased (P < 0.05) total gas production compared with the control except at 6, 9, and 24 h of incubation for 0.10 and 0.15 g/L treatments. The highest total gas production was seen (P < 0.05) in the 0.25 g/L treatment from 24 to 72 h of incubation.
Table 2Effects of different doses of Cordyceps militaris on in vitro cumulative gas production (at 3 to 72 h of incubation) by mixed rumen anaerobic microbial fermentation
Gas production parameters, a, b, and c, for the negative exponential equation GP=a + b(1 – exp−c×time), where GP is gas production (ml/0.1g DM of substrate) of time t; a=gas production from the immediately soluble fraction; b=gas production from the insoluble fraction; c=the fractional rate of gas production per hour; a + b=potential extent of gas production; EGP=effective gas production rate from the cultures, calculated as EGP=a + b[kd/(kd + kp)], where kd (k) is a gas production rate constant, and kp is a passage rate constant assumed to be 0.05h−1.
Means (n=3) with different superscripts in the same rows are different (P<0.05).
0.53
<0.001
<0.001
0.061
a–d Means (n = 3) with different superscripts in the same rows are different (P < 0.05).
1 Gas production parameters, a, b, and c, for the negative exponential equation GP = a + b(1 – exp−c×time), where GP is gas production (ml/0.1 g DM of substrate) of time t; a = gas production from the immediately soluble fraction; b = gas production from the insoluble fraction; c = the fractional rate of gas production per hour; a + b = potential extent of gas production; EGP = effective gas production rate from the cultures, calculated as EGP = a + b[kd/(kd + kp)], where kd (k) is a gas production rate constant, and kp is a passage rate constant assumed to be 0.05 h−1.
Table 3 shows the effects of C. militaris on methane and hydrogen gas production. The addition of C. militaris reduced methane production linearly (P < 0.05) from 24 to 72 h, but a linear reduction of hydrogen gas production was seen only at 24 h of incubation. The largest reduction of methane production relative to the control was seen at 24 h of incubation, showing reductions of 14.1, 22.0, 24.9, 39.7, and 40.9% for 0.10, 0.15, 0.20, 0.25, and 0.30 g/L treatments, respectively.
Table 3Effects of different doses of Cordyceps militaris on methane (CH4) and hydrogen (H2) gas production (at 3 to 72 h of incubation) in supernatant of growing mixed rumen anaerobic microorganisms
A linear reduction of the concentration of ammonia-N by the addition of C. militaris was seen at 12 and 24 h of incubation (Table 4). Total VFA concentration was linearly increased (P < 0.05) by the addition of C. militaris from 24 to 72 h (Table 4), and corresponding decreases of pH were seen. At all levels of C. militaris addition at 24 h of incubation (Figure 1), the molar proportion of acetate was decreased (P < 0.05) compared with the control and that of propionate was increased (P < 0.05) in the 0.20 to 0.30 g/L treatments. This led to corresponding decreases in acetate:propionate ratio as the addition of C. militaris increased.
Table 4Effects of different doses of Cordyceps militaris on pH value, ammonia-N, and total VFA production (at 3 to 72 h of incubation) in supernatant of growing mixed rumen anaerobic microorganisms
Figure 1Influence of different doses of Cordyceps militaris on total VFA (○), and the molar proportion (%) of acetate (shaded bars), propionate (open bars), and acetate:propionate ratio (A:P ratio, ●) in supernatant of growing mixed rumen anaerobic microorganisms after a 24-h incubation. Lowercase letters indicate statistical significance; means (n = 3) with different letters are significantly different (P < 0.05). Color version available in the online PDF.
Figure 2 shows the effects of C. militaris on microbial populations in culture fluid after 24 h of incubation. The numbers of total and cellulolytic bacteria in the supernatant significantly increased (P < 0.05) at the highest dose level of C. militaris compared with the control. Significant decreases (P < 0.05) in the number of live protozoa and anaerobic fungi were seen in the 0.25 and 0.30 g/L treatments compared with the control, whereas numbers of dead protozoa remained similar between the treatments.
Figure 2Influence of different doses of Cordyceps militaris on the populations of total bacteria (×109 cfu/mL, shaded bars), cellulolytic bacteria (×106 cfu/mL, solid bars), anaerobic fungi (×103 cfu/mL, open bars), live protozoa (×102 cfu/mL, ○), and dead protozoa (×103 cfu/mL, ●) in supernatant of growing mixed rumen anaerobic microorganisms after a 24-h incubation. Lowercase letters indicate statistical significance; means (n = 3) with different letters are significantly different (P < 0.05). Color version available in the online PDF.
Real-time PCR analysis indicated that C. militaris significantly affected abundance of cellulolytic bacteria (R. albus, R. flavefaciens, and F. succinogenes), ciliate protozoa, and methanogenic archaea (Figure 3). The addition of C. militaris significantly decreased (P < 0.05) the abundance of R. albus in the 0.25 and 0.30 g/L treatments at 24 and 48 h of incubation, and for the 0.10, 0.20, and 0.30 g/L treatments at 12 h of incubation. Supplementation with C. militaris also decreased the abundance of F. succinogenes at 24 h except in the 0.15 and 0.20 g/L treatments but decreased responses were not shown at 12 and 48 h of incubation. On the other hand, R. flavefaciens in the 0.15, 0.25, and 0.30 g/L treatments was significantly increased (P < 0.05) at 24 h of incubation, and increased responses were shown for the 0.10 and 0.15 g/L treatments only at 12 and 48 h of incubation, respectively. A significant decrease (P < 0.05) in the abundance of ciliate protozoa was evident at 24 h of incubation when C. militaris was added at a level greater than 0.15 g/L. At 48 h of incubation, reductions in the abundance of ciliate protozoa were seen only in the 0.25 and 0.30 g/L treatments. The effects of C. militaris addition on the abundance of methanogenic archaea were inconsistent. The 0.10 and 0.30 g/L treatments at 12 h and the 0.25 g/L treatment at 24 h of incubation decreased the abundance of methanogenic archaea; however, at 48 h of incubation, addition of C. militaris, except at the 0.25 g/L level, increased the abundance of methanogenic archaea.
Figure 3Influence of different doses of Cordyceps militaris on the relative quantification analysis of methanogenic archaea (●), ciliate protozoa (○), and cellulolytic bacteria: Ruminococcus albus (shaded bars), Ruminococcus flavefaciens (solid bars), and Fibrobacter succinogenes (open bars) in supernatant of growing mixed rumen anaerobic microorganisms after (A) 12-h, (B) 24-h, and (C) 48-h incubations. Lowercase letters indicate statistical significance; means (n = 3) with different letters are significantly different (P < 0.05). Color version available in the online PDF.
In general, in vitro ruminal anaerobic microbial fermentation was strongly affected by the addition of dried C. militaris. The addition of C. militaris increased cumulative and potential gas production, but reduced production of methane and hydrogen gas. Supplementation with C. militaris appeared to accelerate the fermentation process, especially in the early stages of incubation, as shown by accelerated rates of cumulative gas production (Table 2). Total gas production was closely related to the digestion of fermentation substrates, VFA production, and microbial activity and growth (
). In the present study, although we observed a lag time between gas production and the responses of pH and total VFA production, a positive correlation between gas production and total VFA production was found (R2 = 0.63, P < 0.001). The increases in total gas production in response to the addition of C. militaris, which is highly nutritious, might be due to the increased activity of related microbes. It is true that the control vials contained less nutrients (N and carbohydrates) than did the treatment vials, but the supply of N in the control vials would seem unlikely to be limited during the early incubation periods, as shown by the similar ammonia-N concentrations between treatments (Table 4). Furthermore, ammonia-N concentration at 12 and 24 h was lower for the treatments than for the control. The difference in supply of carbohydrates would likely be minimal because of the high level of CP (76%) in C. militaris. This suggests that stimulatory responses to C. militaris might have been from an adverse effect on protozoan population and a positive effect on bacterial population rather than from differences in the supply of major nutrients. The numbers of total and cellulolytic bacteria were increased by the addition of C. militaris (Figure 2).
Methane produced by enteric fermentation in ruminants not only represents a severe loss of feed energy for the animals but also has an ecological impact. Therefore, reducing methane production could have significant economic and environmental benefits. In the present study, addition of C. militaris decreased methane production linearly from 24 to 72 h of incubation with a maximum reduction of 40.9% observed for the highest level of C. militaris at 24 h of incubation (Table 3).
screened 93 plant extracts for their potential to inhibit in vitro methanogenesis and ciliate protozoa using buffalo rumen liquor, and reported that 20 extracts abated methane production by more than 25%, accompanied by a sharp decline in methanogen numbers. Some plant species showing a more pronounced effect are rich in saponins (Sapindus mukorossi), tannins (Terminalia chebula, Populus deltoids, Mangifera indica, and Psidium guajava), or essential oils (Syzygium aromaticum and Allium sativum). In the RUMEN-UP project (Rumen Metabolism Enhanced Naturally Using Plants; http://www.rowett.ac.uk/rumen_up/index.html), potential candidates were selected from 500 different plant species based on their ability to inhibit methane production by 15 to 27% without a detrimental effect on total VFA production or feed digestibility. The plant species selected were the Italian plumeless thistle (Carduus pycnocephalus, 30% inhibition), the Chinese peony (Paeonia lactiflora, 8–53%), the European aspen (Populus tremula, 25%), the sweet cherry (Prunus avium, 20%), goat willow (Salix caprea, 30%), English oak (Quercus pedunculata, 25%), and Sikkim rhubarb (Rheum nobile, 25%). The application of these candidate species to ruminant livestock is still in the early stage and many points still need to be clarified (
Our findings cannot be directly compared with numerous methane-suppressing agents reported in the literature because this is the first study to show that C. militaris can suppress methane emission. However, the modes of action of C. militaris appear similar to those of monensin and secondary plant metabolites (saponins) because the reduction of methane production in response to the addition of C. militaris was accompanied by a decrease in live protozoan population (Figure 2) and abundance of ciliate protozoa (Figure 3). It has been reported that monensin and saponin affect methanogens indirectly by suppressing ciliate protozoa (
In the present study, a substantial reduction in methane production did not result in a corresponding decrease in the abundance of methanogenic archaea (Figure 3), as was observed in the study of
. It has been reported that decreases in methanogen populations may not necessarily lead to a reduction in methane production, at least within a short period of time (
). The discrepancy between the production of methane and the dynamics of the methanogen population might be partly attributable to the insensitivity of some ruminal methanogens to C. militaris.
In the present study, although the adverse effects of C. militaris on protozoa were similar to those of monensin and saponin, the increase in numbers of cellulolytic bacteria resulting from addition of C. militaris (Figure 2) was different from increases due to additions of monensin and sarsaponin, in which cellulolytic bacteria numbers were reduced. It has been suggested that the main reason for the methane-suppressing effects of sarsaponin might be the inhibition of H2-producing bacteria such as cellulolytic bacteria (
). In the present study, C. militaris increased R. flavefaciens, whereas F. succinogenes and R. albus were decreased (Figure 3). The reasons for the different responses within cellulolytic bacteria populations to the addition of C. militaris are not clear.
It is also interesting to note that total VFA increased but ammonia-N concentration decreased as the supplementation level of C. militaris increased (Table 4). Volatile fatty acids are the end products of rumen microbial fermentation and represent the main supply of metabolizable energy for ruminants. Therefore, an increase in VFA production would be nutritionally favorable for the animal. In the present study, the addition of C. militaris increased total VFA in the culture fluid. Molar proportion of acetate and acetate:propionate ratio decreased (P < 0.05) and propionate increased as C. militaris increased (Figure 1). Similar results were obtained for monensin (
), both of which shifted the proportions of VFA toward higher propionate and decreased acetate. The decreased acetate:propionate ratio reflects both the reduced production of methane and the redirection of hydrogen from methane to propionate (
). Similar to this, in the present study, a linear reduction of the concentration of ammonia-N, coupled with a decreased protozoan population, was seen following addition of C. militaris.
Conclusions
Dried C. militaris has the ability to partly inhibit methane production in in vitro microbial fermentations. This compound stimulated mixed ruminal microorganism fermentation and a change in fermentation products, and it decreased methane and hydrogen gas production. Further research is necessary to establish the long-term efficacy of C. militaris to inhibit methanogenesis and improve animal performance.
Acknowledgements
This research was supported by Bio-industry Technology Development Program of Food & Rural Affairs in Ministry of Agriculture (Sejong, Korea), and Cooperative Research Program for Agriculture Science & Technology Development of Rural Development Administration (Jeonju, Korea).
References
Ahn Y.J.
Park S.J.
Lee S.G.
Shin S.C.
Choi D.H.
Cordycepin: selective growth inhibitor derived from liquid culture of Cordyceps militaris against Clostridium spp.
Bacterial and protozoal interactions with ruminal fungi.
in: Akin D.E. Ljungdahl L.G. Wilson J.R. Harris P.J. Microbial and Plant Opportunities to Improve Lignocellulose Utilization by Ruminants. Elsevier,
New York, NY1981: 311-324
Development and use of competitive PCR assays for the rumen cellulolytic bacteria: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens..
Lee, H. G. 2006. Composition for cultivating Cordyceps and cultivating process using thereof. AJU International Law & Patent Group, assignee. Korea Pat. No. 1006442430000.
Improving ruminant production and reducing methane emissions from ruminants by strategic supplementation. EPA/400/1-91/004. US Environmental Protection Agency,
Washington, DC1991
Rapid and specific detection of hydroxyl radical using an ultraweak chemiluminescence analyzer and a low-level chemiluminescence emitter: Application to hydroxyl radical-scavenging ability of aqueous extracts of food constituents.
in: Itabashi H. Ushida K. Yano H. Sasaki Y. Rumen Microbes and Digestive Physiology in Ruminants. Japan Scientific Societies Press,
Tokyo1997: 209-220 (and S. Karger AG, Basel, Switzerland)
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