Effects of Cordyceps militaris Mycelia on In vitro Rumen Microbial Fermentation

Effects of Cordyceps militaris mycelia on rumen microbial fermentation were determined by measuring in vitro gas production, cellulose digestion and VFA concentrations. C. militaris mycelia was added to buffered rumen fluid with final concentrations of 0.00, 0.10, 0.15, 0.20, 0.25 and 0.30 g/L and incubation times were for 3, 6, 9, 12, 24, 36, 48 and 72 h. At all incubation times, the gas production showed a quadratic increase with the supplementation of C. militaris mycelia; maximum responses were seen with 0.25 g/L supplementation. However, the gas production was significantly lower for the 0.30 g/L supplementation than for the 0.25 g/L supplementation from 9 h to 72 h incubation. The cellulose filter paper (FP) digestion showed a quadratic increase, as did the gas production except at 3 h incubation. The concentration of total VFA was significantly increased by the supplementation of C. militaris mycelia compared with the control treatment; the highest response was also seen with 0.25 g/L supplementation. This was true for responses in the concentration of acetic and propionic acids. As opposed to other responses, the responses of pH to the supplementation of C. militaris mycelia showed a quadratic decrease from 3 h to 36 h incubation. In conclusion, C. militaris mycelia alter the mixed rumen microbial fermentation with increases in the production of gas and VFA, and cellulose FP digestion. (


INTRODUCTION
Cordyceps species are medical fungi well known for their pharmacological actions such as immunomodulatory (Koh et al., 2002;Yu et al., 2003), anti-inflammatory (Yu et al., 2004a, b), antitumor (Nakamura et al., 1999), antifungal (Kneifel et al., 1977) and antibacterial (Ahn et al., 2000) activities, and contain biologically active components such as nucleosides (cordycepin; 3'-deoxyadenosine, and adenosine), polysaccharides and ergosterol (Li et al., 2006).The typical Coryceps used in oriental medicine was Chinese C. sinensis which forms a fruiting body using the larva of a moth as the host.Because natural C. sinensis is rare and expensive, however, techniques for artificial cultivation of Cordyceps species other than the natural Cordyceps have been developed and, thus commercial products of Cordyceps are now widely available.C. militaris is a related species of C. sinensis commonly used as a substitute of the natural Cordyceps (Li et al., 2006).
Although the pharmacological actions of Cordyceps may also affect livestock beneficially, the application of Cordyceps in livestock has received little attention.Recently, however, it has been reported that oral dose of a hot-water extract of mycelia from C. sinensis in an attempt to substitute for antibiotic growth promoter improved body weight gain and immune system in broiler chicks (Koh et al., 2003).The efforts to find biologically active materials that can improve animal health are certainly welcomed to livestock producers since demand for alternative strategies for enhancing livestock production to the same or higher level obtained with antibiotic growth promoters has markedly being increased (Wallace, 2004;Al-Mamun1 et al., 2007;Li et al., 2008).In addition, as long as the biologically active components in Cordyceps can be transferred to the end products of livestock such as milk, meat and eggs, an increase of the market value of livestock products would be expected.
The application of Cordyceps to ruminants is not simple as to mono-gastric animal because of the digestion of rumen microorganisms and the antibacterial and antifungal effects of Cordyceps.Therefore, the objectives of the present study were to examine effects of C. militaris mycelia on rumen microbial fermentation by measuring in vitro gas production, cellulose digestion, and VFA production.

Sample preparation
Dried C. militaris mycelia cultured on a medium composed of corn gluten, soybean protein, beer yeast and corn steep liquor (culturing method and medium composition are patent pending in Korea) were obtained from EuGene Bio Farm (Hwaseong City, Gyeonggi Province, Korea).The mycelia are composed of 761.6 g CP, 122.4 g crude fat, 9.6 g ether extract, 32.1 g crude ash, 74.3 g nitrogen free extract, and 1.64 g cordycepin per kg of dry matter.The manufacturer reported that C. militaris mycelia used in the present study contained about 2.3 times more cordycepin (1.6 mg/g DM) than C. militaris traditionally cultured on faunal pupae (0.7 mg/g DM).

In vitro batch fermentation
The anaerobic culture techniques of Hungate (1966) were carried out for all incubations with rumen fluid from a 515 kg Korean native steer (Hanwoo) fed a basal diet consisting of rice straw and concentrates mixed at a ratio of 4:6 (fresh weight).The steer was housed in an individual metabolic stall and given the diet at 1.75% of body weight.Rumen fluid was collected via rumen cannulae before the morning feeding into a vacuum flask that was flushed with O 2 -free CO 2 , and squeezed through four layers of cheese cloths into an Erlenmeyer flask in an anaerobic glove box.The fluid was then mixed with buffer (pH 6.9) (containing 292 mg of K 2 HPO 4 , 240 mg of KH 2 PO 4 , 480 mg of (NH 4 ) 2 SO 4 , 480 mg of NaCl, 100 mg of MgSO 4 ⋅7H 2 O, 64 mg of CaCl 2 ⋅2H 2 O, 4,000 mg of Na 2 CO 3 , and 600 mg of cysteine hydrochloride in l,000 ml of O 2 -free distilled water) at a ratio of 1:2.After mixing, 30 ml of diluted rumen fluid was transferred to 60 ml serum bottles containing 750 mg of Whatman No. 1 cellulose filter paper (FP) as a sole carbon source.Weighed amounts of dried C. militaris mycelia were added to achieve final concentrations of 0.00, 0.10, 0.15, 0.20, 0.25 and 0.30 g/L.The bottles (three replicates per treatment) were sealed with butyl rubber stoppers and aluminum caps, and placed in an incubator at 38°C for 3, 6, 9, 12, 24, 36, 48 and 72 h without shaking.

Sampling and analysis
At each sampling time, gas production, cellulose filter paper degradation and pH were determined.Gas production (ml/0.1 g substrate) was determined using a water displacement apparatus (Fedorak and Hrudey, 1983) and pH was determined in culture fluid immediately after the vials had been opened.The concentration of VFA was determined only at 24 h incubation, and for analysis of VFA, 1 ml of 25% meta-phosphoric acid was added to 5 ml of fermentation fluid, centrifuged (10,000×g for 10 min at 4°C) and supernatant was stored at -30°C until analyzed.
VFA analysis was carried out by a gas chromatograph (model GC-14B, Shimadzu Co. Ltd.) using a Thermon-3000 5% Shincarbon A column (1.6 m×3.2 mm i.d., 60 to 80 mesh, Shinwakako) and flame ionization detector (column temperature = 130°C, injector and detector temperature = 200°C).The carrier gas (N 2 ) flow rate was 50 ml/min.Filter paper digestibility was measured by the method of Lee et al. (2004).Residual particulate substrates including adherent microbial biomass were separated from supernatant by centrifugation (3,000 rpm for 20 min) and then treated with 0.15 ml of sodium dodecyl sulphate solution and boiled (100°C) for 1 h to remove adherent rumen mixed microbial biomass.The pellet was rinsed three times with absolute alcohol at 60°C and twice with running distilled water, and was dried to a constant weight at 78°C for 24 h.

Statistical analysis
Data obtained from the experiment were analyzed using the SAS (1996) software package and differences were tested by Duncan's multiple range test, and then P value less than 0.05 was considered significant.Linear, quadratic and cubic responses to C. militaris mycelia level were tested by orthogonal contrast.

RESULTLS
The cumulative in vitro gas production at different incubation times for the treatments is shown in Table 1.At all incubation times, the gas production showed a quadratic increase with the supplementation of C. militaris mycelia; maximum responses were seen with 0.25 g/L supplementation from 9 h to 72 h incubation.After incubation for 24 h, the gas production from the 0.25 g/L supplementation was about 50% higher than that from the control treatment.But the gas production was significantly lower for the 0.30 g/L supplementation than for the 0.25 g/L supplementation from 9 h to 72 h incubation.
The effects of the supplementation of C. militaris mycelia on the cellulose FP digestion by mixed rumen microorganisms are shown in Table 2.The cellulose FP digestion showed a quadratic trend, as did the gas production except at 3 h incubation.At all incubation times, the 0.25 g/L supplementation significantly increased cellulose FP digestion compared with the control treatment.After incubation for 24 h, the cellulose FP digestion for the 0.25 g/L supplementation was about 36% higher than that for the control treatment.The cellulose FP digestion was numerically higher for the 0.25 g/L supplementation than for other treatments at all incubation times, although the significant difference was only seen at 72 h incubation.
The effects of the supplementation of C. militaris mycelia on the concentration of VFAs after 24 h incubation are given in Table 3.The concentration of total VFA was significantly increased by the supplementation of C. militaris mycelia compared with the control treatment; the highest response was also seen with 0.25 g/L supplementation.This is true for the responses in the concentration of acetic and propionic acids.But no significant difference was seen in the concentration of butyric and valeric acids among treatments.
As opposed to other responses, the responses of pH to the supplementation of C. militaris mycelia showed a quadratic decrease from 3 h to 36 h incubation (Table 4).The pH was numerically lower for the 0.25 g/L supplementation than for other treatments at all incubation times, although the significant difference was only seen at 36 h incubation.

DISCUSSION
Increasing public concern about antibiotic resistance and residues in animal products has resulted in the search for alternatives, such as prebiotics, probiotics, and other feed additives.Cordyceps species and its constituents have potential as natural products for its various pharmacological   actions on human health and still extensive work is being carried out to define the pharmacological efficiency of bioactive materials in Cordyceps (Li et al., 2006).
Cordycepin, one of the markers for quality control of Cordyceps was shown to have a selective growth-inhibiting activity towards harmful intestinal bacteria without adverse effects on lactic acid-producing bacteria (Ahn et al., 2000;Yeon et al., 2007) or even with an increase in Lactobacillus sp.(Koh et al., 2003), though the mechanisms underlying the inhibiting or promoting effects are not known.In the present study, the addition of C. militaris mycelia to rumen fluid inoculum caused a marked increase in gas production, cellulose FP degradation and VFA production, suggesting that major changes in the microbial population had occurred in response to the addition of C. militaris.It has been reported that gas production is positively related to microbial protein synthesis (Krishnamoorthy et al., 1991).Therefore, it is tempting to speculate that the increases of cellulose FP digestion and gas production might be due to increased bacteria numbers, the most active organisms for fiber digestion in the rumen (Lee et al., 2004).Protozoa and fungi are also involved in fiber digestion (Lee et al., 2004), but their role in the present study might be excluded or even suppressed since cordycepin was shown to have adverse effects on protozoa (Trigg et al., 1971) and fungi (Kneifel et al., 1977).At 24 h incubation, the production of gas and total VFA and cellulose FP digestion increased linearly with the addition of C. militaris mycelia.However, the addition of 0.3 g/L of C. militaris mycelia reduced the production of gas and total VFA and cellulose FP digestion compared with the 0.25 g/L supplementation.A plateau in the response, rather than a decrease, had been expected.Clearly, this finding needs to be confirmed but, taking the results at face value, it implies that a further increase in the activity of rumen microorganisms could not be expected to supplementing C. militaris mycelia beyond 0.25 g/L.
The production of gas and total VFA measured in the present study was within ranges reported in earlier studies (Doane et al., 1997;Getachew et al., 2004;Lee et al., 2004).In vitro gas methods primarily measure digestion of soluble and insoluble carbohydrates by rumen microorganisms (Menke and Steingass, 1988), and there are linear correlations between the volume of gas and moles of VFA produced and the mass of fiber digested (Calabrò et al., 2001).Indeed, the relationship between gas production and cellulose FP digested in the present study was linear (r 2 = .85,p<0.001; data not shown), and the gas production was also related to VFA production (r 2 = 0.89, p<0.01; data not shown).The calculated amount of total VFA production based on the culture volume of 30 ml on the 0.25 g/L supplementation at 24 h incubation was 3.66 mmol, which can be converted to 0.013 mmol/mg cellulose FP digested (the amount of cellulose FP digested was 281 mg at 24 h incubation), values that agree well with the estimate (0.01 mmol total VFA/mg NDF digested) reported by Doane et al. (1997).But the value for the control treatment was about half of that for the 0.25 g/L supplementation, implying that rumen fluid inoculum might contain a low level of microbial population.The maximum amount of C. militaris mycelia supplemented (0.30 g/L supplementation) was 9 mg.This degree of nutrients supply over the control treatment implies that the large difference in the production of gas and VFA would be very unlikely to be a result of carbohydrates supplied from C. militaris mycelia.
Alternatives for growth promoting antibiotics for ruminants must ensure that they have no detrimental effect on the basic functions of the fermentation, such as fiber digestion and VFA production (Wallace, 2004).In this connection, C. militaris mycelia used in the present study appears to fulfill these criteria as it increased the fiber digestion and volatile production.But, whether these effects were induced directly by increased bacteria numbers or other mechanisms, such as antiprotozoal activity were involved remains to be investigated.

Table 1 .
Effects of supplement levels (g/L) of Cordyceps militaris on in vitro cumulative gas production (ml/0.1 g DM substrate) by

Table 2 .
Effects of supplement levels (g/L) of Cordyceps militaris on in vitro digestibilities (%) of cellulose filter paper by mixed rumen SEM = Standard error of the mean.a,b,c, d, eMeans within a row with different superscripts differ significantly (p<0.05).

Table 3 .
Effects of supplement levels (g/L) of Cordyceps militaris on the concentrations (mmol/L) of volatile fatty acids SEM = Standard error of the mean.a, b, c Means within a row with different superscripts differ significantly (p<0.05).

Table 4 .
Effects of supplement levels (g/L) of Cordyceps militaris on pH Supplement levels (g/L) of C. militaris = Standard error of the mean.a, b, c Means within a row with different superscripts differ significantly (p<0.05). SEM