Application of grifola frondosa ferment in reducing methane emission of dairy cows

Abstract

The application provides application of a grifola ferroxida ferment in preparation of a feed additive for reducing methane emission of a dairy cow. The grifola ferroxida ferment is obtained by liquid submerged fermentation or liquid-solid two-phase fermentation with grifola ferroxida as an object. After the grifola ferroxida ferment is fed to the dairy cow, the activity of a fiber-decomposing bacterial group can be enhanced, the feed digestion rate is improved, the abundance of some bacteria in a rumen is improved, the activity composition of a rumen microbial population is changed, and the methane emission of the dairy cow is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial fermentation and dairy cattle breeding, and particularly relates to application of Grifola frondosa fermentation product in reducing methane emission of dairy cattle. BACKGROUND

[0002] Grifola frondosa, also known as Grifola frondosa, is a kind of rare edible fungus, which is cultivated in Zhejiang, Hebei, Fujian, Sichuan and other places in China. Its fruiting body is rich in protein, amino acid, fat, crude fiber, carbohydrate, trace element and has multiple physiological activities.

[0003] At present, the main way to obtain Grifola frondosa is artificial cultivation of fruiting bodies, but artificial cultivation of Grifola frondosa has long cycle, low production efficiency, high labor intensity, and is limited by season and environment, and is easy to be damaged by diseases and pests, and the quality and yield are unstable. The use of liquid or solid state fermentation method to produce Grifola frondosa mycelium or fermentation product can overcome the shortcomings of traditional fruiting body cultivation. As a feed or feed additive, Grifola frondosa fruiting body or fruiting body extract is mainly used, and the use of Grifola frondosa fermentation product has not been reported. For example, the patent application with publication number CN105519815A discloses a feed additive for improving chicken immunity, which is prepared from the following raw materials by weight: bone meal 50-100 parts, lysozyme 1-10 parts, Grifola frondosa polysaccharide 1-10 parts. The patent application with publication number CN107307196A discloses a feed additive for improving lambing rate of dairy goats, which comprises the following raw materials: Enteromorpha extract, Grifola frondosa extract, medium-chain triglyceride, aescin, copper sulfate, pterin monoglutamic acid, ornithine, antioxidant enzyme, papain, hemicellulase, lecithin and plant salt.

[0004] At present, there is no related report on the use of Grifola frondosa fermentation product to reduce methane emission of dairy cattle.

[0005] Methane emission of ruminants is a problem that attracts much attention and is difficult to solve. Methane emission reduces feed conversion rate for the animal itself, wastes energy, and generally speaking, the energy of methane gas accounts for about 5% to 15% of the total energy of feed; for the environment, it exacerbates global warming. Methane can destroy the ozone layer, although its concentration in the atmosphere is low, but its greenhouse effect is 84 times that of carbon dioxide, and it cannot be used by plants and participate in material circulation. Therefore, it is of great social significance and economic benefit to develop a feed additive that can be used to reduce methane emission of ruminants, especially dairy cattle. SUMMARY

[0006] The application provides application of Grifola ferments in reducing methane emission of dairy cows, and feeding the dairy cows with the Grifola ferments as feed additives can significantly increase total gas production of the dairy cows and reduce methane emission.

[0007] The specific technical solutions are as follows:

[0008] The application provides application of Grifola ferments in preparing feed additives for reducing methane emission of dairy cows.

[0009] Further, the Grifola ferments are obtained through liquid submerged fermentation or liquid-solid two-phase fermentation of Grifola fungi.

[0010] Further, the Grifola fungi is Grifola frondosa MKL0-005, the preservation number is CCTCC NO: M 20231063, and the preservation time is June 19, 2023.

[0011] Further, the culture medium for the liquid submerged fermentation is 1-3% glucose, 5-7% sucrose, 1-2% proteose peptone, 2-3% soybean powder, 0.5-0.7% yeast extract, 0.05-0.07% KH2PO4 and 0.05-0.07% MgSO4, in terms of mass percentage.

[0012] Further, the culture medium for the liquid-solid two-phase fermentation is corn powder 50-60%, soybean meal or soybean cake powder 25-30% and bran 10-25%, and the water content is 65%, in terms of mass percentage.

[0013] Further, the Grifola ferments contain 15-20% Grifola mycelium, in terms of mass percentage.

[0014] The application further provides a method for reducing methane emission of dairy cows, which comprises: feeding the dairy cows with Grifola ferments as feed additives mixed with basic daily rations; and the Grifola ferments are obtained through liquid submerged fermentation or liquid-solid two-phase fermentation of Grifola fungi.

[0015] Further, the addition amount of the Grifola ferments is 0.1-0.3% in terms of dry weight of the basic daily rations.

[0016] Compared with the prior art, the application has the following beneficial effects:

[0017] The Grifola ferments are obtained through liquid submerged fermentation or liquid-solid two-phase fermentation of Grifola fungi, and feeding the dairy cows with the Grifola ferments can not only enhance the activity of fiber-decomposing bacterial groups and improve the feed digestion rate, but also increase the abundance of some bacteria in the rumen, change the activity composition of the rumen microbial population and reduce the methane emission of the dairy cows. DETAILED DESCRIPTION

[0018] The application will be further described in conjunction with specific examples. The following examples are only specific embodiments of the application, but the protection scope of the application is not limited thereto.

[0019] Example 1 Preparation of Fermented Grifola Ferment Feed Additive

[0020] 1. Fermentation broth

[0021] (1) Grifola frondosa strain: Grifola frondosa MKL0-005, preservation number CCTCC NO: M20231063, preservation time June 19, 2023, and preservation unit China Center for Type Culture Collection.

[0022] (2) Seed culture:

[0023] The plate or slant test tube culture medium is PDA.

[0024] The medium formula for shake flask seed culture is: 1.5% glucose, 1.5% sucrose, 0.5% peptone, 0.2% yeast extract, 0.06% KH2PO4. Cultured at 28°C, 150 rpm for about 4 days.

[0025] (3) Liquid submerged fermentation expansion culture

[0026] The medium for expansion culture is: 3% glucose, 6.5% sucrose, 1.0% peptone, 3% soybean powder, 0.6% yeast extract, 0.06% KH2PO4, 0.05% MgSO4.

[0027] The expansion culture conditions are: temperature 28°C, stirring speed 200 rpm, air flow per minute about 1:1 (v / v), fermentation for 4 days.

[0028] After seed culture and expansion culture are completed, Grifola frondosa liquid fermentation broth is obtained. The broth is centrifuged and dried, and it is found that there are about 50 grams of mycelium per liter of broth.

[0029] (4) Liquid-solid two-phase fermentation

[0030] The solid medium formula is: corn flour 60%, soybean meal or soybean cake powder 30%, bran 10%; water content 65%.

[0031] The solid culture conditions are: after the medium is autoclaved (121°C, 0.5 to 1 hour), inoculate 10% liquid fermentation broth, mix evenly, and spread thinly to 5 cm in a sterile environment at 24°C and humidity 80% for 5 days.

[0032] (5) Preparation of feed additive from fermentation product of Grifola frondosa

[0033] The liquid fermentation broth of Grifola frondosa is mixed with solid auxiliary materials (such as corn flour) at a ratio of 200 g / L, dried at low temperature to a moisture content of less than 10%, and ground (40 mesh) to obtain the liquid fermentation additive HY of Grifola frondosa.

[0034] The liquid-solid fermentation product is dried and ground (40 mesh) to obtain the solid fermentation additive HG of Grifola frondosa.

[0035] (6) Quality detection of fermentation product of Grifola frondosa

[0036] Ergosterols are components of fungal cell membranes, and their content in mycelium is relatively stable. Ergosterol, a biomarker of fungi, was selected as a detection index. The ergosterol content in the separated pure mycelium of Grifola frondosa, the liquid fermentation additive HY of Grifola frondosa, and the solid fermentation additive HG of Grifola frondosa were detected, and the results are shown in Table 1.

[0037] Table 1: Ergosterol content in fermentation product of Grifola frondosa

[0038] Sample Ergosterol (mg / kg) Grifola frondosa mycelium 2859.6 Grifola frondosa liquid ferment additive HY 564.3 Grifola frondosa solid ferment additive HG 608.1

[0039] According to the above determination results, the ergosterol content in the solid fermentation additive HY and the solid fermentation additive HG of Grifola frondosa is 19.7% and 21.2% of the ergosterol content in the pure mycelium of Grifola frondosa, i.e., HY and HG contain about 20% of the mycelium of Grifola frondosa.

[0040] Example 2: In vitro gas production test of rumen fluid of dairy cows

[0041] 1. Test design

[0042] Six rumen fistulated Holstein cows (lactation days = 120 ± 11, milk yield = 27.6 ± 3.3 kg) were raised in individual cowsheds with free access to water, and were fed and milked twice a day at 6:30 and 18:30. The cows were fed with a basic daily ration of TMR (Table 2).

[0043] Table 2: Nutritional composition of basic daily ration

[0044]

[0045] Note: The premix mainly contains various metal ions and vitamins.

[0046] Ruminal fluid was collected from fistulated Holstein cows in the morning after feeding and was placed in a vacuum bottle flushed with anoxic carbon dioxide. The ruminal fluid was filtered through four layers of dry gauze and mixed with pre-warmed (39°C) buffer at a 1:2 ratio (buffer:rumen fluid, v:v) under continuous flow of carbon dioxide to ensure an anaerobic environment. After mixing, 150 ml of the buffered rumen fluid was transferred to a 200 ml glass bottle. Among them, 2 grams of Grifola frondosa solid fermentation additive HG prepared in Example 1 was added to the rumen fluid of each of the three cows in the experimental group (Table 2). The bottle was sealed with a silicone plug, with an airtight collection bag, and incubated in a shaking water bath at 39°C, moving 40-50 times per minute, for 24 hours. The total gas production in 24 hours was determined by collecting gas with a gas collection bag, and the 24-hour rumen in vitro gas composition, including methane, carbon dioxide and hydrogen, was measured by high-efficiency gas chromatography.

[0047] The pH of the leachate was immediately determined by a pH meter; the ammonia nitrogen content (NH3-H) of the leachate was determined by a phenol-sodium hypochlorite colorimetric method; and the contents of volatile fatty acids such as acetic acid, propionic acid and butyric acid were determined by a gas chromatograph.

[0048] 2. Test results

[0049] 2.1 Effect of Grifola frondosa fermentation additive on in vitro rumen gas production and gas composition

[0050] The test results are shown in Table 3.

[0051] Table 3 Effect of Grifola frondosa fermentation additive on in vitro rumen gas production and gas composition

[0052]

[0053] Note: * indicates a significant difference (p≤0.05) compared with the control group.

[0054] The test results show that the addition of Grifola frondosa fermentation additive HG significantly increases the total gas production and carbon dioxide production, significantly reduces the methane production, and has no significant effect on hydrogen production. Among them, the methane production is reduced by 12.6% (37.02±4.05 vs 32.35±3.76). The test results show that Grifola frondosa fermentation has a significant effect on regulating in vitro rumen gas.

[0055] The Grifola frondosa fermentation additive also significantly reduces the pH of the rumen fluid, which is related to its promotion of rumen microbial flora activation and improvement of digestion rate. The increase in total gas production and carbon dioxide production is also attributed to this.

[0056] At the same time, it is noted that although the Grifola frondosa fermentation additive increases the concentration of ammonia nitrogen in the rumen, it does not reach a significant level.

[0057] In summary, the addition of G. frondosa fermentation product can promote the activity of some rumen bacterial groups, thus accelerating the digestion of feed in the rumen, which is reflected in the increase of total gas production and carbon dioxide production in the rumen. At the same time, the addition of G. frondosa fermentation product can inhibit the activity of methane-producing bacterial groups, which is reflected in the significant decrease of methane production. Therefore, it is speculated that the addition of G. frondosa fermentation product can accelerate the digestion rate of feed in the rumen and improve the utilization efficiency of feed in the rumen.

[0058] 2.2 Effect of G. frondosa fermentation product on rumen fermentation parameters in vitro

[0059] The experimental results are shown in Table 4.

[0060] Table 4 Effect of G. frondosa fermentation product on rumen fermentation in vitro

[0061]

[0062] Note: * indicates a significant difference (p≤0.05) compared with the control group.

[0063] As can be seen from the experimental results in Table 4, the addition of G. frondosa fermentation product has a higher volatile fatty acid production, and significantly reduces the rumen pH. From the proportion of volatile fatty acids, it can be seen that the addition of G. frondosa fermentation product makes the rumen fermentation type tend to propionic acid fermentation, which is also a major reason for the decrease of rumen methane. The production of propionic acid competitively obtains the hydrogen required for methane production.

[0064] Therefore, from the current results, it is concluded that G. frondosa fermentation product has the effect of reducing rumen methane production, which may be related to the change of rumen fermentation parameters; and the change of rumen fermentation parameters is due to the change of the activity composition of rumen microbial population.

[0065] In order to further explain how G. frondosa fermentation product affects the microbial population in the rumen of dairy cows and whether it has the effect of improving milk yield, the test of Example 3 is carried out.

[0066] Example 3: Dairy cow feeding test - milk yield and rumen microbial population analysis

[0067] 1. Test design

[0068] With a breeding company in Anhui 100 lactating cows (lactation days = 142 ± 13, milk production = 30.6 ± 4.3 kg) as test animals, each adult cow daily feeding 10 kg of concentrate (daily concentrate formula: corn 52%, wheat bran 5%, corn bran 5%, soybean meal 8%, cottonseed meal 12%, rapeseed meal 8%, DDGS 3%, stone powder 2%, magnesium oxide 0.3%, urea 1%, salt 0.7%, baking soda 1%, calcium hydrogen phosphate 1%, premix 1%, etc.). During the test, according to the management system of the breeding farm, the milk production, feed intake, feces, health status of each group of cows were recorded. The test period was 3 months.

[0069] 100 cows were randomly divided into 2 groups, 50 cows in each group. Among them: control group (C), without any addition in the diet; test group (HG), 40 grams of Grifola frondosa solid fermentation additive HG was added in the diet every day.

[0070] In the test group, 3 cows were randomly selected, 2 hours after morning feeding, the rumen contents were collected by using a rumen vacuum pump before the test started for 14 days, after the test started for 14 days and after the test ended for 7 days; total nucleic acid was extracted from the rumen contents by magnetic bead method, the dried DNA precipitate was resuspended in TE buffer and stored at -20℃ until analysis; measure A260 / 280 ratio to determine the concentration and purity of DNA; dilute the DNA sample to 10 ng / μl for qPCR amplification quantification, count the number of yellow rumen coccus, white rumen coccus, succinic acid-producing filamentous bacillus, cellulolytic butyric acid arc, bovine streptococcus, prevotella, amylophilic rumen bacillus, starch succinic acid monosodium, ruminococcus denman and mcsweeney, 2006.

[0071] The specific primer sequence for qPCR is shown in Table 5.

[0072] Table 5 Primers for rumen microbial qPCR analysis

[0073]

[0074] 2、Test results

[0075] 2.1 Daily average milk production of cows

[0076] During the test, no abnormal health of the animals was found. Due to the gradual warming of the weather, the overall milk production has a downward trend.

[0077] The test results of the daily average milk production of cows are shown in Table 6.

[0078] Table 6 Effect of Grifola frondosa fermentation additive on milk production of cows

[0079]

[0080]

[0081] Note: Different letters indicate significant differences (p < 0.05).

[0082] As shown in Table 5, the daily average milk production of dairy cows can be significantly improved by adding 40 grams of Grifola frondosa solid fermentation additive per day. Among them, compared with the control group, the daily milk production of the test group increased by 6.5%, 11.8% and 11.3% in the first, second and third months, respectively.

[0083] As can be seen from the test data, the Grifola frondosa solid fermentation additive can take effect in the first month, and reach the stable period in the second month. Although the overall milk production showed a downward trend due to weather factors, the effect of the additive was still maintained.

[0084] 2.2 Changes in rumen microbial population

[0085] The test results of the changes in rumen microbial flora before and after feeding Grifola frondosa fermentation additive are shown in Table 7.

[0086] Table 7 Changes in rumen microbial population

[0087]

[0088] As shown in Table 7, the abundance of the measured microbial population increased after feeding the Grifola frondosa fermentation additive, and all reached a significant level (except for Butyrivibrio fibrisolvens); after stopping feeding, there was still a certain lag effect.

[0089] There are a large number of cellulose-decomposing microorganisms in the rumen, mainly including white rumen coccus, yellow rumen coccus, amylophilic rumen bacillus, Butyrivibrio fibrisolvens and bovine streptococcus, etc. The abundance of these flora significantly increased after feeding the Grifola frondosa fermentation additive, suggesting that the Grifola frondosa fermentation additive improved the feed digestion rate by enhancing the activity of fiber-decomposing flora.

[0090] Among the cultured bacteria in the rumen, bacteria with high fumarate reduction activity include succinogenes filamentous bacterium, amylosuccinomonas and ruminococcus lunatus, etc. These bacteria can compete with methanogens for hydrogen and formic acid, reducing the production of methane in the rumen. Feeding the Grifola frondosa fermentation additive significantly increased the abundance of these bacteria, suggesting that the Grifola frondosa fermentation additive reduced the methane emission of dairy cows by changing the activity composition of the rumen microbial population.

Claims

1. The application of Grifola frondosa fermented product in the preparation of feed additives that reduce methane emissions from dairy cows, characterized in that, The maitake mushroom fermentation product is obtained by liquid deep fermentation or liquid-solid two-phase fermentation of maitake mushroom fungus; the maitake mushroom fungus is maitake ( Grifola frondosa MKL-005, with accession number CCTCC NO: M 20231063, and accession date June 19, 2023; the liquid deep fermentation medium, by weight percentage, consists of 1-3% glucose, 5-7% sucrose, 1-2% peptone, 2-3% soybean flour, 0.5-0.7% yeast extract, 0.05-0.07% KH2PO4, and 0.05-0.07% MgSO4; the liquid-solid two-phase fermentation medium, by weight percentage, consists of 50-60% corn flour, 25-30% soybean meal or soybean cake flour, and 10-25% wheat bran; the moisture content is 60-65%; and the Grifola frondosa fermentation product contains 15-20% Grifola frondosa mycelium by weight percentage.

2. A method for reducing methane production in dairy cows, characterized in that, include: Maize fermented product was used as a feed additive and mixed with the basal diet before being fed to dairy cows. The maitake mushroom fermented product is obtained by liquid deep fermentation or liquid-solid two-phase fermentation of maitake mushroom fungus; the amount of maitake mushroom fermented product added is 0.02-0.5% based on the dry weight of the basal diet; The preparation method of Grifola frondosa fermented feed additive is as follows: (1) Grifola frondosa fungus strain: Grifola frondosa ( Grifola frondosa MKL-005, accession number CCTCC NO: M20231063, accession date June 19, 2023, deposited by China Center for Type Culture Collection; (2) Seed culture: The culture medium in plates or slant tubes is PDA; The culture medium for shake-flask seed culture was formulated as follows: 1.5% glucose, 1.5% sucrose, 0.5% peptone, 0.2% yeast extract, and 0.06% KH2PO4; cultured at 28℃ and 150 rpm for 4 days. (3) Liquid submerged fermentation for large-scale culture: The culture medium for expanded culture consisted of: 3% glucose, 6.5% sucrose, 1.0% peptone, 3% soybean flour, 0.6% yeast extract, 0.06% KH2PO4, and 0.05% MgSO4. The conditions for scaling up the culture were: temperature 28℃, stirring speed 200 rpm, aeration rate of 1:1 per minute, v / v, and fermentation for 4 days. After seed culture and large-scale culture are completed, the liquid fermentation liquid of Grifola frondosa is obtained; the liquid is dried by centrifugation, and each liter of liquid contains about 50 grams of mycelium; (4) Liquid-solid two-phase fermentation: The solid culture medium formula is: 60% corn flour, 30% soybean meal or soybean cake powder, and 10% wheat bran; moisture content 65%. Solid culture conditions are as follows: after autoclaving at 121℃ for 0.5 to 1 hour, the culture medium is cooled, inoculated with 10% liquid fermentation broth, mixed well, spread thinly at 5 cm, and cultured in a sterile environment at 24℃ and 80% humidity for 5 days. (5) Preparation of feed additives from fermented maitake mushrooms: The liquid fermentation broth of Grifola frondosa is mixed with solid excipients at a ratio of 200g / L, dried at low temperature until the moisture content is below 10%, and then pulverized at 40 mesh to obtain Grifola frondosa liquid fermentation additive. Alternatively, the liquid-solid two-phase fermentation product can be dried and pulverized at 40 mesh to obtain the Grifola frondosa solid fermentation additive.

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