Application of ginger leaf extract in preparation of medicine or product for reducing methane emission of ruminant rumen

Abstract

The invention discloses an application of a ginger leaf extract in preparation of a medicine or a product for reducing methane emission of ruminant rumen. It is found for the first time that the ginger leaf extract can inhibit generation of ruminant rumen methane. The invention not only provides a technology and a method for treatment and value improvement of agricultural waste ginger leaves, but also provides a new technology for methane emission reduction of ruminant rumen.

Description

Technical Field

[0001] The present invention belongs to the technical field of comprehensive utilization of agricultural wastes and application of plant extracts in ruminants, and particularly relates to the application of ginger leaf extract in the preparation of drugs or products for reducing rumen methane emissions in ruminants. Background Art

[0002] In China, the planting area of ginger is large, and a large amount of ginger leaves are produced every year, which cannot be effectively processed and are piled up and rotted, causing great harm to the ecological environment. Ginger leaves have various applications in traditional medicine, daily health care and agriculture. Ginger leaves have medicinal values such as promoting blood circulation to remove blood stasis, warming the middle-jiao to dispel cold, and promoting digestion. It also has edible and daily health care effects such as making tea, extracting aromatic oil, repelling mosquitoes and insects, and beauty and skin care. In addition, it can also be used to make organic fertilizers, etc. However, the above-mentioned ways have limited use of ginger leaves. Therefore, new application methods and application ways need to be developed to deal with a large amount of ginger leaves that cannot be effectively processed. At present, the research and application of ginger leaf extract mainly focus on anti-tumor, antioxidant and antibacterial effects, and no application of ginger leaf extract in ruminant breeding has been found. Summary of the Invention

[0003] Object of the Invention: The object of the present invention is to provide the application of ginger leaf extract in the preparation of drugs or products for reducing rumen methane emissions in ruminants, and it does not affect the feed degradation rate as a feed additive.

[0004] Technical Solution: The application of the ginger leaf extract of the present invention in the preparation of drugs or products for reducing rumen methane emissions in ruminants.

[0005] Further, the ginger leaf extract is an aqueous extract or an ethanol extract.

[0006] Further, the concentration of the ethanol solution is 25% - 100%.

[0007] Further, the ethanol concentration is 50% - 100%.

[0008] Further, the final concentration of the ginger leaf active substance extract is 0.2 - 1.6 mg / mL.

[0009] Further, the final concentration of the ginger leaf active substance extract is 0.8 - 1.6 mg / mL.

[0010] A feeding method for reducing rumen methane emissions in ruminants according to the present invention, wherein the method is to add ginger leaf extract additionally to normal feed.

[0011] Further, the ginger leaf extract includes one or a combination of more of an aqueous extract or an ethanol extract.

[0012] Furthermore, the ethanol extract includes a ginger leaf extract obtained by extracting with an ethanol solution having an ethanol concentration of 25% to 100%.

[0013] Furthermore, the dosage of the ginger leaf extract is 25 g / head / day.

[0014] Beneficial effects: Compared with the prior art, the present invention has the following prominent and significant advantages: The present invention discloses for the first time that the ginger leaf extract can effectively reduce the rumen methane emission of ruminants, provides a new application way for the ginger leaf, an agricultural waste that cannot be effectively treated, and improves the added value. The present invention also discloses a feeding method for reducing the rumen methane emission of ruminants, and this method has no negative impact on the feed intake and body weight. Therefore, the present invention can effectively treat the agricultural waste ginger leaf, and at the same time, the ginger leaf extract can be used for the breeding of ruminants. Specific embodiments

[0015] The following will describe the implementation scheme of the present invention in detail in conjunction with the embodiments. The following embodiments are only used to illustrate the present invention. Those not specified in the embodiments are carried out according to the conventional conditions or the conditions recommended by the manufacturer. Those reagents or instruments not specified in the manufacturer are all conventional products that can be obtained through commercial purchase.

[0016] The detection method of methane and hydrogen contents in the present invention refers to the literature (Effect of nitrooxy compounds with different molecular structures on the rumen methanogenesis, metabolic profile, and methanogenic community. Current Microbiology, 2017). The detection method of volatile fatty acid content in rumen fluid refers to the literature (Jin W, Meng ZX, Wang J, Cheng YF, Zhu WY. Effect of nitrooxy compounds with different molecular structures on the rumen methanogenesis, metabolic profile, and methanogenic community. Current Microbiology, 2017). The detection method of methane production refers to the literature (Hristov AN, Oh J, Giallongo F, Frederick T, Weeks H, Zimmerman PR, Harper MT, Hristova RA, Zimmerman RS, Branco AF. The use of an automated system (GreenFeed) to monitor enteric methane and carbon dioxide emissions from ruminant animals. Journal of Visualized Experiments, 2015). The calculation of methane emissions refers to (Wang R, Cao YR, Zhang XM, Zhang F, Tian X, Zhong RZ, Tan ZL, Wang M. Relationship between daily variations of methane emissions and eructation peaks in dairy cows measured with an automated head-chamber system. Animal Feed Science and Technology, 2023).

[0017] The determination of feed dry matter refers to "Determination of Moisture in Feed, GBT 6435 - 2014".

[0018] Example 1: Experiment on the inhibition of rumen in vitro fermentation methane production by ginger leaf extract

[0019] 1. Preparation of ginger leaf extract

[0020] The purchased dried ginger leaves were cleaned, dried in an oven at 60 °C, pulverized, passed through a 60-mesh sieve, and stored in the dark. 0.25 g of ginger leaf powder was added to a 10 mL centrifuge tube, and 4 mL of deionized water or ethanol solution (the concentrations of ethanol aqueous solutions were 25%, 50%, 75%, and 100% respectively) was added. A rotary shaker was used to shake and extract at 35 r / min at room temperature for 4 hours. Centrifuge for 10 minutes (1660 g), and take the supernatant. Using a nitrogen blower, the extract was removed, and the sample was collected when the liquid was opaque under normal pressure and room temperature conditions. The sample after removing the extract was placed in a freeze dryer and freeze-dried at -80 °C for 48 hours to obtain ginger leaf extract.

[0021] 2. Rumen in vitro fermentation experiment

[0022] The experimental animals were housed in the dairy cow experimental base of the College of Animal Science and Technology, Nanjing Agricultural University (Nanjing, Jiangsu). Three lactating Holstein cows with permanent rumen fistulas, with an average body weight of 613 ± 35 kg. On the day of the experiment, rumen fluid was collected half an hour before morning feeding and quickly placed in a thermos bottle pre-filled with carbon dioxide and brought back to the laboratory. The collected fresh rumen fluid was filtered through four layers of gauze, and the rumen fluid and buffer were mixed at a ratio of 1:2 (v / v) to prepare a mixed artificial rumen culture solution. The composition of the buffer is shown in Table 1. The mixed culture solution was dispensed into 50 mL fermentation bottles with a volume of 120 mL. The temperature was maintained at 39 °C throughout the process, and carbon dioxide was continuously introduced to maintain an anaerobic environment. 0.5 g of substrate (corn 20.4%, DDGS 5.2%, soybean meal 14.4%, alfalfa 21.2%, rice straw 12.9%, corn silage 25.8%, premix 0.1%) and different doses of ginger leaf extract were pre-added to the fermentation bottles. All fermentation bottles were fermented at 39 °C and 80 r / min for 24 hours. Five experimental groups were set for the ginger leaf extract extracted by each extraction method: 1) Control group: no addition; 2) Treatment group: 10 mg of ginger leaf extract, 0.2 mg / mL (final concentration); 3) Treatment group: 20 mg of ginger leaf extract, 0.4 mg / mL (final concentration); 4) Treatment group: 40 mg of ginger leaf extract, 0.8 mg / mL (final concentration); 5) Treatment group: 80 mg of ginger leaf extract, 1.6 mg / mL (final concentration). Methane and hydrogen determination: First, use a pressure conversion instrument to measure the gas production in the fermentation bottle, and then use an Agilent 8860 gas chromatograph to measure. Chromatographic column (J&W HP-PLOT Molesieve, Agilent), column temperature 80 °C; TCD detector temperature 200 °C; carrier gas nitrogen.

[0023] Table 1 Composition of buffer (1 L)

[0024]

[0025] Preparation of specific solutions:

[0026] (1) Trace element solution (Solution A)

[0027]

[0028]

[0029] Add deionized water to 100 mL

[0030] (2) Buffer solution (Solution B)

[0031] NH 4 HCO 3 4.0 g

[0032] NaHCO 3 35.0 g

[0033] Add deionized water to 1000 mL

[0034] (3) Macroelement solution (Solution C)

[0035]

[0036] (4) 0.1% Resazurin solution (Solution D)

[0037] Add 100 mg of resazurin to 100 mL of deionized water.

[0038] (5) Reducing agent solution (Solution E)

[0039]

[0040] Table 2. Effects of water-extracted ginger leaf extract on total gas production, hydrogen, methane and pH of in vitro rumen fermentation

[0041]

[0042] Groups labeled with different letters a, b, c, d are significantly different (P < 0.05).

[0043] Table 3. Effects of 25% ethanol ginger leaf extract on total gas production, hydrogen, methane and pH of in vitro rumen fermentation

[0044]

[0045]

[0046] Groups labeled with different letters a, b, c, d are significantly different (P < 0.05).

[0047] Table 4. Effects of 50% ethanol ginger leaf extract on total gas production, hydrogen, methane and pH of in vitro rumen fermentation

[0048]

[0049] Groups labeled with a, b, c, d are significantly different (P < 0.05).

[0050] Table 5. Effects of 75% ethanol ginger leaf extract on total gas production, hydrogen, methane and pH of in vitro rumen fermentation

[0051]

[0052] Groups labeled with a, b, c, d are significantly different (P < 0.05).

[0053] Table 6. Effects of 100% ethanol ginger leaf extract on total gas production, hydrogen, methane and pH of in vitro rumen fermentation

[0054]

[0055]

[0056] Groups labeled with a, b, c, d are significantly different (P < 0.05).

[0057] The results are shown in Tables 2 - 6. The water extract of ginger leaf and ethanol extracts with different concentrations significantly inhibited methane production during in vitro rumen fermentation (P < 0.05). The water extract and 25% ethanol extract had no significant effect on total gas production (P > 0.05), but the ethanol extracts of 50% - 100% reduced the total gas production (P < 0.05). All extracts had no significant effect on pH (P > 0.05).

[0058] Example 2 Preparation of ginger leaf extract and animal feeding experiment

[0059] 1. Collection and pretreatment of ginger leaves

[0060] Collect the ginger leaves of Qingzhou Zhugen ginger (Qingzhou, Shandong), dry them at 50 °C for 12 h, and pulverize them through a 2 - mesh sieve.

[0061] 2. Preparation of extract

[0062] Reflux extraction was performed 2 times with 6-fold volume of 75% ethanol for 1.5 hours each time at 75 °C under negative pressure to recover the alcohol. Then, the medicinal residues were extracted with 6-fold volume of water for 1.5 hours once, combined with the alcohol extraction concentrate, and concentrated to a relative density D = 1.1. Spray drying was carried out in a centrifugal spray drying tower, controlling the inlet air temperature at 135 °C and the temperature inside the tower at 80 °C. The product moisture was controlled to be ≤8, and the average extraction rate was 22.08% (prepared by Weinan Andaxin Biotechnology Co., Ltd., Weinan, Shaanxi).

[0063] 3. Animal experiments

[0064] The test site of this example: The Hu sheep test and demonstration base of Huzhou Academy of Agricultural Sciences, Zhejiang (Huzhou, Zhejiang). Test time: December 8, 2024 - January 11, 2025.

[0065] For the original data of this example, Excel (2021) was used to preliminarily process the original data. SPSS 22.0 was used for data analysis. Taking the corresponding production data obtained before the start of the test as covariates, a multivariate model in the general linear model was used for significance testing to evaluate the differences between the two groups. When P < 0.05, it indicates a significant difference.

[0066] In this experiment, 20 healthy Hu sheep (purchased from the Hu sheep test and demonstration base of Huzhou Academy of Agricultural Sciences, Zhejiang) were selected. A completely randomized block design was adopted, with 2 treatment groups, namely the control group and the ginger leaf extract group. First, the 20 Hu sheep were divided into 2 blocks according to the principles of similar parity (5.80 ± 0.61, 5.66 ± 0.29, P = 0.851), body weight (50.10 ± 1.16 kg, 49.44 ± 1.12 kg, P = 0.691), and feed intake (2.43 ± 0.11 kg, 2.48 ± 0.16 kg, P = 0.793). Each block had 10 sheep, and the sheep in each block were randomly assigned to 2 treatment groups, with 10 sheep in each treatment group. The Hu sheep in the control group were fed a basal diet, and the Hu sheep in the ginger leaf extract group were fed a basal diet + 25 g / head / day of ginger leaf extract. The pre-trial period was 1 week, and the formal trial period was 4 weeks. During the trial period, the Hu sheep were fed in individual pens, with free access to food and water. The feeding management was carried out according to the daily management of the sheep farm, and the sheep were fed twice a day (09:00, 17:00).

[0067] 4. Effects of feeding ginger leaf extract on the growth performance and methane production of Hu sheep

[0068] Dry matter intake and collection of dietary samples: During the experiment, on the last two days of each week, the dry matter intake of Hu sheep was measured, and the feeding amount and remaining feed amount were recorded. Dry matter intake = (feeding amount - remaining feed amount) × dry matter content (%). The average dry matter intake was the average value of the dry matter intake measured over four weeks. On the last two days of each week during the experimental period, 500 g of feed samples were collected at 09:10 and 17:10 respectively, and the collected feeds were mixed. The freshly collected feed samples were dried in an oven at 65 °C for 48 h to obtain air-dried samples, which were crushed through a 40-mesh sieve. The feed samples that could not pass through the sieve were ground by themselves and then sieved again, and were stored for subsequent determination of nutrient components.

[0069] Methane determination: The AHC rumen methane determination equipment (Subtropical Agricultural Ecology Institute, Chinese Academy of Sciences, Changsha, Hunan) was used for methane determination. Methane determination was carried out in the last week of the experiment. Before the formal determination began, Hu sheep were adapted to the AHC system for 7 days, and then continuously measured for 6 days. Data were collected at 3:00, 6:00, 9:00, 12:00, 15:00, 18:00, 21:00, and 24:00 every day. For each measurement, the measurement of all Hu sheep was completed within 1.5 h. Each Hu sheep was measured for 5 minutes, and then background gas measurement was carried out for 2 minutes. The methane emissions in Table 7 are the data after deducting the background gas.

[0070] The effects of adding ginger leaf extract to the diet on the production performance and methane production of Hu sheep are shown in Table 7. Compared with the control group, feeding ginger leaf extract had no significant effect on the average dry matter intake and body weight of Hu sheep (P>0.05), but significantly reduced methane emissions (g / d, P = 0.018, a decrease of 36.71%) and methane emissions / average dry matter intake (g / (kg / d), P = 0.030, a decrease of 33.62%).

[0071] Table 7 Effects of adding ginger leaf extract to the diet on the production performance and methane production of Hu sheep

[0072]

[0073] 5. Effects of feeding ginger leaf extract on rumen fermentation parameters of Hu sheep

[0074] On the last day of the experiment, fresh rumen fluid of Hu sheep was collected using an oral collector 3 hours after morning feeding. The pH value was immediately measured using a portable pH meter (HI-9024C, HANNA Instruments, USA), and 5 ml of rumen fluid was stored in a -20 °C refrigerator for measuring rumen fermentation parameters. The concentration of VFAs in the supernatant was measured using a gas chromatograph (model GC-14B, manufactured by Shimadzu, Japan). The parameters of the capillary column used were 30 m × 0.32 mm × 0.25 mm, and the detection conditions were: the column temperature was 110 °C, and the temperatures of the vaporization chamber and the detection chamber were set at 180 °C.

[0075] The effects of adding ginger leaf extract to the diet on the rumen fermentation parameters of Hu sheep are shown in Table 8. Adding ginger leaf extract had no significant effect on the rumen fermentation parameters (P > 0.05).

[0076] Table 8 Effects of adding ginger leaf extract to the diet on the rumen fermentation parameters of Hu sheep

[0077]

[0078] Under the conditions of this experiment, adding ginger leaf extract (25 g / head / day) to the diet had no significant negative effects on the dry matter intake, body weight, and rumen fermentation parameters of Hu sheep, and reduced methane emissions.

Claims

1. Application of ginger leaf extract in the preparation of drugs or products for reducing rumen methane emissions in ruminants.

2. The use according to claim 1, characterized in that: The ginger leaf extract is a water extract or an ethanol extract.

3. The use according to claim 2, characterized in that: The ethanol concentration is 25% to 100%.

4. The use according to claim 2, characterized in that: The ethanol concentration is 50% to 100%.

5. The use according to claim 1, characterized in that: The final concentration of the ginger leaf extract is 0.2-1.6 mg / mL.

6. The use according to claim 1, characterized in that: The final concentration of the ginger leaf extract is 0.8-1.6 mg / mL.

7. A feeding method for reducing rumen methane emission in ruminants, characterized in that: The method comprises the steps of additionally adding ginger leaf extract to normal feed.

8. The method according to claim 7, characterized in that The ginger leaf extract comprises one or more combinations of water extracts or ethanol extracts.

9. The method according to claim 8, characterized in that The ethanol extract comprises a ginger leaf extract obtained by extracting with an ethanol solution with an ethanol concentration of 25% to 100%.

10. The method according to claim 7, characterized in that The dosage of the ginger leaf extract is 25 g / head / day.

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