Application of bacillus subtilis peptide

Abstract

The invention discloses an application of bacillus subtilis peptide in reducing methane emission of ruminants. The invention finds that the methane emission of ruminants can be effectively reduced by adding the bacillus subtilis peptide into the feed.

Description

Technical Field

[0001] The present invention relates to the use of bacitracin. Background Art

[0002] Methane is an important component of greenhouse gases, and ruminants are an important source of methane production. How to safely and efficiently reduce methane emissions from ruminants is very important for promoting the sustainable development of the livestock industry and environmental friendliness.

[0003] Bacitracin is an antibacterial peptide with a clear amino acid sequence produced by Bacillus subtilis. Bacitracin can effectively inhibit Gram-positive bacteria such as Clostridium perfringens, Staphylococcus aureus, and Streptococcus pyogenes. Bacitracin is a feed additive with strong antibacterial effect, high stability, low cytotoxicity, low drug resistance, which can protect the intestinal immune barrier and improve growth performance.

[0004] CN116004412A discloses a method for producing bacitracin using a mixed strain producing sublancin, and it discloses that bacitracin is an immune defense peptide.

[0005] CN117379403A discloses the application of a substance including (-)-Vinigrol and Bacitracin in products for treating autoimmune diseases.

[0006] Currently, there is no relevant report on the use of bacitracin in reducing methane emissions from ruminants. Summary of the Invention

[0007] The purpose of the present invention is to provide a new use of bacitracin. The present invention discovers that adding bacitracin to feed can effectively reduce the methane production of ruminants.

[0008] The present invention achieves the above purpose through the following technical solutions.

[0009] The present invention provides the use of bacitracin in reducing methane emissions from ruminants.

[0010] According to the use of the present invention, preferably, the content of bacitracin in the feed is 0.01 - 0.8 wt%.

[0011] According to the use of the present invention, preferably, the content of bacitracin in the feed is 0.02 - 0.6 wt%.

[0012] According to the use of the present invention, preferably, the content of bacitracin in the feed is 0.04 - 0.5 wt%.

[0013] For the use according to the present invention, preferably, the ruminant is selected from one or more of bovidae, ovidae, camelidae, and cervidae.

[0014] For the use according to the present invention, preferably, the feed comprises bacitracin and a basal feed;

[0015] In the basal feed, the crude protein content is 5-20 wt%, the calcium content is 0.3-1.5 wt%, and the phosphorus content is 0.1-0.8 wt%.

[0016] For the use according to the present invention, preferably, the net energy for milk production of the basal feed is 3-12 MJ / kg.

[0017] For the use according to the present invention, preferably, the methane is produced by gastrointestinal fermentation of the ruminant.

[0018] For the use according to the present invention, preferably, the methane emission is reduced by more than 10 vol%.

[0019] For the use according to the present invention, preferably, the methane emission is reduced by more than 15 vol%.

[0020] Bacitracin is an antibacterial peptide that has an inhibitory effect on Gram-positive bacteria and is usually added to feed as an antibacterial agent. The present invention unexpectedly finds that adding bacitracin to feed can significantly reduce the methane emissions of ruminants. Detailed embodiments

[0021] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0022] Bacitracin (CAS No. 1405-87-4, English name: Bacitracin) is an antibacterial peptide with excellent antibacterial properties. The present invention unexpectedly finds that bacitracin can effectively reduce the methane emissions of ruminants. Therefore, the present invention provides a use of bacitracin in reducing the methane emissions of ruminants.

[0023] The content of subtilin in the feed can be 0.01 - 0.8 wt%; preferably 0.02 - 0.6 wt%; more preferably 0.04 - 0.5 wt%. In some embodiments, the content of subtilin in the feed is 0.02 - 0.03 wt%. In some other embodiments, the content of subtilin in the feed is 0.13 - 0.16 wt%. In still some other embodiments, the content of subtilin in the feed is 0.2 - 0.5 wt%; preferably 0.4 - 0.5 wt%. The present invention finds that if the content of subtilin in the feed is too small, the effect of reducing methane emissions is limited; if the content is too large, the feed intake of ruminants will be reduced, which is also not conducive to reducing methane emissions.

[0024] The ruminants of the present invention can be selected from one or more of Bovidae, Caprinae, Camelidae, Cervidae. Preferably, the ruminants are selected from one or more of Bovidae, Caprinae. More preferably, the ruminants are selected from one or more of dairy cows, beef cattle, sheep, goats. Most preferably, the ruminants are dairy cows or beef cattle. For example, Holstein cows, Jersey cows, water buffalo, yellow cattle, Simmental cattle, Angus cattle, Wagyu cattle, etc. Also, for example, dairy cows in the lactation period.

[0025] Methane can be produced by the fermentation of the gastrointestinal tract of ruminants.

[0026] The emission of methane can be reduced by more than 10 vol%; preferably, the emission of methane can be reduced by more than 15 vol%; more preferably, the emission of methane can be reduced by more than 20 vol%. In some embodiments, the emission of methane can be reduced by more than 22 vol%.

[0027] The feed of the present invention comprises subtilin and a basal feed.

[0028] In the basal feed, the content of crude protein can be 5 - 20 wt%; preferably 8 - 15 wt%; more preferably 10 - 13 wt%.

[0029] In the basal feed, the calcium content can be 0.3 - 1.5 wt%; preferably 0.5 - 1 wt%; more preferably 0.55 - 0.7 wt%.

[0030] In the basal feed, the phosphorus content can be 0.1 - 0.8 wt%; preferably 0.2 - 0.6 wt%; more preferably 0.25 - 0.4 wt%.

[0031] The net energy for milk production of the basal feed can be 3 - 12 MJ / kg; preferably 5 - 10 MJ / kg; more preferably 6 - 8 MJ / kg.

[0032] In addition to bacitracin, the feed of the present invention may also contain a basal feed. The basal feed includes corn silage, beet granules, brewer's grains, soybean meal, rolled corn, corn, calcium hydrogen phosphate, and baking soda. Preferably, the basal feed may also contain additives. More preferably, the feed is composed of the above components.

[0033] The content of corn silage can be 20-40 parts by weight; preferably 25-35 parts by weight; more preferably 30-32 parts by weight. The corn silage is preferably whole-plant corn silage.

[0034] The content of beet granules can be 1-10 parts by weight; preferably 2-8 parts by weight; more preferably 4-6 parts by weight.

[0035] The content of brewer's grains can be 1-10 parts by weight; preferably 2-8 parts by weight; more preferably 4-5 parts by weight.

[0036] The content of soybean meal can be 5-25 parts by weight; preferably 8-20 parts by weight; more preferably 10-15 parts by weight.

[0037] The content of rolled corn can be 15-35 parts by weight; preferably 20-30 parts by weight; more preferably 25-28 parts by weight.

[0038] The content of corn can be 15-35 parts by weight; preferably 20-30 parts by weight; more preferably 22-25 parts by weight.

[0039] The content of calcium hydrogen phosphate can be 0.1-1.5 parts by weight; preferably 0.3-1 part by weight; more preferably 0.5-0.7 part by weight.

[0040] The content of baking soda can be 0.1-0.5 part by weight; preferably 0.15-0.3 part by weight; more preferably 0.17-0.2 part by weight.

[0041] The additives may include vitamin A, vitamin D, vitamin E, Cu, Fe, Mn, Zn, Se, I, Co, and Mg.

[0042] In each kilogram of the basal feed, the content of vitamin A can be 200,000-300,000 IU; preferably 220,000-280,000 IU; more preferably 250,000-270,000 IU.

[0043] In each kilogram of the basal feed, the content of vitamin D can be 300,000-400,000 IU; preferably 320,000-380,000 IU; more preferably 340,000-360,000 IU.

[0044] In each kilogram of the basal feed, the content of vitamin E can be 800 - 1500 IU; preferably 900 - 1300 IU; more preferably 1000 - 1100 IU.

[0045] In each kilogram of the basal feed, the content of Cu can be 0.3 - 4 g; preferably 0.5 - 3 g; more preferably 1 - 2 g.

[0046] In each kilogram of the basal feed, the content of Fe can be 1 - 10 g; preferably 3 - 8 g; more preferably 5 - 7 g.

[0047] In each kilogram of the basal feed, the content of Mn can be 1 - 10 g; preferably 3 - 8 g; more preferably 4 - 6 g.

[0048] In each kilogram of the basal feed, the content of Zn can be 0.5 - 7 g; preferably 1 - 5 g; more preferably 3 - 4 g.

[0049] In each kilogram of the basal feed, the content of Se can be 0.005 - 0.05 g; preferably 0.008 - 0.03 g; more preferably 0.01 - 0.02 g.

[0050] In each kilogram of the basal feed, the content of I can be 0.01 - 0.15 g; preferably 0.02 - 0.1 g; more preferably 0.05 - 0.08 g.

[0051] In each kilogram of the basal feed, the content of Co can be 0.005 - 0.05 g; preferably 0.008 - 0.03 g; more preferably 0.01 - 0.02 g.

[0052] In each kilogram of the basal feed, the content of Mg can be 30 - 80 g; preferably 40 - 60 g; more preferably 50 - 55 g.

[0053] Preparation Example 1

[0054] (1) Preparation of trace element solution: Weigh 13.2 g of CaCl₂·H₂O, 10.0 g of MnCl₂·4H₂O, 8.0 g of FeCl₃·6H₂O and 1.0 g of CoCl₂·6H₂O, dissolve them with distilled water and make up the volume to 100 mL.

[0055] (2) Preparation of buffer solution: Weigh 35.0 g of NaHCO₃ and 4.0 g of NH₄HCO₃, dissolve them with distilled water and make up the volume to 1000 mL.

[0056] (3) Preparation of macroelement solution: Weigh 6.2 g of KH₂PO₄, 5.7 g of Na₂HPO₄ and 0.6 g of MgSO₄·7H₂O, dissolve them with distilled water and make up the volume to 1000 mL.

[0057] (4) Preparation of resazurin solution: Dissolve 100 mg of resazurin in distilled water and make up the volume to 100 mL.

[0058] (5) Preparation of reducing solution: Dissolve 2.0 mL of 1 mol / L NaOH solution and 312.5 mg of Na2S·9H2O in 47.5 mL of distilled water.

[0059] (6) Preparation of culture medium: Preheat the mixture of 0.12 mL of trace element solution, 237.00 mL of buffer solution, 237.00 mL of macroelement solution, 1.22 mL of resazurin solution and 474.00 mL of distilled water in a water bath to 39 °C, and pass CO2 until saturated. When the color of the mixture changes from dark blue to light pink (about 1 h), add the reducing solution and mix well. At this time, the color of the mixture changes to light yellow or colorless, and the culture medium is obtained.

[0060] Example 1

[0061] Select 3 Holstein cows with rumen fistulas, mix the rumen fluid after 2 h of morning feeding, and then filter it through four layers of gauze and put it into a thermos bottle filled with CO2 for standby.

[0062] Divide the culture bottles (volume 120 mL) into six groups, with 6 culture bottles in each group, and put 1 g of fermentation substrate into each culture bottle; among them, the fermentation substrate in the first group of culture bottles is the basal substrate, and the fermentation substrates in the second to sixth groups of culture bottles are composed of bacitracin and the basal substrate. The contents of bacitracin in the fermentation substrates of the second to sixth groups of culture bottles are 0.01 wt%, 0.02 wt%, 0.04 wt%, 0.08 wt% and 0.16 wt% of the fermentation substrate respectively. The basal substrate is composed of 10 parts by weight of alfalfa, 70 parts by weight of corn and 20 parts by weight of soybean meal.

[0063] Mix the taken rumen fluid and culture medium at a volume ratio of 1:2, and then use a continuous pipettor to dispense them into culture bottles, 60 mL for each culture bottle. During the dispensing process, continuously pass CO2 to ensure anaerobic conditions. Place the culture bottles containing the fermentation substrate, rumen fluid and culture medium in an AGRS-III type 64-channel microbial fermentation micro gas production automatic recording device and software system for in vitro batch culture. After continuously passing CO2 into the fermentation bottle for 5 s, immediately cover the bottle cap. Connect the culture bottle to the sensor corresponding to the recording device and continuously culture at 39 °C. Use a gas collection bag to collect the gas generated by fermentation. After 24 h of fermentation, quickly take out the corresponding culture bottle from the culture environment and terminate the fermentation in an ice bath.

[0064] Calculate the methane production and methane content using the following formula:

[0065] Methane production = Methane% × (Gas production + 60 mL)

[0066] Methane content = (Gas production + 60 mL) × Methane percentage / Gas production;

[0067] Among them, Methane percentage represents the proportion of methane in the gas in the gas collection bag, with the unit of vol%; Gas production represents the volume of the gas collected in the gas collection bag, with the unit of mL; 60 mL is the volume of the original gas in the culture bottle.

[0068] The methane production and methane content of the first to sixth groups are shown in Table 1.

[0069] Table 1

[0070]

[0071] Example 2

[0072] Select 120 healthy Holstein lactating cows. From the 1st day to the 8th day, feed them with basal diet every day, and measure the methane emissions of each cow as the initial value. According to the parity, days in milk, and initial methane emissions, divide them into 6 groups, with 20 cows in each group, ensuring that there is no difference in the initial methane production among groups.

[0073] From the 9th day to the 30th day, the 1st group is fed with basal diet, and the 2nd to 6th groups are fed with the diet composed of bacitracin and basal diet. The contents of bacitracin in the diets fed to the 2nd to 6th groups are 0.02 wt%, 0.04 wt%, 0.1 wt%, 0.2 wt%, and 0.5 wt% of the diet respectively. From the 23rd day, measure the methane production of cows every day as the experimental value. Use the GreenFeed system to measure the methane production. The average methane production of each cow per day is shown in Table 3.

[0074] The composition of the basal diet is shown in Table 2:

[0075] Table 2

[0076] Raw Material Composition Content (parts by weight) Whole-plant Corn Silage 30.0 Beet Granules 4.5 Brewer's Grains 5 Soybean Meal 10.7 Pressed Corn 26.55 Corn 22.12 Calcium Hydrogen Phosphate 0.5 Baking Soda 0.17 Additive —

[0077] Among them, the additives include vitamin A, vitamin D, vitamin E, Cu, Fe, Mn, Zn, Se, I, Co, and Mg. In every kilogram of basal diet, it contains 250,000 IU of vitamin A, 340,000 IU of vitamin D3, 1000 IU of vitamin E, 1 g of Cu, 5 g of Fe, 4 g of Mn, 3 g of Zn, 0.01 g of Se, 0.05 g of I, 0.01 g of Co, and 50 g of Mg.

[0078] In the basal diet, the crude protein content is 11.74 wt%, the calcium content is 0.56 wt%, and the phosphorus content is 0.29 wt%. The net energy for milk production is 6.56 MJ / kg.

[0079] Table 3

[0080] First Group Second Group Third Group Fourth Group Fifth Group Sixth Group Methane (g / d) 478 413 397 426 405 371

[0081] The present invention is not limited to the above embodiments, and any variations, improvements, or substitutions that can be conceived by those skilled in the art without departing from the essence of the present invention fall within the scope of the present invention.

Claims

1. Use of a subtilisin peptide in reducing methane emissions from ruminants.

2. The use according to claim 1, characterized in that The content of the subtilisin in the feed is 0.01-0.8 wt%.

3. The use according to claim 1, characterized in that The content of the subtilisin in the feed is 0.02-0.6 wt%.

4. The use according to claim 1, characterized in that The content of the subtilisin in the feed is 0.04-0.5wt%.

5. The use according to claim 1, characterized in that: The ruminant is selected from one or more of bovines, ovines, camelids, and deer.

6. The use according to any one of claims 2 to 5, characterized in that: The feed comprises subtilisin and basic feed; The basic feed has a crude protein content of 5-20 wt%, a calcium content of 0.3-1.5 wt%, and a phosphorus content of 0.1-0.8 wt%.

7. The use according to claim 6, characterized in that The net energy of milk production of the basic feed is 3 to 12 MJ / kg.

8. The use according to claim 1, characterized in that The methane is produced by fermentation in the gastrointestinal tract of the ruminant.

9. The use according to claim 1, characterized in that The methane emission is reduced by more than 10 vol%.

10. The use according to claim 1, characterized in that The methane emission is reduced by more than 15 vol%.

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