Preparation and application of carbon emission reduction composition sustained release preparation for ruminant livestock farm

Abstract

The invention belongs to the field of biological feed, and discloses application of P.megatherium in preparation of an additive for inhibiting generation of greenhouse gas in animal waste. The P.megatherium is preserved in the Guangdong Microbial Culture Collection Center on April 18, 2025, and the preservation number is GDMCC NO: 66160. According to the present invention, the P.megatherium has efficient enzyme production capacity, and can efficiently decompose cellulose after the P.megatherium is planted in the rumen so as to significantly reduce the carbon emission of ruminants. Meanwhile, the probiotics with various enzyme production characteristics and propionibacterium are further compounded, so that the obtained intestinal probiotic composition has an obvious synergistic effect, and carbon emission is obviously reduced. Finally, a composition containing a fermentation product, an intestinal probiotic composition and a microcapsule with an insect grease slow-release function acts on the intestinal tract, and carbon emission of ruminants can be remarkably reduced.

Description

Technical Field

[0001] This application relates to the field of biofeed, specifically to a novel use of Priestella megaterium, an intestinal probiotic composition, and an emission reduction composition. Background Technology

[0002] Priestella megaterium is mostly used for soil remediation, such as its contribution to plant growth in saline-alkali land, or for the decomposition of pollutants; relevant literature can be found in the following materials:

[0003] The publication number is CN120536293A, and the subject is a species of Priestella megaterium and its application in the treatment of pig farm manure. It describes the application of Priestella megaterium in reducing the wastewater in pig farms.

[0004] The applicant previously filed a patent application, publication number CN120836648A, which relates to a method for efficiently preparing earthworm functional liquid using endogenous probiotics and enzymes in the earthworm gut, and the earthworm functional liquid itself. This method utilizes the efficient protease production capacity of Priestella megaterium to decompose earthworm proteins to produce small peptides.

[0005] Further research on Priestella megaterium revealed its significant role in aquaculture, leading to this invention. Summary of the Invention

[0006] One of the objectives of this application is to provide a new use for *Priestella megaterium*, which has a highly efficient enzyme-producing capacity and can effectively reduce carbon emissions in ruminants when it colonizes the intestines.

[0007] Generally, carbon emissions from ruminants mainly originate from the rumen and intestines. The probiotics of this invention are primarily used to reduce carbon emissions generated in the intestines.

[0008] Meanwhile, by further combining various probiotics with enzyme-producing characteristics and Propionibacterium acnes, the present invention yields an intestinal probiotic composition with a more significant carbon emission reduction function.

[0009] Finally, the present invention also provides a composition containing fermentation products, a gut probiotic composition, and microcapsules with insect oil slow-release function, which can significantly reduce carbon emissions in ruminants when applied to the intestines.

[0010] To achieve the above objectives, this application provides the use of *Priestella megaterium* in the preparation of intestinal probiotics or feed additives; the intestinal probiotics or feed additives are intestinal probiotics or feed additives used to reduce carbon emissions in ruminants; the *Priestella megaterium* was deposited at the Guangdong Provincial Center for Microbial Culture Collection on April 18, 2025, with accession number: GDMCC NO: 66160.

[0011] In addition, the present invention provides an intestinal probiotic composition for reducing carbon emissions in ruminants, comprising Propionibacterium catarrhalis, Bacillus belye XY1, and Priestella megaterium; wherein the live bacteria ratio of Propionibacterium catarrhalis, Bacillus belye XY1, and Priestella megaterium is 1~5:1~5:1~5.

[0012] In addition, the present invention also provides a slow-release formulation of a composition for reducing carbon emissions from ruminants, comprising the following components in parts by weight:

[0013] 20-30 parts of Aspergillus niger fermentation product and / or Aspergillus oryzae fermentation product;

[0014] One to two parts of the intestinal probiotic composition as described above; wherein the live bacteria content of the intestinal probiotic composition is 1.1 × 10⁻⁶. 9 ~1.5×10 9 cfu / g, preferably 1.3 × 10⁻⁶ 9 ~1.5×10 9 cfu / g.

[0015] 1-5 portions of sustained-release microcapsules;

[0016] The sustained-release microcapsules comprise a shell material composed of sodium alginate, chitosan, and tannic acid, as well as insect oil encapsulated within the shell material.

[0017] In the above-mentioned sustained-release formulation, the weight ratio of the shell material of the sustained-release microcapsule to the insect oil is 10~15:20~40.

[0018] In the above-mentioned sustained-release formulation, the method for preparing the sustained-release microcapsules is as follows:

[0019] Step 1: Heat chitosan, insect oil, emulsifier, and water to 50-60℃ and stir to form an emulsion;

[0020] Step 2: Add the sodium alginate aqueous solution to the emulsion from Step 1 and stir. After stirring, homogenize.

[0021] Step 3: Add tannic acid dropwise to the system in Step 2 to solidify sodium alginate and chitosan. Let it stand to form layers, remove the supernatant to obtain microcapsule powder and dry it.

[0022] In the above-mentioned sustained-release formulation, the mass ratio of chitosan to sodium alginate is 1:0.8~1.2; the mass ratio of chitosan to tannic acid is 1:0.05~0.15; the stirring speed is 500~1000 rpm; the homogenization speed is 6000~10000 rpm; and the homogenization time is 3~5 min.

[0023] In the above-mentioned sustained-release formulation, the emulsifier is Tween-80 or Tween-60; the amount of the emulsifier is 2-5% of the insect oil; and the insect oil is black soldier fly larvae oil.

[0024] Furthermore, the present invention also provides the use of slow-release formulations of the compositions described above in the preparation of feed additives or feeds.

[0025] Finally, the present invention also provides a ruminant feed, which is supplemented with the intestinal probiotic composition as described above or a slow-release formulation of the composition as described above.

[0026] Beneficial effects

[0027] Compared with the prior art, this application has at least the following advantages:

[0028] The *Priestella megaterium* strain of this invention has a highly efficient enzyme-producing capacity, and when it colonizes the intestines, it can effectively reduce carbon emissions in ruminants.

[0029] This invention further combines various probiotics with enzyme-producing properties and Propionibacterium to obtain an intestinal probiotic composition that has a more significant function in reducing carbon emissions.

[0030] The present invention also describes a composition containing fermentation products, intestinal probiotics, and microcapsules with insect oil slow-release function that acts on the intestines, which can significantly reduce carbon emissions in ruminants. Detailed Implementation

[0031] The present application will be further described below with reference to embodiments, but this does not constitute any limitation on the present application. Any limited modifications made within the scope of the claims of the present application shall still be within the scope of the claims of the present application.

[0032] To illustrate the technical content of this application in detail, the following description is provided in conjunction with the embodiments.

[0033] The preparation method of the inclusion body is as follows:

[0034] Step 1: Heat 5g chitosan, 30g black soldier fly larvae oil, 1.5g Tween-60, and water to 60℃ and stir at 800rpm to form an emulsion;

[0035] Step 2: Add 50g of sodium alginate aqueous solution (10wt%) to the emulsion in Step 1 and stir. Maintain the temperature at 60℃. After stirring, homogenize at 8000rpm for 4min.

[0036] Step 3: Add 0.5g of tannic acid to the system in Step 2, stir continuously for 30min to solidify sodium alginate and chitosan, let stand to form layers, remove the supernatant to obtain microcapsule powder and dry it.

[0037] The Bacillus velezensis XY1 involved in this invention is derived from pigs and was deposited at the Guangdong Provincial Microbial Culture Collection Center on November 14, 2023, with accession number GDMCC NO: 64017 and classified as Bacillus velezensis; the Priestia megaterium was deposited at the Guangdong Provincial Microbial Culture Collection Center on April 18, 2025, with accession number GDMCC NO: 66160 and classified as Priestia megaterium; the Propionibacterium velezensis (HZ-P-35) was derived from Xi'an Mixianer Biotechnology Co., Ltd.

[0038] The Aspergillus oryzae fermentation product was obtained from Guangzhou Huiwang Biotechnology Co., Ltd., and its specification is protease activity ≥1000U / g.

[0039] Experimental Groups:

[0040] Blank group: No product processing added.

[0041] Product A (Test Group A): *Priscilla megaterium*; viable count: 1.45 × 10⁻⁶ 9 cfu / g;

[0042] Product B (Experimental Group B): *Priscilla megaterium*, *Bacillus belyssus* XY1, and *Propionibacterium tumefaciens*; *Priscilla megaterium*, *Bacillus belyssus* XY1, and *Propionibacterium tumefaciens* were activated separately, and the activated bacteria were mixed. The live bacteria ratio of *Priscilla megaterium*, *Bacillus belyssus* XY1, and *Propionibacterium tumefaciens* was approximately 1:1:1; the total live bacteria count was 1.42 × 10⁻⁶. 9 cfu / g;

[0043] Product C (Experimental Group C): Priestella megaterium, Bacillus belye XY1, Propionibacterium acnes and Aspergillus oryzae fermentation products;

[0044] Priestella megaterium, Bacillus belye XY1, and Propionibacterium tumefaciens were activated separately. The activated bacteria were then mixed, with the viable ratio of Priestella megaterium, Bacillus belye XY1, and Propionibacterium tumefaciens approximately 1:1:1; the total viable count was 1.35 × 10⁻⁶. 9The total weight of *Priestella megaterium*, *Bacillus belyssus* XY1, and *Propionibacterium micranthum*, and the weight ratio of *Aspergillus oryzae* fermentation products was 25:1.5.

[0045] Product D (Experimental Group D): Priestella megaterium, Bacillus belye XY1, Propionibacterium micranthum, Aspergillus oryzae fermentation products and inclusions.

[0046] *Priscilla megaterium*, *Bacillus belyssus* XY1, and *Propionibacterium tumefaciens* were activated separately. The activated bacteria were then mixed, resulting in a viable ratio of approximately 1:1:1 between *Priscilla megaterium*, *Bacillus belyssus* XY1, and *Propionibacterium tumefaciens*; the total viable count was 1.32 × 10⁻⁶. 9 The total weight of *Priestella megaterium*, *Bacillus belyssus* XY1, and *Propionibacterium micranthum*, and the weight ratio of *Aspergillus oryzae* fermentation products and inclusions were 25:1.5:3.

[0047] Product E (Experimental Group E): Priestella megaterium, Aspergillus oryzae fermentation products and inclusions.

[0048] Priestella megaterium; viable count 1.45 × 10⁻⁶ 9 The weight ratio of CFU / g of fermentation products and inclusions of *Priestella megaterium* and *Aspergillus oryzae* was 25:1.5:3.

[0049] Performance Test 1: In Vitro Fecal Test

[0050] 1. Sample collection: Take 10 kg of manure from each of the large-scale cattle farm and the small-to-medium-scale sheep farm, put them into sterile bags, and store them in the refrigerator for later use.

[0051] 2. Preparation of solid-state mixed fermentation manure materials: A certain amount (1000g) of cow manure and sheep manure were added to a micro-mixer, respectively. A certain amount of product (A, B, C, D, E) was added at 1% of each product, adjusting the moisture content to 40-45% and the carbon-to-nitrogen ratio to 22:1. The mixtures were thoroughly stirred to obtain cow and sheep manure mixtures, which were then placed into anaerobic fermentation bags and labeled as cow manure mixture treatment group (T1) and sheep manure mixture treatment group (T2). A blank control group of cow and sheep manure mixtures was also set up. Each treatment group had 30 replicates. The mixtures were transferred to a controlled environment room for static fermentation at a temperature of 35℃ and a humidity of 55% for 3 days. The indicators to be measured were then determined. Note: The blank control group did not add any products; the operation method was the same as the treatment groups.

[0052] 3. Indicator testing methods:

[0053] (1) CO2 gas detection: A pump-type CO2 gas detector is used. Pump-type CO2 gas detector (brand: PuLiTong; model: PLT300) range: 0-5000PPM;

[0054] (2) CH4 gas detection: Gas from the anaerobic fermentation bag was collected using sterile gas collection bags and tested according to the method of the National Ecological and Environmental Standard of the People's Republic of China (HJ1331-2023). Methane gas value is expressed in ppm, and 1 μmol / mol is equivalent to 1 ppm.

[0055] 4. Results analysis: The test results are shown in Table 1 and Table 2.

[0056] Table 1. Evaluation of CO2 and CH4 gases in the anaerobic fermentation bags of the cow manure mixture treatment group (T1) (unit: ppm)

[0057] Group <![CDATA[CO2 gas]]> <![CDATA[CH4 gas]]> Blank group +++++++ +++++++ Group A ++++ ++++ Group B +++++ +++++ Group C +++ +++ Group D + + Group E ++ ++

[0058] Note: All data are expressed as mean ± standard deviation. Methane gas values ​​are expressed in ppm, where 1 μmol / mol is equivalent to 1 ppm. 0-5 ppm is indicated by "+", 5-10 ppm by "++", 10-30 ppm by "+++", 30-50 ppm by "++++", 50-70 ppm by "+++++", 80-100 ppm by "++++++", and >100 ppm by "+++++++".

[0059] Table 2. Evaluation of CO2 and CH4 gases in anaerobic fermentation bags of sheep manure mixture treatment group (T2) (unit: ppm)

[0060] Group <![CDATA[CO2 gas]]> <![CDATA[CH4 gas]]> Blank group ++++++ ++++++ Group A +++++ +++++ Group B ++++ +++ Group C +++ +++ Group D + + Group E ++ +

[0061] Note: All data are expressed as mean ± standard deviation. Methane gas values ​​are expressed in ppm, where 1 μmol / mol is equivalent to 1 ppm. 0-5 ppm is indicated by "+", 5-10 ppm by "++", 10-30 ppm by "+++", 30-50 ppm by "++++", 50-70 ppm by "+++++", 80-100 ppm by "++++++", and >100 ppm by "+++++++".

[0062] Aquaculture experiment

[0063] A pilot-scale application demonstration was conducted at a large-scale (open-style) dairy farm in Longgui Town, Wujiang District, Shaoguan City. The experiment was divided into an experimental group and a control group. One dairy farm with 1000 cows was selected as the experimental group, and another farm with approximately 1000 cows was selected as the control group. All cows in both farms were multiparous cows from the same batch, fed the same self-prepared feed, and allowed free access to feed. The experiment lasted 30 days. Before the start of the experiment, sterile gas collection bags were used to collect gas from the front, middle, and rear areas of both dairy farms, as well as the fan vents. Thirty gas bags (capacity: 1L / bag) were collected from each area and fan vent as the control group. After the experiment, gas was collected at the same points using the same method. All samples were sent back to the laboratory for testing and evaluation on the same day. Data are expressed as mean ± standard deviation.

[0064] Product application effect evaluation:

[0065] (1) CO2 gas reduction rate (degree of reduction): (CO2 data of blank group - CO2 data of experimental group) / CO2 data of blank group × 100%.

[0066] (2) CH4 gas reduction rate (degree of reduction): (CH4 data of blank group - CH4 data of experimental group) / CH4 data of blank group × 100%.

[0067] The results are shown in Table 3.

[0068] Table 3. Assessment of CO2 and CH4 gases in large-scale dairy farms (unit: %)

[0069] Group <![CDATA[CO2 gas]]> <![CDATA[CH4 gas]]> experimental group 56.42±4.86% 74.30±5.54%

[0070] Note: All data are expressed as mean ± standard deviation. Methane gas values ​​are expressed in ppm, where 1 μmol / mol is equivalent to 1 ppm.

[0071] A pilot-scale application demonstration was conducted at a large-scale (open-style) black goat farm in Wushi Town, Qujiang District, Shaoguan City. The experiment was divided into an experimental group and a control group. One shed with 800 black goats was used as the experimental group, and another shed with approximately 800 goats was used as the control group. All goats in both sheds were from the same batch, fed the same self-prepared feed, and had free access to feed. The experiment lasted 30 days. Before the start of the experiment, sterile gas collection bags were used to collect gas from the front, middle, and rear areas of both goat sheds, as well as the fan vents. Thirty gas bags (capacity: 1L / bag) were collected from each area and fan vent as the control group. After the experiment, gas was collected at the same points using the same method. All samples were sent back to the laboratory for testing and evaluation on the same day. Data are expressed as mean ± standard deviation.

[0072] Product application effect evaluation:

[0073] (1) CO2 gas reduction rate (degree of reduction): (CO2 data of blank group - CO2 data of experimental group) / CO2 data of blank group × 100%

[0074] (2) CH4 gas reduction rate (degree of reduction): (CH4 data of blank group - CH4 data of experimental group) / CH4 data of blank group × 100%.

[0075] The results are shown in Table 4:

[0076] Table 4. Assessment of CO2 and CH4 gases in large-scale black goat farms (unit: %)

[0077] Group <![CDATA[CO2 gas]]> <![CDATA[CH4 gas]]> experimental group 58.16±4.47% 78.22.±6.09

[0078] Note: All data are expressed as mean ± standard deviation.

[0079] As can be seen from the above experiments, the sustained-release formulation of the present invention has good emission reduction effect.

[0080] Summarize:

[0081] 1. This invention is the first to demonstrate through in vitro experiments that *Priscilla megaterium* has an emission reduction effect, especially in the treatment of cow manure, where its effect is superior to that of compound bacteria.

[0082] 2. This invention demonstrates that the combined use of *Priestella megaterium*, *Bacillus belye* XY1, and *Propionibacterium tumefaciens* has a more significant emission reduction effect. This significant effect is reflected in the treatment of sheep manure and the combined use with *Aspergillus oryzae* fermentation products and inclusions.

[0083] 3. Animal experiments have demonstrated that the product of this invention has a good emission reduction effect.

[0084] The embodiments presented herein are merely selected implementations based on combinations of all possible embodiments. The appended claims should not be limited to the embodiments described herein. Some numerical ranges used in the claims include sub-ranges within them, and variations within these ranges should also be covered by the appended claims.

Claims

1. Use of *Priestella megaterium* in the preparation of an additive for inhibiting greenhouse gas production in animal feces; said *Priestella megaterium* was deposited at the Guangdong Provincial Center for Microbial Culture Collection on April 18, 2025, with accession number: GDMCC NO: 66160.

2. The use according to claim 1, characterized in that, The greenhouse is otherwise filled with methane or carbon dioxide.

3. A probiotic composition for reducing carbon emissions in ruminants, characterized in that, It is composed of Propionibacterium tumefaciens, Bacillus belye XY1, and Priestella megaterium; the live bacteria ratio of Propionibacterium tumefaciens, Bacillus belye XY1, and Priestella megaterium is 1~5:1~5:1~5.

4. A sustained-release formulation of a composition for reducing carbon emissions from ruminants, characterized in that, Includes the following components by weight: 20-30 parts of Aspergillus niger fermentation product and / or Aspergillus oryzae fermentation product; One to two parts of the intestinal probiotic composition as described in claim 2; wherein the live bacteria content of the intestinal probiotic composition is 1.1 × 10⁻⁶. 9 ~1.5×10 9 cfu / g; 1-5 portions of sustained-release microcapsules; The sustained-release microcapsules comprise a shell material composed of sodium alginate, chitosan, and tannic acid, as well as insect oil encapsulated within the shell material.

5. The sustained-release formulation of the composition according to claim 4, characterized in that, The weight ratio of the shell material to insect oil in the sustained-release microcapsules is 10~15:20~40.

6. The sustained-release formulation of the composition according to claim 5, characterized in that, The method for preparing the sustained-release microcapsules is as follows: Step 1: Heat chitosan, insect oil, emulsifier, and water to 50-60℃ and stir to form an emulsion; Step 2: Add the sodium alginate aqueous solution to the emulsion from Step 1 and stir. After stirring, homogenize. Step 3: Add tannic acid dropwise to the system in Step 3 to solidify sodium alginate and chitosan. Let it stand to form layers, remove the supernatant to obtain microcapsule powder and dry it.

7. The sustained-release formulation of the composition according to claim 6, characterized in that, The mass ratio of chitosan to sodium alginate is 1:0.8~1.2; the mass ratio of chitosan to tannic acid is 1:0.05~0.15; the stirring speed is 500~1000 rpm; the homogenization speed is 6000~10000 rpm; and the homogenization time is 3~5 min.

8. The sustained-release formulation of the composition according to claim 6, characterized in that, The emulsifier is Tween-80 or Tween-60; the amount of the emulsifier is 2-5% of the insect oil; the insect oil is black soldier fly larvae oil.

9. Use in the preparation of an additive for inhibiting greenhouse gas production in animal feces using a sustained-release formulation of the composition as described in any one of claims 4 to 8.

10. An additive for inhibiting the production of greenhouse gases in animal feces, characterized in that, The formulation contains the intestinal probiotic composition as described in claim 2 or the sustained-release formulation of the composition as described in any one of claims 4 to 8.