CN116531436A

CN116531436A - Biological agent for ruminant rumen regulation and preparation method thereof

Info

Publication number: CN116531436A
Application number: CN202310566428.1A
Authority: CN
Inventors: 王诚; 王玲; 孙华; 王杰; 王峰; 仉弦; 徐吉荣; 王宁; 周磊
Original assignee: Shandong Jianyuan Biological Technology Co ltd
Current assignee: Shandong Jianyuan Biological Technology Co ltd
Priority date: 2023-05-19
Filing date: 2023-05-19
Publication date: 2023-08-04
Anticipated expiration: 2043-05-19
Also published as: CN116531436B

Abstract

The invention discloses a biological agent for ruminant rumen regulation and a preparation method thereof, belonging to the technical field of bioengineering. The biological agent is prepared from bean dreg-orange peel zymolyte, saccharomyces cerevisiae bacterial liquid, bacillus subtilis bacterial liquid and enterococcus faecium bacterial liquid according to the weight ratio of (10-50) g:1mL:0.1 mL:0.1 mixing the mL to obtain the mixture; the preparation method of the bean dreg-orange peel zymolyte comprises the following steps: crushing fresh orange peel, uniformly mixing with fresh bean dregs, and standing at room temperature to obtain a mixture; and adding pepsin and cellulase into the mixture for enzymolysis, inactivating enzyme after the enzymolysis is finished, centrifuging, taking supernatant, and concentrating to obtain bean dreg-orange peel zymolyte. According to the invention, the waste bean dregs and orange peel raw materials are subjected to enzymolysis, so that the obtained enzymolysis product can obviously improve the rumen fermentation level of ruminants regulated and controlled by saccharomyces cerevisiae, and further improve the milk yield and milk quality of ruminants.

Description

Biological agent for ruminant rumen regulation and preparation method thereof

Technical Field

The invention relates to the technical field of bioengineering, in particular to a biological agent for ruminant rumen regulation and control and a preparation method thereof.

Background

Ruminant rumen is a complex anaerobic fermentation system that occupies an important place in ruminant nutrition research. The feeding of ruminant diets mostly comes from rumen fermentation end products, especially short chain fatty acids and microbial proteins, which directly affect the nutritional feeding of ruminants. Short chain fatty acids provide the main source of metabolic energy for the host, while microbial proteins are the main source of metabolic amino acids and synthetic amino acids in milk for the host body. Therefore, it is desired to improve the utilization efficiency and productivity of ruminant nutrition by studying rumen fermentation regulation.

With the importance of animal product safety and awareness of resistance to antibiotics, feed microorganisms are gradually started to increase rumen fermentation in ruminants. At present, mainly accepted feed microorganisms at home and abroad mainly comprise bacillus (Bacillus subtilis), enterococcus (Enterococcus faecium), lactobacillus (Lactobacillus plantarum), pediococcus (Pediococcus) and streptococcus (Streptococcus infantarius) and saccharomycetes (Saccharomyces cerevisiae) and the like. The microorgan preparation for ruminant is mainly microorgan, and the most studied microorgan is Saccharomyces cerevisiae, but the application effect of Saccharomyces cerevisiae additive is unstable, and it has positive effect and no effect. However, it has been reported that the use of Saccharomyces cerevisiae improves the milk yield and milk quality of Holstein cows, so that Saccharomyces cerevisiae is a currently mainly used microorganism for regulating rumen even if its effect is unstable. Therefore, how to improve the effect of the saccharomyces cerevisiae, regulate and control the rumen fermentation level of ruminants more effectively, and maintain high milk yield is a problem to be solved.

Disclosure of Invention

Aiming at the prior art, the invention aims to provide a biological agent for regulating and controlling rumen of ruminant and a preparation method thereof. The invention uses cheap bean dregs and orange peel as raw materials for enzymolysis, the obtained enzymolysis product can obviously improve the rumen fermentation level of ruminants regulated by Saccharomyces cerevisiae, and further improve the milk yield and milk quality of ruminants.

In order to achieve the above purpose, the invention adopts the following technical scheme:

in a first aspect of the present invention, there is provided a biological agent for ruminant rumen control, comprising the following raw materials:

bean dreg-orange peel zymolyte, saccharomyces cerevisiae bacterial liquid, bacillus subtilis bacterial liquid and enterococcus faecium bacterial liquid; the ratio of the addition amounts of the bean dreg-orange peel zymolyte, the saccharomyces cerevisiae bacterial liquid, the bacillus subtilis bacterial liquid and the enterococcus faecium bacterial liquid is (10-50) g:1mL:0.1 mL:0.1 mL; the bean dreg-orange peel zymolyte is obtained by enzymolysis of bean dreg and orange peel with pepsin and cellulase;

the Saccharomyces cerevisiae bacterial liquid is prepared from the following components with the preservation number of CCTCC NO: culturing Saccharomyces cerevisiae (Saccharomyces cerevisiae) of M2016460;

the bacillus subtilis bacterial liquid is prepared from the bacterial liquid with the preservation number of CICC NO: culturing bacillus subtilis (Bacillus subtilis) of 10732;

the enterococcus faecium bacterial liquid is prepared from the bacterial liquid with the preservation number of CICC NO:20430 enterococcus faecium (Enterococcus Faecium).

Preferably, the brewThe concentration of the wine yeast liquid is more than or equal to 10 ⁸ cfu/mL; the concentration of the bacillus subtilis bacterial liquid is more than or equal to 2 x 10 ⁸ cfu/mL; the concentration of the enterococcus faecium bacterial liquid is more than or equal to 2 x 10 ⁸ cfu/mL。

In a second aspect of the present invention, there is provided a method for preparing a biological agent for ruminant rumen modulation, the method comprising: uniformly mixing bean dreg-orange peel zymolyte, saccharomyces cerevisiae bacterial liquid, bacillus subtilis bacterial liquid and enterococcus faecium bacterial liquid to obtain a biological preparation for ruminant rumen regulation;

the bean dreg-orange peel zymolyte is prepared by the following method:

(1) Crushing fresh orange peel, uniformly mixing with fresh bean dregs, and standing at room temperature to obtain a mixture;

(2) And (3) adding pepsin and cellulase into the mixture obtained in the step (1) for enzymolysis, inactivating enzyme after the enzymolysis is finished, centrifuging, taking supernatant, and concentrating to obtain the bean dreg-orange peel zymolyte.

Preferably, in the step (1), the water content of the bean dregs is 50-70wt%; the water content of the orange peel is 15-25wt%; preferably, the mass ratio of the bean dregs to the orange peel is (1-9): 1.

preferably, in the step (1), the standing time is 6-12 hours.

Preferably, in the step (2), the mass ratio of the pepsin to the cellulase is 1:1, a step of; the addition amount of the pepsin accounts for 3-7wt% of the total mass of the bean dregs and the orange peel.

Preferably, in the step (2), the enzymolysis temperature is 30-40 ℃ and the enzymolysis time is 24-48 h.

Preferably, in step (2), the concentration is to: the relative density is 1.15-1.25 measured at 50 ℃.

In a third aspect of the invention, there is provided the use of a biological agent for ruminant rumen control in at least one of the following 1) to 3):

1) Promoting rumen ammoniacal nitrogen metabolism;

2) The propionic acid content is improved;

3) Improving milk yield and milk quality.

The invention has the beneficial effects that:

(1) The invention adopts cheap bean dregs and orange peel raw materials for fermentation, does not need to add plant extracts or reagents with high price or complex extraction process, and utilizes pepsin and cellulase for enzymolysis to prepare an enzymolysis product, thereby obviously improving the rumen fermentation level of ruminants regulated by Saccharomyces cerevisiae and further improving the milk yield and milk quality of ruminants.

(2) The biological agent has no toxic or side effect, simple preparation method, low cost and long-term use; can promote ammonia nitrogen metabolism of rumen, increase propionic acid content, and increase milk yield and milk quality.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As introduced in the background art section, the yeast is low in price and easy to obtain, so that the milk yield and the milk quality of the Holstein lactating cows are improved, but the saccharomyces cerevisiae has unstable effect in the application of regulating and controlling the rumen of ruminants, and has a positive effect report and an effect report.

Based on the above, the invention aims to provide a biological agent for ruminant rumen regulation and a preparation method thereof. In the early stage of research, the effect of the saccharomyces cerevisiae used alone is unstable, the effect of the saccharomyces cerevisiae is improved to a certain extent after the bacillus subtilis and the enterococcus faecium are added, and various cheap raw materials such as bean dregs, corncobs and wheat bran are tested for reducing the cost, and are subjected to enzymolysis and then are matched with the saccharomyces cerevisiae, the bacillus subtilis and the enterococcus faecium for use, but the effect is not improved too much. In the test process, the inventor mistakenly mixes the orange peel broken slag with the bean dreg raw material subjected to standing treatment, and finds that the orange peel is removed after the bean dreg is subjected to enzymolysis treatment, and continues the test, and unexpectedly finds that the effect of regulating and controlling the rumen of ruminants is improved, and the milk yield of Holstein lactation cows is also improved. Therefore, the inventor takes bean dregs and orange peel as raw materials, the preparation method is continuously improved, and the finally obtained biological preparation can obviously promote rumen ammoniacal nitrogen metabolism, improve propionic acid content, and improve milk yield and milk quality.

In the invention, the time of standing after mixing the bean dregs and the orange peel cannot be too long, the protein of the bean dregs can deteriorate, and the bean dregs can only be used as fertilizer for crops. Subsequent researches show that the standing time is controlled to be 6-12 hours, and the orange peel is acidic, so that natural fermentation can be carried out when the orange peel and the bean dregs are mixed and stood, the standing time is strictly controlled, the biological agent cannot be prepared when the time is too long, the bean dregs are easy to excessively ferment, and the orange peel is easy to grow hair. According to the invention, the bean dregs and the orange peel are mixed and kept stand for several hours, so that more beneficial bacteria can be obtained through natural fermentation, an acidic environment can be formed, then pepsin and cellulase are added, enzymolysis is carried out in the acidic environment, the active ingredients obtained after the enzymolysis of the bean dregs and the orange peel can obviously improve the capability of the saccharomyces cerevisiae for regulating and controlling the rumen of ruminants, and as the bean dregs and the orange peel are waste, the cost of biological agents can be greatly reduced, and the waste can be effectively utilized.

In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

Description:

the strain deposit number of the saccharomyces cerevisiae (Saccharomyces cerevisiae) is: cctccc NO: m2016460, from China center for type culture Collection.

The deposit number of the bacillus subtilis is CICC NO:10732; enterococcus faecium has a deposit number of CICC NO:20430, all from China center for type culture Collection of microorganisms.

Tests show that the variety of orange peel has little influence on test results, and the orange peel used in the invention is poncirus trifoliata peel.

The test materials used in the examples of the present invention are all conventional in the art and are commercially available.

Example 1: preparation of bacterial liquid

Saccharomyces cerevisiae (the inoculation amount accounts for 10% of the total mass of the PDA culture medium) is subjected to shaking culture at 30deg.C and 120r/min to obtain strain concentration of 10 ⁸ cfu/mL, the saccharomyces cerevisiae bacteria liquid is obtained.

Bacillus subtilis (the inoculation amount accounts for 15% of the total mass of the LB culture medium) is subjected to shake culture at 37 ℃ and 165r/min by using the LB culture medium until the bacterial concentration is 2×10 ⁸ cfu/mL to obtain bacillus subtilis bacterial liquid.

Enterococcus faecium (inoculum size is 15% of total mass of MRS culture medium) is subjected to static culture at 37deg.C until bacterial concentration is 2×10 ⁸ cfu/mL to obtain enterococcus faecium bacterial liquid.

Example 2

(1) 400g of fresh orange peel (with the water content of about 22 wt%) is crushed to 10-20 meshes, the crushed orange peel is added into 2kg of fresh bean dregs (with the water content of about 60 wt%) and mixed uniformly, and the mixture is stood for 9 hours at room temperature (25 ℃) to obtain the mixture.

(2) Adding 120g pepsin and 120g cellulase into the mixture, performing enzymolysis at 35 ℃ for 36 hours, inactivating enzyme at 110 ℃ for 2 minutes after the enzymolysis is completed, centrifuging, collecting supernatant, and concentrating to a relative density of 1.20 (measured at 50 ℃) to obtain bean dreg-orange peel zymolyte.

The bean dreg-orange peel zymolyte, the saccharomyces cerevisiae bacterial liquid prepared in the example 1, the bacillus subtilis bacterial liquid prepared in the example 1 and the enterococcus faecium bacterial liquid prepared in the example 1 are prepared according to the following weight ratio of 30g:1mL:0.1 mL:0.1 And (3) mixing the materials uniformly to obtain the biological preparation for ruminant rumen regulation.

Example 3

(1) 1kg of fresh orange peel (water content about 25 wt%) was pulverized, then added to 2kg of fresh okara (water content about 70 wt%) and mixed uniformly, and left to stand at room temperature for 6 hours to obtain a mixture.

(2) Adding 90g of pepsin and 90g of cellulase into the mixture obtained in the step (1), carrying out enzymolysis for 24 hours at 37 ℃, inactivating enzyme at 110 ℃ for 2 minutes after the enzymolysis is finished, centrifuging, taking supernatant, and concentrating until the relative density is 1.15 (measured at 50 ℃), thus obtaining the bean dreg-orange peel zymolyte.

The bean dreg-orange peel zymolyte, the saccharomyces cerevisiae bacterial liquid prepared in the example 1, the bacillus subtilis bacterial liquid prepared in the example 1 and the enterococcus faecium bacterial liquid prepared in the example 1 are prepared according to the following weight ratio of 10g:1mL:0.1 mL:0.1 And (3) mixing the materials uniformly to obtain the biological preparation for ruminant rumen regulation.

Example 4

(1) 300g of fresh orange peel (with the water content of about 16 wt%) is crushed, then added into 2.7kg of fresh bean dregs (with the water content of about 50 wt%) and mixed uniformly, and then left to stand for 12 hours at room temperature to obtain a mixture.

(2) And (3) adding 210g of pepsin and 210g of cellulase into the mixture obtained in the step (1), carrying out enzymolysis for 48 hours at 32 ℃, inactivating enzyme at 110 ℃ for 2 minutes after the enzymolysis is finished, centrifuging, taking supernatant, and concentrating to a relative density of 1.25 (measured at 50 ℃) to obtain the bean dreg-orange peel zymolyte.

The bean dreg-orange peel zymolyte, the saccharomyces cerevisiae bacterial liquid prepared in the example 1, the bacillus subtilis bacterial liquid prepared in the example 1 and the enterococcus faecium bacterial liquid prepared in the example 1 are prepared according to 50g:1mL:0.1 mL:0.1 And (3) mixing the materials uniformly to obtain the biological preparation for ruminant rumen regulation.

Comparative example 1

The saccharomyces cerevisiae bacterial liquid prepared in example 1, the bacillus subtilis bacterial liquid prepared in example 1 and the enterococcus faecium bacterial liquid prepared in example 1 are mixed according to the weight ratio of 1mL:0.1 mL:0.1 And (3) mixing the materials uniformly to obtain the biological preparation for ruminant rumen regulation.

Comparative example 2

2kg of fresh okara (water content: about 60% by weight) was allowed to stand at room temperature for 9 hours. Adding 100g pepsin and 100g cellulase, performing enzymolysis at 35deg.C for 36 hr, inactivating enzyme at 110deg.C for 2min, centrifuging, collecting supernatant, concentrating to relative density of 1.20 (measured at 50deg.C), and obtaining bean dreg hydrolysate.

The bean dreg zymolyte, the saccharomyces cerevisiae bacterial liquid prepared in the example 1, the bacillus subtilis bacterial liquid prepared in the example 1 and the enterococcus faecium bacterial liquid prepared in the example 1 are prepared according to the following weight ratio of 30g:1mL:0.1 mL:0.1 And (3) mixing the materials uniformly to obtain the biological preparation for ruminant rumen regulation.

Comparative example 3

400g of fresh orange peel (with the water content of about 22 wt%) is crushed to 10-20 meshes and kept stand for 9h at room temperature (25 ℃). Adding 20g pepsin and 20g cellulase, performing enzymolysis at 35deg.C for 36 hr, inactivating enzyme at 110deg.C for 2min, centrifuging, collecting supernatant, concentrating to relative density of 1.20 (measured at 50deg.C), and obtaining pericarpium Citri Tangerinae zymolyte.

Orange peel zymolyte, saccharomyces cerevisiae bacterial liquid prepared in example 1, bacillus subtilis bacterial liquid prepared in example 1 and enterococcus faecium bacterial liquid prepared in example 1 are prepared according to the following weight ratio of 30g:1mL:0.1 mL:0.1 And (3) mixing the materials uniformly to obtain the biological preparation for ruminant rumen regulation.

Comparative example 4

(1) Adding 100g of pepsin and 100g of cellulase into 2kg of fresh bean dregs (the water content is about 60 wt%) for enzymolysis for 36h at 35 ℃, inactivating enzyme at 110 ℃ for 2min after the enzymolysis is finished, centrifuging, taking supernatant, and concentrating to a relative density of 1.20 (measured at 50 ℃) to obtain bean dreg zymolyte;

crushing 400g of fresh orange peel (with the water content of about 22 wt%) to 10-20 meshes, adding 20g of pepsin and 20g of cellulase, carrying out enzymolysis for 36h at 35 ℃, carrying out enzyme deactivation at 110 ℃ for 2min after the enzymolysis is finished, centrifuging, taking supernatant, and concentrating to the relative density of 1.20 (measured at 50 ℃), thus obtaining the orange peel enzymatic hydrolysate.

(2) The bean dreg zymolyte and the orange peel zymolyte are processed according to a proportion of 5: and mixing the materials according to the mass ratio of 1 to obtain a mixed zymolyte. Mixing the zymolyte, the saccharomyces cerevisiae bacterial liquid prepared in the example 1, the bacillus subtilis bacterial liquid prepared in the example 1 and the enterococcus faecium bacterial liquid prepared in the example 1 according to the weight ratio of 30g:1mL:0.1 mL:0.1 And (3) mixing the materials uniformly to obtain the biological preparation for ruminant rumen regulation.

Comparative example 5

400g of sun-dried orange peel (with the water content of about 6 wt%) is crushed to 10-20 meshes, 2kg of dried bean dregs (with the water content of about 6 wt%) is crushed and uniformly mixed to obtain a bean dregs-orange peel mixture.

The bean dreg-orange peel mixture, the saccharomyces cerevisiae bacterial liquid prepared in example 1, the bacillus subtilis bacterial liquid prepared in example 1 and the enterococcus faecium bacterial liquid prepared in example 1 are mixed according to the weight ratio of 30g:1mL:0.1 mL:0.1 And (3) mixing the materials uniformly to obtain the biological preparation for ruminant rumen regulation.

Comparative example 6

Reflux-extracting soybean powder with 80% ethanol for 2 times, each time for 1.5h, wherein the feed liquid ratio of 80% ethanol to soybean powder is 16:1, filtering the extract to obtain the soybean extract.

Pulverizing orange peel to 10-20 meshes, adding distilled water (feed-liquid ratio is 10:1), heating to 100deg.C, extracting for 2 times, extracting 30 mm each time, filtering, and concentrating to relative density of 1.20 (measured at 50deg.C), to obtain orange peel extract.

Mixing soybean extract and pericarpium Citri Tangerinae extract at a ratio of 5:1, and mixing to obtain a mixed extract. The extract, the Saccharomyces cerevisiae bacterial liquid prepared in example 1, the Bacillus subtilis bacterial liquid prepared in example 1 and the enterococcus faecium bacterial liquid prepared in example 1 are mixed according to 30g:1mL:0.1 mL:0.1 And (3) mixing the materials uniformly to obtain the biological preparation for ruminant rumen regulation.

Test examples

110 healthy Holstein cows with a weight of about 600Kg and a lactation period of about 60 days are selected and randomly divided into 11 groups for free-range. The blank group was fed with a total mixed ration (daily average lactation yield: 26.5.+ -. 2.5 Kg), the control group was fed with a total mixed ration+Saccharomyces cerevisiae, examples 2 to 4 and comparative examples 1 to 6 were fed with a total mixed ration (composition see Table 1) +biological agents prepared in each group, respectively, and 200mg/Kg was added.

TABLE 1 composition and nutrient levels of fully mixed ration

Note that: each kilogram of premix comprises: VA800000IU, VD700000IU, VE10000IU, fe 1600mg,Cu 1500mg,Zn 10000mg,Mn 3500mg,Se 80mg,I120mg,Co 50mg.

Pre-testing period 10d, testing period 20d, free feeding, recording milk yield every day, and calculating average value; milk produced in each group was removed daily, and milk cream rate (%) and milk protein (%) were measured by a Milkway-3 rapid milk ingredient analyzer. Setting the measured instant temperature of milk between 20-30 ℃, detecting each sample three times, and taking an average value; the milk fat percentage (%) and the milk protein (%) of the milk produced on average per day were calculated. The results obtained are shown in Table 2.

Table 2 milk production statistics of cows

Experiments on ammoniacal nitrogen and volatile fatty acids were performed with reference to the method specified in the 3.3 assay methods in daily ration, influence of addition of different yeast cultures on the performance of dairy cows in mid-and later-lactation, blood biochemical index and rumen fermentation (Bichuan, 2018):

on the last day of the test, after fasting 8h, 200mL of rumen fluid content was collected from the oral cavity with an inserted gastric pouring tube. The rumen fluid collected by filtration is filtered by using four layers of gauze, the filtered rumen fluid is centrifuged for 15min at 4000r/min, 1mL of supernatant is taken and added into a sample bottle, 4.5mL of HCL (hydrogen chloride) is added into the sample bottle, and the solution is uniformly mixed for measuring ammonium nitrogen. Simultaneously, 4mL of the centrifuged supernatant was removed by a pipette and added to the sample bottle, followed by mixing with lmL% metaphosphoric acid, and preserving at-20℃for measurement of Volatile Fatty Acids (VFA).

And (3) placing a sample stored at the temperature of minus 20 ℃ at room temperature for melting, taking 0.4mL of mixed solution into a 10mL test tube by pipetting, sequentially adding 2 mL of A solution and 2 mL of B solution into the test tube, shaking uniformly, standing for 10 minutes, and carrying out colorimetric comparison under the condition of 700nm in wavelength by using a 722 spectrophotometer.

And (3) manufacturing a standard curve: accurately weighing 0.382g of ammonia chloride, dissolving the ammonia chloride by using 0.2M hydrochloric acid solution, and fixing the volume to 100 mL, wherein the nitrogen content is lmg/mL. And diluting the 10mL preservation solution to 100 mL by using distilled water, and taking the solution as a working solution, namely the solution with the nitrogen content of 10mg/100 mL. Sequentially taking 0,1,2,4 and 6 mL of working solution, respectively placing the working solution and the working solution into 50 mL volumetric flasks, respectively adding 10,9,8,6 and 4mL of distilled water, then using 0.2M hydrochloric acid to fix the volume, taking 0.4mL of each solution into a l 0mL test tube, and sequentially adding the solution A and the solution B to carry out color comparison. And (3) taking absorbance as an independent variable, taking the nitrogen content of the solution as a dependent variable, and taking a standard curve to obtain a regression formula.

A liquid A; 0.089g of sodium nitrosoferricyanide was weighed and dissolved in 100 mL of 14% sodium salicylate solution.

And (2) liquid B: 1.2g of sodium hydroxide was weighed and dissolved in 100 mL distilled water to give a 0.3M sodium hydroxide solution, and then 2 mL sodium hypochlorite solution was added.

The test uses an internal standard method to measure volatile fatty acid, and the internal standard used in the test is crotonic acid. Centrifuging rumen fluid 20 mL at 4000r/min for 15min, adding 4mL rumen supernatant into 1mL mixed solution prepared from 25% metaphosphoric acid and formic acid at 3:l, mixing, standing for 40min, collecting 1mL mixed solution, and adding 2g acidic adsorbent (Na ₂ SO ₄ ：50% H ₂ S0 ₄ : diatomaceous earth 30:1:20) and 40mL crotonic acid solution (solution CH ₃ Cl ₃ ) Shaking, standing until the mixture is clear, and measuring by a machine. The measurement was performed using a Shimadzu GC-7A gas chromatograph. The chromatographic column is a stainless steel column with the inner diameter of 3mm and the length of 2 m; the column temperature is 150 ℃, the vaporization chamber temperature is 230 ℃, the air pressure is 0.35kg/cm, the flow is 140mL/min, and the hydrogen pressure is 1.2 kg/cm ² The flow rate was 14mL/min. N (N) ₂ The flow rate of the carrier is 55mL/min, and the sample injection amount is L mu L. The results obtained are shown in Table 3.

TABLE 3 rumen acid content and Ammonia Nitrogen content statistics

As can be seen from tables 2 to 3, the biological agents of the groups 1 to 3 of examples not only can effectively reduce ammoniacal nitrogen in the rumen of cows, but also can effectively adjust the content of volatile fatty acids, and the acetic acid/propionic acid ratio is significantly reduced. And the milk yield and the milk quality are obviously improved. The invention shows that the enzyme hydrolysis product obtained by mixing and fermenting the bean dregs and the orange peel and then carrying out the enzyme hydrolysis can obviously improve the capability of the saccharomyces cerevisiae in regulating and controlling the rumen fermentation level.

The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the same, but rather, various modifications and variations may be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A biological agent for ruminant rumen regulation, which is characterized by comprising the following raw materials:

2. The biological agent according to claim 1, wherein the concentration of the Saccharomyces cerevisiae bacteria liquid is not less than 10 ⁸ cfu/mL; the concentration of the bacillus subtilis bacterial liquid is more than or equal to 2 x 10 ⁸ cfu/mL; the concentration of the enterococcus faecium bacterial liquid is more than or equal to 2 x 10 ⁸ cfu/mL。

3. The method of preparing a biological agent according to claim 1 or 2, characterized in that the method of preparing is: uniformly mixing bean dreg-orange peel zymolyte, saccharomyces cerevisiae bacterial liquid, bacillus subtilis bacterial liquid and enterococcus faecium bacterial liquid to obtain a biological preparation for ruminant rumen regulation;

the bean dreg-orange peel zymolyte is prepared by the following method:

4. The preparation method of claim 3, wherein in the step (1), the water content of the bean dregs is 50-70wt%; the water content of the orange peel is 15-25wt%.

5. The preparation method of claim 3, wherein in the step (1), the mass ratio of the bean dregs to the orange peel is (1-9): 1.

6. the method according to claim 3, wherein in the step (1), the standing time is 6 to 12 hours.

7. The method according to claim 3, wherein in the step (2), the mass ratio of pepsin to cellulase is 1:1, a step of; the addition amount of the pepsin accounts for 3-7wt% of the total mass of the bean dregs and the orange peel.

8. The preparation method of claim 3, wherein in the step (2), the enzymolysis temperature is 30-40 ℃ and the enzymolysis time is 24-48 h.

9. The method according to claim 3, wherein in the step (2), the concentration is carried out at 50℃to a relative density of 1.15 to 1.25.

10. Use of the biological agent for ruminant rumen control according to claim 1 or 2 in at least one of the following 1) to 3):

1) Promoting rumen ammoniacal nitrogen metabolism;

2) The propionic acid content is improved;

3) Improving milk yield and milk quality.