Preparation method of compound microbial agent and pasture fermented feed

Abstract

The invention provides a preparation method of a compound microbial agent and a pasture fermented feed, and belongs to the technical field of feed fermentation. The complex microbial inoculant disclosed by the invention comprises lactobacillus acidophilus, bacillus siamensis, bacillus amyloliquefaciens and pichia kluyveri. The lactobacillus acidophilus can produce acid and resist acid, and has better antibacterial performance; the bacillus siamensis can degrade lignin and reduce the content of neutral detergent fibers and acid detergent fibers in the fermented feed; the bacillus amyloliquefaciens produces cellulase and amylase with higher activity; the pichia kluyveri can increase the content of crude protein in the fermented feed and improve the nutritional performance of the feed. The four strains have a synergistic effect and jointly ferment forage feed, infectious microbes can be effectively inhibited, the fermented forage feed has excellent color, smell and texture, the palatability of the feed is improved, the appetite of animals is stimulated, and a good growth promoting effect is achieved; the fermented forage feed disclosed by the invention is high in nutritional quality and relatively high in feed utilization rate.

Description

Technical Field

[0001] This invention relates to the field of feed fermentation technology, and in particular to a method for preparing a compound microbial agent and fermented forage feed. Background Technology

[0002] Forage grasses refer to herbaceous plants primarily used by livestock. They are the most important material basis for developing animal husbandry and are known as the "staple food of animal husbandry." The importance of forage grasses as the basic feed for ruminants is the material foundation for the sustainable development of animal husbandry. The efficient utilization of forage grass resources is directly related to the yield, quality, safety, and economic benefits of livestock products. However, forage grass production has obvious seasonal and regional characteristics. During peak forage grass production seasons (such as summer and autumn), forage grass yields are high, but its high water content makes it difficult to store directly. Traditional forage grass preservation methods mainly include silage and hay. Silage is made by chopping up green forage grass with high water content, filling it into sealed silos or bags, and then allowing it to undergo lactic acid fermentation. While silage can preserve nutrients relatively well, its success is highly dependent on the environment. If the sealing and temperature are not properly controlled, it is prone to contamination by miscellaneous bacteria, leading to butyric acid fermentation, mold, and other problems, resulting in nutrient loss and even the production of toxins. Hay is made by harvesting green forage grass and then drying it under natural or artificial conditions. During the drying process, due to weather uncertainties and mechanical damage, a large amount of dry matter and water-soluble vitamins are lost, greatly reducing the nutritional value of hay.

[0003] Microbial fermentation technology, as a green and safe biotechnology, has been widely used in the feed industry. Its core is to improve feed quality by utilizing the metabolic activities of beneficial microorganisms. Early research focused on using single-strain fermentation. However, single-strain fermentation is difficult to handle the complex fermentation environment of forage. For example, lactic acid bacteria can produce acid rapidly, but their ability to decompose cellulose is weak; yeast can improve palatability, but its acid-producing ability is limited. Compound strains are composed of two or more beneficial microorganisms with synergistic effects. Although there are precedents for fermenting forage feed with compound strains, the strain combinations in existing technologies are mostly conventional pairings, such as simple lactic acid bacteria + yeast. There is a lack of effective synergy between the strains, and even antagonism exists. They cannot effectively degrade cellulose and hemicellulose, resulting in little improvement in feed digestibility. Key nutrients such as crude protein and vitamins are severely lost during fermentation. In addition, the fermentation cycle of existing microbial agents is too long, usually requiring more than 30 days, resulting in low fermentation efficiency.

[0004] alfalfa Medicago sativa L .Alfalfa, especially, is known as the "King of Forage" and is one of the most widely cultivated and important legume forages worldwide. It holds an irreplaceable strategic position in animal husbandry, serving as a high-quality source of roughage in the diets of ruminants such as dairy cows, beef cattle, and sheep. As a legume, alfalfa contains a relatively high amount of organic acid salts and proteins. This means that during fermentation, more lactic acid needs to be produced to significantly lower its pH value, increasing the difficulty of inhibiting harmful microorganisms. However, existing compound microbial agents often lack specificity and are general-purpose fermentation agents, resulting in unsatisfactory fermentation effects on alfalfa. Therefore, it is necessary to develop a novel compound microbial agent and fermentation method that can specifically overcome the bottlenecks in alfalfa silage fermentation and achieve high-quality fermentation. Summary of the Invention

[0005] In view of this, the present invention provides a method for preparing compound microbial agents and fermented forage feed to solve the above problems.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a compound microbial inoculant comprising the following components in parts by volume: 10-20 parts of Lactobacillus acidophilus culture, 18-26 parts of Bacillus sicca culture, 30-40 parts of Bacillus amyloliquefaciens culture, and 24-32 parts of Pichia pastoris culture.

[0007] Preferably, the effective viable cell count in the *Lactobacillus acidophilus* culture, *Bacillus sicca* culture, *Bacillus amyloliquefaciens* culture, and *Kluyveromyces oryzae* culture is independently 1 × 10⁻⁶. 9 ~1×10 10 cfu / mL.

[0008] This invention also provides a method for preparing the aforementioned composite microbial agent, comprising inoculating Lactobacillus acidophilus, Bacillus sicca, Bacillus amyloliquefaciens, and Pichia pastoris into a culture medium and culturing until the effective viable count reaches 1×10⁻⁶. 9 ~1×10 10 cfu / mL, mix to obtain the final product.

[0009] The present invention also provides a method for fermenting forage feed, comprising the following steps: (1) Dry the cut forage grass until the moisture content is 55-65%, then crush it and mix it with corn flour to obtain fermentation raw material; (2) Mix the compound microbial agent with water at a volume ratio of 1:0.8~1.2 to obtain a bacterial suspension; then spray the bacterial suspension onto the fermentation raw materials, pack and seal the raw materials, and ferment at 26~30℃ for 8~12 days to obtain the final product.

[0010] Preferably, the forage includes alfalfa and Sudan grass, with a fresh weight ratio of alfalfa to Sudan grass of 1:1.8~2.2.

[0011] Preferably, the corn flour accounts for 6-10% of the total fresh weight of the forage.

[0012] Preferably, the spraying amount of the bacterial suspension is 40~60mL / kg of fermentation raw material.

[0013] Preferably, the alfalfa is harvested from the initial flowering stage to the full flowering stage, and the Sudan grass is harvested from the early heading stage to the full heading stage.

[0014] Preferably, the forage is dried and then shredded into pieces with a length of 2-3 cm.

[0015] The present invention also provides forage feed prepared by the fermentation method described above.

[0016] By adopting the above technical solution, the present invention has the following beneficial effects: (1) Lactobacillus acidophilus, Bacillus sicca, Bacillus amyloliquefaciens and Kluyveromyces synergistically ferment pasture feed, which can effectively inhibit miscellaneous bacteria. The fermented pasture feed has excellent color, aroma and texture, improves the palatability of the feed, stimulates the appetite of animals, and plays a good role in promoting growth. The fermented pasture feed has high nutritional quality and improves feed utilization.

[0017] (2) The fermentation quality of silage made by mixing alfalfa and Sudan grass in a ratio of 1:1.8~2.2 is the best, with a sensory evaluation of high quality and high nutritional value. Detailed Implementation

[0018] The present invention provides a compound microbial inoculant comprising the following components in parts by volume: 10-20 parts of Lactobacillus acidophilus bacterial solution, 18-26 parts of Bacillus sicca bacterial solution, 20-30 parts of Bacillus amyloliquefaciens bacterial solution, and 24-32 parts of Kluyveromyces oryzae bacterial solution; In the compound microbial agent of the present invention, the volume fraction of Lactobacillus acidophilus bacterial solution is 10-20 parts, preferably 13-18 parts, and more preferably 15 parts; The volume fraction of Bacillus sicca bacterial suspension is 18-26 parts, preferably 20-24 parts, and more preferably 22 parts; The volume fraction of Bacillus amyloliquefaciens bacterial solution is 30-40 parts, preferably 33-37 parts, and more preferably 35 parts; The volume fraction of the Kluyveromyces oryzae culture is 24 to 32 parts, preferably 26 to 30 parts, and more preferably 28 parts.

[0019] In this invention, the effective viable cell count in the *Lactobacillus acidophilus* culture, *Bacillus sicca* culture, *Bacillus amyloliquefaciens* culture, and *Pichia pastoris* culture is independently 1 × 10⁻⁶. 9 ~1×10 10cfu / mL, preferably 3~8×10 9 cfu / mL, more preferably 5 × 10⁻⁶ 9 cfu / mL.

[0020] In this invention, *Lactobacillus acidophilus* is capable of producing and resisting acid, exhibits good antibacterial properties, and can promote the decomposition of complex carbohydrates such as cellulose and hemicellulose; *Bacillus sicca* can degrade lignin, reducing the content of neutral detergent fiber and acid detergent fiber in fermented feed; *Bacillus amyloliquefaciens* can produce fatty acids such as lactic acid and acetic acid, and produces cellulase and amylase with high activity; *Kluyveromyces kluyveromyces* can increase the crude protein content in fermented feed, thereby improving the nutritional performance of the feed.

[0021] The four components work synergistically to ferment forage, effectively inhibiting unwanted bacteria. The fermented forage has excellent color, aroma, and texture, improving palatability, stimulating animal appetite, and promoting growth. Fermented forage also has high nutritional quality, improving feed utilization.

[0022] This invention also provides a method for preparing fermented forage feed, comprising the following steps: (1) Dry the cut forage grass until the moisture content is 65-75%, then crush it and mix it with corn flour to obtain fermentation raw material; (2) Mix the compound microbial agent with water at a volume ratio of 1:0.8~1.2 to obtain a bacterial suspension; then spray the bacterial suspension onto the fermentation raw materials, pack and seal the raw materials, and ferment at 26~30℃ for 8~12 days to obtain the final product.

[0023] In this invention, the harvested forage is dried, pulverized, and mixed with corn flour to obtain fermentation raw material. The forage includes alfalfa and Sudan grass. The alfalfa is harvested from the initial flowering stage to the full flowering stage, and the Sudan grass is harvested from the early heading stage to the full heading stage. The fresh weight ratio of alfalfa to Sudan grass is 1:1.8~2.2, preferably 1:1.9~2.1, and more preferably 1:2. After harvesting, the alfalfa and Sudan grass are dried until the moisture content is 55~65%, preferably 58~62%, and more preferably 60%. Then, they are pulverized into segments with a length of 2~3 cm. The pulverized forage is mixed evenly with corn flour to obtain fermentation raw material. The mass of the corn flour is 6~10% of the total fresh weight of the forage, preferably 7~9%, and more preferably 8%.

[0024] In this invention, the composite microbial agent is mixed with water to obtain a bacterial suspension; then the bacterial suspension is sprayed onto the fermentation raw material, bagged, sealed, and compacted for fermentation. The volume ratio of the composite microbial agent to water is 1:0.8~1.2, preferably 1:0.9~1.1, more preferably 1:1, to obtain the bacterial suspension. The bacterial suspension is then sprayed onto the fermentation raw material at a rate of 40~60 mL / kg, preferably 45~55 mL / kg, more preferably 50 mL / kg; then the raw material is bagged, sealed, and compacted for fermentation. The fermentation temperature is 26~30℃, preferably 27~29℃, more preferably 28℃; the fermentation time is 8~12 days, preferably 9~11 days, more preferably 10 days. The product is obtained after fermentation.

[0025] The present invention also provides forage feed prepared according to the fermentation method described herein.

[0026] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0027] The strains used in this invention are sourced from the following sources: Lactobacillus acidophilus was purchased from the China Center for Type Culture Collection, accession number CCTCC No: M2012305; Bacillus amyloliquefaciens was purchased from the China General Microbiological Culture Collection Center, with accession number CGMCC No. 22508; The Siamese Bacillus was purchased from the China Center for Type Culture Collection, accession number CCTCC NO:M20221169; The Kluyveromyces oryzae was purchased from the China Center for Type Culture Collection, accession number CCTCCNO:M2022488.

[0028] The culture medium preparation method in this embodiment of the invention is as follows: MRS liquid medium: 10.00g peptone, 5.00g yeast extract, 10.00g beef extract, 20.00g glucose, 2.00g dipotassium hydrogen phosphate, 1.00g diammonium hydrogen citrate, 5.00g sodium acetate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 1mL Tween 80, bring the volume to 1000mL with distilled water, adjust the pH to 6.4, add 18.0g agar to the solid medium, and sterilize at 121℃ for 20min.

[0029] LB medium: 10g peptone, 5g yeast extract, 10g NaCl, bring the volume to 1000mL with distilled water, adjust the pH to 7.0, add 10.0g agar to the solid medium, and sterilize at 121℃ for 20min.

[0030] PAD medium: Wash, peel, and cut potatoes into pieces. Take 200g of potatoes, add water and boil for 30 minutes. Filter through 6 layers of gauze, collect the filtrate, add 20g of glucose, and make up to 1000mL with distilled water. Adjust the pH to 5.6. Add 15.0g of agar to the solid medium and sterilize at 121℃ for 20 minutes.

[0031] YEPD medium: 10g yeast extract, 20g peptone, 20g glucose, bring to a final volume of 1000mL with distilled water, adjust the pH to 6.0, add 15.0g agar to the solid medium, and sterilize at 121℃ for 20min.

[0032] Example 1. Preparation of bacterial culture Preparation of Lactobacillus acidophilus bacterial culture: Lactobacillus acidophilus was inoculated onto MRS slant agar and cultured at 32℃ for 24 h to obtain a slant culture. The slant culture was then inoculated into MRS liquid agar and cultured for 48 h to obtain a seed culture. The seed culture was then inoculated into MRS liquid agar at a 6% inoculum for expansion culture until the effective viable count reached 5 × 10⁻⁶. 10 Fermentation was stopped after reaching cfu / mL, and the product was set aside for later use.

[0033] Preparation of Bacillus sicca bacterial culture: Bacillus sicca was inoculated onto LB slant agar and cultured at 28°C for 24 h to obtain a slant culture. This slant culture was then inoculated into LB liquid agar and cultured for 48 h to obtain a seed culture. The seed culture was then inoculated into LB liquid agar at a 6% inoculum for expansion culture until the viable cell count reached 5 × 10⁻⁶. 10 Fermentation was stopped after reaching cfu / mL, and the product was set aside for later use.

[0034] Preparation of Bacillus amyloliquefaciens bacterial culture: Bacillus amyloliquefaciens was inoculated onto PAD slant medium and cultured at 28℃ for 24 h to obtain a slant culture. The slant culture was then inoculated into PAD liquid medium and cultured for 48 h to obtain a seed culture. The seed culture was then inoculated into PAD liquid medium at an inoculation rate of 6% for expansion culture until the effective viable count reached 5 × 10⁻⁶. 10 Fermentation was stopped after reaching cfu / mL, and the product was set aside for later use.

[0035] Preparation of Kluyveromyces yeast culture: Kluyveromyces yeast was inoculated onto PAD slant medium and cultured at 28℃ for 24 h to obtain a slant culture. The slant culture was then inoculated into PAD liquid medium and cultured for 48 h to obtain a seed culture. The seed culture was then inoculated into PAD liquid medium at a 6% inoculation rate for expansion culture until the effective viable count reached 5 × 10⁻⁶ cells / year. 10 Fermentation was stopped after reaching cfu / mL, and the product was set aside for later use.

[0036] Example 2 Take 10 kg of Lactobacillus acidophilus culture, 18 kg of Bacillus sicca culture, 30 kg of Bacillus amyloliquefaciens culture, and 24 kg of Kluyveromyces yeast culture prepared in Example 1, mix them evenly, and obtain a compound microbial agent.

[0037] The preparation steps of fermented forage feed are as follows: alfalfa is harvested from the initial flowering stage to the full flowering stage, and Sudan grass is harvested at the early heading stage. The alfalfa and Sudan grass are mixed at a fresh weight ratio of 1:1.8, dried until the moisture content is 55%, crushed into segments with a length of 2-3 cm, and then 6% of the total fresh weight of the forage is added to corn flour. The mixture is then mixed evenly to obtain the fermentation raw material.

[0038] The compound microbial agent was mixed with water at a volume ratio of 1:0.8 to obtain a bacterial suspension. The bacterial suspension was then sprayed onto the fermentation raw material at a rate of 40 mL / kg. The raw material was then bagged, sealed, and compacted, and fermented at 26℃ for 12 days to obtain the final product.

[0039] Example 3 Take 15 kg of Lactobacillus acidophilus culture, 22 kg of Bacillus sicca culture, 35 kg of Bacillus amyloliquefaciens culture, and 28 kg of Kluyveromyces yeast culture prepared in Example 1, mix them evenly, and obtain a compound microbial agent.

[0040] The preparation steps of fermented forage feed are as follows: alfalfa is harvested from the initial flowering stage to the full flowering stage, and Sudan grass is harvested at the early heading stage. The alfalfa and Sudan grass are mixed at a fresh weight ratio of 1:2, dried until the moisture content is 60%, crushed into segments with a length of 2-3 cm, and then 8% of the total fresh weight of the forage is added to the mixture. The mixture is then mixed evenly to obtain the fermented raw material.

[0041] The compound microbial agent is mixed with water at a volume ratio of 1:1 to obtain a bacterial suspension. The bacterial suspension is then sprayed onto the fermentation raw material at a rate of 50 mL / kg. The raw material is then bagged, sealed, and compacted, and fermented at 28℃ for 10 days to obtain the final product.

[0042] Example 4 Take 20 kg of Lactobacillus acidophilus culture, 26 kg of Bacillus sicca culture, 40 kg of Bacillus amyloliquefaciens culture, and 32 kg of Kluyveromyces yeast culture prepared in Example 1, mix them evenly, and obtain a compound microbial agent.

[0043] The preparation steps of fermented forage feed are as follows: alfalfa is harvested from the initial flowering stage to the full flowering stage, and Sudan grass is harvested at the early heading stage. The alfalfa and Sudan grass are mixed at a fresh weight ratio of 1:2.2, dried until the moisture content is 65%, and crushed into segments with a length of 2-3 cm. Then, corn flour of 10% of the total fresh weight of the forage is added and mixed evenly to obtain fermented raw materials.

[0044] The compound microbial agent was mixed with water at a volume ratio of 1:1.2 to obtain a bacterial suspension. The bacterial suspension was then sprayed onto the fermentation raw material at a rate of 60 mL / kg. The raw material was then bagged, sealed, and compacted, and fermented at 30°C for 8 days to obtain the final product.

[0045] Comparative Example 1 Unlike Example 2, no Lactobacillus acidophilus bacterial solution was added to the compound microbial agent.

[0046] Comparative Example 2 Unlike Example 2, no Bacillus sicca bacterial solution was added to the compound microbial agent.

[0047] Comparative Example 3 Unlike Example 2, no Bacillus amyloliquefaciens liquid was added to the compound microbial agent.

[0048] Comparative Example 4 Unlike Example 2, no Kluyveromyces yeast culture was added to the compound microbial agent.

[0049] Comparative Example 5 Unlike Example 2, the compound microbial agent did not contain Lactobacillus acidophilus or Bacillus sicca bacterial solution.

[0050] Comparative Example 6 Unlike Example 2, Bacillus subtilis culture was used instead of Kluyveromyces culture. Bacillus subtilis was purchased from the China General Microbiological Culture Collection Center, accession number CGMCC No. 21218.

[0051] Comparative Example 7 Unlike Example 2, the amount of Lactobacillus acidophilus bacterial solution added was 30 kg. Comparative Example 8 Unlike Example 2, the fresh weight ratio of alfalfa to Sudan grass was 1:4.

[0052] Comparative Example 9 Unlike Example 2, the fresh weight ratio of alfalfa to Sudan grass was 1:0.5.

[0053] Experimental Example 1. Sensory quality evaluation The fermented feeds of the examples and comparative examples were rated based on their color, odor, texture, moisture, and pH value after fermentation. Seven professional reviewers conducted the evaluation, and each indicator was scored in four levels. The specific scoring criteria are shown in Table 1. The average score after removing the lowest and highest scores for each item is shown in Table 1.

[0054] Table 1 Sensory Evaluation Standards for Silage

[0055] Table 2. Grade Scoring Results of Fermented Forage Feed in Different Groups

[0056] Sensory evaluation is a preliminary assessment of the odor, color, and texture of silage. High-quality silage has a well-preserved stem and leaf structure, a color similar to the raw material, a distinct aroma, and no mold growth.

[0057] As shown in Table 1, the fermented forage feed of the present application examples has better odor, color and texture, and the sensory evaluation is excellent. The total score is better than that of comparative examples 1 to 7, indicating that the co-fermentation of forage feed with Lactobacillus acidophilus liquid, Bacillus sicca liquid, Bacillus amyloliquefaciens liquid and Kluyveromyces oryzae can improve the sensory quality of fermented forage. Deleting or replacing any strain, or changing the proportion of each bacterial liquid, will reduce the sensory quality of fermented forage feed.

[0058] 2. Nutritional composition analysis First, the fermented forage feed of each group was dried at a constant temperature of 55℃, crushed and passed through a 0.5mm sieve, and weighed to obtain the dry matter (DM) content. Based on the dry matter content, the crude protein (CP), neutral detergent fiber (NDF), and acid detergent fiber (ADF) in the dry matter were determined. The crude protein (CP) content was determined by the Kjeldahl method. The neutral detergent fiber (NDF) and acid detergent fiber (ADF) content were determined by the Van Soest method. The results of each group are shown in Table 3.

[0059] Table 3 Nutritional quality of fermented forage feed in different groups

[0060] The dry matter content determines the energy density and nutrient concentration of fermented forage feed. Fermented forage feed with less than 25% dry matter is prone to butyric acid production, leading to Clostridium contamination and affecting palatability. Conversely, fermented forage feed with more than 30% dry matter undergoes more thorough fermentation, inhibiting the growth of harmful microorganisms and ensuring feed safety. Crude protein is crucial for assessing nutritional value; its content is positively correlated with the nutritional value of forage—higher crude protein content equates to higher nutritional value. The ratio of ADF (acid detergent fiber) to NDF (neutral detergent fiber) primarily reflects feed digestibility and nutritional value. Specifically, higher ADF indicates lower digestibility, while higher NDF indicates greater feed intake. An imbalance in this ratio can lead to abnormal rumen fermentation and decreased milk fat percentage in ruminants (such as dairy cows). The ADF to NDF ratio should be maintained between 0.6 and 0.8 to ensure rumen health.

[0061] As shown in Table 3, the fermented forage feed prepared in the embodiments of the present invention has higher dry matter and crude protein than the comparative example, lower ADF (acid detergent fiber) and NDF (neutral detergent fiber), and the ratio of ADF to NDF is moderate, which can ensure the rumen health of ruminants.

[0062] 3. Different groups The experiment employed a single-factor variable control approach, selecting 120 Simmental cattle of similar health, body condition, weight (320±3.01) kg, and age (12±1) months as experimental animals. A randomized design was used to divide them into 12 different treatment groups, each containing 10 samples. Diet formulation: The control group was fed a standard basal diet, while the experimental groups received a basal diet supplemented with 50% fermented forage. The basal diet was formulated according to the beef cattle feeding standards (NY / T 815—2004). The basal diet consisted of 64% corn, 9% dried distillers' grains, 16% soybean meal, 8% wheat bran, 0.8% limestone powder, 1% sodium bicarbonate, 0.2% salt, and 1% premix. The premix provided 5000 IU of vitamin A, 10000 IU of vitamin D, 500 IU of vitamin E, 160 mg of iron, 80 mg of zinc, 20 mg of copper, 0.8 mg of selenium, and 2.5 mg of iodine per kilogram of feed.

[0063] The experiment was conducted in March 2024 at Yurun Livestock Co., Ltd. in Longxi County, with a pre-trial period of 7 days and a formal trial period of 90 days. During the pre-trial period, the health status of the experimental cattle herd was observed, and cattle with poor appetite or disease were replaced in a timely manner. Numbering, weighing, grouping, and deworming were carried out. The experimental cattle were housed in individual pens, fattened in stalls, and fed with TMR (Total Mixed Ration) twice a day (08:30 and 17:30), with free access to feed and water. The daily feed intake and uneaten feed were recorded during the trial period.

[0064] On days 1 and 90 of the experiment, the weight of the cattle in a fasting state was measured, and the initial and final weights were recorded. The dry matter intake of the cattle was recorded daily to calculate the average daily feed intake. The average daily weight gain of each cattle was calculated. The average daily feed intake of the cattle was recorded daily to calculate the average daily feed intake, and the average value was calculated. The results are shown in Table 4.

[0065] Average daily feed intake (kg) = Total feed consumption / Number of trial days Average daily weight gain = (final weight - initial weight) / number of days in the trial Feed conversion ratio = Total feed intake / Total weight gain Table 4. Growth performance of beef cattle in each group

[0066] As shown in Table 4, the net weight gain of beef cattle in the embodiments was higher than that in the comparative example, and the feed conversion ratio was lower than that in the comparative example. This indicates that the fermented forage feed of the embodiments of the present invention can improve the appetite of beef cattle and has a high feed utilization rate, which can effectively promote the growth of beef cattle.

[0067] As can be seen from the above embodiments, the present invention provides a method for preparing compound microbial agents and fermented forage feed. The fermented forage feed prepared by the present invention has high quality and nutritional value, high feed utilization rate, and can effectively promote the growth of beef cattle.

[0068] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A compound microbial agent, characterized in that, The components include the following volume fractions: 10-20 parts of Lactobacillus acidophilus culture, 18-26 parts of Bacillus sicca culture, 30-40 parts of Bacillus amyloliquefaciens culture, and 24-32 parts of Bacillus kluyveromyces culture.

2. The compound microbial agent according to claim 1, characterized in that, The effective viable cell count in Lactobacillus acidophilus culture, Bacillus sicca culture, Bacillus amyloliquefaciens culture, and Pichia pastoris culture was independently 1 × 10⁻⁶. 9 ~1×10 10 cfu / mL.

3. The method for preparing the composite microbial agent according to claim 1 or 2, characterized in that, Lactobacillus acidophilus, Bacillus sicca, Bacillus amyloliquefaciens, and Pichia pastoris were inoculated into the culture medium and cultured until the effective viable count reached 1×10⁻⁶. 9 ~1×10 10 cfu / mL, mix to obtain the final product.

4. A method for preparing fermented forage feed, characterized in that, Includes the following steps: (1) Dry the cut forage grass until the moisture content is 55-65%, then crush it and mix it with corn flour to obtain fermentation raw material; (2) Mix the compound microbial agent described in claim 1 or 2 with water at a volume ratio of 1:0.8~1.2 to obtain a bacterial suspension; then spray the bacterial suspension onto the fermentation raw material, pack it into a bag, seal and compact it, and ferment it at 26~30℃ for 8~12 days to obtain the product.

5. The preparation method according to claim 4, characterized in that, The forage grasses include alfalfa and Sudan grass, with a fresh weight ratio of alfalfa to Sudan grass of 1:1.8 to 2.

2.

6. The preparation method according to claim 5, characterized in that, The corn flour accounts for 6-10% of the total fresh weight of the forage.

7. The preparation method according to claim 4, characterized in that, The spraying amount of the bacterial suspension is 40~60mL / kg of fermentation raw material.

8. The preparation method according to claim 5, characterized in that, The alfalfa was harvested from the initial flowering stage to the full flowering stage, and the Sudan grass was harvested from the early heading stage to the full heading stage.

9. The preparation method according to claim 4, characterized in that, After drying, the hay is shredded into pieces 2-3 cm in length.

10. Fermented forage feed prepared according to any one of claims 4 to 9.

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