Composite acid-resistant bacterial agent additive and application thereof in complete feed for lactating cows

Abstract

The application provides a composite acid-resistant bacterial agent additive and application thereof in full-price feed of lactating cows, and belongs to the technical field of microbial feed additives. The composite acid-resistant bacterial agent additive comprises 30-50 parts of modified apple pomace, 20-40 parts of palm cake, 25-35 parts of rapeseed meal, 5-15 parts of sunflower oil, 5-10 parts of kudzu flower extract, 3-5 parts of composite acid-resistant bacterial agent and 1-3 parts of medical stone. The acid-resistant composite bacterial agent contains lactobacillus plantarum, neurospora crassa and acid-resistant saccharomyces cerevisiae, directly colonizes the small intestine, inhibits and kills pathogenic bacteria, secretes enzymes to degrade anti-nutritional factors, produces carotenoids to promote lactation, and improves feed palatability. The modified apple pomace is prepared by screw extrusion-alkali treatment-composite enzymolysis, the fiber structure is destroyed, the porous structure provides a colonization site for bacterial bodies, the bacterial activity is improved, and the incidence of cows is reduced. The kudzu flower extract has antioxidant and lactation-promoting effects, the sunflower oil and the carotenoids produced by bacteria have synergistic antioxidant effects, and the immune ability, milk yield and milk quality of cows are improved.

Description

Technical Field

[0001] This invention belongs to the field of microbial feed additive technology, and in particular relates to a compound acid-resistant bacteria additive and its application in complete feed for lactating dairy cows. Background Technology

[0002] In recent years, my country's dairy farming industry has accelerated its transformation towards large-scale, intensive, and standardized operations, with a steady increase in the milk yield per lactating cow. However, the prevention and control of metabolic diseases, the improvement of feed conversion efficiency, and the implementation of antibiotic-free farming under high-yield farming models have become core issues restricting the high-quality development of the industry. To meet the energy requirements of high-yield dairy cows, the proportion of concentrate in the diet has generally increased to 40% to 60%, which easily leads to a large accumulation of volatile fatty acids (VFA) in the rumen, frequent pH fluctuations, and an incidence of subacute rumen acidosis (SARA) of 15% to 30%. This directly results in decreased fiber digestibility, reduced milk fat percentage, increased somatic cell count, and even induces secondary diseases such as laminitis and liver abscess, causing significant economic losses to farms.

[0003] Probiotic preparations, by regulating the rumen microbiota, improving internal environmental homeostasis, and enhancing nutrient degradation efficiency, possess multiple benefits including promoting growth, improving quality, and reducing disease. They represent a core technological pathway to replace antibiotics in feed, and their application in ruminant farming continues to expand. The core mechanism of exogenous probiotics is to supplement functional microorganisms, optimize the microbial community structure, and strengthen metabolic pathways, thereby achieving a dual enhancement of rumen digestive function and immune barrier. Currently, single or compound probiotic products such as lactic acid bacteria, Bacillus, and Saccharomyces cerevisiae are widely used in complete dairy cow feed; however, these strains cannot stably colonize and continuously metabolize in the rumen, resulting in poor milk yield and quality. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a compound acid-resistant bacteria additive and its application in the complete feed for lactating dairy cows.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a compound acid-resistant bacteria additive, comprising the following components in parts by weight: 30-50 parts modified apple pomace, 20-40 parts palm meal, 25-35 parts rapeseed meal, 5-15 parts sunflower seed oil, 5-10 parts kudzu flower extract, 3-5 parts compound acid-resistant bacteria agent, and 1-3 parts maifanite.

[0006] Preferably, the compound acid-resistant bacteria agent includes Lactobacillus plantarum, acid-resistant Saccharomyces cerevisiae, and Neurospora crassa. The mass ratio of *Lactobacillus plantarum*, acid-resistant *Saccharomyces cerevisiae*, and *Neurospora crassa* is 2-4:1-3:2-4; the viable counts of *Lactobacillus plantarum*, acid-resistant *Saccharomyces cerevisiae*, and *Neurospora crassa* are all ≥0.5-1.5 × 10⁻⁶. 9 CFU / g.

[0007] Preferably, the method for preparing the modified apple pomace includes the following steps: (1) The apple pomace is extruded and expanded to obtain pretreated apple pomace; (2) The pretreated apple pomace and alkaline solution are mixed to obtain the secondary treated apple pomace; (3) The apple pomace that has undergone secondary treatment is adjusted to pH 4.8-5.0 with citric acid, enzymatically hydrolyzed with a compound enzyme preparation, and spray-dried to obtain modified apple pomace.

[0008] Preferably, the moisture content of the apple pomace in step (1) is 20-30%; the temperature of the extrusion and puffing feeding section is 70-90℃, the temperature of the compression section is 110-130℃, the temperature of the extrusion section is 110-130℃, the screw speed is 160-200 r / min, the working pressure inside the machine is 1.0-1.2 MPa, and the diameter of the discharge die is 8-10 mm.

[0009] Preferably, the alkaline solution in step (2) comprises 1%~2% calcium hydroxide and 0.5%~1% sodium hydroxide, the mass ratio of the pretreated apple pomace to the alkaline solution is 1:8~10, and the mixing time is 24~48 h.

[0010] Preferably, the compound enzyme preparation comprises 30-40 U / g cellulase and 90-100 U / g pectinase, the mass ratio of the secondary treated apple pomace to the compound enzyme preparation is 1:12-18, the enzymatic hydrolysis temperature is 40-60℃, and the enzymatic hydrolysis time is 2-4 h.

[0011] This invention also provides a method for preparing the aforementioned composite acid-resistant bacteria additive, comprising the following steps: (1) The modified apple pomace, palm meal, rapeseed meal, sunflower seed oil, kudzu flower extract and maifanite were mixed to obtain a mixture; (2) Mix the mixed materials and the compound acid-resistant bacteria agent, ferment them, and obtain the compound acid-resistant bacteria agent additive.

[0012] Preferably, the fermentation temperature is 30~35℃ and the fermentation time is 36~60h.

[0013] This invention also provides the application of the aforementioned compound acid-resistant bacteria additive in the preparation of complete feed for lactating dairy cows.

[0014] This invention also provides the application of the aforementioned compound acid-resistant bacteria additive in improving milk yield and milk quality in dairy cows.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The acid-resistant compound microbial agent of this invention possesses acid-resistant properties that enable the strains to resist the low pH of the rumen and the highly acidic environment of the abomasum. Entering the small intestine as live bacteria, they rapidly recover and proliferate, competitively inhibiting pathogens such as *Escherichia coli* and *Salmonella*. Metabolism produces organic acids such as lactic acid and acetic acid, lowering the intestinal pH, constructing an acidic barrier, inhibiting the growth of harmful microorganisms, and simultaneously promoting the absorption of minerals such as calcium and phosphorus. Secreted proteases and amylases assist in the decomposition of anti-nutritional factors in feed, improving nutrient utilization. Among them, *Lactobacillus plantarum* possesses extremely high growth rate and acid-producing capacity, regulating pH, inhibiting putrefactive bacteria activity, significantly reducing ammonia nitrogen production, minimizing protein decomposition losses, and efficiently decomposing phytic acid, thereby increasing the content and absorption efficiency of nutrients. *Neurospora crassa* has a strong ability to produce carotenoids, which can act on the mammary gland cells of dairy cows, stimulating their growth, improving their lactation capacity, and thus increasing milk yield and quality. *Neurospora crassa* can also effectively decompose cellulose, improving its absorption efficiency. Lactobacillus plantarum can produce phytase, which can effectively decompose phytic acid in palm meal and rapeseed meal, improving the digestibility and absorption of minerals. Acid-resistant brewer's yeast can decompose cellulose and increase protein content under high acidity fermentation, producing aromas of alcohol and fruit, as well as volatile fatty acids, which significantly stimulates the appetite of livestock and poultry and improves palatability.

[0016] This invention modifies apple pomace. During the modification process, under the combined effects of shearing, compression, and heat from screw extrusion, the hydrogen bonds between cellulose molecules break, the crystalline regions disintegrate, and insoluble dietary fiber is converted into soluble fiber. Pectin molecules are moderately degraded, and the material undergoes instantaneous pressure release through the die to form a porous and loose structure, significantly improving digestibility. Simultaneously, the contact area of ​​the apple pomace is increased, providing more contact sites for subsequent mixing with alkali solutions and enzyme preparations, thus improving modification efficiency. The alkali solution penetrates deep into the pores and intercellular spaces of the apple pomace fibers, dissolving / degrading the pectin clogging the pores, further loosening the fiber pore structure, allowing deep impurities within the pores to be fully exposed and washed away. Intramolecular and intermolecular hydrogen bonds in cellulose molecules break, and the ester bonds between hemicellulose and lignin hydrolyze and dissolve, reducing fiber crystallinity and increasing porosity. Simultaneously, calcium ions can complex and precipitate with tannins, efficiently removing astringent and anti-nutritional factors from the apple pomace. Further enzymatic hydrolysis using cellulase and pectinase further breaks down and degrades the structure of cellulose and hemicellulose, generating small-molecule sugars and other substances that increase the palatability of apple pomace. The large surface area and porous structure of the modified apple pomace provide more space for bacterial colonization and survival, enhancing the activity and metabolic capacity of the strains, reducing the content of pathogenic bacteria in the gut, and thus further reducing the incidence of disease in dairy cows.

[0017] The kudzu flower extract of this invention contains isoflavones, which can inhibit the rapid release of ammonia nitrogen in the rumen, improve nitrogen utilization, enhance antioxidant properties, and also have estrogen-like activity, which can help increase milk yield and milk protein content. Sunflower seed oil contains vitamin E, which can synergistically work with carotenoids produced by Neurospora crassa to provide antioxidant benefits and enhance the immunity of dairy cows. Detailed Implementation

[0018] This invention provides a compound acid-resistant bacteria additive, comprising the following components in parts by weight: 30-50 parts modified apple pomace, 20-40 parts palm meal, 25-35 parts rapeseed meal, 5-15 parts sunflower seed oil, 5-10 parts kudzu flower extract, 3-5 parts compound acid-resistant bacteria agent, and 1-3 parts maifanite.

[0019] In this invention, the amount of modified apple pomace is preferably 35-45 parts, more preferably 40 parts; the preparation method of the modified apple pomace includes the following steps: (1) The apple pomace is extruded and expanded to obtain pretreated apple pomace; (2) The pretreated apple pomace and alkaline solution are mixed to obtain the secondary treated apple pomace; (3) The apple pomace that has undergone secondary treatment is adjusted to pH 4.8-5.0 with citric acid, enzymatically hydrolyzed with a compound enzyme preparation, and spray-dried to obtain modified apple pomace.

[0020] In this invention, the moisture content of the apple pomace is 20-30%, preferably 22-28%, and more preferably 25%; the temperature of the extrusion and puffing feeding section is 70-90℃, preferably 80℃; the temperature of the compression section is 110-130℃, preferably 120℃; the temperature of the extrusion section is 110-130℃, preferably 120℃; the screw speed is 160-200 r / min, preferably 180 r / min; the internal working pressure is 1.0-1.2 MPa, preferably 1.1 MPa; the diameter of the discharge die is 8-10 mm, preferably 9 mm; the alkaline solution includes 1%-2% calcium hydroxide and 0.5%-1% sodium hydroxide, the concentration of calcium hydroxide is preferably 1.5%, and the concentration of sodium hydroxide is preferably 0.8%; the mass ratio of the pretreated apple pomace to the alkaline solution is 1:8-10, preferably 1:9; the mixing time is 24-48 h, preferably 36 h. The compound enzyme preparation comprises 30-40 U / g cellulase and 90-100 U / g pectinase, wherein the concentration of cellulose is preferably 35 U / g and the concentration of pectinase is 95 U / g; the mass ratio of the secondary treated apple pomace to the compound enzyme preparation is 1:12-18, preferably 1:15; the enzymatic hydrolysis temperature is 40-60℃, preferably 50℃; and the enzymatic hydrolysis time is 2-4 h, preferably 3 h.

[0021] In this invention, the preferred amount of palm meal is 25-35 parts, more preferably 30 parts; the preferred amount of rapeseed meal is 28-32 parts, more preferably 30 parts; the preferred amount of sunflower seed oil is 8-12 parts, more preferably 10 parts; the preferred amount of the compound acid-resistant bacteria agent is 4 parts; the compound acid-resistant bacteria agent includes *Lactobacillus plantarum*, acid-resistant *Saccharomyces cerevisiae*, and *Neurospora crassa*; the mass ratio of *Lactobacillus plantarum*, acid-resistant *Saccharomyces cerevisiae*, and *Neurospora crassa* is 2-4:1-3:2-4, preferably 3:2:3; the viable counts of *Lactobacillus plantarum*, acid-resistant *Saccharomyces cerevisiae*, and *Neurospora crassa* are all ≥0.5-1.5 × 10⁻⁶. 9 CFU / g.

[0022] This invention also provides a method for preparing the aforementioned composite acid-resistant bacteria additive, comprising the following steps: (1) The modified apple pomace, palm meal, rapeseed meal, sunflower seed oil, kudzu flower extract and maifanite were mixed to obtain a mixture; (2) Mix the mixed materials and the compound acid-resistant bacteria agent, ferment them, and obtain the compound acid-resistant bacteria agent additive.

[0023] In this invention, the fermentation temperature is 30~35℃, preferably 33℃; the fermentation time is 36~60h, preferably 48h.

[0024] This invention also provides the application of the aforementioned compound acid-resistant bacteria additive in the preparation of complete feed for lactating dairy cows.

[0025] This invention also provides the application of the aforementioned compound acid-resistant bacteria additive in improving milk yield and milk quality in dairy cows.

[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] *Lactobacillus plantarum* S23Y, accession number CCTCC NO: M2023646, was purchased from the China Center for Type Culture Collection. *Saccharomyces cerevisiae* YR201705, accession number CGMCC No. 15315, was purchased from the China General Microbiological Culture Collection Center. *Lactobacillus plantarum* R6, accession number CGMCC No. 30096, was purchased from the China General Microbiological Culture Collection Center. *Neurospora crassa* (catalog number: HZB121874) was purchased from Wuhan Gray Algae Biotechnology Co., Ltd. *Pueraria lobata* flower extract was purchased from Fufeng Sinote Biotechnology Co., Ltd.

[0028] Example 1

[0029] A compound acid-resistant bacterial agent additive comprises the following components in parts by weight: 40 parts modified apple pomace, 30 parts palm meal, 30 parts rapeseed meal, 10 parts sunflower seed oil, 8 parts kudzu flower extract, and 4 parts compound acid-resistant bacterial agent (Lactobacillus plantarum (Lactobacillus plantarum S23Y and Lactobacillus plantarum R6 in a mass ratio of 1:1), acid-resistant Saccharomyces cerevisiae YR201705, and Neurospora crassa in a mass ratio of 3:2:3, with a viable count of 1×10⁻⁶ for each strain). 9 (CFU / g) and 2 portions of Maifan stone.

[0030] The method for preparing the modified apple pomace is as follows: (1) Adjust the final moisture content of apple pomace to 28%, feed the apple pomace into a single screw extruder, and control the temperature of the cylinder in sections: feeding section 80℃, compression section 120℃, extrusion section 120℃; screw speed 180r / min; internal working pressure 1.1MPa; discharge die diameter 9mm to obtain pretreated apple pomace.

[0031] (2) The pretreated apple pomace and alkaline solution (1.5% calcium hydroxide and 0.8% sodium hydroxide) were mixed at a mass ratio of 1:9 for 36 h to obtain the secondary treated apple pomace; (3) The apple pomace that has undergone secondary treatment is adjusted to pH 4.9 with citric acid, and a compound enzyme preparation (35 U / g cellulase and 95 U / g pectinase) is added. The mixture is then enzymatically hydrolyzed at 50°C for 3 hours and spray-dried to obtain modified apple pomace.

[0032] The preparation method of the aforementioned compound acid-resistant bacteria additive is as follows: (1) The modified apple pomace, palm meal, rapeseed meal, sunflower seed oil, kudzu flower extract and maifanite were mixed to obtain a mixture; (2) Mix the mixture with the compound acid-resistant bacteria agent and ferment at 33°C for 48 hours to obtain the compound acid-resistant bacteria agent additive.

[0033] Example 2

[0034] A compound acid-resistant bacteria additive comprises the following components in parts by weight: 30 parts modified apple pomace, 20 parts palm meal, 25 parts rapeseed meal, 5 parts sunflower seed oil, 5 parts kudzu flower extract, and 3 parts compound acid-resistant bacteria (Lactobacillus plantarum (Lactobacillus plantarum S23Y and Lactobacillus plantarum R6 in a 1:1 mass ratio), acid-resistant Saccharomyces cerevisiae YR201705, and Neurospora crassa in a 2:1:2 mass ratio, with each strain having a viable count of 0.5 × 10⁻⁶. 9 (CFU / g) and 1 part Maifan stone.

[0035] The method for preparing the modified apple pomace is as follows: (1) Adjust the final moisture content of apple pomace to 25%, feed the apple pomace into a single screw extruder, and control the temperature of the cylinder in sections: feeding section 70℃, compression section 110℃, extrusion section 110℃; screw speed 160r / min; internal working pressure 1.0MPa; discharge die diameter 8mm to obtain pretreated apple pomace.

[0036] (2) The pretreated apple pomace and alkaline solution (1% calcium hydroxide and 0.5% sodium hydroxide) were mixed at a mass ratio of 1:8 for 24 hours to obtain the secondary treated apple pomace; (3) The apple pomace that has undergone secondary treatment is adjusted to pH 4.8 with citric acid, and a compound enzyme preparation (30 U / g cellulase and 90 U / g pectinase) is added. The mixture is then enzymatically hydrolyzed at 40°C for 2 hours and spray-dried to obtain modified apple pomace.

[0037] The preparation method of the aforementioned compound acid-resistant bacteria additive is as follows: (1) The modified apple pomace, palm meal, rapeseed meal, sunflower seed oil, kudzu flower extract and maifanite were mixed to obtain a mixture; (2) Mix the mixture with the compound acid-resistant bacteria agent and ferment at 30°C for 36 hours to obtain the compound acid-resistant bacteria agent additive.

[0038] Example 3

[0039] A compound acid-resistant bacteria additive comprises the following components in parts by weight: 50 parts modified apple pomace, 40 parts palm meal, 35 parts rapeseed meal, 15 parts sunflower seed oil, 10 parts kudzu flower extract, and 5 parts compound acid-resistant bacteria (Lactobacillus plantarum (Lactobacillus plantarum S23Y and Lactobacillus plantarum R6 in a 1:1 mass ratio), acid-resistant Saccharomyces cerevisiae YR201705, and Neurospora crassa in a 4:3:4 mass ratio, with a viable count of 1.5 × 10⁻⁶ for each strain). 9 (CFU / g) and 3 portions of Maifan stone.

[0040] The method for preparing the modified apple pomace is as follows: (1) Adjust the final moisture content of apple pomace to 30%, feed the apple pomace into a single screw extrusion puffing machine, and control the temperature of the cylinder in sections: feeding section 90℃, compression section 130℃, extrusion section 130℃; screw speed 200r / min; internal working pressure 1.2 MPa; discharge die diameter 10 mm to obtain pretreated apple pomace.

[0041] (2) The pretreated apple pomace and alkaline solution (2% calcium hydroxide and 1% sodium hydroxide) were mixed at a mass ratio of 1:10 for 48 h to obtain the secondary treated apple pomace; (3) The apple pomace that has undergone secondary treatment is adjusted to pH 5.0 with citric acid, and a compound enzyme preparation (40 U / g cellulase and 100 U / g pectinase) is added. The mixture is then enzymatically hydrolyzed at 60°C for 4 h and spray-dried to obtain modified apple pomace.

[0042] The preparation method of the aforementioned compound acid-resistant bacteria additive is as follows: (1) The modified apple pomace, palm meal, rapeseed meal, sunflower seed oil, kudzu flower extract and maifanite were mixed to obtain a mixture; (2) Mix the mixture with the compound acid-resistant bacteria agent and ferment at 35°C for 60 hours to obtain the compound acid-resistant bacteria agent additive.

[0043] Comparative Example 1

[0044] Replace “modified apple pomace” in Example 1 with “apple pomace”.

[0045] Comparative Example 2

[0046] Remove the compound acid-resistant bacteria agent from Example 1.

[0047] Comparative Example 3

[0048] Remove the kudzu flower extract from Example 1.

[0049] Comparative Example 4

[0050] Remove the sunflower seed oil from Example 1.

[0051] Experimental Example 1

[0052] A dairy cow feed comprises a basal diet and a compound acid-resistant bacterial agent additive. The basal diet consists of 30 parts corn silage, 6 parts alfalfa hay, 6 parts sheepgrass, 6 parts oat hay, 6 parts ryegrass, 10 parts corn stalks, 10 parts wheat bran, 8 parts corn distillers grains, 8 parts rumen-protected protein, 1 part limestone powder, 1 part dicalcium phosphate, 1 part salt, and 1 part premix (each kilogram of the premix includes 0.4 g magnesium, 28 mg zinc, 16 mg manganese, 6 mg copper, 0.24 mg cobalt, 0.8 mg iodine, and 0.23 mg selenium, 7300 IU vitamin A, 1700 IU vitamin D, and 121 mg vitamin E). The compound acid-resistant bacterial agent additive is added at 1.5% of the basal diet. After the basal diet and the compound acid-resistant bacterial agent additive are mixed evenly, they are pelleted into feed pellets using a pellet mill.

[0053] The compound acid-resistant bacterial additives prepared in Examples 1-3 and Comparative Examples 1-4 were respectively made into finished feeds according to the above method, and animal feeding trials were conducted.

[0054] (1) Experimental design

[0055] The experiment selected 140 healthy mid-lactation Holstein dairy cows with similar milk production and lactation days, and randomly divided them into 7 groups of 20 cows each. Group 1 was fed the feed prepared with the compound acid-resistant bacteria additive from Example 1; Group 2 was fed the feed prepared with the compound acid-resistant bacteria additive from Example 2; Group 3 was fed the feed prepared with the compound acid-resistant bacteria additive from Example 3; Group 4 was fed the feed prepared with the compound acid-resistant bacteria additive from Comparative Example 1; Group 5 was fed the feed prepared with the compound acid-resistant bacteria additive from Comparative Example 2; Group 6 was fed the feed prepared with the compound acid-resistant bacteria additive from Comparative Example 3; and Group 7 was fed the feed prepared with the compound acid-resistant bacteria additive from Comparative Example 4.

[0056] (2) Feeding and management

[0057] In the experiment, all dairy cows were housed in groups with free access to water and were fed three times a day.

[0058] (3) Detection of test indicators

[0059] ①After 30 days of feeding, the average feed intake, disease incidence and milk production during the feeding period were measured.

[0060] Common diseases in dairy cows during lactation include metritis, ovarian cysts, mastitis, mammary ulcers, and diarrhea. The morbidity rate is calculated by counting the number of affected cows / 20. 100%. All experimental cows were milked three times a day. The milk yield was automatically recorded when the cows were using the herringbone milking station. During milking, the milking workers recorded the milk yield of all cows. The sum of the milk yields from the morning and evening milking stations was the milk yield of each cow for the day.

[0061] The experimental results are shown in Table 1.

[0062] Table 1. Average feed intake, disease incidence, and milk yield of dairy cows in different treatment groups.

[0063] As shown in Table 1, the compound acid-resistant bacteria additive provided by this invention, when added to the basal diet, can effectively increase the average feed intake and average weight gain of dairy cows, reduce the incidence of disease, and also improve the milk yield and overall health of dairy cows. Dairy cows fed feed containing unmodified apple pomace and those without the compound acid-resistant bacteria additive showed significantly reduced palatability and growth rate. Dairy cows fed feed without kudzu flower extract, the compound acid-resistant bacteria additive, and sunflower seed oil experienced decreased immunity and increased morbidity, thus affecting milk yield.

[0064] ② The collected milk samples were tested for milk protein, milk fat and lactose within 48 hours using a milk composition analyzer.

[0065] The experimental results are shown in Table 2.

[0066] Table 2. Milk quality from dairy cows in different treatment groups

[0067] As shown in Table 2, compared with Comparative Examples 1-4, the addition of modified apple pomace, acid-resistant compound bacterial agent, kudzu flower extract and sunflower seed oil in Examples 1-3 can significantly increase the content of milk fat, milk protein and lactose. The components in the compound acid-resistant bacterial agent additive work synergistically to meet the nutritional needs of dairy cows during lactation, ensuring that dairy cows absorb sufficient nutrition and significantly improving milk quality.

[0068] ③ Antioxidant capacity of dairy cows

[0069] Blood samples were collected 3 hours after morning feeding. 10 ml of blood was collected from the tail vein of dairy cows using a heparin sodium anticoagulant tube. Immediately after collection, the samples were centrifuged at 3000 rpm and 4°C for 15 min to prepare serum samples. The serum samples were aliquoted and stored at -20°C for the determination of blood physiological and biochemical indicators.

[0070] Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) were detected using an enzyme-linked immunosorbent assay (ELISA) reader according to the methods provided by the kits from Nanjing Jiancheng Company.

[0071] The experimental results are shown in Table 3.

[0072] Table 3 Antioxidant capacity of dairy cows in different treatment groups

[0073] As shown in Table 3, compared with Comparative Examples 1-4, the addition of modified apple pomace, acid-resistant compound microbial agent, kudzu flower extract and sunflower seed oil in Examples 1-3 can significantly improve the total antioxidant capacity of dairy cows and significantly reduce the malondialdehyde concentration.

[0074] As can be seen from the above embodiments and experimental examples, the compound acid-resistant bacteria additive of the present invention can significantly improve the average feed intake, milk yield, milk quality and antioxidant capacity of dairy cows, and reduce the incidence of diseases.

[0075] 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 acid-resistant bacteria additive, characterized in that, It includes the following ingredients in parts by weight: 30-50 parts modified apple pomace, 20-40 parts palm meal, 25-35 parts rapeseed meal, 5-15 parts sunflower seed oil, 5-10 parts kudzu flower extract, 3-5 parts compound acid-resistant bacteria agent, and 1-3 parts maifanite.

2. The composite acid-resistant bacteria additive according to claim 1, characterized in that, The compound acid-resistant bacteria agent includes Lactobacillus plantarum, acid-resistant Saccharomyces cerevisiae, and Neurospora crassa. The mass ratio of *Lactobacillus plantarum*, acid-resistant *Saccharomyces cerevisiae*, and *Neurospora crassa* is 2-4:1-3:2-4; the viable counts of *Lactobacillus plantarum*, acid-resistant *Saccharomyces cerevisiae*, and *Neurospora crassa* are all ≥0.5-1.5 × 10⁻⁶. 9 CFU / g.

3. The composite acid-resistant bacteria additive according to claim 1, characterized in that, The method for preparing the modified apple pomace includes the following steps: (1) The apple pomace is extruded and expanded to obtain pretreated apple pomace; (2) The pretreated apple pomace and alkaline solution are mixed to obtain the secondary treated apple pomace; (3) The apple pomace that has undergone secondary treatment is adjusted to pH 4.8-5.0 with citric acid, enzymatically hydrolyzed with a compound enzyme preparation, and spray-dried to obtain modified apple pomace.

4. The composite acid-resistant bacteria additive according to claim 3, characterized in that, The moisture content of the apple pomace in step (1) is 20-30%; the temperature of the extrusion and puffing feeding section is 70-90℃, the temperature of the compression section is 110-130℃, the temperature of the extrusion section is 110-130℃, the screw speed is 160-200 r / min, the working pressure inside the machine is 1.0-1.2 MPa, and the diameter of the discharge die is 8-10 mm.

5. The composite acid-resistant bacteria additive according to claim 3, characterized in that, The alkaline solution in step (2) includes 1%~2% calcium hydroxide and 0.5%~1% sodium hydroxide. The mass ratio of the pretreated apple pomace to the alkaline solution is 1:8~10, and the mixing time is 24~48 h.

6. The composite acid-resistant bacteria additive according to claim 3, characterized in that, The compound enzyme preparation includes 30-40 U / g cellulase and 90-100 U / g pectinase. The mass ratio of the secondary treated apple pomace to the compound enzyme preparation is 1:12-18. The enzymatic hydrolysis temperature is 40-60℃ and the enzymatic hydrolysis time is 2-4 h.

7. The method for preparing the composite acid-resistant bacteria additive according to any one of claims 1 to 6, characterized in that, Includes the following steps: (1) The modified apple pomace, palm meal, rapeseed meal, sunflower seed oil, kudzu flower extract and maifanite were mixed to obtain a mixture; (2) Mix the mixed materials and the compound acid-resistant bacteria agent, ferment them, and obtain the compound acid-resistant bacteria agent additive.

8. The preparation method according to claim 7, characterized in that, The fermentation temperature is 30~35℃, and the fermentation time is 36~60h.

9. The application of the compound acid-resistant bacteria additive according to any one of claims 1 to 6 in the preparation of complete feed for lactating dairy cows.

10. The application of the compound acid-resistant bacteria additive according to any one of claims 1 to 6 in improving milk yield and milk quality in dairy cows.

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