Animal feed processed with high-protein leaf grass and method for preparing the same

Abstract

The application discloses animal feed processed by high-protein leaf grass and a preparation method thereof, and belongs to the technical field of animal feed preparation. The preparation method comprises the following steps: mixing feed raw materials and immobilized fermentation bacterial agents to obtain fermentation raw materials, and sequentially performing aerobic fermentation treatment and anaerobic fermentation treatment on the fermentation raw materials to obtain fermentation mixed liquor; after the fermentation mixed liquor is mixed and stirred with oxidized sodium alginate, the precipitate is collected through centrifugal collection, and the precipitate is subjected to hot air drying treatment to obtain the animal feed. The tannin content of the prepared animal feed is effectively reduced, the crude protein content is relatively high, the loss of viable bacterial count is relatively low, the feeding performance is good, and the fermentation preparation of the animal feed by the leaf grass has achieved a phased result.

Description

Technical Field

[0001] This invention belongs to the field of animal feed preparation technology, specifically relating to an animal feed processed from high-protein leafy grass and its preparation method. Background Technology

[0002] Rumex acetosa, also known as protein grass or amino acid grass, is a perennial herbaceous plant belonging to the genus Rumex in the Polygonaceae family. It is a plant used for both medicinal and edible purposes. Its most prominent advantages lie in its strong resilience and wide adaptability. It is extremely cold-hardy, able to survive winters in temperatures as low as -30°C. Regarding soil adaptability, it is tolerant of saline-alkali soils, poor soil conditions, and drought, growing normally in saline-alkali, alkaline, and arid areas, making it an ideal crop for saline-alkali land improvement and marginal land utilization. In terms of yield, it grows rapidly and has strong regeneration capabilities. Under suitable water and fertilizer conditions, it can be harvested 5-6 times a year in northern regions and even more times in warmer southern regions. Rumex acetosa is rich in a variety of nutrients, especially protein, which far exceeds that of conventional forage grasses and most grain and oil crops, making it a highly promising source of plant protein feed. Protein is a core indicator of feed value, and the protein content in rumex acetosa is significantly higher than that of traditional forage grasses such as alfalfa and ryegrass. In terms of amino acid composition, leafy greens contain 18 kinds of amino acids, among which aspartic acid and glutamic acid, which are related to protein and sugar metabolism, are present in high levels. Leafy greens are rich in vitamins, including vitamin C, vitamin E, β-carotene, folic acid, pantothenic acid, and other vitamins, which can meet the nutritional needs of livestock and poultry for multiple vitamins, reducing the amount of vitamin additives used in feed. Regarding mineral elements, leafy greens contain a complete range of macro- and micro-elements in high amounts, not only meeting the needs of livestock and poultry for bone development and physiological metabolism, but also playing an important role in improving the quality of livestock and poultry products and enhancing the body's stress resistance, particularly with elements such as potassium and selenium. In addition to basic nutrients, leafy greens also contain a variety of functional bioactive substances, such as superoxide dismutase, chlorogenic acid, isoflavones, plant polyphenols, and emodin. These active substances have physiological functions such as antioxidation, anti-inflammation, antibacterial, and immune regulation. In feed, they can replace antibiotics, improve the intestinal health of livestock and poultry, and enhance the body's immunity, aligning with the current development trend of antibiotic-free farming. Dietary fiber is also an important component of leafy grasses, which can promote gastrointestinal motility in livestock and poultry, improve the intestinal microecological environment, prevent digestive tract diseases, and have a positive effect on rumen health in ruminants and intestinal development in monogastric animals.

[0003] Although leafy grass has great potential for feed, there are many technical bottlenecks in direct fresh feeding or simple processing, which limit its large-scale application. These are mainly reflected in the following aspects: (1) Extremely high water content, making preservation and storage difficult. The water content of fresh leafy grass can reach more than 80%, which is much higher than that of conventional forage grasses such as corn stalks and alfalfa. After harvesting, it is very easy to rot and deteriorate at room temperature, especially in the high-temperature season. It will rot after 1-2 days, making it impossible to store for a long time or transport it over long distances, which seriously restricts its industrial application. If natural drying is used to make hay, due to the high water content of fresh grass, the drying cycle is long, and it is very easy to mold and deteriorate on rainy days. Moreover, the loss of nutrients during the drying process is serious, and the degradation of protein and vitamins is large. If mechanical drying is used, the energy consumption is high and the cost increases significantly, making it economically uncompetitive. (2) High content of anti-nutritional factors, affecting nutrient utilization. One of the biggest commonalities of plants in the genus Rumex in the family Lycaceae is that their leaves contain tannins. Tannins have an astringent taste, which can significantly reduce feed palatability and lead to a decrease in feed intake in livestock and poultry. At the same time, tannins can combine with proteins and digestive enzymes to form complexes, reducing protein digestibility and enzyme activity, and affecting the absorption and utilization of nutrients.

[0004] Microbial fermentation can improve feed quality, increase nutritional value, and extend shelf life by reducing anti-nutritional factors. It can also regulate the animal's microecological balance, promoting growth, preventing diarrhea, and enhancing immunity. However, fermented feed is not a product to be prepared immediately; it requires storage. Therefore, post-processing often involves high-temperature drying. High-temperature drying quickly reduces moisture, effectively preventing spoilage and bloating caused by excessive microbial fermentation. However, high-temperature drying can thermally destroy and inactivate probiotics in fermented feed, hindering their positive effects on digestion and immunity in animals.

[0005] Therefore, it is of great significance to further improve the application of leafy grass in the feed industry by finding a way to prepare it into fermented feed while avoiding the defects caused by high-temperature drying in post-processing. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention utilizes a mixed bacterial strain comprising Aspergillus niger, Candida utilis, and Lactobacillus reuteri to ferment and prepare an animal feed primarily composed of leafy grasses, yielding a fermentation broth. This broth is then mixed with sodium alginate oxidase and finally dried using hot air to obtain the final animal feed, thus solving the technical problems mentioned in the background art. Specifically, the technical solution of this invention includes the following: One objective of this invention is to provide a method for preparing animal feed using high-protein leafy greens, the method comprising the following steps: Feed ingredients and immobilized fermentation agents are mixed at a weight ratio of 1:0.07~0.1 to obtain fermentation base material. The fermentation base material is then subjected to aerobic fermentation at 34~35℃ for 14~17h and anaerobic fermentation at 30~32℃ for 7h~10h to obtain fermentation mixture. The fermentation mixture and sodium alginate were mixed at a weight ratio of 1:0.05~0.06 and stirred at 20~25℃ for 6~12 hours. The precipitate was collected by centrifugation and then dried with hot air at 60~70℃ for 10~13 hours to obtain the animal feed.

[0007] Furthermore, the feed ingredients are obtained by mixing enzymatically hydrolyzed leafy grass, soybean meal, and wheat bran in a weight ratio of 1:0.1~0.2:0.2~0.3.

[0008] Furthermore, the method for preparing the enzymatically hydrolyzed leafy greens includes the following steps: After being crushed, the leafy grass is mixed with cellulase at a weight ratio of 1:0.002~0.003 to obtain the enzymatically hydrolyzed leafy grass.

[0009] Furthermore, the conditions for the mixed enzymatic hydrolysis include an enzymatic hydrolysis pH of 5-5.5, an enzymatic hydrolysis temperature of 35-40℃, and an enzymatic hydrolysis time of 60-70 min.

[0010] Furthermore, the preparation method of the immobilized fermentation agent includes the following steps: Pretreated rice husks are obtained by steam explosion treatment of rice husks; Pre-treated rice husks, OD 600 Aspergillus niger solution with an OD of 0.7 600 The concentration of Candida utilis culture was 0.7 and the OD value was 0.7. 600 After the Lactobacillus reuteri bacterial solution at a concentration of 0.7 was mixed and adsorbed, urea was added and mixed and stirred to obtain the immobilized fermentation agent.

[0011] Furthermore, the conditions for the steam explosion include a steam temperature of 170~200℃, a steam pressure of 0.8~1.4MPa, and a processing time of 300~450s.

[0012] Furthermore, the OD 600 The 0.7% Aspergillus niger suspension was prepared by inoculating Aspergillus niger strain CICC 40048 into PDA liquid medium and culturing to OD. 600 To achieve a concentration of 0.7, other culture media can be used for cultivation, or commercially available bacterial solutions can be purchased directly. There are no special requirements for the culture medium, as long as it can produce a bacterial solution.

[0013] Furthermore, the OD 600The 0.7% concentration of *Candida utilis* culture was prepared by inoculating *Candida utilis* strain CICC 1314 into malt extract broth and culturing to OD200. 600 To achieve a concentration of 0.7, other culture media can be used for cultivation, or commercially available bacterial solutions can be purchased directly. There are no special requirements for the culture medium, as long as it can produce a bacterial solution.

[0014] Furthermore, the OD 600 The 0.7% concentration of *Lactobacillus reuteri* bacterial culture was obtained by inoculating *Lactobacillus reuteri* strain CICC 6121 onto MRS liquid medium and culturing to OD. 600 To achieve a concentration of 0.7, other culture media can be used for cultivation, or commercially available bacterial solutions can be purchased directly. There are no special requirements for the culture medium, as long as it can produce a bacterial solution.

[0015] Furthermore, the conditions for the mixed adsorption include a temperature of 10~15℃, a stirring speed of 50r / min, and an adsorption time of 10h~12h.

[0016] Furthermore, the pretreated rice husk: OD 600 Aspergillus niger solution with an OD value of 0.6~0.8: 600 Candida utilis culture with an OD value of 0.6~0.8: 600 The weight ratio of Lactobacillus reuteri bacterial solution (0.6-0.8) to urea is 1:6-9:4-6:3-5:0.08-0.1.

[0017] Furthermore, the mixing and stirring conditions include a temperature of 20~25℃, a stirring speed of 100r / min, and an adsorption time of 10h~12h.

[0018] Furthermore, the preparation method of the oxidized sodium alginate includes the following steps: Sodium alginate and sodium periodate are mixed in a weight ratio of 1:0.5~0.6 and reacted in a light-protected environment at 25~30℃ for 40~50 min to obtain the oxidized sodium alginate.

[0019] The second objective of this invention is to provide an animal feed processed from high-protein leafy grasses.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention uses a mixed strain of Aspergillus niger, Candida utilis, and Lactobacillus reuteri as a microbial agent for fermenting and preparing animal feed mainly composed of leafy grass. Aspergillus niger decomposes the macromolecular polysaccharides, proteins, and anti-nutritional factors remaining in leafy grass into small molecule sugars, small peptides, and free amino acids through various hydrolytic enzymes, providing directly usable basic nutrient substrates for yeast and lactic acid bacteria. Candida utilis uses the small molecule substrates to synthesize proteins and secretes growth factors such as vitamins, which directly improves the protein content and nutritional balance of the feed and provides growth factors for lactic acid bacteria, compensating for the insufficient synthesis capacity of lactic acid bacteria. Lactobacillus reuteri converts sugars into lactic acid, lowers the pH of the system, inhibits the reproduction of miscellaneous bacteria, reduces the ineffective consumption of nutrients by miscellaneous bacteria, and creates a low-pollution and stable metabolic environment for the Aspergillus niger enzyme system and yeast. Next, rice husks were used as an immobilization carrier. Through steam explosion pretreatment, the tightly packed cellulose and hemicellulose were torn apart and layered, forming a porous structure. Oxidation treatment then oxidized and cleaved the vicinal diol structures in the fiber structure, generating functional aldehyde groups. Urea was then added, and a Schiff base reaction occurred between the amino and aldehyde groups. Through physical adsorption and chemical bonding, the immobilization effect on the bacterial strain was synergistically improved. Immobilization treatment resulted in a much higher cell density per unit volume compared to free fermentation, leading to faster substrate conversion, a shorter fermentation cycle, higher yield, and better protection of the fermentation activity of the bacterial strain, thus improving fermentation efficiency. However, poor fermentation efficiency was observed. This may be because although the cell density on the carrier increased after immobilization, the high cellulose content of the leafy grass resulted in a slow release of active ingredients, potentially leading to poor utilization of the leafy grass by the bacteria on the carrier. Therefore, pretreatment of the leafy grass with cellulase before fermentation significantly improved the fermentation efficiency. Sodium alginate that has undergone a certain degree of oxidation treatment is then added to the fermentation mixture. The amino groups on the sodium alginate structure can cross-link with amino acids, proteins, and other amino-containing substances in the fermentation mixture, increasing the number of high-temperature resistant imine bonds and improving the high-temperature protection effect. Finally, animal feed is prepared by hot air drying. The tannin content of this animal feed is not only effectively reduced, but the crude protein content is also high, and the loss of viable bacteria is low. The fermentation of leafy grass to prepare animal feed has achieved phased results. Detailed Implementation

[0021] The technical solution of the present invention will be clearly and completely described below through embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Unless otherwise stated, all raw materials and reagents used in this invention are commercially available or can be prepared by known methods.

[0023] Preparation Example 1 The preparation process of Aspergillus niger liquid is as follows: Aspergillus niger strain CICC 40048 was streaked onto LB agar plates and treated in a constant temperature shaker at 28°C and 80 rpm for 24 h. Then, two loops of the culture were inoculated into 1 L of sterilized PDA liquid medium using a sterilized inoculation loop and cultured at 28°C until OD (dose elapsed). 600 Remove the culture at 0.7°C and store in a refrigerator at 5°C until use.

[0024] Preparation Example 2 The preparation process of Candida utilis culture is as follows: The strain of *Candida utilis*, CICC 1314, was streaked onto LB agar plates and treated for 24 h at 80 rpm in a shaker at 28 °C. Then, two loops of the culture were inoculated into 1 L of sterilized malt extract broth and cultured at 28 °C until OD (dose elapsed). 600 Remove the culture at 0.7°C and store in a refrigerator at 5°C until use.

[0025] Preparation Example 3 The preparation process of Lactobacillus reuteri bacterial culture is as follows: Lactobacillus reuteri strain CICC 6121 was streaked onto LB agar plates and treated in a shaker at 30°C for 24 h at 80 rpm. Then, two loops of the culture were inoculated into 1 L of sterilized MRS liquid medium and cultured at 30°C until OD (dose elapsed). 600 Remove the culture at 0.7°C and store in a refrigerator at 5°C until use.

[0026] Preparation Example 4 The preparation process of immobilized fermentation inoculum is as follows: Rice husks were repeatedly rinsed with deionized water to remove surface impurities, and then dried in an oven at 55°C for 12 hours. Subsequently, the rice husks were placed in a steam explosion device, heated to 170°C, and the pressure was adjusted to 0.8 MPa for 300 seconds. After natural cooling to room temperature, the pretreated rice husks were obtained. One part by weight of the pretreated rice husks, six parts by weight of the *Aspergillus niger* liquid obtained in Preparation Example 1, four parts by weight of the *Candida utilis* liquid obtained in Preparation Example 2, and five parts by weight of the *Lactobacillus reuteri* liquid obtained in Preparation Example 3 were mixed and adsorbed at 10°C and a speed of 50 r / min for 10 hours. Then, 0.08 parts by weight of urea were added, and the temperature was raised to 20°C. The mixture was then stirred at 100 r / min for 10 hours. The immobilized fermentation agent was then collected by filtration.

[0027] Preparation Example 5 The preparation process of immobilized fermentation inoculum is as follows: Rice husks were repeatedly rinsed with deionized water to remove surface impurities, and then dried in an oven at 55°C for 12 hours. Subsequently, the rice husks were placed in a steam explosion device, heated to 180°C, and the pressure was adjusted to 1 MPa for 350 seconds. After natural cooling to room temperature, the pretreated rice husks were obtained. One part by weight of the pretreated rice husks, seven parts by weight of the *Aspergillus niger* liquid obtained in Preparation Example 1, five parts by weight of the *Candida utilis* liquid obtained in Preparation Example 2, and four parts by weight of the *Lactobacillus reuteri* liquid obtained in Preparation Example 3 were mixed and adsorbed at 10°C and a speed of 50 r / min for 11 hours. Then, 0.08 parts by weight of urea were added, and the temperature was raised to 20°C. The mixture was then stirred at 100 r / min for 11 hours. The immobilized fermentation agent was then collected by filtration.

[0028] Preparation Example 6 The preparation process of immobilized fermentation inoculum is as follows: Rice husks were repeatedly rinsed with deionized water to remove surface impurities, and then dried in an oven at 55°C for 12 hours. Subsequently, the rice husks were placed in a steam explosion device, heated to 190°C, and the pressure was adjusted to 1.2 MPa for 400 seconds. After natural cooling to room temperature, the pretreated rice husks were obtained. One part by weight of the pretreated rice husks, eight parts by weight of the *Aspergillus niger* liquid obtained in Preparation Example 1, six parts by weight of the *Candida utilis* liquid obtained in Preparation Example 2, and three parts by weight of the *Lactobacillus reuteri* liquid obtained in Preparation Example 3 were mixed and adsorbed at 15°C and a speed of 50 r / min for 12 hours. Then, 0.09 parts by weight of urea were added, and the temperature was raised to 25°C. The mixture was then stirred at a speed of 100 r / min for 12 hours. The immobilized fermentation agent was then collected by filtration.

[0029] Preparation Example 7 The preparation process of immobilized fermentation inoculum is as follows: Rice husks were repeatedly rinsed with deionized water to remove surface impurities, and then dried in an oven at 55°C for 12 hours. Subsequently, the rice husks were placed in a steam explosion device, heated to 200°C, and the pressure was adjusted to 1.4 MPa for 450 seconds. After natural cooling to room temperature, the pretreated rice husks were obtained. One part by weight of the pretreated rice husks, nine parts by weight of the *Aspergillus niger* liquid obtained in Preparation Example 1, six parts by weight of the *Candida utilis* liquid obtained in Preparation Example 2, and three parts by weight of the *Lactobacillus reuteri* liquid obtained in Preparation Example 3 were mixed and adsorbed at 15°C and a speed of 50 r / min for 12 hours. Then, 0.1 parts by weight of urea were added, and the temperature was raised to 25°C, and the mixture was stirred at a speed of 100 r / min for 12 hours. The immobilized fermentation agent was then collected by filtration.

[0030] Preparation Example 8 The preparation process of immobilized fermentation inoculum is as follows: Rice husks were repeatedly rinsed with deionized water to remove surface impurities, and then dried in an oven at 55°C for 12 hours. Subsequently, the rice husks were placed in a steam explosion device, heated to 210°C, and the pressure was adjusted to 1.6 MPa for 500 seconds. After natural cooling to room temperature, the pretreated rice husks were obtained. One part by weight of the pretreated rice husks, nine parts by weight of the *Aspergillus niger* liquid obtained in Preparation Example 1, six parts by weight of the *Candida utilis* liquid obtained in Preparation Example 2, and three parts by weight of the *Lactobacillus reuteri* liquid obtained in Preparation Example 3 were mixed and adsorbed at 15°C and a speed of 50 r / min for 12 hours. Then, 0.1 parts by weight of urea were added, and the temperature was raised to 25°C, and the mixture was stirred at a speed of 100 r / min for 12 hours. The immobilized fermentation agent was then collected by filtration.

[0031] Preparation Example 9 The preparation process of immobilized fermentation inoculum is as follows: Rice husks were repeatedly rinsed with deionized water to remove surface impurities, and then dried in an oven at 55°C for 12 hours. Subsequently, the rice husks were placed in a steam explosion device, heated to 200°C, and the pressure was adjusted to 1.4 MPa for 450 seconds. After natural cooling to room temperature, the pretreated rice husks were obtained. One part by weight of the pretreated rice husks, nine parts by weight of the *Aspergillus niger* liquid obtained in Preparation Example 1, six parts by weight of the *Candida utilis* liquid obtained in Preparation Example 2, and three parts by weight of the *Lactobacillus reuteri* liquid obtained in Preparation Example 3 were mixed and adsorbed at 15°C and a rotation speed of 50 r / min for 12 hours. Then, one part by weight of urea was added, and the temperature was raised to 25°C, and the mixture was stirred at a rotation speed of 100 r / min for 12 hours. The immobilized fermentation agent was then collected by filtration.

[0032] Preparation Example 10 The preparation process of enzymatically hydrolyzed leafy greens is as follows: After repeatedly rinsing the edible leafy grass with clean water to remove surface impurities, it was dried in a 55℃ oven for 12 hours. Then, it was removed, cut, and pulverized into granules using a grinder. One part by weight of the granulated edible leafy grass and 0.002 parts by weight of cellulase were added to 20 parts by weight of deionized water and stirred continuously at 200 rpm. The temperature was then adjusted to 35℃, and the pH was adjusted to 5 with acid. After adjustment, the mixture was stirred for 60 minutes for enzymatic hydrolysis. After hydrolysis, the temperature was raised to 90℃ for 10 minutes to inactivate the enzyme, yielding the enzymatically hydrolyzed edible leafy grass.

[0033] Preparation Example 11 The preparation process of enzymatically hydrolyzed leafy greens is as follows: After repeatedly rinsing the edible leafy grass with clean water to remove surface impurities, it was dried in a 55℃ oven for 12 hours. Then, it was removed, cut, and pulverized into granules using a grinder. One part by weight of the granulated edible leafy grass and 0.003 parts by weight of cellulase were added to 20 parts by weight of deionized water and stirred continuously at 200 rpm. The temperature was then adjusted to 40℃, and the pH was adjusted to 5.5 with acid. After adjustment, the mixture was stirred for 70 minutes for enzymatic hydrolysis. After hydrolysis, the temperature was raised to 90℃ for 10 minutes to inactivate the enzyme, yielding the enzymatically hydrolyzed edible leafy grass.

[0034] Preparation Example 12 The preparation method of sodium oxidized alginate includes the following steps: One part by weight of sodium alginate was added to six parts by weight of anhydrous ethanol and mixed evenly. Then, 0.5 parts by weight of sodium periodate was dissolved in six parts by weight of deionized water and stirred until dissolved. The dispersed sodium alginate and dissolved sodium periodate were then mixed and placed in the dark. The temperature was controlled at 25°C, and the mixture was stirred at 150 r / min for 40 min. After the reaction was completed, 4 times the amount of ethylene glycol was added to the sodium periodate and the mixture was stirred for another 30 min to quench the oxidation reaction and obtain a mixed solution. Finally, the mixed solution was poured into a dialysis bag with a molecular weight cutoff of 1 kDa and dialyzed for 48 h. Then, it was freeze-dried at -40°C for 4 h to obtain oxidized sodium alginate.

[0035] Preparation Example 13 The preparation method of sodium oxidized alginate includes the following steps: One part by weight of sodium alginate was added to six parts by weight of anhydrous ethanol and mixed evenly. Then, 0.6 parts by weight of sodium periodate was dissolved in six parts by weight of deionized water and stirred until dissolved. The dispersed sodium alginate and dissolved sodium periodate were then mixed and placed in the dark. The temperature was controlled at 30°C, and the mixture was stirred at 150 rpm for 50 min. After the reaction was complete, 4 times the amount of ethylene glycol was added to the sodium periodate mixture, and stirring was continued for 30 min to quench the oxidation reaction and obtain a mixed solution. Finally, the mixed solution was poured into a dialysis bag with a molecular weight cutoff of 1 kDa and dialyzed for 48 h. Then, it was freeze-dried at -40°C for 4 h to obtain oxidized sodium alginate.

[0036] Preparation Example 14 The preparation method of sodium oxidized alginate includes the following steps: One part by weight of sodium alginate was added to six parts by weight of anhydrous ethanol and mixed evenly. Then, one part by weight of sodium periodate was dissolved in six parts by weight of deionized water and stirred until dissolved. The dispersed sodium alginate and dissolved sodium periodate were then mixed and placed in the dark. The temperature was controlled at 30°C, and the mixture was stirred at 150 rpm for 60 min. After the reaction was complete, 4 times the amount of ethylene glycol was added to the sodium periodate mixture, and stirring was continued for 30 min to quench the oxidation reaction and obtain a mixed solution. Finally, the mixed solution was poured into a dialysis bag with a molecular weight cutoff of 1 kDa and dialyzed for 48 h. Then, it was freeze-dried at -40°C for 4 h to obtain oxidized sodium alginate.

[0037] Example 1 A method for preparing animal feed using high-protein leafy greens, specifically including the following steps: One part by weight of enzymatically hydrolyzed leafy grass obtained in Preparation Example 10, 0.1 parts by weight of soybean meal, and 0.2 parts by weight of wheat bran were mixed and stirred evenly to obtain feed ingredients. One part by weight of feed ingredients and 0.07 parts by weight of immobilized fermentation agent obtained in Preparation Example 4 were weighed and mixed evenly to obtain fermentation base material. The temperature of the fermentation base material was controlled at 34°C. Then, it was subjected to aerobic fermentation under open conditions for 14 hours. After aerobic fermentation, it was transferred to a sealed tank and subjected to anaerobic fermentation at 30°C for 7 hours to obtain fermentation mixture. One part by weight of fermentation mixture and 0.05 parts by weight of sodium alginate obtained in Preparation Example 12 were weighed and mixed, and the temperature was controlled at 20°C. Then, it was stirred at 400 r / min for 6 hours. After stirring, the precipitate was separated and collected by centrifugation at 6000 r / min. The precipitate was placed in a hot air dryer and dried at 60°C and 5 m / s for 10 hours to obtain animal feed.

[0038] Example 2 A method for preparing animal feed using high-protein leafy greens, specifically including the following steps: One part by weight of enzymatically hydrolyzed leafy grass obtained in Preparation Example 10, 0.1 parts by weight of soybean meal, and 0.25 parts by weight of wheat bran were mixed and stirred evenly to obtain feed ingredients. One part by weight of feed ingredients and 0.08 parts by weight of immobilized fermentation agent obtained in Preparation Example 5 were weighed and mixed evenly to obtain fermentation base material. The temperature of the fermentation base material was controlled at 34°C. Then, it was subjected to aerobic fermentation under open conditions for 15 hours. After aerobic fermentation, it was transferred to a sealed tank and subjected to anaerobic fermentation at 30°C for 8 hours to obtain fermentation mixture. One part by weight of fermentation mixture and 0.05 parts by weight of sodium alginate obtained in Preparation Example 12 were weighed and mixed, and the temperature was controlled at 20°C. Then, it was stirred at 400 r / min for 8 hours. After stirring, the precipitate was separated and collected by centrifugation at 6000 r / min. The precipitate was placed in a hot air dryer and dried at 60°C and 5 m / s for 11 hours to obtain animal feed.

[0039] Example 3 A method for preparing animal feed using high-protein leafy greens, specifically including the following steps: One part by weight of enzymatically hydrolyzed leafy grass obtained in Preparation Example 11, 0.15 parts by weight of soybean meal, and 0.25 parts by weight of wheat bran were mixed and stirred evenly to obtain feed ingredients. One part by weight of feed ingredients and 0.09 parts by weight of immobilized fermentation agent obtained in Preparation Example 6 were weighed and mixed evenly to obtain fermentation base material. The temperature of the fermentation base material was controlled at 35°C. Then, it was subjected to aerobic fermentation under open conditions for 16 hours. After aerobic fermentation, it was transferred to a sealed tank and subjected to anaerobic fermentation at 31°C for 9 hours to obtain fermentation mixture. One part by weight of fermentation mixture and 0.06 parts by weight of sodium alginate obtained in Preparation Example 13 were weighed and mixed, and the temperature was controlled at 25°C. Then, it was stirred at 400 r / min for 10 hours. After stirring, the precipitate was separated and collected by centrifugation at 6000 r / min. The precipitate was placed in a hot air dryer and dried at 65°C and 5 m / s for 12 hours to obtain animal feed.

[0040] Example 4 A method for preparing animal feed using high-protein leafy greens, specifically including the following steps: One part by weight of enzymatically hydrolyzed leafy grass obtained in Preparation Example 11, 0.2 parts by weight of soybean meal, and 0.3 parts by weight of wheat bran were mixed and stirred evenly to obtain feed ingredients. One part by weight of feed ingredients and 0.1 parts by weight of immobilized fermentation agent obtained in Preparation Example 7 were weighed and mixed evenly to obtain fermentation base material, and the temperature of the fermentation base material was controlled at 35°C. Then, it was subjected to aerobic fermentation under open conditions for 17 hours. After aerobic fermentation, it was transferred to a sealed tank and subjected to anaerobic fermentation at 32°C for 10 hours to obtain fermentation mixture. One part by weight of fermentation mixture and 0.06 parts by weight of sodium alginate obtained in Preparation Example 13 were weighed and mixed, and the temperature was controlled at 25°C. Then, it was stirred at 400 r / min for 12 hours. After stirring, the precipitate was separated and collected by centrifugation at 6000 r / min. The precipitate was placed in a hot air dryer, and the hot air temperature was controlled at 70°C and the air velocity at 5 m / s. After hot air drying for 13 hours, it was taken out to obtain animal feed.

[0041] Comparative Example 1 A method for preparing animal feed using high-protein leafy greens, specifically including the following steps: One part by weight of enzymatically hydrolyzed leafy grass obtained in Preparation Example 11, 0.2 parts by weight of soybean meal, and 0.3 parts by weight of wheat bran were mixed and stirred evenly to obtain feed ingredients. One part by weight of feed ingredients and 0.1 parts by weight of immobilized fermentation agent obtained in Preparation Example 8 were weighed and mixed evenly to obtain fermentation base material. The temperature of the fermentation base material was controlled at 35°C. Then, it was subjected to aerobic fermentation under open conditions for 17 hours. After aerobic fermentation, it was transferred to a sealed tank and subjected to anaerobic fermentation at 32°C for 10 hours to obtain fermentation mixture. One part by weight of fermentation mixture and 0.06 parts by weight of sodium alginate obtained in Preparation Example 13 were weighed and mixed, and the temperature was controlled at 25°C. Then, it was stirred at 400 r / min for 12 hours. After stirring, the precipitate was separated and collected by centrifugation at 6000 r / min. The precipitate was placed in a hot air dryer and dried at 70°C and 5 m / s for 13 hours to obtain animal feed.

[0042] Comparative Example 2 A method for preparing animal feed using high-protein leafy greens, specifically including the following steps: One part by weight of enzymatically hydrolyzed leafy grass obtained in Preparation Example 11, 0.2 parts by weight of soybean meal, and 0.3 parts by weight of wheat bran were mixed and stirred evenly to obtain feed ingredients. One part by weight of feed ingredients and 0.1 parts by weight of immobilized fermentation agent obtained in Preparation Example 9 were weighed and mixed evenly to obtain fermentation base material, and the temperature of the fermentation base material was controlled at 35°C. Then, it was subjected to aerobic fermentation under open conditions for 17 hours. After the aerobic fermentation was completed, it was transferred to a sealed tank and subjected to anaerobic fermentation at 32°C for 10 hours to obtain fermentation mixture. One part by weight of fermentation mixture and 0.06 parts by weight of sodium alginate obtained in Preparation Example 13 were weighed and mixed, and the temperature was controlled at 25°C. Then, it was stirred at 400 r / min for 12 hours. After stirring, the precipitate was separated and collected by centrifugation at 6000 r / min. The precipitate was placed in a hot air dryer, and the hot air temperature was controlled at 70°C and the air velocity at 5 m / s. After hot air drying for 13 hours, it was taken out to obtain animal feed.

[0043] Comparative Example 3 A method for preparing animal feed using high-protein leafy greens, specifically including the following steps: Weigh 1 part by weight of the fermentation mixture prepared in Example 4 and 0.06 parts by weight of the sodium alginate oxidized in Example 14, mix them, and control the temperature at 25°C. Then, stir at 400 r / min for 12 h. After stirring, separate and collect the precipitate using a centrifuge at 6000 r / min. Place the precipitate in a hot air dryer, control the hot air temperature at 70°C and the air velocity at 5 m / s, and dry it for 13 h to obtain animal feed.

[0044] Comparative Example 4 A method for preparing animal feed using high-protein leafy greens, specifically including the following steps: One part by weight of enzymatically hydrolyzed leafy grass obtained in Preparation Example 11, 0.2 parts by weight of soybean meal, and 0.3 parts by weight of wheat bran were mixed and stirred evenly to obtain feed ingredients. One part by weight of feed ingredients and 0.1 parts by weight of immobilized fermentation agent obtained in Preparation Example 7 were weighed and mixed evenly to obtain fermentation base material. The temperature of the fermentation base material was controlled at 35°C. Then, it was subjected to aerobic fermentation under open conditions for 20 hours. After the aerobic fermentation was completed, it was transferred to a sealed tank and subjected to anaerobic fermentation at 32°C for 10 hours to obtain fermentation mixture. One part by weight of fermentation mixture and 0.06 parts by weight of sodium alginate obtained in Preparation Example 13 were weighed and mixed, and the temperature was controlled at 25°C. Then, it was stirred at 400 r / min for 12 hours. After stirring, the precipitate was separated and collected by centrifugation at 6000 r / min. The precipitate was placed in a hot air dryer and dried at 70°C and 5 m / s for 13 hours to obtain animal feed.

[0045] Comparative Example 5 A method for preparing animal feed using high-protein leafy greens, specifically including the following steps: One part by weight of enzymatically hydrolyzed leafy grass obtained in Preparation Example 11, 0.2 parts by weight of soybean meal, and 0.3 parts by weight of wheat bran were mixed and stirred evenly to obtain feed ingredients. One part by weight of feed ingredients and 0.1 parts by weight of immobilized fermentation agent obtained in Preparation Example 7 were weighed and mixed evenly to obtain fermentation base material, and the temperature of the fermentation base material was controlled at 35°C. Then, it was subjected to aerobic fermentation under open conditions for 17 hours. After the aerobic fermentation was completed, it was transferred to a sealed tank and subjected to anaerobic fermentation at 32°C for 15 hours to obtain fermentation mixture. One part by weight of fermentation mixture and 0.06 parts by weight of sodium alginate obtained in Preparation Example 13 were weighed and mixed, and the temperature was controlled at 25°C. Then, it was stirred at 400 r / min for 12 hours. After stirring, the precipitate was separated and collected by centrifugation at 6000 r / min. The precipitate was placed in a hot air dryer, and the hot air temperature was controlled at 70°C and the air velocity at 5 m / s. After hot air drying for 13 hours, it was taken out to obtain animal feed.

[0046] Comparative Example 6 A method for preparing animal feed using high-protein leafy greens, specifically including the following steps: Weigh 1 part by weight of the fermentation mixture obtained in Example 4 and 0.06 parts by weight of the sodium alginate oxidized obtained in Preparation Example 13, mix them, and control the temperature at 25°C. Then, stir at 400 r / min for 12 h. After stirring, separate and collect the precipitate by centrifuging at 6000 r / min. Place the precipitate in a hot air dryer, control the hot air temperature at 90°C and the air velocity at 5 m / s, and dry it for 13 h to obtain animal feed.

[0047] Comparative Example 7 A method for preparing animal feed using high-protein leafy greens, specifically including the following steps: One part by weight of leafy grass, 0.2 parts by weight of soybean meal, 0.3 parts by weight of wheat bran, and 15 parts by weight of water were mixed and stirred evenly to obtain feed ingredients. One part by weight of feed ingredients and 0.1 parts by weight of the immobilized fermentation agent obtained in Preparation Example 7 were weighed and mixed evenly to obtain fermentation base material. The temperature of the fermentation base material was controlled at 35°C. Then, it was subjected to aerobic fermentation under open conditions for 17 hours. After aerobic fermentation, it was transferred to a sealed tank and subjected to anaerobic fermentation at 32°C for 10 hours to obtain fermentation mixture. One part by weight of fermentation mixture and 0.06 parts by weight of sodium alginate obtained in Preparation Example 13 were weighed and mixed, and the temperature was controlled at 25°C. Then, it was stirred at 400 r / min for 12 hours. After stirring, the precipitate was separated and collected by centrifuge at 6000 r / min. The precipitate was placed in a hot air dryer and dried at 70°C and 5 m / s for 13 hours to obtain animal feed.

[0048] The total viable counts of Aspergillus niger, Candida utilis, and Lactobacillus reuteri in the fermentation mixtures obtained in Examples 1-4 and Comparative Examples 1-7 were recorded as m1. Then, the total viable counts of Aspergillus niger, Candida utilis, and Lactobacillus reuteri in the hot-air dried animal feed were recorded as m2. The total viable counts of the bacteria were calculated according to the formula: bacterial survival rate (%) = (m1-m2) / m1×100%. The results are shown in Table 1 below. Table 1. Bacterial cell survival status

[0049] As can be seen from Table 1 above, even though the modification by the present invention can weaken the damage to the activity of the strain caused by high temperature during feed drying, the modification effect is limited. When the hot air drying temperature is too high, the modification method of the present invention cannot effectively protect the activity of the strain.

[0050] The crude protein content of the animal feeds obtained in Examples 1-4, Comparative Examples 1-5 and Comparative Example 7 was determined by a fully automatic Kjeldahl nitrogen analyzer, the neutral detergent fiber content was determined by a semi-automatic fiber analyzer, and the tannin content was measured according to NY / T1600-2008. The results are shown in Table 2 below.

[0051] Table 2. Animal feed analysis results

[0052] The following conclusions can be drawn from Table 2 above: (1) Through Examples 1 to 4, it can be found that the animal feed prepared by the present invention has a high crude protein content, the neutral detergent fiber content is weakened, and the tannin content is significantly reduced.

[0053] (2) Comparative Example 1 shows that the crude protein content is poor, the neutral detergent fiber content is high, and the tannin content is high. This indicates that when the steam explosion intensity exceeds a certain limit, it may cause excessive damage to the structure of the rice husk, resulting in a decrease in the ability to support the strains and hindering subsequent fermentation.

[0054] (3) Comparative Example 2 shows that the crude protein content is poor, the neutral detergent fiber content is high, and the tannin content is high. This indicates that although urea can promote Schiff base cross-linking, due to the alkalinity of urea itself, if the dosage is too high, the overall pH may become alkaline, which may damage the activity of the strain and be detrimental to subsequent fermentation.

[0055] (4) Comparative Examples 4 and 5 show that the crude protein content is poor, the neutral detergent fiber content is high, and the tannin content is high. This indicates that there are strict requirements for the time of aerobic fermentation and anaerobic fermentation in this system. If the time is too long, the nutrients will be over-consumed, which will not be conducive to improving the quality of the feed.

[0056] (5) Comparative Example 7 shows that the crude protein content is poor, the neutral detergent fiber content is high, and the tannin content is high. This indicates that although the bacterial density on the carrier is increased after immobilization treatment, the leafy grass has a high cellulose content and the active ingredients in the leafy grass are released slowly, which may lead to poor efficiency of the bacteria on the carrier in utilizing the leafy grass, thus hindering fermentation.

[0057] The animal feeds obtained in Examples 1-4 and Comparative Example 3 were fed to cattle and pigs respectively, with the feed amount being 2.5% of body weight. The feeding was carried out for 60 days, and the average daily weight gain was then calculated. The results are shown in Table 3 below.

[0058] Table 3 Feeding conditions

[0059] The following conclusions can be drawn from Table 3 above: (1) It can be seen from Examples 1 to 4 that the animal feed prepared by the present invention has good feeding performance, but the feeding performance of ruminant cattle is poor. This may be due to the low content of neutral detergent fiber in the animal feed prepared by the present invention. Fiber is one of the key substances for ruminants to maintain normal rumen peristalsis, fermentation mode and pH stability. If the content is too low, the rumen wall lacks effective stimulation, the peristalsis slows down and the number of contractions decreases, which may easily lead to rumen impaction, forestomach atony and other adverse effects, which are not conducive to the feeding and development of cattle.

[0060] (2) Comparative Example 3 shows that the feeding performance was poor. This indicates that the purpose of sodium alginate oxidation is to build aldehyde groups and then cross-link with the amino structure in the fermentation mixture to form high-temperature resistant imine bonds. However, excessive oxidation will lead to the production of too many aldehyde groups. Excessive aldehyde groups may lead to excessive cross-linking with the fermentation mixture. Although this is beneficial to protect the activity of the strain, it is not conducive to the release of nutrients, resulting in poor feeding performance of the feed.

[0061] The embodiments described above provide a detailed explanation of the technical solutions and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Various changes and modifications can be made to the present invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed.

Claims

1. A method for preparing animal feed using high-protein leafy greens, characterized in that, The preparation method includes the following steps: Feed ingredients and immobilized fermentation agents are mixed at a weight ratio of 1:0.07~0.1 to obtain fermentation base material. The fermentation base material is then subjected to aerobic fermentation at 34~35℃ for 14~17h and anaerobic fermentation at 30~32℃ for 7h~10h to obtain fermentation mixture. The fermentation mixture and sodium alginate were mixed at a weight ratio of 1:0.05~0.06 and stirred at 20~25℃ for 6~12 hours. The precipitate was collected by centrifugation and then dried with hot air at 60~70℃ for 10~13 hours to obtain the animal feed.

2. The method for preparing animal feed using high-protein leafy greens according to claim 1, characterized in that, The feed ingredients are obtained by mixing enzymatically hydrolyzed leafy grass, soybean meal, and wheat bran in a weight ratio of 1:0.1~0.2:0.2~0.

3.

3. The method for preparing animal feed using high-protein leafy greens according to claim 2, characterized in that, The method for preparing enzymatically hydrolyzed leafy greens includes the following steps: After being crushed, the leafy grass is mixed with cellulase at a weight ratio of 1:0.002~0.003 to obtain the enzymatically hydrolyzed leafy grass.

4. The method for preparing animal feed using high-protein leafy greens according to claim 3, characterized in that, The conditions for the mixed enzymatic hydrolysis include a hydrolysis pH of 5-5.5, a hydrolysis temperature of 35-40℃, and a hydrolysis time of 60-70 min.

5. The method for preparing animal feed using high-protein leafy greens according to claim 1, characterized in that, The method for preparing the immobilized fermentation agent includes the following steps: Pretreated rice husks are obtained by steam explosion treatment of rice husks; Pre-treated rice husks, OD 600 Aspergillus niger solution with an OD of 0.7 600 The concentration of Candida utilis culture was 0.7 and the OD value was 0.

7. 600 After the Lactobacillus reuteri bacterial solution at a concentration of 0.7 was mixed and adsorbed, urea was added and mixed and stirred to obtain the immobilized fermentation agent.

6. The method for preparing animal feed using high-protein leafy greens according to claim 5, characterized in that, The conditions for the steam explosion include a steam temperature of 170~200℃, a steam pressure of 0.8~1.4MPa, and a processing time of 300~450s.

7. The method for preparing animal feed using high-protein leafy greens according to claim 5, characterized in that, The pretreated rice husk: OD 600 Aspergillus niger solution with an OD value of 0.6~0.8: 600 Candida utilis culture with an OD value of 0.6~0.8: 600 The weight ratio of Lactobacillus reuteri bacterial solution (0.6-0.8) to urea is 1:6-9:4-6:3-5:0.08-0.

1.

8. The method for preparing animal feed using high-protein leafy greens according to claim 1, characterized in that, The preparation method of the oxidized sodium alginate includes the following steps: Sodium alginate and sodium periodate are mixed in a weight ratio of 1:0.5~0.6 and reacted in a light-protected environment at 25~30℃ for 40~50 min to obtain the oxidized sodium alginate.

9. An animal feed processed from high-protein leafy greens, characterized in that, The animal feed is prepared by any one of the methods described in claims 1 to 8 for preparing animal feed using high-protein leafy grass.

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