A compound feed additive and its preparation method

Abstract

This invention discloses a compound feed additive and its preparation method, belonging to the field of feed technology. The preparation method includes the following steps: mixing a bacterial liposome suspension, a polysaccharide extract, modified steviol glycosides, and an emulsifier in a weight ratio of 1:0.03–0.4:0.1–0.2:0.01, and stirring at 30°C for 20–24 hours to obtain a microcapsule emulsion; the microcapsule emulsion is then spray-dried to obtain the compound feed additive. This invention uses Bacillus subtilis and Bifidobacterium animalis as the core strains in the compound feed additive, preparing them into a bacterial liposome suspension, then mixing and reacting it with the polysaccharide extract, modified steviol glycosides, and emulsifier, and finally spray-drying it at high temperature to obtain a compound feed additive that improves feed intake and weight gain.

Description

Technical Field

[0001] This invention belongs to the field of feed technology, specifically relating to a compound feed additive and its preparation method. Background Technology

[0002] Animal husbandry is a crucial sector of agriculture, serving as a primary source of protein-rich foods such as meat, eggs, and milk. Feed is a core component supporting the development of animal husbandry, providing the material foundation for its growth. Within the feed industry, feed additives are an indispensable part of modern animal husbandry, playing a vital role in improving animal production performance, feed efficiency, and promoting animal health. Feed additives are substances that maintain animal health and promote growth and development. Many types of feed additives are commonly used in livestock and poultry, including vitamins, trace minerals, microecological agents, functional additives, and antifungal agents, each with different functions. Vitamins are small molecules that promote metabolism, encompassing a wide variety and broadly categorized into water-soluble and fat-soluble vitamins. They primarily function as coenzymes in various biochemical reactions. Mineral trace elements include iron, manganese, zinc, copper, boron, molybdenum, cobalt, iodine, and selenium. These elements mainly exist in salt form. Although used in small quantities, they are essential for the normal growth, development, and life maintenance of livestock and poultry. Some participate in catalytic reactions in the body as coenzymes, while others form unstable complexes with hormone precursors, which are beneficial for hormone production and prolonging hormone effects. Still others act as enzyme activators. Microecological feed additives mainly consist of probiotics and prebiotics. Probiotics are microorganisms that help animals grow and develop, prevent diseases, and maintain health. Prebiotics in feed additives are organic substances that are not digested and absorbed by the host but can selectively promote the metabolism and proliferation of beneficial bacteria in the body, thereby improving the host's health. Functional feed additives refer to additives with specific functions, commonly including growth promoters, antibacterial agents, and anthelmintics. Antifungal agents are additives that prevent feed from becoming moldy. In intensive livestock and poultry farming, due to high stocking densities, animals are highly susceptible to respiratory or digestive diseases. Therefore, using antibiotics to control bacterial infections and reduce disease incidence remains a common practice. While antibiotics have played a crucial role in the development of the livestock industry, their long-term and excessive use has led to numerous problems, including increased antibiotic resistance in pathogens, dysbiosis of the digestive tract flora, and weakened immunity. They can even harm human health through the food chain. Therefore, gradually reducing or even prohibiting the use of antibiotics in feed additives has become the mainstream development goal for feed additives.

[0003] Patent CN118648648A discloses a feed additive containing Lactobacillus acidophilus, its preparation method, and its application. This invention utilizes Lactobacillus acidophilus, plant extracts, and hawthorn powder to prepare a natural, safe, and efficient feed additive. By adjusting the ratio of plant extracts, the preparation method, and the number of live Lactobacillus acidophilus, the feed additive is prepared. When added to animal feed, it can not only improve the intestinal health and immunity of livestock and poultry, but also increase the feed conversion rate and reduce the feed-to-meat ratio.

[0004] Patent CN117882798A discloses a feed additive based on buckwheat extract for improving the intestinal health of broilers. This invention uses buckwheat extract, Bacillus subtilis and proline as raw materials. Through the synergistic effect of the three, it inhibits pathogenic bacteria in the intestine, promotes the growth of beneficial anaerobic bacteria, and produces organic acids such as lactic acid, thereby reducing the pH value of the intestine and indirectly inhibiting the growth and reproduction of harmful and putrefactive bacteria in the intestine, correcting intestinal flora imbalance, reducing the production of enterotoxins, increasing the levels of immunoglobulins and antibodies, and enhancing the cellular and humoral immune functions of broilers.

[0005] Using microorganisms to prepare feed additives can improve the balance of intestinal flora, promote the growth of beneficial bacteria, and effectively inhibit the proliferation of pathogenic bacteria. However, in practice, feed additives are often added to feed in the form of finished products. Methods for preparing these finished products include freeze-drying, hot air circulating drying and pulverization, and high-temperature spray drying. Freeze-drying has a good protective effect on preserving the performance of live bacteria, but freeze-drying equipment is expensive and the operation method is complex, making it unsuitable for large-scale production in the short term. High-temperature drying, on the other hand, offers significant cost advantages and simplifies the process. However, a consequence is that high temperatures can cause premature death of microbial strains, easily leading to the loss of the beneficial properties of these strains in the feed additive.

[0006] Therefore, researching a method to reduce the loss of microbial strain activity during the preparation of feed additives at high temperatures, thereby maximizing the beneficial effects of microbial strains entering the animal body with the feed additives, is of great significance for the processing of finished feed additives containing microorganisms and for improving their economic value. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention uses Bacillus subtilis and Bifidobacterium animalis as core strains in a compound feed additive. These strains are prepared into bacterial liposome suspensions, which are then reacted with polysaccharide extracts, modified steviol glycosides, and emulsifiers. Finally, the mixture is spray-dried at high temperature to obtain the compound feed additive, thereby solving the technical problems mentioned in the background art. Specifically, the technical solution of this invention includes the following:

[0008] One objective of this invention is to provide a method for preparing a compound feed additive, the method comprising the following steps:

[0009] Bacterial liposome suspension, polysaccharide extract, modified steviol glycosides and emulsifier were mixed in a weight ratio of 1:0.3-0.4:0.1-0.2:0.01 and stirred at 30°C for 20-24 hours to obtain microcapsule emulsion.

[0010] Microencapsulated emulsions are spray-dried to obtain compound feed additives.

[0011] Furthermore, the preparation method of the bacterial liposome suspension includes the following steps:

[0012] Phospholipids, water-soluble polysaccharides, EDC hydrochloride and 4-dimethylaminopyridine were mixed in a weight ratio of 1:0.4 to 0.5:0.5:0.05 and reacted at 30°C for 22 to 26 hours to obtain modified phospholipids.

[0013] Modified phospholipids, cholesterol, and chloroform were mixed and stirred to obtain a mixture, which was then distilled under reduced pressure to obtain liposome films.

[0014] Liposome membranes, microbial inoculant powders, and buffer solutions were sequentially ultrasonically dispersed and hydrated to obtain a bacterial-loaded liposome suspension.

[0015] Furthermore, the phospholipid includes phosphatidylserine, and the phospholipid needs to have an amino group in its structure for subsequent condensation reaction with the carboxyl group on the water-soluble polysaccharide, thereby achieving hydrophilic modification of the phospholipid.

[0016] Furthermore, the water-soluble polysaccharide includes carboxymethyl chitosan.

[0017] Furthermore, the conditions for vacuum distillation include a vacuum degree of -0.09 MPa and a temperature of 50°C.

[0018] Furthermore, the microbial inoculant powder is composed of Bifidobacterium animalis powder and Bacillus subtilis powder in a weight ratio of 1:0.3 to 0.5.

[0019] Furthermore, the live bacteria content of both the Bifidobacterium animalis powder and the Bacillus subtilis powder is 20 billion CFU / g.

[0020] Furthermore, the buffer solution comprises a phosphate buffer solution with a pH of 6.5.

[0021] Furthermore, the weight ratio of the modified phospholipid:cholesterol:chloroform:microbial inoculant powder:buffer solution is 10-15:3-4:4000-5000:1-1.5:7000-8000.

[0022] Furthermore, the conditions for ultrasonic dispersion include an ultrasonic power of 200W, an ultrasonic temperature of 25℃, and an ultrasonic time of 10min.

[0023] Furthermore, the conditions for the hydration treatment include a temperature of 35°C, a rotation speed of 100 r / min, and a time of 130 min.

[0024] Furthermore, the polysaccharide extract includes chitosan oligosaccharides with a molecular weight of 1700 Da.

[0025] Furthermore, the preparation method of the modified steviol glycoside includes the following steps:

[0026] Stevioside, sodium periodate, and deionized water were mixed in a weight ratio of 1:0.5-0.6:60-70 and protected from light. The mixture was then oxidized at 25-30°C for 4-5 hours to obtain a reaction solution. The reaction solution was then subjected to dialysis and freeze-drying to obtain modified steviol glycosides.

[0027] Furthermore, the steviol glycosides include ribobadiol A.

[0028] Furthermore, the dialysis process includes using a dialysis bag with a molecular weight cutoff of 1000 Da and dialysis with distilled water for 48 hours.

[0029] Furthermore, the freeze-drying process includes pre-freezing at -30°C for 2 hours, followed by vacuum drying at -40°C for 24 hours.

[0030] Furthermore, the emulsifier includes Tween 20, Tween 40, or Tween 80.

[0031] Furthermore, the spray drying conditions include an inlet air temperature of 140℃~150℃, a feed rate of 15mL / min, and an outlet air temperature of 80℃.

[0032] The second objective of this invention is to provide a compound feed additive.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] This invention uses Bacillus subtilis and Bifidobacterium animalis as the core strains in a compound feed additive. Bacillus subtilis can deplete free oxygen in the gastrointestinal tract through biological oxygen depletion, thereby reducing oxygen concentration and promoting the growth and reproduction of beneficial anaerobic microorganisms in the intestine. Furthermore, Bacillus subtilis can secrete active substances such as antimicrobial peptides to inhibit the reproduction of pathogenic bacteria. Bifidobacterium animalis can lower the intestinal pH by producing acetic acid and lactic acid, inhibiting the reproduction of pathogenic bacteria. The two work synergistically to improve the microecological environment in animals, promoting intestinal development and antagonizing pathogenic bacteria. Next, phospholipids are modified using hydrophilic, water-soluble polysaccharides to obtain modified phospholipids. This hydrophilic modification improves the stability of the strains encapsulated in liposomes. The modified phospholipids are then combined with cholesterol to prepare liposomes. Through low-temperature hydration, the strains diffuse into the liposome carrier, where they are encapsulated to form a bacterial liposome suspension, reducing the impact of the external environment on the activity of the strains. Then, steviol glycosides with an ortho-diol structure are oxidized to form functional aldehyde groups, thus obtaining modified steviol glycosides. After mixing the modified steviol glycosides, polysaccharide extract, bacterial liposome suspension, and emulsifier, the aldehyde groups on the modified steviol glycosides and the amino groups on the polysaccharide extract undergo covalent cross-linking condensation to form heat-resistant imine bonds, resulting in a microcapsule emulsion. The microcapsule emulsion is then spray-dried to obtain a compound feed additive. The synergistic mechanism of the heat-resistant imine bonds and liposomes effectively mitigates the loss of viable bacteria caused by high temperatures during drying. Upon entering the animal body, the imine bonds and liposomes gradually decompose and release the bacterial strain in the acidic environment of the stomach, thereby exerting their effects. Furthermore, the polysaccharide extract with heat-resistant imine bonds has a simple structure and can be directly used as a carbon source by the strain, improving the strain's metabolic activity and thus promoting the strain's regulation of the animal's intestines. Steviosides can increase the animal's food intake, improve the survival rate of live bacteria through the synergistic protection of imine bonds and liposomes, and the nutritional value of the polysaccharide extract with imine bonds and steviosides themselves synergistically promote the animal's growth and development. Detailed Implementation

[0035] 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.

[0036] Unless otherwise stated, all raw materials and reagents used in this invention are commercially available or can be prepared by known methods.

[0037] Bifidobacterium animalis and Bacillus subtilis were purchased from Shandong Yihao Biotechnology Co., Ltd.

[0038] Preparation Example 1:

[0039] The preparation method of bacterial liposome suspension specifically includes the following steps:

[0040] Four parts by weight of carboxymethyl chitosan were added to 100 parts by weight of dichloromethane and dispersed using ultrasonic power at 300W until homogeneous. The mixture was then kept warm in a 25°C water bath. Next, five parts by weight of EDC hydrochloride and 0.5 parts by weight of 4-dimethylaminopyridine were added to the incubated carboxymethyl chitosan and stirred for 30 minutes to obtain an activation solution. Then, 10 parts by weight of phosphatidylserine were added to the activation solution, and the mixture was heated to 30°C and stirred for 22 hours. After the reaction was completed, the resulting reaction solution was poured into a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed with distilled water until all residues such as dichloromethane were completely removed, yielding a dialysate. The dialysate was dried to obtain modified phospholipids. Ten parts by weight of the modified phospholipids and four parts by weight of cholesterol were weighed and added to a flask, followed by the addition of 4000 parts by weight of chloroform. The mixture was stirred until completely dispersed and homogeneous. The flask was then evacuated using a vacuum pump until a vacuum level of -0.09 MPa was reached. Subsequently, a 50°C flow rate was applied. The mixture was subjected to vacuum distillation at ℃ until a liposome film was formed. 7000 parts by weight of phosphate buffer solution (pH 6.5) and 1 part by weight of microbial inoculant powder (composed of *Bifidobacterium animalis* powder and *Bacillus subtilis* powder, each with a viable count of 20 billion CFU / g, in a weight ratio of 1:0.3) were weighed and mixed thoroughly. This mixture was then added to the liposome film and mixed. The mixture was first placed in an ultrasonic disperser with an ultrasonic power set to 200W and a water temperature controlled at 25℃ for 10 minutes of ultrasonic dispersion. After treatment, the mixture was removed and then placed in a 35℃ water bath with magnetic stirring at 100 rpm for 130 minutes to obtain a bacterial-loaded liposome suspension. This suspension was then stored at 5℃ for later use.

[0041] Preparation Example 2:

[0042] The preparation method of bacterial liposome suspension specifically includes the following steps:

[0043] 4.5 parts by weight of carboxymethyl chitosan were added to 100 parts by weight of dichloromethane and dispersed using ultrasonic power at 300W until homogeneous. The mixture was then kept warm in a 25°C water bath. Next, 5 parts by weight of EDC hydrochloride and 0.5 parts by weight of 4-dimethylaminopyridine were added to the incubated carboxymethyl chitosan and stirred for 30 minutes to obtain an activation solution. Then, 10 parts by weight of phosphatidylserine were added to the activation solution, and the mixture was heated to 30°C and stirred for 24 hours. After the reaction was completed, the resulting reaction solution was poured into a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed with distilled water until all residues such as dichloromethane were completely removed, yielding a dialysate. The dialysate was dried to obtain modified phospholipids. 12 parts by weight of the modified phospholipids and 3.5 parts by weight of cholesterol were weighed and added to a flask, followed by 4300 parts by weight of chloroform. The mixture was stirred until completely dispersed and homogeneous. The flask was then evacuated using a vacuum pump until a vacuum level of -0.09 MPa was reached. Subsequently, a 50... The mixture was subjected to vacuum distillation at ℃ until a liposome film was formed. 7500 parts by weight of phosphate buffer solution (pH 6.5) and 1.2 parts by weight of microbial inoculant powder (composed of *Bifidobacterium animalis* powder and *Bacillus subtilis* powder, each with a viable count of 20 billion CFU / g, in a weight ratio of 1:0.4) were weighed and mixed thoroughly. This mixture was then added to the liposome film and mixed. The mixture was first placed in an ultrasonic disperser with an ultrasonic power of 200W and a water temperature of 25℃ for 10 minutes of ultrasonic dispersion. After dispersion, the mixture was removed and hydrated in a 35℃ water bath at 100 rpm with magnetic stirring for 130 minutes to obtain a bacterial-loaded liposome suspension. This suspension was then stored at 5℃ for later use.

[0044] Preparation Example 3:

[0045] The preparation method of bacterial liposome suspension specifically includes the following steps:

[0046] Five parts by weight of carboxymethyl chitosan were added to 100 parts by weight of dichloromethane and dispersed using ultrasonic power at 300W until homogeneous. The mixture was then kept warm in a 25°C water bath. Next, five parts by weight of EDC hydrochloride and 0.5 parts by weight of 4-dimethylaminopyridine were added to the incubated carboxymethyl chitosan and stirred for 30 minutes to obtain an activation solution. Then, 10 parts by weight of phosphatidylserine were added to the activation solution, and the mixture was heated to 30°C and stirred for 26 hours. After the reaction was completed, the resulting reaction solution was poured into a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed with distilled water until all residues such as dichloromethane were completely removed, yielding a dialysate. The dialysate was dried to obtain modified phospholipids. 15 parts by weight of the modified phospholipids and 3 parts by weight of cholesterol were weighed and added to a flask, followed by 4500 parts by weight of chloroform. The mixture was stirred until completely dispersed and homogeneous. The flask was then evacuated using a vacuum pump until a vacuum level of -0.09 MPa was reached, and subsequently heated at 50°C. Vacuum distillation was performed until a liposome film was formed. 8000 parts by weight of phosphate buffer solution (pH 6.5) and 1.5 parts by weight of microbial inoculant powder (composed of *Bifidobacterium animalis* powder and *Bacillus subtilis* powder, each with a viable count of 20 billion CFU / g, in a weight ratio of 1:0.5) were weighed and mixed thoroughly. This mixture was then added to the liposome film and mixed. The mixture was first placed in an ultrasonic disperser with an ultrasonic power of 200W and a water temperature of 25℃ for 10 minutes of ultrasonic dispersion. After dispersion, the mixture was removed and then placed in a 35℃ water bath with magnetic stirring at 100 rpm for 130 minutes to obtain a bacterial-loaded liposome suspension. This suspension was then stored at 5℃ for later use.

[0047] Preparation Example 4:

[0048] The preparation method of bacterial liposome suspension specifically includes the following steps:

[0049] Weigh 15 parts by weight of phosphatidylserine and 3 parts by weight of cholesterol and add them to a flask. Then add 4500 parts by weight of chloroform and stir until completely dispersed and uniform. Then, use a vacuum pump to evacuate the flask until the vacuum degree reaches -0.09 MPa. Then, perform vacuum distillation at 50°C until a liposome film is formed. Weigh 8000 parts by weight of phosphate buffer solution with pH 6.5 and 1.5 parts by weight of microbial agent powder (composed of Bifidobacterium animalis powder and Bacillus subtilis powder, both with a viable bacterial content of 20 billion CFU / g, in a weight ratio of 1:0.5) and stir evenly. Then, add all of them to the above liposome film and mix. First, place it in an ultrasonic disperser with the ultrasonic power set to 200W and the water temperature controlled at 25°C, and then ultrasonically disperse for 10 minutes. After processing, the suspension was removed and then hydrated in a water bath at 35°C with a magnetic stirring speed of 100 r / min for 130 min to obtain a bacterial liposome suspension, which was then stored at 5°C for later use.

[0050] Preparation Example 5:

[0051] The preparation method of bacterial liposome suspension specifically includes the following steps:

[0052] In Preparation Example 3, phosphatidylserine was replaced with phosphatidylcholine, and the rest of the preparation process was the same as in Preparation Example 3.

[0053] Preparation Example 6:

[0054] The preparation method of bacterial liposome suspension specifically includes the following steps:

[0055] Weigh 15 parts by weight of the modified phospholipid obtained in Preparation Example 3, 3 parts by weight of cholesterol, and 1.5 parts by weight of microbial inoculant powder (composed of Bifidobacterium animalis powder and Bacillus subtilis powder, each with a viable bacterial content of 20 billion CFU / g, in a weight ratio of 1:0.5) and add them together to a flask. Then add 4500 parts by weight of chloroform and stir until completely dispersed and uniform to form a mixture. Then, use a vacuum pump to evacuate the flask until the vacuum degree reaches -0.09 MPa. Subsequently, perform vacuum distillation at 50°C until a liposome film is formed. Weigh 8000 parts by weight of phosphate buffer solution with pH 6.5 and add it to the above liposome film and mix. First, place it in an ultrasonic disperser with an ultrasonic power set to 200W and a water temperature controlled at 25°C, and then ultrasonically disperse for 10 minutes. After processing, the suspension was removed and then hydrated in a water bath at 35°C with a magnetic stirring speed of 100 r / min for 130 min to obtain a bacterial liposome suspension, which was then stored at 5°C for later use.

[0056] Preparation Example 7:

[0057] The preparation method of modified steviol glycosides specifically includes the following steps:

[0058] One part by weight of ribobadiol A was weighed and added to 60 parts by weight of deionized water. The mixture was stirred until completely dispersed and then protected from light. Next, 0.5 parts by weight of sodium periodate was added and stirred, and the oxidation reaction was carried out at 25°C for 4 hours. After the reaction was complete, one part by weight of ethylene glycol was added and the reaction was continued with stirring for another hour. The resulting reaction solution was poured into a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed with distilled water for 48 hours. The dialysate was placed in a freeze dryer, pre-frozen at -30°C for 2 hours, then evacuated to a vacuum degree of 30 Pa, and further cooled to -40°C for vacuum drying for 24 hours to obtain modified steviol glycosides.

[0059] Preparation Example 8:

[0060] The preparation method of modified steviol glycosides specifically includes the following steps:

[0061] One part by weight of ribobadiol A was weighed and added to 65 parts by weight of deionized water. The mixture was stirred until completely dispersed and then protected from light. Next, 0.5 parts by weight of sodium periodate was added and stirred, and the oxidation reaction was carried out at 30°C for 4 hours. After the reaction was complete, one part by weight of ethylene glycol was added and the reaction was continued with stirring for another hour. The resulting reaction solution was poured into a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed with distilled water for 48 hours. The dialysate was placed in a freeze dryer, pre-frozen at -30°C for 2 hours, then evacuated to a vacuum degree of 30 Pa, and further cooled to -40°C for vacuum drying for 24 hours to obtain modified steviol glycosides.

[0062] Preparation Example 9:

[0063] The preparation method of modified steviol glycosides specifically includes the following steps:

[0064] One part by weight of ribobadiol A was weighed and added to 70 parts by weight of deionized water. The mixture was stirred until completely dispersed and then protected from light. Next, 0.6 parts by weight of sodium periodate was added and stirred, and the oxidation reaction was carried out at 30°C for 5 hours. After the reaction was complete, one part by weight of ethylene glycol was added and the reaction was continued with stirring for 1 hour. The resulting reaction solution was poured into a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed with distilled water for 48 hours. The dialysate was placed in a freeze dryer, pre-frozen at -30°C for 2 hours, then evacuated to a vacuum degree of 30 Pa, and further cooled to -40°C for vacuum drying for 24 hours to obtain modified steviol glycosides.

[0065] Preparation Example 10:

[0066] The preparation method of modified steviol glycosides specifically includes the following steps:

[0067] One part by weight of ribobadiol A was weighed and added to 70 parts by weight of deionized water. The mixture was stirred until completely dispersed and then protected from light. Next, one part by weight of sodium periodate was added and stirred, and the oxidation reaction was maintained at 30°C for 7 hours. After the reaction was complete, one part by weight of ethylene glycol was added and the reaction was continued with stirring for 1 hour. The resulting reaction solution was poured into a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed with distilled water for 48 hours. The dialysate was placed in a freeze dryer, pre-frozen at -30°C for 2 hours, then evacuated to a vacuum of 30 Pa, and further cooled to -40°C for vacuum drying for 24 hours to obtain modified steviol glycosides.

[0068] Preparation Example 11:

[0069] The preparation method of modified steviol glycosides specifically includes the following steps:

[0070] One part by weight of ribobadiol A was weighed and added to 60 parts by weight of deionized water. The mixture was stirred until completely dispersed and then protected from light. Next, 0.3 parts by weight of sodium periodate was added and stirred, and the oxidation reaction was carried out at 25°C for 2 hours. After the reaction was complete, one part by weight of ethylene glycol was added and the reaction was continued with stirring for another hour. The resulting reaction solution was poured into a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed with distilled water for 48 hours. The dialysate was placed in a freeze dryer, pre-frozen at -30°C for 2 hours, then evacuated to a vacuum degree of 30 Pa, and further cooled to -40°C for vacuum drying for 24 hours to obtain modified steviol glycosides.

[0071] Preparation Example 12:

[0072] The preparation method of modified sodium alginate specifically includes the following steps:

[0073] One part by weight of sodium alginate was weighed and added to 70 parts by weight of deionized water. The mixture was stirred until completely dispersed and then protected from light. Next, 0.6 parts by weight of sodium periodate was added and stirred, and the oxidation reaction was carried out at 30°C for 5 hours. After the reaction was complete, one part by weight of ethylene glycol was added and the reaction was continued with stirring for another hour. The resulting reaction solution was poured into a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed with distilled water for 48 hours. The dialysate was placed in a freeze dryer, pre-frozen at -30°C for 2 hours, then evacuated to a vacuum degree of 30 Pa, and further cooled to -40°C for vacuum drying for 24 hours to obtain modified sodium alginate.

[0074] Example 1:

[0075] A method for preparing a compound feed additive specifically includes the following steps:

[0076] Weigh 1 part by weight of the bacterial liposome suspension obtained in Preparation Example 1, 0.3 parts by weight of chitosan oligosaccharide (molecular weight 1700 Da), 0.1 parts by weight of the modified steviol glycoside obtained in Preparation Example 7, and 0.01 parts by weight of Tween 20 and add them to a flask for mixing. Then place the flask in a water bath and heat it to 30°C. Then stir continuously at 200 r / min for 20 h to obtain a microcapsule emulsion. First, electrically heat the inlet air temperature of the spray dryer. When the inlet air temperature reaches 140°C and the outlet air temperature reaches 80°C, start feeding the microcapsule emulsion into the spray dryer at a feed rate of 15 mL / min for spray drying. Then collect the powder, seal and store it to obtain a compound feed additive.

[0077] Example 2:

[0078] A method for preparing a compound feed additive specifically includes the following steps:

[0079] One part by weight of the bacterial liposome suspension obtained in Preparation Example 2, 0.35 parts by weight of chitosan oligosaccharide (molecular weight 1700 Da), 0.15 parts by weight of the modified steviol glycoside obtained in Preparation Example 8, and 0.01 parts by weight of Tween 40 were weighed and added to a flask and mixed. The mixture was then placed in a water bath and heated to 30°C. The mixture was then stirred continuously at 200 r / min for 22 h to obtain a microcapsule emulsion. The inlet air temperature of the spray dryer was electrically heated until it reached 145°C and the outlet air temperature reached 80°C. The microcapsule emulsion was then fed into the spray dryer at a feed rate of 15 mL / min for spray drying. The powder was then collected, sealed, and stored to obtain a compound feed additive.

[0080] Example 3:

[0081] A method for preparing a compound feed additive specifically includes the following steps:

[0082] Weigh 1 part by weight of the bacterial liposome suspension obtained in Preparation Example 3, 0.4 parts by weight of chitosan oligosaccharide (molecular weight 1700 Da), 0.2 parts by weight of the modified steviol glycoside obtained in Preparation Example 9, and 0.01 parts by weight of Tween 80 and add them to a flask for mixing. Then place the flask in a water bath and heat it to 30°C. Then stir continuously at 200 r / min for 24 h to obtain a microcapsule emulsion. First, electrically heat the inlet air temperature of the spray dryer. When the inlet air temperature reaches 150°C and the outlet air temperature reaches 80°C, start feeding the microcapsule emulsion into the spray dryer at a feed rate of 15 mL / min for spray drying. Then collect the powder, seal and store it to obtain a compound feed additive.

[0083] Comparative Example 1:

[0084] A method for preparing a compound feed additive specifically includes the following steps:

[0085] The bacterial liposome suspension in Example 3 was replaced with the bacterial liposome suspension obtained in Preparation Example 4, and the rest of the preparation process was the same as in Example 3.

[0086] Comparative Example 2:

[0087] A method for preparing a compound feed additive specifically includes the following steps:

[0088] The bacterial liposome suspension in Example 3 was replaced with the bacterial liposome suspension obtained in Preparation Example 5, and the rest of the preparation process was the same as in Example 3.

[0089] Comparative Example 3:

[0090] A method for preparing a compound feed additive specifically includes the following steps:

[0091] The bacterial liposome suspension in Example 3 was replaced with the bacterial liposome suspension obtained in Preparation Example 6, and the rest of the preparation process was the same as in Example 3.

[0092] Comparative Example 4:

[0093] A method for preparing a compound feed additive specifically includes the following steps:

[0094] The modified steviol glycosides in Example 3 were replaced with the modified steviol glycosides obtained in Preparation Example 10, and the rest of the preparation process was the same as in Example 3.

[0095] Comparative Example 5:

[0096] A method for preparing a compound feed additive specifically includes the following steps:

[0097] The modified steviol glycosides in Example 3 were replaced with the modified steviol glycosides obtained in Preparation Example 11, and the rest of the preparation process was the same as in Example 3.

[0098] Comparative Example 6:

[0099] A method for preparing a compound feed additive specifically includes the following steps:

[0100] The modified steviol glycosides in Example 3 were replaced with the modified sodium alginate obtained in Preparation Example 12. The rest of the preparation process remained the same as in Example 3. It was found that the overall viscosity was high during the preparation process, making it difficult to spray dry, and the preparation failed. This may be because although sodium alginate also has a vicinal diol structure, it easily forms a viscous liquid after being dissolved and dispersed in water, resulting in high viscosity and making it impossible to spray dry.

[0101] Comparative Example 7:

[0102] A method for preparing a compound feed additive specifically includes the following steps:

[0103] First, the inlet air temperature of the spray dryer is electrically heated. When the inlet air temperature reaches 150°C and the outlet air temperature reaches 80°C, the bacterial liposome suspension obtained in Preparation Example 3 is fed into the spray dryer at a feed rate of 15 mL / min for spray drying. Then, the powder is collected, sealed and stored to obtain the compound feed additive.

[0104] Comparative Example 8:

[0105] A method for preparing a compound feed additive specifically includes the following steps:

[0106] Replacing sodium alginate with chitosan oligosaccharide in Example 3, while maintaining the same preparation process as in Example 3, revealed that the solution exhibited a viscous colloidal state during preparation, making spray drying difficult and resulting in preparation failure. This may be because sodium alginate readily forms a viscous colloid upon contact with water, leading to a high solution viscosity that prevents spray drying.

[0107] Comparative Example 9:

[0108] A method for preparing a compound feed additive specifically includes the following steps:

[0109] One part by weight of the bacterial liposome suspension obtained in Preparation Example 3, 0.4 parts by weight of chitosan oligosaccharide (molecular weight 1700 Da), 0.2 parts by weight of the modified steviol glycoside obtained in Preparation Example 9, and 0.01 parts by weight of Tween 80 were weighed and added to a flask and mixed. The mixture was then placed in a water bath and heated to 30°C. The mixture was then stirred continuously at 200 r / min for 24 h to obtain a microcapsule emulsion. The inlet air temperature of the spray dryer was electrically heated until it reached 170°C and the outlet air temperature reached 80°C. The microcapsule emulsion was then fed into the spray dryer at a feed rate of 15 mL / min for spray drying. The powder was then collected, sealed, and stored to obtain a compound feed additive.

[0110] The compound feed additives obtained in Examples 1-3, Comparative Examples 1-5, Comparative Examples 7, and Comparative Example 9 were mixed with a daily diet (the daily diet consisted of corn, soybean meal, wheat bran, and premix in a weight percentage ratio of 65%:20%:5%:10%). The premix contained 6250 IU of vitamin A, 1.25 mg of vitamin B1, 6 mg of vitamin B2, 3 mg of vitamin B6, 0.03 mg of vitamin B12, 1500 IU of vitamin D3, 50 IU of vitamin E, 2.5 mg of vitamin K3, 0.25 mg of folic acid, 15 mg of niacin, and 12.5 mg of [unspecified ingredient] per kilogram of the diet. D-pantothenic acid, 0.3 mg biotin, 500 mg choline, 6.85 mg copper, 63.5 mg zinc, 69.5 mg iron, 0.08 mg iodine, 72.5 mg manganese, and 0.35 mg selenium were mixed in a ratio of 1:100 to form a feed. Thirty-three pigs with an average weight of 20.87 ± 0.82 kg were randomly divided into 10 groups of 3 pigs each and fed the mixed feed. A control group fed only the diet was used. The feeding was carried out continuously for 30 days. The final weight and total feed intake were recorded on the 30th day. The average daily weight gain and average daily feed intake were then calculated. The results are shown in Table 1 below.

[0111] Table 1. Growth performance of pigs

[0112] Source of materials Average daily weight gain (g) Average daily feed intake (g) Example 1 568.12±25.03 1421.03±22.74 Example 2 620.09±33.41 1469.55±19.73 Example 3 592.06±21.88 1445.34±23.61 Comparative Example 1 469.35±16.09 1350.04±21.23 Comparative Example 2 470.62±15.94 1351.51±19.85 Comparative Example 3 461.35±13.88 1343.94±18.09 Comparative Example 4 478.51±19.74 1365.62±18.91 Comparative Example 5 458.09±16.27 1339.01±19.25 Comparative Example 7 453.22±14.08 1328.02±15.13 Comparative Example 9 485.07±20.44 1379.65±22.08 control group 439.37±18.85 1314.09±28.11

[0113] The following conclusions can be drawn from the test structure in Table 1 above:

[0114] (1) As can be seen from Examples 1 to 3, when the compound feed additive prepared by the present invention is used in combination with the diet, it not only increases the pig's feed intake, but also makes the pig's weight gain performance good.

[0115] (2) Comparative Example 1 shows that when the prepared compound feed additive is used in combination with the diet, the weight gain of pigs is poor. This may be because when the liposomes prepared by vacuum distillation of phosphatidylserine and cholesterol are used to encapsulate the strain, the stability of the liposome membrane may be poor, which may lead to leakage of the strain during spray drying, thus affecting the performance of the compound feed additive.

[0116] (3) Comparative Example 2 shows that when the prepared compound feed additive is used in combination with the diet, the weight gain of pigs is poor. This may be because phosphatidylcholine lacks functional groups that can condense with carboxymethyl chitosan in its structure, resulting in modification failure and thus affecting the performance of the compound feed additive.

[0117] (4) Comparative Example 3 shows that when the prepared compound feed additive is used in combination with the diet, the weight gain of pigs is poor. This may be because if the strain is directly mixed with modified phospholipids and cholesterol and evaporated under reduced pressure to prepare liposomes, and then hydrated to form a suspension of bacterial liposomes, on the one hand, the organic reagents may have a destructive effect on the activity of the strain, resulting in more live bacteria dying, and on the other hand, the high temperature of the reduced pressure evaporation may cause the strain to die, thus affecting the performance of the compound feed additive.

[0118] (5) Comparative Example 4 shows that when the prepared compound feed additive is used in combination with the diet, the weight gain of pigs is poor. This may be because although oxidation treatment can give Rebaudioside A the functional aldehyde group, the structure of Rebaudioside A may be severely damaged due to excessive oxidation, which may result in the loss of its role as a sweetener to stimulate appetite, resulting in poor feed intake and slow weight gain.

[0119] (6) Comparative Example 5 shows that when the prepared compound feed additive is used in combination with the diet, the weight gain of pigs is poor. This may be because although oxidation treatment can give Rebaudioside A the functional aldehyde group, the amount of oxidant used in this system is low and the oxidation treatment time is short. This may result in fewer aldehyde groups generated by the cleavage of the ortho-dihydroxy group on Rebaudioside A, resulting in poor high temperature protection effect and more live bacteria may die, thus affecting the performance of the compound feed additive.

[0120] (7) Comparative Example 7 shows that when the prepared compound feed additive is used in combination with the diet, the weight gain of pigs is poor. This may be because the protective effect of liposome encapsulation alone makes it difficult to retain the activity of live bacteria during high-temperature drying, thus affecting the performance of the compound feed additive.

[0121] (8) Comparative Example 9 shows that the prepared compound feed additive has more charring phenomenon and more serious powder sticking to the wall. When used in combination with the diet, the weight gain of pigs is poor. This may be because, on the one hand, when the temperature is too high, the emulsion dries quickly and softens at the same time, making it easy to stick to the inner surface of the cyclone collector. On the other hand, excessively high temperature may cause the surface moisture of the droplets to evaporate instantly to form a hard shell, and the internal moisture cannot escape. The local temperature rises and then charring occurs, resulting in poor performance of the compound feed additive.

[0122] 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 a compound feed additive, characterized in that, The preparation method includes the following steps: Bacterial liposome suspension, polysaccharide extract, modified steviol glycoside and emulsifier were mixed in a weight ratio of 1:0.03~0.4:0.1~0.2:0.01 and stirred at 30℃ for 20h~24h to obtain microcapsule emulsion; Microencapsulated emulsions are spray-dried to obtain compound feed additives; The preparation method of the bacterial liposome suspension includes the following steps: Phospholipids, water-soluble polysaccharides, EDC hydrochloride and 4-dimethylaminopyridine were mixed in a weight ratio of 1:0.4~0.5:0.5:0.05 and reacted at 30℃ for 22h~26h to obtain modified phospholipids; Modified phospholipids, cholesterol, and chloroform were mixed and stirred to obtain a mixture, which was then distilled under reduced pressure to obtain liposome films. Liposome membranes, microbial agent powders, and buffer solutions were sequentially ultrasonically dispersed and hydrated to obtain a bacterial-loaded liposome suspension. The preparation method of the modified steviol glycosides includes the following steps: Stevioside, sodium periodate, and deionized water were mixed in a weight ratio of 1:0.5~0.6:60~70 and protected from light. The mixture was then oxidized at 25℃~30℃ for 4h~5h to obtain a reaction solution. The reaction solution was then subjected to dialysis and freeze-drying to obtain modified steviol glycosides.

2. The method for preparing a compound feed additive according to claim 1, characterized in that, The phospholipids include phosphatidylserine.

3. The method for preparing a compound feed additive according to claim 1, characterized in that, The water-soluble polysaccharide includes carboxymethyl chitosan.

4. The method for preparing a compound feed additive according to claim 1, characterized in that, The microbial inoculant powder is composed of Bifidobacterium animalis powder and Bacillus subtilis powder in a weight ratio of 1:0.3~0.

5.

5. The method for preparing a compound feed additive according to claim 1, characterized in that, The polysaccharide extract includes chitosan oligosaccharides with a molecular weight of 1700 Da.

6. The method for preparing a compound feed additive according to claim 1, characterized in that, The steviol glycosides include ribobadiol A.

7. A compound feed additive prepared by the method described in any one of claims 1 to 6.