Preparation method and application of selenium-enriched probiotic and chitosan oligosaccharide composite formula animal feed

Abstract

The invention discloses a preparation method and application of selenium-enriched probiotic and chitosan oligosaccharide composite formula animal feed, and relates to the technical field of animal feed preparation. According to the preparation method and application of the selenium-rich probiotic and chitosan oligosaccharide composite formula animal feed, candida utilis, lactobacillus acidophilus, streptococcus thermophilus and lactobacillus rhamnosus are added into a culture medium according to the proportion of 3%, selected mixed strains are inoculated into an MRS culture medium to be cultured, and selenium-rich mixed probiotics are obtained; and stirring and mixing with the prepared feed mixture, granulating by a granulator, and drying by hot air to obtain the required compound formula animal feed. According to the method, the plates are coated with the prepared mixed strains respectively, the strains with red bacterial colonies on the plates with high sodium selenite mass concentration are sodium selenite high-tolerance strains, so that the high-tolerance strains are screened out, the selenium content of the prepared animal feed is basically maintained at a certain level, and the quality of the animal feed is improved. And inappetence and even damage to gastrointestinal organs of animals are avoided.

Description

Technical Field

[0001] The invention relates to the technical field of animal feed preparation, and in particular to a method for preparing a composite formula animal feed of selenium-enriched probiotics and chitosan oligosaccharide and application thereof. Background Art

[0002] Animals maintain their lives and produce products by taking in various nutrients from plant feeds. The main nutrients in feeds include proteins, fats, carbohydrates, minerals and vitamins, which have different functions in animals. Understanding the relationship between animals and plants and their nutritional composition is crucial to optimizing feed configuration and improving animal productivity.

[0003] Selenium is an essential trace nutrient for animals. It plays an important role in animal health and disease by converting into selenoproteins (such as glutathione peroxidase, iodinated tyrosine deiodinase, etc.) in the body, such as improving immunity, antioxidant capacity, anti-tumor and regulating metabolism. Probiotics can maintain the balance of gastrointestinal microbial flora, prevent the proliferation of pathogenic microorganisms in the intestine, slow down intestinal inflammation and infection, improve the digestive system and stimulate the host immune system.

[0004] Studies have shown that selenium or probiotics can be added to piglet diets as anti-heat stress additives to reduce the adverse effects of heat stress on piglet health and production performance. However, there is currently no targeted screening for the introduction of selenium into animal feed preparation and the direct use of selenium-rich probiotics. In the process of animal feed preparation, some selenium-rich probiotics may cause colony tolerance if added too much because edible fiber such as grains and distiller's grains contains selenium, which in turn causes loss of appetite in the animals eating the feed and even damage to the gastrointestinal organs. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a method for preparing a composite formula animal feed of selenium-enriched probiotics and chitosan oligosaccharides and its application, which solves the problems raised in the above-mentioned background technology.

[0006] To achieve the above objectives, the present invention is implemented by the following technical scheme: a method for preparing a composite animal feed of selenium-enriched probiotics and chitosan oligosaccharide and its application, comprising the following specific steps:

[0007] S1: Candida utilis, Lactobacillus acidophilus, Streptococcus thermophilus and Lactobacillus rhamnosus are added to YEPD culture medium at a ratio of 3%, respectively, and cultured at an ambient temperature of 37°C for 24 hours. The obtained Candida utilis culture, Lactobacillus acidophilus culture, Lactobacillus acidophilus culture, Streptococcus thermophilus culture and Lactobacillus rhamnosus culture are stored at 4°C for standby use. The Candida utilis culture, Lactobacillus acidophilus culture, Lactobacillus acidophilus culture, Streptococcus thermophilus culture and Lactobacillus rhamnosus culture stored at 4°C are inoculated into a slant culture medium, cultured at 20-30°C for 24 hours, activated 5 times in a cycle, inoculated into a cultivation culture medium, and cultured at 40°C for 24 hours to obtain a mixed strain;

[0008] S2: The mixed strains were inoculated into MRS medium, and cultured in a shake flask at 37°C for 12 hours. After three passages, five plates containing sodium selenite were prepared, and the final mass concentration of sodium selenite in each plate was 20, 40, 60, 80, and 100 g / m, respectively. 50 uL of the mixed strain suspension was drawn and applied to the above plates, respectively. After culturing for 24-48 hours, the color change of the colonies was observed. The colonies on the plates containing sodium selenite generally showed red. The strains that showed red colonies on the plates with higher sodium selenite mass concentrations were sodium selenite high-tolerance strains. The red probiotics were selected for re-culture, and inorganic selenium was added to the culture medium to control the selenium concentration at 3-6 mg / kg. The selected mixed strains were inoculated into MRS medium for culture to obtain selenium-enriched mixed probiotics.

[0009] S3: Selenium-rich mixed probiotics are inoculated into MRS culture medium, and methionine, serine, chitosan oligosaccharides and protein chrysanthemum decoction are added during the culture to obtain selenium-rich probiotic mixed chitosan oligosaccharide culture medium. After adsorbing with dolyl fiber, it is dried and packaged at 30°C, stirred and mixed with the prepared feed mixture, granulated by a granulator and then hot-air dried to obtain the required compound formula animal feed.

[0010] A further improvement of the technical solution of the present invention is that the chitosan oligosaccharide in S3 comprises the following specific steps:

[0011] S3-1: After the fish and shrimp shells are initially cleaned, decalcification, deproteinization and decolorization are carried out in sequence to obtain granular chitin, and an appropriate amount of NaOH with a concentration of 40% to 45% is weighed, and the chitin is refluxed and circulated at an ambient temperature of 110°C to complete the deacetylation. After the reflux is completed, the chitin is circulated and cleaned until it is neutral, and then placed in a 100°C oven for drying to obtain chitosan oligosaccharides.

[0012] A further improvement of the technical solution of the present invention is that the specific steps of decalcification in T1 are:

[0013] S3-11: Weigh an appropriate amount of discarded shrimp and crab shells, wash them, dry them, and grind them in a grinder. Soak the shell crushed material in HCl at room temperature, wash and filter them after multiple cycles until the acid-base is neutral. Select a small amount of the filtered product and titrate it with EDTA to determine the Ca 2+ , using calcein as an indicator, when calcein exhibits yellow-green fluorescence or fluorescent state, the cleaning and filtration are circulated again until no fluorescence or color change occurs, and the decalcified shell crushed material is retained.

[0014] A further improvement of the technical solution of the present invention is that the specific steps of deproteinization in T1 are:

[0015] S3-12: The decalcified shells are added into 6% diluted alkali, crushed, boiled and soaked. After the protein and fat are decomposed, stir and keep warm for 1 hour.

[0016] A further improvement of the technical solution of the present invention is that the specific steps of the decolorization treatment in T1 are:

[0017] S3-13: After boiling and soaking, the decomposed shells were added with 5% KMnO4 and soaked again. After soaking for 3 hours, the purple color was washed off with NaHCO3, filtered and dried in an oven at 100°C to obtain chitin.

[0018] A further improvement of the technical solution of the present invention is that the preparation of the feed mixture comprises the following specific steps:

[0019] S3-21: Weigh edible fiber, clean it, soak it in a 5% acetic acid solution at an ambient temperature of 60° C., soak it for 48 hours, then crush and filter it to obtain acidified fiber;

[0020] S3-22: Weigh appropriate amounts of edible fiber, wheat bran, second flour, soybean meal, puffed soybeans, cottonseed cake, and fish meal, mix them, spray appropriate amount of water, and steam them in a steamer for 40 minutes to obtain a mixture;

[0021] S3-23: Mix appropriate amounts of SiO2, FeO3, CaO, MgO and NaHCO3 to obtain a mineral mixture;

[0022] S2-24: Beat the acidified fiber obtained from S3-21 and the mixture to mix evenly, roll it out and flatten it, add salt and vitamin additives, knead it into a ball, repeat several times, put it into a fermentation tank, seal it, and sun-dry it for 12-16 days. Take out the fermented material and mix it evenly with the mineral mixture of S3-23, add 4 times the amount of hot water and stir, filter it after stirring evenly, and obtain the feed mixture.

[0023] A further improvement of the technical solution of the present invention is that the pH value of the acidified fiber obtained after the acidified fiber in S3-21 is soaked in 5% acetic acid is controlled to be 2.8±0.2.

[0024] A further improvement of the technical solution of the present invention is that the chitosan obtained in S3-13 has an ash content of ≤4%, a moisture content of ≤4%, and is white or gray in color.

[0025] The present invention also provides the use of the above-mentioned animal feed in the preparation of feed food that promotes digestion and absorption or improves the high temperature resistance of intestinal flora or is low in fat and cholesterol.

[0026] Compared with the prior art, the invention has the following beneficial effects: the mixed strains prepared are coated on the above plates respectively, and the strains whose colonies are red only on the plates with a higher sodium selenite mass concentration are the strains with high tolerance to sodium selenite, thereby screening out the strains with high tolerance, so that the selenium content of the prepared animal feed is basically maintained at a certain level, avoiding excessive selenium content, and avoiding loss of appetite of the animals eating the feed and even damage to the gastrointestinal organs;

[0027] Selenium-rich probiotics can reduce the adverse effects of heat stress on animal health and production performance, improve production performance, enhance antioxidant capacity, regulate intestinal flora, and achieve the benign diversification of animal production performance, antioxidant capacity, blood selenium content, thyroid hormone content and fecal flora under high temperature conditions;

[0028] Selenium-rich probiotics and chitosan oligosaccharides can react with gastric acid in the animal's stomach to form a gel, which can absorb bile acid and cholesterol and excrete them through feces, thereby reducing the cholesterol content in the blood. This meets the current demand for functional foods, such as low-fat, low-cholesterol foods, and provides a new feed preparation direction for low-fat, low-cholesterol animal breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the process for preparing a composite animal feed of selenium-enriched probiotics and chitosan oligosaccharides;

[0030] Figure 2 It is a schematic diagram of the preparation process of chitosan oligosaccharide in the preparation method of selenium-enriched probiotics and chitosan oligosaccharide compound formula animal feed;

[0031] Figure 3 This is a schematic diagram of the feed mixture preparation process in the method for preparing a compound formula animal feed of selenium-enriched probiotics and chitosan oligosaccharides;

[0032] Figure 4 It is a line graph of the growth of experimental piglets in Example 1;

[0033] Figure 5 It is a line graph of the growth of experimental piglets in Example 2. DETAILED DESCRIPTION

[0034] Various exemplary embodiments, features and aspects of the present application will be described in detail below with reference to the accompanying drawings. The same reference numerals in the accompanying drawings represent elements with the same or similar functions. Although various aspects of the embodiments are shown in the accompanying drawings, the drawings are not necessarily drawn to scale unless otherwise specified.

[0035] The word “exemplary” is used exclusively herein to mean “serving as an example, example, or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.

[0036] In addition, in order to better illustrate the present application, numerous specific details are provided in the specific examples below. It should be understood by those skilled in the art that the present application can also be implemented without certain specific details. In some examples, methods, means, and elements well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present application.

[0037] The present invention provides a method for preparing a composite animal feed of selenium-enriched probiotics and chitosan oligosaccharide, comprising the following specific steps:

[0038] S1: Candida utilis, Lactobacillus acidophilus, Streptococcus thermophilus and Lactobacillus rhamnosus are added to YEPD culture medium at a ratio of 3%, respectively, and cultured at an ambient temperature of 37°C for 24 hours. The obtained Candida utilis culture, Lactobacillus acidophilus culture, Lactobacillus acidophilus culture, Streptococcus thermophilus culture and Lactobacillus rhamnosus culture are stored at 4°C for standby use. The Candida utilis culture, Lactobacillus acidophilus culture, Lactobacillus acidophilus culture, Streptococcus thermophilus culture and Lactobacillus rhamnosus culture stored at 4°C are inoculated into a slant culture medium, cultured at 20-30°C for 24 hours, activated 5 times in a cycle, inoculated into a cultivation culture medium, and cultured at 40°C for 24 hours to obtain a mixed strain;

[0039] S2: The mixed strains were inoculated into MRS medium, and cultured in a shake flask at 37°C for 12 hours. After three passages, five plates containing sodium selenite were prepared, and the final mass concentration of sodium selenite in each plate was 20, 40, 60, 80, and 100 g / m, respectively. 50 uL of the mixed strain suspension was drawn and applied to the above plates, respectively. After culturing for 24-48 hours, the color change of the colonies was observed. The colonies on the plates containing sodium selenite generally showed red. The strains that showed red colonies on the plates with higher sodium selenite mass concentrations were sodium selenite high-tolerance strains. The red probiotics were selected for re-culture, and inorganic selenium was added to the culture medium to control the selenium concentration at 3-6 mg / kg. The selected mixed strains were inoculated into MRS medium for culture to obtain selenium-enriched mixed probiotics.

[0040] S3: Selenium-rich mixed probiotics are inoculated into MRS culture medium, and methionine, serine, chitosan oligosaccharides and protein chrysanthemum decoction are added during the culture to obtain selenium-rich probiotic mixed chitosan oligosaccharide culture medium. After adsorbing with dolyl fiber, it is dried and packaged at 30°C, stirred and mixed with the prepared feed mixture, granulated by a granulator and then hot-air dried to obtain the required compound formula animal feed.

[0041] The chitosan oligosaccharide in S3 comprises the following specific steps:

[0042] S3-1: After the fish and shrimp shells are initially cleaned, decalcification, deproteinization and decolorization are carried out in sequence to obtain granular chitin, and an appropriate amount of NaOH with a concentration of 40% to 45% is weighed, and the chitin is refluxed and circulated at an ambient temperature of 110°C to complete the deacetylation. After the reflux is completed, the chitin is circulated and cleaned until it is neutral, and then placed in a 100°C oven for drying to obtain chitosan oligosaccharides.

[0043] The specific steps of decalcification in T1 are:

[0044] S3-11: Weigh an appropriate amount of discarded shrimp and crab shells, wash them, dry them, and grind them in a grinder. Soak the shell crushed material in HCl at room temperature, wash and filter them after multiple cycles until the acid-base is neutral. Select a small amount of the filtered product and titrate it with EDTA to determine the Ca 2+ , using calcein as an indicator, when calcein exhibits yellow-green fluorescence or fluorescent state, the cleaning and filtration are circulated again until no fluorescence or color change occurs, and the decalcified shell crushed material is retained.

[0045] The specific steps of deproteinization in T1 are:

[0046] S3-12: The decalcified shells are added into 6% diluted alkali, crushed, boiled and soaked. After the protein and fat are decomposed, stir and keep warm for 1 hour.

[0047] The specific steps of the decolorization treatment in T1 are:

[0048] S3-13: After boiling and soaking, the decomposed shells were added with 5% KMnO4 and soaked again. After soaking for 3 hours, the purple color was washed off with NaHCO3, filtered and dried in an oven at 100°C to obtain chitin.

[0049] The feed mixture preparation comprises the following specific steps:

[0050] S3-21: Weigh edible fiber, clean it, soak it in a 5% acetic acid solution at an ambient temperature of 60° C., soak it for 48 hours, then crush and filter it to obtain acidified fiber;

[0051] S3-22: Weigh appropriate amounts of edible fiber, wheat bran, second flour, soybean meal, puffed soybeans, cottonseed cake, and fish meal, mix them, spray appropriate amount of water, and steam them in a steamer for 40 minutes to obtain a mixture;

[0052] S3-23: Mix appropriate amounts of SiO2, FeO3, CaO, MgO and NaHCO3 to obtain a mineral mixture;

[0053] S2-24: Beat the acidified fiber obtained from S3-21 and the mixture to mix evenly, roll it out and flatten it, add salt and vitamin additives, knead it into a ball, repeat several times, put it into a fermentation tank, seal it, and sun-dry it for 12-16 days. Take out the fermented material and mix it evenly with the mineral mixture of S3-23, add 4 times the amount of hot water and stir, filter it after stirring evenly, and obtain the feed mixture.

[0054] The pH value of the acidified fiber obtained by soaking the acidified fiber in S3-21 in 5% acetic acid is controlled to be 2.8±0.2.

[0055] The chitosan obtained in S3-13 has an ash content of ≤4%, a moisture content of ≤4%, and is white or gray in color.

[0056] Example 1: In this example, the specific benefits of the selenium-enriched probiotics and chitosan oligosaccharides of the present application in helping animals establish a gastrointestinal microbial community system that is beneficial to the host, such as stimulating the body's specific and nonspecific immunity, diarrhea, growth, and feed utilization, are discussed;

[0057] The experimental subjects selected 48 30-day-old weaned piglets from a three-way crossbreed and randomly divided them into four groups. All pigs were raised in the same pig house with the room temperature controlled at around 21°C. The pigs were ventilated and disinfected daily during the raising period. The experimental period was set to 30 days. For the convenience of analysis, the four groups were set as a blank control group, a sodium selenite group, a probiotic group, and a test group.

[0058] The blank control group was fed with a basal diet;

[0059] The sodium selenite group was fed a basal diet supplemented with 0.3 mg / kg of selenium in sodium selenite;

[0060] The probiotic group was fed a basic diet with probiotics added; (the number of viable bacteria in the probiotic group was the same as the number of viable bacteria in the selenium-enriched probiotics in the experimental group)

[0061] The experimental group was fed with the feed prepared as follows, and the feed amount was the same as the basic diet: Candida utilis, Lactobacillus acidophilus, Streptococcus thermophilus and Lactobacillus rhamnosus were added to the YEPD medium at a ratio of 3%, respectively, and cultured at an ambient temperature of 37°C for 24 hours. The obtained Candida utilis culture, Lactobacillus acidophilus culture, Lactobacillus acidophilus culture, Streptococcus thermophilus culture and Lactobacillus rhamnosus culture were stored at 4°C for use. The Candida utilis culture, Lactobacillus acidophilus culture, Lactobacillus acidophilus culture, Streptococcus thermophilus culture and Lactobacillus rhamnosus culture stored at 4°C were inoculated into the slant medium, cultured at 20-30°C for 24 hours, and after 5 cycles of activation, they were inoculated into the planting medium and cultured at 40°C for 24 hours to obtain a mixed strain; the mixed strain was inoculated into the MRS medium, cultured in a shake flask at 37°C for 12 hours, and after 3 passages, ... , prepare five plates containing sodium selenite, so that the final mass concentration of sodium selenite in each plate is 20, 40, 60, 80, and 100 g / m, respectively, draw 50 uL of the mixed bacterial suspension, apply it to the above plates respectively, observe the color change of the colony after culturing for 24-48 hours, select the red probiotics for re-culturing, add inorganic selenium to the culture medium, control the selenium concentration at 3-6 mg / kg, inoculate the selected mixed bacteria in MRS culture medium for cultivation, and obtain selenium-enriched mixed probiotics; the selenium-enriched mixed probiotics are inoculated in MRS culture medium, and methionine, serine, chitosan oligosaccharide and protein chrysanthemum decoction are added during the culture to obtain selenium-enriched probiotics mixed chitosan oligosaccharide culture solution, after adsorption with dolyfiber, dry and package at 30°C, mix with the prepared feed mixture, granulate by a granulator and then dry with hot air to obtain the required composite formula animal feed;

[0062] After 30 days, the average daily weight gain, average daily feed intake, feed-to-meat ratio and diarrhea rate of the piglets were measured. The results showed that compared with the blank control group, the weight loss of the experimental group, sodium selenite group and probiotic group increased by 15.99% (P < 0.05), 14.78% (P > 0.05) and 15.85% (P > 0.05), respectively.

[0063] Compared with the blank control group, the average daily weight gain of the experimental group, sodium selenite group and probiotics group increased by 22.21% (P < 0.05), 18.88% (P < 0.05) and 21.10% (P < 0.05), respectively;

[0064] There was no significant difference between the experimental group and the sodium selenite group and the probiotic group, and the feed-to-meat ratio of the experimental group was significantly lower than that of the sodium selenite group and the probiotic group; the diarrhea rate of the experimental group and the probiotic group was significantly lower than that of the sodium selenite group and the control group, and there was no significant difference in the diarrhea rate between the experimental group and the probiotic group;

[0065] Therefore, the compound formula animal feed prepared in the present application has no significant difference from the probiotic group at normal temperature, and has a positive effect on stimulating specific and nonspecific immunity, diarrhea, growth, and feed utilization of piglets;

[0066] Example 2: The composite animal feed under normal temperature has a positive effect on stimulating specific and nonspecific immunity, diarrhea, growth, and feed utilization, but there is not much difference with the probiotic group. However, the study found that heat stress under high temperature environment can cause serious harm to the production of farms. It can significantly affect the balance of gastrointestinal microecology of animals, reduce immunity, and destroy the body's antioxidant defense system.

[0067] The adverse effects caused by heat stress, such as decreased appetite, weight loss, decreased feed utilization, excessive morbidity and mortality, etc., cause huge economic losses to the breeding industry.

[0068] In order to explore the effect of selenium-enriched probiotics and chitosan oligosaccharide compound animal feed on

[0069] The experimental subjects selected 48 30-day-old weaned piglets from a three-way crossbreed and randomly divided them into four groups. All pigs were raised in the same pig house with the room temperature controlled at around 21°C. The pigs were ventilated and disinfected daily during the raising period. The experimental period was set to 30 days. For the convenience of analysis, the four groups were set as a blank control group, a sodium selenite group, a probiotic group, and a test group.

[0070] The blank control group was fed with a basal diet;

[0071] The sodium selenite group was fed a basal diet supplemented with 0.3 mg / kg of selenium in sodium selenite;

[0072] The probiotic group was fed a basic diet with probiotics added; (the number of viable bacteria in the probiotic group was the same as the number of viable bacteria in the selenium-enriched probiotics in the experimental group)

[0073] The experimental group was fed with the feed prepared as follows, and the feed amount was the same as the basic diet: Candida utilis, Lactobacillus acidophilus, Streptococcus thermophilus and Lactobacillus rhamnosus were added to the YEPD medium at a ratio of 3%, respectively, and cultured at an ambient temperature of 37°C for 24 hours. The obtained Candida utilis culture, Lactobacillus acidophilus culture, Lactobacillus acidophilus culture, Streptococcus thermophilus culture and Lactobacillus rhamnosus culture were stored at 4°C for use. The Candida utilis culture, Lactobacillus acidophilus culture, Lactobacillus acidophilus culture, Streptococcus thermophilus culture and Lactobacillus rhamnosus culture stored at 4°C were inoculated into the slant medium, cultured at 20-30°C for 24 hours, and after 5 cycles of activation, they were inoculated into the planting medium and cultured at 40°C for 24 hours to obtain a mixed strain; the mixed strain was inoculated into the MRS medium, cultured in a shake flask at 37°C for 12 hours, and after 3 passages, ... , prepare five plates containing sodium selenite, so that the final mass concentration of sodium selenite in each plate is 20, 40, 60, 80, and 100 g / m, respectively, draw 50 uL of the mixed bacterial suspension, apply it to the above plates respectively, observe the color change of the colony after culturing for 24-48 hours, select the red probiotics for re-culturing, add inorganic selenium to the culture medium, control the selenium concentration at 3-6 mg / kg, inoculate the selected mixed bacteria in MRS culture medium for cultivation, and obtain selenium-enriched mixed probiotics; the selenium-enriched mixed probiotics are inoculated in MRS culture medium, and methionine, serine, chitosan oligosaccharide and protein chrysanthemum decoction are added during the culture to obtain selenium-enriched probiotics mixed chitosan oligosaccharide culture solution, after adsorption with dolyfiber, dry and package at 30°C, mix with the prepared feed mixture, granulate by a granulator and then dry with hot air to obtain the required composite formula animal feed;

[0074] After 30 days, the average daily weight gain, average daily feed intake, feed-to-meat ratio and diarrhea rate of the piglets were measured. The results showed that compared with the blank control group, the weight gain of the experimental group, sodium selenite group and probiotic group increased by 9.99% (P < 0.05), 6.78% (P > 0.05) and 6.85% (P > 0.05), respectively.

[0075] Compared with the blank control group, the average daily weight gain of the experimental group, sodium selenite group and probiotics group increased by 18.21% (P < 0.05), 9.88% (P < 0.05) and 10.10% (P < 0.05), respectively;

[0076] There were significant differences between the experimental group and the sodium selenite group and the probiotic group. The feed-to-meat ratio of the experimental group was significantly lower than that of the sodium selenite group and the probiotic group. The diarrhea rate of the experimental group and the probiotic group was significantly lower than that of the sodium selenite group and the control group. There was a significant difference in the diarrhea rate between the experimental group and the probiotic group.

[0077] Therefore, adding selenium-rich probiotics and chitosan oligosaccharide compound formula animal feed to the diet can improve the production performance of piglets under high temperature conditions, reduce the diarrhea rate of piglets, help animals establish a gastrointestinal microbial community system that is beneficial to the host, and have a beneficial effect on diarrhea, growth, and stimulation of the body's specific and nonspecific immunity under high temperature environments.

[0078] Example 3: The piglets of the control group in Example 1 and Example 2 and the piglets of the blank control group were placed at room temperature. After 30 days of continuous testing, the two control groups were 43.06 mg / 100 ml lower than the corresponding control group, and the cholesterol content in the leg muscle and pectoral muscle was 5.08% and 4.96% lower than that in the control group, respectively. There was no significant difference in weight gain and feed efficiency between the control groups of Example 1 and Example 2. It is reasonable to believe that selenium-enriched probiotics and chitosan oligosaccharides can react with gastric acid in the stomach to form a gel, and the gel can be kept from decomposing within the pH range in the intestine. The gel has an adsorption effect and can adsorb bile acid and cholesterol, which are excreted through feces, thereby reducing the cholesterol content in the blood, which meets the current demand for functional foods such as low-fat and low-cholesterol foods.

[0079] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0080] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a composite animal feed of selenium-enriched probiotics and chitosan oligosaccharide, characterized in that: The specific steps include: S1: Candida utilis, Lactobacillus acidophilus, Streptococcus thermophilus and Lactobacillus rhamnosus are added to YEPD culture medium at a ratio of 3%, respectively, and cultured at an ambient temperature of 37°C for 24 hours. The obtained Candida utilis culture, Lactobacillus acidophilus culture, Lactobacillus acidophilus culture, Streptococcus thermophilus culture and Lactobacillus rhamnosus culture are stored at 4°C for standby use. The Candida utilis culture, Lactobacillus acidophilus culture, Lactobacillus acidophilus culture, Streptococcus thermophilus culture and Lactobacillus rhamnosus culture stored at 4°C are inoculated into a slant culture medium, cultured at 20-30°C for 24 hours, activated 5 times in a cycle, inoculated into a cultivation culture medium, and cultured at 40°C for 24 hours to obtain a mixed strain; S2: The mixed strains were inoculated into MRS medium, and cultured in a shake flask at 37°C for 12 hours. After three passages, five plates containing sodium selenite were prepared, and the final mass concentration of sodium selenite in each plate was 20, 40, 60, 80, and 100 g / m, respectively. 50 uL of the mixed strain suspension was drawn and applied to the above plates, respectively. After culturing for 24-48 hours, the color change of the colony was observed, and the red probiotics were selected for re-culture. Inorganic selenium was added to the culture medium, and the selenium concentration was controlled at 3-6 mg / kg. The selected mixed strains were inoculated into MRS medium for culture to obtain selenium-enriched mixed probiotics. S3: Selenium-rich mixed probiotics are inoculated into MRS culture medium, and methionine, serine, chitosan oligosaccharides and protein chrysanthemum decoction are added during the culture to obtain selenium-rich probiotic mixed chitosan oligosaccharide culture medium. After adsorbing with dolyl fiber, it is dried and packaged at 30°C, stirred and mixed with the prepared feed mixture, granulated by a granulator and then hot-air dried to obtain the required compound formula animal feed.

2. The method for preparing the composite formula animal feed of selenium-enriched probiotics and chitosan oligosaccharide according to claim 1, characterized in that: The chitosan oligosaccharide in S3 comprises the following specific steps: S3-1: After the fish and shrimp shells are initially cleaned, decalcification, deproteinization and decolorization are carried out in sequence to obtain granular chitin, and an appropriate amount of NaOH with a concentration of 40% to 45% is weighed, and the chitin is refluxed and circulated at an ambient temperature of 110°C to complete the deacetylation. After the reflux is completed, the chitin is circulated and cleaned until it is neutral, and then placed in a 100°C oven for drying to obtain chitosan oligosaccharides.

3. The method for preparing the composite formula animal feed of selenium-enriched probiotics and chitosan oligosaccharide according to claim 1, characterized in that: The specific steps of decalcification in T1 are: S3-11: Weigh an appropriate amount of discarded shrimp and crab shells, wash them, dry them, and grind them in a grinder. Soak the shell crushed material in HCl at room temperature, wash and filter them after multiple cycles until the acid-base is neutral. Select a small amount of the filtered product and titrate it with EDTA to determine the Ca 2+ , using calcein as an indicator, when calcein exhibits yellow-green fluorescence or fluorescent state, the cleaning and filtration are circulated again until no fluorescence or color change occurs, and the decalcified shell crushed material is retained.

4. The method for preparing the composite formula animal feed of selenium-enriched probiotics and chitosan oligosaccharide according to claim 3, characterized in that: The specific steps of deproteinization in T1 are: S3-12: The decalcified shells are added into 6% diluted alkali, crushed, boiled and soaked. After the protein and fat are decomposed, stir and keep warm for 1 hour.

5. The method for preparing the composite formula animal feed of selenium-enriched probiotics and chitosan oligosaccharide according to claim 1, characterized in that: The specific steps of the decolorization treatment in T1 are: S3-13: After boiling and soaking, the decomposed shells were added with 5% KMnO4 and soaked again. After soaking for 3 hours, the purple color was washed off with NaHCO3, filtered and dried in an oven at 100°C to obtain chitin.

6. The method for preparing the composite formula animal feed of selenium-enriched probiotics and chitosan oligosaccharide according to claim 1, characterized in that: The feed mixture preparation comprises the following specific steps: S3-21: Weigh edible fiber, clean it, soak it in a 5% acetic acid solution at an ambient temperature of 60° C., soak it for 48 hours, then crush and filter it to obtain acidified fiber; S3-22: Weigh appropriate amounts of edible fiber, wheat bran, second flour, soybean meal, puffed soybeans, cottonseed cake, and fish meal, mix them, spray appropriate amount of water, and steam them in a steamer for 40 minutes to obtain a mixture; S3-23: Mix appropriate amounts of SiO2, FeO3, CaO, MgO and NaHCO3 to obtain a mineral mixture; S2-24: Beat the acidified fiber obtained from S3-21 and the mixture to mix evenly, roll it out and flatten it, add salt and vitamin additives, knead it into a ball, repeat several times, put it into a fermentation tank, seal it, and sun-dry it for 12-16 days. Take out the fermented material and mix it evenly with the mineral mixture of S3-23, add 4 times the amount of hot water and stir, filter it after stirring evenly, and obtain the feed mixture.

7. The method for preparing the composite formula animal feed of selenium-enriched probiotics and chitosan oligosaccharide according to claim 6, characterized in that: The pH value of the acidified fiber obtained by soaking the acidified fiber in S3-21 in 5% acetic acid is controlled to be 2.8±0.

2.

8. The method for preparing the composite formula animal feed of selenium-enriched probiotics and chitosan oligosaccharide according to claim 1, characterized in that: The chitosan obtained in S3-13 has an ash content of ≤4%, a moisture content of ≤4%, and is white or gray in color.

9. Use of the animal feed according to claim 1 in preparing feed food that promotes digestion and absorption or improves the high temperature resistance of intestinal flora or is low in fat and cholesterol.

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