Vinasse biological feed as well as preparation method and application thereof

Abstract

The invention discloses a vinasse biological feed as well as a preparation method and application thereof. Belongs to the technical field of vinasse biological feed. The vinasse biological feed is prepared from the following components in parts by weight: 60 to 75 parts of vinasse, 10 to 15 parts of corn flour, 8 to 12 parts of soybean meal, 3 to 5 parts of wheat bran, 0.3 to 0.8 part of a sulfhydrylation modified chitosan intercalated montmorillonite compound, 0.05 to 0.15 part of a compound enzyme preparation, 0.1 to 0.3 part of a compound microbial agent and 1 to 2 parts of a premix. In the preparation process of the vinasse biological feed, enzymolysis pretreatment is combined with an aerobic and anaerobic double-fermentation process, so that the content of crude protein in the vinasse biological feed is increased, and crude fibers are effectively degraded.

Description

Technical Field

[0001] This invention belongs to the field of distillers' grains biological feed technology, specifically, it relates to a distillers' grains biological feed, its preparation method, and its application. Background Technology

[0002] Protein is the material basis for animal life activities and a core element in livestock and poultry farming costs. Distillers' grains, as a major byproduct of the brewing or fuel ethanol industry, retain most of the protein and unfermented fiber components of the original grain, but due to its characteristics of "high crude fiber, high anti-nutritional factors, and easy mold growth," it has long been regarded as a low-value feed ingredient.

[0003] To enhance the feed value of distillers' grains, microbial fermentation technology is widely adopted in the industry. However, conventional fermentation processes often face challenges such as low cellulose degradation efficiency and high risk of mycotoxin residues. In particular, existing technologies have not been able to fundamentally resolve the inherent contradiction of competitive adsorption between toxins and nutrients from a physicochemical perspective.

[0004] To address the issues of high crude fiber content and low protein conversion rate in distillers' grains, existing technologies primarily focus on multi-strain synergistic fermentation. For example, Chinese patent application CN108902446A discloses a high-protein distillers' grains feed and its preparation method, which specifically discloses the use of *Proteus vulgaris*, *Trichoderma reesei*, and *Aspergillus niger* for stepwise fermentation of distillers' grains, successively degrading lignin and cellulose / hemicellulose.

[0005] While the above-mentioned technical solutions improve the degradation rate of crude fiber to some extent through the combination of microbial strains, they rely solely on the natural secretion and action of extracellular enzymes of microorganisms. According to the principles of enzymology, cellulase is easily inactivated by heat, acid and product inhibition in complex fermentation environments, and lacks the assistance of nanoscale carriers, making it difficult to penetrate the dense cell wall structure. As a result, the overall conversion efficiency still has a ceiling. Furthermore, the above-mentioned technical solutions do not have a blocking mechanism against mycotoxins, and if the fermentation process is not properly controlled, secondary toxin contamination can easily occur.

[0006] Chinese patent application CN107801839A discloses a method for preparing fermented pig feed with added mycotoxin adsorbents, which uses a composite adsorbent composed of modified montmorillonite, yeast cell wall powder, and carbon-coated montmorillonite. This method utilizes the high specific surface area and cation exchange capacity of layered silicate montmorillonite to adsorb toxins by expanding the interlayer spacing through intercalation agents.

[0007] The drawback of the above technical solution is that the surfactant modification used is a non-specific hydrophobic modification, and the interlayer domains of montmorillonite mainly rely on physical adsorption. This broad-spectrum adsorption mechanism cannot distinguish between toxin molecules and small molecule nutrients such as vitamins and amino acids in feed at the molecular level, resulting in the adsorption of a large amount of nutrients while adsorbing toxins, causing nutrient loss.

[0008] In conclusion, developing a bio-feed that can effectively enhance the degradation of crude protein and crude fiber in distillers' grains bio-feed has extremely important application value. Summary of the Invention

[0010] To address the deficiencies in the aforementioned technical solutions, the present invention aims to provide a bio-feed made from distiller's grains, its preparation method, and its application.

[0011] To achieve the above objectives, the present invention provides a bio-feed made from distillers' grains, which, by weight, comprises the following raw materials: 60-75 parts distillers' grains, 10-15 parts corn flour, 8-12 parts soybean meal, 3-5 parts wheat bran, 0.3-0.8 parts thiolated modified chitosan-intercalated montmorillonite complex, 0.05-0.15 parts compound enzyme preparation, 0.1-0.3 parts compound microbial agent, and 1-2 parts premix.

[0012] The lees are fresh baijiu lees, a byproduct of solid-state brewing, with a water content of 55%-65% and a pH value of 3.5-4.5; The corn flour is whole corn flour with a starch content of ≥65%; The soybean meal is dehulled soybean meal with a crude protein content of ≥43%; The compound enzyme preparation is composed of cellulase, xylanase and laccase in an activity ratio of 2:1:0.5. The cellulase activity in the compound enzyme preparation is ≥10000 U / g; The compound microbial agent is composed of Candida utilis, Bacillus subtilis and Saccharomyces cerevisiae, in a live cell ratio of 2:1:1, with an effective live cell count ≥1.0×10^8 CFU / g.

[0013] The premix is ​​composed of compound vitamins and compound organic trace elements; The complex vitamins include vitamin A, vitamin B, vitamin C, vitamin D, and vitamin C. The composite organic trace elements include a mixture of lysine iron, glycine zinc, and methionine manganese.

[0014] The thiol-modified chitosan-intercalated montmorillonite complex was prepared by the following method: 1% acetic acid solution was added to a reaction vessel, followed by chitosan raw material with a degree of deacetylation >85%, and the mixture was stirred to dissolve. Chitosanase was added, and a controlled enzymatic hydrolysis reaction was carried out at pH 5.0-5.5 and a temperature of 37°C for 4-6 hours. After the reaction, the temperature was raised to 90°C to inactivate the enzyme preparation. The pH was adjusted to 8.0 to precipitate the mixture, which was then filtered, washed with ethanol, and dried to obtain low molecular weight chitosan. Low molecular weight chitosan was dissolved in a 1% acetic acid solution. High-purity nitrogen was continuously introduced into the reaction system to remove oxygen. Then, mercaptoacetic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide were added in sequence. The mixture was stirred and reacted at room temperature in the dark for 4-6 hours. The reaction product was precipitated with anhydrous ethanol, centrifuged, and washed repeatedly with 70% ethanol to remove residual reagents. The product was then freeze-dried under vacuum to obtain low molecular weight mercapto-substituted chitosan. Subsequently, a sodium-based montmorillonite aqueous dispersion with a mass fraction of 2%-3% was prepared and ultrasonically treated at a frequency of 40kHz for 30 minutes to exfoliate the layers. Low molecular weight thiolated chitosan was dissolved in deionized water and slowly added dropwise to the montmorillonite dispersion under a constant temperature water bath of 60℃ and high-speed stirring at 500-800rpm. After the addition was completed, the reaction was stirred for another 6 hours to carry out cation exchange. Finally, the reaction solution was centrifuged at 4000rpm for 15 minutes and the supernatant was discarded. The precipitate was washed with deionized water until neutral, dried to constant weight in a vacuum drying oven at 60℃, and pulverized through a 200-mesh sieve to obtain a grayish-white thiolated chitosan-intercalated montmorillonite composite powder.

[0015] Furthermore, the low molecular weight chitosan obtained during the preparation of the thiolized modified chitosan intercalated montmorillonite composite has a molecular weight of 5-10 kDa. Furthermore, in the preparation process of the thiolated modified chitosan intercalated montmorillonite composite, the molar ratio of low molecular weight chitosan, thioglycolic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide is controlled to be 1:1.5:1.8:1.8. Furthermore, in the preparation process of the thiolated modified chitosan intercalated montmorillonite composite, the mass ratio of low molecular weight thiolated chitosan to sodium montmorillonite is controlled at 0.3:1.

[0016] A method for preparing a bio-feed from distiller's grains includes the following steps: Step A: Put the distiller's grains, corn flour, soybean meal, and wheat bran into a mixer and stir evenly; add an appropriate amount of water to adjust the total moisture content of the mixture to 60%; then add the compound enzyme preparation, mix evenly, and then transport it to the pretreatment tank for enzymatic hydrolysis at 45°C for 4 hours to destroy the plant cell wall structure. Step B: After the temperature of the enzymatically hydrolyzed material in Step A drops below 35°C, evenly sprinkle in the low molecular weight thiolized chitosan intercalated montmorillonite complex, and stir vigorously for 15-20 minutes. Utilize the active thiol groups on its surface to inhibit miscellaneous bacteria and adsorb mycotoxins in the substrate. Then add the premix and the composite microbial agent activated with warm water, and mix evenly again. Step C: Loosely pile the mixture on the fermentation bed, controlling the material layer thickness to 30-40cm, and carry out aerobic fermentation for 24 hours at 30-35℃ to promote the growth of Bacillus subtilis and consume oxygen; then transfer the aerobic fermented material into a sealed fermentation bag or fermentation tank, compact and seal it, and continue anaerobic fermentation for 48-72 hours at 30-35℃, using Candida utilis to convert non-protein nitrogen into cell protein and accumulate organic acids; Step D: After fermentation, the wet base feed is sent into an airflow dryer, and the inlet air temperature is controlled to be no higher than 60°C for low-temperature rapid drying, so that the moisture content is reduced to below 12%. The dried material is then crushed, sieved, and vacuum-packed to prepare a high-protein, low-fiber biological feed with selective mycotoxin adsorption function.

[0017] Application of a type of distillers' grains biological feed in animal husbandry.

[0018] The beneficial effects of this invention are: 1. This application introduces a thiol-modified chitosan-intercalated montmorillonite complex, using low molecular weight thiolized chitosan to intercalate and modify montmorillonite. Through the hydrophobic microenvironment introduced by thiol and the molecular sieve effect constructed by specific interlayer spacing, the absorption and capture of hydrophobic toxins are achieved. At the same time, the principle of polar repulsion is used to repel hydrophilic nutrients, thereby achieving an extremely high selective adsorption coefficient and solving the problem that traditional montmorillonite adsorbents will absorb nutrients at the same time. 2. The technical solution of this application, in the preparation process of distillers' grains biological feed, creatively increases the crude protein content in the distillers' grains biological feed and effectively improves the degradation of crude fiber by combining enzymatic pretreatment with aerobic and anaerobic dual fermentation process, and prepares a thiol-modified chitosan-intercalated montmorillonite complex as a stable carrier for the enzyme. It not only breaks the physical barrier of plant cell walls, but also prolongs the enzyme's action time through the carrier protection effect, fundamentally overcoming the pain points of difficult fiber degradation and low protein conversion rate in conventional fermentation. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The illustrative embodiments and descriptions of this invention are for explanation only and are not intended to limit the invention. Furthermore, regarding numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] Example 1 A type of bio-feed made from distillers' grains, by weight, comprises the following: 70 parts distillers' grains, 12 parts corn flour, 10 parts soybean meal, 4 parts wheat bran, 0.5 parts thiolated modified chitosan intercalated montmorillonite complex, 0.1 parts compound enzyme preparation, 0.2 parts compound microbial agent, and 1.5 parts premix.

[0024] The lees are fresh baijiu lees, a byproduct of solid-state brewing, with a water content of 55%-65% and a pH value of 3.5-4.5; The corn flour is whole corn flour with a starch content of ≥65%; The soybean meal is dehulled soybean meal with a crude protein content of ≥43%; The compound enzyme preparation is composed of cellulase, xylanase and laccase in an activity ratio of 2:1:0.5. The cellulase activity in the compound enzyme preparation is ≥10000 U / g; The compound microbial agent is composed of Candida utilis, Bacillus subtilis and Saccharomyces cerevisiae, in a live cell ratio of 2:1:1, with an effective live cell count ≥1.0×10^8 CFU / g.

[0025] The premix is ​​composed of compound vitamins and compound organic trace elements; The complex vitamins include vitamin A, vitamin B, vitamin C, vitamin D, and vitamin C. The composite organic trace elements include a mixture of lysine iron, glycine zinc, and methionine manganese.

[0026] The thiol-modified chitosan-intercalated montmorillonite complex was prepared by the following method: 1% acetic acid solution was added to a reaction vessel, followed by chitosan raw material with a degree of deacetylation >85%, and the mixture was stirred to dissolve. Chitosanase was added, and a controlled enzymatic hydrolysis reaction was carried out at pH 5.0-5.5 and a temperature of 37°C for 4-6 hours. After the reaction, the temperature was raised to 90°C to inactivate the enzyme preparation. The pH was adjusted to 8.0 to precipitate the mixture, which was then filtered, washed with ethanol, and dried to obtain low molecular weight chitosan. Low molecular weight chitosan was dissolved in 1% acetic acid solution. High-purity nitrogen was continuously introduced into the reaction system to remove oxygen. Then, mercaptoacetic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide were added sequentially. The mixture was stirred and reacted at room temperature in the dark for 4-6 hours to form amide bonds between the carboxyl groups of mercaptoacetic acid and the amino groups of chitosan. The reaction product was precipitated with anhydrous ethanol, centrifuged, and washed repeatedly with 70% ethanol to remove residual reagents. Then, it was freeze-dried under vacuum to obtain low molecular weight mercapto-substituted chitosan.

[0027] Subsequently, a sodium-based montmorillonite aqueous dispersion with a mass fraction of 2%-3% was prepared and ultrasonically treated at a frequency of 40kHz for 30 minutes to exfoliate the layers. Low molecular weight thiolated chitosan was dissolved in deionized water and slowly added dropwise to the montmorillonite dispersion under a constant temperature water bath of 60℃ and high-speed stirring at 500-800rpm. After the addition was completed, the reaction was stirred for another 6 hours to carry out cation exchange. Finally, the reaction solution was centrifuged at 4000rpm for 15 minutes and the supernatant was discarded. The precipitate was washed with deionized water until neutral, dried to constant weight in a vacuum drying oven at 60℃, and pulverized through a 200-mesh sieve to obtain a grayish-white thiolated chitosan-intercalated montmorillonite composite powder.

[0028] Furthermore, the low molecular weight chitosan obtained during the preparation of the thiolized modified chitosan intercalated montmorillonite composite has a molecular weight of 5-10 kDa. Furthermore, in the preparation process of the thiolated modified chitosan intercalated montmorillonite composite, the molar ratio of low molecular weight chitosan, thioglycolic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide is controlled to be 1:1.5:1.8:1.8. Furthermore, in the preparation process of the thiolated modified chitosan intercalated montmorillonite composite, the mass ratio of low molecular weight thiolated chitosan to sodium montmorillonite is controlled at 0.3:1.

[0029] A method for preparing a bio-feed from distiller's grains includes the following steps: Step A: Put the distiller's grains, corn flour, soybean meal, and wheat bran into a mixer and stir evenly; add an appropriate amount of water to adjust the total moisture content of the mixture to 60%; then add the compound enzyme preparation, mix evenly, and then transport it to the pretreatment tank for enzymatic hydrolysis at 45°C for 4 hours to destroy the plant cell wall structure. Step B: After the temperature of the enzymatic hydrolysate from Step A drops below 35°C, evenly sprinkle in the thiol-modified chitosan-intercalated montmorillonite complex and stir for 15-20 minutes. Utilize the active thiol groups on its surface to inhibit miscellaneous bacteria and adsorb mycotoxins in the substrate. Then add the premix and the composite microbial agent activated with warm water, and mix evenly again. Step C: Loosely pile the mixture on the fermentation bed, controlling the material layer thickness to 30-40cm, and carry out aerobic fermentation for 24 hours at 30-35℃ to promote the growth of Bacillus subtilis and consume oxygen; then transfer the aerobic fermented material into a sealed fermentation bag or fermentation tank, compact and seal it, and continue anaerobic fermentation for 48-72 hours at 30-35℃, using Candida utilis to convert non-protein nitrogen into cell protein and accumulate organic acids; Step D: After fermentation, the wet base feed is sent into an airflow dryer, and the inlet air temperature is controlled to be no higher than 60°C for low-temperature rapid drying, so that the moisture content is reduced to below 12%. The dried material is then crushed, sieved, and vacuum-packed to prepare a high-protein, low-fiber biological feed with selective mycotoxin adsorption function.

[0030] Example 2 A type of bio-feed made from distillers' grains, by weight, comprises the following raw materials: 60 parts distillers' grains, 10 parts corn flour, 8 parts soybean meal, 3 parts wheat bran, 0.3 parts thiol-modified chitosan-intercalated montmorillonite complex, 0.05 parts compound enzyme preparation, 0.1 parts compound microbial agent, and 1 part premix. The lees are fresh baijiu lees, a byproduct of solid-state brewing, with a water content of 55%-65% and a pH value of 3.5-4.5; The corn flour is whole corn flour with a starch content of ≥65%; The soybean meal is dehulled soybean meal with a crude protein content of ≥43%; The preparation methods of the thiol-modified chitosan-intercalated montmorillonite complex and the preparation method of a distiller's grains biological feed in Example 2 are the same as those in Example 1.

[0031] Example 3 A type of bio-feed made from distillers' grains, by weight, comprises the following raw materials: 65 parts distillers' grains, 11 parts corn flour, 9 parts soybean meal, 3.5 parts wheat bran, 0.4 parts thiolated modified chitosan intercalated montmorillonite complex, 0.08 parts compound enzyme preparation, 0.15 parts compound microbial agent, and 1.2 parts premix. The lees are fresh baijiu lees, a byproduct of solid-state brewing, with a water content of 55%-65% and a pH value of 3.5-4.5; The corn flour is whole corn flour with a starch content of ≥65%; The soybean meal is dehulled soybean meal with a crude protein content of ≥43%; The preparation methods of the thiol-modified chitosan-intercalated montmorillonite complex and the preparation method of a distiller's grains biological feed in Example 3 are the same as those in Example 1.

[0032] Example 4 A type of bio-feed made from distillers' grains, by weight, comprises the following raw materials: 72 parts distillers' grains, 14 parts corn flour, 11 parts soybean meal, 4.5 parts wheat bran, 0.6 parts thiolated modified chitosan intercalated montmorillonite complex, 0.12 parts compound enzyme preparation, 0.25 parts compound microbial agent, and 1.8 parts premix. The lees are fresh baijiu lees, a byproduct of solid-state brewing, with a water content of 55%-65% and a pH value of 3.5-4.5; The corn flour is whole corn flour with a starch content of ≥65%; The soybean meal is dehulled soybean meal with a crude protein content of ≥43%; The preparation methods of the thiol-modified chitosan-intercalated montmorillonite complex and the preparation method of a distiller's grains biological feed in Example 4 are the same as those in Example 1.

[0033] Example 5 A type of bio-feed made from distillers' grains, by weight, comprises the following raw materials: 75 parts distillers' grains, 15 parts corn flour, 12 parts soybean meal, 5 parts wheat bran, 0.8 parts thiol-modified chitosan-intercalated montmorillonite complex, 0.15 parts compound enzyme preparation, 0.3 parts compound microbial agent, and 2 parts premix. The lees are fresh baijiu lees, a byproduct of solid-state brewing, with a water content of 55%-65% and a pH value of 3.5-4.5; The corn flour is whole corn flour with a starch content of ≥65%; The soybean meal is dehulled soybean meal with a crude protein content of ≥43%; The preparation methods of the thiol-modified chitosan-intercalated montmorillonite complex and the preparation method of a distiller's grains biological feed in Example 5 are the same as those in Example 1.

[0034] Comparative Example 1 This comparative example is based on Example 1, but differs from Example 1 in that: equal amounts of low molecular weight chitosan, thioglycolic acid, and sodium montmorillonite used in the preparation of the thiolated modified chitosan intercalated montmorillonite complex are used as separate raw materials and are directly added to the feed matrix in a physical mixing manner in step B of a method for preparing a distillers' grains biological feed.

[0035] Comparative Example 2 This comparative example is based on Example 1, but differs from Example 1 in that the thiolation modification operation is omitted in the preparation of the thiolated modified chitosan intercalated montmorillonite complex; instead, unmodified low molecular weight chitosan and sodium-based montmorillonite are directly intercalated and assembled, and the prepared low molecular weight chitosan intercalated montmorillonite complex is added to the feed in step B of a method for preparing distillers' grains biological feed.

[0036] Comparative Example 3 This comparative example is based on Example 1, but differs from Example 1 in that: in the preparation of the low molecular weight thiolated chitosan intercalated montmorillonite complex, the enzymatic preparation of low molecular weight chitosan in step 1 is omitted; instead, ordinary commercial chitosan raw materials with a molecular weight of 200-300 kDa are directly used for subsequent thiolation modification and intercalation assembly.

[0037] Comparative Example 4 This comparative example is based on Example 1, but differs from Example 1 in that the mass ratio of low molecular weight thiolated chitosan to sodium montmorillonite during the preparation of the thiolated modified chitosan intercalated montmorillonite complex is adjusted to 0.1:1.

[0038] Comparative Example 5 This comparative example is based on Example 1, but differs from Example 1 in that the mass ratio of low molecular weight thiolated chitosan to sodium montmorillonite in the intercalation assembly process is adjusted to 1:1 during the preparation of the thiolated modified chitosan intercalated montmorillonite complex.

[0039] Comparative Example 6 This comparative example is based on Example 1, but differs from Example 1 in that: during the preparation of the thiol-modified chitosan-intercalated montmorillonite composite, the reaction environment conditions were changed; during the thiol modification reaction, high-purity nitrogen was not introduced for protection, but air was continuously introduced into the reaction system for stirring.

[0040] Comparative Example 7 This comparative example is based on Example 1, but differs from Example 1 in that the enzymatic pretreatment process in step A is omitted in the preparation method of distillers' grains biological feed. Specifically, after mixing distillers' grains, corn flour, soybean meal, and wheat bran and adjusting the moisture content, no compound enzyme preparation is added, and no enzymatic hydrolysis is performed at 45°C for 4 hours. Instead, the process proceeds directly to the subsequent step B.

[0041] Test case 1. Determination of feed crude protein to crude fiber conversion efficiency: Referring to GB / T 6432-2018 "Determination of Crude Protein in Feed" and GB / T 6434-2006 "Determination of Crude Fiber in Feed", 500g of each of the finished feed samples prepared in Examples 1-5 and Comparative Examples 1-7 were taken and pulverized through a 40-mesh sieve. The dry basis crude protein content (%) and crude fiber content (%) were determined respectively, and compared with the unfermented raw material matrix (fresh distiller's grains + auxiliary material mixture) for calculation. Crude protein enhancement rate = (Crude protein content after fermentation - Unfermented raw material matrix) / Unfermented raw material matrix * 100%; Crude fiber degradation rate = (Crude fiber content before fermentation - Unfermented raw material matrix) / Unfermented raw material matrix * 100%.

[0042] The test results are shown in Table 1: As shown in Table 1, the embodiments of the present invention exhibit superior performance advantages in both conventional feed nutrient composition and conversion efficiency tests. Specifically, the crude protein content of Example 1 is as high as 21.87%, an increase of 44.45% compared to the raw material; the crude fiber content is reduced to 12.13%, and the degradation rate is as high as 40.60%. This fully demonstrates that the technical solution of this application successfully breaks through the physical barrier of the cell wall of distillers' grains and creatively solves the problem of low fermentation conversion efficiency.

[0043] In contrast, in the preparation method of distillers' grains biological feed, the enzymatic pretreatment process in step A was omitted in Comparative Example 7. The crude protein content increased by only 9.58%, and the crude fiber degradation rate was only 6.71%. This indicates that relying solely on microbial fermentation cannot destroy the lignin-cellulose crystal structure in distillers' grains. Cellulase must be introduced for pretreatment in the preparation process of distillers' grains biological feed to break down the cell wall barrier and release the encapsulated nutrients for microbial use.

[0044] The crude protein enhancement rates of Comparative Examples 1, 3, and 5 were 22.52%, 21.66%, and 25.36%, respectively, and the crude fiber degradation rates were all below 28%. The possible reasons are: the simple physical mixing of Comparative Example 1 cannot protect the enzyme protein; the large-molecule chitosan of Comparative Example 3 cannot enter the interlayer of montmorillonite due to steric hindrance and can only coat the surface; and the excessively high intercalation ratio of Comparative Example 5 may have blocked the interlayer pores, resulting in structural defects, which reduces the effective contact between the enzyme and the substrate, thus limiting the fermentation efficiency.

[0045] Although the conversion efficiency of Comparative Example 6 was better than that of other comparative examples, it was still lower than that of Example 1. The possible reason is that although nitrogen was not introduced, which led to the partial oxidation of thiol groups and weakened the antibacterial and carrier functions of the system, it was not completely lost and could still play a part in maintaining the stability of the system and assisting fermentation.

[0046] Referring to the detection methods in GB 5009.209-2016 "National Food Safety Standard - Determination of Zearalenone in Food" and GB 5009.124-2016 "National Food Safety Standard - Determination of Amino Acids in Food", an in vitro biomimetic digestion model was constructed. A simulated small intestinal fluid with pH 6.5 was prepared, and standard concentrations of zearalenone (representing a hydrophobic toxin, concentration 500 μg / L) and L-lysine (representing a hydrophilic nutrient, concentration 500 mg / L) were added. 0.1 g each of the thiol-modified chitosan-intercalated montmorillonite composite materials prepared in Example 1 and Comparative Examples 1-6 were accurately weighed and placed in 50 mL of the simulated solution. The mixture was shaken at 150 r / min for 2 hours at 37℃ in a constant-temperature shaker, and the supernatant was collected by centrifugation. The residual concentrations of zearalenone and L-lysine in the supernatant were determined by high-performance liquid chromatography (HPLC), and the adsorption rate was calculated. The selective adsorption coefficient was calculated as the ratio of (zearalenone adsorption rate / L-lysine adsorption rate); the blank control was ordinary montmorillonite.

[0047] The test results are shown in Table 2: Table 2 The in vitro simulation test data in Table 2 fully demonstrate the excellent selective adsorption effect of the thiol-modified chitosan-intercalated montmorillonite complex in the distillers' grains biomaterial of this application. In Comparative Example 2, the adsorption rate of zearalenone dropped sharply to 65.43%, while the loss rate of L-lysine surged to 32.12%, even higher than the 25.43% of the blank control, resulting in a selective adsorption coefficient of only 2.04. The possible reason is that ordinary chitosan without hydrophobic groups (i.e., thiol modification) is hydrophilic, and its insertion into the interlayer exacerbates the non-specific adsorption of hydrophilic amino acids. In contrast, the technical solution of this application successfully regulates the hydrophilic-hydrophobic balance of the interlayer microenvironment by introducing thiol groups, achieving the repulsion of hydrophilic nutrients and the absorption and capture of hydrophobic toxins. Comparative Example 6 contains some thiol groups, thus exhibiting a superior technical effect compared to other comparative examples.

[0048] The difference in Comparative Example 4 is that the mass ratio of low molecular weight thiolated chitosan to sodium montmorillonite was adjusted to 0.1:1. Because the low molecular weight thiolated chitosan was insufficient, the interlayer spacing was limited, resulting in an improvement in the selective adsorption coefficient, but it was far less than that in Example 1. The difference in Comparative Example 5 is that the mass ratio of low molecular weight thiolated chitosan to sodium montmorillonite was adjusted to 1:1. The higher mass ratio of low molecular weight thiolated chitosan may have caused pore blockage due to excessive polymer, resulting in an adsorption rate of zearalenone that was even lower than that of the blank control, and thus a low selective adsorption coefficient. This also indirectly shows that the 0.3:1 mass ratio selected in this application is an inventive choice.

[0049] The adsorption performance of Comparative Example 3 was similar to that of Comparative Example 1, but both were far lower than that of Example 1. This indicates that macromolecules cannot truly enter the interlayer and can only remain on the surface. They cannot construct an interlayer domain with size sieving function and cannot achieve selective adsorption.

[0050] Feeding trial of growing-finishing pigs: The experimental animals were healthy three-way crossbred pigs that were about 70 days old, weighed about 30 kg, and had the same genetic background. Two pigs were used in each group.

[0051] Blank control group: fed a basic corn-soybean meal diet without any modified adsorbents and enzymes, with the same proportion of ordinary unfermented distillers grains added.

[0052] Treatment group: 20% of the biological feed prepared according to the corresponding Example 1 and Comparative Examples 1-7 on an air-dry matter basis was added to the basal diet to replace an equal amount of the basal diet.

[0053] Feeding and management: The pre-feeding period was 7 days and the trial period was 45 days. During the trial, the pigs had free access to feed and water, and the environmental conditions such as temperature, humidity and ventilation were kept consistent in all groups.

[0054] Growth performance measurement indicators: Average daily weight gain: At the beginning and end of the experiment, each pig was weighed after fasting for 12 hours to calculate the average daily weight gain over the entire period. Average daily feed intake: Accurately record the amount of feed fed and the amount of leftover feed in each pen every day, and calculate the average daily feed intake for the whole period; Material weight ratio: The ratio of total material consumption to total weight gain over the entire period.

[0055] The test results are shown in Table 3: Table 3 As shown in Table 3, the average daily weight gain of the Example 1 group reached 917g, which was significantly higher than the 723g of the blank control group; the feed conversion ratio was reduced to 2.50. These data fully verify that the feed of this application has excellent palatability and high absorption rate.

[0056] Based on the data from Comparative Example 2 in Table 2, which showed a lysine loss rate as high as 32.12%, it can be inferred that although the adsorbent adsorbed some toxins, it also locked in the relevant amino acids in the feed due to the lack of hydrophobic repulsion of thiol groups. As a result, the animals did not fully utilize the high protein content, thus limiting their growth.

[0057] Furthermore, the test results of Example 6 were still not as good as those of Example 1. The possible reason is that the role of highly active free thiol groups is not only to regulate adsorption polarity. After entering the animal intestine, they can also enhance the residence time of feed in the intestine through disulfide bond interaction with mucin and play a role in inhibiting bacteria and repairing damaged mucosa. However, the lack of nitrogen protection led to the oxidation and failure of some thiol groups, which weakened this biological function.

[0058] The daily weight gain of control group 7 was the lowest, even lower than that of the blank control. This may be related to its high crude fiber content shown in Table 1. The possible reason is that excessive crude fiber not only dilutes energy, but also easily causes physical irritation to the pig's intestines, thus making it more prone to diarrhea, which leads to restricted growth and results in a weight gain that is even lower than that of the blank control.

[0059] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A type of biological feed made from distiller's grains, characterized in that, By weight, its composition is as follows: 60-75 parts distiller's grains, 10-15 parts corn flour, 8-12 parts soybean meal, 3-5 parts wheat bran, 0.3-0.8 parts thiol-modified chitosan-intercalated montmorillonite complex, 0.05-0.15 parts compound enzyme preparation, 0.1-0.3 parts compound microbial agent, and 1-2 parts premix.

2. The bio-feed from distiller's grains according to claim 1, characterized in that, The distillers' grains biological feed comprises, by weight, the following components: 70 parts distillers' grains, 12 parts corn flour, 10 parts soybean meal, 4 parts wheat bran, 0.5 parts thiol-modified chitosan-intercalated montmorillonite complex, 0.1 parts compound enzyme preparation, 0.2 parts compound microbial agent, and 1.5 parts premix.

3. The bio-feed from distiller's grains according to claim 1, characterized in that, The lees are fresh baijiu lees, a byproduct of solid-state brewing, with a water content of 55%-65% and a pH value of 3.5-4.5; The corn flour is whole corn flour with a starch content of ≥65%.

4. The bio-feed from distiller's grains according to claim 1, characterized in that, The compound enzyme preparation is composed of cellulase, xylanase and laccase in an activity ratio of 2:1:0.

5.

5. The bio-feed from distiller's grains according to claim 1, characterized in that, The compound microbial agent is composed of Candida utilis, Bacillus subtilis and Saccharomyces cerevisiae, compounded in a live cell ratio of 2:1:1, with an effective live cell count ≥1.0×10^8 CFU / g.

6. The bio-feed from distiller's grains according to claim 1, characterized in that, The premix is ​​composed of compound vitamins and compound organic trace elements; The complex vitamins include vitamin A, vitamin B, vitamin C, vitamin D, and vitamin C. The composite organic trace elements include a mixture of lysine iron, glycine zinc, and methionine manganese.

7. The bio-feed from distiller's grains according to claim 1, characterized in that, The thiol-modified chitosan-intercalated montmorillonite composite was prepared by the following method: acetic acid solution was added to a reaction vessel, chitosan raw material was added, and the mixture was stirred to dissolve; chitosanase was added to carry out a controlled enzymatic hydrolysis reaction; after the reaction was completed, the temperature was raised to inactivate the enzyme preparation; after adjusting the pH, the mixture was filtered, washed with ethanol, and dried to obtain low molecular weight chitosan. Low molecular weight chitosan was dissolved in acetic acid solution; nitrogen gas was continuously introduced into the reaction system; then mercaptoacetic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added in sequence, and the reaction was stirred at room temperature in the dark. The reaction product was precipitated with anhydrous ethanol, centrifuged, washed and then freeze-dried under vacuum to obtain low molecular weight mercapto-substituted chitosan. Subsequently, a sodium-based montmorillonite aqueous dispersion was prepared and ultrasonically treated for 30 minutes. Low molecular weight thiolated chitosan was dissolved in deionized water and slowly added dropwise to the montmorillonite dispersion under high-speed stirring. After the addition was complete, the reaction was stirred continuously. Finally, the reaction solution was cooled for 15 minutes, and the supernatant was discarded. The precipitate was washed with deionized water until neutral, vacuum dried to constant weight, pulverized, and sieved to obtain the thiolated modified chitosan intercalated montmorillonite composite powder.

8. The bio-feed from distiller's grains according to claim 6, characterized in that, The low molecular weight chitosan obtained during the preparation of the thiol-modified chitosan intercalated montmorillonite composite has a molecular weight of 5-10 kDa.

9. A bio-feed from distiller's grains according to claim 6, characterized in that, In the preparation of the thiol-modified chitosan intercalated montmorillonite composite, the molar ratio of low molecular weight chitosan, thioglycolic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide is controlled at 1:1.5:1.8:1.

8.

10. A bio-feed from distiller's grains according to claim 6, characterized in that, In the preparation of the thiol-modified chitosan-intercalated montmorillonite composite, the mass ratio of low molecular weight thiolized chitosan to sodium-based montmorillonite is controlled at 0.3:

1.

11. A method for preparing a bio-feed from distiller's grains according to any one of claims 1-10, characterized in that, The preparation method includes the following steps: Step A: Put the distillers' grains, corn flour, soybean meal, and wheat bran into a mixer and stir evenly; add water to adjust the total moisture content of the mixture to 60%; then add the compound enzyme preparation, mix evenly, and transfer to a pretreatment tank for enzymatic hydrolysis at 45°C for 4 hours. Step B: After the temperature of the enzymatic hydrolysate from Step A drops below 35°C, evenly sprinkle in the low molecular weight thiolized chitosan intercalated montmorillonite complex, stir for 15-20 minutes, then add the premix and the composite microbial agent activated with warm water, and mix evenly again. Step C: Loosely pile the mixed materials on the fermentation bed, control the material layer thickness to 30-40cm, and carry out aerobic fermentation for 24 hours at 30-35℃; then transfer the aerobic fermented material into a sealed fermentation bag or fermentation tank, compact and seal it, and continue anaerobic fermentation for 48-72 hours at 30-35℃. Step D: After fermentation, the wet base feed is sent to an airflow dryer to reduce the moisture content to below 12%; the dried material is then crushed, sieved, and vacuum-packed to obtain a type of distillers' grains biological feed.

12. The use of the distillers' grains biological feed as described in claim 11 in animal husbandry.

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