A method for grading and sterilizing SPF animal feed and its application in preserving heat-sensitive nutrients in SPF animal feed.

Abstract

This invention belongs to the field of feed processing technology, and particularly relates to a method for grading and sterilizing SPF animal feed and its application in preserving the heat-sensitive nutrients in SPF animal feed. A method for grading and sterilizing SPF animal feed includes the following steps: classifying the SPF animal feed raw materials to be sterilized into two categories based on their heat sensitivity; subjecting the heat-resistant base raw materials to high-temperature and high-pressure steam sterilization; subjecting the vitamin premix to encapsulation and / or carrier adsorption treatment; subjecting the enzyme preparation to low-temperature steam sterilization and spraying with an enzyme stabilizer solution; adding the probiotic preparation to the heat-resistant base raw materials; and mixing the treated raw materials under aseptic conditions to obtain the graded and sterilized SPF animal feed. This invention implements a process of grading, preservation, and then mixing, which can increase the vitamin A retention rate to over 80% and the phytase activity retention rate to over 75%, while ensuring that the sterility meets the requirements for SPF grade.

Description

Technical Field

[0001] This invention belongs to the field of feed processing technology, and particularly relates to a method for grading and sterilizing SPF animal feed and its application in preserving the heat-sensitive nutrients in SPF animal feed. Background Technology

[0002] SPF (Specific Pathogen Free) miniature pigs are irreplaceable experimental animal models in biomedical research, preclinical drug evaluation, and xenotransplantation. Maintaining the SPF status of the pig herd, that is, ensuring that they do not carry specific pathogenic microorganisms such as porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), foot-and-mouth disease virus (FMDV), and porcine circovirus type 2 (PCV2), is the core guarantee of the quality of experimental animals.

[0003] As the primary source of nutrition for SPF (Special Positive Growth) miniature pigs, the microbiological safety of feed directly impacts the health and maintenance of the SPF herd. Common contaminants in feed ingredients (corn, soybean meal, fishmeal, etc.) include Salmonella, E. coli, and molds; some viruses have also been detected in feed ingredients. Therefore, SPF miniature pig farms must sterilize feed before feeding.

[0004] Currently, major SPF (Special Feed Forage) pig facilities both domestically and internationally generally use high-pressure steam sterilization to process feed, typically at 121°C for 15 to 20 minutes. While this method effectively kills microorganisms in the feed, it also severely damages heat-sensitive nutrients.

[0005] Currently, SPF pig farms primarily employ a "whole feed sterilization" model for feed sterilization. The general steps are as follows: the pre-formulated feed (including all raw materials and additive premixes) is placed entirely into a sterilization container, then placed in a high-pressure steam sterilizer, and sterilized at 121°C, 0.12 MPa for 15 to 20 minutes. After sterilization, the feed is allowed to cool to room temperature, then vacuum-packed before use.

[0006] To compensate for the loss of vitamins during sterilization, some farms add excessive amounts of vitamins to their feed formulations. This means that when preparing feed, the amount of vitamins added is increased by 30% to 50% above the standard recommended amount, in order to ensure that the residual amount of vitamins after sterilization still meets the minimum nutritional requirements of the animals.

[0007] Significant vitamin loss: Fat-soluble vitamins are heat-sensitive. At 121°C, the retention rate of vitamin A acetate is typically only 40% to 60%, vitamin D3 50% to 70%, vitamin E 50% to 65%, and vitamin K3 has the lowest retention rate, usually not exceeding 30%. Among water-soluble vitamins, vitamin C is almost completely decomposed at high temperatures, and vitamin B1 has a retention rate of approximately 50% to 70%. This means that sterilized feed cannot actually meet the normal nutritional needs of SPF miniature pigs, and long-term feeding will lead to vitamin deficiencies, manifested as stunted growth, decreased immunity, and reduced reproductive performance.

[0008] Enzyme activity is almost completely lost: Functional enzymes such as phytase and protease are often added to SPF miniature pig feed. Phytase can degrade phytic acid in feed, promoting phosphorus absorption and utilization; protease can assist in the digestion of protein in feed. However, the optimal temperature for these enzymes is usually 37 to 55°C. Under high-pressure sterilization at 121°C, enzyme activity is almost completely lost, with a retention rate usually not exceeding 10%. This means that the enzymes added to the feed formulation lose their function after sterilization, resulting in a complete waste of the added cost.

[0009] Probiotics cannot withstand high temperatures: As live microorganisms, probiotics (such as lactobacilli, bifidobacteria, and Bacillus subtilis) cannot survive under high-temperature sterilization conditions. If probiotics are sterilized together with feed, the number of live bacteria will drop to zero, completely losing their expected functions of regulating intestinal flora and enhancing immunity.

[0010] Over-addition strategy is a temporary solution: While over-addition of vitamins adopted by some farms can make up for losses to some extent, this method has fundamental flaws: First, it increases feed costs; second, the amount of loss is unstable between different batches due to differences in the uniformity of sterilization equipment temperature, and the residual amount fluctuates greatly after over-addition, making it impossible to control precisely; third, it is completely ineffective in solving the problem of loss of enzyme preparations and probiotics.

[0011] Radiation sterilization faces obstacles to widespread adoption: some facilities also use it. 60 Co-γ ray irradiation sterilization is an alternative to autoclaving. This method causes less damage to nutrients, and enzyme activity retention can reach over 90%. However, the cost of radiation sterilization is approximately 5 to 8 times that of autoclaving, and the approval process for irradiation facilities is complex and safety regulations are strict, making it difficult for ordinary aquaculture enterprises to build their own facilities or obtain stable services.

[0012] Therefore, developing a technical solution that can ensure sterility while preserving heat-sensitive nutrients under conventional high-pressure steam sterilization conditions has significant technical value and market implications. Summary of the Invention

[0013] The technical problem to be solved by this invention is: how to preserve heat-sensitive nutrients such as vitamins, enzymes and probiotics to the greatest extent while sterilizing SPF animal feed to ensure microbial safety, and overcome the serious damage to nutrition caused by the existing "whole feed uniform sterilization" model.

[0014] The core idea of ​​this invention is to abandon the crude "one-pot sterilization" approach and instead implement a systematic process of "graded and classified processing, followed by protection and then mixing" based on the different temperature tolerances of various raw materials. Experiments have shown that this process can increase the vitamin A retention rate from the conventional 40%~60% to over 80%, and the phytase activity retention rate from less than 10% to over 75%, while ensuring that the sterility meets SPF requirements.

[0015] This invention provides a method for grading and sterilizing SPF animal feed, comprising the following steps: (1) The SPF animal feed ingredients to be sterilized are divided into two categories according to their heat sensitivity: heat-resistant basic ingredients and heat-sensitive ingredients. The heat-sensitive ingredients include vitamin premixes, enzyme preparations, and probiotic preparations; (2) The heat-resistant basic raw material described in step (1) is subjected to high-temperature and high-pressure steam sterilization; (3) The vitamin premix described in step (1) is subjected to encapsulation and / or carrier adsorption treatment; The encapsulation includes one or more of microcapsule encapsulation and β-cyclodextrin encapsulation; (4) The enzyme preparation described in step (1) is sterilized by low-temperature steam, and an enzyme stabilizer solution is sprayed after sterilization; (5) The probiotic preparation described in step (1) is added to the heat-resistant base raw material after step (2) in the form of lyophilized powder by spraying. Among them, while spraying probiotics, a prebiotic mixture solution is sprayed into the heat-resistant base raw materials; (6) The vitamin premix, enzyme preparation, mixed probiotic preparation and heat-resistant basic raw material after the treatment in steps (3) to (5) are mixed under sterile conditions to obtain graded sterilized SPF animal feed.

[0016] Furthermore, the heat-resistant basic raw materials mentioned in step (1) include cereal raw materials and protein raw materials.

[0017] Furthermore, the conditions for the high-temperature and high-pressure steam sterilization process in step (2) are: sterilization temperature of 121 °C, sterilization pressure of 0.12 MPa, and sterilization time of 15 minutes.

[0018] Furthermore, the vitamin A in the vitamin premix in step (3) is encapsulated using a gelatin-glycoside process. The specific process is as follows: vitamin A acetate is mixed with gelatin, sucrose and water in a certain proportion, stirred in a 60 ℃ water bath to form an oil-in-water emulsion, and then spray-dried in a spray drying tower to obtain vitamin A microcapsules. The proportions of vitamin A acetate, gelatin, sucrose, and water were as follows: 50 g vitamin A acetate, 10 g gelatin, 15 g sucrose, and 25 g water. The vitamin D3 in the vitamin premix in step (3) is encapsulated using β-cyclodextrin. The specific process is as follows: β-cyclodextrin is dissolved in 50 °C warm water to make a saturated solution, vitamin D3 is added and stirred to form an inclusion complex, and after cooling and crystallization, it is dried to obtain a powdered inclusion complex. In step (3), the water-soluble B vitamins in the vitamin premix are adsorbed using stone powder or zeolite powder as solid carriers.

[0019] Furthermore, the vitamin premix described in step (3) also includes a method for adding antioxidants, the specific process of which is as follows: Vitamin E is formulated as dl-α-tocopherol acetate as the active ingredient, with 0.1% propyl gallate added as an antioxidant. Treatment of Vitamin K3 and other vitamins: After the vitamin components have undergone their respective protective treatments, they are mixed into a vitamin premix, and a complex antioxidant system is added during the mixing process. The composite antioxidant system consists of: 0.015% ethoxyquinoline, 0.01% BHT (2,6-di-tert-butyl-p-cresol) and 0.05% ascorbic acid phosphate.

[0020] Furthermore, the enzyme preparations mentioned in step (4) include phytase, protease, and non-starch polysaccharide enzymes; The parameters for low-temperature steam sterilization of the enzyme preparation are: 115℃ for 3 minutes; The enzyme stabilizer solution consists of: calcium chloride 5 g / L, glycerol 50 mL / L, and sorbitol 30 g / L. For every kilogram of enzyme preparation premix, 2 mL of stabilizer solution is added.

[0021] Furthermore, the probiotic preparation mentioned in step (5) includes one or more of Lactobacillus plantarum, Bifidobacterium infantis, and Bacillus subtilis; The probiotic preparation has a live bacteria count ≥ 1 × 10⁻⁶. 10 CFU / g; Among them, the coefficient of variation (CV) of spray uniformity does not exceed 8%; Furthermore, the prebiotic mixed solution consists of 0.5% fructooligosaccharides, 0.3% yeast β-glucan, and 0.2% inulin.

[0022] Furthermore, it also includes a dual-indicator evaluation of the sterilized feed, which includes an aseptic effect index and a nutritional preservation index.

[0023] Furthermore, the sterility effect is indicated by the total bacterial count and the detection rate of specific pathogenic microorganisms; Furthermore, nutritional preservation is measured by vitamin retention rate and enzyme activity retention rate.

[0024] This invention provides a method for preserving heat-sensitive nutrients in SPF animal feed.

[0025] Compared with the prior art, the present invention has the following beneficial effects: 1) Significant Vitamin Preservation: Through comprehensive protection methods such as microencapsulation, carrier adsorption, and the addition of antioxidants, the preservation rate of vitamin A after sterilization is increased from the conventional 40% to 60% to over 80%. This fundamentally solves the problem of animal nutritional deficiencies caused by vitamin loss in SPF feed, and significantly reduces feed costs while ensuring feed sterility and compliance.

[0026] 2) Breakthrough in enzyme sterilization process: By reducing the sterilization temperature of enzyme preparations from 121℃ to 115℃ and shortening the time from 15 minutes to 3 minutes, and with the addition of stabilizers, the phytase activity retention rate has been increased from less than 10% in the conventional way to more than 75%, making it possible to add enzyme preparations to SPF feed for functional purposes.

[0027] 3) Probiotic function is fully preserved: By adding probiotics separately after sterilization and combining them with prebiotics for protection, the number of live probiotics in the feed is stably maintained at 10. 8 With a CFU / g or higher, the expected effects of probiotics in regulating gut health and enhancing immune function are ensured.

[0028] 4) Fills the gap in systematic processes for SPF feed nutrient preservation: For the first time, a systematic process is proposed that involves "raw materials being divided into four categories, each employing its optimal sterilization or protection strategy, and finally being aseptically mixed in a centralized manner." This ensures that both the sterility and nutritional content of the feed are met, providing a standardized and industrially viable technical pathway for SPF animal feed production. Feed prepared according to this invention has been applied on a large scale, and SPF miniature pig herds have not shown vitamin deficiencies after feeding, exhibiting stable growth performance.

[0029] (5) The dual-indicator evaluation system provides a basis for industry standardization: it lists aseptic effect and nutritional preservation as core indicators of feed quality control and provides a quantitative scoring method, which provides a scientific tool for establishing industry standards and regulating the SPF feed market. Detailed Implementation

[0030] The experimental animals used in the following examples, comparative examples, and experimental cases are as follows: Experimental animal breed and specifications: SPF grade Bama miniature pigs, weaned piglets at 21±1 days of age, initial IBW 5.15±0.38 kg. Healthy weaned piglets with similar weight and genetic background were selected, with 10 piglets per group (half male and half female, the males were castrated), and randomly assigned to groups, and housed in individual pens in SPF grade isolated or barrier environments.

[0031] Feeding method: Free access to feed was adopted, with feeding twice daily at 08:30 and 16:30, using powdered feed. The amount of feed given and any uneaten feed was recorded daily to calculate the daily feed intake. Nipple drinkers were provided for free access to water. The trial period lasted 8 weeks (56 days, including a 3-day pre-trial period), with the ambient temperature controlled at 25±2℃, relative humidity at 60±10%, and a 12h:12h light cycle.

[0032] Observation and recording: Fecal condition was observed and recorded twice daily, morning and evening. The diarrhea rate was calculated based on the fecal index score (0-3 points, ≥2 points were considered diarrhea). Individuals were weighed after fasting for 12 hours on day 0 (start) and day 56 (end) of the experiment, and the amount of food consumed was recorded in columns.

[0033] Example 1 Feed ingredient processing 1. Classification and pretreatment of feed ingredients The raw materials for compound feed to be processed are divided into the following four categories according to their differences in heat sensitivity, and then packaged and stored separately: The first category is heat-resistant basic raw materials: these refer to cereal and protein raw materials. Specifically, these include corn, wheat, soybean meal, puffed soybean meal, fishmeal, and fermented soybean meal. These raw materials can withstand high temperatures of 121℃ without significant nutritional damage and are suitable for conventional high-pressure steam sterilization.

[0034] The second category is heat-sensitive vitamin premixes: these refer to mixtures of various vitamins added to feed. Specifically, they include fat-soluble vitamins (vitamin A, vitamin D3, vitamin E, vitamin K3) and water-soluble vitamins (vitamin B1, vitamin B2, vitamin B6, vitamin B1). 12 (Ingredients include niacin, pantothenic acid, folic acid, and biotin). These ingredients are highly sensitive to temperature and must undergo special protective treatment before they can participate in sterilization or be excluded from high-temperature sterilization.

[0035] The third category is heat-sensitive enzyme preparations: these refer to functional enzyme preparations added to feed. Specifically, these include phytase premixes, proteases, and non-starch polysaccharide enzymes. These enzyme proteins rapidly denature and become inactive at 121°C, requiring sterilization parameters with lower temperatures.

[0036] The fourth category is heat-sensitive probiotic preparations: these refer to live bacteria preparations added to feed. The probiotic strains used in this invention are all selected from commercially available strains that are publicly available through public channels, requiring no special preservation. Specifically, they are: *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum CGMCC 1.2158 (i.e., Lactobacillus plantarum ST-III, publicly deposited at the China General Microbiological Culture Collection Center), ATCC 8014, or similar commercially available strains can be used; Bifidobacterium infantis ( Bifidobacterium longum subsp. infantis ATCC 15697 or similar commercially available strains can be used; Bacillus subtilis ( Bacillus subtilis CGMCC 1.1086, ATCC 6633, or similar commercially available strains can be used. All of the above strains are feed additive strains permitted for use in the food safety catalog and can be routinely purchased from publicly available collection centers such as ATCC, CGMCC, and DSMZ, or from commercial suppliers, such as *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum The strain has the accession number CGMCC 1.2158. It was purchased from BNCC (Beina Biotechnology), and is distributed by Hebei Pinke Biotechnology Co., Ltd. (a primary distributor of BNCC). This strain is publicly deposited at the China General Microbiological Culture Collection Center (CGMCC), and those skilled in the art can obtain it through CGMCC's standard ordering procedures. Furthermore, this strain is also available as a freeze-dried powder product in the domestic market from qualified commercial suppliers (such as the Shanghai Preservation Biotechnology Center), and is a commonly used commercial strain in the feed additive industry.

[0037] Bifidobacterium infantis ( Bifidobacterium longum subsp. infantis The strain, with accession number ATCC15697, was purchased from Ruizhi Magic Cube Biotechnology Co., Ltd. This strain is publicly deposited at the American Center for Type Culture Collection (ATCC), and several domestic biological reagent distributors (such as Shanghai Xuanya Biotechnology Co., Ltd.) offer spot distribution services for this strain. The strain's origin can be traced back to ATCC 15697. Those skilled in the art can obtain it through the ATCC website or domestic distributors.

[0038] Bacillus subtilis ( Bacillus subtilisThe strain, with accession number CGMCC 1.1086, was purchased from Shandong Liyang Biotechnology Co., Ltd. This strain has been publicly deposited at the China General Microbiological Culture Collection Center (CGMCC). Furthermore, this strain has achieved commercial mass production at the feed grade, and several feed additive companies in Shandong and Guangdong provinces have long-term supply of Bacillus subtilis freeze-dried powder. Those skilled in the art can order it through CGMCC or purchase it directly from qualified feed additive suppliers. This information is readily available to those skilled in the art without any inventive effort. Such live bacteria are completely intolerant of high temperatures and must be added separately after sterilization.

[0039] 2. Routine sterilization treatment of heat-resistant base materials After mixing the first type of heat-resistant basic raw materials, the mixture is placed in a high-temperature resistant sterilization container (such as a high-temperature resistant plastic bag or a stainless steel sterilization tray), and then placed in a high-pressure steam sterilizer. Sterilization is performed according to the following parameters: sterilization temperature 121℃, sterilization pressure 0.12MPa, and sterilization time 15 minutes (using ordinary soybean meal as a standard reference; these conditions ensure that the total bacterial count is reduced to ≤10). 4 CFU / g, Salmonella not detected).

[0040] After sterilization, the sterilized raw materials are immediately transferred to the drying process and dried with hot air at 60°C until the moisture content does not exceed 12%. The dried sterilized raw materials are then temporarily stored in a clean area for later mixing and use.

[0041] 3. Separate protective treatment of heat-sensitive vitamins 1) Protection of Vitamin A: A gelatin-glycoside encapsulation process was employed. Vitamin A acetate (VA content ≥ 500,000 IU / g) was mixed with gelatin, sucrose, and water in a specific ratio (50 g vitamin A acetate, 10 g gelatin, 15 g sucrose, 25 g water), and emulsified in a 60°C water bath to form an oil-in-water emulsion. The emulsion was then spray-dried in a spray drying tower (inlet temperature 180°C, outlet temperature 80°C) to obtain vitamin A microcapsules. The microcapsule particle size ranged from 50 to 150 μm, with an optimal range of 80-120 μm; the encapsulation efficiency was not less than 90%. The synergistic effect of gelatin and sucrose at this specific ratio enables the glass transition temperature (Tg) of the microcapsule wall material to reach 78.5 ℃, which is significantly higher than the subsequent sterilization temperature of 121 ℃. This ensures that the wall material does not crack during sterilization, effectively isolates oxygen and moisture, and can increase the vitamin A retention rate from the conventional 40% to 60% to over 80% under subsequent sterilization conditions.

[0042] 2) Protection of Vitamin D3: A β-cyclodextrin encapsulation process was used. At 50°C, the solubility of β-cyclodextrin in water is approximately 5.62 g / 100 mL. Procedure: 56.2 g of β-cyclodextrin was added to 1000 mL of purified water and stirred in a 50°C water bath to prepare a saturated solution. 5.6 g of vitamin D3 crystals (the molar ratio of β-cyclodextrin to vitamin D3 was approximately 2:1) were added to this saturated solution. The mixture was kept at 50°C and mechanically stirred at 300–500 rpm for 1 hour to ensure complete embedding of vitamin D3 molecules into the β-cyclodextrin cavity. After stirring, the mixture was allowed to cool naturally to room temperature and then refrigerated at 4°C for 12 hours to allow complete crystallization of the inclusion complex. The precipitate was filtered and washed 2-3 times with a small amount of cold purified water. It was then vacuum dried at 45°C until the water content was ≤5% to obtain vitamin D3-β-cyclodextrin inclusion complex powder. After β-cyclodextrin encapsulation, the thermal stability of vitamin D3 can be increased by about 3 times and the light stability by about 5 times.

[0043] 3) Protection by Vitamin E: Raw material selection and antioxidant addition: dl-α-tocopherol acetate was selected as the active ingredient, as this form has higher thermal stability than free tocopherol. 100g of dl-α-tocopherol acetate (content ≥50%) was weighed and 0.1g of propyl gallate (0.1% of the vitamin E mass) was added. The mixture was stirred thoroughly to dissolve, forming a vitamin E oil phase containing antioxidants.

[0044] Emulsion preparation: Prepare the wall material solution—dissolve 10g of gelatin and 15g of sucrose in 100mL of water and stir in a 60℃ constant temperature water bath until completely dissolved. Slowly add the vitamin E oil phase containing antioxidants to the wall material aqueous solution and emulsify for 5–10 minutes using a high-speed shear emulsifier at 10,000–15,000 r / min to form a homogeneous O / W emulsion.

[0045] Spray drying and microencapsulation: The obtained emulsion is spray dried and granulated in a spray drying tower (inlet air temperature 180±5℃, outlet air temperature 80±5℃), and the microcapsule powder is collected and passed through a 60-mesh sieve to obtain the vitamin E microcapsule product.

[0046] 4) Protection of Vitamin K3 and other vitamins: After all vitamin components (including vitamin A microcapsules, vitamin D3-β-cyclodextrin inclusion complex, vitamin E microcapsules, vitamin K3 and all B vitamins) have undergone their respective protection treatments, they are mixed into a vitamin premix, and a complex antioxidant system is added during the mixing process.

[0047] The composite antioxidant system consists of (based on the total mass of the vitamin premix): 0.015% ethoxyquinoline (150 mg / kg), 0.01% BHT (100 mg / kg), and 0.05% ascorbic acid phosphate (500 mg / kg). All vitamin components are weighed according to the formula and added to a mixer. The composite antioxidant system is pre-dissolved in a small amount of anhydrous ethanol and evenly sprayed onto a carrier (defatted rice bran or limestone powder). After drying, it is added to the vitamin mixture in powder form according to the formula. The mixture is then mixed in a V-type or double-cone mixer for 15–20 minutes (coefficient of variation of mixing uniformity ≤ 5%), thus achieving synergistic protection of vitamin K3 and other vitamins. Adding the composite antioxidant system (0.015% ethoxyquinoline, 0.01% BHT (2,6-di-tert-butyl-p-cresol), and 0.05% ascorbic acid phosphate) to the entire vitamin premix effectively prevents oxidative degradation of the vitamin premix during sterilization and subsequent storage.

[0048] 5) Protection of water-soluble B vitamins: Use stone powder or zeolite powder as a solid carrier to adsorb and carry the B vitamin solution on the surface of the carrier particles. Increasing the specific surface area is beneficial for heat dissipation and reduces local overheating damage.

[0049] 6) Post-protection treatment: All vitamin premixes treated as described above shall be vacuum dried at a temperature not exceeding 60 °C until the moisture content meets the standard (all vitamin premixes treated with the above protection (including vitamin A microcapsules, vitamin D3-β-cyclodextrin inclusion complex, vitamin E microcapsules, and B vitamin adsorbent carriers) shall be vacuum dried at a temperature not exceeding 60 °C until the moisture content is ≤7%). Moisture content determination shall be carried out in accordance with GB / T (According to 6435-2014), after sealing and packaging, it may participate in subsequent mixing and sterilization with the base raw materials (if the vitamin premix participates in high-temperature sterilization, it shall be placed in a high-barrier aluminum foil composite bag (PA / Al / PE three-layer structure) and vacuum-packed (vacuum degree ≤ -0.08MPa) or nitrogen-filled packaged (residual oxygen content after replacement ≤1%). The sealed small package shall be placed independently in a heat-resistant sterilization container and placed in a high-pressure steam sterilizer together with the Class A base raw materials, and sterilized at 121℃ for 15min. After sterilization, the vitamin small package shall be removed, and after breaking the bag in the clean area, the sterilized vitamin premix shall be added to the sterilized base raw materials for mixing), or it may not participate in sterilization (if the vitamin premix does not participate in high-temperature sterilization, it shall be protected and sealed in packaging and temporarily stored in a cool and dry place, and directly enter the final aseptic mixing process, and be mixed together with the sterilized and cooled base raw materials, enzyme preparations, etc. in the clean area) directly enter the final mixing process. For carotenoids (such as lutein and zeaxanthin) that are naturally present in feed or added to it, microcapsules can also be prepared using the method described in 1) above to protect them and increase their preservation rate to over 80%.

[0050] Experimental results showed that, after the above-mentioned graded protection treatment, compared with the control group which only classified the vitamins without microencapsulation or carrier protection, the vitamin A retention rate jumped from about 62% to 83%, and the vitamin E retention rate jumped from about 65% to 80%, proving that this protection step is the key to achieving nutrient preservation.

[0051] 4. Low-temperature, high-speed sterilization process for enzyme preparations For Class III heat-sensitive enzyme preparations (phytase, protease, and non-starch polysaccharide enzymes), sterilization parameters with reduced temperature are used alone.

[0052] The enzyme premix was individually bagged and placed in a sterilizer. The sterilization temperature was set to 115 °C and the sterilization time was 3 minutes. The product was sterilized with *Bacillus stearothermophilus* (…). Geobacillus stearothermophilus ATCC 7953) spores were used as a biological indicator for sterilization kinetics validation. At 115 °C, the spore D value was 0.65 min, and the F0 value reached 2.8 min after 3 minutes of treatment, indicating that sterilization kinetics can be achieved with an initial spore count of 10-1. 6 6-log killing of CFU / g, achieving a sterility assurance level of SAL≤10. -6 These conditions have been verified to meet the aseptic requirements of SPF feed (total bacterial count meets standards, pathogenic microorganisms are negative), while significantly reducing the loss of enzyme proteins due to heat denaturation.

[0053] After sterilization, the enzyme premix was immediately removed from the sterilizer and rapidly cooled to below 25 °C. Then, an enzyme stabilizer solution was sprayed on. The stabilizer solution formulation was: 5 g / L calcium chloride, 50 mL / L glycerol, and 30 g / L sorbitol. The spray addition was 2 mL of stabilizer solution per kilogram of enzyme premix. After adding the stabilizer, the mixture was thoroughly mixed. This stabilizer formulation was optimized through orthogonal experiments. Calcium chloride stabilizes the enzyme protein conformation, while glycerol and sorbitol synergistically protect enzyme activity through preferential hydration.

[0054] Under sterilization conditions of 115 °C for 3 minutes, the phytase activity retention rate can reach over 65%, and the protease activity retention rate can reach over 60%. Further addition of a stabilizer solution can increase the enzyme activity retention rate by 10 to 15 percentage points, ultimately achieving a phytase retention rate of over 75%.

[0055] As an alternative, if facility conditions permit, enzyme preparations can be used instead of steam sterilization. 60 Co-γ ray irradiation sterilization (dose 5 kGy) or ethylene oxide (EO) sterilization can be used as alternative sterilization methods, and the enzyme activity preservation rate can reach more than 90% under these methods.

[0056] 5. Post-sterilization addition process of probiotics For the fourth category of heat-sensitive probiotic preparations, they do not participate in any high-temperature sterilization process at all.

[0057] After the basic feed sterilized in step 2 has cooled to below 40°C (below the upper limit of the heat resistance of probiotics, 45°C), the probiotic premix is ​​added to the basic feed by spraying.

[0058] Probiotic strain selection: All strains used are commercially available strains that do not require special preservation. The acid resistance requirement is a survival rate of no less than 60% in simulated gastric juice at pH 2.5; the bile salt resistance requirement is a survival rate of no less than 50% in 0.3% ox bile salts. Representative strains are: *Lactobacillus plantarum* CGMCC 1.2158 (ST-Ⅲ strain), *Bifidobacterium infantis* ATCC 15697, and *Bacillus subtilis* CGMCC 1.1086. All of the above strains meet the requirements of national food safety standards GB 7300.9 or GB / T 35890, etc., and can be directly purchased by those skilled in the art from the China General Microbiological Culture Collection Center, the American Center for Type Culture Collection, or qualified commercial suppliers. The strain information is completely public and does not involve any special preservation procedures.

[0059] Spray addition process: Dissolve the lyophilized probiotic powder (probiotic premix) in sterile saline solution, ensuring a viable count of not less than 1×10⁻⁶. 10 CFU / g. The bacterial solution was evenly sprayed onto the cooled feed surface in a mist form using a small pressure sprayer (atomization pressure 0.3 MPa, nozzle diameter 0.5 mm). The feed was continuously stirred during spraying to ensure uniform distribution. The coefficient of variation (CV) for uniformity of spray addition did not exceed 8%. The final feed contained 10 live probiotics. 8 Up to 10 9 CFU / g.

[0060] Synergistic Protection with Prebiotics: A prebiotic mixture is sprayed into the feed simultaneously with the probiotic spray. The prebiotic ratio is 0.5% fructooligosaccharides, 0.3% yeast β-glucan, and 0.2% inulin (by feed weight). Prebiotics provide a carbon source for probiotics during storage and after they enter the intestines, enhancing the long-term stability of viable bacterial counts. Storage tests have verified that after adding this prebiotic combination, the viable bacterial count remains at 10 for 21 days at 4°C. 8 The CFU / g level was above 10 in the control group without added prebiotics, and it dropped to 10 in 7 days. 8 Below CFU / g.

[0061] Storage conditions: Feed with added probiotics should be stored at 4 ℃ for 21 days; if stored at -20 ℃, the shelf life can be extended to 30 days. The feed should be used within the shelf life to ensure that the number of live probiotics meets the intestinal health regulation requirements of SPF miniature pigs.

[0062] 6. Aseptic mixing and packaging In a clean area (with a cleanliness level of 10,000), the vitamin premix that has been protected in step 3 (if it has not been sterilized, add it at this time), the enzyme premix that has been sterilized at low temperature in step 4, and the feed components for which probiotics have been added in step 5 are all put into a sterile mixer and mixed thoroughly.

[0063] The mixing time is determined based on the mixer type and load capacity (WLDH series horizontal ribbon mixer, model WLDH-1, total volume 1.0 m³). 3 The mixing capacity is 400-600 kg per cycle, with a loading factor of 0.5. This equipment was purchased from Changzhou Bailian Technology Co., Ltd., with equipment serial number WL-20240315-001. The parts of the equipment that come into contact with the materials are made of 304 stainless steel. The standard for acceptance is that the coefficient of variation (CV) of the mixing uniformity does not exceed 5% (measured using the zinc tracer method according to GB / T 5918-2008).

[0064] After mixing, the feed is vacuum-packed or nitrogen-filled in a clean area. The vacuum level for vacuum packaging is no less than -0.08 MPa; the oxygen content inside nitrogen-filled packaging does not exceed 2%. Packaging specifications can be designed from 1 to 10 kg / bag according to the needs of the farming scale.

[0065] 7. Establish a dual-indicator evaluation system for determining whether sterilized feed is qualified. Aseptic performance indicators are primarily based on total bacterial count and the detection rate of specific pathogens. The total bacterial count should not exceed 1 × 10⁻⁶. 4 CFU / g, the detection method is plate counting (according to GB / T 13093-2006, nutrient agar medium, 37 ℃ incubation for 48 hours); fungi should be undetectable (not exceeding 10 CFU / g), the detection method is according to GB / T 13092-2006, Bengal red medium, 28 ℃ incubation for 5 days. Salmonella should be undetectable in 25 g of sample, the detection method is according to GB / T 13091-2018; specific SPF pathogens (PRRSV, CSFV, FMDV, PCV2) should be negative, the detection method is real-time PCR (nucleic acid extraction using KingFisher Flex system, amplification using ABI QuantStudio 5 real-time PCR instrument, primers and probes according to OIE Terrestrial Animal Diagnostic Manual).

[0066] Nutritional preservation indicators: Vitamin preservation rate and enzyme activity preservation rate are the core indicators. The contents of vitamins A, D3, and E were determined using high-performance liquid chromatography (HPLC). HPLC detection conditions for vitamins A, D3, and E: Chromatography system: Agilent 1260 Infinity II with DAD detector; Chromatographic column: C18 reversed-phase column (such as Agilent Zorbax SB-C18, 250mm × 4.6mm, 5μm, or equivalent); Mobile phase: Vitamin A: methanol-water (95:5, v / v), isocratic elution; Vitamin D3: methanol-water (98:2), or separated by normal phase chromatography using a hexane-isopropanol system; Vitamin E: methanol-water (96:4). Detection wavelengths: Vitamin A (325nm), Vitamin D3 (265nm), Vitamin E (280nm); Flow rate: 1.0 mL / min; Column temperature: 25℃ (Vitamin A), 30℃ (Vitamin D3, E); Injection volume: 20 μL; (2) Sample pretreatment methods Take approximately 100g of feed sample and crush it through a 40-mesh sieve. Weigh 1.0g of sample (accurate to 0.001g) into a 100mL brown volumetric flask, add 0.1g of ascorbic acid, 30mL of anhydrous ethanol and 10mL of 50% KOH solution, saponify in an 80℃ water bath for 30min, cool and extract three times with petroleum ether, combine the extracts and wash with water until neutral, dehydrate with anhydrous sodium sulfate and evaporate to dryness at 40℃, make up to 10mL with methanol, filter through a 0.45μm microporous membrane and inject for analysis.

[0067] (3) Methodological verification Linear range: Vitamin A: 0.5–100 μg / mL (r≥0.999); Vitamin D3: 0.2–50 μg / mL (r≥0.999); Vitamin E: 1.0–200 μg / mL (r≥0.999); Precision: Intra-batch RSD ≤ 3%, Inter-batch RSD ≤ 5%; Recovery rate: 95%–105%; Detection limits: Vitamin A 0.1 mg / kg, Vitamin D3 0.05 mg / kg, Vitamin E 0.5 mg / kg. The retention rate was calculated based on the measured values ​​of unsterilized feed from the same batch. Enzyme activity assay: The activity of non-starch polysaccharide enzymes such as xylanase and β-glucanase was determined according to GB / T standards. The DNS (3,5-dinitrosalicylic acid) colorimetric method was used, with xylan (birch xylan) or barley β-glucan as substrates (1% concentration, w / v). The reaction was carried out in a specific buffer system at 37℃ for 30 min (xylanase pH 5.3 with acetate buffer; β-glucanase pH 5.5 with acetate buffer). The reaction was terminated by boiling water bath. After color development with DNS reagent, the absorbance was measured at 540 nm. Definition of enzyme activity unit: The amount of enzyme required to release 1 μmol of reducing sugar (calculated as xylose or glucose) per minute is defined as 1 enzyme activity unit (U). The retention rates of various vitamins should meet the following standards: Vitamin A retention rate not less than 80%, Vitamin D3 retention rate not less than 75%, Vitamin E retention rate not less than 75%, Vitamin K3 retention rate not less than 70%, Vitamin B1 retention rate ≥ 85%, Vitamin B2 retention rate ≥ 85%, Vitamin B6 retention rate ≥ 85%, Folic acid (B9) retention rate ≥ 80%, and Vitamin B2 retention rate ≥ 85%. 12 The retention rate is ≥80%, with niacin and pantothenic acid retention rates both ≥85%. Detection methods for B vitamins: Vitamin B1 is determined by fluorescence method according to GB / T 14700-2018; Vitamin B2 is determined by HPLC method according to GB / T 14701-2002; Vitamin B6, niacin, and pantothenic acid are determined by HPLC methods according to their respective national standards. The phytase activity retention rate is not less than 60% (based on the enzyme activity determination value of the same batch of unsterilized feed); the protease activity retention rate is ≥60% (based on the control of the same batch of unsterilized feed), and the detection method is according to SB / T 10317-1999. The xylanase and β-glucanase activity retention rates are ≥55% (again based on the unsterilized control), and the detection method is according to the DNS colorimetric method in the national standard. The viable count of probiotics is not less than 1×10⁻⁶. 8 CFU / g.

[0068] Overall Score: Overall Score = 0.4 × Aseptic Performance Score + 0.3 × Vitamin Preservation Score + 0.3 × Enzyme Activity Preservation Score. A comprehensive score of 85 or higher is considered a qualified product. The aseptic performance score and the scores for each preservation item are calculated by multiplying the pass rate of each individual indicator (number of compliant items divided by the total number of items) by 100.

[0069] 8. The numerical range and optimal parameters are shown in Table 1. Table 1 Numerical range and optimal parameters

[0070] Comparative Example 1 Existing technology "all-material unified sterilization" mode All feed ingredients (without sorting or preservative treatment) were mixed and then directly autoclaved at 121°C for 15 minutes. Post-sterilization testing showed a total bacterial count of 6.2 × 10⁻⁶. 3 CFU / g; Vitamin A retention rate 46%, Vitamin E retention rate 53%, Vitamin K3 retention rate 42%, Vitamin B1 retention rate 38%; Phytase activity retention rate 9%; Probiotic live bacteria count 0. Overall score 55 points, unqualified. SPF miniature pig feeding trials showed that this feed led to a significant decrease in daily weight gain and a diarrhea rate as high as 29.2%.

[0071] Comparative Example 2 Only sorting and sterilization, vitamins are not protected. Raw materials were categorized by heat resistance and heat sensitivity, but the vitamin premix was not microencapsulated or protected by a carrier. It was directly mixed with the base raw materials and then sterilized at 121 °C for 15 minutes. The result showed a vitamin A retention rate of approximately 62%, which was better than Comparative Example 1. This was due to the crucial factor of "categorization." Comparative Example 2 separated the heat-sensitive vitamin premix from the heat-resistant base raw materials, avoiding the hydrolysis and oxidation reactions that would occur when vitamins were directly mixed with a large amount of water-containing base raw materials and subjected to 121 °C steam sterilization. In contrast, Comparative Example 1 involved directly mixing all raw materials (including vitamins) without categorization and then sterilizing the whole mixture. This exposed the vitamins to a triple destructive factor of high temperature, high humidity, and metal ions (catalyzed by trace amounts of iron and copper ions in the base raw materials), resulting in extremely low vitamin A and E retention rates. However, simply classifying the vitamins without microencapsulation or carrier protection is insufficient to guarantee nutritional quality. This is because the vitamin premix itself still suffers from thermal degradation and auto-oxidation at 121°C. The retention rate of Comparative Example 2 (VA≈62%, VE≈65%) is significantly lower than the 80% standard set by this invention, resulting in a comprehensive score of 68, which is still unacceptable. This demonstrates that protective treatment is another indispensable process step after classification. The vitamin E retention rate was approximately 65%, and the comprehensive score was also 68, which is still unacceptable. This proves that simple classification is insufficient to guarantee nutritional quality.

[0072] The results are shown in Table 2: Table 2 Test Results

[0073] Comparative Example 3 Enzyme preparations are subjected to uniform sterilization after the addition of stabilizers. The enzyme premix was sprayed with stabilizers (5 g / L calcium chloride, 50 mL / L glycerol, and 30 g / L sorbitol), then mixed with the base ingredients, and sterilized at 121 °C for 15 minutes. The results showed that the phytase retention rate was only 18%, and the protease retention rate was approximately 12%. This indicates that the stabilizers cannot withstand high temperatures of 121 °C, and low-temperature sterilization parameters must be used in conjunction with them to effectively protect enzyme activity.

[0074] Comparative Example 4 All feed is sterilized at low temperature. All feed (including basic ingredients, vitamins, and enzymes) was uniformly sterilized at 115 °C for 15 minutes in an attempt to protect heat-sensitive components. While the vitamin retention rate increased to approximately 70% and the enzyme activity retention rate to approximately 30%, the total bacterial count only decreased to 4.8 × 10⁻⁶. 4 The CFU / g concentration was positive for Salmonella in some batches, indicating that the sterility did not meet the SPF requirements. This proves that the basic raw materials still require standard high-temperature sterilization, and sterilization parameters must be set in stages.

[0075] Comparative Example 5 Enzyme preparations are sterilized by irradiation; all other components are sterilized using conventional methods. The basic raw materials and unprotected vitamins were mixed and sterilized at 121°C for 15 minutes; enzyme preparations were used alone. 60 Sterilization by 5 kGy Co-γ irradiation (92% enzyme activity retention); probiotics were added later (mixed after the sterilized base material cooled to below 40 °C). Results: Although the enzyme activity index was excellent, the vitamin index was still severely substandard, resulting in a comprehensive score of 71, which is unacceptable. This indicates that under the main heat sterilization process, the vitamin preservation problem must be systematically addressed; cold substitution in a single step cannot compensate for the overall nutritional loss.

[0076] The above comparative examples clearly demonstrate that the systematic approach of hierarchical classification, synergistic protection, and parameter differentiation in Example 1 is indispensable for simultaneously achieving microbial safety and nutrient preservation.

[0077] Experimental Example 1 SPF Miniature Piglet Weaning Feed Complete Sterilization and Nutritional Preservation Process 1. A batch of SPF Bama miniature pig weaning compound feed was divided into a heat-resistant group and a heat-sensitive group by weight. The heat-resistant group consisted of 60 parts corn, 25 parts extruded soybean meal, 5 parts fish meal, 1.5 parts dicalcium phosphate, and 1.0 part limestone powder; the heat-sensitive group consisted of 0.1 parts vitamin premix, 0.05 parts phytase premix, and 0.1 parts probiotic premix.

[0078] 2. After mixing, the heat-resistant raw materials were placed in high-temperature resistant plastic bags, vacuum-packed, and then placed in a high-pressure steam sterilizer for sterilization at 121℃ and 0.12 MPa for 15 minutes. After sterilization, they were dried with hot air at 60℃ until the moisture content was 10.5%. The total bacterial count of the heat-resistant raw materials after sterilization was 8.7 × 10⁻⁶. 2 CFU / g, Salmonella was not detected in 25 g, which is within the acceptable range.

[0079] 3. Preservative treatment of vitamin premix: 50 g of vitamin A acetate was emulsified with 10 g of gelatin, 15 g of sucrose, and 25 g of water in a 60 ℃ water bath, and spray-dried to obtain VA microcapsules with a particle size distribution ranging from 60 to 140 μm (average about 100 μm) and an encapsulation rate of 92%. Vitamin D3 was encapsulated using β-cyclodextrin, and vitamin E was encapsulated using dl-α-tocopherol acetate with the addition of 0.1% propyl gallate. Water-soluble B vitamins were adsorbed and carried by litmus paper as a carrier. All treated vitamins were vacuum dried at 55 ℃ for 4 hours and then sealed in packaging. The premixed vitamin premix was mixed with the sterilized base material (because the vitamins are well preserved, they can also be sterilized together with the base material; this example uses a co-sterilization process, and the results show that the preservation rate still meets the standard. If a higher preservation rate is desired, it can be added after sterilization).

[0080] 4. The enzyme preparation premix was sterilized separately at 115 °C for 3 minutes. After sterilization, it was immediately cooled to 22 °C, and then sprayed with an enzyme stabilizer solution (calcium chloride 5 g / L, glycerol 50 mL / L, sorbitol 30 g / L) at an addition rate of 2 mL / kg, and mixed thoroughly. The phytase activity retention rate after sterilization was 69%, which increased to 76% after adding the stabilizer.

[0081] 5. After the feed to be sterilized has cooled to 37°C, add the freeze-dried Lactobacillus plantarum CGMCC 1.2158 powder (purchased from an open depository, with a viable count of 1.2 × 10⁻⁶). 11 CFU / g was dissolved in sterile physiological saline to obtain a viable count of 1×10⁻⁶. 10 A bacterial suspension of CFU / mL was evenly sprayed onto the feed surface using a pressure sprayer with an atomization pressure of 0.3 MPa, while a prebiotic mixture (containing 0.5% fructooligosaccharides, 0.3% yeast β-glucan, and 0.2% inulin) was sprayed at the same time.

[0082] 6. In a Class 10,000 clean area, mix all components for 15 minutes using a sterile conical mixer. The coefficient of variation (CV) for mixing uniformity is 3.2% (zinc tracer method). Vacuum package (vacuum degree -0.08 MPa), 1 kg per package.

[0083] 7. Dual-indicator test results: Total bacterial count was 8.5 × 10⁻⁶. 3 CFU / g (standard ≤10) 4(Qualified); fungi not detected; Salmonella not detected in 25 g; PRRSV, CSFV, FMDV, and PCV2 fluorescent quantitative PCR tests were all negative; Vitamin A preservation rate was 83% (standard ≥80%, qualified); Vitamin D... 3 The retention rates were as follows: Vitamin E retention rate was 78% (standard ≥75%, qualified); Vitamin K3 retention rate was 80% (standard ≥75%, qualified); Vitamin B1 retention rate was 72% (standard ≥70%, qualified); Vitamin B1 retention rate was 76% (standard ≥75%, qualified); Vitamin B2 retention rate was 79% (standard ≥75%, qualified); Phytase activity retention rate was 75%, i.e., 3750 FTU / kg (standard ≥60%, qualified); Probiotic live bacteria count was 9.5 × 10⁻⁶. 8 CFU / g (standard ≥10) 8 (Qualified). The overall score was 92 points, and the product was deemed qualified. All testing methods in each test case were equivalent to those in Example 1.

[0084] Experimental Example 2 Comparative verification with conventional whole-material sterilization Take the same batch of SPF miniature pig weaning compound feed and divide it into two equal portions, A and B.

[0085] Part A was processed according to the graded sterilization and nutrient preservation process of Experiment Example 1, while Part B was processed according to the traditional whole-material uniform sterilization mode: all raw materials were mixed, without any protective treatment, and directly placed into a high-pressure steam sterilizer for sterilization at 121°C and 0.12MPa for 15 minutes.

[0086] Two sterilized feed samples were tested for various indicators. Vitamin A retention rate: Sample A 83%, Sample B 46%. Vitamin E retention rate: Sample A 80%, Sample B 53%. Phytase activity retention rate: Sample A 75%, Sample B 9%. Probiotic viable count: Sample A 9.5 × 10⁻⁶. 8 CFU / g, B portion is 0.

[0087] The overall score for sample A is 92 (pass), and the overall score for sample B is 55 (fail).

[0088] Two groups of 10 weaned Bama miniature pigs (4 weeks old, weighing about 4.5 kg) were fed with feed A and feed B respectively for 8 weeks.

[0089] Results: Pigs fed feed A had an average daily weight gain of 342 g, a feed conversion ratio of 2.28 g / g, and a diarrhea incidence rate of 8.3% within 8 weeks. Blood vitamin A and vitamin E levels were within the normal reference range. Pigs fed feed B had an average daily weight gain of only 258 g, a feed conversion ratio of 2.95 g / g, and a diarrhea incidence rate of 29.2% within 8 weeks. After 8 weeks, blood vitamin A and vitamin E levels were below the lower limit of the normal reference range. After the 8-week experiment, 6 piglets were randomly selected from each group. 5 mL of fasting blood was collected from the anterior vena cava into a lithium heparin anticoagulant tube. Plasma was separated by centrifugation at 3000 rpm for 10 min and stored at -80℃ for later analysis. The plasma concentrations of vitamin A and vitamin E were determined by high-performance liquid chromatography (HPLC) using an Agilent 1260 Infinity II with a DAD detector. Sample pretreatment: 200 μL of plasma was added to 200 μL of anhydrous ethanol-n-hexane (1:1) mixture for extraction by shaking. After centrifugation at 10000 rpm for 5 min, the supernatant was collected, dried under N2, and reconstituted with 100 μL of methanol. Chromatographic conditions: C18 column (150 mm × 4.6 mm, 5 μm), mobile phase methanol-water (95:5, v / v), flow rate 1.0 mL / min, detection wavelength 325 nm (vitamin A), 280 nm (vitamin E), injection volume 20 μL. The normal reference range for Bama miniature pigs is: plasma vitamin A ≥ 0.20 mg / L, vitamin E ≥ 0.50 mg / L. The differences in all growth performance indicators between the two groups were statistically significant (P < 0.05).

[0090] Experimental Example 3 Feeding effect and gut microbiota analysis The feeding trial in Example 2 was extended to 16 weeks, and 16S rDNA high-throughput sequencing analysis of gut microbiota was performed on fecal samples from piglets after week 16. At the end of week 16 of the experiment, approximately 2g of fresh feces from each piglet in each group was collected using sterile fecal collection tubes, immediately flash-frozen in liquid nitrogen, and stored at -80℃. Total DNA was extracted from the feces using the QIAamp Fast DNA Stool Mini Kit, and the concentration and purity were determined by NanoDrop. DNA integrity was confirmed by 1% agarose gel electrophoresis. PCR amplification of the V3-V4 hypervariable region of the 16S rRNA gene was performed using universal primers 338F (5'-ACTCCTACGGGAGGCAGCA-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The amplified products were recovered by gel extraction via 2% agarose gel electrophoresis, quantified using the Quant-iT PicoGreen dsDNAAssay Kit, and after equimolar mixing, paired-end sequencing was performed on an Illumina MiSeq PE300 platform. The data from the feed underwent quality control (DADA2 denoising) and ASV clustering using the QIIME2 workflow. Species annotation was performed based on the SILVA 138 database, and the relative abundance and Shannon diversity index at each taxonomic level were calculated. LEfSe analysis was used to identify differentially expressed bacterial genera among the groups (LDA>3, P<0.05). Results showed that in the feed group (Part A), the relative abundance of *Lactobacillus* in the piglets' intestines increased from 4.2% to 15.8%, and *Bifidobacterium* from 2.1% to 9.5%, while the abundance of potentially pathogenic *Escherichia coli* / *Shigella* decreased from 3.6% to 0.8%; the Shannon diversity index was 3.8. In the sterilized whole-feed control group (Part B), the abundance of *Lactobacillus* decreased to 1.1%, *Bifidobacterium* to 0.8%, and *Enterobacteria* to 12.3%, with the Shannon index decreasing to 2.4, indicating a severe imbalance in the intestinal flora. This embodiment demonstrates, from a microecological perspective, the advantages of the probiotic-prebiotic post-addition process of the present invention in actively regulating the intestinal health of SPF animals.

[0091] Test Example 4 1. Orthogonal experiment of temperature and time for low-temperature sterilization of enzyme preparations Phytase premix from the same batch was divided into multiple equal portions and autoclaved under different combinations of temperature and time. Four temperature levels (110℃, 115℃, 118℃, and 121℃) and five time levels (2 minutes, 3 minutes, 5 minutes, 10 minutes, and 15 minutes) were used, resulting in 20 orthogonal experiments. Each group had three replicates. Immediately after sterilization, the phytase activity retention rate and total bacterial count were measured for each group.

[0092] Results: Regarding sterilization effectiveness, when the sterilization temperature was not lower than 115 °C and the sterilization time was not lower than 3 minutes, the total bacterial count in all treatment groups could be reduced to 10. 3Salmonella was not detected at CFU / g, meeting the aseptic requirements for SPF feed. The sterilization effect at 110℃ was unstable across different time groups, with one out of three replicates showing a slight exceedance of the total bacterial count.

[0093] Regarding enzyme activity preservation, the 110 ℃ × 3 minutes group had the highest preservation rate (approximately 78%), but the sterilization effect was unstable; the 115 ℃ × 3 minutes group had a preservation rate of 69% (the highest among the groups with stable sterilization effect); the preservation rate of the 115 ℃ × 5 minutes group dropped to approximately 55%; the preservation rate of the 118 ℃ × 3 minutes group was approximately 52%; and the preservation rate of the 121 ℃ × 15 minutes group was only 9%.

[0094] Considering both sterilization effectiveness and enzyme activity preservation, 115 °C for 3 minutes is the optimal balance point. This result has been validated using sterilization kinetic data from biological indicators.

[0095] 2. Optimization Experiment of Vitamin A Microcapsule Wall Material Ratio 50 g of vitamin A acetate and 25 g of water were fixed. The ratio of gelatin to sucrose was changed according to Table 3 to prepare different microcapsules. The encapsulation efficiency and the vitamin A retention rate after sterilization at 121 ℃ for 15 min were determined. The glass transition temperature (Tg) of the microcapsule wall material was determined by differential scanning calorimetry (DSC).

[0096] Table 3. Gelatin to Sucrose Ratio

[0097] The results showed that while sucrose alone achieved a high encapsulation rate, its total glutaraldehyde (Tg) was only 52.3 °C, lower than the sterilization temperature (121 °C), causing the wall material to melt and crack at high temperatures. Gelatin alone also resulted in a low encapsulation rate and insufficient Tg. The formula of 10 g gelatin and 15 g sucrose provided in Example 1 achieved a Tg of 78.5 °C, significantly higher than the Tg of conventional spray-dried microcapsules (typically 50–70 °C), ensuring the integrity of the wall material during sterilization, which is crucial for high vitamin A retention.

[0098] 3. Sterilization kinetics and aseptic assurance validation of enzyme preparations Commercially available industrial-grade phytase premix was used as the matrix (phytase premix purchased from Jinan Bestjie Biotechnology Co., Ltd., product batch number PY-10000 (heat-resistant type), phytase activity ≥1.0×10⁻⁶). 4 U / g, feed grade, 25kg / drum packaging, production batch number BNJ20240125. Protease was purchased from Shandong Longchang Animal Health Products Co., Ltd., product batch number LC-PT5000, feed grade. Xylanase and β-glucanase compound preparation was purchased from Guangdong Yiduoli Biotechnology Co., Ltd., product batch number YDL-NSP202312), inoculated with *Bacillus stearothermophilus* (…). G. stearothermophilusATCC 7953) spore suspension, to achieve an initial spore concentration of 1.5 × 10⁻⁶. 6 CFU / g. The number of surviving spores was determined after treatment at 115 ℃, 118 ℃, and 121 ℃ for different times, and the D value and F0 value were calculated. The results are as follows: At 115 °C, the D value was 0.65 min, and after 3 minutes of treatment (F0 = 2.8 min), the spore viability count decreased to <10 CFU / g; at 118 °C, the D value was 0.42 min; and at 121 °C, the D value was 0.25 min. The 6-log sterilization effect achieved by treatment at 115 °C for 3 minutes meets the internationally recognized sterilization assurance level (SAL ≤ 10). -6 Under the same conditions, the phytase activity retention rate remained at 69%, far superior to the 9% of the 121℃ control. This provides a solid basis for the microbial safety of setting low-temperature short-time sterilization parameters.

[0099] 4. Application of SPF feed during the laying period of laying hens and protection against carotenoids In SPF laying hen feed, lutein extract is added as a coloring, heat-sensitive ingredient in addition to vitamins. Lutein microcapsules were prepared according to step 3 of Experimental Example 1: Lutein extract (10%) was mixed with gelatin, sucrose (approximately 10% gelatin and 15% sucrose, based on a total volume of core material + wall material + water of approximately 100g. This ratio resulted in a wall material Tg of 78.5℃, the highest Tg formulation), and 0.1% rosemary extract antioxidant. The mixture was then spray-dried (inlet air temperature 180±5℃, outlet air temperature 80±5℃, feed rate 15-20mL / min, atomizer speed 20000r / min, spray tower pressure -0.01MPa slight negative pressure operation. The collected microcapsule powder was sieved through a 60-mesh sieve, sealed, and dried at 4℃). The microcapsules were then protected with the vitamin premix, followed by sterilization with the base ingredients (121℃, 15 minutes). The enzyme preparation was sterilized at low temperature, and the probiotics were added afterward, following the same procedure as in Experiment 1.

[0100] Post-sterilization testing: Total bacterial count in feed 5.2 × 10⁻⁶ 3 CFU / g, Salmonella, and specific viruses were all negative. Lutein retention rate reached 81%, while the control group (using uniformly sterilized whole feed) had a lutein retention rate of only 41%. Both feeds were fed to SPF laying hens for 4 weeks. The Roche colorimetric fan grade of the egg yolk in the invention group was 9.2, while the control group was only 5.8. There was no significant difference in egg production rate and fertilization rate. This demonstrates that this process can be extended to SPF poultry feed containing heat-sensitive colorants.

[0101] 5. Verification of the protective effect of compound enzyme preparations The compound enzyme premix (containing 7500 FTU / g phytase, 5000 U / g acidic protease, and 10000 U / g xylanase) was sterilized at 115 °C for 3 minutes and sprayed with the same stabilizer formulation according to step four. A control group was also sterilized at 121 °C for 15 minutes. The activity retention rates of each enzyme were measured. Results: After treatment at 115 °C, the retention rates of phytase, acidic protease, and xylanase were 76%, 68%, and 73%, respectively; while the control groups showed retention rates of 9%, 5%, and 12%, respectively. This indicates that low-temperature short-time sterilization combined with the stabilizer has a significant protective effect on the multi-enzyme system.

[0102] 6. Probiotic-prebiotic synergistic storage stability test Feed containing *Lactobacillus plantarum* CGMCC 1.2158 was prepared according to the process in Example 1, and the prebiotic combination was added. A control group without prebiotics was also included. The two feed groups were stored at 4 ℃ and 25 ℃, respectively, and samples were taken at 0, 7, 14, 21, and 30 days to determine the viable bacterial count. The results are shown in Table 4. Table 4. Changes in viable bacterial count over 30 days (CFU / g)

[0103] The prebiotic group still had a viable bacteria count higher than 1×10⁻⁶ after 21 days of storage at 4°C. 8 The CFU / g level met the requirements; however, the level in the group without prebiotics dropped to 3.1 × 10⁻⁶ on day 7. 8 The CFU / g level had fallen below the target by day 14. Prebiotic synergistic protection is a key factor in maintaining the stability of live bacteria during long-term storage.

[0104] The above examples, comparative examples, and experimental examples demonstrate that the present invention, through a complete chain system solution of "raw material classification - vitamin microcapsule protection - low-temperature sterilization of enzyme preparations and synergistic effect of stabilizers - post-addition of probiotics and protection of prebiotics - dual-indicator quality control", breaks through the bottleneck of existing technologies that cannot simultaneously achieve sterility and nutrition, and has significant application value in the production of SPF animal feed.

[0105] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for grading and sterilizing SPF animal feed, characterized in that, Includes the following steps: (1) The SPF animal feed ingredients to be sterilized are divided into two categories according to their heat sensitivity: heat-resistant basic ingredients and heat-sensitive ingredients. The heat-sensitive ingredients include vitamin premixes, enzyme preparations, and probiotic preparations; (2) The heat-resistant basic raw material described in step (1) is subjected to high-temperature and high-pressure steam sterilization; (3) The vitamin premix described in step (1) is subjected to encapsulation and / or carrier adsorption treatment; The encapsulation includes one or more of microcapsule encapsulation and β-cyclodextrin encapsulation; (4) The enzyme preparation described in step (1) is sterilized by low-temperature steam, and an enzyme stabilizer solution is sprayed after sterilization; (5) The probiotic preparation described in step (1) is added to the heat-resistant base raw material after step (2) in the form of lyophilized powder by spraying. Among them, while spraying probiotics, a prebiotic mixture solution is sprayed into the heat-resistant base raw materials; (6) The vitamin premix, enzyme preparation, mixed probiotic preparation and heat-resistant basic raw material after the treatment in steps (3) to (5) are mixed under sterile conditions to obtain graded sterilized SPF animal feed.

2. The method according to claim 1, characterized in that, The heat-resistant basic raw materials mentioned in step (1) include cereal raw materials and protein raw materials.

3. The method according to claim 1, characterized in that, The conditions for the high-temperature and high-pressure steam sterilization process in step (2) are: sterilization temperature of 121 ℃, sterilization pressure of 0.12 MPa, and sterilization time of 15 minutes.

4. The method according to claim 1, characterized in that, The vitamin A in the vitamin premix in step (3) is encapsulated using a gelatin-glycoside process. The specific process is as follows: vitamin A acetate is mixed with gelatin, sucrose and water in a certain proportion, stirred in a 60 ℃ water bath to form an oil-in-water emulsion, and then spray-dried in a spray drying tower to obtain vitamin A microcapsules. The proportions of vitamin A acetate, gelatin, sucrose, and water were as follows: 50 g vitamin A acetate, 10 g gelatin, 15 g sucrose, and 25 g water. The vitamin D3 in the vitamin premix in step (3) is encapsulated using β-cyclodextrin. The specific process is as follows: β-cyclodextrin is dissolved in 50 °C warm water to make a saturated solution, vitamin D3 is added and stirred to form an inclusion complex, and after cooling and crystallization, it is dried to obtain a powdered inclusion complex. In step (3), the water-soluble B vitamins in the vitamin premix are adsorbed using stone powder or zeolite powder as solid carriers.

5. The method according to claim 4, characterized in that, The vitamin premix mentioned in step (3) also includes a treatment method for adding antioxidants, the specific process of which is as follows: Vitamin E is formulated as dl-α-tocopherol acetate as the active ingredient, with 0.1% propyl gallate added as an antioxidant. Treatment of Vitamin K3 and other vitamins: After the vitamin components have undergone their respective protective treatments, they are mixed into a vitamin premix, and a complex antioxidant system is added during the mixing process. The composite antioxidant system consists of: 0.015% ethoxyquinoline, 0.01% BHT (2,6-di-tert-butyl-p-cresol) and 0.05% ascorbic acid phosphate.

6. The method according to claim 1, characterized in that, The enzyme preparations mentioned in step (4) include phytase, protease, and non-starch polysaccharide enzyme; The parameters for low-temperature steam sterilization of the enzyme preparation are: 115℃ for 3 minutes; The enzyme stabilizer solution consists of: calcium chloride 5 g / L, glycerol 50 mL / L, and sorbitol 30 g / L. For every kilogram of enzyme preparation premix, 2 mL of stabilizer solution is added.

7. The method according to claim 1, characterized in that, The probiotic preparation mentioned in step (5) includes one or more of Lactobacillus plantarum, Bifidobacterium infantis, and Bacillus subtilis; The probiotic preparation has a live bacteria count ≥ 1 × 10⁻⁶. 10 CFU / g; Among them, the coefficient of variation (CV) of spray uniformity does not exceed 8%; The prebiotic mixture consists of 0.5% fructooligosaccharide, 0.3% yeast β-glucan, and 0.2% inulin.

8. The method according to claim 1, characterized in that, It also includes a dual-index evaluation of the sterilized feed, which includes an aseptic effect index and a nutritional preservation index.

9. The method according to claim 8, characterized in that, The sterility effect is measured by the total bacterial count and the detection rate of specific pathogenic microorganisms. The nutritional preservation rate is measured by the vitamin preservation rate and enzyme activity preservation rate.

10. The application of the method according to any one of claims 1-9 in preserving heat-sensitive nutrients in SPF animal feed.

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