Animal feed containing ginseng and preparation method thereof

Abstract

The invention relates to the technical field of animal feeds, and discloses a ginseng-containing animal feed and a preparation method thereof.The preparation method comprises the steps that a ginseng extract, clostridium butyricum, xylooligosaccharide and a yeast culture are subjected to in-vitro pre-integration through a symbiotic fermentation technology; preparing fermented composite powder through a yeast fermentation stage, a symbiotic culture stage and a drying and curing stage; the fermentation composite powder is respectively prepared into a quick release component and a slow release component by adopting a segmented release embedding technology, so that directional function exertion in different regions of the intestinal tract is realized; and finally, granulating and forming to obtain the animal feed with double antioxidant functions. Ginseng macromolecular saponin is converted into rare small molecular saponin through microbial fermentation, and the antioxidant activity is improved by 3-10 times; the colonization rate of the probiotics is increased by 2-5 times through the pre-adaptive synergistic process, deep integration of exogenous oxidation resistance and endogenous oxidation resistance is achieved, and the oxidation resistance, intestinal health and production performance of animals are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of animal feed technology, and more specifically, to an animal feed containing ginseng and a method for preparing the same. Background Technology

[0002] In intensive livestock and poultry farming, animals are often kept at high densities and under environmental stress, leading to widespread oxidative stress. This manifests as excessive free radical production, lipid peroxidation, and reduced antioxidant enzyme activity, severely impacting animal growth performance and product quality. Current antioxidant feeds primarily utilize single antioxidants such as vitamin E and vitamin C, which have limitations including short-lasting effects and the need for continuous, high-dose supplementation. Furthermore, traditional methods focus solely on exogenous antioxidant supplementation, neglecting the endotoxin release and inflammatory oxidative stress caused by gut microbiota dysbiosis.

[0003] Ginseng is rich in antioxidant active substances such as ginsenosides, organic selenium can activate the glutathione peroxidase system, and Clostridium butyricum can produce butyric acid to improve intestinal health. However, in existing technologies, these functional components are mostly used independently or in simple combinations, failing to achieve deep biological integration. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides an animal feed containing ginseng and a method for preparing the same.

[0005] A method for preparing animal feed containing ginseng, comprising the following steps: Step 1: Yeast fermentation stage: Yeast was inoculated into a culture medium containing ginseng extract, xylooligosaccharides, corn steep liquor, and yeast extract, and aerobic fermentation was carried out at 28-32℃ for 12-18 hours to obtain a yeast culture medium containing yeast metabolites and yeast cell wall polysaccharides. Step 2: Symbiotic Cultivation Stage In the later stage of yeast fermentation, Clostridium butyricum spores were inoculated into the yeast culture medium, and sodium selenite was added as a selenium source. The culture was continued at 35-37℃ for 8-12 hours under micro-anaerobic conditions. The ginseng macromolecular saponins were converted into rare small molecule saponins by microbial glycosidases, and the microbial enrichment and transformation of selenium was achieved to obtain a symbiotic fermentation broth. Step 3: Drying and curing: The symbiotic fermentation broth is dried by spray drying or freeze drying to remove moisture, thereby obtaining symbiotic fermentation composite powder; Step 4: Segmented release and embedding: The symbiotic fermentation composite powder is divided into a fast-release component and a slow-release component. The fast-release component is encapsulated with sodium carboxymethyl cellulose or low-viscosity sodium alginate, while the slow-release component is encapsulated with medium-high viscosity sodium alginate, chitosan or resistant starch. Step 5: Granulation and molding: The fast-release and slow-release components are mixed and then added to feed carrier raw materials for pelleting to obtain animal feed containing ginseng.

[0006] Preferably, the culture medium comprises: 2-5% ginseng extract or ginseng powder, 3-8% xylooligosaccharides, 2-4% corn steep liquor, 1-2% yeast extract, 0.2-0.5% potassium dihydrogen phosphate, and pH adjusted to 6.0-7.0.

[0007] Preferably, the yeast is *Saccharomyces cerevisiae* or *Candida utilis*, with a strain activity ≥10. 8 CFU / g; The *Clostridium butyricum* strain is *Clostridium butyricum*, with a strain viability ≥10. 7 CFU / g, inoculation amount is 106-108 CFU / mL.

[0008] Preferably, during the biotransformation process in the symbiotic culture stage, ginseng macromolecular saponins Rg1, Rb1, and Re are converted into small molecule rare saponins Rh2, Rg3, compound K, and protopanaxadiol-type saponins, with an enzymatic conversion rate of 30-60%.

[0009] Preferably, the amount of sodium selenite added is such that the selenium content of the final product reaches 0.2-0.5 mg / kg, and the conversion rate of inorganic selenium to organic selenium reaches 70-85%.

[0010] Preferably, the rapid-release component accounts for 40% by weight of the symbiotic fermentation composite powder, and the slow-release component accounts for 60% by weight.

[0011] Preferably, the rapid-release component dissolves 10-20% in simulated gastric fluid within 30 minutes and accumulates 80-95% dissolves in simulated small intestinal fluid within 30-60 minutes; the sustained-release component dissolves ≤15% in simulated gastric fluid and the pre-small intestine environment within 2 hours and accumulates 75-90% release in simulated colonic fluid within 4-8 hours.

[0012] Preferably, the weight ratio of the core material to the wall material when the rapid-release component is encapsulated is 3-4:1, and the weight ratio of the core material to the wall material when the slow-release component is encapsulated is 2-3:1.

[0013] Preferably, the encapsulation process of the sustained-release component adopts ion gelation encapsulation, in which the composite powder is mixed with sodium alginate aqueous solution to form a suspension, which is then dropped into calcium chloride solution for cross-linking for 30-60 minutes to form gel microspheres.

[0014] An animal feed containing ginseng prepared by the above method, the feed contains rare ginsenosides, organic selenium chelate complex, Clostridium butyricum live spores and yeast cell wall polysaccharides, wherein the number of live Clostridium butyricum is ≥105 CFU / g.

[0015] The beneficial effects of this invention are as follows: Breakthrough enhancement of antioxidant effect: Through microbial enzymatic hydrolysis and transformation, the antioxidant activity of ginsenosides is increased by 3-10 times (the IC50 value decreased from 150-200 μg / mL of the original ginseng extract to 15-50 μg / mL using DPPH free radical scavenging rate determination), and the bioavailability is increased by 5-20 times (verified by in vitro simulated digestion test and Caco-2 cell transport test, the transport rate increased from 2-4% of the raw material to 15-25%).

[0016] The direct antioxidant effect of ginsenosides and the enzymatic antioxidant effect of organic selenium form a dual defense, with the total antioxidant capacity (determined by ABTS method) increasing by 50-80% compared with traditional single antioxidants (negative control group T-AOC: 4.2±0.8 U / mL serum, positive control group T-AOC: 8.5±1.2 U / mL serum, experimental group T-AOC: 14.2±1.8 U / mL serum, P<0.01), and the antioxidant duration is extended to 24-48 hours (verified by cellular antioxidant stress model, the cell survival rate maintenance time after H2O2 treatment is extended by more than 2 times).

[0017] Significant improvement in probiotic synergistic efficiency: Through in vitro symbiotic culture, Clostridium butyricum and prebiotics establish a synergistic relationship before entering the intestines, increasing the probiotic colonization rate by 2-5 times (verified by fecal microbiota detection in animal experiments; 16S rRNA high-throughput sequencing showed that the number of Clostridium butyricum increased from 104-105 CFU / g in the control group to 106-107 CFU / g in the experimental group, and the relative abundance increased from 0.5% to 2.8%), and butyric acid production increased by 30-50% (determined by gas chromatography of fecal short-chain fatty acid content; butyric acid concentration increased from 5-8 mmol / kg in the control group to 8-15 mmol / kg in the experimental group), effectively improving the technical challenge of low synergistic efficiency between probiotics and prebiotics.

[0018] Deep realization of intestinal health regulation: Through a precise segmented release strategy, rapid absorption of antioxidants is achieved in the proximal small intestine (the serum ginsenoside metabolite reaches a peak of 15.6±2.3 ng / mL 2-4 hours after administration), and effective colonization and microbial regulation of probiotics are achieved in the large intestine (the intestinal villus height increased from 450±25μm in the control group to 520±30μm in the experimental group, the crypt depth decreased from 220±15μm to 185±12μm, and the villus height / crypt depth ratio increased by 15-25%). A three-dimensional defense effect of "exogenous antioxidants + enhanced endogenous antioxidants + reduced oxidative stress sources" is established. The serum endotoxin level decreased from 85±12 EU / mL in the control group to 48±8 EU / mL in the experimental group, a reduction of 40-60% (measured by the Limulus Amebocyte Lysate (LAL) assay). Attached Figure Description

[0019] Figure 1 This invention provides a comparison of ginsenoside conversion rates. Figure 2 This is the time-varying curve of butyric acid production according to the present invention; Figure 3 This is the release curve of the ginsenoside rapid release component of the present invention; Figure 4 This is a comparison of the release curves of the sustained-release components of the present invention; Figure 5 This is a bar chart showing the probiotic protective effect of the present invention; Figure 6 This is a heat map showing the release time distribution of the present invention. Detailed Implementation

[0020] The subject matter described herein will now be discussed with reference to exemplary embodiments. It should be understood that these embodiments are discussed only to enable those skilled in the art to better understand and implement the subject matter described herein, and changes may be made to the function and arrangement of the elements discussed without departing from the scope of this specification. Various processes or components may be omitted, substituted, or added as needed in the examples. Furthermore, some features described in the examples may be combined in other examples.

[0021] Example 1 This embodiment presents a method for preparing animal feed containing ginseng, comprising the following steps: Step 1: Yeast fermentation stage: Yeast was inoculated into a culture medium containing ginseng extract, xylooligosaccharides, corn steep liquor, and yeast extract, and aerobic fermentation was carried out at 30°C for 15 hours to obtain a yeast culture medium containing yeast metabolites and yeast cell wall polysaccharides. The culture medium consists of: 3% ginseng extract, 5% xylooligosaccharides, 3% corn steep liquor, 1.5% yeast extract, and 0.35% potassium dihydrogen phosphate, with the pH adjusted to 6.5. The yeast is *Saccharomyces cerevisiae*, with a strain viability ≥10. 8 CFU / g; The *Clostridium butyricum* strain is *Clostridium butyricum*, with a strain viability ≥10. 7 CFU / g, inoculation amount is 107 CFU / mL.

[0022] Step 2: Symbiotic Cultivation Stage In the later stage of yeast fermentation, Clostridium butyricum spores were inoculated into the yeast culture medium, and sodium selenite was added as a selenium source. The culture was continued at 36°C for 10 hours under micro-anaerobic conditions. Microbial glycosidases were used to convert ginseng macromolecular saponins into rare small molecule saponins, and at the same time, the microbial enrichment and transformation of selenium was achieved to obtain a symbiotic fermentation broth. During the biotransformation process in the symbiotic culture stage, ginseng macromolecular saponins Rg1, Rb1, and Re are converted into small molecule rare saponins Rh2, Rg3, compound K, and protopanaxadiol-type saponins, with an enzymatic conversion rate of 45%. The addition of sodium selenite resulted in a final product selenium content of 0.35 mg / kg, and the conversion rate of inorganic selenium to organic selenium reached 78%.

[0023] Step 3: Drying and curing: The symbiotic fermentation broth was spray-dried to obtain symbiotic fermentation composite powder.

[0024] Step 4: Segmented release and embedding: The symbiotic fermentation composite powder is divided into a fast-release component and a slow-release component. The fast-release component is encapsulated with sodium carboxymethyl cellulose, and the slow-release component is encapsulated with medium-high viscosity sodium alginate and chitosan. The fast-release component accounts for 40% by weight of the symbiotic fermentation compound powder, and the slow-release component accounts for 60% by weight. The rapid-release component dissolves by 15% within 30 minutes in simulated gastric fluid and reaches a cumulative dissolution of 88% within 45 minutes in simulated small intestinal fluid; the sustained-release component dissolves by ≤15% within 2 hours in simulated gastric fluid and the pre-small intestine environment and reaches a cumulative release of 78% within 6 hours in simulated colonic fluid. When the fast-release component is encapsulated, the weight ratio of the core material to the wall material is 3.5:1, and when the slow-release component is encapsulated, the weight ratio of the core material to the wall material is 2.5:1. The encapsulation process for the sustained-release component employs ion gelation encapsulation, in which the composite powder is mixed with an aqueous solution of sodium alginate to form a suspension, which is then dropped into a calcium chloride solution for cross-linking for 45 minutes to form gel microspheres.

[0025] Step 5: Granulation and molding: The fast-release and slow-release components are mixed and then added to feed carrier raw materials for pelleting to obtain animal feed containing ginseng.

[0026] Example 2 The difference between this embodiment and Embodiment 1 is that: Step 1: Carry out aerobic fermentation at 28℃ for 12 hours; The culture medium consists of: 2% ginseng powder, 3% xylooligosaccharides, 2% corn steep liquor, 1% yeast extract, and 0.2% potassium dihydrogen phosphate, with the pH adjusted to 6.0. The yeast strain is *Candida utilis*, with a viability ≥10-1. 8 CFU / g; The *Clostridium butyricum* strain is *Clostridium butyricum*, with a strain viability ≥10. 7 CFU / g, inoculation amount is 106 CFU / mL.

[0027] Step 2: Symbiotic Cultivation Stage In the later stage of yeast fermentation, Clostridium butyricum spores were inoculated into the yeast culture medium, and sodium selenite was added as a selenium source. The culture was then continued for 8 hours under micro-anaerobic conditions at 35°C. During the biotransformation process in the symbiotic culture stage, ginseng macromolecular saponins Rg1, Rb1, and Re are converted into small molecule rare saponins Rh2, Rg3, compound K, and protopanaxadiol-type saponins, with an enzymatic conversion rate of 30%. The addition of sodium selenite resulted in a final product selenium content of 0.2 mg / kg, and the conversion rate of inorganic selenium to organic selenium reached 70%.

[0028] Step 3: Drying and curing: The symbiotic fermentation broth was freeze-dried to remove moisture, resulting in a symbiotic fermentation composite powder.

[0029] Step 4: Segmented release and embedding: The symbiotic fermentation composite powder is divided into a fast-release component and a slow-release component. The fast-release component is encapsulated with low-viscosity sodium alginate, while the slow-release component is encapsulated with medium- and high-viscosity sodium alginate and resistant starch. The rapid-release component dissolves by 10% within 30 minutes in simulated gastric fluid and by a cumulative dissolution of 80% within 30 minutes in simulated small intestinal fluid; the sustained-release component dissolves by ≤15% within 2 hours in simulated gastric fluid and the pre-small intestine environment and by a cumulative release of 75% within 4 hours in simulated colonic fluid. When the fast-release component is encapsulated, the weight ratio of the core material to the wall material is 3:1, and when the slow-release component is encapsulated, the weight ratio of the core material to the wall material is 2:1. The encapsulation process for the sustained-release component employs ion gelation encapsulation, in which the composite powder is mixed with an aqueous solution of sodium alginate to form a suspension, which is then dropped into a calcium chloride solution for cross-linking for 30 minutes to form gel microspheres.

[0030] Example 3 The difference between this embodiment and Embodiment 1 is that: Step 1: Carry out aerobic fermentation at 32℃ for 18 hours; The culture medium consists of: 5% ginseng extract, 8% xylooligosaccharides, 4% corn steep liquor, 2% yeast extract, 0.5% potassium dihydrogen phosphate, and pH adjusted to 7.0. Clostridium butyricum is a strain with an activity ≥10 7 CFU / g, inoculation amount is 108 CFU / mL.

[0031] Step 2: Symbiotic Cultivation Stage In the later stage of yeast fermentation, Clostridium butyricum spores were inoculated into the yeast culture medium, and sodium selenite was added as a selenium source. The culture was then continued at 37°C for 12 hours under micro-anaerobic conditions. During the biotransformation process in the symbiotic culture stage, ginseng macromolecular saponins Rg1, Rb1, and Re are converted into small molecule rare saponins Rh2, Rg3, compound K, and protopanaxadiol-type saponins, with an enzymatic conversion rate of 60%. The addition of sodium selenite resulted in a final product selenium content of 0.5 mg / kg, and the conversion rate of inorganic selenium to organic selenium reached 85%.

[0032] Step 4: Segmented release and embedding: The rapid-release component dissolves by 20% within 30 minutes in simulated gastric fluid and reaches a cumulative dissolution of 95% within 60 minutes in simulated small intestinal fluid; the sustained-release component dissolves by ≤15% within 2 hours in simulated gastric fluid and the pre-small intestine environment and reaches a cumulative release of 90% within 8 hours in simulated colonic fluid. When the fast-release component is encapsulated, the weight ratio of the core material to the wall material is 4:1, and when the slow-release component is encapsulated, the weight ratio of the core material to the wall material is 3:1. The encapsulation process for the sustained-release component employs ion gelation encapsulation, in which the composite powder is mixed with an aqueous solution of sodium alginate to form a suspension, which is then dropped into a calcium chloride solution for cross-linking for 60 minutes to form gel microspheres.

[0033] Example 4 This embodiment presents an animal feed containing ginseng prepared by the method of Example 1. The feed contains rare ginsenosides, organic selenium chelate complex, Clostridium butyricum live spores and yeast cell wall polysaccharides, wherein the number of live Clostridium butyricum is ≥105 CFU / g.

[0034] Example 5 This embodiment presents a method for preparing animal feed containing ginseng.

[0035] The preparation method includes three main stages: symbiotic fermentation, staged release and encapsulation, and granulation. 1. Symbiotic fermentation stage Step 1: Yeast Fermentation Stage Yeast (Saccharomyces cerevisiae or Candida utilis, strain viability ≥10^8 CFU / g) was inoculated into a culture medium containing xylooligosaccharides and ginseng extract. The culture medium consisted of: 2-5% ginseng extract (ginsenoside content ≥80mg / g) or ginseng powder (below 40 mesh, ginsenoside content ≥30mg / g), 3-8% xylooligosaccharides (degree of polymerization 2-10, purity ≥90%), 2-4% corn steep liquor (dry matter content ≥50%), 1-2% yeast extract (protein content ≥45%), and 0.2-0.5% potassium dihydrogen phosphate (analytical grade). The pH was adjusted to 6.0-7.0 with distilled water (measured using a pH meter).

[0036] Aerobic fermentation was carried out in a constant temperature incubator at 28-32℃ for 12-18 hours, with an aeration rate controlled at 0.5-1.0 vvm (volume of air / volume of culture medium / minute) and a stirring speed of 150-200 rpm. Yeast rapidly proliferated using nutrients such as xylooligosaccharides, and produced metabolic products such as B vitamins, organic acids, amino acids, and small peptides through cellular respiration. During fermentation, the yeast cell wall autolyzed, releasing cell wall polysaccharides such as β-glucan (molecular weight 10-100 kDa) and mannan oligosaccharides (degree of polymerization 2-6). Simultaneously, yeast respiration consumed dissolved oxygen in the culture environment, reducing the oxygen content from the initial 6-8 mg / L to 1-2 mg / L (measured using a dissolved oxygen meter), thus creating a microanaerobic environment.

[0037] The product obtained in this step is a yeast culture medium containing live yeast cells (107-108 CFU / mL, determined by plate counting), yeast metabolites, yeast cell wall polysaccharides, and residual nutrient substrate. This culture medium provides a micro-anaerobic environment and nutritional basis for subsequent Clostridium butyricum inoculation.

[0038] Step 2: Symbiotic Cultivation Stage During the later stage of yeast fermentation (14-16 hours of fermentation), Clostridium butyricum spores (strain viability ≥10^7 CFU / g, inoculation amount 10^6-10^8 CFU / mL) were inoculated into the yeast culture medium, and a selenium source (sodium selenite Na2SeO3, analytical grade, the amount added was such that the selenium content of the final product reached 0.2-0.5 mg / kg, which was determined by atomic fluorescence spectrometry) was added at the same time.

[0039] Safety Precautions: Sodium selenite is toxic. Wear protective gloves and safety glasses when handling it to avoid direct skin contact and inhalation of dust. Operate in a well-ventilated environment. Sodium selenite may produce toxic selenium dioxide gas upon contact with acidic substances; therefore, pH adjustments should be made slowly to avoid abrupt pH changes.

[0040] The culture environment was adjusted to a microanaerobic state (dissolved oxygen 1-2 mg / L, measured using a dissolved oxygen meter), the temperature was maintained at 35-37℃, and the pH was controlled at 6.5-7.5 (adjusted slowly using sodium hydroxide or hydrochloric acid to avoid drastic pH changes). Culture was continued for 8-12 hours, with the stirring speed reduced to 50-80 rpm. During this period, the following biotransformation process occurred: (1) Enzymatic hydrolysis of ginsenosides: Yeast cells and germinating Clostridium butyricum secrete glycosidase (EC 3.2.1.21) and β-glucosidase (EC 3.2.1.86) to hydrolyze ginsenoside macromolecular molecules (Rg1 molecular weight 801.01 Da, Rb1 molecular weight 1109.29 Da, Re molecular weight 947.15 Da) into small molecule rare saponins (Rh2 molecular weight 622.87 Da, Rg3 molecular weight 785.00 Da, compound K molecular weight 622.87 Da, protopanaxadiol type saponin molecular weight 460.70 Da). The enzymatic hydrolysis conversion rate reached 30-60% (determined by high performance liquid chromatography), and the concentration of rare saponins produced was 0.8-2.5 mg / mL. (2) Microbial transformation of selenium: Microbial cells convert inorganic selenium (sodium selenite) into selenoamino acids (such as selenomethionine and selenocysteine), selenium polysaccharides and other organic selenium compounds, and combine with microbial cell proteins and cell wall polysaccharides to form microbial-selenium chelate complexes. The conversion rate of inorganic selenium to organic selenium reaches 70-85% (the organic selenium form is determined by liquid chromatography-mass spectrometry LC-MS). (3) Formation of symbiotic fermentation products: Ginseng enzymatic hydrolysis products, organic selenium chelate complex, Clostridium butyricum spores, yeast metabolites, and residual xylooligosaccharides are mixed and distributed in the culture medium to form a symbiotic fermentation broth containing a variety of active ingredients.

[0041] The product obtained in this step is a symbiotic fermentation broth containing rare ginsenosides, organic selenium chelate complex, Clostridium butyricum live spores (106-107 CFU / mL, determined by anaerobic plate counting method), and yeast metabolites, with a total solids content of 12-18% (determined by loss on drying method).

[0042] Step 3: Drying and curing Moisture in the symbiotic fermentation broth is removed using spray drying or freeze drying processes. Spray drying: Use a centrifugal spray dryer (such as Wuxi Huaqiang LPG-5 or equivalent), with an inlet air temperature of 180-220℃, an outlet air temperature of 80-100℃, an atomization pressure of 0.15-0.25 MPa, a feed rate of 15-25 mL / min, and a drying time of 30-60 seconds. If a dedicated spray dryer is unavailable, a small laboratory spray dryer (such as Shanghai Yacheng YC-015) can be used for batch drying. Freeze-drying: First, freeze the fermentation broth at -40℃ or below for 12-24 hours (using a regular ultra-low temperature freezer is sufficient). Then, under a vacuum of ≤50 Pa (using a vacuum pump and vacuum drying oven), sublime drying at a temperature below -10℃ for 24-48 hours. If a freeze dryer is unavailable, the frozen sample can be placed in a vacuum drying oven and dried under reduced pressure at room temperature for 72-96 hours.

[0043] Equipment alternative: For small-batch production, a conventional oven can be used for low-temperature drying at 60-80℃, with the drying time extended to 8-12 hours. However, the temperature must be strictly controlled to protect live bacteria and active ingredients.

[0044] The moisture content was controlled below 8% (using the Karl Fischer moisture determination method). During the drying process, Clostridium butyricum maintained its activity in spore form (spore survival rate ≥95%, determined by plate counting method after heat activation), and rare ginsenosides and organoselenium chelate complexes maintained chemical stability (content loss ≤5%, determined by HPLC and LC-MS, respectively).

[0045] The product obtained in this step is a pale yellow to brownish-yellow symbiotic fermentation complex powder containing live butyric acid Clostridium butyricum spores (≥10^6 CFU / g, determined by anaerobic plate counting method), rare ginsenosides (content increased by 3-10 times compared to the original ginseng extract, determined by HPLC), organic selenium chelate complex (selenium content 0.2-0.5 mg / kg, determined by atomic fluorescence spectrometry), yeast cell wall polysaccharides, residual xylooligosaccharides, etc. The powder particle size is 10-50 μm (determined by laser particle size analyzer), the bulk density is 0.4-0.6 g / mL (determined by bulk density method), the moisture content is ≤8%, and the water activity aw is ≤0.6.

[0046] 2. Segmented release and encapsulation stage Step 4: Preparation of rapid release components Take 40% by weight of the symbiotic fermentation composite powder, and select sodium carboxymethyl cellulose (CMC-Na, viscosity 800-1200 cP, viscosity of 1% aqueous solution at 25℃, measured by rotational viscometer) or low-viscosity sodium alginate (viscosity 300-500 cP, viscosity of 1% aqueous solution at 25℃) as the embedding wall material, and embed it at a weight ratio of core material: wall material = 3-4:1.

[0047] Encapsulation process: A fluidized bed coating process is adopted, in which the composite powder is fluidized in a fluidized bed (inlet air temperature 60-80℃, inlet air volume 50-80 m³ / h), and the coating liquid (aqueous solution with wall material concentration of 8-12%) is sprayed through a two-fluid nozzle for coating, with atomization pressure of 0.1-0.2 MPa, coating liquid flow rate of 5-15 mL / min, and coating time of 60-120 minutes; or a spray coating process is adopted, in which coating and drying are carried out simultaneously in a spray drying tower, with inlet air temperature of 120-150℃ and outlet air temperature of 60-80℃.

[0048] After coating, the particle size is controlled at 100-300μm (measured by laser particle size analyzer), the coating efficiency is ≥85% (observed by microscopic observation of coating integrity), and the coating thickness is 5-15μm (measured by scanning electron microscopy SEM).

[0049] The release behavior of the prepared rapid-release microparticles in the in vitro dissolution test was as follows: 10-20% dissolved within 30 minutes in simulated gastric fluid (pH 1.2, 0.1 mol / L HCl solution, 37℃), and 80-95% cumulatively dissolved within 30-60 minutes in simulated small intestinal fluid (pH 6.8, phosphate buffer, 37℃) (measured by basket method, 100 rpm, UV spectrophotometry).

[0050] The product obtained in this step is: smooth-surfaced spherical rapid-release microparticles containing rare ginsenosides and organic selenium chelate complexes, which are rapidly released in the pre-small intestine for absorption by the body.

[0051] Step 5: Preparation of sustained-release components Take 60% by weight of the symbiotic fermentation composite powder, and select medium-high viscosity sodium alginate (molecular weight 200-400 kDa, viscosity 800-1500 cP, viscosity of 1% aqueous solution at 25℃), chitosan (molecular weight 50-150 kDa, degree of deacetylation 85-95%, determined by nuclear magnetic resonance) or resistant starch (RS3 type, resistant starch content ≥60%) as the composite embedding wall material, and embed it at a weight ratio of core material: wall material = 2-3:1.

[0052] Encapsulation process: Ion gelation encapsulation is adopted, in which the composite powder is mixed with sodium alginate aqueous solution (concentration 2-4%) to form a suspension, which is then dripped into calcium chloride solution (concentration 2-5%) through a needle (diameter 0.5-1.0 mm). Crosslinking is carried out at room temperature for 30-60 minutes to form gel microspheres. Then, the microspheres are washed three times with distilled water to remove free calcium ions. The amount of water used for each wash is 5-10 times the volume of the microspheres, and the washing time is 5-10 minutes. Alternatively, a composite coating process can be adopted, in which the first layer of coating is carried out with chitosan acetate solution (concentration 1-2%, pH 4-5), and after drying, the second layer of coating is carried out with sodium alginate solution. Finally, the microspheres are crosslinked and cured in calcium chloride solution.

[0053] Waste treatment and recycling: Washing wastewater contains free calcium ions and a small amount of sodium alginate. Calcium ions can be recovered through sodium carbonate precipitation, and sodium alginate can be recovered after concentration of the supernatant, with a recovery rate of 60-70%. Coating wastewater can be separated and recycled into polymer wall materials through ultrafiltration membrane (molecular weight cutoff 10 kDa), with a recovery rate of 50-60%. All selenium-containing waste should be collected centrally and treated by professional hazardous waste treatment institutions, and must not be discharged indiscriminately.

[0054] After encapsulation, the particle size was controlled at 200-500 μm (measured by laser particle size analyzer), the encapsulation efficiency was ≥90% (measured by drug loading method), the coating thickness was 20-50 μm (measured by scanning electron microscopy SEM), and the crosslinking degree was 70-85% (measured by FTIR infrared spectroscopy).

[0055] The release behavior of the prepared sustained-release granules in simulated digestive fluids in vitro was as follows: ≤15% dissolution occurred within 2 hours in simulated gastric juice and pre-small intestine (pH 1.2-6.8) environment; in simulated colonic fluid (pH 7.0-8.0, containing 10 U / mL β-glucanase and 5 U / mL cellulase), release began under the action of intestinal flora enzymes, and the cumulative release reached 75-90% within 4-8 hours (measured by ultraviolet spectrophotometry using the paddle method at 50 rpm).

[0056] The product obtained in this step is a sustained-release granule with intestinal-targeted release characteristics, containing live butyric acid bacillus spores, yeast cell wall polysaccharides, and xylooligosaccharides, which are released directionally in the large intestine environment to achieve probiotic colonization.

[0057] 3. Granulation and molding stage Step 6: Mixing and Granulation Mix the fast-release microparticles and slow-release granules at a weight ratio of 2:3, and mix for 20-30 minutes (10-20 rpm) using a V-type mixer or double cone mixer until uniformly distributed. Add feed carrier ingredients: soybean meal powder (protein content ≥44%, passed through a 40-mesh sieve), corn flour (crude protein content ≥8%, passed through a 40-mesh sieve), and wheat bran (crude fiber content ≤12%, passed through a 20-mesh sieve), totaling 60-80% of the total weight; binder: starch paste (concentration 5-8%) or molasses (solids content ≥75%), accounting for 2-3% of the total weight; antioxidant protectant: vitamin E (α-tocopherol content ≥98%) or BHT (2,6-di-tert-butyl-4-methylphenol, purity ≥99%), accounting for 0.1-0.2% of the total weight.

[0058] Pelletizing process: A wet pelletizing process is used. All powdered materials are first mixed evenly in a high-speed mixer pellet mill (mixing speed 200-300 rpm, mixing time 3-5 minutes). Then, the binder is slowly added (addition time 5-10 minutes), and mixing and pelleting continue (mixing speed 400-600 rpm, pelleting time 10-15 minutes) until suitable pellets are formed. The obtained wet pellets are dried in a fluidized bed dryer (inlet air temperature 60-80℃, drying time 30-60 minutes) to reduce the moisture content to ≤10% (using the Karl Fischer moisture determination method). The dried pellets are sieved and sized to produce pelleted feed with a diameter of 2-4 mm (particle size distribution determined using a standard sieve).

[0059] The product obtained in this step is: uniform-looking, free-flowing pelleted feed or premix, containing both fast-release and slow-release functional components, with a pellet hardness of 3-8N (measured using a pellet hardness tester), a friability of ≤1.5% (measured using a friability tester), and a bulk density of 0.5-0.8g / mL.

[0060] Experimental verification Experiment 1: Verification of the effect of symbiotic fermentation technology on the conversion and antioxidant activity enhancement of ginsenosides 1. Experimental Objective The study verified that symbiotic fermentation technology can significantly improve the bioconversion rate of ginsenosides and greatly enhance the antioxidant activity of the product compared with physical mixing, proving that microbial enzymatic transformation is a key technical path to improve antioxidant effects.

[0061] 2. Preparation of experimental samples Experimental group: The symbiotic fermentation product was prepared according to steps 1 and 2 of Example 5, including a yeast fermentation stage (28-32℃, 12-18 hours) and a symbiotic culture stage (35-37℃, 8-12 hours), and a fermentation complex transformed by microorganisms was obtained.

[0062] Control group 1: Physical mixing group, in which equal amounts of ginseng extract, sodium selenite, Clostridium butyricum spores and xylooligosaccharides were simply physically mixed without fermentation.

[0063] Control group 2: Ginseng extract alone, used as raw material control.

[0064] 3. Experimental conditions HPLC detection conditions: Agilent C18 column (4.6×250mm, 5μm), mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution gradient elution, detection wavelength was 203nm, and column temperature was 30℃.

[0065] Antioxidant activity assay: DPPH free radical scavenging method, reaction temperature 25℃, reaction time 30 minutes, detection wavelength 517nm.

[0066] 4. Experimental Procedure Step 1: Determination of ginsenoside content. Take 1g of each sample from the experimental group and the control group, extract with 70% ethanol, and detect the content of Rg1, Rb1, Re (prototype saponins) and Rh2, Rg3 and compound K (rare saponins) by HPLC.

[0067] Step 2: Antioxidant activity determination. Prepare sample solutions of different concentrations (10, 25, 50, 100, 200 μg / mL), react with DPPH solution, determine the free radical scavenging rate, and calculate the IC50 value.

[0068] Step 3: Conversion rate calculation, conversion rate (%) = (rare saponin content / total saponin content) × 100%.

[0069] 5. Experimental Results Results of ginsenoside composition and content detection (Table 1): Results of antioxidant activity assay (Table 2): Figure 1 For: Comparison of ginsenoside conversion rates.

[0070] 6. Analysis and Summary Experimental results showed that the co-fermentation technology increased the conversion rate of ginsenosides from 7.8% in the physical mixing group to 52.4%, a 6.7-fold increase. Regarding antioxidant activity, the IC50 value of the experimental group was 8.7 times higher than that of the raw material group and 4.2 times higher than that of the physical mixing group, demonstrating that microbial enzymatic transformation is a key technological step in enhancing antioxidant effects. The significant increase in antioxidant activity was mainly due to the abundant generation of rare saponins Rh2, Rg3, and compound K.

[0071] Experiment 2: Verification of the effect of pre-adaptation co-culture on improving probiotic colonization efficiency 1. Experimental Objective The study verified that the pre-adaptive synergistic relationship established through symbiotic fermentation can significantly improve the colonization efficiency of Clostridium butyricum and butyric acid production in the intestinal environment, demonstrating the key role of yeast culture as a nutrient carrier for initiation.

[0072] 2. Preparation of experimental samples Experimental group: The symbiotic fermentation composite powder prepared according to Example 5 contains pre-adapted Clostridium butyricum, yeast culture, and xylooligosaccharide complex.

[0073] Control Group 1: Direct Mixing Group. Clostridium butyricum spores (strain activity ≥10^7 CFU / g), yeast culture (containing β-glucan, mannan oligosaccharides, and B vitamins), and xylooligosaccharides (degree of polymerization 2-10, purity ≥90%) were physically mixed in the same proportions as the experimental group, without symbiotic fermentation. Specific composition: Clostridium butyricum spores 30%, yeast culture 40%, xylooligosaccharides 30% (by weight).

[0074] Control group 2: Clostridium butyricum group, with only Clostridium butyricum spores added.

[0075] 3. Experimental conditions In vitro intestinal simulation system: simulates the small intestinal environment (pH 6.8, 37℃, microaerophilic conditions) and the colonic environment (pH 7.2, 37℃, anaerobic conditions).

[0076] Detection methods: viable bacteria count was determined by anaerobic plate counting, short-chain fatty acid content was determined by gas chromatography, and bacterial community structure was analyzed by 16S rRNA gene sequencing.

[0077] 4. Experimental Procedure Step 1: Inoculate each group of samples (containing 10⁶-10⁸ CFU / mL of bacteria) into simulated intestinal culture medium and culture them under small intestine and colon conditions, respectively.

[0078] Step 2: Take samples at regular intervals (6, 12, 24, 48, 72 hours) to test the number of viable Clostridium butyricum and the yield of butyric acid.

[0079] Step 3: 16S rRNA gene sequencing was used to analyze changes in bacterial community structure and to calculate the relative abundance of Clostridium butyricum.

[0080] Step 4: Detect the expression level of the key enzyme gene for butyryl synthesis (butyryl-CoA dehydrogenase) by qPCR.

[0081] 5. Experimental Results Results of Clostridium butyricum colonization efficiency test (Table 3): Butyric acid yield test results (Table 4): Figure 2 For example: Butyric acid production over time curve.

[0082] 6. Analysis and Summary Experimental results showed that pre-adaptation synergistic culture significantly improved the colonization efficiency of Clostridium butyricum. After 72 hours, the viable cell count in the experimental group was 3.7 times higher than that in the direct mixing group and 5.8 times higher than that in the single-strain group. Regarding butyric acid production, the experimental group showed a yield increase of over 100% compared to control group 1 and over 200% compared to control group 2 at all time points. qPCR detection indicated that the expression level of butyric acid synthase gene in the experimental group was 2.8 times higher than that in control group 1, demonstrating that the pre-adaptation synergistic relationship not only improved the colonization ability of the strain but also enhanced its metabolic activity.

[0083] Experiment 3: Validation of the effect of segmented release and encapsulation technology on intestinal targeted release 1. Experimental Objective The study validated that the segmented release encapsulation technology can achieve precise release of functional components in different regions of the intestine, with rapid-release components being released rapidly in the anterior small intestine and sustained-release components being released directionally in the large intestine, demonstrating the technological advantages of matching the spatiotemporal distribution of the intestine.

[0084] 2. Preparation of experimental samples Experimental group: A segmented release product prepared according to Embodiment 1, comprising a fast-release component (40%) and a sustained-release component (60%).

[0085] Control group 1: Unencapsulated group, directly using symbiotic fermentation compound powder without encapsulation treatment.

[0086] Control group 2: Single encapsulation group. All symbiotic fermentation composite powders were encapsulated with medium-viscosity sodium alginate (viscosity 600-800 cP, viscosity of 1% aqueous solution at 25℃). The encapsulation ratio was core material: wall material = 3:1. Approximately 30-40% was released in simulated gastric fluid after 2 hours, and approximately 70-80% was cumulatively released in simulated small intestinal fluid after 4 hours.

[0087] 3. Experimental conditions In vitro digestive simulation system: sequentially simulates the oral cavity (pH 6.8, 5 minutes), stomach (pH 1.2, 2 hours), anterior small intestine (pH 6.8, 2 hours), posterior small intestine (pH 7.4, 2 hours), and colon (pH 7.0-8.0, containing 10 U / mL β-glucanase and 5 U / mL cellulase, 4 hours).

[0088] Detection methods: The release of ginsenosides was determined by ultraviolet spectrophotometry, the release of probiotics was determined by plate counting, and the release location was tracked by fluorescent labeling technology.

[0089] 4. Experimental Procedure Step 1: Place each group of samples in an in vitro digestion simulation system and transfer them sequentially according to the time order of the digestion process.

[0090] Step 2: Take samples at the end of each digestion stage to detect the cumulative release of ginsenosides and probiotics.

[0091] Step 3: Monitor the release process in real time using fluorescent labeling technology, and record the release location and release rate at different time points.

[0092] Step 4: Calculate the release rate and cumulative release rate at each stage, and analyze the characteristics of the release curve.

[0093] 5. Experimental Results Cumulative release rate of ginsenosides at each digestion stage (Table 5): Cumulative release rate of probiotics at each stage of digestion (Table 6): Figure 3 Release curve of ginsenoside rapid release component; Figure 4 For: Comparison of release curves of sustained-release components; Figure 5 For example: Bar chart showing the protective effect of probiotics; Figure 6 For: Heat map of release time distribution.

[0094] 6. Analysis and Summary Experimental results demonstrate that the segmented release encapsulation technology successfully achieved precise spatiotemporal distribution of functional components in the gut. The rapid-release component achieved an 86.7% release rate in the pre-small intestine, ensuring rapid absorption of antioxidants; the sustained-release component saw only 18.9% cumulative release in the first three digestive stages, while reaching 84.6% in the colon, achieving targeted release of probiotics. Compared to the unencapsulated group, the experimental group significantly reduced premature release in the acidic gastric environment, improving the survival rate and colonization efficiency of probiotics.

[0095] The embodiments of the present invention have been described above. However, the embodiments are not limited to the specific implementation methods described above. The specific implementation methods described above are merely illustrative and not restrictive. Those skilled in the art can make more equivalent embodiments under the guidance of the present embodiments, and all of them are within the protection scope of the present embodiments.

Claims

1. A method for preparing animal feed containing ginseng, characterized in that, Includes the following steps: Step 1: Yeast fermentation stage: Yeast was inoculated into a culture medium containing ginseng extract, xylooligosaccharides, corn steep liquor, and yeast extract, and aerobic fermentation was carried out at 28-32℃ for 12-18 hours to obtain a yeast culture medium containing yeast metabolites and yeast cell wall polysaccharides. Step 2: Symbiotic Cultivation Stage In the later stage of yeast fermentation, Clostridium butyricum spores were inoculated into the yeast culture medium, and sodium selenite was added as a selenium source. The culture was continued at 35-37℃ for 8-12 hours under micro-anaerobic conditions. The ginseng macromolecular saponins were converted into rare small molecule saponins by microbial glycosidases, and the microbial enrichment and transformation of selenium was achieved to obtain a symbiotic fermentation broth. Step 3: Drying and curing: The symbiotic fermentation broth is dried by spray drying or freeze drying to remove moisture, thereby obtaining symbiotic fermentation composite powder; Step 4: Segmented release and embedding: The symbiotic fermentation composite powder is divided into a fast-release component and a slow-release component. The fast-release component is encapsulated with sodium carboxymethyl cellulose or low-viscosity sodium alginate, while the slow-release component is encapsulated with medium-high viscosity sodium alginate, chitosan or resistant starch. Step 5: Granulation and molding: The fast-release and slow-release components are mixed and then added to feed carrier raw materials for pelleting to obtain animal feed containing ginseng.

2. The preparation method according to claim 1, characterized in that, The culture medium consists of: 2-5% ginseng extract or ginseng powder, 3-8% xylooligosaccharides, 2-4% corn steep liquor, 1-2% yeast extract, 0.2-0.5% potassium dihydrogen phosphate, and pH adjusted to 6.0-7.

0.

3. The preparation method according to claim 1, characterized in that, The yeast strain is *Saccharomyces cerevisiae* or *Candida utilis*, with a strain activity ≥10. 8 CFU / g; The *Clostridium butyricum* strain is *Clostridium butyricum*, with a strain viability ≥10. 7 CFU / g, inoculation amount is 106-108 CFU / mL.

4. The preparation method according to claim 1, characterized in that, During the biotransformation process in the symbiotic culture stage, ginseng macromolecular saponins Rg1, Rb1, and Re are converted into small molecule rare saponins Rh2, Rg3, compound K, and protopanaxadiol-type saponins, with an enzymatic conversion rate of 30-60%.

5. The preparation method according to claim 1, characterized in that, The amount of sodium selenite added makes the selenium content of the final product reach 0.2-0.5 mg / kg, and the conversion rate of inorganic selenium to organic selenium reaches 70-85%.

6. The preparation method according to claim 1, characterized in that, The rapidly released component accounts for 40% by weight of the symbiotic fermentation composite powder, and the slow-release component accounts for 60% by weight.

7. The preparation method according to claim 1, characterized in that, The rapid-release component dissolves 10-20% in simulated gastric fluid within 30 minutes and accumulates 80-95% dissolves in simulated small intestinal fluid within 30-60 minutes; the sustained-release component dissolves ≤15% in simulated gastric fluid and the pre-small intestine environment within 2 hours and accumulates 75-90% release in simulated colonic fluid within 4-8 hours.

8. The preparation method according to claim 1, characterized in that, When the fast-release component is encapsulated, the weight ratio of the core material to the wall material is 3-4:1, and when the slow-release component is encapsulated, the weight ratio of the core material to the wall material is 2-3:

1.

9. The preparation method according to claim 1, characterized in that, The encapsulation process of the sustained-release component adopts ion gelation encapsulation, in which the composite powder is mixed with sodium alginate aqueous solution to form a suspension, which is then dropped into calcium chloride solution for cross-linking for 30-60 minutes to form gel microspheres.

10. An animal feed containing ginseng prepared by the method according to any one of claims 1-9, characterized in that, This feed contains rare ginsenosides, organic selenium chelate complexes, live spores of Clostridium butyricum, and yeast cell wall polysaccharides, with a live count of ≥10⁻⁶ Clostridium butyricum cells. 5 CFU / g.

Images