Multi-enzyme synergistic section fermentation artemisia pig additive and application thereof

Abstract

The present application belongs to the technical field of traditional Chinese medicine microbial fermentation and livestock feed additives, and particularly relates to a multi-enzyme synergistic segmented fermentation of wormwood pig additive and application thereof. The present application takes de-cortexing Artemisia argyi as the core, and is compounded with auxiliary materials such as Poria cocos and hawthorn, adopts aerobic liquid, anaerobic liquid and solid bacteria substance enrichment three-stage bacteria enzyme synergistic fermentation, and is matched with glycyrrhizic flavone-malt extract powder fermentation inducer, so as to efficiently degrade the crude fiber of wormwood, enrich total flavones, chlorogenic acid and wormwood polysaccharide small molecule active substances, solve the defects of poor palatability, low digestion and utilization rate and applicability to only fattening pigs of original wormwood, and greatly broaden the application range of wormwood in pig breeding. The product of the present application has multiple functions of feeding promotion, anti-inflammatory and anti-diarrhea, antioxidant, immune enhancement and pig skin improvement. The raw materials of the present application are cheap, the fermentation process can be continuously produced in industry, there is no antibiotic residue, the present application is suitable for the green and antibiotic-free pig breeding industry in China, and has high popularization and application value.

Description

Technical Field

[0001] This invention belongs to the technical field of traditional Chinese medicine microbial fermentation and livestock and poultry feed additives. Specifically, it relates to a multi-strain enzyme synergistic segmented fermentation Artemisia argyi pig additive and its application, particularly to a segmented microbial enzyme synergistic fermentation Artemisia argyi preparation, its preparation method, and its application in the entire pig breeding cycle of weaned piglets, growing piglets, fattening pigs, and breeding sows. Background Technology

[0002] I. Shortcomings in the Application of Raw Artemisia argyi Artemisia argyi is a traditional Chinese medicinal herb, rich in flavonoids, chlorogenic acid, artemisia polysaccharides, polyphenols, and triterpenoids. It possesses multiple effects including anti-inflammatory, antibacterial, antioxidant, and immune-boosting properties. It also contains abundant crude protein, vitamins, and minerals, making it a high-quality natural feed ingredient. However, native delinted Artemisia argyi has significant application limitations: 1) The crude fiber content is as high as 25.36%, with many anti-nutritional factors. It is difficult for pigs to degrade and absorb, and the active substances are wrapped in the fiber structure, resulting in extremely low bioavailability.

[0003] 2) The original Artemisia argyi has a spicy and bitter smell, poor palatability, and the feed intake of piglets and sows is likely to decrease.

[0004] 3) The release of active ingredients is limited. Adding them alone can only inhibit bacteria and cannot simultaneously promote growth, resist stress, and improve the reproductive health of sows.

[0005] 4) It has a narrow scope of application. Raw mugwort is only used in small quantities for fattening pigs. Weaned piglets have fragile intestines and cannot tolerate it. Long-term feeding of sows has a weak effect on improving their coat and skin. It is difficult to cover the entire pig farming stage.

[0006] II. Limitations of Existing Fermented Traditional Chinese Medicine Feed Additives Existing fermented traditional Chinese medicine preparations for pigs (such as Gui Tu Ling produced by Guangdong Boxincheng Biotechnology Co., Ltd.) are mostly developed for a single growth stage. Among them, Gui Tu Ling is only for growing piglets weighing 35kg and weaned piglets at 25 days old; while tonic herbs such as tortoise shell and dodder focus on promoting weight gain and slightly reducing mortality, lacking anti-inflammatory, antidiarrheal, antioxidant, and sow skin conditioning functions, and have no ability to adapt to the entire growth cycle.

[0007] Furthermore, existing fermentation processes mostly employ single-stage liquid or single-stage fermentation, failing to differentiate between the metabolic needs of aerobic and anaerobic microorganisms. This leads to antagonism between microbial strains, incomplete degradation of macromolecular fibers, and low dissolution rates of active ingredients. Some patents use single-strain microorganisms without synergistic enzyme reactions, failing to simultaneously decompose cellulose and release flavonoid polysaccharides. Moreover, the absence of fermentation inducers results in insufficient enzyme production by probiotics, leading to limited efficacy of fermentation products that can only promote growth in piglets and are unsuitable for fattening pigs or sows. Additionally, existing fermented traditional Chinese medicine products often have complex formulations and high raw material costs. While Artemisia argyi (mugwort) is abundant and inexpensive in Qichun and Nanyang, there is a lack of mature, efficient, segmented fermentation processes suitable for the entire pig life cycle, limiting the large-scale adoption of Artemisia argyi in Wuhan's livestock industry.

[0008] III. Summary of the shortcomings of existing technologies: 1) Raw Artemisia argyi has excessive fiber content, poor palatability, and low utilization rate of active ingredients, so it is only used in small quantities in fattening pigs and has a narrow scope of application.

[0009] 2) Traditional single fermentation processes result in antagonistic microbial communities, insufficient fiber degradation, and low release of functional substances such as flavonoids and chlorogenic acid.

[0010] 3) Commercially available fermented Chinese herbal medicine additives are only suitable for a single pig herd and cannot cover the entire life cycle of weaned piglets, piglets, fattening pigs, and sows.

[0011] 4) The lack of a fermentation induction system results in low secretion of probiotic hydrolytic enzymes, leading to weak synergistic effects of stomach health, anti-inflammation, and anti-oxidation.

[0012] 5) The raw material costs of similar fermented Chinese medicine products are relatively high, and the input-output ratio of large-scale breeding is insufficient.

[0013] To address the aforementioned shortcomings, this invention designs and develops a multi-strain enzyme synergistic segmented fermentation additive for Artemisia argyi used in pigs. Through a three-stage fermentation process—aerobic-anaerobic-solid-state—and synergistic effect induced by licorice flavonoids and malt extract powder, it significantly degrades crude fiber, enriches small molecule active substances, and significantly improves palatability. It can be adapted to various types of pigs at all stages, broadening the application scenarios of Artemisia argyi in pig farming, and simultaneously achieving multiple values ​​such as promoting growth, preventing diarrhea, anti-oxidation, and sow health care. Summary of the Invention

[0014] To address the shortcomings of existing technologies, this invention provides a multi-strain enzyme synergistic segmented fermentation additive for Artemisia argyi used in pigs and its application, mainly involving the following objectives: 1) A segmented bacterial enzyme synergistic fermentation process is provided to degrade the crude fiber of Artemisia argyi, significantly increase the dissolution of total flavonoids, chlorogenic acid and Artemisia argyi polysaccharides, and solve the problems of difficult digestion and low activity of raw Artemisia argyi.

[0015] 2) Through the synergistic effect of compound probiotics, compound enzymes and fermentation inducers, bacterial antagonism is eliminated, and the effects of strengthening the stomach and promoting appetite, anti-inflammatory and anti-diarrheal, and antioxidant are enhanced.

[0016] 3) Fermented mugwort products can be adapted to weaned piglets, growing piglets, fattening pigs, and breeding sows, covering the entire pig life cycle and greatly expanding the scope of application of mugwort in pig farming.

[0017] 4) Compared with existing single-stage fermented Chinese medicine, it achieves multiple effects such as promoting growth, preventing diarrhea, enhancing immunity, and repairing sow skin, thereby improving the overall breeding income of pig farms.

[0018] 5) Provide a growth-promoting feed additive that uses readily available raw materials, can be continuously industrialized through fermentation processes, is suitable for green and antibiotic-free pig farming, and can replace antibiotics.

[0019] To achieve the above objectives, the technical solution created by this invention is implemented as follows: This invention first provides a multi-strain enzyme synergistic segmented fermentation additive for Artemisia argyi used in pigs, which is prepared by fermentation of fermentation substrate through three-stage synergistic fermentation of bacteria and enzymes.

[0020] Specifically, the activity indicators of the fermented mugwort pig additive are: total flavonoids ≥215mg / g, chlorogenic acid ≥74μg / g, total polysaccharides ≥43mg / g, crude fiber reduced to below 21.48%, and pH stable at 4.4-4.5.

[0021] Specifically, the fermentation base material includes, by weight: 60-160 parts of de-flossed Artemisia argyi, 5-20 parts of Poria cocos, 3-16 parts of red dates, 6-24 parts of hawthorn, and 2-12 parts of dandelion.

[0022] Preferably, the fermentation base material includes, by weight: 120-160 parts of de-flossed Artemisia argyi, 10-20 parts of Poria cocos, 6-16 parts of jujube, 12-24 parts of hawthorn, and 4-12 parts of dandelion.

[0023] Preferably, the fermentation base material includes, by weight: 60-80 parts of de-flossed Artemisia argyi, 5-10 parts of Poria cocos, 3-8 parts of jujube, 6-12 parts of hawthorn, and 2-6 parts of dandelion.

[0024] Further preferred, the fermentation base material includes, by weight: 74 parts of de-fleshed Artemisia argyi, 8 parts of Poria cocos, 5 parts of jujube, 9 parts of hawthorn, and 4 parts of dandelion.

[0025] Further preferred, the fermentation base material includes, by weight: 78 parts of de-fleshed Artemisia argyi, 9 parts of Poria cocos, 3 parts of jujube, 6 parts of hawthorn, and 4 parts of dandelion.

[0026] The formulation principle of the fermentation base is as follows: Artemisia argyi is the main ingredient, providing core anti-inflammatory and antioxidant activities of flavonoids and polysaccharides; hawthorn and Poria cocos invigorate the spleen and promote digestion, and promote intestinal peristalsis; red dates replenish qi and improve palatability. Dandelion clears heat and inhibits bacteria, and works synergistically with Artemisia argyi to reduce the diarrhea rate of piglets and fattening pigs.

[0027] Specifically, the multi-strain enzyme synergistic segmented fermentation Artemisia argyi pig additive is prepared by fermentation of fermentation base material through a fermentation strain enzyme synergistic system composed of compound probiotics and compound enzymes.

[0028] Specifically, the compound probiotics include Bacillus coagulans, Lactobacillus plantarum, Clostridium butyricum, Saccharomyces cerevisiae, and Aspergillus niger; the compound enzymes include cellulase and hemicellulase, with an optimal mass ratio of 1:1, and their main function is to degrade the Artemisia argyi fiber skeleton in advance.

[0029] The mass ratio of each bacterium in the compound probiotic is as follows: Bacillus coagulans: Saccharomyces cerevisiae = 1~2:1 (preferably 2:1); Lactobacillus plantarum: Clostridium butyricum = 1~2:1 (preferably 1.5:1); Aspergillus niger is used alone for solid-state fermentation; the mass ratio of cellulase and hemicellulase in the compound enzyme is 1~2:1.

[0030] Specifically, the enzyme activity of cellulase is 5000-20000 U / g, and the enzyme activity of hemicellulase is 7000-25000 U / g.

[0031] Specifically, the fermentation includes three stages: S1 aerobic liquid fermentation, S2 anaerobic liquid fermentation, and S3 solid microbial enrichment. During the fermentation process, a fermentation inducer composed of glycyrrhizin flavonoids and malt extract powder is added. Its main functions are: glycyrrhizin flavonoids activate the oxidative metabolism of the microorganisms and promote flavonoid production; malt extract powder contains maltose, polypeptides, and vitamins, and takes into account both carbon and nitrogen induction to enhance the synergistic enzyme production capacity of yeast and Bacillus.

[0032] Preferably, the compound probiotics are added in three stages: S1 aerobic liquid fermentation, S2 anaerobic liquid fermentation, and S3 solid-state microbial enrichment, specifically as follows: 1) Aerobic stage (aerobic liquid fermentation): Add Bacillus coagulans and Saccharomyces cerevisiae. The optimal ratio (mass ratio) of the two is 2:1. Their main functions are to secrete proteases and cellulases to decompose macromolecular fibers and proteins. 2) Anaerobic stage (anaerobic liquid fermentation): Add Lactobacillus plantarum and Clostridium butyricum. The optimal ratio (mass ratio) of the two is 1.5:1. The main functions are: to produce lactic acid and short-chain fatty acids, regulate intestinal pH, and inhibit harmful bacteria. 3) Solid segment (solid microbial enrichment): Aspergillus niger is added, and its main function is to enrich complex hydrolytic enzymes and release active substances from the residue in the secondary release process.

[0033] Specifically, during the fermentation process, the added fermentation inducer is a compound of licorice flavonoids and malt extract powder in a mass ratio of 1:1 to 4 (preferably 1:4).

[0034] Furthermore, the present invention also provides a preparation process for the multi-strain enzyme synergistic segmented fermentation of Artemisia argyi pig additive (i.e., a three-segment multi-strain enzyme synergistic fermentation preparation process), comprising the following steps: S1. Aerobic liquid fermentation: The fermentation substrate I is crushed and passed through an 80-100 mesh sieve (preferably a 90 mesh sieve). The obtained powder is extracted with water to prepare suspension I. Then, Bacillus coagulans and Saccharomyces cerevisiae are inoculated, and a fermentation inducer is added. Aerobic fermentation is carried out for 18-24 h (preferably 21 h) under stirring conditions of 200-400 rpm (preferably 300 rpm). After centrifugation, aerobic fermentation broth and aerobic residue are obtained. S2. Anaerobic liquid fermentation: The fermentation substrate II is crushed and passed through a 50-mesh sieve to obtain fine powder. It is then extracted with water to prepare suspension II. A compound enzyme is added. Then, a mixed anaerobic bacteria consisting of Lactobacillus plantarum and Clostridium butyricum is inoculated. The mixture is sealed and anaerobic fermented for 60-70 h (preferably 65 h). After centrifugation, the anaerobic fermentation liquid and anaerobic residue are obtained. S3. Solid microbial enrichment: Mix all the residues from steps S1 and S2, add grain flour and bran to make a solid substrate; then inoculate with Aspergillus niger, ferment aerobically for 20-30 h, dry at 70-80℃ to constant weight, pulverize, and sieve to obtain functional microbial material. S4. Finished product compounding: Mix the aerobic fermentation liquid from step S1 and the anaerobic fermentation liquid from step S2 (specifically, mix at 700~800 rpm for 10~15 min), add functional bacteria, stir at high speed, dry at low temperature, and pulverize to obtain fermented mugwort pig additive.

[0035] Specifically, in step S1, the components and weight parts of fermentation substrate I are as follows: 60-80 parts of de-flossed Artemisia argyi, 5-10 parts of Poria cocos, 3-8 parts of jujube, 6-12 parts of hawthorn, and 2-6 parts of dandelion.

[0036] Specifically, in step S1, during extraction, the powder-to-water ratio is 1:4 to 1:7, preferably 1:4 to 1:6.

[0037] Specifically, in step S1, the extraction conditions are: extraction at 60-80°C for 3-5 hours, followed by cooling to 30-40°C; preferably, extraction at 70°C for 4 hours, followed by cooling to 35°C.

[0038] Specifically, in step S1, the inoculum size of Bacillus coagulans is 1×10⁻⁶. 7 ~2.5×10 7 CFU / mL (preferably 1.8 × 10⁻⁶)7 CFU / mL, 1.6×10 7 (CFU / mL), the inoculum size of *Saccharomyces cerevisiae* was 1.5 × 10⁻⁶. 7 ~2.5×10 7 CFU / mL (preferably 2.3 × 10⁻⁶) 7 CFU / mL, 2.0×10 7 (CFU / mL).

[0039] Specifically, in step S1, the mass ratio of Bacillus coagulans to Saccharomyces cerevisiae is 1~2:1 (preferably 2:1).

[0040] Specifically, in step S1, the fermentation inducer is a compound of glycyrrhizin and malt extract powder, with a mass ratio of 1:1 to 4 (preferably 1:4). The amount of fermentation inducer added is 3% to 5% of the total volume of suspension I in step S1 (preferably 4% of the total volume of suspension I).

[0041] Specifically, in step S1, centrifugation is performed at a speed of 9000 rpm for 10-15 minutes.

[0042] Specifically, in step S2, the components and weight parts of fermentation substrate II are as follows: 60-80 parts of de-flossed Artemisia argyi, 5-10 parts of Poria cocos, 3-8 parts of jujube, 6-12 parts of hawthorn, and 2-6 parts of dandelion.

[0043] Specifically, in step S2, during extraction, the powder-to-water ratio is 1:4 to 1:5, preferably 1:4.5.

[0044] Specifically, in step S2, the extraction conditions are as follows: extraction at 85-95°C for 1-2 hours, followed by cooling to 30-40°C; preferably, extraction at 90°C for 1.5 hours, followed by cooling to 35°C.

[0045] Specifically, in step S2, the amount of the compound enzyme added is 0.2-0.8% of the mass of suspension II, preferably 0.5% of the mass of suspension II; the cellulase activity is 5000-20000 U / g, and the hemicellulase activity is 7000-25000 U / g; the mass ratio of cellulase to hemicellulase in the compound enzyme is 1-2:1 (preferably 1:1).

[0046] Specifically, in step S2, the mixed anaerobic bacteria consisting of *Lactobacillus plantarum* and *Clostridium butyricum* have a mass ratio of 1~2:1 (preferably 1.5:1), and the inoculum size is 3×10⁻⁶. 7 ~5×10 7 CFU / mL (preferably 3.5 × 10⁻⁶) 7 CFU / mL, 4.2×10 7 (CFU / mL).

[0047] Specifically, in step S3, the grain flour in the solid matrix accounts for 20%–30% of the total weight of the residue, and the bran accounts for 5%–10% of the total weight of the residue; the inoculum size of Aspergillus niger is 5 × 10⁻⁶. 7 ~7×10 7 CFU / g (preferably 6×10) 7 CFU / g, 5.8×10 7 CFU / g).

[0048] Specifically, in step S3, the aerobic fermentation temperature is 35~45℃ and the time is 20~30 h. Preferably, the aerobic fermentation temperature is 40℃ and the time is 25 h.

[0049] Specifically, in step S4, the speed of high-speed stirring is 100-200 rpm, and the time is 1-2 hours, preferably 1.5 hours.

[0050] Specifically, in step S4, the mass ratio of the aerobic fermentation broth, the anaerobic fermentation broth, and the functional microbial material is 1:1:(0.1~0.5).

[0051] Furthermore, this invention also provides a multi-strain enzyme synergistic segmented fermentation Artemisia argyi pig additive prepared using the above-described process, the core indicators of which are: After fermentation, the crude fiber content decreased from 25.36% to 21.48%; the total flavonoids content was 215.70 mg / g, chlorogenic acid was 74.58 μg / g, and the total polysaccharides were 43.03 mg / g; the finished product pH was 4.45; the product has a natural fermented aroma and no pungent or spicy taste of native mugwort.

[0052] Furthermore, based on a general inventive concept, the present invention also provides the application of the aforementioned multi-strain enzyme synergistic segmented fermentation Artemisia argyi pig additive in livestock breeding, feed preparation, or veterinary drug preparation.

[0053] Specifically, the feed is poultry or livestock feed.

[0054] Furthermore, based on a general inventive concept, the present invention also provides the application of the aforementioned multi-enzyme synergistic segmented fermentation Artemisia argyi pig additive in improving the growth performance of piglets.

[0055] Furthermore, based on a general inventive concept, the present invention also provides the application of the aforementioned multi-strain enzyme synergistic segmented fermentation Artemisia argyi pig additive in improving the antioxidant performance of fattening pigs.

[0056] Furthermore, based on a general inventive concept, the present invention also provides the application of the aforementioned multi-strain enzyme synergistic segmented fermentation Artemisia argyi pig additive in improving the skin condition of pregnant / lactating sows or reducing the incidence of inflammation in pregnant / lactating sows.

[0057] Furthermore, based on a general inventive concept, the present invention also provides the application of the aforementioned multi-strain enzyme synergistic segmented fermentation Artemisia argyi pig additive in pig farming.

[0058] Specifically, when applying this method, the proportion (by weight) of the feed added is 1% to 3%; the preferred addition proportion (by weight) for weaned piglets is 1%, for growing piglets it is 1.5%, for fattening pigs it is 2%, and for breeding sows it is 2% to 3%.

[0059] Specifically, when applied, the fermented mugwort pig additive is adapted to the entire growth cycle of pigs: reducing the mortality rate of weaned piglets and alleviating weaning stress diarrhea; increasing the average daily weight gain of growing pigs and reducing the feed conversion ratio; promoting feed intake in fattening pigs and increasing the gross profit of fattening pig farming; improving the skin condition of pregnant / lactating sows and reducing the incidence of inflammation in pregnant / lactating sows.

[0060] Specifically, the fermented mugwort swine additive has the following antioxidant and immune-enhancing activities: After feeding fattening pigs with 2% (by weight) for 60 days, serum superoxide dismutase (SOD) activity increased by ≥28% compared with the control group, malondialdehyde (MDA) content decreased by ≥31% compared with the control group, and total antioxidant capacity (T-AOC) increased by ≥25% compared with the control group. After feeding weaned piglets with 1% (by weight) of the additive for 46 days, the serum immunoglobulin IgG content increased by ≥22% compared with the control group, the IgA content increased by ≥18% compared with the control group, and the serum interleukin-2 (IL-2) level increased by ≥20% compared with the control group.

[0061] This invention uses de-flossed Artemisia argyi as the core ingredient, combined with auxiliary ingredients such as Poria cocos and hawthorn. It employs a three-stage fermentation process involving aerobic liquid, anaerobic liquid, and solid microbial enrichment, along with licorice flavonoids-malt extract fermentation inducer. This process efficiently degrades the crude fiber of Artemisia argyi and enriches total flavonoids, chlorogenic acid, and small molecule active substances such as Artemisia argyi polysaccharides. It overcomes the shortcomings of native Artemisia argyi, such as poor palatability, low digestibility and utilization, and its applicability only to fattening pigs, thus significantly expanding the scope of application of Artemisia argyi in pig farming. This invention's product can be added to the daily diet of weaned piglets, growing piglets, fattening pigs, and breeding sows at a dosage of 1% to 3%. It has multiple effects, including stimulating appetite and promoting growth, anti-inflammatory and antidiarrheal effects, antioxidant effects, enhancing immunity, and improving the coat of sows. Pig farm trials have shown that compared with single-stage fermented Chinese medicine, this invention is more effective in promoting weight gain, reducing mortality, and controlling diarrhea. When added at a dosage of 2%, fattening pigs can generate an additional 31.8 yuan per head. The raw materials are inexpensive, the fermentation process can be industrialized for continuous production, and there are no antibiotic residues. It is suitable for the green and antibiotic-free pig farming industry nationwide and has extremely high promotion and application value.

[0062] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Fermentation significantly enhances the active ingredients, addressing the shortcomings of using native mugwort. The three-stage bacterial enzyme synergy and inducer synergistic degradation of Artemisia argyi fiber significantly increases total flavonoids, chlorogenic acid, and polysaccharides, reduces crude fiber, eliminates bitter odor, and greatly improves palatability. Native Artemisia argyi can only be added in small amounts to fattening pig feed, while the fermentation products can be safely fed to piglets and sows, covering the entire life cycle and breaking through the limitations of raw material application.

[0063] 2. Multi-functional collaboration, with the following features: ① Weaned piglets: Reduce mortality and alleviate weaning stress diarrhea; ② Growing piglets: Increase daily weight gain and reduce feed conversion ratio; ③ Fattening pigs: Increase feed intake, promote weight gain, significantly reduce diarrhea rate, and increase gross profit per pig; ④ For breeding sows: Improves rough skin, reduces dead skin shedding, and reduces the incidence of skin and systemic inflammation.

[0064] ⑤ It integrates growth promotion, diarrhea prevention, anti-oxidation, and reproductive health care, with a wider range of functions.

[0065] 3. Significantly resists oxidation and delays oxidative damage in the body. The total flavonoids (215.70 mg / g), chlorogenic acid (74.58 μg / g), and Artemisia argyi polysaccharides (43.03 mg / g) abundant in the fermentation products of this invention are transformed from macromolecular bound states to free small molecule states after three-stage synergistic fermentation, resulting in a significant improvement in bioavailability. Pig farm trials have confirmed that adding 2% of the product increases serum SOD activity by 31.6%, GSH-Px activity by 26.4%, reduces MDA content by 34.2%, and increases T-AOC by 27.8% in fattening pigs. This indicates that the product of this invention can effectively scavenge free radicals and reduce lipid peroxidation damage, significantly outperforming unfermented Artemisia argyi and single-stage fermentation products.

[0066] 4. Enhance the body's immunity and reduce the risk of disease. After microbial degradation, the molecular weight of Artemisia argyi polysaccharides in the fermentation products is reduced to small molecule fragments of 5-50 kDa, making them more easily recognized and absorbed by intestinal mucosal immune cells. Experiments have shown that in weaned piglets, the 1% addition group showed a 24.7% increase in serum IgG, a 19.3% increase in IgA, and a 15.8% increase in IgM; a 18.6% increase in peripheral blood lymphocyte transformation rate; a 22.4% increase in serum IL-2 and a 17.9% increase in IFN-γ; while pro-inflammatory factors IL-6 and TNF-α decreased by 28.3% and 24.1%, respectively. This indicates that the product of this invention can bidirectionally regulate immune function—both enhancing humoral and cellular immune responses and inhibiting excessive inflammatory responses, achieving a synergistic effect of "strengthening the body's resistance and eliminating pathogens."

[0067] 5. Segmented fermentation avoids microbial antagonism and maximizes the utilization rate of active substances. Separate aerobic, anaerobic, and solid-state fermentation processes to match the metabolic environments of different microbial communities, avoiding competition for nutrients and inhibition of enzyme production by single mixed fermentation microbial communities; secondary solid-state enrichment of functional bacteria in the residue greatly improves the utilization rate of raw materials, and all fermentation waste liquid is reused without waste.

[0068] 6. The economic benefits of aquaculture are outstanding. Fattening pig trial: Adding 2% of the product to the diet increased the gross profit per pig by 31.8 yuan, which is higher than the 1% addition group (18.3 yuan) and the 3% addition group (29.5 yuan). The input-output ratio is optimal. The product can reduce mortality by 4.1% and has a stronger anti-stress effect.

[0069] 7. Raw materials are inexpensive and readily available, facilitating industrialized production. Qi Ai (Artemisia argyi) is in ample supply from the two major domestic industrial belts, while Poria cocos and hawthorn are both conventional medicinal and edible raw materials; the three-stage fermentation can be carried out continuously in separate tanks, with controllable temperature and speed, no need for high-cost organic solvents, and no antibiotics or heavy metal residues, meeting the national standards for antibiotic-free aquaculture. Detailed Implementation

[0070] To better understand the present invention, the present invention will be further described below with reference to the embodiments. However, the content of the present invention is not limited to the following embodiments. All changes or equivalent substitutions that do not depart from the concept of the present invention are included within the protection scope of the present invention.

[0071] The raw materials and equipment described in this invention can all be selected from commercially available products. The bacteria used in this invention are: Bacillus coagulans CICC strain preservation number 10069; Saccharomyces cerevisiae CICC strain preservation number 1406; Lactobacillus plantarum CICC strain preservation number 20272; Clostridium butyricum CICC strain preservation number 24498; Aspergillus niger CICC strain preservation number 40294, all of which were purchased from the China Industrial Microbial Culture Collection Center.

[0072] The cellulase activity was 5000-20000 U / g, and the hemicellulase activity was 7000-25000 U / g. They were purchased from Shandong Longket Enzyme Preparation Co., Ltd.

[0073] Example 1 Preferred formulation Example 1 provides a multi-strain enzyme synergistic segmented fermentation additive for Artemisia argyi used in pigs, which is prepared by three-stage synergistic fermentation of fermentation substrate.

[0074] Furthermore, the multi-strain enzyme synergistic segmented fermentation Artemisia argyi pig additive is prepared by fermentation of fermentation substrate through a fermentation strain enzyme synergistic system composed of compound probiotics and compound enzymes.

[0075] The compound probiotics include Bacillus coagulans, Lactobacillus plantarum, Clostridium butyricum, Saccharomyces cerevisiae, and Aspergillus niger; the compound enzymes include cellulase and hemicellulase, whose main function is to degrade the Artemisia argyi fiber skeleton in advance.

[0076] Furthermore, the preparation process of the multi-strain enzyme synergistic segmented fermentation of Artemisia argyi pig additive (i.e., the three-segment multi-strain enzyme synergistic fermentation preparation process) includes the following specific steps: S1. Aerobic liquid fermentation: The fermentation substrate I is crushed and passed through a 90-mesh sieve. The obtained powder is extracted with water to prepare suspension I. Then, Bacillus coagulans and Saccharomyces cerevisiae are inoculated, and a fermentation inducer is added. Aerobic fermentation is carried out for 21 hours under stirring at 300 rpm. After centrifugation (centrifuging at 9000 rpm for 15 minutes), aerobic fermentation broth and aerobic residue are obtained. In step S1, the mass fractions of fermentation base I and each component are as follows: 74 parts of de-flossed Artemisia argyi, 8 parts of Poria cocos, 5 parts of red dates, 9 parts of hawthorn, and 4 parts of dandelion (it should be noted that in actual production, the mass fractions here can be in kilograms or grams, without special limitation. In Example 1, kilograms are preferred).

[0077] In step S1, the powder-to-water ratio during extraction is 1:6.

[0078] In step S1, the extraction conditions are: extraction at 70°C for 4 hours, followed by cooling to 35°C.

[0079] In step S1, the inoculum size of Bacillus coagulans is 1.6 × 10⁻⁶. 7 CFU / mL, Saccharomyces cerevisiae inoculum size 2.0 × 10⁻⁶ 7 CFU / mL.

[0080] In step S1, the fermentation inducer is a compound of glycyrrhizin flavonoids and malt extract powder (purchased from Beijing Hongrun Baoshun Technology Co., Ltd.), with a mass ratio of 1:4. The amount of fermentation inducer added is 4.5% of the total volume of suspension I in step S1.

[0081] S2. Anaerobic liquid fermentation: Fermentation substrate II is pulverized and passed through a 50-mesh sieve to obtain fine powder. It is then extracted with water to prepare suspension II. A compound enzyme is added at a concentration of 0.5% of the mass of suspension II. The compound enzyme includes cellulase and hemicellulase, with a mass ratio of 1:1. The cellulase activity is 10,000 U / g, and the hemicellulase activity is 8,000 U / g. Then, a mixed anaerobic bacteria consisting of Lactobacillus plantarum and Clostridium butyricum is inoculated. The mixture is sealed and anaerobic fermented for 65 hours. After centrifugation, the anaerobic fermentation broth and anaerobic residue are obtained. In step S2, the mass fractions of fermentation base II and each component are as follows: 78 parts of de-flossed Artemisia argyi, 9 parts of Poria cocos, 3 parts of red dates, 6 parts of hawthorn, and 4 parts of dandelion (it should be noted that in actual production, the mass fractions here can be in kilograms or grams, without special limitation. In Example 1, kilograms are preferred).

[0082] In step S2, the powder-to-water ratio during extraction is 1:4.5.

[0083] In step S2, the extraction conditions are: extraction at 90°C for 1.5 hours, followed by cooling to 35°C.

[0084] In step S2, a mixed anaerobic bacterium consisting of *Lactobacillus plantarum* and *Clostridium butyricum* was used, with a mass ratio of 1.5:1, and an inoculum size of 4.2 × 10⁻⁶. 7 CFU / mL.

[0085] S3. Solid microbial enrichment: Mix all the residues from steps S1 and S2, add grain powder and bran to make a solid substrate; then inoculate with Aspergillus niger, ferment aerobically at 40℃ for 25 h, dry at 75℃ to constant weight (the specific drying time is 4-8 h), pulverize, and pass through a 300-mesh sieve to obtain functional microbial material. In step S3, cereal flour (specifically corn flour) accounts for 25% of the total weight of the residue, and wheat bran accounts for 8% of the total weight of the residue; the inoculum amount of Aspergillus niger is 5.8 × 10⁻⁶. 7 CFU / g.

[0086] S4. Finished product compounding: Mix the aerobic fermentation liquid from step S1 and the anaerobic fermentation liquid from step S2 (specifically, mix at 750 rpm for 15 min), add functional bacteria, stir at high speed for 1.5 h (150 rpm), dry at low temperature, and pulverize to obtain fermented mugwort pig additive.

[0087] In step S4, the mass ratio of the aerobic fermentation broth, the anaerobic fermentation broth, and the functional microbial material is 1:1:0.1.

[0088] The complex enzyme is composed of cellulase and hemicellulase in a mass ratio of 1:1.

[0089] According to the test results, the core indicators of the product prepared in Example 1 are as follows: after fermentation, the crude fiber content decreased from 25.36% to 21.48%; total flavonoids 215.70 mg / g, chlorogenic acid 74.58 μg / g, total polysaccharides 43.03 mg / g, pH 4.45; the product has a natural fermented aroma and no pungent or spicy taste of native mugwort.

[0090] Example 2: Low-additive suitable piglets The difference between Example 2 and Example 1 is that: In step S1, the mass fractions of fermentation substrate I and each component are as follows: 60 parts of de-flossed Artemisia argyi, 5 parts of Poria cocos, 3 parts of jujube, 6 parts of hawthorn, and 2 parts of dandelion; the amount of fermentation inducer added is 3% of the total volume of suspension I in step S1. In step S1, the aerobic fermentation time is 24 hours, and in step S2, the anaerobic fermentation time is 70 hours. The other parameters are the same as in Example 1.

[0091] The finished product prepared in Example 2 is milder and less irritating, suitable for the diet of weaned piglets, and is added at a dosage of 1% (by weight).

[0092] Example 3: High-content suitable for sows The difference between Example 3 and Example 1 is that: In step S1, the mass fractions of fermentation substrate I and each component are as follows: 80 parts of de-flossed Artemisia argyi, 10 parts of Poria cocos, 8 parts of jujube, 12 parts of hawthorn, and 6 parts of dandelion; the amount of fermentation inducer added is 5% of the total volume of suspension I in step S1. In step S3, aerobic solid-state fermentation is carried out for 30 hours, and the other parameters are the same as in Example 1.

[0093] The finished product prepared in Example 3 has a higher content of active antioxidants and is suitable for sow diets, and should be added at a dosage of 2% to 3% (by weight).

[0094] Comparative Example 1: No fermentation inducer The process was the same as in Example 1, except that the addition of licorice flavonoids and malt extract inducer was omitted. Testing of the prepared product showed that the total flavonoids in the fermentation broth were only 162.4 mg / g, and the chlorogenic acid content was only 61.2 μg / g, indicating a significant decrease in active substances and no significant improvement in feed intake for piglets.

[0095] In Comparative Example 1, the group without fermentation inducer was added to fattening pigs at a dosage of 2% (mass percentage) for 60 days. The serum SOD increased by only 13.3% (88.74 vs 78.32 U / mL), MDA decreased by only 15.5%, and IgG increased by only 11.8%, all of which were significantly lower than those in Example 1 of this invention (P<0.05). This indicates that the licorice flavonoid-malt extract inducer has a key promoting effect on bacterial enzyme production and release of active substances.

[0096] Comparative Example 2: Single Mixed Fermentation (Unsegmented Mixed Fermentation Group) The raw materials used in Example 1 differed from those in Example 1 in that all raw materials and bacterial strains were mixed and fermented simultaneously in one go, without aerobic / anaerobic separation. Testing revealed antagonistic interactions between the bacterial strains, and crude fiber only decreased to 24.12%, resulting in a significantly weaker diarrhea-improving effect compared to Example 1.

[0097] In Comparative Example 2, the non-segmented mixed fermentation group was added to fattening pigs at a dosage of 2% (mass percentage) for 60 days. The serum SOD increased by 15.1% (90.12 vs 78.32 U / mL), MDA decreased by 17.7%, and IgG increased by 14.2%, which were also significantly lower than those in Example 1 of this invention (P<0.05). This indicates that aerobic-anaerobic-solid-state segmented fermentation has irreplaceable process advantages in avoiding strain antagonism and maximizing the release of antioxidant and immune-active substances.

[0098] Application Example 1: Aquaculture Application Trial Data All addition amounts in the following experiments are mass fractions. The fermented mugwort group, fermented mugwort pig additive, and addition group in the following experiments all used the product prepared in Example 1.

[0099] I. Large-scale trial of fattening pigs over 60 days: Experimental groups: basal diet control group, 1% fermented mugwort group, 2% fermented mugwort group, and 3% fermented mugwort group, with 55 animals in each group, with an initial weight of about 60 kg, and an experimental period of 60 days.

[0100] Table 1.

[0101]

[0102] II. Comparison of 35kg growing piglets at 30 days: Each group consisted of 100 animals, with a trial period of 30 days.

[0103] Table 2.

[0104]

[0105] III. Experiment on 25-day-old weaned piglets for 46 days: Each group had 3 replicates, totaling 150 heads.

[0106] Table 3.

[0107]

[0108] IV. Feeding effect on breeding sows: Adding 2% fermented artemisia to the diet of pregnant and lactating sows for two consecutive months resulted in the following: The rate of rough skin and dead skin shedding in sows increased by 68%, and their skin became rosy and shiny; the incidence of respiratory and digestive tract inflammation decreased by 42%, and the postpartum recovery speed was significantly improved.

[0109] V. Antioxidant Performance Test of Fattening Pigs (60 days): The experimental groups were the same as in the 60-day large-scale trial of fattening pigs. Ten pigs were randomly selected from each group, and blood was collected from the anterior vena cava on day 0 and day 60 of the experiment. The serum was collected by centrifugation at 3000 rpm for 15 min and stored at -20℃ for later testing. Serum antioxidant indicators were determined using a kit method (Nanjing Jiancheng Bioengineering Institute).

[0110] Table 4. Effects of fermented Artemisia argyi additive on serum antioxidant indicators of fattening pigs (n=10).

[0111]

[0112] Note: This indicates a significant difference compared to the control group (P<0.05). This indicates a highly significant difference compared to the control group (P<0.01).

[0113] Results analysis: The 1.2% addition group showed a 31.6% increase in SOD activity (P<0.01), a 26.4% increase in GSH-Px (P<0.01), a 34.2% decrease in MDA (P<0.01), and a 27.8% increase in T-AOC (P<0.01) compared to the control group, demonstrating a highly significant antioxidant effect.

[0114] 2. There were no significant differences in any indicators between the 2% unfermented mugwort group and the control group (P>0.05), indicating that the antioxidant active substances in the original mugwort are wrapped by the fiber structure and have extremely low bioavailability.

[0115] 3. Although Comparative Example 1 (without inducer) and Comparative Example 2 (without segmented fermentation) have certain antioxidant effects, they are significantly weaker than Example 1 of the present invention (P<0.05), which proves that the three-segment bacterial enzyme synergy + inducer system has a key synergistic effect on the release of antioxidant active substances.

[0116] 4. There was no significant difference in antioxidant index between the 2% and 3% addition groups (P>0.05). Considering the overall cost, 2% is the optimal addition amount.

[0117] VI. Immunological function test of weaned piglets (46 days): The experimental groups were the same as those for the 46-day weaned piglet experiment. Twelve piglets were randomly selected from each group, and blood was collected from the vena cava on day 0 and day 46 of the experiment. Serum was separated. Immunoglobulin and cytokine levels were measured by ELISA; peripheral blood lymphocyte transformation rate (ConA stimulation) was measured by MTT assay.

[0118] Table 5. Effects of fermented Artemisia argyi additive on serum immunoglobulins in weaned piglets (n=12).

[0119]

[0120] Table 6 Effects of fermented Artemisia argyi additive on serum cytokines and lymphocyte transformation rate in weaned piglets (n=12).

[0121]

[0122] Note: This indicates a significant difference compared to the control group (P<0.05). This indicates a highly significant difference compared to the control group (P<0.01).

[0123] Results analysis: 1. In this invention, the IgG level in the 1% addition group was 24.7% higher than that in the control group (P<0.01), the IgA level was 19.1% higher (P<0.05), and the IgM level was 16.0% higher (P<0.05), indicating that fermented Artemisia argyi polysaccharide can effectively activate the humoral immune function of weaned piglets.

[0124] 2. The pro-inflammatory factor IL-6 decreased by 28.3% (P<0.01), TNF-α decreased by 24.1% (P<0.01), while the anti-inflammatory / immune-enhancing factor IL-2 increased by 22.3% (P<0.01) and IFN-γ increased by 17.8% (P<0.05), indicating that the product of this invention has a "bidirectional immune regulation" function—enhancing specific immune responses while inhibiting excessive inflammatory responses caused by weaning stress.

[0125] 3. The lymphocyte transformation rate increased by 18.7% (P<0.01), indicating that cellular immune function was simultaneously enhanced.

[0126] 4. There were no significant differences in immune indicators between the unfermented Artemisia argyi group and the control group (P>0.05), further confirming that the immunologically active polysaccharides in native Artemisia argyi are encapsulated by the fibrous matrix and cannot be effectively absorbed and utilized by the piglet intestine.

[0127] 5. The immune indicators of the present invention group were significantly better than those of Comparative Example 1 and Comparative Example 2 (P<0.05), proving that the three-stage fermentation + inducer system has an irreplaceable synergistic effect on the release and enrichment of immune active substances.

[0128] VII. Verification of Immune Function in Fattening Pigs (60 Days) The experimental groups were the same as those in the 60-day large-scale trial of fattening pigs. Ten pigs were randomly selected from each group, and blood was collected and serum was separated on the 60th day of the experiment to determine immune indicators.

[0129] Table 7 Effects of fermented Artemisia argyi additive on serum immune indicators of fattening pigs (n=10).

[0130]

[0131] Results analysis: In the 1.2% addition group, IgG increased by 25.3% (P<0.01), IgA increased by 18.4% (P<0.01), IL-2 increased by 21.1% (P<0.01), while IL-6 decreased by 23.9% (P<0.01) and TNF-α decreased by 22.1% (P<0.01).

[0132] 2. This indicates that the product of the present invention also has significant dual effects of immune enhancement and anti-inflammation in the fattening pig stage, which is consistent with the result of a significant reduction in the diarrhea rate of fattening pigs (from 3.09% to 0.62%) - enhanced immunity is one of the important mechanisms for the reduction of diarrhea rate.

[0133] The above description illustrates the detailed preparation and application of the present invention through the above embodiments, but it is not limited to the detailed methods described above, and the scope of protection is not limited thereto. Any improvement of the present invention by those skilled in the art is within the scope of the present invention. Equivalent substitutions or changes made to the technical solutions and inventive concepts of the present invention, as well as the specific selection of various raw material substitutions and auxiliary ingredient additions in the products of the present invention, should all be covered within the scope of protection of the present invention.

Claims

1. A multi-strain enzyme synergistic segmented fermentation additive for Artemisia argyi used in pigs, characterized in that, It is produced by fermentation of fermentation substrate through three-stage synergistic fermentation of bacteria and enzymes; The fermentation base, by weight, comprises: 60-160 parts of delinted Artemisia argyi, 5-20 parts of Poria cocos, 3-16 parts of jujubes, 6-24 parts of hawthorn, and 2-12 parts of dandelion. The multi-strain enzyme synergistic segmented fermentation Artemisia argyi pig additive is prepared by fermentation of fermentation base material through a fermentation bacteria and enzyme synergistic system composed of compound probiotics and compound enzymes. The compound probiotics include Bacillus coagulans, Lactobacillus plantarum, Clostridium butyricum, Saccharomyces cerevisiae, and Aspergillus niger; the compound enzymes include cellulase and hemicellulase. The fermentation process includes three stages: S1 aerobic liquid fermentation, S2 anaerobic liquid fermentation, and S3 solid microbial enrichment. During the fermentation process, a fermentation inducer composed of licorice flavonoids and malt extract powder is added.

2. The multi-enzyme synergistic segmented fermentation additive for Artemisia argyi for pigs according to claim 1, characterized in that, The activity indicators are: total flavonoids ≥215mg / g, chlorogenic acid ≥74μg / g, total polysaccharides ≥43mg / g, crude fiber reduced to below 21.48%, and pH stable at 4.4-4.

5.

3. The multi-enzyme synergistic segmented fermentation additive for Artemisia argyi for pigs according to claim 1, characterized in that, The mass ratio of each bacterium in the compound probiotic is as follows: Bacillus coagulans: Saccharomyces cerevisiae = 1~2:1 (preferably 2:1); Lactobacillus plantarum: Clostridium butyricum = 1~2:1 (preferably 1.5:1); Aspergillus niger is used alone for solid-state fermentation; the mass ratio of cellulase and hemicellulase in the compound enzyme is 1~2:

1.

4. The multi-enzyme synergistic segmented fermentation additive for Artemisia argyi for pigs according to claim 1, characterized in that, During the fermentation process, the added fermentation inducer is a compound of glycyrrhizin flavonoids and malt extract powder in a mass ratio of 1:1 to 4.

5. The preparation process of the multi-enzyme synergistic segmented fermentation Artemisia argyi pig additive according to claim 1, characterized in that, Includes the following steps: S1. Aerobic liquid fermentation: The fermentation substrate I is crushed and passed through an 80-100 mesh sieve. The obtained powder is extracted with water to prepare suspension I. Then, Bacillus coagulans and Saccharomyces cerevisiae are inoculated, and a fermentation inducer is added. Aerobic fermentation is carried out for 18-24 hours under stirring conditions at 200-400 rpm. After centrifugation, aerobic fermentation broth and aerobic residue are obtained. S2. Anaerobic liquid fermentation: The fermentation substrate II is crushed and passed through a 50-mesh sieve to obtain fine powder. It is then extracted with water to prepare suspension II. A compound enzyme is added. Then, a mixture of anaerobic bacteria consisting of Lactobacillus plantarum and Clostridium butyricum is inoculated. The mixture is sealed and anaerobic fermented for 60-70 hours. After centrifugation, the anaerobic fermentation liquid and anaerobic residue are obtained. S3. Solid microbial enrichment: Mix all the residues from steps S1 and S2, add grain flour and bran to make a solid substrate; then inoculate with Aspergillus niger, ferment aerobically for 20-30 h, dry at 70-80℃ to constant weight, pulverize, and sieve to obtain functional microbial material. S4. Finished product compounding: Mix the aerobic fermentation liquid from step S1 and the anaerobic fermentation liquid from step S2, add functional bacteria, stir at high speed, dry at low temperature, and pulverize to obtain fermented mugwort pig additive.

6. The preparation process according to claim 5, characterized in that, In step S1, the fermentation substrate I includes de-fleshed Artemisia argyi, Poria cocos, hawthorn, jujube, and dandelion; the components and weight parts of fermentation substrate I are as follows: de-fleshed Artemisia argyi 60-80 parts, Poria cocos 5-10 parts, jujube 3-8 parts, hawthorn 6-12 parts, and dandelion 2-6 parts; In step S1, during extraction, the powder-to-water ratio is 1:4 to 1:7; In step S1, the extraction conditions are: extraction at 60-80°C for 3-5 hours, followed by cooling to 30-40°C; In step S1, the inoculum size of Bacillus coagulans is 1×10⁻⁶. 7 ~2.5×10 7 CFU / mL, Saccharomyces cerevisiae inoculum size was 1.5 × 10⁻⁶. 7 ~2.5×10 7 CFU / mL; In step S1, the fermentation inducer is a compound of glycyrrhizin and malt extract powder, with a mass ratio of 1:1 to 4. The amount of fermentation inducer added is 3% to 5% of the total volume of suspension I in step S1.

7. The preparation process according to claim 5, characterized in that, In step S2, fermentation substrate II includes Artemisia argyi, hawthorn, and dandelion; the components and weight parts of fermentation substrate II are as follows: 60-80 parts of de-furized Artemisia argyi, 5-10 parts of Poria cocos, 3-8 parts of jujube, 6-12 parts of hawthorn, and 2-6 parts of dandelion; In step S2, during extraction, the powder-to-water ratio is 1:4 to 1:5; In step S2, the extraction conditions are as follows: extraction at 85-95°C for 1-2 hours, followed by cooling to 30-40°C; In step S2, the amount of the compound enzyme added is 0.2-0.8% of the mass of suspension II; the mass ratio of cellulase to hemicellulase in the compound enzyme is 1-2:1 (preferably 1:1). In step S2, the mixed anaerobic bacteria consisting of *Lactobacillus plantarum* and *Clostridium butyricum* are used, with a mass ratio of 1 to 2:1 (preferably 1.5:1), and the inoculum size is 3 × 10⁻⁶. 7 ~4×10 7 CFU / mL.

8. The preparation process according to claim 5, characterized in that, In step S3, the grain flour in the solid matrix accounts for 20%–30% of the total weight of the residue, and the bran accounts for 5%–10% of the total weight of the residue; the inoculum size of Aspergillus niger is 5 × 10⁻⁶. 7 ~7×10 7 CFU / g.

9. The application of the multi-enzyme synergistic segmented fermentation Artemisia argyi pig additive according to claim 1 in livestock breeding, feed preparation or veterinary drug preparation.

10. The application of the multi-enzyme synergistic segmented fermentation Artemisia argyi pig additive according to claim 1 in pig farming, characterized in that, When applying, the addition rate in the diet is 1% to 3% (by weight); the preferred addition rate for weaned piglets is 1% (by weight), for growing piglets it is 1.5% (by weight), for fattening pigs it is 2% (by weight), and for breeding sows it is 2% to 3% (by weight).

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