Cortex magnoliae officinalis fermented feed for feeding cold water fish as well as preparation method and application of cortex magnoliae officinalis fermented feed

Abstract

The invention discloses a mangnolia officinalis fermented feed for feeding cold water fish and a preparation method and application thereof, and the mangnolia officinalis fermented feed comprises the following components in percentage by weight of sturgeon feed: 50-65% of mangnolia officinalis, 10-15% of astragalus membranaceus, 15-10% of scutellaria baicalensis, 10-5% of cortex moutan, 5-10% of rhizoma atractylodis and 30-38% of bacterial liquid. Washing the mangnolia officinalis, the astragalus membranaceus, the scutellaria baicalensis, the cortex moutan and the rhizoma atractylodis, air-drying, mixing and grinding into mixed powder which can pass through a 200-mesh sieve; adding compound probiotics into distilled water, activating, and mixing with the compound enzyme preparation to obtain bacterial liquid; and putting the mixed powder, the bacterial liquid and the sturgeon feed into a biological fermentation bag, and fermenting to obtain the magnolia officinalis fermented feed. The mangnolia officinalis fermented feed disclosed by the invention has a prevention and treatment effect on the sturgeon bloating disease, and can improve the intestinal tract and the digestive ability thereof and enhance the immunity of the organism.

Description

Technical Field

[0001] The present invention belongs to the technical field of fermented feed, and more specifically, the present invention relates to a Magnolia officinalis fermented feed for feeding cold-water fish, a preparation method thereof, and an application thereof. Background Art

[0002] Magnolia officinalis is the dried dry bark, root bark and branch bark of Magnoliaceae plants, mainly containing magnolol and honokiol, alkaloids, and phenylethanoid glycoside components (see Luo HY, Wu HW, Yu XK, Zhang X, Lu YQ, Fan JW, Tang LY, Wang ZJ, 2019. A review of the phytochemistry and pharmacological activities of magnoliae officinalis cortex. Journal of Ethnopharmacology and Xue ZZ, Yan RY, Yang B, 2016. Phenylethanoid glycosides and phenolic glycosides from stem bark of Magnolia officinalis. Phytochemistry), and is used for symptoms such as dampness stagnating in the middle energizer, epigastric fullness and vomiting and diarrhea, food accumulation and qi stagnation, abdominal distension and constipation, etc., and is a commonly used traditional Chinese medicine. Fermenting traditional Chinese medicine is a new technology formed by combining modern biotechnology on the basis of inheriting the fermentation method of traditional Chinese medicine processing science. At present, fermented traditional Chinese medicine has been widely used in the preparation of traditional Chinese medicine to enhance efficacy and reduce toxicity (see Ai Su, Tang Wei, Guo Ruolin, etc. Research progress on microbial fermentation of Chinese herbal medicines and their active substances [J]. China Journal of Chinese Materia Medica). Solid fermentation of traditional Chinese medicine is usually a method of using traditional Chinese medicine or traditional Chinese medicine residues as a medicinal matrix to replace the traditional fermentation nutrient matrix, using the nutrient components in traditional Chinese medicine as a carbon source and a nitrogen source, and fermenting with one or more probiotics (see Chen Keyuan, Gao Yan, Hao Baocheng, etc. Research progress on probiotic fermentation of traditional Chinese medicine [J]. Progress in Veterinary Medicine), and its advantages are that it does not require sterilization of the fermentation substrate, the operation process is relatively simple, and the fermentation cost is relatively low (see CANO Y POSTIGO L O, D A, Guajardo-Flores D, et al. Solid-state fermentation for enhancing the nutraceutical content of agrifood by-products: recent advances and its industrial feasibility[J]. Food Bioscience). Introducing probiotics during the fermentation process can further promote the transformation and release of traditional Chinese medicine components, improve the medicinal efficacy, and produce beneficial metabolites to help alleviate toxic and side effects (see Jia Yanshu. Research on the fermentation process of Astragalus membranaceus with Lactobacillus[D]. Harbin: Heilongjiang University of Chinese Medicine; Qu Qingsong, Li Zhixun, Zhou Qing, et al. Research progress of fermented traditional Chinese medicine and exploration of its "fermentation compatibility" theory[J]. Chinese Herbal Medicines and Bai Zefang, Wang Xuemeng. Research on the mechanism of action of microorganisms in the processing of traditional Chinese medicine[J]. Chemical Engineering Design Communications). At the same time, during the fermentation process, high-activity enzyme preparations have significant effects on improving the nutritional value of raw materials, the vitality of probiotic populations, promoting body growth and digestion, and enhancing immunity (Liu Qian, Luo Guoliang, Guo Li, et al. Optimization of the process for co-fermenting Chinese herbal medicine residues by enzymes and fungi[J]. China Feed). During the fermentation of medicinal materials such as Magnolia officinalis, the interaction between compound enzyme preparations and compound probiotics reduces the fiber and anti-nutritional factor content in the medicinal materials, improves palatability, reduces toxic and side effects, makes the active ingredients of the medicinal materials more easily absorbed and utilized by the body, and enhances the nutritional value (see Zhu Y, Xu Y, Yang Q. Antifungal properties and AFB1 detoxification activity of a new strain of Lactobacillus plantarum[J]. Journal of Hazardous Materials; Peng Xiaokun, Lü Jiangman, Deng Dun, Biological characteristics of Bacillus subtilis and its application progress in pig production[J]. Feed Research; Xue Rong, Gong Jingwen, Qu Lingyun, et al. Research progress and discussion on the processing of toxic traditional Chinese medicine pieces[J]. World Chinese Medicine). Research shows that Magnolia officinalis has antibacterial and anti-inflammatory effects. The volatile oil components in Magnolia officinalis have inhibitory effects on various bacteria and fungi, and can effectively reduce inflammatory reactions. In addition, Magnolia officinalis is widely used to treat gastrointestinal problems such as indigestion. According to research findings, Magnolia officinalis is rich in volatile oils and curcumin, which can promote gastric juice secretion, increase appetite and digestive juice secretion, thereby improving gastrointestinal function. The present invention relates to the positive effect of Magnolia officinalis fermented feed in treating the tympanites disease of sturgeon. Summary of the Invention

[0003] One object of the present invention is to solve at least the above problems and / or deficiencies and to provide at least the advantages described hereinafter.

[0004] To achieve these objects and other advantages in accordance with the present invention, there is provided a Magnolia officinalis fermented feed for feeding cold-water fish, which, by weight percentage of the sturgeon feed, includes: 50-65% of Magnolia officinalis, 10-15% of Astragalus membranaceus, 15-10% of Scutellaria baicalensis, 5-10% of Paeonia suffruticosa, 5-10% of Atractylodes lancea and 30-38% of bacterial liquid.

[0005] A preparation method of a Magnolia officinalis fermented feed for feeding cold-water fish is characterized by comprising the following steps:

[0006] Step 1: Wash, air-dry Magnolia officinalis, Astragalus membranaceus, Scutellaria baicalensis, Paeonia suffruticosa and Atractylodes lancea and then mix and grind them into a mixed powder passing through a 200-mesh sieve;

[0007] Step 2: Add compound probiotics to distilled water, activate them and then mix with a compound enzyme preparation to obtain a bacterial liquid;

[0008] Step 3: Load the mixed powder, the bacterial liquid and the sturgeon feed into a biological fermentation bag together and ferment to obtain the Magnolia officinalis fermented feed.

[0009] Preferably, in the step 2, the activation method of the compound probiotics includes: activating in a shaking incubator at 25-35°C and 120-160 r / min for 12-24 h.

[0010] Preferably, in the step 2, the mass ratio of the probiotics to the compound enzyme preparation is 1:1.

[0011] Preferably, in the step 2, the compound probiotics are one or more of Lactobacillus plantarum, Bacillus subtilis, Saccharomyces cerevisiae or Bacillus licheniformis, wherein the viable count of Lactobacillus plantarum ≥ 1.0×10 9 CFU / g, the viable count of Bacillus subtilis ≥ 1.5×10 9 CFU / g, the viable count of Saccharomyces cerevisiae ≥ 2.0×10 9 CFU / g, the viable count of Bacillus licheniformis ≥ 1.5×10 9 CFU / g; when the compound probiotics include Lactobacillus plantarum, Bacillus subtilis, Saccharomyces cerevisiae and Bacillus licheniformis, the mass ratio of Lactobacillus plantarum, Bacillus subtilis, Saccharomyces cerevisiae and Bacillus licheniformis is 2-2.5:2.5-3:2-3:2-3.

[0012] Preferably, in the second step, the complex enzyme preparation is one or more of cellulase, xylanase, β-glucanase, β-mannanase or protease. Among them, the enzyme activity of cellulase is ≥1000 U / g, the enzyme activity of xylanase is ≥3000 U / g, the enzyme activity of β-glucanase is ≥500 U / g, the enzyme activity of β-mannanase is ≥500 U / g, and the enzyme activity of protease is ≥1000 U / g; when the complex enzyme preparation is a mixture of cellulase, xylanase, β-glucanase, β-mannanase and protease, the mass ratio of cellulase, xylanase, β-glucanase, β-mannanase and protease is 2-2.5:3-4:1-2:1-2:1-2.

[0013] Preferably, in the second step, the concentration of the bacterial liquid is 20-30 wt%, and the dosage of the bacterial liquid accounts for 30-38% of the weight of the sturgeon feed.

[0014] Preferably, in the third step, the fermentation time is 3-5 days.

[0015] Preferably, in the third step, the fermentation temperature is 30-40 °C.

[0016] Preferably, the preparation method of the complex enzyme preparation includes:

[0017] S1. Mix cellulase, xylanase, β-glucanase, β-mannanase and protease according to the mass ratio of 2-2.5:3-4:1-2:1-2:1-2 to obtain a complex enzyme mixture; among them, the enzyme activity of cellulase is ≥1000 U / g, the enzyme activity of xylanase is ≥3000 U / g, the enzyme activity of β-glucanase is ≥500 U / g, the enzyme activity of β-mannanase is ≥500 U / g, and the enzyme activity of protease is ≥1000 U / g;

[0018] S2. Use glucose as a carrier to dilute the complex enzyme mixture to obtain a complex enzyme diluted mixture, and the mass of glucose is 2-20 times the total mass of the complex enzyme mixture;

[0019] S3. Use chitosan-nano-silica composite stabilizer as an enzyme stabilizer, and mix the complex enzyme diluted mixture with the enzyme stabilizer to obtain a complex enzyme preparation; among them, the enzyme stabilizer is 0.5%-5.0% of the mass of the complex enzyme diluted mixture.

[0020] Among them, the preparation method of the chitosan-nano-silica composite stabilizer used in S3 includes:

[0021] S11. Dissolve chitosan in a 3-10 wt% acetic acid solution to obtain a chitosan solution, and the dosage ratio of chitosan to the acetic acid solution is 1 g:50-200 mL;

[0022] S12. Select nano-silica powder with a specific surface area of 220 - 650 m 2 / g and add it to the chitosan solution. After heating to 40 - 80 °C, add polyethylene glycol - 200, stir at a speed of 200 - 400 rpm, and then let it stand for 12 - 24 h to obtain a composite enzyme mixture; the dosage ratio of nano-silica powder, polyethylene glycol - 200, and chitosan is 0.2 - 1:1:0.2 - 0.5;

[0023] S13. Evaporate and concentrate the composite enzyme mixture at a temperature of 120 - 150 °C, wash and dry it to obtain chitosan - nano-silica composite stabilizer.

[0024] An application of the magnolia officinalis fermented feed for feeding cold - water fish as described in claim 1, wherein the magnolia officinalis fermented feed is used for feeding cold - water fish, and the cold - water fish includes sturgeon.

[0025] Preferably, the magnolia officinalis fermented feed is used to prevent and treat sturgeon flatulence disease, improve the intestinal flora of sturgeon, enhance the digestion ability of sturgeon, and strengthen the immunity of the sturgeon body.

[0026] The present invention has at least the following beneficial effects: Compared with ordinary feeds, the magnolia officinalis fermented feed provided by the present invention has higher nutritional value and better palatability. Under the combined action of probiotics and enzyme preparations, new active ingredients are generated during the fermentation process of the magnolia officinalis fermented feed, improving the medicinal effect and nutritional components. Compared with other drugs, the magnolia officinalis fermented feed is green, non - toxic, does not produce drug resistance, has no residue, and does not pollute the environment.

[0027] Meanwhile, magnolia officinalis and other medicinal materials are added to ordinary feeds as feed raw materials for fermentation, which not only expands the feed resources but also increases the resource utilization of magnolia officinalis and other medicinal materials. The operation method of the fermentation technology is simple, the cost is reduced, and using it in animal production can effectively improve economic benefits.

[0028] In the Magnolia officinalis fermented feed of the present invention, the compound enzyme preparation used takes cellulase, xylanase, β-glucanase, and β-mannanase as the main raw materials, and glucose is used as a carrier to dilute the mixture of cellulase, xylanase, β-glucanase, β-mannanase, and protease, and then a chitosan-nanosilica composite stabilizer is used as an enzyme stabilizer to prepare it; the results show that after the compound enzyme preparation prepared with the chitosan-nanosilica composite stabilizer as the enzyme stabilizer is added to the Magnolia officinalis fermented feed and fed to the sturgeon in the prevention group, the enzyme activities of amylase, lipase, and protease in the digestive tract of the sturgeon are significantly increased, further enhancing the digestive ability of the sturgeon intestine to substances and improving the immunity of the sturgeon body; among them, in the preparation process of the chitosan-nanosilica composite stabilizer, dilute acetic acid is used to dissolve chitosan, and then it is mixed and reacted with nanosilica powder with a large specific surface area and polyethylene glycol-200. Polyethylene glycol-200 is used to improve the biocompatibility of nanosilica powder and chitosan, and the large specific surface area characteristics of nanosilica powder are used to increase the loading amount of the compound enzyme. At the same time, the solubility of chitosan decreases in the weakly alkaline environment of the intestine, forming a gel network with nanosilica to achieve the gradient release of the compound enzyme, while protecting the compound enzyme, increasing the retention rate of the compound enzyme in intestinal juice, and the action mechanism of the compound enzyme (cellulase, xylanase, β-glucanase, β-mannanase, and protease) includes degrading the cell wall of the medicinal materials in the Magnolia officinalis fermented feed, releasing nutrients, and reducing the viscosity of chyme, thereby increasing the activities of endogenous enzymes (amylase, lipase, and protease) in the sturgeon intestine.

[0029] Other advantages, objectives, and features of the present invention will be partially reflected by the following description and partially understood by those skilled in the art through the research and practice of the present invention. Brief Description of the Drawings

[0030] Figure 1 Comparison of the behavior and anatomy of the sturgeon in the prevention group, control group, treatment group, and disease group fed with the Magnolia officinalis fermented feed prepared in Example 1;

[0031] Figure 2 Effect of the Magnolia officinalis fermented feed prepared in Example 1 on the survival rate of sturgeon;

[0032] Figure 3 Effect of the Magnolia officinalis fermented feed prepared in Example 1 on the gill filaments and intestine of sturgeon;

[0033] Figure 4 Effect of the Magnolia officinalis fermented feed prepared in Example 1 on the amylase activity of sturgeon;

[0034] Figure 5 Effect of the Magnolia officinalis fermented feed prepared in Example 1 on the lipase activity of sturgeon;

[0035] Figure 6 Effect of Magnolia officinalis fermented feed prepared in Example 1 on protease activity of sturgeon

[0036] Figure 7 Comparison chart of superoxide dismutase (SOD) of sturgeon in the prevention group, control group, treatment group and disease group fed with Magnolia officinalis fermented feed prepared in Example 1

[0037] Figure 8 Comparison chart of catalase (CAT) of sturgeon in the prevention group, control group, treatment group and disease group fed with Magnolia officinalis fermented feed prepared in Example 1

[0038] Figure 9 Comparison chart of glutathione peroxidase (GSH-PX) of sturgeon in the prevention group, control group, treatment group and disease group fed with Magnolia officinalis fermented feed prepared in Example 1

[0039] Figure 10 Comparison chart of malondialdehyde (MDA) of sturgeon in the prevention group, control group, treatment group and disease group fed with Magnolia officinalis fermented feed prepared in Example 1 Detailed implementation mode

[0040] The present invention will be further described in detail below with reference to the accompanying drawings, so that those skilled in the art can implement it according to the description in the specification.

[0041] It should be understood that the terms such as "having", "comprising" and "including" used herein do not exclude the presence or addition of one or more other elements or their combinations.

[0042] For sturgeon flatulence disease, its symptoms are mainly as follows: from the appearance, the body color of the sturgeon is normal, there are tiny bubbles attached to the body surface, the abdomen is swollen and slightly yellow in color (different from ascites disease); from the behavior, the sturgeon loses balance in the water, with the abdomen facing up and the back facing down, floating on the water surface in a backstroke state; from anatomical observation, it is found that the intestine is inflated, there is no or little content in the intestine, and the anus is red and swollen.

[0043] Example 1

[0044] A preparation method of Magnolia officinalis fermented feed, comprising the following steps:

[0045] Step 1: Wash, air-dry 55 kg of Magnolia officinalis, 15 kg of Astragalus membranaceus, 10 kg of Scutellaria baicalensis, 10 kg of Paeonia suffruticosa and 10 kg of Atractylodes lancea, and then mix and grind them into a mixed powder passing through a 200-mesh sieve.

[0046] Step 2: Add the compound probiotics to distilled water. After activation, mix them with the compound enzyme preparation to obtain a bacterial liquid with a concentration of 25%. The activation method is to mix Lactobacillus plantarum, Bacillus subtilis, Saccharomyces cerevisiae, and Bacillus licheniformis at 25°C and activate them in a shaking incubator at a rotation speed of 120 r / min for 12 h. Among them, the preparation method of the compound probiotic preparation is to mix 2.5 g of Lactobacillus plantarum (the viable count is 1.5×10 9 CFU / g), 2.5 g of Bacillus subtilis (the viable count is 1.5×10 9 CFU / g), 2.5 g of Saccharomyces cerevisiae (the viable count is 2.5×10 9 CFU / g), and 2.5 g of Bacillus licheniformis (the viable count is 2.0×10 9 CFU / g); the preparation method of the compound enzyme preparation includes: mixing 2 g of cellulase (enzyme activity is 2500 U / g), 4 g of xylanase (enzyme activity is 3500 U / g), 1 g of β-glucanase (enzyme activity is 2000 U / g), 1 g of β-mannanase (enzyme activity is 2500 U / g), and 2 g of protease (enzyme activity is 2000 U / g); the mass ratio of the compound probiotic preparation to the compound enzyme preparation is 1:1;

[0047] Step 3: Put 100 kg of the mixed powder, 30 kg of the bacterial liquid, and 100 kg of S5 sturgeon feed (produced by Shandong Shengsuo Feed Technology Co., Ltd.) into a biological fermentation bag and continuously ferment at 36°C for 3 days to obtain the magnolia officinalis fermented feed.

[0048] Example 2

[0049] A preparation method of magnolia officinalis fermented feed, comprising the following steps:

[0050] Step 1: Wash 60 kg of magnolia officinalis, 12 kg of astragalus membranaceus, 10 kg of scutellaria baicalensis, 8 kg of moutan cortex, and 10 kg of atractylodes lancea, air-dry them, and then mix and grind them into a mixed powder passing through a 200-mesh sieve;

[0051] Step 2: Add the compound probiotics to distilled water. After activation, mix them with the compound enzyme preparation to obtain a bacterial liquid with a concentration of 25%. The activation method is to mix Lactobacillus plantarum, Bacillus subtilis, and Saccharomyces cerevisiae at 25°C and activate them in a shaking incubator at a rotation speed of 120 r / min for 12 h. Among them, the preparation method of the compound probiotic preparation is to mix 2.5 g of Lactobacillus plantarum (the viable count is 1.5×10 9 CFU / g), 2.5 g of Bacillus subtilis (the viable count is 1.5×10 9 CFU / g), 2.5 g of Saccharomyces cerevisiae (the viable count is 2.5×10 9 CFU / g), and 2.5 g of Bacillus licheniformis (the viable count is 2.0×10 9It is obtained by mixing; the preparation method of the compound enzyme preparation includes: mixing 2 g of cellulase (enzyme activity of 2500 U / g), 4 g of xylanase (enzyme activity of 3500 U / g), 1 g of β-glucanase (enzyme activity of 2000 U / g), 1 g of β-mannanase (enzyme activity of 2500 U / g) and 2 g of protease (enzyme activity of 2000 U / g); the mass ratio of the compound probiotic preparation to the compound enzyme preparation is 1:1;

[0052] Step 3: Put 100 kg of the mixed powder, 35 kg of the bacterial liquid and 100 kg of S5 sturgeon feed into a biological fermentation bag, and continuously ferment at 36 °C for 4 days to obtain the magnolia officinalis fermented feed.

[0053] Example 3

[0054] A preparation method of magnolia officinalis fermented feed includes the following steps:

[0055] Step 1: Wash 65 kg of magnolia officinalis, 10 kg of astragalus membranaceus, 8 kg of scutellaria baicalensis, 11 kg of moutan cortex and 6 kg of atractylodes lancea, air-dry them and then mix and grind them into a mixed powder passing through a 200-mesh sieve;

[0056] Step 2: Add the compound probiotic to distilled water, activate it and then mix it with the compound enzyme preparation to obtain a bacterial liquid with a concentration of 25%. The activation method is to activate Lactobacillus plantarum, Bacillus subtilis and Saccharomyces cerevisiae in a shaking incubator at 25 °C and a rotation speed of 120 r / min for 12 h; among them, the preparation method of the compound probiotic preparation is to mix 2.5 g of Lactobacillus plantarum (live bacteria count of 1.5×10 9 CFU / g), 2.5 g of Bacillus subtilis (live bacteria count of 1.5×10 9 CFU / g), 2.5 g of Saccharomyces cerevisiae (live bacteria count of 2.5×10 9 CFU / g) and 2.5 g of Bacillus licheniformis (live bacteria count of 2.0×10 9 CFU / g); the preparation method of the compound enzyme preparation includes: mixing 2 g of cellulase (enzyme activity of 2500 U / g), 4 g of xylanase (enzyme activity of 3500 U / g), 1 g of β-glucanase (enzyme activity of 2000 U / g), 1 g of β-mannanase (enzyme activity of 2500 U / g) and 2 g of protease (enzyme activity of 2000 U / g); the mass ratio of the compound probiotic preparation to the compound enzyme preparation is 1:1;

[0057] Step 3: Put 100 kg of the mixed powder, 38 kg of the bacterial liquid and 100 kg of S5 sturgeon feed into a biological fermentation bag, and continuously ferment at 36 °C for 5 days to obtain the magnolia officinalis fermented feed.

[0058] Example 4

[0059] In the preparation method of a Magnolia officinalis fermented feed in this embodiment, different from Embodiment 1, the preparation method of the compound enzyme preparation used in Step 2 of this embodiment includes:

[0060] S1. Mix 2 g of cellulase, 4 g of xylanase, 1 g of β-glucanase, 1 g of β-mannanase and 2 g of protease to obtain a compound enzyme mixture; among them, the enzyme activity of cellulase is 2500 U / g, the enzyme activity of xylanase is 3500 U / g, the enzyme activity of β-glucanase is 2000 U / g, the enzyme activity of β-mannanase is 2500 U / g, and the enzyme activity of protease is 2000 U / g;

[0061] S2. Dilute the compound enzyme mixture with 20 g of glucose as a carrier to obtain a compound enzyme diluted mixture;

[0062] S3. Use 1 g of chitosan-nano-silica composite stabilizer as an enzyme stabilizer, and mix the compound enzyme diluted mixture with the enzyme stabilizer to obtain a compound enzyme preparation.

[0063] Among them, the preparation method of the chitosan-nano-silica composite stabilizer used in S3 includes:

[0064] S11. Dissolve 10 g of chitosan in 500 mL of 4 wt% acetic acid solution to obtain a chitosan solution;

[0065] S12. Select 5 g of nano-silica powder with a specific surface area of 300 - 500 m 2 / g and add it to the chitosan solution. After heating to 60 °C, add 2 g of polyethylene glycol-200, stir at 400 rpm, and then stand for 24 h to obtain a compound enzyme mixture solution;

[0066] S13. Evaporate and concentrate at 120 °C, wash and dry to obtain a chitosan-nano-silica composite stabilizer.

[0067] Then, in this embodiment, the mass ratio of the compound enzyme preparation to the compound probiotic preparation is 3.1:1, and the dosage of the compound probiotic preparation in this embodiment is the same as that in Embodiment 1. The methods and process parameters of the remaining steps in this embodiment are the same as those in Embodiment 1.

[0068] Embodiment 5

[0069] In the preparation method of a Magnolia officinalis fermented feed in this embodiment, different from Embodiment 1, the preparation method of the compound enzyme preparation used in Step 2 of this embodiment includes:

[0070] S1. Mix 2 g of cellulase, 4 g of xylanase, 1 g of β-glucanase, 1 g of β-mannanase and 2 g of protease to obtain a composite enzyme mixture; among them, the enzyme activity of cellulase is 2500 U / g, the enzyme activity of xylanase is 3500 U / g, the enzyme activity of β-glucanase is 2000 U / g, the enzyme activity of β-mannanase is 2500 U / g, and the enzyme activity of protease is 2000 U / g;

[0071] S2. Dilute the composite enzyme mixture with 40 g of glucose as a carrier to obtain a diluted composite enzyme mixture;

[0072] S3. Use 2 g of chitosan-nano silica composite stabilizer as an enzyme stabilizer, and mix the diluted composite enzyme mixture with the enzyme stabilizer to obtain a composite enzyme preparation.

[0073] Among them, the preparation method of the chitosan-nano silica composite stabilizer used in S3 includes:

[0074] S11. Dissolve 10 g of chitosan in 500 mL of 3 wt% acetic acid solution to obtain a chitosan solution;

[0075] S12. Select 5 g of nano silica powder with a specific surface area of 300 - 600 m 2 / g and add it to the chitosan solution. After heating to 80 °C, add 4 g of polyethylene glycol-200, stir at 400 rpm, and then let it stand for 24 h to obtain a composite enzyme mixture;

[0076] S13. Evaporate and concentrate at 130 °C, wash and dry to obtain the chitosan-nano silica composite stabilizer.

[0077] Then, in this example, the mass ratio of the composite enzyme preparation to the composite probiotic preparation is 5.2:1, and the dosage of the composite probiotic preparation in this example is the same as that in Example 1. The methods and process parameters of the remaining steps in this example are the same as those in Example 4.

[0078] Comparative Example 1

[0079] In the preparation method of a Magnolia officinalis fermented feed in this comparative example, different from Example 4, the preparation method of the composite enzyme preparation used in step two of this comparative example includes:

[0080] S1. Mix 10 g of cellulase, 10 g of xylanase, 10 g of β-glucanase and 10 g of β-mannanase to obtain a composite enzyme mixture; among them, the enzyme activity of cellulase is 2500 U / g, the enzyme activity of xylanase is 2000 U / g, the enzyme activity of β-glucanase is 3000 U / g, and the enzyme activity of β-mannanase is 3500 U / g;

[0081] S2. Dilute the composite enzyme mixture with 80 g of glucose as a carrier to obtain a diluted composite enzyme mixture;

[0082] S3. Use 3 g of chitosan as an enzyme stabilizer and mix the diluted composite enzyme mixture with the enzyme stabilizer to obtain a composite enzyme preparation.

[0083] In this example, the mass ratio of the composite enzyme preparation to the composite probiotic preparation is 3.1:1, and the dosage of the composite probiotic preparation in this example is the same as that in Example 1. The methods and process parameters of the remaining steps in this example are the same as those in Example 4.

[0084] Comparative Example 2

[0085] In the preparation method of a Magnolia officinalis fermented feed in this comparative example, different from Example 4, the preparation method of the composite enzyme preparation used in the second step of this comparative example includes:

[0086] S1. Mix 10 g of cellulase, 10 g of xylanase, 10 g of β-glucanase, and 10 g of β-mannanase to obtain a composite enzyme mixture; among them, the enzyme activity of cellulase is 2500 U / g, the enzyme activity of xylanase is 2000 U / g, the enzyme activity of β-glucanase is 3000 U / g, and the enzyme activity of β-mannanase is 3500 U / g;

[0087] S2. Dilute the composite enzyme mixture with 80 g of glucose as a carrier to obtain a diluted composite enzyme mixture;

[0088] S3. Use 3 g of nano-silica with a specific surface area in the range of 300 - 500 m 2 / g as an enzyme stabilizer and mix the diluted composite enzyme mixture with the enzyme stabilizer to obtain a composite enzyme preparation.

[0089] In this example, the mass ratio of the composite enzyme preparation to the composite probiotic preparation is 3.1:1, and the dosage of the composite probiotic preparation in this example is the same as that in Example 1. The methods and process parameters of the remaining steps in this example are the same as those in Example 4.

[0090] Comparative Example 3

[0091] In the preparation method of a Magnolia officinalis fermented feed in this comparative example, different from Example 1, the preparation method of the composite enzyme preparation used in the second step of this example includes:

[0092] S1. Mix 10 g of cellulase, 10 g of xylanase, 10 g of β-glucanase, and 10 g of β-mannanase to obtain a composite enzyme mixture; among them, the enzyme activity of cellulase is 2500 U / g, the enzyme activity of xylanase is 2000 U / g, the enzyme activity of β-glucanase is 3000 U / g, and the enzyme activity of β-mannanase is 3500 U / g;

[0093] S2. Dilute the complex enzyme mixture with 80 g of glucose as a carrier to obtain a diluted complex enzyme mixture;

[0094] S3. Use a mixture of 2 g of chitosan and 1 g of nano-silica as an enzyme stabilizer, and mix the diluted complex enzyme mixture with chitosan and nano-silica to obtain a complex enzyme preparation.

[0095] In this example, the mass ratio of the complex enzyme preparation to the complex probiotic preparation is 3.1:1, and the dosage of the complex probiotic preparation in this example is the same as that in Example 1. The methods and process parameters of the remaining steps in this example are the same as those in Example 4.

[0096] 1. Animal experiment

[0097] Healthy and diseased juvenile fish were obtained from the Beichuan Hybrid Sturgeon Seedling Farm in Mianyang City, Sichuan Province. These juvenile fish were 10.5 ± 1 cm in body length and 5 ± 1 g in body weight. The obtained juvenile hybrid sturgeon were acclimated to the new water temperature and water quality for at least half an hour and then raised in a tank at a water temperature of (15 ± 1) °C, pH (8.0 ± 0.5), and dissolved oxygen content of (8.5 ± 0.5 mg / L) for one month. A control group, a prevention group, a disease group, and a treatment group were set up. The control group and the disease group were fed with S5 ordinary sturgeon feed (produced by Shandong Shengsu Feed Technology Co., Ltd.), and the prevention group and the treatment group were fed with the S5 Houpo fermented feed prepared in Example 1. Each group was fed 4 times a day at 9, 13, 17, and 21 o'clock. Among them, the juvenile hybrid sturgeon in the disease group and the treatment group suffered from sturgeon flatulence disease, and the juvenile hybrid sturgeon in the control group and the prevention group were healthy juvenile fish.

[0098] 2. Behavioral analysis

[0099] On the 7th, 14th, 21st, and 28th days, the behavioral responses and changes of sturgeons in each group were observed, including activity, swimming posture, swimming distance, startle response, etc., for later behavioral analysis.

[0100] 3. Anatomical analysis

[0101] After the feeding period ended, the sturgeons in each group were externally observed and then anatomically observed, including observing and recording whether there were any abnormalities or lesions in the internal organs, such as whether the gills were congested and red, and whether the intestines were inflated and swollen, for later analysis of anatomical characteristics.

[0102] 4. Histological analysis

[0103] The tissue samples were preserved in 4% paraformaldehyde. After dehydration with anhydrous ethanol of different concentrations, they were made transparent in a mixed solution of ethanol and xylene at a ratio of 1:1, and then soaked in paraffin solution for 1 hour and embedded using an embedding machine at an embedding temperature of 60°C. After the wax blocks solidified, they were placed in a -20°C refrigerator and frozen overnight. After demolding, steps such as sectioning, dewaxing, staining, and coverslipping were carried out.

[0104] 5. Statistical analysis

[0105] SPSS 26 was used for significance analysis. The data were all expressed as mean ± SD, and Graphpad 9.0 was used for data visualization. One-way ANOVA was used for comparison among multiple groups.

[0106] Anatomical and behavioral characteristics

[0107] We analyzed the behavior of sturgeons fed with Magnolia officinalis fermented feed and ordinary feed. The sturgeons in the blank group and the prevention group had good vitality, swam normally, ate actively, and there were no obvious abnormalities in their swimming states. The sturgeons in the disease group and the treatment group were slow to respond, stagnated and suspended on the water surface, swam in a chaotic and unstable direction or did not swim, and their bellies were facing up in a backstroke state. The difference was that the recovered sturgeons in the treatment group could move normally in the water ( Figure 1 in A). The anatomical characteristics showed that the internal organs of the sturgeons in the control group and the prevention group were normal, there was no abnormal swelling in the abdomen, no small bubbles were attached to the body surface, and there were no injuries to the fins. Compared with the control group, the gills and lungs of the sturgeons in the prevention group were lighter in color, pink, and the gallbladder was more translucent, yellow-green. The abdomen of the sturgeons in the disease group was swollen, and the intestines were significantly inflated. The abdominal swelling and intestinal inflation in the treatment group were not obvious ( Figure 1 in B).

[0108] Survival rate

[0109] After feeding healthy sturgeons and diseased sturgeons for one month, there were obvious changes in the numbers of healthy sturgeons and diseased sturgeons. The numbers of diseased sturgeons (treatment group and disease group) decreased significantly, and the survival numbers of sturgeons fed with Magnolia officinalis fermented feed (treatment group) were significantly higher than those in the disease group ( Figure 2 ). The Magnolia officinalis fermented feed significantly reduced the mortality rate, increased the survival rate by 20%, and at the same time restored the sturgeons to a normal state. The Magnolia officinalis fermented feed has a positive therapeutic effect on the treatment of sturgeons with big belly disease.

[0110] Observation of gill and intestinal tissue structures

[0111] To further observe the effect of Magnolia officinalis fermented feed on the internal organs of diseased sturgeon, the gill filaments and intestines that are prone to attaching air bubbles were observed. It was found that the gill filaments of healthy sturgeon were intact, while those of diseased sturgeon were shed and the structure was incomplete. At the same time, compared with the control group, the gill filaments of the prevention group were longer and denser, while those of the disease group were severely shed. After feeding Magnolia officinalis fermented feed, the gill filament structure of the treatment group was significantly better than that of the disease group( Figure 3 in A). The intestinal villi structure of healthy sturgeon was intact without lesions, while those of diseased sturgeon were severely atrophied. After feeding Magnolia officinalis fermented feed, the structure of long villi was restored and there was no obvious difference from normal sturgeon. Magnolia officinalis fermented feed can alleviate the problem of intestinal aeration.

[0112] Detection of intestinal digestive enzyme activity

[0113] To understand the effect of Magnolia officinalis fermented feed on the digestive function in the intestine of sturgeon, the activities of intestinal digestive enzymes, including amylase, lipase and protease, were measured. Figures 4 - 6 The results showed that compared with the control group, the activity of protease increased significantly, and the activities of amylase and lipase increased first and then decreased, with the highest enzyme activity shown in the prevention group. At the same time, the activities of amylase and lipase in the treatment group and the disease group were significantly lower than those in the prevention group and significantly higher than those in the control group, indicating that Magnolia officinalis fermented feed can improve the activity of digestive enzymes and enhance the digestive ability of sturgeon intestine to substances.

[0114] At the same time, the contents of amylase, lipase and protease in the sturgeon of the prevention group fed with the Magnolia officinalis fermented feed of Examples 4 - 5 and Comparative Examples 1 - 3 were measured respectively, and the following table was obtained:

[0115]

[0116] As can be seen from the above table, in Examples 4 - 5, with chitosan - nano - silica composite stabilizer as the enzyme stabilizer, after the Magnolia officinalis fermented feed prepared from the obtained composite enzyme preparation was fed to the prevention group, the contents of amylase, lipase and protease in the sturgeon intestine were significantly increased compared with those of the sturgeon fed with the feed of Example 1, and were better than those of the Magnolia officinalis fermented feed of Comparative Examples 1 - 3.

[0117] Detection of antioxidant enzyme activity

[0118] Through the observation of the above results, Magnolia officinalis fermented Chinese herbal medicine has a certain positive effect on sturgeon. Therefore, the immune effect of sturgeon was detected through antioxidant indexes (SOD, CAT, MDA and GSH - PX). As Figures 7 - 10As shown, it was found that the content of MDA was significantly decreased, while the activities of SOD, CAT and GSH-PX were significantly increased compared with the control group. Among them, the preventive group had the highest enzyme activities of SOD, CAT and GSH-PX and the lowest content of MDA. Compared with the treatment group, the antioxidant enzyme activities in the diseased group decreased and the content of MDA increased. It is indicated that feeding fermented Magnolia officinalis feed to diseased sturgeons can improve the resistance of sturgeons themselves, and the preventive group has the best effect. In actual production, prevention is more important than treatment. Doing a good job in prevention before actual diseases occur can greatly reduce disease losses.

[0119] The equipment quantities and treatment scales described here are used to simplify the description of the present invention. Applications, modifications and variations of the present invention will be apparent to those skilled in the art.

[0120] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily achieved. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to the specific details and the illustrated examples described herein.

Claims

1. A fermented magnolia bark feed for feeding cold-water fish, characterized in that: According to the weight percentage of the sturgeon feed, the feed comprises: 50-65% of magnolia bark, 10-15% of astragalus, 15-10% of scutellaria, 5-10% of moutan bark, 5-10% of atractylodes and 30-38% of bacterial liquid.

2. A method for preparing a fermented magnolia bark feed for feeding cold-water fish according to claim 1, characterized in that: The following steps are involved: Step 1: Wash and air-dry Magnolia officinalis, Astragalus membranaceus, Scutellaria baicalensis, Cortex Moutan and Atractylodes lancea, and grind them into a mixed powder that passes through a 200-mesh sieve; Step 2: adding the composite probiotics into distilled water, activating the composite probiotics and mixing them with the composite enzyme preparation to obtain a bacterial solution; Step 3: Put the mixed powder, bacterial liquid and sturgeon feed into a biological fermentation bag together, and ferment to obtain Magnolia officinalis fermented feed.

3. The method for preparing the fermented magnolia bark feed for feeding cold-water fish as claimed in claim 2, characterized in that: In the step 2, the activation method of the composite probiotics includes: activating in an oscillating incubator at 25 to 35° C. and 120 to 160 r / min for 12 to 24 hours.

4. The method for preparing the fermented magnolia bark feed for feeding cold-water fish as claimed in claim 2, characterized in that: In the step 2, the mass ratio of the probiotics to the complex enzyme preparation is 1:

1.

5. The method for preparing the fermented magnolia bark feed for feeding cold-water fish according to claim 2, characterized in that: In step 2, the composite probiotics are one or more of Lactobacillus plantarum, Bacillus subtilis, Saccharomyces cerevisiae or Bacillus licheniformis, wherein the number of viable Lactobacillus plantarum is ≥ 1.0 × 10 9 CFU / g, viable Bacillus subtilis count ≥ 1.5×10 9 CFU / g, the number of live bacteria of Saccharomyces cerevisiae ≥ 2.0×10 9 CFU / g, viable count of Bacillus licheniformis ≥1.5×10 9 CFU / g; when the composite probiotics include Lactobacillus plantarum, Bacillus subtilis, Saccharomyces cerevisiae and Bacillus licheniformis, the mass ratio of Lactobacillus plantarum, Bacillus subtilis, Saccharomyces cerevisiae and Bacillus licheniformis is 2-2.5:2.5-3:2-3:2-3.

6. The method for preparing the fermented magnolia bark feed for feeding cold-water fish according to claim 2, characterized in that: In the step 2, the complex enzyme preparation is one or more of cellulase, xylanase, β-glucanase, β-mannanase or protease, wherein the enzyme activity of cellulase is ≥1000U / g, the enzyme activity of xylanase is ≥3000U / g, the enzyme activity of β-glucanase is ≥500U / g, the enzyme activity of β-mannanase is ≥500U / g, and the enzyme activity of protease is ≥1000U / g; when the complex enzyme preparation is a mixture of cellulase, xylanase, β-glucanase, β-mannanase and protease, the mass ratio of cellulase, xylanase, β-glucanase, β-mannanase and protease is 2-2.5:3-4:1-2:1-2:1-2.

7. The method for preparing the fermented magnolia bark feed for feeding cold-water fish according to claim 2, characterized in that: In the step 2, the concentration of the bacterial solution is 20-30wt%, and the amount of the bacterial solution accounts for 30-38% of the weight of the sturgeon feed.

8. The method for preparing the fermented magnolia bark feed for feeding cold-water fish according to claim 2, characterized in that: In the step 3, the fermentation time is 3 to 5 days, and the fermentation temperature is 30 to 40°C.

9. The method for preparing the fermented magnolia bark feed for feeding cold-water fish according to claim 6, characterized in that: The preparation method of the composite enzyme preparation comprises: S1, mixing cellulase, xylanase, β-glucanase, β-mannanase and protease in a mass ratio of 2-2.5:3-4:1-2:1-2:1-2 to obtain a composite enzyme mixture; S2, using glucose as a carrier, diluting the complex enzyme mixture to obtain a complex enzyme diluted mixture, wherein the mass of glucose is 2 to 20 times the total mass of the complex enzyme mixture; S3. Using chitosan-nano-silicon dioxide composite stabilizer as enzyme stabilizer, mixing the composite enzyme dilution mixture with the enzyme stabilizer to obtain a composite enzyme preparation; wherein the enzyme stabilizer accounts for 0.5% to 5.0% of the mass of the composite enzyme dilution mixture.

10. A use of the fermented magnolia bark feed for feeding cold-water fish as claimed in claim 1, characterized in that: The magnolia bark fermented feed is used for feeding cold water fish, and the cold water fish includes sturgeon.

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