Preparation method of traditional Chinese medicine residue metabiotics and application of traditional Chinese medicine residue metabiotics in fish feed

Abstract

The invention relates to a preparation method of traditional Chinese medicine residue metabiotics and application of the traditional Chinese medicine residue metabiotics in fish feed, and belongs to the technical field of feed additives. Beneficial bacteria such as bacillus subtilis, lactobacillus lactis, bifidobacterium longum and saccharomyces cerevisiae are separated from intestinal contents of grass carp and are subjected to enlarged culture, then traditional Chinese medicine residues such as fresh ginger, folium mori, ginseng, fructus lycii, rhizoma curcumae longae, liquorice and mint are added, staged mixed fermentation culture is performed, obtained metabolites are added into fish feed, and the fish feed is obtained. The feeding rate and the growth rate of fishes such as mandarin fish and micropterus salmoides are effectively improved, and the resistance is enhanced.

Description

Technical Field

[0001] The present invention belongs to the technical field of feed additives, and relates to a preparation method of postbiotics from Chinese medicine residues and its application in fish feed. Background Art

[0002] With the rapid development of the global fish farming industry, problems such as environmental pollution, disease prevention and control, and water quality management in the farming process have become difficult problems that the industry urgently needs to solve. The increase in aquaculture density and water quality deterioration are likely to lead to a decline in fish immunity and an increase in the incidence of diseases, especially the spread of infectious diseases such as bacterial and viral infections. In addition, in the process of fish farming, the feed utilization rate and water quality management directly affect the farming efficiency and the health of fish. Therefore, how to effectively improve the immune ability of fish, reduce the occurrence of diseases, optimize the feed utilization rate, and improve the water quality has become the focus of current aquaculture technology research.

[0003] As a biological agent with broad application prospects, probiotics have been widely used in aquaculture. By regulating the intestinal microbial community, probiotics can promote fish digestion and absorption, inhibit the growth of harmful pathogenic bacteria, and enhance the immune ability of fish, thus effectively preventing and controlling diseases of aquatic animals. At the same time, probiotics also have the function of improving water quality and reducing the accumulation of harmful substances such as ammonia nitrogen, and have a positive regulatory effect on the aquaculture environment.

[0004] Due to the single source of probiotics in feed and their poor survival and proliferation ability in complex water environments, the actual application effect is not ideal. In addition, with the increasing application of Chinese medicine in aquaculture, the resource utilization of Chinese medicine residues, as a by-product, has also become a research hotspot. Chinese medicine residues contain rich natural active ingredients, which can not only be used as good feed additives, but also provide more nutrients and beneficial microorganisms for fish through the fermentation process. Therefore, the preparation of fish feed using postbiotics from Chinese medicine residues can not only utilize the resources of Chinese medicine residues, but also effectively increase the types and quantities of probiotics in the feed, enhance their ability to survive and reproduce in water, and provide a healthier growth environment and higher-quality feed source for fish.

[0005] The natural plant components in Chinese medicine residues can not only provide a good growth matrix for probiotics, but also provide additional nutritional support for fish. The postbiotics obtained by this method have good stability and stress resistance, and can survive and play a role in complex water environments for a long time. Summary of the Invention

[0006] The present invention relates to a preparation method of postbiotics from Chinese herbal medicine residues and its application in fish feed, belonging to the technical field of feed additives. In the present invention, beneficial bacteria are isolated from the intestinal contents of grass carp: Bacillus subtilis, Lactobacillus lactis, Bifidobacterium longum, Saccharomyces cerevisiae and Lactobacillus plantarum, and they are expanded and cultured,

[0007] then Chinese herbal medicine residues are added: ginger, mulberry leaves, ginseng, wolfberry fruits, turmeric, licorice and mint, and they are subjected to staged mixed fermentation culture. The obtained metabolites are added to fish feed, effectively improving the feeding rate and growth rate of fish such as mandarin fish and Micropterus salmonides, and enhancing the resistance.

[0008] The object of the present invention can be achieved by the following technical solutions:

[0009] A preparation method of postbiotics from Chinese herbal medicine residues, comprising the following steps:

[0010] (1) Collect the contents from the intestine of grass carp and isolate probiotics: Bacillus subtilis, Lactobacillus lactis, Bifidobacterium longum and Saccharomyces cerevisiae;

[0011] (2) Expand and culture the probiotics separately;

[0012] (3) Stir glucose, yeast extract, tryptone, KH2PO4, K2HPO4, vitamin B group and biotin with deionized water to make a uniform basic medium, add Chinese herbal medicine residues, and adjust the pH to 5.5 - 6.5 with 1M hydrochloric acid solution to make a mixed medium;

[0013] (4) Add the mixed medium to a fermenter, sterilize at 121 °C for 20 min, then cool down to 30 °C, add the Bacillus subtilis fermentation broth, maintain the temperature at 30 °C and the pH value at 7, and ferment for 20 - 30 h;

[0014] (5) Add the Lactobacillus lactis fermentation broth and the Saccharomyces cerevisiae fermentation broth, adjust the temperature to 32 °C, and keep the pH value at 6.0 - 6.5, and continue to ferment for 20 - 30 h;

[0015] (6) Add the Bifidobacterium longum fermentation broth to adjust the temperature to 35 °C and keep the pH value at 6.0, and continue to ferment for 24 - 48 hours to obtain a mixed fermentation broth;

[0016] (7) Filter the mixed fermentation broth through a filter membrane with a pore size of 0.45 μm to remove the solid residues in the fermentation broth, and then perform concentration and freeze-drying.

[0017] As a preferred technical solution of the present invention, the isolation of probiotics in step (1) is as follows: Bacillus subtilis is separated using a Bacillus subtilis selective medium, Lactobacillus lactis is separated using a Lactobacillus lactis selective medium, Bifidobacterium longum is separated using a Bifidobacterium longum selective medium, and Saccharomyces cerevisiae is separated using a yeast growth medium.

[0018] As a preferred technical solution of the present invention, the conditions for the separate scale-up culture in step (2) are as follows: For Bacillus subtilis, use a Bacillus subtilis medium, control the pH value at 7.0, the temperature at 30°C, and scale up the culture until the OD of the Bacillus subtilis fermentation broth is 1.2 - 1.8; for Lactobacillus lactis, use a Lactobacillus lactis medium, control the pH at 5.5 - 6.0, the temperature at 37°C, and scale up the culture until the OD of the Lactobacillus lactis fermentation broth is 1.2 - 1.8; for Bifidobacterium longum, use a Bifidobacterium longum medium, control the pH value at 6.0 - 6.5, the temperature at 37°C, and scale up the culture until the OD of the Bifidobacterium longum fermentation broth is 1.0 - 1.2; for Saccharomyces cerevisiae, use a glucose-yeast extract medium, control the pH at 5.5, the temperature at 30°C, and scale up the culture until the OD of the Saccharomyces cerevisiae fermentation broth is 2.5 - 3.5.

[0019] As a preferred technical solution of the present invention, the Chinese herbal medicine residues in step (3) include: ginger, mulberry leaves, plantain seeds, ginseng, wolfberries, turmeric, licorice, mint, and houttuynia cordata; the mass ratio of ginger, mulberry leaves, plantain seeds, ginseng, wolfberries, turmeric, licorice, mint, and houttuynia cordata is 1:0.8 - 1.5:0.8 - 1.2:0.4 - 0.8:0.4 - 0.8:0.1 - 0.3:0.1 - 0.3:0.1 - 0.3:0.1 - 0.3.

[0020] As a preferred technical solution of the present invention, in the mixed medium of step (3), the addition amount of glucose is 10 - 20 g / L, the addition amount of yeast extract is 5 - 10 g / L, the addition amount of tryptone is 5 - 10 g / L, the addition amount of KH2PO4 is 1 - 2 g / L, the addition amount of K2HPO4 is 1 - 2 g / L, the addition amount of vitamin B group is 50 - 200 mg / L, the addition amount of biotin is 10 - 50 mg / L, and the addition amount of Chinese herbal medicine residues is 2.3 - 4.8 g / L.

[0021] As a preferred technical solution of the present invention, the mass ratio of the Bacillus subtilis fermentation broth to the mixed medium in step (4) is 1:10 - 20.

[0022] As a preferred technical solution of the present invention, the mass ratio of the Bacillus subtilis fermentation broth, Lactobacillus lactis fermentation broth, Bifidobacterium longum fermentation broth, and Saccharomyces cerevisiae fermentation broth in steps (4), (5), and (6) is 1 - 3:2 - 4:1 - 3:2 - 4.

[0023] Furthermore, the postbiotics of the Chinese herbal medicine residues are applied to fish feed.

[0024] The beneficial effects of the present invention:

[0025] (1) The present invention separates Bacillus subtilis, Lactobacillus lactis, Bifidobacterium longum, and Saccharomyces cerevisiae from fish gut contents for fermenting Chinese herbal medicine residues and then uses them for fish feeding, which has significant advantages. First, Bacillus subtilis and Saccharomyces cerevisiae break down complex proteins into small peptides and amino acids, while Saccharomyces cerevisiae produces B vitamins and other growth factors through fermentation, enhancing protein digestion and vitamin content. Lactobacillus lactis and Bifidobacterium longum produce lactic acid and other organic acids during fermentation, improving feed palatability and mineral absorption. Fermentation technology reduces anti-nutritional factors, improves feed utilization rate, and enhances fish growth efficiency.

[0026] (2) Ginger, turmeric, mulberry leaves, and wolfberries are rich in antioxidants and anti-inflammatory substances, which can neutralize free radicals, reduce oxidative stress, and protect the health of fish cells; plantain seeds and houttuynia cordata improve fish immunity, disease resistance, and anti-inflammation; ginseng and licorice provide saponins and flavonoids that can enhance the immune function of fish and improve their resistance to pathogens and environmental stress; ginger and mint increase the feeding rate and digestion efficiency of fish by stimulating the secretion of digestive juices and improving palatability. The present invention provides a wide range of vitamins and trace elements for fish by fermenting these Chinese herbal medicine residues with beneficial bacteria, supporting the comprehensive nutritional needs of fish and improving their growth rate and health level.

[0027] (3) The present invention can precisely control the fermentation conditions at different stages through staged mixed fermentation, optimize the interaction between beneficial bacteria and Chinese herbal medicine residues, and maximize the diversity and functionality of metabolites. Staged fermentation helps to establish a stable microbial community, ensuring that each strain grows and functions under its most suitable conditions and avoiding excessive competition among single strains. Detailed implementation mode

[0028] To further elaborate on the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the following, in combination with embodiments, details the specific implementation mode, structure, features, and effects according to the present invention as follows.

[0029] In the following examples and comparative examples, the yeast extract was purchased from Shandong Pingju Biotechnology Co., Ltd., the tryptone was purchased from Shandong Xinzhuoyuan Biotechnology Co., Ltd., the vitamin B complex was purchased from Fande Biotechnology Co., Ltd., the biotin was purchased from Anhui Weimao Biotechnology Co., Ltd., ginger, mulberry leaves, ginseng, wolfberries, turmeric, licorice, mint, plantain seeds and houttuynia cordata were purchased from Bozhou Nihong Biotechnology Co., Ltd., the Bacillus subtilis medium was purchased from Tai'an Guotaiminan Biotechnology Co., Ltd., the Lactobacillus lactis medium was purchased from Mingguang Guangjulong Experimental Equipment Co., Ltd., the Bifidobacterium longum medium was purchased from Qingdao Haibo Biotechnology Co., Ltd., the glucose-yeast powder medium was purchased from Qingdao Qingyao Bioengineering Co., Ltd., the basic feed was purchased from Shanghai Chuanyong Trading Co., Ltd., the Bacillus subtilis selective medium and the Lactobacillus lactis selective medium were purchased from Qingdao Haibo Biotechnology Co., Ltd., the Bifidobacterium longum selective medium was purchased from Merck Group of Germany, and the yeast growth medium was purchased from Thermo Fisher Scientific Inc.

[0030] Example 1

[0031] A preparation method of postbiotics from Chinese herbal medicine residues, comprising the following steps:

[0032] (1) Collect the contents from the grass carp intestine and isolate probiotics: Bacillus subtilis, Lactobacillus lactis, Bifidobacterium longum and Saccharomyces cerevisiae;

[0033] (2) Expand the culture of the probiotics separately;

[0034] (3) Stir glucose, yeast extract, tryptone, KH2PO4, K2HPO4, vitamin B complex and biotin with deionized water to prepare a basic medium, add Chinese herbal medicine residues, and adjust the pH to 6.0 with 1M hydrochloric acid solution to prepare a mixed medium;

[0035] (4) Add the mixed medium to the fermenter, sterilize at 121 °C for 20 min, then cool down to 30 °C, add the Bacillus subtilis fermentation broth, maintain the temperature at 30 °C and the pH value at 7, and ferment for 25 h;

[0036] (5) Add the Lactobacillus lactis fermentation broth and the Saccharomyces cerevisiae fermentation broth, adjust the temperature to 32 °C, and keep the pH value at 6.2, and continue to ferment for 25 h;

[0037] (6) Add the Bifidobacterium longum fermentation broth to adjust the temperature to 35 °C, keep the pH value at 6.0, and continue to ferment for 36 hours to obtain a mixed fermentation broth;

[0038] (7) Filter the mixed fermentation broth through a filter membrane with a pore size of 0.45 μm to remove the solid residues in the fermentation broth, and then perform concentration and freeze-drying.

[0039] As a preferred technical solution of the present invention, the separated probiotics in step (1) are: using a Bacillus subtilis selective medium to separate Bacillus subtilis, using a Lactobacillus lactis selective medium to separate Lactobacillus lactis, using a Bifidobacterium longum selective medium to separate Bifidobacterium longum, and using a yeast growth medium to separate Saccharomyces cerevisiae.

[0040] The separate expansion culture conditions in step (2) are as follows: For Bacillus subtilis, use a Bacillus subtilis medium, control the pH value at 7.0, the temperature at 30 °C, and expand the culture until the OD of the Bacillus subtilis fermentation broth is 1.5; for Lactobacillus lactis, use a Lactobacillus lactis medium, control the pH at 5.8, the temperature at 37 °C, and expand the culture until the OD of the Lactobacillus lactis fermentation broth is 1.5; for Bifidobacterium longum, use a Bifidobacterium longum medium, control the pH value at 6.2, the temperature at 37 °C, and expand the culture until the OD of the Bifidobacterium longum fermentation broth is 1.1; for Saccharomyces cerevisiae, use a glucose-yeast powder medium, control the pH at 5.5, the temperature at 30 °C, and expand the culture until the OD of the Saccharomyces cerevisiae fermentation broth is 3.0.

[0041] The Chinese herbal medicine residues in step (3) include: ginger, mulberry leaves, plantain seeds, ginseng, wolfberries, turmeric, licorice, mint, and houttuynia cordata; the mass ratio of ginger, mulberry leaves, plantain seeds, ginseng, wolfberries, turmeric, licorice, mint, and houttuynia cordata is 1:1:1:0.6:0.6:0.2:0.2:0.2:0.2.

[0042] In the mixed medium in step (3), the addition amount of glucose is 16 g / L, the addition amount of yeast extract is 8 g / L, the addition amount of tryptone is 8 g / L, the addition amount of KH2PO4 is 1.5 g / L, the addition amount of K2HPO4 is 1.5 g / L, the addition amount of vitamin B group is 125 mg / L, the addition amount of biotin is 30 mg / L, and the addition amount of Chinese herbal medicine residues is 3.5 g / L.

[0043] The mass ratio of the Bacillus subtilis fermentation broth to the mixed medium in step (4) is 1:15.

[0044] In steps (4), (5), and (6), the mass ratio of the Bacillus subtilis fermentation broth, Lactobacillus lactis fermentation broth, Bifidobacterium longum fermentation broth, and Saccharomyces cerevisiae fermentation broth is 2:3:2:3.

[0045] The postbiotics of the Chinese herbal medicine residues are applied to fish feed.

[0046] Example 2

[0047] A preparation method of postbiotics of Chinese herbal medicine residues, comprising the following steps:

[0048] (1) Collecting the contents from the intestine of grass carp and isolating probiotics: Bacillus subtilis, Lactobacillus lactis, Bifidobacterium longum and Saccharomyces cerevisiae;

[0049] (2) Expanding the probiotic culture alone;

[0050] (3) Glucose, yeast extract, tryptone, KH2PO4, K2HPO4, vitamin B complex and biotin were added to deionized water and stirred evenly to prepare a basal medium, and Chinese medicinal residues were added, and the pH was adjusted to 5.5 with 1 M hydrochloric acid solution to prepare a mixed medium;

[0051] (4) adding the mixed culture medium to the fermentation tank, sterilizing at 121°C for 20 min, cooling to 30°C, adding Bacillus subtilis fermentation liquid, maintaining the temperature at 30°C and pH 7, and fermenting for 20-30 h;

[0052] (5) adding Lactobacillus lactis fermentation broth and Saccharomyces cerevisiae fermentation broth, adjusting the temperature to 32° C., maintaining the pH value at 6.0, and continuing the fermentation for 20 h;

[0053] (6) adding Bifidobacterium longum fermentation broth to adjust the temperature to 35° C., maintain the pH value at 6.0, and continue fermenting for 24 hours to obtain a mixed fermentation broth;

[0054] (7) The mixed fermentation broth was filtered through a filter membrane with a pore size of 0.45 μm to remove solid residues in the fermentation broth, and then concentrated and freeze-dried.

[0055] As a preferred technical solution of the present invention, the probiotics isolated in step (1) are: using a Bacillus subtilis selective medium to separate Bacillus subtilis, using a Lactobacillus lactis selective medium to separate Lactobacillus lactis, using a Bifidobacterium longum selective medium to separate Bifidobacterium longum, and using a yeast growth medium to separate Saccharomyces cerevisiae.

[0056] As a preferred technical solution of the present invention, the separate expansion culture conditions in step (2) are as follows: Bacillus subtilis uses a Bacillus subtilis culture medium with a pH value controlled at 7.0 and a temperature of 30°C, and the culture is expanded until the OD of the Bacillus subtilis fermentation liquid is 1.2; Lactobacillus lactis uses a Lactobacillus lactis culture medium with a pH value controlled at 5.5 and a temperature of 37°C, and the culture is expanded until the OD of the Lactobacillus lactis fermentation liquid is 1.2; Bifidobacterium longum uses a Bifidobacterium longum culture medium with a pH value controlled at 6.0 and a temperature of 37°C, and the culture is expanded until the OD of the Bifidobacterium longum fermentation liquid is 1.0; and Saccharomyces cerevisiae uses a glucose-yeast powder culture medium with a pH value controlled at 5.5 and a temperature of 30°C, and the culture is expanded until the OD of the Saccharomyces cerevisiae fermentation liquid is 2.5.

[0057] The Chinese medicine residues described in step (3) include: ginger, mulberry leaves, plantain seeds, ginseng, wolfberries, turmeric, liquorice, mint, and houttuynia cordata; the mass ratio of ginger, mulberry leaves, plantain seeds, ginseng, wolfberries, turmeric, liquorice, mint, and houttuynia cordata is 1:0.8:0.8:0.4:0.4:0.1:0.1:0.1:0.1.

[0058] In the mixed culture medium described in step (3), the addition amount of glucose is 10 g / L, the addition amount of yeast extract is 5 g / L, the addition amount of tryptone is 5 g / L, the addition amount of KH2PO4 is 1 g / L, the addition amount of K2HPO4 is 1 g / L, the addition amount of vitamin B group is 50 mg / L, the addition amount of biotin is 10 mg / L, and the addition amount of Chinese medicine residues is 2.3 g / L.

[0059] The mass ratio of the Bacillus subtilis fermentation broth described in step (4) to the mixed culture medium is 1:10.

[0060] The mass ratio of the Bacillus subtilis fermentation broth, Lactobacillus lactis fermentation broth, Bifidobacterium longum fermentation broth, and Saccharomyces cerevisiae fermentation broth described in step (4), step (5), and step (6) is 1:2:1:2.

[0061] The postbiotics of the Chinese medicine residues are applied to fish feed.

[0062] Example 3

[0063] A preparation method of postbiotics of Chinese medicine residues, comprising the following steps:

[0064] (1) Collect the contents from the grass carp intestine and isolate probiotics: Bacillus subtilis, Lactobacillus lactis, Bifidobacterium longum, and Saccharomyces cerevisiae;

[0065] (2) Separately expand and culture the probiotics;

[0066] (3) Stir glucose, yeast extract, tryptone, KH2PO4, K2HPO4, vitamin B group, and biotin with deionized water to evenly prepare a basal medium, add Chinese medicine residues, and adjust the pH to 6.5 with 1 M hydrochloric acid solution to prepare a mixed culture medium;

[0067] (4) Add the mixed culture medium to a fermenter, sterilize at 121 °C for 20 min, then cool down to 30 °C, add the Bacillus subtilis fermentation broth, maintain the temperature at 30 °C and the pH value at 7, and ferment for 30 h;

[0068] (5) Add the Lactobacillus lactis fermentation broth and Saccharomyces cerevisiae fermentation broth, adjust the temperature to 32 °C, keep the pH value at 6.5, and continue to ferment for 30 h;

[0069] (6) Add the fermentation broth of Bifidobacterium longum, adjust the temperature to 35°C, keep the pH value at 6.0, and continue fermentation for 48 hours to obtain a mixed fermentation broth;

[0070] (7) After filtering the mixed fermentation broth through a filter membrane with a pore size of 0.45 μm to remove the solid residues in the fermentation broth, conduct concentration and freeze-drying.

[0071] The separated probiotics in step (1) are as follows: Bacillus subtilis is separated using a Bacillus subtilis selective medium, Lactobacillus lactis is separated using a Lactobacillus lactis selective medium, Bifidobacterium longum is separated using a Bifidobacterium longum selective medium, and Saccharomyces cerevisiae is separated using a yeast growth medium.

[0072] The individual enlarged culture conditions in step (2) are as follows: For Bacillus subtilis, use a Bacillus subtilis medium, control the pH value at 7.0, the temperature at 30°C, and enlarge the culture until the OD of the Bacillus subtilis fermentation broth reaches 1.8; for Lactobacillus lactis, use a Lactobacillus lactis medium, control the pH at 6.0, the temperature at 37°C, and enlarge the culture until the OD of the Lactobacillus lactis fermentation broth reaches 1.8; for Bifidobacterium longum, use a Bifidobacterium longum medium, control the pH value at 6.5, the temperature at 37°C, and enlarge the culture until the OD of the Bifidobacterium longum fermentation broth reaches 1.2; for Saccharomyces cerevisiae, use a glucose-yeast powder medium, control the pH at 5.5, the temperature at 30°C, and enlarge the culture until the OD of the Saccharomyces cerevisiae fermentation broth reaches 3.5.

[0073] The Chinese herbal medicine residues in step (3) include: ginger, mulberry leaves, plantain seeds, ginseng, wolfberries, turmeric, licorice, mint, and houttuynia cordata; the mass ratio of ginger, mulberry leaves, plantain seeds, ginseng, wolfberries, turmeric, licorice, mint, and houttuynia cordata is 1:1.5:1.2:0.8:0.8:0.3:0.3:0.3:0.3.

[0074] In the mixed medium in step (3), the addition amount of glucose is 20 g / L, the addition amount of yeast extract is 10 g / L, the addition amount of tryptone is 10 g / L, the addition amount of KH2PO4 is 2 g / L, the addition amount of K2HPO4 is 2 g / L, the addition amount of vitamin B group is 200 mg / L, the addition amount of biotin is 50 mg / L, and the addition amount of Chinese herbal medicine residues is 4.8 g / L.

[0075] The mass ratio of the Bacillus subtilis fermentation broth to the mixed medium in step (4) is 1:20.

[0076] As a preferred technical solution of the present invention, the mass ratio of the Bacillus subtilis fermentation broth, Lactobacillus lactis fermentation broth, Bifidobacterium longum fermentation broth, and Saccharomyces cerevisiae fermentation broth in step (4), step (5), and step (6) is 3:4:3:4.

[0077] The postbiotics of the traditional Chinese medicine residue are applied to fish feed.

[0078] Comparative Example 1

[0079] On the basis of Example 1, Bacillus subtilis is not added to the beneficial bacteria, and the mass ratio of Lactobacillus lactis fermentation broth, Bifidobacterium longum fermentation broth, and Saccharomyces cerevisiae fermentation broth is changed to 5:2:3, and the rest is the same as Example 1.

[0080] Comparative Example 2

[0081] On the basis of Example 1, Lactobacillus lactis fermentation broth is not added to the beneficial bacteria, and the mass ratio of Bacillus subtilis fermentation broth, Bifidobacterium longum fermentation broth, and Saccharomyces cerevisiae fermentation broth is changed to 2:5:3, and the rest is the same as Example 1.

[0082] Comparative Example 3

[0083] On the basis of Example 1, Bifidobacterium longum fermentation broth is not added to the beneficial bacteria, and the mass ratio of Bacillus subtilis fermentation broth, Lactobacillus lactis fermentation broth, and Saccharomyces cerevisiae fermentation broth is changed to 2:3:5, and the rest is the same as Example 1.

[0084] Comparative Example 4

[0085] On the basis of Example 1, Saccharomyces cerevisiae fermentation broth is not added to the beneficial bacteria, and the mass ratio of Bacillus subtilis fermentation broth, Lactobacillus lactis fermentation broth, and Bifidobacterium longum fermentation broth is changed to 5:3:2, and the rest is the same as Example 1.

[0086] Comparative Example 5

[0087] On the basis of Example 1, ginger is not added to the traditional Chinese medicine residue, and the mass ratio of mulberry leaf, plantain seed, ginseng, wolfberry fruit, turmeric, licorice, mint, and houttuynia cordata is changed to 2:1:0.6:0.6:0.2:0.2:0.2:0.2, and the rest is the same as Example 1.

[0088] Comparative Example 6

[0089] On the basis of Example 1, mulberry leaf is not added to the traditional Chinese medicine residue, and the mass ratio of ginger, plantain seed, ginseng, wolfberry fruit, turmeric, licorice, mint, and houttuynia cordata is changed to 1:2:0.6:0.6:0.2:0.2:0.2:0.2, and the rest is the same as Example 1.

[0090] Comparative Example 7

[0091] On the basis of Example 1, plantain seed is not added to the traditional Chinese medicine residue, and the mass ratio of ginger, mulberry leaf, ginseng, wolfberry fruit, turmeric, licorice, mint, and houttuynia cordata is changed to 1:1:1.6:0.6:0.2:0.2:0.2:0.2, and the rest is the same as Example 1.

[0092] Comparative Example 8

[0093] Based on Example 1, ginseng is not added to the Chinese medicine residue, and the mass ratio of ginger, mulberry leaf, plantain seed, wolfberry fruit, turmeric, licorice, mint, and houttuynia cordata is changed to 1:1:1:1.2:0.2:0.2:0.2:0.2, and the rest is the same as in Example 1.

[0094] Comparative Example 9

[0095] Based on Example 1, wolfberry fruit is not added to the Chinese medicine residue, and the mass ratio of ginger, mulberry leaf, plantain seed, ginseng, turmeric, licorice, mint, and houttuynia cordata is changed to 1:1:1:0.6:0.8:0.2:0.2:0.2, and the rest is the same as in Example 1.

[0096] Comparative Example 10

[0097] Based on Example 1, turmeric is not added to the Chinese medicine residue, and the mass ratio of ginger, mulberry leaf, plantain seed, ginseng, wolfberry fruit, licorice, mint, and houttuynia cordata is changed to 1:1:1:0.6:0.6:0.4:0.2:0.2, and the rest is the same as in Example 1.

[0098] Comparative Example 11

[0099] Based on Example 1, licorice is not added to the Chinese medicine residue, and the mass ratio of ginger, mulberry leaf, plantain seed, ginseng, wolfberry fruit, turmeric, mint, and houttuynia cordata is changed to 1:1:1:0.6:0.6:0.2:0.4:0.2, and the rest is the same as in Example 1.

[0100] Comparative Example 12

[0101] Based on Example 1, mint is not added to the Chinese medicine residue, and the mass ratio of ginger, mulberry leaf, plantain seed, ginseng, wolfberry fruit, turmeric, licorice, and houttuynia cordata is changed to 1:1:1:0.6:0.6:0.2:0.2:0.4, and the rest is the same as in Example 1.

[0102] Example 13

[0103] Based on Example 1, houttuynia cordata is not added to the Chinese medicine residue, and the mass ratio of ginger, mulberry leaf, plantain seed, ginseng, wolfberry fruit, turmeric, licorice, and mint is changed to 1.2:1:1:0.6:0.6:0.2:0.2:0.2, and the rest is the same as in Example 1.

[0104] Performance Test

[0105] The microbial metabolites prepared in Examples 1 - 3 and Comparative Examples 1 - 13 were mixed with the basic feed at 5% and fed to Micropterus salmonides for the test experiment;

[0106] Before the test, the Micropterus salmonides fries were pre-fed for 15 days to adapt them to the environment. Basic feed was fed at 8:00 and 18:00 every day, and the feeding rate was 2%.

[0107] After the pre-feeding, the Micropterus salmonides fries were randomly divided into 16 groups, with 100 fries in each group. The mass of the Micropterus salmonides was weighed as the initial weight. Except for the different feeds, the other conditions were the same as those in the pre-feeding, and the feeding was carried out for 56 days.

[0108] After the test, the mass of the Micropterus salmonides was measured and recorded as the final weight, and the growth situation and growth indexes of the Micropterus salmonides were detected:

[0109] Survival rate (%) = number of surviving fish / number of initial fish × 100%

[0110] Weight gain rate (%) = (final weight - initial weight) × 100% / initial weight;

[0111] Condition factor (g / cm 3 ) = 100 × fish weight / (fish body length) 3 ;

[0112]

[0113]

[0114] From the above test data, it can be seen that by fermenting ginger, mulberry leaves, ginseng, plantain seeds, wolfberries, turmeric, licorice, mint and houttuynia cordata with Bacillus subtilis, Lactobacillus lactis, Bifidobacterium longum and Saccharomyces cerevisiae, the metabolic products prepared are added to the feed, which can effectively improve the survival rate, weight gain rate and condition factor of Micropterus salmonides.

[0115] The above is only a preferred embodiment of the present invention, and it does not impose any form of limitation on the present invention. Although the present invention has been disclosed above with a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to equivalent embodiments by using the above-disclosed technical content within the scope of the technical solution of the present invention. However, as long as it does not depart from the content of the technical solution of the present invention, any indirect modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing postbiotics from traditional Chinese medicine residues, characterized in that: The following steps are involved: (1) Collecting the contents from the intestine of grass carp and isolating probiotics: Bacillus subtilis, Lactobacillus lactis, Bifidobacterium longum and Saccharomyces cerevisiae; (2) Expanding the probiotic culture alone; (3) Glucose, yeast extract, tryptone, KH2PO4, K2HPO4, vitamin B complex and biotin were added to deionized water and stirred to prepare a basal medium, and Chinese medicinal residues were added, and the pH was adjusted to 5.5-6.5 with 1M hydrochloric acid solution to prepare a mixed medium; (4) adding the mixed culture medium to the fermentation tank, sterilizing at 121°C for 20 min, cooling to 30°C, adding Bacillus subtilis fermentation liquid, maintaining the temperature at 30°C and pH 7, and fermenting for 20-30 h; (5) adding Lactobacillus lactis fermentation broth and Saccharomyces cerevisiae fermentation broth, adjusting the temperature to 32° C., maintaining the pH value at 6.0-6.5, and continuing the fermentation for 20-30 h; (6) adding Bifidobacterium longum fermentation broth to adjust the temperature to 35° C., maintain the pH value at 6.0, and continue fermenting for 24-48 hours to obtain a mixed fermentation broth; (7) The mixed fermentation broth was filtered through a filter membrane with a pore size of 0.45 μm to remove solid residues in the fermentation broth, and then concentrated and freeze-dried.

2. The method for preparing postbiotics from Chinese medicinal residues according to claim 1, characterized in that: The probiotics isolated in step (1) are: using a Bacillus subtilis selective medium to separate Bacillus subtilis, using a Lactobacillus lactis selective medium to separate Lactobacillus lactis, using a Bifidobacterium longum selective medium to separate Bifidobacterium longum, and using a yeast growth medium to separate Saccharomyces cerevisiae.

3. The method for preparing postbiotics from Chinese medicinal residues according to claim 1, characterized in that: The conditions for the separate expanded culture in step (2) are as follows: Bacillus subtilis uses a Bacillus subtilis culture medium, the pH value is controlled at 7.0, the temperature is 30°C, and the culture is expanded until the OD of the Bacillus subtilis fermentation liquid is 1.2-1.8; Lactobacillus lactis uses a Lactobacillus lactis culture medium, the pH value is controlled at 5.5-6.0, the temperature is 37°C, and the culture is expanded until the OD of the Lactobacillus lactis fermentation liquid is 1.2-1.8; Bifidobacterium longum uses a Bifidobacterium longum culture medium, the pH value is controlled at 6.0-6.5, the temperature is 37°C, and the culture is expanded until the OD of the Bifidobacterium longum fermentation liquid is 1.0-1.2; Saccharomyces cerevisiae uses a glucose-yeast powder culture medium, the pH value is controlled at 5.5, the temperature is 30°C, and the culture is expanded until the OD of the Saccharomyces cerevisiae fermentation liquid is 2.5-3.

5.

4. The method for preparing postbiotics from Chinese medicinal residues according to claim 1, characterized in that: The Chinese medicinal residue in step (3) comprises: ginger, mulberry leaves, plantain seeds, ginseng, wolfberry, turmeric, liquorice, mint and houttuynia cordata; the mass ratio of the ginger, mulberry leaves, plantain seeds, ginseng, wolfberry, turmeric, liquorice, mint and houttuynia cordata is 1:0.8-1.5:0.8-1.2:0.4-0.8:0.4-0.8:0.1-0.3:0.1-0.3:0.1-0.3:0.1-0.

3.

5. The method for preparing postbiotics from Chinese medicinal residues according to claim 1, characterized in that: In the mixed culture medium of step (3), the amount of glucose added is 10-20 g / L, the amount of yeast extract added is 5-10 g / L, the amount of trypsin added is 5-10 g / L, the amount of KH2PO4 added is 1-2 g / L, the amount of K2HPO4 added is 1-2 g / L, the amount of B vitamins added is 50-200 mg / L, the amount of biotin added is 10-50 mg / L, and the amount of traditional Chinese medicine residue added is 2.3-4.8 g / L.

6. The method for preparing postbiotics from Chinese medicinal residues according to claim 1, characterized in that: The mass ratio of the Bacillus subtilis fermentation broth to the mixed culture medium in step (4) is 1:10-20.

7. The method for preparing postbiotics from Chinese medicinal residues according to claim 1, characterized in that: The mass ratio of the Bacillus subtilis fermentation broth, the Lactobacillus lactis fermentation broth, the Bifidobacterium longum fermentation broth and the Saccharomyces cerevisiae fermentation broth in step (4), step (5) and step (6) is 1-3:2-4:1-3:2-4.

8. An application of the Chinese medicine residue postbiotics as claimed in claims 1-7, characterized in that: The Chinese medicine residue postbiotics are used in fish feed.

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