Method for preparing lactic acid bacteria liquid from coprophilous fungi and application of lactic acid bacteria liquid

Abstract

The invention relates to the technical field of lactic acid bacteria liquid preparation, in particular to a method for preparing lactic acid bacteria liquid from coprophilous fungi, and the preparation method comprises the following steps: step 1, culture medium preparation: preparing a solid culture medium and a liquid culture medium for preparing the lactic acid bacteria liquid; step 2, excrement collection: selecting a healthy donor for lactic acid bacteria liquid preparation, and collecting excrement of the healthy donor; step 3, preparing coprophilous bacteria liquid: preparing donor faeces to obtain the coprophilous bacteria liquid; 4, preparation of a contrast sample: preparing donor excrement to obtain a coprophilous fungus capsule; according to the invention, Chinese herbal medicines are added into the components of the culture medium, so that the growth and preservation activity of lactic acid bacteria are obviously promoted, the preservation time of lactic acid bacteria liquid prepared by the culture medium containing the Chinese herbal medicine components is longer, and the Chinese herbal medicine components have a certain effect of treating diarrhea; on the basis of increasing the growth variety and quantity of lactic acid bacteria, the lactic acid bacteria liquid can inhibit enteropathogenic bacteria and regulate the balance of intestinal flora.

Description

Technical Field

[0001] The present invention relates to the technical field of lactic acid bacteria liquid preparation, in particular to a method for preparing lactic acid bacteria liquid from fecal bacteria and application thereof. Background Art

[0002] Fecal bacteria play an important role in the body. They form a symbiotic relationship with the donor and participate in the donor's metabolism, nutrient absorption, anti-cancer effect, gastrointestinal immune barrier and other physiological functions. The dynamic balance of intestinal flora includes beneficial bacteria, neutral bacteria and conditional pathogens. Once this balance is broken, it may lead to the occurrence of intestinal and extraintestinal diseases. As an emerging treatment method, fecal transplantation has broad technical prospects, but it also needs further research and improvement to ensure its safety and effectiveness. Lactic acid bacteria have the effect of improving the function of the gastrointestinal tract, mainly facultative anaerobic bacteria. In a state of ecological balance, it can maintain the synthesis of vitamins and proteins, and functions such as digestion, absorption and immunity are very beneficial to the host. Fecal bacteria can be used to prepare lactic acid bacteria liquid through culture medium.

[0003] At present, the culture medium for preparing lactic acid bacteria liquid from fecal bacteria is prepared by chemical components or compounds. The culture medium lacks natural herbs to promote the activity and growth of lactic acid bacteria, the lactic acid bacteria has a short shelf life, and the prepared lactic acid bacteria liquid has poor efficacy. In order to solve the above-mentioned problems, the present invention proposes a method for preparing lactic acid bacteria liquid from fecal bacteria and its application. Summary of the invention

[0004] In view of the problems in the prior art, the present invention provides a method for preparing lactic acid bacteria liquid from fecal bacteria and application thereof.

[0005] The technical solution adopted by the present invention to solve the technical problem is: a method for preparing lactic acid bacteria liquid from fecal bacteria, the preparation method comprising the following steps: Step 1, culture medium preparation: preparing solid culture medium and liquid culture medium for preparing lactic acid bacteria liquid; Step 2, feces collection: selecting healthy donors for lactic acid bacteria liquid preparation and collecting feces from healthy donors; Step 3, fecal microbial liquid preparation: prepare the feces of the donor to obtain the fecal microbial liquid; Step 4, comparison sample preparation: prepare the feces of the donor and obtain fecal bacteria capsules; Step 5, lactic acid bacteria culture: put the solid culture medium into the plate, take the fecal bacteria liquid and put it into the plate, and culture it in the incubator to obtain lactic acid bacteria; Step 6, lactic acid bacteria identification: selecting lactic acid bacteria identification samples from the incubator, preparing a strain culture solution for lactic acid bacteria identification, and determining the type of lactic acid bacteria by gene sequencing; Step 7, culturing lactic acid bacteria liquid: placing lactic acid bacteria in a liquid culture medium, and culturing in an incubator to obtain lactic acid bacteria liquid.

[0006] Specifically, the specific method for preparing the solid culture medium is as follows: weighing each component of the solid culture medium according to the weight proportion of the solid culture medium, dissolving each component of the solid culture medium in deionized water, sterilizing by high pressure, cooling and adjusting the pH, to obtain a solid culture medium; The solid culture medium comprises the following components by weight: 4-6 parts of peptone, 3-4 parts of yeast extract powder, 5-8 parts of beef extract powder, 3-4 parts of glucose, 1-2 parts of dipotassium hydrogen phosphate, 0.5-1 parts of triammonium citrate, 0.1-0.2 parts of magnesium sulfate, 0.1-0.2 parts of manganese sulfate, 0.3-0.8 parts of Tween-80, 0.1-0.2 parts of sodium acetate, 1-3 parts of guava powder, 5-7 parts of applesauce, 3-5 parts of Chinese herbal medicine powder, 0.1-0.2 parts of mupirocin lithium salt, 0.1-0.2 parts of vitamins and 8-13 parts of agar; When the pressure of the high pressure sterilization is 103-137 kPa and the temperature reaches 121-126°C, the sterilization purpose can be achieved by maintaining it for 20-30 minutes, and the pH is adjusted to 6.2-6.5; The specific method for preparing the liquid culture medium is as follows: weighing each component of the liquid culture medium according to the weight of the liquid culture medium, dissolving each component of the liquid culture medium in the feces filtrate, and sterilizing by high pressure, cooling and adjusting the pH to obtain the liquid culture medium; The liquid culture medium comprises, by weight, 4-6 parts of peptone, 3-4 parts of yeast extract powder, 5-8 parts of beef extract powder, 3-4 parts of glucose, 1-2 parts of dipotassium hydrogen phosphate, 1-3 parts of guava powder, 5-7 parts of applesauce, 3-5 parts of Chinese herbal medicine powder, 0.1-0.2 parts of vitamins and 8-13 parts of agar; The high pressure sterilization pressure is 103-137 kPa, the temperature reaches 121-126° C., and is maintained for 20-30 minutes to achieve the purpose of sterilization, and the pH is adjusted to 6.2-6.5.

[0007] Specifically, the healthy donors for feces collection are three kinds of healthy pigs of 28 days, 50 days and 70 days old, and feces are collected to obtain feces A of 28-day-old pig, feces B of 50-day-old pig and feces C of 70-day-old pig, respectively.

[0008] Specifically, the specific method for preparing the fecal bacteria liquid is as follows: feces A is placed in container 1, feces B is placed in container 2, and feces C is placed in container 3, respectively, sterile saline is added to container 1, container 2, and container 3 and sealed, container 1, container 2, and container 3 are placed in a shaker together and shaken, then allowed to stand and precipitate, and the supernatant is taken to obtain fecal bacteria liquid A, fecal bacteria liquid B, and fecal bacteria liquid C for use.

[0009] Specifically, the specific method for preparing the fecal bacteria capsule is as follows: during the preparation of the fecal bacteria liquid, the fecal bacteria in the sediment after standing and settling to obtain the supernatant are collected, the fecal bacteria in the sediment and the freeze-dried protective liquid are added to a mixing container and mixed to obtain the fecal bacteria liquid, the fecal bacteria liquid is cooled, freeze-dried, and powdered, and then the capsule shell is filled to prepare the fecal bacteria capsule; The freeze-dried protective solution comprises, by weight, 1-2 parts of sucrose, 7-11 parts of soluble starch, 6-10 parts of anhydrous solvent, 0.5-2 parts of surfactant, 0.2-0.5 parts of amino acid and 0.5-1 parts of citrate; The fecal bacteria liquid is cooled to -10°C and then freeze-dried. After freeze-drying, it is ground into powder using a grinder, frozen at -75°C for 12-24 hours, and then freeze-dried at a vacuum degree of -30KPa and a drying temperature of -60°C for 24-48 hours.

[0010] Specifically, the specific method for culturing lactic acid bacteria is as follows: solid culture medium is respectively loaded into plate 1, plate 2 and plate 3, fecal bacteria liquid A is put into plate 1, fecal bacteria liquid B is put into plate 2 and fecal bacteria liquid C is put into plate 3, lactic acid bacteria are cultured by plate pouring method, and after coagulation, the plate is inverted and placed in an anaerobic incubator at 35-37°C and cultured for 50-58 hours to obtain lactic acid bacteria A, lactic acid bacteria B and lactic acid bacteria C.

[0011] Specifically, the specific method for culturing the lactic acid bacteria liquid is as follows: liquid culture medium is loaded into culture dish 1, culture dish 2 and culture dish 3, lactic acid bacteria A is added to culture dish 1, lactic acid bacteria B is added to culture dish 2 and lactic acid bacteria C is added to culture dish 3, and placed in an anaerobic incubator at 35-37°C for 50-58h to obtain lactic acid bacteria liquid A, lactic acid bacteria liquid B and lactic acid bacteria liquid C.

[0012] Specifically, the Chinese herbal medicine powder in the solid culture medium component is selected from one or more of malt, hawthorn, Shenqu, Ophiopogon japonicus and Codonopsis pilosula, and the Chinese herbal medicine powder in the liquid culture medium component is selected from one or more of honeysuckle, houttuynia cordata, Artemisia annua, Radix Isatidis and Coptis chinensis, and the Chinese herbal medicine powder is prepared by removing impurities from raw materials, washing, freeze-drying, pulverizing and sieving and sorting; The freeze-drying process for the Chinese herbal medicine powder raw material freezes the cleaned raw material at -75°C for 6-12 hours, and then freeze-dries it at a vacuum degree of -30KPa and a drying temperature of -60°C for 10-14 hours. After freezing, the freeze-dried raw material is crushed by a pulverizer, and then sieved by a screening machine with a mesh size of 300 meshes.

[0013] A method for preparing lactic acid bacteria liquid from fecal bacteria and application of the lactic acid bacteria liquid prepared in preparing functional bacteria for fecal bacteria transplantation.

[0014] Beneficial effects of the present invention: The present invention discloses a method for preparing lactic acid bacteria liquid from fecal bacteria and its application. The present invention adopts fecal feces of healthy donors to obtain fecal bacteria. The fecal bacteria are cultured in solid culture medium and liquid culture medium to prepare lactic acid bacteria liquid. Agar is used as a coagulant in the culture medium to support and protect the culture. The culture medium provides trace elements necessary for the lactic acid bacteria liquid, participates in the metabolic process, provides energy and nutrients required for culture, and promotes cell growth and division. Chinese herbal medicine is added to the culture medium components, which has a significant promoting effect on the growth and preservation activity of lactic acid bacteria, so that the lactic acid bacteria liquid prepared by the culture medium containing Chinese herbal medicine components of the present invention has a longer shelf life, and the Chinese herbal medicine components have a certain effect on treating diarrhea, so that the lactic acid bacteria liquid prepared by the present invention can inhibit intestinal pathogens, improve intestinal flora, and regulate the balance of intestinal flora on the basis of increasing the growth types and number of lactic acid bacteria, which can help improve intestinal health and promote animal growth. In the process of preparing the lactic acid bacteria liquid, the discarded precipitated fecal bacteria are utilized to prepare fecal bacteria capsules with intestinal improvement effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0016] Figure 1 A flow chart of a method for preparing lactic acid bacteria liquid from fecal bacteria and its application provided by the present invention. DETAILED DESCRIPTION

[0017] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0018] like Figure 1 As shown, a method for preparing lactic acid bacteria liquid from fecal bacteria of the present invention comprises the following steps: Step 1, culture medium preparation: preparing solid culture medium and liquid culture medium for preparing lactic acid bacteria liquid; Step 2, feces collection: selecting healthy donors for lactic acid bacteria liquid preparation and collecting feces from healthy donors; Step 3, fecal bacteria liquid preparation: prepare the feces of the donor to obtain fecal bacteria; Step 4, comparison sample preparation: prepare the feces of the donor to obtain fecal bacteria capsule liquid; Step 5, lactic acid bacteria culture: put the solid culture medium into the plate, take the fecal bacteria liquid and put it into the plate, and culture it in the incubator to obtain lactic acid bacteria; Step 6, lactic acid bacteria identification: selecting lactic acid bacteria identification samples from the incubator, preparing a strain culture solution for lactic acid bacteria identification, and determining the type of lactic acid bacteria by gene sequencing; Step 7, culturing lactic acid bacteria liquid: placing lactic acid bacteria in a liquid culture medium, and culturing in an incubator to obtain lactic acid bacteria liquid.

[0019] Specifically, the specific method for preparing the solid culture medium is as follows: weighing each component of the solid culture medium according to the weight proportion of the solid culture medium, dissolving each component of the solid culture medium in deionized water, sterilizing by high pressure, cooling and adjusting the pH, to obtain a solid culture medium; The solid culture medium comprises the following components by weight: 4-6 parts of peptone, 3-4 parts of yeast extract powder, 5-8 parts of beef extract powder, 3-4 parts of glucose, 1-2 parts of dipotassium hydrogen phosphate, 0.5-1 parts of triammonium citrate, 0.1-0.2 parts of magnesium sulfate, 0.1-0.2 parts of manganese sulfate, 0.3-0.8 parts of Tween-80, 0.1-0.2 parts of sodium acetate, 1-3 parts of guava powder, 5-7 parts of applesauce, 3-5 parts of Chinese herbal medicine powder, 0.1-0.2 parts of mupirocin lithium salt, 0.1-0.2 parts of vitamins and 8-13 parts of agar; The high pressure sterilization pressure is 103-137 kPa, the temperature reaches 121-126° C., maintained for 20-30 minutes, and the pH is adjusted to 6.2-6.5; The specific method for preparing the liquid culture medium is as follows: weighing each component of the liquid culture medium according to the weight of the liquid culture medium, dissolving each component of the liquid culture medium in the feces filtrate, and sterilizing by high pressure, cooling and adjusting the pH to obtain the liquid culture medium; The liquid culture medium comprises, by weight, 4-6 parts of peptone, 3-4 parts of yeast extract powder, 5-8 parts of beef extract powder, 3-4 parts of glucose, 1-2 parts of dipotassium hydrogen phosphate, 1-3 parts of guava powder, 5-7 parts of applesauce, 3-5 parts of Chinese herbal medicine powder, 0.1-0.2 parts of vitamins and 8-13 parts of agar; The pressure of the high pressure sterilization is 103-137 kPa, the temperature reaches 121-126° C., maintained for 20-30 minutes, and the pH is adjusted to 6.2-6.5.

[0020] Specifically, the healthy donors for feces collection are three kinds of healthy pigs of 28 days, 50 days and 70 days old, and feces are collected to obtain feces A of 28-day-old pig, feces B of 50-day-old pig and feces C of 70-day-old pig, respectively.

[0021] Specifically, the specific method for preparing the fecal bacteria liquid is as follows: feces A is placed in container 1, feces B is placed in container 2, and feces C is placed in container 3, respectively, sterile saline is added to container 1, container 2, and container 3 and sealed, container 1, container 2, and container 3 are placed in a shaker together and shaken, then allowed to stand and precipitate, and the supernatant is taken to obtain fecal bacteria liquid A, fecal bacteria liquid B, and fecal bacteria liquid C for use.

[0022] Specifically, the specific method for preparing the fecal bacteria capsule is as follows: during the preparation of the fecal bacteria liquid, the fecal bacteria in the sediment after standing and settling to obtain the supernatant are collected, the fecal bacteria in the sediment and the freeze-dried protective liquid are added to a mixing container and mixed to obtain the fecal bacteria liquid, the fecal bacteria liquid is cooled, freeze-dried, and powdered, and then the capsule shell is filled to prepare the fecal bacteria capsule; The freeze-dried protective solution comprises, by weight, 1-2 parts of sucrose, 7-11 parts of soluble starch, 6-10 parts of anhydrous solvent, 0.5-2 parts of surfactant, 0.2-0.5 parts of amino acid and 0.5-1 parts of citrate; The fecal bacteria liquid is cooled to -10°C and then freeze-dried. After freeze-drying, it is ground into powder using a grinder, frozen at -75°C for 12-24 hours, and then freeze-dried at a vacuum degree of -30KPa and a drying temperature of -60°C for 24-48 hours.

[0023] Specifically, the specific method for culturing lactic acid bacteria is as follows: solid culture medium is respectively loaded into plate 1, plate 2 and plate 3, fecal bacteria liquid A is put into plate 1, fecal bacteria liquid B is put into plate 2 and fecal bacteria liquid C is put into plate 3, lactic acid bacteria are cultured by plate pouring method, and after coagulation, the plate is inverted and placed in an anaerobic incubator at 35-37°C and cultured for 50-58 hours to obtain lactic acid bacteria A, lactic acid bacteria B and lactic acid bacteria C.

[0024] Specifically, the specific method for culturing the lactic acid bacteria liquid is as follows: liquid culture medium is loaded into culture dish 1, culture dish 2 and culture dish 3, lactic acid bacteria A is added to culture dish 1, lactic acid bacteria B is added to culture dish 2 and lactic acid bacteria C is added to culture dish 3, and placed in an anaerobic incubator at 35-37°C for 50-58h to obtain lactic acid bacteria liquid A, lactic acid bacteria liquid B and lactic acid bacteria liquid C.

[0025] Specifically, the Chinese herbal medicine powder in the solid culture medium component is selected from one or more of malt, hawthorn, Shenqu, Ophiopogon japonicus and Codonopsis pilosula, and the Chinese herbal medicine powder in the liquid culture medium component is selected from one or more of honeysuckle, houttuynia cordata, Artemisia annua, Radix Isatidis and Coptis chinensis, and the Chinese herbal medicine powder is prepared by removing impurities from raw materials, washing, freeze-drying, pulverizing and sieving and sorting; The freeze-drying process for the Chinese herbal medicine powder raw material freezes the cleaned raw material at -75°C for 6-12 hours, and then freeze-dries it at a vacuum degree of -30KPa and a drying temperature of -60°C for 10-14 hours. After freezing, the freeze-dried raw material is crushed by a pulverizer, and then sieved by a screening machine with a mesh size of 300 meshes.

[0026] A method for preparing lactic acid bacteria liquid from fecal bacteria and application of the lactic acid bacteria liquid prepared in preparing functional bacteria for fecal bacteria transplantation.

[0027] Example 1

[0028] A method for preparing lactic acid bacteria liquid from fecal bacteria and its application, the preparation method comprising the following steps: Step 1, culture medium preparation: preparing solid culture medium and liquid culture medium for preparing lactic acid bacteria liquid; Step 2, feces collection: selecting healthy donors for lactic acid bacteria liquid preparation and collecting feces from healthy donors; Step 3, fecal microbial liquid preparation: prepare the feces of the donor to obtain the fecal microbial liquid; Step 4, comparison sample preparation: prepare the feces of the donor and obtain fecal bacteria capsules; Step 5, lactic acid bacteria culture: put the solid culture medium into the plate, take the fecal bacteria liquid and put it into the plate, and culture it in the incubator to obtain lactic acid bacteria; Step 6, lactic acid bacteria identification: selecting lactic acid bacteria identification samples from the incubator, preparing a strain culture solution for lactic acid bacteria identification, and determining the type of lactic acid bacteria by gene sequencing; Step 7, culturing lactic acid bacteria liquid: placing lactic acid bacteria in a liquid culture medium, and culturing in an incubator to obtain lactic acid bacteria liquid.

[0029] Specifically, the specific method for preparing the solid culture medium is as follows: weighing each component of the solid culture medium according to the weight proportion of the solid culture medium, dissolving each component of the solid culture medium in deionized water, sterilizing by high pressure, cooling and adjusting the pH, to obtain a solid culture medium; The solid culture medium comprises the following components by weight: 4 parts of peptone, 3 parts of yeast extract powder, 8 parts of beef extract powder, 3 parts of glucose, 1 part of dipotassium hydrogen phosphate, 0.5 parts of triammonium citrate, 0.1 parts of magnesium sulfate, 0.2 parts of manganese sulfate, 0.3 parts of Tween-80, 0.1 parts of sodium acetate, 1 part of guava powder, 7 parts of applesauce, 5 parts of Chinese herbal medicine powder, 0.2 parts of mupirocin lithium salt, 0.1 parts of vitamins and 8 parts of agar; The high pressure sterilization pressure is 103 kPa, the temperature reaches 121 ° C, and the sterilization purpose can be achieved by maintaining it for 30 minutes, and the pH is adjusted to 6.5; The specific method for preparing the liquid culture medium is as follows: weighing each component of the liquid culture medium according to the weight of the liquid culture medium, dissolving each component of the liquid culture medium in the feces filtrate, and sterilizing by high pressure, cooling and adjusting the pH to obtain the liquid culture medium; The liquid culture medium comprises, by weight, 4 parts of peptone, 3 parts of yeast extract powder, 8 parts of beef extract powder, 3 parts of glucose, 1 part of dipotassium hydrogen phosphate, 1 part of guava powder, 7 parts of applesauce, 5 parts of Chinese herbal medicine powder, 0.1 parts of vitamins and 8 parts of agar; The high pressure sterilization was carried out at a pressure of 103 kPa, a temperature of 121° C., maintained for 30 minutes, and a pH value of 6.5.

[0030] Specifically, the healthy donor for feces collection is a 28-day-old healthy pig, and feces A of a 28-day-old pig is obtained.

[0031] Specifically, the specific method for preparing the fecal bacteria liquid is as follows: feces A is put into a container 1, sterile physiological saline is added to the container 1 and sealed, the container 1 is placed in a shaker and shaken evenly, and then the container is allowed to stand and precipitate to obtain the supernatant to obtain the fecal bacteria liquid A for later use.

[0032] Specifically, the specific method for preparing the fecal bacteria capsule is as follows: during the preparation of the fecal bacteria liquid, the fecal bacteria in the sediment after standing and settling to obtain the supernatant are collected, the fecal bacteria in the sediment and the freeze-dried protective liquid are added to a mixing container and mixed to obtain the fecal bacteria liquid, the fecal bacteria liquid is cooled, freeze-dried, and powdered, and then the capsule shell is filled to prepare the fecal bacteria capsule; The freeze-dried protective solution comprises, by weight, 2 parts of sucrose, 7 parts of soluble starch, 6 parts of anhydrous solvent, 0.5 parts of surfactant, 0.2 parts of amino acid and 0.5 parts of citrate; The fecal bacteria liquid is cooled to -10°C and then freeze-dried. After freeze-drying, it is ground into powder using a grinder, frozen at -75°C for 12 hours, and then freeze-dried at a vacuum degree of -30KPa and a drying temperature of -60°C for 48 hours.

[0033] Specifically, the specific method for culturing lactic acid bacteria is as follows: solid culture medium is loaded into plate 1, fecal bacteria liquid A is put into plate 1, lactic acid bacteria are cultured by plate pouring method, after coagulation, the plate is inverted and placed in a 37°C anaerobic incubator for 50 hours to obtain lactic acid bacteria A.

[0034] Specifically, the specific method for culturing the lactic acid bacteria liquid is as follows: liquid culture medium is placed in a culture dish 1, lactic acid bacteria A is added to the culture dish 1, and the culture dish is placed in a 37° C. anaerobic incubator for 50 hours to obtain lactic acid bacteria liquid A.

[0035] Specifically, the Chinese herbal medicine powder in the solid culture medium component is a mixture of malt and hawthorn in a ratio of 1:1, and the Chinese herbal medicine powder in the liquid culture medium component is honeysuckle, and the Chinese herbal medicine powder is prepared by removing impurities from raw materials, washing, freeze-drying, crushing and sieving; The freeze-drying process of the Chinese herbal medicine powder raw material freezes the cleaned raw material at -75°C for 6 hours, and then freeze-dries it at a vacuum degree of -30KPa and a drying temperature of -60°C for 14 hours. After freezing, the freeze-dried raw material is crushed by a pulverizer, and then sieved by a screening machine with a mesh size of 300 meshes.

[0036] Example 2

[0037] A method for preparing lactic acid bacteria liquid from fecal bacteria and its application, the preparation method comprising the following steps: Step 1, culture medium preparation: preparing solid culture medium and liquid culture medium for preparing lactic acid bacteria liquid; Step 2, feces collection: selecting healthy donors for lactic acid bacteria liquid preparation and collecting feces from healthy donors; Step 3, fecal microbial liquid preparation: prepare the feces of the donor to obtain the fecal microbial liquid; Step 4, comparison sample preparation: prepare the feces of the donor and obtain fecal bacteria capsules; Step 5, lactic acid bacteria culture: put the solid culture medium into the plate, take the fecal bacteria liquid and put it into the plate, and culture it in the incubator to obtain lactic acid bacteria; Step 6, lactic acid bacteria identification: selecting lactic acid bacteria identification samples from the incubator, preparing a strain culture solution for lactic acid bacteria identification, and determining the type of lactic acid bacteria by gene sequencing; Step 7, culturing lactic acid bacteria liquid: placing lactic acid bacteria in a liquid culture medium, and culturing in an incubator to obtain lactic acid bacteria liquid.

[0038] Specifically, the specific method for preparing the solid culture medium is as follows: weighing each component of the solid culture medium according to the weight proportion of the solid culture medium, dissolving each component of the solid culture medium in deionized water, sterilizing by high pressure, cooling and adjusting the pH, to obtain a solid culture medium; The solid culture medium comprises the following components by weight: 6 parts of peptone, 4 parts of yeast extract powder, 5 parts of beef extract powder, 4 parts of glucose, 2 parts of dipotassium hydrogen phosphate, 1 part of triammonium citrate, 0.2 parts of magnesium sulfate, 0.1 parts of manganese sulfate, 0.8 parts of Tween-80, 0.2 parts of sodium acetate, 3 parts of guava powder, 5 parts of applesauce, 3 parts of Chinese herbal medicine powder, 0.1 parts of mupirocin lithium salt, 0.2 parts of vitamins and 13 parts of agar; The high pressure sterilization pressure is 137 kPa, the temperature reaches 126 ° C, and the sterilization purpose can be achieved by maintaining it for 20 minutes, and the pH is adjusted to 6.2; The specific method for preparing the liquid culture medium is as follows: weighing each component of the liquid culture medium according to the weight of the liquid culture medium, dissolving each component of the liquid culture medium in the feces filtrate, and sterilizing by high pressure, cooling and adjusting the pH to obtain the liquid culture medium; The liquid culture medium comprises, by weight, 6 parts of peptone, 4 parts of yeast extract powder, 5 parts of beef extract powder, 4 parts of glucose, 2 parts of dipotassium hydrogen phosphate, 3 parts of guava powder, 5 parts of applesauce, 3 parts of Chinese herbal medicine powder, 0.2 parts of vitamins and 13 parts of agar; The high pressure sterilization pressure is 137 kPa, the temperature reaches 126° C., and the sterilization purpose can be achieved by maintaining it for 20 minutes, and the pH is adjusted to 6.2.

[0039] Specifically, the healthy donor for feces collection is a 50-day-old healthy pig, and feces B of a 50-day-old pig is obtained.

[0040] Specifically, the specific method for preparing the fecal bacteria liquid is as follows: take feces B into container 2, add sterile physiological saline into container 2 and seal it, put container 2 into a shaker and shake it evenly, then let it stand and precipitate, take the supernatant, and obtain fecal bacteria liquid B for later use.

[0041] Specifically, the specific method for preparing the fecal bacteria capsule is as follows: during the preparation of the fecal bacteria liquid, the fecal bacteria in the sediment after standing and settling to obtain the supernatant are collected, the fecal bacteria in the sediment and the freeze-dried protective liquid are added to a mixing container and mixed to obtain the fecal bacteria liquid, the fecal bacteria liquid is cooled, freeze-dried, and powdered, and then the capsule shell is filled to prepare the fecal bacteria capsule; The freeze-dried protective solution comprises, by weight, 1 part of sucrose, 11 parts of soluble starch, 10 parts of anhydrous solvent, 2 parts of surfactant, 0.5 parts of amino acid and 1 part of citrate; The fecal bacteria liquid is cooled to -10°C and then freeze-dried. After freeze-drying, it is ground into powder using a grinder, frozen at -75°C for 24 hours, and then freeze-dried at a vacuum degree of -30KPa and a drying temperature of -60°C for 24 hours.

[0042] Specifically, the specific method for culturing lactic acid bacteria is as follows: put fecal bacteria liquid B into solid culture medium plate 2, culture lactic acid bacteria by plate pouring method, and after coagulation, invert and place it in a 35°C anaerobic incubator for 58 hours to obtain lactic acid bacteria B.

[0043] Specifically, the specific method for culturing the lactic acid bacteria liquid is as follows: liquid culture medium is placed in a culture dish 2, lactic acid bacteria B is added to the culture dish 2, and the culture dish is placed in a 35° C. anaerobic incubator for 58 hours to obtain lactic acid bacteria liquid B.

[0044] Specifically, the Chinese herbal medicine powder in the solid culture medium component is selected from Ophiopogon japonicus, and the Chinese herbal medicine powder in the liquid culture medium component is selected from a mixture of honeysuckle and coptis chinensis in a ratio of 1:1, and the Chinese herbal medicine powder is prepared by removing impurities from raw materials, washing, freeze-drying, pulverizing, and sieving and sorting; The freeze-drying process of the Chinese herbal medicine powder raw material freezes the cleaned raw material at -75°C for 12 hours, and then freeze-dries it at a vacuum degree of -30KPa and a drying temperature of -60°C for 10 hours. After freezing, the freeze-dried raw material is crushed by a pulverizer, and then sieved by a screening machine with a mesh size of 300 meshes.

[0045] Example 3

[0046] A method for preparing lactic acid bacteria liquid from fecal bacteria and its application, the preparation method comprising the following steps: Step 1, culture medium preparation: preparing solid culture medium and liquid culture medium for preparing lactic acid bacteria liquid; Step 2, feces collection: selecting healthy donors for lactic acid bacteria liquid preparation and collecting feces from healthy donors; Step 3, fecal microbial liquid preparation: prepare the feces of the donor to obtain the fecal microbial liquid; Step 4, comparison sample preparation: prepare the feces of the donor and obtain fecal bacteria capsules; Step 5, lactic acid bacteria culture: put the solid culture medium into the plate, take the fecal bacteria liquid and put it into the plate, and culture it in the incubator to obtain lactic acid bacteria; Step 6, lactic acid bacteria identification: selecting lactic acid bacteria identification samples from the incubator, preparing a strain culture solution for lactic acid bacteria identification, and determining the type of lactic acid bacteria by gene sequencing; Step 7, culturing lactic acid bacteria liquid: placing lactic acid bacteria in a liquid culture medium, and culturing in an incubator to obtain lactic acid bacteria liquid.

[0047] Specifically, the specific method for preparing the solid culture medium is as follows: weighing each component of the solid culture medium according to the weight proportion of the solid culture medium, dissolving each component of the solid culture medium in deionized water, sterilizing by high pressure, cooling and adjusting the pH, to obtain a solid culture medium; The solid culture medium comprises the following components by weight: 5 parts of peptone, 2.5 parts of yeast extract powder, 6 parts of beef extract powder, 3.5 parts of glucose, 1.5 parts of dipotassium hydrogen phosphate, 0.8 parts of triammonium citrate, 0.15 parts of magnesium sulfate, 0.15 parts of manganese sulfate, 0.6 parts of Tween-80, 0.15 parts of sodium acetate, 2 parts of guava powder, 6 parts of applesauce, 4 parts of Chinese herbal medicine powder, 0.15 parts of mupirocin lithium salt, 0.15 parts of vitamins and 10 parts of agar; When the pressure of the high pressure sterilization is 120 kPa and the temperature reaches 123°C, the sterilization purpose can be achieved by maintaining it for 25 minutes, and the pH is adjusted to 6.4; The specific method for preparing the liquid culture medium is as follows: weighing each component of the liquid culture medium according to the weight of the liquid culture medium, dissolving each component of the liquid culture medium in the feces filtrate, and sterilizing by high pressure, cooling and adjusting the pH to obtain the liquid culture medium; The liquid culture medium comprises, by weight, 5 parts of peptone, 2.5 parts of yeast extract powder, 6 parts of beef extract powder, 3.5 parts of glucose, 1.5 parts of dipotassium hydrogen phosphate, 2 parts of guava powder, 6 parts of applesauce, 4 parts of Chinese herbal medicine powder, 0.15 parts of vitamins and 10 parts of agar; The high pressure sterilization pressure is 120 kPa, the temperature reaches 123° C., and the sterilization purpose can be achieved by maintaining it for 25 minutes, and the pH is adjusted to 6.4.

[0048] Specifically, the healthy donors for feces collection are two kinds of healthy pigs of 50 days old and 70 days old, and feces B of 50-day-old pigs and feces C of 70-day-old pigs are obtained respectively.

[0049] Specifically, the specific method for preparing the fecal bacteria liquid is as follows: feces B is placed in container 2 and feces C is placed in container 3 respectively, sterile saline is added to container 2 and container 3 and sealed, container 2 and container 3 are placed in a shaker together and shaken, then allowed to stand and precipitate to obtain the supernatant to obtain fecal bacteria liquid B and fecal bacteria liquid C for use.

[0050] Specifically, the specific method for preparing the fecal bacteria capsule is as follows: during the preparation of the fecal bacteria liquid, the fecal bacteria in the sediment after standing and settling to obtain the supernatant are collected, the fecal bacteria in the sediment and the freeze-dried protective liquid are added to a mixing container and mixed to obtain the fecal bacteria liquid, the fecal bacteria liquid is cooled, freeze-dried, and powdered, and then the capsule shell is filled to prepare the fecal bacteria capsule; The freeze-dried protective solution comprises 1.5 parts of sucrose, 9 parts of soluble starch, 8 parts of anhydrous solvent, 1 part of surfactant, 0.3 parts of amino acid and 0.7 parts of citrate in parts by weight; The fecal bacteria liquid is cooled to -10°C and then freeze-dried. After freeze-drying, it is ground into powder using a grinder, frozen at -75°C for 18 hours, and then freeze-dried at a vacuum degree of -30KPa and a drying temperature of -60°C for 36 hours.

[0051] Specifically, the specific method for culturing lactic acid bacteria is as follows: solid culture medium is loaded into plate 2 and plate 3 respectively, fecal bacteria liquid B is put into plate 2 and fecal bacteria liquid C is put into plate 3, lactic acid bacteria are cultured by plate pouring method, and after coagulation, the plate is inverted and placed in a 36°C anaerobic incubator for 55 hours to obtain lactic acid bacteria B and lactic acid bacteria C.

[0052] Specifically, the specific method for culturing the lactic acid bacteria liquid is as follows: liquid culture medium is loaded into culture dish 2 and culture dish 3, lactic acid bacteria B is added to culture dish 2 and lactic acid bacteria C is added to culture dish 3, and the mixture is placed in a 36° C. anaerobic incubator and cultured for 55 hours to obtain lactic acid bacteria liquid B and lactic acid bacteria liquid C.

[0053] Specifically, the Chinese herbal medicine powder in the solid culture medium component is a mixture of Shenqu and Radix Ophiopogonis in a ratio of 1:1, and the Chinese herbal medicine powder in the liquid culture medium component is a mixture of Flos Lonicerae and Radix Isatidis in a ratio of 1:1, and the Chinese herbal medicine powder is prepared by removing impurities from raw materials, washing, freeze-drying, pulverizing, and sieving and sorting; The freeze-drying process of the Chinese herbal medicine powder raw material freezes the cleaned raw material at -75°C for 9 hours, and then freeze-dries it at a vacuum degree of -30KPa and a drying temperature of -60°C for 12 hours. After freezing, the freeze-dried raw material is crushed by a pulverizer, and then sieved by a screening machine with a mesh size of 300 meshes.

[0054] Comparative Example 1 Except that the solid culture medium and the liquid culture medium are different from those in Example 1, the other conditions of Comparative Example 1 are consistent with those of Example 1. The solid culture medium of Comparative Example 1 includes the following raw materials by weight: 6.5 g peptone, 3.0 g skim milk powder, 14.0 g beef extract, 4.0 g yeast extract, 4.0 g glucose, 8.0 g banana puree, 2.0 g dipotassium hydrogen phosphate, 0.2 g magnesium sulfate, 0.03 g manganese sulfate, 1.5 g calcium carbonate, 3.0 g dry grapefruit peel powder, 0.8 g Tween 80, and 13.0 g agar; the liquid culture medium of Comparative Example 1 includes the following raw materials by weight: 8.0 g peptone, 5.0 g skim milk powder, 10.0 g beef extract, 10.0 g banana puree, 5.0 g dry grapefruit peel powder, 12.0 g sodium humate, 2.0 g sodium carboxymethyl cellulose, 0.3 g magnesium sulfate, and 0.05 g manganese sulfate.

[0055] Table 1

[0056] It can be seen from Table 1 that the bacterial amount and bacterial species number of the lactic acid bacteria liquid prepared by the culture medium used in the present invention are higher than those of the lactic acid bacteria liquid prepared by the culture medium of Comparative Example 1.

[0057] Table 2

[0058] It can be seen from Table 2 that the lactic acid bacteria liquid prepared by the culture medium containing Chinese herbal medicine in the present invention has a longer shelf life than the lactic acid bacteria liquid prepared by the culture medium in Comparative Example 1.

[0059] Experiment 1: 28-day-old piglets were selected and divided into three groups: A, B, and C. Group A piglets were fed daily feed; Group B piglets were fed with fecal bacteria capsules every seven days, three times a day, 10 g per head each time on feeding days; Group C piglets were fed with lactic acid bacteria liquid every seven days, three times a day, 10 ml per head each time on feeding days. The diarrhea and weight gain of the three groups of piglets were recorded during the period of 30-90 days of age.

[0060] Table 3

[0061] It can be seen from Table 3 above that, using the lactic acid bacteria liquid and fecal bacteria capsules prepared by the present invention, the diarrhea rate of piglets in group C using the lactic acid bacteria liquid was the lowest and the weight gain was the largest, and the diarrhea rate of piglets in group B using the fecal bacteria capsules was lower than that of group A and the weight gain was greater than that of group A.

[0062] Experiment 2: Select piglets and divide them into three groups, A, B and C. The three groups of piglets are fed with daily feed during breeding. The breeding test period of the three groups of piglets is 120 days. When diarrhea symptoms appear in piglets in group A during the breeding period, they are fed with fecal bacteria capsules for three consecutive days, three times a day, and 10 g each time; when diarrhea symptoms appear in piglets in group B during the breeding period, the lactic acid bacteria solution in comparative example 1 is fed for three consecutive days, three times a day, and 10 ml each time; when diarrhea symptoms appear in piglets in group C during the breeding period, the lactic acid bacteria solution in Example 2 is fed for three consecutive days, three times a day, and 10 ml each time. The diarrhea and weight gain of the three groups of piglets during the 120-day test period are recorded.

[0063] Table 4

[0064] It can be seen from Table 4 that during the test period, the piglets in Group C using the lactic acid bacteria liquid prepared by the present invention had the lowest diarrhea rate and the lowest feed-to-meat ratio.

[0065] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for preparing lactic acid bacteria liquid from fecal bacteria, characterized in that: The preparation method comprises the following steps: Step 1, culture medium preparation: preparing solid culture medium and liquid culture medium for preparing lactic acid bacteria liquid; Step 2, feces collection: selecting healthy donors for lactic acid bacteria liquid preparation and collecting feces from healthy donors; Step 3, fecal microbial liquid preparation: prepare the feces of the donor to obtain fecal microbial liquid; Step 4, comparison sample preparation: prepare the feces of the donor and obtain fecal bacteria capsules; Step 5, lactic acid bacteria culture: put the solid culture medium into the plate, take the fecal bacteria liquid and put it into the plate, and culture it in the incubator to obtain lactic acid bacteria; Step 6, lactic acid bacteria identification: selecting lactic acid bacteria identification samples from the incubator, preparing a strain culture solution for lactic acid bacteria identification, and determining the type of lactic acid bacteria by gene sequencing; Step 7, culturing lactic acid bacteria liquid: placing lactic acid bacteria in a liquid culture medium, and culturing in an incubator to obtain lactic acid bacteria liquid.

2. A method for preparing lactic acid bacteria liquid from fecal bacteria according to claim 1, characterized in that: The specific method for preparing the solid culture medium is as follows: weighing each component of the solid culture medium according to the weight proportion of the solid culture medium, dissolving each component of the solid culture medium in deionized water, sterilizing by high pressure, cooling and adjusting the pH to obtain the solid culture medium; The solid culture medium comprises the following components by weight: 4-6 parts of peptone, 3-4 parts of yeast extract powder, 5-8 parts of beef extract powder, 3-4 parts of glucose, 1-2 parts of dipotassium hydrogen phosphate, 0.5-1 parts of triammonium citrate, 0.1-0.2 parts of magnesium sulfate, 0.1-0.2 parts of manganese sulfate, 0.3-0.8 parts of Tween-80, 0.1-0.2 parts of sodium acetate, 1-3 parts of guava powder, 5-7 parts of applesauce, 3-5 parts of Chinese herbal medicine powder, 0.1-0.2 parts of mupirocin lithium salt, 0.1-0.2 parts of vitamins and 8-13 parts of agar; The high pressure sterilization pressure is 103-137 kPa, the temperature reaches 121-126° C., maintained for 20-30 minutes, and the pH is adjusted to 6.2-6.5; The specific method for preparing the liquid culture medium is as follows: weighing each component of the liquid culture medium according to the weight of the liquid culture medium, dissolving each component of the liquid culture medium in the feces filtrate, and sterilizing by high pressure, cooling and adjusting the pH to obtain the liquid culture medium; The liquid culture medium comprises, by weight, 4-6 parts of peptone, 3-4 parts of yeast extract powder, 5-8 parts of beef extract powder, 3-4 parts of glucose, 1-2 parts of dipotassium hydrogen phosphate, 1-3 parts of guava powder, 5-7 parts of applesauce, 3-5 parts of Chinese herbal medicine powder, 0.1-0.2 parts of vitamins and 8-13 parts of agar; The pressure of the high pressure sterilization is 103-137 kPa, the temperature reaches 121-126° C., maintained for 20-30 minutes, and the pH is adjusted to 6.2-6.

5.

3. A method for preparing lactic acid bacteria liquid from fecal bacteria according to claim 1, characterized in that: The healthy donors for feces collection are selected from three healthy pigs of 28 days, 50 days and 70 days old, and feces are collected to obtain feces A of 28-day-old pig, feces B of 50-day-old pig and feces C of 70-day-old pig, respectively.

4. A method for preparing lactic acid bacteria liquid from fecal bacteria according to claim 3, characterized in that: The specific method for preparing the fecal bacteria liquid is as follows: feces A is placed in container 1, feces B is placed in container 2, and feces C is placed in container 3, respectively, sterile physiological saline is added to container 1, container 2, and container 3, and sealed, container 1, container 2, and container 3 are placed in a shaker together and shaken evenly, and then allowed to stand and precipitate to obtain the supernatant to obtain fecal bacteria liquid A, fecal bacteria liquid B, and fecal bacteria liquid C for use.

5. A method for preparing lactic acid bacteria liquid from fecal bacteria according to claim 4, characterized in that: The specific method for preparing the fecal bacteria capsule is as follows: during the preparation of the fecal bacteria liquid, the fecal bacteria in the sediment after standing and settling to obtain the supernatant are collected, the fecal bacteria in the sediment and the freeze-dried protective liquid are added to a mixing container and mixed to obtain the fecal bacteria liquid, the fecal bacteria liquid is cooled, freeze-dried, and powdered, and then the capsule shell is filled to prepare the fecal bacteria capsule; The freeze-dried protective solution comprises, by weight, 1-2 parts of sucrose, 7-11 parts of soluble starch, 6-10 parts of anhydrous solvent, 0.5-2 parts of surfactant, 0.2-0.5 parts of amino acid and 0.5-1 parts of citrate; The fecal bacteria liquid is cooled to -10°C and then freeze-dried. After freeze-drying, it is ground into powder using a grinder, frozen at -75°C for 12-24 hours, and then freeze-dried at a vacuum degree of -30KPa and a drying temperature of -60°C for 24-48 hours.

6. A method for preparing lactic acid bacteria liquid from fecal bacteria according to claim 4, characterized in that: The specific method for culturing lactic acid bacteria is as follows: solid culture medium is respectively loaded into plate 1, plate 2 and plate 3, fecal bacteria liquid A is put into plate 1, fecal bacteria liquid B is put into plate 2 and fecal bacteria liquid C is put into plate 3, lactic acid bacteria are cultured by plate pouring method, after coagulation, the plate is inverted and placed in an anaerobic incubator at 35-37°C for 50-58h to obtain lactic acid bacteria A, lactic acid bacteria B and lactic acid bacteria C.

7. The method for preparing lactic acid bacteria liquid from fecal bacteria according to claim 4, characterized in that: The specific method of culturing the lactic acid bacteria liquid is as follows: liquid culture medium is loaded into culture dish 1, culture dish 2 and culture dish 3, lactic acid bacteria A is added to culture dish 1, lactic acid bacteria B is added to culture dish 2 and lactic acid bacteria C is added to culture dish 3, and the mixture is placed in an anaerobic incubator at 35-37° C. and cultured for 50-58 hours to obtain lactic acid bacteria liquid A, lactic acid bacteria liquid B and lactic acid bacteria liquid C.

8. The method for preparing lactic acid bacteria liquid from fecal bacteria according to claim 2, characterized in that: The Chinese herbal medicine powder in the solid culture medium component is a mixture of one or more of malt, hawthorn, Shenqu, Ophiopogon japonicus and Codonopsis pilosula, and the Chinese herbal medicine powder in the liquid culture medium component is a mixture of one or more of honeysuckle, houttuynia cordata, Artemisia annua, Radix Isatidis and Coptis chinensis, and the Chinese herbal medicine powder is prepared by raw material impurity removal, washing, freeze-drying, pulverization and sieving and sorting in sequence; The freeze-drying process for the Chinese herbal medicine powder raw material freezes the cleaned raw material at -75°C for 6-12 hours, and then freeze-dries it at a vacuum degree of -30KPa and a drying temperature of -60°C for 10-14 hours. After freezing, the freeze-dried raw material is crushed by a pulverizer, and then sieved by a screening machine with a mesh size of 300 meshes.

9. Use of a lactic acid bacteria liquid prepared by the method for preparing a lactic acid bacteria liquid from fecal bacteria as described in any one of claims 1 to 8 in preparing functional bacteria for fecal bacteria transplantation.

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