Application of lactobacillus rhamnosus B12.3 and metabolite of lactobacillus rhamnosus B12.3 in promoting growth and reproduction of AKK bacteria

Abstract

The invention relates to application of lactobacillus rhamnosus B12.3 and metabolites thereof in promoting growth and reproduction of AKK bacteria, and belongs to the technical field of microorganisms. The preservation unit of the lactobacillus rhamnosus B12.3 is China General Microbiological Culture Collection Center, the preservation time is December 25, 2024, and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the preservation number of the lactobacillus rhamnosus strain is CGMCC (China General Microbiological Culture Collection Center) NO: 33186, and the lactobacillus rhamnosus strain is classified and named as lactobacillus rhamnosus. The extracellular metabolite of the lactobacillus rhamnosus can effectively promote the growth and reproduction of the AKK bacteria, and also can increase the survival rate of the AKK bacteria in gastric juice and intestinal juice. The discovery provides a certain reference and basis for AKK bacterium culture, and is expected to bring new breakthrough for industrial production of AKK bacteria.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to application of Lactobacillus rhamnosus B12.3 and metabolites thereof in promoting the growth and reproduction of AKK bacteria. Background Art

[0002] Lactic acid bacteria, as a type of bacteria that can produce a large amount of lactic acid during metabolism, are catalase-negative and Gram-positive, are widely present in every corner of nature. Not only are they widely used in many fields such as food industry, pharmaceutical manufacturing and agricultural production due to their recognized safety, but their extracellular metabolites are also often regarded as safe and harmless valuable resources. In addition to the main product lactic acid, other metabolites of lactic acid bacteria, such as exopolysaccharides and bacteriocins, have also attracted great interest from many researchers.

[0003] As an important metabolite of lactic acid bacteria, exopolysaccharide has multiple biological activities. It exhibits strong antioxidant capacity, can effectively remove free radicals in the body, and protect cells from oxidative damage. At the same time, exopolysaccharide also has antibacterial and anti-inflammatory effects, can inhibit the growth of harmful microorganisms, and reduce inflammatory responses. In addition, it also has immunomodulatory effects, can regulate the body's immune function and enhance the body's resistance. In addition to these biological activities, exopolysaccharides are also widely used as thickeners, emulsifiers and stabilizers in the food industry due to their unique physical and chemical properties, providing a strong guarantee for the quality and taste of food.

[0004] On the other hand, some lactic acid bacteria can also produce bacteriocins, which are safe, non-toxic and easy to industrialize. They can inhibit the growth of many harmful microorganisms, so they have broad application prospects in the field of food preservation. By using bacteriocins for food preservation, it is not only possible to extend the shelf life of food, but also to maintain the nutritional content and taste of food, meeting consumers' needs for food safety and health.

[0005] However, despite the many advantages and application values ​​of lactic acid bacteria and their metabolites, they still face some challenges in certain specific fields, such as the industrial production of Akkermansia muciniphila (AKK bacteria). As an intestinal bacterium, AKK bacteria mainly colonizes the intestinal mucosal layer. In recent years, it has shown outstanding effects in regulating the intestinal barrier, controlling weight, anti-cancer and delaying aging. However, due to the high technical requirements of AKK bacteria for isolation, cultivation, preservation, etc., its industrial production has certain difficulties. In response to this problem, the existing technology urgently needs to find an effective method to promote the growth and reproduction of AKK bacteria and improve its survival rate, so as to realize the localized production of AKK bacteria. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the present invention provides the application of Lactobacillus rhamnosus B12.3 and its metabolites in promoting the growth and reproduction of AKK bacteria, and a strain of Lactobacillus rhamnosus is screened from female vaginal secretions. The extracellular metabolites of Lactobacillus rhamnosus can effectively promote the growth and reproduction of AKK bacteria, and can also increase the survival rate of AKK bacteria in gastric juice and intestinal juice. This discovery provides a certain reference and basis for the cultivation of AKK bacteria, and is expected to bring new breakthroughs to the industrial production of AKK bacteria.

[0007] To achieve the above object, the present invention provides the following technical solutions: A rhamnosus lactobacillus B12.3, wherein the rhamnosus lactobacillus B12.3 is isolated from human vaginal secretions, and the preservation unit of the rhamnosus lactobacillus B12.3 is: General Microbiology Center of China Microorganism Culture Collection Administration, the preservation time is: December 25, 2024, the preservation address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the preservation number is CGMCC NO: 33186, and the classification name is: Rhamnosus lactobacillus Lacticaseibacillus rhamnosus .

[0008] The present invention also provides a Lactobacillus rhamnosus metabolite, which is produced by the metabolism of the Lactobacillus rhamnosus B12.3.

[0009] The present invention also provides a method for preparing a Lactobacillus rhamnosus metabolite. The method comprises the following steps: activating Lactobacillus rhamnosus B12.3 and inoculating the activated Lactobacillus rhamnosus B12.3 into a culture medium, anaerobic culturing the culture medium at 37°C for 24 hours to obtain a fermentation liquid, centrifuging the fermentation liquid at 8000 rpm / min to 10000 rpm / min for 5 minutes to 10 minutes to obtain a supernatant, performing secondary precipitation on the supernatant and centrifuging the supernatant at 5000 rpm / min to 8000 rpm / min for 5 minutes to 10 minutes, and freeze-drying the obtained precipitate to obtain the Lactobacillus rhamnosus metabolite.

[0010] Furthermore, the culture medium raw materials of the Lactobacillus rhamnosus B12.3 are: 1L pumpkin juice, 30 g / L glucose, 20 g / L beef powder, 10 g / L yeast powder, 10 g / L milk powder; The pumpkin juice preparation method comprises the following steps: pumpkin and water are mixed and pulped in a mass ratio of 1:1, and the pumpkin juice is obtained by filtering through 100 meshes.

[0011] The present invention also provides the use of the above-mentioned Lactobacillus rhamnosus metabolites in promoting the growth of AKK bacteria. When 0.2g / 100ml-0.8g / 100ml of the Lactobacillus rhamnosus metabolites are added to the culture medium, the growth of AKK bacteria can be promoted when the culture medium is used to culture AKK bacteria.

[0012] The present invention also provides the use of the above-mentioned Lactobacillus rhamnosus metabolites in improving the gastrointestinal fluid tolerance of AKK bacteria, wherein AKK bacteria and the Lactobacillus rhamnosus metabolites are mixed, wherein 2 mg to 12 mg of the Lactobacillus rhamnosus metabolites are added to the bacterial cells obtained per milliliter of AKK bacterial liquid.

[0013] The present invention also provides the use of the above-mentioned Lactobacillus rhamnosus metabolites in preparing an AKK bacteria culture medium, characterized in that the addition amount of the Lactobacillus rhamnosus metabolites in the AKK bacteria culture medium is 0.2 g / 100 ml to 0.8 g / 100 ml.

[0014] The present invention also provides a method for promoting the growth of AKK bacteria, wherein the above-mentioned Lactobacillus rhamnosus metabolites are added to the AKK bacteria culture medium, and the added amount of the Lactobacillus rhamnosus metabolites is 0.2 g / 100 ml to 0.8 g / 100 ml.

[0015] The present invention also provides a method for improving the gastrointestinal survival rate of AKK bacteria, wherein the AKK bacteria are mixed with rhamnosus lactobacillus metabolites, wherein 2 mg to 12 mg of rhamnosus lactobacillus metabolites are added to the bacterial bodies obtained per milliliter of AKK bacterial liquid.

[0016] The present invention also provides a culture medium, which contains the above-mentioned Lactobacillus rhamnosus metabolites, and the added amount of the Lactobacillus rhamnosus metabolites is 0.2g / 100ml-0.8g / 100ml. The culture medium is used for culturing AKK bacteria.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: The present invention successfully screens a strain of Lactobacillus rhamnosus B12.3 from female vaginal secretions. As a microorganism recognized as safe, Lactobacillus rhamnosus B12.3 has high safety, and its metabolites also have good safety. This discovery not only enriches the diversity of microbial resources, but also provides a reliable basis for subsequent research and application. Through a specific preparation method, the extracellular metabolites of Lactobacillus rhamnosus can be obtained, and the metabolites have multiple biological activities, providing a wide space for subsequent applications.

[0018] The extracellular metabolites of Lactobacillus rhamnosus B12.3 in the present invention have the effect of significantly promoting the growth and reproduction of AKK bacteria. By optimizing the culture medium and using raw materials such as pumpkin juice, a culture medium that can enhance the synthesis of extracellular metabolites by Lactobacillus rhamnosus is obtained. The extracellular polysaccharides of Lactobacillus rhamnosus can specifically or non-specifically bind to a variety of proteins to form a "sugar-protein mixture" that is beneficial to the growth of AKK bacteria. The extracellular metabolites of Lactobacillus rhamnosus B12.3 of the present invention provide the necessary energy for the growth and reproduction of AKK bacteria. This discovery provides a new method and idea for the cultivation of AKK bacteria, which is expected to increase the yield and activity of AKK bacteria and bring breakthroughs to industrial production.

[0019] In addition to promoting the growth and reproduction of AKK bacteria, the extracellular metabolites of Lactobacillus rhamnosus B12.3 in the present invention can also significantly enhance the tolerance of AKK bacteria to gastric and intestinal fluids. This may be because the extracellular polysaccharides encapsulate the AKK bacteria, reducing the direct contact between gastrointestinal fluid and AKK bacteria, thereby effectively protecting the AKK bacteria from damage by gastrointestinal fluid. This discovery provides strong support for the survival and application of AKK bacteria in the gastrointestinal environment, and is expected to broaden the application areas of AKK bacteria, such as probiotic products, health care products, etc. At the same time, it also provides new ideas and references for the study of gastrointestinal fluid tolerance of other beneficial microorganisms.

[0020] In summary, the Lactobacillus rhamnosus B12.3 and its extracellular metabolites screened by the present invention have significant advantages in promoting the growth and reproduction of AKK bacteria and increasing its survival rate in gastric juice and intestinal juice, and are expected to bring new breakthroughs in the industrial production and application of AKK bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The colony image and microscopic examination image of Lactobacillus rhamnosus B12.3 grown on MRS plate for 48 hours.

[0022] Figure 2 Phylogenetic tree of Lactobacillus rhamnosus.

[0023] Figure 3 This is the effect of M12.3 on the growth curve of AKK bacteria.

[0024] Figure 4 This is the effect of M12.3 on the tolerance of AKK bacteria to gastric juice and intestinal juice. DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0026] Example 1 The present invention provides a strain of Lactobacillus rhamnosus B12.3, which is isolated from human vaginal secretions. The deposit unit is: General Microbiology Center of China Microbiological Culture Collection Administration, the deposit time is: December 25, 2024, the deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC NO: 33186, and the classification name is: Lactobacillus rhamnosus Lacticaseibacillus rhamnosus .

[0027] The screening and identification of Lactobacillus rhamnosus B12.3 are as follows: Screening: Add 5 ml of saline to the vaginal secretion sample, mix well, and then dilute it 10 times in a gradient. Take 100 μL of the appropriate gradient dilution solution and apply it to MRS solid medium. Culture it in a 37°C anaerobic incubator for 3 days. Select single colonies with different morphology, colonies with stringy or mucus, name them, and purify them by streaking on MRS solid medium. Then, culture them in a 37°C anaerobic incubator for 3 days. Pick a single colony from the purified plate for liquid culture to the logarithmic phase, then store it in 50% anaerobic glycerol and store it in a -80°C refrigerator for long-term storage.

[0028] like Figure 1 As shown in (A), after the strain B12.3 was cultured on MRS solid medium for 48 hours, its colonies were round, white, moist and shiny; Figure 1 (B) It can be seen that bacteria B12.3 is Gram-positive and rod-shaped.

[0029] Identification: 16s rDNA sequencing method was used, and bacterial universal primers 27F and 1492R were selected to perform PCR amplification on bacteria B12.3. The qualified amplified products were sent to Sangon Biotech Co., Ltd. for sequencing after agarose gel electrophoresis. The sequence of bacteria B12.3 is shown in Sequence 1: Submit the sequencing results to EZBiocloud for homology search, find the strain with the greatest homology to the target gene sequence and construct its phylogenetic tree, such as Figure 2 As shown, strain B12.3 is most closely related to Lactobacillus rhamnosus. Comparison analysis in EZ Biocloud shows that the similarity between strain B12.3 and Lactobacillus rhamnosus is 99.80%. Therefore, strain B12.3 can be identified as Lactobacillus rhamnosus and named Lactobacillus rhamnosus B12.3.

[0030] Example 2 In order to increase the yield of metabolites, the present invention optimizes the culture medium of Lactobacillus rhamnosus B12.3 and provides five culture media A to E, which are as follows: Medium A (MRS liquid medium): 20 g / L glucose, 10 g / L peptone, 5 g / L beef powder, 4 g / L yeast powder, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium hydrogen citrate, 5 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, and 1.0 mL / L Tween 80.

[0031] Culture medium B: 1L pumpkin juice, 30 g / L glucose, 20 g / L beef powder, 10g / L yeast powder, 10g / L milk powder (Pumpkin juice preparation method: pumpkin and water are slurried in a homogenizer at a mass ratio of 1:1, and then filtered with a 100-mesh stainless steel filter to obtain pumpkin juice).

[0032] Culture medium C: 1L apple juice, 20 g / L glucose, 20 g / L peptone, 4 g / L yeast powder, 10 g / L egg white (apple juice preparation method: apples and water are slurried in a homogenizer at a mass ratio of 1:1, and then filtered with a 100-mesh stainless steel filter to obtain apple juice).

[0033] Culture medium D: 1L tomato juice, 40 g / L glucose, 10 g / L peptone, 10 g / L beef powder, 5g / L yeast powder, 10g / L egg yolk (tomato juice preparation method: tomatoes and water are slurried in a homogenizer at a mass ratio of 1:1, and then filtered with a 100-mesh stainless steel filter to obtain tomato juice).

[0034] Culture medium E: 1L carrot juice, 30 g / L glucose, 10 g / L peptone, 5 g / L beef powder, 10 g / L yeast powder (carrot preparation method: carrots and water are slurried in a homogenizer at a mass ratio of 1:1, and then filtered with a 100-mesh stainless steel filter to obtain carrot juice).

[0035] The Lactobacillus rhamnosus B12.3 stored in a glycerol tube was inoculated into MRS broth for activation. After activation, it was transferred to a new MRS broth at a ratio of 1%, and cultured anaerobically at 37°C for 12 hours to obtain seed liquid; 1% of the seed liquid was added to the above 5 culture media, and cultured anaerobically at 37°C for 24 hours. After fermentation, the fermentation liquid was centrifuged at 8000rpm / min for 10 minutes, the supernatant was taken, 3 times the volume of anhydrous ethanol was added to the supernatant, and it was allowed to stand at 4°C for 8 hours. Take it out, centrifuge it at 6000rpm / min for 10 minutes, discard the supernatant, collect the precipitate, and freeze-dry it to obtain the Lactobacillus rhamnosus metabolite. After weighing, take about 0.1g of the metabolite to determine its protein content, sugar content and water content.

[0036] The metabolite yields of Lactobacillus rhamnosus B12.3 in different culture media are shown in Table 1: Table 1 Metabolite yields of Lactobacillus rhamnosus B12.3 fermented in different culture media

[0037] As can be seen from Table 1, the culture medium greatly affects the yield of strain metabolites. Culture medium A is MRS liquid culture medium, which is one of the commonly used culture media for lactic acid bacteria culture. The metabolite yield of the strain in MRS culture medium is 0.91g / L, which is relatively low. The metabolite yield in culture medium B is 4.12g / L, which is 4.5 times that of MRS culture medium. In addition, the sugar content of the metabolites produced by the strain cultured in culture medium B accounts for 68.31%, and the protein content accounts for 26.13%, indicating that sugars and proteins are the main components of the metabolites.

[0038] Example 3 The above-mentioned culture medium B is used to culture Lactobacillus rhamnosus B12.3, and the metabolites of Lactobacillus rhamnosus are extracted. The specific steps are as follows: The Lactobacillus rhamnosus B12.3 stored in a glycerol tube was inoculated into MRS broth for activation. After activation, it was transferred into new MRS broth at a ratio of 1%, and anaerobically cultured at 37°C for 12 hours to obtain seed liquid. Take 1% of the seed solution in medium B, culture it anaerobically at 37°C for 24 hours, and obtain the fermentation liquid after the fermentation is completed; The fermentation liquid was centrifuged at 8000 rpm / min~10000 rpm / min for 5 min~10 min, and the supernatant was taken. 3 volumes of anhydrous ethanol were added to the supernatant, and the mixture was allowed to stand at 4°C for 8 hours to precipitate the macromolecular substances in the fermentation product, including polysaccharides and some proteins.

[0039] The mixture was taken out and centrifuged at 5000 rpm / min~8000 rpm / min for 5 min~10 min, the supernatant was discarded, the precipitate was collected, and after freeze-drying, the Lactobacillus rhamnosus metabolite was obtained and named M12.3.

[0040] Example 4 The effects of the metabolite M12.3 of Lactobacillus rhamnosus on the growth of AKK bacteria were explored as follows: After 24 hours of activation, AKK bacteria were inoculated at a ratio of 0.5% into BHI medium containing different concentrations (0, 0.1, 0.2, 0.4, and 0.8 g / 100 mL) of M12.3. Each group of experiments was repeated 3 times and cultured in an anaerobic incubator at 37°C for 32 hours. The OD600 value was measured every 2 hours. The experimental results are shown in the figure. Figure 3 As shown, when the addition amount is 0.4g / 100ml, AKK bacteria enters the logarithmic growth phase the fastest, indicating that the addition amount of Lactobacillus rhamnosus metabolites is most conducive to the growth of AKK bacteria when it is 0.4g / 100ml.

[0041] Embodiment 5: The effects of the Lactobacillus rhamnosus metabolite M12.3 on the gastrointestinal fluid tolerance of AKK bacteria were explored as follows: Preparation of artificial gastric juice: Take 22.5 ml of dilute hydrochloric acid (1M), add about 400 ml of water and 5 g of pepsin, add water to make up to 500 ml, and set aside.

[0042] Preparation of artificial intestinal fluid: Take 500 ml of PBS solution (pH=6.8), add 5 g of pancreatic enzyme, mix well and set aside.

[0043] Expansion of AKK bacteria: Inoculate the activated AKK bacteria at a ratio of 1% into 30 ml of BHI broth medium containing 0.4 g / 100 ml of Lactobacillus rhamnosus metabolite M12.3. After anaerobic culture at 37°C for 16 hours, vortex mix and detect the amount of live bacteria.

[0044] Add 1.1 ml of AKK bacterial solution to a 10 ml sterile centrifuge tube, centrifuge at 4000 rpm / min for 5 min, remove the supernatant to obtain the bacteria, repeat this operation for 5 groups, add different masses of M12.3 to each tube, namely: Group 1 (0 mg of M12.3), Group 2 (2 mg of M12.3), Group 3 (4 mg of M12.3), Group 4 (8 mg of M12.3), Group 5 (12 mg of M12.3), and then add 1.1 ml of artificial gastric juice to each tube, mix well, place incubator at 37°C for 1 hour, aspirate 100 μL for 10-fold gradient dilution, and then spread on the plate to calculate the number of live bacteria.

[0045] After adding 9 ml of artificial intestinal fluid to each group of tubes and incubating again in a 37°C anaerobic incubator for 2 hours, 100 μL was aspirated for 10-fold gradient dilution and then plated to calculate the number of viable bacteria.

[0046] After the AKK bacteria were expanded, the number of live bacteria reached 3.69×10 9 CFU / mL. Figure 4 It can be seen that the number of viable bacteria of AKK bacteria after being treated with gastric juice for 1 hour is 10 8 CFU / mL or less. After intestinal fluid treatment, the amount of live bacteria was reduced to 10 7 The following shows that both gastric juice and intestinal juice can cause damage to AKK bacteria. However, as the amount of M12.3 added increases, the amount of live bacteria in AKK bacteria increases. When the amount of M12.3 added reaches 8 mg / ml, the amount of live bacteria reaches the highest. Subsequently, the amount of live bacteria does not change significantly when the amount of addition continues to increase. This shows that M12.3 can effectively increase the survival rate of AKK bacteria in the intestinal environment.

[0047] In summary, Lactobacillus rhamnosus is a microorganism that is recognized as safe. It itself has high safety, so its metabolites also have good safety.

[0048] The extracellular products of Lactobacillus rhamnosus have multiple biological activities. On the one hand, the extracellular products of Lactobacillus rhamnosus can protect AKK bacteria from a harsh living environment, enhance the adhesion of AKK bacteria, and facilitate the colonization of the strain; on the other hand, they have biological activities such as antioxidant, anti-inflammatory, and anti-cancer activities; in addition, the extracellular products of Lactobacillus rhamnosus can also provide a carbon source for the growth and reproduction of AKK bacteria, thereby promoting the growth of AKK bacteria; and the extracellular metabolites of Lactobacillus rhamnosus can also enhance the tolerance of AKK bacteria to gastrointestinal fluid, which may be because the extracellular polysaccharide of Lactobacillus rhamnosus embeds AKK bacteria, reduces the direct contact between gastrointestinal fluid and AKK bacteria, and thus reduces the damage of gastrointestinal fluid to AKK bacteria.

[0049] The present invention obtains a pumpkin juice culture medium capable of promoting the synthesis of extracellular metabolites by Lactobacillus rhamnosus by optimizing the culture medium, and the nutrients in the pumpkin can promote the growth, reproduction or metabolism of Lactobacillus rhamnosus.

Claims

1. A Lactobacillus rhamnosus B12.3, characterized in that The rhamnosus lactobacillus B12.3 is isolated from human vaginal secretions. The depository of the rhamnosus lactobacillus B12.3 is: General Microbiology Center of China National Committee for the Preservation of Microbiological Cultures, the deposit time is: December 25, 2024, the deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC NO: 33186, and the classification name is: Rhamnosus lactobacillus Lacticaseibacillus rhamnosus .

2. A Lactobacillus rhamnosus metabolite, characterized in that The Lactobacillus rhamnosus metabolite is produced by the metabolism of the Lactobacillus rhamnosus B12.3 described in claim 1.

3. The method for preparing a Lactobacillus rhamnosus metabolite according to claim 2, characterized in that: The activated Lactobacillus rhamnosus B12.3 was inoculated into a culture medium and anaerobically cultured at 37°C for 24 hours to obtain a fermentation broth. The fermentation broth was centrifuged at 8000 rpm / min to 10000 rpm / min for 5 minutes to 10 minutes to obtain a supernatant. The supernatant was subjected to secondary precipitation by centrifugation at 5000 rpm / min to 8000 rpm / min for 5 minutes to 10 minutes. The obtained precipitate was freeze-dried to obtain a Lactobacillus rhamnosus metabolite.

4. The method for preparing a Lactobacillus rhamnosus metabolite according to claim 3, characterized in that: The culture medium raw materials of the Lactobacillus rhamnosus B12.3 are: 1L pumpkin juice, 30 g / L glucose, 20 g / L beef powder, 10 g / L yeast powder, 10 g / L milk powder; The pumpkin juice preparation method comprises the following steps: pumpkin and water are mixed and pulped in a mass ratio of 1:1, and the pumpkin juice is obtained by filtering through 100 meshes.

5. The use of the Lactobacillus rhamnosus metabolites in promoting the growth of AKK bacteria according to claim 2, characterized in that: Adding 0.2g / 100ml-0.8g / 100ml of Lactobacillus rhamnosus metabolites into the culture medium can promote the growth of AKK bacteria when culturing AKK bacteria with the culture medium.

6. The use of the Lactobacillus rhamnosus metabolite according to claim 2 in improving the gastrointestinal fluid tolerance of AKK bacteria, characterized in that: The AKK bacteria and the Lactobacillus rhamnosus metabolites are mixed, wherein 2 mg to 12 mg of the Lactobacillus rhamnosus metabolites are added to the bacterial cells obtained per milliliter of the AKK bacterial liquid.

7. The use of the Lactobacillus rhamnosus metabolites in the preparation of AKK bacteria culture medium according to claim 2, characterized in that: The amount of Lactobacillus rhamnosus metabolites added to the AKK culture medium is 0.2g / 100ml~0.8g / 100ml.

8. A method for promoting the growth of AKK bacteria, characterized in that: The Lactobacillus rhamnosus metabolite of claim 2 is added to the AKK bacteria culture medium, and the added amount of the Lactobacillus rhamnosus metabolite is 0.2 g / 100 ml to 0.8 g / 100 ml.

9. A method for improving the gastrointestinal survival rate of AKK bacteria, characterized in that: The AKK bacteria and the Lactobacillus rhamnosus metabolites are mixed, wherein 2 mg to 12 mg of the Lactobacillus rhamnosus metabolites are added to the bacterial cells obtained per milliliter of the AKK bacterial liquid.

10. A culture medium, characterized in that The culture medium contains the Lactobacillus rhamnosus metabolite according to claim 2, the added amount of the Lactobacillus rhamnosus metabolite is 0.2g / 100ml-0.8g / 100ml, and the culture medium is used for culturing AKK bacteria.

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