Bifidobacterium enrichment agent and use thereof in high-density fermentation

Abstract

A bifidobacterium enrichment agent and a use thereof in high-density fermentation. The components of the bifidobacterium enrichment agent include potassium carbonate, arginine, methionine, serine, vitamin C, vitamin B1, vitamin B5, thymine, and calcium pantothenate. The bifidobacterium enrichment agent is added to a basal medium for high-density fermentation, so that the proliferation performance of bifidobacteria can be significantly improved, and the production efficiency can be improved, providing the possibility of preparing a highly active bifidobacterium fermentation liquor, and also providing support for the subsequent preparation and application of bacterial agents.

Description

A bifidobacterium enrichment agent and its application in high-density fermentation Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to a bifidobacterium enrichment agent and application thereof in high-density fermentation. Background Art

[0002] Bifidobacterium is a probiotic that is widely distributed in the human intestine. It has beneficial effects such as improving intestinal microbiota, regulating intestinal permeability, enhancing intestinal immunity, alleviating irritable bowel syndrome and resisting intestinal inflammation.

[0003] Bifidobacterium has a wide range of applications in feed, food, medicine and other fields. Its application in feed is mainly as an additive to animal feed, especially in livestock and aquaculture. Bifidobacterium can help improve the digestive system function of animals, enhance immunity, improve feed conversion rate and growth performance, reduce the number of harmful bacteria in feed, and improve the quality of meat and protein. In food, it is mainly used in the food manufacturing process, especially dairy products and fermented foods. Bifidobacterium fermented foods can improve the taste of food and increase the nutritional value of products. They can also be used as dietary supplements to provide health benefits to people. In the pharmaceutical industry, it is mainly used as a probiotic preparation to improve the balance of intestinal microecology and prevent or treat some intestinal-related diseases such as diarrhea, constipation, irritable bowel syndrome, etc. At the same time, a large number of probiotic new drug research and development pipelines based on theories such as the brain-gut axis and the liver-gut axis have been laid out.

[0004] The prerequisite for the industrial application of bifidobacteria is to stably and reliably prepare the strain into a highly active bacterial agent, and high-density fermentation can effectively reduce production costs. However, there is currently no public bacterial enhancement agent that can promote high-density fermentation of bifidobacteria.

[0005] In view of this, the present invention is proposed.

[0006] Summary of the Invention

[0007] An object of the present invention is to provide a bifidobacterium enrichment agent.

[0008] Another object of the present invention is to provide a culture medium containing the bifidobacterium enrichment agent.

[0009] Another object of the present invention is to provide the use of the bifidobacterium enrichment agent and the culture medium in high-density fermentation, wherein the enrichment agent and the culture medium can significantly increase the number of viable bacteria in fermentation broths of various bifidobacteria.

[0010] Another object of the present invention is to provide a method for promoting high-density fermentation of bifidobacteria.

[0011] In a first aspect, the present invention provides a bifidobacterium enrichment agent, the components of which include potassium carbonate, arginine, methionine, serine, vitamin C, vitamin B1, vitamin B5, thymine and calcium pantothenate.

[0012] The bifidobacterium growth enhancer of the present invention achieves synergistic cooperation among the components through the scientific proportioning of the components, thereby facilitating the proliferation of bifidobacteria in high-density fermentation. Potassium carbonate and calcium pantothenate can regulate pH, while potassium carbonate decomposes to produce carbon dioxide to maintain an anaerobic environment; vitamin C, as an oxygen scavenger, can remove hydrogen peroxide; arginine, methionine, and serine, as raw materials for protein synthesis, promote bacterial proliferation; and vitamin B1, vitamin B5, and thymine are added as B vitamins and nucleotides required for stimulating the growth of bifidobacteria.

[0013] Specifically, the weight ratio of the components of the above-mentioned bifidobacterium enrichment agent is: potassium carbonate 0.1g~8g: arginine 0.01g~5g: methionine 0.01g~5g: serine 0.01g~5g: vitamin C 0.1g~5g: vitamin B1 0.1g~5g: vitamin B5 0.1g~5g: thymine 0.1g~5g: calcium pantothenate 1mg~100mg.

[0014] Furthermore, the weight ratio of the components of the above-mentioned bifidobacterium enrichment agent is: potassium carbonate 0.1g~6g: arginine 0.01g~5g: methionine 0.01g~5g: serine 0.01g~5g: vitamin C 0.1g~5g: vitamin B1 0.1g~5g: vitamin B5 0.1g~5g: thymine 0.1g~5g: calcium pantothenate 1mg~100mg.

[0015] Furthermore, the weight ratio of the components of the above-mentioned bifidobacterium enrichment agent is: potassium carbonate 0.2g~5g: arginine 0.05g~4g: methionine 0.05g~4g: serine 0.05g~4g: vitamin C 0.2g~4g: vitamin B1 0.2g~4g: vitamin B5 0.2g~4g: thymine 0.2g~4g: calcium pantothenate 10mg~80mg.

[0016] Preferably, the weight ratio of the components of the above-mentioned bifidobacterium enrichment agent is: potassium carbonate 0.4g~3g: arginine 0.1g~3g: methionine 0.1g~3g: serine 0.1g~3g: vitamin C 0.5g~2g: vitamin B1 0.4g~2g: vitamin B5 0.4g~2g: thymine 0.5g~2g: calcium pantothenate 10mg~80mg.

[0017] More preferably, the weight ratio of the components of the above-mentioned bifidobacterium enrichment agent is: potassium carbonate 1g: arginine 0.2g: methionine 0.5g: serine 0.1g: vitamin C1g: vitamin B1 0.6g: vitamin B5 1.3g: thymine 0.6g: calcium pantothenate 20mg.

[0018] Specifically, the concentrations of the components of the above-mentioned bifidobacterium enrichment agent in the culture medium are: potassium carbonate 0.1-8 g / L, arginine 0.01-5 g / L, methionine 0.01-5 g / L, serine 0.01-5 g / L, vitamin C 0.1-5 g / L, vitamin B1 0.1-5 g / L, vitamin B5 0.1-5 g / L, thymine 0.1-5 g / L, and calcium pantothenate 1-100 mg / L.

[0019] Furthermore, the concentrations of the components of the above-mentioned bifidobacterium enrichment agent in the culture medium are: potassium carbonate 0.1-6 g / L, arginine 0.01-5 g / L, methionine 0.01-5 g / L, serine 0.01-5 g / L, vitamin C 0.1-5 g / L, vitamin B1 0.1-5 g / L, vitamin B5 0.1-5 g / L, thymine 0.1-5 g / L, and calcium pantothenate 1-100 mg / L.

[0020] Furthermore, the concentrations of the components of the above-mentioned bifidobacterium enrichment agent in the culture medium are: potassium carbonate 0.2-5 g / L, arginine 0.05-4 g / L, methionine 0.05-4 g / L, serine 0.05-4 g / L, vitamin C 0.2-4 g / L, vitamin B1 0.2-4 g / L, vitamin B5 0.2-4 g / L, thymine 0.2-4 g / L, and calcium pantothenate 10-80 mg / L.

[0021] Preferably, the concentrations of the components of the above-mentioned bifidobacterium enrichment agent in the culture medium are: potassium carbonate 0.4-3 g / L, arginine 0.1-3 g / L, methionine 0.1-3 g / L, serine 0.1-3 g / L, vitamin C 0.5-2 g / L, vitamin B1 0.4-2 g / L, vitamin B5 0.4-2 g / L, thymine 0.5-2 g / L, and calcium pantothenate 10-80 mg / L.

[0022] More preferably, the concentrations of the components of the above-mentioned bifidobacterium enrichment agent in the culture medium are: potassium carbonate 1g / L, arginine 0.2g / L, methionine 0.5g / L, serine 0.1g / L, vitamin C 1g / L, vitamin B1 0.6g / L, vitamin B5 1.3g / L, thymine 0.6g / L, and calcium pantothenate 20mg / L.

[0023] In a second aspect, the present invention provides a culture medium for promoting high-density fermentation of bifidobacteria, wherein the culture medium is prepared by adding the above-mentioned bifidobacterium enrichment agent to a culture medium of bifidobacteria.

[0024] In some embodiments, the components of the above-mentioned bifidobacterium culture medium include: 20-80 g / L glucose, 10-60 g / L yeast extract powder, 10-60 g / L yeast peptone, 2-4 g / L anhydrous sodium acetate, 1-3 g / L ammonium citrate, 1-3 g / L dipotassium hydrogen phosphate, 0.1-5 g / L L-cysteine ​​hydrochloride, 0.2-0.8 g / L magnesium sulfate, 0.01-0.1 g / L manganese sulfate, and 0.5-1.5 mL Tween 80.

[0025] According to some specific embodiments of the present invention, the bifidobacterium culture medium is a modified MRS culture medium.

[0026] According to some specific embodiments of the present invention, the improved MRS medium is obtained by adding 0.5 g / L L-cysteine ​​hydrochloride to the basic MRS medium formula.

[0027] According to some specific embodiments of the present invention, after adding a bifidobacterium enrichment agent to the above-mentioned bifidobacterium culture medium, the components of the culture medium for promoting high-density fermentation of bifidobacteria include: glucose 20-80 g / L, yeast extract powder 10-60 g / L, yeast peptone 10-60 g / L, anhydrous sodium acetate 2-4 g / L, ammonium citrate 1-3 g / L, dipotassium hydrogen phosphate 1-3 g / L, L-cysteine ​​hydrochloride 0.1-5 g / L, magnesium sulfate 0.2-0.8 g / L, manganese sulfate 0.01-0.1 g / L, Tween 80 0.5-1.5 mL, potassium carbonate 0.1-8 g / L, arginine 0.01-5 g / L, methionine 0.01-5 g / L, serine 0.01-5 g / L, vitamin C 0.1-5 g / L, vitamin B1 0.1-5 g / L, vitamin B5 0.1~5g / L, thymine 0.1~5g / L, calcium pantothenate 1~100mg / L.

[0028] More preferably, after adding a bifidobacterium enrichment agent to the above-mentioned bifidobacterium culture medium, the components of the culture medium for promoting high-density fermentation of bifidobacteria include: 55 g / L glucose, 15 g / L yeast extract powder, 20 g / L yeast peptone, 3 g / L anhydrous sodium acetate, 2 g / L ammonium citrate, 2 g / L dipotassium hydrogen phosphate, 0.3 g / L L-cysteine ​​hydrochloride, 0.6 g / L magnesium sulfate, 0.06 g / L manganese sulfate, 0.75 mL Tween 80, 1 g / L potassium carbonate, 0.2 g / L arginine, 0.5 g / L methionine, 0.1 g / L serine, 1 g / L vitamin C, 0.6 g / L vitamin B1, 1.3 g / L vitamin B5, 0.6 g / L thymine, and 20 mg / L calcium pantothenate.

[0029] The preparation method of the culture medium for promoting high-density fermentation of bifidobacteria of the present invention can be as follows: adding the above-mentioned bifidobacterium enrichment agent to the culture medium of bifidobacteria, adjusting the pH of the culture medium to 7.0±0.2, sterilizing at 121°C for 15 minutes, and cooling the sterilized culture medium for later use.

[0030] In a third aspect, the present invention provides the use of the above-mentioned bifidobacterium enrichment agent or culture medium in promoting high-density fermentation of bifidobacteria.

[0031] In some embodiments, the bifidobacterium comprises one or more of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bifidobacterium bifidum and Bifidobacterium animalis.

[0032] In a fourth aspect, the present invention provides a method for promoting high-density fermentation of bifidobacteria, comprising inoculating a bacterial liquid into the above-mentioned culture medium for anaerobic culture.

[0033] The present invention has the following beneficial effects:

[0034] The bifidobacterium enrichment agent provided by the present invention can significantly increase the number of viable bifidobacteria in the fermentation broth. Adding it to the basic culture medium for high-density fermentation can significantly improve the proliferation performance of bifidobacteria and improve production efficiency, making it possible to prepare highly active bifidobacterium fermentation broth and providing support for subsequent bacterial agent preparation and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0036] FIG1 is a graph showing the comparison of viable cell counts in Example 2;

[0037] FIG2 is a graph showing the comparison of viable cell counts in Example 3;

[0038] FIG3 is a graph showing the comparison of viable cell counts in Example 4;

[0039] FIG4 is a graph showing a comparison of viable cell counts in Example 5;

[0040] FIG5 is a graph showing the comparison of viable cell counts in Example 6;

[0041] FIG6 is a graph showing the comparison of viable cell counts in Example 7;

[0042] FIG7 is a graph showing the comparison of viable bacteria counts in Comparative Example 1;

[0043] FIG8 is a graph showing the comparison of viable cell counts in Comparative Example 2;

[0044] FIG9 is a graph showing the comparison of viable bacteria counts in Comparative Example 3. DETAILED DESCRIPTION

[0045] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0046] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0047] Example 1

[0048] This embodiment provides a bifidobacterium enrichment agent, the contents of which in the culture medium are: potassium carbonate 1 g / L, arginine 0.2 g / L, methionine 0.5 g / L, serine 0.1 g / L, vitamin C 1 g / L, vitamin B1 0.6 g / L, vitamin B5 1.3 g / L, thymine 0.6 g / L, and calcium pantothenate 20 mg / L.

[0049] Example 2

[0050] This example is a verification experiment that the bifidobacterium enrichment agent can promote high-density fermentation of Bifidobacterium longum:

[0051] 1. Culture medium preparation:

[0052] Modified MRS medium: 0.5 g / L L-cysteine ​​hydrochloride was added to the basic MRS medium formula as a control.

[0053] Enrichment medium: Add the enrichment agent to the modified MRS medium according to the composition ratio in Example 1. Adjust the pH of the medium to 7.0±0.2, sterilize at 121°C for 15 minutes, cool to 37°C, and place in an anaerobic workstation for later use.

[0054] 2. Transfer 200 mL each of modified MRS medium and enrichment medium to a conical flask. Inoculate each flask with 2% (v / v) activated Bifidobacterium longum (CICC 6068) culture. Incubate anaerobically at 37°C for 18 hours. Determine the viable bacterial count in the fermentation broth using the gradient dilution and pour method to compare the growth-promoting effects of the enrichment agents.

[0055] The results are shown in Figure 1. The number of viable bacteria in the enrichment medium reached 39.97×10 7 CFU / mL, which was 2.01 times the number of viable bacteria in the modified MRS culture medium, and the effect of promoting proliferation was extremely significant (P<0.001).

[0056] Example 3

[0057] This example is a verification experiment that the bifidobacterium enrichment agent can promote high-density fermentation of Bifidobacterium breve:

[0058] 1. Culture medium preparation:

[0059] Modified MRS medium: 0.5 g / L L-cysteine ​​hydrochloride was added to the basic MRS medium formula as a control.

[0060] Enrichment medium: Add the enrichment agent to the modified MRS medium according to the amount in Example 1. Adjust the pH of the medium to 7.0 ± 0.2, sterilize at 121°C for 15 minutes, cool to 37°C, and place in an anaerobic workstation for later use.

[0061] 2. Transfer 200 mL each of modified MRS medium and enrichment medium to a conical flask. Inoculate each flask with 2% (v / v) activated Bifidobacterium breve (CICC 6079) culture. Incubate anaerobically at 37°C for 18 hours. Determine the viable bacterial count in the fermentation broth using the gradient dilution and pour method to compare the growth-promoting effects of the enrichment agents.

[0062] The results are shown in Figure 2. The number of viable bacteria in the enrichment medium was 15×10 5 CFU / mL, which was 1.73 times the number of viable bacteria in the modified MRS culture medium, and the effect of promoting proliferation was extremely significant (P<0.01).

[0063] Example 4

[0064] This example is a verification experiment that the bifidobacterium enrichment agent can promote the high-density fermentation of Bifidobacterium adolescentis:

[0065] 1. Culture medium preparation:

[0066] Modified MRS medium: 0.5 g / L L-cysteine ​​hydrochloride was added to the basic MRS medium formula as a control.

[0067] Enrichment medium: Add the enrichment agent to the modified MRS medium according to the amount in Example 1. Adjust the pH of the medium to 7.0 ± 0.2, sterilize at 121°C for 15 minutes, cool to 37°C, and place in an anaerobic workstation for later use.

[0068] 2. Transfer 200 mL each of modified MRS medium and enrichment medium to a conical flask. Inoculate each flask with 2% (v / v) activated Bifidobacterium adolescentis (CICC 6070) culture. Incubate anaerobically at 37°C for 18 hours. Determine the viable count of the fermentation broth using the gradient dilution and pour method to compare the growth-promoting effects of the enrichment agents.

[0069] The results are shown in Figure 3. The number of viable bacteria in the enrichment medium was 203.67×10 7 CFU / mL, which was 1.37 times the number of viable bacteria in the modified MRS culture medium, and had a very significant effect in promoting proliferation (P<0.001).

[0070] Example 5

[0071] This example is a verification experiment that the bifidobacterium enrichment agent can promote high-density fermentation of Bifidobacterium bifidum:

[0072] 1. Culture medium preparation:

[0073] Modified MRS medium: 0.5 g / L L-cysteine ​​hydrochloride was added to the basic MRS medium formula as a control.

[0074] Enrichment medium: Add the enrichment agent to the modified MRS medium according to the amount in Example 1. Adjust the pH of the medium to 7.0 ± 0.2, sterilize at 121°C for 15 minutes, cool to 37°C, and place in an anaerobic workstation for later use.

[0075] 2. Transfer 200 mL each of modified MRS medium and enrichment medium to a conical flask. Inoculate each flask with 2% (v / v) activated Bifidobacterium bifidum (CICC 6071) culture. Incubate anaerobically at 37°C for 18 hours. Determine the viable bacterial count in the fermentation broth using the gradient dilution and pour method to compare the growth-promoting effects of the enrichment agents.

[0076] The results are shown in Figure 4. The number of viable bacteria in the enrichment medium was 16.8×10 7 CFU / mL, which was 1.33 times the number of viable bacteria in the modified MRS culture medium, and the effect of promoting proliferation was extremely significant (P<0.001).

[0077] Example 6

[0078] This example is a verification experiment that the bifidobacterium enrichment agent can promote high-density fermentation of animal bifidobacteria:

[0079] 1. Culture medium preparation:

[0080] Modified MRS medium: 0.5 g / L L-cysteine ​​hydrochloride was added to the basic MRS medium formula as a control.

[0081] Enrichment medium: Add the enrichment agent to the modified MRS medium according to the amount in Example 1. Adjust the pH of the medium to 7.0 ± 0.2, sterilize at 121°C for 15 minutes, cool to 37°C, and place in an anaerobic workstation for later use.

[0082] 2. Transfer 200 mL each of modified MRS medium and enrichment medium to a conical flask. Inoculate each flask with 2% (v / v) activated Bifidobacterium animalis (CICC 6165) culture. Incubate anaerobically at 37°C for 18 hours. Determine the viable count of the fermentation broth using the gradient dilution and pour method to compare the growth-promoting effects of the enrichment agents.

[0083] The results are shown in Figure 5. The number of viable bacteria in the enrichment medium was 150.67×10 7 CFU / mL, which was 1.49 times the number of viable bacteria in the modified MRS culture medium, and the effect of promoting proliferation was extremely significant (P<0.001).

[0084] Example 7

[0085] High-density fermentation of Bifidobacterium longum:

[0086] 1. Prepare 2 L of Bifidobacterium longum (CCIC 6068) enrichment medium, the components of which are: glucose 55 g / L, yeast extract powder 15 g / L, yeast peptone 20 g / L, anhydrous sodium acetate 3 g / L, ammonium citrate 2 g / L, dipotassium hydrogen phosphate 2 g / L, L-cysteine ​​hydrochloride 0.3 g / L, magnesium sulfate 0.6 g / L, manganese sulfate 0.06 g / L, Tween 80 0.75 mL, potassium carbonate 1 g / L, arginine 0.2 g / L, methionine 0.5 g / L, serine 0.1 g / L, vitamin C 1 g / L, vitamin B1 0.6 g / L, vitamin B5 1.3 g / L, thymidine 0.6 g / L, and calcium pantothenate 20 mg / L. The above culture medium was placed in a 3 L fermenter, sterilized at 121°C for 15 min, cooled to 37°C, and equilibrated to anaerobic conditions with nitrogen. A modified MRS medium was used as a control.

[0087] 2. Inoculate 200 mL of Bifidobacterium longum (CICC 6068) seed solution, adjust the pH to 6.0 ± 0.1 with 25% NaOH, stir at 200 rpm, ferment at 37 °C for 14 h, and take samples to measure the number of viable bacteria.

[0088] The results are shown in Figure 6. After high-density fermentation, the number of viable Bifidobacterium longum obtained from the enrichment medium was 80.3×10 8 CFU / mL, the modified MRS medium was only 5.96×10 8 CFU / mL, the number of viable bacteria in the enrichment medium was 13.47 times that of the modified MRS medium, and the proliferation-promoting effect was extremely significant (P<0.001).

[0089] Example 8

[0090] This embodiment provides a bifidobacterium enrichment agent, the contents of which in the culture medium are: potassium carbonate 1 g / L, arginine 0.2 g / L, methionine 0.01 g / L, serine 5 g / L, vitamin C 1 g / L, vitamin B1 0.6 g / L, vitamin B5 1.3 g / L, thymine 0.6 g / L, and calcium pantothenate 20 mg / L.

[0091] Example 9

[0092] This embodiment provides a bifidobacterium enrichment agent, the contents of which in the culture medium are: potassium carbonate 1 g / L, arginine 0.01 g / L, methionine 5 g / L, serine 0.1 g / L, vitamin C 1 g / L, vitamin B1 0.6 g / L, vitamin B5 1.3 g / L, thymine 0.6 g / L, and calcium pantothenate 20 mg / L.

[0093] Example 10

[0094] This embodiment provides a bifidobacterium enrichment agent, the contents of which in the culture medium are: potassium carbonate 1 g / L, arginine 5 g / L, methionine 0.5 g / L, serine 0.01 g / L, vitamin C 1 g / L, vitamin B1 0.6 g / L, vitamin B5 1.3 g / L, thymine 0.6 g / L, and calcium pantothenate 20 mg / L.

[0095] Example 11

[0096] This embodiment provides a bifidobacterium enrichment agent, the contents of its components in the culture medium are: potassium carbonate 0.1 g / L, arginine 0.2 g / L, methionine 0.5 g / L, serine 0.1 g / L, vitamin C 1 g / L, vitamin B1 0.6 g / L, vitamin B5 1.3 g / L, thymine 0.6 g / L, and calcium pantothenate 100 mg / L.

[0097] Example 12

[0098] This embodiment provides a bifidobacterium enrichment agent, the contents of which in the culture medium are: potassium carbonate 6 g / L, arginine 0.2 g / L, methionine 0.5 g / L, serine 0.1 g / L, vitamin C 1 g / L, vitamin B1 0.6 g / L, vitamin B5 1.3 g / L, thymine 0.6 g / L, and calcium pantothenate 1 mg / L.

[0099] Example 13

[0100] This embodiment provides a bifidobacterium enrichment agent, the contents of its components in the culture medium are: potassium carbonate 1 g / L, arginine 0.2 g / L, methionine 0.5 g / L, serine 0.1 g / L, vitamin C 0.1 g / L, vitamin B1 0.6 g / L, vitamin B5 1.3 g / L, thymine 5 g / L, and calcium pantothenate 20 mg / L.

[0101] Example 14

[0102] This embodiment provides a bifidobacterium enrichment agent, the contents of which in the culture medium are: potassium carbonate 1 g / L, arginine 0.2 g / L, methionine 0.5 g / L, serine 0.1 g / L, vitamin C 1 g / L, vitamin B1 0.1 g / L, vitamin B5 5 g / L, thymine 0.6 g / L, and calcium pantothenate 20 mg / L.

[0103] Example 15

[0104] This embodiment provides a bifidobacterium enrichment agent, the contents of which in the culture medium are: potassium carbonate 1g / L, arginine 0.2g / L, methionine 0.5g / L, serine 0.1g / L, vitamin C 1g / L, vitamin B1 5g / L, vitamin B5 0.1g / L, thymine 0.6g / L, and calcium pantothenate 20mg / L.

[0105] Example 16

[0106] This embodiment provides a bifidobacterium enrichment agent, the contents of its components in the culture medium are: potassium carbonate 1g / L, arginine 0.2g / L, methionine 0.5g / L, serine 0.1g / L, vitamin C 5g / L, vitamin B1 0.6g / L, vitamin B5 1.3g / L, thymine 0.1g / L, and calcium pantothenate 20mg / L.

[0107] Example 17

[0108] High-density fermentation of Bifidobacterium longum:

[0109] The proportion of the bacterial growth agent was the same as that of Example 8, and the other culture medium components and fermentation process were the same as those of Example 7.

[0110] Example 18

[0111] High-density fermentation of Bifidobacterium longum:

[0112] The proportion of the bacterial growth agent was the same as that of Example 9, and the other culture medium components and fermentation process were the same as those of Example 7.

[0113] Example 19

[0114] High-density fermentation of Bifidobacterium longum:

[0115] The bacterial growth agent was the same as that in Example 10, and the other culture medium components and fermentation process were the same as those in Example 7.

[0116] Example 20

[0117] High-density fermentation of Bifidobacterium longum:

[0118] The bacterial growth agent was the same as that in Example 11, and the other culture medium components and fermentation process were the same as those in Example 7.

[0119] Example 21

[0120] High-density fermentation of Bifidobacterium longum:

[0121] The bacterial growth agent was the same as that in Example 12, and the other culture medium components and fermentation process were the same as those in Example 7.

[0122] Example 22

[0123] High-density fermentation of Bifidobacterium longum:

[0124] The bacterial growth agent was the same as that in Example 13, and the other culture medium components and fermentation process were the same as those in Example 7.

[0125] Example 23

[0126] High-density fermentation of Bifidobacterium longum:

[0127] The bacterial growth agent was the same as that in Example 14, and the other culture medium components and fermentation process were the same as those in Example 7.

[0128] Example 24

[0129] High-density fermentation of Bifidobacterium longum:

[0130] The bacterial growth agent was the same as that in Example 15, and the other culture medium components and fermentation process were the same as those in Example 7.

[0131] Example 25

[0132] High-density fermentation of Bifidobacterium longum:

[0133] The bacterial growth agent was the same as that in Example 16, and the other culture medium components and fermentation process were the same as those in Example 7.

[0134] Table 1 Results of high-density fermentation of Bifidobacterium longum

[0135] According to the viable cell count results in Table 1, it can be seen that when the bacterial enrichment agent of the present invention is added to the culture medium, the viable cell count of Bifidobacterium longum in high-density fermentation is 11.86 to 13.47 times that of the modified MRS culture medium, which proves that the bacterial enrichment agent can effectively promote high-density fermentation of Bifidobacterium longum.

[0136] Comparative Example 1

[0137] In this comparative example, arginine 0.2 g / L, methionine 0.5 g / L and serine 0.1 g / L in the bacterial enrichment formulation of Example 1 were replaced with arginine, methionine, serine, valine, isoleucine, phenylalanine, histidine and lysine 0.8 g / L, respectively. The remaining culture medium components and fermentation process were the same as those in Example 2.

[0138] The results are shown in Figure 7. The proliferation effect was as follows: experimental group > methionine group > arginine group > serine group > lysine group > isoleucine group > valine group > phenylalanine group > histidine group > control group. The proliferation effect of the experimental group was significantly better than that of the other control groups.

[0139] According to the results in FIG7 , even when the amount of arginine, methionine or serine is the same, the proliferation promoting effect of the combination is better than that of the combination of arginine, methionine and serine, which proves that arginine, methionine and serine have a synergistic effect.

[0140] Comparative Example 2

[0141] In this comparative example, 0.6 g / L thymine in the bacterial enrichment component of Example 1 was replaced with 0.6 g / L guanine, adenine, cytosine, and uracil, respectively. The remaining culture medium components and fermentation process were the same as those of Example 2.

[0142] The results are shown in Figure 8. The proliferation effect was as follows: experimental group > guanine group > cytosine group > uracil group > adenine group > control group. The proliferation effect of the experimental group was significantly better than that of the other control groups.

[0143] According to the results in FIG8 , it can be concluded that the thymine screened by the present invention is a component that can better promote the proliferation of bifidobacteria when combined with other components.

[0144] Comparative Example 3

[0145] In this comparative example, 0.6 g / L of vitamin B1 and 1.3 g / L of vitamin B5 in the bacterial enhancement agent formula of Example 1 were replaced by 1.9 g / L of vitamin B1, B2, B5, and B6, respectively. The remaining culture medium components and fermentation process were the same as those of Example 2.

[0146] The results are shown in Figure 9 . The proliferation effect was as follows: experimental group > VB5 group > VB1 group > VB6 group > VB2 group > control group. Although all B vitamins have a certain effect on promoting the proliferation of Bifidobacterium, the present invention clearly demonstrated a superior proliferation effect when using equal amounts of vitamin B1, vitamin B5, vitamin B2, or vitamin B6 alone compared to the vitamin B1 and vitamin B5 combination of the present invention. This demonstrates that the combination of vitamin B1 and vitamin B5 employed in the present invention exhibits a synergistic effect.

[0147] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A bifidobacterium enrichment agent, comprising potassium carbonate, arginine, methionine, serine, vitamin C, vitamin B1, vitamin B5, thymine and calcium pantothenate; Preferably, the concentrations of the components of the bifidobacterium enrichment agent in the culture medium are: potassium carbonate 0.1-6 g / L, arginine 0.01-5 g / L, methionine 0.01-5 g / L, serine 0.01-5 g / L, vitamin C 0.1-5 g / L, vitamin B1 0.1-5 g / L, vitamin B5 0.1-5 g / L, thymine 0.1-5 g / L, and calcium pantothenate 1-100 mg / L.

2. The bifidobacterium enrichment agent according to claim 1, wherein The concentrations of the components of the bifidobacterium enrichment agent in the culture medium are: 0.2-5 g / L potassium carbonate, 0.05-4 g / L arginine, 0.05-4 g / L methionine, 0.05-4 g / L serine, 0.05-4 g / L vitamin C, 0.2-4 g / L vitamin B1, 0.2-4 g / L vitamin B5, 0.2-4 g / L thymine, and 10-80 mg / L calcium pantothenate.

3. The bifidobacterium enrichment agent according to claim 2, wherein The concentrations of the components of the bifidobacterium enrichment agent in the culture medium are: potassium carbonate 0.4-3g / L, arginine 0.1-3g / L, methionine 0.1-3g / L, serine 0.1-3g / L, vitamin C 0.5-2g / L, vitamin B1 0.4-2g / L, vitamin B5 0.4-2g / L, thymine 0.5-2g / L, and calcium pantothenate 10-80mg / L.

4. The bifidobacterium enrichment agent according to claim 3, wherein The concentrations of the components of the bifidobacterium enrichment agent in the culture medium are: 1 g / L potassium carbonate, 0.2 g / L arginine, 0.5 g / L methionine, 0.1 g / L serine, 1 g / L vitamin C, 0.6 g / L vitamin B1, 1.3 g / L vitamin B5, 0.6 g / L thymine, and 20 mg / L calcium pantothenate.

5. A culture medium for promoting high-density fermentation of bifidobacteria, wherein: The culture medium contains the bifidobacterium enrichment agent according to any one of claims 1 to 4.

6. The culture medium according to claim 5, wherein The components of the culture medium include: 20-80 g / L of glucose, 10-60 g / L of yeast extract powder, 10-60 g / L of yeast peptone, 2-4 g / L of anhydrous sodium acetate, 1-3 g / L of ammonium citrate, 1-3 g / L of dipotassium hydrogen phosphate, 0.1-5 g / L of L-cysteine ​​hydrochloride, 0.2-0.8 g / L of magnesium sulfate, 0.01-0.1 g / L of manganese sulfate, 0.5-1.5 mL of Tween 80, 0.1-6 g / L of potassium carbonate, 0.01-5 g / L of arginine, 0.01-5 g / L of methionine, 0.01-5 g / L of serine, 0.1-5 g / L of vitamin C, 0.1-5 g / L of vitamin B1, 0.1-5 g / L of vitamin B5, 0.1-5 g / L of thymine, and 1-100 mg / L of calcium pantothenate.

7. The culture medium according to claim 6, wherein The components of the culture medium include: 55 g / L glucose, 15 g / L yeast extract powder, 20 g / L yeast peptone, 3 g / L anhydrous sodium acetate, 2 g / L ammonium citrate, 2 g / L dipotassium hydrogen phosphate, 0.3 g / L L-cysteine ​​hydrochloride, 0.6 g / L magnesium sulfate, 0.06 g / L manganese sulfate, 0.75 mL Tween 80, 1 g / L potassium carbonate, 0.2 g / L arginine, 0.5 g / L methionine, 0.1 g / L serine, 1 g / L vitamin C, 0.6 g / L vitamin B1, 1.3 g / L vitamin B5, 0.6 g / L thymine, and 20 mg / L calcium pantothenate.

8. Use of the bifidobacterium enrichment agent according to any one of claims 1 to 4 or the culture medium according to any one of claims 5 to 7 in promoting high-density fermentation of bifidobacteria.

9. The use according to claim 8, wherein: The bifidobacterium includes one or more of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bifidobacterium bifidum and Bifidobacterium animalis.

10. A method for promoting high-density fermentation of bifidobacteria, wherein: The bifidobacterium bacterial liquid is inoculated into the culture medium according to any one of claims 5 to 7 for anaerobic culture.

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