Bifidobacterium and clostridium butyricum liquid-state combined fermentation medium and fermentation method

Abstract

The invention relates to the technical field of microbial preparation fermentation processes, in particular to a bifidobacterium and clostridium butyricum liquid-state combined fermentation culture medium and a fermentation method. The combined fermentation culture medium comprises a base material culture medium and a supplemented material culture medium, the base material culture medium comprises 5-10 g / L of yeast extract powder, 20-40 g / L of bean flour hydrolysate, 2-5 g / L of glucose, 10-20 g / L of sucrose, 1-5 g / L of compound oligosaccharide, 1-5 g / L of dipotassium phosphate, 1-5 g / L of monopotassium phosphate, 1-3 g / L of magnesium sulfate, 1-5 g / L of calcium carbonate, 1-3 g / L of sodium acetate, 0.1-0.5 g / L of manganese sulfate, 1-3 g / L of potassium citrate and 0.5-5 g / L of deoxidant. The co-fermentation culture medium can meet the requirement of symbiosis of bifidobacterium and clostridium butyricum on nutrient substances, co-fermentation of bifidobacterium and clostridium butyricum is achieved, and the viable count per unit volume is increased.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial preparation fermentation technology, and in particular to a liquid combined fermentation medium of bifidobacterium and Clostridium butyricum and a fermentation method. Background Art

[0002] Bifidobacterium is an obligate anaerobic Gram-positive bacterium that can ferment glucose, use ammonium salts as a nitrogen source, and does not reduce nitrates. The optimum growth temperature is 35-41°C, the minimum growth temperature is 25-28°C, and the maximum growth temperature is 43-45°C. The optimum pH for initial growth is 6.5-7.0. It will not grow below pH 4.5 or above 8.5. An acidic environment (pH ≤ 5.5) is not conducive to bacterial survival. Bifidobacterium can effectively maintain the balance of normal intestinal flora, and also has unique physiological functions in anti-diarrhea, anti-constipation, anti-infection, and anti-tumor.

[0003] Clostridium butyricum, also known as butyric acid bacteria or Clostridium butyricum, is also an obligate anaerobic Gram-positive bacterium. It can utilize common sugars such as glucose, maltose, sucrose, as well as cellulose, glycerol and starch; it cannot utilize galactitol and sorbitol; it cannot produce enzymes that hydrolyze gelatin and casein, and cannot decompose protein. The optimal growth environment pH is 4.0-9.8, and the most suitable culture temperature is 25-37°C. Clostridium butyricum is widely present in environments such as cheese, intestines and soil. It is a normal flora in the intestines of humans and animals. It can inhibit the growth of certain harmful microorganisms in the intestines, and at the same time produce prebiotics, playing an important role in enhancing the body's immunity and regulating the balance of intestinal microecology.

[0004] Bifidobacterium and Clostridium butyricum are both beneficial bacteria in the intestine. The two play basically the same role in the intestine, but the two complement each other and assist each other to better build a healthy intestinal barrier. Although both probiotics are widely recognized by people, there are still some difficulties and problems in the actual production process. Bifidobacterium has strict requirements on the growth environment, which makes it difficult to cultivate strains in production, the production cycle is long, and the number of live bacteria in the product is small. In addition, bifidobacteria also have high requirements for nutritional conditions, the composition of their culture medium is complex, and the cost is high, which is not conducive to industrial mass production. Clostridium butyricum also has the problem of low level of large-scale fermentation. At the same time, products on the market generally have problems such as low number of effective live bacteria, short shelf life, and high price.

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

[0006] In view of the problems existing in the above-mentioned prior art, the present invention provides a liquid combined fermentation medium of bifidobacterium and Clostridium butyricum and a fermentation method. The medium can simultaneously culture bifidobacterium and Clostridium butyricum, and the number of viable bacteria per unit volume of the two strains in the fermentation liquid is high, thereby reducing production costs.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0008] One aspect of the present invention relates to a liquid combined fermentation medium of Bifidobacterium and Clostridium butyricum, comprising: a base medium and a feed medium;

[0009] Wherein, the base medium comprises the following components:

[0010] Yeast extract powder 5-10g / L, soybean flour hydrolyzate 20-40g / L, glucose 2-5g / L, sucrose 10-20g / L, complex oligosaccharide 1-5g / L, dipotassium hydrogen phosphate 1-5g / L, potassium dihydrogen phosphate 1-5g / L, magnesium sulfate 1-3g / L, calcium carbonate 1-5g / L, sodium acetate 1-3g / L, manganese sulfate 0.1-0.5g / L, potassium citrate 1-3g / L, deoxidizer 0.5-5g / L and water;

[0011] The feed medium comprises the following components:

[0012] Glucose 1-5g / L, sucrose 20-30g / L, complex oligosaccharides 1-3g / L and water.

[0013] The liquid combined fermentation medium of bifidobacterium and Clostridium butyricum can meet the symbiotic requirements of bifidobacterium and Clostridium butyricum for nutrients, realize the combined fermentation of the two, increase the number of live bacteria per unit volume, and harvest two strains in one fermentation.

[0014] Another aspect of the present invention also relates to a liquid combined fermentation method of Bifidobacterium and Clostridium butyricum, comprising the following steps:

[0015] (a) sequentially inoculating a seed liquid of Clostridium butyricum and a seed liquid of Bifidobacterium into a base medium in a liquid combined fermentation medium of Bifidobacterium and Clostridium butyricum for a first fermentation culture;

[0016] (b) 2 to 4 hours after inoculating the seed liquid of Clostridium butyricum, adding the feed medium in the liquid combined fermentation medium of Bifidobacterium and Clostridium butyricum to the culture system of the first fermentation culture for feeding;

[0017] (c) After the feeding is completed, a stability promoter is added to the culture system of the first fermentation culture to carry out a second fermentation culture.

[0018] The bifidobacterium and Clostridium butyricum liquid combined fermentation method adopts the fermentation medium, and can obtain bifidobacterium and Clostridium butyricum in one fermentation. The number of live bacteria per unit volume in the bacterial liquid is high, and the production cost is reduced.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The liquid combined fermentation medium of Bifidobacterium and Clostridium butyricum provided by the present invention comprises components combined in a certain ratio, and the components can act synergistically to meet the symbiotic requirements of Bifidobacterium and Clostridium butyricum for nutrients, thereby achieving combined fermentation of the two, increasing the number of viable bacteria per unit volume, and harvesting two strains in one fermentation.

[0021] (2) The liquid co-fermentation method of bifidobacterium and Clostridium butyricum provided by the present invention enables bifidobacterium and Clostridium butyricum to coexist well, which is more conducive to product application, improves product competitiveness, increases the number of effective live bacteria in a unit fermentation liquid, shortens the fermentation time, produces two probiotics in one fermentation, improves equipment utilization, and further reduces the production cost of the product.

[0022] (3) The liquid co-fermentation method of bifidobacterium and Clostridium butyricum provided by the present invention improves the storage stability of bifidobacterium and Clostridium butyricum by adding a stabilizer, prolongs the shelf life of the product, and is also conducive to the stability of product quality. DETAILED DESCRIPTION

[0023] The technical scheme of the present invention will be clearly and completely described below in conjunction with specific embodiments, but it will be appreciated by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work, all belong to the scope of protection of the present invention. If specific conditions are not indicated in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not indicated in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0024] One aspect of the present invention relates to a liquid combined fermentation medium of Bifidobacterium and Clostridium butyricum, comprising: a base medium and a feed medium;

[0025] Wherein, the base medium comprises the following components:

[0026] Yeast extract powder 5-10g / L, soybean flour hydrolyzate 20-40g / L, glucose 2-5g / L, sucrose 10-20g / L, complex oligosaccharide 1-5g / L, dipotassium hydrogen phosphate 1-5g / L, potassium dihydrogen phosphate 1-5g / L, magnesium sulfate 1-3g / L, calcium carbonate 1-5g / L, sodium acetate 1-3g / L, manganese sulfate 0.1-0.5g / L, potassium citrate 1-3g / L, deoxidizer 0.5-5g / L and water;

[0027] The feed medium comprises the following components:

[0028] Glucose 1-5g / L, sucrose 20-30g / L, complex oligosaccharides 1-3g / L and water.

[0029] In some specific embodiments, in the base medium, yeast extract powder can be, for example, but not limited to, any one of 5g / L, 6g / L, 7g / L, 8g / L, 9g / L or 10g / L, or a range between any two of them; soy flour hydrolysate can be, for example, but not limited to, any one of 20g / L, 23g / L, 25g / L, 28g / L, 30g / L, 33g / L, 35g / L, 38g / L or 40g / L, or a range between any two of them; glucose can be, for example, but not limited to, 2g / L, 3g / L , 4g / L or 5g / L, or a range between any two of them; sucrose, for example, may be, but is not limited to, any one of 10g / L, 12g / L, 14g / L, 16g / L, 18g / L or 20g / L, or a range between any two of them; complex oligosaccharides, for example, may be, but are not limited to, any one of 1g / L, 2g / L, 3g / L, 4g / L or 5g / L, or a range between any two of them; dipotassium hydrogen phosphate, for example, may be, but is not limited to, 1g / L, 2g / L, 3g / L, 4g / L or 5g / L, or a range between any two of them. any point value of 1g / L, 2g / L, 3g / L, 4g / L or 5g / L or a range between them; potassium dihydrogen phosphate, for example, may be, but not limited to, any point value of 1g / L, 2g / L, 3g / L, 4g / L or 5g / L or a range between them; magnesium sulfate, for example, may be, but not limited to, any point value of 1g / L, 2g / L or 3g / L or a range between them; calcium carbonate, for example, may be, but not limited to, any point value of 1g / L, 2g / L, 3g / L, 4g / L or 5g / L or a range between them; sodium acetate, for example, may be, but not limited to, any point value of 1g / L, 2g / L, 3g / L, 4g / L or 5g / L or a range between them The content of manganese sulfate may be, for example, but not limited to, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L or 0.5 g / L; the content of potassium citrate may be, for example, but not limited to, 1 g / L, 2 g / L or 3 g / L or a range between two values; the content of oxygen scavenger may be, for example, but not limited to, 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L or 5 g / L or a range between two values.

[0030] In some specific embodiments, in the feed medium, glucose can be, for example, but not limited to, any point value of 1g / L, 2g / L, 3g / L, 4g / L or 5g / L, or a range value between any two of them; sucrose can be, for example, but not limited to, any point value of 20g / L, 22g / L, 24g / L, 26g / L, 28g / L or 30g / L, or a range value between any two of them; complex oligosaccharides can be, for example, but not limited to, any point value of 1g / L, 2g / L or 3g / L, or a range value between any two of them.

[0031] The fermentation medium provided by the present invention is suitable for simultaneous liquid culture of bifidobacterium and Clostridium butyricum, and bifidobacterium and Clostridium butyricum belong to two different species, and the two have different classification status and growth characteristics. Those skilled in the art know that when two different species are cultured simultaneously, there is often a competitive relationship between the two species. If a suitable fermentation medium and a fermentation culture method cannot be provided, it is impossible to ensure that different species can achieve symbiosis in the same culture environment. Based on the growth characteristics of bifidobacterium and Clostridium butyricum, the present invention provides a fermentation medium and a culture method that can achieve symbiosis between the two.

[0032] The components of the bifidobacterium and Clostridium butyricum liquid combined fermentation medium are combined in a certain ratio, and the components can act synergistically to meet the symbiotic requirements of bifidobacteria and Clostridium butyricum for nutrients, achieve combined fermentation of the two, increase the number of live bacteria per unit volume, and harvest two strains in one fermentation.

[0033] The bifidobacterium and Clostridium butyricum liquid combined fermentation medium uses soybean powder hydrolysate as the main fermentation raw material. The soybean powder hydrolysate contains a large amount of oligosaccharides that are beneficial to the proliferation of bifidobacteria and Clostridium butyricum; the soybean powder hydrolysate also contains peptides and amino acids required for the growth of bifidobacteria and Clostridium butyricum. The hydrolysate replaces the traditional fermentation nitrogen source and part of the carbon source.

[0034] The liquid combined fermentation medium of bifidobacterium and Clostridium butyricum is added with complex oligosaccharides, which can promote bacterial proliferation and ensure that the two bacterial bodies can rapidly proliferate at the same time.

[0035] Furthermore, the substrate culture medium comprises the following components:

[0036] Yeast extract powder 6-8g / L, soybean flour hydrolyzate 25-35g / L, glucose 3-4g / L, sucrose 12-18g / L, complex oligosaccharides 2-4g / L, dipotassium hydrogen phosphate 2-4g / L, potassium dihydrogen phosphate 2-4g / L, magnesium sulfate 2-3g / L, calcium carbonate 2-4g / L, sodium acetate 2-3g / L, manganese sulfate 0.2-0.4g / L, potassium citrate 2-3g / L, deoxidizer 1-4g / L and water.

[0037] Furthermore, the feed medium comprises the following components:

[0038] Glucose 2-4g / L, sucrose 22-28g / L, complex oligosaccharides 2-3g / L and water.

[0039] The soy flour hydrolysate mentioned in the present invention is obtained by hydrolyzing defatted soy flour with non-starch polysaccharide enzymes such as cellulase, pectinase, xylanase and mannanase, then degrading with alkaline protease and aminopeptidase, centrifuging and spray drying the supernatant.

[0040] Furthermore, the soy flour hydrolysate has a total sugar content of 15% to 18%, a reducing sugar content of 10% to 12%, a small peptide content of 62% to 68%, and an amino acid content of 16% to 20%.

[0041] The small peptide mentioned in the present invention is a peptide consisting of 2 to 15 amino acids and having a molecular weight below 1000 Daltons.

[0042] Furthermore, the complex oligosaccharide comprises: oligogalactose, rhamnose and sophorose in a mass ratio of (2-5): (1-3): 1 (for example, but not limited to 2:3:1, 3:2.5:1, 4:2:1 or 5:1:1). The present invention utilizes specific types of oligosaccharides to combine in a certain mass ratio to obtain a complex oligosaccharide, which has the effect of promoting bacterial proliferation and ensuring that two bacterial cells can rapidly proliferate at the same time.

[0043] Furthermore, the deoxidizer includes: glucose oxidase and isoascorbic acid in a mass ratio of (2-5):1 (for example, but not limited to 2:1, 3:1, 4:1 or 5:1). The specific deoxidizer has good deoxidation effect and thorough deoxidation, and can provide an anaerobic environment for bacterial growth.

[0044] Furthermore, the enzymatic activity of the glucose oxidase is 5000-10000 U / g, including but not limited to any one of 5000 U / g, 5500 U / g, 6000 U / g, 6500 U / g, 7000 U / g, 7500 U / g, 8000 U / g, 8500 U / g, 9000 U / g, 9500 U / g or 10000 U / g or a range between any two of them.

[0045] Furthermore, the preparation method of the base medium comprises the following steps:

[0046] Mix yeast extract powder, soybean powder hydrolyzate, glucose, sucrose, complex oligosaccharides, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, calcium carbonate, sodium acetate, MnSO4, potassium citrate and water, and sterilize at 115°C for 30 minutes; when the base culture medium cools to room temperature, add the deoxidizer.

[0047] Furthermore, the method for preparing the feed medium comprises the following steps:

[0048] After mixing the components, sterilize at 115°C for 30 min.

[0049] Another aspect of the present invention also relates to a liquid combined fermentation method of Bifidobacterium and Clostridium butyricum, comprising the following steps:

[0050] (a) sequentially inoculating a seed liquid of Clostridium butyricum and a seed liquid of Bifidobacterium into a base medium in a liquid combined fermentation medium of Bifidobacterium and Clostridium butyricum for a first fermentation culture;

[0051] (b) adding the feed medium in the liquid combined fermentation medium of Bifidobacterium and Clostridium butyricum to the culture system of the first fermentation culture for feeding after inoculating the seed liquid of Clostridium butyricum for 2 to 4 hours (for example, it can be but not limited to any one of 2 hours, 3 hours or 4 hours or a range of values ​​between the two);

[0052] (c) After the feeding is completed, a stability promoter is added to the culture system of the first fermentation culture to carry out a second fermentation culture.

[0053] The bifidobacterium and Clostridium butyricum liquid combined fermentation method adopts the fermentation medium, and can harvest bifidobacterium and Clostridium butyricum in one fermentation. The number of live bacteria per unit volume in the bacterial liquid is high, and the production cost is reduced.

[0054] Furthermore, the seed solution concentration of Clostridium butyricum is 0.5×10 6 ~5×10 6 CFU / mL, including but not limited to 0.5×10 6 CFU / mL, 1×10 6 CFU / mL, 2×10 6 CFU / mL, 3×10 6 CFU / mL, 4×10 6 CFU / mL or 5×10 6 CFU / mL or a range of values ​​between them.

[0055] Furthermore, the concentration of the bifidobacterium seed solution is 0.5×10 6 ~5×10 6 CFU / mL, including but not limited to 0.5×10 6 CFU / mL, 1×10 6 CFU / mL, 2×10 6 CFU / mL, 3×10 6 CFU / mL, 4×10 6 CFU / mL or 5×10 6 CFU / mL or a range of values ​​between them.

[0056] Furthermore, the inoculation amount of the seed solution of Clostridium butyricum is 1 vol% to 5 vol%, including but not limited to any point value of 1 vol%, 2 vol%, 3 vol%, 4 vol% or 5 vol% or a range value between any two of them.

[0057] Furthermore, the inoculation amount of the bifidobacterium seed solution is 2 vol% to 10 vol%, including but not limited to any one of 2 vol%, 4 vol%, 6 vol%, 8 vol% or 10 vol% or a range between any two of them.

[0058] The inoculation amount of Bifidobacterium and Clostridium butyricum is controlled within a certain range to ensure that both can obtain sufficient nutrients and increase the number of live bacteria in fermentation.

[0059] Furthermore, the seed solution of Bifidobacterium is inoculated 2 to 4 hours (for example, but not limited to, any one of 2 hours, 3 hours or 4 hours or a range of values ​​between the two) after the seed solution of Clostridium butyricum is inoculated. The growth rate of Clostridium butyricum is slower than that of Bifidobacterium. In order to ensure that a sufficient number of Clostridium butyricum is obtained at the end of the culture, Clostridium butyricum is preferentially inoculated.

[0060] Furthermore, the temperature of the first fermentation culture is 32-35° C., including but not limited to any point value of 32° C., 33° C., 34° C. or 35° C. or a range value between any two of them. This temperature range is conducive to the symbiosis of Clostridium butyricum and Bifidobacterium, and reduces the competition between the two.

[0061] Further, the first fermentation culture is carried out under stirring conditions, and the stirring speed is 200-500 rpm, including but not limited to any point value of 200 rpm, 250 rpm, 300 rpm, 350 rpm, 400 rpm, 450 rpm or 500 rpm or any range value between two of them. Fermentation culture is carried out under this stirring speed, which is conducive to uniform distribution of nutrients and more uniform growth of Clostridium butyricum and Bifidobacterium.

[0062] Furthermore, the first fermentation culture is carried out in a sterile CO2 gas environment. The present invention adopts CO2 ventilation and the addition of a deoxidizer to the culture medium to ensure an anaerobic environment even with a high stirring speed during the fermentation process, so that the bacteria can reproduce normally. High-speed stirring can promote the uniform distribution of nutrients, avoid excessive local nutrient concentration and bacterial aggregation, increase the growth rate of the bacteria, and ensure more uniform bacterial growth.

[0063] Furthermore, the pressure of the first fermentation culture is 0.02-0.04 MPa, including but not limited to any point value of 0.02 MPa, 0.03 MPa or 0.04 MPa or a range value between any two of them.

[0064] Furthermore, the addition amount of the feed medium is 30% to 50% of the volume of the base medium, including but not limited to any point value of 30%, 35%, 40%, 45% or 50% or a range value between any two of them.

[0065] Furthermore, the feeding is carried out for 18 to 24 hours, including but not limited to any point value of 18 hours, 20 hours, 22 hours or 24 hours, or a range value between any two of them.

[0066] Furthermore, during the feeding process, the pH of the culture system of the first fermentation culture is 6.0-6.5, including but not limited to any point value of 6.0, 6.1, 6.2, 6.3, 6.4 or 6.5 or a range of values ​​between any two of them. This pH range is suitable for the growth of both bacteria.

[0067] Furthermore, the addition amount of the stabilizing agent is 1-5 g / L, including but not limited to any point value of 1 g / L, 2 g / L, 3 g / L, 4 g / L or 5 g / L or a range value between any two of them.

[0068] Furthermore, the stabilization promoter includes: N-acetyl-D-glucosamine and cell wall degradation products in a mass ratio of 1:(5-10) (for example, but not limited to 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10). Adding a specific stabilization promoter during the fermentation process can promote cell ripening, improve cell stress resistance, and enhance product stability.

[0069] Furthermore, the cell wall degradation products are obtained by degrading bifidobacteria and Clostridium butyricum with lysozyme, centrifuging and spray-drying the supernatant.

[0070] Furthermore, the temperature of the second fermentation culture is 38-40° C., including but not limited to any point value of 38° C., 39° C. or 40° C. or any range value between the two. Increasing the temperature of the second fermentation can give certain external stimulation to the bacteria, promote the secretion of protective substances by the bacteria, and promote the growth of the two bacteria.

[0071] Furthermore, the second fermentation culture time is 1 to 2 hours, including but not limited to any one of 1 hour, 1.2 hours, 1.4 hours, 1.6 hours, 1.8 hours or 2 hours, or a range between any two of them.

[0072] Furthermore, the second fermentation culture is carried out under stirring conditions, and the stirring speed is 40-60 rpm, including but not limited to any point value of 40 rpm, 45 rpm, 50 rpm, 55 rpm or 60 rpm or any range value between two of them. When the second fermentation culture is carried out, the two bacteria have developed to near maturity, and continuing high-speed stirring will cause damage to the bacteria, so the stirring speed is reduced to avoid damage to the bacteria.

[0073] Furthermore, the liquid combined fermentation method of Bifidobacterium and Clostridium butyricum also includes:

[0074] The culture solution after the second fermentation culture is centrifuged to collect bacterial sludge; the mixed system containing the bacterial sludge, sorbitol, cysteine, PEG6000 and water is freeze-dried to obtain a live bacterial preparation. Sorbitol, cysteine ​​and PEG6000 are freeze-drying protective agents that can reduce the death of bacteria during the freeze-drying process.

[0075] Furthermore, the mass ratio of the bacterial mud, the sorbitol, the cysteine, the PEG6000 and water is 10:1-3:1-2:2-5:5-10.

[0076] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0077] Example 1

[0078] The liquid combined fermentation medium of Bifidobacterium and Clostridium butyricum provided in this embodiment comprises: a base medium and a feed medium;

[0079] Wherein, the substrate culture medium comprises the following components:

[0080] Yeast extract powder 10g / L, soybean powder hydrolyzate 20g / L, glucose 2g / L, sucrose 10g / L, complex oligosaccharides 1g / L, dipotassium hydrogen phosphate 1g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 1g / L, calcium carbonate 1g / L, sodium acetate 1g / L, MnSO4 0.1g / L, potassium citrate 1g / L, deoxidizer 0.5g / L and the balance water.

[0081] The feed medium consists of the following components:

[0082] Glucose 1g / L, sucrose 20g / L, complex oligosaccharides 1g / L and the balance water.

[0083] The deoxidizer includes: glucose oxidase and isoascorbic acid in a mass ratio of 2:1, and the enzyme activity of glucose oxidase is 5000U / g;

[0084] The complex oligosaccharide comprises galacto-oligosaccharide, rhamnose and sophorose in a mass ratio of 2:1:1.

[0085] Example 2

[0086] The liquid combined fermentation medium of Bifidobacterium and Clostridium butyricum provided in this embodiment comprises: a base medium and a feed medium;

[0087] Wherein, the substrate culture medium comprises the following components:

[0088] Yeast extract powder 5g / L, soy flour hydrolyzate 40g / L, glucose 5g / L, sucrose 20g / L, complex oligosaccharides 5g / L, dipotassium hydrogen phosphate 5g / L, potassium dihydrogen phosphate 5g / L, magnesium sulfate 3g / L, calcium carbonate 5g / L, sodium acetate 3g / L, MnSO4 0.5g / L, potassium citrate 3g / L, deoxidizer 5g / L and the balance water.

[0089] The feed medium consists of the following components:

[0090] Glucose 5g / L, sucrose 30g / L, complex oligosaccharides 3g / L and the balance water.

[0091] The deoxidizer includes: glucose oxidase and isoascorbic acid in a mass ratio of 5:1, and the enzyme activity of glucose oxidase is 10000U / g;

[0092] The complex oligosaccharide comprises galacto-oligosaccharide, rhamnose and sophorose in a mass ratio of 5:3:1.

[0093] Example 3

[0094] The liquid combined fermentation medium of Bifidobacterium and Clostridium butyricum provided in this embodiment comprises: a base medium and a feed medium;

[0095] Wherein, the substrate culture medium comprises the following components:

[0096] Yeast extract powder 8g / L, soybean flour hydrolyzate 30g / L, glucose 4g / L, sucrose 20g / L, complex oligosaccharides 3g / L, dipotassium hydrogen phosphate 2g / L, potassium dihydrogen phosphate 2g / L, magnesium sulfate 2g / L, calcium carbonate 4g / L, sodium acetate 1g / L, MnSO4 0.3g / L, potassium citrate 3g / L, deoxidizer 3g / L and the balance water.

[0097] The feed medium consists of the following components:

[0098] Glucose 3g / L, sucrose 25g / L, complex oligosaccharides 2g / L and the balance water.

[0099] The deoxidizer includes: glucose oxidase and isoascorbic acid in a mass ratio of 4:1, and the enzyme activity of glucose oxidase is 8000U / g;

[0100] The complex oligosaccharide comprises galacto-oligosaccharide, rhamnose and sophorose in a mass ratio of 2:2:1.

[0101] Example 4

[0102] The liquid combined fermentation medium of Bifidobacterium and Clostridium butyricum provided in this embodiment comprises: a base medium and a feed medium;

[0103] Wherein, the substrate culture medium comprises the following components:

[0104] Yeast extract powder 8g / L, soy flour hydrolyzate 35g / L, glucose 4g / L, sucrose 20g / L, complex oligosaccharides 5g / L, dipotassium hydrogen phosphate 2g / L, potassium dihydrogen phosphate 2g / L, magnesium sulfate 1g / L, calcium carbonate 5g / L, sodium acetate 3g / L, MnSO4 0.5g / L, potassium citrate 3g / L, deoxidizer 2g / L and the balance water.

[0105] The feed medium consists of the following components:

[0106] Glucose 2g / L, sucrose 30g / L, complex oligosaccharides 3g / L and the balance water.

[0107] The deoxidizer includes: glucose oxidase and isoascorbic acid in a mass ratio of 4:1, and the enzyme activity of glucose oxidase is 7500U / g;

[0108] The complex oligosaccharide comprises galacto-oligosaccharide, rhamnose and sophorose in a mass ratio of 4:2:1.

[0109] Example 5

[0110] The liquid combined fermentation method of Bifidobacterium and Clostridium butyricum provided in this embodiment uses the fermentation medium provided in Example 1, and comprises the following steps:

[0111] S1. Preparation of seed culture medium: Prepare seed culture medium of Bifidobacterium and seed culture medium of Clostridium butyricum respectively, the concentration of seed culture medium is 0.5×10 6 CFU / ml;

[0112] S2, adding the base medium into the fermentation tank, and controlling the liquid volume to about 65%;

[0113] S3, placing feed medium in a feed tank;

[0114] S4, fermentation tank inoculation: add the Clostridium butyricum seed solution in S1 to the fermentation tank at an inoculation rate of 5 vol%, start stirring, control the stirring speed to 200 rpm, and introduce sterile CO2 gas to control the tank pressure to 0.02 MPa, maintain 32°C, and culture for 4 hours, then add the Bifidobacterium seed solution in S1 to the fermentation tank at an inoculation rate of 10 vol%, and continue fermentation and culture under the above conditions;

[0115] S5, fed-batch fermentation: 4 hours after the inoculation of bifidobacteria, feeding was started. During the feeding process, the pH of the fermentation liquid was adjusted to 6.0 with sodium bicarbonate, and the fermentation temperature was controlled at 32°C. The fed-batch fermentation was maintained for 24 hours and then the feeding was stopped;

[0116] S6, stabilizing the cells: adding a cell stabilization promoter at a rate of 1 g / L, raising the fermentation temperature to 38°C, reducing the stirring speed to 50 rpm, maintaining the culture for 1 hour, then stopping the stirring, introducing condensed water to cool the mixture to below 20°C, and ending the fermentation;

[0117] The stabilizing promoter includes: N-acetyl-D-glucosamine and cell wall degradants in a mass ratio of 1:10;

[0118] S7. Collecting live bacterial preparations: passing the culture fluid obtained in S6 into a tubular high-speed centrifuge to collect bacterial sludge by centrifugation. Add 1 part of sorbitol, 2 parts of cysteine, 2 parts of PEG6000 and 5 parts of sterile water to every 10 parts of wet bacterial sludge. Mix well and freeze-dry to obtain live bacterial preparations.

[0119] Example 6

[0120] The liquid combined fermentation method of Bifidobacterium and Clostridium butyricum provided in this embodiment uses the fermentation medium provided in Example 2, and comprises the following steps:

[0121] S1. Preparation of seed culture medium: Prepare seed culture medium of Bifidobacterium and seed culture medium of Clostridium butyricum respectively, the concentration of seed culture medium is 5×10 6 CFU / ml;

[0122] S2, adding the base medium into the fermentation tank, and controlling the liquid volume to about 65%;

[0123] S3, placing feed medium in a feed tank;

[0124] S4, fermentation tank inoculation: add the seed solution of Clostridium butyricum in S1 into the fermentation tank at an inoculation rate of 1 vol%, start stirring, control the stirring speed to 500 rpm, and introduce sterile CO2 gas to control the tank pressure to 0.04 MPa, maintain 35°C, and culture for 2 hours, then add the seed solution of Bifidobacterium d in S1 into the fermentation tank at an inoculation rate of 2 vol%, and continue fermentation and culture under the above conditions;

[0125] S5, fed-batch fermentation: 2 hours after the inoculation of bifidobacteria, feeding was started. During the feeding process, the pH of the fermentation liquid was adjusted to 6.5 with sodium bicarbonate, and the fermentation temperature was controlled at 35°C. The fed-batch fermentation was maintained for 18 hours and then the feeding was stopped;

[0126] S6, stabilizing the cells: adding a cell stabilization promoter at an amount of 5 g / L, raising the fermentation temperature to 40°C, reducing the stirring speed to 50 rpm, maintaining the culture for 2 hours, then stopping the stirring, introducing condensed water to cool down to below 20°C, and ending the fermentation;

[0127] The stabilizing promoter includes: N-acetyl-D-glucosamine and cell wall degradants in a mass ratio of 1:5;

[0128] S7. Collecting live bacterial preparations: passing the culture fluid obtained in S6 into a tubular high-speed centrifuge to collect bacterial sludge by centrifugation. Add 3 parts of sorbitol, 1 part of cysteine, 5 parts of PEG6000 and 10 parts of sterile water to every 10 parts of wet bacterial sludge. Mix well and freeze-dry to obtain live bacterial preparations.

[0129] Example 7

[0130] The liquid combined fermentation method of Bifidobacterium and Clostridium butyricum provided in this embodiment uses the fermentation medium provided in Example 3, and comprises the following steps:

[0131] S1. Preparation of seed culture medium: Prepare seed culture medium of Bifidobacterium and seed culture medium of Clostridium butyricum respectively, the concentration of seed culture medium is 2×10 6 CFU / ml;

[0132] S2, adding the base medium into the fermentation tank, and controlling the liquid volume to about 65%;

[0133] S3, placing feed medium in a feed tank;

[0134] S4, fermentation tank inoculation: add the seed solution of Clostridium butyricum in S1 into the fermentation tank at an inoculation rate of 4 vol%, start stirring, control the stirring speed to 350 rpm, and introduce sterile CO2 gas to control the tank pressure to 0.04 MPa, maintain 35°C, and culture for 2 hours, then add the seed solution of Bifidobacterium d in S1 into the fermentation tank at an inoculation rate of 10 vol%, and continue fermentation and culture under the above conditions;

[0135] S5, fed-batch fermentation: 2 hours after the inoculation of bifidobacteria, feeding was started. During the feeding process, the pH of the fermentation liquid was adjusted to 6.0 with sodium bicarbonate, and the fermentation temperature was controlled at 33°C. The fed-batch fermentation was maintained for 20 hours and then the feeding was stopped;

[0136] S6, stabilizing the cells: adding a cell stabilization promoter at a rate of 4 g / L, raising the fermentation temperature to 38°C, reducing the stirring speed to 50 rpm, maintaining the culture for 2 h, then stopping the stirring, introducing condensed water to cool the mixture to below 20°C, and ending the fermentation;

[0137] The bacterial cell stabilization promoter includes: N-acetyl-D-glucosamine and cell wall degradation products in a mass ratio of 1:8;

[0138] S7. Collecting live bacterial preparations: passing the culture fluid obtained in S6 into a tubular high-speed centrifuge to collect bacterial sludge by centrifugation. Add 3 parts of sorbitol, 2 parts of cysteine, 4 parts of PEG6000 and 8 parts of sterile water to every 10 parts of wet bacterial sludge. Mix well and freeze-dry to obtain live bacterial preparations.

[0139] Example 8

[0140] The liquid combined fermentation method of Bifidobacterium and Clostridium butyricum provided in this embodiment uses the fermentation medium provided in Example 4, and comprises the following steps:

[0141] S1. Preparation of seed culture medium: Prepare seed culture medium of Bifidobacterium and seed culture medium of Clostridium butyricum respectively, the concentration of seed culture medium is 2.5×10 6 CFU / ml;

[0142] S2, adding the base medium into the fermentation tank, and controlling the liquid volume to about 65%;

[0143] S3, placing feed medium in a feed tank;

[0144] S4, fermentation tank inoculation: add the seed solution of Clostridium butyricum in S1 into the fermentation tank at an inoculation rate of 3 vol%, start stirring, control the stirring speed to 300 rpm, and introduce sterile CO2 gas to control the tank pressure to 0.03 MPa, maintain 35°C, and culture for 3 hours, then add the seed solution of Bifidobacterium in S1 into the fermentation tank at an inoculation rate of 6 vol%, and continue fermentation and culture under the above conditions;

[0145] S5, fed-batch fermentation: 3 hours after the inoculation of bifidobacteria, feeding was started. During the feeding process, the pH of the fermentation liquid was adjusted to 6.5 with sodium bicarbonate, and the fermentation temperature was controlled at 32°C. The fed-batch fermentation was maintained for 22 hours and then the feeding was stopped;

[0146] S6, stabilizing the cells: adding a cell stabilization promoter at an amount of 3 g / L, raising the fermentation temperature to 39°C, reducing the stirring speed to 50 rpm, maintaining the culture for 1.5 hours, then stopping the stirring, introducing condensed water to cool down to below 20°C, and ending the fermentation;

[0147] The bacterial cell stabilization promoter includes: N-acetyl-D-glucosamine and cell wall degradation products in a mass ratio of 1:8;

[0148] S7. Collecting live bacterial preparations: passing the culture fluid obtained in S6 into a tubular high-speed centrifuge to collect bacterial sludge by centrifugation. Add 2 parts of sorbitol, 2 parts of cysteine, 3 parts of PEG6000 and 6 parts of sterile water to every 10 parts of wet bacterial sludge. Mix well and freeze-dry to obtain live bacterial preparations.

[0149] Comparative Example 1

[0150] The only difference between this comparative example and Example 4 is that no complex oligosaccharide is added to the base medium and the feed medium, and the fermentation method is the same as that of Example 8.

[0151] Comparative Example 2

[0152] The only difference between this comparative example and Example 4 is that no deoxidizer is added to the base culture medium, and the fermentation method is the same as Example 8.

[0153] Comparative Example 3

[0154] The only difference between this comparative example and Example 8 is that, in step S6, no stabilizing accelerator is added.

[0155] Comparative Example 4

[0156] The difference between this comparative example and Example 4 is that an equal amount of yeast extract powder is used to replace the soy flour hydrolyzate in the base culture medium, and the fermentation method is the same as that of Example 8.

[0157] Comparative Example 5

[0158] The only difference between this comparative example and Example 4 is that no deoxygenating agent is added to the base culture medium, and the only difference between this comparative example and Example 8 is that the stirring speed in step S4 is 20 rpm.

[0159] Experimental example

[0160] The live bacterial preparations formed in the above examples and comparative examples were counted, the number of live spores of Clostridium butyricum was counted, and the number of live bacteria of Bifidobacterium was counted. The counting results are shown in Table 1.

[0161] Table 1

[0162]

[0163] As shown in Table 1, the combined fermentation method provided by the present invention can effectively increase the number of live bacteria of Clostridium butyricum and Bifidobacterium in the live bacteria preparation. By comparing the embodiment with comparative examples 1 and 2, it can be shown that the complex oligosaccharide and the deoxidizer can effectively increase the number of live bacteria in the live bacteria preparation; by comparing the embodiment with comparative example 3, it can be shown that the stabilizer has little effect on the number of live bacteria in the live bacteria preparation; by comparing the embodiment with comparative example 4, it can be shown that the soy flour hydrolyzate is more suitable than yeast extract powder as the fermentation raw material of these two probiotics; by comparing the embodiment with comparative example 5, the use of a deoxidizer is more suitable for anaerobic fermentation than reducing the stirring speed.

[0164] The live bacteria preparations formed in the above Examples 5 to 8 and Comparative Examples 1 to 5 were vacuum packed and placed at 37°C for 1 month and 45°C for 2 weeks, respectively. Then, the live bacteria were counted, the number of live spores of Clostridium butyricum and the number of live bacteria of Bifidobacterium were counted, and the survival rates were calculated respectively. The results are shown in Table 2.

[0165] Table 2

[0166]

[0167] It can be seen from Table 2 that at different storage temperatures, the survival rates of Clostridium butyricum and Bifidobacterium in Comparative Example 4 are much lower than those in all the embodiments and also lower than those in other comparative examples, indicating that the addition of the stabilizer improves the survival rate of the two bacteria in the live bacteria preparation, especially the survival rate at high temperature.

[0168] Although the present invention has been illustrated and described with specific embodiments, it should be appreciated that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Those skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced by equivalents without departing from the spirit and scope of the present invention. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. Therefore, this means that all such replacements and modifications within the scope of the present invention are included in the appended claims.

Claims

1. A liquid combined fermentation medium of Bifidobacterium and Clostridium butyricum, characterized in that: include: Base medium and feed medium; Wherein, the base medium comprises the following components: Yeast extract powder 5-10g / L, soybean flour hydrolyzate 20-40g / L, glucose 2-5g / L, sucrose 10-20g / L, complex oligosaccharide 1-5g / L, dipotassium hydrogen phosphate 1-5g / L, potassium dihydrogen phosphate 1-5g / L, magnesium sulfate 1-3g / L, calcium carbonate 1-5g / L, sodium acetate 1-3g / L, manganese sulfate 0.1-0.5g / L, potassium citrate 1-3g / L, deoxidizer 0.5-5g / L and water; The feed medium comprises the following components: Glucose 1-5g / L, sucrose 20-30g / L, complex oligosaccharides 1-3g / L and water.

2. The liquid combined fermentation medium of Bifidobacterium and Clostridium butyricum according to claim 1, characterized in that: The substrate culture medium comprises the following components: Yeast extract powder 6-8g / L, soybean flour hydrolyzate 25-35g / L, glucose 3-4g / L, sucrose 12-18g / L, complex oligosaccharide 2-4g / L, dipotassium hydrogen phosphate 2-4g / L, potassium dihydrogen phosphate 2-4g / L, magnesium sulfate 2-3g / L, calcium carbonate 2-4g / L, sodium acetate 2-3g / L, manganese sulfate 0.2-0.4g / L, potassium citrate 2-3g / L, deoxidizer 1-4g / L and water; And / or, the feed medium comprises the following components: Glucose 2-4g / L, sucrose 22-28g / L, complex oligosaccharides 2-3g / L and water.

3. The liquid combined fermentation medium of Bifidobacterium and Clostridium butyricum according to claim 1 or 2, characterized in that: The soybean powder hydrolysate has a total sugar content of 15% to 18%, a reducing sugar content of 10% to 12%, a small peptide content of 62% to 68%, and an amino acid content of 16% to 20%.

4. The liquid combined fermentation medium of Bifidobacterium and Clostridium butyricum according to claim 1 or 2, characterized in that: The complex oligosaccharide comprises: oligogalactose, rhamnose and sophorose in a mass ratio of (2-5):(1-3):1; Preferably, the oxygen scavenger comprises: glucose oxidase and isoascorbic acid in a mass ratio of (2-5):1; Preferably, the enzymatic activity of the glucose oxidase is 5000-10000 U / g.

5. A liquid combined fermentation method of Bifidobacterium and Clostridium butyricum, characterized in that: The following steps are involved: (a) sequentially inoculating a seed liquid of Clostridium butyricum and a seed liquid of Bifidobacterium into a base medium in a liquid combined fermentation medium of Bifidobacterium and Clostridium butyricum according to any one of claims 1 to 4 for a first fermentation culture; (b) 2 to 4 hours after inoculating the seed liquid of Clostridium butyricum, adding a feed medium in the liquid combined fermentation medium of Bifidobacterium and Clostridium butyricum according to any one of claims 1 to 4 to the culture system of the first fermentation culture for feeding; (c) After the feeding is completed, a stability promoter is added to the culture system of the first fermentation culture to carry out a second fermentation culture.

6. The liquid combined fermentation method of Bifidobacterium and Clostridium butyricum according to claim 5, characterized in that: The seed solution of Bifidobacterium is inoculated 2 to 4 hours after the seed solution of Clostridium butyricum is inoculated.

7. The liquid combined fermentation method of Bifidobacterium and Clostridium butyricum according to claim 5, characterized in that: Includes at least one of the following technical features: (1) The temperature of the first fermentation culture is 32-35° C.; (2) the first fermentation culture is carried out under stirring conditions at a stirring speed of 200 to 500 rpm; (3) The first fermentation culture is carried out in a sterile CO2 gas environment.

8. The liquid combined fermentation method of Bifidobacterium and Clostridium butyricum according to claim 5, characterized in that: Includes at least one of the following technical features: (1) The amount of the feed medium added is 30% to 50% of the volume of the base medium; (2) the feeding is carried out for 18 to 24 hours; (3) During the feeding process, the pH of the culture system of the first fermentation culture is 6.0 to 6.

5.

9. The liquid combined fermentation method of Bifidobacterium and Clostridium butyricum according to claim 5, characterized in that: The amount of the stabilizing agent added is 1 to 5 g / L; And / or, the stabilization promoter comprises: N-acetyl-D-glucosamine and cell wall degradation products in a mass ratio of 1:(5-10).

10. The liquid combined fermentation method of Bifidobacterium and Clostridium butyricum according to claim 5, characterized in that: Includes at least one of the following technical features: (1) The temperature of the second fermentation culture is 38 to 40° C.; (2) The second fermentation culture time is 1 to 2 hours; (3) The second fermentation culture is carried out under stirring conditions with a stirring speed of 40 to 60 rpm.

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