CN114806920A

CN114806920A - Culture medium of bifidobacterium as well as culture method and application thereof

Info

Publication number: CN114806920A
Application number: CN202210186252.2A
Authority: CN
Inventors: 汤杰; 高合意; 雷登凤; 薛永永; 蔡晓红; 曾飒
Original assignee: Guangzhou Ridgepole Biological Technology Co ltd
Current assignee: Guangzhou Ridgepole Biological Technology Co ltd
Priority date: 2022-02-28
Filing date: 2022-02-28
Publication date: 2022-07-29
Anticipated expiration: 2042-02-28
Also published as: CN114806920B

Abstract

The invention relates to a culture medium of bifidobacterium, a culture method and application thereof, wherein the culture medium of bifidobacterium comprises a carbon source, a nitrogen source, inorganic salt and prebiotics; the prebiotics include fructooligosaccharides, low ester pectins, and inulin. The invention creatively combines fructo-oligosaccharide, low-ester pectin and inulin as prebiotic components to be added into a bifidobacterium culture medium, and the three components are mutually matched to realize synergistic interaction, thereby having unexpected synergistic interaction effect on the aspects of promoting the proliferation of bifidobacterium and improving the yield of thalli. Compared with the commercial culture medium, the bifidobacterium culture medium provided by the invention has a remarkable proliferation promoting effect on bifidobacterium, and has an important application value in the fields of food, medicines or cosmetics.

Description

Culture medium of bifidobacterium as well as culture method and application of culture medium

Technical Field

The invention belongs to the technical field of microbial culture, and relates to a culture medium of bifidobacterium, a culture method and application thereof.

Background

Bifidobacteria, which were originally isolated from the feces of breast-fed infants by Henry Tissier in 1899, are physiological bacteria beneficial to the body, have nutritional and health-care functions, and serve as important physiological bacteria in the human intestinal tract, and the presence and amount of the bifidobacteria are important markers of human health.

The bifidobacterium can provide various nutrient components including lactic acid, acetic acid, vitamins, protease and the like for the intestinal tract of a host, can improve the pH value of the organism and promote the organism to absorb metal ions such as phosphorus, iron, calcium and the like, vitamins and proteins; the bifidobacteria can compete for nutrients and spatial positions with pathogenic bacteria, and can prevent the growth of the pathogenic bacteria through metabolites of the bifidobacteria and production of antibiotics, bacteriocins and the like; the bifidobacterium has the function of immunoregulation, and can stimulate and activate the immune system to generate antibodies and cytokines, thereby better improving the immunity and anti-infection capability of the human body; the bifidobacterium can obviously increase the content and the biological activity of superoxide dismutase in blood, effectively promote the elimination of free radicals in organisms, reduce the toxicity of harmful substances in the organisms by 90 percent, inhibit the peroxidation of plasma lipid and have the effect of resisting the aging of the organisms.

After the newborn is born for hours, the bifidobacteria can be planted in the intestinal tract, however, due to the reasons of age increase, the use of antibiotics, environmental factors and the like, the quantity of the bifidobacteria in the intestinal tract of the human body is gradually reduced, so that the microbial ecological balance in the intestinal tract is caused, and various diseases or aging are caused. Therefore, the bifidobacterium is often used as a core component of a live bacterial preparation and is widely applied to the markets of medical treatment and health care. In recent years, studies have been made on the action of bifidobacterium, and it has been found that bifidobacterium has a function of promoting DNA repair after ultraviolet irradiation, a function of repairing skin barrier and protecting skin against photoaging caused by ultraviolet, and thus it is also very popular in the daily chemical market such as cosmetics, and the demand for bifidobacterium in the market is further increasing.

However, bifidobacterium belongs to obligate anaerobe, is very sensitive to oxygen, has poor tolerance to low pH and is extremely easy to inactivate, and the bifidobacterium has short life span and is difficult to preserve seeds.

For example, CN108085264A discloses an improved bifidobacterium tomato medium and a preparation method thereof. Wherein each liter of bifidobacterium tomato culture solution contains the following components: 9-10g of molasses, 89g of corn flour, 1.00-1.50mL of isomalto-oligosaccharide, 0.4-0.6g of potassium hydrogen phosphate, 0.0050.007 g of manganese sulfate and 1L of tomato juice basic culture medium. The improved bifidobacterium tomato culture medium has the advantages of short culture time, good activity, low cost, simple preparation method and operation, and the like.

CN109112089A discloses a subculture method for increasing the culture concentration of bifidobacterium. The invention finds the growth inertia of the bifidobacterium, improves the concentration of the viable cells by more than 10 times through a characteristic subculture scheme, has important values for preparing high-concentration bifidobacterium leavening agent and probiotic preparation and popularizing and applying the same, has important significance for meeting the requirements of people on high-quality food, and has good social and economic benefits for popularizing and applying the same.

However, the methods for culturing bifidobacteria provided by the prior art still have the problems of slow growth rate and low yield of bifidobacteria, and are difficult to be used for industrial production. Therefore, the development of a method for culturing bifidobacterium, which can increase the growth rate and yield of bifidobacterium, is a problem to be solved by those skilled in the art.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a culture medium of mycobacterium as well as a culture method and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a bifidobacterium culture medium comprising a carbon source, a nitrogen source, inorganic salts and prebiotics;

the prebiotics include fructooligosaccharides, low ester pectins, and inulin.

The invention creatively combines fructo-oligosaccharide, low-ester pectin and inulin as prebiotic components to be added into a bifidobacterium culture medium, and the three components are mutually matched to realize synergistic interaction, thereby having unexpected synergistic interaction effect on the aspects of promoting the proliferation of bifidobacterium and improving the yield of thalli. Compared with the commercial culture medium, the bifidobacterium culture medium provided by the invention has a remarkable proliferation promoting effect on bifidobacterium and has an important application value.

Preferably, the prebiotics comprise 1-5 parts of fructo-oligosaccharide, 1-5 parts of low ester pectin and 1-5 parts of inulin in parts by weight.

Specific examples of the above-mentioned 1 to 5 parts include 1 part, 1.2 parts, 1.5 parts, 1.7 parts, 2 parts, 2.2 parts, 2.5 parts, 2.7 parts, 3 parts, 3.2 parts, 3.5 parts, 3.7 parts, 4 parts, 4.2 parts, 4.5 parts, 4.7 parts, 5 parts and the like.

Preferably, the culture medium of bifidobacterium comprises 10 to 15g/L of carbon source, 30 to 45g/L of nitrogen source, 3 to 5g/L, L-0.1 to 1g/L of inorganic salt, 800.1 to 1g/L of tween and 1 to 5wt percent of prebiotics.

Specific values among the above 10-15g/L are, for example, 10g/L, 10.5g/L, 11g/L, 11.5g/L, 12g/L, 12.5g/L, 13g/L, 13.5g/L, 14g/L, 14.5g/L, 15g/L and the like.

Specific values among the above-mentioned 30 to 45g/L are, for example, 30g/L, 31g/L, 32g/L, 33g/L, 34g/L, 35g/L, 36g/L, 37g/L, 38g/L, 39g/L, 40g/L, 41g/L, 42g/L, 43g/L, 44g/L, 45g/L and the like.

Specific values among the above 3 to 5g/L are, for example, 3g/L, 3.2g/L, 3.4g/L, 3.6g/L, 3.8g/L, 4g/L, 4.2g/L, 4.4g/L, 4.6g/L, 4.8g/L, 5g/L and the like.

Specific values of the above-mentioned 0.1 to 1g/L are, for example, 0.1g/L, 0.2g/L, 0.3g/L, 0.4g/L, 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L, 1g/L and the like.

Specific values among the above 1 to 5 wt% are, for example, 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, 5 wt%, etc.

Preferably, the carbon source comprises glucose.

Preferably, the nitrogen source comprises any one or combination of at least two of liver soaking powder, plant peptone, tryptone or yeast powder, such as combination of liver soaking powder and plant peptone, combination of plant peptone and tryptone, combination of tryptone and tryptone, and the like, and any other combination can be adopted, preferably tryptone.

In comparison with other choices, tryptone or tryptose peptone as a nitrogen source has a more significant effect of promoting the proliferation of bifidobacteria, and tryptone is preferred as a nitrogen source in the present invention from the viewpoint of cost reduction.

Preferably, the inorganic salt includes any one or a combination of at least two of sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ferrous sulfate, manganese sulfate, magnesium sulfate and calcium chloride, for example, a combination of dipotassium hydrogen phosphate and potassium dihydrogen phosphate, a combination of manganese sulfate and magnesium sulfate, a combination of magnesium sulfate and calcium chloride, and the like, and any other combination may be adopted, and preferably a combination of sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ferrous sulfate, manganese sulfate, magnesium sulfate and calcium chloride.

When the culture medium formula contains sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ferrous sulfate, manganese sulfate, magnesium sulfate and calcium chloride, potassium, sodium, iron, magnesium, calcium, phosphorus, sulfur and other elements required for the growth of the bifidobacteria can be provided for the bifidobacteria, and the elements are very important for maintaining the activity of enzyme and regulating the osmotic pressure inside and outside the bacteria, so that the growth and proliferation of the bacteria are promoted.

Preferably, the inorganic salt comprises, by weight, 0.8-1.2 parts of sodium chloride, 0.8-1.2 parts of dipotassium hydrogen phosphate, 0.8-1.2 parts of monopotassium phosphate, 0.008-0.012 parts of ferrous sulfate, 0.002-0.008 parts of manganese sulfate, 0.2-0.8 parts of magnesium sulfate and 0.1-0.2 parts of calcium chloride.

Specific examples of the above-mentioned 0.8 to 1.2 parts include 0.8 part, 0.85 part, 0.9 part, 0.95 part, 1.0 part, 1.05 part, 1.1 part, 1.15 part, 1.2 part and the like.

Specific examples of the above-mentioned 0.008 to 0.012 parts include 0.008 parts, 0.0085 parts, 0.009 parts, 0.0095 parts, 0.010 parts, 0.0105 parts, 0.011 parts, 0.0115 parts, and 0.012 parts.

Specific examples of the above-mentioned 0.002 to 0.008 parts include 0.002 parts, 0.0025 parts, 0.003 parts, 0.0035 parts, 0.004 parts, 0.0045 parts, 0.005 parts, 0.0055 parts, 0.006 parts, 0.0065 parts, 0.007 parts, 0.0075 parts, and 0.008 parts.

Specific examples of the above-mentioned 0.2 to 0.8 parts include 0.2 part, 0.25 part, 0.3 part, 0.35 part, 0.4 part, 0.45 part, 0.5 part, 0.55 part, 0.6 part, 0.65 part, 0.7 part, 0.75 part, 0.8 part and the like.

Specific examples of the above-mentioned 0.1 to 0.2 parts include 0.1 part, 0.11 part, 0.12 part, 0.13 part, 0.14 part, 0.15 part, 0.16 part, 0.17 part, 0.18 part, 0.19 part, 0.2 part and the like.

Preferably, the pH of the culture medium for bifidobacteria is 6.0-7.0, e.g., 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, etc.

When the pH of the medium is within the above range, growth and proliferation of Bifidobacterium are facilitated and the yield of Bifidobacterium obtained by the culture is higher.

In a second aspect, the present invention provides a method for culturing bifidobacteria, which comprises inoculating bifidobacteria to the culture medium for culture of bifidobacteria according to the first aspect.

Preferably, the temperature of the culture is 35-40 ℃, and the time of the culture is 20-30 h.

Specific values of the above-mentioned temperature range of 35-40 ℃ are, for example, 35 ℃, 35.5 ℃, 36 ℃, 36.5 ℃, 37 ℃, 37.5 ℃, 38 ℃, 38.5 ℃, 39 ℃, 39.5 ℃ and 40 ℃.

Specific numerical values of the above-mentioned 20 to 30h are, for example, 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h and the like.

Preferably, the bifidobacterium is inoculated in an amount of 1-3%, e.g. 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%, 2.2%, 2.4%, 2.6%, 2.8%, 3.0% etc.

Preferably, the culturing further comprises feeding, the feeding comprising supplementing a carbon source.

The invention creatively adds a material supplementing process in the culture process, and can obviously promote the bifidobacterium to fully utilize the nutrient components in the culture medium, thereby greatly improving the bacterium supplementing effect and increasing the thallus yield.

Preferably, the feeding is performed when the thalli grow to the middle or later period of logarithmic growth.

The time of feeding has certain influence on the bacterium increasing effect, and when the feeding operation is carried out in the middle or later period of logarithmic growth, the bacterium increasing effect is better, and the thallus yield is higher.

In a third aspect, the present invention provides a use of the method for culturing bifidobacterium according to the second aspect in the preparation of food, medicine or cosmetics.

The recitation of numerical ranges herein includes not only the above-recited values, but also any values between any of the above-recited numerical ranges not recited, and for brevity and clarity, is not intended to be exhaustive of the specific values encompassed within the range.

Compared with the prior art, the invention has the following beneficial effects:

the invention creatively adds the combination of inulin, fructo-oligosaccharide and low-ester pectin as prebiotic components into the culture medium of bifidobacterium, and the three components are mutually matched, thereby having synergistic effect, remarkable bacterium increasing effect and greatly improving the yield of the bifidobacterium. In addition, the selection of the nitrogen source has certain influence on the bacterium increasing effect, and the trypsin is used as the nitrogen source, so that the cost is lower, and the bacterium increasing effect is better than that of other nitrogen sources. In addition, the invention creatively adds a material supplementing process in the culture process, and can obviously promote the bifidobacterium to fully utilize the nutrient components in the culture medium, thereby greatly improving the bacterium increasing effect and increasing the thallus yield. In addition, the time of feeding has certain influence on the bacterium increasing effect, and the feeding operation is preferably carried out in the middle or later period of logarithmic growth. Compared with the commercial culture medium and the conventional culture method, the culture medium and the culture method for bifidobacterium provided by the invention obviously promote the proliferation of bifidobacterium, improve the yield of thalli and have important application value.

Drawings

FIG. 1 is a graph comparing the results for OD600 of examples 1, 2, 3, and 6, where example 1 is pH6.5, example 2 is pH 6.0, example 3 is pH 7.0, and example 6 is pH 5.0.

FIG. 2 is a graph comparing the results of OD600 cultured with different nitrogen sources. Among them, tryptone was used in example 1, liver extract powder was used in example 7, phytone was used in example 8, yeast powder was used in example 9, and tryptone was used in example 10.

Figure 3 is a graph comparing the results of OD600 for different prebiotic cultures. Wherein example 1 is a prebiotic combination, comparative example 4 is fructo-oligosaccharide, comparative example 5 is low ester pectin, and comparative example 6 is inulin.

Detailed Description

To further illustrate the technical means and effects of the present invention, the following further describes the technical solution of the present invention with reference to the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

The following examples and comparative examples relate to fructooligosaccharides obtained from Zhengzhou Wanbo chemical products Co., Ltd., low ester pectins obtained from Shandong Bailong Chuanchuan biological science Co., Ltd., and inulin obtained from Chongqing Jiaowang agricultural development Co., Ltd. The bifidobacteria referred to in the following examples and comparative examples are Bifidobacterium infantis, purchased from the biological species Collection of Committee, Guangdong province, with the accession number CICC 6069.

Example 1

The present embodiment provides a method for culturing bifidobacterium, which comprises the following steps:

the formula of the culture medium is as follows: 36.0g/L of tryptone, 12.0g/L of glucose, 1.0g/L of sodium chloride, 1.0g/L of dipotassium phosphate, 1.0g/L of monopotassium phosphate, 0.01g/L of ferrous sulfate heptahydrate, 0.005g/L of manganese sulfate, 0.5g/L of magnesium sulfate, 0.15g/L, L-cysteine, 0.5g/L of calcium chloride, 800.5 g/L of tween, 1 wt% of inulin, 1 wt% of fructo-oligosaccharide, 1 wt% of low-ester pectin and water as a solvent.

(1) Preparing a culture medium according to a formula, adjusting the pH value to 6.5 by using NaOH aqueous solution, and sterilizing at the high temperature of 121 ℃ for 20min for later use.

(2) Taking out the bifidobacterium cryopreservation tube from a refrigerator at minus 80 ℃, freezing and thawing, sucking 1mL of seed solution, inoculating the seed solution into a seed activation culture medium (commercial MRS culture medium, specification 027312), and standing and culturing at 37 ℃ for 24h to obtain activated bifidobacterium.

(3) Inoculating the activated bifidobacteria obtained in the step (2) into the culture medium prepared in the step (1) in an inoculation amount of 2%, culturing at 37 ℃, supplementing 50 wt% of glucose aqueous solution when the bacteria grow to the middle logarithmic growth phase, wherein the supplementing speed is 50mL/h, and stirring (50rpm, 5min per 50 min) after supplementing. And co-culturing for 24 h.

Example 2

the formula of the culture medium is as follows: 32.0g/L of tryptone, 10.0g/L of glucose, 1.5g/L of sodium chloride, 1.5g/L of dipotassium phosphate, 1.5g/L of monopotassium phosphate, 0.02g/L of ferrous sulfate heptahydrate, 0.005g/L of manganese sulfate, 0.5g/L of magnesium sulfate, 0.10g/L, L g/L of calcium chloride-0.8 g/L of cysteine, 800.2 g/L of tween, 2 wt% of inulin, 0.5 wt% of fructo-oligosaccharide, 1 wt% of low-ester pectin and water as a solvent.

(1) Preparing culture medium according to the formula, adjusting pH to 6.0 with NaOH water solution, and sterilizing at 121 deg.C for 20 min.

(2) Taking out the bifidobacterium cryopreservation tube from a refrigerator at minus 80 ℃, sucking 1mL of seed liquid after freeze thawing, inoculating the seed liquid into a seed activation culture medium (commercial MRS culture medium, specification 027312), and standing and culturing at 37 ℃ for 24h to obtain activated bifidobacterium.

(3) Inoculating the activated bifidobacteria obtained in the step (2) into the culture medium prepared in the step (1) in an inoculation amount of 2%, culturing at 39 ℃, supplementing 50 wt% of glucose aqueous solution when the bacteria grow to the middle logarithmic growth phase, wherein the supplementing speed is 50mL/h, and stirring (50rpm, 5min per 50 min) after supplementing. And co-culturing for 24 h.

Example 3

the formula of the culture medium is as follows: 42.0g/L tryptone, 15.0g/L glucose, 0.8g/L sodium chloride, 1.1g/L dipotassium hydrogen phosphate, 1.1g/L potassium dihydrogen phosphate, 0.02g/L ferrous sulfate heptahydrate, 0.008g/L manganese sulfate, 0.8g/L magnesium sulfate, 0.2g/L, L-calcium chloride-0.3 g/L cysteine, 800.8 g/L tween, 0.8 wt% inulin, 1.2 wt% fructo-oligosaccharide, 1.2 wt% low ester pectin and water as solvent.

(1) Preparing culture medium according to the formula, adjusting pH to 7.0 with NaOH aqueous solution, and sterilizing at 121 deg.C for 20 min.

(3) Inoculating the activated bifidobacteria obtained in the step (2) into the culture medium prepared in the step (1) in an inoculation amount of 2%, culturing at 35 ℃, supplementing 50 wt% of glucose aqueous solution when the bacteria grow to the late logarithmic growth stage, wherein the supplementing speed is 50mL/h, and stirring (50rpm, 5min per 50 min) after supplementing. And co-culturing for 24 h.

Example 4

This example provides a method for culturing Bifidobacterium, which differs from example 1 only in that "a 50 wt% aqueous glucose solution is supplemented when the cells grow to the mid-logarithmic growth phase" is changed to "a 50 wt% aqueous glucose solution is supplemented when the cells grow to the pre-logarithmic growth phase" in step (3), otherwise refer to example 1.

Example 5

This example provides a method for culturing Bifidobacterium, which is different from example 1 only in that no feeding operation is performed in step (3), and an aqueous glucose solution in an amount equal to that added in the feeding operation of example 1 is added directly at the start of culturing, and otherwise refer to example 1.

Example 6

This example provides a method for culturing Bifidobacterium, which is different from example 1 only in that the pH of the culture medium is adjusted from "6.5" to "5.0" and the other components and contents are not changed.

The preparation process is as in example 1.

Example 7

This example provides a method for culturing Bifidobacterium, which differs from example 1 only in that the nitrogen source "tryptone" in the medium is replaced by an equal amount of "liver extract", and otherwise referred to in example 1.

Example 8

This example provides a method for culturing bifidobacteria, which differs from example 1 only in that the nitrogen source "tryptone" in the culture medium is replaced by an equal amount of "phytone", otherwise referred to in example 1.

Example 9

This example provides a method for culturing Bifidobacterium, which differs from example 1 only in that the nitrogen source "tryptone" in the medium is replaced by an equal amount of "yeast powder", and otherwise refers to example 1.

Example 10

This example provides a method for culturing Bifidobacterium, which differs from example 1 only in that the nitrogen source "tryptone" in the medium is replaced by the same amount of "tryptone", and otherwise referred to in example 1.

Comparative example 1

This comparative example provides a method for culturing bifidobacteria which differs from example 1 only in that the prebiotics "inulin 1 wt%, fructo-oligosaccharide 1 wt%, low ester pectin 1 wt%" in the culture medium are replaced with "inulin 1.5 wt%, fructo-oligosaccharide 1.5 wt%" and otherwise referred to example 1.

Comparative example 2

This comparative example provides a method for culturing bifidobacteria which differs from example 1 only in that the prebiotics "inulin 1 wt%, fructo-oligosaccharide 1 wt%, low ester pectin 1 wt%" in the culture medium are replaced with "inulin 1.5 wt%, low ester pectin 1.5 wt%" and otherwise referred to example 1.

Comparative example 3

This comparative example provides a method for the culture of bifidobacteria which differs from example 1 only in that the prebiotics "inulin 1 wt%, fructo-oligosaccharide 1 wt%, low ester pectin 1 wt%" in the culture medium are replaced by "fructo-oligosaccharide 1.5 wt%, low ester pectin 1.5 wt%" and otherwise referred to example 1.

Comparative example 4

This comparative example provides a method for culturing bifidobacteria which differs from example 1 only in that the prebiotics "inulin 1 wt%, fructo-oligosaccharide 1 wt%, low ester pectin 1 wt%" in the culture medium are replaced by "fructo-oligosaccharide 3 wt%", with reference to example 1.

Comparative example 5

This comparative example provides a method for the culture of bifidobacteria which differs from example 1 only in that the prebiotics "inulin 1 wt%, fructo-oligosaccharide 1 wt%, low ester pectin 1 wt%" in the culture medium are replaced by "low ester pectin 3 wt%" and otherwise refer to example 1.

Comparative example 6

This comparative example provides a method for culturing bifidobacteria which differs from example 1 only in that the prebiotics "inulin 1 wt%, fructo-oligosaccharide 1 wt%, low ester pectin 1 wt%" in the culture medium are replaced with "inulin 3 wt%", with reference to example 1.

Comparative example 7

This comparative example provides a method for the culture of bifidobacteria which differs from example 1 only in that the culture medium lacks the prebiotics inulin, fructo-oligosaccharide and low ester pectin, the amount of which is made up with water, otherwise referred to in example 1.

Test example 1

Test groups were: examples 1 to 10, comparative examples 1 to 7.

The enrichment effect of bifidobacteria was evaluated by measuring the OD600 value in the bacterial liquid after the end of the culture.

After the culture was completed, the bacterial solution was centrifuged at 15000rpm, and the cells were collected, dried and weighed to obtain the yield of Bifidobacterium.

The results of the tests for each group are shown in table 1.

In order to make the result more visual, part of the data is arranged into a picture.

TABLE 1

The results show that: the methods for culturing bifidobacteria provided in examples 1 to 3 significantly promote the proliferation of bifidobacteria and produce higher yields of the resulting cells. The bacteria-increasing effect and the cell yield of the culture methods of comparative examples 1 to 7 are lower than those of example 1, which shows that the addition of the prebiotics into the culture medium plays a role in promoting the proliferation of the bifidobacteria, and when the combination of inulin, fructo-oligosaccharide and low-ester pectin is used as the prebiotic component, the bacteria-increasing effect is better than that of the combination of a single component or any two components, thereby proving the synergistic effect of the three components in promoting the proliferation of the bifidobacteria. The culture methods of examples 7 to 9 showed lower enrichment effects and cell yields than those of example 1, indicating that the selection of nitrogen source in the culture medium has a certain effect on the proliferation of bifidobacteria, and the present invention selected tryptone as nitrogen source, which showed better enrichment effects than those of liver extract, phytone and yeast powder. The bacteria-proliferating effect and the cell yield of the culture method of example 10 are relatively close to those of example 1, and it is explained that tryptone is used as the nitrogen source, and the bacterium-proliferating effect is close to that of tryptone, but tryptone is preferable as the nitrogen source in the present invention from the viewpoint of cost reduction. The culture method of example 6 showed lower growth enhancement effect and cell yield than example 1, indicating that the pH of the culture medium had a greater effect on the growth and proliferation of bifidobacteria, and that the culture medium had a pH of 6.0 to 7.0, which resulted in better growth enhancement effect and higher yield. The bacteria increasing effect and the thallus yield of the culture methods of the examples 4-5 are lower than those of the example 1, which shows that the supplement process is added in the culture process, so that the full utilization of the nutrient components in the culture medium by the bifidobacterium can be obviously promoted, the bacteria increasing effect is greatly improved, and the thallus yield is improved. In addition, the time of feeding has certain influence on the bacterium increasing effect, and the feeding operation is preferably carried out in the middle or later period of logarithmic growth. The bifidobacterium prepared by the corresponding culture method can be well applied to food, medicines or cosmetics.

The applicant states that the present invention is described by the above examples to describe a culture medium for bifidobacterium, a culture method and applications thereof, but the present invention is not limited to the above examples, i.e. it is not meant to be construed that the present invention is necessarily limited to the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

Claims

1. A culture medium for bifidobacterium, comprising a carbon source, a nitrogen source, inorganic salts and prebiotics;

the prebiotics include fructooligosaccharides, low ester pectins, and inulin.

2. A culture medium for bifidobacteria according to claim 1 wherein said prebiotic comprises, in parts by weight, 1-5 parts fructooligosaccharide, 1-5 parts pectin oligoester, and 1-5 parts inulin.

3. A culture medium for bifidobacteria according to claim 1 or 2, wherein said culture medium for bifidobacteria comprises, in concentration, 10 to 15g/L carbon source, 30 to 45g/L nitrogen source, 3 to 5g/L, L g/L inorganic salt, 0.1 to 1g/L cysteine, 800.1 to 1g/L tween and 1 to 5 wt.% prebiotics.

4. The culture medium of bifidobacteria according to any of claims 1-3, wherein said carbon source comprises glucose;

preferably, the nitrogen source comprises any one or a combination of at least two of liver soaking powder, plant peptone, tryptone or yeast powder, preferably tryptone.

5. The culture medium for bifidobacteria according to any of claims 1-4, wherein said inorganic salts comprise any one or a combination of at least two of sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ferrous sulfate, manganese sulfate, magnesium sulfate, or calcium chloride, preferably a combination of sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ferrous sulfate, manganese sulfate, magnesium sulfate, and calcium chloride;

6. A culture medium for bifidobacteria according to any of claims 1 to 5, wherein said culture medium for bifidobacteria has a pH of from 6.0 to 7.0.

7. A method for culturing Bifidobacterium, which comprises inoculating Bifidobacterium medium as claimed in any of claims 1 to 6 and culturing.

8. The method for culturing Bifidobacterium according to claim 7, wherein the culturing is carried out at a temperature of 35 to 40 ℃ for 20 to 30 hours;

preferably, the bifidobacterium is inoculated in an amount of 1-3%.

9. The method for culturing Bifidobacterium according to claim 7 or 8, wherein the culturing further comprises feeding, the feeding comprising supplementing a carbon source;

preferably, the feeding is performed when the thalli grow to the middle or late stage of logarithmic growth.

10. Use of a method of culturing bifidobacteria according to any of claims 7-9 for the preparation of a food, pharmaceutical, or cosmetic product.