CN114806920B - Culture medium of bifidobacterium and culture method and application thereof - Google Patents

Culture medium of bifidobacterium and culture method and application thereof Download PDF

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CN114806920B
CN114806920B CN202210186252.2A CN202210186252A CN114806920B CN 114806920 B CN114806920 B CN 114806920B CN 202210186252 A CN202210186252 A CN 202210186252A CN 114806920 B CN114806920 B CN 114806920B
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bifidobacterium
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culture medium
bifidobacteria
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汤杰
高合意
雷登凤
薛永永
蔡晓红
曾飒
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Guangzhou Ridgepole Biological Technology Co ltd
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Abstract

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

Description

Culture medium of bifidobacterium and culture method and application thereof
Technical Field
The invention belongs to the technical field of microorganism culture, and relates to a bifidobacterium culture medium, a culture method and application thereof.
Background
Bifidobacteria were originally isolated from the faeces of breast-fed infants in 1899 by Henry Tissier, which is a physiological bacterium beneficial to the body, has nutrition and health care functions, is an important physiological bacterium in the intestinal tract of the human body, and the existence and quantity of the bifidobacteria are an important sign of the health of the human body.
The bifidobacteria can provide various nutritional ingredients for the intestinal tracts of a host, such as lactic acid, acetic acid, vitamins, protease and the like, 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; bifidobacteria can compete with pathogenic bacteria for nutrients and space positions, and can also prevent the growth of pathogens through metabolites thereof and the production of antibiotics, bacteriocins and the like; the bifidobacterium has the function of immunoregulation, and can generate antibodies and cytokines by stimulating and activating an immune system, so that the immunity and anti-infection capability of a human body are improved better; the bifidobacterium can obviously increase the content and the biological activity of superoxide dismutase in blood, effectively promote the removal of free radicals in the organism, reduce the toxicity of harmful substances in the organism by 90%, inhibit the peroxidation of plasma lipid, and have the efficacy of resisting the aging of the organism.
The bifidobacteria can be planted in the intestinal tract after the neonate is born for a plurality of hours, however, the quantity of the bifidobacteria in the intestinal tract of the human body gradually decreases due to the age increase, the use of antibiotics, environmental factors and the like, so that the ecological balance of microorganisms in the intestinal tract is caused, and various diseases or aging are caused. Therefore, bifidobacteria are often used as core components of live bacteria preparations and widely used in medical and health-care markets. In recent years, the effect of bifidobacteria has been studied deeply, and it has been found that the bifidobacteria have a function of promoting DNA repair after ultraviolet irradiation, and can repair skin barrier and protect skin against photo-aging caused by ultraviolet irradiation, so that the bifidobacteria are also increasingly excellent in daily chemical markets such as cosmetics, and the demand of bifidobacteria in the market is further increased.
However, bifidobacteria are obligate anaerobes, are very sensitive to oxygen, have poor tolerance to low pH, are extremely easy to inactivate, have shorter survival time and are difficult to preserve, and the strict culture conditions limit the culture and amplification of the bifidobacteria, so that great difficulty is brought to the industrialized production of the bifidobacteria, and a plurality of bifidobacteria culture methods are developed for the majority of researchers.
For example CN108085264a discloses an improved bifidobacterium tomato culture medium and a method for preparing the same. Wherein each liter of bifidobacterium tomato culture solution contains the following components: 9-10g of molasses, 89g of corn meal, 1.00-1.50mL of isomaltooligosaccharide, 0.4-0.6g of potassium dihydrogen phosphate, 0.005.0070 g of manganese sulfate and 1L of tomato juice basal medium. The improved bifidobacterium tomato culture medium has the advantages of short culture time, good activity, low cost, simple preparation method operation and the like.
CN109112089a discloses a subculture method for increasing the culture concentration of bifidobacteria. The invention discovers that the bifidobacterium grows inertly and the concentration of living cells is improved by more than 10 times through a characteristic subculture scheme, which has important value for preparing a high-concentration bifidobacterium starter and a probiotic preparation and popularizing and applying the preparation, and has important significance for meeting the requirements of people on high-quality foods, and the popularization and application of the preparation have good social and economic benefits.
However, the bifidobacterium culture method provided in the prior art still has the problems of low bifidobacterium growth speed and low yield, and is difficult to be used for industrial production. Therefore, how to develop a bifidobacterium culture method capable of improving 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 bifidobacteria, a culture method and application thereof.
In order to achieve the aim of the invention, 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, an inorganic salt and a prebiotic;
the prebiotics include fructooligosaccharides, low ester pectins, and inulin.
The invention creatively adds the combination of fructo-oligosaccharide, low-ester pectin and inulin as the prebiotic components into the bifidobacterium culture medium, and the three components are mutually matched and synergistically act, thus having unexpected synergistic effect in the aspects of promoting the proliferation of bifidobacterium and improving the yield of thalli. The bifidobacterium culture medium provided by the invention has remarkable proliferation promoting effect on bifidobacterium compared with the commercial culture medium, and has important application value.
Preferably, the prebiotics comprise, in parts by weight, 1-5 parts of fructo-oligosaccharides, 1-5 parts of low-ester pectin and 1-5 parts of inulin.
Specific values among the above 1 to 5 parts are, for example, 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, etc.
Preferably, the bifidobacterium culture medium comprises 10-15g/L of carbon source, 30-45g/L of nitrogen source, 3-5g/L, L-cysteine of inorganic salt, 0.1-1g/L of tween 80, 0.1-1g/L and 1-5wt% of prebiotics in terms of concentration.
Specific values among the above 10 to 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, etc.
Specific values among the above 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, etc.
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, etc.
Specific values among the above 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, etc.
Specific values in the above 1 to 5wt% are, for example, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt%, 5wt%, etc.
Preferably, the carbon source comprises glucose.
Preferably, the nitrogen source includes any one or a combination of at least two of liver extract powder, plant peptone, tryptone, and yeast powder, for example, a combination of liver extract powder and plant peptone, a combination of plant peptone and tryptone, a combination of tryptone and tryptone, and the like, and any other combination means may be used, and tryptone is preferable.
When tryptone or tryptone is selected as the nitrogen source, the effect of promoting bifidobacterium proliferation is more remarkable than other choices, and in view of cost reduction, the tryptone is preferable as the nitrogen source in the present invention.
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, etc., and any other combination manner is preferable, and sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ferrous sulfate, manganese sulfate, magnesium sulfate and calcium chloride are preferable.
When the culture medium formula contains sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ferrous sulfate, manganese sulfate, magnesium sulfate and calcium chloride, elements such as potassium, sodium, iron, magnesium, calcium, phosphorus, sulfur and the like required by the growth of the bifidobacterium can be provided for the bifidobacterium, and the elements are very important for maintaining the activity of enzymes and regulating the osmotic pressure in and out of the thalli, so that the growth and proliferation of the thalli 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 values among the above 0.8 to 1.2 parts are, for example, 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 values among the above 0.008 to 0.012 parts are, for example, 0.008 parts, 0.0085 parts, 0.009 parts, 0.0095 parts, 0.010 parts, 0.0105 parts, 0.011 parts, 0.0115 parts, 0.012 parts, and the like.
Specific values among the above 0.002 to 0.008 parts are, for example, 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, 0.008 parts and the like.
Specific values among the above 0.2 to 0.8 parts are, for example, 0.2 parts, 0.25 parts, 0.3 parts, 0.35 parts, 0.4 parts, 0.45 parts, 0.5 parts, 0.55 parts, 0.6 parts, 0.65 parts, 0.7 parts, 0.75 parts, 0.8 parts, and the like.
Specific values among the above 0.1 to 0.2 parts are, for example, 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 bifidobacterium medium is between 6.0 and 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 culture medium is within the above range, the growth and proliferation of bifidobacteria are facilitated, and the yield of the cultured bifidobacteria is higher.
In a second aspect, the present invention provides a method of culturing bifidobacteria comprising inoculating bifidobacteria into a bifidobacterium medium as described in the first aspect for culturing.
Preferably, the temperature of the culture is 35-40 ℃, and the time of the culture is 20-30h.
Specific values of the above 35-40deg.C include 35 deg.C, 35.5 deg.C, 36.5 deg.C, 37 deg.C, 37.5 deg.C, 38 deg.C, 38.5 deg.C, 39 deg.C, 39.5 deg.C, 40 deg.C, etc.
Specific values among the above 20 to 30h are, for example, 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h, etc.
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, which comprises feeding a carbon source.
The invention creatively adds the feeding process in the culture process, and can obviously promote the full utilization of the bifidobacteria on the nutrient components in the culture medium, thereby greatly improving the bacteria increasing effect and the thallus yield.
Preferably, the feeding is performed when the bacterial cells grow to mid-log or late-log growth.
The timing of the material supplementing has a certain influence on the bacteria increasing effect, and when the material supplementing operation is carried out in the mid-phase or later-phase of logarithmic growth, the bacteria increasing effect is better, and the thallus yield is higher.
In a third aspect, the present invention provides the use of a method of culturing bifidobacteria as described in the second aspect in the preparation of a food, pharmaceutical or cosmetic product.
The numerical ranges recited herein include not only the recited point values, but also any point values between the recited numerical ranges that are not recited, and are limited to, and for the sake of brevity, the invention is not intended to be exhaustive of the specific point values that the recited range includes.
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, synergistic, the bacteria increasing effect is obvious, and the yield of bifidobacterium is greatly improved. In addition, the selection of the nitrogen source has a certain influence on the bacterial increasing effect, and the invention adopts the pancreatic protein as the nitrogen source, has lower cost and has better bacterial increasing effect compared with other nitrogen sources. In addition, the invention creatively adds a feeding process in the culture process, and can obviously promote the full utilization of the bifidobacteria on the nutrient components in the culture medium, thereby greatly improving the bacteria increasing effect and the thallus yield. In addition, the timing of feeding has a certain influence on the bacterial increasing effect, and feeding operation is preferably performed in the mid-stage or the later stage of logarithmic growth. Compared with the commercial culture medium and the conventional culture method, the bifidobacterium culture medium and the culture method provided by the invention have the advantages that the proliferation of bifidobacterium is obviously promoted, the yield of thalli is improved, and the application value is important.
Drawings
FIG. 1 is a comparative plot of the results of OD600 for 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 of different nitrogen source cultures. Wherein example 1 is tryptone, example 7 is liver meal, example 8 is plant peptone, example 9 is yeast powder, and example 10 is tryptone.
FIG. 3 is a graph comparing the results of OD600 of different prebiotic cultures. Wherein example 1 is a prebiotic combination, comparative example 4 is fructooligosaccharides, comparative example 5 is low ester pectin, and comparative example 6 is inulin.
Detailed Description
In order to further describe the technical means adopted by the present invention and the effects thereof, the following describes the technical scheme of the present invention in combination with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.
The fructooligosaccharides of the following examples and comparative examples were purchased from Zhengzhou Wanbo chemical products Co., ltd, and the low-ester pectin was purchased from Shandong Bailong Chuangyuan Biotech Co., ltd, and the inulin was purchased from Chongqing Jiaowang agricultural development Co., ltd. The bifidobacteria of the following examples and comparative examples are bifidobacteria infantis and are purchased from the collection of biological species of Committee, guangdong, under the accession number CICC6069.
Example 1
The embodiment provides a bifidobacterium culture method, which specifically 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, 0.5g/L of tween 80, 1wt% of inulin, 1wt% of fructo-oligosaccharide, 1wt% of low-ester pectin and water as a solvent.
(1) Preparing a culture medium according to a formula, regulating the pH to 6.5 by using an aqueous solution of NaOH, and sterilizing at a high temperature of 121 ℃ for 20min for later use.
(2) Taking out the bifidobacterium frozen tube from the refrigerator at the temperature of minus 80 ℃, sucking 1mL of seed liquid after freezing and thawing, inoculating the seed liquid into a seed activation culture medium (commercial MRS culture medium, standard 027312), and standing and culturing for 24 hours at the temperature of 37 ℃ to obtain the activated bifidobacterium.
(3) Inoculating the bifidobacterium activated in the step (2) into the culture medium prepared in the step (1) in an inoculum size of 2%, culturing at 37 ℃, and supplementing a 50wt% glucose aqueous solution when the thalli grow to the mid-logarithmic growth phase, wherein the feeding speed is 50mL/h, and stirring (50 rpm, 5min every 50 min) is performed after feeding. Co-culturing for 24h.
Example 2
The embodiment provides a bifidobacterium culture method, which specifically comprises the following steps:
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-cysteine 0.8g/L of calcium chloride, 0.2g/L of tween 80, 2wt% of inulin, 0.5wt% of fructo-oligosaccharide, 1wt% of low-ester pectin and water as a solvent.
(1) Preparing a culture medium according to a formula, regulating the pH to 6.0 by using an aqueous solution of NaOH, and sterilizing at a high temperature of 121 ℃ for 20min for later use.
(2) Taking out the bifidobacterium frozen tube from the refrigerator at the temperature of minus 80 ℃, sucking 1mL of seed liquid after freezing and thawing, inoculating the seed liquid into a seed activation culture medium (commercial MRS culture medium, standard 027312), and standing and culturing for 24 hours at the temperature of 37 ℃ to obtain the activated bifidobacterium.
(3) Inoculating the bifidobacterium activated in the step (2) into the culture medium prepared in the step (1) in an inoculum size of 2%, culturing at 39 ℃, and supplementing a 50wt% glucose aqueous solution when the thalli grow to the mid-logarithmic growth phase, wherein the feeding speed is 50mL/h, and stirring (50 rpm, 5min every 50 min) is performed after feeding. Co-culturing for 24h.
Example 3
The embodiment provides a bifidobacterium culture method, which specifically comprises the following steps:
the formula of the culture medium is as follows: 42.0g/L of tryptone, 15.0g/L of glucose, 0.8g/L of sodium chloride, 1.1g/L of dipotassium phosphate, 1.1g/L of monopotassium phosphate, 0.02g/L of ferrous sulfate heptahydrate, 0.008g/L of manganese sulfate, 0.8g/L of magnesium sulfate, 0.2g/L, L-cysteine 0.3g/L of calcium chloride, 0.8g/L of tween 80, 0.8wt% of inulin, 1.2wt% of fructo-oligosaccharide, 1.2wt% of low-ester pectin and water as a solvent.
(1) Preparing a culture medium according to a formula, regulating the pH to 7.0 by using an aqueous solution of NaOH, and sterilizing at a high temperature of 121 ℃ for 20min for later use.
(2) Taking out the bifidobacterium frozen tube from the refrigerator at the temperature of minus 80 ℃, sucking 1mL of seed liquid after freezing and thawing, inoculating the seed liquid into a seed activation culture medium (commercial MRS culture medium, standard 027312), and standing and culturing for 24 hours at the temperature of 37 ℃ to obtain the activated bifidobacterium.
(3) Inoculating the bifidobacterium activated in the step (2) into the culture medium prepared in the step (1) in an inoculum size of 2%, culturing at 35 ℃, and supplementing a 50wt% glucose aqueous solution when the thalli grow to a late logarithmic growth stage, wherein the supplementing speed is 50mL/h, and stirring (50 rpm, 5min every 50 min) is performed after the supplementing. Co-culturing for 24h.
Example 4
This example provides a bifidobacterium culture method differing from example 1 only in that "the aqueous solution of glucose at 50wt% is added when the cells grow to mid-log growth" in step (3) is changed to "the aqueous solution of glucose at 50wt% is added when the cells grow to early-log growth", and further referring to example 1.
Example 5
This example provides a bifidobacterium cultivation method differing from example 1 only in that the feeding operation is not performed in step (3), and an aqueous glucose solution having the same amount as that in the feeding operation of example 1 is directly added at the start of cultivation, and further, reference is made to example 1.
Example 6
This example provides a bifidobacterium cultivation method differing from example 1 only in that the pH of the medium is adjusted from "6.5" to "5.0", and the other components and contents are unchanged.
The preparation method is described in example 1.
Example 7
This example provides a bifidobacterium cultivation method differing from example 1 only in that the nitrogen source "tryptone" in the medium is replaced by an equivalent amount of "liver meal", otherwise refer to example 1.
Example 8
This example provides a bifidobacterium cultivation method differing from example 1 only in that the nitrogen source "tryptone" in the medium is replaced by an equivalent amount of "phytone", otherwise refer to example 1.
Example 9
This example provides a bifidobacterium cultivation method differing from example 1 only in that the nitrogen source "tryptone" in the medium is replaced by an equal amount of "yeast powder", otherwise refer to example 1.
Example 10
This example provides a bifidobacterium cultivation method differing from example 1 only in that the nitrogen source "tryptone" in the medium is replaced by an equal amount of "tryptone", otherwise refer to example 1.
Comparative example 1
This comparative example provides a bifidobacterium cultivation method differing from example 1 only in that the prebiotics "inulin 1wt%, fructo-oligosaccharides 1wt%, low-ester pectin 1wt%" in the medium were replaced by "inulin 1.5wt%, fructo-oligosaccharides 1.5wt%" and other references to example 1.
Comparative example 2
This comparative example provides a bifidobacterium cultivation method differing from example 1 only in that the prebiotics "inulin 1wt%, fructo-oligosaccharides 1wt%, low-ester pectin 1wt%" in the medium were replaced by "inulin 1.5wt%, low-ester pectin 1.5wt%" and other references to example 1.
Comparative example 3
This comparative example provides a bifidobacterium cultivation method differing from example 1 only in that the prebiotics "inulin 1wt%, fructo-oligosaccharide 1wt%, low ester pectin 1wt%" in the medium were replaced by "fructo-oligosaccharide 1.5wt%, low ester pectin 1.5wt%" and other references example 1.
Comparative example 4
This comparative example provides a bifidobacterium cultivation method differing from example 1 only in that the prebiotics "inulin 1wt%, fructo-oligosaccharides 1wt%, low-ester pectin 1wt%" in the medium were replaced by "fructo-oligosaccharides 3wt%", otherwise refer to example 1.
Comparative example 5
This comparative example provides a bifidobacterium cultivation method differing from example 1 only in that the prebiotics "inulin 1wt%, fructo-oligosaccharides 1wt%, low-ester pectin 1wt%" in the medium were replaced by "low-ester pectin 3wt%", otherwise refer to example 1.
Comparative example 6
This comparative example provides a bifidobacterium cultivation method differing from example 1 only in that the prebiotics "inulin 1wt%, fructo-oligosaccharides 1wt%, low-ester pectin 1wt%" in the medium were replaced by "inulin 3wt%", otherwise refer to example 1.
Comparative example 7
This comparative example provides a bifidobacterium cultivation method which differs from example 1 only in that the lack of prebiotics "inulin, fructo-oligosaccharides and low ester pectin" in the medium is complemented by the amount of water which is lacking, otherwise refer to example 1.
Test example 1
Test group: examples 1-10, comparative examples 1-7.
And judging the bacterial increasing effect of the bifidobacteria by measuring the OD600 value in the bacterial liquid after the culture is finished.
After the culture, the bacterial liquid was centrifuged at 15000rpm, and the bacterial cells were collected, dried and weighed to obtain the yield of bifidobacteria.
The test results of each group are shown in table 1.
To make the result more intuitive, part of the data is organized into a graph.
FIG. 1 is a comparative plot of the results of OD600 for 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 of different nitrogen source cultures. Wherein example 1 is tryptone, example 7 is liver meal, example 8 is plant peptone, example 9 is yeast powder, and example 10 is tryptone.
FIG. 3 is a graph comparing the results of OD600 of different prebiotic cultures. Wherein example 1 is a prebiotic combination, comparative example 4 is fructooligosaccharides, comparative example 5 is low ester pectin, and comparative example 6 is inulin.
TABLE 1
The results show that: the bifidobacterium culture methods provided in examples 1-3 can significantly promote the proliferation of bifidobacteria, and the obtained thallus has higher yield. The microbial enhancement effect and the bacterial 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 to the culture medium plays a role in promoting the proliferation of bifidobacteria, and when the combination of inulin, fructo-oligosaccharide and low-ester pectin is adopted as the prebiotic component, the microbial enhancement effect is better than that of the combination of single component or any two components, and the synergistic effect of the three components in promoting the proliferation of bifidobacteria is proved. The microbial enhancement effect and the bacterial yield of the culture methods of examples 7 to 9 were lower than those of example 1, indicating that the selection of nitrogen source in the culture medium had a certain effect on the proliferation of bifidobacteria, and that the present invention selected tryptone as nitrogen source was better in microbial enhancement effect than hepatic extract, phytone and yeast powder. The effect of increasing the bacterial growth and the yield of the cells in the culture method of example 10 were similar to those in example 1, and it was demonstrated that the effect of increasing the bacterial growth was similar to that of tryptone when tryptone was used as the nitrogen source, but in view of cost reduction, tryptone was preferable as the nitrogen source in the present invention. The culture method of example 6 has a lower bacterial growth effect and lower bacterial yield than example 1, indicating that the pH of the culture medium has a greater effect on the growth and proliferation of bifidobacteria, and the culture medium has a better bacterial growth effect and higher yield when the pH of the culture medium is between 6.0 and 7.0. The culturing methods of examples 4-5 have lower bacteria increasing effect and lower thallus yield than those of example 1, which means that the addition of the material supplementing process in the culturing process can significantly promote the full utilization of the nutrient components in the culture medium by bifidobacteria, thereby greatly improving the bacteria increasing effect and the thallus yield. In addition, the timing of feeding has a certain influence on the bacterial increasing effect, and feeding operation is preferably performed in the mid-stage or the later stage of logarithmic growth. The bifidobacterium prepared by the corresponding culture method can be well applied to foods, medicines or cosmetics.
The applicant states that the present invention is described by way of the above examples as a culture medium for bifidobacteria, and a method of culturing and use thereof, but the present invention is not limited to the above examples, i.e., it is not meant that the present invention must be practiced in dependence upon the above examples. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Claims (13)

1. A bifidobacterium culture medium, characterized in that the bifidobacterium culture medium comprises a carbon source, a nitrogen source, inorganic salts and prebiotics;
the prebiotics comprise, by mass, 1-5 parts of fructo-oligosaccharide, 1-5 parts of low-ester pectin and 1-5 parts of inulin; the nitrogen source is tryptone, and the bifidobacterium is bifidobacterium infantis CICC6069 strain.
2. The bifidobacterium culture medium of claim 1, wherein the bifidobacterium culture medium comprises, in concentration, 10-15g/L of carbon source, 30-45g/L of nitrogen source, 3-5g/L, L-cysteine 0.1-1g/L of inorganic salt, 0.1-1g/L of tween 80 and 1-5wt% of prebiotics.
3. The bifidobacterium culture medium of claim 1 wherein the carbon source includes glucose.
4. The bifidobacterium culture medium of claim 1 wherein the inorganic salt comprises 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.
5. The bifidobacterium culture medium of claim 4 wherein the inorganic salt is a combination of sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ferrous sulfate, manganese sulfate, magnesium sulfate, and calcium chloride.
6. The bifidobacterium culture medium of claim 5, wherein the inorganic salt comprises, in parts 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 potassium dihydrogen 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.
7. The bifidobacterium culture medium of claim 1 wherein the bifidobacterium culture medium has a pH of 6.0 to 7.0.
8. A method of culturing bifidobacteria, comprising inoculating a bifidobacterium, which is a bifidobacterium infantis cic 6069 strain, into a bifidobacterium medium as claimed in any one of claims 1 to 7 for culturing.
9. The method for culturing bifidobacteria as claimed in claim 8 wherein the temperature of the culturing is 35-40 ℃ and the time of the culturing is 20-30 hours.
10. The method for culturing bifidobacteria as claimed in claim 8 wherein the bifidobacteria are inoculated in an amount of 1 to 3%.
11. The method of culturing bifidobacteria of claim 8, further comprising feeding, the feeding comprising feeding a carbon source.
12. The method for culturing bifidobacteria as claimed in claim 11 wherein the feeding is performed when the cells grow to mid-log or late-log growth.
13. Use of a method of culturing bifidobacteria as claimed in any of claims 8 to 12 in the manufacture of a food, pharmaceutical or cosmetic product.
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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004002240A2 (en) * 2002-06-26 2004-01-08 Danisco A/S Use of hydrocolloids as prebiotic food ingredients and method of producing the same
CN101058796A (en) * 2007-04-25 2007-10-24 中国人民解放军第三军医大学第一附属医院 Composite culture medium for bifidobacterium fermentation and preparation method thereof
CN101715816A (en) * 2009-11-18 2010-06-02 内蒙古伊利实业集团股份有限公司 Fermented milk containing probiotics, production method thereof and application thereof
CN105483059A (en) * 2016-01-18 2016-04-13 王军 Method for cultivating bifidobacteria through inulin
CN105962378A (en) * 2016-05-12 2016-09-28 李�浩 Preparation method of active lactobacillus preparation containing hericium polysaccharides
CN109329699A (en) * 2018-09-25 2019-02-15 中北大学 A kind of probiotics seabuckthorn fruit mud jelly and preparation method thereof
CN109913523A (en) * 2019-04-15 2019-06-21 江南大学 A kind of culture medium of nitrogen source that screening suitable Bifidobacterium proliferation
CN110055301A (en) * 2019-05-06 2019-07-26 上海源本食品质量检验有限公司 A method of the culture medium of detection Bifidobacterium and quickly detection count
CN111876352A (en) * 2020-07-28 2020-11-03 科兴生物制药股份有限公司 Oxygen-resistant acid-resistant bifidobacterium infantis and screening method thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9649380B2 (en) * 2009-01-12 2017-05-16 Pfizer Italia S.R.L. Compositions comprising probiotic and prebiotic components and mineral salts, with lactoferrin

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004002240A2 (en) * 2002-06-26 2004-01-08 Danisco A/S Use of hydrocolloids as prebiotic food ingredients and method of producing the same
CN101058796A (en) * 2007-04-25 2007-10-24 中国人民解放军第三军医大学第一附属医院 Composite culture medium for bifidobacterium fermentation and preparation method thereof
CN101715816A (en) * 2009-11-18 2010-06-02 内蒙古伊利实业集团股份有限公司 Fermented milk containing probiotics, production method thereof and application thereof
CN105483059A (en) * 2016-01-18 2016-04-13 王军 Method for cultivating bifidobacteria through inulin
CN105962378A (en) * 2016-05-12 2016-09-28 李�浩 Preparation method of active lactobacillus preparation containing hericium polysaccharides
CN109329699A (en) * 2018-09-25 2019-02-15 中北大学 A kind of probiotics seabuckthorn fruit mud jelly and preparation method thereof
CN109913523A (en) * 2019-04-15 2019-06-21 江南大学 A kind of culture medium of nitrogen source that screening suitable Bifidobacterium proliferation
CN110055301A (en) * 2019-05-06 2019-07-26 上海源本食品质量检验有限公司 A method of the culture medium of detection Bifidobacterium and quickly detection count
CN111876352A (en) * 2020-07-28 2020-11-03 科兴生物制药股份有限公司 Oxygen-resistant acid-resistant bifidobacterium infantis and screening method thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
不同组成的果聚糖对两歧双歧杆菌体外生长的影响;周皓东;周艳;刘丽萍;夏刚强;韦策;张红漫;;食品工业(第06期);第201-204页 *
提高双歧杆菌活力的优化增菌发酵技术研究;何枫媛;葛武鹏;卫伟;朱红;刘锐;;中国乳品工业(第05期);第18-21页 *
植物乳杆菌SCP53生物膜的形成条件;张国丽;彭瑶;魏露;余茜;敖晓琳;刘书亮;赵珂;;食品与发酵工业(04);第12-19页 *
菊粉低聚果糖与蔗糖低聚果糖对双歧杆菌体外增殖的研究;张泽生;张丽;吕晓玲;王霄然;卢亚莉;;中国食品添加剂(第01期);第44-48页 *

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