CN118207115A - Fermentation method of bifidobacterium and preparation method of bifidobacterium fermentation lysate - Google Patents
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The application discloses a fermentation method of bifidobacteria and a preparation method of bifidobacteria fermentation lysate. The fermentation method comprises the following steps: 30g to 40g of glucose is taken as a main material, 8g to 10g of yeast powder, 3g to 5g of peptone, 0.2g to 0.5g of magnesium sulfate, 1.5g to 2.5g of dipotassium hydrogen phosphate, 1.5g to 2.5g of tri-ammonium citrate, 3g to 6g of sodium acetate, 0.03g to 0.06g of manganese sulfate, 0.5g to 1.2g of L-cysteine and 0.8g to 1.5g of tween-80 are taken as ingredients, deionized water is taken as a solvent to prepare an improved MRS culture medium; inoculating bifidobacterium animalis subspecies lactis to a sterilized commercial MRS broth culture medium for activation to obtain an activated strain; inoculating the activated strain into the improved MRS culture medium in a volume ratio of 0.5-2% to obtain a bifidobacterium fermentation product. The fermentation method of bifidobacteria adopts an improved MRS culture medium to perform high-density fermentation culture of animal bifidobacteria and lacto subspecies, and can obtain a bifidobacteria fermentation product of high-density fermentation.
Description
Technical Field
The application belongs to the technical field of cosmetics, and particularly relates to a bifidobacterium fermentation method and a bifidobacterium fermentation lysate preparation method.
Background
Bifidobacteria are an important physiologically beneficial intestinal microorganism. The symbiotic relationship is formed between the plant and the host in the long-term evolution process, plays an important role in maintaining intestinal microecology and human health, and is widely applied to the fields of foods and health care products. The fermentation product obtained by culturing and lysing the bifidobacteria is rich in various active ingredients such as amino acid, vitamin, polypeptide, mineral substances and the like, has biological activities of capturing free radicals, inhibiting lipid peroxidation and the like, has better effects on improving skin microecology and skin functions, and has been widely used for developing related products as a cosmetic raw material with the effects of resisting skin aging, relieving and the like.
However, the preparation of the bifidobacterium lysate still faces the problems of low fermentation density of bifidobacterium, turbid lysate and difficult absorption of macromolecular protein substances.
Disclosure of Invention
The embodiment of the application provides a bifidobacterium fermentation method, which adopts an improved MRS culture medium to perform high-density fermentation culture on animal bifidobacterium subspecies and can obtain a bifidobacterium fermentation product of high-density fermentation.
In a first aspect, the present application provides a fermentation process for bifidobacteria comprising:
30g to 40g of glucose is taken as a main material, 8g to 10g of yeast powder, 3g to 5g of peptone, 0.2g to 0.5g of magnesium sulfate, 1.5g to 2.5g of dipotassium hydrogen phosphate, 1.5g to 2.5g of tri-ammonium citrate, 3g to 6g of sodium acetate, 0.03g to 0.06g of manganese sulfate, 0.5g to 1.2g of L-cysteine, 0.8g to 1.5g of tween-80 are taken as ingredients, deionized water is taken as a solvent to prepare an improved MRS culture medium;
inoculating bifidobacterium animalis subspecies into sterilized commercial MRS broth culture medium for activation to obtain activated strains;
Inoculating the activated strain into the improved MRS culture medium in a volume ratio of 0.5-2% to obtain a bifidobacterium fermentation product.
According to an embodiment of one aspect of the present application, the step of obtaining an improved MRS medium includes:
sterilizing the ingredients at 117-121 ℃ for 20-30 min;
and sterilizing the main material at 117-121 deg.c for 20-30 min.
According to an embodiment of one aspect of the present application, the strain activation step includes inoculating bifidobacterium animalis subspecies in sterilized commercial MRS broth, and statically culturing at 35-38 ℃ for 24-48 hours to activate the bifidobacterium animalis subspecies, and obtaining the activated strain after the commercial MRS broth is turbid.
According to an embodiment of an aspect of the application, the concentration of the activated species is 1X 10 7cfu/mL～1×108 cfu/mL.
According to an embodiment of one aspect of the application, the latin name of bifidobacterium animalis subspecies lactis: bifidobacterium animalis subsp.lactis, accession number of seed: CGMCC 28490; preservation unit: china general microbiological culture collection center (CHINA GENERAL Microbiological Culture Collection Center, CGMCC); address: the korean district North Star, beijing city, part No. 1, no. 3.
According to an embodiment of one aspect of the present application, a fermentation method of bifidobacteria includes:
Inoculating an activated strain into the improved MRS culture medium in a volume ratio of 0.5% -2% of the improved MRS culture medium, controlling the pH value of the improved MRS culture medium inoculated with the activated strain to be 6.0 by using a fermentation temperature of 35-38 ℃, a rotating speed of 150-200 rpm and 10wt.% sodium hydroxide solution to perform fermentation treatment until residual sugar of the improved MRS culture medium inoculated with the activated strain is less than or equal to 5g/L, thereby obtaining a fermentation product in the first stage;
Feeding the fermentation product of the first stage until the residual sugar is 30 g/L-40 g/L, and fermenting under the process conditions that the pH value of the fermentation product of the first stage after feeding is controlled to be 6.0 by using 10wt.% sodium hydroxide solution at the fermentation temperature of 35-38 ℃ and the rotating speed of 150-200 rpm, so that the residual sugar is less than or equal to 5g/L, thereby obtaining the bifidobacterium fermentation product.
In a second aspect, embodiments of the present application provide a method of preparing a bifidobacterium fermentation lysate, comprising:
separating the bifidobacterium fermentation product to obtain wet thalli and fermentation supernatant;
preparing a suspension by using wet thalli and fermentation supernatant;
Crushing the suspension to obtain crushed suspension;
carrying out enzymolysis treatment on the crushed suspension to obtain an enzymolysis product;
and performing ultrafiltration treatment on the enzymolysis product, and collecting permeate to obtain a bifidobacterium fermentation lysate.
According to an embodiment of one aspect of the application, the method for preparing a bifidobacterium fermentation lysate further comprises:
Concentrating the bifidobacterium fermentation lysate to obtain the bifidobacterium fermentation lysate with the Kjeldahl nitrogen content of 0.1-2 wt%.
According to an embodiment of one aspect of the present application, a method for preparing a bifidobacterium fermentation lysate comprises subjecting a bifidobacterium fermentation product to centrifugation to obtain wet thalli and a fermentation supernatant; wherein, the wet thalli is washed by sterile deionized water until the electric conductivity of the mixed solution of the wet thalli and the sterile deionized water is lower than 400 mu s/cm.
According to an embodiment of one aspect of the present application, the step of preparing the suspension includes:
preparing a suspending agent by compounding fermentation supernatant with sterile deionized water accounting for 20-40% of the volume ratio of the deionized water;
Mixing the wet thalli and the suspending agent according to the mass ratio of 1:10-20 to obtain a suspension.
According to an embodiment of one aspect of the application, the preparation method of the bifidobacterium fermentation lysate comprises the steps of crushing a suspension at a temperature of between 4 and 8 ℃ for 3 to 5 times at a pressure of between 1500 and 1900bar to obtain a crushed suspension.
According to an embodiment of one aspect of the application, the enzymatic hydrolysis treatment step comprises:
Maintaining the crushed suspension at 70-80 deg.c and 200-300 rpm for 10-15 min to obtain deactivated suspension;
Cooling the inactivated bacteria suspension to 50-52 ℃, adding lysozyme accounting for 0.1-0.5% of the bacteria suspension in mass, and carrying out enzymolysis treatment on the bacteria suspension by using composite biological protease accounting for 1-2% of the bacteria suspension in mass for 2-4 hours to obtain an enzymolysis treatment product; wherein, the enzyme activity of the lysozyme is 10000-20000U/mg;
and maintaining the enzymolysis product at 90-100 deg.c and 200-300 rpm for 5-10 min to obtain the enzymolysis product.
According to an embodiment of one aspect of the application, the compound biological protease is neutral protease, acid protease and papain with the mass ratio of (0.3-1) to (0.3-1), or acid protease, papain and trypsin with the mass ratio of (0.3-1) to (0.3-1).
According to an embodiment of one aspect of the present application, the step of enzymatic treatment further comprises adding a preservative to the enzymatic hydrolysate to yield a preserved enzymatic hydrolysate.
According to an embodiment of one aspect of the present application, the step of enzymolysis treatment further comprises adding phenoxyethanol accounting for 0.5% -1% of the mass of the enzymolysis product and sodium benzoate accounting for 0.25% -0.5% of the mass of the enzymolysis product.
According to an embodiment of one aspect of the application, the ultrafiltration step comprises ultrafiltration of the enzymatic hydrolysate using an ultrafiltration membrane having a molecular weight cut-off of 3KD, and collecting the permeate as a bifidobacterium fermentation lysate.
In a third aspect, the application provides a bifidobacterium fermentation lysate, which is prepared according to the preparation method of the bifidobacterium fermentation lysate.
In a fourth aspect, embodiments of the application provide a use of a bifidobacterium fermentation lysate for the preparation of a cosmetic.
According to the fermentation method of the bifidobacteria, disclosed by the embodiment of the application, an improved MRS culture medium which is more suitable for the growth of the bifidobacteria lactis of animals is adopted for fermentation culture, so that the bifidobacteria lactis of animals can grow better, and anaerobic fermentation is performed in a high-density fermentation mode in a fermentation step, so that a bifidobacteria fermentation product of the high-density fermentation is obtained.
According to the preparation method of the bifidobacterium fermentation lysate, on the basis of fermentation according to the bifidobacterium fermentation method, wet thalli and fermentation supernatant are obtained by separating the bifidobacterium fermentation product, components in the thalli are completely released through a thalli crushing step, a crushed suspension, namely lysate, is obtained, and protease is degraded into micromolecular peptides through enzymolysis treatment of lysozyme and biological complex enzyme, so that the protease is easier to be absorbed through skin.
Drawings
In order to more clearly illustrate the technical solution of the embodiments of the present application, the drawings that are needed to be used in the embodiments of the present application will be briefly described, and it is possible for a person skilled in the art to obtain other drawings according to these drawings without inventive effort.
FIG. 1 is a schematic flow chart of a fermentation method of bifidobacteria according to an embodiment of the present application;
FIG. 2 is a schematic flow chart of a bifidobacterium fermentation lysate provided by an embodiment of the present application;
FIG. 3 is a protein electrophoresis pattern of lysates prepared according to each of the preparation methods provided in examples 1 to 4 and comparative examples 1 to 2 of the present application.
Reference numerals illustrate:
1. Lane 1, the lane in which the proteins of the bifidobacterium fermentation lysate prepared in example 1 are located;
2. Lane 2, the lane in which the proteins of the bifidobacterium fermentation lysate prepared in example 2 are located;
3. lane 3, the lane in which the proteins of the bifidobacterium fermentation lysate prepared in example 3 are located;
4. Lane 4, the lane where the proteins of the bifidobacterium fermentation lysate prepared in example 4 are;
5. lane 5, lane where protein markers are located;
6. Lane 6, the lane where the proteins of the bifidobacterium fermentation lysate prepared in comparative example 2 are located.
Detailed Description
Features and exemplary embodiments of various aspects of the present application will be described in detail below, and in order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described in further detail below with reference to the accompanying drawings and the detailed embodiments. It should be understood that the particular embodiments described herein are meant to be illustrative of the application only and not limiting. It will be apparent to one skilled in the art that the present application may be practiced without some of these specific details. The following description of the embodiments is merely intended to provide a better understanding of the application by showing examples of the application.
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
The applicant has found in the course of improving the prior art that: bifidobacteria are strictly anaerobic microorganisms, have high requirements on living environment and nutrition, and have low fermentation density due to fermentation. The preparation of lysate firstly requires a large amount of thalli, so the low fermentation density leads to the problems of high subsequent cost and the like. Substances released in cells after cell disruption contain macromolecular proteins and other substances which are difficult to absorb, and the literature reports that small molecular substances with molecular weight less than 500 daltons are easier to absorb transdermally.
In order to solve the problems in the prior art, the embodiment of the application provides a bifidobacterium fermentation method and a bifidobacterium fermentation lysate preparation method. The fermentation method of bifidobacteria provided by the embodiment of the application is first described below.
Fig. 1 shows a schematic flow chart of a fermentation method of bifidobacteria according to an embodiment of the present application. As shown in fig. 1, the present application provides a fermentation method of bifidobacteria, comprising:
s1, preparing an improved MRS culture medium by taking 30-40 g of glucose as a main material, 8-10 g of yeast powder, 3-5 g of peptone, 0.2-0.5 g of magnesium sulfate, 1.5-2.5 g of dipotassium hydrogen phosphate, 1.5-2.5 g of tri-ammonium citrate, 3-6 g of sodium acetate, 0.03-0.06 g of manganese sulfate, 0.5-1.2 g of L-cysteine and 800.8-1.5 g of tween-using deionized water as a solvent as ingredients;
S2, inoculating bifidobacterium animalis subspecies into a sterilized MRS culture medium for activation to obtain activated strains;
s3, inoculating the activated strain into the improved MRS culture medium in a volume ratio of 0.5-2% of the improved MRS culture medium for fermentation to obtain a bifidobacterium fermentation product.
The fermentation method of the bifidobacteria improves the existing MRS culture medium, adopts the improved MRS culture medium which is more suitable for the growth of the bifidobacterium animalis subspecies to carry out the fermentation culture process of the bifidobacterium animalis subspecies, ensures that the bifidobacterium animalis subspecies grow better, and carries out anaerobic fermentation in a high-density fermentation mode in the fermentation step to obtain a bifidobacterium fermentation product of high-density fermentation.
In an embodiment of one aspect of the present application, a fermentation method of bifidobacteria includes:
sterilizing the ingredients at 117-121 ℃ for 20-30 min;
and sterilizing the main material at 117-121 deg.c for 20-30 min.
The improved MRS culture medium adopted in the embodiment of the application is obtained by improvement on the basis of a commercial MRS culture medium, and is more suitable for the growth of bifidobacteria. The method can enable the bifidobacteria to grow more and obtain a large amount of bifidobacteria thalli, and is an effective means for realizing high-density fermentation of the bifidobacteria.
In an embodiment of the application, the high density fermentation in combination with the modified MRS medium yields a fermentation product with a concentration of 1.5X10 10cfu/mL～1.8×1010 cfu/mL of bifidobacterium animalis subspecies.
In an embodiment of one aspect of the present application, a fermentation method of bifidobacteria includes:
Inoculating the bifidobacterium animalis subspecies lactis into a sterilized commercial MRS broth culture medium, performing static culture at 35-38 ℃ for 24-48 hours to activate the bifidobacterium animalis subspecies lactis, and mixing and turbidity the commercial MRS broth culture medium to obtain the activated strain.
In an embodiment of one aspect of the application, the concentration of the activated species is 1X 10 7cfu/mL～1×108 cfu/mL.
In an embodiment of one aspect of the application, the latin name of bifidobacterium animalis subspecies lactis: bifidobacterium animalis subsp.lactis, accession number of seed: CGMCC 28490; preservation unit: china general microbiological culture collection center (CHINA GENERAL Microbiological Culture Collection Center, CGMCC); address: the korean district North Star, beijing city, part No. 1, no. 3.
In an embodiment of one aspect of the present application, a fermentation method of bifidobacteria includes:
S30, inoculating an activated strain into the improved MRS culture medium in a volume ratio of 0.5% -2% of the improved MRS culture medium, controlling the pH value of the improved MRS culture medium inoculated with the activated strain to be 6.0 by using a 10wt.% sodium hydroxide solution at a fermentation temperature of 35-38 ℃ and a rotation speed of 150-200 rpm for fermentation treatment until the residual sugar of the improved MRS culture medium inoculated with the activated strain is less than or equal to 5g/L, and obtaining a fermentation product in the first stage;
S31, feeding the fermentation product of the first stage until the residual sugar is 30 g/L-40 g/L, and fermenting under the process conditions that the fermentation temperature is 35-38 ℃, the rotating speed is 150-200 rpm, and the pH value of the fermentation product of the first stage after feeding is controlled to be 6.0 by 10wt.% sodium hydroxide solution, so that the residual sugar is less than or equal to 5g/L, thereby obtaining the bifidobacterium fermentation product. Wherein S31 is to ferment the fermentation product of the first stage after feeding under the same fermentation culture process conditions.
In an embodiment of one aspect of the application, 50wt.% glucose is used as feed medium, and the fermentation product of the first stage is supplemented with a carbon source (i.e. sugar) to enable further fermentation of the bifidobacteria subjected to the first stage fermentation under the same fermentation culture conditions to obtain more bifidobacteria and thus more bifidobacteria fermented lysate fraction.
In a second aspect, embodiments of the present application provide a method of preparing a bifidobacterium fermentation lysate, comprising:
s4, separating a bifidobacterium fermentation product to obtain wet thalli and fermentation supernatant;
s5, preparing a suspension by using wet thalli and fermentation supernatant;
S6, crushing the suspension to obtain crushed suspension;
s7, carrying out enzymolysis treatment on the crushed suspension to obtain an enzymolysis product;
s8, performing ultrafiltration treatment on the enzymolysis product, and collecting permeate to obtain a bifidobacterium fermentation lysate.
In an embodiment of one aspect of the application, the method for preparing a bifidobacterium fermentation lysate further comprises:
S9, concentrating the bifidobacterium fermentation lysate to obtain the bifidobacterium fermentation lysate with the Kjeldahl nitrogen content of 0.1-2 wt.%.
In an embodiment of one aspect of the present application, the method for determining the kjeldahl nitrogen content is as follows: GB/11891-1989 determination of Kjeldahl nitrogen in water quality.
In an embodiment of one aspect of the application, a method for preparing a bifidobacterium lysed fermentation lysate comprises centrifuging the bifidobacterium fermentation product to obtain wet thalli and a fermentation supernatant; wherein, the wet thalli is washed by sterile deionized water until the electric conductivity of the mixed solution of the wet thalli and the sterile deionized water is lower than 400 mu s/cm.
In an embodiment of one aspect of the application, a method for preparing a bifidobacterium lysed fermentation lysate comprises:
Centrifuging the bifidobacterium fermentation product at 6000-8000 rpm for 20-30 min to obtain wet thalli and fermentation supernatant; the wet thalli is washed by using sterile deionized water until the conductivity of a mixed solution of the wet thalli and the sterile deionized water is lower than 400 mu s/cm, and then the sterile deionized water is removed to obtain the clean wet thalli; fermenting supernatant for later use. Wherein, the wet thalli can be cleaned by using sterile deionized water to remove harmful components in the wet thalli.
In an embodiment of one aspect of the present application, the step of preparing the suspension includes:
S50, compounding fermentation supernatant with sterile deionized water in a volume ratio of 20% -40% of deionized water to prepare a suspending agent;
s51, mixing the wet thalli and the suspending agent according to the mass ratio of 1:10-20 to obtain a suspension.
In the embodiment of the application, the fermentation supernatant is compounded with sterile deionized water to prepare a suspending agent, and the wet thalli are mixed with the suspending agent, so that beneficial components in the fermentation supernatant, such as lactic acid, acetic acid and the like, can be reserved.
In one embodiment of the application, the preparation method of the bifidobacterium lysate fermentation lysate comprises the steps of crushing a suspension at the temperature of 4-8 ℃ for 3-5 times at the pressure of 1500-1900 bar, so as to obtain the crushed suspension. The substances in the bifidobacterium cells are released by the cell disruption step to obtain lysate therein as much as possible.
In an embodiment of one aspect of the present application, the enzymatic hydrolysis treatment step comprises:
S70, maintaining the crushed suspension at 70-80 ℃ and 200-300 rpm for 10-15 minutes to obtain an inactivated suspension;
S71, cooling the inactivated bacteria suspension to 50-52 ℃, adding lysozyme accounting for 0.1-0.5% of the mass of the bacteria suspension and composite biological protease accounting for 1-2% of the mass of the bacteria suspension, and carrying out enzymolysis treatment for 2-4 hours to obtain an enzymolysis treatment product; wherein, the enzyme activity of the lysozyme is 10000-20000U/mg;
s72, maintaining the enzymolysis product at 90-100 ℃ and 200-300 rpm for 5-10 minutes to obtain the enzymolysis product.
In the embodiment of the application, the protein in the crushed suspension is inactivated by the step S70 at the temperature of 70-80 ℃ and maintained at 200-300 rpm for 10-15 minutes to completely inactivate the protein; after the inactivation of the protein, the temperature is reduced to 50-52 ℃, lysozyme and compound biological protease are added for enzymolysis treatment, so that the macromolecular protein is degraded into micromolecular polypeptide or oligopeptide, and the polypeptide is more suitable for skin absorption. Inactivating lysozyme and composite biological protease at high temperature of 90-100 ℃ after enzymolysis treatment is completed, thereby obtaining an enzymolysis product.
In an embodiment of one aspect of the application, the complex biological protease is selected from the group consisting of neutral protease, acid protease, papain, trypsin, and combinations of two or more thereof. It will be appreciated that the complex biological protease may be selected from the group consisting of neutral protease, acid protease, papain, trypsin, and combinations of two, three or four.
In one embodiment of the application, the compound biological protease is neutral protease, acid protease and papain with the mass ratio of (0.3-1) to (0.3-1), or acid protease, papain and trypsin with the mass ratio of (0.3-1) to (0.3-1). Wherein, trypsin can select specification: 4000U/g; the manufacturer: trypsin of the biological sciences of Henghua road, nanning east.
In the embodiment of the application, the crushed bacterial suspension is subjected to enzymolysis treatment by using lysozyme and composite biological protease, so that macromolecular proteins contained in fermentation lysate released after the cells of bifidobacterium are crushed can be degraded into micromolecular peptides. Such as lysozyme, hydrolyzes the beta-1, 4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the bifidobacterium cell wall peptidoglycan, releasing N-acetylglucosamine. The macromolecular protein is degraded into small molecular polypeptide or oligopeptide through enzymolysis treatment by using the compound biological protease, so that the macromolecular protein is easier to be absorbed through skin.
In an embodiment of one aspect of the present application, the step of performing enzymolysis further comprises:
and S73, adding a preservative into the enzymolysis product to obtain the preserved enzymolysis product.
In an embodiment of one aspect of the present application, the step of performing enzymolysis further comprises adding phenoxyethanol accounting for 0.5% -1% of the mass of the enzymolysis product and sodium benzoate accounting for 0.25% -0.5% of the mass of the enzymolysis product to the enzymolysis product so as to prevent the enzymolysis product from spoiling.
In an embodiment of one aspect of the application, the ultrafiltration step comprises ultrafiltration of the enzymatic hydrolysate using an ultrafiltration membrane having a molecular weight cut-off of 3KD, and collecting the permeate as a bifidobacterium fermentation lysate.
In a third aspect, embodiments of the present application provide a bifidobacterium fermentation lysate, prepared according to the preparation method of the bifidobacterium fermentation lysate.
The starting materials/reagents/components not mentioned in the present application are available from commercial sources, and the following are available sources for the starting materials, reagents used in some examples or comparative examples:
Peptone, model: FP318; the manufacturer: angel Yeast Co Ltd;
yeast powder, model: YI01; the manufacturer: angel Yeast Co Ltd;
l-cysteine, model: l804955; the manufacturer: shanghai Miclin Biochemical technologies Co., ltd;
Commercial MRS broth, product number: HB0384-1; purchased from maribo biotechnology limited;
the compound biological protease comprises neutral protease, acid protease and papain with the mass ratio of 2:1:1. Wherein, neutral protease, specification: 5-40 ten thousand U/g; the manufacturer: nanning Donghenghua biological technology Limited liability company; acid protease, specification: 5 ten thousand U/g; the manufacturer: nanning Donghenghua biological technology Limited liability company; papain, specification: 6-240 ten thousand U/g; the manufacturer: nanning Donghenghua biological technology Limited liability company.
Example 1a method of preparing a bifidobacterium fermentation lysate comprising:
S1, preparing an improved MRS culture medium, namely preparing 1L of the improved MRS culture medium by taking 35g of glucose as a main material, 9g of yeast powder, 4g of peptone, 0.35g of magnesium sulfate, 2g of dipotassium hydrogen phosphate, 2g of triammonium citrate, 5g of sodium acetate, 0.05g of manganese sulfate, 1g of L-cysteine and 1g of tween-80 as ingredients and using deionized water as a solvent; wherein, glucose is sterilized for 25 minutes at a sterilization temperature of 117 ℃, and ingredients are sterilized under the same condition and then mixed with other components to prepare an improved MRS culture medium;
S2, a strain activation step, which comprises inoculating bifidobacterium animalis subspecies (Latin name: bifidobacterium animalis subsp. Lactis, strain preservation number: CGMCC 28490) into a sterilized commercial MRS broth culture medium, performing static culture at 37 ℃ for 40 hours for activation, and performing activation after turbidity of the commercial MRS broth culture medium to obtain an activated strain with a strain concentration of 1 multiplied by 10 7 cfu/mL;
s3, a fermentation culture step, which comprises inoculating an activated strain into the improved MRS culture medium in a volume ratio of 1% of the improved MRS culture medium to perform high-density fermentation, wherein the high-density fermentation comprises the following steps:
S30, controlling the process condition of the pH value of the modified MRS culture medium inoculated with the activated strain to be 6.0 by using a sodium hydroxide solution with the fermentation temperature of 37 ℃ and the rotation speed of 200rpm and the concentration of 10wt.% to perform high-density fermentation until the residual sugar of the modified MRS culture medium inoculated with the activated strain is 4g/L, so as to obtain a fermentation product in the first stage;
S31, feeding 50wt.% glucose into the fermentation product of the first stage at a constant speed of 8 g/L.h, feeding until the residual sugar is 30g/L, and fermenting under the process conditions that the fermentation temperature is 37 ℃, the rotating speed is 200rpm, and the pH value of the fermentation product of the first stage after feeding is controlled to be 6.0 by 10wt.% sodium hydroxide solution, wherein the fermentation is finished until the residual sugar is 3g/L, so as to obtain a bifidobacterium fermentation product;
S4, a thallus collecting step, which comprises the steps of centrifugally separating a bifidobacterium fermentation product at 7000rpm for 25 minutes, and collecting wet thallus and fermentation supernatant; washing the wet thalli with sterile deionized water for 2 times until the conductivity of the mixed solution of the wet thalli and the sterile deionized water is 285 mu s/cm, and then removing the sterile deionized water to obtain the wet thalli; fermenting the supernatant for later use to obtain wet thalli and fermentation supernatant;
s5, preparing a suspension, namely preparing the suspension by using wet thalli and fermentation supernatant, wherein the suspension comprises the following steps:
S50, compounding the fermentation supernatant with sterile deionized water according to the volume ratio of 25% of the fermentation supernatant to prepare a suspending agent;
S51, mixing wet thalli and a suspending agent according to a mass ratio of 1:15 to obtain a suspension;
S6, a thallus crushing step, which comprises the steps of crushing the suspension at the temperature of 4 ℃ for 4 times at 1600bar until the suspension is crushed;
s7, performing enzymolysis treatment, namely performing enzymolysis treatment on the crushed suspension, wherein the enzymolysis treatment comprises the following steps:
s70, maintaining the crushed suspension at 75 ℃ and 280rpm for 10 minutes to obtain an inactivated suspension;
S71, cooling the inactivated bacteria suspension to 50 ℃, and respectively adding lysozyme accounting for 0.2% of the mass of the bacteria suspension and composite biological protease accounting for 1.5% of the mass of the bacteria suspension for enzymolysis treatment for 2 hours to obtain an enzymolysis treatment product; wherein, the enzyme activity of the lysozyme is 20000U/mg;
s72, maintaining the enzymolysis product for 5 minutes at 95 ℃ and 280rpm to obtain an enzymolysis product;
S73, performing antiseptic treatment, namely adding an antiseptic into the enzymolysis product to perform antiseptic treatment to obtain an antiseptic enzymolysis product; wherein, the preservative is phenoxyethanol and sodium benzoate, the consumption of phenoxyethanol accounts for 0.5 to 1 percent of the mass of the enzymolysis product, and the consumption of sodium benzoate accounts for 0.25 to 0.5 percent of the mass of the enzymolysis product;
S8, performing ultrafiltration treatment, namely performing ultrafiltration treatment on the anti-corrosion enzymolysis product by using an ultrafiltration membrane with a molecular weight cutoff of 3KD, and collecting permeate to obtain a bifidobacterium fermentation lysate;
s9, concentrating the bifidobacterium fermentation lysate by using a rotary evaporator to obtain the bifidobacterium fermentation lysate with the Kjeldahl nitrogen content of 0.15 wt.%.
Example 2a method of preparing a bifidobacterium fermentation lysate comprising:
S1, preparing an improved MRS culture medium, which comprises the steps of taking 40g of glucose as a main material, taking 10g of yeast powder, 5g of peptone, 0.35g of magnesium sulfate, 2g of dipotassium hydrogen phosphate, 2g of triammonium citrate, 5g of sodium acetate, 0.05g of manganese sulfate, 1g of L-cysteine and 1g of tween-80 as ingredients, and preparing 1L of the improved MRS culture medium by using deionized water as a solvent; wherein, glucose is sterilized for 25 minutes at a sterilization temperature of 117 ℃, and ingredients are sterilized under the same condition and then mixed with other components to prepare an improved MRS culture medium;
S2, a strain activation step, which comprises inoculating bifidobacterium animalis subspecies (Latin name: bifidobacterium animalis subsp. Lactis, strain preservation number: CGMCC 28490) into a sterilized commercial MRS broth culture medium, performing static culture at 37 ℃ for 36 hours for activation, and performing activation after turbidity of the commercial MRS broth culture medium to obtain an activated strain with a strain concentration of 1 multiplied by 10 7 cfu/mL;
s3, a fermentation culture step, which comprises inoculating an activated strain into the improved MRS culture medium in a volume ratio of 1% of the improved MRS culture medium to perform high-density fermentation, wherein the high-density fermentation comprises the following steps:
S30, controlling the process conditions of the modified MRS culture medium inoculated with the activated strain to have a constant pH value of 6.0 by using a fermentation temperature of 37 ℃, a rotating speed of 200rpm and a 10wt.% sodium hydroxide solution, and performing high-density fermentation until the residual sugar of the modified MRS culture medium inoculated with the activated strain is 3g/L, so as to obtain a fermentation product in the first stage;
S31, feeding 50wt.% glucose into the fermentation product of the first stage at a constant speed of 8 g/L.h, feeding until the residual sugar is 30g/L, fermenting under the process conditions that the fermentation temperature is 37 ℃, the rotating speed is 200rpm, and the pH value of the fermentation product of the first stage after the feeding is controlled to be 6.0 by 10wt.% sodium hydroxide solution, and ending the fermentation when the residual sugar is 2g/L to obtain a bifidobacterium fermentation product;
S4, a thallus collecting step, which comprises the steps of centrifugally separating a bifidobacterium fermentation product at 8000rpm for 25 minutes, and collecting wet thallus and fermentation supernatant; washing the wet thalli with sterile deionized water for 2 times until the conductivity of the mixed solution of the wet thalli and the sterile deionized water is 262 mu s/cm, and then removing the sterile deionized water to obtain the wet thalli; fermenting the supernatant for later use to obtain wet thalli and fermentation supernatant;
s5, preparing a suspension, namely preparing the suspension by using wet thalli and fermentation supernatant, wherein the suspension comprises the following steps:
s50, compounding the fermentation supernatant with sterile deionized water in a volume ratio accounting for 28% of the sterile deionized water to prepare a suspending agent;
s51, mixing wet thalli and a suspending agent according to a mass ratio of 1:18 to obtain a suspension;
S6, a thallus crushing step, which comprises the steps of crushing the suspension at the temperature of 4 ℃ for 4 times at 1600bar until the suspension is crushed;
s7, performing enzymolysis treatment, namely performing enzymolysis treatment on the crushed suspension, wherein the enzymolysis treatment comprises the following steps:
s70, maintaining the crushed suspension at 75 ℃ and 280rpm for 10 minutes to obtain an inactivated suspension;
S71, cooling the inactivated bacteria suspension to 50 ℃, and respectively adding lysozyme accounting for 0.2% of the mass of the bacteria suspension and composite biological protease accounting for 1.5% of the mass of the bacteria suspension for enzymolysis treatment for 2 hours to obtain an enzymolysis treatment product; wherein, the enzyme activity of the lysozyme is 20000U/mg;
s72, maintaining the enzymolysis product for 5 minutes at 95 ℃ and 280rpm to obtain an enzymolysis product;
s73, performing antiseptic treatment, namely adding an antiseptic into the enzymolysis product to perform antiseptic treatment to obtain an antiseptic enzymolysis product; wherein the preservative is phenoxyethanol and sodium benzoate, the amount of phenoxyethanol accounts for 0.9% of the mass of the enzymolysis product, and the amount of sodium benzoate accounts for 0.3% of the mass of the enzymolysis product;
S8, performing ultrafiltration treatment, namely performing ultrafiltration treatment on the anti-corrosion enzymolysis product by using an ultrafiltration membrane with a molecular weight cutoff of 3KD, and collecting permeate to obtain a bifidobacterium fermentation lysate;
s9, concentrating the bifidobacterium fermentation lysate by using a rotary evaporator to obtain the bifidobacterium fermentation lysate with the Kjeldahl nitrogen content of 0.15 wt.%.
Example 3a method of preparing a bifidobacterium fermentation lysate comprising:
S1, preparing an improved MRS culture medium, which comprises the steps of taking 40g of glucose as a main material, taking 10g of yeast powder, 4.5g of peptone, 0.35g of magnesium sulfate, 2g of dipotassium hydrogen phosphate, 2g of triammonium citrate, 5g of sodium acetate, 0.05g of manganese sulfate, 1g of L-cysteine and 1g of tween-80 as ingredients, and preparing 1L of the improved MRS culture medium by using deionized water as a solvent; wherein, glucose is sterilized for 20 minutes at a sterilizing temperature of 121 ℃, ingredients are sterilized under the same condition, and the ingredients are mixed with other components after being sterilized to prepare an improved MRS culture medium;
S2, a strain activation step, which comprises inoculating bifidobacterium animalis subspecies (Latin name: bifidobacterium animalis subsp. Lactis, strain preservation number: CGMCC 28490) into a sterilized commercial MRS broth culture medium, performing static culture at 37 ℃ for 40 hours for activation, and performing activation after turbidity of the commercial MRS broth culture medium to obtain an activated strain with a strain concentration of 1 multiplied by 10 7 cfu/mL;
s3, a fermentation culture step, which comprises inoculating an activated strain into the improved MRS culture medium in a volume ratio of 1% of the improved MRS culture medium to perform high-density fermentation, wherein the high-density fermentation comprises the following steps:
S30, controlling the process conditions of the modified MRS culture medium inoculated with the activated strain to have a constant pH value of 6.0 by using a fermentation temperature of 37 ℃, a rotating speed of 200rpm and a 10wt.% sodium hydroxide solution, and performing high-density fermentation until the residual sugar of the modified MRS culture medium inoculated with the activated strain is 3g/L, so as to obtain a fermentation product in the first stage;
S31, feeding 50wt.% glucose into the fermentation product of the first stage at a constant speed of 8 g/L.h, feeding until the residual sugar is 30g/L, fermenting under the process conditions that the fermentation temperature is 37 ℃, the rotating speed is 200rpm, and the pH value of the fermentation product of the first stage after the feeding is controlled to be 6.0 by 10wt.% sodium hydroxide solution, and ending the fermentation when the residual sugar is 3g/L to obtain a bifidobacterium fermentation product;
S4, a thallus collecting step, which comprises the steps of centrifugally separating a bifidobacterium fermentation product at 6500rpm for 25 minutes, and collecting wet thallus and fermentation supernatant; washing the wet thalli with sterile deionized water for 2 times until the conductivity of the mixed solution of the wet thalli and the sterile deionized water is 175 mu s/cm, and then removing the sterile deionized water to obtain the wet thalli; fermenting the supernatant for later use to obtain wet thalli and fermentation supernatant;
s5, preparing a suspension, namely preparing the suspension by using wet thalli and fermentation supernatant, wherein the suspension comprises the following steps:
S50, compounding the fermentation supernatant with sterile deionized water according to the volume ratio of 25% of the fermentation supernatant to prepare a suspending agent;
S51, mixing wet thalli and a suspending agent according to a mass ratio of 1:20 to obtain a suspension;
S6, a thallus crushing step, which comprises the steps of crushing the suspension at the temperature of 4 ℃ for 4 times at 1700bar until the suspension is crushed;
s7, performing enzymolysis treatment, namely performing enzymolysis treatment on the crushed suspension, wherein the enzymolysis treatment comprises the following steps:
S70, maintaining the crushed suspension at 75 ℃ and 280rpm for 12 minutes to obtain an inactivated suspension;
S71, cooling the inactivated bacteria suspension to 51 ℃, and respectively adding lysozyme accounting for 0.3% of the mass of the bacteria suspension and composite biological protease accounting for 1.8% of the mass of the bacteria suspension for enzymolysis treatment for 2 hours to obtain an enzymolysis treatment product; wherein, the enzyme activity of the lysozyme is 20000U/mg;
s72, maintaining the enzymolysis product for 5 minutes at 95 ℃ and 280rpm to obtain an enzymolysis product;
S73, performing antiseptic treatment, namely adding an antiseptic into the enzymolysis product to perform antiseptic treatment to obtain an antiseptic enzymolysis product; wherein the preservative is phenoxyethanol and sodium benzoate, the amount of phenoxyethanol accounts for 0.8% of the mass of the enzymolysis product, and the amount of sodium benzoate accounts for 0.34% of the mass of the enzymolysis product;
S8, performing ultrafiltration treatment, namely performing ultrafiltration treatment on the anti-corrosion enzymolysis product by using an ultrafiltration membrane with a molecular weight cutoff of 3KD, and collecting permeate to obtain a bifidobacterium fermentation lysate;
S9, concentrating the bifidobacterium fermentation lysate by using a rotary evaporator to obtain the bifidobacterium fermentation lysate with the Kjeldahl nitrogen content of 0.14 wt.%.
Example 4 preparation of bifidobacterium fermentation lysate comprising:
S1, preparing an improved MRS culture medium, which comprises the steps of taking 40g of glucose as a main material, taking 10g of yeast powder, 4.5g of peptone, 0.35g of magnesium sulfate, 2g of dipotassium hydrogen phosphate, 2g of triammonium citrate, 5g of sodium acetate, 0.05g of manganese sulfate, 1g of L-cysteine and 1g of tween-80 as ingredients, and preparing 1L of the improved MRS culture medium by using deionized water as a solvent; wherein, glucose is sterilized for 20 minutes at a sterilizing temperature of 121 ℃, ingredients are sterilized under the same condition, and the ingredients are mixed with other components after being sterilized to prepare an improved MRS culture medium;
S2, a strain activation step, which comprises inoculating bifidobacterium animalis subspecies (Latin name: bifidobacterium animalis subsp. Lactis, strain preservation number: CGMCC 28490) into a sterilized commercial MRS broth culture medium, performing static culture at 37 ℃ for 40 hours for activation, and performing activation after turbidity of the commercial MRS broth culture medium to obtain an activated strain with a strain concentration of 1 multiplied by 10 7 cfu/mL;
s3, a fermentation culture step, which comprises inoculating an activated strain into the improved MRS culture medium in a volume ratio of 1% of the improved MRS culture medium to perform high-density fermentation, wherein the high-density fermentation comprises the following steps:
S30, controlling the process conditions of the modified MRS culture medium inoculated with the activated strain to have a constant pH value of 6.0 by using a fermentation temperature of 37 ℃, a rotating speed of 200rpm and a 10wt.% sodium hydroxide solution, and performing high-density fermentation until the residual sugar of the modified MRS culture medium inoculated with the activated strain is 3g/L, so as to obtain a fermentation product in the first stage;
S31, feeding 50wt.% glucose into the fermentation product of the first stage at a constant speed of 8 g/L.h, feeding until the residual sugar is 30g/L, fermenting under the process conditions that the fermentation temperature is 37 ℃, the rotating speed is 200rpm, and the pH value of the fermentation product of the first stage after the feeding is controlled to be 6.0 by 10wt.% sodium hydroxide solution, and ending the fermentation when the residual sugar is 3g/L to obtain a bifidobacterium fermentation product;
S4, a thallus collecting step, which comprises the steps of centrifugally separating a bifidobacterium fermentation product at 6500rpm for 25 minutes, and collecting wet thallus and fermentation supernatant; washing the wet thalli with sterile deionized water for 2 times until the conductivity is 175 mu s/cm, and then removing the sterile deionized water to obtain the wet thalli; fermenting the supernatant for later use to obtain wet thalli and fermentation supernatant;
s5, preparing a suspension, namely preparing the suspension by using wet thalli and fermentation supernatant, wherein the suspension comprises the following steps:
S50, compounding the fermentation supernatant with sterile deionized water according to the volume ratio of 20% of the fermentation supernatant to prepare a suspending agent;
S51, mixing wet thalli and a suspending agent according to a mass ratio of 1:20 to obtain a suspension;
S6, a thallus crushing step, which comprises the steps of crushing the suspension at the temperature of 4 ℃ for 4 times at 1700bar until the suspension is crushed;
s7, performing enzymolysis treatment, namely performing enzymolysis treatment on the crushed suspension, wherein the enzymolysis treatment comprises the following steps:
S70, maintaining the crushed suspension at 75 ℃ and 280rpm for 12 minutes to obtain an inactivated suspension;
S71, cooling the inactivated bacteria suspension to 51 ℃, and respectively adding lysozyme accounting for 0.3% of the mass of the bacteria suspension and composite biological protease accounting for 1.8% of the mass of the bacteria suspension for enzymolysis treatment for 2 hours to obtain an enzymolysis treatment product; wherein, the enzyme activity of the lysozyme is 20000U/mg;
s72, maintaining the enzymolysis product for 5 minutes at 95 ℃ and 280rpm to obtain an enzymolysis product;
S73, performing antiseptic treatment, namely adding an antiseptic into the enzymolysis product to perform antiseptic treatment to obtain an antiseptic enzymolysis product; wherein the preservative is phenoxyethanol and sodium benzoate, the amount of phenoxyethanol accounts for 0.8% of the mass of the enzymolysis product, and the amount of sodium benzoate accounts for 0.34% of the mass of the enzymolysis product;
S8, performing ultrafiltration treatment, namely performing ultrafiltration treatment on the anti-corrosion enzymolysis product by using an ultrafiltration membrane with a molecular weight cutoff of 3KD, and collecting permeate to obtain a bifidobacterium fermentation lysate;
s9, concentrating the bifidobacterium fermentation lysate by using a rotary evaporator to obtain the bifidobacterium fermentation lysate with the Kjeldahl nitrogen content of 0.16 wt.%.
Comparative example 1a process for the preparation of a bifidobacterium fermentation lysate comprising:
S1, preparing a commercial MRS culture medium, which comprises the steps of taking 20g of glucose as a main material, taking 8.0g of beef powder, 4.0g of yeast powder, 10.0g of peptone, 0.2g of magnesium sulfate, 2.0g of dipotassium hydrogen phosphate, 2g of diammonium hydrogen citrate, 5.0g of sodium acetate, 0.04g of manganese sulfate and 1g of tween-80 as ingredients, and preparing 1L of modified MRS culture medium by using deionized water as a solvent; wherein, glucose is sterilized for 25 minutes at a sterilizing temperature of 121 ℃, ingredients are sterilized under the same condition, and the ingredients are mixed with other components after being sterilized to prepare an improved MRS culture medium;
S2, a strain activation step, which comprises inoculating bifidobacterium animalis subspecies (Latin name: bifidobacterium animalis subsp. Lactis, strain preservation number: CGMCC 28490) into a sterilized commercial MRS broth culture medium, performing static culture at 37 ℃ for 40 hours for activation, and performing activation after turbidity of the commercial MRS broth culture medium to obtain an activated strain with a strain concentration of 1 multiplied by 10 7 cfu/mL;
s3, a fermentation culture step, which comprises inoculating an activated strain into the improved MRS culture medium in a volume ratio of 1% of the improved MRS culture medium to perform high-density fermentation, wherein the high-density fermentation comprises the following steps:
S30, controlling the process condition of the pH value of the modified MRS culture medium inoculated with the activated strain to be 6.0 by using a fermentation temperature of 37 ℃ and a rotating speed of 200rpm and 10wt.% of sodium hydroxide solution, performing high-density fermentation treatment, and ending the fermentation until the residual sugar of the modified MRS culture medium inoculated with the activated strain is 4g/L, thus obtaining bifidobacterium fermentation liquor;
s4, a thallus collecting step, which comprises the steps of centrifugally separating bifidobacterium fermentation liquor for 25 minutes at the speed of 6500rpm, and collecting wet thallus and fermentation supernatant; washing the wet thalli with sterile deionized water for 3 times until the conductivity of the mixed solution of the wet thalli and the sterile deionized water is 175 mu s/cm, and then removing the sterile deionized water to obtain the wet thalli; fermenting the supernatant for later use to obtain wet thalli and fermentation supernatant;
s5, preparing a suspension, namely preparing the suspension by using wet thalli and fermentation supernatant, wherein the suspension comprises the following steps:
S50, compounding the fermentation supernatant with sterile deionized water according to the volume ratio of 20% of the fermentation supernatant to prepare a suspending agent;
S51, mixing wet thalli and a suspending agent according to a mass ratio of 1:20 to obtain a suspension;
S6, a thallus crushing step, which comprises the steps of crushing the suspension at the temperature of 4 ℃ for 4 times at 1700bar until the suspension is crushed;
s7, performing enzymolysis treatment, namely performing enzymolysis treatment on the crushed suspension, wherein the enzymolysis treatment comprises the following steps:
S70, maintaining the crushed suspension at 75 ℃ and 280rpm for 12 minutes to obtain an inactivated suspension;
S71, cooling the inactivated bacteria suspension to 51 ℃, and respectively adding lysozyme accounting for 0.3% of the mass of the bacteria suspension and composite biological protease accounting for 1.8% of the mass of the bacteria suspension for enzymolysis treatment for 2 hours to obtain an enzymolysis treatment product; wherein, the enzyme activity of the lysozyme is 20000U/mg;
s72, maintaining the enzymolysis product for 5 minutes at 95 ℃ and 280rpm to obtain an enzymolysis product;
S73, performing antiseptic treatment, namely adding an antiseptic into the enzymolysis product to perform antiseptic treatment to obtain an antiseptic enzymolysis product; wherein the preservative is phenoxyethanol and sodium benzoate, the amount of phenoxyethanol accounts for 0.8% of the mass of the enzymolysis product, and the amount of sodium benzoate accounts for 0.34% of the mass of the enzymolysis product;
S8, performing ultrafiltration treatment, namely performing ultrafiltration treatment on the anti-corrosion enzymolysis product by using an ultrafiltration membrane with a molecular weight cutoff of 3KD, and collecting permeate to obtain a bifidobacterium fermentation lysate;
s9, concentrating the bifidobacterium fermentation lysate by using a rotary evaporator to obtain the bifidobacterium fermentation lysate with the Kjeldahl nitrogen content of 0.11 wt.%.
Comparative example 2a process for the preparation of a bifidobacterium fermentation lysate comprising:
S1, preparing an improved MRS culture medium, which comprises the steps of taking 35g of glucose as a main material, and 9g of yeast powder, 4g of peptone, 0.35g of magnesium sulfate, 2g of dipotassium hydrogen phosphate, 2g of triammonium citrate, 5g of sodium acetate, 0.05g of manganese sulfate, 1g of L-cysteine and 1g of tween-80, and preparing 1L of the improved MRS culture medium by using deionized water as a solvent; wherein, glucose is sterilized for 25 minutes at a sterilization temperature of 117 ℃, and ingredients are sterilized under the same condition and then mixed with other components to prepare an improved MRS culture medium;
S2, a strain activation step, which comprises inoculating bifidobacterium animalis subspecies (Latin name: bifidobacterium animalis subsp. Lactis, strain preservation number: CGMCC 28490) into a sterilized commercial MRS broth culture medium, performing static culture at 37 ℃ for 40 hours for activation, and performing activation after turbidity of the commercial MRS broth culture medium to obtain an activated strain with a strain concentration of 1 multiplied by 10 7 cfu/mL;
s3, a fermentation culture step, which comprises inoculating an activated strain into the improved MRS culture medium in a volume ratio of 1% of the improved MRS culture medium to perform high-density fermentation, wherein the high-density fermentation comprises the following steps:
s30, controlling the process conditions of the modified MRS culture medium inoculated with the activated strain to have a constant pH value of 6.0 by using a fermentation temperature of 37 ℃, a rotating speed of 200rpm and a 10wt.% sodium hydroxide solution, and performing high-density fermentation until the residual sugar of the modified MRS culture medium inoculated with the activated strain is 4g/L, so as to obtain a fermentation product in the first stage;
S31, feeding 50wt.% glucose into the fermentation product of the first stage at a constant speed of 8 g/L.h, feeding until the residual sugar is 30g/L, fermenting under the process conditions that the fermentation temperature is 37 ℃, the rotating speed is 200rpm, and the pH value of the fermentation product of the first stage after the feeding is controlled to be 6.0 by 10wt.% sodium hydroxide solution, and ending the fermentation when the residual sugar is 3g/L to obtain a bifidobacterium fermentation product;
S4, a thallus collecting step, which comprises the steps of centrifugally separating a bifidobacterium fermentation product at 7000rpm for 25 minutes, and collecting wet thallus and fermentation supernatant; washing the wet thalli with sterile deionized water for 2 times until the conductivity is 285 mu s/cm, and then removing the sterile deionized water to obtain the wet thalli; fermenting the supernatant for later use to obtain wet thalli and fermentation supernatant;
s5, preparing a suspension, namely preparing the suspension by using wet thalli and fermentation supernatant, wherein the suspension comprises the following steps:
S50, compounding the fermentation supernatant with sterile deionized water according to the volume ratio of 25% of the fermentation supernatant to prepare a suspending agent;
S51, mixing wet thalli and a suspending agent according to a mass ratio of 1:15 to obtain a suspension;
S6, a thallus crushing step, which comprises the steps of crushing the suspension at the temperature of 4 ℃ for 4 times at 1600bar until the suspension is crushed;
S7, performing microfiltration treatment, namely performing microfiltration treatment on the suspension by using a microfiltration membrane with a molecular weight cutoff of 0.22 mu m, and collecting a permeate to obtain a bifidobacterium zymocyte lysate; performing antiseptic treatment on the bifidobacterium fermentation lysate to obtain an antiseptic bifidobacterium fermentation lysate; wherein, the preservative is phenoxyethanol and sodium benzoate, the phenoxyethanol accounts for 0.5 to 1 percent of the mass of the bifidobacterium fermentation lysate, and the sodium benzoate accounts for 0.25 to 0.5 percent of the mass of the bifidobacterium fermentation lysate;
S8, concentrating, namely performing rotary steaming and concentrating treatment on the bifidobacterium fermentation lysate by using a rotary steaming instrument to obtain the bifidobacterium fermentation lysate with the Kjeldahl nitrogen content of 0.15 wt.%.
In the preparation of the bifidobacterium fermentation lysate of examples 1 to 4 and comparative examples 1 to 2 of the present application, the bifidobacterium concentration in the bifidobacterium fermentation product after the fermentation treatment step was carried out is shown in table 1 below.
TABLE 1 comparison of Bifidobacterium cell concentrations after fermentation
From the fermentation densities of table 1, it can be derived:
According to the bifidobacterium fermentation method, the bifidobacterium animalis is subjected to high-density fermentation culture by adopting the improved MRS culture medium, so that more bifidobacterium thalli can be obtained, namely, higher-density bifidobacterium fermentation can be performed, and the final concentration is as high as 1.6X10 10cfu/mL～1.8×1010 cfu/mL, which is higher than the concentration of 7.2X10 9 cfu/mL of bifidobacterium in comparative example 1 and the concentration of 8.6X10 9 cfu/mL of bifidobacterium in comparative example 2. Thus solving the problem of low fermentation density of bifidobacteria and obtaining a large amount of bifidobacteria thalli.
Test part
1. Product protein molecular weight detection-SDS-PAGE protein electrophoresis step
1. Sample loading
The volume of the solution containing 50. Mu.g of protein was calculated as the loading of the loaded sample for the bifidobacterium fermentation lysates of examples 1-4 and comparative example 2. The sample was removed to a 0.5ml centrifuge tube, and Protein SDS PAGE Loading Buffer (product number 9173, 4× concentration from Dalianbao biological company) of loading buffer was added to a final concentration of 1×. The sample was boiled in boiling water for 5 minutes to denature the protein, and the total volume of the sample was 15. Mu.l. Protein Marker is a color pre-stained protein (available from Biyun biotechnology Co., ltd., molecular weight standard 10-180kD, product number: P0068).
2. Electrophoresis
The preparation method comprises the steps of mixing and then sampling bifidobacterium fermentation lysate prepared in examples 1-4 and comparative example 2 and protein Marker by using BeyoGel TM SDS-PAGE prefabricated gel (Tris-Gly, 4-20%, 12-hole, product number: P0057A) of Biyun biotechnology Co-Ltd and matched electrophoresis buffer SDS-PAGE (Tris-Gly, powder, product number: P0014A), covering an upper cover after the sample is completely sampled, connecting an electrophoresis apparatus, opening a switch of the electrophoresis apparatus, and keeping voltage at 180V after the electrophoresis apparatus is opened, wherein when bromophenol blue indicator migrates to a position 1 cm-2 cm away from the front edge, electrophoresis is stopped for about 0.5-1 hour, wherein the position of the reference sign 1-6 is the electrophoresis starting position, the distance from the electrophoresis starting position is longer, the molecules of the protein are smaller, and the molecules of the protein are larger.
3. Dyeing and decolorizing
After electrophoresis, the power is turned off, the glass plate is taken out, and in the gap between the lower corners of the two long glass plates and the short glass plates, the rubber surface is separated from one glass plate by a knife, then the rubber sheet is supported and put into a large culture dish for dyeing, and 0.25wt.% of coomassie brilliant blue dye liquor is used for dyeing for 2 to 4 hours, and if necessary, the glass plate can be used overnight. Discarding the dyeing liquid, rinsing the glue surface of the pre-prepared glue with distilled water for several times, adding the decoloring liquid, performing diffusion decoloring, and frequently changing the decoloring liquid until the protein bands are clear.
From the protein electrophoresis patterns of examples 1 to 4, protein markers and comparative example 2 shown in FIG. 3, it can be seen that: the protein molecules of the macromolecular proteins of examples 1-4 after enzymolysis treatment by lysozyme and composite biological protease are concentrated at the lower part of the protein gel in lanes 1-4, namely the black shadow part at the lower part of lanes 1-4, which shows that the molecular weight of the protein after enzymolysis treatment is obviously reduced. The protein Marker is layered in lane 5 with different sizes, which indicates that the molecular weight of the protein is different and that a relatively large molecule protein is present. The proteins of comparative example 2 were not enzymatically treated with lysozyme and complex bioprotein, and more protein molecules were distributed in lanes in a continuous tubular shape, as shown by the more black shaded portions in lane 6 of FIG. 3, indicating more macromolecular proteins in the bifidobacterium fermentation lysate without enzymatic treatment.
In the foregoing, only the specific embodiments of the present application are described, and it will be clearly understood by those skilled in the art that, for convenience and brevity of description, the specific working process described above may refer to the corresponding process in the foregoing method embodiment, which is not repeated herein. It should be understood that the scope of the present application is not limited thereto, and any equivalent modifications or substitutions can be easily made by those skilled in the art within the technical scope of the present application, and they should be included in the scope of the present application.
Claims (10)
1. A method of fermenting bifidobacteria, comprising:
30g to 40g of glucose is taken as a main material, 8g to 10g of yeast powder, 3g to 5g of peptone, 0.2g to 0.5g of magnesium sulfate, 1.5g to 2.5g of dipotassium hydrogen phosphate, 1.5g to 2.5g of tri-ammonium citrate, 3g to 6g of sodium acetate, 0.03g to 0.06g of manganese sulfate, 0.5g to 1.2g of L-cysteine, 0.8g to 1.5g of tween-80 are taken as ingredients, deionized water is taken as a solvent to prepare an improved MRS culture medium;
inoculating bifidobacterium animalis subspecies into sterilized commercial MRS broth culture medium for activation to obtain activated strains;
inoculating the activated strain into the improved MRS culture medium in a volume ratio of 0.5-2% of the improved MRS culture medium for fermentation to obtain a bifidobacterium fermentation product.
2. The fermentation process of claim 1, comprising:
Sterilizing the ingredients, wherein the sterilization temperature of the ingredients is 117-121 ℃ and the sterilization time is 20-30 minutes;
Sterilizing the main material at 117-121 deg.c for 20-30 min; and/or
Inoculating the bifidobacterium animalis subspecies lactis into the sterilized commercial MRS broth culture medium, performing static culture at 35-38 ℃ for 24-48 hours to activate the bifidobacterium animalis subspecies lactis, and obtaining activated strains after the commercial MRS broth culture medium is mixed and turbid.
3. The fermentation process of claim 1, wherein the concentration of the activated species is 1x 10 7cfu/mL～1×108 cfu/mL; and/or
Latin name of the bifidobacterium animalis subspecies lactis: bifidobacterium animalis subsp.lactis, accession number of seed: CGMCC 28490; preservation unit: china general microbiological culture Collection center.
4. The fermentation process of claim 1, comprising:
Inoculating the activated strain into the improved MRS culture medium in a volume ratio of 0.5-2% of the improved MRS culture medium, controlling the process conditions of the pH value of the improved MRS culture medium inoculated with the activated strain to be 6.0 by using a fermentation temperature of 35-38 ℃, a rotating speed of 150-200 rpm and 10wt.% sodium hydroxide solution, fermenting until the residual sugar of the improved MRS culture medium inoculated with the activated strain is less than or equal to 5g/L, and obtaining a fermentation product in the first stage;
And (3) feeding the fermentation product of the first stage until the residual sugar is 30 g/L-40 g/L, and fermenting under the process conditions that the fermentation temperature is 35-38 ℃, the rotating speed is 150-200 rpm, and the pH value of the fermentation product of the first stage after feeding is controlled to be 6.0 by 10wt.% sodium hydroxide solution, so that the residual sugar is less than or equal to 5g/L, thereby obtaining the bifidobacterium fermentation product.
5. A method for preparing a bifidobacterium lysed fermentation lysate, comprising:
separating the bifidobacterium fermentation product to obtain wet thalli and fermentation supernatant; the bifidobacterium fermentation product is prepared according to the bifidobacterium fermentation method of any one of claims 1-4;
Preparing a suspension by using the wet thalli and the fermentation supernatant;
crushing the suspension to obtain crushed suspension;
carrying out enzymolysis treatment on the crushed suspension to obtain an enzymolysis product;
And performing ultrafiltration treatment on the enzymolysis product, and collecting permeate to obtain a bifidobacterium fermentation lysate.
6. The method of manufacturing according to claim 5, further comprising:
Concentrating the bifidobacterium fermentation lysate to obtain the bifidobacterium fermentation lysate with the Kjeldahl nitrogen content of 0.1-2 wt.%.
7. The method according to claim 5 or 6, comprising subjecting a bifidobacterium fermentation product to centrifugation to obtain wet cells and a fermentation supernatant; the wet thalli is washed by using sterile deionized water until the conductivity of the mixed solution of the wet thalli and the sterile deionized water is lower than 400 mu s/cm, and the sterile deionized water is removed to obtain the wet thalli; and/or
Compounding the fermentation supernatant with sterile deionized water in a volume ratio of 20-40% to prepare a suspending agent;
Mixing the wet thalli and the suspending agent according to the mass ratio of 1:10-20 to obtain a suspension.
8. The method according to claim 5 or 6, comprising crushing the suspension at 1500bar to 1900bar at 4 ℃ to 8 ℃ for 3 times to 5 times to obtain a crushed suspension.
9. The method according to claim 5 or 6, characterized by comprising:
maintaining the crushed suspension at 70-80 ℃ and 200-300 rpm for 10-15 minutes to obtain an inactivated suspension;
Cooling the inactivated bacteria suspension to 50-52 ℃, adding lysozyme accounting for 0.1-0.5% of the mass of the bacteria suspension and composite biological protease accounting for 1-2% of the mass of the bacteria suspension, and carrying out enzymolysis treatment for 2-4 hours to obtain an enzymolysis treatment product; wherein the enzyme activity of the lysozyme is 10000-20000U/mg;
maintaining the enzymolysis treatment product for 5-10 minutes at the temperature of 90-100 ℃ and the rpm of 200-300 rpm to obtain an enzymolysis product; and/or
The ultrafiltration treatment step comprises the step of carrying out ultrafiltration treatment on the enzymolysis product by using an ultrafiltration membrane with a molecular weight cutoff of 3KD, and collecting a permeate as a bifidobacterium fermentation lysate.
10. The method of claim 9, wherein the complex biological protease is selected from the group consisting of neutral protease, acid protease, papain, trypsin, and combinations of two or more thereof; and/or
And adding a preservative into the enzymolysis product to obtain a preserved enzymolysis product.
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