CN114426989B - Bifidobacterium fermentation lysate, preparation method and application thereof - Google Patents
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Abstract
The invention provides a bifidobacterium fermentation lysate, a preparation method and application thereof, belonging to the technical field of cosmetics. Wherein a bifidobacterium fermentation lysate comprises: the microbial inoculum comprises a filtrate obtained by centrifuging fermentation liquor obtained by performing mixed fermentation on bifidobacterium longum, bifidobacterium animalis and bifidobacterium adolescentis, and a lysate mother liquor obtained by performing enzymolysis on thalli obtained by performing mixed fermentation on the bifidobacterium longum, the bifidobacterium animalis and the bifidobacterium adolescentis. The combination of lysate mother liquor obtained by the thallus enzymolysis and fermentation filtrate can reduce the loss of soluble micromolecules in the production process of products to the maximum extent, and the lysate mother liquor can be used as a cosmetic raw material to ensure that the concentrations of polysaccharide and polypeptide are suitable for skin absorption and nursing, and even can be directly smeared on the surface of skin for use.
Description
Technical Field
The invention belongs to the technical field of cosmetics, and particularly relates to a bifidobacterium fermentation lysate, a preparation method and application thereof.
Background
The bifidobacterium fermentation lysate is a physiological lysate obtained by processing after bifidobacterium fermentation, and the main components of the lysate are cell metabolites, cytoplasmic fragments, polysaccharides, proteins, polypeptides and the like. A large number of in vitro experiments prove that various amino acids, proteins and various molecular media in the bifidobacterium fermentation lysate have the effects of regulating and balancing skin and regulating immune function, are also nutritional molecules of human skin cells, have certain anti-wrinkle, compact, repairing, sun-screening, anti-oxidation and anti-aging effects, can prevent damage caused by external stimulation such as ultraviolet rays and the like, and promote repair of damaged DNA. However, the existing preparation process of bifidobacterium fermentation lysate used as cosmetic raw materials has the problems of low content of functional active ingredients such as proteins and small molecular polypeptides easy to be absorbed by skin and the like, and the quality of the fermentation lysate is influenced.
Disclosure of Invention
In view of the deficiencies of the prior art, it is a first object of the present invention to provide a bifidobacterium fermentation lysate to increase the content of functional active ingredients.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a Bifidobacterium fermentation lysate comprising: the microbial inoculum comprises a filtrate obtained by centrifuging fermentation liquor obtained by performing mixed fermentation on bifidobacterium longum, bifidobacterium animalis and bifidobacterium adolescentis, and a lysate mother liquor obtained by performing enzymolysis on thalli obtained by performing mixed fermentation on the bifidobacterium longum, the bifidobacterium animalis and the bifidobacterium adolescentis.
In some embodiments of the invention, the volume ratio of filtrate to lysate mother liquor is 1: (1-10), for example, the volume ratio of the filtrate to the lysate mother liquor may be 1: 1. 1: 2. 1: 3. 1: 4. 1: 5. 1: 6. 1: 7. 1: 8. 1: 9 or 1: 10, etc.
In some embodiments of the present invention, the mixed fermentation is a fermentation broth obtained by performing primary amplification, secondary amplification and micro-aerobic fermentation on bifidobacterium longum, bifidobacterium animalis and bifidobacterium adolescentis as strains.
In some embodiments of the present invention, the first-stage amplification is to inoculate bifidobacterium longum, bifidobacterium animalis and bifidobacterium adolescentis in a sterile triangular flask, and perform anaerobic culture to prepare a first-stage seed solution. Preferably, the conditions of the first-stage amplification are as follows: the pH value is 6.0-7.0, the culture temperature is 37 ℃, and the culture time is 24-48 hours. For example, the pH can be selected from 6.0, 6.1, 6.2, 6.5, 6.7, 6.8, 7.0, or the like; the culture time may be 24 hours, 27 hours, 30 hours, 32 hours, 35 hours, 40 hours, 42 hours, 46 hours, 48 hours, or the like.
In some embodiments of the present invention, the second amplification is to mix the first seed solution and inoculate the mixture into a fermentation tank, separate the bacteria after amplification culture, and resuspend the separated bacteria to obtain the second seed solution. Preferably, the conditions for the secondary amplification are: the inoculation amount of the primary seed liquid is 1% -2%, the pH value is 6.0-7.0, the culture temperature is 37 ℃, the culture time is 2-3 days, the dissolved oxygen is 0-5%, a TPY liquid culture medium is adopted for carrying out heavy suspension, and the volume ratio of the heavy suspension is 1: (50-100). For example, the amount of the first seed solution inoculated may be 1%, 1.5%, 2%, or the like; the pH can be 6.0, 6.1, 6.2, 6.5, 6.7, 6.8, 7.0, or the like; the culture time can be 2 days, 2.5 days or 3 days, etc.; the dissolved oxygen may be 0%, 1%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, or the like.
In some embodiments of the invention, the micro-aerobic fermentation is a transfer of secondary seed liquid into a fermentation medium for micro-aerobic fermentation. Preferably, the conditions of the micro-aerobic fermentation are as follows: the inoculation amount of the secondary seed liquid is 1-3%, the initial absorbance at 600nm is 0.1-0.2, the temperature is 37 ℃, the pH value is 6.0-7.0, the amplification time is 2-3 days, and the dissolved oxygen is 0% -10%. For example, the amount of inoculation of the secondary seed liquid may be 1%, 1.5%, 2%, 2.5%, 3%, or the like; the initial 600nm absorbance may be 0.1, 0.15, 0.2, etc.; the pH can be 6.0, 6.1, 6.2, 6.5, 6.7, 6.8, 7.0, or the like; the amplification time can be 2 days, 2.5 days, 3 days and the like; the dissolved oxygen may be 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or the like.
In some embodiments of the invention, the fermentation medium comprises: 4-6 g/L of glucose, 8-12 g/L of acid hydrolyzed casein, 400-600 mL/L of characteristic extracting solution, 0.1-0.3 g/L of amino acid, 1-3 g/L of dipotassium phosphate, 0.4-0.6 g/L of magnesium chloride, 0.1-0.2/L of calcium chloride, 0.04-0.06 g/L of manganese sulfate, 4-6 g/L of sodium acetate and 0.4-0.6 g/L of cysteine.
In some embodiments of the present invention, the characteristic extract is one or more of an angelica extract, a rose extract, a lavender extract, a jasmine extract, a rosemary extract, a pseudo-ginseng extract, a polygonum multiflorum extract, a ganoderma extract, an astragalus extract, a dendrobium officinale extract, a medlar extract, a rhodiola rosea extract, a poria cocos extract, a cistanche extract, a kiwi fruit extract, a longan extract, a tomato extract, a carrot extract and an onion extract. The characteristic extracting solution can be combined by more than two kinds in any proportion, and the total adding amount in the fermentation culture medium is maintained to be 400-600 mL/L, for example: the characteristic extracting solution is the combination of rose extracting solution and lavender extracting solution, and the volume ratio of the rose extracting solution to the lavender extracting solution can be (0.01-99.99%): (0.01% -99.99%). The characteristic extracting solution can be extracted by a conventional traditional Chinese medicine extracting method, and the method comprises the following steps: decocting, extracting with ethanol or micronizing, or dissolving corresponding extract in water 。
In some embodiments of the invention, the amino acid is one or a combination of glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine. The amino acids can be combined in any proportion, and the total addition amount in the fermentation medium is maintained to be 0.1-0.3 g/L, for example: the amino acid is a combination of leucine and isoleucine, and the mass ratio of the leucine to the isoleucine can be (0.01% -99.99%): (0.01% -99.99%). The fermentation medium of the invention takes amino acid as a growth factor, and can promote the growth of thalli and increase the content of active substances in bifidobacterium fermentation lysate.
Based on the mixed fermentation, the thallus components of the fermentation liquor obtained after the bifidobacterium longum, the bifidobacterium animalis and the bifidobacterium adolescentis are subjected to secondary amplification and mixed fermentation can ensure extremely high content of effective and active components of lysate for preparing lysate.
In some embodiments of the present invention, the enzymolysis is to resuspend the mixed fermentation thallus and use a protease warm water bath for enzymolysis to obtain a lysate mother liquor.
In some embodiments of the invention, the resuspension is performed by washing the cells with sterile water and resuspending them.
In some embodiments of the invention, the protease is one or more of lysozyme, neutral protease, chymotrypsin and flavourzyme in combination, wherein, the enzyme activity of lysozyme is 20000U/mg, the usage amount is 0.1-0.3% of the lysate mother liquor mass, for example, the usage amount can be 0.1%, 0.15%, 0.2%, 0.25% or 3% and the like; the enzyme activity of the neutral protease is 50U/mg, the using amount is 0.1-0.3% of the mass of lysate mother liquor, for example, the using amount can be 0.1%, 0.15%, 0.2%, 0.25% or 3%; the enzyme activity of the chymotrypsin is 800U/mg, the usage amount is 0.1-0.3% of the lysate mother liquor mass, for example, the usage amount can be 0.1%, 0.15%, 0.2%, 0.25% or 3% and the like; the enzyme activity of the flavourzyme is 20U/mg, the using amount is 0.1-0.3% of the lysate mother liquor, for example, the using amount can be 0.1%, 0.15%, 0.2%, 0.25% or 3%. The total amount of the protease does not exceed 0.2-0.6% of the mass of the lysate mother liquor. The combination of different kinds of proteases affects the small molecular polypeptide composition of the final product and affects the final effect of the product to some extent, so the invention prefers the combination of the proteases, after the protease hydrolysis, most of the large molecular protein in the lysate mother liquor is hydrolyzed into small peptide, and the content of soluble effective active substances is increased.
In some embodiments of the invention, the conditions of the warm water bath are: the temperature is 37-55 ℃, and the time is 16-24 hours. For example, the temperature may be 37 ℃, 38 ℃, 40 ℃, 42 ℃, 45 ℃, 50 ℃ or 55 ℃ or the like; the time may be 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, or the like.
Through the technical scheme, the obtained bifidobacterium fermentation lysate is basically prepared by micro-aerobic fermentation of bifidobacterium longum, bifidobacterium animalis and bifidobacterium adolescentis, is a natural bioactive raw material and comprises components equivalent to natural probiotics.
The second purpose of the invention is to provide a method for preparing bifidobacterium fermentation lysate, which improves the content of effective active ingredients in the lysate through process improvement.
A method for preparing bifidobacterium fermentation lysate uses bifidobacterium longum, bifidobacterium animalis and bifidobacterium adolescentis as strains, and obtains the bifidobacterium fermentation lysate by first-stage amplification, second-stage amplification, microaerobic fermentation, centrifugation, lysate mother liquor preparation and mixing preparation.
In some embodiments of the present invention, the centrifugation is to centrifuge the fermentation liquid obtained by micro-aerobic fermentation to obtain a filtrate and bacteria. Centrifuging to obtain supernatant as fermentation product filtrate; the precipitation, i.e. bifidobacterium thallus, is washed and resuspended, protease is used for enzymolysis to obtain lysate mother liquor, and the respective treatment of filtrate and thallus can increase the content of soluble active substances in the lysate mother liquor, reduce the loss of active components in the filtrate in the enzymolysis process and improve the product quality.
In some embodiments of the invention, the preparation of lysate mother liquor comprises enzyme deactivation of the lysate mother liquor after enzymatic hydrolysis, wherein the enzyme deactivation is performed at a temperature of 90-95 ℃ for 10-12 minutes.
In some embodiments of the invention, the mixing formulation is a mixture of the filtrate and the lysate mother liquor in a volume ratio of 1: (1-10) mixing.
The third purpose of the invention is to provide the application of the bifidobacterium fermentation lysate in skin care products, wherein the skin care products are anti-wrinkle, firming, repairing, sun-screening or anti-aging skin care products, and the bifidobacterium fermentation lysate is used as a preparation raw material of the skin care products.
Compared with the prior art, the invention has the following beneficial effects:
(1) the combination of lysate mother liquor obtained by the thallus enzymolysis and fermentation filtrate can reduce the loss of soluble micromolecules in the production process of products to the maximum extent, and the lysate mother liquor can be used as a cosmetic raw material to ensure that the concentrations of polysaccharide and polypeptide are suitable for skin absorption and nursing, and even can be directly smeared on the surface of skin for use.
(2) The invention improves the composition of the fermentation culture medium, increases the characteristic extracting solution, takes amino acid as a growth factor, improves the mixed fermentation level of bifidobacterium longum, bifidobacterium animalis and bifidobacterium adolescentis and the content of active substances in a final product, and improves the smell and the color of a fermentation lysate.
(3) According to the invention, by the enzymolysis process of the compound protease, macromolecular proteins which are not easy to absorb in the mixed fermentation product of the bifidobacterium longum, the bifidobacterium animalis and the bifidobacterium adolescentis are hydrolyzed to generate a large amount of soluble micromolecular polypeptides which are easy to absorb, so that the problems that protein components in the existing bifidobacterium fermentation product are not easy to absorb by skin and the like are solved, the clarity of a supernatant after a final product is stood is improved, and the quality of the product is improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1: a beta-galactosidase model map of the anti-aging efficacy of the bifidobacterium fermentation lysate of the invention;
FIG. 2 is a schematic diagram: the invention relates to a micronuclear model map of the efficacy of bifidobacterium fermentation lysate in repairing damaged DNA.
Detailed Description
In order to better understand the present invention, the following examples are further provided to clearly illustrate the contents of the present invention, but the contents of the present invention are not limited to the following examples. In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present invention. It will be apparent, however, to one skilled in the art, that the present invention may be practiced without one or more of these specific details.
In all examples shown and discussed herein, any particular value should be construed as merely illustrative, and not limiting. Thus, other examples of the exemplary embodiments may have different values.
In the following examples, Bifidobacterium longum, Bifidobacterium animalis and Bifidobacterium adolescentis were purchased from the culture Collection of microorganisms of Guangdong province. Wherein:
the Chinese name of Bifidobacterium longum is: bifidobacterium longum subsp.Bifidobacterium longumThe strain preservation number is as follows: GDMCC 1.1258, deposit unit: guangdong province microbial strain preservation center, address: experimental building 5 of 100 # large college of junior furious district of Guangzhou city, Guangdong province.
Chinese name of bifidobacterium animalis: bifidobacterium animalis subspecies zoon, latin name:Bifidobacterium animalis subsp. animalisthe strain preservation number is as follows: GDMCC 1.169, depository: guangdong province microbial strain preservation center, address: experimental building 5 of 100 # large college of junior furious district of Guangzhou city, Guangdong province.
Chinese name of bifidobacterium adolescentis: bifidobacterium adolescentis, latin name:Bifidobacterium adolescentisthe strain preservation number is as follows: GDMCC 1.278, deposit unit: guangdong province microbial strain preservation center, address: experimental building 5 of 100 # large college of junior furious district of Guangzhou city, Guangdong province.
In the following examples, the characteristic extracts were obtained by decoction. Illustratively, raw materials are added into water, and the mass ratio of the raw materials to the water is 1: decocting at 100 + -10 deg.C for 2-3 hr, cooling, and filtering to obtain filtrate as extractive solution of corresponding materials.
Example 1
A method of producing a bifidobacterium fermentation lysate comprising the steps of:
(1) respectively inoculating bifidobacterium longum, bifidobacterium animalis and bifidobacterium adolescentis into three sterile triangular flasks containing 100mL of TPY liquid culture medium, wherein the initial pH of the culture medium is 6.5, and performing anaerobic culture at 37 ℃ for 48 hours to obtain a first-stage fermentation seed solution;
(2) adding 5L of TPY liquid culture medium into a fermentation tank, inoculating the primary fermentation seed liquid, wherein the inoculation amount is 1%, the pH value is 6.5, culturing for 2 days at 37 ℃, and resuspending the obtained thallus by using the TPY liquid culture medium, wherein the volume ratio of the thallus to the TPY liquid culture medium is 1: 50 as secondary fermentation seed liquid;
(3) inoculating the secondary seed liquid into 150L fermentation medium (comprising 5g/L glucose, 10g/L acid hydrolyzed casein, 500mL/L angelica root extract, 0.2g/L histidine, 2g/L dipotassium hydrogen phosphate, 0.5g/L magnesium chloride, 0.15/L calcium chloride, 0.05g/L manganese sulfate, 5g/L sodium acetate and 0.5g/L cysteine), wherein the inoculation amount is as follows: 3% of volume percentage, initial absorbance of 600nm of 0.15, rotation speed of 30-60 rpm, pressure of 0.05-0.1 Mpa, temperature of 37 ℃, pH controlled at 6.5, dissolved oxygen not more than 5%, sealing a fermentation container for anaerobic culture for 2 days, and collecting fermentation liquor after fermentation;
(4) Centrifuging the fermentation liquor at 10000rpm, collecting thalli and supernatant to obtain 120L of bifidobacterium fermentation product filtrate, and washing and resuspending the thalli by using 15L of sterile water;
(5) carrying out 50 ℃ warm water bath enzymolysis treatment on the resuspended thalli for 20 hours by using 0.1 percent of each of lysozyme, neutral protease, chymotrypsin and flavourzyme, and carrying out enzyme deactivation and sterilization on the obtained enzymolysis liquid at 90 ℃ for 10 minutes to obtain bifidobacterium fermentation lysate mother liquor;
(6) the lysate mother liquor and the fermentation filtrate are merged and mixed evenly according to the volume ratio of 1:8, and the bifidobacterium fermentation lysate is obtained.
Example 2
A method of producing a bifidobacterium fermentation lysate comprising the steps of:
(1) respectively inoculating bifidobacterium longum, bifidobacterium animalis and bifidobacterium adolescentis into three sterile triangular flasks containing 100mL of TPY liquid culture medium, wherein the initial pH of the culture medium is 6.0, and performing anaerobic culture at 37 ℃ for 36h to obtain a first-stage fermentation seed solution;
(2) adding 5L of TPY liquid culture medium into a fermentation tank, inoculating the primary fermentation seed liquid, wherein the inoculation amount is 2%, the pH value is 6.0, culturing for 2.5 days at 37 ℃, and carrying out heavy suspension on the obtained thallus by using the TPY liquid culture medium, wherein the volume ratio of the thallus to the TPY liquid culture medium is 1: 100 as secondary fermentation seed liquid;
(3) Inoculating the secondary seed liquid into 150L fermentation medium (composed of glucose 4g/L, acid hydrolyzed casein 8g/L, rose extract 600mL/L, proline 0.1g/L, dipotassium hydrogen phosphate 1g/L, magnesium chloride 0.6g/L, calcium chloride 0.1/L, manganese sulfate 0.06g/L, sodium acetate 4g/L, cysteine 0.6 g/L), wherein the inoculation amount is as follows: the volume percentage is 2%, the initial absorbance at 600nm is 0.1, the rotating speed is 30-60 rpm, the pressure is 0.05-0.1 Mpa, the temperature is 37 ℃, the pH is controlled at 6.0, the dissolved oxygen is not more than 5%, the fermentation container is sealed for anaerobic culture for 3 days, and the fermentation broth is collected after the fermentation is finished;
(4) centrifuging the fermentation liquor at 10000rpm to collect thalli and supernatant fluid to obtain 120L of bifidobacterium fermentation product filtrate, and washing and resuspending the thalli by using 15L of sterile water;
(5) carrying out 37 ℃ warm water bath enzymolysis treatment on the resuspended thalli for 24 hours by using 0.15 percent of each of lysozyme, neutral protease, chymotrypsin and flavourzyme, and obtaining bifidobacterium fermentation lysate mother liquor after carrying out enzyme deactivation and sterilization on the obtained enzymolysis liquid at 95 ℃ for 10 minutes;
(6) the lysate mother liquor and the fermentation filtrate are mixed according to the volume ratio of 1: 1, and mixing uniformly to obtain the bifidobacterium fermentation lysate.
Example 3
A method of producing a bifidobacterium fermentation lysate comprising the steps of:
(1) Respectively inoculating bifidobacterium longum, bifidobacterium animalis and bifidobacterium adolescentis into three sterile triangular flasks containing 100mL of TPY liquid culture medium, wherein the initial pH of the culture medium is 7.0, and performing anaerobic culture at 37 ℃ for 24 hours to obtain a primary fermentation seed solution;
(2) adding 5L of TPY liquid culture medium into a fermentation tank, inoculating the primary fermentation seed liquid, wherein the inoculation amount is 1%, the pH value is 7.0, culturing for 3 days at 37 ℃, and resuspending the obtained thallus by using the TPY liquid culture medium, wherein the volume ratio of the thallus to the TPY liquid culture medium is 1: 80, as secondary fermentation seed liquid;
(3) inoculating the secondary seed liquid into 150L fermentation medium (composed of glucose 4g/L, acid hydrolyzed casein 12g/L, lavender extract 400mL/L, cysteine 0.3g/L, dipotassium hydrogen phosphate 3g/L, magnesium chloride 0.4g/L, calcium chloride 0.2/L, manganese sulfate 0.04g/L, sodium acetate 6g/L, cysteine 0.4 g/L), wherein the inoculation amount is as follows: the volume percentage is 1%, the initial absorbance at 600nm is 0.2, the rotating speed is 30-60 rpm, the pressure is 0.05-0.1 Mpa, the temperature is 37 ℃, the pH is controlled at 7.0, the dissolved oxygen is not more than 5%, the anaerobic culture is carried out in a sealed fermentation container for 2.5 days, and the fermentation broth is collected after the fermentation is finished;
(4) centrifuging the fermentation liquor at 10000rpm to collect thalli and supernatant fluid to obtain 120L of bifidobacterium fermentation product filtrate, and washing and resuspending the thalli by using 15L of sterile water;
(5) Carrying out enzymolysis treatment on the resuspended thallus by using 0.2% of lysozyme, 0.1% of neutral protease, 0.2% of chymotrypsin and 0.1% of flavourzyme in a 55 ℃ warm water bath for 16 hours, and carrying out enzyme deactivation and sterilization on the obtained enzymolysis liquid at 92 ℃ for 12 minutes to obtain a bifidobacterium fermentation lysate mother liquor;
(6) the lysate mother liquor and the fermentation filtrate are mixed according to the volume ratio of 1: 10, and mixing uniformly to obtain the bifidobacterium fermentation lysate.
Example 4
The difference between the method for producing a bifidobacterium fermentation lysate of this example and example 1 is that: replacing 500mL/L of radix angelicae extract in the composition of the fermentation medium in the step (3) with: 200mL/L of medlar extract, 300 mL/L of rosemary extract and the other components are unchanged.
Example 5
The difference between the method for producing a bifidobacterium fermentation lysate of this example and example 1 is that: replacing 500mL/L of radix angelicae extract in the composition of the fermentation medium in the step (3) with: 100 mL/L of pseudo-ginseng extract, 200mL/L of kiwi fruit extract, 200mL/L of carrot extract and the like, and the other components are unchanged.
Example 6
This example differs from example 1 in that: replacing 0.2g/L histidine in the composition of the fermentation medium in step (3) with: tryptophan 0.1g/L and arginine 0.1 g/L.
Example 7
This example differs from example 1 in that: replacing 0.2g/L histidine in the composition of the fermentation medium in step (3) with: histidine 0.1g/L, threonine 0.05 g/L, and cysteine 0.05 g/L.
Example 8
This example differs from example 1 in that: in the step (5), 0.3% of lysozyme, 0.1% of neutral protease, 0.1% of chymotrypsin and 0.1% of flavourzyme are adopted.
Example 9
This example differs from example 1 in that: in the step (5), 0.1% of lysozyme, 0.1% of neutral protease, 0.1% of chymotrypsin and 0.3% of flavourzyme are adopted.
Comparative example 1
The comparative example differs from example 1 in the method of producing a bifidobacterium fermentation lysate: the step (1) is as follows: bifidobacterium longum was inoculated into a sterile flask containing 100mL of TPY liquid medium at an initial pH of 6.5 and anaerobically cultured at 37 ℃ for 48 hours as a primary fermentation seed solution.
Comparative example 2
The comparative example differs from example 1 in the method of producing a bifidobacterium fermentation lysate: the step (1) is as follows: bifidobacterium animalis was inoculated into a sterile flask containing 100mL of TPY liquid medium at an initial pH of 6.5, and anaerobically cultured at 37 ℃ for 48 hours to give a first-stage fermentation seed solution.
Comparative example 3
The comparative example bifidobacterium fermentation lysate was prepared by a method different from that of example 1: the step (1) is as follows: inoculating Bifidobacterium adolescentis into a sterile triangular flask containing 100mL of TPY liquid culture medium with initial pH of 6.5, and anaerobically culturing at 37 deg.C for 48 hr to obtain primary fermentation seed solution.
Comparative example 4
The comparative example differs from example 1 in the method of producing a bifidobacterium fermentation lysate: in the step (3), the fermentation medium omits the angelica dahurica extract.
Comparative example 5
The comparative example differs from example 1 in the method of producing a bifidobacterium fermentation lysate: histidine was omitted from the fermentation medium in step (3).
Comparative example 6
The comparative example differs from example 1 in the method of producing a bifidobacterium fermentation lysate: replacing the step (5) with: crushing the heavy suspension thallus by using a high-pressure homogenizer at the pressure of 1000bar and the temperature of 4-10 ℃, sterilizing the obtained crushed liquid at the temperature of 90 ℃ for 10 minutes to obtain the bifidobacterium fermentation lysate mother liquor.
Evaluation of Effect
(ii) cell proliferation assay
Human skin fibroblasts (HDF) were digested to prepare a cell suspension, which was then inoculated into a 96-well plate at 10000 cells per well. Culturing in DMEM medium for 24 hr, changing the culture solution, setting blank group (adding PBS buffer solution without light), control group (adding PBS buffer solution), and experimental group (adding Bifidobacterium fermentation lysate prepared in examples 1-2 and comparative examples 1-6 respectively in an amount of 1% of the cell culture medium), culturing for 24 hr, and irradiating control group and experimental group with UVA (light dose of 5-6J/cm) 2 ) And culturing for 24 h after the illumination is finished. And adding 10 mu L of sterile CCK-8 solution into each well, continuously culturing for 2 h in a dark place, and detecting the absorbance value of 450 nm by using an enzyme-labeling instrument. Cell proliferation rate = experimental group OD value or control group OD value/blank group OD value. The results of cell proliferation rate measurements are shown in Table 1.
TABLE 1 cell proliferation Rate test data
Note: indicates the presence of a protein in the control group,P＜0.05。
as shown by the data in table 1, examples 1-2, comparative example 3, comparative example 5, and comparative example 6 all showed significant differences compared to the control. The 1% bifidobacterium fermentation lysate prepared in examples 1-2 is non-cytotoxic, can reduce photodamage, and can promote human skin fibroblast proliferation.
(II) cellular Reactive Oxygen Species (ROS) inhibition assay
Human skin keratinocytes (Hacat) were digested to prepare a cell suspension, which was then inoculated into a 96-well plate containing 10000-. Culturing in DMEM medium for 24 hr, changing the culture solution, setting blank group (adding PBS buffer solution without light), control group (adding PBS buffer solution), and experimental group (adding Bifidobacterium fermentation lysate prepared in examples 1-2 and comparative examples 1-6 respectively in an amount of 1% of the cell culture medium), culturing for 24 hr, and irradiating control group and experimental group with UVA (light dose of 6-10J/cm) 2 ). Removing the cell culture solution after the illumination is finished, washing the cells for 3 times by PBS, adding 100 muL of 10 mumol/L DCFH-DA into each hole, and placing the cells in CO 2 Incubate in incubator for 20 min. After incubation, PBS is washed for 3 times, serum-free DMEM culture solution is added again, the excitation wavelength is 488nm, the emission wavelength is 525nm, and the ROS content is detected by using a fluorescence microplate reader. Relative level of cellular Reactive Oxygen Species (ROS) = experimental OD value or control OD value/blank OD value. The results are shown in table 2.
TABLE 2 relative cellular Reactive Oxygen Species (ROS) level assay data
Note: indicates the presence of a protein in the control group,P＜0.05。
as shown by the data in table 2, examples 1-2, comparative examples 1-3, comparative example 5, and comparative example 6 all showed significant differences compared to the control. The results of examples 1-2, in which 1% of the bifidobacterium fermentation lysates were lower than those of the control group, indicate antioxidant properties and reduced photodamage.
(III) DPPH radical scavenging Rate test
The bifidobacterium fermentation lysates (concentration: 1%) obtained in examples 1-2 and comparative examples 1-6 were centrifuged, and the supernatants were collected as samples to be tested. Setting blank group (without adding sample to be tested), control group (without adding DPPH), experimental group (with adding sample to be tested and DPPH, 0.1mM DPPH adding amount is 60% of final volume), standing in dark at room temperature for 30min, centrifuging at 4000rpm for 5min, and testing light absorption value at 517 nm. DPPH radical clearance (%) = (1- (experimental OD value-control OD value)/blank OD value) × 100%. The results are shown in Table 3.
TABLE 3 DPPH radical scavenging Rate test data
Note: the delta is shown in comparison with example 1,P＜0.05。
as shown by the data in table 3, the experimental groups all have radical scavenging ability; comparative example 1, comparative example 2, comparative example 3 and comparative example 5 all showed significant differences compared to example 1.
(IV) test for preventing UV-induced immunosuppression (il-10)
Human skin keratinocytes (Hacat) were digested to prepare a cell suspension, which was then inoculated into a 96-well plate containing 10000-. Culturing in DMEM medium for 24 hr, changing the culture solution, setting blank group (adding PBS buffer solution without light), control group (adding PBS buffer solution), and experimental group (adding Bifidobacterium fermentation lysate prepared in examples 1-2 and comparative examples 1-6 respectively in an amount of 1% of the cell culture medium), culturing for 24 hr, and irradiating control group and experimental group with UVB light (light dose of 60-120 mJ/cm) 2 ) Culturing for 6 h after illumination is finished, and recovering cell supernatant for detecting il-10 content (combined immune aspiration)Absorbance at 450nm by the attached method). il-10 relative level = experimental OD value or control OD value/blank OD value. The results are shown in Table 4.
TABLE 4 cytokine iL-10 proliferation Rate test data
Note: denotes the comparison with the control group,P＜0.05。
the data in Table 4 show that example 1 and example 2 show significant differences compared to the control group, indicating that the 1% Bifidobacterium fermentation lysate prepared by the present invention can significantly inhibit the release of cytokine il-10, prevent the inhibition of il-12, and promote the repair of photodamaged DNA.
(V) anti-aging (beta-galactosidase) test
Human skin fibroblasts (HDF) were digested to prepare a cell suspension, which was then inoculated into a 96-well plate at 10000-50000 cells per well. Culturing in DMEM medium for 24 hr, changing the culture solution, setting blank group (adding PBS buffer solution without light), control group (adding PBS buffer solution), and experimental group (adding Bifidobacterium fermentation lysate prepared in examples 1-2 and comparative examples 1-6 respectively in an amount of 1% of the cell culture medium), culturing for 24 hr, and irradiating control group and experimental group with UVA light (light dose of 15-20J/cm) 2 ) After the illumination is finished, beta-galactosidase is added for staining and fixing for 15 minutes, PBS is used for washing, staining solution is added for incubation overnight at 37 ℃, and microscopic counting is carried out (counting 1000 cells). Senescence rate (%) = number of senescent cells/number of statistical cells × 100%. The results are shown in FIG. 1 and Table 5.
TABLE 5 aging Rate test data
Note: indicates the presence of a protein in the control group,P＜0.05。
as can be seen from the results of fig. 1 and table 5, the aging rate after treatment of 1% bifidobacterium fermentation lysate prepared in examples 1 and 2 was significantly decreased, and it was statistically significant compared to the control group, indicating that it had anti-aging efficacy.
(VI) avoidance of DNA Damage (micronucleus) test
Human skin fibroblasts (HDF) were digested to prepare a cell suspension, which was then seeded into cell-slide wells of 6-well plates with 36000-180000 cells per well. Culturing in DMEM medium for 24 hr, changing the culture solution, setting blank group (adding PBS buffer solution without light), control group (adding PBS buffer solution), and experimental group (adding Bifidobacterium fermentation lysate prepared in examples 1-2 and comparative examples 1-6 respectively in an amount of 1% of the cell culture medium), culturing for 24 hr, and irradiating control group and experimental group with UVA light (light dose of 15-20J/cm) 2 ) And culturing for 24 h after the illumination is finished. Cells were stained with giemsa staining solution and counted under a microscope (1000 cells counted). Micronucleus formation rate (%) = micronucleus cell number/statistical cell number × 100%. The results are shown in fig. 2 and table 6.
TABLE 6 micronucleus formation rate test data
Note: denotes the comparison with the control group,P＜0.05。
fig. 2 and table 6 results show that the microkernel formation rate of the 1% bifidobacterium fermentation lysate prepared by adding examples 1-2 is reduced, which is significantly lower than the control level, indicating that it has the efficacy of avoiding DNA damage.
(VII) wrinkle removing and fine line leveling effects (volunteers)
Test object(s): 60 female volunteers of 35-50 years old were recruited. The facial fine lines of the testee are mild or moderate, the skin is slack or mild or moderate, the skin lacks elasticity, the tests can be well matched, and the life regularity is kept during the product trial period; all the contents of the informed consent can be read and understood and signed voluntarily; during the test period, other skin care products were discontinued, no further clinical trials were involved in any other research center, and no use of any cosmetics, drugs and nutraceuticals affecting the outcome was agreed to.
The testing process comprises the following steps: after cleansing the skin of the volunteer, 1mL of the lysate of the bifidobacterium fermentation product prepared by any one of the preparation methods of examples 1-6 was applied to the facial skin once in the morning and once in the evening. Before, 4 weeks and 8 weeks of use, subjects washed their faces and rested for 30 min at a temperature of 21 ± 1 ℃ and a relative humidity of 50% ± 5% RH, data were collected using the clinical facial skin image analysis system VISIA, and skin wrinkles were detected.
Test results and evaluation: after 8 weeks of using the product, the number of face wrinkles of the subject is reduced by 21-29%, and the number of fine lines is reduced by 33-40%. The data show a significant decrease in both the number of facial wrinkles and the number of fine lines. The comprehensive analysis result shows that the product has obvious effect on removing wrinkles and fine lines.
Finally, the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting, and other modifications or equivalent substitutions made by the technical solutions of the present invention by those of ordinary skill in the art should be covered within the scope of the claims of the present invention as long as they do not depart from the spirit and scope of the technical solutions of the present invention.
Claims (8)
1. A method for producing a bifidobacterium fermentation lysate, comprising: bifidobacterium longum, Bifidobacterium animalis and Bifidobacterium adolescentis are taken as strains, and a bifidobacterium fermentation lysate is obtained by primary amplification, secondary amplification, micro-aerobic fermentation, centrifugation, lysate mother liquor preparation and mixed preparation; wherein:
the conditions of the primary amplification are as follows: the pH value is 6.0-7.0, the culture temperature is 37 ℃, and the culture time is 24-48 hours;
the conditions of the secondary amplification are as follows: the inoculation amount of the primary seed liquid is 1% -2%, the pH value is 6.0-7.0, the culture temperature is 37 ℃, the culture time is 2-3 days, the dissolved oxygen is 0-5%, the obtained bacteria are resuspended by using a TPY liquid culture medium, and the volume ratio of the bacteria to the TPY liquid culture medium is 1: (50-100);
The micro-aerobic fermentation is carried out by transferring the secondary seed liquid into a fermentation culture medium, and the conditions of the micro-aerobic fermentation are as follows: the inoculation amount of the secondary seed liquid is 1-3%, the initial absorbance at 600nm is 0.1-0.2, the temperature is 37 ℃, the pH value is 6.0-7.0, the amplification time is 2-3 days, and the dissolved oxygen is not more than 5%; the medium for the micro-aerobic fermentation comprises the following components: 4-6 g/L of glucose, 8-12 g/L of acid hydrolyzed casein, 400-600 mL/L of characteristic extracting solution, 0.1-0.3 g/L of amino acid, 1-3 g/L of dipotassium phosphate, 0.4-0.6 g/L of magnesium chloride, 0.1-0.2/L of calcium chloride, 0.04-0.06 g/L of manganese sulfate, 4-6 g/L of sodium acetate and 0.4-0.6 g/L of cysteine; the characteristic extracting solution is an angelica dahurica extracting solution, and the amino acid is histidine;
the centrifugation is to carry out centrifugal separation on fermentation liquor obtained by micro-aerobic fermentation to obtain filtrate and thalli;
resuspending the thallus, and performing enzymolysis by using protease in a warm water bath to obtain lysate mother liquor; the protease is a combination of lysozyme, neutral protease, chymotrypsin and flavourzyme, and the using amount of the lysozyme, the neutral protease, the chymotrypsin and the flavourzyme is 0.1-0.3% of the mass of the lysate mother liquor;
the mixing preparation is that the volume ratio of the filtrate to the lysate mother liquor is (1-10): 1, mixing.
2. A method for producing a bifidobacterium fermentation lysate, characterized in that: bifidobacterium longum, Bifidobacterium animalis and Bifidobacterium adolescentis are taken as strains, and a bifidobacterium fermentation lysate is obtained by primary amplification, secondary amplification, micro-aerobic fermentation, centrifugation, lysate mother liquor preparation and mixed preparation; wherein:
the conditions of the first-stage amplification are as follows: the pH value is 6.0-7.0, the culture temperature is 37 ℃, and the culture time is 24-48 hours;
the conditions of the secondary amplification are as follows: the inoculation amount of the primary seed liquid is 1% -2%, the pH value is 6.0-7.0, the culture temperature is 37 ℃, the culture time is 2-3 days, the dissolved oxygen is 0-5%, the obtained bacteria are resuspended by using a TPY liquid culture medium, and the volume ratio of the bacteria to the TPY liquid culture medium is 1: (50-100);
the micro-aerobic fermentation is carried out by transferring the secondary seed liquid into a fermentation culture medium, and the conditions of the micro-aerobic fermentation are as follows: the inoculation amount of the secondary seed liquid is 1-3%, the initial absorbance at 600nm is 0.1-0.2, the temperature is 37 ℃, the pH value is 6.0-7.0, the amplification time is 2-3 days, and the dissolved oxygen is not more than 5%; the medium for micro-aerobic fermentation comprises the following components: 4-6 g/L of glucose, 8-12 g/L of acid hydrolyzed casein, 400-600 mL/L of characteristic extracting solution, 0.1-0.3 g/L of amino acid, 1-3 g/L of dipotassium phosphate, 0.4-0.6 g/L of magnesium chloride, 0.1-0.2/L of calcium chloride, 0.04-0.06 g/L of manganese sulfate, 4-6 g/L of sodium acetate and 0.4-0.6 g/L of cysteine; the characteristic extracting solution is rose extracting solution, and the amino acid is proline;
The centrifugation is to carry out centrifugal separation on fermentation liquor obtained by micro-aerobic fermentation to obtain filtrate and thalli;
resuspending the thallus, and performing enzymolysis in a protease warm water bath to obtain lysate mother liquor; the protease is a combination of lysozyme, neutral protease, chymotrypsin and flavourzyme, and the using amount of the lysozyme, the neutral protease, the chymotrypsin and the flavourzyme is 0.1-0.3% of the mass of the lysate mother liquor;
the mixing preparation is that the volume ratio of the filtrate to the lysate mother liquor is (1-10): 1, mixing.
3. A method of producing a bifidobacterium fermentation lysate as claimed in claim 1 or 2 comprising: the first-stage amplification is to inoculate bifidobacterium longum, bifidobacterium animalis and bifidobacterium adolescentis in a sterile triangular flask respectively, and carry out anaerobic culture to prepare a first-stage seed solution; and the second-stage amplification is to mix and inoculate the first-stage seed liquid into a fermentation tank, separate thalli after amplification culture, and resuspend to obtain a second-stage seed liquid.
4. A method of producing a bifidobacterium fermentation lysate as claimed in claim 3 wherein: the enzyme activity of the lysozyme is 20000U/mg; the enzyme activity of the neutral protease is 50U/mg; the enzyme activity of the chymotrypsin is 800U/mg; the enzyme activity of the flavourzyme is 20U/mg.
5. A method of producing a Bifidobacterium fermentation lysate as claimed in claim 4 comprising: the conditions of the warm water bath are as follows: the temperature is 37-55 ℃, and the time is 16-24 hours.
6. A method of producing a Bifidobacterium fermentation lysate as claimed in claim 5 comprising: the lysate mother liquor preparation comprises the step of carrying out enzyme deactivation treatment on the lysate mother liquor after enzymolysis, wherein the temperature of the enzyme deactivation treatment is 90-95 ℃, and the time is 10-12 minutes.
7. Use of the fermentation lysate of Bifidobacterium produced by the method according to claim 1, 2, 4, 5 or 6 for preparing a skin care product with anti-wrinkle, firming, repairing, sunscreen or anti-aging effects, wherein the fermentation lysate of Bifidobacterium is used as a raw material for preparing the skin care product.
8. The use of the bifidobacterium fermentation lysate prepared by the preparation method according to claim 3 in the preparation of skin care products, wherein the skin care products are anti-wrinkle, firming, repairing, sun-screening or anti-aging skin care products, and the bifidobacterium fermentation lysate is used as a preparation raw material of the skin care products.
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| CN116590169B (en) * | 2022-12-15 | 2024-01-30 | 东莞华工创为生物科技有限公司 | Bifidobacterium longum and application thereof |
| CN115725475B (en) * | 2022-12-15 | 2023-08-15 | 广州悦荟化妆品有限公司 | Symbiotic fermentation two-split yeast lysate with effects of tightening, resisting wrinkles, resisting oxidization and repairing and preparation method thereof |
| CN116350563B (en) * | 2023-05-12 | 2023-08-04 | 广东迪美新材料科技有限公司 | Plant co-fermentation composition and preparation method and application thereof |
| CN117363662B (en) * | 2023-10-10 | 2024-05-03 | 广州市英朴司生物科技有限公司 | Saccharomyces cerevisiae fermentation product, preparation method and application thereof |
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