A kind of bifid yeast fermentation product rich in ergothioneine and its preparation method and application

Abstract

The present application relates to the field of fermentation technology, and more particularly to a kind of bifid yeast fermentation product rich in ergothioneine and its preparation method and application, the present application is rich in the preparation method of the bifid yeast fermentation product of ergothioneine, comprising the following steps: the seed liquid of bifidobacterium and the seed liquid of Corynebacterium glutamicum are inoculated in a fermentation medium, fermentation culture, and crude fermentation liquid is obtained; the crude fermentation liquid is cracked, sterilized and filtered to obtain the bifid yeast fermentation product rich in ergothioneine. The bifid yeast fermentation product rich in ergothioneine obtained by the preparation method of the present application has good free radical scavenging (antioxidant), firming anti-wrinkle and moisturizing effects, and is suitable for application in the preparation of cosmetics.

Description

Technical Field

[0001] The present application relates to the field of fermentation technology, and in particular to a bifid yeast fermentation product rich in ergothioneine, and a preparation method and application thereof. Background Art

[0002] The fermentation products of bifid yeast are metabolites obtained after the cultivation, inactivation or decomposition of bifidobacteria. The metabolites of bifid yeast are rich in vitamins, minerals, amino acids, polysaccharide complexes and other ingredients. The fermentation products of bifid yeast are rich in various nutrients and are now widely used as cosmetic raw materials in functional skin care products.

[0003] Existing studies have shown that the fermentation products of bifid yeast have good effects of tightening the skin and reducing wrinkles. Although the fermentation products of bifid yeast have a certain effect of scavenging free radicals, their antioxidant effect is not significant; and because the fermentation products of bifid yeast are rich in various nutrients, if their antioxidant effect is poor, they are prone to corruption and deterioration when used as cosmetic raw materials; therefore, before application, it is often necessary to add a large amount of antioxidants or preservatives to the fermentation products of bifid yeast to preserve the quality. The use of a large amount of antioxidants or preservatives not only increases the production cost, but also easily increases the skin irritation of the fermentation products of bifid yeast when applied in cosmetics, and easily causes the stability of the cosmetic system to decrease during the application process, which is not conducive to actual storage and application. Summary of the invention

[0004] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and provide a kind of bifid yeast fermentation product rich in ergothioneine and its preparation method and application. The bifid yeast fermentation product rich in ergothioneine obtained by the preparation method of the present application has good free radical scavenging (antioxidant), firming anti-wrinkle and moisturizing effects, and is suitable for application in preparing cosmetics.

[0005] To achieve the above purpose, the technical solution adopted by this application is:

[0006] The present application provides a method for preparing a bifid yeast fermentation product rich in ergothioneine, comprising the following steps:

[0007] (1) inoculating a seed solution of Bifidobacterium and a seed solution of Corynebacterium glutamicum into a fermentation medium, and fermenting and culturing the medium to obtain a crude fermentation solution;

[0008] (2) cracking, sterilizing and filtering the crude fermentation broth to obtain a bifid yeast fermentation product rich in ergothioneine.

[0009] Ergothioneine, also known as trimethylol histidine, is a natural antioxidant that can remove active free radicals in cells and protect cells from ultraviolet radiation. When ergothioneine is added to cosmetics as an additive, it can slow down skin aging.

[0010] The application adopts the mode of mixed bacteria fermentation to prepare bifid yeast fermentation product, and the strain of mixed bacteria fermentation, in addition to the bifidobacterium of conventional preparation of bifid yeast fermentation product, also adds the Corynebacterium glutamicum that can specifically produce thioneine of ergot and ferments.In the present application, the applicant is accidentally found through a large number of experiments, and Corynebacterium glutamicum can survive, breed and metabolize with bifidobacterium in fermentation medium, and the fermented liquid obtained is rich in the nutrients of thioneine and bifid yeast, there is good scavenging free radical (antioxidant), firming anti-wrinkle and moisturizing effect, it is suitable for application in preparing cosmetics.

[0011] As a preferred embodiment of the method for preparing the ergothioneine-rich bifid yeast fermentation product of the present application, the bifidobacterium includes Bifidobacterium longum subspecies infantis and / or Bifidobacterium adolescentis.

[0012] As a preferred embodiment of the method for preparing the ergothioneine-rich bifid yeast fermentation product described in the present application, the deposit number of the Corynebacterium glutamicum is CICC20910.

[0013] The present application comprises a mixed fermentation of bifidobacteria and Corynebacterium glutamicum which can produce ergothioneine, and the fermentation product contains both the nutrients of bifidobacteria and ergothioneine, and is a bifid yeast fermentation product containing ergothioneine.

[0014] The present application conducts a large number of screenings on the fermented bacteria, and finds that bifidobacteria (Bifidobacterium longum infantis subspecies, Bifidobacterium adolescentis) can be mixed with Corynebacterium glutamicum for fermentation, and the two bacteria can co-survive in the fermentation medium, making the final product rich in ergothioneine, amino acids and other nutrients.

[0015] The present application adopts a mixed bacteria fermentation process for fermentation, which is simple in process and avoids multiple inputs of bacteria for fermentation, and avoids multiple inoculation and sterilization steps, which is beneficial to reducing production costs and shortening process time.

[0016] As a preferred embodiment of the method for preparing the ergothioneine-rich bifid yeast fermentation product described in the present application, the total inoculation amount of the seed liquid of bifidobacterium and the seed liquid of Corynebacterium glutamicum is 5-8%; the inoculation volume ratio of the seed liquid of bifidobacterium and the seed liquid of Corynebacterium glutamicum is (2-4):1.

[0017] In some specific embodiments, the method for preparing the bifidobacterium seed solution is: inoculating bifidobacteria into a seed culture medium A for cultivation to obtain the bifidobacterium seed solution.

[0018] More preferably, the culture conditions of the bifidobacterium in the seed culture medium A are: culture temperature of 33-40° C., culture time of 36-60 h, and culture method of static culture.

[0019] More preferably, the seed culture medium A is at least one of MRS liquid culture medium, BBL liquid culture medium, and PYG liquid culture medium.

[0020] In some embodiments, the method for preparing the seed liquid of Corynebacterium glutamicum is: inoculating Corynebacterium glutamicum into seed culture medium B for cultivation to obtain the seed liquid of Corynebacterium glutamicum.

[0021] More preferably, the culture conditions of the Corynebacterium glutamicum in the seed culture medium B are: culture temperature of 28-33° C., culture time of 8-16 h, and fermentation shake flask rotation speed of 150-220 rpm.

[0022] More preferably, the seed culture medium B comprises 20-30 g / L of glucose, 3-8 g / L of ammonium sulfate, 20-30 g / L of corn steep liquor, 0.7-1.3 g / L of potassium dihydrogen phosphate, 0.3-0.8 g / L of magnesium sulfate heptahydrate, 8-13 g / L of calcium carbonate and the balance water.

[0023] As a preferred embodiment of the method for preparing the ergothioneine-rich bifid yeast fermentation product described in the present application, the fermentation medium comprises a carbon source, a nitrogen source, an inorganic salt and water;

[0024] The nitrogen source is at least one of glucose, sucrose and fructose syrup; the nitrogen source is at least one of soy peptone, tryptone and corn steep liquor; the inorganic salt is sulfate and / or phosphate.

[0025] As a preferred embodiment of the method for preparing the ergothioneine-rich bifid yeast fermentation product described in the present application, in terms of mass percentage, the amount of the carbon source added is 5-8%, the amount of the nitrogen source added is 0.8-1.2%, the amount of the inorganic salt added is 0.03-0.08%, and water is supplemented to 100% of the total mass of the fermentation medium.

[0026] As a preferred embodiment of the method for preparing the ergothioneine-rich bifid yeast fermentation product described in the present application, the fermentation culture in step (1) includes a first fermentation and a second fermentation;

[0027] The conditions of the first fermentation are: the first fermentation temperature is 34-40° C., the first fermentation time is 45-65 hours, and the first fermentation method is static fermentation;

[0028] The conditions of the second fermentation are as follows: the temperature of the second fermentation is 28-33° C., the time of the second fermentation is 60-80 hours, and the amount of sterile air introduced is 1.0-1.5 vvm.

[0029] The present application adopts a two-stage fermentation process and optimizes the fermentation process to maximize the nutrients in the fermentation product. The obtained bifid yeast fermentation product rich in ergothioneine has excellent antioxidant, firming, anti-wrinkle and moisturizing effects.

[0030] As a preferred embodiment of the method for preparing the ergothioneine-rich bifid yeast fermentation product described in the present application, the cleavage method in the step (2) is high-pressure homogenization treatment, the pressure of the high-pressure homogenization is 5-9MPa, and the time of the high-pressure homogenization treatment is 15-45min.

[0031] In some specific embodiments, the sterilization temperature in step (2) is 80-95° C., and the sterilization time is 15-30 min.

[0032] In some specific embodiments, the filtering method in step (2) is diatom filter filtering.

[0033] The present application provides a bifid yeast fermentation product rich in ergothioneine prepared by the above preparation method.

[0034] The present application provides the use of the above-mentioned bifid yeast fermentation product rich in ergothioneine in the preparation of cosmetics.

[0035] Preferably, the amount of the ergothioneine-rich bifid yeast fermentation product added to the cosmetic is 0.1-30%.

[0036] The bifid yeast fermentation product of the present application is rich in ergothioneine, which is an excellent antioxidant that can improve the stability of the product and avoid product corruption while ensuring the firming, anti-wrinkle and moisturizing effects of the bifid yeast fermentation product, thus affecting its application in cosmetics.

[0037] Compared with the prior art, this application has the following beneficial effects:

[0038] The present application provides a kind of bifid yeast fermentation product rich in thioneine and its preparation method and application, the present application prepares the nutrients containing bifidobacterium by mixed bacteria fermentation of bifidobacterium and Corynebacterium glutamicum that can produce thioneine, and the bifid yeast fermentation product rich in thioneine, the present application adopts the technology of mixed bacteria fermentation to ferment, and technology is simple, avoids repeatedly putting into bacterial strain fermentation, avoids the step of multiple inoculation and sterilization, is conducive to reducing production cost and shortening process time. The present application adopts the technology of two-stage fermentation, and the fermentation process is optimized so that the nutrients in the fermentation product are maximized, and the bifid yeast fermentation product rich in thioneine obtained has good anti-oxidation, firming anti-wrinkle and moisturizing effect, and is suitable for application in preparing cosmetics. DETAILED DESCRIPTION

[0039] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with specific embodiments.

[0040] In the following examples and comparative examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0041] The Corynebacterium glutamicum (CICC20910) used in this application is from Southwest University, and the Bifidobacterium longum subsp. infantis (BNCC371779), Bifidobacterium adolescentis (BNCC186535), Bifidobacterium longum (BNCC341605), and Bifidobacterium breve (BNCC185972) used in this application are from Shangcheng Beina Chuanglian Biotechnology Co., Ltd.

[0042] The MRS liquid culture medium, BBL liquid culture medium and PYG liquid culture medium mentioned in this application are commonly used culture media in the prior art, and their formulas are as follows:

[0043] MRS liquid culture medium: 10 g / L protein, 5 g / L beef powder, 4 g / L yeast powder, 2 g / L glucose, 1 mL / L Tween 80, 2 g / L potassium dihydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate and the balance water.

[0044] BBL liquid culture medium: 15g / L protein, 20g / L glucose, 2g / L yeast extract powder, 0.5g / L soluble starch, 5g / L sodium chloride, 0.5g / L cysteine, 5g / L tomato extract powder, 2g / L liver extract powder, 1g / L Tween 80 and the balance water.

[0045] PYG liquid culture medium: 20 g / L peptone, 5 g / L glucose, 10 g / L yeast powder, 0.5 g / L cysteine, 0.008 g / L calcium chloride, 0.008 g / L magnesium sulfate, 0.04 g / L dipotassium hydrogen phosphate, 0.04 g / L potassium dihydrogen phosphate, 0.4 g / L sodium bicarbonate, 0.08 g / L sodium chloride and balance water.

[0046] The preparation methods of the fermentation medium and the seed medium used in the present application are: uniformly mix the raw materials in the formula, sterilize at 121° C. for 20 minutes, and cool for use.

[0047] The formula of the fish embryo culture medium used in the present application is: 294 mg / L anhydrous calcium chloride, 123 mg / L magnesium sulfate heptahydrate, 63 mg / L sodium bicarbonate, 5.5 mg / L potassium chloride and the balance water.

[0048] Embodiment 1, a kind of bifid yeast fermentation product rich in ergothioneine and preparation method thereof

[0049] The preparation method of the bifid yeast fermentation product rich in ergothioneine of the present embodiment is as follows:

[0050] (1) Inoculate a total inoculum of 6% of the seed liquid of Bifidobacterium longum subsp. infantis and the seed liquid of Corynebacterium glutamicum into a fermentation medium (the inoculum volume ratio of the seed liquid of Bifidobacterium longum subsp. infantis to the seed liquid of Corynebacterium glutamicum is 3:1), and ferment at 37° C. for 55 h, and then ferment at 30° C. with a sterile air supply of 1.2 vvm for 70 h to obtain a crude fermentation liquid.

[0051] (2) The crude fermentation broth was homogenized at 7 MPa for 30 min under high pressure, and then sterilized at 90° C. for 20 min, and filtered through a diatom filter to obtain a bifid yeast fermentation product rich in ergothioneine.

[0052] The formula of the fermentation medium is, by mass percentage, 6% glucose, 1% soy peptone, 0.03% potassium dihydrogen phosphate, 0.02% ammonium sulfate and the balance water.

[0053] The preparation method of the seed liquid of Bifidobacterium longum subsp. infantis is as follows: inoculating the colonies of Bifidobacterium longum subsp. infantis into MRS liquid culture medium, and culturing the culture medium at 37°C for 48 hours to obtain the seed liquid of Bifidobacterium longum subsp. infantis.

[0054] The preparation method of the seed liquid of Corynebacterium glutamicum is as follows: inoculating the colony of Corynebacterium glutamicum into a seed culture medium B (the formula of the seed culture medium B is 25 g / L glucose, 5 g / L ammonium sulfate, 25 g / L corn steep liquor, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 10 g / L calcium carbonate and the balance water), culturing for 12 hours at 30° C. and a fermentation shake flask speed of 200 rpm to obtain the seed liquid of Corynebacterium glutamicum.

[0055] Embodiment 2, a kind of bifid yeast fermentation product containing ergothioneine and preparation method thereof

[0056] The preparation method of the bifid yeast fermentation product rich in ergothioneine of the present embodiment is as follows:

[0057] (1) The seed liquid of Bifidobacterium adolescentis and the seed liquid of Corynebacterium glutamicum were inoculated into the fermentation medium at a total inoculation amount of 6% (the inoculation volume ratio of the seed liquid of Bifidobacterium adolescentis and the seed liquid of Corynebacterium glutamicum was 3:1), and the mixture was allowed to ferment at 37°C for 55 h, and then fermented at 30°C with a sterile air supply of 1.2 vvm for 70 h to obtain a crude fermentation liquid.

[0058] (2) The crude fermentation broth was homogenized at 7 MPa for 30 min under high pressure, and then sterilized at 90° C. for 20 min, and filtered through a diatom filter to obtain a bifid yeast fermentation product rich in ergothioneine.

[0059] Wherein, the formula of the fermentation medium and the method for preparing the seed solution of Corynebacterium glutamicum are the same as those in Example 1.

[0060] The preparation method of the seed liquid of Bifidobacterium adolescentis is as follows: inoculate the colonies of Bifidobacterium adolescentis into MRS liquid culture medium, and culture the culture medium at 37°C for 48 hours to obtain the seed liquid of Bifidobacterium longum subspecies infantis.

[0061] Example 3: A bifid yeast fermentation product rich in ergothioneine and a method for preparing the same

[0062] The preparation method of the bifid yeast fermentation product rich in ergothioneine of the present embodiment is as follows:

[0063] (1) Inoculate a total inoculum of 6% of the seed liquid of Bifidobacterium longum subsp. infantis and the seed liquid of Corynebacterium glutamicum into a fermentation medium (the inoculum volume ratio of the seed liquid of Bifidobacterium longum subsp. infantis to the seed liquid of Corynebacterium glutamicum is 2:1), ferment at 37° C. for 55 h, and then ferment at 30° C. with a sterile air supply of 1.2 vvm for 70 h to obtain a crude fermentation liquid.

[0064] (2) The crude fermentation broth was homogenized at 7 MPa for 30 min under high pressure, and then sterilized at 90° C. for 20 min, and filtered through a diatom filter to obtain a bifid yeast fermentation product rich in ergothioneine.

[0065] Wherein, the formula of the fermentation medium, the preparation method of the seed solution of Bifidobacterium longum subsp. infantis, and the preparation method of the seed solution of Corynebacterium glutamicum are the same as those in Example 1.

[0066] Embodiment 4, a kind of bifid yeast fermentation product containing ergothioneine and preparation method thereof

[0067] The preparation method of the bifid yeast fermentation product rich in ergothioneine of the present embodiment is as follows:

[0068] (1) Inoculate a total inoculum of 6% of the seed liquid of Bifidobacterium longum subsp. infantis and the seed liquid of Corynebacterium glutamicum into a fermentation medium (the inoculum volume ratio of the seed liquid of Bifidobacterium longum subsp. infantis to the seed liquid of Corynebacterium glutamicum is 4:1), let it stand and ferment for 55 h at 37° C., and then ferment for 70 h at 30° C. with a sterile air supply of 1.2 vvm to obtain a crude fermentation liquid.

[0069] (2) The crude fermentation broth was homogenized at 7 MPa for 30 min under high pressure, and then sterilized at 90° C. for 20 min, and filtered through a diatom filter to obtain a bifid yeast fermentation product rich in ergothioneine.

[0070] Wherein, the formula of the fermentation medium, the preparation method of the seed solution of Bifidobacterium longum subsp. infantis, and the preparation method of the seed solution of Corynebacterium glutamicum are the same as those in Example 1.

[0071] Example 5: A bifid yeast fermentation product rich in ergothioneine and a preparation method thereof

[0072] The preparation method of the bifid yeast fermentation product rich in ergothioneine of the present embodiment is as follows:

[0073] (1) Inoculate a total inoculum of 5% of the seed liquid of Bifidobacterium longum subsp. infantis and the seed liquid of Corynebacterium glutamicum into a fermentation medium (the inoculum volume ratio of the seed liquid of Bifidobacterium longum subsp. infantis to the seed liquid of Corynebacterium glutamicum is 2.5:1), and ferment at 34° C. for 45 h, and then ferment at 33° C. with a sterile air supply of 1.0 vvm for 80 h to obtain a crude fermentation liquid.

[0074] (2) The crude fermentation broth was homogenized under high pressure at 5 MPa for 45 min, and then sterilized at 95° C. for 15 min, and filtered through a diatom filter to obtain a bifid yeast fermentation product rich in ergothioneine.

[0075] The formula of the fermentation medium is, by mass percentage, 8% sucrose, 1.2% corn steep liquor, 0.03% ammonium sulfate and the balance water.

[0076] The preparation method of the seed liquid of Bifidobacterium longum subsp. infantis is as follows: inoculating the colonies of Bifidobacterium longum subsp. infantis into PYG liquid culture medium, and culturing the culture medium at 40°C for 60 hours to obtain the seed liquid of Bifidobacterium longum subsp. infantis.

[0077] The preparation method of the seed liquid of Corynebacterium glutamicum is as follows: inoculating the colony of Corynebacterium glutamicum into a seed culture medium B (the formula of the seed culture medium B is 20 g / L glucose, 8 g / L ammonium sulfate, 20 g / L corn steep liquor, 1.3 g / L potassium dihydrogen phosphate, 0.3 g / L magnesium sulfate heptahydrate, 13 g / L calcium carbonate and the balance water), culturing for 8 hours at 28° C. and a fermentation shake flask speed of 220 rpm to obtain the seed liquid of Corynebacterium glutamicum.

[0078] Example 6: A bifid yeast fermentation product rich in ergothioneine and a preparation method thereof

[0079] The preparation method of the bifid yeast fermentation product rich in ergothioneine of the present embodiment is as follows:

[0080] (1) Inoculate a total inoculum amount of 8% (the inoculum volume ratio of the seed liquid of Bifidobacterium longum subsp. infantis to the seed liquid of Corynebacterium glutamicum is 3.5:1) into a fermentation medium, and ferment at 40° C. for 65 h, and then ferment at 28° C. with a sterile air supply of 1.5 vvm for 60 h to obtain a crude fermentation liquid.

[0081] (2) The crude fermentation broth was homogenized at 9 MPa for 15 min, and then sterilized at 80° C. for 30 min. After filtering through a diatom filter, a bifid yeast fermentation product rich in ergothioneine was obtained.

[0082] The formula of the fermentation medium is, in percentage by mass, 5% fructose syrup, 0.8% tryptone, 0.08% dipotassium hydrogen phosphate and the balance water.

[0083] The preparation method of the seed liquid of Bifidobacterium longum subsp. infantis is as follows: inoculating the colonies of Bifidobacterium longum subsp. infantis into a BBL liquid culture medium, and culturing the culture medium at 33° C. for 36 hours to obtain the seed liquid of Bifidobacterium longum subsp. infantis.

[0084] The preparation method of the seed liquid of Corynebacterium glutamicum is as follows: inoculating the colony of Corynebacterium glutamicum into a seed culture medium B (the formula of the seed culture medium B is 30 g / L glucose, 3 g / L ammonium sulfate, 30 g / L corn steep liquor, 0.7 g / L potassium dihydrogen phosphate, 0.8 g / L magnesium sulfate heptahydrate, 8 g / L calcium carbonate and the balance water), culturing for 16 hours at 33° C. and a fermentation shake flask speed of 150 rpm to obtain the seed liquid of Corynebacterium glutamicum.

[0085] Comparative Example 1

[0086] The preparation method of the bifid yeast fermentation product of this comparative example is as follows:

[0087] (1) Inoculate a total inoculum amount of 6% (the inoculum volume ratio of the seed liquid of Bifidobacterium longum subsp. infantis to the seed liquid of Corynebacterium glutamicum is 3:1) into a fermentation medium, ferment at 30° C. with a sterile air flow rate of 1.2 vvm for 70 h, and then ferment at 37° C. for 55 h to obtain a crude fermentation liquid.

[0088] (2) The crude fermentation broth was homogenized at 7 MPa for 30 min, sterilized at 90°C for 20 min, and filtered through a diatom filter to obtain the bifid yeast fermentation product.

[0089] Wherein, the formula of the fermentation medium, the preparation method of the seed solution of Bifidobacterium longum subsp. infantis, and the preparation method of the seed solution of Corynebacterium glutamicum are the same as those in Example 1.

[0090] Comparative Example 2

[0091] The preparation method of the bifid yeast fermentation product of this comparative example is as follows:

[0092] (1) The seed liquid of Bifidobacterium longum subsp. infantis and the seed liquid of Corynebacterium glutamicum were inoculated into a fermentation medium at a total inoculation amount of 6% (the inoculation volume ratio of the seed liquid of Bifidobacterium longum subsp. infantis to the seed liquid of Corynebacterium glutamicum was 3:1), and the fermentation was allowed to stand at 37° C. for 125 h to obtain a crude fermentation liquid.

[0093] (2) The crude fermentation broth was homogenized at 7 MPa for 30 min, sterilized at 90°C for 20 min, and filtered through a diatom filter to obtain the bifid yeast fermentation product.

[0094] Wherein, the formula of the fermentation medium, the preparation method of the seed solution of Bifidobacterium longum subsp. infantis, and the preparation method of the seed solution of Corynebacterium glutamicum are the same as those in Example 1.

[0095] Comparative Example 3

[0096] The preparation method of the bifid yeast fermentation product of this comparative example is as follows:

[0097] (1) Inoculate a total inoculum amount of 6% of the seed solution of Bifidobacterium longum subsp. infantis and the seed solution of Corynebacterium glutamicum (the inoculum volume ratio of the seed solution of Bifidobacterium longum subsp. infantis to the seed solution of Corynebacterium glutamicum is 3:1) into a fermentation medium, and ferment for 125 h at 30° C. and with a sterile air flow rate of 1.2 vvm to obtain a crude fermentation liquid.

[0098] (2) The crude fermentation broth was homogenized at 7 MPa for 30 min, sterilized at 90°C for 20 min, and filtered through a diatom filter to obtain the bifid yeast fermentation product.

[0099] Wherein, the formula of the fermentation medium, the preparation method of the seed solution of Bifidobacterium longum subsp. infantis, and the preparation method of the seed solution of Corynebacterium glutamicum are the same as those in Example 1.

[0100] Comparative Example 4

[0101] The preparation method of the bifid yeast fermentation product of this comparative example is as follows:

[0102] (1) The seed liquid of Bifidobacterium longum subsp. infantis was inoculated into the fermentation medium at an inoculum amount of 6%, and the fermentation was allowed to stand at 37° C. for 55 h, and then fermented at 30° C. with a sterile air flow rate of 1.2 vvm for 70 h to obtain a crude fermentation liquid.

[0103] (2) The crude fermentation broth was homogenized at 7 MPa for 30 min, sterilized at 90°C for 20 min, and filtered through a diatom filter to obtain the bifid yeast fermentation product.

[0104] The formula of the fermentation medium and the preparation method of the seed liquid of Bifidobacterium longum subsp. infantis are the same as those in Example 1.

[0105] Comparative Example 5

[0106] The preparation method of the bifid yeast fermentation product of this comparative example is as follows:

[0107] (1) The seed liquid of Corynebacterium glutamicum was inoculated into a fermentation medium at an inoculum amount of 6%, and the fermentation was allowed to stand at 37° C. for 55 h, and then fermented at 30° C. with a sterile air flow rate of 1.2 vvm for 70 h to obtain a crude fermentation liquid.

[0108] (2) The crude fermentation broth was homogenized at 7 MPa for 30 min, sterilized at 90°C for 20 min, and filtered through a diatom filter to obtain the bifid yeast fermentation product.

[0109] Wherein, the formula of the fermentation medium and the method for preparing the seed solution of Corynebacterium glutamicum are the same as those in Example 1.

[0110] Comparative Example 6

[0111] (1) The seed liquid of Bifidobacterium longum and the seed liquid of Corynebacterium glutamicum were inoculated into a fermentation medium with a total inoculation amount of 6% (the inoculation volume ratio of the seed liquid of Bifidobacterium longum to the seed liquid of Corynebacterium glutamicum was 3:1), and the mixture was allowed to ferment at 37° C. for 55 h, and then fermented at 30° C. with a sterile air supply of 1.2 vvm for 70 h to obtain a crude fermentation liquid.

[0112] (2) The crude fermentation broth was homogenized at 7 MPa for 30 min, sterilized at 90°C for 20 min, and filtered through a diatom filter to obtain the bifid yeast fermentation product.

[0113] Wherein, the formula of the fermentation medium and the method for preparing the seed solution of Corynebacterium glutamicum are the same as those in Example 1.

[0114] The method for preparing the seed liquid of Bifidobacterium longum is as follows: inoculating the colonies of Bifidobacterium longum into MRS liquid culture medium, and culturing the culture medium at 37° C. for 48 hours to obtain the seed liquid of Bifidobacterium longum.

[0115] Comparative Example 7

[0116] (1) The seed liquid of Bifidobacterium breve and the seed liquid of Corynebacterium glutamicum were inoculated into a fermentation medium with a total inoculation amount of 6% (the inoculation volume ratio of the seed liquid of Bifidobacterium breve to the seed liquid of Corynebacterium glutamicum was 3:1), and the fermentation was allowed to stand at 37° C. for 55 h, and then fermented at 30° C. with a sterile air supply of 1.2 vvm for 70 h to obtain a crude fermentation liquid.

[0117] (2) The crude fermentation broth was homogenized at 7 MPa for 30 min, sterilized at 90°C for 20 min, and filtered through a diatom filter to obtain the bifid yeast fermentation product.

[0118] Wherein, the formula of the fermentation medium and the method for preparing the seed solution of Corynebacterium glutamicum are the same as those in Example 1.

[0119] The method for preparing the seed liquid of Bifidobacterium breve is as follows: inoculating the colonies of Bifidobacterium breve into MRS liquid culture medium, and culturing the culture medium at 37° C. for 48 hours to obtain the seed liquid of Bifidobacterium breve.

[0120] Comparative Example 8

[0121] The preparation method of the bifid yeast fermentation product of this comparative example is as follows:

[0122] (1) Inoculate a total inoculum of 6% of the seed liquid of Bifidobacterium longum subsp. infantis and the seed liquid of Corynebacterium glutamicum into a fermentation medium (the inoculum volume ratio of the seed liquid of Bifidobacterium longum subsp. infantis to the seed liquid of Corynebacterium glutamicum is 1:1), and ferment at 37° C. for 55 h, and then ferment at 30° C. with a sterile air supply of 1.2 vvm for 70 h to obtain a crude fermentation liquid.

[0123] (2) The crude fermentation broth was homogenized at 7 MPa for 30 min, sterilized at 90°C for 20 min, and filtered through a diatom filter to obtain the bifid yeast fermentation product.

[0124] Wherein, the formula of the fermentation medium, the preparation method of the seed solution of Bifidobacterium longum subsp. infantis, and the preparation method of the seed solution of Corynebacterium glutamicum are the same as those in Example 1.

[0125] Test Example 1: Ergothioneine Content Test

[0126] The content of ergothioneine in the bifid yeast fermentation products of Examples 1-6 and Comparative Examples 1-8 was detected by high performance liquid chromatography.

[0127] Test method reference: [1] Zhao Yanmin, Lei Zhidong, Liu Chenghang, et al. Determination of ergothioneine content in various fungi by HPLC [J]. Food Research and Development, 2016. 37(2): 117-119. [2] Feng Lulu, E Hengchao, Zhang Yanmei, et al. Research progress on extraction, separation and detection methods of ergothioneine in edible fungi [J]. Journal of Edible Fungi, 2021, 28(1): 115-123.

[0128] HPLC chromatographic conditions are as follows: chromatographic column: Kromasil 100-5NH, (250mm×4.60mm, 5μm); mobile phase: acetonitrile-5mmol / acetic acid (80:20) flow elution; flow rate 1.0mL / min; detection wavelength 254nm; column temperature 30℃; injection volume 20μL.

[0129] The test results are shown in Table 1 below:

[0130] Table 1

[0131] Group Ergothioneine content (g / L) Group Ergothioneine content (g / L) Example 1 2.24 Comparative Example 1 1.36 Example 2 2.18 Comparative Example 2 0.05 Example 3 2.28 Comparative Example 3 1.18 Example 4 2.02 Comparative Example 4 0.00 Example 5 1.96 Comparative Example 5 1.62 Example 6 2.11 Comparative Example 6 1.58 / / Comparative Example 7 1.27 / / Comparative Example 8 1.85

[0132] Test result shows that the bifid yeast fermentation product of the present application embodiment all contains thioneine.Wherein, the content of thioneine in the bifid yeast fermentation product of embodiment 1-6 is higher than that of comparative example 1-8.The content of thioneine of embodiment 1-6 is higher than that of comparative example 1, indicating that the order of changing the first fermentation and the second fermentation can affect the generation amount of thioneine, in the present application, anaerobic fermentation (quiet fermentation) is first adopted, and then aerobic fermentation is adopted to ferment, and the thioneine content of the final product obtained is higher.The content of thioneine of embodiment 1-6 is higher than that of comparative example 2, indicating that only the mode of quiet fermentation is adopted to cultivate, and the growth of Corynebacterium glutamicum is suppressed, and then the content of thioneine produced by the final product is very little.The content of thioneine of embodiment 1-6 is higher than that of comparative example 3, indicating that only the mode of aerobic fermentation is adopted to ferment, and the time of aerobic fermentation is too long, and Corynebacterium glutamicum reproduces in large quantities, and the phenomenon of thalline autolysis is easily occurred in the later stage of fermentation, resulting in the content of thioneine produced by the final product being reduced. The content of thioneine of embodiment 1-6 is higher than comparative example 4,5, only adopts long bifidobacterium infant subspecies to ferment, final product cannot produce thioneine, only uses the seed liquid of Corynebacterium glutamicum to ferment, the initial input concentration of Corynebacterium glutamicum is too large, and a large amount of growth is carried out in the initial stage of fermentation, and there are too many thalli in the late fermentation period, and thalli is easily autolyzed, resulting in the reduction of the thioneine content produced in the final product. The content of thioneine of embodiment 1-6 is higher than comparative example 6,7, illustrating that in the present application, long bifidobacterium infant subspecies and adolescent bifidobacterium are co-fermented with Corynebacterium glutamicum, and the thioneine content of the final product obtained is higher, and is replaced by other bifidobacteria, and the thioneine content produced in the final product is lower. The content of thioneine of embodiment 1-6 is higher than comparative example 8, illustrating that the inoculation ratio of bifidobacterium and Corynebacterium glutamicum affects the content of thioneine in the final product, and in the present application, the inoculation volume ratio of the seed liquid of bifidobacterium and the seed liquid of Corynebacterium glutamicum is preferably (2-4): 1.

[0133] Test Example 2: Stability Test

[0134] Test samples: the bifid yeast fermentation products of Examples 1-6 and Comparative Examples 1-8.

[0135] Test method: 10 mL of sample was placed in a PE bottle, and each test sample was divided into two bottles. One test sample was placed at 45°C for 30 days, and the other test sample was placed in a light stabilization box at (25±2)°C for 30 days to examine the thermal stability and light stability of the test sample. The test results are shown in Table 2 below.

[0136] Table 2

[0137]

[0138]

[0139] Note: "Pass" means that the appearance of the test sample does not change.

[0140] Test result shows, the bifid yeast fermentation product of the present application embodiment has good thermal stability and light stability.In comparative example 1-8, except comparative example 5 and 8, the thermal stability and light stability of the remaining comparative examples are all poor.Comparative example 5 only adopts Corynebacterium glutamicum to ferment, and the nutrient substance of final product is less, so when thioneine content is less, final product still has good stability.The content of thioneine of the final product of comparative example 8 is higher, so final product still has higher thermal stability and light stability.

[0141] Test Example 3: Antioxidant Performance Test - ABST Free Radical Scavenging Ability Test

[0142] Test sample: The fermentation products of the bifid yeast of Examples 1-6 and Comparative Examples 1-8 were prepared into a solution with a mass concentration of 3% using deionized water.

[0143] Test method: Mix 7.4mmol / L ABTS solution with 2.6mmol / L potassium persulfate, place it in the dark for 12h, dilute it 35 times with 95% ethanol, and the absorbance at 734nm is 0.70±0.02. Add 160μL of working solution to each well. Add 40μL of sample solution of different concentrations to the sample well, mix gently, incubate at 25±3℃ for 6min, measure the absorbance at 734nm, replace the sample solution with 40μL of anhydrous ethanol as the blank group; replace the ABTS solution with anhydrous ethanol as the control group, and calculate the scavenging rate of ABTS free radicals according to the following formula.

[0144]

[0145] Among them, A b is the absorbance of the blank group; A s Absorbance of sample group; A c is the absorbance of the control group.

[0146] The scavenging effects of the test samples on ABTS free radicals are shown in Table 3.

[0147] Table 3

[0148]

[0149] Test results show that the bifid yeast fermentation product of the present application has a good antioxidant effect, and the scavenging rate of ABTS free radicals reaches 60%. Wherein, the bifid yeast fermentation product antioxidant effect of embodiment 1 is the best. The antioxidant effect of embodiment 1-6 is better than comparative example 1-3, indicating that the control of fermentation conditions has a great influence on the antioxidant effect of the final product, and the order of the first fermentation and the second fermentation is replaced, or only the first fermentation and the second fermentation are carried out. One of them, the antioxidant effect of the final product is poor. The antioxidant effect of embodiment 1-6 is better than comparative example 4, 5, indicating that only bifidobacterium or Corynebacterium glutamicum is used for fermentation, and the antioxidant effect of the final product is poor; wherein, the content of ergothioneine in comparative example 4 is 0, but comparative example 4 still shows certain antioxidant effect, indicating that ergothioneine has certain correlation with antioxidant effect, but it is not absolute connection, and the fermentation product of bifidobacterium contains antioxidant active substances, so that the final product still shows certain antioxidant performance, and comparative example 4 is only fermented with bifidobacterium, and the final product obtained still has certain antioxidant effect. The antioxidant effect of Examples 1-6 is better than that of Comparative Examples 6-8, indicating that the type of bifidobacterium and the ratio of the two have a certain influence on the antioxidant effect of the final product. In the present application, bifidobacterium is preferably Bifidobacterium longum infantis subspecies and / or Bifidobacterium adolescentis, and the inoculation volume ratio of the seed liquid of bifidobacterium and the seed liquid of Corynebacterium glutamicum is preferably (2-4):1.

[0150] Test Example 4: Firming and Anti-wrinkle Performance Test - Zebrafish Elastin Gene (Eln1) Expression Promotion Test

[0151] Test principle: Elastin in human skin is a protein that allows many tissues in the body to maintain their original shape after stretching and contraction, and allows the skin to return to its original shape after being squeezed. As we age, the elastin fibers in the skin will gradually break and lose, causing wrinkles and loss of elasticity. Compared with human skin, zebrafish skin is highly similar to human skin in structure, except that the epidermis is differentiated epidermal cells and mucus layer instead of stratum corneum. The elastin gene is highly conserved. Elastin in the human body is synthesized by the Eln gene, and the corresponding gene of this gene in zebrafish is Eln1. By testing whether the sample promotes the expression of the Eln1 gene in zebrafish, it can be judged whether the sample promotes the regeneration of elastin and achieves the effect of skin firming.

[0152] Test sample: The bifid yeast fermentation products of Examples 1-6 and Comparative Examples 1-8 were prepared into a solution with a mass concentration of 5% using fish embryo culture medium.

[0153] The test steps are as follows:

[0154] Blank control group: 36 healthy zebrafish 6 days after fertilization were randomly selected and transferred to a 24-well plate, with each well containing 12 zebrafish and 2.5 mL of fish embryo culture medium, i.e., 3 parallel experiments were conducted.

[0155] Experimental group: 36 healthy zebrafish 6 days after fertilization were randomly selected and transferred to a 24-well plate, with each well containing 12 zebrafish and 2.5 mL of the test sample solution (the operation of the experimental group was repeated for the bifid yeast fermentation products of Examples 2-6 and Comparative Examples 1-8, respectively).

[0156] Cultivation process: The blank control group and experimental group were placed in a 28°C incubator for 24 hours. Twelve zebrafish from each well were collected in a 1.5 mL test tube, the solution was removed, 0.5 mL RNAlater solution (Invitrogen; AM7020) was added, and the tube was frozen for storage.

[0157] RNA extraction process: Remove RNAlater solution from the test tube and rinse with PBS solution three times. Place in an ice bath, remove PBS solution, and add 500μL TRIzol reagent (Invitrogen; 15596018). Homogenize the embryos with a pellet pestle and place in an ice bath for 10 minutes. Add 100μL chloroform, vortex for 1 minute, and place in an ice bath for 5 minutes. Centrifuge the sample for 20 minutes, transfer the upper layer solution to a 1.5mL centrifuge tube, add 250μL isopropanol, vortex for 1 minute, place in an ice bath for 10 minutes, and centrifuge the sample for 20 minutes. Remove all supernatant, add 500μL ethanol, vortex for 1 minute, centrifuge the sample for 5 minutes, and place in an ice bath for 10 minutes. Repeat this step 3 times. Remove ethanol, centrifuge the sample for 5 minutes, and air-dry the sample in a fume hood with the lid open for 10 minutes. Add 10μL DNase / RNase-free ultrapure distilled water and heat at 55°C for 15 minutes.

[0158] cDNA synthesis process: Dilute the above RNA sample to 1000 ng / 7 μL with DNase / RNase-free ultrapure distilled water. Use PrimerScript RT kit containing gDNA Eraser (Takara; Cat no.RR047A) to synthesize RNA into cDNA and store frozen.

[0159] Real-time RT-PCR process: Prepare primer mix and real-time PCR master mix for each primer. Dilute cDNA sample 10 times with DNase / RNase-free ultrapure distilled water. Add 9 μL PCR master mix and 1 μL diluted cDNA sample to each well of 96-well PCR plate. Seal the 96-well PCR plate with optical film, centrifuge the plate for 5 minutes, and perform real-time PCR amplification. The commercially available PCR kit uses SYBR Premix Ex Taq (Takara; Cat no. RR420A).

[0160] Data and result calculation: Real-time PCR data were collected. The relative expression of each gene (Eln1) was calculated as the test result. The elastin gene expression promotion rate was calculated by the formula.

[0161]

[0162] Among them, T represents the relative expression level of zebrafish elastin gene in the experimental group; C represents the relative expression level of zebrafish elastin gene in the blank control group.

[0163] The test results are shown in Table 4 below.

[0164] Table 4

[0165]

[0166] The test results show that the bifid yeast fermentation product of the embodiment of the present application can promote the expression of the zebrafish elastin gene (Eln1), and can promote cells to produce more elastin, that is, the bifid yeast fermentation product of the present application has a good firming and anti-wrinkle effect. The firming and anti-wrinkle effect of Examples 1-6 is better than that of Comparative Examples 1-3, indicating that the control of the fermentation conditions has a great influence on the firming and anti-wrinkle effect of the final product. The order of the first fermentation and the second fermentation is replaced, or only one of the first fermentation and the second fermentation is performed, and the firming and anti-wrinkle effect of the final product is poor. The firming and anti-wrinkle effect of Examples 1-6 is better than that of Comparative Examples 4 and 5, indicating that only fermentation with Bifidobacterium or Corynebacterium glutamicum results in a poor firming and anti-wrinkle effect of the final product. The firming and anti-wrinkle effects of Examples 1-6 are better than those of Comparative Examples 6-8, indicating that the type of bifidobacterium and the ratio of the two have a certain influence on the firming and anti-wrinkle effect of the final product. In the present application, bifidobacterium is preferably Bifidobacterium longum subspecies infantis and / or Bifidobacterium adolescentis, and the inoculation volume ratio of the seed liquid of bifidobacterium to the seed liquid of Corynebacterium glutamicum is preferably (2-4):1.

[0167] Test Example 5: Moisturizing Performance Test - Aquaporin 3 (AQP3) Gene Expression Test

[0168] Test principle: Aquaporin (AQP), also known as aquaporin, is a protein (intrinsic membrane protein) located on the cell membrane. It forms "pores" on the cell membrane and can control the flow of water in and out of the cell, just like a "cell water pump". Aquaporin (AQP3) is expressed in the epidermis and basal layer. It is a natural moisturizing factor that maintains the hydration level of the interstitial and intracellular cellular tissues of the skin and may play a key role in skin hydration and moisturizing. The effect of the sample on the expression of the AQP3 gene in keratinocytes was detected through in vitro experiments to evaluate whether the sample has the effect of promoting moisturizing.

[0169] Test sample: The fermentation products of the bifid yeast of Examples 1-6 and Comparative Examples 1-8 were prepared into a solution with a mass concentration of 3% using cell culture fluid.

[0170] Test steps: HaCaT human keratinocytes were inoculated into 6-well plates (200,000 cells / well) and cultured in an incubator at 37°C for 24 hours. After the culture was completed, the original culture medium in the well was discarded, and DMEM culture medium containing 10% serum was added to the negative control group. Cell culture medium containing the test sample was added to each well of the sample group and cultured for 24 hours. After the culture was completed, the supernatant was discarded. The expression of AQP3 mRNA in HaCaT human keratinocytes was detected by qRT-PCR technology, and the relative expression was calculated based on the expression of mRNA in the negative control group. The test results were averaged. The test results are shown in Table 5 below.

[0171] Table 5

[0172]

[0173]

[0174] The test results show that the bifid yeast fermentation product of the present application embodiment can promote the expression of the moisturizing gene (AQP3), and the bifid yeast fermentation product of the present application has a good moisturizing effect. The moisturizing effect of Examples 1-6 is better than that of Comparative Examples 1-3, indicating that the control of the fermentation conditions has a great influence on the moisturizing effect of the final product. The order of the first fermentation and the second fermentation is replaced, or only the first fermentation and the second fermentation are performed. The moisturizing effect of the final product is poor. The moisturizing effect of Examples 1-6 is better than that of Comparative Examples 4 and 5, indicating that only bifidobacterium or Corynebacterium glutamicum is used for fermentation, and the moisturizing effect of the final product is poor. The moisturizing effect of Examples 1-6 is better than that of Comparative Examples 6-8, indicating that the type of bifidobacterium and the ratio of the two have a certain influence on the moisturizing effect of the final product. In the present application, bifidobacterium is preferably Bifidobacterium longum infantis subspecies and / or Bifidobacterium adolescentis, and the inoculation volume ratio of the seed liquid of bifidobacterium and the seed liquid of Corynebacterium glutamicum is preferably (2-4): 1.

[0175] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. A method for preparing a bifid yeast fermentation product rich in ergothioneine, characterized in that, The following steps are involved: (1) inoculating a seed solution of Bifidobacterium and a seed solution of Corynebacterium glutamicum into a fermentation medium, and fermenting and culturing the medium to obtain a crude fermentation solution; (2) cracking, sterilizing and filtering the crude fermentation liquid to obtain a bifid yeast fermentation product rich in thioneine; The bifidobacterium is Bifidobacterium longum subspecies infantis and / or Bifidobacterium adolescentis; The inoculation volume ratio of the bifidobacterium seed solution to the Corynebacterium glutamicum seed solution is (2-4):1; The fermentation culture in step (1) includes a first fermentation and a second fermentation; The conditions of the first fermentation are: the first fermentation temperature is 34-40° C., the first fermentation time is 45-65 hours, and the first fermentation method is static fermentation; The conditions of the second fermentation are as follows: the temperature of the second fermentation is 28-33° C., the time of the second fermentation is 60-80 hours, and the amount of sterile air introduced is 1.0-1.5 vvm.

2. The preparation method according to claim 1, characterized in that The total inoculation amount of the bifidobacterium seed solution and the Corynebacterium glutamicum seed solution is 5-8%.

3. The preparation method according to claim 1, characterized in that: The fermentation medium comprises a carbon source, a nitrogen source, an inorganic salt and water; The nitrogen source is at least one of glucose, sucrose and fructose syrup; the nitrogen source is at least one of soy peptone, tryptone and corn steep liquor; the inorganic salt is sulfate and / or phosphate.

4. The preparation method according to claim 3, characterized in that: In terms of mass percentage, the added amount of the carbon source is 5-8%, the added amount of the nitrogen source is 0.8-1.2%, the added amount of the inorganic salt is 0.03-0.08%, and water is supplemented to 100% of the total mass of the fermentation medium.

5. The preparation method according to claim 1, characterized in that: The cracking method in step (2) is high-pressure homogenization treatment, the pressure of the high-pressure homogenization is 5-9 MPa, and the time of the high-pressure homogenization treatment is 15-45 minutes.

6. the bifid yeast fermentation product rich in ergothioneine prepared by the preparation method as described in any one of claim 1-5.

7. the application of the bifid yeast fermentation product rich in ergothioneine as claimed in claim 6 in preparing cosmetics.

8. The use according to claim 7, characterized in that The addition amount of the bifid yeast fermentation product rich in ergothioneine in the cosmetics is 0.1-30%.

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