Bifidus yeast fermentation product lysate with repairing and anti-inflammatory effects as well as preparation method and application of bifidus yeast fermentation product lysate

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly discloses a bifidus yeast fermentation product lysate with repairing and anti-inflammatory effects as well as a preparation method and application of the bifidus yeast fermentation product lysate. The preparation method of the bifidobacterium fermentation product lysate comprises the following steps: (1) strain activation: inoculating bifidobacterium adolescentis into a first seed culture medium, and carrying out anaerobic culture to obtain an activated bacterial solution; (2) seed liquid culture: inoculating the activated bacterial liquid to a second seed culture medium, and continuously transferring anaerobic culture twice to obtain a third-stage seed liquid; (3) fermentation culture: inoculating the third-stage seed solution into a fermentation tank filled with a fermentation culture medium, and carrying out continuous fermentation culture for three times to obtain a fermented product; and (4) inactivation and decomposition: sterilizing the fermented product, centrifuging, washing, collecting thalli, re-suspending and breaking walls of the thalli, and adding an auxiliary agent to obtain the bifidus yeast fermentation product lysate. The bifidus yeast fermentation product lysate prepared by the preparation method disclosed by the invention is high in active ingredient content and good in stability, and has good anti-inflammatory and repairing effects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial fermentation, in particular to a bifida ferment lysate with repair and anti-inflammatory effects, a preparation method and application thereof. BACKGROUND

[0002] The bifida ferment lysate is a product obtained by fermenting bifidobacterium, inactivating and decomposing, and containing cell cytoplasm fragments, cell wall components, polysaccharide complexes and more active substances such as vitamin B, short polypeptide, amino acid and nucleotide. The bifida ferment lysate has certain anti-wrinkle, firming, repair, sunscreen, antioxidant and anti-aging effects, and can prevent damage caused by external stimuli such as ultraviolet rays and promote repair of damaged DNA. These advantages make the bifida ferment lysate widely used in skin care products and have great market prospects.

[0003] The existing bifida ferment lysate process has the following problems: low fermentation efficiency, long cycle, high energy consumption, low yield and instability of target active ingredients, and large batch differences. The present application solves the problems of low fermentation efficiency and insufficient active ingredients in the prior art. SUMMARY

[0004] In view of the above, it is necessary to provide a bifida ferment lysate with repair and anti-inflammatory effects, a preparation method and application thereof. The bifida ferment lysate prepared by the present application has high active ingredient content, good stability, good anti-inflammatory and repair effects.

[0005] To achieve the above-mentioned purposes, the technical scheme adopted by the present application is as follows:

[0006] A preparation method of a bifida ferment lysate, comprising the following steps:

[0007] (1) Strain activation: inoculate Bifidobacterium adolescentis into a first seed culture medium, and anaerobically culture to obtain an activated bacterial solution;

[0008] (2) Seed liquid culture: inoculate the activated bacterial solution into a second seed culture medium and continuously transfer and anaerobically culture twice to obtain a third-level seed liquid;

[0009] (3) Fermentation culture: inoculate the third-level seed liquid into a fermentation tank containing a fermentation culture medium, and anaerobically culture to obtain a fermentation product;

[0010] The anaerobic fermentation culture includes first anaerobic fermentation, second anaerobic fermentation and third anaerobic fermentation; the first anaerobic fermentation is carried out under the conditions of temperature 36-37℃, pH 6.0-6.2, rotation speed 100-110rpm and fermentation time 8-10h; the second anaerobic fermentation is carried out under the conditions of temperature 38.5-39℃, pH 5.8-6.0, rotation speed 110-115rpm and fermentation time 10-12h; and the third anaerobic fermentation is carried out under the conditions of temperature 35-35.5℃, pH 5.8-6.0, rotation speed 95-100rpm and fermentation time 2-4h.

[0011] (4) Inactivation and decomposition: after sterilization treatment of the fermentation product, centrifugation and washing are carried out to collect the fermentation bacteria, the fermentation bacteria are resuspended with water, wall breaking treatment is carried out, and a cell lysate is obtained, and an auxiliary agent is added into the cell lysate, so that the fermentation product of the Bifidobacterium adolescentis is obtained.

[0012] Further, in step (1), the Bifidobacterium adolescentis is Bifidobacterium adolescentis HH404-1, the classification name of the Bifidobacterium adolescentis HH404-1 is Bifidobacterium adolescentis, which is preserved in Guangdong Microbial Culture Collection Center (GDMCC) located at No. 59, Building 5, 100, Martyrs' Avenue, Guangzhou, and the preservation date is October 22, 2025, and the preservation number is GDMCC No: 67151.

[0013] Further, the first seed culture medium includes the following raw material components in mass percentage: peptone 1.0-1.2%, beef extract 0.8-1.2%, yeast powder 0.4-0.7%, glucose 1.8-2.3%, dipotassium hydrogen phosphate 0.1-0.3%, triammonium citrate 0.1-0.3%, sodium acetate 0.4-0.6%, magnesium sulfate 0.01-0.04%, manganese sulfate 0.003-0.006%, and Tween-80 0.10-0.15%; and the second seed culture medium includes the following raw material components in mass percentage: glucose 2.3-2.7%, peptone 0.8-1.2%, yeast powder 0.3-0.7%, magnesium sulfate 0.01-0.02%, manganese sulfate 0.003-0.006%, sodium acetate 0.4-0.6%, and ammonium citrate 0.13-0.16%.

[0014] Further, in step (3), during the first anaerobic fermentation, the first solution is fed; during the second anaerobic fermentation, the second solution is fed; during the third anaerobic fermentation, the third solution is fed; the first solution comprises the following components in mass percentage: glucose 5-10% and yeast processing product 0.5-1%; the second solution comprises the following components in mass percentage: glucose 5-10%, yeast processing product 1-2% and glutamine 0.1-0.3%; the third solution comprises the following components in mass percentage: trehalose 0.1-0.3% and serine 0.03-0.05%.

[0015] Further, the feeding amount of the first solution is 2-5% of the mass of the initial fermentation material, the feeding amount of the second solution is 4-7% of the mass of the initial fermentation material, and the feeding amount of the third solution is 1-2% of the mass of the initial fermentation material.

[0016] Further, the preparation method of the yeast processing product is as follows: the yeast processing product is obtained by crushing the yeast cell of Saccharomyces cerevisiae at 30-40 MPa to obtain a bacterial solution, adding a compound enzyme to the bacterial solution, and enzymatically hydrolyzing at 45-50℃ and pH 5.0-5.5 for 2-3h; wherein the compound enzyme is prepared by mixing cellulase and β-glucanase at a mass ratio of 1:2.

[0017] Further, in step (3), the fermentation medium comprises the following raw material components in mass percentage: glucose 4.0-5.0%, yeast processing product 1.5-1.8%, peptone 0.8-1.2%, dipotassium hydrogen phosphate 0.4-0.7%, glycyrrhiza extract 0.3-0.5%, manganese sulfate 0.01-0.02%, magnesium sulfate 0.01-0.03%, anhydrous sodium acetate 0.4-0.7%, ammonium citrate 0.12-0.18%, and cysteine 0.1-0.3%.

[0018] Further, in step (4), the cell wall breaking treatment is as follows: the resuspended fermentation bacteria are placed in a high-pressure cell crusher, crushed at 135-140 MPa, repeated twice, and then transferred to an ultrasonic cell crusher, crushed at ice bath, 20-30 Hz, and 130-150 W for 5-10 min to obtain the cell lysate.

[0019] Further, in step (4), the adjuvant is a suspending agent and a preservative.

[0020] Further, the suspending agent is suspending agent SF-1 or a cosmetic grade carbomer, and the amount of the suspending agent added is 1-2% of the mass of the cell lysate; the preservative is p-hydroxyacetophenone, and the amount of the preservative added is 0.1-0.3% of the mass of the solution.

[0021] The application further provides the Schizochytrium ferment product lysate prepared by the preparation method.

[0022] The application further provides application of the Schizochytrium ferment product lysate in preparation of cosmetics.

[0023] Further, the cosmetics are cosmetics with repair and / or anti-inflammatory effects.

[0024] The application has the following beneficial effects:

[0025] 1. In the main fermentation process, the three-stage fermentation control strategy is carried out by adjusting the fermentation parameters, in the fast growth stage of the bacteria, the fermentation conditions are controlled at 36-37℃, pH 6.0-6.2, 100-110 rpm, which can effectively promote the growth of the bacteria and improve the amount of the bacteria; when the growth of the bacteria tends to be stable, the fermentation conditions are controlled at 38.5-39℃, pH 5.8-6.0, 110-115 rpm, which is beneficial to improve the metabolic rate and promote the accumulation of the target metabolites, thereby improving the content of the active ingredients; and in the late fermentation stage, by reducing the temperature and the rotating speed, the synthesis of the by-products can be reduced, the activity is stabilized, and thus the purity and the stability of the product are improved.

[0026] 2. For the three stages of the main fermentation, specific feed is added respectively to match the nutritional requirements of the bacteria in different stages, so that the active ingredient yield and stability are avoided due to insufficient substrate or inhibition. In the early fermentation stage, by adding glucose and yeast treatment, the carbon and nitrogen sources are supplemented, which is beneficial to promote the growth of the bacteria and improve the amount of the bacteria; in the middle fermentation stage, by adding glucose, yeast treatment and glutamine, the product synthesis can be effectively induced, and the accumulation of the active metabolites is promoted. In the late fermentation stage, by adding trehalose and serine, the activity of the bacterial cells can be maintained and the synthesis of the metabolites is promoted.

[0027] 3. The fermentation medium of the application has comprehensive nutrition, which can provide balanced nutrition for the bacteria, activate the related metabolic pathways, improve the fermentation starting force, and significantly improve the bacterial density and metabolic activity, thereby effectively improving the fermentation efficiency and effect.

[0028] 4. By combining twice high-pressure crushing and once ultrasonic crushing, the cell crushing effect can be improved, and by microscopic examination,

[0029] The cell lysis degree reaches more than 97.5%.

[0030] 5. The Bifidobacterium adolescentis screened and separated by the application has good anaerobic fermentation effect, large amount of bacterial growth, and large amount of metabolic product synthesis, and the cell lysate obtained by anaerobic fermentation of the Bifidobacterium adolescentis has high content of active substances and good stability. When the cell lysate is applied to cosmetics, it can effectively repair DNA damage, resist photoaging, resist oxidation, promote barrier repair, resist inflammation, tighten and resist wrinkles, and the like. By controlling the reasonable addition amount, the cell lysate will not cause damage to the organization structure of the cosmetics, will not adversely affect the stability of the cosmetics, and will not cause discoloration, delamination and other quality problems.

[0031] In summary, by optimizing the fermentation medium, combining with the control of the fermentation conditions in stages and the dynamic feeding strategy, the synthesis efficiency of the metabolic product is improved, the target active ingredient content of the fermentation product lysate of the Schizosaccharomyces pombe prepared by the application is high, the stability is good, the cosmetic and skin care effect is excellent, especially the repair and anti-inflammatory effect is good, and the application is suitable for the development of cosmetics. On the other hand, the controllability of the preparation process is improved, thereby the batch difference is reduced, the consistency and reliability of the product are improved, and the product is easy to scale up and automate. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 The figure is a colony chart of the Bifidobacterium adolescentis HH404-1 strain of the application.

[0033] Figure 2 The figure is a microscope chart of the Bifidobacterium adolescentis HH404-1 strain of the application.

[0034] BIOLOGICAL MATERIAL PRESERVATION INFORMATION

[0035] The strain preserved in the application is Bifidobacterium adolescentis HH404-1, which is classified and named as Bifidobacterium adolescentis, preserved in Guangdong Microbial Culture Collection Center (GDMCC) at No. 59 Building, 5th Floor, Guangzhou Xianlie Middle Road 100, with a preservation date of October 22, 2025 and a preservation number of GDMCC No: 67151. DETAILED DESCRIPTION

[0036] In order to make the technical problems, technical solutions and advantages of the application more clear, specific embodiments will be described in detail below.

[0037] In the application, the reagents and consumables used are purchased from conventional reagent manufacturers in the field, and the experimental methods and technical means used are conventional methods and means in the field, unless otherwise specified.

[0038] Firstly, the present application provides a Bifidobacterium adolescentis. The Bifidobacterium adolescentis is obtained by isolation and screening from the feces of a long-lived old man in Bama Longevity Village, Guangxi Zhuang Autonomous Region, and is identified by biochemical identification and molecular biology identification, and the specific process is shown as follows.

[0039] Isolation and screening of the strain

[0040] The Bifidobacterium adolescentis is obtained by isolation and screening from the feces of a long-lived old man in Bama Longevity Village, Guangxi Zhuang Autonomous Region, and the specific isolation and screening method is as follows:

[0041] Sterilely take 25 g of the feces of the long-lived old man, add 225 mL of normal saline, and then dilute the bacterial suspension to 10 -6 , 10 -7 , 10 -8 gradients, and then take 0.2 mL for Henggait anaerobic rolling tube, and place it in a 37°C incubator for culture for 48-72 h, and then select the colonies with smooth, convex, neat edge, white or slightly yellow, soft texture medium and small colonies to inoculate in Bifidobacterium BS liquid medium, and anaerobic culture for 24-48 h, and then perform gram staining and microscopic examination. Select the colonies with Bifidobacterium morphological characteristics, transparent ring around, smooth and neat edge, white or slightly yellow, and inoculate them in Bifidobacterium BS liquid medium, and culture them in aerobic and anaerobic environments at 37°C for 24 h, and perform KOH, hydrogen peroxide enzyme, indole, and glucose metabolism tests. Select the bacteria that grow under anaerobic conditions, do not grow under aerobic conditions, are negative in KOH test, are negative in hydrogen peroxide enzyme test, are negative in indole test, and can metabolize glucose but do not produce gas, to preliminarily determine them as the screened Bifidobacterium. Inoculate the preliminarily screened Bifidobacterium in MRS liquid medium, and select the colonies with good observation results in the above medium to repeatedly streak and purify the culture to screen a Bifidobacterium adolescentis, numbered as: HH404-1, which is preserved in glycerol at -80°C.

[0042] 1. Biochemical identification of the strain

[0043] Referring to the "Berger Bacteria Identification Manual" (9th edition), the morphology and color of the colonies of the strain growing on the surface of the MRS medium are observed, and the colony morphology chart is shown as Figure 1 ; young cultures are selected, smeared, gram stained, and observed under a microscope for bacterial morphology, size, gram staining reaction, and the presence or absence of spores, morphology, and attachment position, and the microscopic examination chart is shown as Figure 2 .

[0044] 3. Molecular biology identification of the strain

[0045] The target strain HH404-1 is taxonomically identified by 16S rDNA sequence analysis.

[0046] Strain HH404-1 was sent to Shanghai Shengong Biotechnology Sequencing Identification Department for sequencing identification, and the sequencing results are as follows:

[0047] GCGGCGTTGCTGATCCGCGATTACTAGCGACTCCGCCTTCATGGAGTCGGGTTGCAGACTCCAATCCGAACTGAGACCGGTTTTAAGGGATCCGCTCCACCTCACAGTGTCGCATCCCGTTGTACCGGCCATTGTAGCATGCGTGAAGCCCTGGACGTAAGGGGCATGATGATCTGACGTCATCCCCACCTTCCTCCGAGTTGACCCCGGCGGTCCCCCGTGAGTTCCCACCACGACGTGCTGGCAACACAGGGCGAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTGAACCCGCCCCGAAGGGAGACCGTATCTCTACGGCTGTCGGGAACATGTCAAGCCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCGCATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTTCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGATGCTTAACGCGTTGGCTCCGACACGGAGACCGTGGAATGGTCCCCACATCCAGCATCCACCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTGACGGCCCAGAGACCTGCCTTCGCCATTGGTGTTCTTCCCGATATCTACACATTCCACCGTTACACCGGGAATTCCAGTCTCCCCTACCGCACTCAAGCCCGCCCGTACCCGGCGCGGATCCACCGTTAAGCGATGGACTTTCACACCGGACGCGACGAACCGCCTACGAGCCCTTTACGCCCAATAATTCCGGATAACGCTTGCACCCTACGAATTACCGCGG

[0048] The sequencing result is analyzed by comparison on the ribosome database (NCBI). The 16S rDNA sequence analysis result shows that the homology of the strain with Bifidobacterium adolescentis (Bifidobacterium adolescentis) in Bifidobacterium is as high as 100%, that is, the strain is Bifidobacterium adolescentis (Bifidobacterium adolescentis).

[0049] The strain is preserved, and the preservation information is specifically as follows: Bifidobacterium adolescentis HH404-1, the classification name of which is Bifidobacterium adolescentis, is preserved in the Guangdong Microbial Culture Collection Center (GDMCC) located at No. 59 Building, 5th Floor, Guangzhou, Guangdong, on October 22, 2025, and the preservation number is GDMCC No: 67151.

[0050] Secondly, the application provides a fermenting product lysate of Schizosaccharomyces.

[0051] The preparation method of the fermenting product lysate of Schizosaccharomyces includes the following steps:

[0052] (1) Strain activation: inoculate Bifidobacterium adolescentis into a first seed culture medium, anaerobically culture, and obtain activated bacteria liquid;

[0053] (2) Seed liquid culture: inoculate the activated bacteria liquid into a second seed culture medium and continuously transfer and anaerobically culture twice to obtain a third-level seed liquid;

[0054] (3) Fermentation culture: inoculate the third-level seed liquid into a fermenter containing a fermentation culture medium, anaerobically ferment and culture, and obtain a fermentation product;

[0055] The fermentation culture includes first-time anaerobic fermentation, second-time anaerobic fermentation and third-time anaerobic fermentation; the first-time anaerobic fermentation is under the conditions of temperature 36-37℃, pH 6.0-6.2, rotation speed 100-110 rpm and fermentation time 8-10 h; the second-time anaerobic fermentation is under the conditions of temperature 38.5-39℃, pH 5.8-6.0, rotation speed 110-115 rpm and fermentation time 10-12; and the third-time anaerobic fermentation is under the conditions of temperature 35-35.5℃, pH 5.8-6.0, rotation speed 95-100 rpm and fermentation time 2-4 h, at which time the residual sugar in the tank is <1 g / L.

[0056] (4) Inactivation and decomposition: after sterilization treatment of the fermentation product, centrifugation, washing and collection of the fermentation bacteria, the fermentation bacteria are resuspended with water, broken wall treatment, and the cell lysate is obtained. Adding an additive to the cell lysate, the cell lysate of the Erlichia ferment product is obtained.

[0057] Preferably, in step (1), the Bifidobacterium adolescentis is Bifidobacterium adolescentis HH404-1, which is classified as Bifidobacterium adolescentis and preserved in Guangdong Microbial Culture Collection Center (GDMCC), located at No. 59, Building 5, 100, Martyrs' Road, Guangzhou, with a preservation date of October 22, 2025 and a preservation number of GDMCC No: 67151.

[0058] Preferably, in step (1), the first seed medium comprises the following raw material components in mass percentage: 1.0-1.2% of proteose peptone, 0.8-1.2% of beef extract, 0.4-0.7% of yeast powder, 1.8-2.3% of glucose, 0.1-0.3% of potassium phosphate dibasic, 0.1-0.3% of triammonium citrate, 0.4-0.6% of sodium acetate, 0.01-0.04% of magnesium sulfate, 0.003-0.006% of manganese sulfate, and 0.10-0.15% of Tween-80.

[0059] For example, in the first seed medium, the mass percentage of proteose peptone can be 1.0%, 1.1% or 1.2%, etc.; the mass percentage of beef extract can be 0.8%, 0.9%, 1.0% or 1.2%, etc.; the mass percentage of yeast powder can be 0.4%, 0.6% or 0.7%, etc.; the mass percentage of glucose can be 1.8%, 2.0%, 2.1% or 2.3%, etc.; the mass percentage of potassium phosphate dibasic can be 0.1%, 0.2% or 0.3%, etc.; the mass percentage of triammonium citrate can be 0.1%, 0.2% or 0.3%, etc.; the mass percentage of sodium acetate can be 0.4%, 0.5% or 0.6%, etc.; the mass percentage of magnesium sulfate can be 0.003%, 0.004% or 0.006%, etc.; the mass percentage of Tween-80 can be 0.10%, 0.12% or 0.15%, etc.

[0060] Preferably, in step (1), the anaerobic culture conditions are 37-38°C for 16-20h. For example, the anaerobic culture temperature can be 37°C or 38°C, and the culture time can be 16h, 18h or 20h.

[0061] Preferably, in step (2), the two continuous anaerobic cultures are specifically as follows: inoculating the activated bacteria solution into the second seed culture medium, and culturing anaerobically at 36-38℃ for 12-16h to obtain a first-stage seed solution; inoculating the first-stage seed solution into the first seed culture medium, and culturing anaerobically at 36-38℃, 95-110rpm, pH 6.0-6.2 for 10-12h to obtain a second-stage seed solution; inoculating the second-stage seed solution into the second seed culture medium, and culturing anaerobically at 36-38℃, 100-110rpm, pH 6.0-6.2 for 10-13h to obtain a third-stage seed solution.

[0062] Preferably, in step (2), the second seed culture medium comprises the following raw material components in mass percentage: glucose 2.3-2.7%, peptone 0.8-1.2%, yeast powder 0.3-0.7%, magnesium sulfate 0.01-0.02%, manganese sulfate 0.003-0.006%, sodium acetate 0.4-0.6%, and ammonium citrate 0.13-0.16%.

[0063] For example, in the second seed culture medium, the mass percentage of glucose can be 2.3%, 2.5% or 2.7%, etc.; the mass percentage of peptone can be 0.8%, 1.0% or 1.2%, etc.; the mass percentage of yeast powder can be 0.3%, 0.5% or 0.7%, etc.; the mass percentage of magnesium sulfate can be 0.01%, 0.015% or 0.02%, etc.; the mass percentage of manganese sulfate can be 0.003%, 0.005% or 0.006%, etc.; the mass percentage of sodium acetate can be 0.4%, 0.5% or 0.6%, etc.; and the mass percentage of ammonium citrate can be 0.13%, 0.15% or 0.16%, etc.

[0064] For example, in step (2), the temperature for anaerobic culture of the activated bacteria solution can be 36℃, 37℃ or 38℃, etc., and the culture time can be 12h, 14h or 16h, etc.; the temperature for anaerobic culture of the first-stage seed solution can be 36℃, 37℃ or 38℃, etc., the rotation speed can be 95rpm, 100rpm or 110rpm, etc., the pH can be 6.0, 6.1 or 6.2, etc., and the culture time can be 10h, 11h or 12h, etc.; the temperature for anaerobic culture of the second-stage seed solution can be 36℃, 37℃ or 38℃, etc., the rotation speed can be 100rpm, 105rpm or 110rpm, etc., the pH can be 6.0, 6.1 or 6.2, etc., and the culture time can be 10h, 12h or 13h, etc.

[0065] Preferably, in step (3), the inoculation amount of the third seed liquid is 5-10%. Illustratively, the inoculation amount of the third seed liquid can be 5%, 7%, 9% or 10%.

[0066] Preferably, in step (3), the fermentation medium comprises the following raw material components in mass percentage: glucose 4.0-5.0%, yeast extract 1.5-1.8%, peptone 0.8-1.2%, potassium phosphate dibasic 0.4-0.7%, glycyrrhiza extract 0.3-0.5%, manganese sulfate 0.01-0.02%, magnesium sulfate 0.01-0.03%, anhydrous sodium acetate 0.4-0.7%, ammonium citrate 0.12-0.18%, cysteine 0.1-0.3%.

[0067] Illustratively, in the fermentation medium, the mass percentage of glucose can be 4.0%, 4.3%, 4.7%, 5.0%, etc., the mass percentage of yeast extract can be 1.5%, 1.7%, 1.8%, etc., the mass percentage of peptone can be 1.5%, 1.7%, 1.8%, etc., the mass percentage of potassium phosphate dibasic can be 0.4%, 0.4%, 0.7%, etc., the mass percentage of glycyrrhiza extract can be 0.3%, 0.4%, 0.5%, etc., the mass percentage of manganese sulfate can be 0.01%, 0.015%, 0.02%, etc., the mass percentage of magnesium sulfate can be 0.01%, 0.02%, 0.03%, etc., the mass percentage of anhydrous sodium acetate can be 0.4%, 0.6%, 0.7%, etc., the mass percentage of ammonium citrate can be 0.12%, 0.16%, 0.18%, etc., and the mass percentage of cysteine can be 0.1%, 0.2%, 0.3%, etc.

[0068] Illustratively, in step (3), the temperature of the first anaerobic fermentation can be 36℃, 36.5℃ or 37℃, the pH can be 6.0, 6.1 or 6.2, the rotation speed can be 100rpm, 105rpm or 110rpm, and the fermentation time can be 8h, 9h or 10h; the temperature of the second anaerobic fermentation can be 38.5℃, 38.7℃ or 39℃, the pH can be 5.8, 5.9 or 6.0, the rotation speed can be 110rpm, 112rpm or 115rpm, and the fermentation time can be 10h, 11h or 12h; the temperature of the third anaerobic fermentation can be 35℃, 35.2℃ or 35.5℃, the pH can be 5.8, 5.9 or 6.0, the rotation speed can be 95rpm, 98rpm or 100rpm, and the fermentation time can be 2h, 3h or 4h.

[0069] Preferably, in step (3), during the first fermentation, the first solution is fed; during the second fermentation, the second solution is fed; during the third fermentation, the third solution is fed; the first solution comprises glucose and yeast extract, the second solution comprises glucose, yeast extract and glutamine, and the third solution comprises trehalose and serine. Specifically, the feeding rate is dynamically adjusted according to online dissolved oxygen (DO) and pH feedback, and when DO rises sharply or pH fluctuates abnormally, the feeding rate is reduced; when DO decreases stably, the feeding rate is increased.

[0070] Preferably, the first solution comprises the following components in mass percentage: glucose 5-10% and yeast extract 0.5-1%; the second solution comprises the following components in mass percentage: glucose 5-10%, yeast extract 1-2% and glutamine 0.1-0.3%; and the third solution comprises the following components in mass percentage: trehalose 0.1-0.3% and serine 0.03-0.05%.

[0071] Illustratively, in the first solution, the mass percentage of glucose can be 5%, 7%, 9%, 10%, etc., and the mass percentage of yeast extract can be 0.5%, 0.8%, 1%, etc.; in the second solution, the mass percentage of glucose can be 5%, 7%, 9%, 10%, etc., and the mass percentage of yeast extract can be 1%, 1.5%, 2%, etc.; the mass percentage of glutamine can be 0.1%, 0.2% or 0.3%, etc.; in the third solution, the mass percentage of trehalose can be 0.15%, 0.2%, 0.3%, etc., and the mass percentage of serine can be 0.03%, 0.04%, 0.05%, etc.

[0072] Preferably, the feeding amount of the first solution is 2-5% of the mass of the initial fermentation material, the feeding amount of the second solution is 4-7% of the mass of the initial fermentation material, and the feeding amount of the third solution is 1-2% of the mass of the initial fermentation material.

[0073] Illustratively, the feeding amount of the first solution can be 2%, 4% or 5% of the mass of the initial fermentation material, the feeding amount of the second solution can be 4%, 6% or 7% of the mass of the initial fermentation material, and the feeding amount of the third solution can be 1%, 1.5% or 2% of the mass of the initial fermentation material.

[0074] Preferably, in step (3), the preparation method of the yeast treatment product is as follows: the Saccharomyces cerevisiae cell bodies are broken at 30-40 MPa to obtain a bacterial solution, a composite enzyme is added to the bacterial solution, and the enzyme is hydrolyzed at 45-50 DEG C and pH 5.0-5.5 for 2-3 h to obtain the yeast treatment product; wherein the composite enzyme is prepared by mixing cellulase and beta-glucanase at a mass ratio of 1:2.

[0075] Illustratively, the breaking pressure of the Saccharomyces cerevisiae cell bodies can be 30 MPa, 34 MPa or 40 MPa, etc., the enzyme hydrolysis temperature of the bacterial solution is 45 DEG C, 47 DEG C or 50 DEG C, etc., the enzyme hydrolysis pH is 5.0, 5.3 or 5.5, etc., and the enzyme hydrolysis time is 2 h, 2.5 h or 3 h, etc.

[0076] Preferably, in step (4), the sterilization treatment can use pasteurization; the centrifugation conditions can be centrifugation at 4 DEG C and 10,000 r / min for 10 min; and the volume ratio of the fermentation cell bodies to water can be 1:1-4, specifically 1:1, 1:2, 1:3 or 1:4, etc.

[0077] Preferably, in step (4), the wall breaking treatment is as follows: the resuspended fermentation cell bodies are placed in a high-pressure cell crusher, broken at 135-140 MPa, repeated twice, and then transferred to an ultrasonic cell crusher, broken at ice bath, 20-30 Hz and 130-150 W for 5-10 min to obtain the cell lysate.

[0078] Illustratively, the pressure of the high-pressure cell crusher can be 135 MPa, 137 MPa, 140 MPa, etc.; the breaking frequency of the ultrasonic cell crusher can be 20 Hz, 25 Hz, 28 Hz or 30 Hz, etc.; the breaking power can be 130 W, 140 W, 145 W or 150 W, etc.; and the breaking time can be 5 min, 8 min or 10 min, etc.

[0079] Illustratively, in step (4), the adjuvant is a suspending agent and a preservative.

[0080] Preferably, the suspending agent is suspending agent SF-1 or a cosmetic-grade carbomer, the amount of the suspending agent added is 1-2% of the mass of the cell lysate; and the preservative is p-hydroxyacetophenone, the amount of the preservative added is 0.1-0.3% of the mass of the solution.

[0081] The application also provides a use of the above-mentioned Schizochytrium sp. fermentation product lysate in the preparation of cosmetics.

[0082] Preferably, the cosmetic is a cosmetic with anti-inflammatory and / or repair effects.

[0083] It is understood that the above-mentioned cosmetic can be used for topical or transdermal administration, and can be produced in any solid, liquid or semi-solid preparation. The preparation includes but is not limited to cream, emulsion, anhydrous composition, aqueous dispersion, aqueous or oily gel, paste, solution, mask, essence, etc.

[0084] It is understood that the cosmetic of the present application can also contain excipients, adjuvants and / or ingredients acceptable in dermatology and / or cosmetology, such as humectants (e.g. glycerin, propylene glycol, butylene glycol, pentylene glycol, caprylyl glycol, lactic acid, urea, sodium hyaluronate), emollients or skin conditioning agents (e.g. dimethicone, glyceryl stearate, caprylic / capric triglyceride, cetyl stearyl alcohol, lecithin, C12-15 alkyl benzoate, squalane, lanolin, behenyl alcohol, tocopherol acetate, panthenol, shea butter, retinol palmitate, retinol), surfactants (e.g. xanthan gum, sodium laureth sulfate, stearic acid, polysorbate 20, polysorbate 80, stearyl alcohol, cetyl alcohol, steareth-2, ceteareth-20, cocamidopropyl betaine), thickening agents, etc.

[0085] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application, and are not used to limit the scope of the present application.

[0086] Example 1

[0087] The present example provides a preparation method of a Schizosaccharomyces ferment product lysate, which comprises the following steps:

[0088] 1. Strain activation

[0089] (1) Preparation of the first seed medium

[0090] The components are weighed according to the following mass percentage: proteose peptone 1.0%, beef extract 1.0%, yeast powder 0.5%, glucose 2.0%, dipotassium phosphate 0.2%, triammonium citrate 0.2%, sodium acetate 0.5%, magnesium sulfate 0.02%, manganese sulfate 0.005%, Tween-80 0.1%; the first seed medium is prepared according to the above formula, and its pH is adjusted to 6.5; the prepared seed medium is divided into screw tubes, sterilized at 115℃ for 30 min, and cooled for standby.

[0091] (2) Activation culture

[0092] Bifidobacterium adolescentis HH404-1 is inoculated into the first seed medium, placed in an anaerobic workstation, and anaerobically cultured at 37℃ for 18 h to obtain an activated bacterial solution.

[0093] 2. Seed expansion culture

[0094] (1) Preparation of the second seed culture medium

[0095] The components are weighed according to the following mass percentage: glucose 2.5%, peptone 1%, yeast powder 0.5%, magnesium sulfate 0.01%, manganese sulfate 0.005%, anhydrous sodium acetate 0.5%, ammonium citrate 0.15%; the second seed culture medium is prepared according to the above formula, and its pH is adjusted to 6.5, and the prepared seed culture medium is divided into screw test tubes, sterilized at 115°C for 30 min, and cooled for standby.

[0096] (2) First-stage seed liquid culture

[0097] The activated bacterial liquid is transferred to a 250 mL silk mouth bottle containing 200 mL of the second seed culture medium at an inoculation amount of 5%, and is placed in an anaerobic workstation for culture at 37°C for 14 h to obtain a first-stage seed liquid.

[0098] (3) Second-stage seed liquid culture

[0099] The first-stage seed liquid is transferred to a 5L seed tank containing 4L of the second seed culture medium at an inoculation amount of 5%, high-purity nitrogen gas (99.99%) is introduced into the seed tank for 10 min after inoculation, the air in the tank is completely replaced, and the dissolved oxygen drops to 0, then it is clamped and pressurized with a clamp, and is cultured at 37°C, 100 rpm, pH 6.0 (controlled by adding ammonia water) for 12 h to obtain a second-stage seed liquid.

[0100] (3) Third-stage seed liquid culture

[0101] The second-stage seed liquid is transferred to a 150L seed tank containing 80L of the second seed culture medium at an inoculation amount of 5%, high-purity nitrogen gas (99.99%) is introduced into the seed tank for 30 min after inoculation, the air in the tank is completely replaced, and the dissolved oxygen drops to 0, then it is pressurized, and is cultured at 37°C, 100 rpm, pH 6.0 (controlled by adding ammonia water) for 12 h to obtain a third-stage seed liquid.

[0102] 3. Fermentation culture

[0103] (1) Preparation of yeast treatment

[0104] The yeast treatment is obtained by adding a composite enzyme prepared by mixing cellulase and β-glucanase at a mass ratio of 1:2 to the bacterial liquid obtained by crushing the Saccharomyces cerevisiae cells at 30MPa, and enzymolysis at 45°C, pH 5.0 for 3h.

[0105] (2) Preparation of licorice extract

[0106] Licorice powder and ethanol were mixed at a material-liquid ratio of 1:8, extracted at 70°C, and extracted for a total of 3 times, each for 30 min. The extract was combined, concentrated, and dried to obtain the licorice extract, which was used as prepared.

[0107] (3) Preparation of fermentation medium

[0108] The following components were weighed according to the mass percentage: glucose 4.5%, yeast extract 1.5%, peptone 1.0%, potassium phosphate dibasic 0.5%, licorice extract 0.4%, manganese sulfate 0.01%, magnesium sulfate 0.01%, anhydrous sodium acetate 0.5%, ammonium citrate 0.15%, and cysteine 0.2%. The glucose was sterilized separately at 115°C for 20 min, and the remaining components were mixed and sterilized at 121°C for 30 min. After sterilization, the mixture was uniformly mixed to obtain the fermentation medium, which was used as prepared.

[0109] (4) Main fermentation

[0110] The third-stage seed liquid was inoculated into a 1.5-ton fermenter containing 1.05 tons of fermentation medium at an inoculation amount of 7%, and high-purity nitrogen gas (99.99%) was introduced for 50 min after inoculation to completely replace the air in the fermenter. After the dissolved oxygen dropped to 0, the pressure was maintained, and then the anaerobic fermentation was performed in three times, wherein:

[0111] The first-time anaerobic fermentation was performed at a temperature of 37°C, a pH of 6.2, a rotation speed of 110 rpm, and a fermentation time of 8 h;

[0112] The second-time anaerobic fermentation was performed at a temperature of 39°C, a pH of 6.0, a rotation speed of 115 rpm, and a fermentation time of 11 h;

[0113] The third-time anaerobic fermentation was performed at a temperature of 35°C, a pH of 5.8, a rotation speed of 100 rpm, and a fermentation time of 4 h. At this time, the residual sugar in the fermenter was less than 1 g / L, and the fermentation product was obtained after stopping the fermentation.

[0114] 4. Inactivation and decomposition

[0115] After the fermentation product was sterilized by pasteurization, centrifugation was performed at 4°C and 10,000 r / min for 10 min, and the fermentation bacteria were collected after being washed with deionized water three times. The fermentation bacteria and water were mixed and resuspended at a volume ratio of 1:4, and then placed in a high-pressure cell disrupter for disruption at 135 Mpa. After repeating twice, the cell lysate was obtained by disruption in an ultrasonic cell disrupter under the conditions of ice bath, 25 Hz, and 140 w for 8 min. The cell lysate was mixed with 1% cosmetic-grade carbomer and 0.2% p-hydroxyphenylacetone to obtain the cell lysate of the Saccharomyces boulardii fermentation product.

[0116] Example 2

[0117] The present embodiment provides a preparation method of a Schizosaccharomyces ferment product lysate, comprising the following steps:

[0118] 1. Strain activation

[0119] (1) Preparation of the first seed culture medium

[0120] The components are weighed according to the following mass percentage: 1.0% of proteose peptone, 1.0% of beef extract, 0.5% of yeast powder, 2.0% of glucose, 0.2% of dipotassium hydrogen phosphate, 0.2% of triammonium citrate, 0.5% of sodium acetate, 0.02% of magnesium sulfate, 0.005% of manganese sulfate, and 0.1% of Tween-80. The first seed culture medium is prepared according to the above formula, and the pH is adjusted to 6.5. The prepared seed culture medium is divided into screw-neck test tubes, sterilized at 115°C for 30 min, and cooled for standby.

[0121] (2) Activation culture

[0122] Bifidobacterium adolescentis HH404-1 is inoculated into the first seed culture medium, and placed in an anaerobic workstation for anaerobic culture at 37°C for 18 h to obtain an activated bacterial solution.

[0123] 2. Seed expansion culture

[0124] (1) Preparation of the second seed culture medium

[0125] The components are weighed according to the following mass percentage: 2.5% of glucose, 1% of proteose peptone, 0.5% of yeast powder, 0.01% of magnesium sulfate, 0.005% of manganese sulfate, 0.5% of anhydrous sodium acetate, and 0.15% of ammonium citrate. The second seed culture medium is prepared according to the above formula, and the pH is adjusted to 6.5. The prepared seed culture medium is divided into screw-neck test tubes, sterilized at 115°C for 30 min, and cooled for standby.

[0126] (2) First-stage seed liquid culture

[0127] The activated bacterial solution is inoculated into a 250 mL screw-neck flask containing 200 mL of the second seed culture medium at an inoculation amount of 5%, and placed in an anaerobic workstation for culture at 37°C for 14 h to obtain a first-stage seed liquid.

[0128] (3) Second-stage seed liquid culture

[0129] The first seed liquid was transferred to a 5L seed tank containing 4L of second seed culture medium at an inoculation amount of 5%, and after inoculation, high-purity nitrogen gas (99.99%) was introduced into the seed tank for 10 minutes to completely replace the air in the tank. After the dissolved oxygen dropped to 0, the tank was clamped and pressurized, and cultured at 37°C, 100 rpm, pH 6.0 (controlled by adding ammonia water) for 12 hours to obtain a second seed liquid.

[0130] (3) Third seed liquid culture

[0131] The second seed liquid was transferred to a 150L seed tank containing 80L of second seed culture medium at an inoculation amount of 5%, and after inoculation, high-purity nitrogen gas (99.99%) was introduced into the seed tank for 30 minutes to completely replace the air in the tank. After the dissolved oxygen dropped to 0, the tank was pressurized, and cultured at 37°C, 100 rpm, pH 6.0 (controlled by adding ammonia water) for 12 hours to obtain a third seed liquid.

[0132] 3. Fermentation culture

[0133] (1) Preparation of yeast treatment

[0134] The yeast cells were broken at 30MPa to obtain a bacterial liquid, and a composite enzyme was added to the bacterial liquid, which was prepared by mixing cellulase and β-glucanase at a mass ratio of 1:2. The enzyme was hydrolyzed at 45°C, pH 5.0 for 3 hours to obtain the yeast treatment, which was ready for use.

[0135] (2) Preparation of licorice extract

[0136] Licorice powder and ethanol were mixed at a solid-liquid ratio of 1:8, and extracted at 70°C. The extraction was repeated for a total of 3 times, each for 30 minutes. The extraction liquid was combined, concentrated, and dried to obtain the licorice extract, which was ready for use.

[0137] (3) Preparation of flow solution

[0138] First solution preparation: 8% glucose and 0.5% yeast treatment were mixed uniformly according to the mass percentage, to obtain the first solution, which was ready for use.

[0139] Second solution preparation: 10% glucose, 1.5% yeast treatment and 0.02% glutamine were mixed uniformly according to the mass percentage, to obtain the second solution, which was ready for use.

[0140] Third solution preparation: 0.2% trehalose and 0.03% serine were mixed uniformly according to the mass percentage, to obtain the third solution, which was ready for use.

[0141] (4) Preparation of fermentation medium

[0142] The components are weighed according to the following mass percentage: glucose 4.5%, yeast processing product 1.5%, proteose peptone 1.0%, potassium phosphate dibasic 0.5%, glycyrrhiza extract 0.4%, manganese sulfate 0.01%, magnesium sulfate 0.01%, anhydrous sodium acetate 0.5%, ammonium citrate 0.15%, cysteine 0.2%. The glucose is sterilized separately under the condition of 115℃ for 20 min, and the rest of the components are mixed and sterilized under the condition of 121℃ for 30 min. After sterilization, they are mixed uniformly to obtain the fermentation medium, which is ready for use.

[0143] (5) Main fermentation

[0144] The third-stage seed liquid is inoculated into a 1.5-ton fermentation tank containing 1.05 tons of fermentation medium at an inoculation amount of 7%, and after inoculation, high-purity nitrogen gas (99.99%) is introduced for 50 min to completely replace the air in the tank, and then the pressure is maintained after the dissolved oxygen drops. Subsequently, anaerobic fermentation is carried out in three times, wherein:

[0145] The first anaerobic fermentation is carried out under the conditions of temperature 37℃, pH 6.2, rotation speed 110 rpm, and fermentation time 8 h, and during the fermentation process, the first solution is added in an amount of 3% of the initial fermentation material mass;

[0146] The second anaerobic fermentation is carried out under the conditions of temperature 39℃, pH 6.0, rotation speed 115 rpm, and fermentation time 11 h, and during the fermentation process, the second solution is added in an amount of 5% of the initial fermentation material mass;

[0147] The third anaerobic fermentation is carried out under the conditions of temperature 35℃, pH 5.8, rotation speed 100 rpm, and fermentation time 4 h, at which time the residual sugar in the tank is <1 g / L, and during the fermentation process, the third solution is added in an amount of 1.5% of the initial fermentation material mass, and after the tank is stopped, the fermentation product is obtained.

[0148] 4. Inactivation and decomposition

[0149] After the fermentation product is sterilized by pasteurization, it is centrifuged at 4℃ and 10,000 r / min for 10 min, washed with deionized water three times, and the fermentation bacteria are collected. The fermentation bacteria and water are mixed and resuspended at a volume ratio of 1:4, then placed in a high-pressure cell disrupter, disrupted at 135 Mpa, repeated twice, and then transferred to an ultrasonic cell disrupter, disrupted under the conditions of ice bath, 25 Hz, and 140 w for 8 min to obtain a cell lysate. To the cell lysate, 1% of a cosmetic-grade carbomer and 0.2% of p-hydroxyphenylacetone are added and mixed uniformly to obtain the cell lysate of the Zymolyase yeast fermentation product.

[0150] Comparative Example 1

[0151] The comparative example 1 provides a preparation method of a fermentation product lysate of a Schizochytrium sp., which is different from the preparation method of the example 1 only in (4) of step 3. The (4) of step 3 of the comparative example 1 is specifically as follows: the third seed liquid is inoculated into a 1.5-ton fermentation tank containing 1.05 tons of fermentation medium at an inoculation amount of 7%, high-purity nitrogen gas (99.99%) is introduced for 50 min after inoculation, the air in the tank is completely replaced, the pressure is maintained after the dissolved oxygen drops to 0, and fermentation is carried out at 37°C, 100 rpm and pH 6.0 for 24 h. At this time, the residual sugar in the tank is <1 g / L, and the fermentation product is obtained after the tank is stopped.

[0152] Comparative example 2

[0153] The comparative example 2 provides a preparation method of a fermentation product lysate of a Schizochytrium sp., which is different from the preparation method of the example 2 only in the main fermentation of (5) of step 3. The (5) of step 3 of the comparative example 2 is specifically as follows: the third seed liquid is inoculated into a 1.5-ton fermentation tank containing 1.05 tons of fermentation medium at an inoculation amount of 7%, high-purity nitrogen gas (99.99%) is introduced for 50 min after inoculation, the air in the tank is completely replaced, the pressure is maintained after the dissolved oxygen drops to 0, and fermentation is carried out at 37°C, 100 rpm and pH 6.0 for 23 h. At this time, the residual sugar in the tank is <1 g / L, and the fermentation product is obtained after the tank is stopped; and the first solution is added within 1-8 h of fermentation, the addition amount is 3% of the mass of the initial fermentation material; the second solution is added within 8-19 h of fermentation, the addition amount is 5% of the mass of the initial fermentation material; and the third solution is added within 19-24 h of fermentation, the addition amount is 1.5% of the mass of the initial fermentation material, and the fermentation product is obtained after the tank is stopped.

[0154] Comparative example 3

[0155] The comparative example 3 provides a preparation method of a fermentation product lysate of a Schizochytrium sp., which is different from the preparation method of the example 2 only in the main fermentation of (5) of step 3. The (5) of step 3 of the comparative example 2 is specifically as follows:

[0156] The third seed liquid is inoculated into a 1.5-ton fermentation tank containing 1.05 tons of fermentation medium at an inoculation amount of 7%, high-purity nitrogen gas (99.99%) is introduced for 50 min after inoculation, the air in the tank is completely replaced, the pressure is maintained after the dissolved oxygen drops to 0, and then anaerobic fermentation is carried out in three times, wherein:

[0157] The first anaerobic fermentation is carried out at a temperature of 37°C, a pH of 6.2, a rotation speed of 110 rpm, and a fermentation time of 8 h; and the first solution is added during the fermentation process, the addition amount is 3% of the mass of the initial fermentation material;

[0158] The second anaerobic fermentation is carried out under the conditions of temperature 39℃, pH 6.0, rotation speed 115 rpm, and fermentation time 11 h, and the first solution is added during the fermentation process, and the amount of the first solution added is 5% of the initial fermentation material mass.

[0159] The third anaerobic fermentation is carried out under the conditions of temperature 35℃, pH 5.8, rotation speed 100 rpm, and fermentation time 4 h, at this time, the residual sugar in the tank is less than 1 g / L, and the first solution is added during the fermentation process, and the amount of the first solution added is 1.5% of the initial fermentation material mass, and the fermentation material is obtained after the tank is stopped.

[0160] Comparative Example 4

[0161] The comparative example 2 provides a preparation method of the Schizochytrium sp. fermentation product lysate, and the preparation method is different from the preparation method of example 2 only in that the fermentation medium preparation in step 3 (4) is different, and the fermentation medium preparation in step 3 (4) of the comparative example 2 is specifically as follows: the components are weighed according to the following mass percentage: glucose 4.5%, yeast powder 1.5%, peptone 1.0%, potassium phosphate dibasic 0.5%, manganese sulfate 0.01%, magnesium sulfate 0.01%, anhydrous sodium acetate 0.5%, ammonium citrate 0.15%, and cysteine 0.2%. The glucose is sterilized separately under the sterilization condition of 115℃ for 20 min, and the other components are mixed and sterilized under the sterilization condition of 121℃ for 30 min. After sterilization, the fermentation medium is uniformly mixed and obtained, and is ready for use.

[0162] Solid content determination of the Schizochytrium sp. fermentation product lysate

[0163] The main source of the solid content in the Schizochytrium sp. fermentation product lysate is the cell wall and cytoplasm of the fermentation product, and therefore, the cell content can be measured by the solid content.

[0164] Solid content detection of the Schizochytrium sp. fermentation product lysate in example 1-2 and comparative example 1-4: 3 g of the corresponding lysate is accurately weighed and placed in an evaporating dish; the evaporating dish is placed in a 105℃ oven for 2 hours, cooled in a desiccator, and weighed; the heating and weighing are repeated until the constant weight is obtained, and the calculation of the solid content in the lysate is as follows:

[0165] Solid content (%) = (mass of the lysate after drying / original mass of the lysate) x 100

[0166] The solid content determination results of each group of lysates are shown in Table 1.

[0167] Table 1 Solid content of each group of lysates

[0168]

[0169] As shown in Table 1, the solid content of the Schizochytrium sp. fermentation product lysate of Example 1 and Example 2 is superior to that of Comparative Examples 1-4, thus it can be inferred that the content of probiotic cells in the Schizochytrium sp. fermentation product lysate prepared by the preparation method of the present application is high.

[0170] Determination of total nitrogen content of the Schizochytrium sp. fermentation product lysate

[0171] The nitrogen content of Example 1-2 and Comparative Examples 1-4 was determined by Kjeldahl method, and the determination results are shown in Table 2.

[0172] Table 2 Total nitrogen content of the lysate of each group

[0173]

[0174] As shown in Table 2, the total nitrogen content of the Schizochytrium sp. fermentation product lysate of Example 1 and Example 2 is above 0.119%, indicating that the content of effective components such as amino acids and polypeptides in the Schizochytrium sp. fermentation product lysate prepared by the preparation method of the present application is high.

[0175] In combination with Table 1 and Table 2, the solid content and total nitrogen content of the Schizochytrium sp. fermentation product lysate of Comparative Example 1 is lower than that of the Schizochytrium sp. fermentation product lysate of Example 1, indicating that the three-stage fermentation control is conducive to the growth of the bacteria and the improvement of the metabolic rate of the bacteria, thus promoting the accumulation of the target metabolites, thereby increasing the amount of bacterial cells and the content of intracellular active components. The solid content and total nitrogen content of the Schizochytrium sp. fermentation product lysate of Comparative Example 2 and Comparative Example 3 is lower than that of the Schizochytrium sp. fermentation product lysate of Example 2, indicating that the three-stage fermentation control combined with the feeding of the specific solution can better match the nutritional and metabolic needs of the bacteria at different stages, thus further promoting the growth of the bacteria and the synthesis of metabolic substances. The solid content and total nitrogen content of the Schizochytrium sp. fermentation product lysate of Comparative Example 4 is lower than that of the Schizochytrium sp. fermentation product lysate of Example 2, indicating that the use of the special yeast treatment product instead of yeast powder in the fermentation medium of the present application can not only effectively improve the nitrogen source utilization rate, but also, in combination with the licorice extract, meet the growth needs of the bacteria and induce the synthesis of metabolites.

[0176] Three, anti-inflammatory efficacy test of the Schizochytrium sp. fermentation product lysate

[0177] 1. Test principle

[0178] Zebrafish embryos and human neutrophils are highly similar in morphology, biochemistry and physiological function. Neutrophils are the first batch of white blood cells to appear at the site of injury or pathogen invasion, and act to clear infection or harmful substances. The model of CuSO4-induced neutrophil aggregation in zebrafish embryo lateral line region was used for testing, and the number of neutrophils in the lateral line region of the fish embryos in the test material treatment group and the model control group was compared, and the neutrophil inhibition rate was calculated to evaluate the soothing effect of the raw material, formula or product.

[0179] 2. Test groups

[0180] The test needs to set up a model control group (CuSO4 working solution), a positive control group (dexamethasone working solution) and 6 test material groups (the test materials are the Schizochytrium ferment lysate of Examples 1-2 and Comparative Examples 1-4, respectively).

[0181] 3. Test method

[0182] Wild-type zebrafish 72 hpf embryos were collected and randomly divided into 8 groups of 24 embryos each. Among them:

[0183] (1) Model control group setting

[0184] Transfer 24 fish embryos to a six-well plate and add 6 mL of fish embryo culture solution. Place the six-well plate in a 28℃±1℃ incubator for 2 hours, then replace it with 6 mL of fish embryo culture solution containing 10 μM CuSO4 copper sulfate and incubate for 2 hours.

[0185] (2) Positive control group setting

[0186] Transfer 24 fish embryos to a six-well plate and add 6 mL of fish embryo culture solution containing 40 μM dexamethasone. Place the six-well plate in a 28℃±1℃ incubator for 2 hours, then replace it with 6 mL of fish embryo culture solution containing 10 μM CuSO4 copper sulfate and incubate for 2 hours.

[0187] (3) 6 test groups setting

[0188] Transfer 24 fish embryos to a six-well plate and add 6 mL of fish embryo culture solution containing 25 g / L test material (the Schizochytrium ferment lysate of Examples 1-2 and Comparative Examples 1-4, respectively). Place the six-well plate in a 28℃±1℃ incubator for 2 hours, then replace it with 6 mL of fish embryo culture solution containing 10 μM CuSO4 copper sulfate and incubate for 2 hours.

[0189] The fixed and dyed fish embryos are placed on their sides and then photographed under a body microscope. The number of neutrophils in the lateral line region of each fish embryo is counted. The neutrophil inhibition rate is calculated according to the number of neutrophils counted, and the calculation formula is as follows:

[0190] Neutrophil inhibition rate = (M-T) / M x 100%

[0191] In the formula:

[0192] T is the average number of neutrophils in the fish embryos of the test group;

[0193] M is the average number of neutrophils in the fish embryos of the model control group.

[0194] 4. Test results

[0195] The results are shown in Table 3 below.

[0196] Table 3: Calculation results of zebrafish neutrophil inhibition rate

[0197]

[0198] As can be seen from Table 3, the relative neutrophil count of the model control group is significantly higher than that of the blank control group, and there is a very significant difference, indicating that the application of CuSO4 can induce damage to the lateral line region of zebrafish embryos and cause neutrophil aggregation. Compared with the model control group, the neutrophil migration of the test group 1 and the test group 2 has an inhibitory effect and a significant difference, indicating that the Schizochytrium fermentum lysate prepared by the application has an improvement effect on the inflammatory response caused by copper sulfate, and the Schizochytrium fermentum lysate has good anti-inflammatory soothing effect. The cell inhibition rates of the test group 1 and the test group 3-6 are lower than that of the test group 2, indicating that the application can effectively promote the metabolism and synthesis of active substances by adopting the three-stage fermentation strategy + staged flow of specific solution + optimization of fermentation medium, thereby improving the yield of active substances and improving the anti-inflammatory effect.

[0199] Four, Schizochytrium fermentum lysate repair efficacy test

[0200] 1. Test principle

[0201] When injured, the skin must quickly regenerate to repair the skin barrier. In the embryonic stage, wound healing is very rapid and does not leave scars; but after the embryonic stage, wound healing needs to go through the steps of coagulation, inflammation, skin regeneration, blood vessel regeneration and the formation of granulation tissue, and finally form scars. In the process of wound repair, zebrafish is the same as humans in all steps except coagulation. Zebrafish wound skin healing is very rapid, followed by the migration of inflammatory cells to the wound to form a granulation tissue composed of macrophages, fibroblasts, blood vessels and collagen. Therefore, the main steps and principles of zebrafish and human wound healing are very consistent, and can be used as a detection screening model for the skin repair efficacy of the human body.

[0202] 2. Test method

[0203] Healthy 3-day-old zebrafish embryos after fertilization were selected.

[0204] 2.1 Injury model

[0205] The zebrafish embryo was anesthetized with a tricaine solution, and the tail fin of the zebrafish embryo was removed under a microscope using an experimental scalpel. The blade was sterilized with 70% alcohol before cutting. The tail cutting needs to be completed within 24 minutes.

[0206] 2.2 Test grouping

[0207] The test needs to set up a model control group (fish embryo culture solution / solvent solution) and a test group (the test substances are the cell lysates of the Schizochytrium sp. fermentation products of Examples 1-2 and Comparative Examples 1-4, respectively). Among them:

[0208] Model control group setting: 24 fish embryos were randomly selected and transferred to a 96-well plate, each well containing 1 fish embryo and 0.2 mL fish embryo culture solution.

[0209] Test group setting: 24 fish embryos were randomly selected and transferred to a 96-well plate, each well containing 1 fish embryo and 0.2 mL of the test solution.

[0210] The above plate was placed in a 28±1°C incubator for 24±1h.

[0211] 2.3 Sample analysis

[0212] The zebrafish was anesthetized with tricaine, and then placed under a body microscope to take a side photo of the tail of the fish embryo. The relative tail fin area of each fish embryo was measured. The tail fin repair rate was calculated according to the tail fin area, and the calculation formula is as follows:

[0213] Tail fin repair rate = (T-M) / M x 100%

[0214] In the formula:

[0215] T—The average value of the relative tail fin area of the fish embryo in the test group;

[0216] M - Model control group fish embryo tail relative tail fin area average.

[0217] 3. Test results

[0218] The results are shown in Table 4 below.

[0219] Table 4 Zebrafish relative tail fin area statistical results table

[0220]

[0221] From Table 4, compared with the model control group, the relative tail fin area of the test group 1 and the test group 2 is significantly improved, and the tail fin repair rate is more than 61.36%, which shows that the prepared Schizochytrium ferment product lysate has a repair effect on tail fin damage and has a tail fin repair ability, thereby indicating that the Schizochytrium ferment product lysate has a repair effect. The tail fin repair rates of the test group 1 and the test group 3-6 are lower than that of the test group 2, which shows that the three-stage fermentation strategy + staged flow addition of specific solution + optimization of fermentation medium adopted in the application can effectively promote the metabolism and synthesis of active substances by the bacteria, improve the yield of active substances, and thus improve the repair effect.

Claims

1. A method of preparing a Schizosaccharomyces fermentations product lysate, characterized by, The method comprises the following steps: (1) strain activation: inoculate Bifidobacterium adolescentis into a first seed culture medium, and perform anaerobic culture to obtain an activated bacterial solution; (2) seed liquid culture: inoculate the activated bacterial solution into a second seed culture medium, and perform continuous twice transfer and anaerobic culture to obtain a third seed liquid; (3) fermentation culture: inoculate the third seed liquid into a fermentation tank containing a fermentation culture medium, and perform anaerobic fermentation culture to obtain a fermentation product; wherein the anaerobic fermentation culture comprises a first anaerobic fermentation, a second anaerobic fermentation and a third anaerobic fermentation; the first anaerobic fermentation is performed under the conditions of a temperature of 36-37℃, a pH of 6.0-6.2, a rotation speed of 100-110 rpm and a fermentation time of 8-10 h; the second anaerobic fermentation is performed under the conditions of a temperature of 38.5-39℃, a pH of 5.8-6.0, a rotation speed of 110-115 rpm and a fermentation time of 10-12 h; and the third anaerobic fermentation is performed under the conditions of a temperature of 35-35.5℃, a pH of 5.8-6.0, a rotation speed of 95-100 rpm and a fermentation time of 2-4 h; (4) inactivation and decomposition: after sterilization treatment of the fermentation product, centrifugal washing is performed to collect fermentation bacteria, the fermentation bacteria are resuspended in water, wall breaking treatment is performed, a cell lysate is obtained, and an additive is added into the cell lysate to obtain the Schizosaccharomyces fermentate cell lysate.

2. The method of claim 1, wherein the Schizosaccharomyces fermentati fermentation product lysate is prepared by, In step (1), the Bifidobacterium adolescentis is Bifidobacterium adolescentis HH404-1, and the preservation number of the Bifidobacterium adolescentis HH404-1 is GDMCC No: 67151.

3. The method of claim 1, wherein the Schizosaccharomyces fermentati fermentation product lysate is prepared by, In step (3), a first solution is added during the first anaerobic fermentation, a second solution is added during the second anaerobic fermentation, and a third solution is added during the third anaerobic fermentation. The first solution comprises the following components in mass percentage: 5-10% of glucose and 0.5-1% of yeast treatment; the second solution comprises the following components in mass percentage: 5-10% of glucose, 1-2% of yeast treatment and 0.1-0.3% of glutamine; and the third solution comprises the following components in mass percentage: 0.1-0.3% of trehalose and 0.03-0.05% of serine.

4. The method of claim 3, wherein the Schizosaccharomyces fermentati fermentation product lysate is prepared by, The addition amount of the first solution is 2-5% of the mass of the initial fermentation material, the addition amount of the second solution is 4-7% of the mass of the initial fermentation material, and the addition amount of the third solution is 1-2% of the mass of the initial fermentation material.

5. The method of claim 3, wherein the Schizosaccharomyces fermentati fermentation product lysate is prepared by, The preparation method of the yeast treatment is as follows: after yeast cell bodies of Saccharomyces cerevisiae are broken at 30-40 MPa to obtain a bacterial solution, a composite enzyme is added into the bacterial solution, and enzymolysis is performed at 45-50℃ and pH 5.0-5.5 for 2-3 h to obtain the yeast treatment; wherein the composite enzyme is prepared by mixing cellulase and beta-glucanase at a mass ratio of 1:

2.

6. The method of claim 1, wherein the Schizosaccharomyces fermentati fermentation product lysate is prepared by, In step (3), the fermentation medium comprises the following raw material components in mass percentage: glucose 4.0-5.0%, yeast extract 1.5-1.8%, peptone 0.8-1.2%, potassium phosphate dibasic 0.4-0.7%, glycyrrhiza extract 0.3-0.5%, manganese sulfate 0.01-0.02%, magnesium sulfate 0.01-0.03%, anhydrous sodium acetate 0.4-0.7%, ammonium citrate 0.12-0.18%, and cysteine 0.1-0.3%.

7. The method of claim 1, wherein the Schizosaccharomyces fermentati fermentation product lysate is prepared by, In step (4), the wall-breaking treatment is as follows: the resuspended fermentation bacteria are placed in a high-pressure cell disrupter, and are disrupted at 135-140 Mpa, and after repeated twice, are transferred into an ultrasonic disrupter, and are disrupted at ice bath, 20-30 Hz, and 130-150 w for 5-10 min, so that the cell lysate is obtained.

8. The method of claim 1, wherein the Schizosaccharomyces fermentati fermentation product lysate is prepared by, In step (4), the adjuvant is a suspending agent and a preservative.

9. The Schizochytrium sp. fermentation product lysate prepared by the preparation method according to any one of claims 1-8.

10. The use of the Schizochytrium sp. fermentation product lysate according to claim 9 in the preparation of cosmetics.

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