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WO2024148658A1 - 一种极端红曲霉高粱发酵产物的制备方法和应用 - Google Patents

一种极端红曲霉高粱发酵产物的制备方法和应用 Download PDF

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Publication number
WO2024148658A1
WO2024148658A1 PCT/CN2023/077547 CN2023077547W WO2024148658A1 WO 2024148658 A1 WO2024148658 A1 WO 2024148658A1 CN 2023077547 W CN2023077547 W CN 2023077547W WO 2024148658 A1 WO2024148658 A1 WO 2024148658A1
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skin
sorghum
monascus
extreme
preparation
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PCT/CN2023/077547
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English (en)
French (fr)
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张超龙
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成都宝鹿生物科技有限公司
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Priority to KR1020237020947A priority Critical patent/KR20240113703A/ko
Publication of WO2024148658A1 publication Critical patent/WO2024148658A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9794—Liliopsida [monocotyledons]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/34—Alcohols
    • A61K8/345—Alcohols containing more than one hydroxy group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/35—Ketones, e.g. benzophenone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/36—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9728—Fungi, e.g. yeasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/005—Preparations for sensitive skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/52—Stabilizers
    • A61K2800/524—Preservatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the invention belongs to the technical field of microbial fermentation, and in particular relates to a preparation method and application of an extreme Monascus sorghum fermentation product.
  • skin is directly exposed to the external environment and is easily affected by sunlight.
  • photoaging is the main external cause of skin aging, and more than 80% of facial aging is caused by ultraviolet rays (UV) in sunlight.
  • UV ultraviolet rays
  • the most important histological difference between photoaged skin and naturally aged skin is that amorphous elastic fibers in the dermis of photoaged skin are excessively accumulated, and collagen fibers show obvious abnormal fractures and structural disorders.
  • the function of aging skin to defend against damage is also significantly weakened: after excessive UV irradiation, the integrity of the skin barrier is destroyed, and the secretory function is significantly reduced, resulting in a significantly increased risk of skin inflammation and even skin malignancies. Therefore, preventing photoaging and preventing its damage to the skin barrier is of great significance for the prevention and treatment of skin-related diseases, and has significant academic value and huge application potential.
  • Oxidative stress plays a central role in the development of skin photoaging.
  • the light energy is absorbed by intracellular pigments, such as NADH-/NADPH, tryptophan or riboflavin.
  • the pigments are activated to form a series of oxidative products and reactive oxygen species, including superoxide anions, hydroxyl radicals, and O2- derived non-free radicals such as H2O2 .
  • superoxide anions including superoxide anions, hydroxyl radicals, and O2- derived non-free radicals
  • H2O2 O2- derived non-free radicals
  • ROS can mediate the activation of RTKs signal cascades and activate multiple apoptosis-related signaling pathways, leading to reduced collagen production and increased synthesis of matrix metalloproteinases (MMPs), ultimately promoting skin aging and causing skin diseases.
  • MMPs matrix metalloproteinases
  • the fermentation products of probiotics and plants are widely proven to have the effects of promoting the development of probiotics and promoting the health of human beings due to their rich content of short-chain fatty acids (SCFAs), alkaloids, active peptides and other metabolites related to human health. It has the functions of maintaining the health of the epithelial barrier, regulating the immune response and improving the body's antioxidant level. In recent years, researchers have shown that this type of fermentation product also has excellent anti-skin photoaging effects.
  • Shin et al. used lactic acid bacteria to ferment Patchouli leaves and proved that it could significantly reduce the levels of MMP-2 and MMP-9, and effectively reduce the degradation of collagen after light exposure; in 2020, Kang et al.
  • the object of the present invention is to provide a preparation method and application of an extreme Monascus sorghum fermentation product.
  • the extreme Monascus sorghum fermentation product of the present invention can be used for skin care.
  • the present invention provides a method for preparing an extreme Monascus sorghum fermentation product, comprising the following steps:
  • the seed liquid of Monascus pubescens YX-1125 was inoculated into sorghum fermentation medium, and aerobic fermentation was carried out in the dark to obtain an extreme Monascus sorghum fermentation product.
  • the deposit number of the Monascus pubescens YX-1125 is: CGMCC 21938;
  • the method for preparing the sorghum fermentation medium comprises the following steps:
  • the mixture is saccharified by saccharifying enzyme and acidified by lactic acid bacteria, and then the solid and liquid are separated, and the liquid component is collected.
  • the liquid component is sterilized to obtain a sorghum fermentation medium.
  • the method further comprises:
  • membrane filtration is performed to collect the filtrate to obtain an extreme Monascus sorghum fermentation product
  • the pore size of the filter membrane used in the membrane filtration is 300 to 5000 Da.
  • the sorghum flour includes red tassel sorghum flour.
  • the particle size of the sorghum flour is 100 mesh; the mass ratio of the sorghum flour to water is 1:(1-20).
  • the saccharification by saccharifying enzyme comprises:
  • the mixture is mixed with ⁇ -amylase and pre-saccharified for 1 to 8 hours to obtain a slurry;
  • the slurry is cooled to 20-80° C. and then mixed with a composite saccharifying enzyme for saccharification for 2-24 hours to obtain a saccharification product.
  • the lactic acid bacteria include Lactobacillus plantarum.
  • the method further comprises sterilizing the filtrate and adding a preservative to the sterilized filtrate;
  • the preservative comprises one or more of p-hydroxyacetophenone, 1,2-hexanediol and potassium sorbate.
  • the present invention also provides a skin external preparation, comprising the extreme Monascus sorghum fermentation product prepared by the preparation method described in the above scheme; the mass percentage of the extreme Monascus sorghum fermentation product in the skin external preparation is 0.5% to 99%.
  • the present invention also provides the use of the extreme Monascus sorghum fermentation product prepared by the preparation method described in the above scheme to prepare a skin external preparation;
  • the skin external preparation is used to inhibit skin damage caused by exogenous stress
  • the skin damage includes: one or more of: skin melanin production, skin microecological imbalance, skin sensitivity, skin inflammation, skin barrier damage, skin elasticity loss and skin wrinkle generation.
  • the exogenous stress includes photoaging.
  • the present invention provides a method for preparing an extreme Monascus sorghum fermentation product, comprising the following steps: inoculating the seed liquid of Monascus pubescens YX-1125 into a sorghum fermentation medium, and performing aerobic fermentation under dark conditions to obtain an extreme Monascus sorghum fermentation product; the Monascus pubescens YX-1125 has a preservation number of CGMCC21938.
  • the extreme Monascus sorghum fermentation product of the present invention is rich in a variety of active ingredients that are beneficial to the skin, and has an effect on the production of skin melanin, imbalance of skin microecology, skin sensitivity, and skin caused by exogenous stresses such as photoaging.
  • the present invention is the first to develop a cosmetic raw material with anti-photoaging and other stress resistance functions based on Monascus pubescens YX-1125, a Maotai liquor brewing microorganism, and provides a scientific basis, which has great economic value and academic significance.
  • Monascus pilosus YX-1125 was deposited in the General Microbiology Center of China Committee for Microbiological Culture Collection on March 29, 2021, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC 21938.
  • FIG1 is a graph showing the effect of 2% by mass of a fermentation product (left) and 5% by mass of a fermentation product (right) on reducing skin red blood streaks;
  • FIG2 is a graph showing the effect of 2% by mass of a fermented product (left) and 5% by mass of a fermented product (right) on reducing skin texture;
  • FIG. 3 is a graph showing the reducing effect of 2% by mass of a fermentation product (left) and 5% by mass of a fermentation product (right) on wrinkles at the corners of the eyes.
  • the present invention provides a method for preparing an extreme Monascus sorghum fermentation product, comprising the following steps:
  • the seed liquid of Monascus pubescens YX-1125 was inoculated into sorghum fermentation medium, and aerobic fermentation was carried out in the dark to obtain an extreme Monascus sorghum fermentation product.
  • the preservation number of the Monascus ruber YX-1125 is: CGMCC 21938.
  • Monascus pilosus YX-1125 is a strain disclosed in the prior art, see (Monascus pilosus YX-1125: An efficient digester for directly treating ultra-high-strength liquor wastewater and producing short-chain fatty acids under multiple-stress conditions. Bioresource Technology. 2021, 331: 125050.).
  • the method for preparing the seed liquid of Monascus pubescens YX-1125 preferably comprises:
  • the temperature of the seed culture is preferably 28 to 40°C; the time of the seed culture is preferably The selected time is 1 to 6 days, and more preferably 3 to 4 days.
  • the seed culture medium uses sterile distilled water as a solvent and includes components with the following concentrations: 50 g/L glucose and 2.5 g/L yeast extract.
  • the ratio of the volume of the seed liquid of Monascus pubescens YX-1125 to the mass of the sorghum fermentation medium is preferably 1 mL:10 g.
  • the preparation method of the sorghum fermentation medium comprises the following steps: mixing sorghum powder and water to obtain a mixture; the mixture is sequentially saccharified by saccharifying enzyme and acidified by lactic acid bacteria, solid-liquid separation is performed, liquid components are collected, and the liquid components are sterilized to obtain the sorghum fermentation medium.
  • the sorghum flour preferably includes red tassel sorghum flour, and more preferably includes red cherry organic sorghum; the red tassel sorghum flour has a higher content of amylopectin and phenolic substances.
  • the particle size of the sorghum flour is preferably 100 mesh; the mass ratio of the sorghum flour to water is preferably 1: (1-20), and more preferably 1: (5-10).
  • the sorghum culture medium is preferably sterilized before use; the sterilization method is preferably high temperature sterilization.
  • the saccharification by saccharifying enzyme preferably comprises: mixing the mixture with ⁇ -amylase, and pre-saccharifying for 1 to 8 hours to obtain a slurry; cooling the slurry to 20 to 80° C. and then mixing it with a composite saccharifying enzyme, and saccharifying for 2 to 24 hours to obtain a saccharification product.
  • the composite saccharifying enzyme is purchased from Shandong Longda Bioengineering Co., Ltd.
  • the pre-saccharification is preferably carried out in a water bath; the pre-saccharification temperature is preferably 50-100°C, more preferably 80°C; the working concentration of the ⁇ -amylase is preferably 50-200U/g, more preferably 100-150U/g; the pre-saccharification is preferably carried out in a rotary shaker, and the rotation speed of the rotary shaker is preferably 50-200rpm, more preferably 100-150rpm.
  • the pre-saccharification time is preferably 2-5h.
  • the temperature of the slurry after cooling is preferably 40-60°C.
  • the working concentration of the composite saccharifying enzyme is preferably 40U/g.
  • the saccharification is preferably carried out in a rotary shaker, and the rotation speed of the rotary shaker is preferably 50-200rpm, more preferably 100-150rpm.
  • the saccharification time is preferably 8-20h.
  • the lactic acid bacteria preferably include Lactobacillus plantarum.
  • the lactic acid bacteria acidification preferably comprises: mixing the saccharification product and lactic acid bacteria, performing acidification, and obtaining an acidified product; the acidification time is preferably 2 to 24 hours, more preferably 8 to 20 hours.
  • the temperature of the aerobic fermentation is preferably 32° C., and the time is preferably 3 days.
  • the present invention preferably further comprises: performing solid-liquid separation on the product of the aerobic fermentation and collecting the liquid component; after removing the ethanol in the liquid component by rotary evaporation, performing membrane filtration and collecting the filtrate to obtain the extreme Monascus sorghum fermentation product; the pore size of the filter membrane used in the membrane filtration is 300 to 5000Da.
  • the function of the solid-liquid separation is to remove the bacterial cells; the method of the solid-liquid separation is preferably centrifugation.
  • the pore size of the filter membrane used in the membrane filtration is preferably 1000Da.
  • the present invention preferably further comprises sterilizing the filtrate and adding a preservative to the sterilized filtrate.
  • the sterilization method is preferably high temperature secondary sterilization or filtration sterilization.
  • the preservative preferably comprises one or more of p-hydroxyacetophenone, 1,2-hexanediol and potassium sorbate.
  • the present invention also provides a skin external preparation, comprising the extreme Monascus sorghum fermentation product prepared by the preparation method described in the above scheme; the mass percentage of the extreme Monascus sorghum fermentation product in the skin external preparation is 0.5% to 99%.
  • the mass percentage of the extreme Monascus sorghum fermentation product in the skin external preparation is preferably 1% to 5%.
  • the present invention also provides the use of the extreme Monascus sorghum fermentation product prepared by the preparation method described in the above scheme to prepare a skin external preparation.
  • the extreme Monascus sorghum fermentation product of the present invention can be used for skin care.
  • the extreme Monascus sorghum fermentation product is used as the active ingredient in the skin external preparation; the active ingredient has an inhibitory effect on metallothioneins, inflammatory factors, intracellular oxygen free radicals, melanin and epidermal harmful microorganisms, and has been proven to be non-toxic through cell toxicology, human patch tests, chicken embryo allantoic membrane tests and animal tests.
  • the skin topical preparation is used to inhibit skin damage caused by exogenous stress;
  • the skin damage preferably includes: one or more of skin melanin production, skin microecological imbalance, skin sensitivity, skin inflammation, skin barrier damage, loss of skin elasticity and skin wrinkle production.
  • the exogenous stress preferably includes photoaging.
  • the skin external preparation preferably includes a facial mask, essence, toner, cream, shampoo, body lotion or intimate parts care solution.
  • Seed solution preparation After a single colony grows on a PDA plate, scrape the spores and hyphae into a liquid seed culture medium and culture at 28°C for 3 days to obtain a seed solution consisting of 50 g/L glucose and 2.5 yeast extract added to sterile distilled water;
  • Strain fermentation Inoculate the seed liquid into the prepared sorghum fermentation medium at a ratio of 1 mL seed liquid/10 g medium, and ferment aerobically at 32°C in the dark for 3 days;
  • the sorghum fermentation medium is prepared by the following steps: crushing red tassel sorghum to a particle size of 100 mesh, adding sterile water, the mass ratio of red tassel sorghum to sterile water is 1:5, adding ⁇ -amylase with a concentration of 100U/g to the mixture of red tassel sorghum and sterile water, pre-saccharifying at a speed of 150rpm in a rotary shaker under 50°C water bath conditions, after 1h of pre-saccharification, cooling the slurry to 60°C, saccharifying with 40U/g of composite saccharifying enzyme at a speed of 100rpm in a rotary shaker for 8h, then adding plant lactobacillus with an effective live bacterial count of 106 cfu/ml for 2h of acidification, removing solids in the acidified product and sterilizing at high temperature to obtain the medium.
  • the extreme Monascus sorghum fermentation product can be used for skin care, and the extreme Monascus sorghum fermentation product is used as the active ingredient in the skin external preparation; the active ingredient has an inhibitory effect on metallothioneins, inflammatory factors, intracellular oxygen free radicals, melanin and epidermal harmful microorganisms, and has been proven to be non-toxic through cell toxicology, human patch tests, chicken embryo allantoic membrane tests and animal tests.
  • 1MTT experiment 24h after cell transfection, cells were inoculated in a 96-well plate and the culture plate was cultured in an incubator. At 0h, 24h, 48h, and 72h, 20 ⁇ l of MTT solution (5mg/ml, Sigma) was added to each well for color development. After incubation for 4h, a certain concentration of fermentation product was added to each well. The absorbance value was detected at 490nm using an enzyme-linked immunosorbent assay to draw a cell growth curve.
  • MTT solution 5mg/ml, Sigma
  • Test method The test animals are kept in individual cages and adapted to the experimental animal room environment for at least 3 days before the test. 24 hours before the test, remove the hair on both sides of the back spine of the test animals, and the hair removal range is about 3cm x 3cm on each side. Take 0.5mL of the sample and apply it to one side 2.5cm x 3cm. 2.5cm of hair was removed from the skin, and the other side of the skin was used as a control. After 4 hours, the hair was removed to observe whether there was any skin irritation. The results showed no irritation.
  • 3HET-CAM test Select 6 intact 9-day-old chicken embryos, and place a certain amount of the test substance in direct contact with the chorioallantoic membrane of the chicken embryo. After a period of action, observe the changes in the blood vessels of the chorioallantoic membrane, and make positive and negative controls at the same time.
  • sorghum hydrolysate was used as raw material to simulate the stress brewing process of Maotai liquor, and the production of extreme Monascus sorghum fermentation products was achieved; in clinical trials, the protection and repair effects of fermentation products on the skin barrier can be comprehensively evaluated through four indicators, namely short-term moisturizing evaluation, long-term moisturizing evaluation, short-term oil control evaluation and long-term oil control evaluation. Two concentrations of 2% and 5% were used to test its effect on the skin barrier. The experimental results showed that in the short-term moisturizing evaluation, the skin moisture content dropped rapidly from an average difference of more than 20 just applied to about 2 after 6 hours.
  • the 2% concentration fermentation liquid has a stronger hydrating ability than the 5% fermentation liquid; in terms of transepidermal water loss (TEWL), the 2% concentration fermentation liquid has the highest TEWL value.
  • the results show that the 2% fermentation liquid has a stronger short-term moisturizing effect.
  • the long-term moisturizing effect although the 5% fermentation liquid showed better skin hydrating ability and higher TEWL value on the second day, the 2% concentration fermentation liquid was better in terms of data after 7 days.
  • 2% fermentation liquid showed a certain oil control effect, and the downward trend was not significant.
  • 5% fermentation liquid has a certain effect of increasing oil content, which may be related to the lower pH value of higher concentration fermentation liquid, and the lower pH value can promote skin oil secretion in the short term.
  • both 2% and 5% fermentation liquids showed significant oil control effects, and the oil control effect was positively proportional to the concentration of fermentation liquid, which shows that the fermentation liquid effectively inhibits the oil secretion of the skin. It is speculated that the reason is that the skin barrier damage is repaired and the balance of epidermal flora is maintained.
  • the fermentation liquid has a good skin barrier protection effect and can effectively maintain the integrity and elasticity of the skin barrier.
  • the 2% concentration fermentation liquid has a stronger long-term skin barrier repair ability
  • the 5% fermentation liquid has a stronger short-term skin barrier repair ability and skin oil control ability.
  • the fermentation liquid has a strong anti-photoaging effect.
  • the fermentation liquid has a good inhibitory effect on various indicators such as skin sensitivity, skin inflammation, skin barrier damage, skin elasticity loss, and skin wrinkle formation caused by photoaging, and can be considered as an excellent potential raw material for skin care products.

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Abstract

本发明提供了一种极端红曲霉高粱发酵产物的制备方法和应用，属于微生物发酵技术领域。将毛红曲霉YX-1125的种子液接种于高粱发酵培养基，于黑暗条件下进行好氧发酵，得到极端红曲霉高粱发酵产物。本发明的极端红曲霉高粱发酵产物对光老化等外源胁迫导致的皮肤黑色素产生、皮肤微生态失衡、皮肤敏感、皮肤炎症、皮肤屏障受损、皮肤弹性丧失和皮肤皱纹产生等各项指标中，均有较好的抑制效果。

Description

一种极端红曲霉高粱发酵产物的制备方法和应用
本申请要求于2023年01月10日提交中国专利局、申请号为“202310036896.8”、发明名称为“一种极端红曲霉高粱发酵产物的制备方法和应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于微生物发酵技术领域，具体涉及一种极端红曲霉高粱发酵产物的制备方法和应用。
背景技术
皮肤会随着时间的推移经历不可逆的老化。并且，皮肤直接暴露于外环境中，容易受到阳光的影响。研究表明，光老化是导致皮肤老化的主要外因，80％以上的面部老化由阳光中的紫外线(UV)引起。光老化皮肤与自然老化皮肤最重要的组织学差异是光老化皮肤真皮组织内无定形弹性纤维呈现过度积累状态，胶原纤维出现明显的异常断裂和结构紊乱。老化的皮肤除美观性下降外，其防御损伤的功能也明显减弱：受过量UV照射后皮肤屏障的完整性被破坏、分泌功能明显下降导致罹患皮肤炎症甚至皮肤恶性肿瘤的风险明显增加。所以，预防光老化，防止其对皮肤屏障的破坏，对防治皮肤相关疾病有重要意义，具有显著的学术价值和巨大的应用潜力。
氧化应激在皮肤光老化的发展过程中扮演了核心角色。当光照射皮肤以后，光能被细胞内色素团，如NADH-/NADPH、色氨酸或核黄素等所吸收，在分子氧存在的情况下，色素团会被激活形成一系列的氧化产物和活性氧，包括超氧阴离子、羟基自由基和O2衍生的非自由基物如H2O2等。在强度过高的UV照射下，胞内产生ROS越来越多并超过胞内氧化还原平衡的调节能力，将导致两个结果：
(1)高水平的ROS将破坏细胞的结构和功能，并导致细胞释放白细胞介素家族IL-1、IL-6、IL-8、IL-10和TNF-α等系列炎症介质，使局部皮肤发生炎症；
(2)ROS可介导激活RTKs信号级联放大，激活多条细胞凋亡相关信号通路，导致胶原生成减少、基质金属蛋白酶(matrixmetalloproteinase，MMPs)合成增多，最终促进皮肤衰老，引起皮肤疾病。
益生菌与植物的发酵产物，又被称为后生元(postbiotics)，因富含短链脂肪酸(SCFAs)、生物碱、活性肽等与人类健康有关的代谢产物，而被广泛证实具有 维护上皮屏障健康、调节免疫应答和提高机体抗氧化水平的功能。近年来，陆续有研究者表明这类发酵产物也具有优秀的抗皮肤光老化效果。2018年Shin等采用乳酸菌发酵藿香叶，证明能明显降低MMP-2和MMP-9的水平，有效减少光照后胶原蛋白的降解；2020年Kang等发现布氏乳杆菌(发现于泡菜中)发酵植物的提取物，可有效抑制阳光诱导的弹性蛋白酶活性和MMPs的表达，促进皮肤胶原蛋白的合成。根据现有文献分析，基于益生菌与植物发酵产物抗皮肤光老化的研究正处于初期探索阶段，其菌种选择还有较大优化空间，关键性代谢产物及其调控方式还不清楚，相关作用机制还不清晰，具有巨大的研究价值。
极端环境下生长的微生物，为了适应生存，经自然选择，通常具有独特的响应机制和代谢产物，以应答因强烈胁迫因子带来的高浓度ROS。从生物技术的角度来看，这种代谢路径和基因表达的可塑性使得极端微生物应用前景广阔，因为它们能够适应不利的环境，并在特定条件下提供高效的酶和丰富的抗氧化代谢产物。作者的极端环境微生物发酵研究工作始于2017年，在国际上首次发现了能够利用尼古丁耐受丝状真菌在高浓度尼古丁胁迫下发酵烟叶生产高活性的糖化酶，在后续研究中，通过代谢分析发现，该极端微生物还大量分泌了有机酸和不饱和脂肪酸等抗氧化和抗炎症代谢产物，以应对环境胁迫带来的ROS损伤。关于极端微生物发酵产物的抗氧化性或抗炎性方面的巨大潜力，尽管国内外学者开展了少量研究，主要集中于环境领域。
但是极端微生物发酵产物用于皮肤健康领域的研究，迄今为止未见有进行研究。
发明内容
有鉴于此，本发明的目的在于提供一种极端红曲霉高粱发酵产物的制备方法和应用，本发明的极端红曲霉高粱发酵产物能够用于皮肤护理。
本发明提供了一种极端红曲霉高粱发酵产物的制备方法，包括以下步骤：
将毛红曲霉YX-1125的种子液接种于高粱发酵培养基，于黑暗条件下进行好氧发酵，得到极端红曲霉高粱发酵产物；
所述毛红曲霉YX-1125的保藏编号为：CGMCC 21938；
所述高粱发酵培养基的制备方法包括以下步骤：
将高粱粉和水混合，得到混合物；
所述混合物依次经过糖化酶糖化和乳酸菌酸化后，固液分离，收集液体组分， 对所述液体组分进行灭菌，得到高粱发酵培养基。
优选的，在所述好氧发酵后，还包括：
对所述好氧发酵的产物进行固液分离，收集液体组分；
旋蒸去除所述液体组分中的乙醇后，进行膜过滤，收集滤液，得到极端红曲霉高粱发酵产物；
所述膜过滤采用的滤膜的孔径为300～5000Da。
优选的，所述高粱粉包括红缨子高粱粉。
优选的，所述高粱粉的粒径为100目；所述高粱粉和水的质量比为1：(1～20)。
优选的，所述糖化酶糖化包括：
将混合物和α-淀粉酶混合，进行1～8h预糖化，得到浆液；
将所述浆液冷却至20～80℃后和复合糖化酶混合，进行2～24h糖化，得到糖化产物。
优选的，所述乳酸菌包括植物乳杆菌。
优选的，在收集滤液后，还包括对所述滤液进行灭菌，在灭菌后的滤液中加入防腐剂；所述防腐剂包括对羟基苯乙酮、1,2-己二醇和山梨酸钾中的一种或几种。
本发明还提供了一种皮肤外用制剂，包括上述方案所述的制备方法制备得到的极端红曲霉高粱发酵产物；所述皮肤外用制剂中极端红曲霉高粱发酵产物的质量百分含量为0.5％～99％。
本发明还提供了上述方案所述的制备方法制备得到的极端红曲霉高粱发酵产物制备皮肤外用制剂的应用；
所述皮肤外用制剂用于抑制外源胁迫导致的皮肤损伤；
所述皮肤损伤包括：皮肤黑色素产生、皮肤微生态失衡、皮肤敏感、皮肤炎症、皮肤屏障受损、皮肤弹性丧失和皮肤皱纹产生中的一种或几种。
优选的，所述外源胁迫包括光老化。
本发明提供了一种极端红曲霉高粱发酵产物的制备方法，包括以下步骤：将毛红曲霉YX-1125的种子液接种于高粱发酵培养基，于黑暗条件下进行好氧发酵，得到极端红曲霉高粱发酵产物；所述毛红曲霉YX-1125的保藏编号为：CGMCC21938。本发明的极端红曲霉高粱发酵产物富含多种有益于皮肤的活性成分，其对光老化等外源胁迫导致的皮肤黑色素产生、皮肤微生态失衡、皮肤敏感、皮肤 炎症、皮肤屏障受损、皮肤弹性丧失和皮肤皱纹产生等各项指标中，均有较好的抑制效果，可被认为是一种安全而优秀的皮肤护理产品原料。本发明率先开发基于毛红曲霉YX-1125这一酱香白酒酿酒微生物的抗光老化等抗逆性功能化妆品原料提供科学依据，具有较大的经济价值与学术意义。
生物保藏说明
毛红曲霉(Monascuspilosus)YX-1125，已于2021年03月29日保藏在中国微生物菌种保藏委员会普通微生物中心，地址北京市朝阳区北辰西路1号院3号，保藏号：CGMCC 21938。
附图说明
图1为质量百分比为2％的发酵产物(左)和质量百分比为5％的发酵产物(右)对皮肤红血丝的降低作用图；
图2为质量百分比为2％的发酵产物(左)和质量百分比为5％的发酵产物(右)对皮肤纹理的降低作用图；
图3为质量百分比为2％的发酵产物(左)和质量百分比为5％的发酵产物(右)对眼角皱纹的降低作用图。
具体实施方式
本发明提供了一种极端红曲霉高粱发酵产物的制备方法，包括以下步骤：
将毛红曲霉YX-1125的种子液接种于高粱发酵培养基，于黑暗条件下进行好氧发酵，得到极端红曲霉高粱发酵产物；
所述毛红曲霉YX-1125的保藏编号为：CGMCC 21938。
在本发明中，毛红曲霉YX-1125是现有技术公开的菌株，具体参见(Monascus pilosus YX-1125:An efficient digester for directly treating ultra-high-strength liquor wastewater and producing short-chain fatty acids under multiple-stress conditions.Bioresource Technology.2021,331:125050.)。
在本发明中，所述毛红曲霉YX-1125的种子液的制备方法优选的包括：
将毛红曲霉YX-1125接种在PDA平板上，于28℃、黑暗条件下培养，至PDA平板上长出单菌落；在PDA平板上长出单菌落之后，将孢子和菌丝刮至液体种子培养基中，于25～45℃条件下进行种子培养，得到种子液；
在本发明中，所述种子培养的温度优选为28～40℃；所述种子培养的时间优 选为1～6d，更优选为3～4d。
在本发明中，所述种子培养基以无菌蒸馏水为溶剂，包括以下浓度的组分：50g/L葡萄糖和2.5g/L酵母浸出物。
在本发明中，所述毛红曲霉YX-1125的种子液的体积和高粱发酵培养基的质量的比例优选为1mL：10g。
在本发明中，所述高粱发酵培养基的制备方法包括以下步骤：将高粱粉和水混合，得到混合物；所述混合物依次经过糖化酶糖化和乳酸菌酸化后，固液分离，收集液体组分，对所述液体组分进行灭菌，得到高粱发酵培养基。
在本发明中，所述高粱粉优选的包括红缨子高粱粉，更优选的包括红樱子有机高粱；所述红缨子高粱粉具有较高含量的支链淀粉和酚类物质。在本发明中，所述高粱粉的粒径优选为100目；所述高粱粉和水的质量比优选为1：(1～20)，更优选为1：(5～10)。
在本发明中，所述高粱培养基在使用前优选的还包括灭菌；所述灭菌的方法优选为高温灭菌法。
在本发明中，所述糖化酶糖化优选的包括：将混合物和α-淀粉酶混合，进行1～8h预糖化，得到浆液；将所述浆液冷却至20～80℃后和复合糖化酶混合，进行2～24h糖化，得到糖化产物。
在本发明中，所述复合糖化酶购自于山东隆大生物工程有限公司。
在本发明中，所述预糖化优选的水浴进行；所述预糖化的温度优选为50～100℃，更优选为80℃；所述α-淀粉酶的工作浓度优选为50～200U/g，更优选为100～150U/g；所述预糖化优选的在旋转摇床中进行，所述旋转摇床的转速优选为50～200rpm，更优选为100～150rpm。在本发明中，所述预糖化的时间优选为2～5h。
在本发明中，所述浆液冷却后的温度优选为40～60℃。在本发明中，所述复合糖化酶的工作浓度优选为40U/g。在本发明中，所述糖化优选的在旋转摇床中进行，所述旋转摇床的转速优选为50～200rpm，更优选为100～150rpm。在本发明中，所述糖化的时间优选为8～20h。
在本发明中，所述乳酸菌优选的包括植物乳杆菌。
在本发明中，所述乳酸菌酸化优选的包括：将糖化产物和乳酸菌混合，进行酸化，得到酸化产物；所述酸化的时间优选为2～24h，更优选为8～20h。
在本发明中，所述好氧发酵的温度优选为32℃，时间优选为3d。
在所述好氧发酵后，本发明优选的还包括：对所述好氧发酵的产物进行固液分离，收集液体组分；旋蒸去除所述液体组分中的乙醇后，进行膜过滤，收集滤液，得到极端红曲霉高粱发酵产物；所述膜过滤采用的滤膜的孔径为300～5000Da。在本发明中，所述固液分离的作用是去除菌体；所述固液分离的方式优选为离心。在本发明中，所述膜过滤采用的滤膜的孔径优选为1000Da。
在收集滤液后，本发明优选的还包括对所述滤液进行灭菌，在灭菌后的滤液中加入防腐剂。在本发明中，所述灭菌的方式优选为高温二次灭菌或过滤除菌法。在本发明中，所述防腐剂优选的包括对羟基苯乙酮、1,2-己二醇和山梨酸钾中的一种或几种。
本发明还提供了一种皮肤外用制剂，包括上述方案所述的制备方法制备得到的极端红曲霉高粱发酵产物；所述皮肤外用制剂中极端红曲霉高粱发酵产物的质量百分含量为0.5％～99％。
在本发明中，所述皮肤外用制剂中极端红曲霉高粱发酵产物的质量百分含量优选为1％～5％。
本发明还提供了上述方案所述的制备方法制备得到的极端红曲霉高粱发酵产物制备皮肤外用制剂的应用。
本发明的极端红曲霉高粱发酵产物能够用于皮肤护理。在本发明中，所述极端红曲霉高粱发酵产物作为所述皮肤外用剂中的活性成分；所述活性成分对金属硫蛋白酶、炎症因子、胞内氧自由基、黑色素和表皮有害微生物均具有抑制作用，且通过细胞毒理、人体斑贴、鸡胚胎尿囊膜试验和动物测试均证明无毒性。
在本发明中，所述皮肤外用制剂用于抑制外源胁迫导致的皮肤损伤；所述皮肤损伤优选的包括：皮肤黑色素产生、皮肤微生态失衡、皮肤敏感、皮肤炎症、皮肤屏障受损、皮肤弹性丧失和皮肤皱纹产生中的一种或几种。
在本发明中，所述外源胁迫优选的包括光老化。
在本发明中，所述皮肤外用剂优选的包括面膜、精华、爽肤水、面霜、洗发水、身体乳或私密部位护理液。
下面将结合本发明中的实施例，对本发明中的技术方案进行清楚、完整地描述。
实施例1
极端红曲霉高粱发酵产物的制备方法：
1、制备孢子：将从毛红曲霉YX-1125接种在PDA平板上，置于28℃、黑暗条件下培养3天；
2、种子液制备：在PDA平板上长出单菌落之后，将孢子和菌丝刮至液体种子培养基中，28℃条件下培养3天，得到种子液，该种子培养液由50g/L葡萄糖和2.5酵母浸出物加入无菌蒸馏水构成；
3、菌株发酵：按照1mL种子液/10g培养基的比例将种子液接种到制备好的高粱发酵培养基中，于32℃、黑暗条件下好氧发酵3天；
所述高粱发酵培养基由以下步骤制备得到：将红缨子高粱粉碎至粒径为100目后加入无菌水，红缨子高粱和无菌水的质量比为1：5，将红缨子高粱和无菌水的混合物中加入浓度为100U/g的α-淀粉酶于50℃水浴条件下在旋转摇床中以150rpm的速度进行预糖化，经过1h的预糖化后，将浆液冷却到60℃，在旋转摇床中以100rpm的速度用为40U/g的复合糖化酶糖化8h，随后加入有效活菌数为106cfu/ml；的植物乳杆菌进行2h酸化，去除酸化产物中的固形物后高温灭菌，既得。
4、发酵产物提取与纯化：固液分离去除菌体后，使用旋蒸去除溶液中的乙醇，再使用1000Da的膜过滤，高温二次灭菌后，加入体积百分浓度为0.1％～0.5％的羟基苯乙酮，即可得到极端红曲霉高粱发酵产物。
极端红曲霉高粱发酵产物能够用于皮肤护理，极端红曲霉高粱发酵产物作为所述皮肤外用剂中的活性成分；所述活性成分为对金属硫蛋白酶、炎症因子，胞内氧自由基、黑色素和表皮有害微生物均具有抑制作用，且通过细胞毒理、人体斑贴、鸡胚胎尿囊膜试验和动物测试均证明无毒性。
其中，①MTT实验：细胞转染24h后，接种细胞于96孔板中，将培养板于培养箱培养。在0h、24h、48h、72h时间点，每孔加20μlMTT液(5mg/ml，Sigma公司)呈色，继续孵育4h后，每孔加入一定浓度发酵产物。使用酶联免疫检测仪于490nm处检测吸光度值，绘制细胞生长曲线图。
②动物急性皮肤刺激试验：《化妆品安全技术规范》2015版；动物数/性别：4支，雌雄各半(雌性未怀孕和未曾产仔)试验方法：试验动物单笼喂养，试验前在实验动物房环境中至少适应3d。试验前24h，将试验动物背部脊椎两侧被毛去除，去毛范围左右各约3cm乘以3cm。取0.5mL样品涂敷与一侧2.5cm乘以 2.5cm去毛皮肤，另一侧皮肤作为对照，4h后去除观察有无皮肤刺激性现象。结果显示无刺激。
③HET-CAM测试：选取6只完好的9日龄鸡胚，将一定量受试物直接与鸡胚尿囊膜接触，作用一段时间之后观察绒毛尿囊膜血管的变化情况，同时做阳性和阴性对照。
利用固定化连续发酵系统以高粱水解液为原料模拟茅台酒胁迫酿造工艺，实现了极端红曲霉高粱发酵产物的生产；在临床试验中，发酵产物对皮肤屏障的保护与修复作用能够通过4项指标进行综合评估，分别为短期保湿评价、长期保湿评价、短期控油评价和长期控油评价。2％和5％两种浓度被用于测试其对皮肤屏障的影响。实验结果表明，在短期保湿评价当中，随着时间的变化肌肤含水量从刚涂抹的平均差值20以上，急速降低为6h以后的2左右，值得注意的是，2％浓度发酵液反而比5％发酵液具有更强的补水能力；而在经表皮水分流失(TEWL)方面，2％浓度发酵液的TEWL值最高，结果表明，2％发酵液拥有更强的短期保湿效果。在长期保湿效果中，虽然5％发酵液在第2天表现出了较好的肌肤补水能力与更高的TEWL值，但是在7天后的数据方面，2％浓度发酵液则更为优秀。在短期控油方面，2％发酵液显示出了一定的控油效果，并下降趋势并不显著，然而值得注意的是，5％发酵液却有一定的增进油脂含量效果，这可能与更高浓度的发酵液pH值更低，而较低的pH值更能在短期内促进皮肤油脂分泌有关。而在长期控油方面，2％和5％的发酵液均表现出了显著的控油效果，且控油效果与发酵液浓度呈正比例效果，这表明发酵液有效抑制了皮肤的油脂分泌，推测原因在于修复了皮肤屏障损伤而维持了表皮菌群平衡。综上，发酵液具有较好的皮肤屏障保护效果，能够有效维护皮肤屏障完整性和弹性，其中，2％浓度发酵液长期皮肤屏障修复能力更强，5％发酵液短期皮肤屏障修复能力和皮肤控油能力更强，该结果暗示不同浓度的发酵液可以用于不同用途的皮肤屏障维护相关护肤产品当中，如图1所示。
通过Antera3D技术，我们测试了2％和5％发酵液对皮肤红血丝、黑色素、皮肤纹理、眼角皱纹、和眼角细纹方面的效果。结果表明，在使用发酵液42天以后，受试者的红血丝(图1)、皮肤纹理(图2)、眼角皱纹(图3)均有不同程度的降低，整体来看2％和5％发酵液均有较好效果，其中5％效果更为明显，但是改善程度不多且与发酵液浓度未成正比例。而黑色素相关效果不明显的可能原 因是，由于未对受试者在使用后的行踪有所限16/21制，较大部分受试者在日常生活中有较长时间的阳光照射经历，同时可以看出，在此情况下的红血丝、皮肤纹理、眼角皱纹的改善，更加证明了该发酵液具有较强的抗光老化效果。综上，该发酵液在光老化导致的皮肤敏感、皮肤炎症、皮肤屏障受损、皮肤弹性丧失和皮肤皱纹产生等各项指标中，具有较好的抑制效果，可被认为是一种优秀的潜在皮肤护理产品原料。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本发明保护范围。

Claims (10)

  1. 一种极端红曲霉高粱发酵产物的制备方法，其特征在于，包括以下步骤：
    将毛红曲霉YX-1125的种子液接种于高粱发酵培养基，于黑暗条件下进行好氧发酵，得到极端红曲霉高粱发酵产物；
    所述毛红曲霉YX-1125的保藏编号为：CGMCC 21938；
    所述高粱发酵培养基的制备方法包括以下步骤：
    将高粱粉和水混合，得到混合物；
    所述混合物依次经过糖化酶糖化和乳酸菌酸化后，固液分离，收集液体组分，对所述液体组分进行灭菌，得到高粱发酵培养基。
  2. 根据权利要求1所述的制备方法，其特征在于，在所述好氧发酵后，还包括：
    对所述好氧发酵的产物进行固液分离，收集液体组分；
    旋蒸去除所述液体组分中的乙醇后，进行膜过滤，收集滤液，得到极端红曲霉高粱发酵产物；
    所述膜过滤采用的滤膜的孔径为300～5000Da。
  3. 根据权利要求1所述的制备方法，其特征在于，所述高粱粉包括红缨子高粱粉。
  4. 根据权利要求1或3所述的制备方法，其特征在于，所述高粱粉的粒径为100目；所述高粱粉和水的质量比为1：(1～20)。
  5. 根据权利要求1所述的制备方法，其特征在于，所述糖化酶糖化包括：
    将混合物和α-淀粉酶混合，进行1～8h预糖化，得到浆液；
    将所述浆液冷却至20～80℃后和复合糖化酶混合，进行2～24h糖化，得到糖化产物。
  6. 根据权利要求1所述的制备方法，其特征在于，所述乳酸菌包括植物乳杆菌。
  7. 根据权利要求2所述的制备方法，其特征在于，在收集滤液后，还包括对所述滤液进行灭菌，在灭菌后的滤液中加入防腐剂；所述防腐剂包括对羟基苯乙酮、1,2-己二醇和山梨酸钾中的一种或几种。
  8. 一种皮肤外用制剂，包括权利要求1～7任意一项所述的制备方法制备得到的极端红曲霉高粱发酵产物；所述皮肤外用制剂中极端红曲霉高粱发酵产物的 质量百分含量为0.5％～99％。
  9. 权利要求1～8任意一项所述的制备方法制备得到的极端红曲霉高粱发酵产物制备皮肤外用制剂的应用；
    所述皮肤外用制剂用于抑制外源胁迫导致的皮肤损伤；
    所述皮肤损伤包括：皮肤黑色素产生、皮肤微生态失衡、皮肤敏感、皮肤炎症、皮肤屏障受损、皮肤弹性丧失和皮肤皱纹产生中的一种或几种。
  10. 根据权利要求9所述的应用，其特征在于，所述外源胁迫包括光老化。
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