WO2021057022A1 - 珍珠制备品及其制备方法和用途 - Google Patents

珍珠制备品及其制备方法和用途 Download PDF

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WO2021057022A1
WO2021057022A1 PCT/CN2020/087099 CN2020087099W WO2021057022A1 WO 2021057022 A1 WO2021057022 A1 WO 2021057022A1 CN 2020087099 W CN2020087099 W CN 2020087099W WO 2021057022 A1 WO2021057022 A1 WO 2021057022A1
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Prior art keywords
freeze
pearl
preparation
solution
powder
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PCT/CN2020/087099
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English (en)
French (fr)
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王菁
杨安全
莫家欢
张丽华
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欧诗漫生物股份有限公司
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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
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A61K8/987—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of species other than mammals or birds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment 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
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • 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
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • 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
    • A61K2800/84—Products or compounds obtained by lyophilisation, freeze-drying

Definitions

  • This application relates to a pearl preparation, its preparation method, and its use.
  • Aging is a special law of nature, and anti-aging is also a technical problem that civilization has always wanted to overcome. Especially in cosmetics and health products, products with anti-aging functions are very popular among consumers. Many scientific research institutions at home and abroad have carried out comprehensive and in-depth research on the mechanism of aging. Combined with the current development technology, anti-aging mechanisms can be roughly divided into seven categories: regulation of extracellular mechanisms, skin whitening function, improvement of stratum corneum barrier function, promotion of cell proliferation, resistance to cell apoptosis, anti-inflammatory effect and antioxidant effect . These seven mechanisms interact with each other with different focuses. At present, in studying the mechanism of anti-aging active substances, one or more of them are selected to prove that they have anti-aging effects.
  • Pearl or pearl, is a hard, smooth product produced by mollusks (mainly oysters).
  • mollusks mainly oysters.
  • pearls are salty, sweet, cold and non-toxic, so you can calm your eyes; pearls are coated on the surface to moisturize and color. Apply hands and feet to remove skin and diarrhea; drop phlegm, remove facial spots, and stop diarrhea; remove children’s convulsions and calm souls; stop seminal turbidity, relieve acne, treat poison, and make gloss and white.
  • the technical purpose of the present invention is to provide a method that can effectively extract and utilize the anti-aging ingredients in pearls, and provide pearl anti-aging products prepared therefrom, in the hope that they can be used in skin anti-aging and anti-inflammatory products.
  • the present invention provides a method for preparing pearl preparations, which includes the following steps:
  • step 4) Fermentation: After the solution obtained in step 3) is sterilized, it is connected to the microbial expansion broth for fermentation culture;
  • Freeze-drying freeze-dry the fermentation broth obtained in step 4) to obtain freeze-dried powder of pearl preparation.
  • the method may further include the steps of washing, refining, and spray drying the pearls.
  • the mixing mass ratio of pearl powder and deionized water can be 1:5-20, and the mixing conditions are: 30-70°C through internal stirring rotor or stirring blade 50-100rpm Stir and mix at speed.
  • step 3 if a weak acid solution is added, the volume ratio of the weak acid solution and the mixed solution is 1:4-10, preferably 1:5, and the weak acid solution is selected from acetic acid, lemon One or more of acid, lactic acid, malic acid, etc.; preferably lactic acid, acetic acid or citric acid.
  • the added microbial expansion culture solution accounts for 1-10% by volume of the solution obtained in step 3), and the microorganisms used are selected from the group consisting of Saccharomyces cerevisiae, grape juice yeast, lactic acid bacteria, and subtilis One or more of Bacillus, Bacillus licheniformis, etc.; preferably, Saccharomyces cerevisiae, Lactobacillus rhamnosus, Bacillus subtilis or Bacillus licheniformis; fermentation culture conditions can be: in the fermentation tank at 25-38°C, pH The value is 7.0 ⁇ 7.5, 300 ⁇ 500r/min, ventilation 3-5L/min and cultivation for 24 ⁇ 72 hours.
  • step 5 after stirring the fermentation broth at 85-100°C for 20-50 minutes for inactivation, the sterile filtrate is obtained by pressure filtration through a 0.22 ⁇ m filter membrane, and the aseptic filtrate is obtained.
  • the culture solution and the lyophilized protective agent are mixed at a volume/mass ratio of 100:0.5-2.
  • the volume unit is ml and the mass unit is gram, using liquid nitrogen or at -70 ⁇ 85°C Pre-freeze for 3-10 hours, then put it into a vacuum freeze-drying bin and freeze-dry for 24 to 72 hours. Finally, freeze-dried powder with a moisture content of 2 to 5% by weight is harvested.
  • the method may further include the step of storing the freeze-dried powder in a brown vial or vacuum-packed plastic bag, preferably at 1-10°C store.
  • the present invention provides a pearl preparation prepared by the above preparation method.
  • the present invention provides a composition comprising at least the aforementioned pearl preparation.
  • the present invention provides the use of the above-mentioned pearl preparation or the above-mentioned composition for preparing an anti-aging product.
  • the anti-aging product is a health care product or a cosmetic.
  • the anti-aging product does not contain preservatives.
  • the pseudo-agglomeration of pearl powder can be broken up, and then further smashed to obtain a small and concentrated small particle size powder with a small particle size distribution range.
  • Retaining part of the calcium in the pearl is beneficial to increase the calcium ion channel of the skin, and is beneficial to the pearl anti-aging polypeptide to cross the skin barrier and act on inflammatory cells and basal fibroblasts;
  • the pearl preparation can effectively inhibit the expression of inflammatory factors bcl-2, caspase-3 and caspase-9, inhibit the production of reactive oxygen species and malondialdehyde, and simultaneously promote the production of glutathione and superoxide dismutase produce.
  • inflammatory factors bcl-2, caspase-3 and caspase-9 inhibit the production of reactive oxygen species and malondialdehyde, and simultaneously promote the production of glutathione and superoxide dismutase produce.
  • Through the mitochondrial anti-aging pathway it can effectively repair senescent cells and prolong cell activity. It has great application prospects in anti-aging cosmetics and health products.
  • Figure 1 shows the electron micrographs of the pearl powder before and after the jet milling treatment (Figure A: before treatment, Figure B: after treatment).
  • Figure 2 shows the particle size distribution of the pearl powder after jet pulverization.
  • Figure 3 shows the SDS-PAGE electrophoresis diagrams of pearl preparations before and after microbial fermentation.
  • Figure A Lane 1 is the pre-fermentation test sample, Lane 2 is the standard protein;
  • Figure B Lanes 1 and 3 are the standard protein, Lane 2 is the fermentation After the test sample.
  • Figure 4 shows the effects of the pearl preparation prepared according to Preparation Example 1 of the present invention on various oxidation indicators in keratinocytes at different concentrations (1*10 -4 g/ml and 1*10 -3 g/ml) ( Figure 4( a): ROS; Fig. 4(b): GSH-Px; Fig. 4(c): MDA; and Fig. 4(d): SOD) influence diagram.
  • Figure 5 shows a schematic diagram of the effect of pearl preparations prepared according to Preparation Example 2 of the present invention on the expression of cytoinflammatory factors at different concentrations (1*10 -4 g/ml and 1*10 -3 g/ml) (swimming lanes) 1: blank, lane 2: UV treatment, lane 3: UV+10 -4 g/ml, lane 4: UV+10 -3 g/ml).
  • Step 1) After the pearls are cleaned, refined and spray dried, they are passed through a jet mill to obtain ultra-fine pearl powder with a particle size of 0.1 to 1 micron;
  • Step 2 Mix 1kg of pearl powder and deionized water at a mass ratio of 1:10 at 30°C through an internal stirring rotor or stirring blade at a speed of 50rpm;
  • Step 3 Add 20% (v/v) lactic acid solution to dissolve and filter;
  • Step 4 After sterilizing the solution obtained after filtering in the previous step, connect to 5% Saccharomyces cerevisiae expansion culture solution, and place it in a fermentation tank at 28°C, pH 7.0, rotation speed 300r/min, and ventilation 3L/min Cultivate for 36 hours;
  • Step 5 After stirring the fermentation broth at 85°C for 20 minutes for inactivation, press filtration through a 0.22 ⁇ m filter membrane to obtain a sterile filtrate;
  • Step 6) Mix the filtrate with the freeze-dried protective agent at a ratio of 100:0.5 (volume/mass ml/g), pre-freeze with liquid nitrogen for 2 hours, and then put it into a vacuum freeze-drying bin for 12 hours to freeze-dry. The freeze-dried powder is finally obtained;
  • Step 7) The lyophilized powder is canned in a brown vial and stored at 4°C.
  • Step 1) After the pearls are cleaned, refined and spray dried, they are passed through a jet mill to obtain ultra-fine pearl powder with a particle size of 0.1-1 micron;
  • Step 2 Stir and mix 1kg of pearl powder and deionized water at a mass ratio of 1:5 at 30°C through an internal stirring rotor or stirring blade at a speed of 50rpm;
  • Step 3 Add 20% (v/v) acetic acid solution to dissolve and filter;
  • Step 4 After sterilizing the solution obtained after filtering in the previous step, insert 5% Saccharomyces cerevisiae expansion culture solution and place it in a fermentation tank at 28°C, pH 6.8, rotation speed 250r/min, ventilation 4L/min, and culture 36 hour;
  • Step 5 After stirring the fermentation broth at 85°C for 20 minutes for inactivation, press filtration through a 0.22 ⁇ m filter membrane to obtain a sterile filtrate;
  • Step 6 Mix the filtrate and the freeze-dried protective agent at a ratio of 100:0.5 (volume/mass), pre-freeze at -80° C. for 3 hours, and then put it into a vacuum freeze-drying bin for 12 hours to freeze-dry. The freeze-dried powder is finally obtained;
  • Step 7) The lyophilized powder is canned in a brown vial and stored at 4°C.
  • Step 1) After the pearls are cleaned, refined and spray dried, they are passed through a jet mill to obtain ultra-fine pearl powder with a particle size of 0.1-1 micron;
  • Step 2 Stir and mix 1kg of pearl powder and deionized water at a mass ratio of 1:5 at 30°C through an internal stirring rotor or stirring blade at a speed of 50rpm;
  • Step 3 Add 20% (v/v) lactic acid solution to dissolve and filter;
  • Step 4 After sterilizing the solution obtained after filtering in the previous step, insert 5% Lactobacillus expansion culture solution and place it in a fermentation tank at 37°C, pH 7.0, rotating speed 300r/min, and ventilating 3L/min for 36 hour;
  • Step 5 After stirring the fermentation broth at 85°C for 20 minutes for inactivation, press filtration through a 0.22 ⁇ m filter membrane to obtain a sterile filtrate;
  • Step 6 Mix the filtrate and the freeze-dried protective agent at a ratio of 100:0.5 (volume/mass), pre-freeze at -80°C for 5 hours, and then put it into a vacuum freeze-drying bin for 12 hours to freeze-dry. The freeze-dried powder is finally obtained;
  • Step 7) The lyophilized powder is canned in a brown vial and stored at 4°C.
  • Step 1) After the pearls are cleaned, refined and spray dried, they are passed through a jet mill to obtain ultra-fine pearl powder with a particle size of 0.3-1 microns;
  • Step 2 Stir and mix 1kg of pearl powder and deionized water at a mass ratio of 1:5 at 30°C through an internal stirring rotor or stirring blade at a speed of 50rpm;
  • Step 3 Pass the excess carbon dioxide into the bottom of the mixed solution for 0.5 hour, and then filter;
  • Step 4) After sterilizing the solution obtained after filtering in the previous step, insert a 5% lactic acid bacteria and Saccharomyces cerevisiae (1:1 mass ratio) mixed expansion culture solution, and place it in a fermentation tank at 37°C, pH 7.0, Cultivate for 36 hours under the rotation speed of 300r/min and ventilation of 3L/min;
  • Step 5 After stirring the fermentation broth at 85°C for 20 minutes for inactivation, press filtration through a 0.22 ⁇ m filter membrane to obtain a sterile filtrate;
  • Step 6 Mix the filtrate and the freeze-dried protective agent at a ratio of 100:0.5 (volume/mass), pre-freeze at -80° C. for 3 hours, and then put it into a vacuum freeze-drying bin for 12 hours to freeze-dry. The freeze-dried powder is finally obtained;
  • Step 7) The lyophilized powder is canned in a brown vial and stored at 4°C.
  • Step 1) After the pearls are cleaned, refined and spray dried, they are passed through a jet mill to obtain ultra-fine pearl powder with a particle size of 0.3-1 microns;
  • Step 2 Stir and mix 1kg of pearl powder and deionized water at a mass ratio of 1:5 at 30°C through an internal stirring rotor or stirring blade at a speed of 50rpm;
  • Step 3 Add 20% (v/v) acetic acid solution to dissolve and filter;
  • Step 4 After sterilizing the solution obtained after filtering in the previous step, insert 5% lactic acid bacteria expansion culture solution, and place it in a fermentation tank at 37°C, pH 7.0, rotation speed 300r/min, ventilation 3L/min, and cultivate 36 hour;
  • Step 5 After stirring the fermentation broth at 85°C for 20 minutes for inactivation, press filtration through a 0.22 ⁇ m filter membrane to obtain a sterile filtrate;
  • Step 6 Mix the filtrate and the freeze-dried protective agent at a ratio of 100:0.5 (volume/mass), pre-freeze for 5 hours under liquid nitrogen, and then put it into a vacuum freeze-drying bin for 12 hours to freeze-dry. The freeze-dried powder is finally obtained;
  • Step 7) The lyophilized powder is canned in a brown vial and stored at 4°C.
  • Step 1) After the pearls are cleaned, refined and spray dried, they are passed through a jet mill to obtain ultra-fine pearl powder with a particle size of 0.3-1 microns;
  • Step 2 Stir and mix 1kg of pearl powder and deionized water at a mass ratio of 1:5 at 30°C through an internal stirring rotor or stirring blade at a speed of 50rpm;
  • Step 3 Add 20% (v/v) lactic acid solution to dissolve and filter;
  • Step 4) After sterilizing the solution obtained after filtering in the previous step, insert 5% lactic acid bacteria expansion culture solution, and place it in a fermentation tank at 37°C, pH 6.5, rotating speed 300r/min, and ventilating 36 at 3L/min. hour;
  • Step 5 After stirring the fermentation broth at 85°C for 20 minutes for inactivation, press filtration through a 0.22 ⁇ m filter membrane to obtain a sterile filtrate;
  • Step 6 Mix the filtrate and the freeze-dried protective agent at a ratio of 100:0.5 (volume/mass), pre-freeze with liquid nitrogen for 3 hours, and then put it into a vacuum freeze-drying bin for 12 hours to freeze-dry. The freeze-dried powder is finally obtained;
  • Step 7) The lyophilized powder is canned in a brown vial and stored at 4°C.
  • Step 1) After the pearls are cleaned, refined and spray dried, they are passed through a jet mill to obtain ultra-fine pearl powder with a particle size of 0.3-1 microns;
  • Step 2 Stir and mix 1kg of pearl powder and deionized water at a mass ratio of 1:5 at 30°C through an internal stirring rotor or stirring blade at a speed of 50rpm;
  • Step 3 Pass the excess carbon dioxide into the bottom of the mixed solution for 0.5 hour, and then filter;
  • Step 4 After sterilizing the solution obtained after filtering in the previous step, insert 5% Bacillus subtilis expansion culture solution and place it in a fermentation tank at 37°C, pH 6.5, rotation speed 300r/min, and ventilation 3L/min Cultivate for 36 hours;
  • Step 5 After stirring the fermentation broth at 85°C for 20 minutes for inactivation, press filtration through a 0.22 ⁇ m filter membrane to obtain a sterile filtrate;
  • Step 6 Mix the filtrate and the freeze-dried protective agent at a ratio of 100:0.5 (volume/mass), pre-freeze with liquid nitrogen, and then put it into a vacuum freeze-drying bin and freeze-dry for 12 hours. The freeze-dried powder is finally obtained;
  • Step 7) The lyophilized powder is canned in a brown vial and stored at 4°C.
  • Step 1) After the pearls are cleaned, refined and spray dried, they are passed through a jet mill to obtain ultra-fine pearl powder with a particle size of 0.3-1 microns;
  • Step 2 Stir and mix 1kg of pearl powder and deionized water at a mass ratio of 1:5 at 30°C through an internal stirring rotor or stirring blade at a speed of 50rpm;
  • Step 3 Add 20% (v/v) acetic acid solution to dissolve and filter;
  • Step 4 After sterilizing the solution obtained after filtering in the previous step, insert 5% Bacillus subtilis expansion culture solution and place it in a fermentation tank at 37°C, pH 6.8, rotation speed 300r/min, and ventilation 3L/min Cultivate for 36 hours;
  • Step 5 After stirring the fermentation broth at 85°C for 20 minutes for inactivation, press filtration through a 0.22 ⁇ m filter membrane to obtain a sterile filtrate;
  • Step 6) Mix the filtrate with the freeze-dried protective agent at a ratio of 100:0.5 (volume/mass), pre-freeze with liquid nitrogen or at -80°C, and then put it into a vacuum freeze-drying bin and freeze-dry for 24 hours. The freeze-dried powder is finally obtained;
  • Step 7) The lyophilized powder is canned in a brown vial and stored at 4°C.
  • Step 1) After the pearls are cleaned, refined and spray dried, they are passed through a jet mill to obtain ultra-fine pearl powder with a particle size of 0.3-1 microns;
  • Step 2 Stir and mix 1kg of pearl powder and deionized water at a mass ratio of 1:5 at 30°C through an internal stirring rotor or stirring blade at a speed of 50rpm;
  • Step 3) Add 15% lactic acid solution to dissolve and filter;
  • Step 4 After sterilizing the solution obtained after filtering in the previous step, insert 5% Bacillus subtilis expansion culture solution and place it in a fermentation tank at 37°C, pH 6.8, rotation speed 300r/min, and ventilation 3L/min Cultivate for 48 hours;
  • Step 4) After stirring for 20 minutes at 85°C for inactivation, press filtration through a 0.22 ⁇ m filter membrane to obtain a sterile filtrate;
  • Step 5 The filtrate and the freeze-dried protective agent are mixed at a ratio of 100:0.5 (volume/mass), pre-freeze at -80°C for 1 hour, and then put into a vacuum freeze-drying bin for freeze-drying for 72 hours. Finally, the lyophilized powder is obtained.
  • Step 6 The lyophilized powder is canned in a brown vial and stored at 4°C.
  • the refractive index is selected as 1.61, and the absorptance is selected as 1; it is determined according to GB/T 19077.1-2008.
  • each group of cells is ultrasonically broken on ice, and the cell homogenate is collected by centrifugation on a centrifuge, and the absorbance of each group is measured according to the instructions of the Nanjing Jiancheng kit, and measured by the BCA protein concentration determination kit Corresponding to the protein concentration of each group, finally calculate the content of ROS, SOD, and MDA in each group.
  • Test method Western blot method to detect the protein expression levels of Bcl-2, Bax, Caspase-3 and Caspase-9
  • the pearl preparation of the present application still has a very significant anti-aging ability at a concentration of 1/10,000, and it acts on the mitochondria through the PI3K/AKT pathway to play an anti-aging effect.

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Abstract

本发明提供一种珍珠制备品及其制备方法和用途。所述珍珠制备品通过以下方法制备：1)将珍珠用气流粉碎机粉碎；2)将粉碎的珍珠粉与去离子水混合得到混合液；3)将混合液用弱酸溶解；4)将步骤3)获得的溶液接入微生物扩培菌液进行发酵培养；以及5)将步骤4)中得到的发酵培养液冻干，即获得珍珠制备品冻干粉。所述珍珠制备品具有非常显著的抗衰能力，并且其通过PI3K/AKT通路作用于线粒体起到抗衰老作用。

Description

珍珠制备品及其制备方法和用途 技术领域
本申请涉及一种珍珠制备品，其制备方法、及其用途。
背景技术
衰老是大自然生物的特殊规律，抗衰也是人类一直想要克服的技术难题。尤其在化妆品和保健品中，具有抗衰老功能的产品深受消费者的欢迎。国内外不少科研单位，就衰老机理展开了全面深入的研究。结合现有的发展技术，抗衰老机制大体可分为七大类：细胞外机制的调节、皮肤美白功能、改善角质层屏障功能、促细胞增殖、抵御细胞凋亡、抗炎作用以及抗氧化作用。这七个机制相互作用，侧重点不同。目前，在研究抗衰活性物的作用机制中都会选择其中的一种或者几种去证明其具备抗衰老作用。
珍珠，或称真珠(Pearl)，是一种由软体动物(主要是牡蛎)生产的硬的、圆滑的产物。在《本草纲目》中特别写道：珍珠味咸甘寒无毒，镇心点目；珍珠涂面，令人润泽好颜色。涂手足，去皮肤逆胪；坠痰，除面斑，止泻；除小儿惊热，安魂魄；止遗精白浊，解痘疗毒令光泽洁白等。鉴于珍珠的以上功能，有必要开发一种方法，其能够有效地提取和利用珍珠中的抗衰老成分。
发明内容
技术目的
本发明的技术目的是提供一种方法，其能够有效地提取和利用珍珠中的抗衰老成分，以及提供由其制备的珍珠抗衰老产品，以期可用于皮肤抗衰老、抗炎产品中。
技术方案
一方面，本发明提供一种珍珠制备品的制备方法，其包括以下步骤：
1)粉碎：将珍珠用气流粉碎机粉碎，获得粒径为小于6微米，优选0.3-1微米的超微细珍珠粉；
2)与水混合：将珍珠粉与去离子水混合得到混合液；
3)弱酸溶解：将过量二氧化碳通入混合液底部，或添加弱酸溶液以使所述混合液溶解；
4)发酵：将步骤3)获得的溶液灭菌后，接入微生物扩培菌液进行发酵培养；
5)冻干：将步骤4)中得到的发酵培养液冻干获得珍珠制备品冻干粉。
根据本发明的具体实施方式，在步骤1)之前，所述方法还可包括对珍珠进行清洗、磨浆、喷雾干燥的步骤。
根据本发明的具体实施方式，在步骤2)中，珍珠粉与去离子水混合质量比可为1:5～20，混合条件为：30～70℃下通过内部搅拌转子或者搅拌叶50～100rpm速度下搅拌混合。
根据本发明的具体实施方式，在步骤3)中，如果添加弱酸溶液，则弱酸溶液和混合液的体积比例为1:4～10，优选为1:5，所述弱酸溶液选自乙酸、柠檬酸、乳酸、苹果酸等中的一种或多种；优选为乳酸、乙酸或柠檬酸。
根据本发明的具体实施方式，在步骤4)中，添加的微生物扩培菌液占步骤3)中获得溶液的1～10体积％，所用的微生物选自酿酒酵母、葡萄汁酵母、乳酸菌、枯草芽孢杆菌、地衣芽孢杆菌等中的一种或多种；优选为酿酒酵母、鼠李糖乳杆菌、枯草芽孢杆菌或地衣芽孢杆菌；发酵培养条件可为：发酵罐中25～38℃下，pH值7.0～7.5、300～500r/min、通风3-5L/min和培养24～72小时。
根据本发明的具体实施方式，在步骤5)中，在85～100℃下搅拌发酵培养液20～50min进行灭活后，通过0.22μm的过滤膜压滤获取无菌过滤液，并将无菌培养液与冻干保护剂以体积/质量比例为100：0.5～2进行混合，在上述体积/质量比例中，体积单位为ml，质量单位为克，采用液氮或者在-70～85℃下进行预冻3～10h，随后放入真空冷冻干燥仓中冻干24～72小时。最终收获水分含量在2～5重量％的冻干粉。
根据本发明的具体实施方式，在步骤5)后，所述方法还可包括将冻干粉置于棕色的西林瓶中或者真空包装的塑料袋中进行储存的步骤，优选在1～10℃下储存。
另一方面，本发明提供一种由上述制备方法制备的珍珠制备品。
另一方面，本发明提供一种组合物，其至少包含上述珍珠制备品。
再一方面，本发明提供上述珍珠制备品或上述组合物用于制备抗衰产品的用途。
根据本发明的具体实施方式，所述抗衰产品为保健品或化妆品。
根据本发明的具体实施方式，所述抗衰产品不含防腐剂。
有益效果
本发明的方法具有以下优点：
1.经过气流粉碎处理，能够打散珍珠粉末的假性团聚，并且再进一步打碎得到粒径分布范围小且集中的小粒径粉体。
2.通过弱酸溶解以温和方式无损地提取珍珠中酸性溶液成分；
3.保留珍珠中的部分钙质，有利于提高皮肤钙离子通道，有利于珍珠抗衰多肽越过皮肤屏障，作用于炎症细胞和基底成纤维细胞；
4.通过微生物分解的方式加以降解，获得小分子多肽，从而在微生物的作用下获得更为丰富的活性混合物，同时，由于多种微生物代谢产物中含有抑菌肽，能够保证珍珠制备品后期不必添加防腐剂；
5.所述珍珠制备品能够有效抑制炎症因子bcl-2、caspase-3和caspase-9的表达，抑制活性氧、丙二醛的产生，并且同时促进谷胱甘肽和超氧化物歧化酶的产生。通过线粒体抗衰途径，有效修护衰老细胞，延长细胞活性，在抗衰老化妆品和保健品中具有很大的应用前景。
附图说明
图1显示经过气流粉碎处理前后(图A：处理前，图B：处理后)的珍珠粉末的电镜图。
图2显示经过气流粉碎处理后的珍珠粉末的粒径分布。
图3显示微生物发酵前后珍珠制备品的SDS-PAGE电泳图，其中，图A：泳道1为发酵前测试样品，泳道2为标准蛋白；图B：泳道1和3为标准蛋白，泳道2为发酵后的测试样品。
图4显示根据本发明的制备实施例1制备的珍珠制备品在不同浓度下(1*10 -4g/ml和1*10 -3g/ml)对于角质细胞中各氧化指标(图4(a)：ROS；图4(b)：GSH-Px；图4(c)：MDA；和图4(d)：SOD)影响的示意图。
图5显示根据本发明的制备实施例2制备的珍珠制备品在不同浓度下(1*10 -4g/ml和1*10 -3g/ml)对于细胞炎症因子表达的影响的示意图(泳道1：空白，泳道2：UV处理，泳道3：UV+10 -4g/ml，泳道4：UV+10 -3g/ml)。
具体实施方式
实验材料：珍珠采购于浙江欧诗漫集团德清生物科技有限公司，角质形成细胞和成纤维细胞采购于中国科学院典型培养物保藏委员会细胞库，抗体均来自于美国Cell Signaling Technology,Inc(CST)。
制备实施例1
步骤1)将珍珠清洗干净，磨浆，喷雾干燥后，过气流粉碎机，获得粒径为0.1～1微米的超微细珍珠粉；
步骤2)将1kg珍珠粉与去离子水按1:10的质量比在30℃下通过内部搅拌转子或者搅拌叶在50rpm速度下搅拌混合；
步骤3)添加20％(v/v)的乳酸液加以溶解并过滤；
步骤4)将上一步骤过滤后得到溶液灭菌后，接入5％的酿酒酵母扩培菌液，置于发酵罐中于28℃，pH值7.0、转速300r/min、通风3L/min下培养36小时；
步骤5)在85℃下搅拌发酵培养液20min进行灭活后，通过0.22μm的过滤膜压滤获得无菌过滤液；
步骤6)将过滤液与冻干保护剂以100:0.5(体积/质量ml/g)比例混合，采用液氮进行预冻2h，随后放入真空冷冻干燥仓中冻干12小时。最终得到冻干粉；
步骤7)将冻干粉罐装于棕色的西林瓶中在4℃下储存。
制备实施例2
步骤1)将珍珠清洗干净，磨浆，喷雾干燥后，过气流粉碎机，获得粒径为0.1～1微米超微细珍珠粉；
步骤2)将1kg珍珠粉与去离子水按1:5的质量比在30℃下通过内部搅拌转子或者搅拌叶在50rpm速度下搅拌混合；
步骤3)添加20％(v/v)的乙酸溶液加以溶解并过滤；
步骤4)将上一步骤过滤后得到溶液灭菌后，接入5％的酿酒酵母扩培菌液，置于发酵罐中28℃，pH值6.8、转速250r/min、通风4L/min培养36小时；
步骤5)在85℃下搅拌发酵培养液20min进行灭活后，通过0.22μm的过滤膜压滤获得无菌过滤液；
步骤6)将过滤液与冻干保护剂以100:0.5(体积/质量)比例混合，采用在-80℃下进行预冻3h，随后放入真空冷冻干燥仓中冻干12小时。最终得到冻干粉；
步骤7)将冻干粉罐装于棕色的西林瓶中在4℃下储存。
制备实施例3
步骤1)将珍珠清洗干净，磨浆，喷雾干燥后，过气流粉碎机，获得粒径为0.1～1微米超微细珍珠粉；
步骤2)将1kg珍珠粉与去离子水按1:5的质量比在30℃下通过内部搅拌转子或者搅拌叶在50rpm速度下搅拌混合；
步骤3)添加20％(v/v)的乳酸溶液加以溶解并过滤；
步骤4)将上一步骤过滤后得到溶液灭菌后，接入5％的乳酸杆菌扩培菌液，置于发酵罐中37℃，pH值7.0、转速300r/min、通风3L/min培养36小时；
步骤5)在85℃下搅拌发酵培养液20min进行灭活后，通过0.22μm的过滤膜压滤获得无菌过滤液；
步骤6)将过滤液与冻干保护剂以100:0.5(体积/质量)比例混合，采用在-80℃下进行预冻5h，随后放入真空冷冻干燥仓中冻干12小时。最终得到冻干粉；
步骤7)将冻干粉罐装于棕色的西林瓶中在4℃下储存。
制备实施例4
步骤1)将珍珠清洗干净，磨浆，喷雾干燥后，过气流粉碎机，获得粒径为0.3～1微米超微细珍珠粉；
步骤2)将1kg珍珠粉与去离子水按1:5的质量比在30℃下通过内部搅拌转子或者搅拌叶在50rpm速度下搅拌混合；
步骤3)将过量二氧化碳通入混合液底部0.5小时，之后过滤；
步骤4)将上一步骤过滤后得到溶液灭菌后，接入5％的乳酸菌和酿酒酵母(1:1质量比)的混合扩培菌液，置于发酵罐中37℃，pH值7.0、转速300r/min、通风3L/min下培养36小时；
步骤5)在85℃下搅拌发酵培养液20min进行灭活后，通过0.22μm的过滤膜压滤获得无菌过滤液；
步骤6)将过滤液与冻干保护剂以100:0.5(体积/质量)比例混合，采用在-80℃下进行预冻3h，随后放入真空冷冻干燥仓中冻干12小时。最终得到冻干粉；
步骤7)将冻干粉罐装于棕色的西林瓶中在4℃下储存。
制备实施例5
步骤1)将珍珠清洗干净，磨浆，喷雾干燥后，过气流粉碎机，获得粒径为0.3～1微米超微细珍珠粉；
步骤2)将1kg珍珠粉与去离子水按1:5的质量比在30℃下通过内部搅拌转子或者搅拌叶在50rpm速度下搅拌混合；
步骤3)添加20％(v/v)的乙酸溶液加以溶解并过滤；
步骤4)将上一步骤过滤后得到溶液灭菌后，接入5％的乳酸菌扩培菌液，置于发酵罐中37℃，pH值7.0、转速300r/min、通风3L/min下培养36小时；
步骤5)在85℃下搅拌发酵培养液20min进行灭活后，通过0.22μm的过滤膜压滤获得无菌过滤液；
步骤6)将过滤液与冻干保护剂以100:0.5(体积/质量)比例混合，采用液氮下进行预冻5h，随后放入真空冷冻干燥仓中冻干12小时。最终得到冻干粉；
步骤7)将冻干粉罐装于棕色的西林瓶中在4℃下储存。
制备实施例6
步骤1)将珍珠清洗干净，磨浆，喷雾干燥后，过气流粉碎机，获得粒径为0.3～1微米超微细珍珠粉；
步骤2)将1kg珍珠粉与去离子水按1:5的质量比在30℃下通过内部搅拌转子或者搅拌叶在50rpm速度下搅拌混合；
步骤3)添加20％(v/v)的乳酸溶液加以溶解并过滤；
步骤4)将上一步骤过滤后得到溶液灭菌后，接入5％的乳酸菌扩培菌液，置于发酵罐中37℃，pH值6.5、转速300r/min、通风3L/min下培养36小时；
步骤5)在85℃下搅拌发酵培养液20min进行灭活后，通过0.22μm的过滤膜压滤获得无菌过滤液；
步骤6)将过滤液与冻干保护剂以100:0.5(体积/质量)比例混合，采用液氮进行预冻3h，随后放入真空冷冻干燥仓中冻干12小时。最终得到冻干粉；
步骤7)将冻干粉罐装于棕色的西林瓶中在4℃下储存。
制备实施例7
步骤1)将珍珠清洗干净，磨浆，喷雾干燥后，过气流粉碎机，获得粒径为0.3～1微米超微细珍珠粉；
步骤2)将1kg珍珠粉与去离子水按1:5的质量比在30℃下通过内部搅拌转子或者搅拌叶在50rpm速度下搅拌混合；
步骤3)将过量二氧化碳通入混合液底部0.5小时，之后过滤；
步骤4)将上一步骤过滤后得到溶液灭菌后，接入5％的枯草芽孢杆菌扩培菌液，置于发酵罐中37℃，pH值6.5、转速300r/min、通风3L/min下培养36小时；
步骤5)在85℃下搅拌发酵培养液20min进行灭活后，通过0.22μm的过滤膜压滤获得无菌过滤液；
步骤6)将过滤液与冻干保护剂以100:0.5(体积/质量)比例混合，采用液氮进行预冻，随后放入真空冷冻干燥仓中冻干12小时。最终得到冻干粉；
步骤7)将冻干粉罐装于棕色的西林瓶中在4℃下储存。
制备实施例8
步骤1)将珍珠清洗干净，磨浆，喷雾干燥后，过气流粉碎机，获得粒径为0.3～1微米超微细珍珠粉；
步骤2)将1kg珍珠粉与去离子水按1:5的质量比在30℃下通过内部搅拌转子或者搅拌叶在50rpm速度下搅拌混合；
步骤3)添加20％(v/v)的乙酸溶液加以溶解并过滤；
步骤4)将上一步骤过滤后得到溶液灭菌后，接入5％的枯草芽孢杆菌扩培菌液，置于发酵罐中37℃，pH值6.8、转速300r/min、通风3L/min下培养36小时；
步骤5)在85℃下搅拌发酵培养液20min进行灭活后，通过0.22μm的过滤膜压滤获得无菌过滤液；
步骤6)将过滤液与冻干保护剂以100:0.5(体积/质量)比例混合，采用液氮或者在-80℃下进行预冻，随后放入真空冷冻干燥仓中冻干24小时。最终得到冻干粉；
步骤7)将冻干粉罐装于棕色的西林瓶中在4℃下储存。
制备实施例9
步骤1)将珍珠清洗干净，磨浆，喷雾干燥后，过气流粉碎机，获得粒径为0.3～1微米超微细珍珠粉；
步骤2)将1kg珍珠粉与去离子水按1:5的质量比在30℃下通过内部搅拌转子或者搅拌叶在50rpm速度下搅拌混合；
步骤3)添加15％的乳酸溶液加以溶解并过滤；
步骤4)将上一步骤过滤后得到溶液灭菌后，接入5％的枯草芽孢杆菌扩培菌液，置于发酵罐中37℃，pH值6.8、转速300r/min、通风3L/min下培养48小时；
步骤4)在85℃下搅拌20min的灭活后，通过0.22μm的过滤膜压滤获取无菌过滤液；
步骤5)过滤液与冻干保护剂进行100:0.5(体积/质量)比例的混合，进行-80℃进行预冻1h，随后放入真空冷冻干燥仓中进行冻干72小时。最终收获得冻干粉。
步骤6)将冻干粉罐装于棕色的西林瓶中进行4℃下储存。
实验实施例1采用扫描电镜法观察气流粉碎前后的珍珠形态
分别将处理干燥后的样品粉末(分别为经气流粉碎前和气流粉碎后的样品粉末)用双面导电胶带黏到洁净铜台上，经离子溅射镀膜后，在扫描电镜上观察照相，加速电压为20kV。实验结果见图1。
从图1中可以看出，气流粉碎前，粉末假性团聚。经过气流粉碎后，能够打散假性团聚，并且再进一步打碎得到粒径分布范围小且集中的小粒径粉体。
实验实施例2采用激光粒度仪法测量气流粉碎后的珍珠粉末粒径分布
操作步骤：
a)分散纳米珍珠粉试样的液体用去离子水；
b)检查溶液有无气泡，有气泡则用超声消除气泡；
c)检查样品池是否干净；
d)折射率选定在1.61，吸收率选定为1；按GB/T 19077.1-2008规定测定。
结果如图2所示，由图2可见，经过粉碎后的珍珠粉末粒径分布范围窄。
实验实施例3经发酵处理前后的珍珠制备品的SDS-PAGE电泳
采用SDS-PAGE电泳对经发酵处理前后的珍珠制备品进行检测，结果如图3A和图3B所示，从图中可以看出经过发酵处理珍珠制备品的分子量范围明显下降，证实通过本发明的方法获得小分子多肽。
实验实施例4在UV衰老模型中，珍珠制备品对角质细胞中各氧化指标的影响
试验方法：紫外照射损伤角质形成细胞后冰上超声破碎各组细胞，在离心机上离心收集细胞匀浆，并按南京建成试剂盒说明书操作检测各组的吸光值，以BCA蛋白浓度测定试剂盒测定相应各组的蛋白质浓度，最后计算出各组的ROS、SOD、MDA的含量。
如附图4所示，将角质形成细胞通过UV照射后，角质细胞中ROS、谷胱甘肽过氧化物酶以及丙二醛和超氧化物歧化酶的含量均发生显著性变化，随后添加0.01％和0.1％(g/ml)浓度的珍珠制备品作用24小时后进行测定，结果显示，珍珠制备品可显著改善氧化指标(有影响*p＜0.1，显著**p＜0.01，非常显著**p＜0.001)。
实验实施例5在UV衰老模型中，珍珠制备品对于细胞炎症因子表达的影响
试验方法：蛋白免疫印迹法(Western blot)检测Bcl-2、Bax、Caspase-3及Caspase-9蛋白表达水平
六孔板中成纤维细胞生长达80％-90％时，收集各组细胞，丢弃上清液，按每孔100μL的量添加RIPA裂解液(含1％PMSF)，静置3min后将液体移至1.5ml离心管中，冰上静置30min然后离心(4℃，12 000r·min-15000r.min)，将上清吸出至另1个新1.5mL离心管中，即为蛋白。将蛋白样品与5×SDS上样缓冲液以4:1比例混合，沸水加热7min，使蛋白充分变性。配 制分离胶和浓缩胶，将制好的凝胶放入电泳槽内，内外槽加入新鲜配制的电泳缓冲液，拔出样梳，加入蛋白样品，上样量为60μg。电泳结束切割凝胶，转膜(100V，30min)。一抗(1:500)封闭，4℃过夜，二抗(1:10 000)孵育1h，ECL发光液A液和B液按照1:1的比例混合均匀，滴至PVDF膜上，使其均匀覆盖，于暗室中曝光并显影，分析结果。
如附图5显示，在不同浓度的珍珠制备品的作用下，Caspase3和Caspase3凋亡因子的表达均明显减少、抑制凋亡因子Bcl-2的表达增加，表明本发明的珍珠制备品通过PI3K/AKT通路具有抑制炎性因子表达的作用。
综上，本申请的珍珠制备品在万分之一的浓度添加量下仍具备非常显著的抗衰能力，并且其通过PI3K/AKT通路作用于线粒体起到抗衰老作用。

Claims (10)

  1. 一种珍珠制备品的制备方法，其包括以下步骤：
    1)粉碎：将珍珠用气流粉碎机粉碎，获得粒径为小于6微米，优选0.3-1微米的超微细珍珠粉；
    2)混合：将珍珠粉与去离子水混合得到混合液；
    3)弱酸溶解：将过量二氧化碳通入混合液底部，或添加弱酸溶液以使所述混合液溶解；
    4)发酵：将步骤3)中获得的溶液灭菌后接入微生物扩培菌液进行发酵培养；以及
    5)冻干：将步骤4)中得到的发酵培养液冻干获得珍珠制备品冻干粉。
  2. 根据权利要求1所述的方法，其中，在步骤1)之前，所述方法还包括对珍珠进行清洗、磨浆、喷雾干燥的步骤。
  3. 根据权利要求1所述的方法，其中，在步骤2)中，所述珍珠粉与所述去离子水混合质量比为1:5～20。
  4. 根据权利要求1所述的方法，其中，在步骤3)中，所述弱酸溶液和所述混合液的体积比例为1:4～10，优选为1:5，
    优选地，所述弱酸溶液选自乙酸、柠檬酸、乳酸、苹果酸中的一种或多种。
  5. 根据权利要求1所述的方法，其中，在步骤4)中，所述微生物扩培菌液占待接入溶液的1～10体积％，
    优选地，所用的微生物选自酿酒酵母、葡萄汁酵母、乳酸菌、枯草芽孢杆菌、地衣芽孢杆菌中的一种或多种，
    优选地，发酵罐培养条件为转速300～500r/min，温度25～38℃，通风3-5L/min，培养时间为24～72h。
  6. 根据权利要求1所述的方法，其中，在步骤5)中，将发酵培养液灭活后，通过过滤膜压滤获取无菌过滤液，并将无菌培养液与冻干保护剂以体积/质量比例为100:0.5～2进行混合，在上述体积/质量比例中，体积单位为 ml，质量单位为克，采用液氮或者在-70～85℃下进行预冻3～10h，随后放入真空冷冻干燥仓中冻干24～72小时。
  7. 根据权利要求1所述的方法，其中，在步骤5)后，所述方法还包括将冻干粉置于棕色的西林瓶中或者真空包装的塑料袋中进行储存的步骤，优选在1～10℃下储存。
  8. 一种珍珠制备品，其根据权利要求1至7任一项所述的方法制备。
  9. 一种组合物，其至少包含权利要求8所述的珍珠制备品。
  10. 一种如权利要求8所述的珍珠制备品或如权利要求9所述的组合物用于制备抗衰产品的用途，优选地，所述抗衰产品为抗衰老化妆品或抗衰老保健品。
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