TWI764304B - Pearl preparation and preparation method and use thereof - Google Patents
Pearl preparation and preparation method and use thereofInfo
- Publication number
- TWI764304B TWI764304B TW109133715A TW109133715A TWI764304B TW I764304 B TWI764304 B TW I764304B TW 109133715 A TW109133715 A TW 109133715A TW 109133715 A TW109133715 A TW 109133715A TW I764304 B TWI764304 B TW I764304B
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- Taiwan
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- pearl
- freeze
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Images
Classifications
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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
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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
-
- 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
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- 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
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- 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
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Polymers & Plastics (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Dermatology (AREA)
- Food Science & Technology (AREA)
- Engineering & Computer Science (AREA)
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Abstract
本發明提供一種珍珠製備品及其製備方法和用途。所述珍珠製備品通過以下方法製備：1）將珍珠用氣流粉碎機粉碎；2）將粉碎的珍珠粉與去離子水混合得到混合液；3）將混合液用弱酸溶解；4）將步驟3）獲得的溶液接入微生物擴培菌液進行發酵培養；以及5）將步驟4）中得到的發酵培養液凍乾，即獲得珍珠製備品凍乾粉。所述珍珠製備品具有非常顯著的抗衰能力，並且其通過PI3K/AKT路徑作用於線粒體起到抗衰老作用。The present invention provides a pearl preparation and its preparation method and use. The pearl preparation is prepared by the following methods: 1) pulverizing the pearls with a jet mill; 2) mixing the pulverized pearl powder with deionized water to obtain a mixed solution; 3) dissolving the mixed solution with weak acid; 4) dissolving step 3 ) The obtained solution is inserted into the microbial expansion culture solution for fermentation culture; and 5) the fermented culture solution obtained in step 4) is freeze-dried, that is, the freeze-dried powder of the pearl preparation is obtained. The pearl preparation has a very significant anti-aging ability, and it acts on mitochondria through the PI3K/AKT pathway to play an anti-aging effect.
Description
本申請涉及一種珍珠製備品，其製備方法、及其用途。 The present application relates to a pearl preparation, its preparation method, and its use.
衰老是大自然生物的特殊規律，抗衰也是人類一直想要克服的技術難題。尤其在化妝品和保健品中，具有抗衰老功能的產品深受消費者的歡迎。國內外不少科研單位，就衰老機理展開了全面深入的研究。結合現有的發展技術，抗衰老機制大體可分為七大類：細胞外機制的調節、皮膚美白功能、改善角質層屏障功能、促細胞增殖、抵禦細胞凋亡、抗炎作用以及抗氧化作用。這七個機制相互作用，側重點不同。目前，在研究抗衰活性物的作用機制中都會選擇其中的一種或者幾種去證明其具備抗衰老作用。 Aging is a special law of natural organisms, and anti-aging is also a technical problem that human beings have always wanted to overcome. Especially in cosmetics and health care products, products with anti-aging function 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 existing 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 apoptosis, anti-inflammatory effect and antioxidant effect. These seven mechanisms interact with each other with different emphases. At present, in the study of the mechanism of action of anti-aging actives, one or several of them are selected to prove that they have anti-aging effects.
珍珠，或稱真珠(Pearl)，是一種由軟體動物(主要是牡蠣)生產的硬的、圓滑的產物。在《本草綱目》中特別寫道：珍珠味甘、鹹、性寒無毒，鎮心點目；珍珠塗面，令人潤澤好顏色。塗手足，去皮膚逆臚；墜痰，除面斑，止瀉；除小兒驚熱，安魂魄；止遺精白濁，解痘療毒令光澤潔白等。鑒於珍珠的以上功能，有必要開發一種方法，其能夠有效地萃取和利用珍珠中的抗衰老成分。 A pearl, or pearl, is a hard, sleek product produced by mollusks, mainly oysters. In the "Compendium of Materia Medica", it is specially written: Pearls are sweet, salty, cold and non-toxic, soothing and eye-catching; Apply on hands and feet, remove skin inversion; drop phlegm, remove facial spots, stop diarrhea; remove fever in children, soothe the soul; stop seminal emission from white turbidity, relieve acne, treat toxins, and make luster white and so on. In view of the above functions of pearls, it is necessary to develop a method that can effectively extract and utilize the anti-aging ingredients in pearls.
本發明的技術目的是提供一種方法，其能夠有效地萃取和利用珍珠中的抗衰老成分，以及提供由其製備的珍珠抗衰老產品，以期可用於皮膚抗衰老、抗炎產品中。 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, so as to be used in skin anti-aging and anti-inflammatory products.
一方面，本發明提供一種珍珠製備品的製備方法，其包括以下步驟：1)粉碎：將珍珠用氣流粉碎機粉碎，獲得粒徑為小於6微米，優選0.3-1微米的超微細珍珠粉；2)與水混合：將珍珠粉與去離子水混合得到混合液；3)弱酸溶解：將過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中之珍珠粉溶解；4)發酵：將步驟3)獲得的溶液滅菌後，接入微生物擴培菌液進行發酵培養；5)凍乾：將步驟4)中得到的發酵培養液凍乾獲得為珍珠製備品之凍乾粉。 In one aspect, the present invention provides a preparation method of a pearl preparation, which comprises the following steps: 1) pulverizing: pulverizing the pearls with a jet mill to obtain ultrafine pearl powder with a particle size of less than 6 microns, preferably 0.3-1 microns; 2) Mix with water: mix pearl powder with deionized water to obtain a mixed solution; 3) Weak acid dissolution: pass excess carbon dioxide into the bottom of the mixed solution, or add a weak acid solution to dissolve the pearl powder in the mixed solution; 4) Fermentation: after sterilizing the solution obtained in step 3), it is inserted into the microbial expansion bacteria solution for fermentation culture; 5) freeze-drying: freeze-drying the fermented culture solution obtained in step 4) to obtain freeze-dried powder of pearl preparations.
根據本發明的具體實施方式，在步驟1)之前，所述方法還可包括對珍珠進行清洗、磨漿、噴霧乾燥的步驟。 According to a specific embodiment of the present invention, before step 1), the method may further include the steps of washing, refining, and spray drying the pearls.
根據本發明的具體實施方式，在步驟2)中，珍珠粉與去離子水混合重量比可為1：5~20，混合條件為：30~70℃下通過內部攪拌子或者攪拌葉於50~100rpm速度下攪拌混合。 According to a specific embodiment of the present invention, in step 2), the mixing weight ratio of pearl powder and deionized water can be 1:5~20, and the mixing conditions are: at 30~70°C, the internal stirrer or stirring blade is used at 50~70°C. Stir and mix at 100 rpm speed.
根據本發明的具體實施方式，在步驟3)中，如果添加弱酸溶液，則弱酸溶液和混合液的體積比例為1：4~10，優選為1：5，所述弱酸溶液選自乙酸、檸檬酸、乳酸、蘋果酸等中的一種或多種；優選為乳酸、乙酸或檸檬酸。 According to a specific embodiment of the present invention, in 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.
根據本發明的具體實施方式，在步驟4)中，添加的微生物擴培菌液占步驟3)中獲得溶液的1~10體積%，所用的微生物選自釀酒酵母、葡萄汁酵母、乳酸菌、枯草芽孢桿菌、地衣芽孢桿菌等中的一種或多種；優選為釀酒酵母、鼠李糖乳桿菌、枯草芽孢桿菌或地衣芽孢桿菌；發酵培養條件可為：發酵罐中25~38℃下，pH值7.0~7.5、300~500r/min、通風3~5L/min和培養24~72小時。
According to a specific embodiment of the present invention, in step 4), the added microorganism expansion bacteria solution accounts for 1 to 10% by volume of the solution obtained in step 3), and the microorganisms used are selected from Saccharomyces cerevisiae, grape juice yeast, lactic acid bacteria, subtilis One or more of Bacillus, Bacillus licheniformis, etc.; preferably Saccharomyces cerevisiae, Lactobacillus rhamnosus, Bacillus subtilis or Bacillus licheniformis; the fermentation culture conditions can be: 25~38 ℃ in the fermentation tank, pH value 7.0 ~7.5, 300~500r/min,
根據本發明的具體實施方式，在步驟5)中，在85~100℃下攪拌發酵培養液20~50分鐘進行滅活後，通過0.22μm的過濾膜壓濾獲取無菌過濾液，並將無菌過濾液與凍乾保護劑以體積/重量比例為100：0.5~2進行混合，在上述體積/重量比例中，體積單位為ml，重量單位為克，採用液態氮或者在-70~-85℃下進行預凍3~10小時，隨後放入真空冷凍乾燥倉中凍乾24~72小時。最終收穫水分含量在2~5重量%的凍乾粉。 According to a specific embodiment of the present invention, in step 5), after stirring the fermentation broth for 20 to 50 minutes at 85 to 100° C. for inactivation, the sterile filtrate is obtained by pressure filtration through a 0.22 μm filter membrane, and the sterile filtration is performed. The liquid and the lyophilized protective agent are mixed in a volume/weight ratio of 100:0.5~2. In the above volume/weight ratio, the volume unit is ml and the weight unit is gram. Liquid nitrogen or at -70~-85°C are used. Pre-freeze for 3-10 hours, and then put it into a vacuum freeze-drying chamber for lyophilization for 24-72 hours. The freeze-dried powder with a moisture content of 2 to 5% by weight is finally harvested.
根據本發明的具體實施方式，在步驟5)後，所述方法還可包括將凍乾粉置於棕色的樣品瓶(vial)中或者真空包裝的塑膠袋中進行儲存的步驟，優選在1~10℃下儲存。 According to a specific embodiment of the present invention, after step 5), the method may further include the step of placing the freeze-dried powder in a brown sample vial (via) or a vacuum-packed plastic bag for storage, preferably at 1- Store at 10°C.
另一方面，本發明提供一種由上述製備方法製備的珍珠製備品。 In another aspect, the present invention provides a pearl preparation prepared by the above preparation method.
另一方面，本發明提供一種組合物，其至少包含上述珍珠製備品。 In another aspect, the present invention provides a composition comprising at least the above-mentioned pearl preparation.
再一方面，本發明提供一種上述珍珠製備品或上述組合物作為抗衰產品的用途。 In another aspect, the present invention provides the use of the above-mentioned pearl preparation or the above-mentioned composition as an anti-aging product.
根據本發明的具體實施方式，所述抗衰產品為保健品或化妝品。 According to a specific embodiment of the present invention, the anti-aging product is a health product or a cosmetic.
根據本發明的具體實施方式，所述抗衰產品不含防腐劑。 According to a specific embodiment of the present invention, the anti-aging product contains no preservatives.
本發明的方法具有以下優點： The method of the present invention has the following advantages:
1.經過氣流粉碎處理，能夠打散珍珠粉末的假性團聚，並且再進一步打碎得到粒徑分佈範圍小且集中的小粒徑粉體。 1. After the jet pulverization process, the pseudo-agglomeration of the pearl powder can be broken up, and further pulverized to obtain a small particle size powder with a small and concentrated particle size distribution.
2.通過弱酸溶解以溫和方式無損地萃取珍珠中酸性溶液成分； 2. The acidic solution components in pearls are extracted in a mild manner and non-destructively through weak acid dissolving;
3.保留珍珠中的部分鈣質，有利於提高皮膚鈣離子通道，有利於珍珠抗衰多肽越過皮膚屏障，作用於發炎細胞和皮膚基底層的成纖維細胞； 3. Retaining part of the calcium in pearls is conducive to improving skin calcium ion channels, and is conducive to pearl anti-aging polypeptides to cross the skin barrier and act on inflammatory cells and fibroblasts in the basal layer of the skin;
4.通過微生物分解的方式加以降解，獲得小分子多肽，從而在微生物的作用下獲得更為豐富的活性混合物，同時，由於多種微生物代謝產物中含有抑菌肽，能夠保證珍珠製備品後期不必添加防腐劑； 4. It is degraded by microbial decomposition to obtain small molecular polypeptides, so as to obtain more abundant active mixtures under the action of microorganisms. At the same time, since various microbial metabolites contain bacteriostatic peptides, it can ensure that pearl preparations do not need to be added later. preservative;
5.所述珍珠製備品能夠有效抑制發炎因子bcl-2、caspase-3和caspase-9的表達，抑制活性氧、丙二醛的產生，並且同時促進麩胱甘肽和超氧化物歧化酶的產生。通過線粒體抗衰途徑，有效修護衰老細胞，延長細胞活性，在抗衰老化妝品和保健品中具有很大的應用前景。 5. 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 expression of glutathione and superoxide dismutase. produce. Through the mitochondrial anti-aging pathway, it can effectively repair senescent cells and prolong cell activity, which has great application prospects in anti-aging cosmetics and health care products.
圖1A和圖1B顯示經過氣流粉碎處理前後(圖1A：處理前，圖1B：處理後)的珍珠粉末的電顯圖。 Figures 1A and 1B show electrographs of pearl powder before and after jet milling (Figure 1A: before treatment, Figure 1B: after treatment).
圖2顯示經過氣流粉碎處理後的珍珠粉末的粒徑分佈。 Figure 2 shows the particle size distribution of the pearl powder after jet milling.
圖3A和圖3B顯示微生物發酵前後珍珠製備品的SDS-PAGE電泳圖，其中，圖3A：泳道1為發酵前測試樣品，泳道2為標準蛋白；圖3B：泳道1和4為標準蛋白，泳道2和3為發酵後的測試樣品。
Figure 3A and Figure 3B show the SDS-PAGE electropherograms of pearl preparations before and after microbial fermentation, wherein Figure 3A:
圖4A至圖4D顯示根據本發明的製備實施例1製備的珍珠製備品在不同濃度下(1*10-4g/ml和1*10-3g/ml)對於角質細胞中各氧化指標(圖4A：ROS；圖4B：GSH-Px；圖4C：MDA；和圖4D：SOD)影響的示意圖。 4A to 4D show the effects of the pearl preparations prepared according to Preparation Example 1 of the present invention on each oxidation index ( Figure 4A: ROS; Figure 4B: GSH-Px; Figure 4C: MDA; and Figure 4D: Schematic representation of the effects of SOD).
圖5顯示根據本發明的製備實施例2製備的珍珠製備品在不同濃度下(1*10-4g/ml和1*10-3g/ml)對於細胞發炎因子表達的影響的示意圖(泳道1：空白，泳道2：UV處理，泳道3：UV+10-4g/ml，泳道4：UV+10-3g/ml)。 5 is a schematic diagram showing the effect of pearl preparations prepared according to Preparation Example 2 of the present invention on the expression of cellular inflammatory factors at different concentrations (1* 10-4 g/ml and 1* 10-3 g/ml) (lane 1: blank, lane 2: UV treatment, lane 3: UV+ 10-4 g/ml, lane 4: UV+ 10-3 g/ml).
實驗材料：珍珠採購於浙江歐詩漫集團德清生物科技有限公司，角質形成細胞和成纖維細胞採購於中國科學院典型培養物保藏委員會細胞庫，抗體均來自於美國Cell Signaling Tcchnology,Inc(CST)。 Experimental materials: Pearls were purchased from Zhejiang Oushiman Group Deqing Biotechnology Co., Ltd., keratinocytes and fibroblasts were purchased from the cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences, and the antibodies were from Cell Signaling Tcchnology, Inc (CST) in the United States.
製備實施例1 Preparation Example 1
步驟1)將珍珠清洗乾淨，磨漿，噴霧乾燥後，過氣流粉碎機，獲得粒徑為0.1~1微米的超微細珍珠粉；步驟2)將1kg珍珠粉與去離子水按1：10的重量比在30℃下通過內部攪拌子或者攪拌葉在50rpm速度下攪拌混合；步驟3)添加20%(v/v)的乳酸液加以溶解並過濾；步驟4)將上一步驟過濾後得到溶液滅菌後，接入5%的釀酒酵母擴培菌液，置於發酵罐中於28℃，pH值7.0、轉速300r/min、通風3L/min下培養36小時；步驟5)在85℃下攪拌發酵培養液20分鐘進行滅活後，通過0.22μm的過濾膜壓濾獲得無菌過濾液；步驟6)將無菌過濾液與凍乾保護劑以100：0.5(體積/重量，即ml/g)比例混合，採用液態氮進行預凍2小時，隨後放入真空冷凍乾燥倉中凍乾12小時。最終得到凍乾粉；步驟7)將凍乾粉罐裝於棕色的樣品瓶中在4℃下儲存。 Step 1) Clean the pearls, grind them, spray dry them, and pass them through a jet mill to obtain ultra-fine pearl powder with a particle size of 0.1-1 micron; Step 2) Mix 1kg of pearl powder with deionized water at a ratio of 1:10 The weight ratio is stirred and mixed at a speed of 50 rpm by an internal stirrer or a stirring blade at 30 ° C; step 3) adding 20% (v/v) lactic acid liquid to dissolve and filter; step 4) filtering the previous step to obtain a solution After sterilization, insert 5% Saccharomyces cerevisiae expansion culture liquid, and place it in a fermenter at 28°C, pH 7.0, rotation speed 300r/min, and ventilation 3L/min for 36 hours; step 5) Stir at 85°C After the fermentation broth was inactivated for 20 minutes, the sterile filtrate was obtained by pressure filtration through a 0.22 μm filter membrane; step 6) the sterile filtrate and the freeze-drying protective agent were in a ratio of 100:0.5 (volume/weight, i.e. ml/g) Mixed, pre-frozen in liquid nitrogen for 2 hours, and then placed in a vacuum freeze-drying chamber for 12 hours. Finally, freeze-dried powder is obtained; Step 7) The freeze-dried powder is canned in a brown sample bottle and stored at 4°C.
製備實施例2 Preparation Example 2
步驟1)將珍珠清洗乾淨，磨漿，噴霧乾燥後，過氣流粉碎機，獲得粒徑為0.1~1微米超微細珍珠粉；步驟2)將1kg珍珠粉與去離子水按1：5的重量比在30℃下通過內部攪拌子或者攪拌葉在50rpm速度下攪拌混合；步驟3)添加20%(v/v)的乙酸溶液加以溶解並過濾；步驟4)將上一步驟過濾後得到溶液滅菌後，接入5%的釀酒酵母擴培菌液，置於發酵罐中28℃，pH值6.8、轉速250r/min、通風4L/min培養36小時；步驟5)在85℃下攪拌發酵培養液20分鐘進行滅活後，通過0.22μm的過濾膜壓濾獲得無菌過濾液；步驟6)將無菌過濾液與凍乾保護劑以100：0.5(體積/重量)比例混合，採用在-80℃下進行預凍3小時，隨後放入真空冷凍乾燥倉中凍乾12小時。最終得到凍乾粉；步驟7)將凍乾粉罐裝於棕色的樣品瓶中在4℃下儲存。 Step 1) Clean the pearls, grind them, spray dry them, and pass them through a jet mill to obtain ultra-fine pearl powder with a particle size of 0.1 to 1 micron; The ratio is stirred and mixed at 50 rpm through an internal stirrer or a stirring blade at 30°C; step 3) add 20% (v/v) acetic acid solution to dissolve and filter; step 4) sterilize the solution obtained after the previous step is filtered Then, insert 5% Saccharomyces cerevisiae expansion bacteria liquid, place it in a fermentation tank at 28°C, pH 6.8, rotating speed 250r/min, and ventilate 4L/min for 36 hours; Step 5) Stir the fermentation broth at 85°C After 20 minutes of inactivation, press filtration through a 0.22 μm filter membrane to obtain a sterile filtrate; step 6) Mix the sterile filtrate and the freeze-drying protective agent in a ratio of 100:0.5 (volume/weight), using a temperature of -80°C. Pre-freeze for 3 hours, followed by lyophilization in a vacuum freeze-drying chamber for 12 hours. Finally, freeze-dried powder is obtained; Step 7) The freeze-dried powder is canned in a brown sample bottle and stored at 4°C.
製備實施例3 Preparation Example 3
步驟1)將珍珠清洗乾淨，磨漿，噴霧乾燥後，過氣流粉碎機，獲得粒徑為0.1~1微米超微細珍珠粉；步驟2)將1kg珍珠粉與去離子水按1：5的重量比在30℃下通過內部攪拌子或者攪拌葉在50rpm速度下攪拌混合；步驟3)添加20%(v/v)的乳酸溶液將超微細珍珠粉加以溶解並過濾；步驟4)將上一步驟過濾後得到溶液滅菌後，接入5%的乳酸桿菌擴培菌液，置於發酵罐中37℃，pH值7.0、轉速300r/min、通風3L/min培養36小時； 步驟5)在85℃下攪拌發酵培養液20分鐘進行滅活後，通過0.22μm的過濾膜壓濾獲得無菌過濾液；步驟6)將過濾液與凍乾保護劑以100：0.5(體積/重量)比例混合，採用在-80℃下進行預凍5小時，隨後放入真空冷凍乾燥倉中凍乾12小時。最終得到凍乾粉；步驟7)將凍乾粉罐裝於棕色的樣品瓶中在4℃下儲存。 Step 1) Clean the pearls, grind them, spray dry them, and pass them through a jet mill to obtain ultra-fine pearl powder with a particle size of 0.1 to 1 micron; Ratio at 30 ℃ through the internal stirring bar or stirring blade at 50rpm speed stirring and mixing; Step 3) Add 20% (v/v) lactic acid solution to dissolve the ultra-fine pearl powder and filter; Step 4) Mix the previous step After the solution is sterilized after filtration, 5% Lactobacillus expansion broth is added, and placed in a fermenter at 37°C, pH 7.0, rotational speed 300r/min, and ventilation 3L/min for 36 hours; Step 5) After stirring the fermentation broth at 85°C for 20 minutes for inactivation, press filtration through a 0.22 μm filter membrane to obtain sterile filtrate; ) in a proportion of 100°C, pre-freeze at -80°C for 5 hours, and then placed in a vacuum freeze-drying chamber for 12 hours of freeze-drying. Finally, freeze-dried powder is obtained; Step 7) The freeze-dried powder is canned in a brown sample bottle and stored at 4°C.
製備實施例4 Preparation Example 4
步驟1)將珍珠清洗乾淨，磨漿，噴霧乾燥後，過氣流粉碎機，獲得粒徑為0.3~1微米超微細珍珠粉；步驟2)將1kg珍珠粉與去離子水按1：5的重量比在30℃下通過內部攪拌子或者攪拌葉在50rpm速度下攪拌混合；步驟3)將過量二氧化碳通入混合液底部0.5小時，之後過濾；步驟4)將上一步驟過濾後得到溶液滅菌後，接入5%的乳酸菌和釀酒酵母(1：1重量比)的混合擴培菌液，置於發酵罐中37℃，pH值7.0、轉速300r/min、通風3L/min下培養36小時；步驟5)在85℃下攪拌發酵培養液20分鐘進行滅活後，通過0.22μm的過濾膜壓濾獲得無菌過濾液；步驟6)將無菌過濾液與凍乾保護劑以100：0.5(體積/重量)比例混合，採用在-80℃下進行預凍3小時，隨後放入真空冷凍乾燥倉中凍乾12小時。最終得到凍乾粉；步驟7)將凍乾粉罐裝於棕色的樣品瓶中在4℃下儲存。 Step 1) Clean the pearls, grind them, spray dry them, and pass them through a jet mill to obtain ultra-fine pearl powder with a particle size of 0.3-1 micron; Than at 30 ℃ through the internal stirrer or stirring blade at 50rpm speed stirring and mixing; Step 3) pass excess carbon dioxide into the bottom of the mixed solution for 0.5 hours, then filter; Step 4) After the previous step is filtered to obtain the solution sterilization, Insert 5% lactic acid bacteria and Saccharomyces cerevisiae (1:1 weight ratio) mixed culture expansion liquid, place in a fermenter at 37° C., pH 7.0, rotating speed 300r/min, and ventilate 3L/min for 36 hours; step 5) After stirring the fermentation broth for 20 minutes at 85°C for inactivation, press filtration through a 0.22 μm membrane to obtain a sterile filtrate; step 6) mix the sterile filtrate and the freeze-drying protective agent at a ratio of 100:0.5 (volume/weight). ) in the same proportion, pre-freeze at -80°C for 3 hours, and then put it into a vacuum freeze-drying chamber for 12 hours of freeze-drying. Finally, freeze-dried powder is obtained; Step 7) The freeze-dried powder is canned in a brown sample bottle and stored at 4°C.
製備實施例5 Preparation Example 5
步驟1)將珍珠清洗乾淨，磨漿，噴霧乾燥後，過氣流粉碎機，獲得粒徑為0.3~1微米超微細珍珠粉； 步驟2)將1kg珍珠粉與去離子水按1：5的重量比在30℃下通過內部攪拌子或者攪拌葉在50rpm速度下攪拌混合；步驟3)添加20%(v/v)的乙酸溶液加以溶解並過濾；步驟4)將上一步驟過濾後得到溶液滅菌後，接入5%的乳酸菌擴培菌液，置於發酵罐中37℃，pH值7.0、轉速300r/min、通風3L/min下培養36小時；步驟5)在85℃下攪拌發酵培養液20分鐘進行滅活後，通過0.22μm的過濾膜壓濾獲得無菌過濾液；步驟6)將過濾液與凍乾保護劑以100：0.5(體積/重量)比例混合，採用液態氮下進行預凍5小時，隨後放入真空冷凍乾燥倉中凍乾12小時。最終得到凍乾粉；步驟7)將凍乾粉罐裝於棕色的樣品瓶中在4℃下儲存。 Step 1) Clean the pearls, grind them, spray dry them, and pass them through a jet mill to obtain ultra-fine pearl powder with a particle size of 0.3-1 micron; Step 2) Mix 1kg of pearl powder and deionized water at a weight ratio of 1:5 at 30°C through an internal stirrer or stirring blade at a speed of 50rpm; Step 3) Add 20% (v/v) acetic acid solution Dissolve and filter; Step 4) After the sterilization of the solution obtained after the previous step is filtered, insert 5% of the lactic acid bacteria expansion bacteria solution, place it in a fermenter at 37 ° C, pH value 7.0, rotating speed 300r/min, ventilation 3L/ Incubate for 36 hours at min; step 5) After stirring the fermentation broth at 85°C for 20 minutes for inactivation, press filtration through a 0.22 μm filter membrane to obtain a sterile filtrate; : 0.5 (volume/weight) ratio, pre-freeze for 5 hours under liquid nitrogen, and then put into a vacuum freeze-drying chamber for lyophilization for 12 hours. Finally, freeze-dried powder is obtained; Step 7) The freeze-dried powder is canned in a brown sample bottle and stored at 4°C.
製備實施例6 Preparation Example 6
步驟1)將珍珠清洗乾淨，磨漿，噴霧乾燥後，過氣流粉碎機，獲得粒徑為0.3~1微米超微細珍珠粉；步驟2)將1kg珍珠粉與去離子水按1：5的重量比在30℃下通過內部攪拌子或者攪拌葉在50rpm速度下攪拌混合；步驟3)添加20%(v/v)的乳酸溶液加以溶解並過濾；步驟4)將上一步驟過濾後得到溶液滅菌後，接入5%的乳酸菌擴培菌液，置於發酵罐中37℃，pH值6.5、轉速300r/min、通風3L/min下培養36小時；步驟5)在85℃下攪拌發酵培養液20分鐘進行滅活後，通過0.22μm的過濾膜壓濾獲得無菌過濾液； 步驟6)將過濾液與凍乾保護劑以100：0.5(體積/重量)比例混合，採用液態氮進行預凍3小時，隨後放入真空冷凍乾燥倉中凍乾12小時。最終得到凍乾粉；步驟7)將凍乾粉罐裝於棕色的樣品瓶中在4℃下儲存。 Step 1) Clean the pearls, grind them, spray dry them, and pass them through a jet mill to obtain ultra-fine pearl powder with a particle size of 0.3-1 micron; The ratio is stirred and mixed at a speed of 50 rpm by an internal stirrer or a stirring blade at 30°C; step 3) adding 20% (v/v) lactic acid solution to dissolve and filter; step 4) sterilizing the solution obtained after filtering the previous step Then, insert 5% lactic acid bacteria expansion bacteria liquid, and place it in a fermenter at 37° C., pH value 6.5, rotation speed 300r/min, and ventilation 3L/min for 36 hours; Step 5) Stir the fermentation broth at 85° C. After 20 minutes of inactivation, the sterile filtrate was obtained by pressure filtration through a 0.22 μm filter membrane; Step 6) Mix the filtrate with the freeze-drying protective agent in a ratio of 100:0.5 (volume/weight), use liquid nitrogen to pre-freeze for 3 hours, and then put it into a vacuum freeze-drying chamber for freeze-drying for 12 hours. Finally, freeze-dried powder is obtained; Step 7) The freeze-dried powder is canned in a brown sample bottle and stored at 4°C.
製備實施例7 Preparation Example 7
步驟1)將珍珠清洗乾淨，磨漿，噴霧乾燥後，過氣流粉碎機，獲得粒徑為0.3~1微米超微細珍珠粉；步驟2)將1kg珍珠粉與去離子水按1：5的重量比在30℃下通過內部攪拌子或者攪拌葉在50rpm速度下攪拌混合；步驟3)將過量二氧化碳通入混合液底部0.5小時，之後過濾；步驟4)將上一步驟過濾後得到溶液滅菌後，接入5%的枯草芽孢桿菌擴培菌液，置於發酵罐中37℃，pH值6.5、轉速300r/min、通風3L/min下培養36小時；步驟5)在85℃下攪拌發酵培養液20分鐘進行滅活後，通過0.22μm的過濾膜壓濾獲得無菌過濾液；步驟6)將過濾液與凍乾保護劑以100：0.5(體積/重量)比例混合，採用液態氮進行預凍，隨後放入真空冷凍乾燥倉中凍乾12小時。最終得到凍乾粉；步驟7)將凍乾粉罐裝於棕色的樣品瓶中在4℃下儲存。 Step 1) Clean the pearls, grind them, spray dry them, and pass them through a jet mill to obtain ultra-fine pearl powder with a particle size of 0.3-1 micron; Than at 30 ℃ through the internal stirrer or stirring blade at 50rpm speed stirring and mixing; Step 3) pass excess carbon dioxide into the bottom of the mixed solution for 0.5 hours, then filter; Step 4) After the previous step is filtered to obtain the solution sterilization, Insert 5% Bacillus subtilis expansion culture solution, and place it in a fermenter at 37°C, pH value 6.5, rotation speed 300r/min, and ventilation 3L/min for 36 hours; Step 5) Stir the fermentation broth at 85°C After 20 minutes of inactivation, press filtration through a 0.22 μm filter membrane to obtain a sterile filtrate; step 6) mix the filtrate and the freeze-drying protective agent in a ratio of 100:0.5 (volume/weight), and use liquid nitrogen to pre-freeze, It was then placed in a vacuum freeze-drying chamber for lyophilization for 12 hours. Finally, freeze-dried powder is obtained; Step 7) The freeze-dried powder is canned in a brown sample bottle and stored at 4°C.
製備實施例8 Preparation Example 8
步驟1)將珍珠清洗乾淨，磨漿，噴霧乾燥後，過氣流粉碎機，獲得粒徑為0.3~1微米超微細珍珠粉；步驟2)將1kg珍珠粉與去離子水按1：5的重量比在30℃下通過內部攪拌子或者攪拌葉在50rpm速度下攪拌混合； 步驟3)添加20%(v/v)的乙酸溶液加以溶解並過濾；步驟4)將上一步驟過濾後得到溶液滅菌後，接入5%的枯草芽孢桿菌擴培菌液，置於發酵罐中37℃，pH值6.8、轉速300r/min、通風3L/min下培養36小時；步驟5)在85℃下攪拌發酵培養液20分鐘進行滅活後，通過0.22μm的過濾膜壓濾獲得無菌過濾液；步驟6)將過濾液與凍乾保護劑以100：0.5(體積/重量)比例混合，採用液態氮或者在-80℃下進行預凍，隨後放入真空冷凍乾燥倉中凍乾24小時。最終得到凍乾粉；步驟7)將凍乾粉罐裝於棕色的樣品瓶中在4℃下儲存。 Step 1) Clean the pearls, grind them, spray dry them, and pass them through a jet mill to obtain ultra-fine pearl powder with a particle size of 0.3-1 micron; The ratio is stirred and mixed at 50 rpm by internal stirring bar or stirring blade at 30 °C; Step 3) Add 20% (v/v) acetic acid solution to dissolve and filter; Step 4) After sterilizing the solution obtained after filtration in the previous step, insert 5% Bacillus subtilis expansion culture liquid, and place it in a fermenter 37°C, pH value 6.8, rotation speed 300r/min, and ventilation 3L/min for 36 hours; Step 5) After stirring the fermentation broth at 85°C for 20 minutes for inactivation, press filtration through a 0.22 μm filter membrane to obtain sterile Filtrate; Step 6) Mix the filtrate with the freeze-drying protective agent in a ratio of 100:0.5 (volume/weight), use liquid nitrogen or pre-freeze at -80°C, and then put it into a vacuum freeze-drying chamber for lyophilization for 24 Hour. Finally, freeze-dried powder is obtained; Step 7) The freeze-dried powder is canned in a brown sample bottle and stored at 4°C.
製備實施例9 Preparation Example 9
步驟1)將珍珠清洗乾淨，磨漿，噴霧乾燥後，過氣流粉碎機，獲得粒徑為0.3~1微米超微細珍珠粉； Step 1) Clean the pearls, grind them, spray dry them, and pass them through a jet mill to obtain ultra-fine pearl powder with a particle size of 0.3-1 micron;
步驟2)將1kg珍珠粉與去離子水按1：5的重量比在30℃下通過內部攪拌子或者攪拌葉在50rpm速度下攪拌混合； Step 2) Stir and mix 1kg of pearl powder and deionized water at a weight ratio of 1:5 at 30°C through an internal stirrer or a stirring blade at a speed of 50rpm;
步驟3)添加15%的乳酸溶液加以溶解並過濾； Step 3) add 15% lactic acid solution to dissolve and filter;
步驟4)將上一步驟過濾後得到溶液滅菌後，接入5%的枯草芽孢桿菌擴培菌液，置於發酵罐中37℃，pH值6.8、轉速300r/min、通風3L/min下培養48小時； Step 4) After sterilizing the solution obtained after filtration in the previous step, insert 5% Bacillus subtilis expansion bacterial solution, and place it in a fermenter at 37 ° C, pH value 6.8, rotation speed 300r/min, and ventilation 3L/min for cultivation 48 hours;
步驟4)在85℃下攪拌20分鐘的滅活後，通過0.22μm的過濾膜壓濾獲取無菌過濾液； Step 4) After stirring at 85° C. for 20 minutes for inactivation, press filtration through a 0.22 μm filter membrane to obtain sterile filtrate;
步驟5)無菌過濾液與凍乾保護劑進行100：0.5(體積/重量)比例的混合，進行-80℃進行預凍1小時，隨後放入真空冷凍乾燥倉中進行凍乾72小時。最終收穫得凍乾粉。 Step 5) The sterile filtrate and the freeze-drying protective agent are mixed in a ratio of 100:0.5 (volume/weight), pre-freeze at -80° C. for 1 hour, and then placed in a vacuum freeze-drying chamber for freeze-drying for 72 hours. The final harvest is lyophilized powder.
步驟6)將凍乾粉罐裝於棕色的樣品瓶中進行4℃下儲存。 Step 6) The lyophilized powder was canned in a brown sample bottle for storage at 4°C.
實驗實施例1 採用掃描電子顯微鏡法觀察氣流粉碎前後的珍珠形態 Experimental Example 1 Observation of pearl morphology before and after jet pulverization by scanning electron microscopy
分別將處理乾燥後的樣品粉末(分別為經氣流粉碎前和氣流粉碎後的樣品粉末)用雙面導電膠帶黏到潔淨銅臺上，經離子濺射鍍膜後，在掃描電子顯微鏡上觀察照相，加速電壓為20kV。實驗結果見圖1。 The processed and dried sample powders (respectively, the sample powders before and after jet pulverization) were adhered to a clean copper table with double-sided conductive tape, and after ion sputtering coating, observed and photographed on a scanning electron microscope. The accelerating voltage is 20kV. The experimental results are shown in Figure 1.
從圖1中可以看出，氣流粉碎前，粉末假性團聚。經過氣流粉碎後，能夠打散假性團聚，並且再進一步打碎得到粒徑分佈範圍小且集中的小粒徑粉體。 It can be seen from Figure 1 that the powders are pseudo-agglomerated before jet milling. After jet pulverization, the pseudo-agglomeration can be broken up, and further pulverized to obtain a small particle size powder with a small particle size distribution range and a concentration.
實驗實施例2 採用鐳射細微性電位儀法測量氣流粉碎後的珍珠粉末粒徑分佈 Experimental Example 2 The particle size distribution of the pearl powder after jet pulverization was measured by the laser micro-potentiometer method
操作步驟： Steps:
ar)分散納米珍珠粉樣品的液體用去離子水；b)檢查溶液有無氣泡，有氣泡則用超音波消除氣泡；c)檢查樣品池是否乾淨；d)折射率選定在1.61，吸收率選定為1；按GB/T 19077.1-2008規定測定。 ar) Use deionized water to disperse the nano pearl powder sample liquid; b) Check the solution for bubbles, and use ultrasonic to eliminate bubbles if there are bubbles; c) Check whether the sample cell is clean; d) The refractive index is selected as 1.61, and the absorptivity is selected as 1; Determined according to GB/T 19077.1-2008.
結果如圖2所示，由圖2可見，經過粉碎後的珍珠粉末粒徑分佈範圍窄。 The results are shown in Figure 2, and it can be seen from Figure 2 that the particle size distribution of the pulverized pearl powder is narrow.
實驗實施例3 經發酵處理前後的珍珠製備品的SDS-PAGE電泳 Experimental Example 3 SDS-PAGE electrophoresis of pearl preparations before and after fermentation
採用SDS-PAGE電泳對經發酵處理前後的珍珠製備品進行檢測，結果如圖3A和圖3B所示，從圖中可以看出經過發酵處理珍珠製備品的分子量範圍明顯下降，證實通過本發明的方法獲得小分子多肽。 SDS-PAGE electrophoresis was used to detect the pearl preparations before and after the fermentation treatment. The results are shown in Figure 3A and Figure 3B. It can be seen from the figures that the molecular weight range of the pearl preparations after fermentation treatment is significantly reduced. Methods to obtain small molecule polypeptides.
實驗實施例4 在UV衰老模型中，珍珠製備品對角質細胞中各氧化指標的影響 Experimental Example 4 In the UV aging model, the effect of pearl preparations on various oxidation indexes in keratinocytes
試驗方法：紫外照射損傷角質形成細胞後，於冰上以超音波破碎各組細胞，在離心機上離心收集細胞勻漿(homogenate)，並按南京建成試劑盒說明書操作檢測各組的吸光值，以BCA蛋白濃度測定試劑盒測定相應各組的蛋白質濃度，最後計算出各組的ROS、SOD、MDA的含量。 Test method: After UV irradiation damaged keratinocytes, the cells of each group were disrupted by ultrasonic wave on ice, and the cell homogenate was collected by centrifugation on a centrifuge. The protein concentration of the corresponding groups was measured with BCA protein concentration assay kit, and the contents of ROS, SOD and MDA in each group were finally calculated.
如圖4A至圖4D所示，將角質形成細胞通過UV照射後，角質細胞中ROS、麩胱甘肽過氧化物酶以及丙二醛和超氧化物歧化酶的含量均發生顯著性變化，隨後添加0.01%和0.1%(g/ml)濃度的珍珠製備品作用24小時後進行測定，結果顯示，珍珠製備品可顯著改善氧化指標(有影響*p<0.1，顯著**p<0.01，非常顯著**p<0.001)。 As shown in Figure 4A to Figure 4D, after keratinocytes were irradiated by UV, the contents of ROS, glutathione peroxidase, malondialdehyde and superoxide dismutase in keratinocytes were significantly changed, followed by After adding 0.01% and 0.1% (g/ml) pearl preparations for 24 hours, the results show that pearl preparations can significantly improve the oxidation index (influenced*p<0.1, significant**p<0.01, very Significant **p<0.001).
實驗實施例5 在UV衰老模型中，珍珠製備品對於細胞發炎因子表達的影響 Experimental Example 5 Influence of pearl preparations on the expression of cellular inflammatory factors in a UV aging model
試驗方法：西方墨點法(Western blot)檢測Bcl-2、Bax、Caspase-3及Caspase-9蛋白表達水準 Test method: Western blot was used to detect the protein expression levels of Bcl-2, Bax, Caspase-3 and Caspase-9.
六孔盤中成纖維細胞生長達80%-90%時，收集各組細胞，丟棄上清液，按每孔100μL的量添加RIPA裂解液(含1% PMSF)，靜置3分鐘後將液體移至1.5ml離心管中，冰上靜置30分鐘然後離心(4℃，12000r/min-15000r/min)，將上清液吸出至另1個新1.5mL離心管中，即為蛋白。將蛋白樣品與5×SDS上樣緩衝液(Loading Buffer)以4：1比例混合，沸水加熱7分鐘，使蛋白充分變性。配製分離膠和濃縮膠，將製好的凝膠放入電泳槽內，內外槽加入新鮮配製的電泳緩衝液，拔出樣本梳，加入蛋白樣品，樣品量為60μg。電泳結束切割凝膠，轉印(100V，30分鐘)。一抗(1：500)封閉，4℃過夜，二抗 (1：10 000)反應1小時，ECL發光液A液和B液按照1：1的比例混合均勻，滴至PVDF膜上，使其均勻覆蓋，於暗室中曝光並顯影，分析結果。 When the growth of fibroblasts in the six-well plate reaches 80%-90%, collect the cells of each group, discard the supernatant, add RIPA lysis buffer (containing 1% PMSF) in an amount of 100 μL per well, and let the liquid stand for 3 minutes. Transfer to a 1.5ml centrifuge tube, let stand on ice for 30 minutes and then centrifuge (4°C, 12000r/min-15000r/min), aspirate the supernatant into another new 1.5mL centrifuge tube, which is the protein. The protein sample was mixed with 5×SDS loading buffer (Loading Buffer) in a ratio of 4:1, and heated in boiling water for 7 minutes to fully denature the protein. Prepare separating gel and stacking gel, put the prepared gel into the electrophoresis tank, add freshly prepared electrophoresis buffer to the inner and outer tanks, pull out the sample comb, and add the protein sample. The sample amount is 60 μg. After electrophoresis, the gel was cut and transferred (100V, 30 minutes). Primary antibody (1:500) blocked, overnight at 4°C, secondary antibody (1:10 000) reacted for 1 hour, the ECL luminescent solution A and B were mixed uniformly according to the ratio of 1:1, dropped onto the PVDF film to make it evenly covered, exposed and developed in the dark room, and the results were analyzed.
如附圖5顯示，在不同濃度的珍珠製備品的作用下，Caspase3和Caspase9凋亡因子的表達均明顯減少、抑制凋亡因子Bcl-2的表達增加，表明本發明的珍珠製備品通過PI3K/AKT路徑具有抑制發炎因子表達的作用。 As shown in Figure 5, under the action of different concentrations of pearl preparations, the expression of Caspase3 and Caspase9 apoptotic factors were significantly reduced, and the expression of apoptosis factor Bcl-2 was inhibited from increasing, indicating that the pearl preparations of the present invention passed PI3K/ The AKT pathway has the effect of inhibiting the expression of inflammatory factors.
綜上，本申請的珍珠製備品在萬分之一的濃度添加量下仍具備非常顯著的抗衰能力，並且其通過PI3K/AKT路徑作用於線粒體起到抗衰老作用。 To sum up, the pearl preparation of the present application still has a very significant anti-aging ability under the addition of 1/10,000 of the concentration, and it acts on mitochondria through the PI3K/AKT pathway to play an anti-aging effect.
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| JP2928095B2 (en) * | 1994-05-23 | 1999-07-28 | 株式会社エフ・エル・アイ | Pearl jewelry cleaner |
| CN1061094C (en) * | 1996-09-06 | 2001-01-24 | 中国科学院植物研究所 | Biological degradation method of fresh water pearl powder |
| KR20120045390A (en) * | 2010-10-29 | 2012-05-09 | 최대성 | Method for manufacturing the fermented pearl powder |
| CN104911239A (en) * | 2014-03-14 | 2015-09-16 | 浙江欧诗漫生物股份有限公司 | Method for enzymolysis separation of pearl active polypeptide from pearl protein |
| CN104558137A (en) * | 2015-01-15 | 2015-04-29 | 浙江欧诗漫生物股份有限公司 | Preparation method of conchiolin as well as water-soluble conchiolin and acid-soluble conchiolin prepared by virtue of preparation method |
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2019
- 2019-09-29 CN CN201910934074.5A patent/CN112569172A/en active Pending
-
2020
- 2020-04-27 WO PCT/CN2020/087099 patent/WO2021057022A1/en active Application Filing
- 2020-09-28 TW TW109133715A patent/TWI764304B/en active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106265412A (en) * | 2016-08-04 | 2017-01-04 | 海南京润珍珠生物技术股份有限公司 | A kind of method utilizing probiotics fermention to prepare Margarita hydrolyzed solution |
Non-Patent Citations (2)
| Title |
|---|
| 期刊 董志華， "水溶性珍珠粉不同制备工艺比较研究"， 湖南中医药导报， 第10卷第12期 ， 2004年12月， 第58-59頁 * |
| 期刊 董志華， "水溶性珍珠粉不同制备工艺比较研究"， 湖南中医药导报， 第10卷第12期 ， 2004年12月， 第58-59頁。 |
Also Published As
| Publication number | Publication date |
|---|---|
| TW202114634A (en) | 2021-04-16 |
| WO2021057022A1 (en) | 2021-04-01 |
| CN112569172A (en) | 2021-03-30 |
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