WO2012024817A1 - 全蛋蛋白肽及其制备方法和应用 - Google Patents

全蛋蛋白肽及其制备方法和应用 Download PDF

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WO2012024817A1
WO2012024817A1 PCT/CN2010/001287 CN2010001287W WO2012024817A1 WO 2012024817 A1 WO2012024817 A1 WO 2012024817A1 CN 2010001287 W CN2010001287 W CN 2010001287W WO 2012024817 A1 WO2012024817 A1 WO 2012024817A1
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whole egg
egg protein
product
peptide
protein peptide
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PCT/CN2010/001287
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English (en)
French (fr)
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邹远东
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武汉九生堂生物工程有限公司
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Application filed by 武汉九生堂生物工程有限公司 filed Critical 武汉九生堂生物工程有限公司
Priority to CN201080068668.XA priority Critical patent/CN103108957B/zh
Priority to JP2013525103A priority patent/JP5927191B2/ja
Priority to PCT/CN2010/001287 priority patent/WO2012024817A1/zh
Priority to US13/818,642 priority patent/US9657086B2/en
Priority to EP10856270.3A priority patent/EP2610349A4/en
Publication of WO2012024817A1 publication Critical patent/WO2012024817A1/zh

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/02Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from eggs
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56Materials from animals other than mammals
    • A61K35/57Birds; Materials from birds, e.g. eggs, feathers, egg white, egg yolk or endothelium corneum gigeriae galli
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/01Hydrolysed proteins; Derivatives thereof
    • A61K38/012Hydrolysed proteins; Derivatives thereof from animals
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • A61P37/02Immunomodulators
    • A61P37/04Immunostimulants
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/465Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from birds
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00Preparation of peptides or proteins
    • C12P21/06Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products

Definitions

  • the invention relates to a preparation method and application of a novel peptide immunological whole egg protein peptide.
  • peptide immunostimulants belong to polypeptide hormones, mainly drugs such as thymosin, thymopentin, immunoglobulin, gamma globulin, human serum albumin, interferon, tumor necrosis factor, interleukin I, and leukocyte mediator. Prime II, interleukin III, etc.
  • drugs such as thymosin, thymopentin, immunoglobulin, gamma globulin, human serum albumin, interferon, tumor necrosis factor, interleukin I, and leukocyte mediator.
  • These peptide immunizing agents are isolated from the gland, tissue or blood of an animal. Most of these peptide drugs are injections, which can only be used in certain patients. During use, they are prone to rejection, allergies and side effects. They must be used with caution and must be used under strict supervision by a doctor.
  • the object of the present invention is to provide a peptide-based immunizing agent whole egg protein peptide and a preparation method thereof.
  • the whole egg protein peptide provided by the present invention is prepared according to the following method: Enzymatic degradation of whole egg protein powder by a composite plant protease to obtain the whole egg protein peptide.
  • the composite plant protease may be composed of papain, ficin and pineapple protease, and the enzyme activity ratio thereof is (100 to 1.2 million) U: (30 to 400,000) U: (560 to 7 million) U.
  • the whole egg protein powder can be prepared from at least one of the following bird eggs: eggs, duck eggs, goose eggs, quail eggs, sparrow eggs, pigeon eggs, spotted eggs and ostrich eggs.
  • the mass fraction of whole egg protein powder to water is 1: 8-10, and the amount of each enzyme required to digest the whole egg protein powder is: papain 5 ⁇ 6 Million U, fig protease 1. 5 ⁇ 2 million U, bromelain 28 ⁇ 3 million U. 5 ⁇
  • the enzyme is catalyzed by a temperature of 48 to 50 ° C, a time of 3 to 4 hours, a pH of 7. 5 ⁇ 8.
  • the enzymatic hydrolysate is also subjected to sterilization and enzyme inactivation treatment, and then the enzymatic hydrolysate is cooled and frozen to obtain the final whole egg protein peptide.
  • the freezing temperature was 4 to 8 ° C for 48 hours; the sterilization and enzyme inactivation conditions were: temperature 100 ° C, time 10 minutes.
  • the enzyme activity unit U refers to the amount of enzyme required to convert 1 micromolar substrate per minute or to convert 1 micromolar of the relevant group under specific conditions (25 ° C, pH 7.0).
  • Enzyme The whole egg protein peptide obtained by the invention contains a polypeptide, an oligopeptide, 20 kinds of amino acids (8 of which are essential amino acids which cannot be synthesized), and a plurality of vitamins (VA, VC, VE, Bl, B2, B6, B12), calcium and a variety of organic and natural trace elements (CPPS, zinc, selenium, magnesium, copper, iron, manganese, etc., which are naturally chelated with proteins).
  • vitamins VA, VC, VE, Bl, B2, B6, B12
  • CPPS organic and natural trace elements
  • the polypeptides and oligopeptides have a relative molecular mass of less than 100 ODA and consist of 2-6 amino acids.
  • the whole egg protein peptide may be present in the form of a concentrate, a lyophilized powder, a spray dried dry powder, a liquid or a solid.
  • Another object of the invention is to provide the use of the whole egg protein peptide.
  • the use of the whole egg protein peptide provided by the present invention is its use in the preparation of products for enhancing immunity.
  • the product for enhancing immunity provided by the present invention the active ingredient thereof comprises the whole egg protein peptide provided by the present invention.
  • the product can be a pharmaceutical or health supplement.
  • the mass ratio of whole egg protein peptide, jujube polysaccharide and anthraquinone polysaccharide may be (900-1200): (8-30): (3-15).
  • An immunopotentiating drug or nutraceutical prepared by using whole egg protein peptide as an active ingredient, and one or more pharmaceutically acceptable carriers may be added to the above-mentioned drug or health care product as needed.
  • the carrier includes conventional diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants and the like in the pharmaceutical field.
  • the immunopotentiating drug can be formulated into various forms such as an oral solution, a tablet, a granule, a capsule, a paste, a film, and the like.
  • the above various dosage forms of the drug can be prepared according to a conventional method in the pharmaceutical field, and the obtained drug has no bitter taste.
  • Enzymatic hydrolysis using whole egg protein powder as a raw material is a remarkable feature of the present invention.
  • the protein scores of the above eggs are close to 100 points.
  • the protein is close to human protein and is easily absorbed and utilized by the body.
  • the choice of whole eggs is a scientific choice. If only egg white protein is selected, the protein and many nutrients in the egg yolk will be lost. If only egg yolk is selected, the protein in the egg white is also lost. Choosing whole eggs means choosing a complete nutrition system. Not only is nutrition comprehensive, but each nutrient can exert synergy with each other.
  • the invention adopts compound plant proteases, namely papain, ficin and pineapple eggs.
  • the white enzyme, the three enzyme scientific formula forms a composite enzyme preparation, and the whole egg protein powder is catalytically degraded to obtain a whole egg protein peptide.
  • the whole egg protein peptide can replace the polypeptide hormone immunosuppressive agent and function as an immunomodulatory agent for the polypeptide hormone immunosuppressive agent.
  • the polypeptide hormone immunosuppressive agent is a medicine and can only be used for a patient, and the product of the invention can be made into an oral polypeptide nutritional immunizing agent, which not only can treat the patient, but also can enhance the immunity to the disease-free person. The role of force, disease prevention and health.
  • This product is an oral preparation that is easier to carry and safe to use than the injection. It does not cause rejection, allergies or other side effects during use, and does not need to be used under the supervision of a doctor. It can be used for many diseases and sub-health states caused by low immune function, immune dysfunction, decreased immune function, impaired immune organs, etc. It can also be used for protein-deficient people, people with many diseases caused by protein deficiency and sub-health status.
  • It can be used to prevent colds, can be used after exercise, postpartum, post-ill, post-operative population, promote "negative nitrogen balance", postpartum recovery, post-mortem recovery, wound healing, liver protection, hangover, appetite recovery, sleep promotion It has a good effect on restoring kidney function, stimulating the phagocytic ability of macrophages, inhibiting the growth of tumor cells, and raising white blood cells.
  • Figure 1 is a flow chart showing the production process of the whole egg protein peptide of the present invention.
  • Example 1 Preparation of whole egg protein peptide oral liquid for enhancing immunity
  • the whole egg protein powder (freeze-dried dry powder or spray-dried dry powder) into the fermenter, add water equivalent to 10 times the mass of the whole egg protein powder, and gradually increase the temperature to 5 CTC, and then enzymatically digest the whole egg protein per gram.
  • the amount of various enzymes required for the powder is: 50,000 U of papain, 20,000 U of fig protease, and 350,000 U of Bromelain.
  • the complex protease is added to the fermenter for enzymatic hydrolysis. The temperature is always maintained at 50 V. After 4 hours of enzymatic hydrolysis, it is sterilized and inactivated by enzyme, then cooled to 60 ° C for barreling, then cooled to room temperature and placed in a freezer.
  • Example 2 Preparation of whole egg protein peptide oral liquid for enhancing immunity containing jujube polysaccharide and lycium polysaccharide
  • Example 3 Preparation of an immune-enhancing whole egg protein peptide oral solution containing jujube polysaccharide and lycium polysaccharide
  • the formulated functional ingredients are composed of: 1000 mg ⁇ 10% of whole egg protein peptide per 100 ml of oral liquid, 15 mg of jujube polysaccharide, 10 mg of medlar polysaccharide, and after filling and sterilizing at 10 CTC for 10 minutes, it is enhanced.
  • Immune oral solution 1000 mg ⁇ 10% of whole egg protein peptide per 100 ml of oral liquid, 15 mg of jujube polysaccharide, 10 mg of medlar polysaccharide, and after filling and sterilizing at 10 CTC for 10 minutes, it is enhanced.
  • Immune oral solution 1000 mg ⁇ 10% of whole egg protein peptide per 100 ml of oral liquid, 15 mg of jujube polysaccharide, 10 mg of medlar polysaccharide, and after filling and sterilizing at 10 CTC for 10 minutes, it is enhanced.
  • Immune oral solution 1000 mg ⁇ 10% of whole egg protein peptide per 100 ml of oral liquid, 15 mg of jujube poly
  • Test sample Oral solution prepared in Example 1
  • the organic nitrogen (%) represents the peptide and amino acid content
  • the determination Refer to GB5009 for the law.
  • mice Kunming mice 18-20g, male and female, a total of 20; animals were provided by the Hubei Medical Laboratory Animal Center.
  • Test drug The oral solution for enhancing immunity prepared in Example 1.
  • test animals were orally administered with 0.3 ml/10 g.b.w. Fasting for 12 hours before gavage, observe for one week.
  • test article is a non-toxic substance.
  • Test method The test drug was administered twice by intragastric administration. After the second gavage for 6 hours, the animals were sacrificed. The thoracic bone marrow was diluted with calf serum and stained with Gimsa. 1000 polychromatic red blood cells were observed under microscope. The number of micronucleated cell formation was recorded and expressed in parts per thousand. The results are shown in Table 4. Among them, the CP group was a cyclophosphamide positive control group.
  • mice Kunming mice 23-25g.
  • Test method After 5 days of continuous gavage, continue feeding for 30 days. The animals were sacrificed and the lateral testicular slices were taken. Each animal counted 1000 structurally intact sperm and calculated the incidence of sperm abnormalities. The results are shown in Table 5. Among them, the CP group was a cyclophosphamide positive control group.
  • PCB Polychlorinated biphenyl
  • the -S 9 positive control group was 3-nitro 9-fluorenone (2.4.7-TNFone), and the +S 9 positive control group was 2-aminopurine (2-AF).
  • each experimental group was returned. The number of colonies did not exceed twice the number of spontaneous reversions; and there was no dose-response relationship, and the test result was negative.
  • Example 6 Detection of immunomodulatory effects of whole egg protein peptide
  • Test animals Kunming mice 18-22g, male and female. (provided by Hubei Medical Laboratory Animal Center)
  • the dose grouping is: control group, digestive distilled water; the concentration of "oral solution prepared in 1" is 1% (ie, lOOOO/lOOml), and the low, medium and high dose groups are respectively lOOmg/kg. b. w, 300mg/kg. b. w, 900mg/kg. b. w mouth sputum sputum; the low, medium and high dose groups are equivalent to 6, 18, 54 times the recommended intake.
  • the stomach was administered for one month, and then various experiments were performed.
  • RESULTS MTT assay (ConA-induced spleen lymphocyte transformation test in mice)
  • One-way ANOVA was performed on the mean values of absorbance differences between ConA wells and ConA wells.
  • ConA induces spleen lymphocyte proliferation
  • DNFB induces DTH group
  • DNFB Dinitrofluorobenzene induces delayed type hypersensitivity
  • Ear swelling method After sensitizing the mice with DNFB, the right ear was attacked with DNFB on the fifth day. After 24 hours, the animals were sacrificed and the left and right ear shells were cut out. The ear pieces with a diameter of 8 mm were removed with a puncher, and weighed, and the degree of DTH was expressed by the difference between the weights of the left and right ears.
  • Blood coagulation method Blood coagulation method.
  • the anti-volume number was calculated according to the level of serum coagulation, and the mean value of each group of antibody was analyzed by one-way ANOVA.
  • Method Semi-in vivo method. Prepare 20% chicken red blood cell suspension; each mouse was intraperitoneally injected with 1 mL of the suspension. After 30 minutes, the animals were sacrificed, laparotomy, 2 mL of normal saline was intraperitoneally injected, and the average was divided into 2 slides, and incubated at 37 °C for 30 min. The cells were rinsed with physiological saline, air-dried, fixed in 1:1 acetone in methanol, stained with 4% Giemsa-phosphate buffer for 3 min, and rinsed with distilled water to dry. 100 macrophages were counted under an oil microscope, and the phagocytosis rate and phagocytic index were calculated as follows:
  • Phagocytosis rate ( %) number of macrophages that phagocytose chicken red blood cells
  • the phagocytic index of each group of mice was calculated according to the carbon concentration in the blood after the injection of ink for 2 min and lOmin.
  • Control group 5 6.609 ⁇ 0.755
  • the whole oral liquid of egg protein peptide 1) can enhance the proliferation ability of mouse spleen lymphocytes induced by ConA; 2) enhance the induction of dinitrofluorobenzene Delayed hair allergy in mice; 3) Increase serum hemolysin content in mice; 4) Enhance the ability of mouse peritoneal macrophages to engulf chicken red blood cell function and carbon clearance.
  • the whole egg protein peptide prepared by the present invention can be considered to have an immunomodulatory effect.
  • the whole egg protein powder is used as a substrate, and a composite plant protease: papain, ficin and bromelain is used as a substrate to form a composite enzyme preparation, and the whole egg protein powder is catalyzed and degraded to obtain a whole egg protein peptide.
  • This product has high biological activity and is a A new type of immunomodulator and immunopotentiator.
  • the whole egg protein peptide of the present invention can be used for the development of immunopharmaceuticals, health foods, functional foods, anti-aging and infant foods, and nutritional supplements for seriously ill patients.
  • the invention can be made into a lyophilized powder by freeze-drying, thereby being made into a capsule or a tablet, and can deeply develop a tumor adjuvant therapeutic drug (enhancing immune function, enhancing radiation resistance of a patient undergoing radiotherapy and chemotherapy, increasing red and white blood cells), and anti-inflammatory drugs.
  • a tumor adjuvant therapeutic drug enhancing immune function, enhancing radiation resistance of a patient undergoing radiotherapy and chemotherapy, increasing red and white blood cells
  • anti-inflammatory drugs enhancing immune function, enhancing radiation resistance of a patient undergoing radiotherapy and chemotherapy, increasing red and white blood cells
  • hepatitis drugs drugs for the prevention and treatment of atypical pneumonia, can also be used as other raw materials or as food additives. No matter what kind of drugs, health products, and functional foods are developed, they will have huge economic and social benefits, and their prospects are very broad.

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Description

全蛋蛋白肽及其制备方法和应用 技术领域
本发明涉及一种新型肽类免疫剂全蛋蛋白肽的制备方法与应用。
背景技术
通常肽类免疫剂都属于多肽激素类, 主要是药物, 如胸腺肽、 胸腺五 肽、 免疫球蛋白、 丙种球蛋白、 人血清白蛋白、 干扰素、 肿瘤细胞坏死因 子、 白细胞介素 I 、 白细胞介素 II 、 白细胞介素 III 等。 这些肽类免疫剂 都是从动物的腺体、 组织或血液中分离提取的。 这些多肽药物大都是针 剂, 只能对一定的病人使用, 使用过程中, 容易产生排异、 过敏和副作用 反应, 须谨慎, 必须在医生严格监督下使用。
发明公开
本发明的目的是提供一种肽类免疫剂全蛋蛋白肽及其制备方法。
本发明所提供的全蛋蛋白肽是按照下述方法制备得到的: 采用复合植 物蛋白酶对全蛋蛋白粉进行酶催化降解, 得到所述全蛋蛋白肽。
其中, 所述复合植物蛋白酶可由木瓜蛋白酶、 无花果蛋白酶和菠萝蛋 白酶组成, 其酶活力比为(100〜120万) U : (30〜40万) U : (560〜700万) U。
所述全蛋蛋白粉可由下述至少一种禽鸟蛋为原料制备得到的: 鸡蛋、 鸭蛋、 鹅蛋、 鹌鹑蛋、 麻雀蛋、 鸽蛋、 斑鸠蛋和鸵鸟蛋。
所述酶催化降解的反应体系中, 全蛋蛋白粉与水的质量份数比为 1 : 8-10, 酶解每克全蛋蛋白粉所需各种酶的量为:木瓜蛋白酶 5〜6万 U,无花 果蛋白酶 1. 5〜2万 U,菠萝蛋白酶 28〜35万 U。 所述酶催化降解的温度为 48〜50°C,时间为 3〜4小时, pH值为 7. 5〜8. 5。
在酶催化降解反应结束后, 还需对酶解液进行灭菌和酶灭活处理, 然 后再对所述酶解液进行冷却、 冷冻处理, 即得到最终的全蛋蛋白肽。 所述 冷冻的温度为 4〜8°C, 时间为 48小时; 所述灭菌和酶灭活的条件为: 温 度 100°C, 时间 10分钟。
本发明中, 酶活力单位 U是指在特定条件下 (25°C, pH7. 0) , 每 1分 钟转化 1微摩尔的底物所需的酶量或转化 1微摩尔的有关基团所需的酶 本发明所得到的全蛋蛋白肽含有多肽, 寡肽, 20种氨基酸 (其中有 8 种是人体必需而又不能合成的氨基酸), 多种维生素(VA、 VC、 VE、 Bl、 B2、 B6、 B12), 钙及多种人体极易吸收的有机天然的各种微量元素(CPPS, 蛋白锌、 硒、 镁、 铜、 铁、 锰等, 这些微量元素与蛋白质天然螯合)。 所述 多肽和寡肽的相对分子质量均在 lOOODa以下, 由 2-6个氨基酸组成。 所述 全蛋蛋白肽可以以浓缩液、 冻干粉、 喷雾干燥干粉、 水剂或固体剂的形式 存在。
本发明的另一个目的是提供所述全蛋蛋白肽的应用。
本发明所提供的全蛋蛋白肽的应用是其在制备增强免疫力的产品中的 应用。
本发明的再一个目的是提供一种用于增强免疫力的产品。
本发明所提供的用于增强免疫力的产品, 其活性成分包括本发明所提 供的全蛋蛋白肽。 所述产品可为药品或保健品。
当然, 还可根据需要在所述产品中加入其它能增强免疫力的物质,如大 枣多糖和枸杞多糖。 所述增强免疫力产品中, 全蛋蛋白肽、 大枣多糖和枸 杞多糖的质量比可为 (900-1200 ) : ( 8-30 ) : ( 3-15 ) 。
以全蛋蛋白肽为活性成分制备的增强免疫力的药物或保健品, 需要的 时候, 在上述药物或保健品中还可以加入一种或多种药学上可接受的载 体。 所述载体包括药学领域常规的稀释剂、 赋形剂、 填充剂、 粘合剂、 湿 润剂、 崩解剂、 吸收促进剂、 表面活性剂、 吸附载体、 润滑剂等。
所述增强免疫力的药物可以制成口服液、 片剂、 颗粒剂、 胶囊、 膏 剂、 膜剂等多种形式。 上述各种剂型的药物均可以按照药学领域的常规方 法制备, 所得到的药物均无苦味。
以全蛋蛋白粉为原料进行酶解是本发明的一个显著特点。 上述蛋类的蛋 白质评分均接近 100分, 其蛋白质与人体蛋白质接近, 容易被人体吸收和利 用。 而选择全蛋是科学的选择, 若仅选择蛋清蛋白质, 蛋黄中的蛋白质和很 多营养素会丢失; 若仅选择蛋黄, 蛋清中的蛋白质也同样丢失, 选择全蛋也 就是选择了一个完整的营养体系, 不仅营养全面, 而且各营养物质彼此可发 挥协同增效作用。
本发明采用复合植物蛋白酶, 即木瓜蛋白酶、 无花果蛋白酶和菠萝蛋 白酶, 三酶科学配方形成复合酶制剂, 对全蛋蛋白粉进行催化降解, 获得 全蛋蛋白肽。 该全蛋蛋白肽可替代多肽激素类免疫剂, 起到多肽激素类免 疫剂的免疫调节作用。 多肽激素类免疫剂是药物, 只能对病人使用, 而本 发明的产品可以制成口服的多肽营养免疫剂, 不仅能对病人起到治疗的效 果, 而且能对无病的人起到增强免疫力、 防病健体的作用。 该产品是一种 口服制剂, 比针剂携带方便、 使用安全, 使用时不会产生排异、 过敏或其 他副作用反应, 不需在医生的监督下使用。 可用于免疫功能低下、 免疫功 能障碍、 免疫功能下降、 免疫器官受损等引起的诸多疾病和亚健康状态, 还可用于蛋白质营养缺乏者, 因蛋白质营养缺乏引起的诸多病症及亚健康 状态的人群, 可用于预防感冒, 可用于运动后、 产后、 病后、 术后人群, 促进 "负氮平衡" , 对产后恢复、 病后恢复、 伤口愈合、 保肝护肝、 醒 酒、 恢复食欲、 促进睡眠、 恢复肾脏功能、 剌激巨噬细胞的吞噬能力、 抑 制肿瘤细胞的生长、 升高白血球等有较好的作用。
附图说明
图 1为本发明制备全蛋蛋白肽的生产工艺流程图。
实施发明的最佳方式
下述实施例中所述实验方法, 如无特殊说明, 均为常规方法; 所述试 剂和材料, 如无特殊说明, 均可从商业途径获得。 实施例 1、 制备增强免疫力的全蛋蛋白肽口服液
将全蛋蛋白粉 (冷冻干燥干粉或喷雾干燥干粉) 放到发酵罐内, 加相 当于全蛋蛋白粉质量 10 倍的水, 逐步将温度升至 5CTC时, 将按酶解每克全 蛋蛋白粉所需各种酶的量为:木瓜蛋白酶 5万 U,无花果蛋白酶 2万 U,菠萝蛋 白酶 35万 U配伍成的复合蛋白酶加入发酵罐中酶解。 温度始终保持在 50 V, 酶解 4小时后, 进行灭菌和酶灭活处理,然后冷却至 60°C进行装桶, 再 冷却至常温时放入冷藏室, 冷冻沉降 2天后过滤, 即获得全蛋蛋白肽浓缩 液。 然后进行配制, 配制的功效成分构成为: 每 100ml口服液中含全蛋蛋白 肽 1000mg ± 10%,配制后灌装并于 10CTC灭菌 10分钟, 即得到增强免疫力的口 服液。 实施例 2、 制备含大枣多糖和枸杞多糖的增强免疫力的全蛋蛋白肽口服 液
将全蛋蛋白粉 (冷冻干燥干粉或喷雾干燥干粉) 放到发酵罐内, 加相 当于全蛋蛋白粉质量 8倍的水, 逐步将温度加温至 48°C时, 按照酶解每克全 蛋蛋白粉需木瓜蛋白酶 6万 U, 无花果蛋白酶 1. 5万 U,菠萝蛋白酶 28万 U, 将 上述三种酶配伍成复合蛋白酶加入发酵罐中进行酶解, 温度始终保持在 48 V, 酶解 3.5小时后, 进行灭菌和酶灭活处理, 然后冷却至 60°C进行装桶, 再冷却至常温时放入冷藏室, 冷冻沉降 2天后过滤, 即获得全蛋蛋白肽浓缩 液。 然后进行配制, 配制的功效成分构成为: 每 100ml口服液中含全蛋蛋白 肽 1000mg ± 10%, 大枣多糖 10mg, 枸杞多糖 5mg, 配制后灌装并于 10CTC灭 菌 10分钟, 即得到增强免疫力的口服液。 实施例 3、 制备含大枣多糖和枸杞多糖的增强免疫力的全蛋蛋白肽口服 液
将全蛋蛋白粉 (冷冻干燥干粉或喷雾干燥干粉) 放到发酵罐内, 加相 当于全蛋蛋白粉质量 8倍的水, 逐步将温度加温至 5CTC时, 按照酶解每克全 蛋蛋白粉需木瓜蛋白酶 6万 U, 无花果蛋白酶 2万 U,菠萝蛋白 32万 U, 将上述 三种酶配伍成复合蛋白酶加入发酵罐中进行酶解, , 温度始终保持在 50 V, 酶解 3小时后, 进行冷却装桶, 再冷却 12 °C时放入冷藏室, 冷冻沉淀 2 天后过滤, 即获得全蛋蛋白肽浓缩液。 然后进行配制, 配制的功效成分构 成为: 每 100ml口服液中含全蛋蛋白肽 1000mg ± 10%, 大枣多糖 15mg, 枸杞 多糖 10mg, 配制后灌装并于 10CTC灭菌 10分钟, 即得到增强免疫力的口服 液。 实施例 4、 增强免疫力的口服液初步稳定性试验
试验样品: 实施例 1制备的口服液
考察指标:
1、 性状: 按常规方法对样品的形状 (含颜色) 及气味进行检査。
2、 含量测定:
对样品中的主要成分, 即以有机氮 (%) 表示肽及氨基酸含量, 测定方 法参照 GB5009。
3、 卫生学及理化指标检査: 按照食品卫生国标 GB4789、 GB5009要求 对产品中的细菌总数、 大肠菌群、 致病菌、 霉菌及酵母和砷、 铅、 汞含量 进行检测。
4、 加速试验:
将三批不同日期生产的样品 (批号分别为 090718、 090720、 090724, 均为玻璃瓶装) 置于相对湿度为 75%的干燥器内 (干燥器底部放置氯化钠饱 和溶液) , 然后将干燥器放入 37°C恒温箱中, 连续加热三个月, 定期抽样 (0、 1、 2、 3月) , 测定相关指标 (结果见表 1-3) 。 在三个月的加速试验 中各项指标均未发生明显变化, 由此可推算出: 本品的室温留样期大于 2 年。
表 1、 批号 090718样品的稳定性结果
0月 1月 2月 3月 项目^ ^^^ί果
性状 淡黄色液体 同左 同左 同左 质量指标 ρΗ 值 5.28 5.27 5.29 5.27
肽及氨基酸 (以有 0.218 0.216 0.216 0.213
机氮表示%)
砷 (mg/kg) <0.1 未测 未测 <0.1 铅 (mg/kg) <0.3 未测 未测 <0.3
¾ (mg/kg) 未检出 未测 未测 未检出 卫生学检测 细菌总数(CFU/ml) <10 <10 <10 <10
大肠菌群 MNP (100ml) <3 <3 <3 <3
霉菌(CFU/ml) 未检出 同左 同左 同左 酵母菌(CFU/ml) 未检出 同左 同左 同左
致病菌 未检出 同左 同左 同左 表 2、 批号 090720样品的稳定性结果
0月 1月 2月 3月 项目^ ^^^ί果 性状 淡黄色液体 同左 同左 同左 质量指标 ρΗ 值 5.29 5.26 5.26 5.27 肽及氨基酸 (以有 0.219 0.216 0.213 0.218 机氮表示%)
砷 (mg/kg) <0.1 未测 未测 <0.1 铅 (mg/kg) <0.3 未测 未测 <0.3
¾ (mg/kg) 未检出 未测 未测 未检出 卫生学检测 细菌总数(CFU/ml) <10 <10 <10 <10 大肠菌群 MNP (100ml) <3 <3 <3 <3 霉菌 (CFU/ml) 未检出 同左 同左 同左 酵母菌 (CFU/ml) 未检出 同左 同左 同左 致病菌 未检出 同左 同左 同左 表 3、 批号 090724样品的稳定性结果
0月 1月 2月 3月 项目^ ^^^ί果
性状 淡黄色液体 同左 同左 同左 质量指标 pH 值 5.27 5.27 5.28 5.27 肽及氨基酸 (以有 0.216 0.214 0.213 0.212 机氮表示%)
砷 (mg/kg) <0.1 未测 未测 <0.1 铅 (mg/kg) <0.3 未测 未测 <0.3
¾ (mg/kg) 未检出 未测 未测 未检出 卫生学检测 细菌总数(CFU/ml) <10 <10 <10 <10 大肠菌群 MNP (100ml) <3 <3 <3 <3 霉菌 (CFU/ml) 未检出 同左 同左 同左 酵母菌 (CFU/ml) 未检出 同左 同左 同左 致病菌 未检出 同左 同左 同左 结论: 本品对热比较稳定, 在常温下可保存两年。 实施例 5、 增强免疫力的口服液的安全性评价试验
1) 急性毒性试验
动物: 昆明种小鼠 18-20g, 雌雄各半, 共 20只; 动物由湖北省医学实 验动物中心提供。
受试药物: 实施例 1制备的增强免疫力的口服液。
给药方式: 受试动物按 0.3ml/10g.b.w灌胃。 灌胃前禁食 12h, 观察一 周。
结果: 小鼠灌胃后无中毒症状, 一周内无死亡, LD50>30ml/kg。 按急 性毒性分级, 该受试品属无毒类物质。
2) 微核试验
动物: 昆明种小鼠, 25-27g。
试验方法: 两次灌胃给予受试药物, 第二次灌胃 6h后, 处死动物, 取 胸骨髓用小牛血清稀释涂片, Gimsa染色, 每只动物显微镜下观察 1000个嗜 多染红细胞。 记录微核细胞形成的数目, 以千分率表示。 结果见表 4。 其 中, CP组为环磷酰胺阳性对照组。
表 4
剂量 动物数 观察细胞数 出现微核细 微核形成率 受试物
(ml/mg) (只) (个) 胞数 (个) (%) 阴性对照组 0 10 10X 1000 21 2.1
2 10 10X 1000 29 2.9
4 10 10X 1000 27 2.7 试验组 6 10 10X 1000 21 2.1
8 10 10X 1000 26 2.6
10 10 10X 1000 24 2.4
CP组
60 8 8X 1000 325 40.6 (mg/kg)
结论: 各实验组与阴性对照组结果比较, 无显著性差异; 表明该受试 产品骨髓微核试验结果为阴性。
3) 小鼠精子畸形试验:
动物: 昆明种小鼠 23-25g。 试验方法: 连续灌胃 5天后, 再继续饲喂 30天。 处死动物, 取两侧副睾 制片。 每只动物计数 1000个结构完整的精子, 计算精子畸形发生率。 结果 见表 5。 其中, CP组为环磷酰胺阳性对照组。
表 5
剂量 动物数 观察细胞数 出现微核细 微核形成率 受试物
(ml/mg) (只) (个) 胞数 (个) ( %) 阴性对照组 0 5 5 X 1000 89 1. 78
5 5 5 X 1000 91 1. 82 试验组 10 5 5 X 1000 99 1. 98
15 5 5 X 1000 97 1. 94
CP组
30 5 5 X 1000 359 7. 18 (mg/kg)
结论: 各实验组与阴性对照组结果比较, 无显著性差异; 表明该受试 产品精子畸形试验结果为阴性。
4 ) Ames试验
采用经鉴定符合生物学要求的鼠伤寒沙门氏菌组氨酸缺陷型 TA97、 TA98、 TA100、 TA102四株菌株进行试验: 采用多氯联苯 (PCB ) 诱导的大 鼠肝匀浆作为体外活化系统 (+S9 ) 。 受试产品分为三个剂量组, 样品稀释 成不同浓度, 灭菌每皿加入 0. 5ml, 使浓度分别为 1 : 10、 1: 100、 1: 1000。 采用掺入法记录两次重复的平行样品结果。 结果见表 6。
表 6
Figure imgf000009_0001
阳性 0.0003 > > 396 25 164 > > 283 对照 5000 3000 3000 3000
注: -S9阳性对照组为 3-硝基 9-芴酮 (2.4.7-TNFone) , +S9阳性对照组为 2-氨基芴 (2-AF) 由上表可知, 各实验组回变菌落数均未超过自发回变数的两倍; 且无 剂量反应关系, 试验结果为阴性。 实施例 6、 全蛋蛋白肽的免疫调节作用检测
1、 试验动物: 昆明种小鼠 18-22g, 雌雄各半。 (湖北省医学实验动 物中心提供)
2、 分组: 剂量分组为: 对照组, 灌胃蒸馏水; "实施 1 制备的口服 液" 的浓度为 1% (即 lOOOmg/lOOml ) , 低、 中、 高剂量组分别以 lOOmg/kg. b. w、 300mg/kg. b. w、 900mg/kg. b. w的口月艮液灌胃; 低、 中、 高剂量组剂量分别相当于人推荐摄入量的 6、 18、 54 倍。 灌胃一个 月, 然后进行各项实验。
3、 实验方法与结果:
3.1 ConA诱导的小鼠脾淋巴细胞转化试验
方法: MTT法 (ConA诱导的小鼠脾淋巴细胞转化试验) 对各组加 ConA 孔与不加 ConA孔吸光度差值的均值进行单因素方差分析。
结果: 见表 7。
从表 7 可见, 组间差距显著, "口服液"低剂量组显著性增强 ConA诱 导的脾淋巴细胞增殖能力。 表 7 "口服液"对小鼠细胞免疫功能的影响
ConA诱导脾淋巴细胞增值 DNFB诱导 DTH 组别
N=10 (OD差值, M+SD) N=9 (左右耳重差量, mg) 对照组 0.229±0.075 16.14± 12.02
低剂量组 0.344±0.055* 26.89±6.64*
中剂量组 0.241±0.013 18.21±6.96
高剂量组 0.24. ±0.097 18.89±7.26
与对照组比较 * P<0.05 注: 取 0.2ml测定 0D57。值
3.2二硝基氟苯 (DNFB) 诱导迟发型变态反应 (DTH)
方法: 耳肿胀法。 用 DNFB 致敏小鼠后, 第五天再用 DNFB 攻击右耳, 24h 后处死动物剪下左右耳壳用打孔器取下直径 8mm 的耳片, 称重, 以左 右耳的重量之差来表示 DTH的程度。
结果: 见表 7。
从表 7可见: "口服液"低剂量组可显著提高小鼠对 DNFB诱发的 DTH 反应。
3. 3 血清溶血素的测定
方法: 血凝法。 根据血清凝聚程度的级别计算出抗体积数,对各组抗体 积数的均值进行单因素方差分析.
结果: 见表 8。
从表 8 可见, 组间差距显著, "口服液"低、 高剂量组可显著性增加血 清溶血素含量。 表 8 "口服液"对小鼠体液免疫功能的影响
组别 N 血清溶血素 (抗体积数) 对照组 10 66.7 ± 11.6
低剂量组 10 90.4 ± 8.40*
中剂量组 10 73.5 ± 17.7
10 80.9 ± 5.09*
Figure imgf000011_0001
3. 4 腹腔巨噬细胞吞噬鸡红细胞实验
方法: 半体内法。 制备 20%的鸡红细胞悬液; 每只鼠腹腔注射 ImL该悬 液, 30min后处死动物, 开腹, 经腹腔注入生理盐水 2mL, 平均分滴于 2片 载玻片上, 37 °C温育 30min; 育毕用生理盐水漂洗, 晾干, 以 1 : 1 的丙酮 甲醇溶液固定, 4% Giemsa-磷酸缓冲液染色 3min, 再用蒸馏水漂洗晾干。 油镜下计数 100个巨噬细胞, 按下式计算吞噬率和吞噬指数:
吞噬率 (% ) =吞噬鸡红细胞的巨噬细胞数議
计数的巨噬细胞数
^ ^ψ-被吞噬的鸡红细胞总数
'、 — ~计数的巨噬细胞数
结果: 见表 9。 从表 9可见, "口服液"低、 高剂量组吞噬率明显高于 对照组; 低剂量组吞噬指数明显高于对照组。 表 9 "口服液"对小鼠腹腔巨噬细胞吞噬功能的影响 组别 N 吞噬百分率 (%) 吞噬指数 对照组 10 77.2±2.15 1.037±0.053 低剂量组 10 81.5±2.76** 1.237±0.145 中剂量组 10 79.3±3.95 1.097±0.130 高剂量组 10 80.2±3.52 1.106±0.095 与对照组比较 * P<0.05 与对照组比较 * * P<0.01 3.5 小鼠碳廓清试验
方法: 依程序规定的方法。 根据注入墨汁 2min、 lOmin 后血中碳浓度 (测吸光值) , 计算出各组小鼠的吞噬指数。
结果: 见表 10。 从表 10 可见, "口服液"低剂量组可显著增强小鼠 碳廓清吞噬指数。 表 10 "口服液"对小鼠碳廓清功能的影响
组别 N 吞噬指数
对照组 5 6.609 ±0.755
低剂量组 10 7.196 ±0.489* 中剂量组 10 5.966±0.670
高剂量组 10 6.152±0.854
与对照组比较 * P〈0.05 免疫调节作用实验结果表明: "全蛋蛋白肽的口服液" 1) 可增强 ConA 诱导的小鼠脾淋巴细胞增殖能力; 2) 增强二硝基氟苯诱导的小鼠迟 发型变态反应; 3) 提高小鼠血清溶血素含量; 4) 增强小鼠腹腔巨噬细胞 吞噬鸡红细胞功能和碳廓清能力。 依据 《保健食品功能学评价程序和检验 方法》 第 3.1 项规定的免疫调节作用的评价方法, 可认为本发明所制备的 全蛋蛋白肽具有免疫调节作用。
工业应用
本发明以全蛋蛋白粉为底物, 采用复合植物蛋白酶: 即木瓜蛋白酶、 无花果蛋白酶和菠萝蛋白酶, 三酶科学配方形成复合酶制剂, 对全蛋蛋白 粉进行催化、 降解, 得到全蛋蛋白肽。 该产品具有较高的生物活性, 是一 种新型的免疫调节剂和免疫增强剂。 本发明的全蛋蛋白肽可用于开发免疫 药剂、 保健食品、 功能食品、 抗衰老和婴幼儿食品及重病患者的营养补充 剂。 本发明通过冷冻干燥制成冻干粉,进而可制成胶囊或片剂,可深度开发 肿瘤辅助治疗药物 (增强免疫功能, 增强放化疗患者抗辐射能力、 升高 红、 白血球) 、 抗炎药物及肝炎治疗药物、 非典型肺炎的防治药物,也可作 为其他药物原料或作为食品添加剂。 无论哪类药物、 保健品、 功能食品的 开发都将产生巨大的经济和社会效益, 其前景非常广阔。

Claims

权利要求
1、 一种制备全蛋蛋白肽的方法, 是采用复合植物蛋白酶对全蛋蛋白粉 进行酶催化降解, 得到所述全蛋蛋白肽; 所述复合植物蛋白酶由木瓜蛋白 酶、 无花果蛋白酶和菠萝蛋白酶组成。
2、 根据权利要求 1所述的方法, 其特征在于: 所述复合植物蛋白酶 中, 木瓜蛋白酶、 无花果蛋白酶和菠萝蛋白酶的酶活力比为(100〜120 万) U : (30—40万) U : (560—700万) U。
3、 根据权利要求 1或 2所述的方法, 其特征在于: 所述全蛋蛋白粉是 由下述至少一种禽鸟蛋为原料制备得到的: 鸡蛋、 鸭蛋、 鹅蛋、 鹌鹑蛋、 麻雀蛋、 鸽蛋、 斑鸠蛋和鸵鸟蛋。
4、 根据权利要求 1-3中任一所述的方法, 其特征在于: 所述酶催化降 解的反应体系中, 全蛋蛋白粉与水的质量份数比为 1 : 8-10; 酶解每克全 蛋蛋白粉所需各种酶的量为: 木瓜蛋白酶 5〜6万 U,无花果蛋白酶 1. 5〜2 万 U,菠萝蛋白酶 28〜35万 U; 所述酶催化降解的温度为 48-50°C, 时间为 3〜4小时, pH值为 7. 5〜8. 5。
5、 根据权利要求 1-4中任一所述的方法, 其特征在于: 所述方法还包 括在酶催化降解反应结束后, 对酶解液进行灭菌和酶灭活处理, 然后再对 所述酶解液进行冷却、 冷冻处理的步骤; 所述冷冻的温度为 4-8°C, 时间 为 48小时; 所述灭菌和灭酶的条件为: 温度 100°C, 时间 10分钟。
6、 权利要求 1-5中任一所述方法制备得到的全蛋蛋白肽。
7、 权利要求 6所述的全蛋蛋白肽在制备增强免疫力产品中的应用。
8、 根据权利要求 7所述的应用, 其特征在于: 所述产品为药品或保健
P
9、 一种增强免疫力的产品, 其活性成分包括权利要求 8所述的全蛋蛋 白肽。
10、 根据权利要求 9所述的产品, 其特征在于: 所述增强免疫力的产品 其活性成分还包括大枣多糖和枸杞多糖; 所述增强免疫力产品中, 全蛋蛋白 肽、 大枣多糖和枸杞多糖的质量比为 (900-1200) : (80-140) : (40- 70) 。
PCT/CN2010/001287 2010-08-24 2010-08-24 全蛋蛋白肽及其制备方法和应用 WO2012024817A1 (zh)

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CN107164448A (zh) * 2017-07-12 2017-09-15 邹远东 一种铁棍怀山药多肽及其制备方法
CN108244031B (zh) * 2017-12-15 2020-08-28 中国水产科学研究院东海水产研究所 一种提高免疫低下模型小鼠非特异性免疫力的方法
CN109744492A (zh) * 2019-01-10 2019-05-14 蕴彤本草(北京)生物科技有限公司 高生物活性全蛋体发酵物的生产方法及其产品与应用
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CN113995071A (zh) * 2021-10-29 2022-02-01 杭州倍欣生物科技有限公司 一种白蛋白肽饮品和制备方法以及在提升血浆白蛋白指标上的应用
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