WO2017076112A1 - 一种具有提升性功能的牡蛎肽及其制备方法和应用 - Google Patents

一种具有提升性功能的牡蛎肽及其制备方法和应用 Download PDF

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WO2017076112A1
WO2017076112A1 PCT/CN2016/097358 CN2016097358W WO2017076112A1 WO 2017076112 A1 WO2017076112 A1 WO 2017076112A1 CN 2016097358 W CN2016097358 W CN 2016097358W WO 2017076112 A1 WO2017076112 A1 WO 2017076112A1
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Prior art keywords
oyster
preparation
protease
peptide
oyster peptide
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PCT/CN2016/097358
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English (en)
French (fr)
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肖小春
翟旭峰
郭晓蕾
娄勇军
马忠华
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无限极（中国）有限公司
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Priority to EP16861381.8A priority Critical patent/EP3231873A4/en
Priority to US15/542,743 priority patent/US20170362629A1/en
Publication of WO2017076112A1 publication Critical patent/WO2017076112A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43509—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from crustaceans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • the invention relates to the field of biotechnology, in particular to an oyster peptide having a lifting function and a preparation method and application thereof.
  • Male sexual dysfunction is a common disease with an incidence of about 10% of adult males. With the acceleration of the pace of modern life, the pressure from work, family, and economy increases, the higher the probability of male sexual dysfunction.
  • Oyster also known as medlar, medlar, and sea bream, is a bivalve mollusc that belongs to the mollusk, the scorpion, and the oyster.
  • Oyster is the world's largest farmed shellfish, mainly distributed along the maritime and tropical oceans, and is also one of the four largest shellfish species in China.
  • "Herbal Supplements” records that oyster meat has the effect of "cooking, main damage, ... raw food in ginger vinegar, main erysipelas, hot after drinking, thirst quenching".
  • the application areas are limited; currently oysters are mainly sold as fresh or dried seafood.
  • an object of the present invention is to provide an oyster peptide having a lifting function and The preparation method and application thereof enable the preparation method to reduce the amount of enzyme used, and the prepared oyster peptide has better effect of improving sexual function and anti-oxidation effect, and can be applied to preparation of related health care products, foods and medicines. in.
  • the present invention provides the following technical solutions:
  • a method for preparing an oyster peptide having a lifting function comprising:
  • Step 1 Grind the oyster meat, calcium salt and water to obtain an oyster meat slurry
  • Step 2 enzymatic hydrolysis of the oyster meat slurry, and centrifuging the supernatant after enzymatic hydrolysis to obtain a crude liquid of the oyster;
  • Step 3 decolorizing and removing impurities from the oyster enzymatic crude liquid to obtain an oyster peptide refining liquid
  • Step 4 The oyster peptide refining solution is concentrated and spray-dried to obtain the oyster peptide.
  • the invention aims at the defects of using the enzyme preparation in the existing enzymatic hydrolysis technology, the target efficacy is not strong, and the protein recovery rate is not high.
  • the calcium salt is used for pretreatment.
  • the addition of calcium ions activates the endogenous enzyme of oyster meat, and the released endogenous enzyme of oyster meat can efficiently hydrolyze oyster protein, thereby reducing the amount of commercial protease and reducing the production cost.
  • synergistic step of enzymatic hydrolysis not only improves the protein recovery rate, but also makes the obtained oyster polypeptide have better efficacy.
  • the calcium salt is food grade calcium chloride, calcium lactate, calcium carbonate, calcium hydrogen phosphate or calcium citrate.
  • the calcium salt is a calcium salt of from 0.1 to 0.3% by mass of the oyster meat.
  • the calcium salt may be 0.1% calcium chloride, 0.3% calcium lactate or 0.2% calcium citrate.
  • the water in the step 1 is 0.5 to 1.0 times the demineralized water of the mass of the oyster meat.
  • the water may be 0.5 times the softening water of the mass of the oyster meat, 0.8 times the softened water of the mass of the oyster meat or 1.0 times the softened water of the mass of the oyster meat.
  • step 2 is:
  • the oyster meat slurry is first digested with a neutral protease or an alkaline protease, and then enzymatically digested with a flavor protease. After enzymatic hydrolysis, the enzyme is inactivated and centrifuged, and the supernatant is lysed to dissolve the crude liquid.
  • step 2 is:
  • the neutral protease is one or more of papain, bromelain, animal proteolytic enzyme, and complex protease Protamex; the neutral protease or alkaline protease is added in an amount of oyster meat slurry. 0.3 to 0.8 ⁇ ; the alkaline protease is one or more of Alcalase alkaline protease, FoodPro Alkaline Protease alkaline protease, trypsin; the flavor protease is added in an amount of 0.5 to 1.0 ⁇ of the oyster meat slurry. .
  • the pH was adjusted to 6.0-7.0 when enzymatically digested with a neutral protease, and the pH was adjusted to 7.5-8.5 when enzymatically digested with an alkaline protease.
  • step 3 is:
  • the obtained filtrate is an oyster peptide refining liquid.
  • step 3 is:
  • Activated carbon was added to the oyster enzymatic crude solution, and the mixture was stirred at 45 to 55 ° C for 0.5 to 1.0 hour, and filtered, and the obtained filtrate was an oyster peptide purified solution.
  • the oyster peptide prepared by the method of the invention consumes no more than 0.8 ⁇ of neutral protease or alkaline protease, and the flavor protease does not exceed 1.0 ⁇ , but does not use the same enzymatic hydrolysis process of calcium salt pretreatment, and consumes neutral protease or alkali.
  • the protease was 1.0 ⁇
  • the flavor protease was also 1.0 ⁇
  • the enzyme preparation was used in a high amount.
  • the enzymatic hydrolysis is carried out in two steps by using the enzyme preparation of the present invention, and the obtained oyster peptide can improve the sexual function and the antioxidant function of the mouse in comparison with the process of enzymatic hydrolysis of other enzyme preparations or the same enzyme preparation of the present invention. The effect is better.
  • the present invention provides an oyster peptide prepared by the preparation method of the present invention, and the use of the oyster peptide in the preparation of an improved sexual function and/or an antioxidant health care product, a food, and a pharmaceutical.
  • the medicine and the health care product are pills, capsules, tablets, powders, granules, oral liquids or ointments.
  • the present invention pretreats the calcium oyster before enzymatic hydrolysis of the oyster meat to activate the release of the endogenous enzyme of the oyster, reduce the enzymatic preparation of the subsequent enzymatic hydrolysis, and cooperate with the oyster endogenous enzyme.
  • the invention discloses a preparation method of an oyster peptide having a lifting function, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the oyster peptides of the present invention, and the preparation methods and applications thereof have been described by way of examples, and it is obvious that the methods and applications described herein can be modified or appropriately modified and combined without departing from the spirit, scope and scope of the present invention. To implement and apply the techniques of the present invention.
  • the method for detecting the antioxidant activity (reducing power, DPPH IC 50 ) of oyster peptide in vitro and the method for testing the ascending function of oyster peptide in male mature mice are as follows:
  • the potassium ferricyanide reduction system was used, and the reducing power of the sample was measured at 700 nm.
  • the prepared sample was serially diluted with protein concentration, and 2 ml of the sample solution was taken in a test tube, and 2 ml of 0.2 mol/L phosphate buffer (PBS, pH 6.6) and 2 ml of 1% (m/V) potassium hexacyanocyanide solution were added.
  • PBS 0.2 mol/L phosphate buffer
  • m/V 1% potassium hexacyanocyanide solution
  • mice Male mature mice, weighing 18-22 g, were randomly divided into blank control group, sample group and comparative sample group, 12 rats in each group, and administered continuously for 28 days.
  • each female was injected subcutaneously with estradiol benzoate (0.02 mg/mouse) to make the females have the same estrus, and the test was carried out at 7-11 pm.
  • estradiol benzoate 0.2 mg/mouse
  • Percentage of capture number of captured animals / total number of animals
  • Percentage of ejaculation number of ejaculation animals / total number of animals
  • the experimental data were analyzed by SPSS19.0 software.
  • the data of each group were expressed as mean.
  • the comparison between groups was analyzed by one-way analysis of variance; when the overall variance of each group was not When the homogeneity of variance was satisfied, the comparison between groups was analyzed by Welch method, and the difference was significant at p ⁇ 0.05.
  • Example 1 Preparation method of oyster peptide of the present invention
  • the oysters are shelled, the meat is cleaned, the meat grinder is minced, the oyster meat minced meat is added with 0.1% calcium chloride and 1.0 times water, and then the oyster meat slurry is obtained by colloid grinding.
  • the oyster peptide refining solution was concentrated to a solid content of 35% or more by vacuum concentration, and spray-dried to obtain oyster peptide sample 1.
  • the oysters are shelled, the meat is cleaned, minced by a meat grinder, and the oyster meat is 0.3% calcium lactate and 0.5 times water, and then the oyster meat slurry is obtained by a colloid mill.
  • the oyster peptide refining solution was concentrated to 45% of solid content by vacuum concentration, and spray-dried to obtain oyster peptide sample 2.
  • the oysters are shelled, the meat is cleaned, minced by a meat grinder, and the oyster meat is 0.2% calcium citrate and 0.8 times water, and then the oyster meat slurry is obtained by a colloid mill.
  • the oyster peptide refining solution was concentrated to 40% of solid content by vacuum concentration, and spray-dried to obtain oyster peptide sample 3.
  • the oysters are shelled, the meat is cleaned, minced by a meat grinder, and the oyster meat is 0.2% calcium citrate and 0.8 times water, and then the oyster meat slurry is obtained by a colloid mill.
  • the oyster peptide refining solution was concentrated to 40% of solid content by vacuum concentration, and spray-dried to obtain oyster peptide sample 4.
  • the oysters are shelled, the meat is cleaned, minced by a meat grinder, and the oyster meat is 0.2% calcium citrate and 0.8 times water, and then the oyster meat slurry is obtained by a colloid mill.
  • the oyster peptide refining solution was concentrated to 40% of solid content by vacuum concentration, and spray-dried to obtain oyster peptide sample 5.
  • the oysters are shelled, the meat is cleaned, minced by a meat grinder, and the oyster meat is 0.2% calcium citrate and 0.8 times water, and then the oyster meat slurry is obtained by a colloid mill.
  • the oyster peptide refining solution was concentrated to 40% of solid content by vacuum concentration, and spray-dried to obtain oyster peptide sample 6.
  • the oysters are shelled, the meat is cleaned, minced by a meat grinder, and the oyster meat is 0.2% calcium citrate and 0.8 times water, and then the oyster meat slurry is obtained by a colloid mill.
  • the oyster peptide refining solution was concentrated to 40% of solid content by vacuum concentration, and spray-dried to obtain oyster peptide sample 7.
  • Example 8 Preparation method of oyster peptide of the present invention
  • the oysters are shelled, the meat is cleaned, minced by a meat grinder, and the oyster meat is 0.2% calcium citrate and 0.8 times water, and then the oyster meat slurry is obtained by a colloid mill.
  • the oyster peptide refining solution was concentrated to 40% of solid content by vacuum concentration, and spray-dried to obtain oyster peptide sample 8.
  • Comparative Example 1 Preparation method of comparative oyster peptide
  • the oysters are shelled, the meat is cleaned, the meat grinder is minced, 1.0 times of water is added to the oyster meat, and then the oyster meat slurry is obtained by colloid grinding.
  • Comparative Example 2 Preparation method of comparative oyster peptide
  • the oysters are shelled, the meat is cleaned, the meat grinder is minced, the oyster meat minced meat is added with 0.1% calcium chloride and 0.8 times water, and then the oyster meat slurry is obtained by colloid grinding.
  • Example 9 Enzymatic protein recovery and in vitro antioxidant activity of different peptide samples
  • Test samples oyster peptide samples 1-3 prepared in Examples 1-3, Comparative Samples 1-2 prepared in Comparative Examples 1-2.
  • the in vitro antioxidant value (reducing power and DDPH clearance) of the oyster peptide prepared by the method of the invention is superior to the comparative sample; the oyster peptide sample has higher reducing power and lower DPPH IC 50 value, which is mainly Because the particular combination of enzyme preparations selected in the present invention and the two-step enzymatic hydrolysis are more favorable for the release of antioxidant activity.
  • Comparative Example 2 has a high protein recovery rate, its in vitro antioxidant value (reducing power and DPPH) is low, and thus it can be proved that the enzyme preparation selected in this patent is an optimal enzyme preparation combination for hydrolyzing oysters to obtain antioxidant activity.
  • the oyster peptide samples 4-8 were tested, and the results showed that the protein recovery rate of the oyster peptide samples 4-8 was about 82%, which was significantly higher than that of the comparative sample 1, and the difference was not obvious from the comparative sample 2.
  • the reducing power of oyster peptide samples 4-8 was about 0.55, which was higher than that of comparative samples 1 and 2; the DDPH clearance rate was not higher than 3.5 mg/ml, which was lower than that of comparative samples 1 and 2; the comprehensive test results show that the present invention
  • the oyster peptide samples provided have higher reductive power and lower DPPH IC 50 values while having higher protein recovery.
  • Example 10 Effect of different oyster peptide samples on mating ability of male mice
  • Test samples oyster peptide samples 1-3 prepared in Examples 1-3, Comparative Samples 1-2 prepared in Comparative Examples 1-2.
  • the oyster peptide samples prepared by this patent had extremely significant differences in the number of captures and the number of ejaculations compared with the blank control (P ⁇ 0.01);
  • the oyster peptide samples prepared by this patent had extremely significant differences in the interval between the capture latency and the ejaculation latency period compared with the blank control (P ⁇ 0.01). ), and significantly increased the percentage of capture and ejaculation percentage; and the percentage of capture, ejaculation percentage is higher than the blank control group.
  • the oyster peptide samples prepared by this patent showed significant improvement in the number of captures and the number of ejaculations compared with the control samples (Comparative Sample 1 and Comparative Sample 2) (P ⁇ 0.01).
  • Significant (P ⁇ 0.01) increased percentage of capture and percentage of ejaculation; and the percentage of capture and ejaculation was higher than that of the control sample group.
  • the oyster peptide samples 4-8 were detected, and the detection results of the oyster peptide samples were significantly improved in the number of captures and the number of ejaculations compared with the above-mentioned comparative samples and the blank control group (P ⁇ 0.01).
  • Significant (P ⁇ 0.01) increased the percentage of capture and ejaculation percentage, and there was also a significant difference in the interval between capture latency and ejaculation latency (P ⁇ 0.01).
  • the oyster peptide sample prepared according to the patented method has good anti-oxidation effect in vitro and has a good improvement effect on the sexual function of the mouse.

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Abstract

本发明公开了一种具有提升性功能的牡蛎肽及其制备方法和应用。该方法在对牡蛎肉进行酶解前先用钙盐进行预处理，用以激活释放牡蛎内源性酶，可减少后续酶解消耗的酶制剂。

Description

一种具有提升性功能的牡蛎肽及其制备方法和应用
本申请要求于2015年11月05日提交中国专利局、申请号为201510750039.X、发明名称为“一种具有提升性功能的牡蛎肽及其制备方法和应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物技术领域，具体涉及一种具有提升性功能的牡蛎肽及其制备方法和应用。
背景技术
男性性功能障碍是一个常见病，发病率约占成年男性的10％。随着现代生活节奏的加快，来自工作、家庭、经济等方面的压力增大，男性发生性功能障碍的概率越高。
牡蛎，又称蚝、蛎黄、海蛎子，是一种双壳类软体动物，属软体动物门，瓣鳃纲，牡蛎科。牡蛎是全球的第一大养殖贝类，主要分布在温带和热带的各大洋沿岸，同时也是我国四大养殖贝类之一。《本草拾遗》记载牡蛎肉具有“煮食，主虚损，……于姜醋中生食之，主丹毒，酒后烦热，止渴”的功效。但由于牡蛎的生产季节强，且有较强的鱼腥味，限制其应用领域；目前牡蛎主要以鲜吃或作为海产干货出售。因此，以牡蛎为原料，结合生物酶解技术制备蛋白肽产品，拓展其应用领域并提高其附加值，具有较大的经济价值和社会意义。但是在目前的以酶解技术制备牡蛎肽的过程中，酶的使用量偏多造成了生产成本高。同时，不同的酶解技术所获得的牡蛎肽也不太相同，这表现在这些多肽的功效作用及其强弱上，而通过不同的酶解技术获得高功效的活性多肽一直是技术人员研究的重点。
发明内容
有鉴于此，本发明的目的在于提供一种具有提升性功能的牡蛎肽及 其制备方法和应用，使得所述制备方法能够减少酶的使用量，并且所制备的牡蛎肽具有效果更优的提高性功能和抗氧化的功效，可应用于相关保健品、食品以及药品的制备中。
为实现上述发明目的，本发明提供如下技术方案：
一种具有提升性功能的牡蛎肽的制备方法，包括：
步骤1、将牡蛎肉、钙盐和水研磨得到牡蛎肉浆液；
步骤2、牡蛎肉浆液酶解，酶解后离心取上清，获得牡蛎酶解粗液；
步骤3、将牡蛎酶解粗液脱色除杂，获得牡蛎肽精制液；
步骤4、牡蛎肽精制液浓缩、喷雾干燥获得所述牡蛎肽。
本发明针对现有酶解技术中使用酶制剂用量偏多以及目标功效不强、蛋白回收率不高的缺陷，在对牡蛎肉进行酶解提取多肽前，先采用钙盐对其进行预处理，以外加钙离子激活牡蛎肉内源性酶，释放的牡蛎肉内源性酶可高效专一水解牡蛎蛋白，进而减少商业蛋白酶的用量，降低生产成本。同时协同后面的酶解步骤，不仅提高了蛋白回收率，还使得所获得的牡蛎多肽具有更佳的功效作用。
其中，作为优选，所述钙盐为食品级氯化钙、乳酸钙、碳酸钙、磷酸氢钙或柠檬酸钙。
作为优选，所述钙盐为牡蛎肉质量的0.1-0.3％的钙盐。
在本发明的一些具体实施例中，所述钙盐可以为0.1％的氯化钙、0.3％的乳酸钙或0.2％的柠檬酸钙。
作为优选，步骤1中所述水为牡蛎肉质量的0.5-1.0倍的软化水。在本发明的一些具体实施例中，所述水可以为牡蛎肉质量的0.5倍的软化水、牡蛎肉质量的0.8倍的软化水或牡蛎肉质量的1.0倍的软化水。
在使用钙盐预处理牡蛎后，进行酶解时均可减少酶制剂的使用量，故常规的酶解均可，但是作为本发明优选，步骤2为：
将牡蛎肉浆液先用中性蛋白酶或碱性蛋白酶酶解，然后再用风味蛋白酶酶解，酶解后灭酶、离心，上清液即牡蛎酶解粗液。
进一步优选地，步骤2为：
将牡蛎肉浆液至于35～45℃下保温搅拌1-2小时，然后加入中性蛋白 酶或碱性蛋白酶，调节pH升温至50～60℃继续水解5-8小时后，调节pH至5.0-5.5，加入风味蛋白酶在50～60℃下继续酶解，2-3小时后灭酶，离心，上清液即牡蛎酶解粗液。
进一步优选地，所述中性蛋白酶为木瓜蛋白酶、菠萝蛋白酶、动物蛋白水解酶、复合蛋白酶Protamex中的一种或多种；所述中性蛋白酶或碱性蛋白酶的加入量为牡蛎肉浆液质量的0.3～0.8‰；所述碱性蛋白酶为Alcalase碱性蛋白酶、FoodPro Alkaline Protease碱性蛋白酶、胰蛋白酶中的一种或多种；所述风味蛋白酶的加入量为牡蛎肉浆液质量的0.5～1.0‰。
其中，对于使用中性蛋白酶酶解时，pH值调整到6.0-7.0；对于使用碱性蛋白酶酶解时，pH值调整到7.5-8.5。
作为优选，步骤3为：
往牡蛎酶解粗液中添加活性炭搅拌脱色，过滤，所得滤液为牡蛎肽精制液。
进一步优选地，步骤3为：
往牡蛎酶解粗液中添加活性炭，45～55℃保温搅拌0.5～1.0小时，过滤，所得滤液为牡蛎肽精制液。
采用本发明方法制备的牡蛎肽，消耗的中性蛋白酶或碱性蛋白酶未超过0.8‰，风味蛋白酶未超过1.0‰，而没有采用钙盐预处理的相同酶解工艺，消耗的中性蛋白酶或碱性蛋白酶为1.0‰，风味蛋白酶也为1.0‰，酶制剂使用量较高。同时，采用本发明酶制剂分两步进行酶解，相比将其他酶制剂或和本发明相同酶制剂一步酶解的工艺，所获得的牡蛎肽在提升小鼠性功能以及体外抗氧化功能上效果更优。
基于上述技术效果，本发明提供了由本发明制备方法制备的牡蛎肽，以及所述牡蛎肽在制备提高性功能和/或抗氧化保健品、食品以及药品中的应用。其中，所述药品和保健品为丸剂、胶囊剂、片剂、粉剂、颗粒剂、口服液或膏剂。
由以上技术方案可知，本发明在对牡蛎肉进行酶解前先用钙盐进行预处理，用以激活释放牡蛎内源性酶，减少后续酶解消耗的酶制剂，同时协同牡蛎内源性酶，采用特定的酶制剂组合分两步进行酶解，制备出 具有较强的抗氧化活性、可显著提高性功能的牡蛎肽。
具体实施方式
本发明公开了一种具有提升性功能的牡蛎肽的制备方法，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明所述牡蛎肽及其制备方法和应用已经通过实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
在具体实施例中涉及到牡蛎肽体外抗氧化活性(还原力、DPPH IC50)的检测方法和牡蛎肽对雄性成熟小鼠提升性功能实验方法参照如下步骤。
1、采用铁氰化钾还原体系，700nm处测定样品的还原力。将制备的样品以蛋白浓度进行梯度稀释，取样品液2ml于试管中，加0.2mol/L磷酸盐缓冲液(PBS，pH 6.6)2ml和1％(m/V)六合铁氰化钾溶液2ml，混合均匀于50℃恒温水浴锅中保温20min，加10％TCA 2ml，充分混匀。取上清液2ml于试管中，加蒸馏水2ml及0.1％的FeCl3溶液0.4ml，充分混匀后静置10min，在波长为700nm处测定其吸光度。每个样品按上述步骤操作，平行测定三次(还原力IC50值指当A700nm＝0.5时，样品的蛋白浓度，单位为mg/ml)。
2、选择雄性成熟小鼠，体重18～22g，随机分为空白对照组、样品组、对比样品组，每组12只，连续给药28d。
实验前48h，每只雌鼠皮下注射苯甲酸雌二醇(0.02mg/只)，使雌鼠动情期相同，试验于晚上7-11时进行。末次给药30min后，将雄性小鼠单独放入笼中5min，使其适应环境，然后每笼加入1只雌鼠，观察记录下列指标：
①雌鼠投入至雄鼠第1次扑捉雌鼠的时间(捕捉潜伏期)；
②20min内雄鼠扑捉雌鼠或爬背的动作次数(扑捉次数)；
③20min内雄鼠扑捉雌鼠或爬背成功后出现舔阴茎的次数(射精次数)；
④自爬背成功出现舔阴茎的时间(射精潜伏期)。
⑤20min内各组的扑捉百分率和射精百分率。
捕捉百分率＝捕捉动物数/动物总数
射精百分率＝射精动物数/动物总数
数据处理：采用SPSS19.0软件对实验数据进行分析，每组实验数据均以均值表示，各组总体方差满足方差齐性条件时，多组间比较采用单因素方差分析；当各组总体方差不满足方差齐性条件时，多组间比较采用Welch法分析，以p＜0.05判断差异具有显著性。
以下就本发明所提供的一种具有提升性功能的牡蛎肽的制备方法做进一步说明。
实施例1：本发明所述牡蛎肽的制备方法
(1)将牡蛎去壳，取肉清洗干净，过绞肉机绞碎，加入牡蛎肉碎肉质量0.1％的氯化钙和1.0倍的水，然后过胶体磨得到牡蛎肉浆液。
(2)将牡蛎肉浆液至于45℃下保温搅拌1小时后，往牡蛎肉浆液内加入其量0.3‰的木瓜蛋白酶，体系pH值调至6.5，在55℃下保温水解6小时，再用0.1mol/L的HCl溶液将体系pH值调至5.5，加入牡蛎肉质量1.0‰的风味蛋白酶继续水解2小时，然后在95℃下加热15min进行灭酶，最后离心，取上清液，得到牡蛎酶解粗液；
(3)往牡蛎酶解粗液加入其质量0.6％的活性炭，在50℃下保温搅拌1.0小时，过滤，所得滤液为牡蛎肽精制液。
(4)采用真空浓缩法将牡蛎肽精制液浓缩至固形物35％以上，进行喷雾干燥，得牡蛎肽样品1。
实施例2：本发明所述牡蛎肽的制备方法
(1)将牡蛎去壳，取肉清洗干净，过绞肉机绞碎，加入牡蛎肉碎肉质量0.3％的乳酸钙和0.5倍的水，然后过胶体磨得到牡蛎肉浆液。
(2)将牡蛎肉浆液至于35℃下保温搅拌2小时后，往牡蛎肉浆液内加入其量0.8‰的Alcalase，体系pH值调至8.0，在60℃下保温水解5小时，再用0.1mol/L的HCl溶液将体系pH值调至5.0，加入牡蛎肉质量0.7‰的风味蛋白酶继续水解2.5小时，然后在95℃下加热15min进行灭酶，最后离心，取上清液，得到牡蛎酶解粗液；
(3)往牡蛎酶解粗液加入其质量0.3％的活性炭，在55℃下保温搅拌0.5小时，过滤，所得滤液为牡蛎肽精制液。
(4)采用真空浓缩法将牡蛎肽精制液浓缩至固形物45％，进行喷雾干燥，得牡蛎肽样品2。
实施例3：本发明所述牡蛎肽的制备方法
(1)将牡蛎去壳，取肉清洗干净，过绞肉机绞碎，加入牡蛎肉碎肉质量0.2％的柠檬酸钙和0.8倍的水，然后过胶体磨得到牡蛎肉浆液。
(2)将牡蛎肉浆液至于40℃下保温搅拌1.5小时后，往牡蛎肉浆液内加入其量0.6‰的木瓜蛋白酶，体系pH调至7.0，在50℃下保温水解8小时，再用0.1mol/L的HCl溶液将体系pH值调至5.3，加入牡蛎肉质量0.5‰的风味蛋白酶继续水解3.0小时，然后在95℃下加热15min进行灭酶，最后离心，取上清液，得到牡蛎酶解粗液；
(3)往牡蛎酶解粗液加入其质量0.5％的活性炭，在55℃下保温搅拌0.7小时，过滤，所得滤液为牡蛎肽精制液。
(4)采用真空浓缩法将牡蛎肽精制液浓缩至固形物40％，进行喷雾干燥，得牡蛎肽样品3。
实施例4：本发明所述牡蛎肽的制备方法
(1)将牡蛎去壳，取肉清洗干净，过绞肉机绞碎，加入牡蛎肉碎肉质量0.2％的柠檬酸钙和0.8倍的水，然后过胶体磨得到牡蛎肉浆液。
(2)将牡蛎肉浆液至于40℃下保温搅拌1小时后，往牡蛎肉浆液内加入其量0.4‰的菠萝蛋白酶，体系pH调至6.0，在52℃下保温水解7小时，再用0.1mol/L的HCl溶液将体系pH值调至5.1，加入牡蛎肉质量0.9‰的风 味蛋白酶继续水解2.0小时，然后在95℃下加热15min进行灭酶，最后离心，取上清液，得到牡蛎酶解粗液；
(3)往牡蛎酶解粗液加入其质量0.5％的活性炭，在55℃下保温搅拌0.7小时，过滤，所得滤液为牡蛎肽精制液。
(4)采用真空浓缩法将牡蛎肽精制液浓缩至固形物40％，进行喷雾干燥，得牡蛎肽样品4。
实施例5：本发明所述牡蛎肽的制备方法
(1)将牡蛎去壳，取肉清洗干净，过绞肉机绞碎，加入牡蛎肉碎肉质量0.2％的柠檬酸钙和0.8倍的水，然后过胶体磨得到牡蛎肉浆液。
(2)将牡蛎肉浆液至于35℃下保温搅拌2小时后，往牡蛎肉浆液内加入其量0.5‰的动物蛋白水解酶，体系pH调至6.5，在50℃下保温水解8小时，再用0.1mol/L的HCl溶液将体系pH值调至5.2，加入牡蛎肉质量0.8‰的风味蛋白酶继续水解2.5小时，然后在95℃下加热15min进行灭酶，最后离心，取上清液，得到牡蛎酶解粗液；
(3)往牡蛎酶解粗液加入其质量0.5％的活性炭，在55℃下保温搅拌0.7小时，过滤，所得滤液为牡蛎肽精制液。
(4)采用真空浓缩法将牡蛎肽精制液浓缩至固形物40％，进行喷雾干燥，得牡蛎肽样品5。
实施例6：本发明所述牡蛎肽的制备方法
(1)将牡蛎去壳，取肉清洗干净，过绞肉机绞碎，加入牡蛎肉碎肉质量0.2％的柠檬酸钙和0.8倍的水，然后过胶体磨得到牡蛎肉浆液。
(2)将牡蛎肉浆液至于45℃下保温搅拌1小时后，往牡蛎肉浆液内加入其量0.7‰的复合蛋白酶Protamex(诺维信)，体系pH调至7.0，在57℃下保温水解6小时，再用0.1mol/L的HCl溶液将体系pH值调至5.4，加入牡蛎肉质量0.5‰的风味蛋白酶继续水解3.0小时，然后在95℃下加热15min进行灭酶，最后离心，取上清液，得到牡蛎酶解粗液；
(3)往牡蛎酶解粗液加入其质量0.5％的活性炭，在55℃下保温搅拌 0.7小时，过滤，所得滤液为牡蛎肽精制液。
(4)采用真空浓缩法将牡蛎肽精制液浓缩至固形物40％，进行喷雾干燥，得牡蛎肽样品6。
实施例7：本发明所述牡蛎肽的制备方法
(1)将牡蛎去壳，取肉清洗干净，过绞肉机绞碎，加入牡蛎肉碎肉质量0.2％的柠檬酸钙和0.8倍的水，然后过胶体磨得到牡蛎肉浆液。
(2)将牡蛎肉浆液至于38℃下保温搅拌1.5小时后，往牡蛎肉浆液内加入其量0.5‰的胰蛋白酶，体系pH调至8.5，在55℃下保温水解7小时，再用0.1mol/L的HCl溶液将体系pH值调至5.5，加入牡蛎肉质量0.6‰的风味蛋白酶继续水解2.0小时，然后在95℃下加热15min进行灭酶，最后离心，取上清液，得到牡蛎酶解粗液；
(3)往牡蛎酶解粗液加入其质量0.5％的活性炭，在55℃下保温搅拌0.7小时，过滤，所得滤液为牡蛎肽精制液。
(4)采用真空浓缩法将牡蛎肽精制液浓缩至固形物40％，进行喷雾干燥，得牡蛎肽样品7。
实施例8：本发明所述牡蛎肽的制备方法
(1)将牡蛎去壳，取肉清洗干净，过绞肉机绞碎，加入牡蛎肉碎肉质量0.2％的柠檬酸钙和0.8倍的水，然后过胶体磨得到牡蛎肉浆液。
(2)将牡蛎肉浆液至于42℃下保温搅拌2小时后，往牡蛎肉浆液内加入其量0.3‰的FoodPro Alkaline Protease碱性蛋白酶，体系pH调至7.5，在53℃下保温水解6小时，再用0.1mol/L的HCl溶液将体系pH值调至5.3，加入牡蛎肉质量0.5‰的风味蛋白酶继续水解2.5小时，然后在95℃下加热15min进行灭酶，最后离心，取上清液，得到牡蛎酶解粗液；
(3)往牡蛎酶解粗液加入其质量0.5％的活性炭，在55℃下保温搅拌0.7小时，过滤，所得滤液为牡蛎肽精制液。
(4)采用真空浓缩法将牡蛎肽精制液浓缩至固形物40％，进行喷雾干燥，得牡蛎肽样品8。
对比例1：对比牡蛎肽的制备方法
(1)将牡蛎去壳，取肉清洗干净，过绞肉机绞碎，加入牡蛎肉碎肉1.0倍的水，然后过胶体磨得到牡蛎肉浆液。
(2)往牡蛎肉浆液内加入其量1.0‰的木瓜蛋白酶和1.0‰的风味蛋白酶，体系pH调至6.5，在55℃下保温水解8小时，然后在95℃下加热15min进行灭酶，最后离心，取上清液，得到牡蛎酶解粗液；
(3)往牡蛎酶解粗液加入其质量0.6％的活性炭，在50℃下保温搅拌1.0小时，过滤，所得滤液为牡蛎肽精制液。
(4)采用真空浓缩法将牡蛎肽精制液浓缩至固形物40％，进行喷雾干燥，得对比样品1。
对比例2：对比牡蛎肽的制备方法
(1)将牡蛎去壳，取肉清洗干净，过绞肉机绞碎，加入牡蛎肉碎肉质量0.1％的氯化钙和0.8倍的水，然后过胶体磨得到牡蛎肉浆液。
(2)将牡蛎肉浆液至于40℃下保温搅拌1.5小时后，往牡蛎肉浆液内加入其量1.0‰的Alcalase和1.0‰的风味蛋白酶，体系pH调至8.0，在50℃下保温水解11小时，然后在95℃下加热15min进行灭酶，最后离心，取上清液，得到牡蛎酶解粗液；
(3)往牡蛎酶解粗液加入其质量0.5％的活性炭，在55℃下保温搅拌0.7小时，过滤，所得滤液为牡蛎肽精制液。
(4)采用真空浓缩法将牡蛎肽精制液浓缩至固形物40％，进行喷雾干燥，得对比样品2。
实施例9：不同肽样品的酶解蛋白回收率和体外抗氧化活性
试验样品：实施例1-3制备的牡蛎肽样品1-3，对比例1-2制备的对比样品1-2。
结果见下表。
表1不同肽样品的酶解蛋白回收率和体外抗氧化活性
样品 蛋白回收率/％ 还原力※ DPPH IC50mg/ml
牡蛎肽样品1 79.95 0.52 3.12
牡蛎肽样品2 82.41 0.56 3.64
牡蛎肽样品3 80.43 0.51 3.05
对比样品1 71.24 0.40 5.61
对比样品2 83.01 0.38 4.99
※当样品固形物浓度为1mg/ml时在A700nm下的吸收值。
由表1数据分析可知，采用本发明的方法对牡蛎肉进行水解，可得到较高的蛋白回收率(79.95-82.41％)，而对比例1加入更大量的酶制剂，蛋白回收率仅为71.24％，主要是因为本专利首先通过添加酶制剂激活剂(钙盐)激活牡蛎肉的内源酶，并在内源酶的最适温度下(35-45℃)水解一段时间，促进牡蛎肉的自溶，从而减少商业酶制剂用量仍可得到较好的水解效率；对比例2中的蛋白回收率高达83.01％，验证了内源酶在水解中的重要性。
同时，采用本发明方法制备的牡蛎肽的体外抗氧化值(还原力和DDPH清除率)均优于对比例样；牡蛎肽样品具有更高还原力和更低的DPPH IC50值，这主要是因为本发明选择的特定的酶制剂组合以及两步酶解水解更有利于抗氧化活性的释放。
对比例2虽然具有较高的蛋白回收率，但其体外抗氧化值(还原力和DPPH)较低，由此可证明本专利选择的酶制剂是水解牡蛎获取抗氧化活性最佳酶制剂组合。
依据本实施例的方法，对牡蛎肽样品4-8进行检测，结果显示，牡蛎肽样品4-8的蛋白回收率在82％左右，显著高于对比样品1，与对比样品2差别不明显，但是牡蛎肽样品4-8的还原力在0.55左右，高于对比样品1和2；DDPH清除率，不高于3.5mg/ml，比对比样品1和2更低；综合试验结果可知，本发明所提供的牡蛎肽样品在具有较高的蛋白回收率的同时，具有更高还原力和更低的DPPH IC50值。
实施例10：不同牡蛎肽样品对雄性小鼠交配能力的影响
试验样品：实施例1-3制备的牡蛎肽样品1-3，对比例1-2制备的对比样品1-2。
结果见表2和表3。
表2不同牡蛎肽样品对雄性小鼠交配能力的影响
Figure PCTCN2016097358-appb-000001
组别 剂量(g/kg) 捕捉次数 捕捉潜伏期(s) 捕捉百分率(％)
空白对照组 - 5.08±1.86 479.50±139.03 75％
牡蛎肽样品1 0.5 13.01±0.98**，ab 35.00±1.70**,ab 100％
牡蛎肽样品2 0.5 12.33±1.47**,ab 32.00±7.39**,ab 100％
牡蛎肽样品3 0.5 11.58±1.38**,ab 30.08±2.42**,ab 100％
对比样品1 0.5 6.42±0.48* 84.42±16.91** 85％
对比样品2 0.5 5.33±0.43 389.00±16.96 80％
注：**示与对照组比较，P<0.01，*示与对照组比较，P<0.05，a示与对比1比较，P<0.01，b示与对比样2比较，P<0.01
表3不同牡蛎肽样品对雄性小鼠交配能力的影响
Figure PCTCN2016097358-appb-000002
组别 剂量(g/kg) 射精次数 射精潜伏期(s) 射精百分率(％)
空白对照组 - 1.50±1.04 1017.33±112.18 25％
牡蛎肽样品1 0.5 5.20±0.00**,ab 100.00±3.05**,b 100％
牡蛎肽样品2 0.5 7.08±1.10**,ab 73.75±9.36**,ab 100％
牡蛎肽样品3 0.5 6.92±0.60**,ab 81.25±58.51**,ab 100％
对比样品1 0.5 3.25±0.33* 137.17±21.74** 90％
对比样品2 0.5 1.92±0.34* 984.33±31.21** 25％
注：**示与对照组比较，P<0.01，*示与对照组比较，P<0.05，a示与对比1比较，P<0.01，b示与对比样2比较，P<0.01
由表2、表3数据分析可知：
与空白对照组相比，采用本专利制备的牡蛎肽样品(牡蛎肽样品1、2、3)在捕捉次数、射精次数的提升上与空白对照相比有极其显著性差异(P<0.01)；
与空白对照组相比，采用本专利制备的牡蛎肽样品(牡蛎肽样品1、2、3)，在捕捉潜伏期、射精潜伏期时间的间隔上与空白对照相比有极其显著性差异(P<0.01)，并明显提高扑捉百分率和射精百分率；且捕捉百分率、射精百分率高于空白对照组。
与对比样品相对，采用本专利制备的牡蛎肽样品(牡蛎肽样品1、2、3)在捕捉次数、射精次数与对照样品(对比样品1和对比样品2)相比有显著性的提升(P<0.01)。并显著(P<0.01)提高扑捉百分率和射精百分率；且捕捉百分率、射精百分率高于对照样品组。
依照本实施例的方法，将牡蛎肽样品4-8检测，各牡蛎肽样品检测结果与上述对比样品以及空白对照组相比，均在捕捉次数、射精次数上有明显提升(P<0.01)，并显著(P<0.01)提高扑捉百分率和射精百分率，在捕捉潜伏期、射精潜伏期时间的间隔上也均具有显著性差异(P<0.01)。
目前，虽然还未见文献直接证明抗氧化与提升性功能具有直接关系，但本发明从抗氧化及提升性功能的两个角度对牡蛎肽活性进行研究，发现二者有一定的相关性，即依本专利方法制备的牡蛎肽样品既具有良好的体外抗氧化功效，同时对小鼠的性功能有良好的改善作用。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种具有提升性功能的牡蛎肽的制备方法，其特征在于，包括：
    步骤1、将牡蛎肉、钙盐和水研磨得到牡蛎肉浆液；
    步骤2、牡蛎肉浆液酶解，酶解后离心取上清，获得牡蛎酶解粗液；
    步骤3、将牡蛎酶解粗液脱色除杂，获得牡蛎肽精制液；
    步骤4、牡蛎肽精制液浓缩、喷雾干燥获得所述牡蛎肽。
  2. 根据权利要求1所述制备方法，其特征在于，所述钙盐为食品级氯化钙、乳酸钙、碳酸钙、磷酸氢钙或柠檬酸钙。
  3. 根据权利要求1所述制备方法，其特征在于，步骤1所述钙盐为牡蛎肉质量的0.1-0.3％的钙盐。
  4. 根据权利要求1所述制备方法，其特征在于，步骤2为：
    将牡蛎肉浆液先用中性蛋白酶或碱性蛋白酶酶解，然后再用风味蛋白酶酶解，酶解后灭酶、离心，上清液即牡蛎酶解粗液。
  5. 根据权利要求4所述制备方法，其特征在于，步骤2为：
    将牡蛎肉浆液至于35～45℃下保温搅拌1-2小时，然后加入中性蛋白酶或碱性蛋白酶，调节pH升温至50～60℃继续水解5-8小时后，调节pH至5.0-5.5，加入风味蛋白酶在50～60℃下继续酶解，2-3小时后灭酶，离心，上清液即牡蛎酶解粗液。
  6. 根据权利要求4所述制备方法，其特征在于，所述中性蛋白酶为木瓜蛋白酶、菠萝蛋白酶、动物蛋白水解酶、复合蛋白酶Protamex中的一种或多种；碱性蛋白酶为Alcalase碱性蛋白酶、FoodPro Alkaline Protease碱性蛋白酶、胰蛋白酶中的一种或多种。
  7. 根据权利要求4所述制备方法，其特征在于，所述中性蛋白酶或碱性蛋白酶的加入量为牡蛎肉浆液质量的0.3～0.8‰。
  8. 根据权利要求4所述制备方法，其特征在于，所述风味蛋白酶的加入量为牡蛎肉浆液质量的0.5～1.0‰。
  9. 权利要求1-8任意一项所述制备方法制备的牡蛎肽。
  10. 权利要求9所述牡蛎肽在制备提高性功能和/或抗氧化保健品、 食品以及药品中的应用。
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