WO2020181833A1 - 一种从富硒食用菌中提取硒蛋白的方法 - Google Patents

一种从富硒食用菌中提取硒蛋白的方法 Download PDF

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WO2020181833A1
WO2020181833A1 PCT/CN2019/120760 CN2019120760W WO2020181833A1 WO 2020181833 A1 WO2020181833 A1 WO 2020181833A1 CN 2019120760 W CN2019120760 W CN 2019120760W WO 2020181833 A1 WO2020181833 A1 WO 2020181833A1
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selenium
selenoprotein
enriched
parts
edible fungi
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PCT/CN2019/120760
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English (en)
French (fr)
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明松
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江苏中农科食品工程有限公司
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Publication of WO2020181833A1 publication Critical patent/WO2020181833A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G18/00—Cultivation of mushrooms
    • A01G18/20—Culture media, e.g. compost
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites

Definitions

  • the invention relates to the field of selenoprotein extraction, in particular to a method for extracting selenoprotein from selenium-rich edible fungi.
  • Selenium is one of the essential trace elements for the human body. Selenium can improve immunity and accelerate the production of immunoglobulin and antibodies. Protect cardiovascular and myocardium, reduce the incidence of cardiovascular disease and angina pectoris. It has a variety of effects in anti-oxidation, anti-aging, anti-cancer and anti-cancer, and has special physiological functions such as reducing heavy metal poisoning. It is hailed by scientists at home and abroad as the "king of anti-cancer, longevity element, fire of life, and natural detoxification.” Agent, the patron saint of the heart”. Many diseases are closely related to the nutritional status of selenium.
  • Edible fungi refer to mushrooms (large fungi) that have large fruit bodies and are edible. Edible fungi not only contain rich protein, polysaccharides and other physiologically active ingredients, but also can improve human immunity, anti-aging, anti-tumor and other abilities. High edible and medicinal value, and strong selenium-enriching ability. Based on the strong selenium-enriching ability of edible fungi, people have studied the method of converting inorganic selenium into selenium protein by edible fungi, thereby making up for the selenium in natural edible fungi. The disadvantage of very low content.
  • the purpose of the present invention is to provide a method for extracting selenoprotein from selenium-enriched edible fungi, so as to solve the problems raised in the background art.
  • a method for extracting selenoprotein from selenium-enriched edible fungi the specific steps are as follows:
  • Step one prepare selenium-enriched edible fungi: prepare selenium-enriched medium, add edible fungus strains to selenium-enriched medium for 3-5 days and add 8-12% selenium in selenium-enriched medium under aseptic conditions Sodium corrosion inhibitor to obtain selenium-rich edible fungi;
  • Step two acid extraction: add selenium-enriched edible fungi into water 4-6 times its weight and keep it at minus 25 to minus 16 degrees Celsius for 180-260 minutes, and then soak in acidic solution for 5-8 hours with infrared radiation. Then keep in the electric field for 10-15 minutes, remove and add a strong alkali solution to adjust the pH to 8-9, to obtain the first mixture;
  • Step 3 Enzymatic hydrolysis: adding 4-9% of the mass of the composite protease to the first mixture and enzymatic hydrolysis at 38-50 degrees Celsius, inactivating the enzyme at a high temperature, and centrifuging the supernatant to obtain the second mixture;
  • Step 4 prepare the crude selenoprotein solution: add petroleum ether 5-8 times the volume of the second mixture at a speed of 2-3 mL/min under ice bath conditions, and then keep it under the adjustment of minus 15 to minus 2 degrees Celsius 4-7.2 hours and supplemented by ultrasound, centrifuge in a high-speed centrifuge, collect the supernatant and filter to obtain the crude selenoprotein solution;
  • Step 5 Prepare the finished product: add the crude selenoprotein solution to an ethanol solution with a mass fraction of 88-94% in a volume of 3-5 times for alcohol precipitation to obtain a precipitate, and then wash the precipitate with absolute ethanol, petroleum ether and acetone in sequence And vacuum drying to obtain the finished product.
  • the selenium-enriched medium in step one includes the following raw materials by weight: 35-42.5 parts of straw powder, 12-16.8 parts of soybean meal, 0.18-0.35 parts of organic selenium, 24.6-27.2 parts of edible fungus leftovers, 6-9 parts of compound amino acids, 14.2-18.6 parts of ferrous sulfate, 5.6-9.2 parts of shrimp shells, 2.5-4.4 parts of gypsum, and 2.8-4.4 parts of heavy calcium carbonate.
  • the straw powder includes at least two of soybean straw powder, corn straw powder, rice straw powder, and sugarcane straw powder, and edible mushroom scraps include shiitake mushroom scraps, pleurotus eryngii scraps, and oyster mushroom scraps , Fungus scraps and enoki mushroom scraps at least three.
  • the intensity of infrared radiation in step two is 180-260W
  • the strong alkali solution includes any one of sodium hydroxide solution and potassium hydroxide solution.
  • step two in step two, the electric field intensity is 15-18V/m, and the voltage is 40-55V.
  • step 4 the temperature of the ice bath is minus 15 to minus 6 degrees Celsius, the ultrasonic power is 120-165W, and the ultrasonic frequency is 19-24KHz.
  • the speed of the high-speed centrifuge in step 4 is 1860-2430 rpm, and the temperature of hollow drying in step 5 is 40-48 degrees Celsius.
  • the invention realizes that selenium is enriched in edible fungi and converted into organic selenium, and then selenoprotein is obtained by enzymatic hydrolysis, centrifugation, filtration and the like.
  • the extraction rate of selenoprotein is high, and the purity of the obtained selenoprotein is high.
  • the finished product can be used in the food processing field to meet the human body's demand for selenium supplementation and has broad application prospects.
  • Example 1 A method for extracting selenoprotein from selenium-enriched edible fungi, the specific steps are as follows: Step one, prepare selenium-enriched edible fungi: prepare a selenium-enriched medium, which includes the following raw materials by weight: straw powder 35 Parts, 12 parts of soybean meal, 0.18 parts of organic selenium, 24.6 parts of edible fungus leftovers, 6 parts of compound amino acids, 14.2 parts of ferrous sulfate, 5.6 parts of shrimp shells, 2.5 parts of gypsum, and 2.8 parts of heavy calcium carbonate, straw powder including soybean straw Mixture of powder and sugar cane straw powder. Edible fungus leftovers include a mixture of shiitake mushrooms, oyster mushrooms and fungus leftovers.
  • Step two acid extraction: add selenium-enriched edible fungi to water 5 times its weight and keep it at minus 22 degrees Celsius for 235 minutes, and then Soak in acidic solution for 6.5 hours and supplemented by 210W infrared radiation, then keep in electric field for 12 minutes, remove and add potassium hydroxide solution to adjust the pH to 8.6 to obtain the first mixture;
  • step three enzymatic hydrolysis: A mixture of 6.5% by mass of the composite protease was added to the mixture and enzymatically hydrolyzed at 44 degrees Celsius, the enzyme was inactivated at a high temperature and the supernatant was centrifuged to obtain the second mixture;
  • Step four preparing the crude selenoprotein solution: Put the second mixture in an ice bath Add petroleum ether 6 times the volume of the
  • Example 2 A method for extracting selenium protein from selenium-enriched edible fungi, the specific steps are as follows: Step one, prepare selenium-enriched edible fungi: prepare a selenium-enriched medium, and add edible fungi to selenium-enriched culture under aseptic conditions Selenium-enriched edible fungus was cultured for 4 days while adding 10% sodium selenite corrosion inhibitor to obtain selenium-enriched edible fungus; Step two, acid extraction: add selenium-enriched edible fungus to water 6 times its weight and below zero Keep at 20 degrees Celsius for 196 minutes, then soak in acidic solution for 7.2 hours with infrared radiation, then keep in electric field for 14 minutes, electric field strength is 16V/m, voltage is 52V, remove and add strong alkali solution to adjust pH value 8.4.
  • Step 3 Enzymatic hydrolysis: add 4-9% of the mass of the composite protease to the first mixture and enzymolyze at 45 degrees Celsius, inactivate the enzyme at a high temperature and centrifuge the supernatant to obtain the second mixture
  • Step 4 prepare the crude selenoprotein solution: add petroleum ether 7.5 times the volume of the second mixture at a speed of 3 mL/min under the ice bath condition of minus 8 degrees Celsius, and then keep it at minus 5 degrees Celsius for 6 hours.
  • the power of the ultrasound is 145W
  • the frequency of the ultrasound is 21KHz
  • centrifugation in a high-speed centrifuge the supernatant is collected and filtered to obtain the crude selenoprotein solution
  • Step 5 the finished product: add the crude selenoprotein solution
  • the ethanol solution with a 90% mass fraction of 5 times its volume is subjected to alcohol precipitation to obtain a precipitate, and then the precipitate is washed with absolute ethanol, petroleum ether and acetone in sequence and dried in vacuum to obtain the finished product.
  • Example 3 A method for extracting selenoprotein from selenium-enriched edible fungi, the specific steps are as follows: Step one, prepare selenium-enriched edible fungi: prepare a selenium-enriched medium, which includes the following raw materials by weight: straw powder 38.4 Parts, 14.7 parts of soybean meal, 0.24 parts of organic selenium, 26.3 parts of edible fungus leftovers, 7.7 parts of compound amino acids, 17.4 parts of ferrous sulfate, 8.5 parts of shrimp shells, 3.8 parts of gypsum, and 3.5 parts of heavy calcium carbonate.
  • Step one prepare selenium-enriched edible fungi: prepare a selenium-enriched medium, which includes the following raw materials by weight: straw powder 38.4 Parts, 14.7 parts of soybean meal, 0.24 parts of organic selenium, 26.3 parts of edible fungus leftovers, 7.7 parts of compound amino acids, 17.4 parts of ferrous sulfate, 8.5 parts of shrimp shells, 3.8 parts of gypsum, and
  • Straw powder includes soybean straw Mixture of powder, corn stalk powder, rice stalk powder and sugarcane stalk powder, edible fungus leftovers including shiitake mushroom leftovers, pleurotus eryngii leftovers, oyster mushroom leftovers, fungus leftovers and enoki mushroom leftovers, under aseptic conditions Add the edible fungi strains to the selenium-enriched medium for 4 days and add sodium selenite corrosion inhibitor of 8.8% by mass of the selenium-enriched medium to obtain the selenium-enriched edible fungus; Step two, acid extraction: the selenium-enriched edible fungus Add 6 times its weight in water and keep it at minus 21 degrees Celsius for 240 minutes, then soak in acidic solution for 6 hours with 235W infrared radiation, and then keep it in an electric field for 10 minutes.
  • Step 4 remove and add potassium hydroxide solution to adjust the pH to 8.6 to obtain the first mixture; Step three, enzymatic hydrolysis: then add 5.6% of its mass composite protease to the first mixture and enzymatically hydrolyze it at 45 degrees Celsius, and kill the enzyme at a high temperature And centrifuge the supernatant to obtain the second mixture; Step 4, prepare the crude selenoprotein solution: add the second mixture to 6.5 times the volume of the second mixture at a rate of 2.4 mL/min under an ice bath at minus 10 degrees Celsius Ether, then kept at minus 11 degrees Celsius for 5.6 hours and supplemented by ultrasound.
  • the power of the ultrasound is 144W and the frequency of the ultrasound is 22KHz.
  • Example 4 A method for extracting selenium protein from selenium-enriched edible fungi, the specific steps are as follows: Step one, prepare selenium-enriched edible fungi: prepare a selenium-enriched medium, and add edible fungi strains to selenium-enriched culture under aseptic conditions Culture medium for 3-5 days while adding 10.5% sodium selenite corrosion inhibitor with selenium-enriched medium quality to obtain selenium-enriched edible fungi; Step two, acid extraction: add selenium-enriched edible fungi into water 6 times its weight and Keep at minus 20 degrees Celsius for 190 minutes, then soak in acidic solution for 6 hours and supplemented by 200W infrared radiation, then keep in electric field for 14 minutes, electric field strength is 16V/m, voltage is 48V, remove and add sodium hydroxide Adjust the pH value of the solution to 8.6 to obtain the first mixture; Step three, enzymatic hydrolysis: add 6.9% of the mass of the composite protease to the first mixture and hydrolyze the enzyme at 45 degrees Celsius, inactivate the
  • Example 5 A method for extracting selenoprotein from selenium-enriched edible fungi, the specific steps are as follows: Step one, prepare selenium-enriched edible fungi: prepare a selenium-enriched medium, which includes the following raw materials by weight: straw powder 42.5 Parts, 16.8 parts of soybean meal, 0.35 parts of organic selenium, 27.2 parts of edible fungus leftovers, 9 parts of compound amino acids, 18.6 parts of ferrous sulfate, 9.2 parts of shrimp shells, 4.4 parts of gypsum and 4.4 parts of heavy calcium carbonate, straw powder including corn stalks Mixture of powder, rice straw powder and sugarcane straw powder, edible fungus leftovers including leftovers of Pleurotus eryngii, leftovers of oyster mushrooms, leftovers of fungus and leftovers of enoki mushrooms.
  • a selenium-enriched medium which includes the following raw materials by weight: straw powder 42.5 Parts, 16.8 parts of soybean meal, 0.35 parts of organic selenium, 27.2 parts of edible
  • Step two acid extraction: add selenium-enriched edible fungi into water 4 times its weight and Keep at minus 20 degrees Celsius for 205 minutes, then soak in acidic solution for 6.5 hours with infrared radiation, then keep in electric field for 12 minutes, electric field strength is 16V/m, voltage is 52V, remove and add strong alkali solution to adjust pH The value is 9, the first mixture is obtained;
  • Step 3 Enzymatic hydrolysis: add 7.7% of the mass of the composite protease to the first mixture and hydrolyze at 44 degrees Celsius, inactivate the enzyme at high temperature and centrifuge the supernatant to obtain the second mixture
  • Step 4 prepare crude selenoprotein solution: add petroleum ether 8 times the volume of the second mixture at a speed of 2 mL/min under ice bath conditions at minus 10 degrees Celsius, and then keep it
  • the finished products of Examples 1-5 were inspected, and the measurement method was to measure the selenium content by microwave digestion-ultraviolet spectrophotometry.
  • the total selenium content was determined by GB5009.93-2007, and the method in Appendix A was used for the determination of selenomethionine (in terms of Se).
  • the mass percentage of organic selenium in total selenium is the percentage of selenomethionine (calculated as Se)/total selenium content; use BG5009.5 Kjeldahl method to detect protein (mass fraction ⁇ 40), see the table 1.
  • the mass fraction of organic selenium in the total selenium is higher than 94%, and the mass fraction of protein is not less than 68, which makes selenium
  • the content is effectively enriched, which is beneficial to the body's absorption.
  • the working principle of the present invention is: in the present invention, edible fungi are planted in a selenium-enriched medium and supplemented with sodium selenite corrosion inhibitor to obtain selenium-enriched edible fungi. The selenium-enriched edible fungi are soaked in an acid solution to make the The structure of the selenium edible fungus becomes loose, which is conducive to the subsequent enzymolysis.
  • the selenium-enriched edible fungus is modified by infrared irradiation and electric field treatment, so that the molecules of the selenium-enriched edible fungus absorb energy and make the selenium protein easier to enzyme Under alkaline conditions, enzymatic hydrolysis and centrifugation, and then petroleum ether supplemented with ultrasonic extraction, to obtain crude selenoprotein solution. Under the vibration of ultrasonic waves, selenoprotein also vibrates continuously to avoid the occurrence of selenoprotein aggregation. Furthermore, the particle size of the selenoprotein is reduced, which is beneficial to the absorption of the human body, and the crude selenoprotein solution is obtained. The crude selenoprotein solution is subjected to alcohol precipitation to obtain the finished product.

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Abstract

一种从富硒食用菌中提取硒蛋白的方法，涉及硒蛋白提取领域，要解决的是如何弥补天然食用菌中的硒含量很低的问题。具体步骤如下：步骤一，制备富硒食用菌；步骤二，酸性提取；步骤三，酶解；步骤四，制备粗硒蛋白液；步骤五，制备成品：将粗硒蛋白液加入其体积3-5倍的质量分数为88-94％的乙醇溶液进行醇析，得到沉淀，再将沉淀依次采用无水乙醇、石油醚和丙酮洗涤并且真空干燥，即得到成品。

Description

一种从富硒食用菌中提取硒蛋白的方法 技术领域
本发明涉及硒蛋白提取领域，具体是一种从富硒食用菌中提取硒蛋白的方法。
 背景技术
硒是人体必需的微量元素之一，硒可以提高免疫力，能加速免疫球蛋白及抗体产生。保护心血管与心肌，降低心血管病与心绞痛的发生率。在抗氧化抗衰老与抗癌防癌等方面有多种作用，并具有降低重金属毒害等特殊生理功能，被国内外科学家们誉为人类“抗癌之王、长寿元素、生命的火种、天然解毒剂、心脏守护神”。多种疾病都与硒营养状况密切相关。
由于硒在地球上分布不均，目前已知有 40 多个国家和地区不同程度缺硒，我国也有 2/3 以上地区缺硒，涉及人口达 7 亿多。直接利用无机硒盐补硒具有一定的毒性，存在较大的安全隐患，为了使人们在正常膳食之外再补充一些硒，科学家们研究了很多良好的补硒食品，例如硒酵母、富硒食用菌、富硒面粉、富硒大蒜、富硒蛋、硒盐等等举不胜举。而食用菌营养丰富、味道鲜美，富硒食用菌是一种颇有前途的富硒保健品。
食用菌是指子实体硕大、可供食用的蕈菌（大型真菌），食用菌不仅含有丰富的蛋白质、多糖及其他生理活性成分,能提高人体免疫力、抗衰老、抗肿瘤等能力,具有很高的食用和药用价值,而且有较强的富硒能力，人们根据食用菌富硒能力强的特点，研究将无机硒被食用菌转化为硒蛋白的方法，从而弥补天然食用菌中的硒含量很低的缺点。
 技术问题
本发明的目的在于提供一种从富硒食用菌中提取硒蛋白的方法，以解决上述背景技术中提出的问题。
技术解决方案
为实现上述目的，本发明提供如下技术方案：
一种从富硒食用菌中提取硒蛋白的方法，具体步骤如下：
步骤一，制备富硒食用菌：制备富硒培养基，在无菌条件下将食用菌菌种加入富硒培养基中培养3-5天同时添加富硒培养基质量8-12%的亚硒酸钠缓蚀剂，得到富硒食用菌；
步骤二，酸性提取：将富硒食用菌加入其重量4-6倍的水中并且在零下25至零下16摄氏度保持180-260分钟，然后在酸性溶液中浸泡5-8小时并且辅以红外线照射，然后在电场中保持10-15分钟，捞出并且加入强碱溶液调节pH值为8-9，得到第一混合物；
步骤三，酶解：再向第一混合物中加入其质量4-9%的复合蛋白酶并且在38-50摄氏度酶解，高温灭酶并且离心分离上清液，得到第二混合物；
步骤四，制备粗硒蛋白液：将第二混合物在冰浴条件下以2-3mL/分钟的速度加入第二混合物体积5-8倍的石油醚，然后在零下15至零下2摄氏度调节下保持4-7.2小时并且辅以超声波，在高速离心机中进行离心，收集上清液并且过滤，得到粗硒蛋白液；
步骤五，制备成品：将粗硒蛋白液加入其体积3-5倍的质量分数为88-94%的乙醇溶液进行醇析，得到沉淀，再将沉淀依次采用无水乙醇、石油醚和丙酮洗涤并且真空干燥，即得到成品。
作为本发明进一步的方案：步骤一中富硒培养基包括以下重量份的原料：秸秆粉末35-42.5份、豆粕12-16.8份、有机硒0.18-0.35份、食用菌下脚料24.6-27.2份、复合氨基酸6-9份、硫酸亚铁14.2-18.6份、虾壳5.6-9.2份、石膏2.5-4.4份和重质碳酸钙2.8-4.4份。
作为本发明进一步的方案：秸秆粉末包括大豆秸秆粉末、玉米秸秆粉末、水稻秸秆粉末和甘蔗秸秆粉末中的至少两种，食用菌下脚料包括香菇下脚料、杏鲍菇下脚料、平菇下脚料、木耳下脚料和金针菇下脚料中的至少三种。
作为本发明进一步的方案：步骤二中红外线照射的强度为180-260W，强碱溶液包括氢氧化钠溶液和氢氧化钾溶液中的任意一种。
作为本发明进一步的方案：步骤二中电场强度为15-18V/m，电压为40-55V。
 作为本发明进一步的方案：步骤四中冰浴的温度为零下15至零下6摄氏度，超声波的功率为120-165W，超声波的频率为19-24KHz。
 作为本发明进一步的方案：步骤四中高速离心机的转速为1860-2430rpm，步骤五中中空干燥的温度为40-48摄氏度。
与现有技术相比，本发明的有益效果是：
 有益效果
本发明实现了将硒富集在食用菌中并且转换为有机硒，再用酶解、离心、过滤等方式得到硒蛋白，硒蛋白的提取率高，得到的硒蛋白纯度高，本发明得到的成品可应用于食品加工领域中，满足人体的补硒需求，具有广阔的应用前景。
 本发明的实施方式
下面结合具体实施方式对本专利的技术方案作进一步详细地说明。
 实施例1 一种从富硒食用菌中提取硒蛋白的方法，具体步骤如下： 步骤一，制备富硒食用菌：制备富硒培养基，富硒培养基包括以下重量份的原料：秸秆粉末35份、豆粕12份、有机硒0.18份、食用菌下脚料24.6份、复合氨基酸6份、硫酸亚铁14.2份、虾壳5.6份、石膏2.5份和重质碳酸钙2.8份，秸秆粉末包括大豆秸秆粉末和甘蔗秸秆粉末的混合物，食用菌下脚料包括香菇下脚料、平菇下脚料和木耳下脚料的混合物，在无菌条件下将食用菌菌种加入富硒培养基中培养3.8天同时添加富硒培养基质量11%的亚硒酸钠缓蚀剂，得到富硒食用菌； 步骤二，酸性提取：将富硒食用菌加入其重量5倍的水中并且在零下22摄氏度保持235分钟，然后在酸性溶液中浸泡6.5小时并且辅以210W的红外线照射，然后在电场中保持12分钟，捞出并且加入氢氧化钾溶液调节pH值为8.6，得到第一混合物； 步骤三，酶解：再向第一混合物中加入其质量6.5%的复合蛋白酶并且在44摄氏度酶解，高温灭酶并且离心分离上清液，得到第二混合物； 步骤四，制备粗硒蛋白液：将第二混合物在冰浴条件下以2.4mL/分钟的速度加入第二混合物体积6倍的石油醚，然后在零下8摄氏度调节下保持5.5小时并且辅以超声波，在高速离心机中进行离心，收集上清液并且过滤，得到粗硒蛋白液； 步骤五，制备成品：将粗硒蛋白液加入其体积4倍的质量分数为91%的乙醇溶液进行醇析，得到沉淀，再将沉淀依次采用无水乙醇、石油醚和丙酮洗涤并且真空干燥，即得到成品。 实施例2 一种从富硒食用菌中提取硒蛋白的方法，具体步骤如下： 步骤一，制备富硒食用菌：制备富硒培养基，在无菌条件下将食用菌菌种加入富硒培养基中培养4天同时添加富硒培养基质量10%的亚硒酸钠缓蚀剂，得到富硒食用菌； 步骤二，酸性提取：将富硒食用菌加入其重量6倍的水中并且在零下20摄氏度保持196分钟，然后在酸性溶液中浸泡7.2小时并且辅以红外线照射，然后在电场中保持14分钟，电场强度为16V/m，电压为52V，捞出并且加入强碱溶液调节pH值为8.4，得到第一混合物； 步骤三，酶解：再向第一混合物中加入其质量4-9%的复合蛋白酶并且在45摄氏度酶解，高温灭酶并且离心分离上清液，得到第二混合物； 步骤四，制备粗硒蛋白液：将第二混合物在零下8摄氏度的冰浴条件下以3mL/分钟的速度加入第二混合物体积7.5倍的石油醚，然后在零下5摄氏度调节下保持6小时并且辅以超声波，超声波的功率为145W，超声波的频率为21KHz，在高速离心机中进行离心，收集上清液并且过滤，得到粗硒蛋白液； 步骤五，制备成品：将粗硒蛋白液加入其体积5倍的质量分数为90%的乙醇溶液进行醇析，得到沉淀，再将沉淀依次采用无水乙醇、石油醚和丙酮洗涤并且真空干燥，即得到成品。
实施例3 一种从富硒食用菌中提取硒蛋白的方法，具体步骤如下： 步骤一，制备富硒食用菌：制备富硒培养基，富硒培养基包括以下重量份的原料：秸秆粉末38.4份、豆粕14.7份、有机硒0.24份、食用菌下脚料26.3份、复合氨基酸7.7份、硫酸亚铁17.4份、虾壳8.5份、石膏3.8份和重质碳酸钙3.5份，秸秆粉末包括大豆秸秆粉末、玉米秸秆粉末、水稻秸秆粉末和甘蔗秸秆粉末的混合物，食用菌下脚料包括香菇下脚料、杏鲍菇下脚料、平菇下脚料、木耳下脚料和金针菇下脚料的混合物，在无菌条件下将食用菌菌种加入富硒培养基中培养4天同时添加富硒培养基质量8.8%的亚硒酸钠缓蚀剂，得到富硒食用菌； 步骤二，酸性提取：将富硒食用菌加入其重量6倍的水中并且在零下21摄氏度保持240分钟，然后在酸性溶液中浸泡6小时并且辅以235W的红外线照射，然后在电场中保持10分钟，电场强度为17V/m，电压为52V，捞出并且加入氢氧化钾溶液调节pH值为8.6，得到第一混合物； 步骤三，酶解：再向第一混合物中加入其质量5.6%的复合蛋白酶并且在45摄氏度酶解，高温灭酶并且离心分离上清液，得到第二混合物； 步骤四，制备粗硒蛋白液：将第二混合物在零下10摄氏度的冰浴条件下以2.4mL/分钟的速度加入第二混合物体积6.5倍的石油醚，然后在零下11摄氏度调节下保持5.6小时并且辅以超声波，超声波的功率为144W，超声波的频率为22KHz，在转速为2160rpm的高速离心机中进行离心，收集上清液并且过滤，得到粗硒蛋白液； 步骤五，制备成品：将粗硒蛋白液加入其体积3倍的质量分数为91%的乙醇溶液进行醇析，得到沉淀，再将沉淀依次采用无水乙醇、石油醚和丙酮洗涤并且在45摄氏度下真空干燥，即得到成品。  实施例4 一种从富硒食用菌中提取硒蛋白的方法，具体步骤如下： 步骤一，制备富硒食用菌：制备富硒培养基，在无菌条件下将食用菌菌种加入富硒培养基中培养3-5天同时添加富硒培养基质量10.5%的亚硒酸钠缓蚀剂，得到富硒食用菌； 步骤二，酸性提取：将富硒食用菌加入其重量6倍的水中并且在零下20摄氏度保持190分钟，然后在酸性溶液中浸泡6小时并且辅以200W的红外线照射，然后在电场中保持14分钟，电场强度为16V/m，电压为48V，捞出并且加入氢氧化钠溶液调节pH值为8.6，得到第一混合物； 步骤三，酶解：再向第一混合物中加入其质量6.9%的复合蛋白酶并且在45摄氏度酶解，高温灭酶并且离心分离上清液，得到第二混合物； 步骤四，制备粗硒蛋白液：将第二混合物在冰浴条件下以2.4mL/分钟的速度加入第二混合物体积6倍的石油醚，然后在零下7摄氏度调节下保持6小时并且辅以超声波，在转速为2250rpm的高速离心机中进行离心，收集上清液并且过滤，得到粗硒蛋白液； 步骤五，制备成品：将粗硒蛋白液加入其体积4倍的质量分数为94%的乙醇溶液进行醇析，得到沉淀，再将沉淀依次采用无水乙醇、石油醚和丙酮洗涤并且在42摄氏度真空干燥，即得到成品。
实施例5 一种从富硒食用菌中提取硒蛋白的方法，具体步骤如下： 步骤一，制备富硒食用菌：制备富硒培养基，富硒培养基包括以下重量份的原料：秸秆粉末42.5份、豆粕16.8份、有机硒0.35份、食用菌下脚料27.2份、复合氨基酸9份、硫酸亚铁18.6份、虾壳9.2份、石膏4.4份和重质碳酸钙4.4份，秸秆粉末包括玉米秸秆粉末、水稻秸秆粉末和甘蔗秸秆粉末的混合物，食用菌下脚料包括杏鲍菇下脚料、平菇下脚料、木耳下脚料和金针菇下脚料的混合物，在无菌条件下将食用菌菌种加入富硒培养基中培养5天同时添加富硒培养基质量9%的亚硒酸钠缓蚀剂，得到富硒食用菌； 步骤二，酸性提取：将富硒食用菌加入其重量4倍的水中并且在零下20摄氏度保持205分钟，然后在酸性溶液中浸泡6.5小时并且辅以红外线照射，然后在电场中保持12分钟，电场强度为16V/m，电压为52V，捞出并且加入强碱溶液调节pH值为9，得到第一混合物； 步骤三，酶解：再向第一混合物中加入其质量7.7%的复合蛋白酶并且在44摄氏度酶解，高温灭酶并且离心分离上清液，得到第二混合物； 步骤四，制备粗硒蛋白液：将第二混合物在零下10摄氏度的冰浴条件下以2mL/分钟的速度加入第二混合物体积8倍的石油醚，然后在零下13摄氏度调节下保持6小时并且辅以超声波，超声波的功率为156W，超声波的频率为22KHz，在高速离心机中进行离心，收集上清液并且过滤，得到粗硒蛋白液； 步骤五，制备成品：将粗硒蛋白液加入其体积3倍的质量分数为91%的乙醇溶液进行醇析，得到沉淀，再将沉淀依次采用无水乙醇、石油醚和丙酮洗涤并且真空干燥，即得到成品。
 对实施例1-5的成品进行检验，测定方法为利用微波消解-紫外分光光度法测定硒含量。
采用GB5009.93-2007测定总硒含量，附录A中方法测定硒代蛋氨酸（以Se计）。有机硒占总硒质量百分比（质量分数≥80）为硒代蛋氨酸（以Se计）含量/总硒含量的百分比； 采用BG5009.5 凯氏定氮法检测蛋白质（质量分数≥40）结果见表1。
表1   原料中的硒含量（μg/g） 成品中的硒含量（μg/g） 硒含量提升倍数 有机硒占总硒的质量分数 蛋白质的质量分数 实施例1 5 16.85 3.37 95.36 65.81 实施例2 1 3.41 3.41 96.84 71.06 实施例3 10 31.8 3.18 94.27 68.41 实施例4 15 46.5 3.1 95.87 70.51 实施例5 20 64.48 3.224 94.22 68.24 从表1中可以看出，实施例1-5的成品中硒含量均有3倍以上的提升，有机硒占总硒质量分数高于94%，蛋白质的质量分数不低于68，使得硒含量得到有效富集，有利于人体的吸收。本发明的工作原理是：本发明将食用菌菌种种植在富硒培养基中并且辅以亚硒酸钠缓蚀剂，得到富硒食用菌，将富硒食用菌用酸性溶液浸泡，使得富硒食用菌的结构变得松散，有利于后续的酶解，辅以红外线照射并且采用电场处理，对富硒食用菌进行改性，使得富硒食用菌的分子吸收能量，使得硒蛋白更容易酶解，在碱性条件下酶解并且离心，再以石油醚辅以超声波提取，得到粗硒蛋白液，在超声波的振动下，硒蛋白也随之不断振动，避免了硒蛋白团聚现象的发生，进而降低了硒蛋白的粒径，有利于人体的吸收，得到粗硒蛋白液，将粗硒蛋白液进行醇析，即可得到成品。
 以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。    此外，应当理解，虽然本说明书按照实施方式加以描述，但并非每个实施方式仅包含一个独立的技术方案，说明书的这种叙述方式仅仅是为清楚起见，本领域技术人员应当将说明书作为一个整体，各实施例中的技术方案也可以经适当组合，形成本领域技术人员可以理解的其他实施方式。 
 

Claims (7)

  1. 一种从富硒食用菌中提取硒蛋白的方法，其特征在于，具体步骤如下：
    步骤一，制备富硒食用菌：制备富硒培养基，在无菌条件下将食用菌菌种加入富硒培养基中培养3-5天同时添加富硒培养基质量8-12%的亚硒酸钠缓蚀剂，得到富硒食用菌；
    步骤二，酸性提取：将富硒食用菌加入其重量4-6倍的水中并且在零下25至零下16摄氏度保持180-260分钟，然后在酸性溶液中浸泡5-8小时并且辅以红外线照射，然后在电场中保持10-15分钟，捞出并且加入强碱溶液调节pH值为8-9，得到第一混合物；
    步骤三，酶解：再向第一混合物中加入其质量4-9%的复合蛋白酶并且在38-50摄氏度酶解，高温灭酶并且离心分离上清液，得到第二混合物；
    步骤四，制备粗硒蛋白液：将第二混合物在冰浴条件下以2-3mL/分钟的速度加入第二混合物体积5-8倍的石油醚，然后在零下15至零下2摄氏度调节下保持4-7.2小时并且辅以超声波，在高速离心机中进行离心，收集上清液并且过滤，得到粗硒蛋白液；
    步骤五，制备成品：将粗硒蛋白液加入其体积3-5倍的质量分数为88-94%的乙醇溶液进行醇析，得到沉淀，再将沉淀依次采用无水乙醇、石油醚和丙酮洗涤并且真空干燥，即得到成品。
  2. 根据权利要求1所述的从富硒食用菌中提取硒蛋白的方法，其特征在于，所述步骤一中富硒培养基包括以下重量份的原料：秸秆粉末35-42.5份、豆粕12-16.8份、有机硒0.18-0.35份、食用菌下脚料24.6-27.2份、复合氨基酸6-9份、硫酸亚铁14.2-18.6份、虾壳5.6-9.2份、石膏2.5-4.4份和重质碳酸钙2.8-4.4份。
  3. 根据权利要求2所述的从富硒食用菌中提取硒蛋白的方法，其特征在于，所述秸秆粉末包括大豆秸秆粉末、玉米秸秆粉末、水稻秸秆粉末和甘蔗秸秆粉末中的至少两种，食用菌下脚料包括香菇下脚料、杏鲍菇下脚料、平菇下脚料、木耳下脚料和金针菇下脚料中的至少三种。
  4. 根据权利要求1所述的从富硒食用菌中提取硒蛋白的方法，其特征在于，所述步骤二中红外线照射的强度为180-260W，强碱溶液包括氢氧化钠溶液和氢氧化钾溶液中的任意一种。
  5. 根据权利要求1所述的从富硒食用菌中提取硒蛋白的方法，其特征在于，所述步骤二中电场强度为15-18V/m，电压为40-55V。
  6. 根据权利要求1或5所述的从富硒食用菌中提取硒蛋白的方法，其特征在于，所述步骤四中冰浴的温度为零下15至零下6摄氏度，超声波的功率为120-165W，超声波的频率为19-24KHz。
  7. 根据权利要求1所述的从富硒食用菌中提取硒蛋白的方法，其特征在于，所述步骤四中高速离心机的转速为1860-2430rpm，步骤五中中空干燥的温度为40-48摄氏度。
     
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