WO2016082327A1 - 一种虫草菌丝体的加工方法 - Google Patents

一种虫草菌丝体的加工方法 Download PDF

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Publication number
WO2016082327A1
WO2016082327A1 PCT/CN2015/071621 CN2015071621W WO2016082327A1 WO 2016082327 A1 WO2016082327 A1 WO 2016082327A1 CN 2015071621 W CN2015071621 W CN 2015071621W WO 2016082327 A1 WO2016082327 A1 WO 2016082327A1
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Prior art keywords
fermentation
mycelium
processing
active ingredient
cordyceps
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PCT/CN2015/071621
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English (en)
French (fr)
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冯敏
沈建
钱一帆
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南京中科药业有限公司
南京中科集团股份有限公司
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Publication of WO2016082327A1 publication Critical patent/WO2016082327A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/062—Ascomycota
    • A61K36/066—Clavicipitaceae
    • A61K36/068—Cordyceps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization

Definitions

  • the invention belongs to the field of biotechnology, and particularly relates to a method for processing Cordyceps mycelium rich in active ingredients.
  • Cordyceps sinensis is a medicinal fungus of the genus Cordyceps. It is mainly produced in the mountains and meadow soils of 4000m ⁇ 5000m above sea level in Qinghai, Cambodia, Sichuan, Yunnan and Guizhou. It is a complex produced by Cordy ceps s inensis sall parasitic on the larvae of the bat family, Hepfalus a rmoricanus devisthu r, and is one of the more commonly used precious Chinese herbal medicines.
  • Cordyceps sinensis contains various active trace elements such as adenosine, Cordyceps polysaccharide, mannitol, protein, amino acid, fatty acid, vitamins and organic selenium, which can improve human immunity, endurance, vitality, anti-fatigue, anti-aging, anti-aging
  • active trace elements such as adenosine, Cordyceps polysaccharide, mannitol, protein, amino acid, fatty acid, vitamins and organic selenium, which can improve human immunity, endurance, vitality, anti-fatigue, anti-aging, anti-aging
  • a variety of medicinal functions such as tumors, treatment of kidney and lung diseases.
  • Cordyceps sinensis has extremely harsh parasitic and special ecological environment, and its natural resources are very rare.
  • artificially excavated and excavated causing tremendous damage to the natural environment, wild resources have shrunk year by year, and ecological damage has intensified.
  • Cordyceps sinensis wild resources medicines have been unable to meet the requirements of people's health care and medication.
  • people began to explore the artificial cultivation of Cordyceps sinensis.
  • the bat moth is cultured in the medium by the Phytophthora sinensis, and the plant produces non-harmful bacteria cultivated in the plateau, low temperature and dilute oxygen environment. It is white, pure and stable.
  • the wild worms of the larvae of the genus Cordyceps sinensis are microscopically observed under the microscope.
  • the mycelium is propagated from the genus Cordyceps bat, which is the closest to the wild natural Cordyceps.
  • the bat moth has been included in the list of Cordyceps sinensis in the 2010 edition of the Pharmacopoeia, and is also one of the fungal species that the State Food and Drug Administration has clearly identified for use in health foods. Its safety and efficacy have been recognized.
  • the bat moth is a Chinese endemic Cordyceps sinensis found in the Qinghai-Tibet Plateau in China and is also known as the Chinese genus.
  • the strain has the characteristics of long fermentation time and easy to be contaminated with bacteria.
  • Cordyceps mycelium extracts Cordyceps polysaccharide by water extraction and alcohol precipitation method or extracts cordycepin by alcohol extraction method. It has also been reported that the active ingredient extracted by supercritical carbon dioxide extraction is also reported. But they are more complicated and cumbersome and have lower yields.
  • Cordyceps mycelium contains a variety of active ingredients, and it is currently recognized that it has cordycepin, cordyceps polysaccharide, cordycepic acid, various low molecular proteins, etc., and it is difficult to fully extract and concentrate it by one method.
  • the unique fermentation, extraction and concentration method can fully concentrate and concentrate the active ingredients without damage. While retaining the basic active ingredients of Cordyceps, it minimizes the amount of consumption and is convenient for consumers to take.
  • the object of the present invention is to overcome the above-mentioned deficiencies and to provide a method for processing Cordyceps mycelium rich in active ingredients.
  • a method for processing Cordyceps mycelium rich in active ingredients comprising the following steps:
  • the mass to volume ratio of the mycelium to the extract is 1:10 ⁇ 30 (kg/L). Then, the membrane with a molecular weight cut off of 2000-4000 Daltons is used for filtration, and the obtained concentrate is centrifuged, the precipitate is discarded, the supernatant is taken, and freeze-dried to obtain a mycelium concentrated freeze-dried powder.
  • the film used is preferably a polyamide film.
  • the ultrasonic temperature is 20 to 30 ° C, and the ultrasonic time is 20 to 75 min.
  • the volume ratio of the ultrasonic power to the extraction tank is 10 to 20 KW/m 3 .
  • the centrifugation is carried out by centrifugation at 5000 to 8000 rpm for 5 to 20 minutes, preferably at 6000 rpm for 10 minutes.
  • the freeze-drying conditions are: freeze-drying for 35 to 40 hours under the condition of a temperature of ⁇ 30 ° C and a vacuum of 5 Pa to 10 Pa.
  • the whole process of extraction, ultrafiltration and concentration of the bat moth is not more than 30 °C.
  • the bat moth of the bat moth used in the present invention can be obtained by fermentation culture of the strain, and the fermentation and culture steps and conditions of the strain are as follows: the volume of the shake flask is 80 ml/250 ml, the temperature is 16-18 ° C, and the shaking speed is 110 ⁇ 160/min, fermentation medium composition: glucose 3 ⁇ 9g, corn flour 2 ⁇ 6g, beef ⁇ 4 ⁇ 7g, silkworm pupa powder 3-4g, fresh milk 60 ⁇ 90ml, adding NH 4 Cl 0.4 ⁇ 0.6g, KH 2 PO 4 0.1-0.2g, MgSO 4 7H 2 O 0.1-0.2g, riboflavin 0.3-0.6g, the initial pH of the fermentation broth is 6.0-7.0, and the pH gradually increases with the fermentation, and the thorn pear is added.
  • the fermentation step and conditions of the preferred strains are as follows: the volume of the shake flask is 80 ml/250 ml, the temperature is 16-18 ° C, the shaker speed is 150/min, and the fermentation medium consists of glucose 5 g, corn flour 5 g, beef bran 5 g, silkworm cocoon.
  • SOD superoxide dismutase
  • the SOD content in the SOD preparation is very unstable, and it is easily oxidized and does not have the corresponding effect.
  • the SOD content of the concentrated lyophilized powder of the bat moth of the present invention is relatively stable, and 24 pieces are placed at normal temperature. After the month, the SOD content can still reach 92% of the original initial detection value, indicating that the SOD is effectively encapsulated in the mycelium during the fermentation of the mycelium, and the inclusions are not damaged during the extraction and concentration process.
  • the invention has the beneficial effects that the invention adopts a special fermentation, extraction and concentration method, can fully enrich and concentrate the active ingredients therein without damage, and the obtained product can significantly enhance immunity and antioxidant capacity. Moreover, while retaining the basic active ingredients of Cordyceps, the dosage is minimized and is convenient for consumers to take.
  • the ability of the mouse to clear the carbon profile by the phagocytic index was significantly higher than that of the control group, and the experimental result was judged to be positive.
  • the phagocytic index a was tested by homogeneity of variance to meet the homogeneity of variance, and multiple experimental groups and one control group were used in one-way analysis of variance (blank).
  • the two-two comparison method of the mean of the groups) is statistically processed.
  • the carbon clearance capacity of the groups of 0.08 g/kg ⁇ bw, 0.15 g/kg ⁇ bw, and 0.46 g/kg ⁇ bw was significantly higher than that of the 0 g/kg ⁇ bw group.
  • Example 1 Forty female Sprague-Dawley rats aged 20 months were randomly divided into four groups, 10 in each group. The samples of Example 1 were administered with low, medium and high doses and distilled water control once daily for 30 days. After continuous administration of the animals for 1 month, the rat heart blood was taken and the SOD was measured.
  • sample solution 10 ⁇ l whole blood was flushed into 0.5 ml normal saline, centrifuged at 2000 r/min for 3 min, the supernatant was discarded, and 0.2 ml of ice-cold double distilled water was added to mix, 95 ml of ethanol was added, shaken for 30 s, layered, upper layer For the SOD extract, the middle layer is hemoglobin precipitate, the lower layer is chloroform, and the supernatant volume is recorded for testing.
  • Standard curve The SOD standard was formulated into a 750 U/ml solution in phosphate buffer solution, and then diluted to 50 times to prepare different amounts of SOD standard solution. The percentage inhibition rate was determined by spectrophotometry at a wavelength of 530 nm. On the ordinate, the SOD activity unit U/ml is a standard curve for the abscissa.
  • Example 2 The product obtained in Example 1 and the standard solution were separately added to 1 ml of pH 7.8 phosphate buffer, 0.1 ml of 10 mmol/L hydroxylamine hydrochloride, 0.2 ml of 7.5 mmol/L xanthine, 0.2 ml of 0.2 ml mg/ml yellow. ⁇ oxidase, 0.49ml of double distilled water, mix, 37 ° C constant temperature water bath for 30min, then add 2.0ml of 0.33% p-aminobenzene sulfonic acid, 2.0ml of 0.1% naphthylamine, mix for 15min, then pour 1 cm of light was passed through the cuvette, zeroed with distilled water, and the SOD value was determined by colorimetry at 530 nm.
  • the freshly prepared bat moth was extracted and concentrated.
  • the temperature of the whole process of production does not exceed 30 °C.
  • the ultrasonic temperature is 25 ° C
  • the ultrasonic time is 50 min
  • the mycelium and the extract are
  • the mass to volume ratio was 1:20 kg/L
  • the volume ratio of ultrasonic power to the extraction tank was 15 KW/m 3 .
  • the polyamide membrane with a molecular weight cut off of 4000 Daltons was used for ultrafiltration.
  • the obtained concentrate was centrifuged at 6000 rpm for 10 minutes, the precipitate was discarded, the supernatant was taken, and the supernatant was at a temperature of ⁇ 30 ° C and a vacuum degree.
  • the mixture was freeze-dried at 5 Pa for 35 hours to obtain a mycelium concentrated lyophilized powder.
  • the mycelium concentrated lyophilized powder obtained in Example 1 was allowed to stand under the conditions of a temperature of 25 ° C and a relative humidity of 60% for 6 months.
  • the state and the content of the active ingredients are shown in Table 4:
  • the freshly prepared bat moth was extracted and concentrated.
  • the whole process temperature does not exceed 30 °C.
  • the polyamide membrane with a molecular weight cut off of 2000 Dalton was used for ultrafiltration, and the obtained concentrate was centrifuged at 6000 rpm for 10 minutes, the precipitate was discarded, and the supernatant was taken, and then the temperature was ⁇ 30 ° C and the vacuum was 10 Pa. After freeze-drying for 40 hours, a mycelium concentrated lyophilized powder was obtained.
  • the mycelium concentrated lyophilized powder obtained in Example 2 was allowed to stand under the conditions of a temperature of 25 ° C and a relative humidity of 60% for 6 months.
  • the state and the content of the active ingredients are shown in Table 6:

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Abstract

一种虫草菌丝体的加工方法，包括下列步骤：培养蝙蝠蛾被毛孢得到菌丝体，用30-50 wt %乙醇超声提取，再用截留分子量为2000-4000道尔顿的膜过滤，离心，弃去沉淀物，取上清液，冷冻干燥得到菌丝体的浓缩冻干粉。

Description

一种富含活性成分的虫草菌丝体加工方法 技术领域
本发明属于生物技术领域，具体涉及一种富含活性成分的虫草菌丝体加工方法。
背景技术
冬虫夏草，别名虫草，为麦角菌科虫草属的药用真菌。主产于青海、西藏、四川、云南、贵州等海拔4000m～5000m的山中、草甸土中。它是由冬虫夏草菌(Cordy ceps s inensis sall)寄生在蝙蝠科昆虫蝙蝠蛾(Hepfalus a rmoricanus oberthu r)幼虫上的子座及幼虫尸体产生的复合体，为较常用的名贵中药材之一。
早在清代，吴仪洛在《本草从新》中对冬虫夏草就有翔实的记载：“冬在土中，身活如老蚕，有毛能动，夏至则毛出土上，连身俱化为草”；其性味功用为：“甘、平”“保肺益肾、止血化痰、以劳嗽”。《中华人民共和国药典》2010年版记载，其主要功效是“补肺益肾，止血，化痰；用于久咳虚喘，劳嗽咯血，阳痿遗精，腰膝酸痛”。现代研究发现，冬虫夏草具有抑制肿瘤、抗癌和显著提高人体免疫功能等独特功效。现代医学研究冬虫夏草含有免疫活性虫草腺苷、虫草多糖、甘露醇、蛋白质、氨基酸、脂肪酸、维生素和有机硒等多种微量元素，具有提高人体免疫力、耐力、活力、抗疲劳、延缓衰老、抗肿瘤、治疗肾和肺部疾病等多种药用功能。
但是，冬虫夏草有着极其苛刻的寄生性和特殊的生态环境，天然资源十分稀少，加之近年来人为地乱采乱挖，给自然环境造成了巨大的破坏，野生资源逐年萎缩，生态破坏愈演愈烈。冬虫夏草野生资源药材已无法满足人们保健和用药的要求。为了扩大虫草的药源，人们开始探索用人工方法培育仿冬虫夏草。20多年来，我国先后有60多个科研单位、数千人开展人工培育仿冬虫夏草的研究。虽然也有成功进行冬虫夏草蝙蝠蛾系统饲养繁育的报道，但这些距离全人工培育以及产业化尚有多个环节的关键技术有待攻克。不过人们采用新型生物技术液体发酵培养获得冬虫夏草菌丝体的技术已获得成功，并且此技术是目前解决虫草药源的唯一途径。20世纪80年代人们为了拓宽冬虫夏草的药源，专家学者们开展了冬虫夏草菌的人工培养，并对其化学成分、药理作用进行了大量的研究工作。经过大量的化学成分、药理作用研究发现：天然虫草以及虫草菌体中含有活性成分腺苷、甘露醇、 生物碱、有机酸、维生素等化学成分，而且二者所含成分十分相近；药理作用研究表明，天然虫草及虫草菌丝制剂均具有良好的免疫调节作用。化学成分及药理作用的研究为天然虫草的替代提供了理论基础，为拓宽天然虫草的资源开辟了有效的途径，使其在药品保健食品的研发和生产展现出广阔的前景。目前已有百令胶囊，金水宝胶囊等多种产品面市，广受消费者欢迎。
蝙蝠蛾被毛孢菌丝体系将虫草蝙蝠蛾被毛孢菌在培养基中培养出来，在实验室中创造高原、低温、稀氧环境培养出来的无有害菌，洁白、纯净、质量稳定的天然野生冬虫夏草的蝙蝠蛾被毛孢菌丝体菌种，在显微镜下可见菌丝体由虫草蝙蝠蛾被毛孢繁殖而成的菌丝体，目前工人效果最接近野生天然虫草。蝙蝠蛾被毛孢已被2010版药典一部列入冬虫夏草名录，同时也是国家食品药品监督管理局明确可用于保健食品的真菌品种之一。其安全性和功效已得到了公认，当前以蝙蝠蛾被毛孢菌丝体为原料的药品和保健食品已有产品面世，在市场上也获得广大消费者的认可与好评。蝙蝠蛾被毛孢系在中国青藏高原所发现并分离鉴定的中国特有虫草种系，故又称作中国被毛孢。但是该菌种有发酵时间长、易染杂菌的特点。
目前虫草菌丝体中有报道通过水提醇沉方法提取其中虫草多糖或通过醇提方法提取其中虫草素，也有报道采用二氧化碳超临界萃取发提取的有效成分。但都较为复杂烦琐且收率较低。而虫草菌丝体中含有多种有效成分，目前比较公认的有虫草素、虫草多糖、虫草酸、各种低分子蛋白质等等，而很难用一种方法将之充分提取浓缩，本发明选用特有的发酵、提取浓缩方法，可充分将其中有效成分富集浓缩且不发生破坏。在保留了虫草的基本有效成分的同时，最大限度地减少了服用量，方便消费者服用。
发明内容
本发明的目的是克服上述不足之处，提供一种富含活性成分的虫草菌丝体加工方法。
本发明的目的是通过以下方式实现的：
一种富含活性成分的虫草菌丝体加工方法，该方法包括以下步骤：
取蝙蝠蛾被毛孢菌丝体，以质量百分比浓度为30～50％的乙醇溶液为提取液进行超声提取，菌丝体与提取液的质量体积比为1:10～30(kg/L)，再采用截留分子量为2000～4000道尔顿的膜进行过滤，所得浓缩液进行离心，弃去沉淀物，取上清液，冷冻干燥得到菌丝体浓缩冻干粉。所采用的膜优选为聚酰胺膜。
优选超声温度20～30℃，超声时间为20～75min。上述超声功率与提取罐的体积比为 10～20KW/m3。
所述的离心为在5000～8000rpm转速下离心5～20分钟，优选在6000rpm转速下离心10分钟。
所述的冷冻干燥条件为：温度≤30℃，真空度5Pa～10Pa条件下冷冻干燥35～40小时。蝙蝠蛾被毛孢菌丝体在进行提取、超滤、浓缩全过程全程温度不超过30℃。
本发明使用的蝙蝠蛾被毛孢菌丝体可采用菌种发酵培养方式得到，该菌种发酵培养步骤及条件如下：菌种摇瓶装液量80ml/250ml，温度16～18℃，摇床转速110～160/min，发酵培养基组成：葡萄糖3～9g，玉米粉2～6g，牛肉胨4～7g，蚕蛹粉3～4g，鲜牛奶60～90ml，加入NH4Cl 0.4～0.6g、KH2PO40.1～0.2g、MgSO47H2O 0.1～0.2g、核黄素0.3～0.6g，发酵液初始pH6.0～7.0，随着发酵进行，pH值逐渐升高，补入刺梨汁，保持发酵液pH6.5～7.0，发酵时间126-130小时，得到蝙蝠蛾被毛孢菌丝体。优选菌种发酵培养步骤及条件如下：菌种摇瓶装液量80ml/250ml，温度16～18℃，摇床转速150/min，发酵培养基组成：葡萄糖5g，玉米粉5g，牛肉胨5g，蚕蛹粉3g，鲜牛奶80ml，加入NH4Cl 0.5g、KH2PO40.1g、MgSO47H2O 0.1g、核黄素0.5g，发酵液初始PH6.5，随着发酵进行，pH值逐渐升高，补入刺梨汁，保持发酵液pH6.5～7.0，发酵时间128小时，得到蝙蝠蛾被毛孢菌丝体。
原料蝙蝠蛾被毛孢菌丝体也可采用市售商品。
众所周知，超氧化物歧化酶(SOD)属于人体内自身含有的一种抗氧化酶，是生物体内清除自由基的重要物质，它可对抗与阻断自由基对细胞造成的伤害，而现代生活压力大，各种环境污染，辐射和超量运动都会造成氧自由基的大量生成，所以研究富含SOD的药品及保健品是对人体有极大好处的。
但通常情况下，SOD制剂中的SOD含量非常不稳定，极易被氧化而起不到相应功效，但本发明的蝙蝠蛾被毛孢浓缩冻干粉中SOD含量高较稳定，常温放置24个月后，其SOD含量仍可达到原来起始检测值的92％，说明在菌丝体发酵过程中SOD被有效包裹在菌丝体内，而在提取浓缩过程中，又未对包裹物产生破坏。
与现有技术比较本发明的有益效果：本发明选用特有的发酵、提取、浓缩方法，可充分将其中有效成分富集浓缩且不发生破坏，得到的产品可显著增强免疫力和抗氧化能力，而且，在保留了虫草的基本有效成分的同时，最大限度地减少了服用量，方便消费者服用。
以下通过药效实验对本发明进行阐述：
1、增强免疫力实验
小鼠碳廓清试验：
各剂量组动物连续灌胃1个月后，每鼠尾静脉注入4倍稀释的印度墨汁(0.1mL/10g·bw)，待墨汁注入，立即计时。注入墨汁后2min及10min分别从眼内眦静脉丛取血20μL，并将其加到2mL0.1％Na2CO3溶液中，用721分光光度计在600nm波长处测光密度值，并取胸腺、肝、脾，利用光密度值、肝重和脾重计算吞噬指数a。另计算胸腺/体比、脾/体比。
以吞噬指数表示小鼠碳廓清的能力。受试样品组的吞噬指数显著高于对照组的吞噬指数，可判定该项实验结果阳性。
经口给予小鼠不同剂量的本实施例1样品1个月后，吞噬指数a经方差齐性检验，满足方差齐性要求，用单因素方差分析方法中多个实验组与一个对照组(空白组)间均数的两两比较方法进行统计处理。由表1结果可见，0.08g/kg·bw、0.15g/kg·bw、0.46g/kg·bw组碳廓清能力显著高于0g/kg·bw组。
表1 本发明小鼠碳廓清实验结果表
Figure PCTCN2015071621-appb-000001
2、抗氧化实验
抗氧化酶活力测定法：
选用20月龄SD雌性大鼠40只，随机分为四组，每组10只，分别灌服实施例1样品低、中、高剂量及蒸馏水对照，每日一次，连续30天。各组动物连续灌胃1个月后，取大鼠心脏血，测定其SOD。
样品溶液制备：将10μl全血冲入0.5ml生理盐水，2000r/min离心3min，弃上清，加冰冷的双蒸水0.2ml混匀，加入95％乙醇0.1ml，振荡30s，分层，上层为SOD抽提液，中层为血红蛋白沉淀物，下层为三氯甲烷，记录上清液体积待测。标准曲线：将SOD标准品用磷酸盐缓冲液配制成750U/ml的溶液，再稀释到50倍，配制不同量的SOD标准液，用分光光度法在波长530nm处测定，以百分抑制率为纵坐标，SOD活力单位U/ml为横坐标绘制标准曲线。
测定法：将实施例1得到的产品和标准液分别加入1mlpH7.8磷酸盐缓冲液，0.1ml的10mmol/L盐酸羟胺，0.2ml的7.5mmol/L黄嘌呤，0.2ml的0.2mlmg/ml黄嘌呤氧化酶，0.49ml的双蒸水，混匀后，37℃恒温水浴30min，再分别加入2.0ml的0.33％对氨基苯磺酸，2.0ml的0.1％甲萘胺，混匀15min后，倒入1cm光经比色杯，以蒸馏水调零，530nm处比色测定SOD值。
由表2可以看出，各剂量组大鼠血清SOD活力明显高于对照组(空白组)，说明本样品具有增高血清SOD活力的作用(P＜0.05)
表2 本发明对大鼠血清SOD活力的影响结果表
Figure PCTCN2015071621-appb-000002
注：“*”与对照组比较，P＜0.05。
具体实施方式
以下通过具体实施例进一步说明本发明。但实施例的具体细节仅用于解释本发明，不应理解为对本发明总的技术方案的限定。其中，超氧化物歧化酶(SOD)的含量按照GB/T5009.171-2003标准检测，虫草素的含量按照农业部标准NY/T 2116-2012方法检测。
实施例1
1、菌种发酵培养
取蝙蝠蛾被毛孢菌种(中国科学院微生物研究所)，摇瓶装液量80ml/250ml，温度16～18℃，摇床转速150/min，发酵培养基组成：葡萄糖5g，玉米粉5g，牛肉胨5g，蚕蛹粉3g，鲜牛奶80ml，加入NH4Cl 0.5g、KH2PO40.1g、MgSO47H2O 0.1g、核黄素0.5g，发酵液初始pH6.5，随着发酵进行，pH值逐渐升高，补入刺梨汁，保持发酵液pH6.5～7.0之间，发酵时间128小时，得菌丝体干重为7.6g/L。
2、提取、浓缩、冻干
将新鲜制备的蝙蝠蛾被毛孢菌丝体进行提取、浓缩。生产全过程温度不超过30℃。
具体方法如下：
取蝙蝠蛾被毛孢菌丝体，放入超声提取罐，以质量百分比浓度为40％的乙醇溶液为提取液进行超声提取，超声温度25℃，超声时间为50min，菌丝体与提取液的质量体积比为1:20kg/L，超声功率与提取罐的体积比为15KW/m3。
再采用截留分子量为4000道尔顿的聚酰胺膜进行超滤，所得浓缩液在6000rpm转速下离心10分钟，弃去沉淀物，取上清液，上清液再在温度≤30℃、真空度5Pa条件下冷冻干燥35小时，得到菌丝体浓缩冻干粉。
实施例1得到的蝙蝠蛾被毛孢菌丝体浓缩冻干粉与蝙蝠蛾被毛孢菌丝体有效成分含量对比见表3：
表3
  虫草素 虫草多糖 超氧化物歧化酶
蝙蝠蛾被毛孢菌丝体 0.007％ 0.95％ 16.5u/mi
实施例1浓缩冻干粉 0.48％ 27.6％ 654.3u/mi
对比倍数 68.6 29.0 39.7
将实施例1得到的菌丝体浓缩冻干粉在温度为25℃、相对湿度60％的条件下放置6个月，状态及有效成分的含量见表4：
表4
  虫草素 虫草多糖 超氧化物歧化酶
蝙蝠蛾被毛孢菌丝体 0.002％ 0.63％ 1.25u/mi
实施例1浓缩冻干粉 0.39％ 27.3％ 524.3u/mi
对比倍数 195.0 43.3 419.4
实施例2
1、菌种发酵培养方法同实施例1
2、提取、浓缩、冻干
将新鲜制备的蝙蝠蛾被毛孢菌丝体进行提取、浓缩。生产全程温度不超过30℃。
具体方法如下：
取蝙蝠蛾被毛孢菌丝体，放入超声提取罐，以质量百分比浓度为30％的乙醇溶液作为提取液进行超声提取，超声温度30℃，超声时间为20min，菌丝体与提取液的质量体积比为1:10kg/L，超声功率与提取罐的体积比为20KW/m3。
再采用截留分子量为2000道尔顿的聚酰胺膜进行超滤，所得浓缩液在6000rpm转速下离心10分钟，弃去沉淀物，取上清液，再在温度≤30℃、真空度10Pa条件下冷冻干燥40小时，得到菌丝体浓缩冻干粉。
将实施例2方法得到的蝙蝠蛾被毛孢菌丝体浓缩冻干粉与蝙蝠蛾被毛孢菌丝体有效成分含量对比见表5：
表5
  虫草素 虫草多糖 超氧化物歧化酶
蝙蝠蛾被毛孢菌丝体 0.007％ 0.95％ 16.5u/mi
实施例2浓缩冻干粉 0.47％ 27.3％ 651.3u/mi
对比倍数 67.1 28.7 39.5
将实施例2得到的菌丝体浓缩冻干粉在温度为25℃、相对湿度60％的条件下放置6个月，状态及有效成分的含量见表6：
表6
  虫草素 虫草多糖 超氧化物歧化酶
蝙蝠蛾被毛孢菌丝体 0.002％ 0.63％ 1.25u/mi
实施例2浓缩冻干粉 0.38％ 27.1％ 523.1u/mi
对比倍数 190 43.0 418.5

Claims (7)

  1. 一种富含活性成分的虫草菌丝体加工方法，其特征在于该方法包括以下步骤：
    取蝙蝠蛾被毛孢菌丝体，以质量百分比浓度为30～50％的乙醇溶液为提取液进行超声提取，菌丝体与提取液的质量体积比为1:10～30(kg/L)，再采用截留分子量为2000～4000道尔顿的膜进行过滤，所得浓缩液进行离心，弃去沉淀物，取上清液，冷冻干燥得到菌丝体浓缩冻干粉。
  2. 根据权利要求1所述的富含活性成分的虫草菌丝体加工方法，其特征在于超声温度20～30℃，超声时间为20～75min。
  3. 根据权利要求1所述的富含活性成分的虫草菌丝体加工方法，其特征在于超声功率与提取罐的体积比为10～20KW/m3。
  4. 根据权利要求1所述的富含活性成分的虫草菌丝体加工方法，其特征在于所述的离心为在5000～8000rpm转速下离心5～20分钟。
  5. 根据权利要求1所述的富含活性成分的虫草菌丝体加工方法，其特征在于所述的冷冻干燥条件为：温度≤30℃，真空度5Pa～10Pa条件下冷冻干燥35～40小时。
  6. 根据权利要求1所述的富含活性成分的虫草菌丝体加工方法，其特征在于菌种发酵培养条件如下：温度16～18℃，摇床转速110～160/min，发酵培养基组成：葡萄糖3～9g，玉米粉2～6g，牛肉胨4～7g，蚕蛹粉3～4g，鲜牛奶60～90ml，加入NH4Cl 0.4～0.6g、KH2PO40.1～0.2g、MgSO47H2O 0.1～0.2g、核黄素0.3～0.6g，发酵液初始pH6.0～7.0，随着发酵进行，pH值逐渐升高，补入刺梨汁，保持发酵液pH6.5～7.0，发酵时间126-130小时，得到蝙蝠蛾被毛孢菌丝体。
  7. 根据权利要求6所述的富含活性成分的虫草菌丝体加工方法，其特征在于菌种发酵培养条件如下：温度16～18℃，摇床转速150/min，发酵培养基组成：葡萄糖5g，玉米粉5g，牛肉胨5g，蚕蛹粉3g，鲜牛奶80ml，NH4Cl 0.5g、KH2PO40.1g、MgSO47H2O 0.1g、核黄素0.5g，发酵液初始pH6.5，随着发酵进行，pH值逐渐升高，补入刺梨汁，保持发酵液pH6.5～7.0，发酵时间128小时，得到蝙蝠蛾被毛孢菌丝体。
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