WO2020177421A1 - 一种结合复合酶解的两步式蛹虫草酵素的发酵制备方法 - Google Patents

一种结合复合酶解的两步式蛹虫草酵素的发酵制备方法 Download PDF

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WO2020177421A1
WO2020177421A1 PCT/CN2019/123518 CN2019123518W WO2020177421A1 WO 2020177421 A1 WO2020177421 A1 WO 2020177421A1 CN 2019123518 W CN2019123518 W CN 2019123518W WO 2020177421 A1 WO2020177421 A1 WO 2020177421A1
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cordyceps militaris
fermentation
enzymatic hydrolysis
lactic acid
acid bacteria
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张慜
劳艳艳
阳武雄
易思富
侯丽华
郭小玲
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Jiangnan University
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Jiangnan University
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
    • A23L2/38—Other non-alcoholic beverages
    • A23L2/382—Other non-alcoholic beverages fermented
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
    • A23L2/70—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter
    • A23L2/84—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter using microorganisms or biological material, e.g. enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/113—Acidophilus
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/125—Casei
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/169—Plantarum
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A23V2400/531—Lactis

Definitions

  • the invention relates to a two-step fermentation preparation method of Cordyceps militaris enzyme combined with compound enzymatic hydrolysis, in particular to a method of using brown sugar as a carbon source, using compound enzymatic hydrolysis to promote fermentation of lactic acid bacteria, and then inoculating yeast for rapid secondary fermentation
  • the fermentation method belongs to the technical field of research and development of fermented beverages.
  • Cordyceps militaris is a parasitic fungus and has been listed as a new resource food in 2009. It contains a variety of nutrients, such as protein and amino acids, cordyceps polysaccharides, cordycepic acid (D-mannitol), cordycepin and superoxide dismutation Enzyme (SOD) etc.
  • Ancient book records and modern studies show that the medicinal value of Cordyceps militaris is similar to that of Cordyceps sinensis and is comparable to ginseng and deer antler.
  • cordycepin can be used as a nucleoside analog to disrupt the synthesis of DNA and RNA, thereby causing cancer Apoptosis has many physiological functions such as anti-tumor, lowering blood sugar, immune regulation, antibacterial and anti-inflammatory.
  • Cordyceps militaris is a kind of edible fungus, which is mainly eaten fresh. For example, it is often used in soups in daily family meals. Fresh Cordyceps militaris is not easy to store, and it is easy to grow "white hair". It is prone to browning when exposed to the air. If the fruit bodies of the fresh Cordyceps militaris are properly dried or processed, the storage period of Cordyceps militaris can be prolonged. It can preserve nutrients and greatly enhance its application value. At present, there are many kinds of products processed with Cordyceps militaris as raw materials, such as Cordyceps militaris lozenges, bread, canned food, meat, beverages, Cordyceps militaris wine, etc. Cordyceps militaris products have developed due to their rich nutritional value and unique flavor. Broad prospects.
  • Enzyme is a new type of fermented beverage that originally originated in Japan. It is generally made of vegetables, fruits, grains, beans, edible fungi with the same medicine and food, and Chinese medicine as raw materials, and fermented by lactic acid bacteria, yeasts, acetic bacteria and other probiotics It is a microbial preparation containing a variety of enzymes, vitamins, trace elements, esters, polyphenols and other nutrients. Studies have shown that enzymes have health-care functions such as preventing obesity, anti-oxidation, anti-aging, promoting cell regeneration, and enhancing immunity. Today, they are very popular in Europe, America, Japan, and Taiwan. Although a small number of enzyme products have appeared on the domestic market, the fermentation time is long and the product quality is uneven. Therefore, my country's enzyme industry still has broad room for development.
  • Liu Qin et al. disclosed "a preparation method of wolfberry lemon grapefruit peel enzyme" (Chinese Patent Application No. 201611136927.3), which washes and dries the wolfberry, lemon, and grapefruit peel in a certain ratio and then diced it into a fermentation tank. Then add a certain amount of brown sugar and honey to submerge the fruit, stir evenly, and carry out natural fermentation under sealed conditions. The fermentation time is 30-40 days.
  • the grapefruit peel, lemon and other raw materials used in this method have high cellulose and pectin content and are difficult to be decomposed and utilized by common probiotics.
  • the enzyme produced by this method has a long fermentation cycle and the nutrients are difficult to effectively dissolve.
  • the present invention adopts the method of enzymatic hydrolysis and synergistic inoculation and fermentation, which can decompose the macromolecular substances in Cordyceps militaris, promote the utilization of probiotics, and fully dissolve the nutrients in Cordyceps militaris, thereby improving the fermentation efficiency and shortening Fermentation time, the prepared product has excellent flavor and rich nutrition.
  • the Cordyceps militaris compound enzyme beverage prepared by this method is rich in nutrients and has a variety of health care functions, and it is inoculated with Rhizopus and Monascus for compound fermentation to degrade the toxic and harmful substances in the raw materials, but it does not use enzymatic hydrolysis Technology reduces the efficiency of fermentation and increases the fermentation time.
  • the present invention not only adopts the two-step inoculation fermentation technology, but also uses the method of compound enzymolysis and lactic acid bacteria fermentation to further improve the fermentation efficiency of lactic acid bacteria, so that high-quality Cordyceps militaris enzyme products can be quickly prepared.
  • Lu Liqun et al. disclosed "a rapid fermentation preparation method of barley seedling enzymes" (Chinese Patent Application No. 201510653100.9), which fermented barley seedlings homogenate that has been prepared and sterilized with auxiliary materials by artificial inoculation of yeast, and it only takes 12 hours to The whole fermentation process was completed, and the wheat grass enzyme with high enzyme activity and strong antioxidant capacity was obtained. However, it was only fermented with yeast. Although the process was simplified, the taste of the obtained product was not good. In contrast, the present invention adopts the fermentation method of lactic acid bacteria first and then yeast, using simple raw materials and simple technology not only to quickly complete the entire fermentation process, but also to effectively retain nutrients, and obtain an enzyme product with good taste and nutrition. .
  • Jiang Xiaolu et al. disclosed "a method for producing Cordyceps militaris enzyme based on enzyme-enzyme coupling" (Chinese Patent Application No. 201711263212.9), which uses Cordyceps militaris mycelium or bacterial powder as raw materials, first combined enzymatic hydrolysis for 2 hours, and then inoculated with plant milk Bacillus coupled fermentation for 12 to 48 hours, the liquid Cordyceps militaris enzyme product was obtained.
  • Xu Guohua et al. disclosed "a preparation method of Cordyceps militaris fruit and vegetable enzyme” (Chinese Patent Application No.
  • Cordyceps militaris concentrate is mixed with fruit and vegetable juice with maltitol and xylitol in a ratio of 1:3 ⁇ 5, and inoculated with 3.5 ⁇ 7% of Bifidobacterium adolescentis, Lactobacillus casei, Lactobacillus acidophilus, Streptococcus thermophilus
  • Bifidobacterium adolescentis Lactobacillus casei
  • Lactobacillus acidophilus Streptococcus thermophilus
  • the seven lactic acid bacteria, Lactobacillus rhamnosus, Lactobacillus plantarum and Lactobacillus bulgaricus are fermented for 4-6 days to obtain the product. Zhang Furong et al.
  • a fermented Cordyceps militaris probiotic beverage and its preparation method (Chinese Patent Application No. 201810069589.9), which uses Cordyceps militaris powder as the main raw material, and adds Ganoderma lucidum powder, selenium-enriched yeast powder, and soy protein Powder, chrysanthemum extract and wolfberry extract are mixed to obtain the original solution, and then the original solution is enzymatically hydrolyzed by ⁇ -amylase, glucoamylase, cellulase, pectinase and protease.
  • the raw materials used in the present invention are simple, it is inoculated with lactic acid bacteria and then inoculated with yeast to carry out two-step fermentation, which makes the enzyme product more mellow, and the flavor is more prominent than that of the enzyme obtained by one-step fermentation, and it also saves The process of preparing other Chinese herbal medicines or fruit and vegetable treatment solutions saves costs.
  • the purpose of the present invention is to provide a two-step fermentation preparation method of Cordyceps militaris enzyme combined with complex enzymatic hydrolysis, which can quickly complete the entire fermentation process and greatly shorten the fermentation period by using the complex enzymatic hydrolysis to coordinate lactic acid bacteria fermentation, yeast fermentation and other processes.
  • the raw materials and processes used in this method are very simple, only Cordyceps militaris powder is used as raw materials, no additional fruits, vegetables or Chinese herbal medicines are added, and brown sugar is used as the carbon source in the fermentation process. The cost is low, and the resulting product is sweet and delicious with flavor. It is unique, has good suspension stability, and can well retain cordycepin, an important functional component, which has certain promotion value.
  • Centrifugation Take the Cordyceps militaris fermentation broth after two-step fermentation of lactic acid bacteria and fruit wine yeast and centrifuge at 4200r/min for 10min, and filter to obtain a clear liquid after centrifugation;
  • Sterilization filling sterilize the prepared fermentation broth at 95 ⁇ 100°C for 5 ⁇ 10min; cool it to 85 ⁇ 90°C, pour it into the pre-sterilized glass bottle while it is hot to obtain Cordyceps militaris enzyme product .
  • the pasteurization temperature in the step (2) is 80-90° C., and the time is 10-20 min.
  • the concentration of the citric acid solution is 10% (w/w); the enzyme activity of the cellulase is 50,000 U/g, and the enzyme activity of pectinase is 100,000 U/g, which is suitable
  • the enzymatic hydrolysis temperature is 40 ⁇ 50°C.
  • the inoculation process of the lactic acid bacteria is completed in an ultra-clean table;
  • the lactic acid bacteria are probiotic fermentation bacteria powder, which contains Lactobacillus plantarum, Lactobacillus acidophilus, Bifidobacterium lactis, Lactobacillus casei and Bifidobacterium longum has an amount of 8 billion CFU/g, and its suitable growth temperature is 37 ⁇ 45°C.
  • the inoculation process of the fruit wine yeast is completed in an ultra-clean table; the fruit wine yeast is a "special yeast powder for wine and fruit wine", which needs to be activated with warm water at 35-40°C before use. Inoculate into the fermentation broth at a temperature of 26-30°C.
  • this method adopts inoculation fermentation, which greatly improves the fermentation efficiency, shortens the fermentation time, and obtains products with unique flavor and rich nutrition;
  • the enzymatic hydrolysis of this method is carried out at the same time as the first fermentation.
  • the process is simplified to the greatest extent and the processing time is shortened;
  • this method adopts a two-step fermentation method of inoculating lactic acid bacteria first and then yeast inoculating, which enriches the flavor of the product and makes Cordyceps militaris enzymes have a good taste without adding other fruit and vegetable raw materials;
  • the fermentation process is simple and easy to operate, short fermentation cycle, low production cost, and suitable for large-scale promotion and production.
  • Example 1 A rapid fermentation method of Cordyceps militaris enzyme combined with low-proportion cellulase/pectinase enzyme hydrolysis
  • Cordyceps militaris powder After removing impurities from the dry sample of Cordyceps militaris, it is crushed by a high-speed grinder and passed through a 100-mesh sieve to obtain Cordyceps militaris powder; mix the Cordyceps militaris powder with water 1:10 and mix them evenly with a high-speed homogenizer to obtain Cordyceps militaris slurry; take 300g Cordyceps militaris slurry, add 150g brown sugar, and add 550g distilled water to quantify to 1kg, stir well to dissolve the brown sugar completely, and then pasteurize at 85°C for 10min to obtain the fermentation substrate of Cordyceps militaris; wait for the fermentation substrate to cool To 40 ⁇ 45°C, adjust the pH to 4.80 ⁇ 0.10, add 0.4% cellulase and 0.6% pectinase, and inoculate 1g compound lactic acid bacteria powder, make compound enzymolysis and lactic acid bacteria fermentation at 40°C for 48h at the same time, Then in
  • the resulting product has a rich brown sugar flavor, a clear and transparent reddish brown, sweet and sour, and has a faint mellow fragrance; the electronic nose is used to analyze the changes in flavor components during the fermentation process of the product, and it is found that it is compared with the fermentation liquid phase only inoculated with lactic acid bacteria Compared with the fermentation broth after two-step fermentation of lactic acid bacteria and yeast, the response value of S1 sensor (aromatic compounds) increased by about 131%, and the response value of S5 sensor (terpenoids and esters) increased by about 114%. Step fermentation can make the enzyme flavor more prominent.
  • the physical and chemical indicators and functional components of the product are analyzed: the pH of the final product is 3.60 ⁇ 0.02, the soluble solid content is about 15.6%, the alcohol content is less than 0.5g/100g; the cordycepin content is 45.940 ⁇ 1.538g/mL, and its retention The rate is about 77.68%, indicating that the fermentation process has little effect on cordycepin and has a good retention effect on cordycepin in the product; the hydroxyl radical scavenging rate of the product obtained is 79.27 ⁇ 0.94%, and ferricyanide is used.
  • the reducing power of the enzyme was determined by the potassium method, and the result showed that the absorbance value of the product at 700nm was 0.7396 ⁇ 0.0083, indicating that the product obtained had strong reducing power and certain antioxidant properties.
  • Example 2 A rapid fermentation method of Cordyceps militaris enzyme combined with equal ratio of cellulase/pectinase enzyme hydrolysis
  • Cordyceps militaris powder After removing impurities from the dry sample of Cordyceps militaris, it is pulverized by a high-speed pulverizer and passed through a 100-mesh sieve to obtain Cordyceps militaris powder; mix the Cordyceps militaris powder with water 1:10 and mix them evenly with a high-speed homogenizer to obtain Cordyceps militaris slurry; take 300g Cordyceps militaris slurry, add 150g brown sugar, and add 550g distilled water to quantify to 1kg, stir thoroughly to dissolve the brown sugar completely, and then pasteurize at 85°C for 10min to obtain the fermentation substrate of Cordyceps militaris; wait for the fermentation substrate to cool To 40 ⁇ 45°C, adjust the pH to 4.80 ⁇ 0.10, add 0.5% cellulase and 0.5% pectinase, and inoculate 1g compound lactic acid bacteria powder, make compound enzymolysis and lactic acid bacteria fermentation at 40°C for 48h at the
  • the resulting product has a rich brown sugar flavor, a clear and transparent reddish brown color, a moderately sweet and sour taste, and a faint mellow aroma; the electronic nose is used to analyze the changes in flavor components during the fermentation process of the product, and it is found that it is comparable to the fermentation liquid inoculated with lactic acid bacteria Compared with the fermentation broth after two-step fermentation of lactic acid bacteria and yeast, the response value of S1 sensor (aromatic compounds) increased by about 136%, and the response value of S5 sensor (terpenoids, esters) increased by about 128%. Two-step fermentation can make the enzyme flavor more prominent.
  • the physical and chemical indicators and functional components of the product are analyzed: the pH of the final product is 3.54 ⁇ 0.02, the soluble solid content is about 14.5%, and the alcohol content is ⁇ 0.5g/100g; the cordycepin content of the final product is 58.808 ⁇ 0.116g/mL The retention rate is about 97.87%, indicating that the fermentation process has little effect on cordycepin and has a good retention effect on cordycepin in the product; the hydroxyl radical scavenging rate of the product obtained is 71.46 ⁇ 0.22%, and iron is used.
  • the potassium cyanide method was used to determine the reducing power of the enzyme, and the result showed that the absorbance value of the product at 700nm was 0.8478 ⁇ 0.0056, indicating that the product obtained has strong reducing power and certain antioxidant properties.
  • Example 3 A rapid fermentation method of Cordyceps militaris enzyme combined with high-proportion cellulase/pectinase enzyme hydrolysis
  • Cordyceps militaris powder After removing impurities from the dry sample of Cordyceps militaris, it is crushed by a high-speed grinder and passed through a 100-mesh sieve to obtain Cordyceps militaris powder; mix the Cordyceps militaris powder with water 1:10 and mix them evenly with a high-speed homogenizer to obtain Cordyceps militaris slurry; take 300g Cordyceps militaris slurry, add 150g brown sugar, and add 550g distilled water to quantify to 1kg, stir well to dissolve the brown sugar completely, and then pasteurize at 85°C for 10min to obtain the fermentation substrate of Cordyceps militaris; wait for the fermentation substrate to cool To 40 ⁇ 45°C, adjust the pH to 4.80 ⁇ 0.10, add 0.6% cellulase and 0.4% pectinase, and inoculate 1g compound lactic acid bacteria powder, make compound enzymatic hydrolysis and lactic acid bacteria fermentation simultaneously at 40°C for 48h, Then inactivate the
  • the resulting product has a rich brown sugar flavor, a clear and transparent reddish brown, sweet and sour, and has a faint mellow fragrance; the electronic nose is used to analyze the changes in flavor components during the fermentation process of the product, and it is found that it is compared with the fermentation liquid phase only inoculated with lactic acid bacteria Compared with the two-step fermentation of lactic acid bacteria and yeast, the response value of S1 sensor (aromatic compounds) increased by about 121%, and the response value of S5 sensor (terpenes, esters) increased by about 132%. Step fermentation can make the enzyme flavor more prominent.
  • the physical and chemical indicators and functional components of the product are analyzed: the pH of the final product is 3.53 ⁇ 0.01, the soluble solid content is about 14.9%, and the alcohol content is ⁇ 0.5g/100g; the cordycepin content of the final product is 47.289 ⁇ 1.953g/mL , Its retention rate is 79.95%, indicating that the fermentation process has little effect on cordycepin and has a better retention effect on cordycepin in the product; the hydroxyl radical scavenging rate of the product obtained is 70.31 ⁇ 0.61%, and it is used
  • the potassium ferricyanide method was used to determine the reducing power of the enzyme, and the result showed that the absorbance value of the product at 700nm was 0.8072 ⁇ 0.0027, indicating that the product obtained had strong reducing power and certain antioxidant properties.
  • Comparative example 1 Combining equal proportions of cellulase/pectinase for enzymatic hydrolysis and synergistic one-time fermentation and comparison of the effects of enzymatic hydrolysis and then fermentation
  • Example 2 for the method of combining the same ratio of cellulase/pectinase for enzymatic hydrolysis and one-time fermentation.
  • the specific method of preparing Cordyceps militaris enzyme by enzymatic hydrolysis and then fermentation is as follows: After the dry sample of Cordyceps militaris is removed, it is crushed with a high-speed grinder and passed through a 100-mesh sieve to obtain Cordyceps militaris powder; The powder and water are mutually soluble in 1:10 and mixed evenly with a high-speed homogenizer to obtain Cordyceps militaris pulp; the pH of the resulting Cordyceps militaris pulp is adjusted to 4.80 ⁇ 0.10, 0.5% cellulase and 0.5% pectinase are added, at 50°C Enzymatic hydrolysis in the water bath for 4 hours, after the enzymolysis is completed, inactivate the enzyme in a water bath at a temperature of 90°C for 5 minutes; take 350g of the cordy
  • the enzymatic hydrolysis and one-time fermentation use low-temperature long-term enzymatic hydrolysis, that is, enzymatic hydrolysis at 40°C for 48 hours, which is different from the high-temperature short-term enzymatic hydrolysis in the first enzymolysis followed by fermentation, that is, enzymatic hydrolysis at 50°C. 4h.
  • the flavor of the enzyme obtained by synergistic fermentation with enzymatic hydrolysis is more abundant, and the electronic nose has a higher response value.
  • the response value of the two representative sensors S1 (representing aromatic compounds) increased by about 36%
  • the response value of S5 (representing terpenes and esters) increased by about 33%.
  • the pH and soluble solids of the two final products are not much different, and the alcohol content is also less than 0.5g/100g.
  • the content of cordycepin in the enzyme product after enzymatic hydrolysis and then fermentation is 56.159 ⁇ 0.014mg/L, which is about 18% less than the cordycepin in the first fermentation product of enzymatic hydrolysis and synergy; its hydroxyl radical scavenging rate is 59.58 ⁇ 0.69 %, compared with the enzyme hydrolysis synergistic primary fermentation product, the clearance rate is reduced by about 17%; the potassium ferricyanide method is used to measure the enzyme reducing power, and the result shows that its absorbance at 700nm is 0.6177 ⁇ 0.0086, which is compared with the enzymatic hydrolysis synergistic primary fermentation The reducing power of the product is reduced by about 18%.

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Abstract

一种结合复合酶解的两步式蛹虫草酵素的发酵制备方法，属于发酵饮料的研制与开发技术领域。本发明通过蛹虫草粉和蛹虫草浆的制备、蛹虫草发酵基质的制备、复合酶解协同乳酸菌发酵、酵母菌发酵等过程，再经过离心、过滤、调配、杀菌、灌装制得产品。本发明利用复合酶解协同乳酸菌发酵，再经过酵母菌的二次发酵，可以在2~3天的时间内快速完成整个发酵过程，得到的蛹虫草酵素酸甜可口、风味独特、悬浮稳定性好，并能很好地保留虫草素这一重要活性成分：虫草粉中虫草素的溶出量可达78.21%，溶出虫草素的保存率最高达到了97.78%，具有一定抗氧化性和提高免疫力的功能，符合现代人们对健康、美味的高品质饮料的追求。

Description

一种结合复合酶解的两步式蛹虫草酵素的发酵制备方法 技术领域
本发明涉及一种结合复合酶解的两步式蛹虫草酵素的发酵制备方法，具体涉及一种以红糖为碳源，利用复合酶解促进乳酸菌发酵，然后再接种酵母菌进行二次发酵的快速发酵法，属于发酵饮料的研制与开发技术领域。
 背景技术
蛹虫草是一种寄生真菌，已在2009年被列为新资源食品，其含有多种营养成分，如蛋白质及氨基酸、虫草多糖、虫草酸（D-甘露醇）、虫草素和超氧化物歧化酶（SOD）等。古书记载和现代研究表明，蛹虫草的药用价值与冬虫夏草相似，并可与人参、鹿茸相媲美，其特有的虫草素可以作为一种核苷类似物打乱DNA和RNA的合成，从而使癌细胞凋亡，具有抗肿瘤、降血糖、免疫调节和抑菌消炎等多种生理功能。
在我国，蛹虫草作为一种食用菌，以鲜食为主，如日常家庭饮食中，常常将其用于煲汤。新鲜蛹虫草不易保存，易长“白毛”，将其露置于空气中易发生褐变，若将鲜虫草子实体进行适当的干制或加工处理，既能延长蛹虫草的储存期，又能使营养成分得以保存，大大提升了其应用价值。目前，以蛹虫草为原料进行加工的产品多种多样，如蛹虫草含片、面包、罐头、肴肉、饮料、蛹虫草酒等，蛹虫草制品因其丰富的营养价值和独特的风味，发展前景广阔。
随着经济的发展和生活水平的提高，人们的保健意识也慢慢增强，健康营养又美味的饮料也逐渐成为消费者的新宠。酵素是一种新型发酵饮料，最初起源于日本，一般是以蔬菜、水果、谷物、豆类、药食同源的食用菌和中药等为原料，经过乳酸菌、酵母菌、醋酸菌等益生菌发酵而成的，含有多种酶、维生素、微量元素、酯类和多酚等营养物质的微生物制剂。研究表明，酵素具有预防肥胖、抗氧化、抗衰老、促进细胞新生、增强免疫力等保健功能，如今在欧美、日本以及我国的台湾地区十分流行。尽管国内市场上也出现了少量的酵素产品，但发酵时间长、产品质量参差不齐，因此我国的酵素产业还有广阔的发展空间。
郭红莲等（2018）以枸杞干果为原料，利用枸杞自身固有的多种有益菌常温密封自然发酵45d，得到了含有酯类、醛类、酮类、醇类、酸类、芳香杂环类等风味物质，并具有较强抗氧化性的枸杞酵素。自然发酵虽使这一产品拥有良好的风味和较强的抗氧化性，但发酵时间长，对生产条件要求高，产品品质不易控制。与其相比，本发明采用接种发酵，大大提高了发酵效率，缩短了发酵时间，仅用2~3d就可以完成整个发酵过程，所得的蛹虫草酵素酸甜可口，并对蛹虫草特有的功能性成分虫草素也有较好的保留。
刘勤等公开了“一种枸杞柠檬柚子皮酵素的制备方法”（中国专利申请号201611136927.3），其将一定配比的枸杞、柠檬、柚子皮清洗、晾干后切丁放入发酵罐中，再加入一定量的红糖和蜂蜜淹没水果，搅拌均匀，在密封条件下进行自然发酵，发酵时间为30~40天。该方法中所用的柚子皮，柠檬等原料，纤维素和果胶含量高，难以被常见的益生菌分解利用，则用该法生产的酵素，发酵周期长，营养成分也难以有效溶出。与其相比，本发明采用酶解协同接种发酵的方法，能使蛹虫草中的大分子物质分解，促进益生菌的利用，并使蛹虫草中的营养成分充分溶出，提高了发酵效率，缩短了发酵时间，制得的产品风味优良，营养丰富。
刘忠诚等公开了“一种抗疲劳增强免疫力的蛹虫草复合酵素饮品制备方法”（中国专利申请号：201810158350.9），其先将人参、玛咖、黄精、枸杞、葛根等中药通过浸泡、煎煮、浓缩、灭菌、过滤等步骤得到营养液，再加入红糖混合均匀得培养液，最后加入蛹虫草粉，并接种根霉菌、红曲酶菌种密封发酵3~6个月即得产品。该法制得的蛹虫草复合酵素饮料营养丰富，具有多种保健功能，且其接种了根霉菌和红曲霉菌进行了复合发酵，使原料中的有毒有害物质得到了降解，然而其未使用酶解技术，降低了发酵的效率，增加了发酵时间。与其相比本发明不光采用了两步接种发酵技术，还利用复合酶解与乳酸菌发酵共同进行的方式，进一步提高了乳酸菌的发酵效率，使得能快速制得品质较高的蛹虫草酵素产品。
卢利群等公开了“一种麦苗酵素的快速发酵制备方法”（中国专利申请号201510653100.9），其通过人工接种酵母菌的方式对经过辅料调配和灭菌的大麦苗匀浆进行发酵，仅用12h就完成了整个发酵过程，并得到了高酶活、抗氧化性较强的麦苗酵素，然而其仅用酵母菌发酵，虽简化了工艺，但所得产品的口感欠佳。与其相比，本发明采用了先乳酸菌后酵母菌发酵的方式，利用简单的原料和简捷的工艺不但快速完成了整个发酵过程，还有效保留了营养成分，得到了口感好、营养佳的酵素产品。
王勇等公开了“一种可以提高机体免疫力的蛹虫草饮料及其制备方法”（中国专利申请号：201610097996.1），其所用原料包括鲜观音柴叶、蛹虫草、月季花瓣粉、月季花粉、红参、枸杞子、酵母菌、蛋白酶等。该专利中虽也利用了酶解和接种发酵技术，但由于所用原料繁多，其发酵周期依旧较长，需要在28~30℃下发酵22~24d。与其相比，本发明采用简单的原料，并改进了工艺，先同时进行复合酶解和乳酸菌发酵，再进行酵母菌发酵，2~3d就完成了整个发酵过程，并得到风味独特，营养丰富的产品。
江晓路等公开了“一种基于酶酵耦合生产蛹虫草酵素的方法”（中国专利申请号201711263212.9），其以蛹虫草菌丝体或菌粉为原料，采用先联合酶解2h，再接种植物乳杆菌耦合发酵12~48h的方式，获得了液体蛹虫草酵素产品。徐国华等公开了“一种蛹虫草果蔬酵素的制备方法”（中国专利申请号201810085470.0），其将蛹虫草粉溶于水，加入1~2%几丁质酶酶解后浓缩，然后将得到的蛹虫草浓缩液与加有麦芽糖醇和木糖醇的果蔬汁按1：3~5的比例混合，并接种3.5~7%的青春双歧杆菌、干酪乳杆菌、嗜酸乳杆菌、嗜热链球菌、鼠李糖乳杆菌、植物乳杆菌和保加利亚乳杆菌这七种乳酸菌进行发酵，发酵4~6天即得产品。张芙蓉等公开了“一种发酵蛹虫草益生菌饮料及其制备方法”（中国专利申请号201810069589.9），其以蛹虫草菌粉为主要原料，并加入灵芝菌粉、富硒酵母粉，大豆蛋白粉、菊花提取物和枸杞提取物混合得到原液，然后利用α-淀粉酶、糖化酶、纤维素酶、果胶酶和蛋白酶将原液酶解，得到预处理的原液之后，接种4~6%的青春双歧杆菌、副干酪乳杆菌、鼠李糖乳杆菌和嗜酸乳杆菌四种益生菌，并加入5~20%的木糖醇在37℃下进行恒温发酵40h即得产品。上述方法均采用了先酶解原液后发酵的工艺，而本发明中的第一步发酵采用酶解与发酵同时进行的方式，能在保持产品品质的基础上最大程度节省加工时间。此外本发明虽然所用原料简单，但先接种乳酸菌后接种酵母菌进行了两步发酵，使酵素产品多了一份醇香，风味相比一步发酵所制得的酵素更为突出，同时也省去了制备其它中草药或果蔬处理液的工艺，节省了成本。
 技术问题
本发明的目的是提供一种结合复合酶解的两步式蛹虫草酵素的发酵制备方法，利用复合酶解协同乳酸菌发酵、酵母菌发酵等工艺，可以快速完成整个发酵过程，大大缩短发酵周期。该法所采用的原料和工艺都十分简单，仅以蛹虫草粉为原料，不添加额外的果蔬或中草药，又将红糖作为发酵过程中的碳源，成本较低，所得产品酸甜可口、风味独特、悬浮稳定性好，并能很好地保留虫草素这一重要功能成分，具有一定的推广价值。
 技术解决方案
（1）蛹虫草粉和蛹虫草浆的制备：将蛹虫草干样去杂后，利用电机转速为34000r/min的高速粉碎机粉碎，并过100目筛，得蛹虫草粉；蛹虫草粉与水按照1:10~15的质量比例互溶后，用转速为3000~3600r/min的高速匀浆机间歇搅拌1~2min混匀，每次持续时间不超过10s，得到蛹虫草浆；
（2）蛹虫草发酵基质的制备：以蛹虫草发酵基质的总质量计，其中30~40%为步骤（1）所得的蛹虫草浆，10~20%为作为发酵碳源的红糖，其余为蒸馏水；对配制好的蛹虫草发酵基质进行巴氏杀菌处理；
（3）复合酶解协同乳酸菌发酵：将杀完菌的蛹虫草发酵基质冷却到40~45℃，用柠檬酸溶液调pH至4.80~5.00，以蛹虫草发酵基质的总质量计，加入0.4~0.6%的纤维素酶和0.4~0.6%的果胶酶，并在发酵基质上接种0.1~0.2%的乳酸菌，使复合酶解和乳酸菌同时在40~45℃下酶解并发酵36~48h，然后在85~90℃的水浴锅中灭酶5~10min，获得发酵液；
（4）果酒酵母菌发酵：将步骤（3）所得的发酵液降温至28~32℃，然后接种0.10~0.15%（w/w）的果酒酵母菌，在26~30℃下恒温发酵6~18h；
（5）离心：取经过乳酸菌、果酒酵母菌两步发酵的蛹虫草发酵液进行离心4200r/min，10min，离心后过滤得澄清液；
（6）调配：在步骤（5）离心所得的澄清液中加入稳定剂；
（7）杀菌灌装：调配完的发酵液进行95~100℃，5~10min的杀菌；冷却到85~90℃，趁热灌入已预先灭菌的玻璃瓶内，即得到蛹虫草酵素产品。
所述步骤（2）中的巴氏消毒温度为80~90℃，时间为10~20min。
所述步骤（3）中，柠檬酸溶液浓度为10%（w/w）；所述纤维素酶的酶活为5万U/g，果胶酶的酶活为10万U/g，适宜的酶解温度为40~50℃。
所述步骤（3）中，所述乳酸菌的接种过程在超净台中完成；所述乳酸菌为益生菌发酵菌粉，其中含有植物乳杆菌、嗜酸乳杆菌、乳双歧杆菌、干酪乳杆菌和长双歧杆菌，菌种量达80亿CFU/g，适宜生长温度为37~45℃。
所述步骤（4）中，果酒酵母菌的接种过程在超净台中完成；所述果酒酵母菌为“葡萄酒果酒专用酵母粉”，使用前需先用35~40℃温水进行活化，再将其接种至温度为26~30℃的发酵液中。
所述的稳定剂为黄原胶和羧甲基纤维素钠CMC-Na的复配稳定剂，其中稳定剂的总添加量为0.10~0.20%（w/w），复配比为黄原胶：CMC-Na=2:1。
 有益效果
1、与自然发酵相比，本方法采用接种发酵，大大提高了发酵效率，缩短了发酵时间，并得到了风味独特，营养丰富的产品；
2、与先酶解后发酵相比，本方法的酶解与第一步发酵同时进行，在保证酶解效果和发酵品质的基础上，最大限度地简化了工艺，缩短了加工时间；
3、与一步发酵相比，本方法采用先接种乳酸菌，后接种酵母菌的两步发酵法，丰富了产品的风味，使蛹虫草酵素无需添加其它果蔬原料也能具有优良的口感；
4、综合来看，采用本发酵工艺，操作简单易行，发酵周期短，生产成本低，适于大规模的推广生产。
 本发明的实施方式
下面将结合具体实施例对本发明的技术方案进行进一步的说明。
实施例1：一种结合低比例纤维素酶/果胶酶复合酶解的蛹虫草酵素的快速发酵方法
蛹虫草干样去杂后，利用高速粉碎机粉碎，并过100目筛，得蛹虫草粉；将蛹虫草粉与水1：10互溶，并用高速匀浆机搅拌均匀，得蛹虫草浆；取300g蛹虫草浆液，加入150g红糖，并加550g蒸馏水定量至1kg，充分搅拌，使红糖溶解完全，再经过85℃，10min的巴氏杀菌处理后，即得蛹虫草的发酵基质；等发酵基质冷却至40~45℃，调pH至4.80±0.10，加入0.4%的纤维素酶和0.6%的果胶酶，并接种1g复合乳酸菌粉，使复合酶解与乳酸菌发酵同时在40℃下进行48h，然后在90℃的水浴锅中灭酶5min；待乳酸菌发酵液冷却至30℃，加入1g用10倍温水活化后的果酒酵母，在28℃条件下发酵12h；发酵液进行离心过滤后，加入总量为1g的黄原胶和CMC-Na，两者复配比为黄原胶：CMC-Na=2:1；最后，将调配好的发酵液在100℃下杀菌10min后冷却至85℃进行热灌装，即得蛹虫草酵素。
通过感官、电子鼻检测对产品的风味进行分析。所得产品红糖味浓郁，为清亮透明的红棕色，酸甜适口，并有一股淡淡的醇香味；利用电子鼻对产品发酵过程中风味成分的变化进行了分析，发现与仅接种乳酸菌的发酵液相比，经过乳酸菌和酵母菌两步发酵的发酵液，S1传感器（芳香族化合物类）响应值上升了约131%，S5传感器（萜类、酯类）响应值上升了约114%，可见利用两步发酵能使酵素风味更为突出。
对产品的理化指标和功能性成分进行分析：所得终产品pH为3.60±0.02，可溶性固形物约为15.6%，酒精含量＜0.5g/100g；虫草素含量为45.940±1.538g/mL，其保留率约为77.68%，说明该发酵工艺对虫草素的影响较小，对产品中的虫草素有较好的保留作用；所得产品的羟自由基清除率为79.27±0.94%，并利用了铁氰化钾法测定酵素还原力，结果显示产品在700nm的吸光值为0.7396±0.0083，说明所得产品还原力较强，具有一定的抗氧化性。
实施例2：一种结合等比例纤维素酶/果胶酶复合酶解的蛹虫草酵素的快速发酵方法
蛹虫草干样去杂后，利用高速粉碎机粉碎，并过100目筛，得蛹虫草粉；将蛹虫草粉与水1：10互溶，并用高速匀浆机搅拌均匀，得蛹虫草浆；取300g蛹虫草浆液，加入150g红糖，并加550g蒸馏水定量至1kg，充分搅拌，使红糖溶解完全，再经过85℃，10min的巴氏杀菌处理后，即得蛹虫草的发酵基质；等发酵基质冷却至40~45℃，调pH至4.80±0.10，加入0.5%的纤维素酶和0.5%的果胶酶，并接种1g复合乳酸菌粉，使复合酶解与乳酸菌发酵同时在40℃下进行48h，然后在90℃的水浴锅中灭酶5min；待乳酸菌发酵液冷却至30℃，加入1g用10倍温水活化后的果酒酵母，在28℃条件下发酵12h；发酵液进行离心过滤后，加入总量为1g的黄原胶和CMC-Na（两者复配比为黄原胶：CMC-Na=2:1）；最后，将调配好的发酵液在100℃下杀菌10min后冷却至85℃进行热灌装，即得蛹虫草酵素。
通过感官、电子鼻检测对产品的风味进行分析。所得产品红糖味浓郁，为清亮透明的红棕色，口感酸甜适中，并有一股淡淡的醇香味；利用电子鼻对产品发酵过程中风味成分的变化进行了分析，发现与仅接种乳酸菌的发酵液相比，经过乳酸菌和酵母菌两步发酵的发酵液，S1传感器（芳香族化合物类）响应值上升了约136%，S5传感器（萜类、酯类）响应值上升了约128%，可见利用两步发酵能使酵素风味更为突出。
对产品的理化指标和功能性成分进行分析：所得终产品pH为3.54±0.02，可溶性固形物约为14.5%，酒精含量＜0.5g/100g；终产品的虫草素含量为58.808±0.116g/mL，其保留率约为97.87%，说明该发酵工艺对虫草素的影响很小，对产品中的虫草素有很好的保留作用；所得产品的羟自由基清除率为71.46±0.22%，利用铁氰化钾法测定酵素还原力，结果显示产品在700nm的吸光值为0.8478±0.0056，说明所得产品还原力较强，具有一定的抗氧化性。
实施例3：一种结合高比例纤维素酶/果胶酶复合酶解的蛹虫草酵素的快速发酵方法
蛹虫草干样去杂后，利用高速粉碎机粉碎，并过100目筛，得蛹虫草粉；将蛹虫草粉与水1：10互溶，并用高速匀浆机搅拌均匀，得蛹虫草浆；取300g蛹虫草浆液，加入150g红糖，并加550g蒸馏水定量至1kg，充分搅拌，使红糖溶解完全，再经过85℃，10min的巴氏杀菌处理后，即得蛹虫草的发酵基质；等发酵基质冷却至40~45℃，调pH至4.80±0.10，加入0.6%的纤维素酶和0.4%的果胶酶，并接种1g复合乳酸菌粉，使复合酶解与乳酸菌发酵同时在40℃下进行48h，然后在90℃的水浴锅中灭酶5min；待乳酸菌发酵液冷却至30℃，加入1g用10倍温水活化后的果酒酵母，在28℃条件下发酵12h；发酵液进行离心过滤后，加入总量为1g的黄原胶和CMC-Na（两者复配比为黄原胶：CMC-Na=2:1）；最后，将调配好的发酵液在100℃下杀菌10min后冷却至85℃进行热灌装，即得蛹虫草酵素。
通过感官、电子鼻检测对产品的风味进行分析。所得产品红糖味浓郁，为清亮透明的红棕色，酸甜适口，并有一股淡淡的醇香味；利用电子鼻对产品发酵过程中风味成分的变化进行了分析，发现与仅接种乳酸菌的发酵液相比，经过乳酸菌和酵母菌两步发酵的发酵液，S1传感器（芳香族化合物类）响应值上升了约121%，S5传感器（萜类、酯类）响应值上升了约132%，可见利用两步发酵能使酵素风味更为突出。
对产品的理化指标和功能性成分进行分析：所得终产品pH为3.53±0.01，可溶性固形物约为14.9%，酒精含量＜0.5g/100g；终产品的虫草素含量为47.289±1.953g/mL，其保留率为79.95%，说明该发酵工艺对虫草素的影响较小，对产品中的虫草素有较好的保留作用；所得产品的羟自由基清除率为70.31±0.61%，并利用了铁氰化钾法测定酵素还原力，结果显示产品在700nm的吸光值为0.8072±0.0027，说明了所得产品还原力较强，具有一定的抗氧化性。
为了更好地说明酶解与一次发酵同时进行的效果，增添以下对比例。
对比例1：结合等比例的纤维素酶/果胶酶进行酶解协同一次发酵与先酶解后发酵的效果对比
结合等比例的纤维素酶/果胶酶进行酶解协同一次发酵的方法参见实施例2。相对应的，采用先酶解后发酵的方式进行蛹虫草酵素制备的具体方式如下：蛹虫草干样去杂后，利用高速粉碎机粉碎，并过100目筛，得蛹虫草粉；将蛹虫草粉与水1:10互溶，并用高速匀浆机搅拌均匀，得蛹虫草浆；所得蛹虫草浆调pH至4.80±0.10，加入0.5%的纤维素酶和0.5%的果胶酶，在50℃的水浴锅中酶解4h，完成酶解后在温度为90℃的水浴锅中灭酶5min；取350g酶解后的蛹虫草浆液，加入150g红糖，并加500g蒸馏水定量至1kg，充分搅拌，使红糖溶解完全，再经过85℃，10min的巴氏杀菌处理后，即得蛹虫草的发酵基质；待发酵基质冷却至45℃左右，接种1g复合乳酸菌粉，在40℃条件下发酵48h，然后加入1g用10倍温水活化后的果酒酵母，在28℃条件下发酵12h，即完成发酵；发酵液进行离心过滤后，加入总量为1.5g的黄原胶和CMC-Na，两者复配比为黄原胶：CMC-Na=2:1；最后，将调配好的发酵液在100℃下杀菌5min后冷却至85℃进行热灌装，即得先酶解后发酵的蛹虫草酵素。工艺方面，酶解协同一次发酵采用了低温长时酶解的方式，即在40℃下酶解48h，有别于先酶解后发酵中的高温短时酶解，即在50℃下酶解4h。
采用酶解协同一次发酵所得酵素的风味更为丰富，电子鼻有更高的响应值。与先酶解后发酵相比，具有代表性的两个传感器S1（代表芳香族化合物类）响应值上升了约36%，S5（代表萜类、酯类）响应值上升了约33%。
两者终产品的pH和可溶性固形物相差不大，酒精含量也同样小于0.5g/100g。而先酶解后发酵的酵素产品中，虫草素含量为56.159±0.014mg/L，相比酶解协同一次发酵产品中的虫草素减少了约18%；其羟自由基清除率为59.58±0.69%，相比酶解协同一次发酵产品的清除率降低了约17%；利用铁氰化钾法测定酵素还原力，结果显示其在700nm的吸光值为0.6177±0.0086，相比酶解协同一次发酵产品的还原力降低了约18%。
从以上结果可以看出，同时进行酶解和一步发酵，不但节省了前期酶解预处理的时间，还能增进酵素产品的风味，增加酵素中的虫草素含量，提高其抗氧化性。

Claims (10)

  1. 一种结合复合酶解的两步式蛹虫草酵素的发酵制备方法，其特征在于，步骤如下：
    （1）蛹虫草粉和蛹虫草浆的制备：将蛹虫草干样去杂后，利用电机转速为34000r/min的高速粉碎机粉碎，并过100目筛，得蛹虫草粉；蛹虫草粉与水按照1:10~15的质量比例互溶后，用转速为3000~3600r/min的高速匀浆机间歇搅拌1~2min混匀，每次持续时间不超过10s，得到蛹虫草浆；
    （2）蛹虫草发酵基质的制备：以蛹虫草发酵基质的总质量计，其中30~40%为步骤（1）所得的蛹虫草浆，10~20%为作为发酵碳源的红糖，其余为蒸馏水；对配制好的蛹虫草发酵基质进行巴氏杀菌处理；
    （3）复合酶解协同乳酸菌发酵：将杀完菌的蛹虫草发酵基质冷却到40~45℃，用柠檬酸溶液调pH至4.80~5.00，以蛹虫草发酵基质的总质量计，加入0.4~0.6%的纤维素酶和0.4~0.6%的果胶酶，并在发酵基质上接种0.1~0.2%的乳酸菌，使复合酶解和乳酸菌同时在40~45℃下酶解并发酵36~48h，然后在85~90℃的水浴锅中灭酶5~10min，获得发酵液；
    （4）果酒酵母菌发酵：将步骤（3）所得的发酵液降温至28~32℃，然后接种0.10~0.15%（w/w）的果酒酵母菌，在26~30℃下恒温发酵6~18h；
    （5）离心：取经过乳酸菌、果酒酵母菌两步发酵的蛹虫草发酵液进行离心4200r/min，10min，离心后过滤得澄清液；
    （6）调配：在步骤（5）离心所得的澄清液中加入稳定剂；
    （7）杀菌灌装：调配完的发酵液进行95~100℃，5~10min的杀菌；冷却到85~90℃，趁热灌入已预先灭菌的玻璃瓶内，即得到蛹虫草酵素产品。
  2. 根据权利要求1所述的一种结合复合酶解的两步式蛹虫草酵素的发酵制备方法，其特征在于，所述步骤（2）中的巴氏消毒温度为80~90℃，时间为10~20min。
  3. 根据权利要求1或2所述的一种结合复合酶解的两步式蛹虫草酵素的发酵制备方法，其特征在于，所述步骤（3）中，柠檬酸溶液浓度为10%（w/w）；所述纤维素酶的酶活为5万U/g，果胶酶的酶活为10万U/g，适宜的酶解温度为40~50℃。
  4. 根据权利要求1或2所述的一种结合复合酶解的两步式蛹虫草酵素的发酵制备方法，其特征在于，所述步骤（3）中，所述乳酸菌的接种过程在超净台中完成；所述乳酸菌为益生菌发酵菌粉，其中含有植物乳杆菌、嗜酸乳杆菌、乳双歧杆菌、干酪乳杆菌和长双歧杆菌，菌种量达80亿CFU/g，适宜生长温度为37~45℃。
  5. 根据权利要求3所述的一种结合复合酶解的两步式蛹虫草酵素的发酵制备方法，其特征在于，所述步骤（3）中，所述乳酸菌的接种过程在超净台中完成；所述乳酸菌为益生菌发酵菌粉，其中含有植物乳杆菌、嗜酸乳杆菌、乳双歧杆菌、干酪乳杆菌和长双歧杆菌，菌种量达80亿CFU/g，适宜生长温度为37~45℃。
  6. 根据权利要求1、2或5所述的一种结合复合酶解的两步式蛹虫草酵素的发酵制备方法，其特征在于，所述步骤（4）中，果酒酵母菌的接种过程在超净台中完成；所述果酒酵母菌为“葡萄酒果酒专用酵母粉”，使用前需先用35~40℃温水进行活化，再将其接种至温度为26~30℃的发酵液中。
  7. 根据权利要求3所述的一种结合复合酶解的两步式蛹虫草酵素的发酵制备方法，其特征在于，所述步骤（4）中，果酒酵母菌的接种过程在超净台中完成；所述果酒酵母菌为“葡萄酒果酒专用酵母粉”，使用前需先用35~40℃温水进行活化，再将其接种至温度为26~30℃的发酵液中。
  8. 根据权利要求4所述的一种结合复合酶解的两步式蛹虫草酵素的发酵制备方法，其特征在于，所述步骤（4）中，果酒酵母菌的接种过程在超净台中完成；所述果酒酵母菌为“葡萄酒果酒专用酵母粉”，使用前需先用35~40℃温水进行活化，再将其接种至温度为26~30℃的发酵液中。
  9. 根据权利要求1、2、5、7或8所述的一种结合复合酶解的两步式蛹虫草酵素的发酵制备方法，其特征在于，所述的稳定剂为黄原胶和羧甲基纤维素钠CMC-Na的复配稳定剂，其中稳定剂的总添加量为0.10~0.20%（w/w），复配比为黄原胶：CMC-Na=2:1。
  10. 根据权利要求6所述的一种结合复合酶解的两步式蛹虫草酵素的发酵制备方法，其特征在于，所述的稳定剂为黄原胶和羧甲基纤维素钠CMC-Na的复配稳定剂，其中稳定剂的总添加量为0.10~0.20%（w/w），复配比为黄原胶：CMC-Na=2:1。
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