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WO2016191936A1 - 含有麦角硫因的制品及其制备方法和蕈菌胞外发酵液的用途 - Google Patents

含有麦角硫因的制品及其制备方法和蕈菌胞外发酵液的用途 Download PDF

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Publication number
WO2016191936A1
WO2016191936A1 PCT/CN2015/080269 CN2015080269W WO2016191936A1 WO 2016191936 A1 WO2016191936 A1 WO 2016191936A1 CN 2015080269 W CN2015080269 W CN 2015080269W WO 2016191936 A1 WO2016191936 A1 WO 2016191936A1
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Prior art keywords
liquid
fermentation
fermentation broth
extracellular
article
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PCT/CN2015/080269
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English (en)
French (fr)
Inventor
姜文侠
杨萍
刘琦
张维亚
周涛
梅保良
周子振
李淼
张大龙
Original Assignee
中国科学院天津工业生物技术研究所
天津世纪药业有限公司
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Application filed by 中国科学院天津工业生物技术研究所, 天津世纪药业有限公司 filed Critical 中国科学院天津工业生物技术研究所
Priority to EP15893599.9A priority Critical patent/EP3305274A4/en
Priority to US15/577,571 priority patent/US20180163238A1/en
Priority to PCT/CN2015/080269 priority patent/WO2016191936A1/zh
Priority to CN201580080502.2A priority patent/CN107708443A/zh
Publication of WO2016191936A1 publication Critical patent/WO2016191936A1/zh
Priority to HK18110537.9A priority patent/HK1251122A1/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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • 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
    • A23L2/02—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof containing fruit or vegetable juices
    • 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
    • A23L2/52—Adding ingredients
    • 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
    • A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
    • 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
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/175—Amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
    • A61K8/44—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof
    • 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
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine

Definitions

  • the invention relates to a product containing ergothione and a preparation method thereof, and a use of the extracellular fermentation liquid of the fungus, in particular to a product containing ergothione and a preparation method thereof, and an extracellular fermentation liquid of the fungus Use in the article.
  • L-Ergothioneine is the only known 2-thio-imidazole-amino acid, which is a rare natural chiral amino acid. Maintain system redox balance, detoxification of foreign substances, anti-oxidation damage, prevent radiation damage and prevent cancer.
  • Human blood contains ergothione, which contains about 1-4 mg per 100 mL of blood, but the body itself cannot synthesize ergot thiol, which needs to be taken from food. Ergosine can be synthesized in fungi, and fungi are an important source of ergot sulfur in animal and plant.
  • the ergothione can be prepared by a chemical synthesis method.
  • ergot sulfur is a chiral compound, and chemical synthesis is difficult and costly.
  • the deep fermentation of the mycelium of edible fungi to prepare ergothione has high safety. Through metabolic regulation and deep fermentation control strategy, the content of ergot sulfur in the fermentation product can be greatly improved. This fermentation method is obviously superior.
  • Mycobial enhancement Of ergothioneine by submerged cultivation of edible mushroom mycelia and its application as an Antioxidative compound[J].Food Chemistry,2012,131:247-258.;Huynh NDBao,Yoichi Shinomiya,Hiroaki Ikeda,et al.Preventing discoloration and lipid oxidation in dark muscle of yellowtail by feeding an extract prepared from mushroom(Flammulina Velutipes) cultured medium [J]. Aquaculture, 2009, 295: 243-249.); Wi Young Lee uses a surfactant-containing 70% ethanol solution to extract ergothione from the mycelium of Ganoderma lucidum (Wi Young Lee, Eung-Jun Park, Jin Kwon Ahn.
  • CN 103181933 A and CN 103743825 A also disclose the leaching of ergothione from mycelium, which uses an aqueous phase leaching process, avoiding the danger that organic solvents may pose to the production process and products.
  • CN 103181933 A and CN 103743825 A also disclose the leaching of ergothione from mycelium, which uses an aqueous phase leaching process, avoiding the danger that organic solvents may pose to the production process and products.
  • the object of the present invention is to overcome the deficiencies of the prior art and to provide an article containing ergothione, a preparation method thereof and the use of a bacterium extracellular fermentation broth.
  • the shake flask extracellular fermentation broth contains a certain amount (7 mg/L or more) of ergothione, and by improving the fermentation conditions, the ergot sulfur can be further increased extracellularly.
  • the content in the fermentation broth for example, the content of ergothione in the extracellular fermentation broth fermented in a 75 L tank can be as high as 61.7 mg/L).
  • the present invention provides an article comprising ergothione, wherein the ergot sulfur is obtained by liquid fermentation of a bacterium, and removal of the mycelium in the fermentation broth.
  • the outer fermentation broth and/or the concentrate of the extracellular fermentation broth is provided, and the preparation includes food, cosmetics, and animal feed.
  • the present invention provides a method of preparing the article of the first aspect, the method comprising: subjecting the bacterium to liquid fermentation, removing the mycelium in the fermentation broth, and selectively removing the mycelium The obtained extracellular fermentation broth was concentrated.
  • the present invention provides the use of a concentrate of an extracellular fermentation broth and/or an extracellular fermentation broth obtained by liquid fermentation of sputum in the preparation of an article containing ergothione, the product comprising food, cosmetics and animal feed.
  • the invention utilizes an extracellular fermentation broth rich in ergothione to prepare a product containing ergothione, which is not only sufficient
  • the ergot sulfur in vitro is used, and the process is simpler without leaching.
  • the term "fermentation liquid” as used herein refers to a liquid fermentation product of sputum bacteria (containing mycelium), and "extracellular fermentation broth” means that no hyphae are contained in the fermentation broth.
  • the ergothiocyanate is obtained by liquid fermentation of the bacterium, and the extracellular fermentation broth obtained after removing the mycelium in the fermentation broth and/or The concentrate of the extracellular fermentation broth is provided, and the preparation includes food, cosmetics, and animal feed.
  • the content of ergothione in the extracellular fermentation broth is preferably 7-62 mg/L.
  • the bacillus may be a common bacterium of the genus Tricholomataceae, such as a bacterium of the genus Pleurotus.
  • the bacterium is Pleurotus ostreatus, such as the Pleurotus ostreatus with the preservation number CGMCC No. 6232 (this species has been commonly used on the Chinese Microbial Culture Collection Management Committee on June 15, 2012).
  • the Microbiology Center (referred to as CGMCC) is deposited in CN103734022A).
  • the liquid medium used for the liquid fermentation is not particularly limited, and may contain 10-95 g/L of glycerin, 10-80 g/L of casein, 1-6 g/L of KH 2 PO 4 , 0.2. -5 g/L of MgSO 4 .
  • the inventors of the present invention have found that the addition of a specific amino acid to a liquid medium can further increase the content of ergothione in the extracellular fermentation broth.
  • the liquid medium used for the liquid fermentation contains 10-95 g/L of glycerin, 10-80 g/L of casein, 1-6 g/L of KH 2 PO 4 , 0.2- 5 g/L of MgSO 4 , 3 to 45 mmol/L of methionine and 2 to 45 mmol/L of cysteine. More preferably, the liquid medium used for the liquid fermentation contains 65-95 g/L of glycerin, 40-80 g/L of casein, 2-4 g/L of KH 2 PO 4 and 0.5-2 g/L of MgSO 4 .
  • liquid fermentation using liquid medium containing 75g / L of glycerol, 50g / L casein peptone, 3g / L of KH 2 PO 4 and 1.5g / L of MgSO 4, 14mmol /L of methionine and 7.5 mmol/L of cysteine.
  • the conditions of the liquid fermentation include: the temperature is 19-31 ° C, and the time is The dissolved oxygen saturation is 10-50% for 7-15 days.
  • the conditions for the liquid fermentation include a temperature of 23-28 ° C, a time of 10-13 days, and a dissolved oxygen saturation of 15-40%.
  • Pleurotus ostreatus as a fermentation strain and selection of the above liquid medium and liquid fermentation conditions can increase the content of ergothione in the extracellular fermentation broth to a greater extent.
  • the content of ergot sulfur can be selected in a wide range, for example, the ergothione content is 0.0005 to 50% by weight, preferably 0.001 to 20% by weight based on the total weight of the product.
  • the method of removing the mycelium in the fermentation broth may be a conventional removal method such as solid-liquid separation (e.g., filtration, centrifugation, precipitation).
  • the extracellular fermentation broth may be a liquid phase obtained by solid-liquid separation of a liquid fermentation product.
  • the concentrate of the extracellular fermentation broth may be used to provide ergothione.
  • the concentrate for providing ergothione to the product may be viscous, may be a solution, may be a suspension, an emulsion, or may be in the form of a paste, or may be a powder or a solid of other shapes.
  • the product of the present invention may be various foods, cosmetics and animal feeds which require the addition of ergothione as an additive or an active ingredient (or an active ingredient).
  • the specific form of the article is not particularly limited and may be liquid, solid or paste-like.
  • the food may include a meal (a food that must be ingested in daily life, that is, a staple food), a functional food, a snack food, and the like.
  • a meal a food that must be ingested in daily life, that is, a staple food
  • a functional food a snack food, and the like.
  • the meal may include various cereal staple foods such as wheat, rice, potatoes, sweet potatoes, and the like, such as taro, steamed buns, dumplings, noodles, vermicelli, bread, cakes, and the like.
  • the functional food may be a functional beverage, a capsule, or a tablet.
  • the specific dosage form of the functional food may be a decoction, a pill, a powder, an ointment, an agent, a wine, a granule, an effervescent, an oral solution, a capsule, a tablet or an injection.
  • the functional food is preferably in the form of a functional beverage.
  • the snack food may include dried fruit, puffed food, candy, meat food, beverage, etc., such as potato chips, French fries, shrimp strips, snow cakes, preserved fruit, plum, peanuts, pine nuts, almonds, pistachios, fish fillets, Dried meat, spiced fried meat, juice, etc.
  • the cosmetic may be in various forms of cosmetics, for example, lotion, makeup remover, perfume, cologne, toilet water, lotion, cream, mask, base cream, powder cream, lipstick, toothpaste, tooth powder, bathing Liquid, shampoo Liquid, conditioner, soap, soap, etc.
  • the animal feed may be a variety of feeds and nutritional supplements that provide nutrients to various animals at various stages of growth.
  • the article being a functional beverage, which may also contain sweeteners, thickeners, preservatives, and acidity adjustments.
  • At least one of the agents eg, carboxymethyl cellulose, phospholipid, aspartame, xylitol, flavor, xanthan gum, sucrose, citric acid, etc. will not be repeated here.
  • the amount (amount of use) of the product of the present invention can be selected according to the use of different products and the needs of the user, and the product is exemplified as a functional food. Generally, the product of the present invention is based on the weight of ergothione.
  • the dosage can be from 20 ⁇ g/kg body weight/day to 3 mg/kg body weight/day.
  • a method of preparing the article of the first aspect comprises liquid fermentation of the bacterium, removal of the mycelium in the fermentation broth, and selective extracellular fermentation after removal of the mycelium
  • the solution was concentrated (to obtain a concentrate of the extracellular fermentation broth).
  • the ergot containing sulfur can be obtained by mixing the extracellular fermentation broth obtained by liquid fermentation of the sputum and removing the mycelium in the fermentation broth and/or the concentrate of the extracellular fermentation broth with other components required for preparing the preparation.
  • Various products are Among them, the specific selection or conditions of the fermentation of the bacterium and the liquid are as described above, and will not be described herein.
  • the method may further comprise sequentially performing activation and seed culture of the sputum bacteria before the liquid fermentation, and the activation is for cultivating the preserved species in a suitable medium to restore vitality; the seed culture is In order to obtain more pure and strong cultures, it is possible to obtain a mycelium with vigorous vitality and sufficient inoculation amount.
  • Activation and seed culture can be carried out by methods conventional in the art.
  • the activation can include inoculating the mycelium of the sputum into a PDA plate and incubating at 23-28 ° C for 5-9 days.
  • the seed culture may include inoculating the activated strain into a seed medium (liquid), and culturing at 23-28 ° C for 3-6 days. Both PDA plates and seed media are well known to those skilled in the art and will not be further described herein.
  • the method of liquid fermentation of sputum comprises the following steps:
  • the method of liquid fermentation of sputum bacteria comprises the following steps:
  • the liquid medium further contains tween (e.g., Tween 60 and/or Tween 80).
  • Tween e.g., Tween 60 and/or Tween 80
  • the amount of Tween added may be 0.5-50 g/L, for example, Tween 60: 2-50 g/L (preferably 5-40 g/L, more preferably 5 g/L), and Tween 80: 0.5-40 g/L (preferred) 5-40 g/L, more preferably 10 g/L).
  • Tween can significantly increase the content of ergothione in the extracellular fermentation broth.
  • the extracellular fermentation liquid may be selectively concentrated in an appropriate manner, and the concentration conditions may include a degree of vacuum of 0 to 0.1 MPa.
  • the method may further comprise sterilizing the extracellular fermentation broth (or a concentrate thereof) obtained after removing the mycelium.
  • the other ingredients of the article can then be combined with the sterilized extracellular fermentation broth (or concentrate thereof).
  • the present invention provides the use of a concentrate of an extracellular fermentation broth and/or an extracellular fermentation broth obtained by liquid fermentation of a bacterium, preferably Pleurotus ostreatus, in the preparation of an article containing ergothione.
  • the article includes food, cosmetics, and animal feed.
  • the concentrate of the extracellular fermentation broth and/or the extracellular fermentation broth obtained by liquid fermentation of the sputum may be an extracellular fermentation broth (or a concentrate thereof) as described above and the specific form of the preparation is as described above, no longer Narrate one by one.
  • Pleurotus ostreatus the preservation number is CGMCC No.6232.
  • ergot sulfur reference substance purity ⁇ 98%, Biomol International Inc.
  • PDA medium Becton, Dickinson and Company
  • reagents such as methanol are commercially available chromatographically pure; potassium dihydrogen phosphate, magnesium sulfate purchased from Chinese medicine Group Chemical Reagent Co., Ltd.; corn flour was purchased from Meihekou Xingda Rice Industry Co., Ltd.; soybean meal and bean cake powder were purchased from Beijing Zhongmian Ziguang Biotechnology Co., Ltd.; glycerin was purchased from Tianjin Sailboat Chemical Reagent Technology Co., Ltd.; Casein was purchased from Yanshi City Baijia Industry and Trade Co., Ltd.
  • Example 1 Shake flask fermentation experiment of ergothione
  • Seed medium corn flour 30g / L, soybean cake powder 15g / L, ⁇ -amylase 80U / L, potassium dihydrogen phosphate 3g / L, magnesium sulfate 1.5g / L, the rest is water, sterilized at 121 ° C for 20min, The liquid volume in the 500 mL flask was 150 mL.
  • Liquid medium glycerol 50g / L, casein ⁇ 35g / L, potassium dihydrogen phosphate 3g / L, magnesium sulfate 1.5g / L, the rest is water, sterilized at 121 ° C for 20min, the volume of liquid in a 500mL flask is 150mL.
  • the broth of the PDA slant strain was picked up and seeded, and cultured at 25 ° C, 150 rpm for 4 days to obtain a seed liquid.
  • the seed solution was added to the liquid medium at an inoculation amount of 5% by volume, and cultured at 25 ° C for 15 days at 150 rpm to obtain a mycelial fermentation broth.
  • the fermentation broth of ergothione was filtered with a cloth, and the filtrate was an extracellular fermentation liquid, and the content of ergothione in the extracellular fermentation broth was 7.14 mg/L.
  • the method for determining the content of ergothione in the extracellular fermentation broth is as follows:
  • the extracellular fermentation broth was placed in an ultrafiltration centrifuge tube with a molecular weight cut-off of 3 kDa, centrifuged at 12840 ⁇ g for 10 min, and the permeate was collected to obtain the sample to be tested for the ergothione content of the extracellular fermentation broth.
  • Preparation of reference solution accurately weigh 10mg of ergot sulfur reference substance, add pure water in 25mL volumetric flask A control stock solution having a concentration of 400 mg/L was prepared. Then accurately absorb the appropriate amount of stock solution, add pure water to make a solution with concentrations of 40mg / L, 80mg / L, 120mg / L, 160mg / L and 200mg / L, filtered through a 0.22 ⁇ m microporous membrane, that is, the reference Solution.
  • the column was Agilent Eclipse XDB-C18 (4.6 ⁇ 250 mm, 5 ⁇ m), two columns were used in series; the mobile phase was 1% methanol solution, and the pH of the mobile phase was adjusted to 5.0 using acetic acid-sodium acetate buffer; Wavelength 257 nm; flow rate 0.7 mL/min; column temperature 25 ° C; injection volume 5 ⁇ L.
  • Example 2 Effect of Tween 60 on the content of ergothione in extracellular fermentation broth
  • the shake flask fermentation was carried out in accordance with the method of Example 1, except that 5 g/L, 10 g/L, 20 g/L, and 40 g/L of Tween 60 were additionally added to the liquid medium, respectively, while no Tween 60 was added.
  • the liquid medium was used as a control group.
  • the fermentation of the mycelium of Pleurotus ostreatus was carried out, and the fermentation was completed.
  • the content of ergothione in the extracellular fermentation broth was determined. It was found that the addition of 5g/L, 10g/L, 20g/L and 40g/L of Tween 60 can be improved.
  • Example 3 Effect of Tween 80 on the content of ergothione in extracellular fermentation broth
  • the shake flask fermentation was carried out in accordance with the method of Example 1, except that 5 g/L, 10 g/L, 20 g/L, and 40 g/L of Tween 80 were additionally added to the liquid medium, respectively, while no Tween 80 was added.
  • the liquid medium was used as a control group.
  • the fermentation of the mycelium of Pleurotus ostreatus was carried out, and the fermentation was completed.
  • the content of ergothione in the extracellular fermentation broth was determined. It was found that the addition of 5g/L, 10g/L, 20g/L and 40g/L of Tween 80 could be improved.
  • the content of extracellular ergothione, when 10g/L was added, the highest content of ergothione in the extracellular fermentation broth was 18.72mg/L, which was 114.4% higher than that of the control group (8.73mg/L).
  • Example 4 75L tank fermentation experiment of ergothione
  • Shake flask seed culture medium corn flour 30g / L, bean cake powder 15g / L, ⁇ -amylase 80U / L, potassium dihydrogen phosphate 3g / L, magnesium sulfate 1.5g / L, the rest is water, 1L in a triangular flask The liquid volume was 300 mL and sterilized at 121 ° C for 20 min.
  • Liquid medium of 75L fermenter glycerol 75g / L, casein ⁇ 50g / L, potassium dihydrogen phosphate 3g / L, magnesium sulfate 1.5g / L, methionine 14mmol / L, cysteine 7.5mmol / L, the rest For water, the amount of liquid in the tank was 45.6 L, and sterilization was carried out at 121 ° C for 20 min.
  • the broth of the PDA slant strain was picked up into the seed culture medium, and cultured at 25 ° C, 150 rpm shaker for 4.5 days to obtain a seed liquid.
  • the seed liquid was connected to the fermenter at an inoculation amount of 6.6% by volume, the tank pressure was 0.1 MPa, the culture temperature was 25 ° C, and the dissolved oxygen saturation during the fermentation was controlled to be 30%. After 12.5 days of fermentation, the content of ergothione in the extracellular fermentation broth was 61.7 mg/L.
  • the liquid fermentation was carried out in accordance with the method of Example 4, except that the liquid medium was: glycerin 10 g/L, casein ⁇ 45 g/L, potassium dihydrogen phosphate 2 g/L, magnesium sulfate 0.2 g/L, methionine 25 mmol/L, Cysteine 10mmol / L, the rest is water, the liquid volume is 48L / 75L fermenter, sterilization at 121 ° C for 20min.
  • the liquid medium was: glycerin 10 g/L, casein ⁇ 45 g/L, potassium dihydrogen phosphate 2 g/L, magnesium sulfate 0.2 g/L, methionine 25 mmol/L, Cysteine 10mmol / L, the rest is water, the liquid volume is 48L / 75L fermenter, sterilization at 121 ° C for 20min.
  • the content of ergothione in the obtained extracellular fermentation broth was 20.4 mg/L.
  • the liquid fermentation was carried out in accordance with the method of Example 4, except that the liquid medium was: glycerin 95 g/L, casein ⁇ 80 g/L, potassium dihydrogen phosphate 4.5 g/L, magnesium sulfate 5 g/L, methionine 3 mmol/L, Cysteine 45mmol / L, the rest is water, the liquid volume is 48L / 75L fermenter, sterilization at 121 ° C for 20min.
  • the liquid medium was: glycerin 95 g/L, casein ⁇ 80 g/L, potassium dihydrogen phosphate 4.5 g/L, magnesium sulfate 5 g/L, methionine 3 mmol/L, Cysteine 45mmol / L, the rest is water, the liquid volume is 48L / 75L fermenter, sterilization at 121 ° C for 20min.
  • the content of ergothione in the obtained extracellular fermentation broth was 22.1 mg/L.
  • the liquid fermentation was carried out in accordance with the method of Example 4, except that the liquid medium was: glycerin 50 g/L, casein ⁇ 35 g/L, potassium dihydrogen phosphate 2 g/L, magnesium sulfate 2.5 g/L, methionine 45 mmol/L, Cysteine 2mmol / L, the rest is water, the liquid volume is 48L / 75L fermenter, sterilization at 121 ° C for 20min.
  • the liquid medium was: glycerin 50 g/L, casein ⁇ 35 g/L, potassium dihydrogen phosphate 2 g/L, magnesium sulfate 2.5 g/L, methionine 45 mmol/L, Cysteine 2mmol / L, the rest is water, the liquid volume is 48L / 75L fermenter, sterilization at 121 ° C for 20min.
  • the content of ergothione in the obtained extracellular fermentation broth was 27.2 mg/L.
  • This example is intended to illustrate the preparation of the article (functional beverage) of the present invention.
  • This example is intended to illustrate the preparation of the article (functional beverage) of the present invention.
  • the material obtained in the step (3) is pasteurized at 85 ° C for 30 minutes and hot-filled.
  • This example is intended to illustrate the preparation of the article (functional beverage) of the present invention.
  • Example 4 The extracellular fermentation broth prepared in Example 4 was concentrated under a vacuum of 0.05 MPa for 0.5 hour to obtain a concentrated liquid having a ergothione content of 0.2 mg/mL.
  • step (3) The xanthan gum is fully mixed with sucrose and then dissolved in a small amount of water. After being thoroughly mixed by the colloid mill, the concentrate prepared in step (1), apple juice concentrate, citric acid and purified water are added according to the proportion of the formula. After mixing, add the essence, pass the colloid mill and homogenizer.
  • This example is intended to illustrate the preparation of the article (functional beverage) of the present invention.
  • Example 7 The extracellular fermentation broth prepared in Example 7 was sterilized at 121 ° C for 20 min, and then concentrated under a vacuum of 0.1 MPa for 1 hour to obtain a concentrated liquid.
  • the ergot content in the concentrated solution was 0.3 mg/mL. .
  • the method of the present invention does not require leaching, the process is simple, and the ergothione in the extracellular fermentation broth is fully utilized.

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Abstract

一种含有麦角硫因的制品，所述麦角硫因由蕈菌经液体发酵并将发酵液中的菌丝体去除后获得的胞外发酵液和/或该胞外发酵液的浓缩物提供，所述制品包括食品、化妆品和动物饲料。所述制品的制备方法包括将蕈菌进行液体发酵，除去发酵产物中的菌丝体，并选择性地对除去菌丝体后得到的胞外发酵液进行浓缩。蕈菌经液体发酵获得的胞外发酵液和/或胞外发酵液的浓缩物用于制备含有麦角硫因的制品。

Description

含有麦角硫因的制品及其制备方法和蕈菌胞外发酵液的用途 技术领域
本发明涉及一种含有麦角硫因的制品及其制备方法和蕈菌胞外发酵液的用途，具体地，涉及一种含有麦角硫因的制品及其制备方法以及蕈菌胞外发酵液在制备所述制品中的用途。
背景技术
麦角硫因(L-Ergothioneine，EGT)是至今唯一为人们所知的天然2-硫代咪唑氨基酸(2-thio-imidazole-amino acid)，是一种稀有的天然手性氨基酸，在人体内具有维持体系氧化还原平衡，外源物质解毒，抗氧化损伤，防止辐射性损伤及预防癌症等功效。人的血中含有麦角硫因，每100mL血液约含1-4mg，但是人体自身不能合成麦角硫因，需从食物中摄取。麦角硫因可以在真菌内合成，并且真菌是动植物体麦角硫因的重要来源。
可以使用化学合成法制备麦角硫因，然而，麦角硫因是手性化合物，化学合成的难度大，成本较高。而采用食用菌的菌丝体深层发酵制备麦角硫因，产品的安全性高，通过代谢调控和深层发酵控制策略，可以大幅度提高发酵产物中的麦角硫因含量，这种发酵手段明显优于从猪血、动物组织、食用菌子实体、麦角和谷物中提取麦角硫因的技术。
文献报导蕈菌合成的麦角硫因积累在菌丝体内(Wi Young Lee,Eung-Jun Park,Jin Kwon Ahn.Supplementation of Methionine Enhanced the Ergothioneine Accumulation in the Ganoderma neo-japonicum Mycelia[J].Appl Biochem Biotechnol,2009,158:213-221.；Pramvadee Tepwong,Anupam Giri,Fumito Sasaki,et al.Mycobial enhancement of ergothioneine by submerged cultivation of edible mushroom mycelia and its application as an antioxidative compound[J].Food Chemistry,2012,131:247-258.)，为了测定菌丝体内麦角硫因的含量，或从菌丝体内提取麦角硫因，要用萃取或浸提的方法，将菌丝体内的麦角硫萃取或浸提到细胞外。相关研究多使用有机溶剂萃取麦角硫因，如Huynh N.D.Bao和Pramvadee Tepwong分别使用70％乙醇浸提金针菇、香菇菌丝体内的麦角硫因(Pramvadee Tepwong,Anupam Giri,Fumito Sasaki,et al.Mycobial enhancement of ergothioneine by submerged cultivation of edible mushroom mycelia and its application as an  antioxidative compound[J].Food Chemistry,2012,131:247-258.；Huynh N.D.Bao,Yoichi Shinomiya,Hiroaki Ikeda,et al.Preventing discoloration and lipid oxidation in dark muscle of yellowtail by feeding an extract prepared from mushroom(Flammulina velutipes)cultured medium[J].Aquaculture,2009,295:243-249.)；Wi Young Lee利用含有表面活性剂的70％乙醇溶液浸提黑灵芝菌丝体中的麦角硫因(Wi Young Lee,Eung-Jun Park,Jin Kwon Ahn.Supplementation of Methionine Enhanced the Ergothioneine Accumulation in the Ganoderma neo-japonicum Mycelia[J].Appl Biochem Biotechnol,2009,158:213-221.)；周念波采用pH8.0的NaOH溶液及沸水浸提双孢菇柄中的麦角硫因(周念波,李轶群,殷勤红.氧化铝柱层析从双孢菇菇柄中提取麦角硫因[J].安徽农业科学,2010,38(27):14842-14843.)。CN 103181933A和CN 103743825A也公开了从菌丝体中浸提麦角硫因，其使用水相浸提的方法，避免了有机溶剂可能对生产过程和产品造成的危险。然而，并未有如何利用发酵液经固液分离后得到的液体部分的相关报道。
发明内容
本发明的目的是克服现有技术的不足，提供一种含有麦角硫因的制品及其制备方法与蕈菌胞外发酵液的用途。
基于现有文献的报道，本领域技术人员的常规认知是胞内的麦角硫因不会分泌至胞外，且即便分泌至胞外，含量也会极低(低至无法检测到，或低至没有利用价值)。而本发明的发明人在研究中发现，蕈菌的摇瓶胞外发酵液中含有一定量(7mg/L以上)的麦角硫因，而且通过改善发酵条件，可以进一步提高麦角硫因在胞外发酵液中的含量(例如，75L罐发酵的胞外发酵液中麦角硫因的含量可高达61.7mg/L)。因此，为了实现以上目的，第一方面，本发明提供了一种含有麦角硫因的制品，其中，所述麦角硫因由蕈菌经液体发酵并将发酵液中的菌丝体去除后获得的胞外发酵液和/或该胞外发酵液的浓缩物提供，所述制品包括食品、化妆品和动物饲料。
第二方面，本发明提供了一种制备第一方面所述的制品的方法，该方法包括将蕈菌进行液体发酵，除去发酵液中的菌丝体，并选择性地对除去菌丝体后得到的胞外发酵液进行浓缩。
第三方面，本发明提供了蕈菌经液体发酵获得的胞外发酵液和/或胞外发酵液的浓缩物在制备含有麦角硫因的制品中的用途，所述制品包括食品、化妆品和动物饲料。
本发明利用富含麦角硫因的胞外发酵液制备含有麦角硫因的制品，不仅可以充分 利用菌丝体外的麦角硫因，而且无需浸提，工艺更简单。
本发明的其它特征和优点将在随后的具体实施方式部分予以详细说明。
具体实施方式
以下对本发明的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本发明，并不用于限制本发明。
本发明中，在未做相反说明的情况下，使用的术语“发酵液”是指蕈菌的液体发酵产物(含有菌丝体)；“胞外发酵液”是指发酵液中不含菌丝体的部分，即由蕈菌经液体发酵并将发酵液中的菌丝体去除后获得的产物；“溶解氧饱和度”是表示溶氧含量的一种指标，其值越高，说明溶氧含量越大，溶解氧饱和度(％)＝(溶解氧实测含量/实测条件下溶解氧的饱和含量)×100％。
根据本发明的第一方面，本发明提供的含有麦角硫因的制品中，所述麦角硫因由蕈菌经液体发酵并将发酵液中的菌丝体去除后获得的胞外发酵液和/或该胞外发酵液的浓缩物提供，所述制品包括食品、化妆品和动物饲料。其中，所述胞外发酵液中麦角硫因的含量优选为7-62mg/L。
本发明中，所述蕈菌可以为白蘑科(Tricholomataceae)的常见蕈菌，如侧耳属(Pleurotus)的蕈菌。优选情况下，所述蕈菌为糙皮侧耳(Pleurotus ostreatus)，如保藏编号为CGMCC No.6232的糙皮侧耳(该菌种已于2012年6月15日在中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC)保藏，公开在CN103734022A中)。
本发明中，对所述液体发酵使用的液体培养基没有特别的限制，可以含有10-95g/L的甘油，10-80g/L的酪蛋白胨，1-6g/L的KH2PO4，0.2-5g/L的MgSO4。但是，本发明的发明人发现，如果往液体培养基中添加特定的氨基酸能够进一步提高胞外发酵液中麦角硫因的含量。因此，根据本发明的优选实施方式，所述液体发酵使用的液体培养基含有10-95g/L的甘油，10-80g/L的酪蛋白胨，1-6g/L的KH2PO4，0.2-5g/L的MgSO4，3-45mmol/L的蛋氨酸和2-45mmol/L的半胱氨酸。更优选地，所述液体发酵使用的液体培养基含有65-95g/L的甘油，40-80g/L的酪蛋白胨，2-4g/L的KH2PO4和0.5-2g/L的MgSO4，9-35mmol/L的蛋氨酸和5-20mmol/L的半胱氨酸。根据本发明最优选的实施方式，所述液体发酵使用的液体培养基含有75g/L的甘油，50g/L的酪蛋白胨，3g/L的KH2PO4和1.5g/L的MgSO4，14mmol/L的蛋氨酸和7.5mmol/L的半胱氨酸。
根据本发明的优选实施方式，所述液体发酵的条件包括：温度为19-31℃，时间为 7-15天，溶解氧饱和度为10-50％。根据本发明进一步优选的实施方式，所述液体发酵的条件包括：温度为23-28℃，时间为10-13天，溶解氧饱和度为15-40％。
采用糙皮侧耳作为发酵菌种并选择上述液体培养基和液体发酵的条件能够更大程度地提高胞外发酵液中麦角硫因的含量。
本发明的制品中，麦角硫因的含量可选范围较宽，例如，以制品的总重量为基准，所述麦角硫因的含量为0.0005-50重量％，优选为0.001-20重量％。
去除发酵液中的菌丝体的方法可以为常规的去除方法，例如，固液分离(如：过滤、离心、沉淀)。如前所述，所述胞外发酵液可以为液体发酵产物经固液分离获得的液相。
本发明中，为了提高制品中麦角硫因的含量，可以使用胞外发酵液的浓缩物提供麦角硫因。为制品提供麦角硫因的浓缩物可以呈粘稠状，也可以为溶液，也可以为悬浊液、乳浊液，也可以为膏状，还可以为粉末或其他形状的固体。
获得胞外发酵液涉及的活化、种子培养、液体发酵及液体培养基等的选择还可参见下文的记载，并不受限于此。
本发明的制品可以为各种需要添加麦角硫因作为添加剂或活性成分(或有效成分)的食品、化妆品和动物饲料。对所述制品的具体形态没有特别的限制，可以为液态的，也可以为固态的，还可以为膏状的。
本发明中，所述食品可以包括膳食(日常生活必须摄入的食物，即主食)、功能食品、休闲食品等。
所述膳食可以包括各种谷物类主食，如采用小麦、稻谷、土豆、红薯等制得得食品，如馒头、包子、饺子、面条、粉条、面包、糕点等。
所述功能食品可以为功能饮料，也可以为胶囊，还可以为片剂。事实上，对所述功能食品的具体剂型没有特别的要求，可以为汤剂、丸剂、散剂、膏剂、丹剂、酒剂、冲剂、泡腾剂、口服液剂、胶囊剂、片剂或注射剂等各种常见的口服制剂。考虑到工艺的简化，所述功能食品优选为功能饮料的形式。
所述休闲食品可以包括干果、膨化食品、糖果、肉制食品、饮料等，例如薯片、薯条、虾条、雪饼、果脯、话梅、花生、松子、杏仁、开心果、鱼片、肉干、五香炸肉、果汁等。
本发明中，所述化妆品可以为各种形式的化妆品，例如，化妆水、卸妆水、香水、古龙水、花露水、乳液、霜、面膜、打底霜、粉霜、口红、牙膏、牙粉、沐浴液、洗发 液、护发素、香皂、肥皂等。
本发明中，所述动物饲料可以为处于各种生长阶段的各种动物提供营养的各种饲料和营养补充剂。
根据上述制品的具体形式，本领域技术人员能够对制品中的其他成分进行选择，以所述制品为功能饮料为例，所述制品还可以含有甜味剂、增稠剂、防腐剂和酸度调节剂中的至少一种(如：羧甲基纤维素、磷脂、阿斯巴甜、木糖醇、香精、黄原胶、蔗糖和柠檬酸等)，在此不再一一赘述。
本发明的制品的使用量(服用量)可以根据不同制品的用途和使用者的需求进行选择，以所述制品为功能食品为例，通常地，以麦角硫因的重量计，本发明的制品的服用量可以为20μg/kg体重/天至3mg/kg体重/天。
根据本发明的第二方面，制备第一方面所述的制品的方法包括将蕈菌进行液体发酵，除去发酵液中的菌丝体，并选择性地对除去菌丝体后得到的胞外发酵液进行浓缩(获得胞外发酵液的浓缩物)。通过将蕈菌经液体发酵并将发酵液中的菌丝体去除后获得的胞外发酵液和/或该胞外发酵液的浓缩物与制备制品所需的其他成分混合即可获得含有麦角硫因的各种制品。其中，蕈菌和液体发酵的具体选择或条件如前所述，在此不再赘述。
本发明中，所述方法还可以包括在液体发酵前对蕈菌依次进行活化和种子培养，活化是为了将保藏状态的菌种放入适宜的培养基中培养，使其恢复活力；种子培养是为了得到较多的纯而壮的培养物，即获得活力旺盛、接种数量足够的菌丝体。可以采用本领域常规的方法进行活化和种子培养，例如，所述活化可以包括将蕈菌的菌丝体接种至PDA平板上，23-28℃下培养5-9天。所述种子培养可以包括将活化后的菌种接种至种子培养基(液体)中，23-28℃培养3-6天。PDA平板和种子培养基均为本领域技术人员所熟知，在此不再赘述。
在本发明的优选实施方式中，将蕈菌进行液体发酵的方法包括如下步骤：
(a)将糙皮侧耳的菌种接种至种子培养基，在23-28℃培养3-6天制备种子液，所述种子培养基含有25-40g/L的玉米粉、15-35g/L的豆饼粉、30-80U/L的α-淀粉酶、2-4.5g/L的磷酸二氢钾和0.2-3g/L的硫酸镁；和
(b)将种子液以4-20体积％的接种量接种至液体培养基，在19-31℃(优选23-28℃)培养7-15天(优选10-13天)，培养过程中控制溶解氧饱和度为10-50％(优选为15-40％)，得到糙皮侧耳菌丝体发酵液，所述液体培养基含有65-95g/L的甘油、40-80g/L的酪蛋 白胨、2-4g/L的磷酸二氢钾、0.5-2g/L的硫酸镁、9-35mmol/L的蛋氨酸和5-20mmol/L的半胱氨酸。
在本发明更优选的实施方式中，将蕈菌进行液体发酵的方法包括如下步骤：
(a)将糙皮侧耳的菌种接种至种子培养基，在25℃培养4天制备种子液，所述种子培养基含有30g/L的玉米粉、15g/L的豆饼粉、80U/L的α-淀粉酶、3g/L的磷酸二氢钾和1.5g/L硫酸镁；和
(b)将种子液以5体积％的接种量接种至液体培养基，在25℃培养3天然后将pH调节至6并在31℃下培养12天，培养过程中控制溶解氧饱和度为30％，得到糙皮侧耳菌丝体发酵液，所述液体培养基含有75g/L的甘油、50g/L的酪蛋白胨、3g/L的磷酸二氢钾、1.5g/L的硫酸镁、14mmol/L的蛋氨酸和7.5mmol/L的半胱氨酸。
根据本发明最优选的实施方式，所述液体培养基进一步含有吐温(tween，例如吐温60和/或吐温80)。吐温的添加量可以为0.5-50g/L，例如，吐温60：2-50g/L(优选5-40g/L，更优选5g/L)，吐温80：0.5-40g/L(优选5-40g/L，更优选10g/L)。吐温的添加能够显著地进一步提高胞外发酵液中麦角硫因的含量。
为了获得麦角硫因含量较高的制品，可以选择性地采用适当的方式对胞外发酵液进行浓缩，所述浓缩的条件可以包括：真空度为0-0.1MPa。
本发明中，所述方法还可以包括将除去菌丝体后得到的胞外发酵液(或其浓缩物)进行灭菌处理。然后，可以将制品的其他成分与灭菌后的胞外发酵液(或其浓缩物)混合。
根据本发明的第三方面，本发明提供了蕈菌(优选糙皮侧耳)经液体发酵获得的胞外发酵液和/或胞外发酵液的浓缩物在制备含有麦角硫因的制品中的用途，所述制品包括食品、化妆品和动物饲料。蕈菌经液体发酵获得的胞外发酵液和/或胞外发酵液的浓缩物可以为如上所述的胞外发酵液(或其浓缩物)且所述制品的具体形式如上所述，不再逐一叙述。
以下将通过实施例对本发明进行详细描述。以下实施例中，各种实验器材如下所示：
菌种：糙皮侧耳，保藏编号为CGMCC No.6232。
实验仪器和/或材料：摇床(IS-RDV3，美国精骐有限公司)；75L发酵罐(BIOSTAT D-DCU75，Sartorius)；高效液相色谱仪(Agilent 1260，Agilent Technologies)；水浴锅(TW20，Julabo)；磁力搅拌器(WHMIXdrive6，WIGGENS)及其控制器(MIXcontrol  20，WIGGENS)；电子分析天平(AB204-S，METTLER TOLEDO)；高速台式冷冻离心机(TGL-16M，湘仪离心机仪器有限公司)；真空干燥箱(VOS-60A，STIK)；活塞式真空泵(V610，ChemVak)；超声波清洗机(SB-5200D，宁波新芝生物科技股份有限公司)；0.22μm孔径微孔滤膜(天津博纳艾杰尔科技有限公司)；超滤离心管(0.5mL，3kDa，Millipore)。
主要试剂：麦角硫因对照品(纯度≥98％，Biomol International Inc.)；PDA培养基(Becton，Dickinson and Company)；甲醇等试剂为市售色谱纯；磷酸二氢钾、硫酸镁购自国药集团化学试剂有限公司；玉米粉购自梅河口市兴达米业有限责任公司；豆粕粉、豆饼粉购自北京中棉紫光生物科技有限公司；甘油购自天津市风船化学试剂科技有限公司；酪蛋白胨购自偃师市百家工贸有限公司。
实施例1：麦角硫因的摇瓶发酵实验
种子培养基：玉米粉30g/L、豆饼粉15g/L、α-淀粉酶80U/L、磷酸二氢钾3g/L、硫酸镁1.5g/L，其余为水，于121℃灭菌20min，500mL三角瓶中的装液量为150mL。
液体培养基：甘油50g/L、酪蛋白胨35g/L、磷酸二氢钾3g/L、硫酸镁1.5g/L，其余为水，于121℃灭菌20min，500mL三角瓶中的装液量为150mL。
挑取PDA斜面菌种的菌苔接入种子培养基，25℃、150rpm摇床培养4天，得到种子液。将种子液按5体积％的接种量接入液体培养基，25℃摇床150rpm培养15天，得菌丝体发酵液。
将麦角硫因的发酵液用布过滤，滤液即为胞外发酵液，胞外发酵液中麦角硫因的含量为7.14mg/L。
此外，往液体培养基中额外添加“14mmol/L的蛋氨酸和7.5mmol/L的半胱氨酸”进行摇瓶发酵，得到的胞外发酵液中麦角硫因的含量为9.28mg/L。可见添加氨基酸能够进一步提高胞外发酵液中麦角硫因的含量。
胞外发酵液中麦角硫因的含量测定方法如下：
(1)胞外发酵液中麦角硫因含量测定的前处理
取胞外发酵液置于截留分子量3kDa的超滤离心管中，12840×g离心10min，收集透过液，即得胞外发酵液麦角硫因含量检测的待测样。
(2)HPLC检测
对照品溶液的制备：精密称取麦角硫因对照品10mg，于25mL容量瓶中加纯水配 制成浓度为400mg/L的对照品贮备液。再精密吸取贮备液适量，加纯水制成浓度分别为40mg/L、80mg/L、120mg/L、160mg/L和200mg/L的溶液，经0.22μm微孔滤膜过滤，即得对照品溶液。
定性与定量检测：相同色谱条件下，对麦角硫因对照品溶液与待测样进行HPLC测定，将待测样的色谱图与麦角硫因对照品溶液的色谱图进行对比，根据保留时间确定待测样中的麦角硫因色谱峰。以麦角硫因对照品溶液的浓度与相应的峰面积绘制标准曲线，在对照品溶液与待测样进样量相同的情况下，用外标法定量，计算出待测样中麦角硫因的含量。定义信噪比(样品峰峰高与噪音峰峰高的比值)为3和10时的进样样品浓度为该检测方法的检测限和定量限，分别为10.4μg/L和34.7μg/L。
检测条件：色谱柱为Agilent Eclipse XDB-C18(4.6×250mm，5μm)，两根色谱柱串联使用；流动相为1％甲醇溶液，使用乙酸-乙酸钠缓冲液调节流动相的pH为5.0；检测波长257nm；流速0.7mL/min；柱温25℃；进样量5μL。
实施例2：吐温60对胞外发酵液中麦角硫因的含量的影响
按照实施例1的方法进行摇瓶发酵，不同之处在于分别向液体培养基中额外添加5g/L、10g/L、20g/L和40g/L的吐温60，同时以不添加吐温60的液体培养基作为对照组。对糙皮侧耳菌丝体进行发酵培养，发酵结束，测定胞外发酵液中麦角硫因的含量，发现添加5g/L、10g/L、20g/L和40g/L的吐温60均可提高胞外麦角硫因的含量，其中添加5g/L时，胞外发酵液中麦角硫因的含量最高，达16.24mg/L，与对照组(8.73mg/L)相比提高了86％。
实施例3：吐温80对胞外发酵液中麦角硫因的含量的影响
按照实施例1的方法进行摇瓶发酵，不同之处在于分别向液体培养基中额外添加5g/L、10g/L、20g/L和40g/L的吐温80，同时以不添加吐温80的液体培养基作为对照组。对糙皮侧耳菌丝体进行发酵培养，发酵结束，测定胞外发酵液中麦角硫因的含量，发现添加5g/L、10g/L、20g/L和40g/L的吐温80均可提高胞外麦角硫因的含量，其中添加10g/L时，胞外发酵液中麦角硫因的含量最高，达18.72mg/L，与对照组(8.73mg/L)相比提高了114.4％。
实施例4：麦角硫因的75L罐发酵实验
摇瓶种子培养基：玉米粉30g/L、豆饼粉15g/L、α-淀粉酶80U/L、磷酸二氢钾3g/L、硫酸镁1.5g/L，其余为水，1L三角瓶中的装液量为300mL，121℃灭菌20min。
容积75L发酵罐的液体培养基：甘油75g/L、酪蛋白胨50g/L、磷酸二氢钾3g/L、硫酸镁1.5g/L、蛋氨酸14mmol/L、半胱氨酸7.5mmol/L，其余为水，罐内装液量为45.6L，121℃灭菌20min。
挑取PDA斜面菌种的菌苔接入种子培养基，25℃、150rpm摇床培养4.5天，得到种子液。将种子液按6.6体积％的接种量接入发酵罐，罐压0.1MPa，培养温度25℃，控制发酵过程中溶解氧饱和度为30％。发酵12.5天，测得胞外发酵液中麦角硫因的含量为61.7mg/L。
实施例5
按照实施例4的方法进行液体发酵，不同的是，液体培养基为：甘油10g/L、酪蛋白胨45g/L、磷酸二氢钾2g/L、硫酸镁0.2g/L、蛋氨酸25mmol/L、半胱氨酸10mmol/L，其余为水，装液量为48L/75L发酵罐，121℃灭菌20min。
得到的胞外发酵液中麦角硫因的含量为20.4mg/L。
实施例6
按照实施例4的方法进行液体发酵，不同的是，液体培养基为：甘油95g/L、酪蛋白胨80g/L、磷酸二氢钾4.5g/L、硫酸镁5g/L、蛋氨酸3mmol/L、半胱氨酸45mmol/L，其余为水，装液量为48L/75L发酵罐，121℃灭菌20min。
得到的胞外发酵液中麦角硫因的含量为22.1mg/L。
实施例7
按照实施例4的方法进行液体发酵，不同的是，液体培养基为：甘油50g/L、酪蛋白胨35g/L、磷酸二氢钾2g/L、硫酸镁2.5g/L、蛋氨酸45mmol/L、半胱氨酸2mmol/L，其余为水，装液量为48L/75L发酵罐，121℃灭菌20min。
得到的胞外发酵液中麦角硫因的含量为27.2mg/L。
实施例8
本实施例用来说明本发明的制品(功能饮料)的制备。
(1)量取下列原料：实施例1制备的胞外发酵液800mL、羧甲基纤维素(稳定剂)0.2g、磷脂0.2g，阿斯巴甜(矫味剂)0.5g、木糖醇30g、香精3g，纯净水200mL。
(2)将羧甲基纤维素、磷脂、阿斯巴甜和木糖醇充分混匀后，先用少量的水溶解后再加入实施例1制备的胞外发酵液和剩余的水，过胶体磨两遍，使之混合均匀后加入香精。
(3)将步骤(2)获得的物料灌装，121℃灭菌20分钟。
实施例9
本实施例用来说明本发明的制品(功能饮料)的制备。
(1)量取下列原料：实施例2(添加5g/L吐温60)制备的胞外发酵液800mL、羧甲基纤维素(稳定剂)0.2g、磷脂0.2g，阿斯巴甜(矫味剂)0.5g、木糖醇30g、香精3g，纯净水200mL。
(3)将羧甲基纤维素、磷脂、阿斯巴甜和木糖醇充分混匀后，先用少量的水溶解后再加入实施例2制备的胞外发酵液和剩余的水，过胶体磨两遍，使之混合均匀后加入香精。
(4)将步骤(3)获得的物料经85℃巴氏杀菌30分钟，热灌装。
实施例10
本实施例用来说明本发明的制品(功能饮料)的制备。
(1)将实施例4制备的胞外发酵液，在0.05MPa真空条件下，浓缩0.5小时，制得浓缩液，浓缩液中麦角硫因含量为0.2mg/mL。
(2)分别称取下列原料：步骤(1)制备的浓缩液200mL、苹果浓缩汁(可溶性固形物含量65％)100mL，黄原胶(稳定剂)3g，蔗糖80g、柠檬酸1g、香精3g，加纯净水至1000mL。
(3)黄原胶与蔗糖充分混合均匀后加少量的水溶解，过胶体磨充分混合均匀后，再按配方比例加入步骤(1)制备的浓缩液、苹果浓缩汁，柠檬酸和纯净水，混合均匀后加入香精，过胶体磨和均质机。
(4)将步骤(3)获得的物料进行130℃高温瞬时灭菌10秒。
(5)将步骤(4)处理后的物料进行无菌灌装。
实施例11
本实施例用来说明本发明的制品(功能饮料)的制备。
(1)将实施例7制备的胞外发酵液经121℃灭菌20min后，再在0.1MPa真空条件下，浓缩1小时，制得浓缩液，浓缩液中麦角硫因含量为0.3mg/mL。
(2)分别称取下列原料：步骤(1)获得的浓缩液250mL、大豆分离蛋白(蛋白含量为90重量％)50g，黄原胶(稳定剂)5g，磷脂2g，蔗糖20g、阿斯巴甜0.25g、香精1g，加纯净水至1000mL。
(3)黄原胶、磷脂、大豆分离蛋白、蔗糖充分混合均匀后加少量的水溶解，过胶体磨充分混合均匀后，再按配方比例加入浓缩液、阿斯巴甜、纯净水，混合均匀后加入香精，过胶体磨和均质机。
(4)将步骤(4)获得的物料进行135℃高温瞬时灭菌6秒；
(5)将步骤(5)处理后的物料进行无菌灌装。
从以上实施例可以看出，本发明的方法无需浸提，工艺简单，充分利用了胞外发酵液中的麦角硫因。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。
此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容。

Claims (13)

  1. 一种含有麦角硫因的制品，其特征在于，所述麦角硫因由蕈菌经液体发酵并将发酵液中的菌丝体去除后获得的胞外发酵液和/或该胞外发酵液的浓缩物提供，所述制品包括食品、化妆品和动物饲料。
  2. 根据权利要求1所述的制品，其中，所述蕈菌为糙皮侧耳(Pleurotus ostreatus)。
  3. 根据权利要求1或2所述的制品，其中，所述液体发酵使用的液体培养基含有10-95g/L的甘油，10-80g/L的酪蛋白胨，1-6g/L的KH2PO4和0.2-5g/L的MgSO4，3-45mmol/L的蛋氨酸和2-45mmol/L的半胱氨酸。
  4. 根据权利要求3所述的制品，其中，所述液体发酵使用的液体培养基含有65-95g/L的甘油，40-80g/L的酪蛋白胨，2-4g/L的KH2PO4和0.5-2g/L的MgSO4，9-35mmol/L的蛋氨酸和5-20mmol/L的半胱氨酸。
  5. 根据权利要求3或4所述的制品，其中，所述液体发酵使用的液体培养基还含有0.5-50g/L的吐温。
  6. 根据权利要求1-5中任意一项所述的制品，其中，所述液体发酵的条件包括：温度为19-31℃，时间为7-15天，溶解氧饱和度为10-50％。
  7. 根据权利要求6所述的制品，其中，所述液体发酵的条件包括：温度为23-28℃，时间为10-13天，溶解氧饱和度为15-40％。
  8. 根据权利要求1-7中任意一项所述的制品，其中，以制品的总重量为基准，所述麦角硫因的含量为0.0005-50重量％，优选为0.001-20重量％。
  9. 一种制备权利要求1-8中任意一项所述的制品的方法，该方法包括将蕈菌进行液体发酵，除去发酵液中的菌丝体，并选择性地对除去菌丝体后得到的胞外发酵液进行浓缩。
  10. 根据权利要求9所述的方法，其中，将蕈菌进行液体发酵的方法包括如下步骤：
    (a)将糙皮侧耳的菌种接种至种子培养基，在23-28℃培养3-6天制备种子液，所述种子培养基含有25-40g/L的玉米粉、15-35g/L的豆饼粉、30-80U/L的α-淀粉酶、2-4.5g/L的磷酸二氢钾和0.2-3g/L的硫酸镁；和
    (b)将种子液以4-20体积％的接种量接种至液体培养基，在19-31℃培养7-15天，培养过程中控制溶解氧饱和度为10-50％，得到糙皮侧耳菌丝体发酵液，所述液体培养基含有65-95g/L的甘油、40-80g/L的酪蛋白胨、2-4g/L的磷酸二氢钾、0.5-2g/L的硫酸镁、9-35mmol/L的蛋氨酸和5-20mmol/L的半胱氨酸。
  11. 根据权利要求10所述的方法，其中，所述液体培养基还含有0.5-50g/L的吐温。
  12. 蕈菌经液体发酵获得的胞外发酵液和/或胞外发酵液的浓缩物在制备含有麦角硫因的制品中的用途，所述制品包括食品、化妆品和动物饲料。
  13. 根据权利要求12所述的用途，其中，所述蕈菌为糙皮侧耳。
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