CN108795773A - Moschus trichoderma strain and its fragrance of preparation - Google Patents
Moschus trichoderma strain and its fragrance of preparation Download PDFInfo
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- CN108795773A CN108795773A CN201710291679.8A CN201710291679A CN108795773A CN 108795773 A CN108795773 A CN 108795773A CN 201710291679 A CN201710291679 A CN 201710291679A CN 108795773 A CN108795773 A CN 108795773A
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- 238000011161 development Methods 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- 239000001734 eugenia caryophyllata l. bud oleoresin Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 230000001408 fungistatic effect Effects 0.000 description 1
- ZEMPKEQAKRGZGQ-XOQCFJPHSA-N glycerol triricinoleate Natural products CCCCCC[C@@H](O)CC=CCCCCCCCC(=O)OC[C@@H](COC(=O)CCCCCCCC=CC[C@@H](O)CCCCCC)OC(=O)CCCCCCCC=CC[C@H](O)CCCCCC ZEMPKEQAKRGZGQ-XOQCFJPHSA-N 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 235000008216 herbs Nutrition 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 description 1
- 150000002596 lactones Chemical class 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 239000012263 liquid product Substances 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 150000004702 methyl esters Chemical class 0.000 description 1
- NJTGANWAUPEOAX-UHFFFAOYSA-N molport-023-220-454 Chemical compound OCC(O)CO.OCC(O)CO NJTGANWAUPEOAX-UHFFFAOYSA-N 0.000 description 1
- 238000003541 multi-stage reaction Methods 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 235000011837 pasties Nutrition 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 235000021017 pears Nutrition 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000001738 pogostemon cablin oil Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
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- 235000019605 sweet taste sensations Nutrition 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- MWOOGOJBHIARFG-UHFFFAOYSA-N vanillin Chemical compound COC1=CC(C=O)=CC=C1O MWOOGOJBHIARFG-UHFFFAOYSA-N 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
-
- A—HUMAN NECESSITIES
- A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
- A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
- A24B3/00—Preparing tobacco in the factory
- A24B3/12—Steaming, curing, or flavouring tobacco
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/12—Carboxylic acids; Salts or anhydrides thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B9/00—Essential oils; Perfumes
- C11B9/0007—Aliphatic compounds
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B9/00—Essential oils; Perfumes
- C11B9/0007—Aliphatic compounds
- C11B9/0015—Aliphatic compounds containing oxygen as the only heteroatom
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B9/00—Essential oils; Perfumes
- C11B9/0026—Essential oils; Perfumes compounds containing an alicyclic ring not condensed with another ring
- C11B9/0034—Essential oils; Perfumes compounds containing an alicyclic ring not condensed with another ring the ring containing six carbon atoms
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B9/00—Essential oils; Perfumes
- C11B9/0042—Essential oils; Perfumes compounds containing condensed hydrocarbon rings
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
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- C11B9/00—Essential oils; Perfumes
- C11B9/0042—Essential oils; Perfumes compounds containing condensed hydrocarbon rings
- C11B9/0046—Essential oils; Perfumes compounds containing condensed hydrocarbon rings containing only two condensed rings
- C11B9/0049—Essential oils; Perfumes compounds containing condensed hydrocarbon rings containing only two condensed rings the condensed rings sharing two common C atoms
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- 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
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Abstract
The present invention relates to microbial engineering field more particularly to Moschus trichoderma strains and its fragrance of preparation.Strain provided by the invention is the mould Muscodor sp.W-S-41 of endogenetic fungus Moschus, is rich in a variety of volatile components in cultured products, can be used to prepare natural perfume material.
Description
Technical field
The present invention relates to microbial engineering field more particularly to Moschus trichoderma strains and its fragrance of preparation.
Background technology
Fragrance, essence are of crucial importance to food, cigarette, feed, cosmetics and pharmaceuticals industry.Currently, fragrance, perfume (or spice) in the world
About 15,100,000,000 dollars of the yield of essence, occupies 25% or so food additives market, and increase year by year.The fragrance that produces now,
About 85% product is obtained by chemical synthesis process in essence.But with the fragrance of chemical method synthesis, there are following serious
Defect：First, a large amount of chemical synthesis substance is abused brings harm to people's health；Second is that in being chemically synthesized due to
Lack single-minded substrate, product purity is caused to decline；Three are chemical synthesis in product often containing racemic mixture, such as therefrom extract
Purpose isomers or chiral fragrance will be extremely difficult and costly；Fourth, people are used to eat to chemically synthesized additive
Product, cosmetics etc. are increasingly disliked.As the improvement of people's living standards, being intended to natural, healthy, peace to food additives demand
Entirely, nutrition and multifunctionality.Natural essence fragrance is the fine chemical product and food additives of high value, and originally from natural
The natural perfume material of animal and plant extraction, since raw material is limited, extraction cost is high, much can not meet the demand in market.
Currently, utilization microorganism common in field prepares natural perfume material and mainly passes through：(1) natural perfume is prepared with enzyme engineering
Material, such as have vanillic aldehyde, lactone, ester perfume, leaf-alcohol etc., the limitation of biological enzyme has following reason：1. enzyme is active
It keeps.The activity that height is shown in the environment of enzyme molecule in vivo is environment and other molecules pair because residing for enzyme
Its regulation and control, once leaving that environment, the activity of enzyme will substantially reduce, or even inactivation.2. difficulty prepared by enzyme.Enzyme preparation packet
The technique for including a series of complex such as extraction, refined, immobilization.Presently found biological enzyme has thousands of kinds, almost every in organism
One biochemical reaction has the catalysis of enzyme, but only hundreds of kinds of the enzyme that laboratory is prepared, and is used for industrial commercial enzyme
Only tens kinds (most of is the crude enzyme liquid product containing albumen and other a small amount of enzymes, rather than pure enzyme), are used for chiral compound
Object is split and the enzyme of asymmetric syntheses is a wherein minimum part (only lipase and esterase etc. is several) again, this makes the application of enzyme
Generate significant limitation.3. the stability problem of enzyme.Enzyme is substantially a protein that is chiral, having catalysis.It
Stability by physical environment, chemical environment and bioenvironmental test, the variation of the physical conditions such as heat, ultraviolet light is organic
The variation of the electrochemical conditions such as solvent, pH, putrefaction of microorganism etc. can make enzyme denaturation, be reduced so as to cause activity, even
Inactivation.4. the cost problem of enzyme.The raw materials for production of enzyme are generally from animal and plant and microorganism, so production cost is high, price ratio
Chemical catalyst is expensive, and competitiveness is poor.(2) using natural plants as substrate, such as using sweet osmanthus, water as substrate, the yeast of improvement is added
Bacterium is cultivated, and the cultured products of sweet osmanthus note can be generated, very close with the natural essential oil fragrance that is extracted from sweet osmanthus, but its according to
Large-scale culture is so unable to get to obtain.
Moschus is mould (Muscodor sp.), is named as that aerogenesis is mould before, is a kind of endogenetic fungus for belonging to Xylariaceae,
Separating obtained from xylocinnamomum by Worapong etc. (2001) earliest, people utilize its distinctive morphology, physiology, biochemistry
The mould category endogenetic fungus of new Moschus is found and identified to the feature of aspect and the sequence of rRNA, and being noteworthy characterized by can produce
Raw volatile organic compounds (volatile organic compounds, abbreviation VOCs), these VOCs have extensive raw
Object activity, can inhibit or kill many disease fungus, pathogenetic bacteria and some insects, however different bacterial strains, active effect
It is different.Due to the mould VOCs that can have extensive bacteriostatic activity of Moschus, scientists are detached using chemical analyses means such as GC-MS
Identify the VOCs ingredients of the mould generation of Moschus, it is found that the VOCs ingredients that different strains generate are not quite similar, active effect and its
VOCs ingredients are related, and in the past, what is paid close attention in field is all the mould fungistatic effect of Moschus.
Invention content
In view of this, the technical problem to be solved in the present invention is to provide Moschus trichoderma strain and its fragrance of preparation, this hair
The Moschus enzyme bacterial strain of bright offer can cultivate generation metabolite, have Volatile infochemicals, can be used as perfume material extraction day
Right fragrance.
It is CCTCC NO the present invention provides deposit number：The Moschus enzyme bacterial strain of M 2016759.
The mould Muscodor sp.W-S-41 of Moschus provided by the invention are isolated from Yunnan Province of China Na Banhe acquisition wild states
Under hispid arthraxon, the strain culturing generate metabolite, have Volatile infochemicals, can be used as perfume material extraction natural perfume
Material.
It is CCTCC NO the present invention also provides deposit number：The Moschus enzyme bacterial strain of M 2016759 is in preparing fragrance
Using.
The preservation of Moschus enzyme strain provided by the invention uses PDA solid mediums, inclined-plane culture, 10~30 DEG C of preservation bacterium
Strain.Specifically, step includes：
By inoculation to PDA slant mediums, culture to fresh mycelia is grown, and the stone to sterilize through 3 sub-high pressures is added
Wax floods mycelia, covers freezen protective pipe lid, and under room temperature or 10 DEG C of refrigerators preserve.
Freezen protective liquid can also be used in the preservation of Moschus enzyme strain provided by the invention, carries out freezen protective.Temperature is -80
℃.Specifically, step includes：The fungus block for taking growth animated period, is added autoclaved freezen protective liquid, fungus block is flooded.It will
After freezen protective pipe places 1h at 4 DEG C and -20 DEG C successively, it is transferred in -80 DEG C of ultra low temperature freezers and carries out long-term preservation.
The activation of Moschus enzyme strain provided by the invention also uses PDA solid mediums, 25 DEG C, living under dark condition for 24 hours
Change 7d.
The present invention also provides a kind of preparation method of fragrance, this method culture deposit number is CCTCC NO：M
2016759 Moschus enzyme bacterial strain obtains the culture containing fragrance, the extracted obtained fragrance of culture.
In embodiments of the present invention, culture uses solid culture, and the pH value of the culture medium of the culture is 5.5~6.5, by
Millet, perlite and nutrient solution are made；
The nutrient solution include murphy juice and：
Glucose 10g/L~120g/L；
Defatted soy flour 100g/L~280g/L；
Peptone 20g/L~200g/L；
Potassium dihydrogen phosphate 6g/L~22g/L；
Magnesium sulfate 3g/L~6g/L.
In some specific embodiments, the pH value of the culture medium of the culture is 6.
In some specific embodiments, the preparation method of the culture medium of the culture is：It is added 55 in per 100g raw millets~
65ml murphy juices are heated 14~16 minutes in 120~122 DEG C with autoclave, obtain ripe millet；After cooling with perlite and nutrient solution
Mix the culture medium of system.
The mass ratio of the ripe millet and perlite is 100：(19~21).
Per the ripe millets of 120g and 24~26ml of nutrient solution is added in the mixture of perlite.
The preparation method of the nutrient solution is：0.5~3g of glucose, 5~7g of defatted soy flour, peptone (peptone) 1
~5g, 0.3~0.55g of potassium dihydrogen phosphate, magnesium sulfate 0.15g adjust pH to 6 after being dissolved with 25~50mL murphy juices, obtain nutrition
Liquid.The quality of substance involved in the nutrient solution preparation is only used as example with volume, for mass production purpose, in practical behaviour
It can expand at double in work.
Solid culture (solid-state fermentation, SSF) of the present invention is to refer to without or almost do not having
In the presence of Free water, in having certain humidity solid state substrate, a biological respinse mistake being carried out with one or more microorganisms
Journey.From the point of view of bioprocesses, solid state rheology is the bioprocesses using gas phase as continuous phase, specifically
, one can consider that solid culture is a kind of a kind of culture hand utilized in the case where culture medium is solia particle
Section.There are many notable advantages compared with Liquid Culture for solid culture：Culture medium is more cheap, low energy consumption, input cost are low, more
It is less to be easy to control volume of culture, the waste water of generation.By taking the materials of culture medium as an example, the culture medium for solid culture is more single
Pure, in general, such as grain class, wheat bran, agropyron, large cereal or agricultural product etc. can be used, so culture is former
Expect that cost is more economical.
In embodiments of the present invention, the condition of the culture is dark, and temperature is 23 DEG C~28 DEG C, and the time is 5~10 days.
In some embodiments, the temperature of culture is 25 DEG C, and the time is 7 days.
In embodiments of the present invention, the solvent of the extraction is selected from water, alcohols, acetone, chloroform, ether, benzene or petroleum ether.
In some specific embodiments, the solvent used is extracted as ethanol water or petroleum ether.
Specifically, the volume fraction of ethyl alcohol is 65% in ethanol water.
In embodiments of the present invention, the method for the extraction be refluxing extraction, temperature be 50 DEG C~60 DEG C, the time be 6~
8h；Then extracting solution is collected, after filtering, concentrating, is washed with the ethyl alcohol of 90vol%~100vol%, filters, concentrate again,
Fragrance is made.
The volume fraction of the ethyl alcohol is 95%.
In the extraction, volume-mass ratio of Extraction solvent and culture is 1L：200g.
The temperature of the extraction is preferably 55 DEG C.
After the filtering, filtrate is taken.
The concentration flings to ethyl alcohol using reduced pressure.
The washing mixes for the medicinal extract after concentrating with ethyl alcohol, after freezing (- 20 degrees Celsius) standing 2h, filter again,
Concentration.
The present invention also provides a kind of fragrance, are CCTCC NO by deposit number：The Moschus enzyme bacterial strain of M 2016759 is trained
It supports, including isobutyric acid, methyl isobutyrate, α-guaiene, δ-guaiene, guaiaci lignum -9,11- alkene.
Further include beta-elemene, β-carypohyllene, isoamyl acetate, (-)-β-chamigrene in fragrance of the present invention.
After testing, in fragrance of the present invention, including following volatile ingredient：
Application of the fragrance provided by the invention as the additive of food, drug or cosmetics.
Strain provided by the invention is the mould Muscodor sp.W-S-41 of endogenetic fungus Moschus, is rich in cultured products more
Kind of volatile component, for preparing natural perfume material, overcome microbial technique prepare need during fragrance biological enzyme or
Using plant as the technological deficiency of substrate, the extraction of natural perfume material, Nei Shengzhen of the present invention are realized by the natural sex of raw material
Bacterium is the fungi for not causing apparent disease symptoms in the plant tissue lived on the ground partly, living.Endogenetic fungus passes through solid
Culture --- extraction can must have aromatic natural absolute oil, have the application and development foreground extensively sent out.The present invention has following
Advantage：(1) mild condition some realize the multistep reaction that chemical method in aqueous solution can not possibly be completed, reaction in a mild condition
Safety, reaction carry out under room temperature, normal pressure, and production site and equipment requirement are low；(2) environment well cultivates completion, product separation
The three wastes formed afterwards are few；(3) sterile production；(4) culture device has versatility；(5) it overcomes and makees for a long time from animal and plant
For natural perfume material sole source, existing active constituent content is low, and separation is difficult, climate and animal and plant harm influence, no
The defects of breaking unreal existing natural resources.
Biological deposits explanation
Moschus enzyme, Classification And Nomenclature：Muscodor sp.W-S-41 are deposited on December 16th, 2016 and are preserved in Chinese allusion quotation
Type culture collection (CCTCC), address is：Wuhan, China Wuhan University.Deposit number is CCTCC NO：M 2016759.
Description of the drawings
The specific implementation mode of the present invention is described in further detail below in conjunction with the accompanying drawings；
Fig. 1 shows that the mould Muscodor sp.W-S-41 bacterium colony figures of Moschus, the specially mould Muscodor sp.W-S-41 of Moschus exist
PDA grows 7 days bacterium colony full faces, and white mycelium is radially grown；
Fig. 2 shows the mould Muscodor sp.W-S-41 absolute oil total ion current figures of Moschus, and wherein abscissa is retention time, indulges and sits
Mark is kurtosis；As retention time is recommended, low-boiling compound appearance first.
Specific implementation mode
The present invention provides Moschus trichoderma strain and its fragrance of preparation, those skilled in the art can use for reference present disclosure,
It is suitably modified technological parameter realization.In particular, it should be pointed out that all similar substitutions and modifications carry out those skilled in the art
Say it is it will be apparent that they are considered as being included in the present invention.The method of the present invention and application have passed through preferred embodiment
Be described, related personnel obviously can not depart from the content of present invention, in spirit and scope to methods herein and application into
Row change is suitably changed and is combined, to realize and apply the technology of the present invention.
Material：
Strains tested is the mould Muscodor sp.W-S-41 of Moschus.The mould Muscodor sp.W-S-41 of Moschus.Depositary institution：
China typical culture collection center；Preserving number CCTCC NO：M 2016759；The preservation time：On December 16th, 2016.
Instrument according to the present invention includes superclean bench, constant incubator, electronic balance, pH meter, baking oven, electromagnetism
Stove, micro-wave oven, refrigerator, vacuum drying chamber, MLS3750 types high-pressure sterilizing pot, BS233S type analysis day equality.
The present invention relates to four kinds of culture mediums, respectively PDA culture medium, PDB culture mediums, 2% wort agar culture medium and
Freezen protective culture medium, preparation method difference are as follows：
PDA culture medium (potato dextrose agar)：Potato 200g, glucose 20g, agar 1.5%, tap water
1000mL.It takes quantitative potato to be cut into uniform fritter, adds boiling to boil 20 minutes to potato easily at mashed potatoes, 8 layers of filtered through gauze are added
20g glucose, it is cooling, add tap water to be settled to 1000mL, liquid is added in the agar of triangle bottled 1.5%, encapsulates, 121 DEG C,
15min high pressure sterilizations are for use.
PDB culture mediums (potato dextrose medium)：Potato 200g, glucose 20g, tap water 1000mL.It takes quantitative
Potato is cut into uniform fritter, adds boiling to boil 20 minutes to potato easily at mashed potatoes, 20g glucose is added in 8 layers of filtered through gauze, cold
But, tap water is added to be settled to 1000mL, encapsulate, 121 DEG C, 15min high pressure sterilizations it is for use.
2% wort agar culture medium (malt extract agar, MEA)：In malt leaches powder 20g, agar 20g
Distilled water is added to be settled to 1000mL；Then carry out conventional high-temperature sterilization (1.1 atmospheric pressure, sterilize at 121 DEG C 20min).
Freezen protective culture medium (liquid)：Glucose 10g, yeast extract 1g, casein hydrolysis 0.5g, sour water solution junket egg
White 0.5g, glycerine (glycerine) 180mL.With distilled water by glucose, yeast extract, casein hydrolysis and acid hydrolyzed casein
After dissolving, glycerine is added, finally with distilled water be settled to 1000mL, 121 DEG C, sterilizing 20min.
With reference to embodiment, the present invention is further explained：
Embodiment 1,
1, the separation of Muscodor sp.W-S-41 bacterial strains obtains
Hispid arthraxon under Yunnan Province of China Na Banhe acquisition wild states, the hispid arthraxon tap water rinse of acquisition is clean, it is loyal
Blade of grass sheath sterilizes 30 seconds in 75% (v/v) alcoholic solution, and 10 points of kinds, nothing are then sterilized in 1% (v/v) liquor natrii hypochloritis
Bacterium water rinses 3 times.Leaf sheath is cut into the segment of about 0.6cm long with sterile razor blade, is placed in containing 50 μ gml-1Ampicillin and 50
μg·ml-1On 2% wort agar culture medium (MEA) plate of streptomysin, 25 DEG C of light cultures, after mycelia grows, by mycelia
Top transposing is to PDA culture medium；Isolated bacteria colony white, radially spreads, and 25 DEG C of light cultures connect in PDA culture medium
It is continuous to be inoculated with three times, it is confirmed as single bacterium colony, without other fungies or bacterium syntrophism, it is believed that be to obtain pure culture, be named as
Muscodor sp.W-S-41。。
The bacterial strain is fine and close white on culture dish, radially spreads (Fig. 1), Xylariaceae bacterium occurs after growing the several months
The characteristic feature of strain：Occurs the bacterial strain of black charing mycelia from bacterium colony center.
1.1, the identification of bacterial strain
1.1.1 extracting genome DNA
Bacterial strain Muscodor sp.W-S-41 are seeded on PDA plate 25 DEG C of dark culturings one week.With transfer needle picking
A small amount of mycelia block is put into each 5 of the steel ball of diameter 1mm and 5mm, is impregnated 1 minute in liquid nitrogen in 1.5ml sterile centrifugation tubes,
Then sample is fully ground on JXFSTPRP refrigeration grinding machines (Shanghai Jing Xin Science and Technology Ltd.s).Use fungal gene group
DNA rapid extractions kit (Axygen) extracts Muscodor sp.W-S-41 genomic DNAs.400 μ l AP1 and 4 are added
μ l RNaseA (10mg/ml), vortex oscillation.It is then incubated 20 minutes in 65 DEG C of water-baths, abundant lysed sample.130 μ l are added
AP2 is mixed well, and is placed 5 minutes on ice bath.14,000rpm centrifugations 8 minutes, careful Aspirate supernatant are new to one
In 1.5ml sterile centrifugation tubes, the AP3/E of 1.5 times of volumes is added, carrying out piping and druming with pipette tips is uniformly mixed so as to obtain mixed liquor, first adds 650 μ l
Mixed liquor is added in an adsorption column AC (adsorption column is placed in collecting pipe), and 13,000rpm centrifugations 40 seconds are outwelled in collecting pipe
Waste liquid repeats the above steps until adding all mixed liquors.600 μ l rinsing liquids WB, 12,000rpm centrifugations are added in adsorption column
30 seconds, abandon waste liquid.Adsorption column AC is placed back in collecting pipe, 13,000rpm centrifugations 2 minutes, remaining rinsing of going out as possible
Liquid.It takes out adsorption column AC to be put into new 1.5ml sterile centrifugation tubes, 100 μ l elution buffers is added at the intermediate position of adsorbed film
Liquid EB is placed at room temperature for 5 minutes, 12,000rpm centrifugations 1 minute obtain DNA sample, saved backup at -20 DEG C.
1.1.2 PCR amplification
Ribosomes transcribed spacer (ITS) gene order of bacterial strain Muscodor sp.W-S-41 is measured.Bacterial strain
The PCR forward direction amplimers ITS1-F of the ITS genes of Muscodor sp.W-S-41：TCCGTAGGTGAACCTGCGG, it is reversed to expand
Increase primer I TS4：The pcr amplification reaction condition of TCCTCCGCTTATTGATATGC, ITS gene is：94 DEG C of pre-degenerations 2 minutes；94
DEG C denaturation 30 seconds, 55 DEG C anneal 40 seconds, 72 DEG C extend 50 seconds, 35 times cycle；Last 72 DEG C extend 10 minutes.
PCR reaction systems are 50 μ L, including：
Wherein, 2 × Es Taq MasterMix are purchased from Vazyme companies, contain Es Taq DNA Polymerase, 2x
Es Taq PCR buffer, 3mM MgCl2 and 400 μM of dNTP mix.
1.1.3 the purifying (being purified with AxyPrep DNA Gel Extraction Kit kits) of PCR product
1) Ago-Gel containing target DNA is cut under purple lamp, and gel surface liquid is exhausted with paper handkerchief and is shredded.Meter
Gel weight (recording 1.5ml centrifuge tubes weight in advance) is calculated, the weight is as a gel volume (such as 100mg=100ul body
Product).
2) BufferDE-A of 3 (using 300 μ l in the present invention) gel volumes is added, adds after mixing in 75 DEG C
Heat, interruption mixing (per 2-3min), until gel piece is completely melt (about 6 minutes).Note：BufferDE-A is red liquid.?
During melting agarose gel, it can help to observe whether gel is completely melt.
3) add the BufferDE-B (that is, BufferDE-B of 150 μ l is added) of 0.5 BufferDE-A volume, mixing is equal
It is even.When the DNA fragmentation of separation is less than 400bp, the isopropanol of 1 gel volume need to be added.Note：It is mixed after adding BufferDE-B
Closing object color becomes yellow, mixes well to ensure to form uniform yellow solution.Remarks explanation：According to design of primers site,
NSA3-NLC2 amplified production clip sizes be 950bp, the amplified production segment about 800bp of NSI1-NLB4, other primers
Amplified production size is 200-250bp.
4) mixed liquor in aspiration step 3, being transferred to DNA preparations pipe, (when reset condition, which prepares pipe and is placed in 2ml
(being provided in kit) centrifuge tube) in, 12000g centrifuges 1min.Abandon filtrate.
5) pipe will be prepared and puts back into 2ml centrifuge tubes, added Buffer W1,12000g the centrifugation 30s of 500 μ l, abandon filtrate.
6) pipe will be prepared and puts back into 2ml centrifuge tubes, added Buffer W2,12000g the centrifugation 30s of 700 μ l, abandon filtrate.With same
The method of sample adds the Buffer W2 of 700 μ l washed once again, and 12000g centrifuges 1min.Note：(a) confirm in Buffer
Absolute ethyl alcohol is added in the designated volume pressed in W2concentrate on reagent bottle.(b) Buffer W2 are used to rinse energy twice
Ensure that salinity is completely removed, eliminates the influence to subsequent experimental.
7) pipe will be prepared to put back into 2ml centrifuge tubes, 12000g centrifuges 1min.
8) DNA is prepared pipe to be placed in clean 1.5ml centrifuge tubes (providing in kit), the filtering of pipe is prepared in DNA
Film center adds the Eluent or deionized water of 25-30ul, is stored at room temperature 1min.12000g centrifuges 1min and (collects filtered fluid, filtrate
In contain be exactly PCR amplified production).Eluted dna.
Purified PCR product is sent to Sangon Biotech (Shanghai) Co., Ltd. and is sequenced, following sequence is obtained
Such as SEQ ID NO:Shown in 1.Sequencing result carries out homologous sequence search to further determine that its classification in GenBank databases
Status.The above sequence is uploaded in NCBI and is compared, obtains the classification position of bacterial strain Muscodor sp.W-S-41, the bacterium
Strain is that Moschus is mould (Muscodor sp.), belongs to mycota Fungi, Ascomycota Ascomycota, excrement shell Gammaproteobacteria
Sordariomycetes, carbon angle bacteria mesh Xylariales, Xylariaceae Xylariaceae.
The preservation and preservation of 1.2 Muscodor sp.W-S-41 bacterial strains
The bacterial strain of above-mentioned gained has carried out following preservation：Muscodor sp.W-S-41, depositary institution：Chinese Typical Representative culture
Object collection, preservation address：Wuhan, China Wuhan University；Preservation date：On December 16th, 2016, preserving number：CGMCC NO：
M 2016759。
1.2.1 the room temperature or Cord blood of Muscodor sp.W-S-41 bacterial strains.
In superclean bench, preserves pipe in 2ml sterile cryos and the PDA culture medium for being no more than 1.5ml and melting is added, cover cold
Freeze and preserves pipe lid, slant setting.After to be solidified, the Muscodor sp.W-S-41 inoculations that will preserve to inclined-plane culture
Base, after a couple of days, Muscodor sp.W-S-41 mycelia successfully colonizes in inclined-plane, it is seen that fresh mycelia grows, and is added through 3 height
The paraffin of pressure sterilizing floods mycelia, covers freezen protective pipe lid, and under room temperature or 10 DEG C of refrigerators preserve.In use, with nothing
Bacterium transfer needle picking hyphostroma, is inoculated in tablet PDA culture medium and is activated in 25 DEG C of incubators.
1.2.2 the freezen protective of Muscodor sp.W-S-41 bacterial strains
When Muscodor sp.W-S-41 grow animated period on PDA plate, it is placed on superclean bench, it will be on PDA
Bacterium colony takes 4-5 ferfas blocks, is placed in 2ml sterile cryos and preserves pipe, autoclaved freezen protective liquid is added, by Muscodor
Sp.W-S-41 fungus blocks are flooded.Freezen protective pipe lid is covered, after freezen protective pipe is placed 1h at 4 DEG C and -20 DEG C successively,
It is transferred in -80 DEG C of ultra low temperature freezers and carries out long-term preservation；Or programmed cooling instrument is used, so that it is unlikely to intracellular ice,
Tissue temperature is dropped to and is preserved suitable for -80 DEG C of ultra low temperature freezers.In use, the naturally to thaw in room temperature, picking fungus block therein
It is inoculated in tablet PDA culture medium and is activated.
The mould Muscodor sp.W-S-41 solid cultures of 2 Moschus of embodiment
Solid culture (solid-state fermentation, SSF) of the present invention is to refer to without or almost do not having
In the presence of Free water, in having certain humidity solid state substrate, a biological respinse mistake being carried out with one or more microorganisms
Journey.From the point of view of bioprocesses, solid state rheology is the bioprocesses using gas phase as continuous phase, specifically
, one can consider that solid culture is a kind of a kind of culture hand utilized in the case where culture medium is solia particle
Section.There are many notable advantages compared with Liquid Culture for solid culture：Culture medium is more cheap, low energy consumption, input cost are low, more
It is less to be easy to control volume of culture, the waste water of generation.By taking the materials of culture medium as an example, the culture medium for solid culture is more single
Pure, in general, such as grain class, wheat bran, agropyron, large cereal or agricultural product etc. can be used, so culture is former
Expect that cost is more economical.
The mycelia in pipe, inoculation are preserved with the inclined-planes a small amount of Moschus of sterilizing toothpick picking mould Muscodor sp.W-S-41PDA
In PDA culture medium plate, (25 DEG C, dark for 24 hours) activation culture 7d is placed in constant temperature illumination box.According to 0.1~0.3%
The inoculum concentration of (quality %) is seeded on solid culture culture medium, in the dark under 23~28 DEG C of cultivation temperature culture 5~
Obtain within 10 days the mould Muscodor sp.W-S-41 cultured products of Moschus；Solid culture culture medium consists of the following compositions：Ripe millet
24~26ml of 100g, 19~21g of perlite and nutrient solution；The preparation method of ripe millet is：In the raw millets of 100g be added 55~
65ml murphy juices are heated 14~16 minutes in 120~122 DEG C with autoclave, obtain ripe millet；The preparation method of nutrient solution is：It will
0.5~3g of glucose, 5~7g of defatted soy flour, peptone (peptone) 1~5g, 0.3~0.55g of potassium dihydrogen phosphate, sulfuric acid
Magnesium 0.15g obtains nutrient solution (in the pasty state) with pH to 6 is adjusted after the murphy juice dissolving of 25~50ml.
3 ethanol immersion of embodiment prepares the mould Muscodor sp.W-S-41 natural perfume materials of Moschus
The present embodiment using ethyl alcohol as solvent, the 200g solid culture products of Muscodor sp.W-S-41 mould to Moschus into
Row extraction, method are as follows：Collect solid state rheology object；Add 65% ethyl alcohol of 1L, 55 DEG C of Extracting temperature water-bath；Reflux extraction 8h；It crosses
Obtained medicinal extract is concentrated under reduced pressure in filter；95% ethyl alcohol of 200mL is added, freezing stands 2h, filtering and impurity removing matter；Absolute oil is made in concentration.
To Muscodor sp.W-S-41 absolute oil composition measurements
The absolute oil that endogenetic fungus Muscodor sp. bacterial strains W-S-41 is obtained uses Solid Phase Extraction/gas chromatography/mass spectrometry skill
Art (Solid phase microextraction/Gas chromatograph/Mass spetra, SPME/GC/MS) is analyzed.
GC-MS analysis conditions：Gas chromatograph-mass spectrometer Agilent 6890N/5975B, chem workstation Agilent
G1701DA, chromatographic column Agilent HP-5MS capillary column (5% phenyl methyl siloxanes：30m×0.25mm×0.25μm)；
Chromatographic condition：Carrier gas is helium；Temperature programming：30 DEG C of initial temperature keeps 3min, rises to 220 DEG C with the speed of 5 DEG C/min, keeps
1min；With 3 DEG C of min-1Speed rise to 250 DEG C, keep 1min；With 10 DEG C of min-1Speed rise to 280 DEG C, keep 5min；With
20℃min-1Speed rise to 300 DEG C, keep 10min.290 DEG C of injector temperature, 1 μ L of sample size；Split ratio 5：1；Solvent prolongs
Slow 2min；Mass Spectrometry Conditions：EI ionizing energies 70eV；230 DEG C of ion source temperature；Mass scan range 60-600a.m.u.
It is analyzed by GC/MS, obtains the total ion current figure (Fig. 2) of each ingredient, determined using NIST14 library searching methods each
The chemical composition of component, and by peak area normalization method calculate each component percentage contents, Measurement results combination Fig. 2 and
Table 1.
Table 1：The mould Muscodor sp.W-S-41 absolute oil main components of Moschus
| Serial number | Retention time | Peak height | Peak area | Compound | Content % |
| 1 | 3.5004 | 81101 | 12713459 | Methyl isobutyrate | 4.7149% |
| 2 | 7.6001 | 398876 | 124739366 | Isobutyric acid | 46.2612% |
| 3 | 9.4907 | 55198 | 5944880 | Isoamyl acetate | 2.2047% |
| 4 | 25.2126 | 55998 | 1447819 | Beta-elemene | 0.5369% |
| 5 | 25.949 | 31285 | 947165 | β-carypohyllene | 0.3513% |
| 6 | 26.4067 | 104108 | 2618436 | α-guaiene | 0.9711% |
| 7 | 27.3769 | 114673 | 4194365 | Ledene | 1.5555% |
| 8 | 27.6854 | 350829 | 9017261 | (-)-β-chamigrene | 3.3442% |
| 9 | 27.9341 | 172700 | 5613371 | Guaiaci lignum -9,11- alkene | 2.0818% |
| 10 | 28.0933 | 1314140 | 33892190 | δ-guaiene | 12.5693% |
| 11 | 31.576 | 2228600 | 68513358 | Pogostol | 25.4090% |
Sensory evaluation shows, the wooden fragrance of the mould Muscodor sp.W-S-41 absolute oil homophonies of Moschus, including isobutyric acid, isobutyl
The substances such as sour methyl esters, α-guaiene, δ-guaiene, beta-elemene, β-carypohyllene, isoamyl acetate, (-)-β-chamigrene.
Key component is：(1) isobutyric acid, content ratio 46.2612% are mainly used to produce corresponding ester, and it is fragrant to can be used as synthesis
Smart substrate and solvent；(2) methyl isobutyrate content ratio is 4.7149%, and it is light fruit that substance is adjusted before can smell in absolute oil
Perfume (or spice) has like apricot sweet taste, GB 2760-2007 is defined as the flavorant for allowing to use in apple, pineapple fruity.The U.S. year consumption
Dosage about 2400kg, natural product are present in strawberry, pineapple, strawberry, Kiwi berry, Russian champagne, apple, banana, blueberry, sweet tea
In melon, papaya, baked potato etc..(3) α-guaiene content 0.9711%, δ-guaiene content 12.5693%, the wide leaves of pulse plants
Fragrant Austria's alcohol content 25.4090% and guaiaci lignum -9,11- alkene content 2.0818% constitute the middle tune of absolute oil, and fragrance is light, graceful
Root of Aucklandia lappa Decne flavor, this three tastes substance is present on a small quantity in patchouli oil and other essential oils.(4) beta-elemene content 0.5369%,
There is pungent fennel smell, is naturally occurring in and is present in burley tobaccos tobacco leaf, Turkish tobaccos tobacco leaf, β-carypohyllene 0.3513%, tool
There is the cloves fragrance that Xin Xiang, root of Aucklandia lappa Decne, citrus are fragrant, camphor is fragrant, mild, is naturally occurring in lemon, garden shaddock, nutmeg, pepper, covers basin
In son, blackcurrant, cinnamon leaves oil, clove leaf oil.For allocating the food flavors such as cloves, pepper, nutmeg, citrus, medicinal herbs.?
It can be used for synthesizing other fragrance, such as more valuable for synthesizing acetyl group carypohyllene.This two tastes substance centering tune root of Aucklandia lappa Decne, or
It says that wooden fragrance has Titian effect, makes its more lasting reservation.(5) other substances：Isoamyl acetate, content 2.2047% are
Important solvent can dissolve nitrocellulose, three rosin ester of glycerine, vinyl, coumarone resin, rosin, frankincense, reach horse
Resin, sandarac, castor oil etc..In Japan, this product 80% is used as fragrance, has stronger fruit fragrance, like pears, banana, apple
The fragrance such as fruit.Therefore various edible fruit essence are widely used as.Also there is appropriate application in flavouring essence for tobacco, daily perfume compound for cosmetics；
(-)-β-chamigrene, content 3.3442% are naturally occurring to contain volatile oil in Schisandra chinensis Atractylis lancea rhizome.Also have in absolute oil of the present invention
Volatile materials unknown at present needs subsequently to find and develop and use such as compound ledene.
4 petroleum ether extraction of embodiment prepares the mould Muscodor sp.W-S-41 natural perfume materials of Moschus
The present embodiment using petroleum ether as solvent, the 200g solid culture products of Muscodor sp.W-S-41 mould to Moschus
It is extracted, method is as follows：It is dry to collect the mould Muscodor sp.W-S-41 solid state rheology objects of Moschus；Add 1L petroleum ethers, extraction
55 DEG C of temperature water bath；Reflux extraction 6h；Obtained medicinal extract is concentrated under reduced pressure in filtering；95% ethyl alcohol of 200mL is added, freezing stands 2h,
Filtering and impurity removing matter；Absolute oil is made in concentration.The absolute oil component that the present embodiment obtains is consistent with embodiment 3, key component packet in absolute oil
Include isobutyric acid, methyl isobutyrate, α-guaiene, δ-guaiene, guaiaci lignum -9,11- alkene, beta-elemene, β-carypohyllene, second
The substances such as isoamyl valerate, (-)-β-chamigrene.
It the above is only the preferred embodiment of the present invention, it is noted that those skilled in the art are come
It says, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as
Protection scope of the present invention.
SEQUENCE LISTING
<110>Guizhou Tobacco Industry Co., Ltd
<120>Moschus trichoderma strain and its fragrance of preparation
<130> MP1704720
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 548
<212> DNA
<213> Muscodor sp.
<400> 1
cattacagag ttttctaaac tcccaaccct atgtgaactt acctttgttg cttcggcggc 60
ggaggctacc ctatagggga taccacatag tggttaccct gtagtcccag gtgctagatc 120
gtgctcaatg tcttatcgtc tacgactagc tacccggtgg ccctccccgc cggcggccaa 180
ctaaactctg tttttatggc attctgaatt ataaacttaa taagttaaaa ctttcaacaa 240
cggatctctt ggttctggca tcgatgaaga acgcagcgaa atgcgataag taatgtgaat 300
tgcagaattc agtgaatcat cgaatctttg aacgcacatt gcgcccatta gcattctagt 360
gggcatgcct gttcgagcgt catttcacca cttaagccct gttgcttagc gttgggagcc 420
tacggcactg cccgtagctc cctaaagtga ttggcggagt tggttctcac tctaggcgta 480
gtaaatctat ctcgcctctg tagtggttcc ggcccctgcc gtaaaacccc ctatatcaaa 540
ggttgacc 548
Claims (10)
1. deposit number is CCTCC NO：The Moschus enzyme bacterial strain of M 2016759.
2. deposit number is CCTCC NO：Application of the Moschus enzyme bacterial strain of M 2016759 in preparing fragrance.
3. a kind of preparation method of fragrance, which is characterized in that culture deposit number is CCTCC NO：The Moschus enzyme of M 2016759
Bacterial strain obtains the culture containing fragrance, the extracted obtained fragrance of culture.
4. preparation method according to claim 3, which is characterized in that the culture uses solid culture, the culture
The pH value of culture medium is 5.5~6.5, is made by millet, perlite and nutrient solution；
The nutrient solution include murphy juice and：
Glucose 10g/L~120g/L；
Defatted soy flour 100g/L~280g/L；
Peptone 20g/L~200g/L；
Potassium dihydrogen phosphate 6g/L~22g/L；
Magnesium sulfate 3g/L~6g/L.
5. preparation method according to claim 3, which is characterized in that the condition of the culture is dark, and temperature is 23 DEG C
~28 DEG C, the time is 5~10 days.
6. preparation method according to claim 3, which is characterized in that the solvent of the extraction be selected from water, alcohols, acetone,
Chloroform, ether, benzene or petroleum ether.
7. preparation method according to claim 3, which is characterized in that the method for the extraction is refluxing extraction, and temperature is
50 DEG C~60 DEG C, the time is 6~8h；Then extracting solution is collected, after filtering, concentrating, with the ethyl alcohol of 90vol%~100vol%
Washing is filtered, is concentrated, fragrance is made again.
8. a kind of fragrance, which is characterized in that by deposit number be CCTCC NO：The Moschus enzyme bacterial strain of M 2016759 is made, wherein
Including isobutyric acid, methyl isobutyrate, α-guaiene, δ-guaiene, guaiaci lignum -9,11- alkene, Pogostol.
Further include 9. fragrance according to claim 8, in the fragrance beta-elemene, β-carypohyllene, isoamyl acetate,
(-)-β-chamigrene, ledene.
10. the application of fragrance described in claim 8 or 9 as the additive of food, cigarette, drug or cosmetics.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108795772A (en) * | 2017-04-28 | 2018-11-13 | 贵州中烟工业有限责任公司 | Moschus trichoderma strain and its fragrance of preparation |
| CN108795771A (en) * | 2017-04-28 | 2018-11-13 | 贵州中烟工业有限责任公司 | Moschus trichoderma strain and its fragrance of preparation |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108795772A (en) * | 2017-04-28 | 2018-11-13 | 贵州中烟工业有限责任公司 | Moschus trichoderma strain and its fragrance of preparation |
| CN108795771A (en) * | 2017-04-28 | 2018-11-13 | 贵州中烟工业有限责任公司 | Moschus trichoderma strain and its fragrance of preparation |
| CN108795772B (en) * | 2017-04-28 | 2022-09-20 | 贵州中烟工业有限责任公司 | Musk mould strain and perfume prepared from same |
| CN108795771B (en) * | 2017-04-28 | 2022-09-20 | 贵州中烟工业有限责任公司 | Musk mould strain and perfume prepared from same |
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