CN108795772A - Moschus trichoderma strain and its fragrance of preparation - Google Patents

Moschus trichoderma strain and its fragrance of preparation Download PDF

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CN108795772A
CN108795772A CN201710291648.2A CN201710291648A CN108795772A CN 108795772 A CN108795772 A CN 108795772A CN 201710291648 A CN201710291648 A CN 201710291648A CN 108795772 A CN108795772 A CN 108795772A
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fragrance
culture
moschus
preparation
enzyme
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CN108795772B (en
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胡世龙
刘剑
谢顺萍
陈海江
邹西梅
赵新海
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China Tobacco Guizhou Industrial Co Ltd
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    • 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
    • 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
    • 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
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    • C11B9/00—Essential oils; Perfumes
    • C11B9/0042—Essential oils; Perfumes compounds containing condensed hydrocarbon rings
    • 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
    • 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
    • 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
    • C11B9/0061—Essential oils; Perfumes compounds containing a six-membered aromatic ring not condensed with another ring
    • 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
    • C11B9/0069—Heterocyclic compounds
    • C11B9/0096—Heterocyclic compounds containing at least two different heteroatoms, at least one being nitrogen

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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.N-L-7 of endogenetic fungus Moschus, is rich in a variety of volatile components in cultured products, can be used to prepare natural perfume material.

Description

Moschus trichoderma strain and its fragrance of preparation
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 2016758.
The mould Muscodor sp.N-L-7 of Moschus provided by the invention are isolated under Yunnan Province of China Na Banhe acquisition wild states Eleusine indica, 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 2016758 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 CCTCCNO:M 2016758 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 2016758 is trained It supports, including β-farnesene, Pogostol, β-carypohyllene, α-guaiene and δ-guaiene.
Further include 3- isopropyl -6,8a- dimethyl -1,2,4,5,8,8a- hexahydros Azulene, epoxy water in fragrance of the present invention Calamus alkene, (3aS, 8aS) -6,8a- dimethyl -3- (propyl- 2- second diene) -1,2,3,3a, 4,5,8,8a- octahydro Azulene.
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.N-L-7 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.N-L-7 were preserved in Chinese Typical Representative culture on December 16th, 2016 Object collection (CCTCC), address is：Wuhan, China Wuhan University.Deposit number is CCTCC NO:M 2016758.
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 the mould Muscodor sp.N-L-7 bacterium colony figures of Moschus, and the specially mould Muscodor sp.N-L-7 of Moschus are in PDA The bacterium colony full face of growth 7 days, white mycelium are radially grown；
Fig. 2 shows the mould Muscodor sp.N-L-7 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.N-L-7 of Moschus.Depositary institution：China typical culture collection center；It protects Tibetan CCTCC NO：M 2016758；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.N-L-7 bacterial strains obtains
Eleusine indica under Yunnan Province of China Na Banhe acquisition wild states, the eleusine indica of acquisition is done with tap water rinse Only, eleusine indica blade sterilizes 30 seconds in 75% (v/v) alcoholic solution, then sterilizes 10 in 1% (v/v) liquor natrii hypochloritis Divide kind, rinsed with sterile water 3 times.Blade is cut into the segment of about 0.6cm long with sterile razor blade, is placed in green containing 50 μ gml-1 ammonia benzyls On 2% wort agar culture medium (MEA) plate of mycin and 50 μ gml-1 streptomysins, 25 DEG C of light cultures wait for that mycelia grows Afterwards, by mycelia top transposing to PDA culture medium；Isolated bacteria colony white, radially spreads, 25 DEG C of light cultures, is trained in PDA It supports and is continuously inoculated with three times on base, single bacterium colony is confirmed as, without other fungies or bacterium syntrophism, it is believed that be to obtain pure training It supports, is named as Muscodor sp.N-L-7.
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.N-L-7 are seeded on PDA plate 25 DEG C of dark culturings one week.It is few with transfer needle picking Mycelia block is measured in 1.5ml sterile centrifugation tubes, each 5 of the steel ball of diameter 1mm and 5mm is put into, is impregnated 1 minute in liquid nitrogen, with Afterwards sample is fully ground on JXFSTPRP refrigeration grinding machines (Shanghai Jing Xin Science and Technology Ltd.s).Use fungal genomic DNA Rapid extraction kit (Axygen) extracts Muscodor sp.N-L-7 genomic DNAs.400 μ l AP1 and 4 μ l are added RNaseA (10mg/ml), vortex oscillation.It is then incubated 20 minutes in 65 DEG C of water-baths, abundant lysed sample.130 μ l AP2 are added, It mixes well, is placed 5 minutes on ice bath.14,000rpm centrifugation 8 minutes, careful Aspirate supernatant to a new 1.5ml without In bacterium centrifuge tube, 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 liquors It being added in an adsorption column AC (adsorption column is placed in collecting pipe), the waste liquid in collecting pipe is outwelled in 13,000rpm centrifugations 40 seconds, It repeats the above steps until adding all mixed liquors.600 μ l rinsing liquids WB are added in adsorption column, 12,000rpm centrifuge 30 seconds, Abandon waste liquid.Adsorption column AC is placed back in collecting pipe, 13,000rpm centrifugations 2 minutes, remaining rinsing liquid of going out as possible.It takes Go out adsorption column AC to be put into new 1.5ml sterile centrifugation tubes, 100 μ l elution buffer EB be added at the intermediate position of adsorbed film, 5 minutes are placed at room temperature for, 12,000rpm centrifugations 1 minute obtain DNA sample, saved backup at -20 DEG C.
1.1.2PCR amplification
Ribosomes transcribed spacer (ITS) gene order of bacterial strain Muscodor sp.N-L-7 is measured.Bacterial strain The PCR forward direction amplimers ITS1-F of the ITS genes of Muscodor sp.N-L-7：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.
1.1.3PCR the purifying (being purified with AxyPrep DNA Gel Extraction Kit kits) of 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.N-L-7, 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 1.2Muscodor sp.N-L-7 bacterial strains
The bacterial strain of above-mentioned gained has carried out following preservation：Muscodor sp.N-L-7, 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 2016758。
1.2.1Muscodor the room temperature or Cord blood of sp.N-L-7 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.N-L-7 inoculations that will preserve to inclined-plane culture Base, after a couple of days, Muscodor sp.N-L-7 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.
1.2.2Muscodor the freezen protective of sp.N-L-7 bacterial strains
When Muscodor sp.N-L-7 grow animated period on PDA plate, it is placed on superclean bench, by the bacterium on PDA It falls and takes 4-5 ferfas blocks, be placed in 2ml sterile cryos and preserve pipe, autoclaved freezen protective liquid is added, by Muscodor Sp.N-L-7 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, is turned It moves to and carries out long-term preservation in -80 DEG C of ultra low temperature freezers；Or programmed cooling instrument is used, and so that it is unlikely to intracellular ice, it will Tissue temperature is dropped to be preserved suitable for -80 DEG C of ultra low temperature freezers.In use, the naturally to thaw in room temperature, picking fungus block therein connects Kind is activated in tablet PDA culture medium.
The mould Muscodor sp.N-L-7 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 is preserved with the inclined-planes a small amount of Moschus of sterilizing toothpick picking mould Muscodor sp.N-L-7PDA, is seeded in (25 DEG C, for 24 hours dark) activation culture 7d in PDA culture medium plate, 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.N-L-7 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.N-L-7 natural perfume materials of Moschus
The present embodiment using ethyl alcohol as solvent, the 200g solid culture products of Muscodor sp.N-L-7 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.N-L-7 absolute oil composition measurements
The absolute oil that endogenetic fungus Muscodor sp. bacterial strains N-L-7 is obtained uses Solid Phase Extraction/gas chromatography/mass spectrometry technology (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.N-L-7 absolute oil main components of Moschus
Sensory evaluation shows that the elegant fragrance of a flower of the mould Muscodor sp.N-L-7 absolute oil homophonies of Moschus and root of Aucklandia lappa Decne, including β-gold close The substances such as joyous alkene, Pogostol, β-carypohyllene, α-guaiene and δ-guaiene.Key component is：(1) β-Acacia Alkene, content ratio 15.6601%, there is the green fragrant, fragrance of a flower and with face cream fragrance, it is lasting to retain in being adjusted in preceding reconciliation, main It is naturally occurring in the essential oils such as orange oil, attar of rose, Java Cananga Oil and citrus seed oil, is present in main flume in tobacco leaf application In, it is insoluble in water, is dissolved in ethyl alcohol, ether, chloroform, can be mixed with most of other fragrance；(2) Pogostol (content 1.8537%), α-guaiene (content 0.8366%) and δ-guaiene (content 2.4282%) fragrance are light, graceful Root of Aucklandia lappa Decne flavor be present in absolute oil and adjust, be naturally occurring in patchouli oil and other essential oils.(3) β-carypohyllene 0.3513%, With pungent perfume, the cloves fragrance that root of Aucklandia lappa Decne, citrus is fragrant, camphor is fragrant, mild, it is naturally occurring in lemon, garden shaddock, nutmeg, pepper, covers In basin, blackcurrant, cinnamon leaves oil, clove leaf oil.For allocating the food flavors such as cloves, pepper, nutmeg, citrus, medicinal herbs. It can also be used for synthesizing other fragrance, such as more valuable for synthesizing acetyl group carypohyllene.β-carypohyllene carries wooden fragrance Perfume effect makes its more lasting reservation.(4) other substances：3- isopropyl -6,8a- dimethyl -1,2,4,5,8,8a- hexahydro Azulene (content 27.6447%), epoxy calerene (content 12.0190%), (3aS, 8aS) -6,8a- dimethyl -3- (propyl- 2- second Diene) -1,2,3,3a, 4,5,8,8a- octahydro Azulene (content 23.6556%) do not analyze fragrance and characteristic at present, need follow-up It was found that and developing and using.
4 petroleum ether extraction of embodiment prepares the mould Muscodor sp.N-L-7 natural perfume materials of Moschus
The present embodiment using petroleum ether as solvent, the 200g solid culture products of Muscodor sp.N-L-7 mould to Moschus It is extracted, method is as follows：It is dry to collect the mould Muscodor sp.N-L-7 solid state rheology objects of Moschus；Add 1L petroleum ethers, extraction temperature Spend 55 DEG C of 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, mistake Filter out impurity；Absolute oil is made in concentration.The absolute oil component that the present embodiment obtains is consistent with embodiment 3, and key component includes in absolute oil The substances such as β-farnesene, Pogostol, β-carypohyllene, α-guaiene and δ-guaiene.
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> MP1704719
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 522
<212> DNA
<213> Muscodor sp.
<400> 1
cattacagag ttttctaaac tcccaaccct atgtgaactt acctttgttg cttcggcggc 60
ggaggctacc ctgcggggga ttaccactta gtgattaccc tgcagtccca ggtacatttg 120
ttacccggta gtcatccccg ccggcggcca actaaactct gttttctttg gaattctgaa 180
tcataaactt aattagttaa aactttcaac aacggatctc ttggttctgg catcgatgaa 240
gaacgcagcg aaatgcgata agtaatgtga attgcagaat tcagtgaatc atcgaatctt 300
tgaacgcaca ttgcgcccat tagcattcta gtgggcatgc ctgttcgagc gtcatttcac 360
cacttaagcc ctgttgctta gcgttgggag cctacggcat agcccgtagc tccttaaagt 420
gattggcgga gttggttctc actctaagcg tagtaactat atctcgcttt tgtagtggtt 480
ccggcccctt gccgtaaaac ccccttatat aaagggttga cc 522

Claims (10)

1. deposit number is CCTCC NO:The Moschus enzyme bacterial strain of M 2016758.
2. deposit number is CCTCC NO:Application of the Moschus enzyme bacterial strain of M 2016758 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 bacterium of M2016758 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 2016758 is made, wherein Including β-farnesene, Pogostol, β-carypohyllene, α-guaiene and δ-guaiene.
Further include 3- isopropyl -6,8a- dimethyl -1,2 9. fragrance according to claim 8, in the fragrance, 4,5,8, 8a- hexahydros Azulene, epoxy calerene, (3aS, 8aS) -6,8a- dimethyl -3- (propyl- 2- second diene) -1,2,3,3a, 4,5,8, 8a- octahydro Azulene.
10. the application of fragrance described in claim 8 or 9 as the additive of food, cigarette, drug or cosmetics.
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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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Publication number Priority date Publication date Assignee Title
CN108795773A (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
CN108795773B (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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