CN105713846B - A kind of trichoderma aureoviride bacterial strain and the method with its preparation rose tobacco aromatics - Google Patents
A kind of trichoderma aureoviride bacterial strain and the method with its preparation rose tobacco aromatics Download PDFInfo
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- CN105713846B CN105713846B CN201610240445.6A CN201610240445A CN105713846B CN 105713846 B CN105713846 B CN 105713846B CN 201610240445 A CN201610240445 A CN 201610240445A CN 105713846 B CN105713846 B CN 105713846B
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- 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
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- 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
- C12R2001/885—Trichoderma
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
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Abstract
The invention discloses a kind of trichoderma aureoviride bacterial strain and the methods for preparing rose tobacco aromaticss with it.The bacterial strain is trichoderma aureoviride (Trichoderma aureoviride) HLMM-04 CGMCC No.10839, has cellulase-producing, and then promote cellulose decomposition at the ability of oligosaccharides or monosaccharide.Bacterial strain is passed through into activation, is fermented, filtering, obtained cellulase crude enzyme liquid of dialysing, tobacco aromaticss are made for the Shangri-la Wild Rosa multiflora that ferments, then extracted, filtering, reduced pressure.The Liquid fermentation condition of HLMM-04 bacterial strain is that the initial pH of culture medium is 6.5, and cultivation temperature is 28 DEG C, incubation time 5d.Perfuming cigarette the result shows that, prepared fragrance has abundant fragrance, the effect of the prominent fragrance of a flower.
Description
Technical field
The invention belongs to microbial fermentation and its application fields, and in particular to one plant of trichoderma aureoviride new strains HLMM-04, together
When, the invention further relates to the methods using bacterial strain preparation rose tobacco aromaticss.
Background technique
Flavoring and casing is the key link in cigarette process, is effective promotion cigarette smoking quality, forms the peculiar wind of cigarette
One of effective means of lattice.The tobacco aromaticss added in China tobacco at present are deployed again using conventional method extraction mostly
Gained there is also fragrance dullness, improves the disadvantages of cigarette smoking effect is unobvious although production operation is simple.Utilize micro- life
Object, which ferments to perfume material, can increase the content that tradition extracts a variety of aroma substances in fragrance, and promotion fragrance is rich, into
And produce that fragrance is abundant, cigarette of high-quality, meet requirement of the consumer to cigarette quality.
Trichoderma (Trichoderma spp.) is distributed widely in nature, rotten wood, seed, plant residue, organic fertilizer,
It is outstanding more in soil and air.Trichoderma growing nutrient requires simply, and the speed of growth is fast, highly beneficial for industrialized production.It is yellow
Green trichoderma (Trichoderma aureoviride) is one kind of trichoderma, as a kind of important bio-control factors, to phytopathy
Former antagonism has broad spectrum activity, property mostly processed, moreover it is possible to generate the plant and animal residues in a variety of enzymes decomposition soil, increase soil
Fertility.Have to the report of trichoderma aureoviride focus mostly on soil improvement, biological control and in terms of.In the prior art
There is not yet trichoderma aureoviride is used for the report that rose fermentation prepares tobacco aromaticss.
Summary of the invention
In view of the above-mentioned deficiencies in the prior art, it is an object of the present invention to the new strains of one plant of trichoderma aureoviride are provided, bacterial strain tool
There is cellulase-producing, and then promotes cellulose decomposition at the ability of oligosaccharides or monosaccharide.
The object of the invention is also to provide a kind of methods for preparing tobacco aromaticss using the strain fermentation rose.
The purpose of the present invention is achieved by the following technical programs.
Unless otherwise indicated, percentage of the present invention is mass percent.
A kind of trichoderma aureoviride (Trichoderma aureoviride) HLMM-04 bacterial strain, CGMCC No.10839, tool
There is cellulase-producing, and then promotes cellulose decomposition at the ability of oligosaccharides or monosaccharide.
The method for preparing tobacco aromaticss using the bacterial strain, comprising the following steps:
(1) it by after the activation twice of trichoderma aureoviride HLMM-04 CGMCC No.10839 bacterial strain, is inoculated in equipped with 200mL PDA
In the triangular flask of fluid nutrient medium, at 20~35 DEG C, 1~7d of fermented and cultured under 180rpm；The PDA liquid medium: horse
Bell potato 200g, glucose 20g mend distilled water to 1L, pH5.5~8.5,115 DEG C of high pressure sterilization 15min；
(2) filtering fermentation liquor removes thallus, takes supernatant to be slowly added to sulfate of ammoniac to 80% saturation degree, stood at 4 DEG C
Night is collected by centrifugation sediment and is dissolved in 2mL buffer, then dialysed with buffer, obtains crude enzyme liquid；The buffer
Are as follows: 0.02mol/L Na2HPO4-NaH2PO4, pH 7.3；
(3) Shangri-la Wild Rosa multiflora dried flower piece is carried out after being crushed, sprays trichoderma aureoviride HLMM-04 crude enzyme liquid, fermented,
Crude enzyme liquid additive amount is the 20% of Wild Rosa multiflora quality, and fermentation temperature is 30 DEG C, 2~5h of fermentation time；Add double weight
Water, refluxing extraction 3h, filtering and removing slag, by supernatant be concentrated under reduced pressure to get required tobacco aromaticss.
Further, to prevent fragrance rotten, propylene glycol is added by the 5% of tobacco aromaticss weight.
In step (1) optimum condition of enzyme production be initial pH 6.5,28 DEG C of cultivation temperature, incubation time 5d.
Compared with the existing technology, the invention has the following advantages that
(1) trichoderma aureoviride is applied to tobacco aromaticss preparation field for the first time by the present invention.Using trichoderma aureoviride HLMM-04 bacterial strain
Fermentation liquor treatment Shangri-la Wild Rosa multiflora is protected compared with untreated control in original Shangri-la Wild Rosa multiflora characteristic perfume
While holding, the fragrance of a flower is prominent, the rich increase of fragrance.
(2) present invention is low in cost, easy to operate, is easy to implement large-scale industrial production, application prospect is good.
The explanation of preservation biomaterial
It is common to be deposited in China Committee for Culture Collection of Microorganisms to bacterial strain of the invention on May 22nd, 2015
Microorganism center (CGMCG)；The centre address: city, BeiJing, China, North Star West Road 1, Chaoyang District institute 3, the micro- life of the Chinese Academy of Sciences
Object research institute.The strain classification is named as trichoderma aureoviride (Trichoderma aureoviride) HLMM-04, and deposit number is
CGMCC No.10839。
Specific embodiment
Below by embodiment, the present invention is described in further detail, but embodiment is not to the technology of the present invention side
The restriction of case.
Embodiment 1
1, the suitable condition of enzyme production of trichoderma aureoviride HLMM-04 bacterial strain
Influence of the initial pH of 1.1 culture mediums to HLMM-04 strain enzyme-producing
The pH of fluid nutrient medium is transferred to 5.5,6.0,6.5,7.0,7.5,8.0,8.5 respectively, is shaken at 28 DEG C, 120rpm
After swinging culture 3 days, cellulase activity is measured, shows that optimum initial pH is 6.5.
Influence of 1.2 cultivation temperatures to HLMM-04 strain enzyme-producing
Liquid fermentation medium after inoculation is respectively placed at 20 DEG C, 25 DEG C, 28 DEG C, 32 DEG C, 35 DEG C, 120r/min vibration
After swinging culture 3d, cellulase activity is measured, shows that optimum cultivation temperature is 28 DEG C.
Influence of 1.3 incubation times to HLMM-04 strain enzyme-producing
Inoculated fluid nutrient medium (pH 6.5) is placed in climatic chamber, 28 DEG C, 120r/min shaken cultivation, often
Primary fiber element enzyme activity is measured for 24 hours, and cellulase activity reaches maximum value when culture was by the 5th day.
2, trichoderma aureoviride HLMM-04 crude enzyme liquid preparation method
It is inoculated in after the activation twice of trichoderma aureoviride HLMM-04 bacterial strain equipped with 200mL PDA according to the above optimum results
In the triangular flask of fluid nutrient medium (pH 6.5), at 28 DEG C, fermented and cultured 5d under 180rpm.Filtering removal thallus.Take supernatant
Sulfate of ammoniac is slowly added to 80% saturation degree, is stood overnight at 4 DEG C, sediment is collected by centrifugation and is dissolved in 2mL buffer
(0.02mol/L Na2HPO4-NaH2PO4Buffer, pH 7.3) in, dialysed with same buffer, obtain thick enzyme
Liquid.
3, the method that trichoderma aureoviride HLMM-04 strain fermentation prepares tobacco aromaticss
Shangri-la Wild Rosa multiflora dried flower piece is carried out after being crushed, trichoderma aureoviride HLMM-04 crude enzyme liquid is sprayed, is fermented, slightly
Enzyme solution additive amount is the 20% of Wild Rosa multiflora quality, and fermentation temperature is 30 DEG C, fermentation time 3h；The water of double weight is added,
Supernatant is concentrated under reduced pressure to get rose tobacco aromaticss for refluxing extraction 3h, filtering and removing slag.
To prevent from going bad, by 5% addition propylene glycol of fermentation fragrance weight.
4, product feature and physicochemical property
Gained ferments tobacco aromaticss for rufous stream shape lotion, does not clarify.Product physicochemical property is shown in Table 1.
The physical and chemical index of 1 trichoderma aureoviride HLMM-04 of table fermentation Wild Rosa multiflora fragrance
5, perfuming cigarette is tested
Shangri-la Wild Rosa multiflora fermentation tobacco aromaticss 0.1g is taken uniformly to be sprayed at 50g not after taking sterile water to dilute 50 times
Flavoring Ye Zuzhong is packed into clean hermetic bag, after placing 2h at room temperature, pipe tobacco is dried in 120 DEG C of baking ovens, makes moisture
Content reaches 11%~13%.Treated pipe tobacco and control are put into climatic chamber, in 22 DEG C of temperature, humidity 60%
It is balanced under part for 24 hours, rolls into cigarette by hand, sensory evaluating smoking is carried out by the group of smokeing panel test that the expert that smokes panel test forms.With rose of not fermenting
It extracts fragrance and fermentation of bacillus subtilis rose fragrance is control.Flavoring the result shows that, the yellowish green wood of Shangri-la Wild Rosa multiflora
Mould HLMM-04 fermentation fragrance and control area are obvious, have abundant fragrance, the effect of the prominent fragrance of a flower.Evaluation result is shown in Table 2.
2 Shangri-la Wild Rosa multiflora of table fermentation fragrance smoking result
Claims (2)
1. a kind of preparation method of rose tobacco aromaticss, comprising the following steps:
(1) trichoderma aureoviride (Trichoderma aureoviride) HLMM-04 for being CGMCC No. 10839 by deposit number
After bacterial strain activation twice, it is inoculated in the triangular flask equipped with 200 mL PDA liquid mediums, in initial pH 6.5, cultivation temperature
28 DEG C, fermented and cultured 5d under 180 rpm；The PDA liquid medium: potato 200g, glucose 20g mend distilled water to 1
L, pH5.5~8.5,115 DEG C of 15 min of high pressure sterilization；10839 bacterial strain of trichoderma aureoviride HLMM-04 CGMCC No., tool
There is cellulase-producing, and then promotes cellulose decomposition at the ability of oligosaccharides or monosaccharide；
(2) filtering fermentation liquor removes thallus, takes supernatant to be slowly added to ammonium sulfate to 80% saturation degree, stands overnight at 4 DEG C, from
The heart is collected sediment and is dissolved in 2 mL buffers, then is dialysed with buffer, obtains crude enzyme liquid；The buffer are as follows:
0.02 mol/L Na2HPO4-NaH2PO4 , pH 7.3；
(3) Shangri-la Wild Rosa multiflora dried flower piece is carried out after being crushed, sprays trichoderma aureoviride HLMM-04 crude enzyme liquid, fermentation, thick enzyme
Liquid additive amount is the 20% of Wild Rosa multiflora quality, and fermentation temperature is 30 DEG C, 2~5h of fermentation time；The water of double weight is added,
Supernatant is concentrated under reduced pressure to get required rose tobacco aromaticss for refluxing extraction 3h, filtering and removing slag.
2. rose tobacco aromaticss preparation method according to claim 1, it is characterised in that: add by the 5% of tobacco aromaticss weight
Add propylene glycol.
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