CN105733960B - A kind of trichoderma aureoviride bacterial strain and the method for preparing Dian clove tobacco aromaticss with it - Google Patents
A kind of trichoderma aureoviride bacterial strain and the method for preparing Dian clove tobacco aromaticss with it Download PDFInfo
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- CN105733960B CN105733960B CN201610239786.1A CN201610239786A CN105733960B CN 105733960 B CN105733960 B CN 105733960B CN 201610239786 A CN201610239786 A CN 201610239786A CN 105733960 B CN105733960 B CN 105733960B
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- tobacco aromaticss
- bacterial strain
- dian clove
- trichoderma aureoviride
- fermentation
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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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- 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
- 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
Abstract
The invention discloses a kind of trichoderma aureoviride bacterial strain and the methods for preparing Dian clove 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 Dian clove 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, the miscellaneous gas of prepared fragrance and irritation are reduced, and have the function of improvement throat's comfort.
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 for preparing Dian clove tobacco aromaticss using the bacterial strain.
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.
Dian clove (Luculia pinceana Hook.), also known as hiding cloves, clove leaf, cloves, cloves floral leaf, are evergreen
Shrub or dungarunga, flower, the fruiting period 3-11 month.Decoction is for oral administration, has relieving cough and reducing sputum and other effects.Cure mainly cough, pertussis, chronic branch
Tracheitis etc..
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 Dian clove 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 provide one plant of trichoderma aureoviride new strains, which has
Cellulase-producing, and then promote 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 Dian clove.
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-04CGMCC 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) Dian clove branches and leaves decontamination is cleaned, the water of double weight is added after being crushed, is uniformly mixed, adjusts pH to 6.0；Add
Enter trichoderma aureoviride HLMM-04 crude enzyme liquid, ferment, crude enzyme liquid additive amount is the 20% of Dian clove quality, and fermentation temperature is 30 DEG C, hair
Ferment time 3h；Supernatant is concentrated under reduced pressure to get required tobacco aromaticss for refluxing extraction 3h, filtering and removing slag.
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 Dian clove, compared with untreated control, while original Dian clove feature is kept, miscellaneous gas and irritation subtract
It is few, have the function of improving throat's comfort.
(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 of BeiJing, China Chaoyang District North Star West Road 1 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.
1.2 influence of the cultivation temperature 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
Dian clove branches and leaves decontamination is cleaned, the water of double weight is added after being crushed, is uniformly mixed, adjusts pH to 6.0；It is added yellow
Green trichoderma HLMM-04 crude enzyme liquid, fermentation；Crude enzyme liquid additive amount is the 20% of Dian clove quality, and fermentation temperature is 30 DEG C, when fermentation
Between 3h；Supernatant is concentrated under reduced pressure to get Dian clove 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 liquid, 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 Dian clove fragrance
5, perfuming cigarette is tested
Dian clove fermentation tobacco aromaticss 0.1g is taken uniformly to be sprayed at the non-flavoring leaf group of 50g after taking sterile water to dilute 50 times
In, it is packed into clean hermetic bag, after placing 2h at room temperature, pipe tobacco is dried in 120 DEG C of baking ovens, reaches moisture content
11%~13%.Treated pipe tobacco and control are put into climatic chamber, balanced under the conditions of 22 DEG C of temperature, humidity 60%
For 24 hours, cigarette is rolled by hand, and sensory evaluating smoking is carried out by the group of smokeing panel test that the expert that smokes panel test forms.It is extracted with the Dian clove that do not ferment fragrant
Material and fermentation of bacillus subtilis Dian clove fragrance are control.Flavoring the result shows that, Dian clove trichoderma aureoviride HLMM-04 fermentation is fragrant
Material is obvious with control area, and miscellaneous gas and irritation are reduced, and has the function of improving throat's comfort.Evaluation result is shown in Table 2.
2 Dian clove of table fermentation fragrance smoking result
Claims (3)
1. a method of prepare Dian clove tobacco aromaticss, comprising the following steps:
(1) trichoderma aureoviride HLMM-04 (Trichoderma aureoviride) bacterium for being CGMCC No.10839 by deposit number
After strain activation twice, it is inoculated in the triangular flask equipped with 200mL PDA liquid medium, ferments under 20~35 DEG C, 180rpm
Cultivate 1~7d；The PDA liquid medium: potato 200g, glucose 20g, benefit distilled water to 1L, pH5.5~8.5,
115 DEG C of high pressure sterilization 15min；The trichoderma aureoviride HLMM-04 CGMCC No.10839 bacterial strain has cellulase-producing, into
And promote cellulose decomposition at the ability of oligosaccharides or monosaccharide；
(2) filtering fermentation liquor removes thallus, takes supernatant to be slowly added to sulfate of ammoniac to 80% saturation degree, stands overnight at 4 DEG C, from
The heart is collected sediment and is dissolved in 2mL buffer, then is dialysed with buffer, obtains crude enzyme liquid；The buffer are as follows:
0.02mol/L Na2HPO4-NaH2PO4, pH 7.3；
(3) Dian clove branches and leaves decontamination is cleaned, the water of double weight is added after being crushed, is uniformly mixed, adjusts pH to 6.0；It is added yellow
Green trichoderma HLMM-04 crude enzyme liquid, fermentation, crude enzyme liquid additive amount are the 20% of Dian clove quality, and fermentation temperature is 30 DEG C, when fermentation
Between 3h；Supernatant is concentrated under reduced pressure to get required tobacco aromaticss for refluxing extraction 3h, filtering and removing slag.
2. the method according to claim 1 for preparing Dian clove tobacco aromaticss, it is characterised in that: by tobacco aromaticss weight
5% addition propylene glycol.
3. the method according to claim 1 for preparing Dian clove tobacco aromaticss, it is characterised in that: described in step (1)
Fermentation culture conditions be initial pH 6.5,28 DEG C of cultivation temperature, incubation time 5d.
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| CN103478894A (en) * | 2013-09-27 | 2014-01-01 | 福建中烟工业有限责任公司 | Camellia extract composition with spicy fragrance and application of same to cigarettes |
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| CN104178341A (en) * | 2014-09-04 | 2014-12-03 | 南京标科生物科技有限公司 | Method for preparing microcapsule clove essential oil and application of clove essential oil in tobacco |
| CN105124736A (en) * | 2015-09-02 | 2015-12-09 | 云南中烟工业有限责任公司 | Method of promoting moisturizing and aroma enhancement effects of cigarettes by means of cloves |
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2016
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| CN103266014A (en) * | 2013-05-29 | 2013-08-28 | 江苏中烟工业有限责任公司徐州卷烟厂 | Spicy sweet flue-cured tobacco essence and application thereof |
| CN103478894A (en) * | 2013-09-27 | 2014-01-01 | 福建中烟工业有限责任公司 | Camellia extract composition with spicy fragrance and application of same to cigarettes |
| CN103525553A (en) * | 2013-10-23 | 2014-01-22 | 红云红河烟草(集团)有限责任公司 | Tobacco and spice plant mixing essence and application thereof |
| CN104178341A (en) * | 2014-09-04 | 2014-12-03 | 南京标科生物科技有限公司 | Method for preparing microcapsule clove essential oil and application of clove essential oil in tobacco |
| CN105124736A (en) * | 2015-09-02 | 2015-12-09 | 云南中烟工业有限责任公司 | Method of promoting moisturizing and aroma enhancement effects of cigarettes by means of cloves |
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