CN105733962B - A kind of trichoderma aureoviride HLMM-04 bacterial strain and the method for preparing tobacco aromaticss - Google Patents
A kind of trichoderma aureoviride HLMM-04 bacterial strain and the method for preparing tobacco aromaticss Download PDFInfo
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- CN105733962B CN105733962B CN201610239982.9A CN201610239982A CN105733962B CN 105733962 B CN105733962 B CN 105733962B CN 201610239982 A CN201610239982 A CN 201610239982A CN 105733962 B CN105733962 B CN 105733962B
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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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- 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
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Abstract
The invention discloses a kind of trichoderma aureoviride HLMM-04 bacterial strain and the methods for preparing tobacco aromaticss.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 loquat of fermenting, loguat leaf, lily, hawthorn, sterculia seed mixture, then extracted, filtering, reduced pressure.The Liquid fermentation condition of HLMM-04 bacterial strain be the initial pH6.5 of culture medium, 28 DEG C of cultivation temperature, incubation time 5d.Flavoring the result shows that, prepared fragrance have reduce stimulation, improve throat's comfort and pleasant impression effect.
Description
Technical field
The invention belongs to microbial fermentation and its application fields, and in particular to one plant of trichoderma aureoviride new strains.Meanwhile this hair
The bright method for further relating to that there are the tobacco aromaticss for improving the effect of throat's comfort using bacterial strain preparation.
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 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.
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) loquat, loguat leaf, lily, hawthorn, the sterculia seed 2:1:1:1:2 in mass ratio are mixed, decontamination is cleaned, and is crushed
The water of double weight is added afterwards, is uniformly mixed, adjusts pH to 6.0；Trichoderma aureoviride HLMM-04 crude enzyme liquid, fermentation is added, crude enzyme liquid adds
Dosage is the 5%~20% of plant material quality, and fermentation temperature is 30 DEG C, 2~5h of fermentation time；Refluxing extraction 3h is crossed and is filtered out
Supernatant is concentrated under reduced pressure to get required tobacco aromaticss 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) present invention is compounded loquat, loguat leaf, lily, hawthorn, the sterculia seed by proper proportion, and exploitation, which has, to be changed
The tobacco aromaticss of kind throat's comfort effect, have and reduce throat's stimulation, promote the production of body fluid, the effect of comfortable pleasant impression.
(2) trichoderma aureoviride is applied to tobacco aromaticss preparation field for the first time.Using trichoderma aureoviride HLMM-04 bacterial strain fermentation liquor
Loquat, loguat leaf, lily, hawthorn, sterculia seed compound are handled, compared with non-extractive from fermentative, miscellaneous gas is reduced, and fragrance is more
Add mellow and full improvement result increase to throat's comfort.
(3) 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
Loquat, loguat leaf, lily, hawthorn, the sterculia seed 2:1:1:1:2 in mass ratio are mixed, decontamination is cleaned, and is added after being crushed
Enter the water of double weight, be uniformly mixed, adjusts pH to 6.0.Trichoderma aureoviride HLMM-04 crude enzyme liquid, fermentation is added.Crude enzyme liquid additive amount
It is the 15% of vegetable material (loquat, loguat leaf, lily, hawthorn, sterculia seed mixture) quality, fermentation temperature is 30 DEG C, fermentation
Time 3h.Supernatant is concentrated under reduced pressure to get fermentation 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 fragrance
5, perfuming cigarette is tested
It takes fermentation tobacco aromaticss 0.1g to be uniformly sprayed at the non-flavoring Ye Zuzhong of 50g after taking sterile water to dilute 50 times, is packed into
In clean hermetic bag, after placing 2h at room temperature, pipe tobacco is dried in 120 DEG C of baking ovens, moisture content is made to reach 11%-
13%.Treated pipe tobacco and control are put into climatic chamber, balanced for 24 hours under the conditions of 22 DEG C of temperature, humidity 60%, hand
Work rolls into cigarette, carries out sensory evaluating smoking by the group of smokeing panel test that the expert that smokes panel test forms.Fragrance is extracted with mixing of not fermenting as control.
Flavoring the result shows that, trichoderma aureoviride HLMM-04 ferment fragrance and control area it is obvious, miscellaneous gas is reduced, the increase of throat's comfort, remaining
Taste improves.Evaluation result is shown in Table 2.
The fermentation fragrance smoking result of table 2
Embodiment 2
Embodiment 1 is repeated, have following difference: crude enzyme liquid additive amount is the 5% of Yunnan olive quality, fermentation time 2h.
1, product feature and physicochemical property
Gained ferments tobacco aromaticss for rufous stream shape lotion, does not clarify.Product physicochemical property is shown in Table 3.
The physical and chemical index of 3 trichoderma aureoviride HLMM-04 of table fermentation fragrance
2, perfuming cigarette is tested
Flavoring the result shows that, trichoderma aureoviride HLMM-04 ferment fragrance and control area it is obvious, the rich increase of fragrance, larynx
Portion's comfort increases, and pleasant impression improves.Evaluation result is shown in Table 4.
The fermentation fragrance smoking result of table 4
Embodiment 3
Embodiment 1 is repeated, have following difference: crude enzyme liquid additive amount is the 20% of Yunnan olive quality, fermentation time 5h.
1, product feature and physicochemical property
Gained ferments tobacco aromaticss for rufous stream shape lotion, does not clarify.Product physicochemical property is shown in Table 5.
The physical and chemical index of 5 trichoderma aureoviride HLMM-04 of table fermentation fragrance
2, perfuming cigarette is tested
Flavoring the result shows that, trichoderma aureoviride HLMM-04 ferment fragrance and control area it is obvious, flue gas coordination, throat is comfortable
Property increase, stimulation reduce, pleasant impression improve.Evaluation result is shown in Table 6.
The fermentation fragrance smoking result of table 6
Claims (3)
1. a kind of method for preparing tobacco aromaticss, 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 liquid
In the triangular flask of culture medium, at 20~35 DEG C, 1~7d of fermented and cultured under 180rpm；The PDA liquid medium: potato
200g, glucose 20g mend 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, 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 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) loquat, loguat leaf, lily, hawthorn, the sterculia seed 2:1:1:1:2 in mass ratio are mixed, decontamination is cleaned, and is added after being crushed
Enter the water of double weight, be uniformly mixed, adjusts pH to 6.0；Trichoderma aureoviride HLMM-04 crude enzyme liquid, fermentation, crude enzyme liquid additive amount is added
It is the 5%~20% of plant material quality, fermentation temperature is 30 DEG C, 2~5h of fermentation time；Refluxing extraction 3h, filtering and removing slag will
Supernatant is concentrated under reduced pressure to get required tobacco aromaticss.
2. the method according to claim 1 for preparing tobacco aromaticss, it is characterised in that: to prevent fragrance rotten, used by cigarette
5% addition propylene glycol of fragrance weight.
3. the method according to claim 1 for preparing tobacco aromaticss, it is characterised in that: optimum fermentation in step (1)
Condition of culture be initial pH 6.5,28 DEG C of cultivation temperature, incubation time 5d.
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| CN108753839A (en) * | 2018-05-25 | 2018-11-06 | 颐中（青岛）实业有限公司 | A kind of preparation method for the filter material of cigarette that there is drop evil to protect lung function |
| CN109097404A (en) * | 2018-08-23 | 2018-12-28 | 江西中烟工业有限责任公司 | A kind of preparation and its application of the dry composite fermentation extract of loquat |
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| CN104232689A (en) * | 2014-09-30 | 2014-12-24 | 云南中烟工业有限责任公司 | Tobacco flavor prepared by blending and fermenting lily and tobacco |
| CN104388191A (en) * | 2014-11-06 | 2015-03-04 | 云南中烟工业有限责任公司 | Fruit flavor for cigarettes and application of flavor |
| CN105105318A (en) * | 2015-10-09 | 2015-12-02 | 云南中烟工业有限责任公司 | Aroma brewing flavor for cigarettes and application thereof in improving smoking quality of cigarettes |
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|---|---|---|---|---|
| CN1335388A (en) * | 2000-07-25 | 2002-02-13 | 玉溪红塔烟草(集团)有限责任公司 | Microbe TV-1 and its prepn |
| CN104232689A (en) * | 2014-09-30 | 2014-12-24 | 云南中烟工业有限责任公司 | Tobacco flavor prepared by blending and fermenting lily and tobacco |
| CN104388191A (en) * | 2014-11-06 | 2015-03-04 | 云南中烟工业有限责任公司 | Fruit flavor for cigarettes and application of flavor |
| CN105105318A (en) * | 2015-10-09 | 2015-12-02 | 云南中烟工业有限责任公司 | Aroma brewing flavor for cigarettes and application thereof in improving smoking quality of cigarettes |
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