Trichoderma longibrachiatum strain as well as preparation method and application thereof

Abstract

The invention relates to a trichoderma longibrachiatum strain, the preservation number of which is CGMCC NO: 41364, the trichoderma longibrachiatum strain can be applied to preparation of cigarette perfume, the trichoderma longibrachiatum strain is activated and cultured, when green spores grow out from a slant solid culture medium, sterile water is taken and added into a slant test tube, the spores on the slant solid culture medium are taken down and dissolved in the sterile water of the slant test tube, and the spores on the slant solid culture medium are dissolved in the sterile water of the slant test tube; and diluting the spore suspension, and inoculating the diluted spore suspension into a fermentation culture medium for fermentation. According to the trichoderma longibrachiatum strain provided by the embodiment of the invention, the trichoderma longibrachiatum strain can be applied to the field of preparation of cigarette perfume, the application range of trichoderma is widened, waste tobacco of a cigarette factory can be utilized as the tobacco extracting solution, a new way is provided for recycling of the waste tobacco, and the cigarette perfume prepared from the trichoderma longibrachiatum strain by utilizing tobacco products has the advantages that the cigarette perfume has a good application prospect. The negative influence on the taste caused by reduction of the tar content can be effectively improved, the smoking taste of the cigarette is improved, and the smoking quality of the cigarette is improved.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and in particular to a long-branch Trichoderma strain and a preparation method and application thereof. Background Art

[0002] In the cigarette industry, cigarette products are constantly being improved. For example, the tar content of cigarette products is constantly being reduced to achieve tar reduction and harm reduction. While reducing the tar content, it is bound to affect the smoking taste and aroma of cigarette products, so the aroma and taste of cigarette products must be improved accordingly. The improvement method is often achieved by adding flavors to cigarette products. The addition of flavors can make up for the insufficient taste caused by the reduction in tar content. The flavors currently used in cigarette products include natural flavors and synthetic flavors. Microbial fermentation to prepare flavors is an important way to obtain natural flavors, and compared with synthetic flavors, the aroma of flavors prepared by microbial fermentation is more saturated and the production cycle is short. In the field of microorganisms, Trichoderma is a fungus widely used in agriculture, pharmaceuticals and other industries. There has been no relevant research and discovery on the use of Trichoderma in the preparation of cigarette flavors. Summary of the invention

[0003] The purpose of the present application is to provide a long-branch Trichoderma strain and a preparation method and application thereof. The long-branch Trichoderma strain can prepare cigarette flavors, improve the aroma quality of cigarettes, and thus effectively solve the shortcomings of the prior art.

[0004] To this end, the present application provides a long-branch Trichoderma strain, whose preservation number is CGMCC NO: 41364.

[0005] The present application also provides a method for preparing a long-branch Trichoderma strain, comprising the following steps:

[0006] A. Place tea samples in sterile physiological saline, dilute by shaking, and then spread on YPD solid medium to obtain primary screening strains;

[0007] B. The primary screening strain obtained in step A is activated and cultured in YPD liquid medium, and the mycelium is picked or the bacterial precipitate is obtained by centrifugation and inoculated into the secondary screening fermentation medium for fermentation.

[0008] In some possible implementations, in step B, the YPD liquid culture medium includes, by mass percentage, 2% peptone, 2% glucose, 1% yeast powder and 100 ml of deionized water.

[0009] In some possible implementations, in step A, the YPD solid culture medium includes, by mass percentage, 2% peptone, 2% glucose, 1% yeast powder, 100 ml deionized water and 2% agar.

[0010] In some possible implementations, in step A, the re-screening fermentation medium includes tobacco extract.

[0011] In some possible implementations, the tea sample is a green tea sample.

[0012] In some possible implementations, in the step A, the shaking is performed at 30° C. and on a shaker for 6-8 hours.

[0013] In some possible implementations, in step A, the dilution is carried out by 10 -0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 dilutions were performed in gradients.

[0014] The present application example also provides a use of a long-branch Trichoderma strain in the preparation of cigarette flavors.

[0015] The method for preparing cigarette flavoring material using the long-branch Trichoderma strain comprises:

[0016] The long-branched Trichoderma is activated and cultured to obtain a bacterial liquid and hyphae, which are inoculated into a slant solid culture medium for culture. When green spores grow on the slant solid culture medium, sterile water is added into a slant test tube, the spores on the slant solid culture medium are removed and dissolved in the sterile water in the slant test tube to prepare a spore suspension, which is diluted and inoculated into a fermentation medium for fermentation.

[0017] In some possible implementations, the spore concentration of the diluted spore suspension is 1×10 7 ~1.1×10 7 cfu / ml.

[0018] In some possible implementations, the inoculation amount of the bacterial liquid in the slant solid culture medium is 1 vol%.

[0019] In some possible implementations, the fermentation medium includes tobacco extract.

[0020] In some possible implementations, the fermentation time of the diluted spore suspension in the fermentation medium is 72 hours, and the fermentation is carried out at a rotation speed of 180 r / min.

[0021] The long-branch Trichoderma strain provided in the embodiments of the present application can be used in the preparation of cigarette flavors, broadening the application scope of the genus Trichoderma, and the tobacco extract can also utilize waste tobacco from cigarette factories, providing a new way for the recycling of waste tobacco. The cigarette flavors prepared by the long-branch Trichoderma strain using tobacco products can effectively improve the negative impact of taste caused by the reduction of tar content, improve the smoking taste of cigarettes, and help improve the smoking quality of cigarettes. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The colony morphology of the long-branch Trichoderma strain QF-1 provided in the examples of the present application and the microscopic observation of the colony morphology before and after staining;

[0023] Figure 2 An agarose gel electrophoresis diagram of the ITS gene of the long-branch Trichoderma strain QF-1 provided in the examples of the present application;

[0024] Figure 3 A phylogenetic tree of the long-branched Trichoderma strain QF-1 provided in the examples of the present application;

[0025] Figure 4 This is a graph showing the comparative analysis results of different types of components in the fermentation broth of the long-branch Trichoderma strain QF-1 provided in the examples of the present application and the control group. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0027] The present application example provides a long-branch Trichoderma strain, whose preservation number is CGMCC NO:41364.

[0028] The strain has been deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC), located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is June 14, 2024, the deposit number is CGMCCNO: 41364, and the classification name is Trichodema longibrachiatum.

[0029] The present application also provides a method for preparing a long-branch Trichoderma strain, comprising the following steps:

[0030] A. Place tea samples in sterile physiological saline, dilute by shaking, and then spread on YPD solid medium to obtain primary screening strains;

[0031] B. The primary screening strain obtained in step A is activated and cultured in YPD liquid medium, and the mycelium is picked or the bacterial precipitate is obtained by centrifugation and inoculated into the secondary screening fermentation medium for fermentation.

[0032] In step A, preferably, the tea sample is cut into pieces and placed in 0.9% (wt%) sterile saline to form a mixed solution, in which the volume ratio of the mass of the tea sample to the sterile saline is 10-20 g:100 mL; the mixed solution is shaken in a shaker at 180 r / min at 30°C for 6-8 hours, and then the mixed solution is diluted. After the mixture is diluted, it is spread on a YPD solid culture medium and cultured at 30°C for 24 to 48 hours to obtain the primary screening strains and store them by number.

[0033] Preferably, in step B, the YPD liquid culture medium comprises, by mass percentage, 2% peptone, 2% glucose, 1% yeast powder and 100 ml of deionized water.

[0034] In step A, the YPD solid culture medium includes, by mass percentage, 2% peptone, 2% glucose, 1% yeast powder, 100 ml deionized water and 2% agar.

[0035] In step A, the re-screened fermentation medium includes tobacco extract.

[0036] The tea samples can be green tea samples.

[0037] In step A, dilution was performed by 10 -0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 dilutions were performed in a gradient manner.

[0038] Preferably, for step B, the primary screened strain is activated and cultured with YPD liquid culture medium for 16 hours, the mycelium or the bacterial precipitate obtained by centrifugation is picked up and inoculated into a sterilized re-screened fermentation medium, and the uninoculated re-screened fermentation medium is used as a control. The fermentation is carried out on a shaker at 30°C for 72 hours, and the fermentation liquid produces fragrance, indicating that the produced strain has an aroma-producing effect.

[0039] It should be noted that the tea samples may also be fresh tea leaves.

[0040] The present application example also provides a use of a long-branch Trichoderma strain in the preparation of cigarette flavors.

[0041] That is, the long-branch Trichoderma strain is used to prepare cigarette flavor. Preferably, the method for preparing cigarette flavor using the long-branch Trichoderma strain comprises:

[0042] The long-branched Trichoderma is activated and cultured to obtain a bacterial liquid and hyphae, which are inoculated into a slant solid culture medium for culture. When green spores grow on the slant solid culture medium, sterile water is added into a slant test tube, the spores on the slant solid culture medium are removed and dissolved in the sterile water in the slant test tube to prepare a spore suspension, which is diluted and inoculated into a fermentation medium for fermentation.

[0043] Preferably, the spore concentration of the diluted spore suspension is 1×10 7 ~1.1×10 7 cfu / ml.

[0044] Preferably, the inoculation amount of the bacterial liquid in the slant solid culture medium is 1 vol%.

[0045] Preferably, the fermentation medium comprises tobacco extract.

[0046] Preferably, the fermentation time of the diluted spore suspension in the fermentation medium is 72 hours, and the fermentation is carried out on a shaker at a rotation speed of 180 r / min.

[0047] Schematically, for the primary screening and rescreening of Trichoderma longibrachiatum strains, the tea samples were cut into pieces and placed in 0.9% (wt%) sterile saline, the volume ratio of the tea sample mass to the sterile saline was 10 g:100 mL, and the mixture was shaken in a shaker at 180 r / min at 30°C for 6-8 h, and then subjected to 10 -0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 After gradient dilution, the strains were spread on YPD solid culture medium and cultured at 30℃ for 24 to 48 hours to obtain primary screening strains, which were numbered and preserved. The primary screening strains were activated and cultured with YPD liquid culture medium for 16 hours, and the mycelium was hooked or centrifuged to obtain the bacterial precipitate, which was inoculated into the sterilized secondary screening fermentation medium. After multiple primary and secondary screenings, the fermentation liquid was smelled and the results were comprehensively analyzed. The strain with the most obvious aroma production effect was marked as QF-1.

[0048] For the morphological and molecular biological identification of Trichoderma longifolia strains:

[0049] 1. Colony preparation: Inoculate spores or mycelial fragments of the QF-1 long-branch Trichoderma strain onto YPD solid culture medium, and then insert a sterile glass slide into the culture medium to allow the QF-1 long-branch Trichoderma strain to grow on the glass slide to form colonies.

[0050] 2. Staining and microscopic observation: Stain the slide with QF-1 long-branch Trichoderma strain and observe the morphology of long-branch Trichoderma hyphae and spores. Figure 1 shown.

[0051] 3. Fungal DNA extraction steps:

[0052] (1) Adjust the water bath to 65°C in advance;

[0053] (2) Grind 50-100 mg of macrofungus or 20 mg of dried fruiting bodies into powder. The macrofungus can be mushrooms. Add them to a 1.5 ml centrifuge tube, add 400 μl of Buffer Digestion and 4 μl of β-mercaptoethanol, and shake to mix. Then, place the tube in a water bath at 65°C for 1 h until the cells are completely lysed. During the water bath, invert and mix every 10 min to promote sample lysis. If RNA-free DNA is required, add 20 μl of RNase A (10 mg / ml) after the water bath.

[0054] (3) Add 200 μl Buffer PF, mix thoroughly by inversion, and place at -20°C for 5 min;

[0055] (4) Centrifuge the tube at 10,000 rpm for 5 min at 25°C. Transfer 500-550 μl of the supernatant to another 1.5 ml centrifuge tube. If the supernatant is turbid, add an equal volume of chloroform to mix. Centrifuge the tube at 12,000 rpm and take the supernatant.

[0056] (5) Add an equal volume of isopropanol to the obtained supernatant, mix, and transfer to a new centrifuge tube. Invert 5-8 times to mix thoroughly. Place the centrifuge tube at 25°C for 2-3 minutes, then centrifuge the tube at 10,000 rpm for 5 minutes and discard the supernatant.

[0057] (6) Add 1 ml of 75% ethanol to the centrifuge tube, invert and rinse for 1-3 minutes, centrifuge the tube at 10,000 rpm for 2 minutes, and discard the supernatant;

[0058] (7) Repeat step 6 once;

[0059] (8) Place the centrifuge tube at 25 degrees Celsius for 5-10 minutes to allow the remaining ethanol to evaporate completely;

[0060] (9) The obtained DNA was dissolved in 50-100 μl TE Buffer. The extracted DNA can be used immediately for the next experiment or stored at -20°C. TE Buffer contains 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.

[0061] 4. PCR amplification and sequencing identification: The universal PCR amplification primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') of fungal ITSrDNA were used for amplification. The PCR products were stored at -20°C and sequenced after agarose gel electrophoresis. The sequencing results were compared by BLAST and the NeighborJoining algorithm was used to construct the phylogenetic tree of the target strain. The results are as follows: Figure 3 As shown, the screened strain is in the same branch and has the closest evolutionary distance to the standard strain of Trichoderma genus Trichoderma longibrachiatum, so the screened strain was identified as Trichoderma longibrachiatum and named QF-1.

[0062] The strain has been deposited in the General Microbiology Center (CGMCC) of China Microbiological Culture Collection, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC NO: 41364.

[0063] Preparation of tobacco flavoring using Trichoderma longifolia strain QF-1:

[0064] The long-branch Trichoderma strain QF-1 is activated and cultured to obtain a bacterial liquid and hyphae, the bacterial liquid and hyphae are inoculated into a test tube slant solid culture medium for culture, and green spores grow on the slant solid culture medium, sterile water is added into the slant test tube, spores on the slant solid culture medium are scraped and dissolved in sterile water to prepare a spore suspension, and the spore suspension is inoculated into a fermentation medium at a ratio of 1% (v / v) for fermentation.

[0065] The fermentation medium consisted of 50 g tobacco extract and 50 mL water. The spore concentration of Trichoderma longifolia in the spore suspension was 1 × 10 7 ~1.1×10 7 cfu / ml, the fermentation temperature was 30℃, the fermentation speed was 180rpm / min, the fermentation time was 72h, and the fermentation medium without inoculation of long-branch Trichoderma spores was used as the control group. Three replicates were set for each treatment group. After the fermentation liquid was diluted with alcohol, 10μL was injected into blank cigarettes for sensory evaluation. Compared with the fermentation liquid without bacteria, the aroma quantity and aroma quality of the tobacco extract fermented with bacteria were significantly increased, the sweet aftertaste was enhanced, the impurities were reduced, the irritation was reduced, and the smoking quality of the cigarette was significantly improved.

[0066] As for the fermentation temperature of tobacco extract, the spore suspension was inoculated into the liquid fermentation medium at a ratio of 1% (v / v), the fermentation speed was set to 180 rpm / min, and fermented at 27°C, 30°C, and 33°C for 72h, respectively. After the fermentation, 10 μL was aspirated and injected into blank cigarettes for sensory evaluation. When fermented at 27°C, the aroma volume was increased, the fragrance was prominent, but the irritation was large. The aroma of the fermented liquid at 33°C was not full enough, but the irritation and miscellaneous smell were reduced compared with the original cigarette. When fermented at 30°C, the aroma quality and aroma volume were good, the irritation was small, and the aftertaste was sweet, clean and comfortable, and the application effect was good.

[0067] As for the fermentation speed of tobacco extract, the spore suspension was inoculated into the liquid fermentation medium at a ratio of 1% (v / v), the fermentation temperature was set to 30°C, and the fermentation was carried out at speeds of 120rpm / min, 150rpm / min, 180rpm / min, and 200rpm / min for 72h. After the fermentation, 10μL was taken and injected into blank cigarettes for sensory evaluation. When fermented at 120r / min, the aroma volume was slightly increased, but the irritation was large, there were more miscellaneous gases, and the aftertaste was bitter and spicy. When fermented at 150 and 200rpm / min for 72h, the aroma volume increased, but the aroma quality was not as high as that at 180rpm / min. The fermentation liquid fermented at 180rpm / min had an obvious honey sweet fragrance, supplemented by floral and fruity aromas, rich aroma, and better application effect.

[0068] As for the fermentation inoculum amount of tobacco extract, the fermentation temperature was set at 30°C and the rotation speed was 180rpm / min. The spore suspension was inoculated into the liquid fermentation medium at a ratio of 0.2% (v / v), 0.5%, 0.8%, 1%, 1.2%, and 1.5%, respectively, and fermented for 72 hours. After the fermentation, 10 μL was injected into blank cigarettes for sensory evaluation. With the increase of the inoculum amount, the sensory evaluation score was the highest at 1% (v / v). When the inoculum amount was too small or too large, the fermentation liquid had the problem of insufficient aroma when used in cigarettes for evaluation. At 1% (v / v), the aroma was richer, the aftertaste was comfortable, and the smoke was smooth and harmonious.

[0069] A three-factor three-level orthogonal experiment was formulated based on the three factors of fermentation temperature, fermentation speed and mycelium inoculation amount, and sensory evaluation and result scoring were carried out. The optimal combination conditions for the fermentation of tobacco extract by the long-branch Trichoderma strain QF-1 were 30°C environment, 180rpm / min rotation speed and 1% (v / v) inoculation amount. The rotation speed can be achieved by placing the culture medium in a fermentation tank and rotating it.

[0070] For the detection of aroma components of tobacco extract before and after the optimal fermentation conditions, the fermentation broth of Trichoderma longifolia strain QF-1 and the fermentation broth of the control group were centrifuged and filtered, and 10 μL was injected into blank cigarettes after dilution. Twenty sample cigarettes and 20 reference blank cigarettes were smoked using an RM20H rotary smoking machine according to the Canadian deep smoking mode (HCI) standard requirements. The smoking conditions were: the smoking volume was 55 mL, the duration was 2 s, and the aerosol of the heated cigarette was captured using a Cambridge filter with a diameter of 92 mm. Glue and retain the filter stick. After the suction is completed, transfer the Cambridge filter disc and filter stick that capture the flue gas to a 150mL conical flask, add 50mL of dichloromethane solution respectively, and then put it into a shaker for 40 minutes. Take the obtained extract and add it to the concentration bottle, then add 1mL of 0.204mg / mL phenylethyl acetate (internal standard) to start concentration. When it is concentrated to less than 1mL, use dichloromethane to make up to 1mL, pass it through a 0.22μm filter membrane in a 2.0mL chromatography bottle, and perform GC-MS analysis.

[0071] GC-MS analysis conditions:

[0072] Gas phase detection conditions: chromatographic column HP-5MS (60m×0.25mm×0.25μm); injection port temperature 280℃; carrier gas is high-purity helium; column oven is programmed temperature, starting temperature 50℃ for 1min, increasing to 148℃ at 4℃ / min for 1min, increasing to 172℃ at 2℃ / min for 1min, increasing to 280℃ at 4℃ / min for 5min; split ratio 10:1; flow rate 3.0mL / min.

[0073] Mass spectrometry conditions:

[0074] The ion source temperature was 230°C; the quadrupole temperature was 150°C; the EI ion source was 70 eV; the mass scanning range was 35-550 amu; and the NIST20 search was used.

[0075] The results are calculated as follows:

[0076] Aroma component content calculation method: Aroma component content = (component peak area * internal standard content) / internal standard peak area, the results are shown in Table 1 and Figure 4 .

[0077]

[0078]

[0079]

[0080] Table 1: Analysis results of aroma components of Trichoderma longifolia QF-1 fermentation broth after application in cigarettes

[0081] Note: The substance content (μg / g) in the table refers to the mass of the chemical substance measured in each gram of tobacco sample. "-" indicates that the content of volatile aroma components is extremely low or absent. The blank group is the sample obtained by smoking a blank cigarette without fermentation liquid, the control group is the sample obtained by smoking a cigarette without fermentation liquid of QF-1 bacteria, and the QF-1 group is the sample obtained by smoking a cigarette with fermentation liquid of QF-1 bacteria.

[0082] From Table 1 and Figure 4 It can be seen that after the QF-1 fermentation liquid is applied to cigarettes, the aroma components of alcohols, ketones, phenols, hydrocarbons, heterocyclics and other substances increase, while esters and acids decrease. The aroma content of QF-1 and the untreated control is higher than that of blank cigarettes. The total amount of aroma substances in the QF-1 group is the highest, which is 483.890μg / g, an increase of 41.4% over the control group. Among the alcohol substances, 2-furanmethanol increases by 138.5% compared with the control, which has a slight sweet and aromatic smell. Trans-geraniol and nerol have a fresh and sweet floral fragrance. The aldehyde substances increase the most, a total increase of 100.4% compared with the control, among which 5-hydroxymethylfurfural usually has a sweet, caramel-like aroma and is often used in baked goods. , coffee and caramel. 5-methyl-2-furaldehyde has sweet aromas such as almonds, nuts, roasted aroma and smoky aroma. It is mainly used in the preparation of flavors such as cocoa, nuts, caramel, bread and coffee. Its content is significantly increased compared with blank cigarettes. Both aldehydes may contribute to the outstanding sweet aroma of the QF-1 fermentation group. Ketones mostly have floral and fruity aromas, which can improve the taste of cigarettes and coordinate with the aroma of tobacco. After the QF-1 fermentation liquid is applied to cigarettes, the contents of DDMP and cyclopentenone substances are increased, and three ketone substances such as 3-ethyl-2-cyclopentene-1-one are newly added. It has been reported that furanones, pyrones and cyclopentenone compounds are important sources of roasted sweet aroma in the water-soluble component fraction of smoke particulate matter.

[0083] Among the phenolic substances, 4-methylguaiacol has been newly added to the QF-1 group. Studies have shown that 4-methylguaiacol (4-MG) is an important flavor compound of Luzhou-flavor liquor, contributing fruity and sweet, floral and smoky flavors to the liquor. Although the content of ester substances has decreased, scopoletin has been added to enhance the sweet aroma of tobacco leaves. Among the olefin substances, nodules have been newly added, which have woody, amber and ambergris-type aromas. There are more indoles in the heterocyclic class, and the smell itself is described as a complex mixture of soil, floral and animal scents, and may present a soft floral scent at low concentrations.

[0084] The flavors prepared by fermentation with tobacco extract as the fermentation substrate and inoculation with the long-branched Trichoderma strain QF-1 are better compatible with tobacco when applied to cigarettes. The aroma components measured by using a smoking machine to smoke and collect the particle phase are closer to the aroma felt by the smokers when they smoke. The increase in ketones and the sweet fragrance felt by smelling the fermentation liquid also indicate that QF-1 degrades the organic macromolecules in the crude tobacco concentrate during the fermentation process and forms a unique aroma style. This may make the ratio of chemical components in the fermentation liquid more coordinated. Its application in cigarettes not only increases the aroma volume, makes the smoke smooth and soft, but also gives the cigarettes a unique honey-sweet style.

[0085] It should be noted that the phrases "one embodiment", "an embodiment", "an exemplary embodiment", "some embodiments", etc. mentioned in the specification indicate that the described embodiments may include certain features, structures or characteristics, but not every embodiment may include the certain features, structures or characteristics. In addition, such phrases do not necessarily refer to the same embodiment. In addition, when describing certain features, structures or characteristics in conjunction with an embodiment, it is within the knowledge of those skilled in the art to implement such features, structures or characteristics in conjunction with other embodiments, whether explicitly or not explicitly described.

[0086] It should be easily understood that “on,” “above,” and “over” in the present disclosure should be interpreted in the broadest manner, so that “on” not only means “directly on something,” but also includes the meaning of “on something” with intervening features or layers therebetween, and “above” or “over” not only includes the meaning of “above” or “over,” but also may include the meaning of “above” or “over something” with no intervening features or layers therebetween (i.e., directly on something).

[0087] In addition, spatially relative terms, such as "below," "below," "beneath," "above," "above," etc., may be used herein for ease of description to describe the relationship of one element or feature relative to other elements or features as shown in the figures. Spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation shown in the figures. The device may have other orientations (rotated 90 degrees or at other orientations), and the spatially relative descriptors used herein may likewise be interpreted accordingly.

[0088] It should be noted that, in this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A long-branch Trichoderma strain, characterized in that Its deposit number is CGMCC NO:41364.

2. The method for preparing a long-branch Trichoderma strain according to claim 1, characterized in that: The steps include: A. Place tea samples in sterile physiological saline, dilute by shaking, and then spread on YPD solid medium to obtain primary screening strains; B. The primary screening strain obtained in step A is activated and cultured in YPD liquid medium, and the mycelium is picked or the bacterial precipitate is obtained by centrifugation and inoculated into the secondary screening fermentation medium for fermentation.

3. The method for preparing the long-branch Trichoderma strain according to claim 2, characterized in that: In step B, the YPD liquid culture medium includes, by mass percentage, 2% peptone, 2% glucose, 1% yeast powder and 100 ml of deionized water.

4. The method for preparing the long-branch Trichoderma strain according to claim 2, characterized in that: In step A, the YPD solid culture medium includes, by mass percentage, 2% peptone, 2% glucose, 1% yeast powder, 100 ml deionized water and 2% agar.

5. The method for preparing the long-branch Trichoderma strain according to claim 2, characterized in that: In the step A, the re-screening fermentation medium includes tobacco extract.

6. The method for preparing the long-branch Trichoderma strain according to claim 2, characterized in that: The tea samples are green tea samples.

7. The method for preparing the long-branch Trichoderma strain according to claim 2, characterized in that: In the step A, the shaking is performed at 30° C. and on a shaker for 6-8 hours.

8. The method for preparing the long-branch Trichoderma strain according to claim 2, characterized in that: In step A, the dilution was carried out by 10 -0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 dilutions were performed in gradients.

9. Use of the long-branch Trichoderma strain according to claim 1 in preparing cigarette flavors.

10. The use according to claim 9, characterized in that: The method for preparing cigarette flavoring material using the long-branch Trichoderma strain comprises: The long-branched Trichoderma is activated and cultured to obtain a bacterial liquid and hyphae, which are inoculated into a slant solid culture medium for culture. When green spores grow on the slant solid culture medium, sterile water is added into a slant test tube, the spores on the slant solid culture medium are removed and dissolved in the sterile water in the slant test tube to prepare a spore suspension, which is diluted and inoculated into a fermentation medium for fermentation.

11. The use according to claim 10, characterized in that: The spore concentration of the diluted spore suspension is 1×10 7 ~1.1×10 7 cfu / ml.

12. The use according to claim 10, characterized in that: The inoculation amount of the bacterial liquid in the slant solid culture medium is 1 vol%.

13. The use according to claim 10, characterized in that: The fermentation medium includes tobacco extract.

14. The use according to claim 10, characterized in that: The fermentation time of the diluted spore suspension in the fermentation medium is 72 hours, and the fermentation is carried out at a rotation speed of 180 r / min.

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