A kind of fragrant golden ear strain and its application

Abstract

The invention discloses a fragrance-producing golden ear strain and a use thereof. The golden ear strain or its fermentation liquid is used as a tobacco biological flavoring and added into cigarettes or cigars. The golden ear strain has good coordination with tobacco, can enrich the tobacco fragrance, reduce impurities and irritation, and increase the sweetness.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a fragrant golden fungus strain and application thereof. Background Art

[0002] There is a close relationship between the aroma components of tobacco leaves and their sensory quality. Microbial metabolism can produce many aroma-related substances on the surface of tobacco leaves. Selecting appropriate microorganisms and fermentation conditions during tobacco fermentation can significantly increase the aroma, reduce green odor, and improve quality. Under the existing tobacco raw material conditions, the use of appropriate fermentation methods can improve the quality of tobacco leaves and thus improve the industrial availability of tobacco leaves.

[0003] As people pursue green food, natural flavors produced by microbial fermentation have attracted much attention due to their naturalness and greenness. There are many types of tobacco flavors, but the types of flavors produced and applied by microbial fermentation are relatively few; therefore, screening more flavor-producing strains and producing natural tobacco flavors are important for maintaining the taste of cigarettes and increasing the aroma of cigarettes.

[0004] my country is rich in bacterial strain resources, and Golden Ear (Naematelia aurantialba) is an important edible and medicinal bacterial strain resource in my country. Rationally developing and utilizing my country's natural bacterial resources, screening for aroma-producing Golden Ear strains (Tremella aurantia), and using them for tobacco fermentation to enhance flavor, improve tobacco leaf quality, and increase tobacco leaf availability are of great significance to enhancing the use value of domestic tobacco leaves and developing and producing high-quality Chinese cigarettes and Chinese cigars. Summary of the invention

[0005] In order to improve the current poor performance of natural aging fermentation of tobacco leaves, such as low efficiency and long time, the present invention screened and obtained an aroma-producing strain from the fruiting body of Tremella fuciformis. The strain has good universality for tobacco fermentation and quality improvement, and has good aroma-enhancing and quality-improving effects on the treatment of flue-cured tobacco leaves, cigar tobacco leaves and tobacco waste.

[0006] The present invention provides a golden ear (Tremella aurantia), which has been deposited in the China General Microbiological Culture Collection Center on October 28, 2021, with a deposit number of CGMCC No. 23831, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0008] The first aspect of the present invention is to provide a Tremella aurantia strain, which is deposited in China General Microbiological Culture Collection Center with a deposit number of CGMCC No.23831.

[0009] The second aspect of the present invention is to provide the use of the Tremella fuciformis described in the first aspect of the present invention in tobacco plant tissue fermentation.

[0010] The third aspect of the present invention is to provide a method for preparing Tremella fuciformis fermented liquid, comprising the following steps:

[0011] (1) providing a Tremella fuciformis strain; the Tremella fuciformis strain has a deposit number of CGMCC No.23831;

[0012] (2) inoculating the above strain into a tobacco extract medium at an inoculum volume ratio of 2-5%, and culturing in a constant temperature shaker at 28-30° C. and 150-200 rpm for 5-7 days to obtain a seed solution;

[0013] (3) Inoculate the seed liquid in a tobacco extract medium at a volume ratio of 2-5%, and culture in a constant temperature shaker at 28-30° C. and 150-200 rpm for 5-7 days to obtain a fermentation liquid.

[0014] The fourth aspect of the present invention is to provide the use of the seed liquid or fermentation liquid described in the third aspect of the present invention in tobacco plant tissue fermentation; the specific method of use is: add the above-mentioned fermentation liquid or seed liquid to the tobacco plant tissue according to 5-30% of the weight of the tobacco plant tissue, and ferment it at 25-45°C for 5-30 days.

[0015] A fifth aspect of the present invention is to provide a tobacco biological flavoring, characterized in that the preparation method comprises the following steps:

[0016] (1) providing a Tremella fuciformis strain; the deposit number of the Tremella fuciformis strain is CGMCC No.23831;

[0017] (2) inoculating the bacterial strain into a tobacco extract medium at an inoculum volume ratio of 1-3%, and culturing in a constant temperature shaker at 28-30° C. and 150-200 rpm for 5-7 days to obtain a seed solution;

[0018] (3) taking the seed liquid at an inoculum amount of 1-3% by volume and inoculating it into a tobacco extract medium, and culturing it in a constant temperature shaker at 28-30° C. and 150-200 rpm for 5-7 days to obtain a fermentation liquid;

[0019] (4) Centrifuging the seed liquid or fermentation liquid at 5000-10000 rpm for 10-20 min, taking the supernatant; pre-freezing the supernatant at -80°C for 24 h, and then vacuum freeze-drying the supernatant to obtain a tobacco bio-flavor.

[0020] The sixth aspect of the present invention is to provide the use of the tobacco biological flavoring described in the fifth aspect of the present invention in tobacco raw materials or finished cigarettes, wherein the tobacco raw materials include tobacco leaves, tobacco shreds, stem shreds, sheets, rolling papers or filters; the finished cigarettes include cigarettes, cigars or electronic cigarettes.

[0021] The tobacco plant tissues described in the present invention include but are not limited to tobacco leaves, tobacco shreds, and shredded tobacco stems.

[0022] The beneficial effects of the present invention are:

[0023] The present invention inoculates the screened aroma-producing golden ear strain or its fermentation liquid onto the surface of tobacco leaves and ferments them under certain conditions, which can significantly improve the aroma quality and aroma quantity of the tobacco leaves and increase the sweetness.

[0024] The yellow ear strain or its fermentation liquid is used as a tobacco biological flavoring, added to cigarettes or cigars, and has good coordination with tobacco, can enrich the tobacco flavor, reduce impurities and irritation, and increase sweetness. The preparation process of the tobacco biological flavoring is simple, the raw material source is abundant, the cost is low, and it is easy to use. The tobacco flavoring is used for flavoring and adding to tobacco raw materials or finished cigarettes, which can meet people's demand for green and healthy cigarette products, and therefore has significant industrial application value and broad prospects for promotion and application. DETAILED DESCRIPTION

[0025] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0026] Implementation Example 1

[0027] Enrichment, purification and screening of strains:

[0028] Take golden ear fruiting bodies (Sichuan Tibetan area), soak them in 10% 84 disinfectant for 4-5 minutes, and clean them with sterile distilled water. Cut soybean-sized slices from the cleaned golden ear fruiting bodies with a sterile scalpel and place them on a tobacco extract solid culture medium containing 1% chloramphenicol. Place the culture dish in a constant temperature and humidity box at 28°C and 75% relative humidity for 5-7 days. After the mycelium grows out, use a sterile inoculation shovel to intercept soybean-sized mycelium with agar blocks at 4 weeks and the center of the culture dish, place it in a tobacco extract culture medium containing 1% chloramphenicol, place it in a shaking table at 28°C and 150rpm for 5-7 days, and screen the aroma-producing culture by smelling the aroma. After the culture is gradient diluted, it is coated on a tobacco extract solid culture medium to obtain a single colony, and the single colony is inoculated into a tobacco extract culture medium, and the aroma-producing culture is screened by smelling the aroma. The culture with aroma was diluted in gradient and spread on tobacco extract solid medium to obtain Tremella aurantia strain, which was numbered as SCT-F3.

[0029] The tobacco extract culture medium is: 15.0 g of tobacco leaf powder, 100 mL of water, ultrasonic extraction for 30 minutes, filtration and water supplementation to 100 mL, 5.0 g of glucose, 2.0 g of KH2PO4, 0.2 g of MgSO4, 900 mL of distilled water, natural pH; 18 g of agar; sterilization at 115°C for 20 minutes.

[0030] Implementation Example 2

[0031] 18S rRNA molecular identification and biological characteristics of the strain:

[0032] The 18S rRNA region of the strain was amplified by PCR using primers NS1 (5'-GTAGTCATATGCTTGTCTC-3') and NS8 (5'-TCCGCAGGTTCACCTACGGA-3'), and then sequenced after agarose gel electrophoresis to obtain the 18S rRNA sequence of the strain.

[0033] The culture morphology of the Tremella fuciformis strain is described as follows:

[0034] The surface of the colony on potato dextrose agar medium is dry, the hyphae are large, fluffy and opaque, and are firmly bonded to the medium. The front of the colony is white and the back is yellow.

[0035] The sequence was compared with the gene sequences of some strains registered in GenBank for similarity, and combined with the results of morphological identification, it was shown that the strain SCT-F3 belonged to Tremella aurantia.

[0036] Implementation Example 3

[0037] Inoculate the golden ear in the tobacco extract medium, place it in a constant temperature shaker at 28°C, 200rpm, and culture for 7 days. Take the seed liquid at a volume ratio of 4%, inoculate it in the tobacco extract medium, and culture it in a constant temperature shaker at 28°C, 150rpm for 7 days. Spray the culture solution: water: tobacco leaves at a mass ratio of 1:1:8 evenly on the surface of the flue-cured tobacco leaves, place it in a constant temperature and humidity chamber at 25°C and 75% relative humidity for 10 days, and dry the treated tobacco leaves at 60°C to a moisture content of 12% to obtain fermented tobacco leaves.

[0038] The tobacco extract culture medium is the same as that in Example 1.

[0039] After the fermented tobacco leaves have balanced their moisture, they are cut into strips and proofed. Professional smokers evaluate the sensory quality of the tobacco leaves. The results show that compared with the unfermented samples, the sensory quality of the fermented tobacco leaves is significantly improved, specifically: the aroma quality and aroma volume are improved, the smoke is soft, the impurities and irritation are significantly reduced, and the sweetness is improved. It has been determined that the content of aromatic compounds such as diacetyl, linalool, phenylethyl alcohol, terpineol, isobutyl alcohol, methyl isovalerate, methyl 3-furancarboxylate, methyl benzoate, ethyl benzoate, methyl p-methylbenzoate, methyl cinnamate, ethyl anisate, nerolidol, damasconone, 2-furfural, solanone and neophytadiene in the fermented tobacco leaves has increased.

[0040] Implementation Example 4

[0041] Inoculate the golden ear in the tobacco extract medium, place it in a constant temperature shaker at 28°C and 200rpm, and culture it for 7 days. Take the seed liquid at a volume ratio of 5%, inoculate it in the tobacco extract medium, and culture it in a constant temperature shaker at 28°C and 150rpm for 5 days. Spray the culture solution: water: tobacco leaves at a mass ratio of 1.5:1.5:7 evenly on the surface of domestic cigar tobacco leaves, and place it in a constant temperature and humidity chamber at 30°C and 65% relative humidity for 10 days. After 10 days, add the fermentation medium, and continue to ferment the cigar tobacco leaves at 40°C for 10 days to obtain fermented tobacco leaves.

[0042] The tobacco extract culture medium is the same as that in Example 1.

[0043] After the fermented cigar tobacco leaves have balanced moisture, they are rolled and the sensory quality is evaluated by professional smoking testers. The results show that compared with the unfermented samples, the sensory quality of the fermented tobacco leaves is significantly improved, specifically: the maturity of the cigar tobacco leaves is significantly improved, the aroma quality and aroma volume are improved, the smoke is soft, and the impurities and irritation are significantly reduced. It has been determined that the content of aromatic compounds such as diacetyl, linalool, phenylethyl alcohol, terpineol, isobutanol, methyl isovalerate, methyl 3-furancarboxylate, methyl benzoate, nerolidol, damasconone, 2-furfural and neophytadiene in the fermented tobacco leaves has increased.

[0044] Implementation Example 5

[0045] Inoculate Tremella fuciformis in a tobacco extract medium, place it in a constant temperature shaker at 30°C, 200rpm, and culture for 7 days. Take 2% of the seed liquid by volume, inoculate it in a tobacco extract medium, place it in a constant temperature shaker at 30°C, 150rpm and culture for 5 days. Centrifuge the fermentation liquid at 5000rpm for 20min, and take the supernatant. Prefreeze the fermentation liquid supernatant at -20°C for 24h, and then freeze-dry it in vacuum to obtain a tobacco bio-flavor.

[0046] The tobacco powder extract culture medium is: 15.0 g tobacco powder plus 100 mL warm water, extraction at 150 rpm for 2 h, ultrasonic extraction for 30 min, filtration and water replenishment to 100 mL, 2.0 g glucose, 1.0 g KH2PO4, 900 mL distilled water, pH 6.5-7.0; 18 g agar; sterilization at 105°C for 30 min.

[0047] After the tobacco bio-flavor was dissolved in 30% ethanol, it was added to cigarette tobacco at a ratio of 0.05%, 0.10% and 0.20% by weight of tobacco, and sensory evaluation was performed. The results showed that compared with the blank sample, the sensory quality of the cigarettes with tobacco flavor added was significantly improved, and the evaluation results are shown in Table 1. After measurement, it was found that the main volatile aroma substances in the tobacco bio-flavor, in addition to nicotine, neophytadiene, megastigmatrienone and other volatile aroma components of tobacco, also added isobutanol, isopentanol, 2-methylbutanol, methyl isovalerate, methyl 3-furancarboxylate, methyl benzoate, ethyl benzoate, methyl p-methylbenzoate, methyl cinnamate, ethyl anisate, nerolidol, (-)-eucalyptus alcohol and other volatile aroma components.

[0048] Table 1 Results of the cigarette flavoring test of tobacco biological flavor (fermented yellow ear)

[0049]

[0050] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of changes or replacements within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims. It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further explain various possible combinations. In addition, the various different embodiments of the present invention can also be combined arbitrarily, as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A Tremella aurantia strain, deposited in China General Microbiological Culture Collection Center, with the deposit number: CGMCC No.23831.

2. Use of Tremella fuciformis as claimed in claim 1 in tobacco plant tissue fermentation.

3. A method for preparing Tremella fuciformis fermented liquid, characterized in that: The following steps are involved: (1) providing a Tremella fuciformis strain; the Tremella fuciformis strain has a deposit number of CGMCC No.23831; (2) inoculating the above strain into a tobacco extract medium at an inoculum volume ratio of 2-5%, and culturing in a constant temperature shaker at 28-30° C. and 150-200 rpm for 5-7 days to obtain a seed solution; (3) Inoculate the seed liquid in a tobacco extract medium at a volume ratio of 2-5%, and culture in a constant temperature shaker at 28-30° C. and 150-200 rpm for 5-7 days to obtain a fermentation liquid.

4. Use of the seed liquid or fermentation liquid as claimed in claim 3 in tobacco plant tissue fermentation.

5. The use according to claim 4, characterized in that The fermentation liquid or seed liquid is added to the tobacco plant tissue according to 5-30% of the weight of the tobacco plant tissue, and fermented at 25-45° C. for 5-30 days.

6. A tobacco biological flavoring, characterized in that: The preparation method comprises the following steps: (1) providing a Tremella fuciformis strain; the Tremella fuciformis strain has a deposit number of CGMCC No.23831; (2) inoculating the bacterial strain into a tobacco extract medium at an inoculum volume ratio of 1-3%, and culturing in a constant temperature shaker at 28-30° C. and 150-200 rpm for 5-7 days to obtain a seed solution; (3) taking the seed liquid and inoculating it in an inoculum amount of 1-3% by volume into a tobacco extract medium, and culturing it in a constant temperature shaker at 28-30° C. and 150-200 rpm for 5-7 days to obtain a fermentation liquid; (4) Centrifuging the seed liquid or fermentation liquid at 5000-10000 rpm for 10-20 min, taking the supernatant; pre-freezing the supernatant at -80°C for 24 h, and then vacuum freeze-drying the supernatant to obtain a tobacco bio-flavor.

7. Use of the tobacco biological flavoring according to claim 6 in tobacco raw materials or finished cigarettes, wherein the tobacco raw materials include tobacco leaves, tobacco shreds, stem shreds, sheets, rolling papers or filters; and the finished cigarettes include cigarettes, cigars or electronic cigarettes.

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