Debaryomyces hansenii and application of preparation of debaryomyces hansenii in cigar core tobacco leaves

Abstract

The invention provides application of debaryomyces hansenii and a preparation of the debaryomyces hansenii in cigar core tobacco leaves, and belongs to the technical field of microorganisms, the debaryomyces hansenii is named as Debaryomyces hansenii Z3, and the preservation number of the debaryomyces hansenii is CCTCC (China Center for Type Culture Collection) NO: M 20242187. The strain is high in tolerance and wide in pH tolerance range, can tolerate high temperature during cigar core tobacco leaf fermentation, can rapidly produce gas, and shortens the cigar core tobacco leaf fermentation time. Meanwhile, the tobacco leaves fermented by the strain are more balanced in conventional chemical components and richer in aroma component content, sensory evaluation shows that cigar tone is improved, smoke is oily, irritation is obviously improved, aftertaste is comfortable, the effect of improving the quality of cigar core tobacco leaves is obvious, and the strain has great application potential.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to application of a strain of Debaryomyces hansenii and a preparation thereof in cigar core tobacco leaves. Background Art

[0002] Cigar core tobacco fermentation is an important process in cigar processing, but after fermentation, cigar core tobacco still faces problems such as lack of aroma, strong flavor, obvious foreign smell, and strong bitterness, resulting in a lot of room for improvement in the quality of cigar core tobacco. Affected by factors such as temperature, humidity, and microorganisms, fermentation is an important way to improve the intrinsic quality of cigar core tobacco, which is of great significance to improving the quality of cigars.

[0003] Microbial fermentation is an important method to improve the quality of cigar core tobacco leaves. At present, most of the research on cigar core tobacco leaf fermentation uses bacteria such as Bacillus. Although the quality of cigar core tobacco leaves after fermentation has been improved to a certain extent, the effect is not ideal. At the same time, due to the special conditions such as the special temperature and humidity during the fermentation of cigar core tobacco leaves and the low water activity of the tobacco leaves themselves, it is difficult for bacterial microorganisms to play a role in the fermentation process of cigar core tobacco leaves. Yeast not only has good aroma and quality improvement properties, but is also very suitable for growth and reproduction under the special fermentation conditions of cigar core tobacco leaves. Therefore, it has important potential in improving the fermentation quality of cigar core tobacco leaves.

[0004] There are a lot of yeasts in the fermentation process of the cigar core tobacco leaves themselves, but the current exploration and utilization of yeast resources in the cigar core tobacco leaves is not sufficient, most of them focus on bacteria, and the application of yeast in the quality improvement of cigar core tobacco leaves is still rare. Therefore, the exploration of yeast in a cigar core tobacco leaf for use in its fermentation process not only ensures the safety of the microbial strains, but also explores the high-quality microbial germplasm resources in the cigar leaves, which is of great significance for improving the quality of cigar core tobacco leaves. Summary of the invention

[0005] The first object of the present invention is to provide a Debaryomyces hansenii Z3, which has been deposited in the China Center for Type Culture Collection (CCTCC) on October 14, 2024, with a deposit number of CCTCC NO: M20242187, and the deposit address is Wuhan University. To solve the problems in the prior art that the fermentation time is long, the cigar melody and the oiliness of the smoke need to be improved.

[0006] The second object of the present invention is to provide a microbial preparation.

[0007] The third object of the present invention is to provide the use of Debaryomyces hansenii and its preparation in cigar filler tobacco leaves.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is:

[0009] A strain of Debaryomyces hansenii, named Debaryomyces hansenii Z3, with a deposit number of CCTCC NO:M 20242187.

[0010] The beneficial effect of the above technical scheme is that the Debaryomyces hansenii Z3 of the present invention is separated and screened from cigar core tobacco leaves, has the advantages of strong pH tolerance, can withstand a temperature environment of 40°C, and has high gas production efficiency. After fermenting cigar core tobacco leaves with the bacteria, the cigar's tone can be improved, the aroma can be enriched, the irritation can be reduced, and the aftertaste is comfortable.

[0011] To achieve the above object, the present invention provides a technical solution adopted by a microbial preparation:

[0012] A microbial preparation comprises Debaryomyces hansenii Z3.

[0013] Preferably, the microbial preparation is the fermentation broth of Debaryomyces hansenii Z3.

[0014] Further preferably, the microbial preparation is obtained by fermenting Debaryomyces hansenii Z3 in a liquid culture medium, and the concentration of Debaryomyces hansenii Z3 is ≥1.0×10 7 CFU / mL.

[0015] The beneficial effects of the above technical solution are: on the one hand, the Debaryomyces hansenii Z3 and the microbial preparation of the present invention can significantly shorten the fermentation time of cigar core tobacco leaves, which is beneficial to improving industrial production efficiency and saving production costs; on the other hand, the microbial preparation can improve the quality of cigar core tobacco leaves after fermentation.

[0016] To achieve the above objectives, the present invention provides the use of Debaryomyces hansenii Z3 in cigar core tobacco leaves, the application including shortening the fermentation time of cigar core tobacco leaves, and improving the cigar mellowness and smoke oiliness.

[0017] The present invention is a pioneering invention. The beneficial effect of the above technical solution is that Debaryomyces hansenii Z3 and its preparation can effectively shorten the fermentation time of cigar core tobacco leaves, and the fermented tobacco leaves can enhance the cigar's flavor, with the characteristics of oily smoke, rich aroma, reduced irritation and comfortable aftertaste.

[0018] Preferably, 50 to 70 mL of bacterial solution is sprayed on every 500 g of tobacco leaves, and the concentration of the bacterial solution is 1.0×10 7 ~1.0×10 9 CFU / mL.

[0019] Further preferably, after spraying the bacterial solution, the cigar filler tobacco leaves are fermented for 15 days at a constant temperature of 35±2° C. and a humidity of 75±5%. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a colony morphology diagram of the Debaryomyces hansenii strain Z3 in Example 2 of the present invention;

[0021] Figure 2 This is the phylogenetic tree of the Debaryomyces hansenii strain Z3 in Example 2 of the present invention. DETAILED DESCRIPTION

[0022] The present invention mainly aims at the technical problem that the fermentation effect of bacterial microorganisms on cigar core tobacco leaves in the prior art is not ideal, and provides a strain of Debaryomyces hansenii and an application of a preparation thereof in cigar core tobacco leaves. The strain is an endophyte of cigar core tobacco leaves, has strong adaptability to cigar core tobacco leaves, and has strong tolerance. It can still survive well at a pH of 9 and can tolerate a temperature environment of 40°C, so that the fermentation time of tobacco leaves can be shortened and the quality of tobacco leaves after fermentation can be improved.

[0023] The technical solution of the present invention is further described below in conjunction with specific embodiments.

[0024] Unless otherwise specified, the terms used in the present invention are commonly used terms in the relevant field, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the reagents and products used are also commercially available.

[0025] Preparation of YPD solid culture medium: prepare according to the ratio of 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and 15 g / L agar, and sterilize at 115°C for 30 min.

[0026] Preparation of YPD liquid culture medium: prepare according to the ratio of 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose, and sterilize at 115°C for 30 min.

[0027] Preparation of PDA medium: Select the potato dextrose agar (PDA) medium (Cat. No. 021050) from Guangdong Huankai Microbiological Technology Co., Ltd., configure it at 40.1 g / L, and sterilize it at 115°C for 30 min.

[0028] Biological materials: The samples of cigar tobacco leaves screened for bacteria are shown in Table 1. The research object of fermented cigar filler tobacco leaves is the tobacco leaves raw materials from Yunnan production area.

[0029] Table 1 List of cigar tobacco grades after screening

[0030]

[0031] 1. Specific Examples of Debaryomyces hansenii Z3 and Its Preparations

[0032] Example 1

[0033] This example is the screening and biological characteristics analysis of Debaryomyces hansenii Z3, and the specific implementation operations are as follows:

[0034] Weigh 25 g of cigar tobacco leaf sample and homogenize it in 225 mL of phosphate buffer for 3 min using a slapping homogenizer to obtain a concentration of 10 -1 The bacterial suspension was diluted in gradients to obtain dilutions of different concentrations. 100 μL of the appropriate gradient dilution was applied to the YPD solid medium, evenly spread and sealed with a sealing film, and then placed in a constant temperature and humidity incubator at 28°C for 24 hours. Single colonies with yeast characteristics were selected and purified on the YPD solid medium. Sensory evaluation experts performed sensory smelling on the solid plates of microorganisms with different colony morphologies to screen out yeasts with better aroma production. Finally, a yeast strain Z3 with better aroma production was obtained, and its pH tolerance, temperature tolerance, fermentation ability, etc. were studied.

[0035] The results are shown in Tables 2 to 4. Yeast Z3 has strong pH tolerance and can still survive well at pH 9; it can tolerate a temperature environment of 40°C; it can produce gas after 24 hours of fermentation, and its fermentation ability is strongest at 48 hours.

[0036] Table 2 pH tolerance of yeast Z3

[0037]

[0038] Table 3 Temperature tolerance of yeast Z3

[0039]

[0040] Table 4 Fermentation ability of yeast strain Z3 in YPD liquid medium

[0041]

[0042] Note: “-” means no gas production; “+” means the amount of gas in the Dulbecco’s tubules ≤ 1 / 4; “++” means 1 / 4<the amount of gas in the Dulbecco’s tubules ≤ 1 / 2; “+++” means 1 / 2<the amount of gas in the Dulbecco’s tubules ≤ 3 / 4; “++++” means 3 / 4<the amount of gas in the Dulbecco’s tubules ≤ 1.

[0043] Example 2

[0044] This example is the identification of Debaryomyces hansenii Z3, and the specific implementation operations are as follows:

[0045] 1) Morphological identification

[0046] The yeast strain Z3 obtained by the above screening was inoculated onto a YPD solid culture medium plate and cultured at 28°C for 2 days before observing the colony morphology. Figure 1 As shown, the colonies are milky white in color, smooth but opaque, moist and easy to pick up.

[0047] 2) Molecular biological identification

[0048] The yeast strain Z3 was sent to BGI for ITS sequencing, and the ITS nucleotide sequence of the yeast strain Z3 was measured as shown in SEQ.No 1. The sequencing results were submitted to the NCBI database for comparison and strain identification. The identification results showed that the ITS sequence of the yeast strain Z3 was 100% similar to the ITS sequence of Debaryomyces hansenii (accession number PP112075.1), and the phylogenetic tree was constructed using MEGA7.0 software. The phylogenetic tree is shown in Figure 2 As shown. Figure 2 It can be seen that yeast strain Z3 belongs to the Debaryomyces hansenii yeast evolutionary branch. Combining morphological characteristics and ITS sequence analysis, strain Z3 was identified as Debaryomyces hansenii, classified and named Debaryomyces hansenii Z3, and was deposited in the China Center for Type Culture Collection (CCTCC) on October 14, 2024, with the deposit number CCTCC NO: M 20242187, and the deposit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0049] Example 3

[0050] This example is a method for preparing a microbial preparation of Debaryomyces hansenii Z3, and the specific implementation operations are as follows:

[0051] The single colony of the above-mentioned Debaryomyces hansenii Z3 was inoculated into a YPD liquid culture medium and cultured for 24 h in a horizontal shaker at 28°C. The stirring speed during the culture process was 180 rpm to accelerate the growth of Debaryomyces hansenii Z3 to obtain a microbial preparation of Debaryomyces hansenii Z3. The preparation was a liquid preparation, in which the concentration of Debaryomyces hansenii Z3 was 1.0×10 8 CFU / mL.

[0052] 2. Specific examples of the use of the Debaryomyces hansenii Z3 and its preparation in cigar tobacco fermentation

[0053] Example 4

[0054] This example is the application of Debaryomyces hansenii Z3, and the specific implementation operations are as follows:

[0055] The blank control group of cigar tobacco raw tobacco leaves were fermented in a constant temperature of 37 ° C and 80% humidity fermentation room, the tobacco was turned once a day, and the window was opened for ventilation for 30 minutes. The cigar core tobacco leaves of the strain inoculation group (treatment group) were fermented: the prepared microbial preparation was evenly sprayed on the surface of the cigar core tobacco leaves, 50 mL of bacterial solution was sprayed on every 500g of tobacco leaves, 3 replicates were set, and the fermentation was carried out in a constant temperature of 37 ° C and 80% humidity fermentation room, the tobacco was turned once a day, and the ventilation was 30 minutes.

[0056] The results showed that it took 30 days for the control group to reach a usable state, while it only took 15 days for the tobacco fermented with the Hansen Debaryomyces strain Z3 to reach a usable state. This shows that the strain can significantly shorten the fermentation time of cigar core tobacco leaves, which is beneficial to industrial production.

[0057] The conventional chemical components of cigar core tobacco leaves were determined using a continuous flow analyzer. The water-soluble sugar test adopted the "YC / T159-2019 Continuous Flow Method for Determination of Water-soluble Sugar in Tobacco and Tobacco Products", the nicotine test adopted the "YC / T 468-2021 Continuous Flow Method for Determination of Total Alkaloids in Tobacco and Tobacco Products (Potassium Thiocyanate)", and the total nitrogen test adopted the "YC / T 161-2002 Continuous Flow Method for Determination of Total Nitrogen in Tobacco and Tobacco Products".

[0058] The results are shown in Table 5: after treatment with Debaryomyces hansenii Z3, the contents of total nicotine, total sugar and total nitrogen were all reduced. Total nicotine, total sugar and total nitrogen are three important factors affecting the taste of cigar tobacco. The direct effects of the three on the taste quality of cigar tobacco and the indirect effects caused by the correlation between them are the key to determining the taste quality of cigar tobacco. It can be seen that treatment with Debaryomyces hansenii Z3 has a significant effect on improving the quality of cigar core tobacco leaves.

[0059] Table 5 Changes of conventional chemical components in tobacco leaves after treatment with Debaryomyces hansenii Z3

[0060]

[0061] The method for detecting the content of neutral aroma substances is as follows: accurately weigh 25g of the sample, place the sample in a 1000mL round-bottom flask, add 25g NaCl and 350mL deionized water, and place it in a constant temperature electric heating mantle for heating. Add 50mL of dichloromethane to a 250mL round-bottom flask, heat it in a constant temperature water bath, maintain the water temperature at 60°C, connect two round-bottom flasks with a simultaneous distillation extractor, distill and extract for 2.5h, collect the liquid in the 250mL flask, dry it with anhydrous sodium sulfate, transfer it to a concentration bottle, and use a rotary evaporator to concentrate it to 1mL in a 40°C water bath to obtain tobacco essential oil for GC-MS analysis. GC-MS conditions: the chromatographic column is DB-5MS, the carrier gas is helium, the inlet temperature is 280°C; the carrier gas is high-purity helium; the column oven is programmed temperature rise; the split ratio is 5:1; the flow rate is 1.0mL / min. 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 NIST 17 search was used.

[0062] The results showed that 35 aroma substances were screened out, as shown in Table 6. According to common functional groups, they can be divided into eight categories: esters, ketones, aldehydes, alcohols, phenols, acids, olefins and nitrogen-containing heterocyclics. Nitrogen-containing heterocyclics include two alkaloids, mesamine and isonicotine, and two aroma components, 3-acetylpyridine and indole. The contents of mesamine and isonicotine in the treatment group samples were lower than the CK value. In addition to nitrogen-containing heterocyclics, esters, ketones, aldehydes, alcohols, phenols, acids and olefins were compared. The contents of esters, ketones and aldehydes in the treatment group samples were higher than those in the control group. The contents of β-ionone and (1-hexyl-1H-indol-3-yl)-1-naphthyl ketone in the carotenoid degradation products were higher in the treatment group samples than in the control group. The contents of acetophenone, benzyl alcohol, phenylethanol, benzaldehyde and phenylacetaldehyde in the aromatic amino acid degradation products, phytol and phyton in the chlorophyll degradation products, and cedarene and β-cadinene in the treatment group were also higher than those in the control group samples. In summary, from the data of neutral aroma components, it can be seen that the treatment group has more types of neutral aroma components and more components with increased content, and the reduction of alkaloids such as isonicotine is beneficial to human health, so the results of the treated group samples after fermentation are better than those of the control group.

[0063] Table 6 Content of neutral aroma components in cigar filler tobacco leaves (unit: μg / g)

[0064]

[0065]

[0066]

[0067] The sensory quality evaluation was conducted by a panel of 8 cigar sensory evaluators, and the sensory quality of the treatment group and control group samples was evaluated using the evaluation method described in "GB 15269.4-2011 Cigar Part 4: Sensory Technical Requirements".

[0068] The results are shown in Table 7: The sensory evaluation scores also show that the treatment group is better than the control group. The experts agree that the treatment group has a higher total score, a more pronounced tone, richer aroma, significantly improved irritation compared to CK, and a comfortable aftertaste. The results of the smoking test show that the treatment group, fermented with Hansen Debaryomyces Z3, has a significantly improved aroma. Its aroma, miscellaneous odor, irritation and aftertaste are better than those of the control group, and has the characteristics of oily smoke.

[0069] Table 7 Cigar sensory quality evaluation table

[0070]

Claims

1. A strain of Debaryomyces hansenii, characterized in that Debaryomyces hansenii Debaryomyces hansenii Z3, the accession number is CCTCC NO: M 20242187.

2. A microbial preparation, characterized in that: Debaryomyces hansenii Debaryomyces hansenii Z3.

3. The microbial preparation according to claim 2, characterized in that The microbial preparation is the fermentation broth of Debaryomyces hansenii Z3.

4. The microbial preparation according to claim 3, characterized in that The microbial preparation is obtained by fermenting the Debaryomyces hansenii Z3 in a liquid culture medium, and the concentration of the Debaryomyces hansenii Z3 is ≥1.0×10 7 CFU / mL.

5. Use of the Debaryomyces hansenii Z3 according to claim 1 or the microbial preparation according to claim 2 in cigar filler tobacco leaves, characterized in that: The application includes shortening the fermentation time of the cigar core tobacco leaves and improving the cigar mellowness and smoke oiliness.

6. The use according to claim 5, characterized in that: Every 500 g of tobacco leaves was sprayed with 50-70 mL of bacterial solution, the concentration of which was 1.0×10 7 ~1.0×10 9 CFU / mL.

7. The use according to claim 6, characterized in that: After spraying the bacterial liquid, the cigar core tobacco leaves are fermented for 15 days at a constant temperature of 35±2° C. and a humidity of 75±5%.

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