Reconstituted tobacco leaves prepared by fermentation with Hansenula sporogenes HO-1

Abstract

The present invention discloses a reconstituted tobacco leaf prepared by fermenting Hansenia sporangioides HO-1, specifically a reconstituted tobacco concentrate prepared by fermenting seed liquid prepared by Hansenia sporangioides HO-1, belonging to the technical field of tobacco preparation. Hansenia sporangioides is Hansenia sporangioides HO-1, the base sequence of which is shown in SEQ ID NO.1. It has been deposited in the General Microbiological Center of the China Microorganism Culture Collection Committee on July 27, 2023, with a deposit number of CGMCC No.28023, and its classification name is Hanseniaspora opuntiae. The present invention proves that it can effectively increase the key tobacco flavor substances such as esters, alcohols and ketones through the selection of a specific Hansenia sporangioides strain, thereby achieving the purpose of improving quality and enhancing flavor, and can effectively improve the quality of the prepared reconstituted tobacco leaf.

Description

Technical Field

[0001] The invention belongs to the technical field of tobacco preparation, relates to a technology for preparing reconstituted tobacco leaves by a papermaking method, and specifically relates to a reconstituted tobacco leaf prepared by fermenting Hansenula sporogenes HO-1. Background Art

[0002] Hanseniaspora opuntiae is taxonomically classified as belonging to the Eukaryota, Fungi, Ascomycota, Saccharomyces, Saccharomyces, Saccharomyces, and Genus Hanseniaspora. Hanseniaspora opuntiae is a non-Saccharomyces yeast isolated from naturally harvested grapes and other berries. It has the ability to produce ethanol, cause berry spoilage, and protect plants from fungal diseases.

[0003] The production of reconstituted tobacco leaves using the papermaking method is an important technology in the current tobacco industry. Adding an appropriate amount of reconstituted tobacco leaves to cigarettes can, on the one hand, maximize the savings in tobacco raw materials and effectively reduce cigarette costs; on the other hand, it can, to a certain extent, adjust and improve the physical properties and chemical composition of cigarettes according to people's wishes or requirements, effectively reducing the amount of tar and other harmful substances in cigarettes, stabilizing the quality of cigarette products, and thus helping to improve the inherent quality of cigarettes. It is an important technical measure to reduce and improve the undesirable components of tobacco. Given that reconstituted tobacco leaves using the papermaking method use tobacco materials (tobacco stems, tobacco dust, and broken tobacco leaves, etc.) as the main raw materials, after water extraction and concentration, the solid part is processed into a sheet base, and the liquid part is formed into a concentrate (i.e., reconstituted tobacco concentrate), which is further proportioned to form a coating liquid and coated on the sheet base to form a tobacco product. In the process of forming the concentrate, some macromolecular substances are dissolved in water and retained, which increases the viscosity of the concentrate, affects the coating rate, and is a precursor of the inhaled miscellaneous gas; the aroma components in the concentrate have a low boiling point and a large amount of volatilization loss, which reduces the inhalation quality of the papermaking method reconstituted tobacco leaves, resulting in the reconstituted tobacco leaves having insufficient aroma quality compared with natural tobacco leaves.

[0004] In order to improve the aroma quality of reconstituted tobacco leaves, researchers have made many attempts in production technology and exogenous addition of flavors and fragrances. For example, Zhu Hongqin et al. studied the effects of drying time and temperature in the drying process of reconstituted tobacco leaves on aroma components ("Effects of Drying Temperature and Time on Aroma Components of Reconstituted Tobacco Leaves by Papermaking", Zhu Hongqin et al., Journal of Henan Agricultural University); Ning Yong et al. added agarwood components to reconstituted tobacco leaves for aroma enhancement ("Research on the Application of Agarwood Components in Reconstituted Tobacco Leaves by Papermaking", Ning Yong et al., Forest Products Industry). The above methods can have a certain effect on preserving and improving the aroma quality of reconstituted tobacco leaves, but have little effect on improving the problem of poor original aroma quality of reconstituted tobacco leaves.

[0005] Studies have shown that microbial fermentation technology has potential application prospects in enhancing the flavor of different tobacco products. For example, Du Fei et al. screened out aroma-producing yeast and aroma-producing Aspergillus from Maotai liquor lees and Fuzhuan tea. The products of the two fermented with green tea powder as raw material have tea aroma, wine aroma, honey sweet aroma and special aroma, which significantly improve the sensory quality of tobacco products ("Isolation and Identification of Two Aroma-Producing Microorganisms and Research on Flavoring Tobacco with Their Fermentation Broth", Du Fei et al., "Journal of Mountain Agriculture and Biology"); Ye Jianbin et al. used kombucha to ferment tobacco extract and obtained a characteristic sour-flavored tobacco extract ("Preparation of Characteristic Sour-Fragrance Tobacco Extract by Kombucha Fermentation", Ye Jianbin et al., "Journal of Food and Biotechnology").

[0006] It can be seen that the development of microbial strains suitable for flavoring tobacco products is of great significance for improving the defect of insufficient aroma of tobacco concentrate and ultimately improving the quality of reconstituted tobacco leaves. Summary of the Invention

[0007] The present invention aims to provide a technology for preparing reconstituted tobacco leaves by fermenting Hansenula sporangiophora HO-1. By selecting a specific Hansenula sporangiophora, the tobacco concentrate is fermented with Hansenula cactus sporangiophora HO-1 to effectively increase the content of key tobacco flavor substances such as esters, alcohols, and ketones, thereby achieving the purpose of improving the quality and flavor of the reconstituted tobacco leaves.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] A reconstituted tobacco leaf prepared by fermenting Hansenia sporophora HO-1, wherein the reconstituted tobacco leaf is prepared by a papermaking method, specifically by fermenting seed liquid prepared by Hansenia sporophora cactus HO-1 to produce a reconstituted tobacco concentrate; the Hansenia sporophora cactus HO-1 has a base sequence as shown in SEQ ID NO.1, is classified as Hanseniaspora opuntiae, and was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms on July 27, 2023, with a deposit number of CGMCC No. 28023, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] Preferably, the preparation steps of the Hansenula sporeans HO-1 seed solution are as follows:

[0011] A single colony of Hansenula cacti HO-1 was inoculated into a YPD liquid culture medium, and cultured in a shaking incubator at a temperature of 25-35° C., a pH of 3-5, and a rotation speed of 200 r / min for 24 hours to obtain a seed solution.

[0012] Preferably, the fermentation is carried out as follows: adjusting the OD600 of the Hansenula cacti HO-1 seed solution to 1, inoculating it into 80% concentration gradient reconstituted tobacco concentrate at a volume ratio of 1:5, and fermenting at 25°C and pH 4 for 24 hours.

[0013] Preferably, the 80% concentration gradient reconstituted tobacco concentrate is prepared by mixing reconstituted tobacco leaf concentrate, YPD liquid culture medium and water in a volume ratio of 8:1:1.

[0014] Preferably, the YPD liquid culture medium comprises the following components: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and the solvent is water.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] By selecting a specific strain of Hansenula sporangiophora, the present invention amplifies Hansenula cacti HO-1 to prepare a seed solution. This solution, when fermented into a reconstituted tobacco concentrate to produce papermaking reconstituted tobacco leaves, effectively increases the content of key tobacco flavoring substances, including esters, alcohols, ketones, heterocycles, and other compounds, thereby enhancing the smoking experience. Because the present invention can effectively improve tobacco smoking quality in a short period of time, at low cost and with high efficiency, coupled with its relatively simple operational process, the present invention demonstrates significant application value and potential in the field of cigarette production. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the bacterial morphology of Hansenula cacti HO-1 under the streaking line on the plate;

[0018] Figure 2 This is a 40x microscope morphology of Hansenula cacti HO-1;

[0019] Figure 3 Construct a phylogenetic tree for Hansenula sporangiophora;

[0020] Figure 4 Figure 1 is a graph showing the results of determining the optimal growth temperature and culture medium pH of Hansenula cacti HO-1, where A is a statistical graph showing the bacterial concentration of cells with an initial concentration of OD600 = 0.2 after culturing at different temperatures for 24 hours, and B is a statistical graph showing the bacterial concentration of cells with an initial concentration of OD600 = 0.2 after culturing at different pH values ​​for 24 hours;

[0021] 5A to 5D The following are the changes in the content and types of aroma components of reconstituted tobacco concentrates with different dilution concentrations after fermentation by Hansenula cactus spore yeast HO-1. The red curve is after fermentation and the black curve is before fermentation. Figure 5A It is a 40% concentration of reconstituted tobacco concentrate. Figure 5BIt is a 60% concentration of reconstituted tobacco concentrate. Figure 5C It is 80% reconstituted tobacco concentrate. Figure 5D It is undiluted 100% reconstituted tobacco concentrate;

[0022] Figure 6 The figure shows the changes in aroma substances in 80% diluted reconstituted tobacco concentrate fermented by Hansenula spores HO-1 under optimal conditions, where CK is the group not inoculated with Hansenula spores HO-1, and HO-1 is the group inoculated with Hansenula spores HO-1. DETAILED DESCRIPTION

[0023] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0024] The reagents involved in the embodiments of the present invention were all purchased from commercial channels, and the methods not mentioned are conventional experimental methods and will not be described in detail here.

[0025] YPD liquid medium formula: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, solvent is water.

[0026] Example 1 Identification of Hansenula cacti HO-1:

[0027] 1. Isolation and cultivation of strains:

[0028] The strain is derived from Kyoho grapes grown in the Pingdu grape production garden in Shandong Province. The specific method of obtaining it is as follows:

[0029] Weigh 100 g of grapes and add 30 ml of sterile saline. Incubate on a shaker at 200 rpm at room temperature for 5 min. Use a sterile pipette to aspirate 500 μl of the solution onto YPD medium. After spreading, incubate at 30°C overnight. Select single colonies with round, opaque milky white color and smooth edges and incubate them in 3 ml of YPD liquid medium at 30°C for 24 h.

[0030] 2. Screening and identification of strains:

[0031] Use an inoculation loop to dip the cultured bacterial solution and draw a line on the YPD culture plate. After 48 hours of growth, observe the bacterial morphology (see Figure 1100 μl of bacterial solution with OD600 of 0.2 was inoculated into 3 ml of YPD medium and cultured at 30°C and 200 rpm for 24 h. The bacterial solution was dropped onto a glass slide and pressed. The bacterial cell morphology was observed under a 40x objective lens. The cells showed different morphologies. Single cells were mostly oval, but some cells were connected head to tail to form rods (see Figure 2 ).

[0032] After the YPD liquid culture was completed, Qingdao Weilai Biotechnology Co., Ltd. carried out sequence determination, and the 26SrDNA sequence of the strain was shown as SEQ ID NO.1. The sequencing results were compared with the sequences in the NCBI nucleic acid sequence database, and the species information with the greatest similarity to the sequence of the species to be tested was obtained, which was the identification result. Based on the neighbor-joining method, a phylogenetic tree was constructed to analyze the evolutionary relationship of species (see Figure 3 ), identified it as Hanseniaspora opuntiae, and named it Hanseniaspora opuntiae HO-1.

[0033] Example 2 Cultivation of Hansenula sporeifera HO-1:

[0034] 1. Determination of optimal growth temperature and culture medium pH:

[0035] The temperature conditions were set at 25°C, 28°C, 30°C, 32°C, and 35°C, and the pH conditions were set at pH 4, pH 5, pH 6, pH 7, and pH 8. The cactus spore Hansenula HO-1 bacteria with an initial OD600 of 0.2 were inoculated into the above-mentioned culture media at different temperatures and pH values ​​and cultured. The bacterial concentrations were measured after 24 hours of culture. The results are shown in Table 1. Figure 4 A and Figure 4 B in.

[0036] Depend on Figure 4 As shown in A, the growth rate of bacteria varies at different culture temperatures. In the temperature range of 25℃-35℃, the growth rate first increases and then decreases, and the growth rate is the highest at 28℃. Figure 4 As shown in Figure B, the growth ability of the bacteria varies under different culture medium pH conditions, and the bacteria grows fastest at pH 4. In summary, the optimal growth temperature and pH conditions for Hansenula cacti HO-1 are 25°C and pH 4, respectively.

[0037] 2. Preparation of Hansenula sporeifera HO-1 seed solution:

[0038] A single colony of Hansenula sporogenes HO-1 was inoculated into YPD liquid culture medium using an inoculation loop, and cultured in a shaking incubator at 200 rpm / min at 25-35° C. (optimal 25° C.), pH 3-5 (optimal pH 4) for 24 hours to obtain a seed solution.

[0039] Example 3 Application of Hansenula sporeifera HO-1 in flavoring tobacco products:

[0040] The reconstituted tobacco concentrate KY-7 produced by Zhejiang China Tobacco Industrial Co., Ltd. was used as the test sample.

[0041] 1. Effect of reconstituted tobacco concentrate concentration on fermentation of Hansenula sporangiophora HO-1:

[0042] The reconstituted tobacco concentrate was diluted to 40% (V) with water and YPD liquid medium according to the corresponding volume ratio. 水 : VYPD :V 浓缩液 =5:1:4), 60% (V 水 :V YPD :V 浓缩液 =3:1:6), 80% (V 水 :V YPD :V 浓缩液 =1:1:8), the seed solution prepared in Example 2 was adjusted to OD600=1 and added to the same volume of the tobacco concentrates with different concentration gradients and undiluted reconstituted tobacco concentrate (concentration gradient of 100%) at a volume ratio of 1:5. Fermentation was carried out at 25°C and pH 4. After fermentation for 24 hours, the content and type of the aroma components produced were detected by the following GC-MS / MS method.

[0043] The specific procedure was to take 2 mL of reconstituted tobacco concentrate (pre- and post-fermentation) at different concentrations and thoroughly mix it with an equal volume of ethyl acetate, then sonicate at 40 kHz for 10 minutes. The system was then removed and centrifuged at 4000 rpm for 10 minutes to separate the organic solvent and aqueous phases. After 1 mL of the ethyl acetate supernatant was filtered through a 0.22 μm membrane, the filtrate was transferred to a vial for GC-MS / MS analysis.

[0044] The specific conditions for GC-MS / MS are as follows:

[0045] The chromatographic column was a capillary column HP-5MS (30m×0.25mm×0.25μm). The initial temperature of the chromatographic column was set to 40°C, maintained for 2 minutes, then heated to 185°C at 5°C / min, maintained for 1 minute, and then heated to 240°C at 2°C / min, maintained at this temperature for 10 minutes, for a total of 60.5 minutes. Carrier: helium, column flow rate 1mL / min, inlet temperature 260°C. Mass spectrometry conditions: EI source, 70eV, ion source temperature 230°C, mass spectrometry transfer line temperature 270°C, quadrupole temperature 150°C, mass spectrometry scanning range 40-450m / z. The NIST 11.0 standard spectral library was used for retrieval analysis, and the results of each concentration gradient are shown in Figure 2. Figures 5A-5D .

[0046] Depend on Figures 5A-5D It can be seen that when the reconstituted tobacco concentrate was fermented for 24 hours at a concentration gradient of 40%, 60% and 80%, multiple new peaks different from those in the control group appeared. The reconstituted tobacco concentrate had fewer new peaks at a concentration gradient of 100% (perhaps the high concentration of tobacco concentrate is not conducive to microbial growth), indicating that multiple new substances were produced in the diluted reconstituted tobacco concentrate fermented by Hansenula cacti spores HO-1. In addition, the abundance of some substances in the fermented reconstituted tobacco concentrate showed an increasing change, indicating that their concentrations in the reconstituted tobacco concentrate increased after fermentation by Hansenula cacti spores HO-1. From the perspective of saving subsequent concentration time, the 80% concentration gradient was selected as the optimal fermentation concentration of the reconstituted tobacco concentrate.

[0047] 2. Effects of Hansenula cacti HO-1 fermentation under optimal fermentation conditions on aroma compounds in reconstituted tobacco concentrate:

[0048] Select 80% concentration gradient of tobacco concentrate (V 水 :V YPD :V 浓缩液 =1:1:8), the one inoculated with Hansenula sporogenes HO-1 was designated HO-1, and the reconstituted tobacco concentrate after replacing HO-1 with an equal volume of water was designated CK. The seed solution prepared in Example 2 was adjusted to OD600 = 1 and inoculated at 20% of the volume of the reconstituted tobacco concentrate in the HO-1 group. Fermentation was carried out at 25°C and pH 4 for 24 hours. The above experiment was repeated three times, and the content of aroma components in the PK-1 group and the CK group was measured each time and statistical analysis was performed. The specific operation was as follows:

[0049] 5 mL of the reconstituted tobacco concentrate from the HO-1 or CK group was placed in a 20 mL headspace vial and extracted at 60°C for 30 min using a 50 / 30 μm DVB / CAR / PDMS extraction tip. The extract was then immediately inserted into the inlet of a gas chromatograph for GC-MS / MS analysis.

[0050] The specific conditions for GC-MS / MS are as follows:

[0051] The chromatographic column was a DB-5MS capillary column (30 m × 0.25 mm × 0.25 μm). The column temperature was initially set at 45°C and held for 3 min. The temperature was then increased at 3°C / min to 180°C, held for 1 min, then increased at 5°C / min to 250°C and held at this temperature for 5 min, for a total of 68 min. Carrier gas was helium, column flow rate was 1 mL / min, and the inlet temperature was 230°C. Mass spectrometry conditions included an EI source at 70 eV, ion source temperature at 230°C, mass spectrometer transfer line temperature at 270°C, quadrupole temperature at 150°C, and a mass spectrometer scan range of 35–550 m / z. The NIST 17.0 standard spectral library was used for search and analysis. The contents of esters, alcohols, ketones, acids, aldehydes, phenols, heterocyclic compounds, and other volatile aroma components were determined for both the HO-1 and CK groups. The results are shown in Tables 1-7.

[0052] Table 1 Ester content in the two groups of concentrates (μg / mL)

[0053]

[0054]

[0055] Esters are important aroma compounds that impart rich floral and fruity aromas to tobacco. Table 1 shows that tobacco concentrate fermented with Hansenula cacti spore yeast HO-1 contains nine new ester aroma compounds, most of which are ethyl esters. Phenylethyl acetate, which has a honey aroma and is a key flavoring, is the most abundant. Furthermore, ethyl hexadecanoate, found in apricots, blackcurrants, and sour cherries, imparts a fruity and creamy aroma. Ethyl acetate and ethyl linoleate are important flavor compounds in Chinese liquor, imparting sweet fruity and caramelized aromas, respectively.

[0056] Table 2 Alcohol content in the two groups of concentrates (μg / mL)

[0057]

[0058]

[0059] As shown in Table 2, with the exception of propylene glycol, 3-furyl alcohol, and linalool, the content of most alcohol compounds in the tobacco concentrate fermented with Hansenula sporangium HO-1 increased to varying degrees compared to the CK control. The most significant increase was in phenylethanol, which increased by 15.34-fold. Phenylethanol has a sweet, rose-like, fruity aroma. Its increased content significantly improves the mainstream smoke of the tobacco concentrate and enhances the flavor of the tobacco. In addition to the increase in existing alcohol compounds, six new alcohol aroma compounds were added: isobutanol, n-butanol, isopentanol, 2-methylbutanol, farnesol, and 3-deoxy-17B-estradiol. Among these, isobutanol, n-butanol, and isopentanol are important aroma components of tea and wine aromas. When combined with ester aroma compounds, these compounds enrich the aroma.

[0060] Table 3 Ketone content in the two groups of concentrates (μg / mL)

[0061]

[0062]

[0063] As shown in Table 3, compared with the CK group, in the tobacco concentrate fermented with Hansenula sporogenes HO-1, the levels of 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one and 4-oxoisophorone decreased. However, the levels of most ketone compounds, including damascone, solanone, and megastigmatrienone, increased. Solanone had the highest content of all ketone aroma compounds, with the largest increase, from 65.71 μg / mL to 92.14 μg / mL. Solanone is a degradation product of tobacco sibirica trienes and has a sweet and fresh carrot-like aroma. It enhances the natural aroma of tobacco, making the smoke fuller, mellower, and more delicate. In addition, the tobacco concentrate fermented by Hansenula sporangiophora HO-1 contains three new ketone aroma substances, namely 2,3-butanedione, 3-hydroxy-2-butanone and geranyl acetone. Among them, 2,3-butanedione and 3-hydroxy-2-butanone have a creamy aroma and are widely used in alcohol, dairy products, etc. Geranyl acetone has the aroma of raw pears, apples, and tropical fruits, and is widely used in cosmetics, perfumes, medicine, tobacco and other industries.

[0064] Table 4 Acid content in the two groups of concentrates (μg / mL)

[0065]

[0066] As shown in Table 4, acetic acid, an acidic aroma compound, was detected in the tobacco concentrate fermented by Hansenula sporangiophora HO-1, while nonanoic acid and palmitic acid were absent compared with the CK group.

[0067] Table 5 Aldehyde content in the two groups of concentrates (μg / mL)

[0068]

[0069]

[0070] As shown in Table 5, the content of most aldehyde aroma compounds in the tobacco concentrate fermented with Hansenula sporeensis HO-1 decreased, including phenylacetaldehyde, benzaldehyde, 2,4-dimethylbenzaldehyde, 5-hydroxymethylfurfural, and isovaleraldehyde. The reduction of furfural aroma compounds helps reduce the bitterness and off-flavors of tobacco.

[0071] Table 6 Phenolic content in the two groups of concentrates (μg / mL)

[0072]

[0073] As shown in Table 6, the total amount of phenolic aroma compounds in the tobacco concentrate fermented with Hansenula spores HO-1 decreased, primarily due to reductions in 5-vinyl-2-methoxyphenol and ethyl maltol. Furthermore, two new phenolic compounds, 2,4-di-tert-butylphenol and 2-methoxyphenol, were found in the tobacco concentrate fermented with Hansenula spores HO-1.

[0074] Table 7 Contents of heterocyclic and other substances in the two groups of concentrates (μg / mL)

[0075]

[0076]

[0077] As shown in Table 7, the total contents of most heterocyclic and other substances in the tobacco concentrate fermented by Hansenula sporangiophora HO-1 were reduced, and three new aroma components, γ-cedrene and cedrene, were added.

[0078] The statistical results of the total contents of volatile aroma components such as esters, alcohols, ketones, acids, aldehydes, phenols, heterocyclics and others in the HO-1 and CK groups are shown in Tables 8 and Figure 6 .

[0079] Table 8 Content of aroma substances in the two groups of concentrates (μg / mL)

[0080]

[0081] From Table 8 and Figure 6It can be seen that after fermentation with Hansenula sporangiophora HO-1, the content of aroma components such as esters, alcohols and ketones in the concentrate was significantly increased compared with the unfermented CK group. After calculation, the increase was 361.12%, 327.29% and 34.98% respectively, which was a significant increase. The overall quality of the concentrate fermented with Hansenula sporangiophora HO-1 was improved.

[0082] 3. Preparation and smoking evaluation of papermaking reconstituted tobacco

[0083] By volume ratio V 浓缩液 :V YPD :V 水 =8:1:1. Reconstituted tobacco concentrate, YPD liquid culture medium, and water are mixed to prepare a reconstituted tobacco concentrate with an 80% concentration gradient. The OD600 of Hansenula cacti spore-forming yeast HO-1 seed liquid is adjusted to 1, and the mixture is inoculated into the tobacco concentrate at a volume ratio of 1:5. The mixture is fermented at 25°C and pH 4 for 24 hours. The fermented tobacco concentrate is vacuum concentrated at 50°C to a sugar content of 50% Brix, and then evenly coated on a tobacco sheet at a coating rate of 39%. The mixture is quickly dried at 90°C to a humidity of 12%-15%. The coated tobacco sheet is rehumidified at 20°C and 60% humidity to a moisture content of 12%, thereby obtaining reconstituted tobacco leaves by a papermaking process.

[0084] The papermaking-processed reconstituted tobacco leaves prepared above were shredded to make single-ingredient cigarettes, designated as the HO-1 group. 80% concentration gradient tobacco concentrate was not inoculated with Hansenula cacti spores HO-1, and other operations were the same. The single-ingredient cigarettes made by shredding the reconstituted tobacco leaves prepared above were designated as the CK group. Seven qualified smoking experts smoked the single-ingredient cigarettes of the above two groups and scored the cigarettes for characteristics such as aroma, sweetness, permeability, off-flavor, irritation, aftertaste and odor. The results are shown in Table 9.

[0085] Table 9 Sensory evaluation results of two groups of reconstituted tobacco leaves

[0086]

[0087]

[0088] As shown in Table 9, the papermaking reconstituted tobacco prepared from tobacco concentrate fermented with Hansenula sporangiophora HO-1 has a relatively strong floral and fruity aroma, with improved aroma quality and aroma quantity, better coordination between smoke, enhanced burnt sweetness and permeability, weakened off-flavors, reduced irritation, and a cleaner aftertaste.

[0089] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0090] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A reconstituted tobacco leaf prepared by fermentation with Hansenula sporogenes HO-1, characterized in that: The reconstituted tobacco leaf is made by a papermaking method, specifically by fermenting seed liquid prepared with Hansenia sporaopuntiae HO-1 to reconstitute tobacco concentrate; the Hansenia sporaopuntiae HO-1 is classified and named Hansenia sporaopuntiae, and was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms on July 27, 2023, with a deposit number of CGMCC No. 28023, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The reconstituted tobacco leaf prepared by fermentation with Hansenula sporogenes HO-1 according to claim 1, characterized in that: The base sequence of the Hansenula cacti HO-1 is shown in SEQ ID NO.

1.

3. The reconstituted tobacco leaf prepared by fermentation with Hansenula sporogenes HO-1 according to claim 1, characterized in that: The preparation steps of the Hansenula sporeifera HO-1 seed solution are as follows: A single colony of Hansenula cacti HO-1 was inoculated into a YPD liquid culture medium, and cultured in a shaking incubator at a temperature of 25-35° C., a pH of 3-5, and a rotation speed of 200 r / min for 24 hours to obtain a seed solution.

4. The reconstituted tobacco leaf prepared by fermentation with Hansenula sporogenes HO-1 according to claim 1, characterized in that: The specific fermentation operation is: adjusting the OD600 of Hansenula cacti HO-1 seed liquid to 1, inoculating it into 80% concentration gradient reconstituted tobacco concentrate at a volume ratio of 1:5, and fermenting it at 25°C and pH 4 for 24 hours.

5. The reconstituted tobacco leaf prepared by fermentation with Hansenula sporogenes HO-1 according to claim 4, characterized in that: The 80% concentration gradient reconstituted tobacco concentrate is prepared by mixing reconstituted tobacco leaf concentrate, YPD liquid culture medium and water in a volume ratio of 8:1:

1.

6. The reconstituted tobacco leaf prepared by fermentation with Hansenula sporogenes HO-1 according to claim 5, characterized in that: The YPD liquid culture medium comprises the following components: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and the solvent is water.

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