Reconstituted tobacco prepared by fermentation of Guiyuan Mongolian Yarrowia yeast

Abstract

The invention discloses a reconstituted tobacco prepared by a papermaking method, and particularly relates to a reconstituted tobacco prepared by fermenting a seed solution prepared by Meyrowia guilliermondii MG-1 into a reconstituted tobacco concentrated solution, belonging to the technical field of tobacco preparation. The invention discloses a Yarrowia guilliermondii MG-1, which is preserved in China General Microbiological Culture Collection Center (CGMCC) on July 27, 2023, the preservation number is CGMCC No.28024, and the Yarrowia guilliermondii MG-1 is classified and named as Yarrowia guilliermondii. The Yarrowia guilliermondii MG-1 has the advantages that the Yarrowia guilliermondii MG-1 has the advantages that the Yarrowia guilliermondii MG-1 can be The selection of specific yeast strains proves that the yeast strains can effectively increase ketones, alcohols, acids, phenols, heterocyclic rings and other tobacco key flavor substances, the purpose of improving quality and aroma is achieved, and the quality of the prepared reconstituted tobacco can be effectively improved.

Description

Technical Field

[0001] The invention belongs to the technical field of tobacco preparation, and particularly relates to preparing reconstituted tobacco leaves by fermenting reconstituted tobacco concentrate with Meyer yeast MG-1. Background Art

[0002] Reconstituted tobacco refers to the by-products (tobacco stems, broken leaves, tobacco dust, etc.) produced during the cigarette processing process, which are made into sheet or filamentary regeneration products by physical, chemical, biological and other methods, and can be used in the leaf group formula of cigarettes. Compared with traditional tobacco leaves, reconstituted tobacco leaves have significant advantages such as low tar content, strong plasticity, and good combustibility, which can provide effective support for reducing tar and reducing harm and enhancing the characteristic style of cigarette products. However, the raw materials for processing reconstituted tobacco leaves mostly come from tobacco waste resources, and the quality of the raw materials is relatively low. In addition, the extraction, concentration and drying of the reconstituted tobacco leaf processing process will cause the loss of some volatile aroma substances. Therefore, reconstituted tobacco leaves generally have problems such as poor aroma quality, obvious stimulation, and poor comfort, which limits their addition and use in high-quality cigarette products.

[0003] The main production processes of reconstituted tobacco include papermaking, thick pulping, and roller pressing. The papermaking method processes tobacco materials into reconstituted tobacco through extraction, concentration, pulping, coating, and drying. It has the characteristics of high uniformity, good filling, and low tar content. It is one of the mainstream technologies for the production of reconstituted tobacco in the tobacco industry. Reconstituted tobacco concentrate is a tobacco-soluble substance obtained by water extraction, filtration, and concentration of tobacco raw materials in the process of preparing reconstituted tobacco by the papermaking method. It can be coated on the tobacco substrate and dried to prepare reconstituted reconstituted tobacco. Since the reconstituted tobacco concentrate contains a large amount of aroma components, soluble sugars, proteins, and structural polysaccharides with smaller particle sizes, its quality directly affects or even determines the quality of reconstituted tobacco. Based on this, researchers have conducted many explorations in improving the quality of reconstituted tobacco concentrate. For example, Wang Runan et al. studied the effects of storage conditions and temperature on the quality of concentrate, and found that storage at 50°C for 5 to 6 hours can effectively increase its soluble sugar content and achieve the stable existence of various aroma components ("The Effect of Storage Conditions on the Quality of Concentrates in the Production Process of Reconstituted Tobacco Leaves", Wang Runan et al., "Journal of Light Industry"); Ji Yawen et al. used microorganisms to ferment tobacco stem extracts, which reduced the soluble sugar content in the extracts and improved the aroma quality ("Study on the Effect and Regulation of Sugar Compounds on the Quality of Reconstituted Tobacco Leaves"); Ma Ke et al. treated the concentrates with biological enzymes, which effectively improved the aroma quality, coordination, irritation and aftertaste of the reconstituted tobacco leaves ("Study on the Effect of Concentrates Treated with Different Enzymes on the Quality of Guizhou Reconstituted Tobacco Leaves", Ma Ke et al., "Journal of Light Industry").

[0004] Many microorganisms in nature have the ability to secrete aromatic substances, and have development potential in improving the concentrated aroma quality of reconstituted tobacco leaves. Therefore, screening microbial resources with strong aroma production ability and rich aroma quality levels is of great significance to improving the quality of reconstituted tobacco leaves. Summary of the invention

[0005] In view of the above problems, the present invention selects specific yeast and ferments the tobacco concentrate by using Meyer yeast guillerie MG-1 to effectively increase the key tobacco flavor substances such as ketones, alcohols, acids and phenols, thereby achieving the purpose of improving quality and enhancing flavor, and can effectively improve the quality of reconstituted tobacco leaves.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A reconstituted tobacco leaf prepared by fermentation of Meyerozyma guilliermondii, wherein the reconstituted tobacco leaf is prepared by a papermaking method, specifically by fermenting seed liquid prepared by Meyerozyma guilliermondii MG-1 to reconstitute tobacco concentrate; the Meyerozyma guilliermondii MG-1 is classified and named Meyerozyma guilliermondii, and was deposited in the General Microbiology Center of China Microbiological Culture Collection on July 27, 2023, with a deposit number of CGMCC No. 28024, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] Preferably, the preparation steps of the Meyer yeast MG-1 seed solution are as follows:

[0009] A single colony of Meyersinia guilliermondii MG-1 was inoculated into a YPD liquid culture medium, and cultured in a shaking incubator at a temperature of 22-30°C, a pH of 6-8, and a rotation speed of 150 r / min for 20 hours to obtain a seed solution.

[0010] Preferably, the specific operation of the fermentation is: adjusting the OD600 of the Meyer yeast MG-1 seed liquid to 2, inoculating it into a 60% concentration gradient of reconstituted tobacco concentrate at a volume ratio of 1:19, and fermenting it at 25°C and pH 7 for 24 hours.

[0011] Preferably, the reconstituted tobacco concentrate with a concentration gradient of 60% is prepared by mixing reconstituted tobacco leaf concentrate, YPD liquid culture medium and water in a volume ratio of 6:1:3.

[0012] Preferably, the YPD liquid culture medium comprises: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and the solvent is water.

[0013] Through the above technical scheme, it can be known that the present invention can effectively increase ketones, alcohols, acids, phenols, heterocycles and other tobacco key flavor substances and improve its smoking effect by selecting specific yeast, amplifying Meyer yeast MG-1 to prepare seed liquid, and fermenting reconstituted tobacco concentrate to prepare papermaking reconstituted tobacco leaves. Since the present invention can improve the smoking quality of tobacco in a short time, at a lower cost and with higher efficiency, and its operation process is relatively simple, the present application shows good application value and application prospects in the field of cigarette preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : The bacterial morphology of Meyer yeast MG-1 under the streak line on the plate;

[0015] Figure 2 : A 40x microscope morphology of Meyer yeast MG-1;

[0016] Figure 3 For: Phylogenetic tree of Meyer yeast MG-1;

[0017] Figure 4 : The results of determining the optimal growth temperature and medium pH of Meyersinia guilliermondii MG-1, wherein A is a statistical diagram of bacterial concentrations after culturing at different temperatures for 24 hours with an initial concentration of OD600=0.2, and B is a statistical diagram of bacterial concentrations after culturing at different pH values ​​for 24 hours with an initial concentration of OD600=0.2;

[0018] Figure 5 is a graph showing the content and type of aroma components of reconstituted tobacco concentrates of different dilution concentrations after fermentation by Meyer yeast MG-1, curve b is after fermentation, curve a is before fermentation, wherein A is a 40% concentration of reconstituted tobacco concentrate, B is a 60% concentration of reconstituted tobacco concentrate, and C is an 80% concentration of reconstituted tobacco concentrate;

[0019] Figure 6 The figure is: the change of aroma substances in 60% concentration of reconstituted tobacco concentrate fermented by Guillermo MG-1 under optimal conditions, wherein CK is the group without inoculation of Guillermo MG-1, and MG-1 is the group inoculated with Guillermo MG-1. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present invention are described clearly and completely below. 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 creative work are within the scope of protection of the present invention.

[0021] The reagents involved in the embodiments of the present invention are all purchased from commercial channels, and the methods not mentioned are conventional experimental methods and will not be described in detail here.

[0022] YPD liquid culture medium formula: 10g / L yeast extract, 20g / L peptone, 20g / L glucose, the solvent is water.

[0023] YPD solid culture medium formula: 10g / L yeast extract, 20g / L peptone, 20g / L glucose, 15g / L agar powder.

[0024] Example 1 Identification of Meyer yeast MG-1

[0025] 1. Isolation and cultivation of strains

[0026] The strain is derived from the surface of freshly picked Kyoho grapes in Qingdao, Shandong. The specific method of obtaining it is as follows:

[0027] Take 30 fresh grapes and put them into a sterile bag, add 50ml sterilized saline to rinse, and shake with an oscillator for 5 minutes. Dip the above mixture with a sterile inoculation loop in a clean bench, separate and streak on YPD solid culture medium, select single colonies with uniform texture, pure white, smooth edges, and smooth circular distribution of single colonies, culture in YPD liquid, enrich overnight, centrifuge the bacterial liquid at 5000rpm in an ultracentrifuge, discard the supernatant, retain the bacterial body, add 30% glycerol, and store at -80℃ for later use.

[0028] 2. Screening and identification of strains

[0029] Use an inoculation loop to dip the cultured bacteria and draw a line on the YPD culture plate. After 48 hours of growth, observe the morphology of the bacteria (see Figure 1 Take a certain amount of bacterial solution to adjust OD600 = 0.2, take 100 μL and inoculate it into 3 mL YPD medium, culture it at 30℃ and 200 rpm for 24 hours, then take the bacterial solution and make it into a pressed sheet. Observe the bacterial cell morphology under a 40x objective lens. It was observed that the bacteria were distributed in a single cell, the cell morphology was oblate, and some cells were accompanied by budding reproduction (see Figure 2 ).

[0030] 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 in 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 between species and genera (see Figure 3), and determined it to be Meyerozyma guilliermondii, and named Meyerozyma guilliermondii MG-1.

[0031] Example 2 Cultivation of Meyer yeast MG-1

[0032] 1. Determination of optimal growth temperature and medium pH

[0033] The temperature conditions were set at 22°C, 25°C, 28°C, 30°C, 32°C, and 35°C, and the pH conditions were set at pH4, pH5, pH6, pH7, and pH8. The bacteria with an initial OD600 of 0.2 were inoculated into the above-mentioned culture media at different temperatures and pH values ​​for culture. After 24 hours of culture, the OD600 values ​​of the bacteria were measured. The results are shown in Table 1. Figure 4 A and Figure 4 B.

[0034] Depend on Figure 4 A shows that the growth rate of bacteria varies at different culture temperatures. In the temperature range of 22℃-35℃, the growth rate first increases and then decreases, and the growth ability is strongest at 25℃. Figure 4 B shows that the growth ability of the bacteria gradually increases with the increase of pH in the range of pH 4 to pH 7, while under alkaline pH 8 conditions, the growth ability of the bacteria decreases. The optimal culture temperature and pH of Meyer yeast MG-1 are 25°C and pH 7, respectively.

[0035] 2. Preparation of Meyer yeast MG-1 seed stock

[0036] A single colony of Mycobacterium guilliermondii MG-1 was inoculated into YPD liquid culture medium using an inoculation loop, and cultured in a shaking incubator at 22-30°C (optimal 25°C), pH 6-8 (optimal pH 7), and 150 rpm / min for 20 hours to obtain a seed solution.

[0037] Example 3 Preparation of Reconstituted Tobacco by Fermentation of Reconstituted Tobacco Concentrate by Meyer yeast MG-1

[0038] The reconstituted tobacco concentrate provided by Yunnan China Tobacco Reconstituted Tobacco Co., Ltd. was used as the test sample.

[0039] 1. Effect of reconstituted tobacco concentrate concentration on fermentation of Meyer yeast MG-1

[0040] The seed liquid prepared in Example 2 was adjusted to OD600 = 2, 1 mL was transferred to 19 mL of sterile tobacco concentrate, and cultured with shaking at 150 rpm / min at 30°C. Every 48 h, 5 smellers (who can provide professional qualification certificates) smelled 3 times (5 min / time) and scored. The results are shown in Table 1, where the total score is the sum of aroma quality, aroma quantity and irritation.

[0041] Table 1

[0042]

[0043] As shown in Table 1, in 100% tobacco concentrate, it takes 144 hours of fermentation to produce a strong aroma. However, a long fermentation cycle is not conducive to the practical application of the strain, and the study found that too high a soluble solid content in the tobacco concentrate will limit the growth of microorganisms. Therefore, the tobacco concentrate was diluted and its effect on the production of aroma substances was analyzed. The details are as follows:

[0044] 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) concentration gradient.

[0045] The seed solution prepared in Example 2 was adjusted to OD600=2, and was added to the tobacco concentrates of different concentration gradients at a volume ratio of 1:19. Fermentation was carried out at 25°C and pH 7. After fermentation for 24 hours, the content and type of the aroma components produced were detected according to the following GC-MS / MS method.

[0046] The specific operation is as follows: 2 mL of reconstituted tobacco concentrate with different concentrations before and after fermentation was taken and mixed with an equal volume of ethyl acetate, and then ultrasonicated at 40kHz for 10 minutes. After the system was taken out, it was centrifuged at 4000rpm / min for 10 minutes to separate the organic solvent phase and the aqueous phase. After 1 mL of the ethyl acetate supernatant solution was passed through a 0.22um membrane, the filtrate was placed in a sample injection bottle for GC-MS / MS detection.

[0047] The specific conditions of GC-MS / MS are as follows: the chromatographic column is a capillary column HP-5MS (30m×0.25mm×0.25μm), the initial temperature of the chromatographic column is set to 40°C, maintained for 2 min, heated to 185°C at 5°C / min, maintained for 1 min, and then heated to 240°C at 2°C / min, maintained at this temperature for 10 min, for a total of 60.5 min. Carrier: helium, column flow rate 1mL / min, injection port temperature 260°C. Mass spectrometry conditions: EI source, 70eV, ion source temperature 230°C, mass spectrometry transmission 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 Table 1. Figures 5A-5C .

[0048] Depend on Figures 5A-5C It can be seen that compared with the uninoculated CK group, the peaks of various aroma components in the reconstituted tobacco concentrate fermented by Meyer yeast MG-1 increased significantly, and at least 4 new peaks with significant abundance appeared, indicating that the content of aroma substances in the concentrate increased significantly after fermentation, and new aroma substances were produced. From the analysis of the difference in the abundance of aroma components of reconstituted tobacco concentrates of different concentrations, the abundance difference of each aroma component before and after fermentation of 60% reconstituted tobacco concentrate was more significant, especially the abundance difference of aroma components detected at 35-45min was greater, which indicates that the fermentation of Meyer yeast MG-1 has the greatest impact on the increase in the content of aroma components in 60% reconstituted tobacco concentrate.

[0049] 2. Effect of fermentation of Meyer yeast MG-1 under optimal fermentation conditions on aroma substances in reconstituted tobacco concentrate

[0050] Select 60% concentration gradient of reconstituted tobacco concentrate (V 水 :V YPD :V 浓缩液 =3:1:6), the reconstituted tobacco concentrate inoculated with Meyer yeast MG-1 is recorded as MG-1, and the reconstituted tobacco concentrate after replacing MG-1 with an equal volume of water is recorded as CK, the seed liquid prepared in Example 2 is adjusted to OD600=2, and the added amount is 5% of the volume of the reconstituted tobacco concentrate, and fermented for 48h at 25°C and pH7. The above experiment was repeated 3 times, and the content of aroma components in the MG-1 group and the CK group was detected each time and statistical analysis was performed. The specific operation is as follows:

[0051] 5 mL of the reconstituted tobacco concentrate from the MG-1 group or the CK group was placed in a 20 mL headspace bottle, extracted at 60 °C for 30 min using a 50 / 30 μm DVB / CAR / PDMS extraction head, taken out, and immediately inserted into the injection port of a gas chromatograph for GC-MS / MS detection;

[0052] The specific conditions of GC-MS / MS are as follows: the chromatographic column is a capillary column DB-5MS (30m×0.25mm×0.25μm), the initial temperature of the chromatographic column is set to 45℃, maintained for 3min, heated to 180℃ at 3℃ / min, maintained for 1min, then heated to 250℃ at 5℃ / min, maintained at this temperature for 5min, a total of 68min. Carrier: helium, column flow rate 1mL / min, injection port temperature 230℃. Mass spectrometry conditions: EI source, 70eV, ion source temperature 230℃, mass spectrometry transmission line temperature 270℃, quadrupole temperature 150℃, mass spectrometry scanning range 35-550m / z. The NIST 17.0 standard spectral library was used for retrieval analysis, and the contents of volatile aroma components such as ketones, alcohols, aldehydes, esters, acids, phenols, heterocyclics and others in the two groups of MG-1 and CK were determined respectively. The results are shown in Table 2-8.

[0053] Table 2 Ketone aroma substances content in two groups of concentrates (μg / mL)

[0054]

[0055]

[0056] As shown in Table 2, a total of 15 ketone substances were detected in the tobacco concentrate after fermentation by quaternary yeast MG-1, among which the contents of 9 ketone substances were significantly increased compared with the unfermented CK group, especially solanone, one of the main aroma components of tobacco, which increased from 42.66 μg / mL to 58.97 μg / mL, an increase of 38.23%; in addition, two new ketone aroma components, dehydrodihydro-β-ionone and geranyl acetone, were added after fermentation by quaternary yeast MG-1. Dehydrodihydro-β-ionone is a ketone compound composed of 13 carbons, has a monocyclic terpene skeleton, has the smell of violet, is an important natural aroma volatile compound, and geranyl acetone is also a natural flavor with fruity aroma.

[0057] Table 3 Content of alcohol aroma substances in the two groups of concentrates (μg / mL)

[0058]

[0059]

[0060] As shown in Table 3, a total of 11 alcohol aroma components were detected in the tobacco concentrate after fermentation by quaternary yeast MG-1, among which the contents of 5 aroma components increased, especially the content of phenylethanol, which can effectively improve the aroma of cigarettes and enhance the fineness of smoke, increased from 1.66 μg / mL to 12.56 μg / mL, an increase of 656.63%; in addition, 5 new alcohol aroma components were added after fermentation by quaternary yeast MG-1, namely (2R, 3R)-(-)-2,3-butanediol, phytol, isobutanol, isopentanol and 2-methylbutanol. Among them, isopentanol is one of the important aroma components of tea and wine, and can be mixed with ester aroma to make the aroma of cigarettes richer.

[0061] Table 4 Aldehyde aroma substance content in the two groups of concentrates (μg / mL)

[0062]

[0063] As shown in Table 4, the aldehyde aroma components of the tobacco concentrate fermented by Meyer yeast MG-1 did not change much, among which the contents of furfural, benzaldehyde and isovaleraldehyde decreased significantly, and the content of phenylacetaldehyde with a sweet-green smell increased by 14.13%.

[0064] Table 5 Content of ester aroma substances in the two groups of concentrates (μg / mL)

[0065]

[0066] As shown in Table 5, only one ester aroma component, dihydroactinol, was detected in the tobacco concentrate fermented by MG-1 yeast. Its content increased by 17.12% compared with the unfermented CK group. Dihydroactinol is a natural aroma substance with the fresh taste of kiwi fruit and is widely used in the tobacco industry.

[0067] Table 6 Content of acidic aroma substances in the two groups of concentrates (μg / mL)

[0068]

[0069] As shown in Table 6, a total of four acid aroma components were detected in the tobacco concentrate fermented by Meyer yeast MG-1, among which oleic acid and stearic acid were newly added components, and the contents of acetic acid and palmitic acid were significantly increased compared with the unfermented CK group, with the increases being 144.83% and 239.27%, respectively.

[0070] Table 7 Content of phenolic aroma substances in the two groups of concentrates (μg / mL)

[0071]

[0072] As shown in Table 7, two phenolic aroma components were detected in the tobacco concentrate after fermentation with Meyer yeast MG-1, namely 5-vinyl-2-methoxyphenol and 2,4-di-tert-butylphenol. Although there was no increase in phenolic aroma components, the total phenolic content increased by 33.62%.

[0073] Table 8 Contents of heterocyclic and other aroma substances in the two groups of concentrates (μg / mL)

[0074]

[0075]

[0076] As shown in Table 8, the content of aroma substances including pyridine, pyrrole, anthracene, naphthalene, etc. was detected in the tobacco concentrate after fermentation by guillerie yeast MG-1. In addition, three new components, 1,2-dihydro-1,5,8-trimethylnaphthalene, γ-cedrene and cerberene, were added. Among them, γ-cedrene and cerberene belong to macrocyclic diterpenoid compounds. γ-cedrene has woody fragrance and is an important woody fragrance. Cerberene can give tobacco roasted aroma, sweetness, cocoa flavor and light floral aroma, and it has also been reported to have a physiological effect of reducing the harm of smoking.

[0077] The statistical results of the total contents of volatile aroma components such as ketones, alcohols, aldehydes, esters, acids, phenols, heterocyclics and others in the MG-1 and CK groups are shown in Tables 9 and Figure 6 .

[0078] Table 9 Content of aroma substances in two groups of concentrates (μg / mL)

[0079]

[0080] From Table 9 and Figure 6 It can be seen that after fermentation with Guillermo Meyer yeast MG-1, the contents of ketones, alcohols, acids, phenols, heterocyclic rings and other aroma components in the concentrate were significantly increased compared with the unfermented CK group. After calculation, the increases were 26.92%, 567.34%, 349.47%, 33.62% and 47.44%, respectively. The increase was obvious. The overall quality of the concentrate after fermentation with Guillermo Meyer yeast MG-1 was improved.

[0081] 3. Preparation and smoking evaluation of papermaking reconstituted tobacco

[0082] By volume ratio V 浓缩液 :V YPD :V 水=6:1:3. Reconstituted tobacco concentrate, YPD liquid culture medium and water are mixed to obtain a reconstituted tobacco concentrate with a concentration gradient of 60%, the MG-1 seed solution of Myers guillermonis prepared in Example 2 is adjusted to OD600=2, inoculated into the tobacco concentrate at a volume ratio of 1:19, and fermented for 24 hours at 25° C. and pH 7; the fermented tobacco concentrate is vacuum concentrated to a sugar content of 50% Brix at 50° C., and then evenly coated on a tobacco sheet base at a coating rate of 39%, and quickly dried at 90° C. to a humidity of about 12%, and the coated tobacco sheet base is rehumidified at 20° C. and 60% humidity to a moisture content of 12.5% ​​to obtain papermaking reconstituted tobacco.

[0083] The papermaking reconstituted tobacco leaves prepared as above were cut into shreds to make single-ingredient cigarettes, recorded as MG-1 group; the 60% concentration gradient tobacco concentrate was not inoculated with Meyer yeast MG-1, and other operations were the same. The single-ingredient cigarettes made by cutting the prepared reconstituted tobacco leaves were recorded as CK group. Seven experts with smoking evaluation qualifications evaluated the single-ingredient cigarettes of the above two groups, and scored the characteristics of the cigarettes such as aroma, sweetness, permeability, miscellaneous odor, irritation, aftertaste and odor. The results are shown in Table 10.

[0084] Table 10 Sensory evaluation results of two groups of reconstituted tobacco leaves

[0085]

[0086] As shown in Table 10, the aroma quality and layering of the papermaking reconstituted tobacco prepared from tobacco concentrate fermented by Guillermo Meyer yeast MG-1 are enhanced, the sweet fruity sweetness is prominent, the impurities and irritation are reduced, the smoke comfort and permeability are significantly improved, and the sense of pleasure is better.

[0087] 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.

[0088] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be 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 will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A reconstituted tobacco leaf prepared by fermentation of Meyer yeast, characterized in that: The reconstituted tobacco leaves are made by a papermaking method, specifically by fermenting seed liquid prepared with Meyerozyma guilliermondii MG-1 to reconstitute tobacco concentrate; the Meyerozyma guilliermondii MG-1 is classified and named Meyerozyma guilliermondii, and was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on July 27, 2023, with a deposit number of CGMCC No. 28024, 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 according to claim 1, characterized in that: The preparation steps of the Meyer yeast MG-1 seed solution are as follows: A single colony of Meyersinia guilliermondii MG-1 was inoculated into a YPD liquid culture medium, and cultured in a shaking incubator at a temperature of 22-30°C, a pH of 6-8, and a rotation speed of 150 r / min for 20 hours to obtain a seed solution.

3. The reconstituted tobacco according to claim 1, characterized in that: The specific operation of the fermentation is: adjusting the OD600 of the guillier yeast MG-1 seed liquid to 2, inoculating it into the reconstituted tobacco concentrate with a concentration gradient of 60% at a volume ratio of 1:19, and fermenting it for 24 hours at 25° C. and pH 7.

4. The reconstituted tobacco leaf according to claim 3, characterized in that: The 60% concentration gradient reconstituted tobacco concentrate is prepared by mixing reconstituted tobacco leaf concentrate, YPD liquid culture medium and water in a volume ratio of 6:1:

3.

5. The reconstituted tobacco according to claim 2 or 4, characterized in that: The components of the YPD liquid culture medium are: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and the solvent is water.

Images