Yarrowia guilliermondii MG-1 and application thereof

Abstract

The invention discloses Yarrowia guilliermondii MG-1 and application thereof, and belongs to the technical field of microorganisms. The strain is preserved in the China General Microbiological Culture Collection Center on July 27, 2023, the preservation number is CGMCC No.28024, and the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Yard, West Beichen Road, Chaoyang District, Beijing. The strain can effectively utilize nutritional ingredients such as protein and reducing sugar in tobacco to generate ketones, alcohols, acids, phenols, heterocyclic rings and other key tobacco flavor substances, so that the purposes of improving quality and enhancing aroma are achieved, and the quality of the prepared reconstituted tobacco can be effectively improved.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and more particularly to a strain of Meyer yeast and its application in improving the aroma of tobacco concentrate. 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 sheets or filaments by physical, chemical, biological and other methods. They can be used in the leaf group formula of cigarettes. [1] . 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 their raw materials is relatively low. In addition, the extraction, concentration, and drying of reconstituted tobacco leaves 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 tobacco leaf flakes 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 tobacco leaf flakes. 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 tobacco leaf flakes. 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 tobacco flakes ("Study on the Effect of Concentrates Treated with Different Enzymes on the Quality of Guizhou Tobacco Flakes", 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 provides a strain of Meyer yeast MG-1 and its application, which can achieve the effect of improving quality and enhancing aroma.

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

[0007] A strain of Meyerozyma guilliermondii MG-1, classified and named Meyerozyma guilliermondii, was deposited in the General Microbiology Center of China Microorganism Culture Collection on July 27, 2023, with the deposit number 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] Another object of the present invention is to provide the use of the above-mentioned Meyer yeast MG-1 in improving the aroma of tobacco concentrate.

[0009] Another object of the present invention is to provide a flavored tobacco concentrate, specifically, the flavored tobacco concentrate is prepared by using the above-mentioned Meyer yeast MG-1 to prepare seed liquid, and then fermenting the seed liquid to regenerate tobacco concentrate to prepare the flavored tobacco concentrate.

[0010] Preferably, the specific preparation steps of the seed solution are as follows: a single colony of Meyeriella guilliermondii MG-1 is 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.

[0011] Preferably, the specific operation of the fermentation is: adjusting the OD600 of the Meyer yeast MG-1 seed liquid to 2, inoculating it into the reconstituted tobacco concentrate at a volume ratio of 1:20, and fermenting it at 25° C. and pH 7 for 24 hours.

[0012] Preferably, in the flavored tobacco concentrate, the content of soluble reducing sugar and protein is reduced, and the total amount of flavor components is increased by 89.26%, among which ketones, alcohols, acids, phenols, heterocycles and other flavor components are significantly increased.

[0013] Through the above technical scheme, it can be known that the present invention discloses a strain of Meyer yeast, which can effectively utilize nutrients such as protein and reducing sugar in tobacco to produce ketones, alcohols, acids, phenols, heterocycles and other key tobacco flavor substances, thereby achieving the purpose of improving quality and enhancing flavor, and can effectively improve the quality of the prepared reconstituted tobacco leaves. 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;

[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 : The results of the analysis of the ability of Meyer yeast guilliermonensis MG-1 to utilize the soluble reducing sugar and protein in the reconstituted tobacco concentrate, wherein A is the soluble reducing sugar content before and after the reconstituted tobacco concentrated fermentation, B is the soluble protein concentration before and after the reconstituted tobacco concentrated fermentation, CK is the group without inoculation of Meyer yeast guilliermonensis MG-1, and MG-1 is the group inoculated with Meyer yeast guilliermonensis MG-1;

[0019] Figure 6 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;

[0020] Figure 7 The figure is: the change of aroma substances in the reconstituted tobacco concentrate with a concentration gradient of 60% fermented by Guillermo MG-1 under the 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

[0021] 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.

[0022] 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.

[0023] YPD liquid culture medium formula: 10g / L yeast extract, 20g / L peptone, 20g / L glucose, the solvent is water.

[0024] YPD solid culture medium formula: 10g / L yeast extract, 20g / L peptone, 20g / L glucose, 15g / L agar powder.

[0025] Example 1 Identification of Meyer yeast MG-1

[0026] 1. Isolation and cultivation of strains

[0027] The strain is derived from the surface of freshly picked Kyoho grapes in Qingdao, Shandong. The specific method of obtaining it is as follows:

[0028] 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.

[0029] 2. Screening and identification of strains

[0030] 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 ).

[0031] 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.

[0032] Example 2 Cultivation of Meyer yeast MG-1

[0033] 1. Determination of optimal growth temperature and medium pH

[0034] 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.

[0035] 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.

[0036] 2. Preparation of Meyer yeast MG-1 seed stock

[0037] 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.

[0038] Example 3 Application of Meyer yeast MG-1 in reconstituted tobacco concentrate

[0039] The reconstituted tobacco concentrate provided by Yunnan China Tobacco Reconstituted Tobacco Co., Ltd. was used as the test sample.

[0040] 1. Analysis of the ability of Meyer yeast MG-1 to utilize soluble reducing sugars and proteins

[0041] 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 for 72 h as the MG-1 group. The reconstituted tobacco concentrate after replacing MG-1 with an equal volume of water was recorded as the CK group. After the culture was completed, 5 smellers (who could provide professional qualification certificates) smelled it 3 times (5 min / time) and found that a new aroma that was significantly different from the original reconstituted tobacco concentrate was presented.

[0042] The DNS method was used to determine the soluble reducing sugar content in the reconstituted tobacco concentrate before and after fermentation. Specifically, the reconstituted tobacco concentrate of the above-mentioned MG-1 group or CK group was diluted 200 times with water, 2 ml was taken and added to 3 ml of DNS reagent, and after boiling in a water bath for 10 minutes, 20 ml of water was added to terminate the reaction, and the OD value was read at a wavelength of OD540 nm. The soluble reducing sugar concentration in the reconstituted tobacco concentrate before and after fermentation was calculated using a standard curve formed by the OD value of the glucose standard solution at different concentrations and its concentration value (the method refers to the light industry standard QB 2583-2003). The results are shown in the figure. Figure 5 A.

[0043] The Bradford method was used to determine the soluble protein content in the reconstituted tobacco concentrate before and after fermentation. Specifically, the reconstituted tobacco concentrate of the MG-1 group or CK group was diluted 100 times with water, and 20 μl was added to 200 μl Bradford reaction solution. After reacting in the dark for 5 minutes, the reading was taken at OD 595nm. The soluble protein concentration in the reconstituted tobacco concentrate before and after fermentation was calculated using the standard curve formed by the OD value of the BCA standard solution at different concentrations and its concentration value (the method refers to the Bradford protein concentration determination method of Shenggong Biology (kit product number NO.C503041)). The results are shown in Figure 5 B.

[0044] Depend on Figure 5 A-5B shows that after fermentation for 72 hours, the content of soluble reducing sugar in the reconstituted tobacco concentrate decreased from 299.68 mg / ml to 209.53 mg / ml, a decrease of 30.08%; the concentration of soluble protein decreased from 9.38 mg / ml to 7.44 mg / ml, a decrease of 20.68%.

[0045] This indicates that Mycobacterium guillierensis MG-1 can not only tolerate reconstituted tobacco concentrate but also utilize its soluble reducing sugars, proteins and other nutrients for growth and fermentation.

[0046] 2. Effect of reconstituted tobacco concentrate concentration on fermentation of Meyer yeast MG-1

[0047] 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.

[0048] Table 1

[0049]

[0050] 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 excessive soluble solids content in leaf paste 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:

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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 6A-6C .

[0055] Depend on Figures 6A-6C 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.

[0056] 3. Effect of fermentation of Meyer yeast MG-1 under optimal fermentation conditions on aroma substances in reconstituted tobacco concentrate

[0057] 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:

[0058] 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;

[0059] 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°C, maintained for 3 min, heated to 180°C at 3°C / min, maintained for 1 min, then heated to 250°C at 5°C / min, maintained at this temperature for 5 min, for a total of 68 min. Carrier: helium, column flow rate 1mL / min, inlet temperature 230°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 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 and phenols in the two groups of MG-1 and CK were determined, respectively. The results are shown in Tables 2 and Figure 7 .

[0060] Table 2 Content of aroma substances in the two groups of concentrates (μg / mL)

[0061]

[0062] From Table 2 and Figure 7 It can be seen that after being treated 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.

[0063] 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.

[0064] 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 strain of Meyer yeast MG-1, characterized in that The strain was classified and named Meyerozyma guilliermondii. It was deposited in the General Microbiology Center of China Culture Collection Administration on July 27, 2023, with the deposit number CGMCC No.28024. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the Meyer yeast MG-1 as claimed in claim 1 in improving the aroma of tobacco concentrate.

3. A flavored tobacco concentrate, characterized in that: The flavored tobacco concentrate is prepared by using the Meyer yeast MG-1 described in claim 1 to prepare seed liquid, and then fermenting the seed liquid to regenerate tobacco concentrate.

4. A flavored tobacco concentrate according to claim 3, characterized in that: The specific preparation steps of the 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.

5. The flavored tobacco concentrate according to claim 3, characterized in that: The specific operation of the fermentation is: adjusting the OD600 of the quaternary yeast MG-1 seed liquid to 2, inoculating it into the reconstituted tobacco concentrate at a volume ratio of 1:20, and fermenting it for 24 hours at 25° C. and pH 7.

6. The flavored tobacco concentrate according to claim 3, characterized in that: In the flavored tobacco concentrate, the contents of soluble reducing sugar and protein are reduced; the total amount of flavor components is increased by 89.26%, among which ketones, alcohols, acids, phenols, heterocycles and other flavor components are significantly increased.

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