Pichia kudriavzevii PK-1 and application thereof

Abstract

The invention discloses pichia kudriavzevii PK-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.28021, 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 esters, alcohols, ketones, acids, 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 Pichia kudriavzevii and application thereof in improving the aroma of tobacco concentrate. Background Art

[0002] Reconstituted tobacco, also known as tobacco leaf flakes, is mainly made by processing tobacco stems, tobacco dust and other tobacco processing byproducts produced during the cigarette processing process into reconstituted thin shreds. Its color and taste are similar to natural tobacco leaves and can be added to the leaf group formula of cigarettes. Reconstituted tobacco has the characteristics of low density, good filling, high shred rate and fast burning speed. Adding a proper amount of reconstituted tobacco to cigarettes can effectively improve the combustion performance of cigarettes and reduce the tar and nicotine content. However, the raw materials of reconstituted tobacco are mostly surplus resources produced in the cigarette processing process. Its quality is still somewhat different from that of natural tobacco. It has defects such as low content of aroma components, heavy impurities and excessive irritation. There are limitations in its application in high-end cigarettes.

[0003] Papermaking is one of the mainstream technologies for producing reconstituted tobacco. It mainly extracts the solid phase of tobacco raw materials such as tobacco leaves and tobacco stems into tobacco extracts, which are then concentrated into tobacco concentrates. The remaining residues are mixed with auxiliary materials such as wood pulp fibers to make a sheet base. The tobacco concentrate is coated on the sheet base and then dried to prepare reconstituted tobacco sheets. Reconstituted tobacco concentrate contains a large amount of aroma components, water-soluble sugars, proteins, etc., and its quality directly determines the quality of reconstituted tobacco. Therefore, how to improve the aroma quality of reconstituted tobacco concentrate is the key to developing high-quality reconstituted tobacco raw materials.

[0004] At present, the methods for tobacco flavoring are mainly divided into physical methods, chemical methods and microbial fermentation methods. Microbial fermentation flavoring has the advantages of being green and safe, high-quality flavoring substances and short reaction cycle. It is a research hotspot in the tobacco industry. Tuo Youpeng et al. selected superior microorganisms from aged tobacco leaves and applied them to tobacco fermentation, which improved the aroma quality and aroma quantity of tobacco leaves and reduced the irritation and foreign smell of tobacco leaves ("Research on Flue-cured Tobacco Fermentation Using Biotechnology", Tuo Youpeng et al., "Food and Fermentation Technology"); Yan Hongyang et al. selected a terrestrial Pichia yeast from the surface of grapes, and its fermentation liquid had many characteristic aroma components of tobacco leaves, which could effectively improve the aroma quality of tobacco ("Isolation and Identification of Aroma-Producing Strains and Application of Their Fermentation Products in Cigarette Flavoring", Yan Hongyang et al., "Journal of Light Industry"); Du Fei et al. selected brewer's yeast and Aspergillus niger from Maotai liquor lees and Fuzhuan tea, respectively, and after spraying their fermentation liquid in green tea powder on tobacco leaf slices, a variety of aroma-causing components were added, and the sensory quality of tobacco leaves was effectively improved ("Isolation and Identification of Two Aroma-Producing Microorganisms and Research on Tobacco Flavoring with Their Fermentation Liquid", Du Fei et al., "Journal of Mountain Agriculture and Biology").

[0005] It can be seen that 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

[0006] In view of the above problems, the present invention provides a strain of Pichia kudriavzevii PK-1 and application thereof, which can achieve the effect of improving quality and enhancing aroma.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A strain of Pichia kudriavzevii PK-1, classified and named Pichia kudriavzevii Pichiakudriavzevii, was deposited in the General Microbiology Center of China Microorganism Culture Collection on July 27, 2023, with the deposit number CGMCC No.28021, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] Another object of the present invention is to provide the use of the above-mentioned Pichia kudriavzevii PK-1 in improving the aroma of tobacco concentrate.

[0010] 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 Pichia kudriavzevii PK-1 to prepare seed liquid, and then fermenting the seed liquid to regenerate tobacco leaf concentrate to prepare the flavored tobacco concentrate.

[0011] Preferably, the specific preparation steps of the seed solution are as follows: a single colony of Pichia kudriavzevii PK-1 is inoculated into a YPD liquid culture medium, and cultured in a shaking incubator at a temperature of 22-32° C., a pH of 5-7, and a rotation speed of 180 r / min for 22 hours to obtain a seed solution.

[0012] Preferably, the specific operation of the fermentation is: adjusting the OD600 of the Pichia kudriavzevii PK-1 seed liquid to 2, inoculating it into the reconstituted tobacco concentrate at a volume ratio of 1:19, and fermenting it for 24 hours at 25° C. and pH 5.

[0013] Preferably, in the flavored tobacco concentrate, the total amount of flavor components is increased by 1.75 times, among which esters, alcohols, ketones, acids, heterocycles and other flavor components are significantly increased.

[0014] It can be seen from the above technical scheme that the present invention discloses a strain of Pichia kudriavzevii, which can effectively utilize nutrients such as protein and reducing sugar in tobacco to generate esters, alcohols, ketones, acids, 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

[0015] Figure 1 : The bacterial morphology of Pichia kudriavzevii PK-1 under the streaking line on the plate;

[0016] Figure 2 : 40 times microscope morphology of Pichia kudriavzevii PK-1;

[0017] Figure 3 For: Phylogenetic tree of Pichia kudriavzevii;

[0018] Figure 4 : The results of determining the optimal growth temperature and medium pH of Pichia kudriavzevii PK-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;

[0019] Figure 5 : The results of the analysis of the ability of Pichia kudriavzevii PK-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 Pichia kudriavzevii PK-1, PK-1 is the group inoculated with Pichia kudriavzevii PK-1;

[0020] Fig. 6 is a graph showing the content and type of aroma components of reconstituted tobacco concentrates of different dilution concentrations after fermentation by Pichia kudriavzevii PK-1, wherein curve a is after fermentation, and curve b is before fermentation, wherein A is a 60% concentration reconstituted tobacco concentrate, B is an 80% concentration reconstituted tobacco concentrate, and C is an undiluted 100% concentration reconstituted tobacco concentrate;

[0021] Figure 7 Figure 3: Changes in aroma substances in 80% diluted reconstituted tobacco concentrate fermented by Pichia kudriavzevii PK-1 under optimal conditions. CK is the group not inoculated with Pichia kudriavzevii PK-1, and PK-1 is the group inoculated with Pichia kudriavzevii PK-1. DETAILED DESCRIPTION

[0022] 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.

[0023] 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.

[0024] YPD liquid culture medium formula: 10g / L yeast extract, 20g / L peptone, 20g / L glucose, the solvent is water.

[0025] YPD solid culture medium formula: 10g / L yeast extract, 20g / L peptone, 20g / L glucose, 15g / L agar powder.

[0026] Example 1 Identification of Pichia kudriavzevii PK-1

[0027] 1. Isolation and cultivation of strains

[0028] The strain is derived from Guangxi's special-flavor liquor Daqu (purchased from the Huashifu liquor cake brand), and the specific method of obtaining it is as follows:

[0029] Put the solid Daqu into a sterile bag and break it, take 20g, add 50ml sterilized physiological saline, and shake it with an oscillator for 5min. Take 100μl of the above liquid and spread it on a YPD plate, and culture it overnight at 30℃. The next day, select the colonies with regular edges, smooth, moist, opaque milky white surface, and easy to pick up. After enrichment culture in YPD liquid culture medium for 22h, store it in 30% glycerol at -80℃ for use.

[0030] 2. Screening and identification of strains

[0031] Use an inoculation loop to dip the cultured bacteria and draw a line on the YPD culture plate. After 24 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 out the bacterial solution and make it into pressed sheets. Observe the morphology of bacterial cells under a 40x objective lens. The cells are oval, and some of the cells have convex buds on one side, showing budding reproduction (see Figure 2 ), based on the morphological phenotype results, it was speculated to be yeast.

[0032] The genomic DNA of the strain was further extracted and sequenced by Qingdao Weilai Biotechnology Co., Ltd. The 26S rDNA sequence of the strain is shown in SEQ ID NO.1. It was sequenced and compared with the 26S rDNA sequences of multiple known fungal species in the NCBI database by Blast analysis. A phylogenetic tree was constructed based on the Neighbor-Joining method (see Figure 3), and identified it as Pichia kudriavzevii, and named it Pichia kudriavzevii PK-1.

[0033] Example 2 Cultivation of Pichia kudriavzevii PK-1

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

[0035] 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.

[0036] After 24 hours of culture, there were significant differences in the growth ability of the Y-1 strain under different temperature and pH conditions. Figure 4 A shows that the growth rate of bacteria varies at different culture temperatures. At 22℃-35℃, the growth rate is fast at first and then slows down. The growth rate of bacteria is the best at 25℃. Figure 4 B shows that when the Y-1 strain is cultured at a medium temperature of 30°C and different pH conditions within the range of pH 4 to pH 8, its growth rate first increases and then gradually decreases with the increase of medium pH, and the bacterial growth ability is strongest under the medium pH 5 condition. From the above results, it can be concluded that the optimal culture conditions for the Y-1 strain are a temperature of 25°C and a medium pH of 5.

[0037] 2. Preparation of Pichia kudriavzevii PK-1 Seed Liquid

[0038] A single colony of Pichia kudriavzevii PK-1 was inoculated into YPD liquid culture medium using an inoculation loop, and cultured in a shaking incubator at 22-32°C (optimal 25°C), pH 5-7 (optimal pH 5), and 180 rpm / min for 22 h to obtain a seed solution.

[0039] Example 3 Application of Pichia kudriavzevii PK-1 in reconstituted tobacco concentrate

[0040] The reconstituted tobacco concentrate provided by Yunnan China Tobacco Reconstituted Tobacco Co., Ltd. was used as the test sample.

[0041] 1. Analysis of the ability of Pichia kudriavzevii PK-1 to utilize soluble reducing sugars and proteins

[0042] 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 at 30°C with shaking at 150 rpm / min for 72 h as the PK-1 group. The reconstituted tobacco concentrate after replacing PK-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.

[0043] 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 PK-1 group or CK group was diluted 200 times with water, 2 ml was taken and added with 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.

[0044] 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 PK-1 group or CK group was diluted 100 times with water, 20 μl of the dilution was taken, 200 μl of Bradford reaction solution was added, and the reaction was carried out in the dark for 5 minutes, and 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.

[0045] Depend on Figure 5 A-5B shows that after 72h of fermentation, the content of soluble reducing sugar in the reconstituted tobacco concentrate decreased from 299.68mg / ml to 245.73mg / ml, a decrease of 18.00%; the concentration of soluble protein decreased from 9.38mg / ml to 7.09mg / ml, a decrease of 24.41%.

[0046] This indicates that Pichia kudriavzevii PK-1 can not only tolerate reconstituted tobacco concentrate but also utilize its soluble reducing sugars, proteins and other nutrients for growth and fermentation.

[0047] 2. Effect of Reconstituted Tobacco Leaf Concentrate Concentration on Fermentation of Pichia kudriavzevii PK-1

[0048] Since high concentrations of reconstituted tobacco concentrate can inhibit the growth of microbial cells, the effect of dilution concentration on the fermentation of Pichia kudriavzevii PK-1 was investigated.

[0049] The reconstituted tobacco concentrate was diluted to 60% (V) with water and YPD liquid medium according to the corresponding volume ratio. 水 :V YPD :V 浓缩液 =3:1:6), 80% (V 水 :V YPD :V 浓缩液 =1:1:8) concentration gradient, the seed solution prepared in Example 2 was adjusted to OD600=2, and was added to the tobacco concentrates with different concentration gradients and undiluted (concentration gradient of 100%) at an inoculum volume ratio of 1:19, and fermented at 25°C and pH 5. After fermentation for 24 hours, the content and type of the aroma components produced were detected according to the following GC-MS / MS method.

[0050] 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.

[0051] 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 .

[0052] Depend on Figures 6A-6CIt can be seen that compared with the uninoculated CK group, the peaks of various aroma components of the reconstituted tobacco concentrate fermented by Pichia kudriavzevii PK-1 increased significantly, and at least 3 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 abundance of aroma components of reconstituted tobacco concentrates with different concentrations, the content of most aroma components in the fermented tobacco concentrate with a concentration gradient of 80% was higher than that in the fermented tobacco with a concentration gradient of 60%. Compared with the diluted tobacco concentrate, the undiluted tobacco concentrate had less increase in the abundance and variety of aroma substances after fermentation. In summary, the concentration gradient of 80% is the optimal fermentation concentration for reconstituted tobacco concentrate.

[0053] 3. Effect of Pichia kudriavzevii PK-1 fermentation under optimal fermentation conditions on aroma substances in reconstituted tobacco concentrate

[0054] Select 80% concentration gradient of tobacco concentrate (V 水 :V YPD :V 浓缩液 =1:1:8), the one inoculated with Pichia kudriavzevii PK-1 was recorded as PK-1, the reconstituted tobacco concentrate after replacing PK-1 with an equal volume of water was recorded as CK, the seed liquid prepared in Example 2 was adjusted to OD600=2, the addition amount was 5% of the volume of the reconstituted tobacco concentrate, and fermented for 24 hours at 25°C and pH 5. The above experiment was repeated 3 times, and the content of aroma components in the PK-1 group and the CK group was detected each time and statistical analysis was performed. The specific operation is as follows:

[0055] 5 mL of the reconstituted tobacco concentrate from the PK-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;

[0056] 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 esters, alcohols, ketones, acids, aldehydes, phenols, heterocyclics and others in the two groups of PK-1 and CK were determined, respectively. The results are shown in Tables 1 and Figure 7 .

[0057] Table 1 Content of aroma substances in two groups of concentrates (μg / mL)

[0058]

[0059] From Table 1 and Figure 7 It can be seen that after fermentation with Pichia kudriavzevii PK-1, the contents of esters, alcohols, ketones, acids, heterocyclic rings and other aroma components in the concentrate were significantly increased compared with the unfermented CK group. After calculation, the increases were 194.00%, 1215.35%, 65.67%, 274.18% and 58.75%, respectively. The increase was obvious. The overall quality of the concentrate after fermentation with Pichia kudriavzevii PK-1 was improved.

[0060] 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.

[0061] 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 Pichia kudriavzevii PK-1, characterized in that The yeast was classified and named Pichia kudriavzevii. It was deposited in the General Microbiology Center of China Culture Collection on July 27, 2023. The deposit number is CGMCC No.28021. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Application of Pichia kudriavzevii PK-1 as claimed in claim 1 in improving the aroma of tobacco concentrate.

3. A flavored tobacco concentrate, characterized in that: The Pichia kudriavzevii PK-1 described in claim 1 is used to prepare seed liquid, and the seed liquid is then fermented to regenerate tobacco leaf concentrate to prepare flavored 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 Pichia kudriavzevii PK-1 was inoculated into YPD liquid culture medium, and cultured in a shaking incubator at a temperature of 22-32°C, a pH of 5-7, and a rotation speed of 180 r / min for 22 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 Pichia kudriavzevii PK-1 seed liquid to 2, inoculating it into the reconstituted tobacco concentrate at a volume ratio of 1:19, and fermenting it for 24 hours at 25° C. and pH 5.

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 1.75 times, among which esters, alcohols, ketones, acids, heterocycles and other flavor components are significantly increased.

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