Application of sclerotium penicillium in promoting the aging of flue-cured tobacco

Abstract

The application discloses application of sclerotinia penicillium in promoting flue-cured tobacco aging. The method for promoting flue-cured tobacco aging is as follows: (1) washing and drying harvested flue-cured tobacco leaves to obtain dried tobacco leaves; (2) diluting sclerotinia penicillium spore powder with water to obtain a sclerotinia penicillium spore powder diluent; then uniformly spraying the sclerotinia penicillium spore powder diluent on the surface of the dried tobacco leaves to ensure that the spore powder and the tobacco leaves are in full contact, and then placing the tobacco leaves in an environment with a temperature of 20-30 DEG C and a humidity of 60-70% for cultivation to promote tobacco aging. The method can effectively shorten the aging period of flue-cured tobacco, improve the aroma substance basis of flue-cured tobacco, thereby reducing the production cost of flue-cured tobacco and improving the quality stability of flue-cured tobacco.

Description

Technical Field

[0001] This invention belongs to the field of tobacco aging technology, and specifically relates to the application of Penicillium sclerotiorum in promoting the aging of flue-cured tobacco. Background Technology

[0002] Flue-cured tobacco is an important economic crop, and aging is a crucial step in its processing, directly affecting the aroma, taste, and overall quality of the tobacco leaves. Traditional aging methods rely mainly on natural fermentation and artificially controlled conditions. While these methods can improve tobacco quality to some extent, their effects are inconsistent and difficult to control. Therefore, finding a more effective method to promote flue-cured tobacco aging and improve leaf quality has become an urgent need in the industry.

[0003] In recent years, with the continuous development of biotechnology, the application of microbial agents in agriculture has gradually attracted attention. Existing research has shown that microbial agents have the following four advantages in the aging process of flue-cured tobacco: First, when microbial agents are sprayed during the aging process, the microorganisms, through protein degradation and absorption of different trace elements, synthesize various types of bioactive enzymes. These enzymes can accelerate the transformation and decomposition of organic matter in tobacco leaves, thereby shortening the aging time. Second, while promoting the aging of flue-cured tobacco, microbial agents can also degrade macromolecules in tobacco leaves, such as carbohydrates and proteins, producing aroma precursors such as alcohols, esters, and various organic acids. These aroma precursors are further transformed during subsequent aging processes, forming… The unique aroma of flue-cured tobacco treated with microbial agents is more intense, longer-lasting, and offers a wider variety of aroma types. Third, during the aging process, microbial agents can regulate the content of major chemical components in tobacco leaves, such as reducing nicotine, protein, and amino acid content, resulting in a more harmonious and balanced chemical composition and thus improving the overall quality of the tobacco. Fourth, the application of microbial agents allows beneficial microorganisms to occupy ecological niches, inhibits the growth and reproduction of harmful microorganisms, and improves the storage environment, reducing humidity and temperature—conditions conducive to mold growth—thereby slowing down the mold process. However, current applications of microbial agents in flue-cured tobacco aging have drawbacks, including environmental hazards and safety concerns related to the tobacco itself.

[0004] *Penicillium sclerotiorum* belongs to the phylum Ascomycota, class Eurotiomycetes, order Eurotiales, family Trichocomaceae, and genus *Penicillium*. This fungus was first isolated from the air in 1935 and is widely distributed in various environments, such as air and soil. It has been widely used in medicine, agriculture, and the food industry. In previous studies in our laboratory, this endophytic fungus was first isolated from purslane and its positive effects on Solanaceae crops have been demonstrated. However, there are currently no studies on its application in the aging of flue-cured tobacco. Summary of the Invention

[0005] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide the application of Penicillium sclerotiorum in promoting the aging of flue-cured tobacco.

[0006] Another object of the present invention is to provide a method for promoting the aging of flue-cured tobacco using Penicillium sclerotiorum.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] Application of Penicillium sclerotiorum in promoting the aging of flue-cured tobacco.

[0009] The Penicillium sclerotiorum mentioned is Penicillium sclerotiorum SCAUMCX01.

[0010] A method for promoting the aging of flue-cured tobacco using Penicillium sclerotiorum includes the following steps:

[0011] (1) Pretreatment of tobacco leaves: The harvested flue-cured tobacco leaves are washed and dried to obtain dried tobacco leaves;

[0012] (2) Inoculation and aging treatment: Dilute the Penicillium spore powder with water to obtain a diluted Penicillium spore powder solution; then spray it evenly on the surface of the dried tobacco leaves obtained in step (1) (to ensure that the spore powder is in full contact with the tobacco leaves), and then place the inoculated tobacco leaves in an environment with a temperature of 20-30℃ and a humidity of 60%-70% to promote the aging of tobacco leaves (promote the transformation of organic matter and the generation of aroma substances in tobacco leaves).

[0013] In step (1), select tobacco leaves with appropriate maturity, wash and dry them to ensure that there are no impurities and moisture on the surface of the tobacco leaves, and then mix the tobacco leaf samples to unify the initial moisture content.

[0014] The drying process described in step (1) can be achieved by conventional natural air drying.

[0015] The moisture content of the dried tobacco leaves described in step (1) is 10% to 13%.

[0016] The Penicillium sclerotiorum mentioned in step (2) is Penicillium sclerotiorum.

[0017] SCAUMCX01.

[0018] The Penicillium sclerotiorum spore powder mentioned in step (2) can be prepared by conventional methods in the art, such as activating the strain, producing spores through solid-state fermentation, and then making spore powder; preferably, it is prepared by referring to the method described in Chinese Patent (Patent No.: 202210053325.0, entitled "A method for producing spores by solid-state fermentation of Penicillium sclerotiorum and its application").

[0019] The number of spores in the Penicillium sclerotiorum spore powder mentioned in step (2) is (2~2.5)×10 9 pcs / g; preferably 2.214×10 9 per g.

[0020] The Penicillium spore powder mentioned in step (2) is diluted with water to a ratio of 100 to 10,000.

[0021] The amount of Penicillium spore powder dilution mentioned in step (2) is 20±2% of the tobacco leaf mass.

[0022] The cultivation time described in step (2) is 12 to 24 months; preferably 12 months.

[0023] In step (2), during the aging process, Penicillium sclerotiorum produces specific enzymes and metabolites that promote the conversion of organic matter and the generation of aroma substances in tobacco leaves.

[0024] The promotion of tobacco aging in step (2) is to increase the content of aroma substances in tobacco leaves; wherein the aroma substances include at least one of 2-azhexane, glycerol, 9-hydroxy-4,7-mega-stigmadien-3-one, stearic acid, N,N-dimethyloctamide and n-nonacosane.

[0025] The method for promoting the aging of flue-cured tobacco using Penicillium sclerotiorum includes the following steps after step (2):

[0026] (3) Dry the tobacco leaves after the alcohol treatment in step (2) to remove excess moisture, and then package and store them.

[0027] The drying process described in step (3) can be carried out using conventional methods.

[0028] The present invention has the following advantages and effects compared with the prior art:

[0029] (1) In this invention, Penicillium sclerotiorum is activated and prepared into a fungal agent, which is then applied to the surface of flue-cured tobacco leaves to be aged. This can effectively shorten the aging cycle of flue-cured tobacco and improve the aroma material basis of flue-cured tobacco.

[0030] (2) Penicillium sclerotiorum has a high metabolic capacity and growth rate, and can complete the aging process of tobacco leaves in a short time, shortening the aging cycle by at least 30% compared with traditional methods.

[0031] (3) During the aging process, Penicillium sclerotiorum can stably produce specific enzymes and metabolites, making the aging process of tobacco leaves more stable and controllable, thereby improving the quality stability of flue-cured tobacco and providing an effective way for the development and utilization of microbial agents.

[0032] (4) The present invention is simple to operate and can be processed in batches. It can be sprayed directly before and after tobacco leaves are re-dried.

[0033] (5) Due to the shortened aging cycle and improved quality stability, the present invention can reduce the production cost of flue-cured tobacco and improve economic benefits. Attached Figure Description

[0034] Figure 1 This is a chromatogram of the volatile substances in processed tobacco leaves.

[0035] Figure 2 This is a diagram of an OPLS-DA (Orthogonal Partial Least Squares Discriminant Analysis) model constructed using aroma-producing substance data with SIMCA14.0 software.

[0036] Figure 3 This is a graph showing the results of 200 permutation tests on the constructed OPLS-DA model.

[0037] Figure 4 This is a graph showing the results of screening for differential metabolites in the aroma of flue-cured tobacco. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed under conventional experimental conditions. Unless otherwise specified, the reagents and raw materials used in the present invention are all commercially available.

[0039] The *Penicillium sclerotiorum* involved in this embodiment of the invention is derived from *Penicillium sclerotiorum* SCAUMCX01, deposited at the Guangdong Provincial Microbial Culture Collection Center, accession number GDMCC No. 60249, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on October 11, 2017. This strain has been disclosed in Chinese Patent (Patent No.: 201711279355.9, entitled: *Penicillium sclerotiorum* and its application in the preparation of anti-*Ralstonia solanacearum* drugs).

[0040] Example 1: Preparation of Penicillium sclerotiorum agent

[0041] The method described in Chinese Patent (Patent No.: 202210053325.0, entitled "A Method for Sporulation of Penicillium sclerotiorum by Solid Fermentation and Its Application") is as follows:

[0042] (1) Activation of strains

[0043] The *Penicillium sclerotinia* strain was inoculated onto potato dextrose agar (PDA) medium and incubated at 28°C in a constant temperature incubator until the mycelium was abundant, thus completing the strain activation. The PDA medium formula is as follows: 200g potato, 15-20g glucose, 15-20g agar, distilled water added to 1000mL, and the pH adjusted to 7.0.

[0044] (2) Solid-state fermentation

[0045] Activated *Penicillium sclerotiorum* colonies were collected, punched into mycelial cakes, and inoculated into PDB (potato broth) medium. The culture was shaken for 7 days at 28℃ and 150 rpm to obtain the *Penicillium sclerotiorum* fermentation broth. The PDB liquid medium formula is as follows: 200g potato, 15-20g glucose, distilled water added to 1000mL, and pH adjusted to 7.0.

[0046] Commercially available rice was packed into spawn bags (edible mushroom cultivation bags, 18cm*36cm, 11.4cm diameter at the opening), 200g per bag, and sterilized at 121℃ for 20 minutes. After the sterilized rice cooled, 50mL of Penicillium sclerotiorum fermentation broth was poured into each bag, and the bags were sealed with spawn cap rings. The bags were then incubated under suitable conditions (25-27℃) for 3 days. Once the mycelium had fully grown on the surface of the rice solid culture medium, the rings were removed, and the rice was kneaded evenly. Fermentation continued until day 7 to obtain the solid fermentation product.

[0047] (3) Preparation of spore powder

[0048] The solid fermentation product was poured into a pure aqueous solution containing 1‰ (volume fraction) Tween 80, and the spores were washed away by rubbing. The rice residue was filtered out using gauze, retaining the filtrate containing the spores. The filtrate was transferred to a centrifuge tube and centrifuged at 3000g / rpm for 2 minutes. The supernatant was discarded, and this step was repeated until all the filtrate was processed. The collected precipitate was placed in a freeze dryer and dried for 2 days to obtain Penicillium sclerotinia spore powder, i.e., the Penicillium sclerotinia inoculum.

[0049] Example 2: Application of different dilution concentrations of Penicillium sclerotiorum inoculum to the quality of flue-cured tobacco aging.

[0050] The Penicillium sclerotium spore powder prepared in Example 1 (2.214 × 10⁻⁶) 9 The *Penicillium sclerotium* agent (per gram) was diluted with water at three concentration gradients: 100 times, 1000 times, and 10000 times. The experiment was then divided into three treatment groups with different dilutions and one control group: ① Control group (CK), containing raw tobacco leaves without any fungal species; ② *Penicillium sclerotium* agent diluted 100 times; ③ *Penicillium sclerotium* agent diluted 1000 times; ④ *Penicillium sclerotium* agent diluted 10000 times. Each group had three parallel experiments (named CK_1, CK_2, CK_3, etc.). The effects of different dilutions of *Penicillium sclerotium* agent on the aging of flue-cured tobacco were then analyzed. The specific treatment steps are as follows:

[0051] 1. Pretreatment of tobacco leaves before aging

[0052] (1) After the flue-cured tobacco is harvested, select tobacco leaves with moderate maturity, wash them, and air dry them naturally to ensure that there are no impurities or moisture on the surface of the tobacco leaves. Then mix the tobacco leaf samples evenly to unify the initial moisture content (moisture content of 10% to 13%) and store them at 4℃.

[0053] (2) Pack into different containers according to the same weight;

[0054] (3) Add different concentrations of Penicillium sclerotiorum agent to the tobacco leaves after packaging in step (2) at 20% of the tobacco leaf mass (the proportion of Penicillium sclerotiorum agent dilution to the tobacco leaf mass), mix evenly, and ensure that the spore powder is in full contact with the tobacco leaves;

[0055] (4) Place the inoculated tobacco leaves in a constant temperature and humidity environment of 20-30℃ and 60%-70% for aging for 1 year.

[0056] 2. Aroma Analysis of Tobacco Leaves

[0057] The tobacco leaves after the above aging treatment were analyzed to compare the content of major aroma components and major differential metabolites under different concentrations of treatment. The extraction and qualitative / quantitative analysis methods for aroma compounds are as follows:

[0058] (1) Sample processing

[0059] The aged tobacco leaves were ground and passed through a 40-mesh sieve to obtain tobacco powder. Then, 2g of tobacco powder (accurate to 0.01g) was accurately weighed and the aroma components in the tobacco powder sample were extracted and collected using a simultaneous distillation extraction device (after distillation, water was removed with anhydrous sodium sulfate).

[0060] (2) Aroma compounds were extracted using gas chromatography-mass spectrometry (GC / MS).

[0061] The GC / MS conditions are as follows:

[0062] Chromatographic conditions: Instrument model: SHIMADZUGCMS-TQ8040; Column model: DB-5MS; Capillary column: 30m x 0.25mm x 0.25um; Carrier gas: He, 13.6mL / min; Injector temperature: 260℃; Injection volume: 1uL; Split ratio: 20:1; Temperature program: Initial temperature 40℃, hold time 3min; then increase temperature by 7℃ / min to 220℃, hold for 5min; finally increase temperature by 7℃ / min to 280℃, hold for 15min.

[0063] Mass spectrometry conditions: TQ series MS transfer line temperature: 250℃; ionization mode: EI source; ionization energy: 70eV; ion source temperature: 200℃; scan time: 3min; scan range: 50-600Da.

[0064] (3) Analysis of test results

[0065] The mass spectrometry information of the detected volatile components was qualitatively determined by combining it with the NIST14 standard mass spectrometry database, and the internal standard method was used for quantitative analysis of each component. The detection results are shown in Table 1.

[0066] Table 1 Quantitative Analysis of Volatile Components

[0067]

[0068]

[0069] According to Table 1, the amount of aroma compounds in the treated tobacco leaves was generally increased compared to the control group. In particular, the content of neophytadiene, an aroma precursor affecting the flavor of the tobacco leaves, increased significantly, indicating that treatment with Penicillium sclerotinia can degrade and transform high-molecular-weight substances, promote the formation of small-molecule flavor compounds, and play a positive role in improving the quality of tobacco leaves and enabling them to develop a unique aroma and flavor. The chromatographic results of volatile substances are as follows: Figure 1 As shown.

[0070] 3. Screening of differential metabolites after treatment with different concentrations of bacterial agents

[0071] (1) Model building

[0072] An OPLS-DA (Orthogonal Partial Least Squares Discriminant Analysis) model was constructed using SIMCA 14.0 software based on the aroma compound data. The results are shown in [Figure 1]. Figure 2 The model results show a clear separation trend between the control group and each treatment group, with the independent variable fit index (R²) being high. 2 x The value was 0.527, and the dependent variable fit index (R²) was... 2 y The model prediction index (Q) is 0.952. 2 The R-squared value is 0.89, indicating a prediction rate of 89% for the model. 2 and Q 2 A value greater than 0.5 indicates that the model fit is acceptable.

[0073] (2) Model validity verification

[0074] The above model underwent 200 permutation tests, and the results are as follows: Figure 3 As shown, Q 2 The intersection of the regression line with the vertical axis is less than 0, indicating that the model is not overfitting and the model validation is effective. Therefore, the result can be used for the identification and analysis of the aroma of flue-cured tobacco.

[0075] (3) Screening of differential metabolites

[0076] Aroma components that differentiated between the control (CK) and the inoculum were screened using VIP > 1 (projected significance value) and P < 0.05. The results are as follows: Figure 4 As shown, a total of 7 differential aroma compounds were identified: 2-azhexane, glycerol, 9-hydroxy-4,7-megaladien-3-one, stearic acid, N,N-dimethyloctamide, 6-ethyl-5,6-dihydro-2H-pyran-2-one, and n-nonacosane. These include ketones (3), alcohols (1), alkanes (1), organic acids (1), and other compounds (1). Among these, 6 compounds showed an increase in content: 2-azhexane, glycerol, 9-hydroxy-4,7-megaladien-3-one, stearic acid, N,N-dimethyloctamide, and n-nonacosane. One compound showed a decrease in content: 6-ethyl-5,6-dihydro-2H-pyran-2-one.

[0077] Glycerol is a key substance in promoting a smoother aroma in flue-cured tobacco. Its addition helps balance the components in the tobacco, making the smoke smoother, reducing irritation, and thus improving the smoking experience. It also promotes the absorption of active ingredients in the tobacco, enhancing the overall quality of the cigarette. 9-Hydroxy-4,7-mega-stigmadien-3-one and 2-azhexanecycloone are natural aroma components in tobacco leaves. Increased ketone content imparts unique aroma characteristics to flue-cured tobacco, improving its aroma quality. Stearic acid, as a non-volatile organic acid, can impart a waxy, greasy, and smooth aromatic flavor to the smoke. The presence of stearic acid makes the smoke smoother and reduces irritation. Ketones are important products of the Maillard reaction between sugars and amino acids in tobacco leaves. This reaction continues during the curing, aging, processing, and combustion of flue-cured tobacco, producing a rich variety of aroma compounds with a low aroma threshold and good aroma quality. These compounds can blend seamlessly with the tobacco itself, giving cigarettes a rich and unique aroma profile. Glycerol can keep tobacco moist, preventing it from drying out and breaking, thereby improving its hygroscopicity and softness, allowing smokers to experience a better taste and comfort. Meanwhile, organic acids have important effects on the growth, nitrogen metabolism, smoke quality, and aroma formation of flue-cured tobacco.

[0078] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for promoting the aging of flue-cured tobacco using Penicillium sclerotiorum, characterized in that, Includes the following steps: (1) Pretreatment of tobacco leaves: The harvested flue-cured tobacco leaves are washed and dried to obtain dried tobacco leaves; (2) Inoculation and alcohol treatment: Dilute the spore powder of Penicillium sclerotiorum with water to obtain a diluted solution of Penicillium sclerotiorum spore powder; then spray it evenly on the surface of the dried tobacco leaves obtained in step (1), and then place the inoculated tobacco leaves in an environment with a temperature of 20-30℃ and a humidity of 60%-70% to promote the alcoholization of tobacco leaves. The *Penicillium sclerotium* mentioned in step (2) is *Penicillium sclerotium* (… Penicillium sclerotiorum )SCAUMCX01; The promotion of tobacco leaf aging in step (2) is to increase the content of aroma substances in tobacco leaves; wherein, the aroma substances include at least one of 2-azhexane, glycerol, 9-hydroxy-4,7-mega-stigmadien-3-one, stearic acid, N,N-dimethyloctamide and n-nonacosane.

2. The method according to claim 1, characterized in that: The number of spores in the Penicillium sclerotiorum spore powder mentioned in step (2) is 2 to 2.5 × 10⁻⁶. 9 pcs / g; The Penicillium spore powder mentioned in step (2) is diluted with water to a ratio of 100 to 10,000 times; The amount of Penicillium spore powder dilution mentioned in step (2) is 20±2% of the tobacco leaf mass.

3. The method according to claim 1, characterized in that: The drying process described in step (1) is natural air drying; The moisture content of the dried tobacco leaves described in step (1) is 10% to 13%.

4. The method according to claim 1, characterized in that: The cultivation time described in step (2) is 12 to 24 months.

5. The method according to claim 1, characterized in that, The following steps are included after step (2): (3) Dry the tobacco leaves after the alcohol treatment in step (2) to remove excess moisture, and then package and store them.

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