Mildew critical point detection and identification method for alcoholized tobacco leaves and application

Abstract

The invention discloses a mildewing critical point detection and identification method for alcoholized tobacco leaves and an application of the mildewing critical point detection and identification method. The problem that in the prior art, fermentation environment regulation and control cannot be carried out based on alcoholized tobacco leaf critical points is mainly solved. The method comprises the following steps: respectively collecting strip samples from different storage environments and grouping, preparing an original bacterial suspension, obtaining a single bacterial colony, carrying out morphological analysis on the bacterial colony and individuals, carrying out rDNA-ITS sequence analysis on the bacterial colony, determining a mildew-causing dominant bacterial colony according to microscopic examination and rDNA-ITS sequence analysis results, and determining the influence of different culture temperatures on the growth of the mold. And determining the influence of different culture humidity on the growth of the mold, and determining the tobacco leaf mildewing critical point. Microflora characteristics under different storage conditions are analyzed, suitable growth conditions of mildewing dominant strains are determined under different growth temperature and humidity conditions, a tobacco leaf mildewing critical point is determined based on tobacco leaf quality analysis, and then suitable fermentation conditions of core raw materials are selected according to the tobacco leaf mildewing critical point; and the anti-mildew effect in the tobacco mellowing process can be achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of tobacco leaf processing, and in particular to a method for detecting and identifying a critical point of mildew in aged tobacco leaves and its application. Background Art

[0002] The redried tobacco leaves must be stored for 2 to 3 years for aging or fermentation before they can be directly used in cigarette production. The process of tobacco leaf aging mainly includes three aspects: chemical changes, enzyme catalysis and microbial action. Among them, microorganisms, especially bacteria, play a vital role in the smoking quality of tobacco leaves during the aging process of tobacco leaves. In addition, during the storage process, storage environmental factors (such as temperature, humidity, etc.), the content of tobacco chemical components (such as total sugar, protein, starch, biological enzymes, etc.), tobacco moisture and pH, etc., are interconnected, promoted and restricted by the microorganisms on the surface of tobacco leaves, forming a specific ecosystem for tobacco leaf aging.

[0003] As tobacco leaves have a strong hygroscopicity, the probability of them becoming moldy increases with the storage time. Once mold occurs, fungi will quickly absorb nutrients from the leaves, metabolize blue, green and black pigments, and release unpleasant odors. This will not only cause serious economic problems, but also bring serious health problems.

[0004] The existing storage mildew prevention and insecticide technology known to the inventor, especially the application of atmosphere conditioning maintenance, temperature and humidity control, mechanical control and other methods, is bound to change the original tobacco leaf aging environment, and has a greater impact on tobacco leaf microorganisms, affecting the aging effect of tobacco leaves. In particular, the current tobacco leaf storage and maintenance emphasizes accelerating the aging process of tobacco leaves, and adopts warming, humidification and other means to regulate the aging process, which is bound to increase the risk of tobacco leaf mildew. In the conventional storage and maintenance process, the traditional tobacco leaf mildew identification or detection mainly relies on artificial sense organs, and usually adopts the methods of seeing, pinching, smelling and other methods to judge the mildew degree of tobacco leaves. It is often that tobacco leaves have been mildewed before they are processed, and the losses caused are huge, and some microorganisms have a tendency to multiply in large quantities before the tobacco leaf mildew is discovered, and the tobacco leaves may be near the critical point of mildew, but it has already caused a greater impact on the quality of tobacco leaves, and the mildew trend cannot be discovered in time by artificial means.

[0005] The information disclosed in this background technology section is only used to deepen the understanding of the background technology of the present disclosure, and should not be regarded as acknowledging or suggesting in any form that the information constitutes the prior art known to those skilled in the art. Summary of the invention

[0006] Based on long-term practical research, the inventors found that the fungi that cause stored tobacco leaves to mildew mainly include Aspergillus, Penicillium, Rhizopus, Mucor, etc. These fungi mainly belong to arid or semi-arid storage fungi, and their composition is related to the tobacco variety, storage environment, management methods and storage time.

[0007] In view of at least one of the above technical problems, the present disclosure provides a method and application for detecting and identifying the critical point of mildew in aged tobacco leaves, which mainly solves the problem that the prior art cannot accurately identify the critical point of mildew in aged tobacco leaves and cannot implement fermentation environment regulation based on the critical point.

[0008] According to one aspect of the present disclosure, a method for detecting the critical point of mildew of aged tobacco leaves is provided, comprising the following steps: (1) Collect tobacco samples to be tested from different storage environments and group them; (2) Weigh each tobacco leaf sample to be tested and place them in a container containing 0.9% sterile saline under aseptic operation conditions. Then, shake them at 28-32°C and separate them to obtain the original bacterial suspension corresponding to each group of tobacco leaves. (3) The original bacterial suspension was stirred at 10 -2 , 10 -3 , 10 -4 , 10 -5 After gradient dilution, spread on PDA plate culture medium and culture at a constant temperature of 28-32°C for at least 72 hours until a single colony is obtained and then frozen; (4) Picking spores of a single colony from the PDA plate culture medium and inoculating them in the middle of the culture medium, and inverting and culturing them at a constant temperature of 27 to 29° C. for at least 5 days, observing and recording the morphological characteristics of the colony and changes in the culture medium, and performing microscopic examination of the hyphae; (5) Inoculate the strain of the single colony isolated in step (3) into the center of the corresponding PDA plate culture medium and culture it at a constant temperature of 28-32°C, record the morphological characteristics of the colony and perform rDNA-ITS sequence analysis; (6) Obtaining the dominant mold-causing bacterial flora based on the microscopic examination and the rDNA-ITS sequence analysis results; (7) The strains of the dominant mold-causing bacteria were inoculated into DG18 plate culture medium, and after constant temperature culture at 27-29°C for 6-8 days, the obtained bacterial cake was transferred to the center of a new DG18 plate culture medium under aseptic operation conditions, and the colony diameters were measured after culture at 15°C, 20°C, 25°C, 30°C, 35°C, and 40°C for 6-8 days; (8) The strains of the dominant mold-causing bacteria were inoculated into DG18 plate culture medium, and the bacterial cakes obtained after culturing at a constant temperature of 27 to 29° C. for 6 to 8 days were transferred to the center of a new DG18 plate culture medium under aseptic operation conditions, and were placed in sealed containers with 51% calcium chloride, 60% potassium carbonate, 70% sodium nitrite, 82% ammonium sulfate, and 90% lead sulfate as relative humidity control materials at 27 to 29° C. for 6 to 8 days, and then the colony diameters were measured; (9) Place each tobacco leaf sample to be tested in a culture dish and sterilize at 120-122° C. for 15-25 min. Inoculate the single colony strain isolated in step (3) onto the surface of the sterilized tobacco leaf, and culture under the temperature and humidity combination culture conditions described in steps (7) and (8). Regularly observe the moldy effect of the strain on the tobacco leaf, and determine the moldy critical point of the tobacco leaf under different culture conditions including microbial biomass, temperature, humidity, and culture time based on the changes in tobacco leaf quality.

[0009] In some embodiments of the present disclosure, in step (1), the tobacco slice samples to be tested are collected under sterile conditions and the sampling is repeated at least twice.

[0010] In some embodiments of the present disclosure, in the step (2), glass beads are added into the container before the shaking operation; the frequency of the shaking is 170 to 190 r / min.

[0011] In some embodiments of the present disclosure, in step (4), during microscopic examination, after lactic acid carbolic acid solution is dripped onto the glass slide, the hyphae with spores at the edge of the colony are placed in the lactic acid carbolic acid solution on the glass slide, and after the hyphae are evenly dispersed, a cover glass is placed accordingly.

[0012] In some embodiments of the present disclosure, in step (5), the rDNA-ITS sequence analysis includes the following steps: (51) Use the Fungal Genomic DNA Rapid Extraction Kit to extract the corresponding fungal DNA; (52) PCR amplification of fungal DNA was performed using ITS1 and ITS4 universal primers; (53) The product bands were observed by agarose gel electrophoresis and the PCR products were sequenced; (54) The sequencing results were compared using BLAST homology in the NCBI database, and a phylogenetic tree was constructed.

[0013] In some embodiments of the present disclosure, in step (5), during PCR amplification, the pre-denaturation is carried out at 95°C for 15 minutes, the denaturation is carried out at 94°C for 50 seconds, the annealing is carried out at 60°C for 1 minute, the extension is carried out at 72°C for 1.5 minutes, and after 35 cycles, the extension is carried out at 72°C for 6 minutes.

[0014] In some embodiments of the present disclosure, step (7) and step (8) are respectively repeated at least twice.

[0015] In some embodiments of the present disclosure, in step (9), the moldy effect is classified into mold grades, and the quality of tobacco leaves at each mold grade is evaluated and determined based on the microbial characteristics of tobacco leaves, chemical components of tobacco leaves, key aroma components, and sensory quality evaluation.

[0016] According to another aspect of the present disclosure, a method for controlling mildew prevention of aged tobacco leaves is provided. Based on the mildew critical point obtained as described above, corresponding tobacco samples are taken and placed in culture dishes, and cultured in an incubator for 2 months under different temperature and humidity combinations corresponding to the mildew critical point. Samples are taken regularly and tobacco leaf quality analysis is performed, and tobacco leaf aging conditions are controlled according to the culture conditions corresponding to the best quality.

[0017] One or more technical solutions provided in the embodiments of the present application have at least any of the following technical effects or advantages: By analyzing the characteristics of microbial communities of the core raw materials in the warehouse under different storage conditions, the suitable growth conditions of the dominant mold-causing bacteria can be accurately and reliably determined under different growth temperature and humidity conditions. The mildew critical point of tobacco leaves can be identified and determined based on the tobacco leaf quality analysis. Then, the suitable fermentation conditions for the core raw materials can be selected based on the mildew critical point of tobacco leaves, so as to achieve the effect of accurately preventing and controlling mildew during the tobacco leaf aging process. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the purification result of sample 1 mechanically regulated in an embodiment of the present application.

[0019] Figure 2 This is the purification result of sample 2 with gas conditioning in one embodiment of the present application. DETAILED DESCRIPTION

[0020] In order to better understand the technical solution of the present application, the above technical solution will be described in detail below in conjunction with the accompanying drawings and specific implementation methods. Example

[0021] In order to solve the problem that the trend of tobacco leaf mildew cannot be detected in time during the existing tobacco leaf aging, this example discloses a method for detecting and identifying the critical point of tobacco leaf mildew during aging, which specifically includes the following steps: (1) Collect tobacco samples to be tested from different storage environments and group them.

[0022] In this example, in order to clarify the community characteristics of microorganisms of the core raw materials under different storage conditions, 1 to 2 core raw material tobacco strips were selected as research objects, and 5 kg of alcoholized tobacco strip samples were collected from storage environments of different origins. In order to avoid other strains during the sampling process and affect the experimental results, in this example, protective measures were adopted when sampling tobacco strips to achieve aseptic collection to avoid external infection of the samples. In addition, considering that the abundance and diversity of microorganisms will be affected by sample collection operations, etc., resulting in deviations in the results, therefore, in order to avoid adverse effects on the stability and reproducibility of the data, the sampling in this example was repeated at least twice. The collected samples were grouped and marked.

[0023] In this embodiment, tobacco strips produced in Yunnan and cured by mechanical control are collected as test tobacco strip sample 1, and tobacco strips produced in Yunnan and cured by controlled atmosphere control are collected as test tobacco strip sample 2.

[0024] (2) Preparation of original bacterial suspension: After the samples were collected and grouped, the microorganisms were separated. In this embodiment, 10 g of tobacco leaf samples were weighed for each group of samples and aseptically placed in a conical flask containing 90 ml of 0.9% sterile saline. To facilitate the separation of the strains from the tobacco leaves, two glass beads were added to the conical flask, and the conical flask was placed in a shaker and shaken at 180 r / min for 30 min at 30°C. Thus, the original bacterial suspension corresponding to each group of tobacco leaf was obtained.

[0025] (3) Obtaining a single colony: Based on the original bacterial suspension corresponding to each group of samples, a single colony is obtained from the original bacterial suspension to achieve microbial purification. Specifically, in this embodiment, the original bacterial suspension of each group is respectively 10 -2 , 10 -3 , 10 -4 , 10 -5 Perform gradient dilutions and spread on PDA plate culture medium, and culture in a 30°C constant temperature incubator for at least 72 hours. Purify for two generations until a single colony is obtained, number it, and store it in a 4°C refrigerator for future use.

[0026] (4) Morphological analysis of colonies and individuals: Spores of a single colony were picked from the PDA plate culture medium and dotted in the middle of the culture medium. The culture medium was inverted and cultured in a constant temperature and humidity incubator at (28±1)℃ for at least 5 days. The colonies were then observed and analyzed, and the morphological characteristics of the colonies and changes in the culture medium were recorded. Photos were taken with a camera for record keeping, and the characteristics of the colonies were analyzed accordingly.

[0027] See also Figure 1, is the purification result of sample 1 mechanically regulated sample. Figure 1 (a) and Figure 1 The two bacteria shown in (b) are the same color, both green, but Figure 1 (a) Compared with Figure 1 (b) The colonies are small and small white colonies appear. Figure 1 The colony in (a) grows more compactly. The surface of the colonies of both molds is uneven, with a bulge in the center, and the organization is similar to a carpet. Figure 1 (c) The colony is white with light yellow in the middle, and the hyphae appear loose.

[0028] See also Figure 2 , which is the purification result of the sample regulated by gas conditioning. The three molds have different colors and are easier to distinguish than the molds in the mechanically regulated sample. Figure 2 (a) The colony is yellowish brown in color, with a prominent center and smooth surroundings. The colony is spreading and growing with uneven edges and loose hyphae. Figure 2 In (b), the outermost circle of the colony is white, while the rest is black. The surface is relatively flat, the edges are relatively neat, the hyphae are relatively compact, and the surface shape is similar to concentric rings. Figure 2 The outer circle of the colony shown in (c) is white, the middle is light red, and the very center is white. The surface is uneven, the edges are uneven, the hyphae are loose, and the tissue shape is cotton-like.

[0029] In addition, in this embodiment, in order to analyze the morphological characteristics of individual colonies, lactic acid carbolic acid solution was added to the slide glass, and then the mycelium with spores at the edge of the colony was placed in the lactic acid carbolic acid solution on the slide glass, and after the mycelium was evenly dispersed, a cover glass was placed on the corresponding surface for microscopic examination.

[0030] (5) rDNA-ITS sequence analysis of the colonies: The strains of the single colonies isolated in step (3) were inoculated into the center of the PDA plate culture medium and cultured in a 30°C constant temperature incubator. The morphological characteristics of the colonies were observed and recorded and photographed. Then rDNA-ITS sequence analysis was performed.

[0031] In this embodiment, rDNA-ITS sequence analysis includes the following steps: (51) The corresponding fungal DNA was extracted using a fungal genomic DNA rapid extraction kit.

[0032] (52) ITS1 and ITS4 universal primers were used to perform PCR amplification of fungal DNA. During the PCR amplification, the DNA was pre-denatured at 95°C for 15 min, denatured at 94°C for 50 s, annealed at 60°C for 1 min, and extended at 72°C for 1.5 min. After 35 cycles, the DNA was extended at 72°C for 6 min.

[0033] (53) The product bands were observed by agarose gel electrophoresis and the PCR products were sequenced.

[0034] (54) The sequencing results were compared using BLAST homology comparison in the NCBI database, and a phylogenetic tree was constructed using MEGA software.

[0035] This enables PCR amplification of the ITS gene sequence and phylogenetic tree analysis, thereby achieving the purpose of microbial identification.

[0036] In this embodiment, the mechanically regulated samples are numbered JX1, JX2, and JX3, and the gas-controlled samples are numbered QT1, QT2, and QT3, respectively. The sample detection and identification results are shown in Table 1.

[0037] .

[0038] (6) Determine the dominant mold-causing bacterial community based on the results of microscopic examination and rDNA-ITS sequence analysis.

[0039] (7) Determine the effect of different culture temperatures on mold growth.

[0040] In this example, in order to explore the difference in mold growth under different temperature conditions, the strains of the dominant mold-causing bacteria determined in step (6) were inoculated into the DG18 plate culture medium, and the DG18 plate culture medium was placed in a constant temperature incubator at 28°C for 7 days. The cultured bacterial cake with a diameter of 5 mm was aseptically transferred to the center of a new DG18 plate culture medium, and the colony diameter was measured after culturing for 7 days at 15°C, 20°C, 25°C, 30°C, 35°C, and 40°C. In order to avoid data result deviation caused by processing operations, in this example, the operation was repeated three or more times to perform repeated experiments.

[0041] Among them, in this embodiment, when measuring the diameter of the colony, the actual colony diameter = the average colony diameter - 5mm. When 5mm≤diameter<40mm, it means that the colony can grow; when 20mm≤diameter<30mm, it means that the colony grows well; when 30mm≤diameter<40mm, it means that the colony grows well; when 40mm≤diameter<50mm, it means that the colony grows very well and the hyphae are vigorous; when the diameter is ≥50mm, it means that the colony grows very well and its hyphae are vigorous, so as to explore and define the influence of environmental temperature on the growth of the dominant mold-causing bacteria.

[0042] (8) Determine the effect of different culture humidity on mold growth: In this example, in order to explore the difference in mold growth under different humidity conditions, the strains of the dominant mold-causing bacteria determined in step (6) were inoculated into DG18 plate culture medium, and the DG18 plate culture medium was cultured in a constant temperature incubator at 28°C for 7 days. The cultured bacterial cake with a diameter of 5 mm was aseptically transferred to the center of a new DG18 plate culture medium, and placed in a sealed box with 51% calcium chloride, 60% potassium carbonate, 70% sodium nitrite, 82% ammonium sulfate, and 90% lead sulfate as relative humidity control materials for culture at 28°C. In this example, the sealed box used a sealed fresh-keeping box, thereby using saturated solutions of different salts as relative humidity control materials, and the colony diameter was measured after 7 days of culture. Similarly, in order to avoid data result deviation caused by processing operations, in this example, the operation was repeated more than twice to perform repeated experiments.

[0043] Among them, in this embodiment, when measuring the diameter of the colony, the actual colony diameter = the average colony diameter - 5mm. When 5mm≤diameter<40mm, it means that the colony can grow; when 20mm≤diameter<30mm, it means that the colony grows well; when 30mm≤diameter<40mm, it means that the colony grows well; when 40mm≤diameter<50mm, it means that the colony grows very well and the hyphae are vigorous; when the diameter is ≥50mm, it means that the colony grows very well and its hyphae are vigorous, so as to explore the influence of environmental temperature and humidity on the growth of the dominant mold-causing bacteria.

[0044] In this embodiment, the growth conditions of the colonies of mold F, A, and X at different temperatures under controlled atmosphere control are shown in Table 2, and the growth conditions at different humidities are shown in Table 4; the growth conditions of the colonies of P and A at different temperatures under mechanical control are shown in Table 3, and the growth conditions at different humidities are shown in Table 5.

[0045] .

[0046] As shown in Table 2, for F bacteria, at 25℃, its average growth length is the smallest, which can be regarded as no growth; the average growth length under the temperature conditions of 15℃, 20℃, 30℃, and 35℃ has a small fluctuation range; the average growth length is the largest at 40℃, indicating that among these six temperatures, 40℃ is the temperature condition for the most active growth of this bacteria, and 25℃ is the temperature condition for the slowest growth of this bacteria. The average growth length of A bacteria increases with the increase of temperature under the temperature conditions of 15℃, 20℃, and 30℃; the average growth length reaches the minimum value under the temperature condition of 25℃; under the temperature conditions of 30℃, 35℃, and 40℃, the average growth length of A bacteria shows a trend of decreasing first and then increasing, and under the temperature condition of 35℃, the average growth length decreases. As the temperature further increases, A bacteria resumes the state of active growth; among these six temperature conditions, 40℃ is the temperature condition for the most active growth of this bacteria, and 25℃ is the temperature condition for the slowest growth of this bacteria. X bacteria under the temperature conditions of 15℃, 20℃, 30℃, and 35℃. The average growth length increases with the increase of temperature; at 40℃, the average growth length decreases; at 25℃, the average growth length reaches the lowest point; among these six temperature conditions, 30℃ is the most active temperature condition for the growth of this fungus, and 25℃ is the slowest temperature condition for the growth of this fungus. According to the data analysis in the table, the temperature that can best inhibit the growth of mold in controlled atmosphere is 25℃.

[0047] .

[0048] From the data in Table 3, it can be seen that for P bacteria, the average growth length of P bacteria shows a trend of increasing first and then decreasing with the increase of temperature; when the temperature continues to rise to 40℃, the average growth length of P bacteria reaches the highest point; among these six temperature conditions, 25℃ and 40℃ are the temperature conditions for the active growth of this bacteria, and 15℃ and 35℃ are the temperature conditions for the slowest growth of this bacteria. The average growth length of A bacteria shows an increasing trend with the increase of temperature; 30℃ and 35℃ are the temperature conditions for the most active growth of this bacteria, and 15℃ is the temperature condition for the slowest growth of this bacteria. From the analysis of the data in the table, it can be obtained that the temperature that can best inhibit the growth of mechanically regulated mold should be 25℃.

[0049] .

[0050] From the data in Table 4, it can be seen that for F bacteria, the average growth value of F bacteria shows a trend of first decreasing and then increasing with the increase of humidity. Under the humidity condition of 70%, the average growth length of F bacteria is the smallest; under these five humidity conditions, 70% is the humidity condition under which this kind of bacteria grows the slowest. The average growth length of A bacteria shows a trend of first increasing and then decreasing with the increase of humidity; under the humidity condition of 80%, the average growth length of A bacteria reaches the minimum value; under these five humidity conditions, 80% is the humidity condition under which this kind of bacteria grows the slowest. The average growth length of X bacteria shows a trend of first decreasing, then increasing, and then decreasing with the increase of humidity; under the humidity conditions of 60%, 80%, and 90%, the average growth length of X bacteria is relatively low. According to the data analysis in the table, the humidity that can best inhibit the growth of mold in controlled atmosphere should be 70%.

[0051] .

[0052] From the data in Table 5, it can be seen that for P bacteria, the average growth length of P bacteria shows a trend of first increasing, then decreasing, and then increasing with the increase of humidity. Under the humidity conditions of 50% and 70%, P bacteria grows the slowest. The average growth length of A bacteria shows a trend of first increasing and then decreasing with the increase of humidity. Under the humidity conditions of 50%, 60% and 70%, A bacteria grows the slowest. According to the data analysis in the table, the humidity that can best inhibit the growth of mechanically regulated mold should be 60%.

[0053] (9) Determine the critical point of tobacco leaf mold.

[0054] In order to comprehensively verify the microbial culture temperature and humidity conditions determined in step (7) and step (8), it is determined whether the microbial community growth and development rules in the culture medium can be maintained when attached to the tobacco leaves. Therefore, in this embodiment, each tobacco sample is placed in a culture dish and sterilized in an autoclave at 121°C for 20 minutes to obtain a surface-sterile tobacco sample. Then, the strain of the single colony isolated in step (3) is inoculated onto the surface of the tobacco leaf, and cultured under the temperature and humidity combination culture conditions determined in steps (7) and (8) for suitable colony growth. The moldy effect of the strain on the tobacco leaves is regularly observed every 12 hours, and the moldy effect is classified into moldy grades to determine the relationship between each moldy grade and the quality of the tobacco leaves.

[0055] Specifically, in this embodiment, according to the experiments in step (7) and step (8), it can be determined that under the same humidity conditions, the temperature that can best inhibit the growth of both mechanically regulated and gas-controlled molds is 25°C; under the same temperature conditions, the humidity that can best inhibit the growth of gas-controlled molds should be 70%, and the humidity that can best inhibit the growth of mechanically regulated molds should be 60%. Combined with the actual storage environment and storage time, in this embodiment, under the temperature condition of 25°C and the humidity condition of 60%, the temperature gradient of 28°C, 30°C, and 32°C and the humidity gradient of 60%, 65%, and 70% are set to analyze the growth status of molds under different combination culture conditions.

[0056] In this embodiment, the tobacco leaf quality at each mildew level is evaluated based on the tobacco leaf microbial characteristics, tobacco leaf chemical composition, key aroma components, and sensory quality evaluation, and the influence of microbial species and fermentation conditions on the tobacco leaf chemical composition and sensory quality is determined. Based on the changes in tobacco leaf quality, the tobacco leaf mildew critical point under different culture conditions including microbial biomass, temperature, humidity, and culture time is determined.

[0057] In addition, this example also discloses a method for controlling the mildew prevention of aged tobacco leaves, which is based on the above-determined mildew critical point, uses the microbial community obtained by sampling to carry out directional fermentation of the raw materials, and sets different combinations of temperature, humidity and fermentation time according to the temperature, humidity and moisture status of the tobacco leaves under the storage conditions, and performs response surface optimization analysis. Specifically, the corresponding tobacco samples are placed in culture dishes, and different temperature and humidity combinations are selected according to the conditions corresponding to the mildew critical point. The culture dishes are placed in the corresponding culture environment under each temperature and humidity combination condition and cultured for 2 months. During the culture process, samples are taken every 7 days, and the samples after sampling are sealed in time and stored in the refrigerator, and the tobacco leaf quality analysis is performed.

[0058] After sampling, the tobacco leaf microbial characteristics, tobacco leaf chemical composition, and key aroma components of the sampled samples were tested, and a sensory quality evaluation was performed to determine the key aroma components of the tobacco leaf style characteristics and the sensory evaluation results as well as the response surface analysis results, and to determine the tobacco leaf fermentation conditions that can reduce the irritation and impurities of the tobacco leaves and improve the aroma quality and aroma quantity before the mildew critical point. The actual tobacco leaf aging conditions are controlled by the tobacco leaf fermentation culture conditions corresponding to the optimal quality, so as to achieve the effect of preventing mildew in the aging tobacco leaves.

[0059] Although some preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present application.

[0060] Obviously, those skilled in the art can make various changes and modifications to the present application without departing from the spirit and scope of the inventive concept. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application is also intended to include these modifications and variations.

Claims

1. A method for detecting and identifying the critical point of mildew in aged tobacco leaves, characterized in that: The steps include: (1) Collect tobacco samples to be tested from different storage environments and group them; (2) Weigh each tobacco leaf sample to be tested and place them in a container containing 0.9% sterile saline under aseptic operation conditions. Then, shake them at 28-32°C and separate them to obtain the original bacterial suspension corresponding to each group of tobacco leaves. (3) The original bacterial suspension was stirred at 10 -2 , 10 -3 , 10 -4 , 10 -5 After gradient dilution, spread on PDA plate culture medium and culture at a constant temperature of 28-32°C for at least 72 hours until a single colony is obtained and then frozen; (4) Picking spores of a single colony from the PDA plate culture medium and inoculating them in the middle of the culture medium, and inverting and culturing them at a constant temperature of 27 to 29° C. for at least 5 days, observing and recording the morphological characteristics of the colony and changes in the culture medium, and performing microscopic examination of the hyphae; (5) Inoculate the strain of the single colony isolated in step (3) into the center of the corresponding PDA plate culture medium and culture it at a constant temperature of 28-32°C, record the morphological characteristics of the colony and perform rDNA-ITS sequence analysis; (6) Obtaining the dominant mold-causing bacterial flora based on the microscopic examination and the rDNA-ITS sequence analysis results; (7) The strains of the dominant mold-causing bacteria were inoculated into DG18 plate culture medium, and after constant temperature culture at 27-29°C for 6-8 days, the obtained bacterial cake was transferred to the center of a new DG18 plate culture medium under aseptic operation conditions, and the colony diameters were measured after culture at 15°C, 20°C, 25°C, 30°C, 35°C, and 40°C for 6-8 days; (8) The strains of the dominant mold-causing bacteria were inoculated into DG18 plate culture medium, and the bacterial cakes obtained after culturing at a constant temperature of 27 to 29° C. for 6 to 8 days were transferred to the center of a new DG18 plate culture medium under aseptic operation conditions, and were placed in sealed containers with 51% calcium chloride, 60% potassium carbonate, 70% sodium nitrite, 82% ammonium sulfate, and 90% lead sulfate as relative humidity control materials at 27 to 29° C. for 6 to 8 days, and then the colony diameters were measured; (9) Place each tobacco leaf sample to be tested in a culture dish and sterilize at 120-122° C. for 15-25 min. Inoculate the single colony strain isolated in step (3) onto the surface of the sterilized tobacco leaf, and culture under the temperature and humidity combination culture conditions described in steps (7) and (8). Regularly observe the moldy effect of the strain on the tobacco leaf, and determine the moldy critical point of the tobacco leaf under different culture conditions including microbial biomass, temperature, humidity, and culture time based on the changes in tobacco leaf quality.

2. The method for detecting and identifying the critical point of mildew of aged tobacco leaves according to claim 1, characterized in that: In step (1), the tobacco samples to be tested are collected under sterile conditions and the sampling is repeated at least twice.

3. The method for detecting and identifying the critical point of mildew of aged tobacco leaves according to claim 1, characterized in that: In the step (2), glass beads are added into the container before the shaking operation; the frequency of the shaking is 170 to 190 r / min.

4. The method for detecting and identifying the critical point of mildew of aged tobacco leaves according to claim 1, characterized in that: In step (4), during microscopic examination, lactic acid carbolic acid solution is dripped onto the glass slide, and then the mycelium with spores at the edge of the colony is placed in the lactic acid carbolic acid solution on the glass slide, and after the mycelium is evenly dispersed, a cover glass is placed accordingly.

5. The method for detecting and identifying the critical point of mildew of aged tobacco leaves according to claim 1, characterized in that: In step (5), the rDNA-ITS sequence analysis includes the following steps: (51) Use the Fungal Genomic DNA Rapid Extraction Kit to extract the corresponding fungal DNA; (52) PCR amplification of fungal DNA was performed using ITS1 and ITS4 universal primers; (53) The product bands were observed by agarose gel electrophoresis and the PCR products were sequenced; (54) The sequencing results were compared using BLAST homology in the NCBI database, and a phylogenetic tree was constructed.

6. The method for detecting and identifying the critical point of mildew of aged tobacco leaves according to claim 5, characterized in that: In the step (5), the PCR amplification is performed in sequence by pre-denaturation at 95°C for 15 minutes, denaturation at 94°C for 50 seconds, annealing at 60°C for 1 minute, and extension at 72°C for 1.5 minutes. After 35 cycles, the amplification is performed by extension at 72°C for 6 minutes.

7. The method for detecting and identifying the critical point of mildew of aged tobacco leaves according to claim 5, characterized in that: The step (7) and the step (8) are respectively repeated at least twice.

8. The method for detecting and identifying the critical point of mildew of aged tobacco leaves according to claim 5, characterized in that: In the step (9), the moldy effect is classified into mold grades, and the quality of the tobacco leaves at each mold grade is evaluated and determined based on the tobacco leaf microbial characteristics, tobacco leaf chemical components, key aroma components, and sensory quality evaluation.

9. A method for preventing mildew of aged tobacco leaves, characterized in that: Based on the mold critical point obtained in step (8) of claim 1, the corresponding tobacco leaf samples are placed in a culture dish and cultured in an incubator for 2 months under different temperature and humidity combinations corresponding to the mold critical point. Samples are taken regularly and tobacco leaf quality analysis is performed, and the tobacco leaf aging conditions are controlled according to the culture conditions corresponding to the best quality.

Images