Method for evaluating mildew-causing capability of mildew in cigar tobacco leaves

Abstract

The invention relates to a method for evaluating the mildew-causing capability of mildew in cigar tobacco leaves, and belongs to the technical field of mildew-causing capability evaluation. According to the method, a systematic evaluation system comprising four indexes of visual evaluation, olfaction evaluation, colony counting evaluation and analysis and evaluation of volatile components in mildewed tobacco leaves is constructed by carrying out separation and back inoculation infection on various moulds in the cigar tobacco leaves, and the scientific and systematic evaluation method for the mildew-causing ability of the moulds in the cigar tobacco leaves is established. According to the method disclosed by the invention, objective and accurate evaluation indexes, namely, a colony counting result and an analysis and evaluation index of volatile components in mildewed tobacco leaves, are provided for the mildewing ability of the mould; and subjective sensory evaluation indexes, namely visual and olfactory evaluation, are used for assistance, and the evaluation indexes are set objectively and reasonably. In addition, the method converts the evaluation result of qualitative description into quantitative evaluation, so that the sensory evaluation result is clearer and more objective.

Description

Technical Field

[0001] The invention relates to a method for evaluating the mildew-causing ability of molds in cigar tobacco leaves, and belongs to the technical field of mildew-causing ability evaluation of molds. Background Art

[0002] Tobacco raw materials and tobacco products are easily affected by environmental factors during the growth, storage and aging process, and are prone to mildew. Once mildew occurs, tobacco raw materials and tobacco products will directly lose their value, which has gradually made mildew one of the thorny issues plaguing the global tobacco industry.

[0003] Compared with traditional cigarettes, the mold in cigar tobacco leaves has the characteristics of many types, wide distribution, strong fertility and latency, which leads to the risk of mold in all links of cigar tobacco production and processing. In addition, the unique processing technology of cigar tobacco leaves, such as high moisture content of cigar tobacco products, many high-humidity process conditions, long residence time of cigar tobacco leaves for transfer, poor tools and environmental hygiene conditions, and products in a non-sealed system, make its production theoretically face a greater risk of mold. Especially in the fermentation process of cigar tobacco leaves, since the action of enzymes and microorganisms requires a high temperature and humidity environment, and cigar tobacco leaves contain rich substances, mold is very easy to breed. In addition to causing the quality of tobacco leaves to decrease, causing waste of high-quality tobacco resources, and generating huge economic losses, the mold of cigar tobacco leaves may also produce mycotoxins and directly affect the quality and safety of cigar tobacco leaves (posing a potential threat to the health of consumers). Therefore, it is very important to prevent and control the mold of cigar tobacco leaves and products.

[0004] However, at present, when controlling cigar tobacco leaves and products, the isolated mold is defined as the main mold only through microbial isolation and culture. However, there are many types of molds (filamentous fungi) in cigar tobacco leaves, some of which even play a positive role in the sensory quality style characteristics of cigar tobacco leaves, while some play a negative role in the sensory quality style characteristics. Therefore, this method is extremely unscientific. Therefore, it is of great significance to establish a method that can scientifically and systematically evaluate the mildew-causing ability of molds in cigar tobacco leaves for the prevention and control of mildew in cigar tobacco leaves and products. Summary of the invention

[0005] The purpose of the present invention is to provide a method for evaluating the mildew-causing ability of molds in cigar tobacco leaves, and to provide a scientific and systematic method for evaluating the mildew-causing ability of molds in cigar tobacco leaves for the prior art.

[0006] In order to achieve the above-mentioned object, a technical scheme of a method for evaluating the mildew-causing ability of mold in cigar tobacco leaves in the present invention is:

[0007] A method for evaluating the mildew-causing ability of mold in cigar tobacco leaves, wherein the mold in the cigar tobacco leaves is separated and then inoculated, and then the mildew-causing ability of the mold is evaluated, which includes the following steps:

[0008] (1) Determine the limit range and individual score of key evaluation indicators for the mold-causing ability of mold on cigar tobacco leaves; the key evaluation indicators include visual evaluation, olfactory evaluation, colony count evaluation, and volatile component analysis evaluation in moldy tobacco leaves;

[0009] (2) Determine the weights of key evaluation indicators for the mildew-causing ability of fungi on cigar tobacco leaves and obtain a comprehensive score calculation formula for the mildew-causing ability of fungi;

[0010] (3) Determine the grade and value range of the mold-causing ability of mold on cigar tobacco leaves;

[0011] (4) Evaluate the sample to be tested to obtain the limit range and individual score of the key evaluation index, and obtain the comprehensive score of the mold-causing ability according to the calculation formula of step (2), and determine the level of the mold-causing ability according to the mold-causing ability strength assignment range within which the comprehensive score falls.

[0012] The beneficial effect of the above technical solution is that the method for evaluating the mildew-causing ability of mold in cigar tobacco leaves of the present invention is a pioneering invention. The present invention separates a variety of molds in cigar tobacco leaves, and then carries out back infection, constructs a systematic evaluation system including four indicators of visual evaluation, olfactory evaluation, colony count evaluation, and volatile component analysis evaluation in moldy tobacco leaves, and establishes a scientific and systematic evaluation method for the mildew-causing ability of mold in cigar tobacco leaves. In the method of the present invention, the mildew-causing ability of molds has both objective and accurate evaluation indicators, namely, colony count results and volatile component analysis evaluation indicators in moldy tobacco leaves, and is assisted by subjective sensory evaluation indicators, namely, visual and olfactory evaluation, and the evaluation indicators are set objectively and reasonably. Moreover, the method of the present invention converts the evaluation results of qualitative description into quantitative evaluation, so that the sensory evaluation results are clearer and more objective.

[0013] Specifically, the most intuitive and quickest way to judge the degree of mildew of cigar tobacco leaves in the current industry is usually to look with the eyes and smell with the nose. The number of mildewed tobacco leaves seen with the eyes corresponds to the mildewed area in the visual evaluation; the size of the mildewed smell smelled with the nose corresponds to the mildewed taste and degree in the olfactory evaluation, and these two indicators have the advantages of simple and quick judgment, so these two are selected as key evaluation indicators. In addition, colony count evaluation is the most commonly used method in the current industry to judge the degree of mildew of cigar tobacco leaves. It is often based on GB 4789.15-2016 operation, and the number of plate colonies is counted to roughly judge the degree of mildew of tobacco leaves, but there is no standard for qualitatively judging the degree of mildew by the number of colonies. The present invention, based on a large amount of experimental data from previous studies, can derive a standard for qualitatively judging the degree of mildew by the number of colonies. And this indicator is operated according to GB 4789.15-2016, has strong operability and scientificity, and also makes up for the subjectivity of the visual evaluation indicator, so this indicator is selected as the key evaluation indicator. On the basis of the first three key evaluation indicators, the musty odor substance analysis is detected by GC-IMS, which has the advantages of simple operation, short time consumption and high sensitivity, increases the rigor of the evaluation method of the present invention, and also makes up for the subjectivity of the olfactory evaluation indicator. Therefore, this indicator is selected as the key evaluation indicator.

[0014] As a further improvement, the limited range and individual score of the visual evaluation are as follows: if the moldy area is between 0% and 20%, the score is 1; if the moldy area is between 20% and 40%, the score is 2; if the moldy area is between 40% and 60%, the score is 3; if the moldy area is between 60% and 80%, the score is 4; if the moldy area is between 80% and 100%, the score is 5.

[0015] As a further improvement, the olfactory evaluation is performed based on the strength of the moldy odor after mildew.

[0016] As a further improvement, the restricted range and individual scores of the olfactory evaluation are as follows: the musty degree is scored as 0-20%, with a score of 1; the musty degree is scored as 20-40%, with a score of 2; the musty degree is scored as 40-60%, with a score of 3; the musty degree is scored as 60-80%, with a score of 4; the musty degree is scored as 80-100%, with a score of 5.

[0017] As a further improvement, colony count evaluation was performed according to GB 4789.15-2016.

[0018] As a further improvement, the limit range and individual score of the colony count evaluation are as follows: mold content is 0-1×10 2 CFU / g, score is 1; mold content is 1×10 2 -1×10 3 CFU / g, score is 2; mold content is 1×103 -1×10 4 CFU / g, score is 3; mold content is 1×10 4 -1×10 6 CFU / g, score is 4; mold content is 1×10 6 CFU / g and above, the score is 5.

[0019] As a further improvement, gas phase ion mobility spectrometer was used to analyze and evaluate the volatile components in the moldy tobacco leaves.

[0020] As a further improvement, the restricted range and individual scores of the analysis and evaluation of volatile components in the moldy tobacco leaves are as follows: the moldy marker components are analyzed by the normalization method, and the relative content of the moldy marker components is 0-12%, and the score is 1; the relative content of the moldy marker components is 12%-16%, and the score is 2; the relative content of the moldy marker components is 16%-17.5%, and the score is 3; the relative content of the moldy marker components is 17.5%-19%, and the score is 4; the relative content of the moldy marker components is 19%-20%, and the score is 5.

[0021] As a further improvement, the marker components of mildew are 2-ethylhexanol, 2-methylpropanol, furfural, E-2-pentenal, 3-methyl-2-butenal-D, 3-pentanone, 2-butanone, and 2,5-dimethylthiophene.

[0022] As a further improvement, the calculation formula in step (2) is 4 times the sum of the products of the individual scores of each key evaluation indicator and their corresponding weights; the calculation formula is as follows: A = (0.2×visual evaluation index score+0.25×olfactory evaluation index score+0.25×colony evaluation index score+0.3×volatile component analysis evaluation index score in moldy tobacco leaves)×4, where A is the comprehensive score of the mold-causing ability of mold.

[0023] Specifically, there are adjustments based on the average distribution of weights. First of all, visual and olfactory evaluations are subjective, and the objective reflection of visual evaluation corresponds to the colony count evaluation, and the objective reflection of olfactory evaluation corresponds to the analysis and evaluation of volatile components in moldy tobacco leaves. Therefore, the overall principle of subjective weight is lower than objective weight is followed. In addition, in the actual evaluation of cigar tobacco leaves, more attention is paid to smell and odor substances. Therefore, the principle that the olfactory weight is higher than the visual weight, and the analysis and evaluation of volatile components in moldy tobacco leaves is higher than the colony count evaluation is also followed. The weight of the calculation formula in the evaluation method of the present invention is determined according to these two principles.

[0024] As a further improvement, the grade and value range of the mold-causing ability of the mold on the cigar tobacco leaves in step (3) are as follows: 17≤A≤20, extremely strong mold-causing ability; 12≤A<17, relatively strong mold-causing ability; 8≤A<12, medium mold-causing ability; 6≤A<8, relatively weak mold-causing ability; 4≤A<6, extremely weak mold-causing ability.

[0025] Specifically, extremely weak mold-causing ability means that when the fungus appears on cigar leaves, there is no need to pay much attention to the fungus; relatively weak mold-causing ability means that when the fungus appears on cigar leaves, a small amount of attention can be given; medium mold-causing ability means that when the fungus appears on cigar leaves, a certain degree of attention can be given; relatively strong mold-causing ability means that when the fungus appears on cigar leaves, a high degree of attention can be given; and extremely strong mold-causing ability means that when the fungus appears on cigar leaves, extremely high attention should be given. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The figure is an operation flow chart of an embodiment of the present invention. DETAILED DESCRIPTION

[0027] The existing technology for the prevention and control of cigar tobacco leaves and products only defines the isolated mold as the main mold through microbial isolation and cultivation, which is extremely unscientific. The present invention separates a variety of molds in cigar tobacco leaves, uses artificial mildew tests for back infection, and constructs a systematic evaluation system including four indicators of visual evaluation, olfactory evaluation, colony count evaluation, and volatile component analysis and evaluation in moldy tobacco leaves, and establishes a scientific and systematic evaluation method for the mold-causing ability of mold in cigar tobacco leaves. In the method of the present invention, the mold-causing ability of mold has both objective and accurate evaluation indicators, namely, colony count results and volatile component analysis and evaluation indicators in moldy tobacco leaves, and is assisted by subjective sensory evaluation indicators, namely, visual and olfactory evaluation, and the evaluation indicators are set objectively and reasonably. Moreover, the method of the present invention converts the evaluation results of qualitative description into quantitative evaluation, so that the sensory evaluation results are clearer and more objective.

[0028] The present invention is further described below in conjunction with specific implementation methods, but the protection scope of the present invention is not limited thereto; unless otherwise specified, various reagents, instruments, etc. used in the examples are commercially available products.

[0029] In the following examples, the volatile components in the moldy tobacco leaves were analyzed and evaluated by using a gas phase ion mobility spectrometer (GC-IMS) to determine the volatile organic components in the cigar tobacco leaves. The specific operation is as follows:

[0030] (1) Sample preparation: Accurately weigh 1 g of sample and place it in a 20 mL headspace vial. Incubate at 80°C for 15 min before injecting. Each sample was measured in triplicate.

[0031] (2) Headspace injection conditions: incubation temperature: 80°C; incubation time: 15 min; injection volume: 500 μL; splitless injection; incubation speed: 500 r / min; injection needle temperature: 85°C. GC conditions: column temperature: 60°C; carrier gas: high-purity nitrogen (purity ≥ 99.999%); programmed pressure increase: initial flow rate 2.0 mL / min maintained for 2 min, linearly increased to 10.0 mL / min within 8 min, linearly increased to 100.0 mL / min within 10 min, and then maintained for 10 min, chromatographic run time: 30 min; injection port temperature: 80°C.

[0032] (3) IMS conditions: ionization source: tritium source (3H); migration tube length: 98 mm; electric field strength: 500 V / cm; migration tube temperature: 45°C; drift gas: high-purity nitrogen (purity ≥ 99.999%), flow rate: 150.0 mL / min; positive and negative ion modes: positive ion.

[0033] (4) Data processing: A mixed standard of six ketones (the six ketones are: 2-butanone, 2-pentanone, 2-hexanone, 2-heptanone, 2-octanone and 2-nonanone (all analytically pure)) was detected to establish a calibration curve of retention time and retention index. The retention index of the substance was then calculated by the retention time of the target substance. The GC retention index database (NIST 2020) and IMS migration time database built into the VOCal software were used for retrieval and comparison, and the target substance was qualitatively analyzed. The Reporter, Gallery Plot and Dynamic PCA plug-ins in the VOCal data processing software were used to generate three-dimensional spectra, two-dimensional spectra, difference spectra, fingerprint spectra and PCA graphs of volatile components for comparison of volatile organic compounds between samples. The compounds that increased significantly in the volatile compounds in the tobacco leaves after moldy were selected as the marker components of tobacco leaf moldy. The compounds that increased significantly were 2-ethylhexanol, 2-methylpropanol, furfural, E-2-pentenal, 3-methyl-2-butenal-D, 3-pentanone, 2-butanone, and 2,5-dimethylthiophene.

[0034] A specific embodiment of a method for evaluating the mildew-causing ability of mold in cigar tobacco leaves of the present invention:

[0035] The present invention takes 8 kinds of molds obtained after isolating molds from moldy tobacco leaves in two cigar tobacco producing areas in China as the research objects, carries out back-inoculation, and then determines the key evaluation indexes of the mold-causing ability of the molds on cigar tobacco leaves, establishes the scoring criteria and single-phase weights of the key evaluation indexes, and calculates the comprehensive score of the mold-causing ability of the molds to determine the strength of the mold-causing ability of the molds on cigar tobacco leaves. The specific operation process is as follows: Figure 1 As shown, the specific implementation operations are as follows:

[0036] Example 1

[0037] The mold used in the method for evaluating the mold-causing ability of mold in cigar tobacco leaves in this embodiment is from Trichoderma longipes in the Hubei production area, and the specific process is as follows:

[0038] 1. Fungal infection

[0039] (1) Tobacco leaf pretreatment

[0040] Take the tobacco leaves for aseptic treatment and sterilize them at 121°C for 30 minutes. After sterilization, use sterile scissors to cut the tobacco leaves into small pieces of about 3 cm in length.

[0041] (2) Preparation of fungal spore suspension

[0042] The spore suspension was obtained by plate culture method. The specific operation was as follows: the mold to be tested was inoculated into the PDA plate, placed in a 28°C incubator for 5 days, and then the surface of the plate was washed with 5 mL of PBS buffer to obtain a mixture of mycelium and spores, which was filtered through 4 layers of sterile gauze to obtain the spore suspension stock solution. The spores were counted using a hemocytometer and the spore suspension was diluted to 10 according to the counting results. 6 The concentration of CFU / g.

[0043] (3) Fungal spraying and cultivation

[0044] Take a 5g sterilized cigar tobacco leaf piece and place it on the surface of a sterile bag, take 3.5mL of the diluted spore suspension and spray it on the surface of the tobacco leaf, and use sterile tweezers to turn the tobacco leaf while spraying to ensure that the spore suspension is sprayed evenly. After the tobacco leaf fully absorbs the spore suspension, use tweezers to place the tobacco leaf piece in a sterile triangular bottle, seal it and place it in a constant temperature and humidity incubator at 28°C and RH=75% to observe the mildew process of the tobacco leaf in the triangular bottle, stop the culture after 5 days, and take pictures to record the mildew state of the tobacco leaf.

[0045] 2. Determine the key evaluation indicators of the mold-causing ability of mold on cigar tobacco leaves

[0046] The key evaluation indicators are: visual evaluation, olfactory evaluation, colony count evaluation, and musty odor substance analysis evaluation.

[0047] 3. Determine the limit range and individual score of key evaluation indicators for the mold-causing ability of mold on cigar tobacco leaves

[0048] The scope of the key evaluation indicators and the individual scores are as follows:

[0049] (1) Visual evaluation

[0050] According to the mildew situation, mildew is divided into five levels: mild (mildew area 0%-20%), mild (mildew area 20%-40%), moderate (mildew area 40%-60%), severe (mildew area 60%-80%), and severe (mildew area 80%-100%). Seven experts scored the mildew area, and then took the average score as the measured analysis value of this indicator of the mold. The mild mildew area level is scored as 1; the mild mildew area level is scored as 2; the moderate mildew area level is scored as 3; the severe mildew area level is scored as 4; the severe mildew area level is scored as 5.

[0051] (2) Olfactory evaluation

[0052] According to the size of the moldy smell after mold, the moldy smell is divided into five levels: lighter (the moldy smell is lighter, almost no moldy smell, the moldy smell is scored as 0-20%), mild (the moldy smell is lighter, but the moldy smell can be smelled, the moldy smell is scored as 20%-40%), moderate (the moldy smell is slightly pungent, and the moldy smell can be obviously smelled, the moldy smell is scored as 40%-60%), heavier (the moldy smell is more pungent and the moldy smell is larger, the moldy smell is scored as 60%-80%), and severe (the moldy smell is very pungent and the moldy smell is very obvious, the moldy smell is scored as 80%-100%). 7 experts scored the moldy smell, and then took the average score as the measured analysis value of this indicator of the mold. The moldy odor level is relatively light, with a score of 1; the moldy odor level is mild, with a score of 2; the moldy odor level is moderate, with a score of 3; the moldy odor level is relatively heavy, with a score of 4; and the moldy odor level is heavy, with a score of 5.

[0053] (3) Colony count evaluation

[0054] According to GB 4789.15-2016, the sample dilution was plated for 5 days, and the colony count results were counted. Based on this result, the number of colonies of the mold on the moldy cigar tobacco leaves was evaluated. The mold content was divided into low (mold content between 0-1×10 2 CFU / g range), small amounts (mold content within 1×10 2 -1×10 3 CFU / g range), medium (mold content is within 1×10 3 -1×10 4 CFU / g range), more (mold content is 1×10 4 -1×10 6 CFU / g range), more (mold content within 1×10 6The mold count result level is 1 if it is less, 2 if it is a small amount, 3 if it is medium, 4 if it is more, and 5 if it is a lot.

[0055] (4) Analysis and evaluation of volatile components in moldy tobacco leaves

[0056] The specific operations for analyzing and evaluating the volatile components in moldy tobacco leaves are as described in the specific implementation method.

[0057] The normalization method was used to analyze the marker components of mildew, and the compounds that increased significantly in tobacco leaves after mildew were selected as the marker components of tobacco leaf mildew. The significantly increased compounds were 2-ethylhexanol, 2-methylpropanol, furfural, E-2-pentenal, 3-methyl-2-butenal-D, 3-pentanone, 2-butanone, and 2,5-dimethylthiophene. If the relative content of the marker components of mildew is 0-12%, the mildew-causing ability of this mold is level 1, and the score is 1; if the relative content of the marker components of mildew is 12%-16%, the mildew-causing ability of this mold is level 2, and the score is 2; if the relative content of the marker components of mildew is 16%-17.5%, the mildew-causing ability of this mold is level 3, and the score is 3; if the relative content of the marker components of mildew is 17.5%-19%, the mildew-causing ability of this mold is level 4, and the score is 4; if the relative content of the marker components of mildew is 19%-20%, the mildew-causing ability of this mold is level 5, and the score is 5.

[0058] 4. Determine the weights of key evaluation indicators for the mildew-causing ability of fungi on cigar tobacco leaves and calculate the comprehensive score of the mildew-causing ability of fungi

[0059] (1) Determination of weights

[0060] Visual evaluation: 0.2; olfactory evaluation: 0.25; colony count evaluation: 0.25; volatile component analysis evaluation in moldy tobacco leaves: 0.3.

[0061] (2) Calculation of the comprehensive score of mold-causing ability

[0062] According to the individual scores and weights of key indicators, the comprehensive score of mold-causing ability (A) is calculated:

[0063] A=(0.2×visual evaluation index score+0.25×olfactory evaluation index score+0.25×colony evaluation index score+0.3×volatile component analysis evaluation index score in moldy tobacco leaves)×4.

[0064] The individual mold-causing ability evaluation scores of Trichoderma longipes and the comprehensive score of mold-causing ability are shown in Table 1.

[0065] Table 1 Trichoderma longipes

[0066]

[0067] 5. Determine the level and value range of the mold-causing ability of mold on cigar tobacco leaves, and determine the level of the mold-causing ability of mold according to the comprehensive score of the mold-causing ability

[0068] The grades and value ranges of the mold-causing ability of mold on cigar tobacco leaves are as follows: 17≤A≤20, extremely strong mold-causing ability; 12≤A<17, relatively strong mold-causing ability; 8≤A<12, medium mold-causing ability; 6≤A<8, relatively weak mold-causing ability; 4≤A<6, extremely weak mold-causing ability.

[0069] As shown in Table 1, the comprehensive score (A) of the mildew-causing ability of Trichoderma longipes is 10.6. According to the grade and value range of the mildew-causing ability of the fungus on cigar tobacco leaves, its mildew-causing ability is medium.

[0070] In summary, the mold-causing ability of Trichoderma longibrachiatum is moderate, which means that a certain degree of attention should be paid when this fungus appears on cigar tobacco leaves.

[0071] Example 2

[0072] The mold used in the method for evaluating the mold-causing ability of mold in cigar tobacco leaves in this embodiment is from Aspergillus versicolor produced in Hubei Province. The specific process is as follows:

[0073] The specific operation process is as described in Example 1, and the single mold-causing ability evaluation score of Aspergillus versicolor and the comprehensive score of the mold-causing ability are shown in Table 2.

[0074] Table 2 Aspergillus versicolor

[0075]

[0076] As shown in Table 2, the comprehensive score (A) of the mildew-causing ability of Aspergillus versicolor is 16.6. According to the grade and value range of the mildew-causing ability of the fungus on cigar tobacco leaves, its mildew-causing ability is relatively strong.

[0077] In summary, Aspergillus versicolor has a strong mold-causing ability, which means that when this fungus appears on cigar tobacco leaves, it should be given greater attention.

[0078] Example 3

[0079] The mold used in the method for evaluating the mold-causing ability of mold in cigar tobacco leaves in this embodiment is from Aspergillus monteritus produced in Hubei Province. The specific process is as follows:

[0080] The specific operation process is as described in Example 1, and the single mold-causing ability evaluation score of Aspergillus monteritus and the comprehensive score of mold-causing ability are shown in Table 3.

[0081] Table 3 Aspergillus monteritus

[0082]

[0083] As shown in Table 3, the comprehensive score (A) of the mold-causing ability of Aspergillus monteritus is 15.4. According to the grade and value range of the mold-causing ability of the fungus on cigar tobacco leaves, its mold-causing ability is relatively strong.

[0084] In summary, Aspergillus monteritus has a strong mold-causing ability, which means that when this fungus appears on cigar tobacco leaves, it should be given greater attention.

[0085] Example 4

[0086] The mold used in the method for evaluating the mold-causing ability of mold in cigar tobacco leaves in this embodiment is from Aspergillus pseudoglaucus from the Hubei production area, and the specific process is as follows:

[0087] The specific operation process is as described in Example 1, and the single mold-causing ability evaluation score of Aspergillus pseudoglaucus and the comprehensive score of mold-causing ability are shown in Table 4.

[0088] Table 4 Pseudo-gray green Aspergillus

[0089]

[0090]

[0091] As shown in Table 4, the comprehensive score (A) of the mildew-causing ability of Aspergillus spp. is 19. According to the grade and value range of mildew-causing ability of the fungus on cigar tobacco leaves, its mildew-causing ability is extremely strong.

[0092] In summary, Pseudocaogenes has a very strong mold-causing ability, which means that when this fungus appears on cigar tobacco leaves, it should be given great attention.

[0093] Example 5

[0094] The fungus used in the method for evaluating the mold-causing ability of fungi in cigar tobacco leaves in this embodiment is from the rope-shaped blue fungus produced in Sichuan. The specific process is as follows:

[0095] The specific operation process is as described in Example 1, and the single mold-causing ability evaluation score of the rope-shaped blue fungus and the comprehensive score of the mold-causing ability are shown in Table 5.

[0096] Table 5 Rope-shaped blue bacteria

[0097]

[0098]

[0099] As shown in Table 5, the comprehensive score (A) of the mildew-causing ability of the rope-shaped blue fungus is 11.6. According to the grade and value range of the mildew-causing ability of the fungus on cigar tobacco leaves, the mildew-causing ability is medium.

[0100] In summary, the fungus's mildew-causing ability is moderate, which means that a certain degree of attention should be paid when this fungus appears on cigar tobacco leaves.

[0101] Example 6

[0102] The mold used in the method for evaluating the mold-causing ability of mold in cigar tobacco leaves in this embodiment is from Penicillium chrysogenum in the Sichuan production area, and the specific process is as follows:

[0103] The specific operation process is as described in Example 1, and the single mold-causing ability evaluation score of Penicillium chrysogenum and the comprehensive score of the mold-causing ability are shown in Table 6.

[0104] Table 6 Penicillium chrysogenum

[0105]

[0106] As shown in Table 6, the comprehensive score (A) of the mildew-causing ability of Penicillium chrysogenum is 15.2. According to the grade and value range of mildew-causing ability of the fungus on cigar tobacco leaves, the mildew-causing ability is relatively strong.

[0107] In summary, the mold-causing ability of Penicillium chrysogenum is relatively strong, which means that when this fungus appears on cigar tobacco leaves, it should be given greater attention.

[0108] Example 7

[0109] The mold used in the method for evaluating the mold-causing ability of mold in cigar tobacco leaves in this embodiment is from Aspergillus shevarium in the Sichuan production area, and the specific process is as follows:

[0110] The specific operation process is as described in Example 1, and the single mold-causing ability evaluation score of Aspergillus shevarium and the comprehensive score of mold-causing ability are shown in Table 7.

[0111] Table 7 Aspergillus shevarium

[0112]

[0113] As shown in Table 7, the comprehensive score (A) of the mildew-causing ability of Aspergillus shevarium is 6.4. According to the grade and value range of mildew-causing ability of mildew fungi on cigar tobacco leaves, the mildew-causing ability is relatively weak.

[0114] In summary, the mold-causing ability of Aspergillus shevarium is relatively weak, which means that when this fungus appears on cigar tobacco leaves, it can be given a little attention.

[0115] Example 8

[0116] The mold used in the method for evaluating the mold-causing ability of mold in cigar tobacco leaves in this embodiment is from Aspergillus polydorum produced in Sichuan. The specific process is as follows:

[0117] The specific operation process is as described in Example 1, and the single mold-causing ability evaluation score of Aspergillus polymorpha and the comprehensive score of the mold-causing ability are shown in Table 8.

[0118] Table 8 Aspergillus poly

[0119]

[0120]

[0121] As shown in Table 8, the comprehensive score (A) of the mold-causing ability of Aspergillus polymorpha is 11. According to the grade and value range of the mold-causing ability of molds on cigar tobacco leaves, the mold-causing ability is medium.

[0122] In summary, the mold-causing ability of Aspergillus polymorpha is moderate, which means that a certain degree of attention should be paid when this fungus appears on cigar tobacco leaves.

[0123] Based on the above embodiments, it can be seen that the 8 kinds of molds selected by the present invention are based on the previous existing data and actual conditions, and 8 kinds of molds with different mold-causing abilities in tobacco are selected. First, the mold-causing abilities of these 8 kinds of molds are divided into 4 levels. The mold-causing ability is extremely strong for Aspergillus pseudogrey green; the mold-causing abilities are relatively strong for Aspergillus versicolor, Aspergillus monteritus, and Penicillium chrysogenum; the mold-causing abilities are medium for Trichoderma longiflorum, Blue-shaped fungi, and Aspergillus polydophorus; the mold-causing ability is relatively weak for Aspergillus shevarium. Then, the grades are divided according to the range of the scores of these 8 kinds of molds. The lowest 4 points are the lowest score of the control group, which is 4, and the highest 20 points are the highest score of the experimental group, which is 20. The division standard has the characteristics of being consistent with reality.

[0124] Comparative Example 1

[0125] This comparative example is a control treatment, in which the same amount of sterile water was sprayed on the cigar tobacco leaves, and the remaining operations were as described in Example 1. The single mold-causing ability evaluation score and the comprehensive score of the mold-causing ability of the control treatment are shown in Table 9.

[0126] Table 9 Control treatment

[0127]

[0128]

[0129] As shown in Table 9, the comprehensive score (A) of the mold-causing ability of the control treatment is 4. According to the level and value range of the mold-causing ability of mold on cigar tobacco leaves, the mold-causing ability is extremely weak.

[0130] In summary, the mold-causing ability of the control treatment is extremely weak, indicating that this cigar tobacco leaf does not require much attention.

[0131] In summary, the present invention separates a variety of molds in cigar tobacco leaves, uses artificial mildew test for back infection, constructs a system evaluation system including visual evaluation, olfactory evaluation, colony count evaluation, and volatile component analysis and evaluation in moldy tobacco leaves, and establishes a scientific and systematic evaluation method for the mildew-causing ability of molds in cigar tobacco leaves. Specifically, based on a large amount of research data in the early stage, the present invention uses the national standard GB 4789.15-2016 to count colonies and GC-IMS to analyze the volatile components of moldy tobacco leaves, and displays the subjective and fuzzy evaluation results in a qualitative mildew grade assignment range. The experimental design and experimental result data analysis are objective and reasonable, and the experimental results are repeatable and verifiable. The four indicators set by the present invention are based on experimental data, complement each other, verify each other, and have rigorous logic; the molds used in the selected 8 embodiments are also typical mildew-causing strains on cigar tobacco leaves in China, and have different mildew-causing abilities, which are representative.

[0132] Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for evaluating the mildew-causing ability of mold in cigar tobacco leaves, characterized in that: After the mold is isolated from the cigar tobacco leaves, they are inoculated and then evaluated for their mold-causing ability, which includes the following steps: (1) Determine the limit range and individual score of the key evaluation indicators of the mold-causing ability of mold on cigar tobacco leaves; the key evaluation indicators include visual evaluation, olfactory evaluation, colony count evaluation, and volatile component analysis evaluation in moldy tobacco leaves; (2) Determine the weights of key evaluation indicators for the mildew-causing ability of fungi on cigar tobacco leaves, and obtain the calculation formula for the comprehensive score of the mildew-causing ability of fungi; (3) Determine the grade and value range of the mold-causing ability of mold on cigar tobacco leaves; (4) Evaluate the samples to be tested to obtain the limit range and individual scores of the key evaluation indicators, and obtain the comprehensive score of the mold-causing ability according to the calculation formula in step (2). Determine the level of the mold-causing ability according to the range of the mold-causing ability strength within which the comprehensive score falls.

2. The method for evaluating the mildew-causing ability of mold in cigar tobacco leaves according to claim 1, characterized in that: The limited range and individual scores of the visual evaluation are as follows: if the moldy area is between 0% and 20%, the score is 1; if the moldy area is between 20% and 40%, the score is 2; if the moldy area is between 40% and 60%, the score is 3; if the moldy area is between 60% and 80%, the score is 4; if the moldy area is between 80% and 100%, the score is 5.

3. The method for evaluating the mildew-causing ability of mold in cigar tobacco leaves according to claim 1, characterized in that: The olfactory evaluation was performed based on the strength of the moldy odor after mildew.

4. The method for evaluating the mildew-causing ability of mold in cigar tobacco leaves according to claim 3, characterized in that: The restricted range and individual scores of the olfactory evaluation are as follows: the musty degree is scored as 0-20%, with a score of 1; the musty degree is scored as 20-40%, with a score of 2; the musty degree is scored as 40-60%, with a score of 3; the musty degree is scored as 60-80%, with a score of 4; the musty degree is scored as 80-100%, with a score of 5.

5. The method for evaluating the mildew-causing ability of mold in cigar tobacco leaves according to claim 1, characterized in that: The colony count evaluation was performed according to GB4789.15-2016.

6. The method for evaluating the mildew-causing ability of mold in cigar tobacco leaves according to claim 5, characterized in that: The limit range and individual score of the colony count evaluation are as follows: mold content is 0-1×10 2 CFU / g, score is 1; mold content is 1×10 2 -1×10 3 CFU / g, score is 2; mold content is 1×10 3 -1×10 4 CFU / g, score is 3; mold content is 1×10 4 -1×10 6 CFU / g, score is 4; mold content is 1×10 6 CFU / g and above, the score is 5.

7. The method for evaluating the mildew-causing ability of mold in cigar tobacco leaves according to claim 1, characterized in that: The volatile components in moldy tobacco leaves were analyzed and evaluated using gas phase ion mobility spectrometry.

8. The method for evaluating the mildew-causing ability of mold in cigar tobacco leaves according to claim 7, characterized in that: The restricted scope and individual scores of the analysis and evaluation of volatile components in the moldy tobacco leaves are as follows: the moldy marker components are analyzed by the normalization method, and the relative content of the moldy marker components is 0-12%, and the score is 1; the relative content of the moldy marker components is 12%-16%, and the score is 2; the relative content of the moldy marker components is 16%-17.5%, and the score is 3; the relative content of the moldy marker components is 17.5%-19%, and the score is 4; the relative content of the moldy marker components is 19%-20%, and the score is 5.

9. The method for evaluating the mildew-causing ability of mold in cigar tobacco leaves according to claim 8, characterized in that: The characteristic ingredients of mildew are 2-ethylhexanol, 2-methylpropanol, furfural, E-2-pentenal, 3-methyl-2-butenal-D, 3-pentanone, 2-butanone and 2,5-dimethylthiophene.

10. The method for evaluating the mildew-causing ability of mold in cigar tobacco leaves according to any one of claims 1 to 9, characterized in that: The calculation formula in step (2) is 4 times the sum of the product of the individual scores of each key evaluation index and its corresponding weight; the calculation formula is as follows: A=(0.2×visual evaluation index score+0.25×olfactory evaluation index score+0.25×colony evaluation index score+0.3×moldy tobacco leaf volatile component analysis evaluation index score)×4, where A is the comprehensive score of the mold-causing ability of mold.

11. The method for evaluating the mildew-causing ability of mold in cigar tobacco leaves according to claim 10, characterized in that: The grades and value ranges of the mold-causing ability of the mold on the cigar tobacco leaves in step (3) are as follows: 17≤A≤20, extremely strong mold-causing ability; 12≤A<17, relatively strong mold-causing ability; 8≤A<12, medium mold-causing ability; 6≤A<8, relatively weak mold-causing ability; 4≤A<6, extremely weak mold-causing ability.

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