Method for evaluating effectiveness of enzyme preparation in improving tobacco leaf quality and application of enzyme preparation

Abstract

The present invention belongs to the field of tobacco products and relates to a method for evaluating the effectiveness of an enzyme preparation in improving the quality of tobacco leaves, including: applying the enzyme preparation to tobacco leaves of a certain grade; detecting the tobacco leaves of the certain grade, the treated tobacco leaves of the certain grade and the tobacco leaves of a higher grade by Raman spectroscopy to obtain Raman spectral curves; selecting a Raman shift range of 800-1800 cm ‑1 on the curve, connecting the two ends to obtain a straight line segment; subtracting the intensity corresponding to the straight line segment from the intensities corresponding to multiple Raman shifts on the curve segment to obtain a data set I R , dividing by the maximum intensity I max on the curve segment to obtain a data set I; calculating the first root mean square error of the data set I between the treated tobacco leaves of the certain grade and the tobacco leaves of the certain grade, and the second root mean square error of the data set I between the treated tobacco leaves of the certain grade and the tobacco leaves of a higher grade; if the former is greater than the latter, it is effective in improving the quality of tobacco leaves, otherwise it is ineffective. The present invention also relates to the application of the enzyme preparation in improving the quality of tobacco leaves. The method of the present invention can quickly and accurately evaluate the effectiveness of the enzyme preparation in improving the quality of tobacco leaves.

Description

Technical Field

[0001] The present invention belongs to the field of tobacco products, and specifically relates to a method for evaluating the effectiveness of enzyme preparations in improving the quality of tobacco leaves, and also relates to the application of enzyme preparations in improving the quality of tobacco leaves. Background Art

[0002] The number of microorganisms on the surface of tobacco leaves is large and diverse, and it plays a very important role in the aging process of tobacco leaves. In recent years, applying enzyme preparations or commercial enzyme preparations fermented from microbial strains isolated from the surface of tobacco leaves to tobacco leaf processing and production has become one of the research hotspots at home and abroad. The enzyme preparation acts on the surface of tobacco leaves to degrade macromolecular substances in tobacco leaves such as starch, cellulose, lignin, protein, pectin, etc. into various small molecules such as monosaccharides, amino acids, and organic acids. These small molecules further react or degrade into aroma components such as alcohols, aldehydes, acids, and esters, so as to achieve the purpose of increasing the content of aroma substances in tobacco leaves, improving the smoking quality of tobacco leaves, and shortening the aging time. At present, the treatment effect of enzyme preparations mainly depends on sensory quality evaluation. However, sensory quality evaluation requires organizing a large amount of manpower and material resources, and the evaluation speed is slow, and the evaluation results cannot be obtained directly and quickly. Therefore, there is an urgent need for a method to quickly and accurately evaluate the effectiveness of enzyme preparations in improving the quality of tobacco leaves. Summary of the Invention

[0003] One of the purposes of the present invention is to provide a method for evaluating the effectiveness of enzyme preparations in improving the quality of tobacco leaves, which can quickly and accurately evaluate whether the enzyme preparation has an effect on improving the quality of tobacco leaves. Another purpose of the present invention is to provide the application of enzyme preparations in improving the quality of tobacco leaves.

[0004] To achieve the above purpose, the first aspect of the present invention provides a method for evaluating the effectiveness of enzyme preparations in improving the quality of tobacco leaves, including the following steps:

[0005] Applying the enzyme preparation to tobacco leaves of a certain grade to obtain the treated tobacco leaves of the grade;

[0006] Respectively detecting the tobacco leaves of the grade, the treated tobacco leaves of the grade, and the tobacco leaves of a higher grade by Raman spectroscopy to obtain the Raman spectral curves of each tobacco leaf;

[0007] Select a curve segment with a Raman shift of 800 cm -1 -1800 cm -1 on the Raman spectral curve, and connect the two end points of the curve segment to obtain a straight line segment; select multiple Raman shifts on the curve segment, and subtract the Raman intensity corresponding to the corresponding Raman shift on the straight line segment from the Raman intensity corresponding to the multiple Raman shifts to obtain a Raman parameter set I R , and divide the Raman parameter set I R by the maximum Raman intensity value I max, obtain the Raman parameter set I of each tobacco leaf;

[0008] Calculate the first root mean square error of the Raman parameter set I between the processed tobacco leaves of a certain grade and those of the same grade, and the second root mean square error of the Raman parameter set I between the processed tobacco leaves of a certain grade and those of a higher grade;

[0009] If the first root mean square error > the second root mean square error, the enzyme preparation is effective in improving the quality of tobacco leaves, otherwise it is ineffective.

[0010] In any embodiment of the first aspect of the present invention, the first root mean square error or the second root mean square error is calculated according to the following formula:

[0011]

[0012] Wherein,

[0013] NRMES represents the first root mean square error or the second root mean square error;

[0014] N represents the total number of parameters in the Raman parameter set I, or the number of Raman shifts selected on the curve segment;

[0015] i represents the order of the parameters in the Raman parameter set I, or the order of the Raman shifts selected on the curve segment;

[0016] represents the i-th parameter in the Raman parameter set I of the processed tobacco leaves of a certain grade;

[0017] represents the i-th parameter in the Raman parameter set I of the tobacco leaves of a certain grade or a higher grade;

[0018] Preferably, N ≥ 500, and more preferably, N is selected from 500 - 1000.

[0019] In any embodiment of the first aspect of the present invention, the conditions of the Raman spectrum include one or more of the following:

[0020] A. Use a Fourier transform Raman spectrometer;

[0021] B. Detect using an InGaAs detector cooled in liquid nitrogen;

[0022] C. The laser wavelength is 1064 nm;

[0023] D. The laser power is 10 - 100 mw, preferably 50 mw;

[0024] E. The microscope magnification is ≥ 50 times, preferably 50 times;

[0025] F. The test spectrum is superimposed ≥300 times, preferably 300 times.

[0026] In any embodiment of the first aspect of the present invention, before Raman spectroscopy detection, each tobacco leaf is shredded, crushed, and sieved to obtain particles of each tobacco leaf for detection;

[0027] Preferably, the particle size of the particles of each tobacco leaf is 150 - 210 μm, preferably 165 - 198 μm.

[0028] In any embodiment of the first aspect of the present invention, the enzyme preparation is applied to the graded tobacco leaves through the following steps:

[0029] The enzyme preparation is coated or sprayed on the surface of the graded tobacco leaves and enzymatically hydrolyzed in a sealed environment;

[0030] Preferably, the mass of the enzyme preparation is 1% - 10% of the mass of the graded tobacco leaves, more preferably 3%;

[0031] Preferably, the enzymatic hydrolysis is carried out at 25°C - 45°C, more preferably at 35°C or 40°C;

[0032] Preferably, the enzymatic hydrolysis is carried out for 5 - 25 hours, more preferably for 10 hours, 12 hours or 15 hours;

[0033] Preferably, inactivation is carried out after the enzymatic hydrolysis.

[0034] In any embodiment of the first aspect of the present invention, the higher - grade tobacco leaf is a tobacco leaf that is at least one grade higher than the graded tobacco leaf, preferably a tobacco leaf that is one grade higher than the graded tobacco leaf.

[0035] In any embodiment of the first aspect of the present invention, the graded tobacco leaf is a tobacco leaf of B3F grade.

[0036] In any embodiment of the first aspect of the present invention, the higher - grade tobacco leaf is a tobacco leaf of B2F grade.

[0037] The second aspect of the present invention relates to the application of an enzyme preparation in improving the quality of tobacco leaves; wherein, the enzyme preparation is prepared from one or more of the following strains:

[0038] (1) Bacillus koreensis YLA - 2, which was deposited on December 20, 2021 at the China General Microbiological Culture Collection Center (CGMCC), deposit number CGMCC No.24132;

[0039] (2) Bacillus subtilis YLG - 2, which was deposited on December 20, 2021 at the China General Microbiological Culture Collection Center (CGMCC), deposit number CGMCC No.24133;

[0040] (3) Bacillus soloensis 5-1, which was deposited on January 5, 2022 at the China General Microbiological Culture Collection Center (CGMCC), with the deposit number CGMCC No. 24250.

[0041] In any embodiment of the first or second aspect of the present invention, the enzyme preparation is prepared by the following steps:

[0042] Fermenting the strain to obtain a fermentation product;

[0043] Separating the solid and liquid of the fermentation product and collecting the liquid phase;

[0044] Filtering the liquid phase through an ultrafiltration membrane with a cut-off molecular weight of 80-200 KD (preferably 100-150 KD), and collecting the permeate;

[0045] Concentrating the permeate through an ultrafiltration membrane with a cut-off molecular weight of 5-50 KD (preferably 10-30 KD), and collecting the concentrate;

[0046] Drying the concentrate, and then mixing the dried product with water to obtain the enzyme preparation.

[0047] In any embodiment of the second aspect of the present invention, the enzyme preparation is prepared from Bacillus subtilis YLG-2 and Bacillus soloensis 5-1 by the following steps:

[0048] Preparing the enzyme preparation from Bacillus subtilis YLG-2 and Bacillus soloensis 5-1 respectively according to the aforementioned steps;

[0049] Mixing the two enzyme preparations in equal mass.

[0050] In any embodiment of the first or second aspect of the present invention, in the steps of preparing the enzyme preparation, one or more of the following are characterized:

[0051] a. Fermenting the strain by the following steps:

[0052] Picking the strain and inoculating it into the first medium, culturing at 28°C to 39°C (such as 35°C, 37°C) for 4 to 14 h (such as 6 h, 8 h, 10 h, 12 h) to obtain a culture;

[0053] Inoculating the culture into the second medium, culturing at 28°C to 39°C (such as 35°C, 37°C) for 12 to 26 h (such as 16 h, 18 h, 24 h) to obtain a fermentation product;

[0054] Preferably, the mass of the culture is 1-5 wt% of the mass of the second medium, such as 3 wt%, 4 wt%;

[0055] Preferably, the first culture medium and the second culture medium are selected from LB culture medium and beef extract culture medium;

[0056] Preferably, the culturing is carried out in a shaker, and the rotation speed of the shaker is preferably 100 - 200 rpm, such as 180 rpm;

[0057] b. Solid - liquid separation is carried out by centrifugation, the centrifugation speed is 7000 - 9000 rpm, and the centrifugation time is 10 - 20 minutes;

[0058] c. Solid - liquid separation is carried out at 3℃ - 6℃;

[0059] d. Drying is carried out by pressure spray drying method;

[0060] e. The dried product is mixed with water at a ratio of 1:40 - 60 g / mL.

[0061] In the present invention, the tobacco leaves of B3F grade and B2F grade are the graded tobacco leaves obtained according to the tobacco leaf grading method well - known to those skilled in the art.

[0062] The beneficial effects achieved by the present invention:

[0063] 1. The method of the present invention uses Raman spectroscopy under specific operating conditions to respectively detect the graded tobacco leaves, the graded tobacco leaves after enzyme preparation treatment, and the higher - grade tobacco leaves, performs specific data processing on the Raman spectroscopy curves of various tobacco leaves to obtain the Raman parameter set I of various tobacco leaves, and respectively calculates the standard root - mean - square error of the Raman parameter set I of the graded tobacco leaves after enzyme preparation treatment and the Raman parameter set I of the graded tobacco leaves and the higher - grade tobacco leaves. By comparing the magnitudes of the two standard root - mean - square errors, the effectiveness of the enzyme preparation in improving the quality of tobacco leaves can be quickly and accurately evaluated.

[0064] 2. The specific enzyme preparation prepared by the present invention can significantly improve the quality of tobacco leaves. Description of the Drawings

[0065] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to the specific embodiments of the present invention in conjunction with the drawings, where:

[0066] Figure 1 It is a schematic diagram of an embodiment of the method for evaluating the effectiveness of the enzyme preparation in improving the quality of tobacco leaves of the present invention;

[0067] Figure 2 It is the Raman spectroscopy curve of each tobacco leaf in Embodiment 1 of the present invention;

[0068] Figure 3 It is the Raman spectroscopy curve of each tobacco leaf in Embodiment 2 of the present invention;

[0069] Figure 4This is the Raman spectrum curve of each tobacco leaf in Example 3 of the present invention. Detailed implementation manners

[0070] The following will clearly and completely describe the implementation schemes of the present invention in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. The description of at least one exemplary embodiment below is actually only illustrative and in no way limits the present invention and its application or use. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0071] Figure 1 This is a schematic diagram of an embodiment of the method for the present invention to evaluate the effectiveness of enzyme preparations in improving the quality of tobacco leaves.

[0072] A method for evaluating the effectiveness of enzyme preparations in improving the quality of tobacco leaves includes the following steps:

[0073] Step S1: Apply the enzyme preparation to the tobacco leaves of a certain grade to obtain the treated tobacco leaves of the grade.

[0074] Step S2: Detect the tobacco leaves of the grade, the treated tobacco leaves of the grade, and the tobacco leaves of a higher grade respectively by Raman spectroscopy to obtain the Raman spectrum curves of each tobacco leaf.

[0075] Step S3: Select a curve segment with a Raman shift of 800 cm -1 -1800 cm -1 on the Raman spectrum curve, and connect the two endpoints of the curve segment to obtain a straight line segment; select multiple Raman shifts on the curve segment, and subtract the Raman intensity corresponding to the corresponding Raman shift on the straight line segment from the Raman intensity corresponding to the multiple Raman shifts to obtain a Raman parameter set I R , and divide the Raman parameter set I R by the maximum Raman intensity value I max on the Raman spectrum curve segment to obtain the Raman parameter set I of each tobacco leaf.

[0076] Step S4: Calculate the first standard root mean square error of the Raman parameter sets I of the treated tobacco leaves of the grade and the tobacco leaves of the grade, and the second standard root mean square error of the Raman parameter sets I of the treated tobacco leaves of the grade and the tobacco leaves of a higher grade.

[0077] Step S5: If the first standard root mean square error > the second standard root mean square error, then the enzyme preparation is effective in improving the quality of tobacco leaves; otherwise, the enzyme preparation is ineffective in improving the quality of tobacco leaves.

[0078] In some embodiments of the present invention, the first standard root mean square error or the second standard root mean square error is calculated according to the following formula:

[0079]

[0080] Among them,

[0081] NRMES represents the first standard root mean square error or the second standard root mean square error;

[0082] N represents the total number of parameters in the Raman parameter set I, or represents the number of Raman shifts selected on the curve segment;

[0083] i represents the order of the parameters in the Raman parameter set I, or represents the order of the Raman shifts selected on the curve segment;

[0084] represents the i-th parameter in the Raman parameter set I of the processed graded tobacco leaves;

[0085] represents the i-th parameter in the Raman parameter set I of the graded tobacco leaves or higher-grade tobacco leaves;

[0086] Preferably, N≥500, and more preferably, N is selected from 500-1000.

[0087] In some embodiments of the present invention, the conditions of the Raman spectrum include one or more of the following:

[0088] A. Using a Fourier transform Raman spectrometer;

[0089] B. Detecting by using an InGaAs detector cooled in liquid nitrogen;

[0090] C. The laser wavelength is 1064nm;

[0091] D. The laser power is 10-100mw, preferably 50mw;

[0092] E. The microscope magnification is ≥50 times, preferably 50 times;

[0093] F. The test spectrum is superimposed ≥300 times, preferably 300 times.

[0094] In some embodiments of the present invention, before detecting by Raman spectrum, each tobacco leaf is shredded, crushed, and sieved to obtain particles of each tobacco leaf for detection;

[0095] Preferably, the particle size of the particles of each tobacco leaf is 150-210μm, preferably 165-198μm.

[0096] In some embodiments of the present invention, the enzyme preparation is applied to the graded tobacco leaves through the following steps:

[0097] Apply the enzyme preparation on the surface of the graded tobacco leaves by coating or spraying, and carry out enzymatic hydrolysis in a sealed environment;

[0098] Preferably, the mass of the enzyme preparation is 1%-10% of the mass of the graded tobacco leaves, more preferably 3%.

[0099] Preferably, the enzymatic hydrolysis is carried out at 25°C - 45°C, more preferably at 35°C or 40°C.

[0100] Preferably, the enzymatic hydrolysis is carried out for 5 - 25 hours, more preferably for 10 hours, 12 hours or 15 hours.

[0101] Preferably, inactivation is carried out after the enzymatic hydrolysis.

[0102] In some embodiments of the present invention, the higher-grade tobacco leaves are tobacco leaves that are at least one grade higher than the graded tobacco leaves, preferably tobacco leaves that are one grade higher than the graded tobacco leaves.

[0103] In some embodiments of the present invention, the graded tobacco leaves are tobacco leaves of the B3F grade.

[0104] In some embodiments of the present invention, the higher-grade tobacco leaves are tobacco leaves of the B2F grade.

[0105] The materials used in the following examples include:

[0106] LB medium comprises 10 g / L of tryptone, 5 g / L of yeast extract, 5 g / L of sodium chloride, and the pH value is 7.5.

[0107] Beef extract medium comprises 3 g / L of beef extract, 10 g / L of peptone, 5 g / L of sodium chloride, and the pH value is 7.0.

[0108] Bacillus koreensis YLA-2, screened from Yunnan Malong C3F-2014 tobacco leaves, the 16sRNA identification result is Bacillus koreensis, and it was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms (CGMCC) on December 20, 2021, with the deposit number CGMCC No. 24132.

[0109] Bacillus amyloliquefaciens 18-1, screened from Fujian Longyan Sanming YLC2YCB-1-2019 cut tobacco, the 16sRNA identification result is Bacillus amyloliquefaciens, and it was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms (CGMCC) on January 5, 2022, with the deposit number CGMCC No. 24249.

[0110] Bacillus subtilis YLG-2 was screened from the B2F-2014 tobacco leaves in Zhumadian, Henan. The 16sRNA identification result was Bacillus subtilis. It was preserved in the China General Microbiological Culture Collection Center (CGMCC) on December 20, 2021, with the preservation number CGMCC No. 24133;

[0111] Solibacillus silvestris 5-1 was screened from the C3F-2019 cut tobacco leaves in Ningyuan, Yongzhou, Hunan. The 16sRNA identification result was Solibacillus silvestris. It was preserved in the China General Microbiological Culture Collection Center (CGMCC) on January 5, 2022, with the preservation number CGMCC No. 24250.

[0112] Example 1

[0113] 1. Preparation of enzyme preparation:

[0114] (1) Pick the strain of Bacillus koreensis YLA-2 and inoculate it into 50 mL of LB medium. Culture it at 37 °C and a shaker speed of 180 rpm for 8 h; inoculate the obtained product into the beef extract medium at a ratio of 4 wt%, and culture it at 37 °C and a shaker speed of 180 rpm for 16 h to obtain the FA fermentation broth;

[0115] (2) Centrifuge the FA fermentation broth at 8000 rpm at 4 °C for 15 min to obtain the supernatant;

[0116] (3) Filter the supernatant through a 100 KD ultrafiltration membrane, collect the permeate, and then concentrate it through a 10 KD ultrafiltration membrane to collect the retentate;

[0117] (4) Dry the retentate by pressure spray drying to obtain the FA enzyme dry powder;

[0118] (5) Take 2 g of the FA enzyme dry powder and mix it evenly with 100 mL of pure water to obtain the FA enzyme preparation.

[0119] 2. Tobacco leaves treated with FA enzyme preparation:

[0120] Take 200 g of B3F tobacco leaves produced in Youxi, Fujian in 2019, spread them out flat in a stainless-steel square tray, and use an enzyme addition device to evenly apply the FA enzyme preparation to the surface of the tobacco leaves. The application mass of the FA enzyme preparation is 3% of the mass of the tobacco leaves. After mixing the tobacco leaves, put them into a self-sealing bag and seal it, and place it at 40 °C for reaction for 12 h. After the reaction, the tobacco leaves are loaded onto a plate and inactivated in an oven at 130 °C for 1 min, and then balanced in a constant temperature and humidity box at 22 °C and a relative humidity of 60% for 48 h for standby.

[0121] 3. Sample pretreatment:

[0122] The B3F tobacco leaves and B2F tobacco leaves produced in Youxi, Fujian in 2019 and the tobacco leaves treated with the above FA enzyme preparation were respectively cut into cut tobacco, ground into powder, and particles with a particle size of 165 - 198 μm were selected by sieving as test samples, and placed in an environment with a temperature of 22°C and a relative humidity of 60% for 48 hours of equilibration, and sealed for later use.

[0123] 4. Sample detection:

[0124] The above samples were respectively subjected to Raman spectroscopy detection using a Raman spectrometer. The Nicolet iS50R Raman spectrometer was selected, and an InGaAs detector cooled in liquid nitrogen was used to detect and collect Raman signals. The laser wavelength of the spectrometer was 1064 nm, the laser power was 50 mw, the microscope magnification was 50 times, and the test spectrum was superimposed 300 times to obtain the Raman spectral curves of each sample as Figure 2 shown. FA enzyme represents the tobacco leaves treated with the FA enzyme preparation, control 1 represents B3F tobacco leaves, and control 2 represents B2F tobacco leaves.

[0125] 5. Data processing and calculation:

[0126] Data processing was performed on the Raman spectral curves of each sample. The Raman spectral curve segment with a Raman shift of 800 cm -1 -1800 cm -1 was selected. The two endpoints of the Raman spectral curve segment were connected to obtain a straight line segment. N Raman shifts were selected on the Raman spectral curve segment. The Raman intensity corresponding to the N Raman shifts was subtracted from the Raman intensity corresponding to the corresponding Raman shift on the straight line segment to obtain a set of Raman peak numbers I R , and then the maximum peak I -1 -1800 cm -1 on the Raman spectral curve segment with a Raman shift of 800 cm max was taken. Each Raman peak in the set of Raman peak numbers I R was divided by I max to obtain the set of Raman parameter numbers I of the sample:

[0127] I i = I Ri / I max

[0128] where I i is the i-th Raman parameter in the set of Raman parameter numbers I of the sample, and I Ri is the corresponding i-th Raman peak in the set of Raman peak numbers I R .

[0129] Calculate the normalized root mean square error (NRMSE) of the Raman parameter sets of the tobacco leaves treated with the FA enzyme preparation and the B3F tobacco leaves, as well as the normalized root mean square error (NRMSE) of the Raman parameter sets of the tobacco leaves treated with the FA enzyme preparation and the B2F tobacco leaves according to the following formula.

[0130]

[0131] Among them, N represents the total number of parameters in the Raman parameter set I, and also represents the number of Raman shifts selected in the Raman shift range of 800 cm -1 -1800 cm -1 The order of the Raman shift selected in the Raman shift range of 800 cm -1 -1800 cm -1 Interval; i represents the parameter order in the Raman parameter set I of the tobacco leaves treated with the FA enzyme preparation, and also represents the Raman shift order selected in the range of 800 cm Represents the i-th Raman parameter in the Raman parameter set I of the tobacco leaves treated with the FA enzyme preparation; Represents the i-th Raman parameter in the Raman parameter set I of the B3F tobacco leaves or B2F tobacco leaves. N is taken as 1000.

[0132] The NRMSE of the Raman parameter sets of the tobacco leaves treated with the FA enzyme preparation and the B3F tobacco leaves is 0.4232%, and the NRMSE of the Raman parameter sets of the tobacco leaves treated with the FA enzyme preparation and the B2F tobacco leaves is 0.2418%. The tobacco leaves treated with the FA enzyme preparation are closer to the B2F tobacco leaves, indicating that the treatment with the FA enzyme preparation has an effect on improving the quality of tobacco leaves.

[0133] Example 2

[0134] 1. Preparation of enzyme preparation:

[0135] (1) Pick the Bacillus amyloliquefaciens 18-1 strain and inoculate it into 50 mL of LB medium, and culture it at 35 °C and a shaking speed of 180 rpm for 10 h; inoculate the obtained fermentation product into the beef extract medium at a ratio of 4 wt%, and culture it at 35 °C and a shaking speed of 180 rpm for 24 h to obtain the FB fermentation broth;

[0136] (2) Centrifuge the FB fermentation broth at 4 °C at a speed of 8000 rpm for 15 min to obtain the supernatant;

[0137] (3) Filter the supernatant through a 150 KD ultrafiltration membrane, collect the permeate, and then concentrate it through a 30 KD ultrafiltration membrane to collect the concentrate;

[0138] (4) Dry the concentrate by pressure spray drying to obtain the FB enzyme dry powder;

[0139] (5) Take 2 g of the FB enzyme dry powder and mix it evenly with 100 mL of pure water to obtain the FB enzyme preparation.

[0140] 2. Tobacco leaves treated with FB enzyme preparation:

[0141] Take 200 g of B3F tobacco leaves produced in Youxi, Fujian in 2019, spread them out flat in a stainless-steel square tray, and use an enzyme addition device to evenly apply the FB enzyme preparation to the surface of the tobacco leaves. The application mass of the FB enzyme preparation is 3% of the mass of the tobacco leaves. After mixing the tobacco leaves evenly, put them into a self-sealing bag and seal it, and place it at 40 °C for reaction for 12 h. After the reaction is completed, the tobacco leaves are loaded into a tray and inactivated in an oven at 130 °C for 1 min, and then balanced in a constant temperature and humidity box at 22 °C and a relative humidity of 60% for 48 h for standby.

[0142] 3. Sample pretreatment:

[0143] Cut the B3F tobacco leaves and B2F tobacco leaves produced in Youxi, Fujian in 2019 and the tobacco leaves treated with the above FB enzyme preparation into cut tobacco, grind them into powder, and sieve to select particles with a particle size of 165 - 198 μm as test samples, and place them in an environment at 22 °C and a relative humidity of 60% to balance for 48 hours, and seal for standby.

[0144] 4. Sample detection:

[0145] Use a Raman spectrometer to perform Raman spectroscopy detection on each of the above samples respectively. The operating conditions of the Raman spectrometer are the same as item 4 in Example 1, and the Raman spectral curves of each sample are obtained as Figure 3 shown. FB enzyme represents the tobacco leaves treated with the FB enzyme preparation, control 1 represents B3F tobacco leaves, and control 2 represents B2F tobacco leaves.

[0146] 5. Data processing and calculation:

[0147] Refer to item 5 in Example 1 for data processing and calculation. N is taken as 1000.

[0148] The NRMSE of the Raman parameter dataset of the tobacco leaves treated with the FB enzyme preparation and B3F tobacco leaves is 0.1187%, and the NRMSE of the Raman parameter dataset of the tobacco leaves treated with the FB enzyme preparation and B2F tobacco leaves is 0.8930%. The tobacco leaves treated with the FB enzyme preparation are closer to B3F tobacco leaves, indicating that the treatment with the FB enzyme preparation has basically no effect on improving the quality of tobacco leaves.

[0149] Example 3

[0150] 1. Preparation of enzyme preparation.

[0151] (1) Pick up the Bacillus subtilis YLG-2 strain and inoculate it into 50 mL of LB medium. Culture it at 37 °C with a shaking speed of 180 rpm for 6 h; inoculate the obtained fermentation product into the beef extract medium at a ratio of 3 wt%, and culture it at 37 °C with a shaking speed of 180 rpm for 18 h to obtain the FC fermentation broth;

[0152] Pick up the Bacillus edaphicus 5-1 strain and inoculate it into 50 mL of LB medium. Culture it at 37 °C with a shaking speed of 180 rpm for 12 h; inoculate the obtained fermentation product into the beef extract medium at a ratio of 4 wt%, and culture it at 37 °C with a shaking speed of 180 rpm for 24 h to obtain the FD fermentation broth;

[0153] (2) Centrifuge the FC fermentation broth and the FD fermentation broth at 4 °C at a speed of 8000 rpm for 15 min to obtain the FC supernatant and the FD supernatant;

[0154] (3) Filter the FC supernatant and the FD supernatant through a 150 KD ultrafiltration membrane respectively, collect the permeate, and then concentrate it through a 10 KD ultrafiltration membrane to collect the FC concentrate and the FD concentrate;

[0155] (4) Dry the FC concentrate and the FD concentrate by the pressure spray drying method respectively to obtain the FC enzyme dry powder and the FD enzyme dry powder;

[0156] (5) Take 1 g of the FC enzyme dry powder and 1 g of the FD enzyme preparation dry powder and mix them evenly with 100 mL of pure water to obtain the FCD enzyme preparation.

[0157] 2. Tobacco leaves treated with the FCD enzyme preparation:

[0158] Take 200 g of B3F tobacco leaves produced in Youxi, Fujian in 2019, spread them out flat in a stainless-steel square plate, and use an enzyme addition device to evenly apply the FCD enzyme preparation to the surface of the tobacco leaves. The application mass of the FCD enzyme preparation is 3% of the mass of the tobacco leaves. Mix the tobacco leaves evenly and put them into a self-sealing bag and seal it, and place it at 35 °C for reaction for 12 h. The reacted tobacco leaves are loaded into a plate and inactivated in an oven at 130 °C for 1 min, and then balanced in a constant temperature and humidity box at 22 °C and a relative humidity of 60% for 48 h for standby.

[0159] 3. Sample pretreatment:

[0160] Cut the B3F tobacco leaves and B2F tobacco leaves produced in Youxi, Fujian in 2019 and the tobacco leaves treated with the above FCD enzyme preparation into shredded tobacco, grind them into powder, and sieve to select particles with a particle size of 165 - 198 μm as the test samples. Place them in an environment at 22 °C and a relative humidity of 60% for 48 hours to balance, and seal for use.

[0161] 4. Sample detection:

[0162] The Raman spectra of the above samples were detected separately using a Raman spectrometer. The operating conditions of the Raman spectrometer were the same as item 4 in Example 1, and the Raman spectral curves of each sample were obtained as Figure 4 shown. FCD enzyme refers to the tobacco leaves treated with the FCD enzyme preparation, Control 1 refers to B3F tobacco leaves, and Control 2 refers to B2F tobacco leaves.

[0163] 5. Data processing and calculation:

[0164] Data processing and calculation were carried out with reference to item 5 in Example 1. N was taken as 1000.

[0165] The NRMSE of the Raman parameter dataset of the tobacco leaves treated with the FCD enzyme preparation and B3F tobacco leaves was 0.4696%, and the NRMSE of the Raman parameter dataset of the tobacco leaves treated with the FCD enzyme preparation and B2F tobacco leaves was 0.1250%. The tobacco leaves treated with the FCD enzyme preparation were closer to B2F tobacco leaves, indicating that the treatment with the FCD enzyme preparation had an effect on improving the quality of tobacco leaves.

[0166] Taking the three examples together, the NRMSE of the Raman parameter dataset of the tobacco leaves treated with the FCD enzyme preparation and B3F tobacco leaves was the largest. At the same time, the NRMSE of the Raman parameter dataset of the tobacco leaves treated with the FCD enzyme preparation and B2F tobacco leaves was the smallest. It can be seen that the order of the effect of enzyme preparations on improving the quality of tobacco leaves from high to low is: FCD enzyme preparation > FA enzyme preparation > FB enzyme preparation.

[0167] Test Example 1 Sensory Evaluation

[0168] The tobacco leaves treated with the FA enzyme preparation, the tobacco leaves treated with the FB enzyme preparation, the tobacco leaves treated with the FCD enzyme preparation, and B3F tobacco leaves were cut into cut tobacco, and cigarettes were made according to the same method using the same cigarette sleeves and left to stand for 24 hours for standby. Nine professional personnel scored the fragrance style, sweet taste, aroma quality, aroma quantity, off-flavor, fineness, concentration, strength, irritation, and aftertaste indexes of the cigarettes according to the standard method of YC / T 415-2011, and the average value was taken. A difference of 0.5 was considered a difference of one grade. Then, the style characteristic evaluation score, aroma characteristic score, smoke characteristic score, taste characteristic score, quality characteristic evaluation score, and sensory quality total score were calculated according to the following formula recognized by those skilled in the art; the results are shown in Table 1.

[0169] Style characteristic evaluation score = fragrance style × 0.60 + sweet taste × 0.40

[0170] Aroma characteristic score = aroma quality × 0.40 + aroma quantity × 0.30 + off-flavor × 0.30

[0171] Smoke characteristic score = fineness × 0.40 + concentration × 0.30 + strength × 0.30

[0172] Taste characteristic score = irritation × 0.45 + aftertaste × 0.55

[0173] Quality characteristic evaluation score = aroma characteristic × 0.55 + smoke characteristic × 0.20 + taste characteristic × 0.25

[0174] Comprehensive sensory quality score = style characteristic evaluation × 0.40 + quality characteristic evaluation × 0.60

[0175] Table 1 Results of sensory quality evaluation

[0176]

[0177]

[0178] As can be seen from Table 1, compared with B3F tobacco leaves, the comprehensive sensory quality scores of the tobacco leaves treated with FA enzyme preparation and the tobacco leaves treated with FCD enzyme preparation are significantly improved. The main manifestations are that the scores of indexes such as flavor style, sweet taste, aroma quality, aroma quantity, off-flavor, fineness, irritation, and aftertaste are significantly improved. Moreover, the sensory quality score of the tobacco leaves treated with FCD enzyme preparation is higher; the sensory quality score of the tobacco leaves treated with FB enzyme preparation is equivalent to that of B3F tobacco leaves; the above shows that the order of the effects of enzyme preparations on improving the sensory quality of tobacco leaves from high to low is: FCD enzyme preparation > FA enzyme preparation > FB enzyme preparation, which verifies the accuracy of the method for evaluating the effect of enzyme preparation on treating tobacco leaves in the present invention.

[0179] Test Example 2 Routine Chemical Composition Analysis

[0180] According to the standard method of YC / T 159-2002, the total sugar and reducing sugar contents of the tobacco leaves treated with FA enzyme preparation, the tobacco leaves treated with FB enzyme preparation, the tobacco leaves treated with FCD enzyme preparation, and B3F tobacco leaves were analyzed by continuous flow analysis method, and the results are shown in Table 2.

[0181] Table 2 Results of total sugar and reducing sugar analysis

[0182] Sample Total sugar Reducing sugar B3F tobacco leaves 24.39 22.98 Tobacco leaves treated with FA enzyme preparation 24.84 23.47 Tobacco leaves treated with FB enzyme preparation 24.41 22.53 Tobacco leaves treated with FCD enzyme preparation 25.03 23.67

[0183] As can be seen from Table 2, the total sugar and reducing sugar of the tobacco leaves treated with FA and FCD enzymes have been improved to a certain extent, and the reducing sugar of FB has decreased slightly.

[0184] In summary, the results of evaluating the effect of enzyme preparation on treating tobacco leaves by the method of the present invention are consistent with the results of manual sensory evaluation, and the conventional chemical components also show that the total sugar and reducing sugar of the tobacco leaves treated with FA and FCD enzymes have increased. Therefore, the method of the present invention can quickly and accurately evaluate the treatment effect of enzyme preparation, can significantly improve the work efficiency of enzyme preparation screening, and objectively and fairly evaluate the treatment effect of enzyme preparation.

[0185] Obviously, the above embodiments are merely examples given for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to exhaustively list all the implementation manners here. And the obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.

Claims

1. A method for evaluating the effectiveness of an enzyme preparation in improving the quality of tobacco leaves, comprising the following steps: Applying the enzyme preparation to tobacco leaves of a certain grade to obtain the treated tobacco leaves of that grade; Respectively detecting the tobacco leaves of that grade, the treated tobacco leaves of that grade, and the tobacco leaves of a higher grade by Raman spectroscopy to obtain the Raman spectral curves of each tobacco leaf; Select a curve segment with a Raman shift of 800 cm -1 -1800 cm -1 on the Raman spectrum curve, and connect the two endpoints of the curve segment to obtain a straight line segment; select multiple Raman shifts on the curve segment, and subtract the Raman intensity corresponding to the corresponding Raman shift on the straight line segment from the Raman intensities corresponding to the multiple Raman shifts to obtain the Raman parameter set I R , and use the Raman parameter set I R to divide by the maximum Raman intensity value I max on the Raman spectrum curve segment to obtain the Raman parameter set I of each tobacco leaf; Calculating the first root mean square error of the Raman parameter set I between the treated tobacco leaves of that grade and the tobacco leaves of that grade, and the second root mean square error of the Raman parameter set I between the treated tobacco leaves of that grade and the tobacco leaves of a higher grade; If the first root mean square error > the second root mean square error, the enzyme preparation is effective in improving the quality of tobacco leaves, otherwise the enzyme preparation is ineffective in improving the quality of tobacco leaves.

2. The method according to claim 1, wherein The first root mean square error or the second root mean square error is calculated according to the following formula: Wherein, NRMES represents the first root mean square error or the second root mean square error; N represents the total number of parameters in the Raman parameter set I; i represents the parameter order in the Raman parameter set I; represents the i-th parameter in the Raman parameter set I of the processed flue-cured tobacco leaves of a certain grade; The i-th parameter in the Raman parameter set I representing tobacco leaves of a certain grade or higher grade.

3. The method according to claim 2, wherein N≥500。 4. The method according to claim 1, wherein The conditions of the Raman spectroscopy include one or more of the following: A. Using a Fourier transform Raman spectrometer; B. Detecting with an InGaAs detector cooled in liquid nitrogen; C. The laser wavelength is 1064nm; D. The laser power is 10 - 100mw; E. The microscope magnification is ≥50 times; F. The test spectrum is superimposed ≥300 times.

5. The method according to claim 4, wherein, The laser power is 50mw.

6. The method according to claim 4, wherein The microscope magnification is 50 times.

7. The method according to claim 4, wherein The test spectrum is superimposed 300 times.

8. The method according to claim 1, wherein Before detecting with Raman spectroscopy, each tobacco leaf is shredded, crushed, and sieved to obtain the particles of each tobacco leaf for detection.

9. The method according to claim 8, wherein The particle size of the particles of each tobacco leaf is 150 - 210μm.

10. The method according to claim 1, wherein, The enzyme preparation is applied to the tobacco leaves of a certain grade through the following steps: Coating or spraying the enzyme preparation on the surface of the tobacco leaves of a certain grade and enzymolyzing in a sealed environment.

11. The method according to claim 10, wherein, The mass of the enzyme preparation is 1% - 10% of the mass of the tobacco leaves of a certain grade.

12. The method according to claim 10, wherein, Enzymolyzing at 25°C - 45°C.

13. The method according to claim 10, wherein, Enzymolyzing for 5 - 25 hours.

14. The method according to claim 10, wherein, Inactivating after enzymolyzing.

15. The method according to claim 1, wherein The tobacco leaves of a higher grade are tobacco leaves at least one grade higher than the tobacco leaves of that grade.

16. The method according to claim 1, wherein The tobacco leaves of that grade are tobacco leaves of the B3F grade.

17. The method according to claim 1, wherein, The tobacco leaves of a higher grade are tobacco leaves of the B2F grade.

18. The method according to any one of claims 1 to 17, wherein The enzyme preparation is prepared through the following steps: Fermenting the strain to obtain a fermentation product; Separating the solid and liquid of the fermentation product and collecting the liquid phase; Filtering the liquid phase with an ultrafiltration membrane with a cut-off molecular weight of 80 - 200KD and collecting the permeate; Concentrating the permeate with an ultrafiltration membrane with a cut-off molecular weight of 5 - 50KD and collecting the concentrate; Drying the concentrate, and then mixing the dried product with water to obtain the enzyme preparation.

19. The method according to claim 18, wherein, In the steps of preparing the enzyme preparation, one or more of the following are characterized: a. Fermenting the strain through the following steps: Picking the strain and inoculating it into the first medium, culturing at 28°C - 39°C for 4 - 14h to obtain a culture; Inoculating the culture into the second medium, culturing at 28°C - 39°C for 12 - 26h to obtain a fermentation product; b. Separating the solid and liquid by centrifugation, with a centrifugation speed of 7000 - 9000rpm and a centrifugation time of 10 - 20 minutes; c. Separating the solid and liquid at 3°C - 6°C; d. Drying is carried out by the pressure spray drying method; e. The dried product is mixed with water at a ratio of 1:40 - 60 g / mL.

20. The method according to claim 19, wherein, In item a, the mass of the culture is 1 - 5 wt% of the mass of the second culture medium.

21. The method according to claim 19, wherein, In item a, the first culture medium and the second culture medium are selected from LB culture medium and beef extract culture medium.

22. The method according to claim 19, wherein In item a, the cultivation is carried out in a shaker, and the rotation speed of the shaker is 100 - 200 rpm.

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