Reconstituted tobacco preparation method based on compound enzyme technology

Abstract

The invention discloses a reconstituted tobacco preparation method based on a compound enzyme technology, which comprises the following steps: carrying out surface impurity removal pretreatment on a reconstituted tobacco sheet base to obtain a standby sheet base; dissolving cellulase and pectinase in water according to a certain mass ratio to prepare an enzyme solution; the preparation method comprises the following steps: spraying a citric acid-disodium hydrogen phosphate buffer solution to the surface of a standby film base, then spraying an enzyme solution to the surface of the standby film base, carrying out an enzymatic hydrolysis reaction for 3-5 h under the conditions that the temperature is 40-50 DEG C and the humidity is 50-70%, and then drying the enzymatic hydrolysis film base for 30-50 min at the temperature of 70-80 DEG C. According to the method, a collaborative regulation system of cellulase and pectinase is constructed, the enzymolysis process is optimized, oriented improvement of the sour and sweet taste of the reconstituted tobacco is achieved, the aroma richness is remarkably enhanced, the overall acceptability is greatly improved, the technical problems that in a traditional process, the flavor is single, and enzymolysis regulation is extensive are solved, and the quality of the reconstituted tobacco is improved. And a green and efficient standardized solution is provided for quality upgrading of the reconstituted tobacco.

Description

Technical Field

[0001] The present invention relates to the field of reconstituted tobacco processing, and more particularly to a method for preparing reconstituted tobacco based on composite enzyme technology. Background Art

[0002] Reconstituted tobacco, as a core technology for the circular economy of the tobacco industry, can reduce tar content while improving resource utilization by reusing tobacco waste. However, the traditional reconstituted tobacco production process has the following defects: (1) the product flavor is single, and the natural ingredients are not fully utilized, making it difficult to meet consumers' demand for high-quality tobacco products; (2) there are obvious deficiencies in the application of biotechnology, such as the limited effect of fermentation with a single strain on improving the sweet and sour taste, the extensive enzyme treatment conditions leading to excessive or insufficient degradation of ingredients, and the lack of systematic analysis of the enzymatic hydrolysis pathway, which seriously restricts the improvement of the quality of reconstituted tobacco.

[0003] In the field of enzyme processing technology, although single-enzyme treatment with cellulase and pectinase has been shown to disrupt tobacco cell walls to a certain extent, releasing soluble sugars and phenolic substances, related research is still in its infancy. Specifically, single enzymatic hydrolysis has limited effect on improving the sweet and sour taste, aroma richness, and overall acceptability of reconstituted tobacco leaves. Under single-cellulase treatment, only 20-30% of the embedded sour substances are released, and the sweetness is not improved. Under single-pectinase treatment, the release rate of sweet substances is ≤40%, and the excessive sourness disrupts the balance.

[0004] Therefore, developing a technology that utilizes composite enzymatic hydrolysis technology to provide targeted improvement in the quality of reconstituted tobacco leaves has become an urgent problem to be solved in the tobacco processing field. Summary of the Invention

[0005] One object of the present invention is to provide a new technical solution for a method for preparing reconstituted tobacco leaves by utilizing composite enzymatic hydrolysis technology to provide a targeted improvement in the quality of reconstituted tobacco leaves.

[0006] According to a first aspect of the present invention, a method for preparing reconstituted tobacco leaves based on compound enzyme technology is provided.

[0007] The method for preparing reconstituted tobacco leaves based on compound enzyme technology comprises the following steps:

[0008] Reconstituted tobacco leaf pretreatment: pre-treating the surface of the reconstituted tobacco leaf base to remove impurities and obtain a spare leaf base;

[0009] Preparation of enzyme solution: dissolve cellulase and pectinase in water at a certain mass ratio to prepare enzyme solution;

[0010] Enzymatic hydrolysis treatment: first spray citric acid-disodium hydrogen phosphate buffer with a pH value of 4.8 onto the surface of the spare film base, then spray the enzyme solution on the surface of the spare film base, and carry out enzymatic hydrolysis reaction for 3-5 hours at a temperature of 40-50°C and a humidity of 50-70% to obtain an enzymatic hydrolysis film base, and then dry the enzymatic hydrolysis film base at 70-80°C for 30-50 minutes to obtain reconstituted tobacco leaves.

[0011] Optionally, the reconstituted tobacco leaf pretreatment further includes the step of cutting the reconstituted tobacco leaf base into 5 cm×5 cm squares.

[0012] Optionally, the surface impurity removal pretreatment in the reconstituted tobacco leaf pretreatment is achieved by air separation.

[0013] Optionally, the spare substrate is equilibrated for 48 hours in an environment with a temperature of 22±1° C. and a relative humidity of 60±2% before being subjected to the enzymatic hydrolysis treatment.

[0014] Optionally, the enzyme solution is prepared as follows:

[0015] 200,000 U / g of cellulase and 50,000 U / g of pectinase were dissolved in water at a mass ratio of 1:2-3:1 to prepare an enzyme solution.

[0016] Optionally, the preparation method of the citric acid-disodium hydrogen phosphate buffer in the enzymatic hydrolysis treatment is as follows:

[0017] According to a volume ratio of 100:107, 0.1 mol / L citric acid solution was added to 0.1 mol / L disodium hydrogen phosphate solution to obtain a reserve buffer solution, which was then filtered through a 0.45 μm microporous filter membrane to obtain a citric acid-disodium hydrogen phosphate buffer solution with a pH value of 4.8.

[0018] Optionally, the enzyme solution in the enzymatic hydrolysis treatment is sprayed on the surface of the spare film base until the content of cellulase relative to the spare film base is 60-360 U / g, and the content of pectinase relative to the spare film base is 30 U / g.

[0019] The method for preparing reconstituted tobacco leaves based on composite enzyme technology of the present application constructs a synergistic regulation system of cellulase and pectinase, optimizes the enzymatic hydrolysis process, and achieves a targeted improvement in the sourness and sweetness of the reconstituted tobacco leaves, a significant enhancement in the richness of the aroma, and a substantial improvement in the overall acceptance. It solves the technical problems of single flavor and extensive enzymatic hydrolysis regulation in traditional processes, and provides a green, efficient, and standardized solution for the quality upgrade of reconstituted tobacco leaves.

[0020] Further features and advantages of the present invention will become apparent from the following detailed description of exemplary embodiments of the present invention. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present invention will now be described in detail. It should be noted that the relative arrangement of components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention unless otherwise specifically stated.

[0022] The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses.

[0023] Technologies, methods, and equipment known to ordinary technicians in the relevant art may not be discussed in detail, but where appropriate, the technologies, methods, and equipment should be considered part of the specification.

[0024] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not limiting. Therefore, other examples of the exemplary embodiments may have different values.

[0025] The present application provides a method for preparing reconstituted tobacco leaves based on compound enzyme technology, comprising the following steps:

[0026] Pretreatment of reconstituted tobacco leaves: The reconstituted tobacco leaf base is pretreated to remove impurities from the surface to obtain a spare leaf base.

[0027] The reconstituted tobacco leaf pretreatment also includes the step of cutting the reconstituted tobacco leaf base into 5 cm×5 cm squares to ensure that the contact area between the enzyme solution and the leaf base medium is consistent and the reaction is sufficient and uniform.

[0028] Surface impurity removal during reconstituted tobacco leaf pretreatment can be achieved through air separation. This air separation removes impurities from the surface of the reconstituted tobacco leaf base, which not only allows for targeted screening of small particles of impurities, but also prevents breakage of brittle reconstituted tobacco leaves and fiber breakage that could affect their structural integrity.

[0029] Before enzymatic hydrolysis, the spare film substrate was balanced in an environment with a temperature of 22±1°C and a relative humidity of 60±2% for 48 hours to ensure uniform moisture distribution of the spare film substrate and provide stable substrate conditions for subsequent enzymatic hydrolysis reactions.

[0030] Preparation of enzyme solution: dissolve cellulase and pectinase in water at a certain mass ratio to prepare enzyme solution.

[0031] The enzyme solution preparation can be specifically as follows:

[0032] 200,000 U / g of cellulase and 50,000 U / g of pectinase were dissolved in water at a mass ratio of 1:2-3:1 to prepare an enzyme solution.

[0033] Enzymatic hydrolysis treatment: first spray citric acid-disodium hydrogen phosphate buffer with a pH value of 4.8 onto the surface of the spare film base, then spray the enzyme solution on the surface of the spare film base, and carry out enzymatic hydrolysis reaction for 3-5 hours at a temperature of 40-50°C and a humidity of 50-70% to obtain an enzymatic hydrolysis film base, and then dry the enzymatic hydrolysis film base at 70-80°C for 30-50 minutes to obtain reconstituted tobacco leaves.

[0034] The temperature and humidity conditions of the above enzymatic hydrolysis reaction can simultaneously maintain high activity of cellulase (optimum pH is 4.5-5.5) and pectinase (optimum pH is 3.5-4.5), with an enzyme activity retention rate of >80%.

[0035] The drying process described above terminates the enzymatic hydrolysis reaction, thereby obtaining the final reconstituted tobacco leaf product. By inactivating the enzyme protein at high temperature, the enzymatic hydrolysis reaction is terminated, ensuring a stable flavor content.

[0036] The preparation method of the citric acid-disodium hydrogen phosphate buffer in the enzymatic hydrolysis treatment can be as follows:

[0037] Add 0.1 mol / L citric acid solution to 0.1 mol / L sodium hydrogen phosphate solution at a volume ratio of 100:107 to prepare a buffer solution. Filter the buffer solution through a 0.45 μm microporous filter to obtain a citric acid-sodium hydrogen phosphate buffer solution with a pH of 4.8. This filtration procedure can extend the shelf life of the buffer solution.

[0038] The enzyme solution in the enzymatic hydrolysis treatment is sprayed on the surface of the spare sheet base until the content of cellulase relative to the spare sheet base is 60-360 U / g, and the content of pectinase relative to the spare sheet base is 30 U / g.

[0039] This application utilizes the synergistic action of two enzymes: ① cellulase dissociates microfibril bundles, exposing the pectin coating; ② pectinase degrades galacturonic acid chains, releasing embedded organic acids (malic acid, citric acid) and reducing sugars (glucose, fructose). This process increases the sourness intensity of reconstituted tobacco leaves to -8--12 (a 40%-60% increase compared to the blank), increases the sweetness intensity to 0.25-0.35 (a 300-400% increase), improves sensory acceptance (≥7.5), and increases the water-soluble sugar content of the enzymatic hydrolysis product by ≥0.2mg / g and the total organic acid content by ≥2.20mg / g.

[0040] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. The materials and reagents used are all commercially available unless otherwise specified. The equipment used in the experiments are all well known to those skilled in the art unless otherwise specified.

[0041] Example 1

[0042] The impurities on the surface of the reconstituted tobacco leaf base 1 were removed by air separation; the reconstituted tobacco leaves were cut into 5 cm × 5 cm squares using a shear knife; and the reconstituted tobacco leaves were then laid flat in a constant temperature and humidity environment (22 ± 1 ° C, relative humidity 60 ± 2%) for 48 hours.

[0043] Measure 500 mL of 0.1 mol / L citric acid solution and slowly add 535 mL of 0.1 mol / L disodium hydrogen phosphate solution while stirring to prepare a standby buffer solution with a pH value of 4.8. Then filter the standby buffer solution through a 0.45 μm microporous filter membrane to obtain a citric acid-disodium hydrogen phosphate buffer solution.

[0044] Cellulase (200,000 U / g) and pectinase (50,000 U / g) were dissolved in water at a mass ratio of 2:1 to prepare an enzyme solution.

[0045] First, spray citric acid-disodium hydrogen phosphate buffer onto the surface of the spare film base, and then spray the enzyme solution onto the surface of the spare film base until the cellulase concentration is 240U / g of the spare film base and the pectinase concentration is 30U / g of the spare film base. At a temperature of 40°C and a humidity of 60%, perform enzymatic hydrolysis for 4 hours to obtain an enzymatic hydrolysis film base.

[0046] The enzymatic hydrolyzed sheet was dried at 80° C. for 40 min to obtain a finished reconstituted tobacco leaf.

[0047] The finished reconstituted tobacco leaf obtained in Example 1 was tested, and the water-soluble sugar content was 0.48 mg / g, and the total organic acid content was 8.76 mg / g.

[0048] Comparative Example 1

[0049] The impurities on the surface of the reconstituted tobacco leaf base 1 were removed by air separation; the reconstituted tobacco leaves were cut into 5 cm × 5 cm squares using a shear knife; and the reconstituted tobacco leaves were then laid flat in a constant temperature and humidity environment (22 ± 1 ° C, relative humidity 60 ± 2%) for 48 hours.

[0050] Measure 500 mL of 0.1 mol / L citric acid solution and slowly add 535 mL of 0.1 mol / L disodium hydrogen phosphate solution while stirring to prepare a standby buffer solution with a pH value of 4.8. Then filter the standby buffer solution through a 0.45 μm microporous filter membrane to obtain a citric acid-disodium hydrogen phosphate buffer solution.

[0051] Dissolve cellulase (200,000 U / g) in water to prepare enzyme solution.

[0052] First, spray citric acid-disodium hydrogen phosphate buffer onto the surface of the spare film base, and then spray the enzyme solution onto the surface of the spare film base until the cellulase concentration is 240U / g spare film base. Under the conditions of temperature of 40°C and humidity of 60%, perform enzymatic hydrolysis for 4 hours to obtain an enzymatic hydrolysis film base.

[0053] The enzymatic hydrolyzed sheet was dried at 80° C. for 40 min to obtain a finished reconstituted tobacco leaf.

[0054] The finished reconstituted tobacco leaf obtained in Comparative Example 1 was tested, and the water-soluble sugar content was 0.18 mg / g, and the total organic acid content was 5.02 mg / g.

[0055] Comparative Example 2

[0056] The impurities on the surface of the reconstituted tobacco leaf base 1 were removed by air separation; the reconstituted tobacco leaves were cut into 5 cm × 5 cm squares using a shear knife; and the reconstituted tobacco leaves were then laid flat in a constant temperature and humidity environment (22 ± 1 ° C, relative humidity 60 ± 2%) for 48 hours.

[0057] Measure 500 mL of 0.1 mol / L citric acid solution and slowly add 535 mL of 0.1 mol / L disodium hydrogen phosphate solution while stirring to prepare a standby buffer solution with a pH value of 4.8. Then filter the standby buffer solution through a 0.45 μm microporous filter membrane to obtain a citric acid-disodium hydrogen phosphate buffer solution.

[0058] Pectinase (50,000 U / g) was dissolved in water at a mass ratio of 2:1 to prepare an enzyme solution.

[0059] First, spray citric acid-disodium hydrogen phosphate buffer onto the surface of the spare film base, and then spray the enzyme solution onto the surface of the spare film base until the pectinase concentration is 30U / g of the spare film base. Under the conditions of temperature of 40°C and humidity of 60%, perform enzymatic hydrolysis for 4 hours to obtain an enzymatic hydrolysis film base.

[0060] The enzymatic hydrolyzed sheet was dried at 80° C. for 40 min to obtain a finished reconstituted tobacco leaf.

[0061] The finished reconstituted tobacco leaf obtained in Comparative Example 2 was tested, and the water-soluble sugar content was 0.32 mg / g, and the total organic acid content was 7.25 mg / g.

[0062] Comparative Example 3

[0063] The impurities on the surface of the reconstituted tobacco leaf base 1 were removed by air separation; the reconstituted tobacco leaves were cut into 5 cm × 5 cm squares using a shear knife; and the reconstituted tobacco leaves were then laid flat in a constant temperature and humidity environment (22 ± 1 ° C, relative humidity 60 ± 2%) for 48 hours.

[0064] Measure 500 mL of 0.1 mol / L citric acid solution and slowly add 535 mL of 0.1 mol / L disodium hydrogen phosphate solution while stirring to prepare a standby buffer solution with a pH value of 4.8. Then filter the standby buffer solution through a 0.45 μm microporous filter membrane to obtain a citric acid-disodium hydrogen phosphate buffer solution.

[0065] Spray citric acid-disodium hydrogen phosphate buffer onto the surface of the spare substrate, and then spray water in an amount equal to the enzyme solution sprayed in Example 1, and place it at a temperature of 40° C. and a humidity of 60% for 4 hours to obtain a treated substrate.

[0066] The treated sheets were dried at 80° C. for 40 min to obtain finished reconstituted tobacco leaves.

[0067] The finished reconstituted tobacco leaf obtained in Comparative Example 3 was tested, and the water-soluble sugar content was 0.15 mg / g, and the total organic acid content was 4.11 mg / g.

[0068] Example 2

[0069] Use air separation to remove impurities on the surface of the reconstituted tobacco leaf base 2; use a shear knife to cut the reconstituted tobacco leaves into 5cm×5cm squares; then lay the reconstituted tobacco leaves flat in a constant temperature and humidity environment (22±1℃, relative humidity 60±2%) for 48 hours.

[0070] Measure 500 mL of 0.1 mol / L citric acid solution and slowly add 535 mL of 0.1 mol / L disodium hydrogen phosphate solution while stirring to prepare a standby buffer solution with a pH value of 4.8. Then filter the standby buffer solution through a 0.45 μm microporous filter membrane to obtain a citric acid-disodium hydrogen phosphate buffer solution.

[0071] Cellulase (200,000 U / g) and pectinase (50,000 U / g) were dissolved in water at a mass ratio of 1:1 to prepare an enzyme solution.

[0072] First, spray citric acid-disodium hydrogen phosphate buffer onto the surface of the spare film base, and then spray the enzyme solution onto the surface of the spare film base until the cellulase concentration is 120U / g of the spare film base and the pectinase concentration is 30U / g of the spare film base. Under the conditions of temperature of 40°C and humidity of 50%, carry out enzymatic hydrolysis reaction for 5 hours to obtain an enzymatic hydrolysis film base.

[0073] The enzymatic hydrolyzed sheet was dried at 75° C. for 50 min to obtain a finished reconstituted tobacco leaf.

[0074] The finished reconstituted tobacco leaf obtained in Example 2 was tested, and the water-soluble sugar content was 0.42 mg / g, and the total organic acid content was 7.54 mg / g.

[0075] Comparative Example 4

[0076] Use air separation to remove impurities on the surface of the reconstituted tobacco leaf base 2; use a shear knife to cut the reconstituted tobacco leaves into 5cm×5cm squares; then lay the reconstituted tobacco leaves flat in a constant temperature and humidity environment (22±1℃, relative humidity 60±2%) for 48 hours.

[0077] Measure 500 mL of 0.1 mol / L citric acid solution and slowly add 535 mL of 0.1 mol / L disodium hydrogen phosphate solution while stirring to prepare a standby buffer solution with a pH value of 4.8. Then filter the standby buffer solution through a 0.45 μm microporous filter membrane to obtain a citric acid-disodium hydrogen phosphate buffer solution.

[0078] Dissolve cellulase (200,000 U / g) in water to prepare enzyme solution.

[0079] First, spray citric acid-disodium hydrogen phosphate buffer onto the surface of the spare film base, and then spray the enzyme solution onto the surface of the spare film base until the cellulase concentration is 120U / g of the spare film base. Under the conditions of temperature of 40°C and humidity of 50%, perform enzymatic hydrolysis for 5 hours to obtain an enzymatic hydrolysis film base.

[0080] The enzymatic hydrolyzed sheet was dried at 75° C. for 50 min to obtain a finished reconstituted tobacco leaf.

[0081] The finished reconstituted tobacco leaf obtained in Comparative Example 4 was tested, and the water-soluble sugar content was 0.17 mg / g, and the total organic acid content was 4.86 mg / g.

[0082] Comparative Example 5

[0083] Use air separation to remove impurities on the surface of the reconstituted tobacco leaf base 2; use a shear knife to cut the reconstituted tobacco leaves into 5cm×5cm squares; then lay the reconstituted tobacco leaves flat in a constant temperature and humidity environment (22±1℃, relative humidity 60±2%) for 48 hours.

[0084] Measure 500 mL of 0.1 mol / L citric acid solution and slowly add 535 mL of 0.1 mol / L disodium hydrogen phosphate solution while stirring to prepare a standby buffer solution with a pH value of 4.8. Then filter the standby buffer solution through a 0.45 μm microporous filter membrane to obtain a citric acid-disodium hydrogen phosphate buffer solution.

[0085] Dissolve pectinase (50,000 U / g) in water to prepare enzyme solution.

[0086] First, spray citric acid-disodium hydrogen phosphate buffer onto the surface of the spare film base, and then spray the enzyme solution onto the surface of the spare film base until the pectinase concentration is 30U / g of the spare film base. Under the conditions of temperature of 40°C and humidity of 50%, perform enzymatic hydrolysis for 5 hours to obtain an enzymatic hydrolysis film base.

[0087] The enzymatic hydrolyzed sheet was dried at 75° C. for 50 min to obtain a finished reconstituted tobacco leaf.

[0088] The finished reconstituted tobacco leaf obtained in Comparative Example 5 was tested, and the water-soluble sugar content was 0.30 mg / g, and the total organic acid content was 7.01 mg / g.

[0089] Comparative Example 6

[0090] Use air separation to remove impurities on the surface of the reconstituted tobacco leaf base 2; use a shear knife to cut the reconstituted tobacco leaves into 5cm×5cm squares; then lay the reconstituted tobacco leaves flat in a constant temperature and humidity environment (22±1℃, relative humidity 60±2%) for 48 hours.

[0091] Measure 500 mL of 0.1 mol / L citric acid solution and slowly add 535 mL of 0.1 mol / L disodium hydrogen phosphate solution while stirring to prepare a standby buffer solution with a pH value of 4.8. Then filter the standby buffer solution through a 0.45 μm microporous filter membrane to obtain a citric acid-disodium hydrogen phosphate buffer solution.

[0092] Spray citric acid-disodium hydrogen phosphate buffer onto the surface of the spare substrate, and then spray water in an amount equal to the enzyme solution sprayed in Example 2. Leave it at a temperature of 40° C. and a humidity of 50% for 5 hours to obtain a treated substrate.

[0093] The treated sheets were dried at 75° C. for 50 min to obtain finished reconstituted tobacco leaves.

[0094] The finished reconstituted tobacco leaf obtained in Comparative Example 6 was tested, and the water-soluble sugar content was 0.14 mg / g, and the total organic acid content was 4.09 mg / g.

[0095] Example 3

[0096] Use air separation to remove impurities on the surface of the reconstituted tobacco leaf base 3; use a shear knife to cut the reconstituted tobacco leaves into 5cm×5cm squares; then lay the reconstituted tobacco leaves flat in a constant temperature and humidity environment (22±1℃, relative humidity 60±2%) for 48 hours.

[0097] Measure 500 mL of 0.1 mol / L citric acid solution and slowly add 535 mL of 0.1 mol / L disodium hydrogen phosphate solution while stirring to prepare a standby buffer solution with a pH value of 4.8. Then filter the standby buffer solution through a 0.45 μm microporous filter membrane to obtain a citric acid-disodium hydrogen phosphate buffer solution.

[0098] Cellulase (200,000 U / g) and pectinase (50,000 U / g) were dissolved in water at a mass ratio of 2:1 to prepare an enzyme solution.

[0099] The citric acid-disodium hydrogen phosphate buffer solution is first sprayed on the surface of the standby sheet base, and then the enzyme solution is sprayed on the surface of the standby sheet base, until the cellulase is 240 U / g of the standby sheet base and the pectinase is 30 U / g of the standby sheet base. Under the conditions of a temperature of 45℃ and a humidity of 50%, the enzymolysis reaction is carried out for 4.5 h, and the enzymolysis sheet base is obtained.

[0100] The enzymolysis sheet base is dried at 75℃ for 50 min, and the reconstituted tobacco product is obtained.

[0101] The reconstituted tobacco product obtained in Example 3 is detected, and the water-soluble sugar content is 0.38 mg / g, and the total organic acid content is 6.31 mg / g.

[0102] Comparative Example 7

[0103] The surface impurities of the reconstituted tobacco sheet base 3 are removed by air selection, the reconstituted tobacco is cut into a square of 5 cm x 5 cm by using a shearing knife, and then the reconstituted tobacco is laid flat in a constant temperature and humidity environment (22±1℃, relative humidity 60±2%) for 48 h.

[0104] 500 mL of 0.1 mol / L citric acid solution is measured, 535 mL of 0.1 mol / L disodium hydrogen phosphate solution is slowly added, and stirring is performed while adding, to prepare a standby buffer solution with a pH value of 4.8. Then, the standby buffer solution is filtered through a 0.45 μm microporous filter membrane to obtain the citric acid-disodium hydrogen phosphate buffer solution.

[0105] The cellulase (200,000 U / g) is dissolved in water to prepare an enzyme solution.

[0106] The citric acid-disodium hydrogen phosphate buffer solution is first sprayed on the surface of the standby sheet base, and then the enzyme solution is sprayed on the surface of the standby sheet base, until the cellulase is 240 U / g of the standby sheet base. Under the conditions of a temperature of 45℃ and a humidity of 50%, the enzymolysis reaction is carried out for 4.5 h, and the enzymolysis sheet base is obtained.

[0107] The enzymolysis sheet base is dried at 75℃ for 50 min, and the reconstituted tobacco product is obtained.

[0108] The reconstituted tobacco product obtained in Comparative Example 7 is detected, and the water-soluble sugar content is 0.15 mg / g, and the total organic acid content is 4.62 mg / g.

[0109] Comparative Example 8

[0110] The surface impurities of the reconstituted tobacco sheet base 3 are removed by air selection, the reconstituted tobacco is cut into a square of 5 cm x 5 cm by using a shearing knife, and then the reconstituted tobacco is laid flat in a constant temperature and humidity environment (22±1℃, relative humidity 60±2%) for 48 h.

[0111] Take 500 mL of 0.1 mol / L citric acid solution, slowly add 535 mL of 0.1 mol / L disodium hydrogen phosphate solution, stir while adding, to prepare a standby buffer solution with a pH value of 4.8, then filter the standby buffer solution through a 0.45 μm microporous filter to obtain a citric acid-disodium hydrogen phosphate buffer solution.

[0112] Dissolve pectinase (50,000 U / g) in water to prepare an enzyme solution.

[0113] First, spray the citric acid-disodium hydrogen phosphate buffer solution onto the surface of the standby sheet base, then spray the enzyme solution onto the surface of the standby sheet base, so that the pectinase is 30 U / g of the standby sheet base substrate, and carry out enzymatic reaction at a temperature of 45°C and a humidity of 50% for 4.5 h to obtain an enzymatic sheet base.

[0114] Dry the enzymatic sheet base at 75°C for 50 min to obtain the finished product of the reconstituted tobacco sheet.

[0115] Detect the finished product of the reconstituted tobacco sheet obtained in Comparative Example 8, and the water-soluble sugar content is 0.28 mg / g, and the total organic acid content is 6.11 mg / g.

[0116] Comparative Example 9

[0117] Remove the surface impurities of the reconstituted tobacco sheet by air selection; cut the reconstituted tobacco sheet into 5 cm x 5 cm squares using a shear cutter; then place the reconstituted tobacco sheet flat in a constant temperature and humidity environment (22±1°C, relative humidity 60±2%) for 48 h.

[0118] Take 500 mL of 0.1 mol / L citric acid solution, slowly add 535 mL of 0.1 mol / L disodium hydrogen phosphate solution, stir while adding, to prepare a standby buffer solution with a pH value of 4.8, then filter the standby buffer solution through a 0.45 μm microporous filter to obtain a citric acid-disodium hydrogen phosphate buffer solution.

[0119] Spray the citric acid-disodium hydrogen phosphate buffer solution onto the surface of the standby sheet base, then spray an equal amount of water as the enzyme solution sprayed in Example 3, and place it at a temperature of 45°C and a humidity of 50% for 4.5 h to obtain a treated sheet base.

[0120] Dry the treated sheet base at 75°C for 50 min to obtain the finished product of the reconstituted tobacco sheet.

[0121] Detect the finished product of the reconstituted tobacco sheet obtained in Comparative Example 9, and the water-soluble sugar content is 0.12 mg / g, and the total organic acid content is 4.06 mg / g.

[0122] Example 4

[0123] The impurities on the surface of the reconstituted tobacco leaf base were removed by air separation. The reconstituted tobacco leaves were cut into 5 cm × 5 cm squares using shears. The reconstituted tobacco leaves were then laid flat in a constant temperature and humidity environment (22 ± 1 ° C, relative humidity 60 ± 2%) for 48 hours.

[0124] Measure 500 mL of 0.1 mol / L citric acid solution and slowly add 535 mL of 0.1 mol / L disodium hydrogen phosphate solution while stirring to prepare a standby buffer solution with a pH value of 4.8. Then filter the standby buffer solution through a 0.45 μm microporous filter membrane to obtain a citric acid-disodium hydrogen phosphate buffer solution.

[0125] Cellulase (200,000 U / g) and pectinase (50,000 U / g) were dissolved in water at a mass ratio of 2:1 to prepare an enzyme solution.

[0126] First, spray citric acid-disodium hydrogen phosphate buffer onto the surface of the spare film base, and then spray the enzyme solution onto the surface of the spare film base until the cellulase concentration is 240U / g of the spare film base and the pectinase concentration is 30U / g of the spare film base. Under the conditions of temperature of 40°C and humidity of 50%, the enzymatic hydrolysis reaction is carried out for 5 hours to obtain an enzymatic hydrolysis film base.

[0127] The enzymatic hydrolyzed sheet was dried at 70° C. for 50 min to obtain a finished reconstituted tobacco leaf.

[0128] The finished reconstituted tobacco leaf obtained in Example 4 was tested, and the water-soluble sugar content was 0.39 mg / g, and the total organic acid content was 7.12 mg / g.

[0129] Comparative Example 10

[0130] The impurities on the surface of the reconstituted tobacco leaf base were removed by air separation. The reconstituted tobacco leaves were cut into 5 cm × 5 cm squares using shears. The reconstituted tobacco leaves were then laid flat in a constant temperature and humidity environment (22 ± 1 ° C, relative humidity 60 ± 2%) for 48 hours.

[0131] Measure 500 mL of 0.1 mol / L citric acid solution and slowly add 535 mL of 0.1 mol / L disodium hydrogen phosphate solution while stirring to prepare a standby buffer solution with a pH value of 4.8. Then filter the standby buffer solution through a 0.45 μm microporous filter membrane to obtain a citric acid-disodium hydrogen phosphate buffer solution.

[0132] Dissolve cellulase (200,000 U / g) in water to prepare enzyme solution.

[0133] The citric acid-disodium hydrogen phosphate buffer solution is first sprayed on the surface of the standby sheet base, and then the enzyme solution is sprayed on the surface of the standby sheet base, until the cellulase is 240 U / g of the standby sheet base substrate. Under the conditions of a temperature of 40℃ and a humidity of 50%, the enzymolysis reaction is carried out for 5h, and the enzymolysis sheet base is obtained.

[0134] The enzymolysis sheet base is dried at 70℃ for 50min, and the reconstituted tobacco product is obtained.

[0135] The reconstituted tobacco product obtained in the comparative example 10 is detected, and the water-soluble sugar content is 0.16 mg / g, and the total organic acid content is 4.69 mg / g.

[0136] Comparative example 11

[0137] The surface impurities of the reconstituted tobacco sheet base 4 are removed by air selection, the reconstituted tobacco is cut into 5cm×5cm squares by using a shear cutter, and then the reconstituted tobacco is laid flat in a constant temperature and humidity environment (22±1℃, relative humidity 60±2%) for 48h.

[0138] 500mL of 0.1mol / L citric acid solution is measured, 535mL of 0.1mol / L disodium hydrogen phosphate solution is slowly added, and stirring is carried out while adding, to prepare a standby buffer solution with a pH value of 4.8. Then, the standby buffer solution is filtered through a 0.45μm microporous filter to obtain the citric acid-disodium hydrogen phosphate buffer solution.

[0139] The pectinase (500,000 U / g) is dissolved in water according to a mass ratio of 2:1 to prepare an enzyme solution.

[0140] The citric acid-disodium hydrogen phosphate buffer solution is first sprayed on the surface of the standby sheet base, and then the enzyme solution is sprayed on the surface of the standby sheet base, until the pectinase is 30 U / g of the standby sheet base substrate. Under the conditions of a temperature of 40℃ and a humidity of 50%, the enzymolysis reaction is carried out for 5h, and the enzymolysis sheet base is obtained.

[0141] The enzymolysis sheet base is dried at 70℃ for 50min, and the reconstituted tobacco product is obtained.

[0142] The reconstituted tobacco product obtained in the comparative example 11 is detected, and the water-soluble sugar content is 0.29 mg / g, and the total organic acid content is 6.51 mg / g.

[0143] Comparative example 12

[0144] The surface impurities of the reconstituted tobacco sheet base 4 are removed by air selection, the reconstituted tobacco is cut into 5cm×5cm squares by using a shear cutter, and then the reconstituted tobacco is laid flat in a constant temperature and humidity environment (22±1℃, relative humidity 60±2%) for 48h.

[0145] Take 500 mL of 0.1 mol / L citric acid solution, slowly add 535 mL of 0.1 mol / L disodium hydrogen phosphate solution, stir while adding, prepare a standby buffer solution with pH value of 4.8, then filter the standby buffer solution through a 0.45 μm microporous filter to obtain a citric acid-disodium hydrogen phosphate buffer solution.

[0146] Spray the citric acid-disodium hydrogen phosphate buffer solution onto the surface of the standby sheet base, then spray an equal amount of water as the enzyme solution sprayed in Example 4, and place it in a temperature of 40℃ and humidity of 50% for 5h to obtain a treated sheet base.

[0147] Dry the treated sheet base at 70℃ for 50min to obtain a reconstituted tobacco product.

[0148] The reconstituted tobacco product obtained in Comparative Example 12 was detected, and the water-soluble sugar content was 0.13 mg / g, and the total organic acid content was 4.07 mg / g.

[0149] The reconstituted tobacco product was subjected to sensory evaluation by taste analyzer combined with sensory evaluation certified by ISO8586-2012, and the evaluation indexes were sourness intensity, sweetness intensity, sour-sweet balance, aroma richness, and overall acceptance, and a 0-10 point system was used, each expert evaluated independently, and the average score was calculated. The results are as follows:

[0150] The scores of sourness, sweetness, sour-sweet balance, aroma richness, and overall acceptance of the reconstituted tobacco product of Example 1 were -20.56, 0.15, 4.21, 4.38, and 4.37, respectively.

[0151] The scores of sourness, sweetness, sour-sweet balance, aroma richness, and overall acceptance of the reconstituted tobacco product of Comparative Example 1 were -20.84, 0.12, 3.97, 4.12, and 4.15, respectively.

[0152] The scores of sourness, sweetness, sour-sweet balance, aroma richness, and overall acceptance of the reconstituted tobacco product of Comparative Example 2 were -20.96, 0.14, 4.03, 4.21, and 4.24, respectively.

[0153] The scores of sourness, sweetness, sour-sweet balance, aroma richness, and overall acceptance of Comparative Example 3 were -25.56, 0.07, 3.52, 3.44, and 3.51, respectively.

[0154] The scores of sourness, sweetness, sour-sweet balance, aroma richness, and overall acceptance of the reconstituted tobacco product of Example 2 were -21.11, 0.15, 4.09, 4.31, and 4.33, respectively.

[0155] The sourness, sweetness, sour-sweet balance, aroma richness, and overall acceptability scores of the reconstituted tobacco product of Comparative Example 4 were -20.87, 0.13, 4.34, 4.09, and 4.12, respectively;

[0156] The sourness, sweetness, sour-sweet balance, aroma richness, and overall acceptability scores of the reconstituted tobacco product of Comparative Example 5 were -20.98, 0.14, 4.26, 4.33, and 4.40, respectively;

[0157] The sourness, sweetness, sour-sweet balance, aroma richness and overall acceptance scores of the reconstituted tobacco product of Comparative Example 6 were -23.73, 0.03, 3.40, 3.42 and 3.49, respectively.

[0158] The sourness, sweetness, sour-sweet balance, aroma richness, and overall acceptability scores of the reconstituted tobacco product of Example 3 were -20.79, 0.14, 4.08, 4.26, and 4.21, respectively;

[0159] The sourness, sweetness, sour-sweet balance, aroma richness, and overall acceptability scores of the reconstituted tobacco product of Comparative Example 7 were -21.09, 0.11, 3.78, 4.00, and 4.02, respectively;

[0160] The sourness, sweetness, sour-sweet balance, aroma richness, and overall acceptability scores of the reconstituted tobacco product of Comparative Example 8 were -21.16, 0.13, 3.83, 4.09, and 4.11, respectively;

[0161] The sourness, sweetness, sour-sweet balance, aroma richness and overall acceptance scores of the reconstituted tobacco product of Comparative Example 9 were -23.51, 0.04, 3.30, 3.41 and 3.46, respectively.

[0162] The sourness, sweetness, sour-sweet balance, aroma richness, and overall acceptability scores of the reconstituted tobacco product of Example 4 were -22.20, 0.14, 3.87, 4.13, and 4.18, respectively;

[0163] The sourness, sweetness, sour-sweet balance, aroma richness, and overall acceptability scores of the reconstituted tobacco product of Comparative Example 10 were -22.50, 0.13, 3.65, 3.88, and 3.97, respectively;

[0164] The sourness, sweetness, sour-sweet balance, aroma richness, and overall acceptability scores of the reconstituted tobacco product of Comparative Example 11 were -22.63, 0.14, 3.71, 3.97, and 4.14, respectively;

[0165] The sourness, sweetness, sour-sweet balance, aroma richness and overall acceptance scores of the reconstituted tobacco product of Comparative Example 12 were -25.32, 0.05, 3.2, 3.38 and 3.45, respectively.

[0166] The sensory improvements achieved after co-modulation were consistent across different types of reconstituted tobacco leaf base, demonstrating the stability of the complex enzyme co-modulation process across diverse raw materials. This makes the method suitable for promotion as a standardized pretreatment technology, eliminating the need for complex adjustments based on raw material differences and effectively reducing production process complexity and costs.

[0167] Although some specific embodiments of the present invention have been described in detail by way of examples, it should be understood by those skilled in the art that the above examples are for illustration only and are not intended to limit the scope of the present invention. It should be understood by those skilled in the art that modifications may be made to the above embodiments without departing from the scope and spirit of the present invention. The scope of the present invention is defined by the appended claims.

Claims

1. A method for preparing reconstituted tobacco leaves based on compound enzyme technology, characterized in that: The steps include: Reconstituted tobacco leaf pretreatment: pre-treating the surface of the reconstituted tobacco leaf base to remove impurities and obtain a spare leaf base; Preparation of enzyme solution: dissolve cellulase and pectinase in water at a certain mass ratio to prepare enzyme solution; Enzymatic hydrolysis treatment: first spray citric acid-disodium hydrogen phosphate buffer with a pH value of 4.8 onto the surface of the spare film base, then spray the enzyme solution on the surface of the spare film base, and carry out enzymatic hydrolysis reaction for 3-5 hours at a temperature of 40-50°C and a humidity of 50-70% to obtain an enzymatic hydrolysis film base, and then dry the enzymatic hydrolysis film base at 70-80°C for 30-50 minutes to obtain reconstituted tobacco leaves.

2. The method for preparing reconstituted tobacco leaves based on compound enzyme technology according to claim 1, characterized in that: The reconstituted tobacco leaf pretreatment further comprises the step of cutting the reconstituted tobacco leaf base into 5 cm×5 cm squares.

3. The method for preparing reconstituted tobacco using compound enzyme technology according to claim 1, characterized in that: The surface impurity removal pretreatment in the reconstituted tobacco leaf pretreatment is achieved by air separation.

4. The method for preparing reconstituted tobacco leaves based on composite enzyme technology according to claim 1, characterized in that: The spare film base was equilibrated for 48 hours at a temperature of 22±1°C and a relative humidity of 60±2% before enzymatic hydrolysis.

5. The method for preparing reconstituted tobacco based on composite enzyme technology according to claim 1, characterized in that: The enzyme solution is prepared as follows: 200,000 U / g of cellulase and 50,000 U / g of pectinase were dissolved in water at a mass ratio of 1:2-3:1 to prepare an enzyme solution.

6. The method for preparing reconstituted tobacco leaves based on compound enzyme technology according to claim 1, characterized in that: The preparation method of the citric acid-disodium hydrogen phosphate buffer in the enzymatic hydrolysis treatment is as follows: According to a volume ratio of 100:107, 0.1 mol / L citric acid solution was added to 0.1 mol / L disodium hydrogen phosphate solution to obtain a reserve buffer solution, which was then filtered through a 0.45 μm microporous filter membrane to obtain a citric acid-disodium hydrogen phosphate buffer solution with a pH value of 4.

8.

7. The method for preparing reconstituted tobacco based on compound enzyme technology according to claim 1, characterized in that: The enzyme solution in the enzymatic hydrolysis treatment is sprayed on the surface of the spare sheet base until the content of cellulase relative to the spare sheet base is 60-360 U / g, and the content of pectinase relative to the spare sheet base is 30 U / g.