A bio-enzymatic hydrolyzed fruit extract, and a preparation method and application thereof

Abstract

The present application relates to the technical field of tobacco flavor, in particular to a kind of biological enzymatic fruit extract and its preparation method and application.This method includes the following steps: fruit is soaked with ethylene solution with a concentration of 1~100mg / L, and after completion, it is taken out and dried; polygalacturonase and cellulase are used for enzymatic hydrolysis treatment to obtain a first enzymatic hydrolysis solution; the first enzymatic hydrolysis solution is subjected to solid-liquid separation to obtain solid phase and liquid phase; the solid phase is soaked with ethylene solution with a concentration of 450~550mg / L, and after completion, it is taken out and dried, and then added back to the liquid phase; xylanase is added to the liquid phase for enzymatic hydrolysis treatment to obtain a second enzymatic hydrolysis solution; the second enzymatic hydrolysis solution is extracted with a solvent. The present application mainly obtains small molecular polysaccharides with a molecular weight of more than 3500. The enzymatic hydrolysate is used as a cigarette flavor, which not only enriches the aroma quality and aroma amount of cigarette smoking, reduces irritation and miscellaneous gas, but also improves the moisture retention capacity of cigarette and enhances the sweet and moist feeling of smoking.

Description

Technical Field

[0001] The present invention relates to the technical field of tobacco flavorings, in particular to a bio-enzymatic hydrolysis fruit extract and a preparation method and application thereof. Background Art

[0002] As a core component of cigarette formulations, tobacco flavoring plays a vital role in enriching cigarette aroma and improving draw quality. Fresh fruit, with its abundant moisture, rich aroma, and high sugar content, is a high-quality natural tobacco flavoring. Using fruit to create natural flavorings can impart a unique and natural aroma to cigarettes, softening smoke and improving flavor.

[0003] Extracts can be obtained by extracting fruits with solvents. Adding fruit extracts to cigarettes can enhance the aroma quality and quantity of cigarettes and improve the taste comfort, but it usually brings some negative effects to the oral cavity. It is speculated that this may be related to the fact that they contain macromolecular substances such as cellulose, hemicellulose, pectin, and protein.

[0004] In the prior art, enzymatic hydrolysis is typically used to convert these cellulose and pectin compounds into small molecules closely related to tobacco's characteristic aroma components. For example, patent publication number CN113755244A discloses a method for preparing a red date tobacco flavor, which includes extracting sorghum liquor, pre-treating red dates, enzymatic hydrolysis, hot reflux extraction, flocculation, vacuum-assisted segmented separation, and preparing the red date tobacco flavor. The enzymatic hydrolysis process involves adding a complex enzyme consisting of pectinase, cellulase, and papain, and performing enzymatic hydrolysis at 40°C to 60°C for 2 to 6 hours. This enzymatic hydrolysis disrupts cell walls, improving the dissolution rate of active ingredients while promoting the conversion of starch, protein, and other substances, increasing the content of reducing sugars and other substances in the product. This addresses the problem of residual protein, pectin, starch, and polysaccharide macromolecules in current red date flavor preparations.

[0005] In the existing technology, most of the focus is on degrading cellulose, hemicellulose, pectin, etc. into small molecular reducing sugars as much as possible to reduce the irritation and impurities when smoking cigarettes. However, reducing sugars have a small molecular weight and limited ability to bind water, resulting in insufficient moisture retention. When applied to cigarettes as flavorings, on the one hand, it is not conducive to the long-term storage of cigarettes, and on the other hand, it causes the cigarette smoke to be dry and lack of sweetness when smoking. Summary of the Invention

[0006] The present invention aims to solve the above problems and provides a bio-enzymatic hydrolysis fruit extract and a preparation method and application thereof.

[0007] The technical solution to the problem of the present invention is to first provide a method for preparing fruit extract by bioenzymatic hydrolysis, comprising the following steps:

[0008] S1. Soaking the fruit with ethylene solution with a concentration of 1-100 mg / L, and then taking out and drying after soaking; then using polygalacturonase and cellulase to enzymatically treat the fruit to obtain a first enzymatic hydrolysate;

[0009] S2. Solid-liquid separation is performed on the first enzymatic hydrolysate to obtain solid phase and liquid phase; the solid phase is soaked with ethylene solution with a concentration of 450-550 mg / L, and then taking out and drying after soaking, and adding back to the liquid phase; xylanase is added to the liquid phase for enzymatic treatment to obtain a second enzymatic hydrolysate;

[0010] S3. The second enzymatic hydrolysate is extracted with a solvent to obtain a fruit extract.

[0011] The fruit cell wall is mainly composed of pectin, cellulose and hemicellulose, and a small amount of protein, and the degradation of the cell wall components is mainly achieved by the degradation of the corresponding cell wall polysaccharides by cell wall enzymes. Pectin methyl esterase (PME) and polygalacturonase (PG) are the main enzymes for degrading pectin polysaccharides, cellulase (CS) is the main enzyme for degrading cellulose polysaccharides, and xylanase (Xyl) is an important hemicellulose polysaccharide degrading enzyme.

[0012] Studies have shown that ethylene can promote the activity of various cell wall enzymes and accelerate the degradation of cell walls. On this basis, the inventors have found through experiments that within a low concentration range (not more than 100 mg / L), ethylene can promote the activity of pectin methyl esterase, polygalacturonase and cellulase, and exceeding the concentration range will cause reverse inhibition. Meanwhile, within the above range, as the ethylene concentration increases, the activity of pectin methyl esterase and polygalacturonase increases, but the activity of cellulase first increases and then decreases. Relatively high concentration (about 500 mg / L) of ethylene promotes the activity of xylanase, and too low or too high concentration will cause reverse inhibition. In addition, only the increase of polygalacturonase activity promotes the conversion of cell wall components to small molecular components with a molecular weight of less than 3500 and increases the content of soluble sugar; pectin methyl esterase acts on the methoxyl group of pectin molecules and provides a substrate for polygalacturonase, and has no direct effect on pectin solubilization; even if the activity of cellulase and xylanase increases, the enzymatic products are still mainly large molecules with a molecular weight of more than 3500.

[0013] The present application utilizes this characteristic to perform ethylene-assisted enzymatic hydrolysis on fruits, by controlling the concentration of ethylene, less promoting polygalacturonase activity, and greater promoting cellulase and xylanase activity, to mainly obtain small molecular polysaccharides with a molecular weight of 3500 or more. The enzymatic hydrolysate of the present application has a much smaller molecular weight than the enzymatic substrates such as pectin, cellulose and hemicellulose, and can effectively reduce the suction irritability and miscellaneous odor; compared with oligosaccharides and monosaccharides, the molecular weight is slightly larger, and therefore has higher viscosity, film-forming property, water-swellable property, and is more excellent in terms of moisture retention and stability. Using the enzymatic hydrolysate as a cigarette flavor not only enriches the cigarette smoking aroma quality and quantity, reduces the irritability and miscellaneous odor, but also improves the moisture retention capacity of the cigarette and enhances the sweet and moist feeling during smoking.

[0014] In step S1, an ethephon solution with a concentration of 1-100 mg / L is used. For polygalacturonase, the higher the concentration of ethephon, the greater the activity. For cellulase, the higher the concentration of ethephon, the activity first increases and then decreases. Therefore, it is preferred to use an ethephon concentration that makes the cellulase activity peak, and to expand the activity gap between polygalacturonase and cellulase. Therefore, as a preferred embodiment of the present application, an ethephon solution with a concentration of 20-30 mg / L is used, such as 20 mg / L, 22.5 mg / L, 25 mg / L, 27.5 mg / L, 30 mg / L, and preferably 25 mg / L, at which the activity gap between the two enzymes is maximized.

[0015] In step S2, a concentration of 450-550 mg / L is used, such as 450 mg / L, 480 mg / L, 500 mg / L, 520 mg / L, 550 mg / L, and preferably 550 mg / L.

[0016] The promotion and inhibition of ethylene are also related to temperature. Within a temperature range of 10-50℃, the activity of cellulase first decreases and then increases with increasing temperature, and the activity of polygalacturonase first increases and then decreases and then increases with increasing temperature. The soaking temperature and the enzymatic hydrolysis temperature are preferably controlled within a temperature range in which the cellulase activity is large and the polygalacturonase activity is small. In addition, in order to ensure the efficiency of enzymatic hydrolysis, the enzymatic hydrolysis temperature should not be too low. Therefore, as a preferred embodiment of the present application, the soaking temperature is 10-30℃, such as 10℃, 15℃, 20℃, 25℃, 30℃, and preferably 10℃. The enzymatic hydrolysis temperature is 40-50℃, such as 40℃, 42℃, 45℃, 48℃, 50℃, and preferably 45℃.

[0017] The activity of the xylanase increases with the increase of temperature in the range of 10-60°C. As a preferred embodiment of the present application, the soaking temperature in step S2 is 50-60°C, such as 50°C, 52°C, 55°C, 58°C, 60°C, and preferably 60°C. Meanwhile, in order to ensure the enzymatic hydrolysis efficiency, as a preferred embodiment of the present application, the enzymatic hydrolysis temperature in step S2 is 45-55°C, such as 45°C, 48°C, 50°C, 52°C, 55°C, and preferably 50°C.

[0018] The amount of enzyme and the enzymatic hydrolysis time in steps S1 and S2 also need to be limited. Too much amount of enzyme and too long enzymatic hydrolysis time will lead to over-hydrolysis, and too little amount of enzyme and too short enzymatic hydrolysis time will lead to under-hydrolysis. Therefore, as a preferred embodiment of the present application, the total mass concentration of polygalacturonase and cellulase in step S1 is 0.5%-1.5%, such as 0.5%, 0.8%, 1.0%, 1.2%, 1.5%, and preferably 1.0%. The mass ratio of polygalacturonase to cellulase is 2:(0.8-1.2), such as 2:0.8, 2:0.9, 2:1, 2:1.1, 2:1.2, and preferably 2:1.

[0019] As a preferred embodiment of the present application, the mass concentration of xylanase in step S2 is 0.01%-0.03%, such as 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, and preferably 0.02%.

[0020] As a preferred embodiment of the present application, the enzymatic hydrolysis time in steps S1 and S2 is independently 30-90 min, such as independently 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, and preferably 60 min.

[0021] The enzymatic hydrolysis mainly degrades the cell wall of the fruit, and after the enzymatic hydrolysis is completed, the effective components including volatile aroma substances in the fruit cells can be quickly and effectively extracted by extraction. The solvent used for extraction is not limited, and one or more of water, low-concentration ethanol, and ethyl acetate can be used.

[0022] Generally in the prior art, the added enzymes are inactivated before extraction. In the present application, the enzyme activity is reduced and the extraction efficiency is improved by adding acid and high temperature. Generally, the optimum pH of pectinase is 2.5-6.0, the optimum pH of cellulase is 4.5-6.5, and the optimum pH of xylanase is 3.5-6.5. Therefore, as a preferred embodiment of the present application, the pH of the secondary enzymatic hydrolysis solution is adjusted to 1.0-3.0, and then extracted at 50-80°C. The pH can be 1.0, 1.5, 2.0, 2.5, 3.0, and preferably 2.0. The extraction temperature can be 50°C, 60°C, 70°C, 80°C, and preferably 80°C. As a preferred embodiment of the present application, hydrochloric acid is used to adjust the pH.

[0023] In some embodiments, to further improve the extraction efficiency, as a preferred embodiment of the present application, ultrasonic assisted extraction is used. The ultrasonic power is 100-300W, such as 100W, 150W, 200W, 250W, 300W, and preferably 200W. The ultrasonic time is 20-60min, such as 20min, 30min, 40min, 50min, 60min, and preferably 40min.

[0024] In some embodiments, to improve the purity and facilitate storage, as a preferred embodiment of the present application, after extraction, centrifugation is performed at 6000-10000r / min for 15-25min, and the supernatant is filtered through a filter membrane. The filter membrane pore size is 800D-1000D. The speed can be 6000r / min, 7000r / min, 8000r / min, 9000r / min, 10000r / min, and preferably 8000r / min. The centrifugation time can be 15min, 18min, 20min, 22min, 25min, and preferably 20min. The filter membrane pore size can be 800D, 900D, 1000D, and preferably 1000D.

[0025] Another object of the present application is to provide a biological enzyme hydrolyzed fruit extract prepared by the above method. The extract is rich in small molecular weight polysaccharides with a molecular weight of 3500-8000 and volatile aroma components of the fruit, and has good aroma and moisturizing properties.

[0026] Another object of the present application is to provide an application of a biological enzyme hydrolyzed fruit extract in cigarettes. The extract is added to cigarette tobacco, so that the cigarette tobacco can provide a rich aroma, low irritation, and sweet and moist smoke taste to the user when heated and smoked.

[0027] As a preferred embodiment of the present application, the mass ratio of the extract to the tobacco shred is 0.01% to 0.1%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, and preferably 0.05%.

[0028] Advantages of the present application:

[0029] The present application provides a biological enzymatic fruit extract, a preparation method and application thereof. By controlling the concentration of ethylene, the activity of polygalacturonase is promoted less, and the activity of cellulase and xylanase is promoted more, so as to mainly obtain small molecular polysaccharides with a molecular weight of more than 3500. The enzymatic product is used as a tobacco flavor, which not only enriches the aroma quality and amount of tobacco smoking, reduces irritation and miscellaneous odor, but also improves the moisture retention capacity of the tobacco and enhances the sweet and moist feeling of smoking. DETAILED DESCRIPTION

[0030] The following is a detailed embodiment of the present application, and the technical solutions of the present application are further described, but the present application is not limited to these embodiments. Embodiment 1

[0031] A biological enzymatic plum extract is prepared by the following steps:

[0032] S1. Fresh and undamaged plums are selected, washed, pitted and sliced, and then vacuum freeze-dried for standby.

[0033] A container containing an ethephon solution with a concentration of 25 mg / L is placed in a water bath, and the water bath temperature is controlled at 10°C. The plum slices are soaked in the ethephon solution at 10°C for 1 hour, and then taken out and dried with a cold air flow at 10°C until the surface moisture content is 10%.

[0034] According to the mass fraction, 1 part of dried plum slices and 20 parts of water are uniformly mixed, and then a mixed enzyme of polygalacturonase and cellulase with a mass ratio of 2:1 is added, and the mass concentration of the mixed enzyme in the system is controlled at 1.0%. The mixture is stirred uniformly. Then the obtained mixed system is sent into a water bath, and the water bath temperature is controlled at 45°C. The enzymolysis is carried out under neutral conditions for 60 min to obtain a first enzymolysis solution.

[0035] S2. The first enzymolysis solution is filtered to separate solid phase and liquid phase.

[0036] The container containing the ethylene solution with a concentration of 500 mg / L is placed in a water bath, the temperature of which is controlled at 60℃, and is preheated at 60℃ for 30 min. Then the solid phase is immersed in the above-mentioned ethylene solution, taken out after being immersed at 60℃ for 1 h, and dried with a gas flow at 60℃ until the surface moisture content is 10%.

[0037] The solid phase is re-added to the liquid phase, and then xylanase is added, with the mass concentration of xylanase in the system being controlled at 0.02%, and stirred uniformly. Then the obtained mixed system is sent into a water bath, the temperature of which is controlled at 50℃, and enzymolysis is carried out under neutral conditions for 60 min, to obtain a secondary enzymolysis liquid.

[0038] S3. Hydrochloric acid is added to the secondary enzymolysis liquid, and the pH of the secondary enzymolysis liquid is adjusted to 2.0. Then water with a volume 10 times that of the secondary enzymolysis liquid is added, and extraction is carried out by using an ultrasonic-assisted method, with the ultrasonic power being 200 W, the ultrasonic temperature being 80℃, and the ultrasonic time being 40 min. After the extraction is completed, centrifugation is carried out at 8000 r / min for 20 min, and the supernatant is filtered through a 1000D filter membrane to obtain an extract liquid. The extract liquid is concentrated under reduced pressure at 0.5 Mpa and 55℃, and the concentration is stopped until the relative density is 1.3 g / mL, to obtain an extract. Example 2

[0039] This example is basically the same as Example 1, except that in step S1, an ethylene solution with a concentration of 20 mg / L is used. Example 3

[0040] This example is basically the same as Example 1, except that in step S1, an ethylene solution with a concentration of 30 mg / L is used. Example 4

[0041] This example is basically the same as Example 1, except that in step S1, an ethylene solution with a concentration of 100 mg / L is used. Example 5

[0042] This example is basically the same as Example 1, except that in step S2, an ethylene solution with a concentration of 450 mg / L is used. Example 6

[0043] This example is basically the same as Example 1, except that in step S2, an ethylene solution with a concentration of 550 mg / L is used. Example 7

[0044] This example is basically the same as Example 1, except that in step S1, the immersion temperature is 20℃.

[0045] That is, in step S1, the container containing the ethephon solution with a concentration of 25 mg / L was placed in a water bath, the water bath temperature was controlled at 20°C, and preheated at 20°C for 30 min. Then the plum slices were soaked in the above ethephon solution, taken out after soaking at 20°C for 1 h, and dried with air flow at 20°C to a surface moisture content of 10%. Example 8

[0046] This example is basically the same as Example 1, except that in step S1, the soaking temperature is 30°C. Example 9

[0047] This example is basically the same as Example 1, except that in step S2, the soaking temperature is 50°C.

[0048] That is, in step S2, the container containing the ethephon solution with a concentration of 500 mg / L was placed in a water bath, the water bath temperature was controlled at 50°C, and preheated at 50°C for 30 min. Then the solid phase was soaked in the above ethephon solution, taken out after soaking at 50°C for 1 h, and dried with air flow at 50°C to a surface moisture content of 10%. Example 10

[0049] This example is basically the same as Example 1, except that in step S2, the soaking temperature is 80°C. Example 11

[0050] This example is basically the same as Example 1, except that in step S2, the enzymolysis temperature is 60°C. Example 12

[0051] This example is basically the same as Example 1, except that step S3 is different.

[0052] S3. 10 times the volume of water was directly added to the secondary enzymolysis solution, and extraction was carried out using the ultrasonic assisted method, the ultrasonic power was 200 W, the ultrasonic temperature was 80°C, and the ultrasonic time was 40 min. After extraction, centrifugation was carried out at 8000 r / min for 20 min, the supernatant was filtered through a 1000D filter membrane to obtain the extract. The extract was concentrated under reduced pressure at 0.5 MPa and 55°C, and concentrated to a relative density of 1.3 g / mL to obtain the extract. Example 13

[0053] This example is basically the same as Example 1, except that the enzymolysis step in step S1 is different.

[0054] In step S1, 1 part of dried plum pieces and 20 parts of water are mixed according to mass fraction, and then a mixed enzyme of polygalacturonase and cellulase with a mass ratio of 2:1 is added to control the mass concentration of the mixed enzyme in the system at 1.0%, and stirred uniformly. Then a pH buffer solution is added to control the pH of the system at 5. The obtained mixed system is then sent into a water bath, the water bath temperature is controlled at 45°C, and the enzyme is hydrolyzed under acidic conditions for 60 min to obtain a first enzyme hydrolysis liquid. Example 14

[0055] This example is basically the same as example 1, and the difference is only that the enzyme hydrolysis step in step S2 is different.

[0056] In step S2, the solid phase is re-added to the liquid phase, and then xylanase is added to control the mass concentration of the xylanase in the system at 0.02%, and stirred uniformly. Then a pH buffer solution is added to control the pH of the system at 5. The obtained mixed system is then sent into a water bath, the water bath temperature is controlled at 50°C, and the enzyme is hydrolyzed for 60 min to obtain a second enzyme hydrolysis liquid. Example 15

[0057] An application of a biological enzyme-hydrolyzed plum extract in cigarettes.

[0058] The extract prepared in example 1 is selected, and diluted with deionized water to a concentration of 10% to obtain a diluent. According to mass fraction, 1 part of the diluent is sprayed onto 20 parts of cigarette tobacco, and naturally dried. Example 16

[0059] A biological enzyme-hydrolyzed apple extract is prepared by the following steps:

[0060] S1. Fresh and undamaged apples are selected, cored and cut into pieces after washing, and then vacuum freeze-dried for standby. A container containing an ethylene solution with a concentration of 1 mg / L is placed in a water bath, the water bath temperature is controlled at 15°C, and preheated at 15°C for 60 min. Then the apple pieces are soaked in the above-mentioned ethylene solution, taken out after 2h of 15°C soaking, and dried with 15°C airflow to a surface moisture content of 8%. According to mass fraction, 1 part of dried apple pieces and 30 parts of water are mixed uniformly, and then a mixed enzyme of polygalacturonase and cellulase with a mass ratio of 2:1.2 is added to control the mass concentration of the mixed enzyme in the system at 1.5%, and stirred uniformly. The obtained mixed system is then sent into a water bath, the water bath temperature is controlled at 40°C, and the enzyme is hydrolyzed under neutral conditions for 90 min to obtain a first enzyme hydrolysis liquid.

[0061] S2. The primary enzymatic hydrolysis liquid is filtered using a filter screen to separate solid phase and liquid phase. A container containing an ethephon solution with a concentration of 480 mg / L is placed in a water bath, and the water bath temperature is controlled at 55°C. The solid phase is immersed in the ethephon solution at 55°C for 2 hours, and then taken out and dried at 55°C until the surface moisture content is 8%. The solid phase is added back to the liquid phase, and xylanase is added to the system, with the mass concentration of the xylanase controlled at 0.03%. The mixture is stirred uniformly, and then placed in a water bath. The water bath temperature is controlled at 45°C, and the enzymatic hydrolysis is performed under neutral conditions for 90 minutes to obtain a secondary enzymatic hydrolysis liquid.

[0062] S3. Hydrochloric acid is added to the secondary enzymatic hydrolysis liquid to adjust the pH of the secondary enzymatic hydrolysis liquid to 1.0. Then, 10 times the volume of water is added to the secondary enzymatic hydrolysis liquid, and the extraction is performed at 50°C for 80 minutes. After the extraction is completed, the mixture is centrifuged at 6000 r / min for 30 minutes, and the supernatant is filtered through an 800D filter membrane to obtain an extract.

[0063] An application of a biological enzymatic hydrolysis apple extract in cigarettes.

[0064] The prepared extract is diluted with deionized water to a concentration of 10% to obtain a dilution liquid. According to the mass parts, 1 part of the dilution liquid is sprayed onto 10 parts of cigarette tobacco, and then naturally dried.

[0065] Comparative Example 1

[0066] This comparative example is basically the same as Example 1, except that in step S1, an ethephon solution with a concentration of 0 mg / L is used, i.e., the ethephon solution is replaced with an equal mass of deionized water.

[0067] Comparative Example 2

[0068] This comparative example is basically the same as Example 1, except that in step S1, an ethephon solution with a concentration of 200 mg / L is used.

[0069] Comparative Example 3

[0070] This comparative example is basically the same as Example 1, except that in step S2, an ethephon solution with a concentration of 200 mg / L is used.

[0071] Comparative Example 4

[0072] This comparative example is basically the same as Example 1, except that in step S2, an ethephon solution with a concentration of 700 mg / L is used.

[0073] Comparative Example 5

[0074] The comparative example is basically the same as example 1, the difference is that the ethylene glycol in steps S1, S2 is replaced by the same mass of deionized water.

[0075] Extract polysaccharide detection

[0076] To the extract prepared in the examples and comparative examples, 3 times the volume of anhydrous ethanol was added and stirred, and then placed at 4℃ for 12h to obtain the precipitate. The polysaccharide in the precipitate was analyzed.

[0077] The precipitate was redissolved with deionized water, and the supernatant was taken. The molecular weight and distribution of the polysaccharide in the supernatant were detected by high performance gel permeation chromatography technology using a TSKgel GMPWXL high performance gel permeation chromatography column and a TSK guard column PWXL high performance gel chromatography protection column in series. The mobile phase was 0.1mol / L Na2SO4 aqueous solution, the column temperature was 50℃, the flow rate was 0.5mL / min, and the differential refractive index detector was used. The chromatographic column was calibrated with dextran standard substance for molecular weight determination, and the injection volume was 25μL. The weight fraction of the component with a molecular weight of 3500~8000 (including 3500 and 8000) was calculated, and the weight fraction of the component with a molecular weight of less than 3500 was calculated. The results are shown in Table 1.

[0078] Table 1.

[0079]

[0080] As shown in Table 1, by comparing Example 1 and Comparative Example 5, it can be seen that after the fruit is soaked with ethephon and then subjected to enzymolysis, the enzymolysis efficiency can be improved, the content of low molecular weight oligosaccharides in the product can be increased, and the content of polysaccharides with a molecular weight of 3500-8000 can be greatly increased. Based on Comparative Example 1, by comparing Comparative Example 1 with Examples 1-4 and Comparative Example 2, it can be seen that the concentration of ethephon has an effect on the activities of polygalacturonase and cellulase. When the concentration of ethephon is within the range of 100 mg / L, it greatly promotes the activity of cellulase, significantly increases the content of polysaccharides with a molecular weight of 3500-8000, and slightly promotes the activity of polygalacturonase, slightly increases the content of low molecular weight oligosaccharides. By comparing Example 1 with Examples 5-6 and Comparative Examples 3-4, it can be seen that the concentration of ethephon has an effect on the activity of xylanase. Too high or too low a concentration of ethephon can reduce the activity of xylanase, greatly affect the content of polysaccharides with a molecular weight of 3500-8000 in the product, and slightly affect the content of low molecular weight oligosaccharides in the product. By comparing Example 1 with Examples 7-8, it can be seen that the soaking temperature has an effect on the effect of ethephon on polygalacturonase and cellulase. As the temperature rises, the activity of cellulase decreases, the activity of polygalacturonase increases, the content of polysaccharides with a molecular weight of 3500-8000 decreases, and the content of low molecular weight oligosaccharides increases. By comparing Example 1 with Examples 9-10, it can be seen that the soaking temperature has an effect on the effect of ethephon on xylanase. As the temperature decreases, the activity of xylanase is too low to affect the content of polysaccharides and oligosaccharides. However, too high a temperature can reduce the activity of xylanase. By comparing Example 1 with Examples 12-14, it can be seen that the pH value of the system during the enzymolysis process and the extraction process also has an effect on the enzyme activity. Generally, enzymolysis and extraction under acidic conditions can improve the enzyme activity. However, under the same acidic conditions, the activity of polygalacturonase is increased to a greater extent than the activity of cellulase, which slightly increases the content of polysaccharides and greatly increases the content of oligosaccharides in Example 13, and reduces the proportion of polysaccharides in the extract.

[0081] Moisture retention capacity detection

[0082] After the extracts in the examples and comparative examples were respectively diluted to 10% with deionized water, they were respectively sprayed onto equal amounts of tobacco shreds, and the tobacco shreds were sent into a constant temperature and humidity environment with a temperature of 20℃ and a relative humidity of 40% for 48h of balancing.

[0083] Accurately weigh 5 g (accurate to 0.0001 g) of the balanced tobacco and tobacco products, and determine the initial moisture content according to the method of national standard YCT 31-1996; meanwhile, accurately weigh 5 g (accurate to 0.0001 g) of the balanced same tobacco and tobacco products, and use the continuous tobacco moisture content measuring device to determine the change of the mass of the tobacco sample with time in a constant environment (20℃, relative humidity 40%), and the results are shown in Table 2.

[0084] Table 2.

[0085]

[0086] As shown in Table 2, and in combination with Table 1, it can be seen from the comparison of the examples and the comparative examples that the extract with a relatively high content of polysaccharides with a molecular weight of 3500-8000 has a slower decrease in the moisture content of the tobacco shreds, and has a better effect of moisture retention for the tobacco shreds.

[0087] The specific embodiments described herein are merely illustrative of the spirit of the present application. Those skilled in the art of the present application can make various modifications or supplements to the described specific embodiments or use similar ways to replace, but will not deviate from the spirit of the present application or exceed the scope defined by the appended claims.

Claims

1. A method for preparing fruit extract by bioenzymatic hydrolysis, characterized in that: The following steps are involved: S1. The fruit is soaked in a 1-100 mg / L ethephon solution, and then dried after soaking; the fruit is then enzymatically hydrolyzed with polygalacturonase and cellulase to obtain a primary hydrolyzate; in step S1, the soaking temperature is 10-30°C, and the hydrolysis temperature is 40-50°C; the enzymatic hydrolysis condition is neutral, or a pH buffer solution is added to control the pH of the system to 5; S2. The primary hydrolyzate was subjected to solid-liquid separation to obtain a solid phase and a liquid phase; the solid phase was soaked in an ethephon solution at a concentration of 450~550mg / L, and after soaking, the solid phase was removed and dried, and re-added to the liquid phase; xylanase was then added to the liquid phase for enzymatic hydrolysis to obtain a secondary hydrolyzate; In step S2, the soaking temperature is 50-60°C, the enzymatic hydrolysis temperature is 45-55°C; the enzymatic hydrolysis condition is neutral, or a pH buffer solution is added to control the pH of the system to 5; S3. The secondary enzymatic hydrolysate is extracted with a solvent to obtain a fruit extract; in step S3, the pH of the secondary enzymatic hydrolysate is adjusted to 1.0-3.0, and then the extract is performed at 50-80°C.

2. The method for preparing fruit extract by bioenzymatic hydrolysis according to claim 1, wherein: In step S1, an ethephon solution with a concentration of 20-30 mg / L is used.

3. The method for preparing fruit extract by bioenzymatic hydrolysis according to claim 1, wherein: In step S1, the total mass concentration of polygalacturonase and cellulase is 0.5%-1.5%, and the mass ratio of polygalacturonase to cellulase is 2:(0.8-1.2).

4. The method for preparing fruit extract by bioenzymatic hydrolysis according to claim 1, wherein: In step S2, the mass concentration of xylanase is 0.01% to 0.03%.

5. The method for preparing fruit extract by bioenzymatic hydrolysis according to claim 1, wherein: Ultrasonic-assisted extraction was used with an ultrasonic power of 100-300 W and an ultrasonic time of 20-60 min.

6. A bio-enzymatic fruit extract obtained by the method according to any one of claims 1 to 5.

7. Use of the bio-enzymatically hydrolyzed fruit extract according to claim 6 in cigarettes.

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