Method for extracting polysaccharide by reutilizing tobacco leaf residues and application

Abstract

The invention relates to the technical field of polysaccharide extraction, in particular to a method for extracting polysaccharide by reusing tobacco leaf residues and application. Comprising the following steps: soaking the tobacco leaf residues obtained by supercritical extraction in a water bath, adding a disodium hydrogen phosphate-citric acid buffer solution to adjust the pH value to 4.5-5.5, adding a compound enzyme preparation, heating in a water bath for enzymolysis, boiling for inactivation, cooling, extracting by adopting a flash extraction method, filtering, concentrating under reduced pressure to obtain a concentrated solution, adding 95% ethanol for alcohol precipitation, centrifuging, taking a precipitate, and drying to obtain the tobacco leaf residues. And adding water to redissolve, adding papain to perform enzymolysis, inactivating, cooling to room temperature, centrifuging to remove the precipitate, dialyzing the crude polysaccharide solution, concentrating, and freeze-drying to obtain the refined tobacco polysaccharide. According to the method, the supercritical extraction tobacco leaf residues are used as raw materials to extract the polysaccharide, so that the extraction efficiency and the extraction rate are improved, the use of an organic solvent can be avoided, and resource waste and environmental pollution are reduced.

Description

Technical Field

[0001] This invention relates to the field of polysaccharide extraction technology, specifically to a method and application for extracting polysaccharides from tobacco residue. Background Technology

[0002] Tobacco, as an indispensable pillar of the global economy, is not only a core raw material in the cigarette manufacturing industry but also widely used in the high-end development of tobacco flavorings and fragrances. Among these applications, the extraction of tobacco essential oils using supercritical CO2 technology, as an important flavor enhancer, significantly improves the aroma and taste of cigarettes and enhances tobacco quality, thus gaining widespread use in the tobacco industry. However, it is worth noting that while this process efficiently extracts tobacco essential oils, it also generates a large amount of tobacco leaf residue resources that have not yet been fully developed and recycled. As an indispensable byproduct of the tobacco processing industry chain, these residues contain rich bioactive components and have significant potential application value. Therefore, the in-depth development and comprehensive utilization of tobacco leaf residues are of great significance to the sustainable development of the tobacco industry.

[0003] In the application of supercritical CO2 extraction technology to tobacco oil production, this technology effectively extracts oils and volatile components from tobacco leaves due to its superior extraction capabilities. Simultaneously, non-volatile, CO2-insoluble components such as polysaccharides and proteins are retained in the extracted tobacco residue. This phenomenon not only reveals the limitations of pretreatment steps such as defatting and decolorization in traditional polysaccharide extraction methods, but also opens up an innovative path—using the tobacco residue after supercritical CO2 extraction as a novel and efficient raw material for polysaccharide extraction, thereby achieving full utilization of this valuable resource. Summary of the Invention

[0004] The purpose of this invention is to provide a method and application for extracting polysaccharides from tobacco residue, aiming to realize the resource utilization of tobacco residue obtained by supercritical extraction in industrial production and improve the added value and competitiveness of the tobacco industry.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0006] A method for extracting polysaccharides from tobacco residue includes the following steps:

[0007] S1. Add the tobacco residue obtained by supercritical extraction to a set amount of water and soak it in a water bath for 30 to 60 minutes.

[0008] S2. Add disodium hydrogen phosphate-citric acid buffer to adjust the pH to 4.5-5.5, add the compound enzyme preparation, and heat in a water bath at 45-50℃ for 1-2 hours for enzymatic hydrolysis.

[0009] S3. Boil for 5-10 minutes to inactivate the tobacco leaves. When the temperature is cooled to 55-60℃, use flash extraction for 1 minute. Filter the mixture and add 45-50℃ warm water (set mass) to the tobacco residue. Repeat the extraction 2-3 times. After filtration, combine the filtrates and concentrate under reduced pressure to obtain the concentrate.

[0010] S4. Add 4-5 times the mass of 95% ethanol to the concentrate, precipitate at 4 ℃ for 24-36 h, centrifuge, take the precipitate, add 2-3 times the mass of 95% ethanol and wash 2-3 times to obtain the crude polysaccharide precipitate of tobacco leaves.

[0011] S5. Add water of a set mass to reconstitute the crude polysaccharide, add papain for 1 h of enzymatic hydrolysis, inactivate, cool to room temperature, centrifuge to remove the precipitate, dialyze the crude polysaccharide solution, concentrate, and freeze dry to obtain refined tobacco polysaccharide.

[0012] Furthermore, in step S1, add 10-15 times the weight of water and soak in a water bath at 45-50℃ for 30-60 minutes.

[0013] Furthermore, in step S2, the mass ratio of cellulase to pectinase in the compound enzyme preparation is 1:1, and the compound enzyme preparation with a mass percentage of 1%-1.5% of tobacco residue is added.

[0014] Furthermore, in step S3, 8 to 10 times the mass of warm water at 45 to 50°C is added to the tobacco residue.

[0015] Further, in step S4, the specific steps are as follows: add 4 to 5 times the mass of 95% ethanol to the concentrate, precipitate at 4 ℃ for 24 to 36 h, centrifuge at 5000 to 6000 r / min for 8 to 12 min, take the precipitate, add 2 to 3 times the mass of 95% ethanol and wash 2 to 3 times to obtain the crude polysaccharide precipitate of tobacco leaves.

[0016] Further, in step S5, the specific steps are as follows: add 10 times the mass of water to reconstitute the crude polysaccharide, add 1% by mass of 20 mg / mL papain, enzymatically hydrolyze in a 50 ℃ water bath for 1 h, raise the temperature to boiling, inactivate for 8-10 min, cool to room temperature, centrifuge at 6000-8000 r / min for 5-10 min to remove the precipitate, dialyze the crude polysaccharide solution using a dialysis bag with a molecular weight of 3500 Da for 48-72 h, concentrate, and freeze-dry to obtain refined tobacco polysaccharide.

[0017] One application is the use of tobacco polysaccharides extracted from any of the above methods in cigarettes.

[0018] Furthermore, the tobacco polysaccharide is prepared into a 10% (w / w) aqueous solution and sprayed evenly onto the tobacco shreds at a ratio of 0.5-3% (w / w) by weight.

[0019] The beneficial effects of this invention are:

[0020] 1. This invention uses supercritical CO2 extraction of tobacco residue as raw material for polysaccharide extraction, which can eliminate the defatting and decolorization steps in the traditional polysaccharide extraction process, improve extraction efficiency and extraction rate, and avoid the use of organic solvents, thereby reducing resource waste and environmental pollution.

[0021] 2. Compared with traditional polysaccharide extraction methods, this invention uses flash extraction technology for the extraction of polysaccharides from tobacco residues by supercritical CO2 extraction, which has the advantages of small solvent consumption, short extraction time, high extraction efficiency, mild extraction conditions, simple operation and easy industrial production.

[0022] 3. This invention uses enzymatic hydrolysis to break down the cell walls and remove proteins from the raw materials, which can increase extraction efficiency, shorten extraction time, and avoid the use of organic solvents and damage to the polysaccharide structure, thus having the advantages of being green and environmentally friendly. Detailed Implementation

[0023] The technical solutions of the present invention will be described in detail below through embodiments. The following embodiments are merely exemplary and can only be used to explain and illustrate the technical solutions of the present invention, and should not be construed as limiting the technical solutions of the present invention.

[0024] This application provides a method for extracting polysaccharides from tobacco residue, comprising the following steps:

[0025] S1. Take 100-150 g of tobacco residue obtained from supercritical extraction, add 10-15 times the weight of water, and soak in a water bath at 45-50℃ for 30-60 min.

[0026] S2. Adjust the pH to 4.5-5.5 by adding disodium hydrogen phosphate-citric acid buffer, add 1%-1.5% by weight of compound enzyme preparation (cellulase and pectinase in a 1:1 mass ratio), and heat in a water bath at 45-50℃ for 1-2 hours for enzymatic hydrolysis.

[0027] S3. Boil to inactivate for 5-10 minutes. When cooled to 55-60℃, extract for 1 minute using flash extraction (220 V, 2000 W). Filter and add 8-10 times the mass of 45-50℃ warm water to the tobacco residue. Repeat the extraction 2-3 times. After filtration, combine the filtrates. Concentrate under reduced pressure to 1 / 5-1 / 8 of the filtrate mass.

[0028] S4. Add 4-5 times the mass of 95% ethanol to the concentrate, precipitate at 4 ℃ for 24-36 h, centrifuge at 5000-6000 r / min for 8-12 min, take the precipitate, add 2-3 times the mass of 95% ethanol and wash 2-3 times to obtain the crude polysaccharide precipitate of tobacco leaves.

[0029] S5. Add 10 times the mass of water to reconstitute the crude polysaccharide, add 1% of 20 mg / mL papain, and enzymatically hydrolyze in a 50 ℃ water bath for 1 h. Then raise the temperature to boiling and inactivate for 8-10 min. Cool to room temperature and centrifuge at 6000-8000 r / min for 5-10 min to remove the precipitate. Dialyze the crude polysaccharide solution using a dialysis bag with a molecular weight of 3500 Da for 48-72 h, concentrate, and freeze-dry to obtain refined tobacco polysaccharide.

[0030] This application also relates to the application of tobacco polysaccharides in cigarettes. The obtained tobacco polysaccharides were prepared into a 10% (w / w) polysaccharide aqueous solution. A certain mass of tobacco shreds from Xiangxian County, Henan Province, was placed in an environment with a temperature of (22±1) ℃ and a relative humidity of (60%±2%) for 48 h to equilibrate. The samples were then divided into two groups, and the polysaccharide aqueous solution and distilled water were evenly sprayed on each group at a tobacco shred mass ratio of 0.5%. The distilled water group served as a blank control group. Both groups of samples were further equilibrated in an environment with a temperature of (22±1) ℃ and a relative humidity of (60%±2%) for 48 h. A certain amount of tobacco shreds was weighed from each group and then placed in a glove box with a temperature of 22±1 ℃ and a relative humidity of 40±2% to examine its dehumidification process. The tobacco shreds samples were tested after 24, 48, and 72 h to obtain the equilibrium moisture content of the polysaccharide group and the control group. Each group of samples was subjected to three parallel tests to examine its physical moisture retention performance.

[0031] Example 1 Enzymatic hydrolysis

[0032] Take 100 g of tobacco residue obtained from supercritical extraction, add 1200 g of water, and soak in a 50°C water bath for 40 min; add disodium hydrogen phosphate-citric acid buffer to adjust the pH to 5, add 1.2 g of compound enzyme preparation (cellulase and pectinase (mass ratio 1:1), and heat in a 50°C water bath for 2 h for enzymatic hydrolysis.

[0033] Example 2 Preparation of crude polysaccharides from tobacco leaves

[0034] Boil for 8 min to inactivate the polysaccharide. After cooling to 60℃, extract for 1 min using flash extraction (220 V, 2000 w). Filter and add 1000 g of 50℃ warm water to the tobacco residue for repeated extraction twice. Combine the filtrates after filtration. Concentrate under reduced pressure to 500 g. Add 2000 g of 95% ethanol to the concentrate and precipitate at 4℃ for 24 h. Centrifuge at 5000 r / min for 10 min, collect the precipitate, and wash three times with 3 times its weight of 95% ethanol to obtain 4.6 g of crude tobacco polysaccharide precipitate.

[0035] Example 3 Preparation of refined tobacco polysaccharides

[0036] The crude polysaccharide was reconstituted with 46 g of water, and 0.046 g of 20 mg / mL papain was added. The mixture was enzymatically hydrolyzed in a 50 ℃ water bath for 1 h, then heated to boiling and inactivated for 8 min. After cooling to room temperature, the mixture was centrifuged at 6000 r / min for 8 min to remove the precipitate. The crude polysaccharide solution was dialyzed for 72 h using a dialysis bag with a molecular weight of 3500 Da. After concentration and freeze-drying, 4.3 g of refined tobacco polysaccharide was obtained.

[0037] Example 4 Physical Moisture Retention Performance Test

[0038] Take 2 g of refined tobacco polysaccharide and add 18 g of distilled water to prepare a polysaccharide aqueous solution with a mass fraction of 10%. Take 40 g of Xiangxian tobacco shreds from Henan Province and place them in an environment with a temperature of (22±1)℃ and a relative humidity of (60±2)% for 48 h to equilibrate. Then divide them into two groups (20 g in each group). Spray polysaccharide aqueous solution and distilled water evenly at a tobacco shred mass ratio of 0.5%. The distilled water group serves as a blank control group. Both groups of samples continue to equilibrate in an environment with a temperature of (22±1)℃ and a relative humidity of (60%±2%) for 48 h. Weigh 6.0 g of tobacco shreds from each group and place them in a glove box with a temperature of 22±1℃ and a relative humidity of 40±2% to investigate their dehumidification process. Test the tobacco shreds samples placed for 24, 48 and 72 h respectively to obtain the moisture content of the polysaccharide group and the control group (Table 1). Each group of samples was tested in three parallel experiments. As shown in Table 1, after 72 hours of storage, the moisture content of the control group tobacco decreased from 12.5±0.1% to 7.6±0.2%, with a change rate of 39.2%. In contrast, after 72 hours of storage, the moisture content of the polysaccharide group tobacco decreased from 13.4±0.1% to 11.1±0.2%, with a change rate of 17.2%, which was much lower than that of the control group. This indicates that the prepared tobacco polysaccharide has excellent physical moisture retention properties for tobacco.

[0039] Table 1. Changes in equilibrium moisture content of tobacco shreds after spraying with polysaccharide aqueous solution and distilled water (%)

[0040] Time / h sample 0 h 24 h 48 h 72 h control group 12.5±0.1 9.8±0.2 8.5±0.2 7.6±0.3 polysaccharide group 13.4±0.1 12.8±0.1 11.9±0.1 11.1±0.2

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for extracting polysaccharides from tobacco residue, characterized in that, Includes the following steps: S1. Add the tobacco residue obtained by supercritical extraction to a set amount of water and soak it in a water bath for 30 to 60 minutes. S2. Add disodium hydrogen phosphate-citric acid buffer to adjust the pH to 4.5-5.5, add the compound enzyme preparation, and heat in a water bath at 45-50℃ for 1-2 hours for enzymatic hydrolysis. S3. Boil for 5-10 minutes to inactivate the tobacco leaves. When the temperature is cooled to 55-60℃, use flash extraction for 1 minute. Filter the mixture and add 45-50℃ warm water (set mass) to the tobacco residue. Repeat the extraction 2-3 times. After filtration, combine the filtrates and concentrate under reduced pressure to obtain the concentrate. S4. Add 4-5 times the mass of 95% ethanol to the concentrate, precipitate at 4 ℃ for 24-36 h, centrifuge, take the precipitate, add 2-3 times the mass of 95% ethanol and wash 2-3 times to obtain the crude polysaccharide precipitate of tobacco leaves. S5. Add water of a set mass to reconstitute the crude polysaccharide, add papain for 1 h of enzymatic hydrolysis, inactivate, cool to room temperature, centrifuge to remove the precipitate, dialyze the crude polysaccharide solution, concentrate, and freeze dry to obtain refined tobacco polysaccharide.

2. The method for extracting polysaccharides from tobacco residue according to claim 1, characterized in that, In step S1, add 10-15 times the weight of water and soak in a water bath at 45-50℃ for 30-60 minutes.

3. The method for extracting polysaccharides from tobacco residue according to claim 1, characterized in that, In step S2, the mass ratio of cellulase to pectinase in the compound enzyme preparation is 1:1, and 1%-1.5% of the compound enzyme preparation of tobacco residue is added.

4. The method for extracting polysaccharides from tobacco residue according to claim 1, characterized in that, In step S3, add 8 to 10 times the weight of warm water at 45 to 50°C to the tobacco residue.

5. The method for extracting polysaccharides from tobacco residue according to claim 1, characterized in that, In step S4, the specific steps are as follows: add 4 to 5 times the mass of 95% ethanol to the concentrate, precipitate at 4 ℃ for 24 to 36 h, centrifuge at 5000 to 6000 r / min for 8 to 12 min, take the precipitate, add 2 to 3 times the mass of 95% ethanol and wash 2 to 3 times to obtain the crude polysaccharide precipitate of tobacco leaves.

6. The method for extracting polysaccharides from tobacco residue according to claim 1, characterized in that, In step S5, the specific steps are as follows: add 10 times the mass of water to reconstitute the crude polysaccharide, add 1% of 20 mg / mL papain, enzymatically hydrolyze in a 50 ℃ water bath for 1 h, raise the temperature to boiling, inactivate for 8-10 min, cool to room temperature, centrifuge at 6000-8000 r / min for 5-10 min to remove the precipitate, dialyze the crude polysaccharide solution using a dialysis bag with a molecular weight of 3500 Da for 48-72 h, concentrate, and freeze-dry to obtain refined tobacco polysaccharide.

7. An application characterized in that, The application of tobacco polysaccharides extracted according to any one of claims 1 to 6 in cigarettes.

8. The application according to claim 7, characterized in that, Prepare a 10% (w / w) aqueous solution of tobacco polysaccharide and spray it evenly onto the tobacco shreds at a ratio of 0.5-3% (w / w) of tobacco shreds.