Preparation method of sargassum fusiforme extract and application of sargassum fusiforme extract in cigarettes

Abstract

The invention discloses a preparation method of a sargassum fusiforme extract and application of the sargassum fusiforme extract in cigarettes, and the preparation method comprises the following steps: washing sargassum fusiforme with clear water to remove silt, salt and the like, then transferring the sargassum fusiforme into low-temperature rolling-freeze-drying combined equipment for treatment, and crushing the sargassum fusiforme; mixing the sargassum fusiforme powder with a sodium citrate solution in proportion, and performing ultrasonic pretreatment to obtain a sargassum fusiforme suspension; adjusting the pH value of the suspension by using 0.08 mol / L citric acid-sodium citrate buffer solution, placing the suspension in a constant-temperature oscillation enzymolysis tank, and adding a compound enzyme preparation; raising the temperature of the material liquid after enzymolysis for enzyme deactivation, and centrifuging after the liquid is cooled to take supernate; and carrying out ultrafiltration on the supernatant by using an ultrafiltration membrane, and carrying out vacuum concentration and drying treatment on the ultrafiltrate. Efficient release and enrichment of active ingredients are achieved through the synergistic effect of ultrasonic-assisted wall breaking, compound enzyme system degradation, membrane treatment and the like, meanwhile, the extract is applied to cigarettes, the cigarettes are endowed with unique seaweed faint scent and sweetness, irritation is reduced, and aftertaste is improved.

Description

Technical Field

[0001] This invention relates to the field of interdisciplinary technology of biotechnology and tobacco products, and in particular to a method for preparing Sargassum fusiforme extract and its application in cigarettes. Background Technology

[0002] Sargassum fusiforme, a plant belonging to the Sargassum family of the Phaeophyta phylum, is rich in active ingredients such as seaweed polysaccharides, polyphenols, betaine, and mannitol. It possesses various physiological functions, including antioxidant, anti-inflammatory, lipid-lowering, and immune-enhancing effects. Its unique chemical composition makes it a potential source of natural tobacco additives. Enzymatic hydrolysis is a green and efficient extraction technology that has been developed in recent years. By destroying the cell wall structure of plants through biological enzymatic hydrolysis, active ingredients are released, which can significantly improve extraction efficiency and product activity. However, there are no reports on the application of Sargassum fusiforme extract prepared by enzymatic hydrolysis in the cigarette industry, although traditional Sargassum fusiforme extract has been tried in the cigarette industry.

[0003] Patent CN1961761A discloses the application of Sargassum fusiforme water extract in tobacco, indicating that it can improve cigarette quality, promote salivation, and relieve phlegm and cough. Patent CN1962699B introduces the extraction of Sargassum fusiforme polysaccharides and their application in tobacco, indicating that they can reduce certain harmful components in mainstream cigarette smoke.

[0004] The extracts of Sargassum fusiforme obtained by traditional water extraction methods have large molecular weights and limited biological activity, resulting in less than ideal application effects in cigarettes. Furthermore, problems such as insufficient cell wall breaking efficiency of single enzyme systems and difficulty in obtaining dedicated enzyme sources remain unresolved. Therefore, developing an efficient and stable composite enzymatic hydrolysis process and applying it to the cigarette industry has significant innovative value and application potential. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing Sargassum fusiforme extract and its application in cigarettes. By using ultrasonic-assisted cell wall disruption, combined enzyme degradation, membrane treatment and other synergistic effects, the active ingredients can be efficiently released and enriched. At the same time, the extract is applied to cigarettes to give them a unique seaweed aroma and sweetness, reduce irritation and improve the aftertaste.

[0006] The above-mentioned technical objective of the present invention is achieved through the following technical solution: A method for preparing Sargassum fusiforme extract includes the following steps: S1. Raw material pretreatment: The seaweed is first rinsed with clean water 3 to 5 times to remove mud, sand, salt and other substances, and then transferred to a low temperature roller pressing-freeze drying equipment for processing. After being crushed, seaweed powder is obtained. S2. Preparation of Sargassum fusiforme suspension: Sargassum fusiforme powder was mixed with 0.03-0.06% (w / v) sodium citrate solution at a material-to-liquid ratio of 1:20-1:30, and then pretreated with ultrasound at 300-350W and 45 kHz for 15-20 min to obtain Sargassum fusiforme suspension. S3. Enzymatic hydrolysis reaction: First, adjust the pH of the suspension to 6.0~7.0 with 0.08 mol / L citrate-sodium citrate buffer, then place it in a constant temperature shaking enzymatic hydrolysis vessel, add the compound enzyme preparation, and enzymatically hydrolyze at 35~45℃ and 120~130 rpm for 3~4 h. S4. Enzyme inactivation and solid-liquid separation: Heat the enzymatically hydrolyzed liquid to 85~95℃ to inactivate the enzyme for 10~15 min. After cooling the liquid, centrifuge at 6000~8000 rpm for 15~20 min and collect the supernatant. S5. Purification and Concentration: The supernatant is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 8000~10000 Da. The ultrafiltrate is then concentrated under reduced pressure and dried to obtain Sargassum fusiforme extract.

[0007] Further: In S1, the washed Sargassum fusiforme is first placed in a roller press and rolled three times at 0~5℃ to initially rupture the cell walls. Then it is transferred to a freeze dryer and freeze-dried at -30~-25℃ and vacuum degree ≤15Pa for 6~8 hours to remove moisture. Finally, it is pulverized to 70~80 mesh using a temperature-controlled ultrafine pulverizer to obtain Sargassum fusiforme powder, which is then sealed and stored in an aluminum foil bag containing a desiccant.

[0008] Furthermore: the rolling pressure of the roller pressing equipment is 5~8 MPa, and it is left to stand for 10 minutes after each rolling. The temperature inside the temperature-controlled ultrafine pulverizer is 28~32℃.

[0009] Furthermore, in S3, the complex enzyme preparation is based on alginate lyase as the core, supplemented by enzyme preparations such as β-glucanase, fucoidanase and esterase, thereby forming a binary, ternary or quaternary multi-component enzyme system.

[0010] Furthermore, the binary enzyme system consists of alginate lyase and β-glucanase, with an enzyme activity ratio of 4:1 to 6:1 and a total enzyme addition of 70 to 90 U / g of raw material.

[0011] Furthermore, the ternary enzyme system consists of alginate lyase, β-glucanase, and fucoidanase, with an enzyme activity ratio of 6:2:1 to 8:3:1, and a total enzyme addition of 100 to 120 U / g of raw material.

[0012] Furthermore, the quaternary enzyme system consists of alginate lyase, β-glucanase, fucoidanase, and esterase, with an enzyme activity ratio of 8:2:1:0.5 to 10:3:1:1, and a total enzyme addition of 110 to 130 U / g of raw material.

[0013] Furthermore: In S5, the operating parameters for the vacuum concentration are: vacuum degree 0.06 MPa~0.08 MPa, temperature 45~55℃; the drying process is spray drying and its operating parameters are: air inlet 190℃, air outlet 85℃, spray pressure 0.2 MPa.

[0014] The present invention also provides an application of Sargassum fusiforme extract in cigarettes. Sargassum fusiforme extract is dissolved in purified water or 50%~70% ethanol aqueous solution to prepare a solution with a mass concentration of 1%~10%, and then added at an amount of 0.01%~0.5% of the weight of cigarette tobacco, and evenly sprayed or coated onto the tobacco.

[0015] In summary, the present invention has the following beneficial effects: Firstly, this invention constructs a multi-enzyme system with alginate lyase as the core, which can simultaneously degrade alginate, β-glucan and intracellular fucoidan in Sargassum fusiforme cell walls. At the same time, esterase can convert ester aroma precursors, realizing one-step extraction of multiple active ingredients. Secondly, the conditions of this invention are mild and protect the activity. It adopts a neutral buffer system with pH 6.0~7.0 and low-temperature enzymatic hydrolysis at 35~40℃, which avoids the destruction of active ingredients by acid-base method and high temperature method. Fucoidan, ester aroma and other components are retained at a high rate, and the bioactivity and application value of the product are significantly improved. Thirdly, this invention allows for flexible selection of binary, ternary, or quaternary enzyme systems based on different target products, supplemented by ultrasonic and ultrafiltration membrane treatments, to adapt to the synergistic extraction of polysaccharide-aroma components in the tobacco field. Furthermore, the buffer system and enzymatic hydrolysis parameters do not require significant adjustments, making the operation simple and easy to scale up industrially. Attached Figure Description

[0016] Figure 1 This is a flowchart of the preparation process of the present invention; Figure 2 This is a table showing the composition and application of the complex enzyme system of this invention. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to the accompanying drawings.

[0018] In the description of this invention, it should be understood that the terms "upper", "lower", "left", "right", "front", "rear", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0019] Example 1, referring to Figure 1-2 A method for preparing Sargassum fusiforme extract includes the following steps: S1. Raw material pretreatment: The seaweed is first rinsed with clean water 3-5 times to remove mud, sand, salt and other substances. Then, the washed seaweed is placed in a roller press with a pressure of 5-8 MPa and rolled 3 times at 0℃. After each roller press, it is left to stand for 10 minutes to allow the cell walls to break down initially. Then, it is transferred to a freeze dryer and freeze-dried at -25℃ and vacuum degree ≤15Pa for 6-8 hours to remove moisture. Finally, it is pulverized to 70 mesh in a temperature-controlled ultra-micro pulverizer with an internal temperature of 28-32℃. Then, it is sealed and stored in an aluminum foil bag containing desiccant. After pulverization, seaweed powder is obtained. S2. Preparation of Sargassum fusiforme suspension: Sargassum fusiforme powder was mixed with 0.03 (w / v) sodium citrate solution at a material-to-liquid ratio of 1:20, and then pretreated with ultrasound at 300W and 45 kHz for 15 min to obtain Sargassum fusiforme suspension. S3. Enzymatic hydrolysis reaction: First, adjust the pH of the suspension to 6.0 with 0.08 mol / L citrate-sodium citrate buffer, then place it in a constant temperature shaking enzymatic hydrolysis vessel, add a complex enzyme preparation with alginate lyase as the core, supplemented by β-glucanase, fucoidanase and esterase, etc., forming a binary enzyme system, in which the enzyme activity ratio of alginate lyase and β-glucanase is 4:1, and the total enzyme amount is 70~90U / g raw material, and then enzymatically hydrolyze at 35℃ and 120rpm for 3~4h. S4. Enzyme inactivation and solid-liquid separation: Heat the enzymatically hydrolyzed liquid to 85℃ for 10 min to inactivate the enzyme. After cooling the liquid, centrifuge at 6000 rpm for 15 min and take the supernatant. S5. Purification and Concentration: The supernatant is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 8000 Da to remove large molecular impurities and undegraded fibrous materials. The filtrate is collected and then concentrated under reduced pressure at a vacuum of 0.06 MPa to 0.08 MPa and a temperature of 45 to 55°C until it becomes a paste. Then, it is spray-dried under operating parameters of 190°C inlet air, 85°C outlet air, and 0.2 MPa spray pressure to obtain Sargassum fusiforme extract.

[0020] Example 2, a method for preparing Sargassum fusiforme extract, comprising the following steps: S1. Raw material pretreatment: The seaweed is first rinsed with clean water 3-5 times to remove mud, sand, salt and other substances. Then, the washed seaweed is placed in a roller press with a pressure of 5-8 MPa and rolled 3 times at 5℃. After each roller press, it is left to stand for 10 minutes to allow the cell walls to break down initially. Then, it is transferred to a freeze dryer and freeze-dried at -30℃ and vacuum degree ≤15Pa for 6-8 hours to remove moisture. Finally, it is pulverized to 80 mesh in a temperature-controlled ultra-micro pulverizer with an internal temperature of 28-32℃. Then, it is sealed and stored in an aluminum foil bag containing desiccant. After pulverization, seaweed powder is obtained. S2. Preparation of Sargassum fusiforme suspension: Sargassum fusiforme powder was mixed with 0.05 (w / v) sodium citrate solution at a material-to-liquid ratio of 1:30, and then pretreated with ultrasound at 350W and 45 kHz for 20 min to obtain Sargassum fusiforme suspension. S3. Enzymatic hydrolysis reaction: First, adjust the pH of the suspension to 6.5 with 0.08 mol / L citrate-sodium citrate buffer, then place it in a constant temperature shaking enzymatic hydrolysis vessel, add a ternary complex enzyme preparation composed of alginate lyase, β-glucanase and fucoidanase in a ratio of 6:2:1~8:3:1, the total enzyme amount is 100~120U / g raw material, and then enzymatically hydrolyze at 37℃ and 120rpm for 3~4h. S4. Enzyme inactivation and solid-liquid separation: Heat the enzymatically hydrolyzed liquid to 90℃ for 15 min to inactivate the enzyme. After cooling the liquid, centrifuge at 8000 rpm for 20 min and take the supernatant. S5. Purification and Concentration: The supernatant is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 10000 Da to remove large molecular impurities and undegraded fibrous materials. The filtrate is collected and then concentrated under reduced pressure at a vacuum of 0.06 MPa to 0.08 MPa and a temperature of 45 to 55°C until it becomes a paste. Then, it is spray-dried under the working parameters of 190°C inlet air, 85°C outlet air, and 0.2 MPa spray pressure to obtain Sargassum fusiforme extract.

[0021] Example 3, a method for preparing Sargassum fusiforme extract, comprising the following steps: S1. Raw material pretreatment: The seaweed is first rinsed with clean water 3-5 times to remove mud, sand, salt and other substances. Then, the washed seaweed is placed in a roller press with a pressure of 5-8 MPa and rolled 3 times at 3℃. After each roller press, it is left to stand for 10 minutes to allow the cell walls to break down initially. Then, it is transferred to a freeze dryer and freeze-dried at -28℃ and vacuum degree ≤15Pa for 6-8 hours to remove moisture. Finally, it is pulverized to 75 mesh in a temperature-controlled ultra-micro pulverizer with an internal temperature of 28-32℃. Then, it is sealed and stored in an aluminum foil bag containing desiccant. After pulverization, seaweed powder is obtained. S2. Preparation of Sargassum fusiforme suspension: Sargassum fusiforme powder was mixed with 0.04 (w / v) sodium citrate solution at a material-to-liquid ratio of 1:20, and then pretreated with ultrasound at 320W and 45 kHz for 18 min to obtain Sargassum fusiforme suspension. S3. Enzymatic hydrolysis reaction: First, adjust the pH of the suspension to 7.0 with 0.08 mol / L citrate-sodium citrate buffer, then place it in a constant temperature shaking enzymatic hydrolysis vessel, add alginate lyase, β-glucanase, fucoidanase and esterase in a quaternary enzyme system with an enzyme activity ratio of 8:2:1:0.5~10:3:1:1, the total enzyme amount is 110-130 U / g raw material, and then enzymatically hydrolyze at 45℃ and 130 rpm for 3~4 hours. S4. Enzyme inactivation and solid-liquid separation: Heat the enzymatically hydrolyzed liquid to 95℃ for 10 min to inactivate the enzyme. After cooling the liquid, centrifuge at 7000 rpm for 18 min and take the supernatant. S5. Purification and Concentration: The supernatant is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 8000 Da to remove large molecular impurities and undegraded fibrous materials. The filtrate is collected and then concentrated under reduced pressure at a vacuum of 0.06 MPa to 0.08 MPa and a temperature of 45 to 55°C until it becomes a paste. Then, it is spray-dried under operating parameters of 190°C inlet air, 85°C outlet air, and 0.2 MPa spray pressure to obtain Sargassum fusiforme extract.

[0022] Sargassum extract is dissolved in purified water or 50%~70% ethanol aqueous solution to prepare a solution with a mass concentration of 1%~10%. Then, it is added at a rate of 0.01%~0.5% of the weight of cigarette tobacco and sprayed or coated evenly onto the tobacco.

[0023] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make inventive modifications to this embodiment as needed, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A method for preparing Sargassum fusiforme extract, characterized in that, Includes the following steps: S1. Raw material pretreatment: The seaweed is first rinsed with clean water 3 to 5 times to remove mud, sand, salt and other substances, and then transferred to a low temperature roller pressing-freeze drying equipment for processing. After being crushed, seaweed powder is obtained. S2. Preparation of Sargassum fusiforme suspension: Sargassum fusiforme powder was mixed with 0.03-0.06% (w / v) sodium citrate solution at a material-to-liquid ratio of 1:20-1:30, and then pretreated with ultrasound at 300-350W and 45 kHz for 15-20 min to obtain Sargassum fusiforme suspension. S3. Enzymatic hydrolysis reaction: First, adjust the pH of the suspension to 6.0~7.0 with 0.08 mol / L citrate-sodium citrate buffer, then place it in a constant temperature shaking enzymatic hydrolysis vessel, add the compound enzyme preparation, and enzymatically hydrolyze at 35~45℃ and 120~130 rpm for 3~4 h. S4. Enzyme inactivation and solid-liquid separation: Heat the enzymatically hydrolyzed liquid to 85~95℃ to inactivate the enzyme for 10~15 min. After cooling the liquid, centrifuge at 6000~8000 rpm for 15~20 min and collect the supernatant. S5. Purification and Concentration: The supernatant is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 8000~10000 Da. The ultrafiltrate is then concentrated under reduced pressure and dried to obtain Sargassum fusiforme extract.

2. The method for preparing Sargassum fusiforme extract according to claim 1, characterized in that: In S1, the washed Sargassum fusiforme is first placed in a roller press and rolled three times at 0~5℃ to initially rupture the cell walls. Then it is transferred to a freeze dryer and freeze-dried at -30~-25℃ and vacuum degree ≤15Pa for 6~8 hours to remove moisture. Finally, it is pulverized to 70~80 mesh using a temperature-controlled ultrafine pulverizer to obtain Sargassum fusiforme powder, which is then sealed and stored in an aluminum foil bag containing a desiccant.

3. The method for preparing Sargassum fusiforme extract according to claim 2, characterized in that: The rolling pressure of the roller pressing equipment is 5~8 MPa, and it is left to stand for 10 minutes after each rolling. The temperature inside the temperature-controlled ultrafine pulverizer is 28~32℃.

4. The method for preparing Sargassum fusiforme extract according to claim 1, characterized in that: In S3, the complex enzyme preparation is based on alginate lyase as the core, supplemented by enzyme preparations such as β-glucanase, fucoidanase and esterase, thereby forming a binary, ternary or quaternary multi-component enzyme system.

5. The method for preparing Sargassum fusiforme extract according to claim 4, characterized in that: The binary enzyme system consists of alginate lyase and β-glucanase, with an enzyme activity ratio of 4:1 to 6:1 and a total enzyme addition of 70 to 90 U / g of raw material.

6. The method for preparing Sargassum fusiforme extract according to claim 4, characterized in that: The ternary enzyme system consists of alginate lyase, β-glucanase, and fucoidanase, with an enzyme activity ratio of 6:2:1 to 8:3:1, and a total enzyme addition of 100 to 120 U / g of raw material.

7. The method for preparing Sargassum fusiforme extract according to claim 4, characterized in that: The quaternary enzyme system consists of alginate lyase, β-glucanase, fucoidanase, and esterase, with an enzyme activity ratio of 8:2:1:0.5 to 10:3:1:1, and a total enzyme addition of 110 to 130 U / g of raw material.

8. The method for preparing Sargassum fusiforme extract according to claim 1, characterized in that: In S5, the operating parameters for vacuum concentration are: vacuum degree 0.06 MPa~0.08 MPa, temperature 45~55℃; the drying process is spray drying and its operating parameters are: air inlet 190℃, air outlet 85℃, spray pressure 0.2 MPa.

9. The application of Sargassum fusiforme extract in cigarettes, characterized in that: Sargassum extract is dissolved in purified water or 50%~70% ethanol aqueous solution to prepare a solution with a mass concentration of 1%~10%. Then, it is added at a rate of 0.01%~0.5% of the weight of cigarette tobacco and sprayed or coated evenly onto the tobacco.

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