Carriers, immobilized enzymes prepared therefrom, and tobacco extracts

Abstract

This application relates to the field of enzyme engineering technology, and particularly to a carrier, an immobilized enzyme prepared therefrom, and a tobacco extract. The preparation method includes the following steps: providing a hydrophobic silica suspension and a sol, the sol comprising sodium alginate and gelatin; mixing the hydrophobic silica suspension and the sol to prepare a mixture; adding the mixture dropwise to a calcium chloride solution for hardening treatment, collecting a first gel bead; placing the first gel bead in a glutaraldehyde solution for a cross-linking reaction, collecting a second gel bead; and drying the second gel bead to prepare the carrier. This application uses a specific process to prepare the carrier, the resulting carrier can be used to immobilize enzymes, and the resulting immobilized enzyme has good ethanol tolerance, making it suitable for the enzymatic hydrolysis of the original tobacco extract solution.

Description

Technical Field

[0001] This application relates to the field of enzyme engineering technology, and in particular to carriers, immobilized enzymes prepared therefrom, and tobacco extracts. Background Technology

[0002] Tobacco extracts are key ingredients in tobacco flavoring, typically extracted directly from raw tobacco leaves or waste materials generated during their processing. By enriching the aroma components of tobacco leaves, tobacco extracts can significantly enhance the fullness, sweetness, and complexity of the smoke. However, tobacco extract systems are complex, and the large molecules they contain, such as pectin and protein, can easily produce irritating off-flavors and unpleasant tastes during combustion, severely impacting cigarette quality.

[0003] Currently, researchers are improving the extraction process through methods such as extraction optimization, physical membrane retention, alcohol precipitation purification, bio-fermentation, and bio-enzyme treatment. Among these, bio-enzyme treatment shows significantly better results than other methods, making it a research hotspot in recent years. However, after treating samples with free enzymes, complete separation can be difficult, which can negatively impact the taste of tobacco extracts.

[0004] To address this issue, researchers began employing enzyme immobilization methods. However, since tobacco extracts are often extracted using solvent extraction methods, a large amount of residual ethanol remains (60-80% by volume). Therefore, the ethanol tolerance of most immobilized enzyme carriers, such as chitosan, polyacrylamide gel, and agarose gel, is insufficient. Thus, improving the quality of tobacco extracts urgently requires a new carrier with high ethanol tolerance. Summary of the Invention

[0005] Based on this, one or more embodiments of this application provide a carrier, an immobilized enzyme prepared therefrom, and a tobacco extract. The technical solutions include the following:

[0006] One or more embodiments of this application provide a method for preparing a carrier, the method comprising the following steps:

[0007] A hydrophobic silica suspension and a sol are provided, wherein the sol includes sodium alginate and gelatin;

[0008] The hydrophobic silica suspension and the sol are mixed to prepare a mixture;

[0009] The mixture was added dropwise to a calcium chloride solution for hardening, and the first gel beads were collected.

[0010] The first gel bead was placed in a glutaraldehyde solution to undergo a cross-linking reaction, and the second gel bead was collected.

[0011] The second gel beads are dried to prepare a carrier.

[0012] In some embodiments of this application, the hydrophobic silica suspension satisfies one or more of the following conditions:

[0013] (1) The concentration of hydrophobic silica in the hydrophobic silica suspension is 20~30 wt%;

[0014] (2) The hydrophobic groups carried by the hydrophobic silica in the hydrophobic silica suspension include triethoxysilyl groups;

[0015] Optionally, the preparation steps of the hydrophobic silica suspension include:

[0016] Hydrophobic silica and water are mixed and ultrasonicated to prepare the hydrophobic silica suspension.

[0017] Optionally, the ultrasound conditions include a power density of 0.5~1.5 W / cm². 2 The time is 20-40 minutes.

[0018] In some embodiments of this application, the sol comprises 1-5 wt% sodium alginate and 1-5 wt% gelatin;

[0019] Optionally, the preparation steps of the sol include:

[0020] The sodium alginate, the gelatin, and water are mixed, heated, and stirred to prepare the sol.

[0021] Optionally, the heating and stirring conditions include a temperature of 45~65℃ and a time of 1~3h.

[0022] In some embodiments of this application, the preparation method satisfies one or more of the following conditions:

[0023] (1) The volume ratio of the hydrophobic silica suspension to the sol is 1:1 to 1:4; and,

[0024] (2) Mix at 35~45℃ and 8000~12000rpm for 8~12min.

[0025] In some embodiments of this application, the preparation method satisfies one or more of the following conditions:

[0026] (1) The calcium chloride solution comprises 2 to 3 parts calcium chloride and 45 to 55 parts water by mass;

[0027] (2) The hardening treatment time is 1~3 hours;

[0028] (3) During the process of adding the mixture, the vertical distance between the dropper head and the surface of the calcium chloride solution is 15±2cm, and the dripping speed is controlled at 1~2 drops / s;

[0029] (4) The first gel beads are washed and then placed in the glutaraldehyde solution for cross-linking reaction;

[0030] (5) The concentration of glutaraldehyde in the glutaraldehyde solution is 1.5~2.5 wt%;

[0031] (6) The cross-linking reaction time is 1~3h; and,

[0032] (7) The drying temperature is 20~28℃.

[0033] One or more embodiments of this application provide a carrier prepared by the preparation method described above.

[0034] One or more embodiments of this application provide an immobilized enzyme, the immobilized enzyme comprising the aforementioned carrier and the enzyme;

[0035] Optionally, the enzyme includes an acidic protease.

[0036] One or more embodiments of this application provide a method for preparing the immobilized enzyme described above, the method comprising:

[0037] The carrier was prepared using the method described above;

[0038] An enzyme solution containing the carrier and the enzyme is mixed and then subjected to an immobilization reaction to prepare an immobilized enzyme.

[0039] Optionally, the immobilization reaction meets the following conditions: temperature 34~36℃, shaking speed 110~130rpm, and time 3~5h;

[0040] Optionally, the amount of enzyme solution used for each 0.15g of the carrier is 8-12mL;

[0041] Optionally, the concentration of the enzyme in the enzyme solution is 0.5~1wt%;

[0042] Optionally, the enzyme in the enzyme solution includes acidic protease;

[0043] Optionally, the solvent for the enzyme solution includes citrate buffer;

[0044] Optionally, the pH of the citrate buffer solution is 2 to 4.

[0045] One or more embodiments of this application provide a method for preparing a tobacco extract, the method comprising:

[0046] To prepare tobacco extract, the tobacco extract stock solution and the immobilized enzyme as described in claim 7 are mixed, enzymatically hydrolyzed, and the enzyme is inactivated.

[0047] Optionally, the immobilized enzyme is added based on an enzyme activity of 400-600 U per 1g of the initial reaction system.

[0048] Optionally, the enzymatic hydrolysis conditions include: a temperature of 30~40℃, a shaking speed of 140~160rpm, and a time of 2~3h;

[0049] Optionally, the tobacco extract stock solution is diluted 3 to 8 times before being mixed with the immobilized enzyme.

[0050] One or more embodiments of this application provide a tobacco extract, which is prepared by the preparation method described above.

[0051] One or more embodiments of this application provide the use of the tobacco extract in the preparation of cigarettes.

[0052] Compared with traditional technologies, this application has the following advantages: This application uses a specific process to prepare the carrier, and the resulting carrier can be used to immobilize enzymes. The immobilized enzymes have good tolerance to ethanol and are suitable for the enzymatic hydrolysis of tobacco extract stock solution. Detailed Implementation

[0053] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0055] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0056] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0057] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0058] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0059] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0060] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0061] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0062] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0063] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0064] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0065] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0066] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0067] In this application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass-volume percentage.

[0068] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0069] Tobacco extracts / mousses contain high concentrations of ethanol due to their extraction methods. Traditional carrier materials are unsuitable for direct enzyme immobilization in tobacco extracts due to their low ethanol tolerance. The main reason is insufficient hydrophobicity of the carriers, leading to swelling, softening, and even breakage in high-ethanol environments, resulting in weak enzyme immobilization and reduced loading. Furthermore, weak immobilization further leads to the release of free enzymes, increasing their residue in the tobacco extract system. Therefore, innovative designs of carrier materials capable of withstanding high ethanol concentrations while forming stable immobilized enzyme systems are essential to meet the development needs of the tobacco extract industry. This application proposes a novel method to construct an immobilized carrier system with high ethanol tolerance.

[0070] A first aspect of this application provides a method for preparing a carrier, the method comprising the following steps:

[0071] A hydrophobic silica suspension and a sol are provided, wherein the sol includes sodium alginate and gelatin;

[0072] The hydrophobic silica suspension and the sol are mixed to prepare a mixture;

[0073] The mixture was added dropwise to a calcium chloride solution for hardening, and the first gel beads were collected.

[0074] The first gel bead was placed in a glutaraldehyde solution to undergo a cross-linking reaction, and the second gel bead was collected.

[0075] The second gel beads are dried to prepare a carrier.

[0076] In some embodiments of this application, the hydrophobic silica suspension satisfies one or more of the following conditions:

[0077] (1) The concentration of hydrophobic silica in the hydrophobic silica suspension is 20~30wt%, for example 20wt%, 21wt%, 22wt%, 23wt%, 24wt%, 25wt%, 26wt%, 27wt%, 28wt%, 29wt%, 30wt%;

[0078] (2) The hydrophobic groups carried by the hydrophobic silica in the hydrophobic silica suspension include triethoxysilyl groups;

[0079] Optionally, the preparation steps of the hydrophobic silica suspension include:

[0080] Hydrophobic silica and water are mixed and ultrasonicated to prepare the hydrophobic silica suspension.

[0081] Optionally, the ultrasound conditions include a power density of 0.5~1.5 W / cm². 2 (For example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5 W / cm) 2 The time is 20-40 minutes (e.g., 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 minutes).

[0082] In some embodiments of this application, the sol comprises 1-5 wt% (e.g., 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, 5 wt%) of sodium alginate and 1-5 wt% (e.g., 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, 5 wt%) of gelatin;

[0083] Optionally, the preparation steps of the sol include:

[0084] The sodium alginate, the gelatin, and water are mixed, heated, and stirred to prepare the sol.

[0085] Optionally, the heating and stirring conditions include: a temperature of 45~65℃ (e.g., 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65℃) and a time of 1~3h (e.g., 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3h).

[0086] In some embodiments of this application, the preparation method satisfies one or more of the following conditions:

[0087] (1) The volume ratio of the hydrophobic silica suspension to the sol is 1:1 to 1:4 (1:1, 1:1.5, 1.2, 1:2.5, 1:3, 1:3.5, 1:4); and,

[0088] (2) Mix at 35~45℃ (e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45℃) and 8000~12000rpm (e.g., 8000, 8500, 9000, 9500, 10000, 10500, 11000, 11500, 12000rpm) for 8~12min (e.g., 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12min).

[0089] In some embodiments of this application, the preparation method satisfies one or more of the following conditions:

[0090] (1) The calcium chloride solution comprises, by mass, 2 to 3 parts (e.g., 2, 2.2, 2.4, 2.6, 2.8, or 3 parts) of calcium chloride and 45 to 55 parts (e.g., 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 parts) of water;

[0091] (2) The hardening treatment time is 1~3h (e.g., 1, 1.5, 2, 2.5, 3h);

[0092] (3) During the process of adding the mixture, the vertical distance between the dropper head and the surface of the calcium chloride solution is 15±2cm, and the dripping speed is controlled at 1~2 drops / s;

[0093] (4) The first gel beads are washed and then placed in the glutaraldehyde solution for cross-linking reaction;

[0094] (5) The concentration of glutaraldehyde in the glutaraldehyde solution is 1.5~2.5wt% (1.5wt%, 1.6wt%, 1.7wt%, 1.8wt%, 1.9wt%, 2wt%, 2.1wt%, 2.2wt%, 2.3wt%, 2.4wt%, 2.5wt%).

[0095] (6) The cross-linking reaction time is 1~3h (e.g., 1, 1.5, 2, 2.5, 3h); and,

[0096] (7) The drying temperature is 20~28℃ (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28℃).

[0097] A second aspect of this application provides a carrier prepared by the aforementioned preparation method.

[0098] A third aspect of this application provides an immobilized enzyme, the immobilized enzyme comprising the aforementioned carrier and enzyme;

[0099] Optionally, the enzyme includes an acidic protease.

[0100] A fourth aspect of this application provides a method for preparing the immobilized enzyme, the method comprising:

[0101] The carrier was prepared using the method described above;

[0102] An enzyme solution containing the carrier and the enzyme is mixed and then subjected to an immobilization reaction to prepare an immobilized enzyme.

[0103] Optionally, the immobilization reaction satisfies the following conditions: temperature of 34~36℃ (e.g., 34, 34.5, 35, 35.5, 36℃), shaking speed of 110~130rpm (e.g., 110, 115, 120, 125, 130rpm), and time of 3~5h (e.g., 3, 3.5, 4, 4.5, 5h).

[0104] Optionally, the amount of enzyme solution used for each 0.15g of the carrier is 8 to 12 mL (e.g., 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12 mL).

[0105] Optionally, the concentration of the enzyme in the enzyme solution is 0.5~1wt% (e.g., 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1wt%).

[0106] Optionally, the enzyme in the enzyme solution includes acidic protease;

[0107] Optionally, the solvent for the enzyme solution includes citrate buffer;

[0108] Optionally, the pH of the citrate buffer solution is 2 to 4 (e.g., 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4).

[0109] A fifth aspect of this application provides a method for preparing a tobacco extract, the method comprising:

[0110] To prepare tobacco extract, the tobacco extract stock solution and the immobilized enzyme as described in claim 7 are mixed, enzymatically hydrolyzed, and the enzyme is inactivated.

[0111] Optionally, the immobilized enzyme is added at a rate of 400-600 U (e.g., 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600 U) per 1g of the initial reaction system after enzymatic hydrolysis.

[0112] Optionally, the enzymatic hydrolysis conditions include: a temperature of 30-40℃ (e.g., 30, 32, 34, 36, 38, 40℃), a shaking speed of 140-160 rpm (e.g., 140, 145, 150, 155, 160 rpm), and a time of 2-3 h (e.g., 2, 2.2, 2.4, 2.6, 2.8, 3 h).

[0113] Optionally, the tobacco extract stock solution is diluted 3 to 8 times (e.g., 3, 4, 5, 6, 7, 8) before being mixed with the immobilized enzyme.

[0114] A fifth aspect of this application provides a tobacco extract prepared by the aforementioned preparation method.

[0115] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0116] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0117] In the following example:

[0118] Citrate buffer solution at pH 3, Shanghai Yuanye Biotechnology Co., Ltd.

[0119] Acidic protease, 50 U / mg, Shanghai Yuanye Biotechnology Co., Ltd.

[0120] The tobacco extract stock solution is the conventional Henan tobacco extract, which was prepared in-house. Reference: Journal of Yunnan Agricultural University (Natural Science), 2019, 34(1): 63-69, “Optimization of preparation process and study of chemical composition of Zimbabwe tobacco extract”.

[0121] Sodium alginate (SA), Sinopharm Chemical Reagent Co., Ltd.

[0122] Gelatin (GE), Sinopharm Chemical Reagent Co., Ltd.

[0123] Hydrophilic silica, model AEROSIL 200, Shanghai Muying Trading Co., Ltd.

[0124] Octyltriethoxysilane (OTES), Wuhan Kanos Technology Co., Ltd.

[0125] Example 1

[0126] Step 1, SiO2 hydrophobic modification treatment: Take hydrophilic SiO2 (AEROSIL 200), add octyltriethoxysilane (OTES) at a mass ratio of 1:10 (SiO2:OTES), disperse in ethanol to form a mixed system; place in a shaker at 70℃ for 24h to allow the hydrophobic groups of OTES to cover the SiO2 surface through chemical bonding; after the reaction, separate by centrifugation, wash twice with anhydrous ethanol to remove unreacted OTES, and finally dry to obtain hydrophobic SiO2.

[0127] Step 2, Particle Dispersion: Take hydrophobic SiO2, add 3 times its mass of deionized water, and use 1W / cm³... 2 The power density ultrasonic treatment lasted for 30 minutes to form a uniform and stable hydrophobic SiO2 suspension for later use.

[0128] Step 3, Mixture Preparation: Take 1 part each of commercial sodium alginate (SA) and gelatin (GE) in a beaker, add 25 times the amount of deionized water, heat at 55℃ and stir at low speed for 2 hours to obtain a uniformly mixed sol. Slowly pour 0.5 parts of the hydrophobic SiO2 suspension prepared in Step 2 into 1 part (volume part) of the sol, and shear at high speed at 10,000 rpm for 10 minutes at 40℃, carefully controlling the stirring rhythm to reduce bubble generation, to obtain a uniform mixture A.

[0129] Step 4, Preparation of Reaction Solution: Simultaneously prepare 3 portions of commercial calcium chloride (CaCl2) and add 49 times its mass of water to obtain calcium chloride solution B. Prepare an acidic protease solution C with a concentration of 1 wt% using citrate buffer at pH=3. Dilute the original tobacco extract solution 5 times to obtain tobacco extract treatment solution D.

[0130] Step 5, Degassing: Place mixture A from step 3 and calcium chloride solution B from step 4 separately in an ultrasonic machine and sonicate for 30 minutes at a frequency of 20 kHz and a power density >0.5 W / cm³. 2 .

[0131] Step 6, Hardening: Use a 1mL dropper to draw up mixture A and drop it into calcium chloride solution B. The vertical distance between the dropper tip and the liquid surface should be 15±2cm. The dripping speed should be controlled at 1 drop / s. After the addition is finished, let the gel beads stand in calcium chloride solution B to harden for 2 hours. Filter and collect gel beads E.

[0132] Step 7, Cleaning: Clean gel beads E with deionized water to remove calcium chloride solution from the surface. Repeat cleaning twice.

[0133] Step 8, crosslinking: Place the cleaned gel beads E into a solution containing 2wt% glutaraldehyde and crosslink for 2 hours, then dry at room temperature to prepare gel beads F.

[0134] Step 9: Immobilize the enzyme: Mix 0.15g of gel beads F with 10mL of enzyme solution C, place in a constant temperature shaker at 35℃ and 120rpm for 4h to immobilize the enzyme and obtain the protease immobilization system, i.e., the immobilized enzyme.

[0135] Step 10: Sample treatment: Add immobilized acidic protease to tobacco extract treatment solution D at a rate of 500 U / g enzyme activity (i.e., 500 U per 1g of enzymatic hydrolysis system), and place in a shaker at 35℃ and 150 rpm for 2.5 h for enzymatic hydrolysis.

[0136] Step 11, Inactivation: After the treatment is completed, place the sample in a 100℃ water bath for 10 minutes to inactivate the enzyme.

[0137] Example 2

[0138] Step 1, SiO2 hydrophobic modification treatment: Take hydrophilic SiO2 (AEROSIL 200), add octyltriethoxysilane (OTES) at a mass ratio of 1:10 (SiO2:OTES), disperse in ethanol to form a mixed system; place in a shaker at 70℃ for 24h to allow the hydrophobic groups of OTES to cover the SiO2 surface through chemical bonding; after the reaction, separate by centrifugation, wash twice with anhydrous ethanol to remove unreacted OTES, and finally dry to obtain hydrophobic SiO2.

[0139] Step 2, Particle Dispersion: Take hydrophobic SiO2, add 3 times its mass of deionized water, and use 1W / cm³... 2 The power density ultrasonic treatment lasted for 30 minutes to form a uniform and stable hydrophobic SiO2 suspension for later use.

[0140] Step 3, Mixture Preparation: Take 1 part each of commercial sodium alginate (SA) and gelatin (GE) in a beaker, add 30 times the amount of deionized water, heat at 55℃ and stir at low speed for 2 hours to obtain a uniformly mixed sol. Slowly pour 1 part of the hydrophobic SiO2 suspension prepared in Step 2 into 4 parts (volume parts) of the sol, and shear at high speed at 10,000 rpm for 10 minutes at 40℃, carefully controlling the stirring rhythm to reduce bubble generation, to obtain a uniform mixture A.

[0141] Step 4, Preparation of Reaction Solution: Simultaneously prepare 3 portions of commercial calcium chloride (CaCl2) and add 49 times its mass of water to obtain calcium chloride solution B. Prepare an acidic protease solution C with a concentration of 1 wt% using citrate buffer at pH=3. Dilute the original tobacco extract solution 5 times to obtain tobacco extract treatment solution D.

[0142] Step 5, Degassing: Place mixture A from step 3 and calcium chloride solution B from step 4 separately in an ultrasonic machine and sonicate for 30 minutes at a frequency of 20 kHz and a power density >0.5 W / cm³. 2 .

[0143] Step 6, Hardening: Use a 1mL dropper to draw up mixture A and drop it into calcium chloride solution B. The vertical distance between the dropper tip and the liquid surface should be 15±2cm. The dripping speed should be controlled at 2 drops / s. After the addition is finished, let the gel beads stand in calcium chloride solution B to harden for 2 hours. Filter and collect gel beads E.

[0144] Step 7, Cleaning: Clean gel beads E with deionized water to remove calcium chloride solution from the surface. Repeat cleaning twice.

[0145] Step 8, crosslinking: Place the cleaned gel beads E into a solution containing 2wt% glutaraldehyde and crosslink for 2 hours, then dry at room temperature to prepare gel beads F.

[0146] Step 9: Immobilize the enzyme: Mix 0.15g of gel beads F with 10mL of enzyme solution C, place in a constant temperature shaker at 35℃ and 120rpm for 4h to immobilize the enzyme and obtain the protease immobilization system, i.e., the immobilized enzyme.

[0147] Step 10: Sample treatment: Add immobilized acidic protease to tobacco extract treatment solution D at a concentration of 500 U / g enzyme activity, and incubate at 35°C on a shaker at 150 rpm for 2.5 h.

[0148] Step 11, Inactivation: After the treatment is completed, place the sample in a 100℃ water bath for 10 minutes to inactivate the enzyme.

[0149] Comparative Example 1

[0150] Compared to the examples, this comparative example directly treated the tobacco extract with free enzymes, without using enzyme immobilization on a high-ethanol-resistant carrier. Specifically:

[0151] Step 1: Preparation of materials: Prepare an acidic protease solution C with a concentration of 1 wt% using citrate buffer at pH=3. Dilute the original tobacco extract solution 5 times to obtain tobacco extract treatment solution D.

[0152] Step 2, Sample treatment: Add the above-mentioned acidic protease enzyme solution C to the tobacco extract treatment solution D at an enzyme activity concentration of 500 U / g, and place it in a shaker at 35℃ and 150 rpm for 2.5 h for enzymatic hydrolysis.

[0153] Step 3, Inactivation: After the enzymatic hydrolysis is completed, the obtained enzymatic hydrolysis product is placed in a 100℃ water bath for 10 minutes to inactivate the enzyme, and then cooled to obtain the treated tobacco extract.

[0154] Comparative Example 2

[0155] Compared to the examples, this comparative example used other immobilized enzymes to treat tobacco extracts, without using an enzyme immobilized on a high-ethanol-resistant carrier. Compared to the immobilized enzymes in the examples, the other immobilized enzymes used in this comparative example did not have a hydrophobic SiO2 suspension added during preparation. Specifically:

[0156] Step 1: Preparation of Immobilized Enzyme Carrier: Take 1 part each of commercial sodium alginate (SA) and gelatin (GE) in a beaker, add 25 times the volume of deionized water, heat at 55°C and stir at low speed for 2 hours to obtain a uniformly mixed sol. Simultaneously, prepare 3 parts of commercial calcium chloride (CaCl2) and add 49 times the volume of water to obtain calcium chloride solution B. Referring to steps 6-8 in the examples, drop the sol into calcium chloride solution B to prepare the immobilized enzyme carrier, denoted as gel bead F'.

[0157] Step 2, Preparation of reaction solution: Same as in Example 1, prepare enzyme solution C with a concentration of 1 wt% acidic protease using citrate buffer at pH=3. Simultaneously, dilute the original tobacco extract solution appropriately to obtain tobacco extract treatment solution D.

[0158] Step 3: Immobilize the enzyme: Mix 0.15g of gel beads F' with 10mL of enzyme solution B, and place the mixture in a constant temperature shaker at 35℃ and 120rpm for 4h to immobilize the enzyme.

[0159] Step 4: Sample processing: Add immobilized acidic protease to tobacco extract processing solution D at an enzyme activity of 500 U / g, and incubate at 35°C on a shaker at 150 rpm for 2.5 h.

[0160] Step 5, Inactivation: After the treatment is completed, place the sample in a 100℃ water bath for 10 minutes to inactivate the enzyme.

[0161] Result comparison test method:

[0162] The tolerance of the immobilized enzyme carriers was evaluated by detecting their swelling rate, mechanical strength, and carrier loss rate. Changes in protein content and amino acid content in conventional Henan tobacco extract were simultaneously measured.

[0163] (1) Carrier swelling rate: The carrier was placed in 75% (v / v) ethanol and kept at 30℃ for 24h. The volume / volume change before and after swelling was calculated (swelling rate = (volume after swelling - initial volume) / initial volume × 100%).

[0164] (2) Mechanical strength: The carrier was placed in 75% (v / v) ethanol for 24 hours and the compressive strength of the carrier at 70% (v / v) ethanol concentration was measured by a compressive strength tester.

[0165] (3) Carrier loss rate: The mass loss rate of the carrier after standing in 75% (v / v) ethanol at 30°C was determined (weighed after 72 hours and the percentage of weight loss was calculated). Loss rate = (initial mass - mass after standing) / initial mass × 100%.

[0166] (4) Protein content determination: Coomassie brilliant blue method was used.

[0167] (5) Determination of amino acid increase: Referring to standard YC / T 282—2009 "Determination of Free Amino Acids in Tobacco Leaves - Amino Acid Analyzer Method", the amino acid content was determined using a fully automated amino acid analyzer (Wang Haiqing 2024). The amino acid increase rate was calculated using the following formula:

[0168]

[0169] In the formula: D0—amino acid content before enzymatic hydrolysis (μg / g); D1—amino acid content after enzymatic hydrolysis (μg / g).

[0170] Table 1

[0171]

[0172] As shown in the table above, this application can effectively improve the effect of immobilized enzymes in Henan tobacco extract, decompose proteins, increase amino acid content, and improve taste quality. The effect of Example 1 is not only better than that of free enzyme treatment but also surpasses that of ordinary carrier treatment, demonstrating a significant advantage in the production of tobacco extracts to improve cigarette taste. Comparative Example 1 uses free protease to treat Henan tobacco extract without using a carrier for immobilization, thus requiring less modification to production equipment. Comparative Example 2 uses commercially available carrier immobilization technology; the carrier material is readily available, and the production cost is slightly higher, but it effectively improves the quality of Henan tobacco extract, demonstrating high practical value.

[0173] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0174] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing a carrier, characterized in that, The preparation method includes the following steps: A hydrophobic silica suspension and a sol are provided, wherein the sol includes sodium alginate and gelatin; The hydrophobic silica suspension and the sol are mixed to prepare a mixture; The mixture was added dropwise to a calcium chloride solution for hardening, and the first gel beads were collected. The first gel bead was placed in a glutaraldehyde solution to undergo a cross-linking reaction, and the second gel bead was collected. The second gel beads are dried to prepare a carrier.

2. The method for preparing the carrier according to claim 1, characterized in that, The hydrophobic silica suspension satisfies one or more of the following conditions: (1) The concentration of hydrophobic silica in the hydrophobic silica suspension is 20~30 wt%; (2) The hydrophobic groups carried by the hydrophobic silica in the hydrophobic silica suspension include triethoxysilyl groups; Optionally, the preparation steps of the hydrophobic silica suspension include: Hydrophobic silica and water are mixed and ultrasonicated to prepare the hydrophobic silica suspension. Optionally, the ultrasound conditions include a power density of 0.5~1.5 W / cm². 2 The time is 20-40 minutes.

3. The method for preparing the carrier according to claim 1, characterized in that, The sol comprises 1-5 wt% sodium alginate and 1-5 wt% gelatin; Optionally, the preparation steps of the sol include: The sodium alginate, the gelatin, and water are mixed, heated, and stirred to prepare the sol. Optionally, the heating and stirring conditions include a temperature of 45~65℃ and a time of 1~3h.

4. The method for preparing the carrier according to claim 1, characterized in that, The preparation method satisfies one or more of the following conditions: (1) The volume ratio of the hydrophobic silica suspension to the sol is 1:1 to 1:4; and, (2) Mix at 35~45℃ and 8000~12000rpm for 8~12min.

5. The method for preparing the carrier according to any one of claims 1 to 4, characterized in that, The preparation method satisfies one or more of the following conditions: (1) The calcium chloride solution comprises 2 to 3 parts calcium chloride and 45 to 55 parts water by mass; (2) The hardening treatment time is 1~3 hours; (3) During the process of adding the mixture, the vertical distance between the dropper head and the surface of the calcium chloride solution is 15±2cm, and the dripping speed is controlled at 1~2 drops / s; (4) The first gel beads are washed and then placed in the glutaraldehyde solution for cross-linking reaction; (5) The concentration of glutaraldehyde in the glutaraldehyde solution is 1.5~2.5 wt%; (6) The cross-linking reaction time is 1~3h; (7) The drying temperature is 20~28℃.

6. A carrier, characterized in that, The carrier is prepared by the preparation method according to any one of claims 1 to 5.

7. An immobilized enzyme, characterized in that, The immobilized enzyme comprises the carrier and enzyme as described in claim 6; Optionally, the enzyme includes an acidic protease.

8. The method for preparing the immobilized enzyme according to claim 7, characterized in that, The preparation method includes: The carrier is prepared by the preparation method according to any one of claims 1 to 5; An enzyme solution containing the carrier and the enzyme is mixed and then subjected to an immobilization reaction to prepare an immobilized enzyme. Optionally, the immobilization reaction meets the following conditions: temperature 34~36℃, shaking speed 110~130rpm, and time 3~5h; Optionally, the amount of enzyme solution used for each 0.15g of the carrier is 8-12mL; Optionally, the concentration of the enzyme in the enzyme solution is 0.5~1wt%; Optionally, the enzyme in the enzyme solution includes acidic protease; Optionally, the solvent for the enzyme solution includes citrate buffer; Optionally, the pH of the citrate buffer solution is 2 to 4.

9. A method for preparing tobacco extract, characterized in that, The preparation method includes: To prepare tobacco extract, the tobacco extract stock solution and the immobilized enzyme as described in claim 7 are mixed, enzymatically hydrolyzed, and the enzyme is inactivated. Optionally, the immobilized enzyme is added based on an enzyme activity of 400-600 U per 1g of the initial reaction system. Optionally, the enzymatic hydrolysis conditions include: a temperature of 30~40℃, a shaking speed of 140~160rpm, and a time of 2~3h; Optionally, the tobacco extract stock solution is diluted 3 to 8 times before being mixed with the immobilized enzyme.

10. A tobacco extract, characterized in that, The tobacco extract is prepared by the preparation method according to claim 9.

11. The use of the tobacco extract of claim 10 in the preparation of cigarettes.

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