Essence as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of essence preparation, and particularly relates to essence and a preparation method and application thereof. According to the method, tobacco stems are used as a core raw material, a compound enzyme system composed of alpha-amylase, saccharifying enzyme, pullulanase and protease is adopted for enzymolysis treatment, after enzyme deactivation treatment is conducted on enzymatic hydrolysate, alanine, glycine and aspartic acid are added into the enzymatic hydrolysate to serve as amino acid substrates, and the amino acid amino acid is obtained. The amino acid substrate and reducing sugar in the enzymatic hydrolysate are promoted to be subjected to Maillard reaction, finally, flavor products with typical baking flavor and represented by pyrazine, furan and pyrrole are obtained, and the total content of flavor substances with the characteristics is remarkably increased. Through detection, the total content of volatile flavor components in the essence prepared by the invention can reach 2.76-3.88 mu g / mL, the total OAV value is 3.45-10.1, and compared with the prior art, the perceptible concentration of effective aroma substances is improved in a leap-over manner.

Description

Technical Field

[0001] This application belongs to the field of flavor preparation technology, specifically relating to a method for preparing roasted flavorings using tobacco stem extract concentrate as raw material through Maillard reaction. Background Technology

[0002] Tobacco stems are one of the main wastes generated during tobacco processing. For a long time, they have been directly discarded or simply burned, resulting not only in resource waste but also environmental pollution. In fact, tobacco stems contain abundant nutrients and functional substances, including proteins, polysaccharides, alkaloids, and various aroma precursors (such as carotenoids, polyphenols, and fatty acid derivatives). These components give tobacco stems extremely high comprehensive utilization value, making them an important potential resource for the development of a circular economy in the tobacco industry.

[0003] Currently, the comprehensive utilization of tobacco stems has developed into several mature technological pathways, among which the most widely used on a large scale is the processing of reconstituted tobacco leaves. This technology, through processes such as physical crushing and chemical extraction, transforms tobacco stems into reconstituted tobacco leaves with uniform texture and stable performance, significantly improving the utilization rate of tobacco resources. Besides reconstituted tobacco leaves, the application of tobacco stems in the preparation of natural flavorings has attracted much attention. This technology mainly includes key steps such as raw material pretreatment, solvent extraction, enzymatic hydrolysis, Maillard reaction, and separation and purification. The enzymatic hydrolysis process degrades the large-molecule polysaccharides and proteins in tobacco stems into small-molecule sugars, amino acids, and other active substances, providing sufficient precursors for subsequent aroma generation. The Maillard reaction, through the interaction of reducing sugars and amino acids, generates compounds with typical tobacco aromas. Because these tobacco flavorings are directly derived from tobacco raw materials, they have significant advantages such as aroma characteristics closely resembling those of raw tobacco, good compatibility, natural origin, and high safety. They can be widely used in cigarette flavoring, e-cigarette liquid blending, and other fields, effectively compensating for the insufficient aroma of natural tobacco leaves and improving the aroma quality of tobacco products and the consumer smoking experience.

[0004] In the evaluation system of tobacco flavorings, the Oral Ability Value (OAV) is a core indicator for defining the aroma intensity and application value of a flavoring. It is defined as the ratio of the concentration of a certain aroma compound to its olfactory threshold. A higher OAV value indicates a greater contribution of the aroma compound to the overall aroma of the flavoring, resulting in stronger aroma intensity and greater recognizability. When the OAV value is less than 1, the aroma of the compound is difficult for the human olfactory system to perceive, and it cannot effectively play its flavoring role. Currently, the overall OAV value of tobacco stem-based natural tobacco flavorings prepared using existing technologies is generally low, mostly around 1, making it difficult to meet the high-end and personalized flavoring needs of tobacco products. Therefore, how to improve the OAV value of natural tobacco flavorings prepared from tobacco stems has become a core technical challenge that the tobacco industry urgently needs to overcome. Summary of the Invention

[0005] The purpose of this application is to provide a fragrance with a high OAV value, which is achieved through the following technical solution:

[0006] A method for preparing a flavoring includes the following steps: enzymatically hydrolyzing a tobacco stem extract with a complex enzyme to obtain an enzymatic hydrolysate; adding a mixture of amino acids to the enzymatic hydrolysate to induce a Maillard reaction between the amino acids and reducing sugars in the enzymatic hydrolysate to obtain the flavoring; wherein the complex enzyme includes α-amylase, saccharifying enzyme, pullulanase, and protease; wherein the mixed amino acids include alanine, glycine, and aspartic acid; and wherein the enzymes in the enzymatic hydrolysate are inactivated before adding the mixed amino acids to the enzymatic hydrolysate.

[0007] Preferably, the Maillard reaction conditions are a temperature of 110-130℃, a pH of 5.0-8.0, and a time of 1.5-2.5h; the inactivation conditions are a temperature of 90-100℃ and a time of 10-20min.

[0008] Preferably, during the Maillard reaction, the water activity is controlled by adding glycerol, and the amount of glycerol added is 30-100% of the mass of the tobacco stem extract.

[0009] Preferably, the enzymatic hydrolysis conditions are a temperature of 50-80℃, a pH of H5.0, and a time of 1-2.5h.

[0010] Preferably, based on the mass of tobacco stem extract added, the amount of α-amylase added is 50-400 u / g, the amount of saccharifying enzyme added is 50-200 u / g, the amount of pullulanase added is 50-200 u / g, and the amount of protease added is 50-300 u / g.

[0011] Preferably, the molar ratio of the mixed amino acids to the total reducing sugars in the enzymatic hydrolysate is (0.05-0.5):1.

[0012] Preferably, the molar ratio of alanine, glycine and aspartic acid is (1-2):1:1.

[0013] A flavoring, prepared using any one of the preparation methods described above.

[0014] Preferably, the total content of volatile components in the fragrance is 2.76-3.88 μg / mL, and the total OAV value is 3.45-10.1.

[0015] The application of the aforementioned flavoring in cigarette manufacturing.

[0016] Compared with the prior art, this application has the following beneficial effects:

[0017] This application utilizes tobacco stems as the core raw material and employs a complex enzyme system consisting of α-amylase, saccharifying enzyme, pullulanase, and protease for enzymatic hydrolysis. After inactivating the enzymes in the hydrolysate, alanine, glycine, and aspartic acid are added as amino acid substrates to promote Maillard reactions between the amino acid substrates and reducing sugars in the hydrolysate. This ultimately yields flavor products with typical roasted aromas, represented by pyrazine (contributing a roasted sweetness), furan (imparting fruity and roasted aromas), and pyrrole (enhancing aroma complexity). The total content of these characteristic flavor compounds is significantly increased. Testing shows that the total content of volatile flavor components in the flavoring prepared in this application reaches 2.76-3.88 μg / mL, with a total OAV value of 3.45-10.1. Compared to existing technologies, this represents a significant leap in the perceptible concentration of effective aroma compounds. In scenarios requiring high aroma, such as heated cigarettes, it can significantly enhance aroma intensity and enrich the layers of tobacco aroma. Furthermore, by adjusting the amount of raw materials added to the compound enzyme and mixed amino acids, the Maillard reaction and enzymatic hydrolysis reaction parameters, and designing the enzyme inactivation step, this application prepared a fragrance with an OAV value as high as 10.1, which is about 5.6 times the OAV value of conventional similar fragrances, demonstrating unexpected technical effects. Detailed Implementation

[0018] The present application will now be further described by way of specific embodiments. Those skilled in the art will be able to implement the present application based on these descriptions. Furthermore, the embodiments of the present application described below are generally only a part of the embodiments of the present application, and not all of the embodiments. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present application without inventive effort should fall within the scope of protection of the present application.

[0019] Example 1

[0020] A method for preparing a fragrance, specifically including the following steps:

[0021] (1) Complex enzymatic hydrolysis of tobacco stem extract: A certain amount of concentrated tobacco stem extract was placed in a constant temperature water bath reactor, and a complex enzyme system consisting of α-amylase, saccharifying enzyme, pullulanase, and protease was added. The amounts of each enzyme added were: α-amylase 50 U / g, saccharifying enzyme 100 U / g, pullulanase 75 U / g, and protease 300 U / g. Under thorough stirring, the reaction was carried out at a constant temperature of 65℃ for 2.5 hours to achieve the targeted degradation of macromolecules in the tobacco stem extract. After the reaction was completed, the enzymes were inactivated by heating at 95℃ for 10 minutes, and then rapidly cooled to room temperature. Analysis showed that the starch content in the enzymatically hydrolyzed system decreased by 21.28% compared to before treatment, indicating that polysaccharides were effectively hydrolyzed into smaller sugar molecules. The protein content decreased by 22.18%, while the total free amino acid content increased by 8.11%, indicating that the protease effectively catalyzed protein hydrolysis, releasing more aroma precursors. The reducing sugar content reached 10.02%, providing sufficient substrate for the subsequent Maillard reaction. This enzymatic hydrolysis process significantly increased the content of aroma precursors in tobacco stem extract and enhanced the reactivity of the system.

[0022] (2) Preparation of flavor base material by directional Maillard reaction: Accurately weigh 50g of the above enzymatic hydrolysate and add 15g of glycerol as a water activity control agent to effectively reduce the free water content in the system, slow down the non-enzymatic browning rate, and improve the selectivity of Maillard reaction and the stability of aroma components. Finely adjust the pH of the reaction system to 5.0 with saturated sodium carbonate solution. This weakly acidic environment is conducive to the formation of tobacco's characteristic roasted and caramel sweet aroma. According to the optimal reaction ratio of amino acids and reducing sugars in the Maillard reaction, control the molar ratio of amino acid mixture to reducing sugar to 0.05:1. Accurately add 0.04g of alanine, 0.04g of glycine, and 0.06g of aspartic acid according to the formula. These three amino acids contribute malty aroma, sweet aroma, and caramel-meat aroma, respectively. Their synergistic effect can simulate the aroma profile of natural tobacco during pyrolysis. After the mixture is thoroughly stirred, transfer it to a heat-resistant glass container and place it in a constant temperature oven at 110℃ for 1.5 hours. During the reaction, the system gradually undergoes browning, generating a large amount of melanoidins and volatile aroma compounds, exhibiting a rich aroma of roasted tobacco, nuts, and caramel. After the reaction is completed and the system is naturally cooled to room temperature, a brownish-red flavoring with a mellow aroma is obtained.

[0023] Example 2

[0024] A method for preparing a fragrance, specifically including the following steps:

[0025] (1) Enzymatic hydrolysis of tobacco stem extract: A certain amount of concentrated tobacco stem extract was placed in a constant temperature water bath reactor, and a complex enzyme system consisting of α-amylase, saccharifying enzyme, pullulanase, and protease was added. The amounts of each enzyme added were: α-amylase 50 U / g, saccharifying enzyme 100 U / g, pullulanase 75 U / g, and protease 200 U / g. Under thorough stirring, the reaction was carried out at 60℃ for 2 hours to achieve the directional degradation of macromolecules in the tobacco stem extract. After the reaction, the enzymes were inactivated by heating at 95℃ for 10 minutes and then rapidly cooled to room temperature. The results showed that the starch content in the system decreased by 16.8% after enzymatic hydrolysis compared to before treatment, indicating that polysaccharides were effectively hydrolyzed into small molecule sugars; the protein content decreased by 16.48%, while the total amount of free amino acids increased by 6.76%, indicating that the protease effectively catalyzed the hydrolysis of proteins and released more aroma precursors; the reducing sugar content reached 9.43%, providing sufficient substrate for the subsequent Maillard reaction. This enzymatic hydrolysis process significantly increases the content of aroma precursors in tobacco stem extract and enhances the reactivity of the system.

[0026] (2) Preparation of flavor base material by directional Maillard reaction: Accurately weigh 50g of the above enzymatic hydrolysate and add 15g of glycerol as a water activity control agent to effectively reduce the free water content in the system, slow down the non-enzymatic browning rate, and improve the selectivity of Maillard reaction and the stability of aroma components. Finely adjust the pH of the reaction system to 7.0 with saturated sodium carbonate solution. This weakly acidic environment is conducive to the formation of tobacco's characteristic roasted and caramel sweet aroma. According to the optimal reaction ratio of amino acids and reducing sugars in the Maillard reaction, control the molar ratio of amino acid mixture to reducing sugar to 0.3:1. Accurately add 0.26g of alanine, 0.22g of glycine, and 0.39g of aspartic acid according to the formula. These three amino acids contribute malty aroma, sweet aroma, and caramel-meat aroma, respectively. Their synergistic effect can simulate the aroma profile of natural tobacco during pyrolysis. After the mixture is thoroughly stirred, transfer it to a heat-resistant glass container and place it in a constant temperature oven at 120℃ for 2 hours. During the reaction, the system gradually undergoes browning, generating a large amount of melanoidins and volatile aroma compounds, exhibiting a rich aroma of roasted tobacco, nuts, and caramel. After the reaction is completed and the system is naturally cooled to room temperature, a brownish-red flavoring with a mellow aroma is obtained.

[0027] Example 3

[0028] A method for preparing a fragrance, specifically including the following steps:

[0029] (1) Enzymatic hydrolysis of tobacco stem extract: A certain amount of concentrated tobacco stem extract was placed in a constant temperature water bath reactor, and a complex enzyme system consisting of α-amylase, saccharifying enzyme, pullulanase, and protease was added. The amounts of each enzyme added were: α-amylase 50 U / g, saccharifying enzyme 50 U / g, pullulanase 50 U / g, and protease 200 U / g. Under thorough stirring, the reaction was carried out at 70℃ for 1.5 hours to achieve the directional degradation of macromolecules in the tobacco stem extract. After the reaction, the enzymes were inactivated by heating at 95℃ for 10 minutes and then rapidly cooled to room temperature. The results showed that the starch content in the system decreased by 15.08% after enzymatic hydrolysis, indicating that polysaccharides were effectively hydrolyzed into small molecule sugars; the protein content decreased by 14.81%, while the total amount of free amino acids increased by 7.16%, indicating that the protease effectively catalyzed the hydrolysis of proteins and released more aroma precursors; the reducing sugar content reached 11.22%, providing sufficient substrate for the subsequent Maillard reaction. This enzymatic hydrolysis process significantly increases the content of aroma precursors in tobacco stem extract and enhances the reactivity of the system.

[0030] (2) Preparation of flavor base material by directional Maillard reaction: Accurately weigh 50g of the above enzymatic hydrolysate and add 25g of glycerol as a water activity control agent to effectively reduce the free water content in the system, slow down the non-enzymatic browning rate, and improve the selectivity of Maillard reaction and the stability of aroma components. Finely adjust the pH of the reaction system to 8.0 with saturated sodium carbonate solution. This weakly acidic environment is conducive to the formation of tobacco's characteristic roasted and caramel sweet aroma. According to the optimal reaction ratio of amino acids and reducing sugars in the Maillard reaction, control the molar ratio of amino acid mixture to reducing sugar to be 0.5:1. Accurately add 0.76g of alanine, 0.32g of glycine, and 0.57g of aspartic acid according to the formula. These three amino acids contribute malty aroma, sweet aroma, and caramel-meat aroma, respectively. Their synergistic effect can simulate the aroma profile of natural tobacco during pyrolysis. After the mixture is thoroughly stirred, transfer it to a heat-resistant glass container and place it in a constant temperature oven at 110℃ for 2.5 hours. During the reaction, the system gradually undergoes browning, generating a large amount of melanoidins and volatile aroma compounds, exhibiting a rich aroma of roasted tobacco, nuts, and caramel. After the reaction is completed and the system is naturally cooled to room temperature, a brownish-red flavoring with a mellow aroma is obtained.

[0031] Example 4

[0032] A method for preparing a fragrance, specifically including the following steps:

[0033] (1) Complex enzymatic hydrolysis of tobacco stem extract: A certain amount of concentrated tobacco stem extract was placed in a constant temperature water bath reactor, and a complex enzyme system consisting of α-amylase, saccharifying enzyme, pullulanase, and protease was added. The amounts of each enzyme added were: α-amylase 400 U / g (based on substrate dry weight), saccharifying enzyme 100 U / g, pullulanase 100 U / g, and protease 300 U / g. Under thorough stirring, the reaction was carried out at a constant temperature of 80℃ for 2.5 hours to achieve the targeted degradation of macromolecules in the tobacco stem extract. After the reaction was completed, the enzymes were inactivated by heating at 95℃ for 10 minutes, and then rapidly cooled to room temperature. Analysis showed that the starch content in the enzymatically hydrolyzed system decreased by 20.78% compared to before treatment, indicating that polysaccharides were effectively hydrolyzed into smaller sugar molecules. The protein content decreased by 25.02%, while the total free amino acid content increased by 14.61%, indicating that the protease effectively catalyzed protein hydrolysis, releasing more aroma precursors. The reducing sugar content reached 13.83%, providing sufficient substrate for the subsequent Maillard reaction. This enzymatic hydrolysis process significantly increased the content of aroma precursors in tobacco stem extract and enhanced the reactivity of the system.

[0034] (2) Preparation of flavor base material by directional Maillard reaction: Accurately weigh 50g of the above enzymatic hydrolysate and add 50g of glycerol as a water activity control agent to effectively reduce the free water content in the system, slow down the non-enzymatic browning rate, and improve the selectivity of Maillard reaction and the stability of aroma components. Finely adjust the pH of the reaction system to 8.0 with saturated sodium carbonate solution. This weakly acidic environment is conducive to the formation of tobacco's characteristic roasted and caramel sweet aroma. According to the optimal reaction ratio of amino acids and reducing sugars in the Maillard reaction, control the molar ratio of amino acid mixture to reducing sugar to 0.5:1. Accurately add 1.23g of alanine, 0.52g of glycine, and 0.92g of aspartic acid according to the formula. These three amino acids contribute malty aroma, sweet aroma, and caramel-meat aroma, respectively. Their synergistic effect can simulate the aroma profile of natural tobacco during pyrolysis. After the mixture is thoroughly stirred, transfer it to a heat-resistant glass container and place it in a constant temperature oven at 130℃ for 2.5 hours. During the reaction, the system gradually undergoes browning, generating a large amount of melanoidins and volatile aroma compounds, exhibiting a rich aroma of roasted tobacco, nuts, and caramel. After the reaction is completed and the system is naturally cooled to room temperature, a brownish-red flavoring with a mellow aroma is obtained.

[0035] Comparative Example 1

[0036] A certain amount of tobacco stem concentrate was weighed, and 50 U / g of α-amylase, 50 U / g of saccharifying enzyme, 50 U / g of pullulanase, and 200 U / g of protease were added. The mixture was enzymatically hydrolyzed at 70℃ for 1.5 h. No enzyme inactivation treatment was performed after hydrolysis. The starch content decreased by 15.08%, the protein content decreased by 14.81%, the reducing sugar content was 12.22%, and the amino acid content increased by 7.16%. 50 g of the hydrolysate was taken, 25 g of glycerol was added, and the pH was adjusted to 8 with saturated sodium carbonate solution. Alanine 0.76 g, glycine 0.32 g, and aspartic acid 0.57 g were added at an amino acid to reducing sugar molar ratio of 0.5:1. The mixture was reacted at 110℃ for 2.5 h to obtain the flavoring.

[0037] Comparative Example 2

[0038] A certain amount of tobacco stem concentrate was weighed, and 400 U / g of α-amylase, 100 U / g of saccharifying enzyme, 100 U / g of pullulanase, and 300 U / g of protease were added. The mixture was enzymatically hydrolyzed at 80℃ for 2.5 h. No enzyme inactivation treatment was performed after hydrolysis. The starch content decreased by 24.78%, the protein content decreased by 19.72%, the reducing sugar content was 19.83%, and the amino acid content increased by 14.61%. 50 g of the hydrolysate was taken, 50 g of glycerol was added, and the pH was adjusted to 8 with saturated sodium carbonate solution. Alanine 1.23 g, glycine 0.52 g, and aspartic acid 0.92 g were added at an amino acid to reducing sugar molar ratio of 0.5:1. The mixture was reacted at 130℃ for 2.5 h to obtain the flavoring.

[0039] Performance testing

[0040] This performance test analyzed the volatile aroma components in the fragrances prepared in Examples 1-4 and Comparative Examples 1-2. Qualitative and quantitative evaluation was performed using solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS). 5 mL of the reacted fragrance sample was placed in a 20 mL headspace vial, and 2 μL of a 0.827 μg / mL n-octanol internal standard solution (methanol solvent) was accurately added. The sample was equilibrated in a 50°C water bath for 20 min, followed by headspace adsorption extraction using an SPME extraction head (e.g., DVB / CAR / PDMS50 / 30 μm) for 30 min. After extraction, the extraction head was quickly inserted into the gas chromatograph inlet, and thermal desorption was performed at 250°C for 3 min to completely release the target compounds, allowing them to enter the chromatographic system for separation and mass spectrometry detection. Semi-quantitative analysis of detected volatile components was performed using the internal standard method to calculate the relative content of each component. The odor activity value (OAV) of each aroma substance (OAV = concentration / perceived threshold) was calculated using an aroma threshold database, with OAV ≥ 1 used as the criterion for determining key aroma contributors. The total content and total OAV value of flavor compounds in the samples from each example were finally summarized, and the results are shown in the table below.

[0041] Table 1. Total content of volatile components and total OAV value in the fragrances of Examples 1-4 and Comparative Examples 1-2

[0042]

[0043] Observation of the table above reveals that the total flavor product content (2.76–3.88 μg / mL) and total OAV value (3.45–10.1) of Examples 1 to 4 are significantly higher than those of Comparative Example 1 (0.56 μg / mL, OAV 1.64) and Comparative Example 2 (1.00 μg / mL, OAV 1.79). Among them, the total flavor product content of Example 1 reaches 3.35 μg / mL, and the total OAV value is 10.1, which is the highest among all groups, demonstrating outstanding technical effects. In contrast, the two comparative examples did not adopt the compound enzyme ratio, amino acid dosage, and key enzyme inactivation step of this application, resulting in insufficient flavor substance generation and significantly lower sensory contributions. The above data indicate that this invention, by precisely controlling the amount of raw materials added and introducing an enzyme inactivation process, effectively improves the yield and harmony of key flavor components in the flavoring, thereby achieving substantial progress and reproducible technical advantages in improving the quality of tobacco flavorings.

Claims

1. A method of preparing a fragrance, comprising the steps of: The tobacco stem extract is subjected to enzymolysis by a complex enzyme to obtain an enzymolysis solution; mixed amino acids are added to the enzymolysis solution to promote Maillard reaction between the amino acids and reducing sugars in the enzymolysis solution to obtain a flavor essence; characterized in that the complex enzyme comprises alpha-amylase, glucoamylase, pullulanase and protease; the mixed amino acids comprise alanine, glycine and aspartic acid; and the enzyme in the enzymolysis solution is inactivated before the mixed amino acids are added to the enzymolysis solution.

2. A method of preparing a flavouring according to claim 1, characterised in that, The Maillard reaction is carried out at a temperature of 110-130℃, a pH value of 5.0-8.0 and for a time of 1.5-2.5h; and the inactivation is carried out at a temperature of 90-100℃ for a time of 10-20min.

3. A method of preparing a flavouring according to claim 1, characterised in that, During the Maillard reaction, the water activity is controlled by adding glycerol, and the amount of the glycerol added is 30-100% of the mass of the tobacco stem extract.

4. The method of claim 1, wherein the flavor is prepared by the steps of: The enzymolysis is carried out at a temperature of 50-80℃, a pH value of 5.0 and for a time of 1-2.5h.

5. The method for preparing a fragrance according to claim 1, characterized in that, The alpha-amylase is added in an amount of 50-400u / g, the glucoamylase is added in an amount of 50-200u / g, the pullulanase is added in an amount of 50-200u / g and the protease is added in an amount of 50-300u / g, all based on the mass of the tobacco stem extract.

6. The method of claim 1, wherein the flavor is prepared by the steps of: The total molar ratio of the mixed amino acids to the reducing sugars in the enzymolysis solution is (0.05-0.5):

1.

7. The method of claim 1, wherein the flavor is prepared by the steps of: The molar ratio among alanine, glycine and aspartic acid is (1-2):1:

1.

8. A flavouring characterised in that, The flavor essence is prepared by the preparation method of any one of claims 1-7.

9. A flavouring according to claim 8, characterised in that, The total content of volatile components in the flavor essence is 2.76-3.88μg / mL, and the total OAV value is 3.45-10.

1.

10. The flavor essence of claim 9 in the preparation of cigarettes.

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