Preparation method and application of Kyoho grape extract

Abstract

The invention relates to a preparation method and application of a Kyoho grape extract, and belongs to the technical field of extracts. The preparation method of the Kyoho grape extract comprises the following steps: grinding Kyoho grape pulp into pulp, performing enzymolysis reaction by adopting pectinase to obtain fruit pulp enzymatic hydrolysate, performing enzyme deactivation on the enzymatic hydrolysate, mixing with an ethanol water solution, performing ultrasonic extraction, filtering, centrifuging, concentrating and drying an extracting solution to obtain the Kyoho grape extract. The Kyoho grapes are extracted by adopting a specific optimal process, and the Kyoho grape extract prepared by the method contains various active ingredients such as polyphenol and flavone with relatively high content, has good antioxidant activity and can be used for remarkably improving the flavor quality of the grape extract. When the grape essence is applied to cigarette flavor raw materials, the aroma quality, the aroma richness, the fineness and the softness of cigarettes can be improved, the irritation is reduced, offensive odor is reduced, and the grape essence has typical sweet aroma of grapes, can be coordinated with smoke and is wide in market prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of extracts, and in particular to a preparation method and application of a Kyoho grape extract. Background Art

[0002] Grapes, a perennial deciduous vine belonging to the genus Vitis in the family Vitaceae, are rich in nutrients such as carbohydrates, amino acids, organic acids, trace elements, and anthocyanins, making them highly sought after by consumers. However, most of the bioactive compounds in grapes are located intracellularly, requiring appropriate extraction methods to disrupt the cell structure and promote the diffusion of intracellular active substances into the solvent. Currently, conventional extraction methods, including Soxhlet reflux extraction and maceration extraction, suffer from drawbacks such as long extraction times, low extraction efficiency, and high energy consumption. Consequently, green extraction has attracted increasing attention, focusing on shortening extraction times and improving yields. Ultrasound-assisted extraction can preserve the integrity of heat-sensitive compounds and reduce extraction time, while enzymatic extraction offers mild reaction conditions and can increase the yield of target compounds. However, the effects of different extraction processes on the active components in grape pulp extracts remain unclear, and their effectiveness is also uncertain.

[0003] Natural extracts possess unique flavors that cannot be replaced by synthetic flavorings, making them indispensable ingredients in cigarette flavorings. Grape extracts contain a variety of active substances and are highly sought after as food additives and flavor enhancers in the food and tobacco industries. Currently, research on grape extracts primarily focuses on stems, skins, seeds, and grape pomace, while relatively little research has focused on the edible grape pulp. Only Heidari et al. used ultrasonic extraction to extract phenolic compounds from grape pulp and used them in the production of soft candy.

[0004] In recent years, there have been reports on the use of grape extracts in cigarettes. Among them, the patent "A method for preparing a raisin extract for tobacco and its application in cigarettes" (application number CN201510450486.3) soaks raisins in water and crushes them into raisin pulp, then extracts and concentrates them with ethanol to obtain raisin extract. It is found that the raisin extract can increase the aroma and fruity flavor, mask the impurities, and has the effects of sweetening and softening the smoke. In addition, the patent "A method for improving and purifying raisin extract and its application" (application number CN201710541488.2) first uses biological agents to hydrolyze and ferment frozen raisins, and then combines alcohol precipitation, membrane separation and other technologies to obtain its extract. It is found that the raisin extract can reduce irritation, reduce impurities, and improve quality. However, the above extraction methods still have problems such as low extraction efficiency and high energy consumption.

[0005] Research has shown that ultrasound-assisted enzymatic extraction (UAE) combines the advantages of both ultrasound-assisted and enzymatic extraction methods, offering broad application prospects for the extraction of bioactive plant compounds. However, currently, the main methods for extracting grape pulp are Soxhlet reflux extraction and ultrasonic extraction. No reports have been found using UAE to prepare Kyoho grape pulp extract, and the effects of this extraction method on extract quality and its application in cigarettes are unclear. Summary of the Invention

[0006] To address the gaps in the prior art, the present invention aims to provide a Kyoho grape extract and a preparation method thereof, which can significantly increase the bioactive substances and antioxidant activity of the extract, enhance its flavor quality, and significantly improve the aroma quality, aroma richness, and delicate softness of cigarettes, reduce irritation, and reduce impurities.

[0007] This invention innovatively develops a method for preparing Kyoho grape extract, which contains high levels of active ingredients such as polyphenols and flavonoids, exhibits excellent antioxidant properties, and improves the flavor of the extract. Furthermore, this method is time-efficient and simple, and the extraction solvent is recyclable, making it easily scalable for industrial production and practical.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a method for preparing a Kyoho grape extract, comprising the following steps:

[0010] (1) grinding the pulp into pulp, mixing it with pectinase to perform enzymatic hydrolysis reaction, and obtaining pulp enzymatic hydrolyzate;

[0011] (2) inactivating the enzyme in the enzymatic solution and performing ultrasonic extraction with an ethanol aqueous solution to obtain an extract;

[0012] (3) filtering and centrifuging the extract to obtain a supernatant and a residue;

[0013] (4) The supernatant was concentrated by rotary evaporation and dried to obtain Kyoho grape extract.

[0014] Preferably, the pectinase comprises polygalacturonase or polygalacturonate lyase.

[0015] Preferably, the mass ratio of the pectinase to the pulp sample is (0.52):(50100).

[0016] Preferably, the temperature of the enzymatic hydrolysis reaction is 40-70°C, such as 40°C, 50°C, 60°C or 70°C.

[0017] Preferably, the enzymatic hydrolysis reaction time is 30-120 min, such as 30 min, 60 min, 90 min or 120 min.

[0018] Preferably, the pH value of the enzymatic hydrolysis reaction is 3.0-6.0, such as 3.0, 4.0, 5.0 or 6.0.

[0019] Preferably, the time for inactivating the enzyme in the boiling water bath is 10-30 min, such as 10 min, 20 min or 30 min.

[0020] Preferably, the extraction solvent for the ultrasonic extraction is a 40-80% ethanol aqueous solution, such as 40%, 50%, 60%, 70% or 80%.

[0021] Preferably, the extraction temperature of the ultrasonic extraction is 25-40°C, such as 25°C, 28°C, 31°C, 34°C, 37°C or 40°C.

[0022] Preferably, the extraction time of the ultrasonic extraction is 10-20 min, for example, 10 min, 12 min, 14 min, 16 min, 18 min or 20 min.

[0023] Preferably, the ultrasonic extraction has an ultrasonic frequency of 10-100 kHz, such as 10 kHz, 28 kHz, 40 kHz, 50 kHz, and 60 kHz.

[0024] Preferably, the ultrasonic amplitude of the ultrasonic extraction is 50-100%, for example, 50% (150W), 60% (180W), 70% (210W), 80% (240W), 90% (270W) or 100% (300W), etc.

[0025] Preferably, the rotation speed of the centrifugal treatment is 4000-8000 rpm, such as 4000 rpm, 5000 rpm, 6000 rpm, 7000 rpm or 8000 rpm.

[0026] Preferably, the centrifugal treatment time is 10-20 min, such as 10 min, 12 min, 14 min, 16 min, 18 min or 20 min.

[0027] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0028] Preferably, the concentration comprises any one or a combination of at least two of rotary evaporation, precipitation or recrystallization.

[0029] Preferably, the drying comprises any one of freeze drying, boiling drying or spray drying, or a combination of at least two thereof.

[0030] In a second aspect, the present invention provides a Kyoho grape extract prepared by the preparation method described in the first aspect.

[0031] The Kyoho grape extract produced by the above method is a brown liquid with a characteristic grape aroma. Its yield is ≥19.0%, and its total polyphenol content is ≥18.0 mg GAE / g, total flavonoids content is ≥3.2 mg RE / g, total anthocyanins ≥3.2 mg / g, and proanthocyanidins ≥16.0 mg / g. This grape extract exhibits excellent antioxidant activity by inhibiting ABTS and DPPH free radicals.

[0032] In a third aspect, the present invention provides a fragrance raw material containing Kyoho grape extract, wherein the fragrance raw material comprises the grape extract with antioxidant efficacy described in the second aspect, propylene glycol and water.

[0033] The present invention creatively applies Kyoho grape extract to tobacco flavor raw materials, which can improve the aroma quality, aroma richness and delicate softness of cigarettes, reduce irritation, and reduce foreign smells. It has the typical sweet and mellow aroma of grapes, can be coordinated with smoke, and has broad market prospects.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) Grape pulp mainly contains macromolecular polysaccharides such as pectin, and its active substances such as polyphenols and flavonoids are mainly located in the pectin layer of the cell wall. The enzymatic hydrolysis of Kyoho grape pulp using specific pectinase is beneficial to the decomposition of macromolecular polysaccharides such as pectin, reducing the viscosity of the dissolved matter, and thus promoting the dissolution of cell bioactive compounds. At the same time, the use of cellulase or β-glucosidase may release excess monosaccharides (such as glucose), which will have a certain impact on the flavor of the product. Pectinase is a single enzyme preparation that is only used to degrade pectin and has little effect on the flavor. In addition, pectinase is low in cost and simple to operate, which can reduce the difficulty of quality control in production operations. In addition, ultrasound-assisted extraction can induce periodic compression and expansion of the solvent through the cavitation effect of ultrasound, forming a high-energy characteristic microjet phenomenon, which can destroy the cell wall structure and promote the dissolution and migration of target components. At the same time, ultrasound can inactivate polyphenol oxidase during the extraction process, thereby effectively retaining the active substances in the grape extract. Ultrasonic-assisted enzymatic extraction can combine the advantages of ultrasonic-assisted extraction and enzymatic extraction, and shows significant advantages in the preparation of Kyoho grape extract and its actual use in cigarettes.

[0036] (2) The Kyoho grapes used are readily available, the preparation process is simple, the extraction time is short, the production cost is low, and the feasibility is strong. In addition, this method can obtain a high-yield and high-quality Kyoho grape pulp extract with a yield of ≥19.0%, a total polyphenol content of ≥18.0 mg GAE / g, a total flavonoid content of ≥3.2 mg RE / g, a total anthocyanin content of ≥3.2 mg / g, and a proanthocyanidin content of ≥16.0 mg / g, and has a unique grape aroma. In addition, the grape extract also has good antioxidant activity.

[0037] Applying Kyoho grape extract to tobacco flavor raw materials can significantly improve the aroma quality, aroma richness and delicate softness of cigarettes, reduce irritation and reduce impurities. It has the typical sweet and mellow aroma of grapes, can be coordinated with the smoke, and has broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 These are the results of a single-factor experiment using different Kyoho grape extracts.

[0039] Figure 2 Response surface design results for different Kyoho grape extracts.

[0040] Figure 3 This is the result of the antioxidant activity of different Kyoho grape extracts.

[0041] Figure 4 This is the total ion map of volatile components of different Kyoho grape extracts.

[0042] Figure 5 These are the analysis results of volatile components of different Kyoho grape extracts. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below with reference to specific embodiments. Kyoho grapes: purchased from the Dazeshan planting base in Pingdu City, Qingdao; 1,1-diphenyl-2-trinitrophenylhydrazine, 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid, and pectinase: purchased from Shanghai Yuanye Biotechnology Co., Ltd. (product number: S10007, Aspergillus niger, 5000U / g); Folin phenol reagent, rutin, ascorbic acid V C : purchased from Beijing Solebow Technology Co., Ltd.; 2-octanol: purchased from Maclean Biotechnology Co., Ltd.; C7-C40 saturated normal alkanes: purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0044] Example 1

[0045] A method for preparing Kyoho grape extract, the specific process is as follows:

[0046] (1) Mature Kyoho grapes were peeled and deseeded, and the pulp was ground into a pulp using a mortar and pestle and mixed uniformly to obtain a Kyoho grape pulp sample;

[0047] (2): The pulp sample was added to 60% ethanol for ultrasonic extraction with a solid-liquid ratio of 1:5. The yield was used as the evaluation index. Under the condition of an ultrasonic frequency of 40 kHz, the ultrasonic time (10-50 min), ultrasonic temperature (10-50 ° C), and ultrasonic amplitude (10-90%) were optimized;

[0048] (3): The crude extract of Kyoho grapes was filtered and centrifuged at a speed of 4000 rpm, a centrifugal time of 20 min, and a centrifugal temperature of 4°C. After centrifugation, the supernatant was concentrated to a small amount by rotary evaporation, placed in a refrigerator at -80°C, and freeze-dried to obtain the Kyoho grape extract;

[0049] (4): Single factor optimization results are shown in Figure 1 The study found that the yield of Kyoho grape extract increased with the increase of extraction time, reaching its maximum at 20 minutes, and then showed a downward trend. Similarly, the extraction yield also showed a trend of first increasing and then decreasing with the continuous increase of ultrasonic amplitude, among which the yield reached its maximum when the ultrasonic amplitude was 70%. In addition, the ultrasonic temperature also showed a similar trend, with the highest yield at a temperature of 30°C.

[0050] (5): Based on the single-factor optimization results, the response surface design Box-Benhnken experiment was used to further optimize the ultrasonic extraction parameters, in which the ultrasonic time was 10 min, 20 min, and 30 min, the ultrasonic amplitude was 50% (power 150 W), 70% (power 210 W), and 90% (power 270 W), and the ultrasonic temperature was 20 °C, 30 °C, and 40 °C.

[0051] (6): The obtained different Kyoho grape crude extracts were filtered and centrifuged at a speed of 4000 rpm, a centrifugal time of 20 min, and a centrifugal temperature of 4°C. After centrifugation, the supernatant was concentrated to a small amount by rotary evaporation, placed in a -80°C refrigerator, and freeze-dried to obtain different Kyoho grape extracts. The yield, total phenol content, and total flavonoid content in the grape extracts were used as evaluation indicators.

[0052] (7): Table 1 shows the response surface optimization results of ultrasound-assisted extraction, in which the yield was 8.47-15.55%, the total phenolic content was 7.22-14.58 mg GAE / g, and the total flavonoid content was 1.68-3.16 mg RE / g.

[0053] Table 1 Response surface experimental design and results

[0054]

[0055] (8): The effects of ultrasonic time, ultrasonic amplitude and extraction temperature on yield, total phenol content and total flavonoid content were analyzed by Design-Expert 13 statistical software. The variance analysis and regression model of the model are shown in Table 2.

[0056] Table 2 Analysis of variance of response surface regression simulation

[0057] *P≤0.05 is significant; **P≤0.01 is extremely significant.

[0058] The coefficients of variation of the yield, total phenol content, and total flavonoid content models were 1.03, 1.95, and 2.32, respectively, indicating high repeatability and validity. According to the results of variance analysis, except for the interaction between ultrasonic amplitude and extraction temperature, the other factors had a very significant effect on the yield. Factors A, B, and A 2 and B 2 is an important variable affecting the total phenol content, while A, C, AB, AC, A 2 、B 2 and C 2 is an important variable affecting the total flavonoid content. Therefore, the quadratic polynomial regression model for yield, total phenolic content, and total flavonoid content is as follows: Yield =15.40-0.76A+0.594B+0.201C+2.33AB+0.53AC+0.013BC-1.14A 2 -2.11B 2 -1.62C 2 Total phenol content =13.77-1.55A+0.131B+0.051C+2.04AB-0.265AC+0.138BC-1.03A 2 -1.57B 2 +0.12C 2 Total flavonoids content =3.05-0.225A-0.009B-0.141C+0.195AB-0.105AC+0.03BC-0.611A 2 -0.358B 2 +0.117C 2

[0059] R of the models for yield, total phenolic content and total flavonoid content 2The results were 0.9982, 0.9920, and 0.9913, respectively, with P < 0.001 and the lack-of-fit term P > 0.05, indicating that the data are reliable. The order of influence of the three factors on yield and total phenol content was ultrasonic time > ultrasonic amplitude > extraction temperature, while the order of influence of the three factors on total flavonoid content was ultrasonic time > extraction amplitude > ultrasonic power.

[0060] Response surface analysis is shown in Figure 2 , we can see that the curve is flat and the contour line shape tends to be circular. Except for the interaction terms AB and AC, the interaction effects of other factors are weak, which is consistent with the variance analysis.

[0061] Design-Expert 13 statistical software predicted the optimal ultrasonic conditions to be 16.12 minutes, 67.56% ultrasonic amplitude, and 28.48°C. However, adjustments were made in practice. The optimal ultrasonic conditions were set to 16 minutes, 70% ultrasonic amplitude, and 28°C. The resulting grape extract had an extraction yield of 14.94%, 14.38 mg GAE / g, and 2.98 mg RE / g, respectively. These results differed from all predicted values ​​by less than 5%, demonstrating the reliability and accuracy of the method.

[0062] Example 2

[0063] A method for preparing Kyoho grape extract, the specific process is as follows:

[0064] (1): Mature Kyoho grapes were peeled and deseeded, and the pulp was ground into a pulp using a mortar and pestle and mixed evenly to obtain a pulp sample. 0.5% pectinase was added to the pulp, and the pH value was adjusted to 4. The pulp was placed at 50°C for 1 h to obtain Kyoho grape pulp hydrolyzate. The pectinase activity was 5×10 4 U / g;

[0065] (2): The enzymatic hydrolysate was heated at 100°C for 15 min to inactivate the enzyme, and after cooling, 60% ethanol was added for ultrasonic treatment. The solid-liquid ratio was 1:5. The ultrasonic extraction conditions were based on the response surface analysis results, that is, under the conditions of ultrasonic frequency of 40 kHz, ultrasonic time of 16 min, temperature of 28°C, amplitude of 70% (power of 280 W) to obtain the crude extract of Kyoho grapes;

[0066] (3): The crude extract of Kyoho grapes was filtered and centrifuged at a speed of 4000 rpm, a centrifugal time of 20 min, and a centrifugal temperature of 4°C. After centrifugation, the supernatant was concentrated to a small amount by rotary evaporation, placed in a -80°C refrigerator, and freeze-dried to obtain the Kyoho grape extract, which was recorded as UAEE.

[0067] Example 3

[0068] A method for preparing Kyoho grape extract, the specific process is as follows:

[0069] (1) Mature Kyoho grapes were peeled and deseeded, and the pulp was ground into a pulp using a mortar and pestle and mixed uniformly to obtain a Kyoho grape pulp sample;

[0070] (2): The pulp sample was added with 60% ethanol for ultrasonic extraction with a solid-liquid ratio of 1:5. The ultrasonic extraction conditions were based on the response surface analysis results, i.e., the ultrasonic frequency was 40 kHz, the ultrasonic time was set to 16 min, the temperature was 28 °C, the amplitude was 70% (power was 280 W), and the crude extract of Kyoho grapes was obtained;

[0071] (3): The crude extract of Kyoho grapes was filtered and centrifuged at a speed of 4000 rpm, a centrifugal time of 20 min, and a centrifugal temperature of 4°C. After centrifugation, the supernatant was concentrated to a small amount by rotary evaporation, placed in a -80°C refrigerator, and freeze-dried to obtain the Kyoho grape extract, which was recorded as UAE.

[0072] Example 4

[0073] A method for preparing Kyoho grape extract, the specific process is as follows:

[0074] (1): Mature Kyoho grapes were peeled and deseeded, and the pulp was ground into a pulp using a mortar and pestle and mixed evenly to obtain a pulp sample. 0.5% pectinase was added to the pulp, and the pH value was adjusted to 4. The pulp was placed at 50°C for 1 h to obtain Kyoho grape pulp hydrolyzate. The pectinase activity was 5×10 4 U / g;

[0075] (2): The enzymatic hydrolysate was heated at 100°C for 15 min to inactivate the enzyme, and after cooling, 60% ethanol was added for reflux extraction. The solid-liquid ratio was 1:5, the extraction time was 3 h, the temperature was 60°C, and the extraction was repeated once to obtain a crude extract of Kyoho grapes;

[0076] (3): The crude extract of Kyoho grapes was filtered and centrifuged at a speed of 4000 rpm, a centrifugal time of 20 min, and a centrifugal temperature of 4°C. After centrifugation, the supernatant was concentrated to a small amount by rotary evaporation, placed in a -80°C refrigerator, and freeze-dried to obtain the Kyoho grape extract, which was recorded as EAE.

[0077] Comparative Example 1

[0078] A method for preparing Kyoho grape extract, the specific process is as follows:

[0079] (1) Mature Kyoho grapes were peeled and deseeded, and the pulp was ground into a pulp using a mortar and pestle and mixed uniformly to obtain a Kyoho grape pulp sample;

[0080] (2): The pulp sample was added with 60% ethanol for reflux extraction, with a solid-liquid ratio of 1:5, an extraction time of 3 h, a temperature of 60°C, and one extraction to obtain a crude extract of Kyoho grapes;

[0081] (3): The crude extract of Kyoho grapes was filtered and centrifuged at a speed of 4000 rpm, a centrifugal time of 20 min, and a centrifugal temperature of 4°C. After centrifugation, the supernatant was concentrated to a small amount by rotary evaporation, placed in a -80°C refrigerator, and freeze-dried to obtain the Kyoho grape extract, which was recorded as SRE.

[0082] The extract sample obtained in Example 24 was subjected to a comparative test with the sample in Comparative Example 1.

[0083] 1. Comparison of bioactive ingredients

[0084] The total phenol content in the extracts of each embodiment and comparative example was determined by the Folin-phenol colorimetric method; the total flavonoid content in the extracts was determined by the aluminum chloride colorimetric method; the total anthocyanidin content in the extracts was determined by the pH differential method; and the proanthocyanidin content in the extracts was determined by the vanillin-hydrochloric acid method.

[0085] Table 3 Bioactive components of different Kyoho grape extracts

[0086] a-c : Different letters in the same column indicate significant differences.

[0087] As shown in Table 3, the yields of grape extracts obtained by the four extraction methods ranged from 13.43% to 19.34%, with UAEE achieving the highest yield. Furthermore, the contents of total polyphenols, total polyphenols, total anthocyanins, and proanthocyanidins in UAEE, UAE, and EAE were all significantly increased compared to SRE. Among them, the UAEE-extracted sample had the highest content of the four bioactive compounds, with total phenols, total flavonoids, total anthocyanins, and proanthocyanidins reaching 18.21 mg GAE / g, 3.56 mg RE / g, 3.51 mg / g, and 16.24 mg / g, respectively. Compared to the SRE obtained in the control example, the contents of the four active substances increased by 25.85%, 25.79%, 134.00%, and 8.85%, respectively. These results demonstrate that UAEE can combine the advantages of ultrasonic and enzymatic extraction to significantly improve the yield and content of bioactive compounds in grape extracts.

[0088] 2. Comparison of antioxidant activity

[0089] 2.1 The antioxidant activity of the extracts from each example and comparative example was evaluated using the DPPH free radical scavenging ability.

[0090] Mix 0.5 mL of extract samples of varying concentrations with 1 mL of 0.1 mmol / L DPPH ethanol solution. Incubate in the dark for 30 minutes. Measure absorbance at 517 nm using a microplate reader. Perform triplicate replicates for each sample. Ascorbic acid (Vc) serves as a positive control, with 1 mL of anhydrous ethanol replacing the DPPH solution. A blank control is prepared with 0.5 mL of ethanol replacing the sample solution.

[0091] The DPPH free radical scavenging rate was calculated according to the following formula:

[0092]

[0093] Where: A2 is the absorbance of the sample group; A1 is the absorbance of the control group; A0 is the absorbance of the blank group.

[0094] 2.2 The antioxidant activity of the extracts from each example and comparative example was evaluated using the ABTS free radical scavenging ability.

[0095] Mix a 7.4 mmol / L ABTS solution with a 3.8 mmol / L potassium persulfate solution overnight at 25°C in the dark. Take an appropriate amount of the overnight mixture and dilute it with PBS (pH 7.4) to an absorbance of 0.70 ± 0.02 at 734 nm. Mix 100 μL of extract samples of varying concentrations with 1 mL of the ABTS dilution, incubate at room temperature in the dark for 15 minutes, and measure the absorbance at 734 nm using a microplate reader. Ascorbic acid (Vc) was used as a positive control, and the DPPH solution was replaced with 1 mL of anhydrous ethanol. The blank control was treated with 0.5 mL of ethanol solution instead of the sample solution.

[0096] The ABTS free radical scavenging rate was calculated according to the following formula:

[0097]

[0098] Where: A2 is the absorbance of the sample group; A1 is the absorbance of the control group; A0 is the absorbance of the blank group.

[0099] The antioxidant results of the extracts in each embodiment and comparative example are as follows Figure 3As shown, all four grape extracts possessed excellent DPPH and ABTS free radical scavenging abilities, with scavenging abilities significantly increasing with increasing extract concentration. Specifically, UAEE, EAE, and UAE exhibited significantly higher DPPH and ABTS free radical scavenging abilities compared to SRE. However, different grape extracts exhibited stronger ABTS free radical scavenging abilities. At a sample concentration of 4 mg / mL, the ABTS free radical scavenging rates of UAEE, EAE, UAE, and SRE were 57.73%, 46.94%, 50.63%, and 45.38%, respectively. Clearly, UAEE possessed the highest free radical scavenging rate of the four extracts. In summary, the grape extract obtained by ultrasound-enzyme-assisted extraction possessed the greatest antioxidant capacity among the different extraction methods.

[0100] 3. Comparison of volatile components

[0101] HS-SPME-GCMS was used to determine the volatile components in the extracts of each example and comparative example.

[0102] Solid phase microextraction conditions: solid phase microextraction head: 50 / 30μm solid phase microextraction head; extraction temperature 80℃; preheating equilibrium time 30min; extraction time 30min; desorption time 5min; extraction head aging temperature 250℃; extraction head aging time 40min; extraction head aging purge gas is nitrogen, and the purity is not less than 99.999%.

[0103] Chromatographic conditions: HP-5MS column (30m×0.25mm×0.25μm); injection port temperature 250℃; temperature program: initial temperature 50℃, hold for 3min, increase to 200℃ at a rate of 3℃ / min, hold for 3min, increase to 230℃ at a rate of 6℃ / min, hold for 1min.

[0104] Mass spectrometry conditions: EI ionization energy 70 eV; transfer line temperature 260°C; ion source temperature 230°C; quadrupole temperature 150°C; mass scan range 30-550 amu.

[0105] Qualitative analysis: Through the NIST 20 spectral library search, based on the RI calculated from the retention time of volatile components and n-alkanes, substances with a matching degree of more than 80 were selected and combined with manual spectrum analysis to complete the qualitative analysis of volatile components.

[0106] The RI calculation formula is as follows:

[0107]

[0108] Where: ta, t b , t b+1They represent the retention time (min) of the component, the retention time (min) of the previous normal alkane of the component, and the retention time (min) of the next normal alkane, respectively, where b+1>a>b.

[0109] Quantitative analysis: All volatile components were semi-quantitatively analyzed using the internal standard method.

[0110] The content of volatile components is calculated according to the following formula:

[0111]

[0112] Where: M i Represents the concentration of the component to be tested; N x 、N i and M x They represent the peak area of ​​2-octanol, the concentration of 2-octanol (50 μg / kg), and the peak area of ​​the component to be tested, respectively.

[0113] Table 4 Volatile components of different Kyoho grape extracts

[0114] -: not detected; a-d : Different letters in the same row indicate significant differences.

[0115] The total ion diagram of volatile components in the extracts of each embodiment and comparative example is as follows: Figure 4 As shown, a total of 86 volatile components were identified in the four grape extracts, including 12 alcohols, 31 aldehydes, 10 ketones, 24 esters, 6 acids, 1 phenol, and 2 other compounds (Table 4). UAEE had the highest number of volatile components, with 69 identified, compared to only 59, 55, and 57 volatile compounds in SRE, EAE, and UAE, respectively. These results indicate that ultrasound-enzyme-assisted extraction can increase the volatile content of grape extracts.

[0116] The volatile component analysis results of the extracts of each embodiment and comparative example are as follows: Figure 5 As shown in Figure 2, the four grape extracts contained 48 common volatile components, of which only two unique volatile components were identified in SRE, SEE and UAE, while 16 unique volatile components were found in UAEE ( Figure 5 AB). In addition, UAEE contained the highest volatile compound content, which could reach 1366.09±20.44μg / kg. The four grape extracts were mainly composed of aldehydes and esters, but the content of volatile components of different types was significantly different ( Figure 5C). Compared with the control example SRE, the relative content of aldehydes in EAE and UAEE was significantly reduced, but the ester content was significantly increased ( Figure 5 D). The difference is that the relative content of esters in UAE decreased. In summary, the different treatments significantly altered the types and content of volatile compounds in the extracts, with ultrasound-enzyme-assisted treatment demonstrating a significant improvement in the flavor quality of the extract. These findings provide theoretical basis and technical support for the application of grape extracts.

[0117] 4. Comparison of cigarette flavoring effects

[0118] The industry standard "YC / T 497-2014 Sensory Evaluation Method for Chinese-Style Cigarettes" was used as an evaluation reference, and the judges of the smoking evaluation panel evaluated the flavoring effects of the cigarettes of each embodiment and comparative example from the aspects of aroma characteristics, comfort characteristics, and aroma style characteristics.

[0119] The sensory evaluation method is as follows: Finished cut tobacco is equilibrated at 22°C and 60% relative humidity for 48 hours. Six grams of the equilibrated cut tobacco are then injected into the cigarettes using a flavoring injector at a rate of 0.2%. The control is the same cigarette treated with the same amount of propylene glycol sprayed under the same conditions. The panelists' scores are the average of their responses.

[0120] Table 5 Sensory evaluation results of different Kyoho grape extracts in cigarettes

[0121]

[0122] Table 5 shows the sensory evaluation results of the extracts from each example and comparative example. Compared to the cigarettes in the control group, Examples 2-4 all enhanced the fresh, sweet, and fruity aroma of Kyoho grapes, while also increasing sweetness and smoothness, improving the quality and volume of the cigarette aroma, and reducing irritation and off-flavors, resulting in a softer and more refined smoke. Among them, UAEE demonstrated the best sensory effects, achieving the highest efficacy in enhancing the quality, volume, and richness of the cigarette aroma, as well as improving oral smoothness. These studies demonstrate that Kyoho grape pulp extracts prepared using this extraction method have broad market prospects as a cigarette flavoring ingredient.

[0123] Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the claims. Any person skilled in the art may make several possible changes and modifications without departing from the concept of the present application. Therefore, the scope of protection of the present application shall be based on the scope defined by the claims of the present application.

Claims

1. A method for preparing Kyoho grape extract, characterized in that: The preparation method comprises: (1) Grinding Kyoho grape pulp into pulp, mixing with pectinase for enzymatic hydrolysis, and obtaining pulp enzymatic hydrolyzate; (2) inactivating the enzyme in the enzymatic hydrolyzate and performing ultrasonic extraction with an ethanol aqueous solution to obtain an extract; (3) Filtering and centrifuging the extract to obtain a supernatant and a residue; (4) Concentrate and dry to obtain Kyoho grape extract.

2. The preparation method according to claim 1, characterized in that The pectinase includes polygalacturonase or polygalacturonate lyase.

3. The preparation method according to claim 1, characterized in that The pH value of the enzymatic hydrolysis reaction is 3.0-6.0, the temperature is 40-70° C., and the reaction time is 30-120 min.

4. The preparation method according to claim 1, characterized in that The extraction temperature of the ultrasonic extraction is 25-40° C., and the extraction time is 10-20 min.

5. The preparation method according to claim 1, characterized in that The ultrasonic extraction has an ultrasonic frequency of 10-100 kHz and a power of 150-270 W.

6. A Kyoho grape extract prepared by the preparation method according to claim 1.

7. A fragrant raw material containing the Kyoho grape extract according to claim 6.

8. The incense raw material according to claim 7, characterized in that The aroma raw materials include grape extract and diluent.

9. Use of the Kyoho grape extract according to claim 6 in cigarettes.

10. Use of the Kyoho grape extract according to claim 6 in tobacco flavor raw materials.

Images