A method for preparing flavorings by low-temperature enzymatic fermentation coupled with supercritical CO2 extraction

Abstract

The application belongs to the technical field of natural perfume extraction and essence preparation, and particularly relates to a method for preparing essence by low-temperature enzyme fermentation coupled with supercritical CO2 extraction. The method takes rose petals, perilla leaves and grapefruit peels as raw materials, releases aroma precursor substances through the synergistic low-temperature enzymolysis of pectinase, cellulase and other enzymes, introduces non-brewing yeast and esterification bacteria for step-by-step anaerobic fermentation to generate multi-level composite aroma components, and then performs freeze-drying and three-stage gradient supercritical CO2 extraction to fractionally enrich different polar aroma components, and finally mixes and stabilizes the components to obtain essence products. The method constructs a complete process flow from precursor release to structure construction and then to component extraction, and is suitable for the fields of tobacco essence, food essence and the like.

Description

Technical Field

[0001] This invention belongs to the field of natural fragrance extraction and flavor preparation technology, specifically relating to a flavor preparation method of low-temperature enzyme fermentation coupled with supercritical CO2 extraction. Background Technology

[0002] Tobacco flavorings are important components in cigarette products used to improve and enhance aroma, harmonize the natural flavor of tobacco, and improve overall flavor quality. Their sources mainly include natural plant extracts, thermally reacted flavorings, synthetic flavorings, and fermentation products. Especially in recent years, with the increasingly prominent consumer trend towards "natural, green, and safe" products, research and development of tobacco flavorings prepared from natural plants has continued to heat up. However, a series of technical bottlenecks still exist in the existing preparation processes of natural flavorings, restricting their widespread application in the tobacco industry.

[0003] Currently, the main methods for preparing natural tobacco flavorings include steam distillation, solvent extraction, ultrasound-assisted extraction, and some enzymatic treatments. Steam distillation is simple, but the high temperature required easily leads to the degradation or loss of some heat-sensitive volatile components, resulting in a disrupted aroma spectrum and a more singular flavor profile. Solvent extraction, while highly efficient, often requires organic solvents such as ethanol, ethyl acetate, and petroleum ether, leading to solvent residues, environmental pollution, and difficulties in post-processing, making it difficult to meet the safety and purity requirements of tobacco additives. While ultrasound-assisted or microwave extraction improves diffusion rates, practical applications still face challenges such as high energy consumption, poor selectivity of aroma substances, and difficulty in separating impurities.

[0004] On the other hand, fermentation technology has been gradually introduced into the field of flavor preparation in recent years. It utilizes microbial metabolism or enzymatic reactions to convert precursors such as sugars, amino acids, and fatty acids in plant raw materials into a series of aroma-active secondary metabolites. This method has advantages such as low temperature and mildness, diverse conversion pathways, and rich and natural aromas. However, in practical applications, fermentation conditions are greatly affected by factors such as temperature, time, pH, and the activity of microbial / enzyme strains, resulting in insufficient reaction stability and a tendency to generate excessive impurities or off-flavor components. Furthermore, the concentration of aroma substances in the fermentation broth is low, making extraction and separation difficult, resulting in low processing efficiency and limited industrialization capabilities.

[0005] In the purification of aroma components, supercritical CO2 extraction technology is considered an ideal solution that is efficient, green, and leaves low residues. Because it is carried out at relatively low temperatures and under anaerobic conditions, it can maximize the preservation of the activity and integrity of aroma molecules, while also possessing strong selectivity and low energy consumption. However, in practice, supercritical CO2 extraction is suitable for pre-treated samples that have already been enriched or purified to a high degree. When directly applied to the original fermentation broth or complex plant mixtures, it still suffers from problems such as low extraction efficiency, unbalanced aroma composition, and low yields of some polar components, making it difficult to fully meet the requirements of tobacco flavorings for aroma complexity and concentration.

[0006] In summary, existing tobacco flavoring preparation technologies generally suffer from the following prominent problems: significant heat loss of aroma substances during extraction, resulting in unstable aroma profiles; numerous residual impurities in the preparation system, making purification difficult; relatively thin aroma layers, insufficient naturalness and longevity; and low efficiency, demanding process conditions, or factors hindering large-scale production. Especially when applied to cigarettes, flavorings must not only meet the requirements of complex flavors, distinct layers, and high integration with cigarette smoke, but also possess good thermal stability and safety.

[0007] Therefore, there is an urgent need to develop a new method for flavor preparation that can efficiently generate aroma components under mild conditions while taking into account selective extraction and flavor integrity, in order to overcome the above-mentioned technical bottlenecks and meet the tobacco industry's demand for high-quality natural flavors. Summary of the Invention

[0008] The purpose of this invention is to provide a method for preparing flavorings by coupling low-temperature enzymatic fermentation with supercritical CO2 extraction, which improves the extraction efficiency and flavor retention of aroma substances and solves the problems of severe aroma loss, single flavor level and difficulty in purification in the prior art.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] A method for preparing flavorings using low-temperature enzymatic fermentation coupled with supercritical CO2 extraction, comprising the following steps:

[0011] (1) Mix rose petals, perilla leaves and grapefruit peel and then crush them. Mix the crushed raw materials with deionized water to obtain a mixture. Add pectinase, cellulase, β-glucosidase and flavor protease to the mixture and hydrolyze it to obtain an enzymatic hydrolysate.

[0012] (2) Non-Saccharomyces cerevisiae and esterifying bacteria were inoculated into the enzymatic hydrolysate in sequence, and fermentation was carried out to obtain the fermentation broth;

[0013] (3) Centrifuge the fermentation broth to separate the supernatant, concentrate the supernatant and freeze-dry it to obtain freeze-dried powder;

[0014] (4) Compress the lyophilized powder into tablets (e.g., to a thickness of 2-4 mm), place them in a supercritical CO2 extraction vessel, and extract them under three-stage gradient conditions to obtain the extract;

[0015] (5) Combine the extracts to obtain a compound fragrance base, add rosemary extract to the compound fragrance base to obtain the fragrance.

[0016] Furthermore, the mass ratio of rose petals, perilla leaves, and pomelo peel in step (1) is (3-6):(1-2):1.

[0017] Further, in step (1), the amount of pectinase added to the mixture is 60-100 U / mL, the amount of cellulase added to the mixture is 80-120 U / mL, the amount of β-glucosidase added to the mixture is 15-25 U / mL, and the amount of flavor protease added to the mixture is 20-30 U / mL.

[0018] Further, the enzymatic hydrolysis in step (1) is carried out at a temperature of 35-40°C, a pH value of 5.0-5.5, and a time of 2-4 hours.

[0019] This invention boasts a significant advantage in the selection of raw material substrates for constructing a fundamental flavor profile. Rose petals, perilla leaves, and grapefruit peel represent floral, herbal, and citrus notes respectively in their flavor structure, exhibiting excellent complementarity and harmonizing potential. Furthermore, their naturally abundant glycosides, terpenes, and aldehydes and ketones are important precursors for forming complex tobacco aromas. Rose petals, rich in linalool and geraniol, can be converted into typical floral aroma molecules; perilla provides perillaldehyde and perillyl alcohol, forming the core framework of the herbal notes; and grapefruit peel contains naringin and limonene, constructing a volatile and penetrating top note layer. In terms of enzyme selection, pectinase and cellulase synergistically work on the complex structure of plant cell walls, breaking down cell walls to release aroma precursors from the intercellular spaces; β-glucosidase hydrolyzes glycosidic bonds to release bound terpenes and phenolic compounds; and flavor proteases further release flavor peptides and short-chain nitrogenous compounds, forming a base flavor support. This strategy, which employs multi-enzyme synergy and gentle enzymatic hydrolysis, achieves efficient release of aroma-active substances and pre-construction of substrate flavor structures while ensuring structural integrity, providing highly reactive aroma precursors for subsequent fermentation.

[0020] Further, the inoculation concentration of the non-Saccharomyces cerevisiae in step (2) is (0.5–1.5) × 10⁻⁶. 7 CFU / mL, the inoculation concentration of esterifying bacteria is (5–10) × 10⁻⁶. 6 CFU / mL.

[0021] Further, in step (2), after inoculating with non-Saccharomyces cerevisiae, fermentation is carried out at 24-28°C for 24-36 hours, and after inoculating with esterified bacteria, fermentation is carried out at 20-22°C for 48-72 hours.

[0022] During the fermentation stage, the present invention first inoculates non-Saccharomyces yeast (such as Kluyveromyces) with strong ester-producing ability and high metabolic activity towards polyols and terpenes, which can rapidly generate fruity and floral aroma molecules with top notes, such as benzyl acetate and linalool acetate. Subsequently, esterifying bacteria (such as Hansenula d'Barry yeast) are introduced to carry out anaerobic fermentation at a lower temperature. This enzyme has a strong ability to produce long-chain fatty acid esters and lactones, which can supplement the roundness and viscosity of the mid- and base notes, and improve the "lingering aroma" and smoothness of the smoke. The two bacteria work synergistically and operate in stages, avoiding interference from competition for metabolite substrates or the generation of toxic byproducts. More importantly, this model naturally transforms highly active precursors into a complex and natural aroma system through a microbial enzymatic pathway, ensuring the authenticity and high stability of the aroma source, and avoiding flavor overload and loss of layers caused by continuous fermentation of a single strain.

[0023] Furthermore, the centrifugal separation temperature in step (3) is 4 to 10°C.

[0024] Furthermore, the conditions for the three-stage gradient extraction in step (4) are as follows:

[0025] First stage: Pressure 10-14 MPa, temperature 38-42℃, no entrainer, extraction time 30-45 minutes;

[0026] Second stage: pressure 20-25 MPa, temperature 43-47℃, entrainer is anhydrous ethanol, the volume of entrainer accounts for 3-7% of the total volume of CO2 and entrainer, extraction time 40-60 minutes;

[0027] The third stage: pressure 30-35 MPa, temperature 48-52℃, the entrainer is a mixture of ethanol and water, the volume ratio of ethanol to water is 7-9:1, the volume of the entrainer accounts for 3-7% of the total volume of CO2 and entrainer, and the extraction time is 50-70 minutes.

[0028] Further, the combined extract in step (5) is specifically as follows: first, the extracts obtained in the second and third stages are subjected to reduced pressure treatment to remove the entrainer solvent, and the residual total mass of ethanol and water is controlled to be no higher than 0.5%. Then, the extracts from the first, second and third stages are mixed in a volume ratio of 6-8: 1.5-2.5: 0.5-1 to obtain a composite fragrance base.

[0029] Further, the added rosemary extract in step (5) is 0.008 to 0.015% of the complex fragrance base.

[0030] In terms of aroma component extraction, this invention employs a three-stage gradient supercritical CO2 extraction process, systematically solving the problems of poor selectivity and aroma imbalance inherent in traditional single-stage extraction. The first stage extracts non-polar volatile components under low pressure, mild temperature, and without entrainers, maximizing the retention of top aroma compounds such as limonene and linalool. The second stage introduces a low concentration of ethanol as an entrainer, improving the affinity for moderately polar components (such as phenols and alcohol esters). The third stage further expands the dissolution window of polar components through an ethanol-water mixture, enabling the efficient extraction of components such as coumarins, lactones, and esterified compounds. After the three-stage fractions are blended in proportion, not only is a comprehensive extraction of aroma components achieved, but a gradual aroma progression from light to sweet to deep woody notes is also constructed, resulting in higher integration with the natural aroma of tobacco and better stability. Furthermore, CO2, as a green and residue-free solvent, not only provides good protection for heat-sensitive components but also does not affect the purity of the flavoring after volatilization, laying a solid foundation for producing safe, natural, and tobacco-compatible flavorings.

[0031] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0032] This invention organically integrates a mild enzymatic hydrolysis system with a phased, targeted fermentation strategy, enabling the full release and biosynthetic transformation of potential aroma precursors in plant raw materials. Furthermore, a three-stage supercritical CO2 extraction process is employed to sequentially enrich target aroma substances according to their polarity differences. This method not only effectively enhances the top notes and mid-to-base notes of the fragrance but also significantly improves the overall aroma's layering and harmony. Compared to unoptimized raw material replacement schemes, enzyme substitution schemes, microbial fermentation mode variations, and single-stage extraction methods, this invention demonstrates superior performance in multiple key performance dimensions, including top note release, aroma configuration stability, and persistence. This indicates that the synergistic design between each step is highly targeted and effective, with significant and stable technical results, making it suitable for the development of cigarette and natural flavor products where high aroma quality is required. Detailed Implementation

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Unless otherwise specified, all raw materials used in the embodiments are commercially available products. The following is an illustrative example:

[0035] Pectinase was purchased from Wuhan AmyJet Scientific, ≥20 U / mg.

[0036] Cellulase was purchased from Wuhan AmyJet Technology Co., Ltd., ≥45 U / mg.

[0037] β-glucosidase was purchased from Isehisa (Jiangsu) Biotechnology, ≥30 U / mg.

[0038] Flavor protease was purchased from the Bio-Equipment Network (Lanxu brand), ≥20 U / mg.

[0039] The non-Saccharomyces cerevisiae (Kluyveromyces) was purchased from the China General Microbiological Culture Collection Center, CGMCC 2.4330.

[0040] The esterified bacteria (Hansøe de Barry yeast) was purchased from the China General Microbiological Culture Collection Center, CGMCC 2.3966.

[0041] Rosemary extract was purchased from Nanjing Dausif Biotechnology Co., Ltd.

[0042] α-Amylase was purchased from Guangzhou Kangda Biological Products Co., Ltd., ≥600KNU / kg.

[0043] The saccharifying enzyme was purchased from Qingdao Stan Biotechnology Co., Ltd., at a concentration of 1000 U / g.

[0044] Papain was purchased from Nanning Pangbo Biotechnology Co., Ltd. at a concentration of 3.5 million U / g.

[0045] Lactobacillus brevis was purchased from the China General Microbiological Culture Collection Center, CGMCC 1.2028.

[0046] Lactobacillus plantarum was purchased from the China General Microbiological Culture Collection Center, CGMCC 1.16089.

[0047] The brewing yeast was purchased from the China General Microbiological Culture Collection Center, CGMCC 2.3973.

[0048] Example 1

[0049] This embodiment provides a method for preparing flavorings using low-temperature enzymatic fermentation coupled with supercritical CO2 extraction, comprising the following steps:

[0050] (1) Weigh 500 g of dried rose petals, 200 g of perilla leaves and 100 g of grapefruit peel, mix them and grind them into fine powder below 70 mesh. Mix the resulting powder with deionized water at a ratio of 1:5 (g:mL). Adjust the pH of the mixture to 5.2 using 0.1 mol / L citrate-sodium citrate buffer. Heat the mixture to 38°C and add enzyme preparations to the mixture according to the following ratio: pectinase 80 U / mL, cellulase 100 U / mL, β-glucosidase 20 U / mL and flavor protease 25 U / mL. Keep the mixture at a constant temperature for 3 hours. After the enzymatic hydrolysis is completed, heat the mixture to 75°C and keep it at that temperature for 10 minutes to inactivate the enzyme. Cool the mixture to 26°C for later use.

[0051] (2) Transfer the above enzymatic hydrolysate to a sterilized 10 L fermenter, and inoculate it with non-Saccharomyces cerevisiae at an inoculation concentration of 1.0 × 10⁻⁶. 7 The culture was fermented anaerobicly at 26°C for 30 hours at a concentration of CFU / mL; then inoculated with esterifying bacteria at a concentration of 8.0 × 10⁻⁶. 6 CFU / mL, and continued anaerobic fermentation at 21℃ for 60 hours.

[0052] (3) Place the fermentation broth in a refrigerated centrifuge and centrifuge at 6°C and 4000 rpm for 10 minutes. Collect the supernatant and transfer it to a rotary evaporator to concentrate it to 20% of the original volume. Then freeze-dry it to obtain a powdered freeze-dried fragrance base powder.

[0053] (4) Compress the lyophilized powder into round sheets with a thickness of about 3 mm, and place them in a supercritical CO2 extraction vessel for three-stage extraction:

[0054] First stage: Pressure 12 MPa, temperature 40℃, no entrainer, extraction time 40 minutes, collect the first fraction;

[0055] Second stage: pressure 23 MPa, temperature 45℃, entrainer is anhydrous ethanol, the volume of entrainer accounts for 5% of the total volume of CO2 and entrainer, extraction for 50 minutes, and the second fraction is collected.

[0056] Third stage: pressure 33 MPa, temperature 50℃, the entrainer is a mixture of ethanol and water in a volume ratio of 9:1, the volume of the entrainer accounts for 5% of the total volume of CO2 and entrainer, extraction for 60 minutes, and the third fraction is collected.

[0057] The second and third fractions were separately de-entrained in a rotary evaporator until the total residual ethanol / water content was less than 0.5% (w / w). The three fractions were then mixed in a volume ratio of 7:2:1 to obtain a compound flavor base.

[0058] (5) Add rosemary extract to the compound fragrance base at an amount of 0.01% of the mass of the compound fragrance base. After mixing evenly, the fragrance is obtained. The fragrance is then packaged under nitrogen protection and stored at 4°C away from light.

[0059] Example 2

[0060] This embodiment provides a method for preparing flavorings using low-temperature enzymatic fermentation coupled with supercritical CO2 extraction, comprising the following steps:

[0061] (1) Weigh 450 g of dried rose petals, 150 g of perilla leaves and 100 g of grapefruit peel, mix them and grind them into fine powder less than 60 mesh. Mix the resulting powder with deionized water at a ratio of 1:6 (g:mL). Use 0.1 mol / L lactic acid solution to slowly add dropwise to adjust the pH of the mixture to 5.0. Heat to 37°C and add enzyme preparations to the mixture: pectinase 90 U / mL, cellulase 110 U / mL, β-glucosidase 18 U / mL and flavor protease 22 U / mL. Keep at a constant temperature for 2.5 hours for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, heat the mixture to 72°C and keep it at that temperature for 10 minutes for enzyme inactivation treatment. Then cool it to 25°C for later use.

[0062] (2) Transfer the above enzymatic hydrolysate to a sterilized 10 L fermenter, and first inoculate it with non-Saccharomyces cerevisiae at an inoculation concentration of 1.2 × 10⁻⁶. 7 The culture was fermented anaerobicly at 27°C for 36 hours at a concentration of CFU / mL; subsequently, it was inoculated with esterifying bacteria at a concentration of 6.0 × 10⁻⁶. 6 CFU / mL, and continue anaerobic fermentation at 20℃ for 72 hours.

[0063] (3) Place the fermentation broth in a refrigerated centrifuge and centrifuge at 4°C and 3800 rpm for 12 minutes. Collect the supernatant and transfer it to a rotary evaporator to concentrate it to 25% of its original volume. Then freeze-dry it to obtain a powdered freeze-dried fragrance base powder.

[0064] (4) Compress the freeze-dried powder into round tablets with a thickness of 2.5 mm, and place them into a supercritical CO2 extraction vessel for three-stage extraction:

[0065] First stage: Pressure 11 MPa, temperature 39℃, no entrainer, extraction time 35 minutes, collect the first fraction;

[0066] Second stage: pressure 22 MPa, temperature 46℃, entrainer is anhydrous ethanol, the volume of entrainer accounts for 4% of the total volume of CO2 and entrainer mixture, extraction time 45 minutes, and the second fraction is collected.

[0067] Third stage: pressure 32 MPa, temperature 51℃, the entrainer is a mixture of ethanol and water in a volume ratio of 9:1, the volume of the entrainer accounts for 4% of the total volume of CO2 and entrainer, the extraction time is 55 minutes, and the third fraction is collected.

[0068] The second and third extracts were placed in a rotary evaporator to remove entrainer solvents until the ethanol / water residue was controlled below 0.5% (w / w). The three fractions were then mixed in a volume ratio of 6:2.5:1 to obtain the compound fragrance base.

[0069] (5) Add rosemary extract to the compound fragrance base at a concentration of 0.012% of the mass of the compound fragrance base. After mixing evenly, the fragrance product is obtained. The fragrance is packaged under nitrogen protection and stored at 4°C in the dark.

[0070] Comparative Example 1

[0071] The difference between this comparative example and Example 1 is that in step (1), dried rose petals, perilla leaves, and grapefruit peel were replaced with dried loquat, ophiopogon japonicus, and acai berries, respectively.

[0072] Comparative Example 2

[0073] The difference between this comparative example and Example 1 is that in step (1), pectinase, β-glucosidase, and flavor protein are replaced with α-amylase, saccharifying enzyme, and papain, respectively.

[0074] Comparative Example 3

[0075] The difference between this comparative example and Example 1 is that step (2) involves transferring the above enzymatic hydrolysate to a sterilized 10 L fermenter and inoculating it with *Lactobacillus brevis* at a concentration of 1.0 × 10⁻⁶. 9 The culture was fermented anaerobicly at 32°C for 28 hours at a concentration of CFU / mL; then inoculated with *Lactobacillus plantarum* at a concentration of 1.0 × 10⁻⁶. 8 The concentration of CFU / mL was increased, and anaerobic fermentation was continued at 32℃ for 24 hours; then, *Saccharomyces cerevisiae* was inoculated at an inoculum concentration of 1.0 × 10⁻⁶ CFU / mL. 7 CFU / mL, anaerobic fermentation at 30℃ for 48 hours.

[0076] Comparative Example 4

[0077] The difference between this comparative example and Example 1 is as follows: the supercritical CO2 three-stage extraction process in step (4) is as follows: first stage: pressure 30 MPa, temperature 50℃, entrainer is 6% ethanol-water mixture (volume ratio 9:1), extraction for 50 minutes, and collection of the first fraction; second stage: pressure 23 MPa, temperature 45℃, entrainer is 5% anhydrous ethanol, extraction for 50 minutes, and collection of the second fraction; third stage: pressure 10 MPa, temperature 38℃, no entrainer, extraction time 30 minutes, and collection of the third fraction; the first and second fractions are respectively de-entrained in a rotary evaporator until the total residual amount of ethanol / water is less than 0.5% (w / w), and then the three fractions are mixed in a volume ratio of 7:2:1 to obtain a composite fragrance base.

[0078] Performance testing

[0079] The flavorings prepared in Examples 1-2 and Comparative Examples 1-4 were subjected to performance tests.

[0080] The total volatile matter content should be tested according to GB / T 11538-2006, and the test method is as follows:

[0081] Accurately weigh 1.00 g of fragrance sample and place it in a 500 mL round-bottom flask. Add 200 mL of deionized water and connect the volatile oil extractor. Heat to boiling and maintain a gentle boil for 2 hours. After cooling, read the volume of volatile oil. Transfer the collected volatile oil to a pre-dried and weighed glass weighing bottle (denoted as m0). Dry the bottle in a vacuum drying oven at 40℃ until constant weight and weigh it (denoted as m1). Calculate the total volatile matter content using the following formula: Total volatile matter content (mg / g) = ((m1-m0)×1000) / sample mass (g).

[0082] For thermal stability, refer to GB / T 21296-2015. Take 100mg of fragrance and place it in a sealed glass tube. Heat it in an oven at 200℃ for 30min. After cooling, determine the total amount of volatile substances and calculate the retention rate (%) = (content after heating / content before heating) × 100%.

[0083] Sensory evaluation was conducted in accordance with GB 5606.4-2005. Flavoring was evenly sprayed onto blank tobacco (fluff-cured type) at an addition rate of 0.1 wt% to prepare cigarette samples. A 10-person evaluation panel conducted blind tests in an environment with a temperature of (22±1)℃ and a humidity of (60±5)%. The samples were scored from four dimensions: aroma characteristics (30 points), irritation (30 points), aftertaste (20 points), and off-flavors (20 points), and the average value was taken.

[0084] The test results are shown in Table 1.

[0085] Table 1. Fragrance performance test results

[0086] Total volatile substances (mg / g) Thermal stability retention rate (%) Sensory rating (points) Example 1 58.7 94.2 92.5 Example 2 56.2 92.7 90.3 Comparative Example 1 41.3 85.6 76.8 Comparative Example 2 38.6 79.3 80.2 Comparative Example 3 49.8 88.1 83.5 Comparative Example 4 44.5 86.4 81.0

[0087] The above results indicate that the total volatile matter content, thermal stability, and sensory score of the flavorings prepared in Examples 1-2 of this invention are significantly better than those in the comparative examples. In Comparative Example 1, the replacement of raw materials resulted in insufficient characteristic aroma precursors and a significant decrease in total volatile matter. In Comparative Example 2, the enzyme replacement reduced the decomposition effect on plant cell walls, significantly decreasing aroma release and thermal stability. In Comparative Example 3, the replacement of bacterial strains led to the production of acidic substances during lactic acid bacteria fermentation, which accelerated decomposition at high temperatures, resulting in decreased stability. Although Comparative Example 4 employed a three-stage extraction method, the unreasonable sequence of extraction parameters led to insufficient extraction of some aroma components, resulting in lower total volatile matter content and sensory scores compared to the examples, and an overall mediocre flavor profile.

[0088] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing flavorings using low-temperature enzymatic fermentation coupled with supercritical CO2 extraction, comprising the following steps: (1) Mix rose petals, perilla leaves and grapefruit peel and then crush them. Mix the crushed raw materials with deionized water to obtain a mixture. Add pectinase, cellulase, β-glucosidase and flavor protease to the mixture and hydrolyze it to obtain an enzymatic hydrolysate. (2) Non-Saccharomyces cerevisiae and esterifying bacteria were inoculated into the enzymatic hydrolysate in sequence, and fermentation was carried out to obtain the fermentation broth; (3) Centrifuge the fermentation broth to separate the supernatant, concentrate the supernatant and freeze-dry it to obtain freeze-dried powder; (4) Compress the lyophilized powder into tablets, place them in a supercritical CO2 extraction vessel, and extract them under three-stage gradient conditions to obtain the extract; (5) Combine the extracts to obtain a complex fragrance base, add rosemary extract to the complex fragrance base to obtain the fragrance; The conditions for three-stage gradient extraction are: First stage: Pressure 10-14 MPa, temperature 38-42℃, no entrainer, extraction time 30-45 minutes; Second stage: pressure 20-25 MPa, temperature 43-47℃, entrainer is anhydrous ethanol, the volume of entrainer accounts for 3-7% of the total volume of CO2 and entrainer, extraction time 40-60 minutes; The third stage: pressure 30-35 MPa, temperature 48-52℃, the entrainer is a mixture of ethanol and water with a volume ratio of 7-9:1, the volume of the entrainer accounts for 3-7% of the total volume of CO2 and the entrainer, and the extraction time is 50-70 minutes.

2. The flavor preparation method according to claim 1, characterized in that, The mass ratio of rose petals, perilla leaves and pomelo peel in step (1) is (3-6):(1-2):

1.

3. The flavor preparation method according to claim 1, characterized in that, In step (1), the amount of pectinase added to the mixture is 60-100 U / mL, the amount of cellulase added to the mixture is 80-120 U / mL, the amount of β-glucosidase added to the mixture is 15-25 U / mL, and the amount of flavor protease added to the mixture is 20-30 U / mL.

4. The flavor preparation method according to claim 1, characterized in that, The enzymatic hydrolysis in step (1) is carried out at a temperature of 35-40°C, a pH of 5.0-5.5, and a time of 2-4 hours.

5. The method for preparing flavoring according to claim 1, characterized in that, The inoculation concentration of the non-Saccharomyces cerevisiae in step (2) is (0.5–1.5) × 10⁻⁶. 7 CFU / mL, the inoculation concentration of esterifying bacteria is (5–10) × 10⁻⁶. 6 CFU / mL.

6. The flavor preparation method according to claim 1, characterized in that, Step (2) After inoculating with non-Saccharomyces cerevisiae, ferment at 24-28°C for 24-36 hours, and after inoculating with esterified bacteria, ferment at 20-22°C for 48-72 hours.

7. The flavor preparation method according to claim 1, characterized in that, The centrifugal separation temperature in step (3) is 4 to 10°C.

8. The method for preparing flavoring according to claim 7, characterized in that, The specific steps of step (5) are as follows: first, the extracts obtained in the second and third stages are subjected to reduced pressure treatment to remove the entrainer solvent, and the total residual mass of ethanol and water is controlled to be no higher than 0.5%. Then, the extracts from the first, second and third stages are mixed in a volume ratio of 6-8: 1.5-2.5: 0.5-1 to obtain a composite fragrance base.

9. The method for preparing flavoring according to claim 1, characterized in that, The amount of rosemary extract added in step (5) is 0.008 to 0.015% of the complex fragrance base.