Essence preparation method by coupling low-temperature enzyme fermentation with supercritical CO2 extraction

Abstract

The invention belongs to the technical field of natural perfume extraction and essence preparation, and particularly relates to an essence preparation method by coupling low-temperature enzyme fermentation with supercritical CO2 extraction. According to the method, rose petals, perilla leaves and shaddock peels are used as raw materials, aroma precursor substances are released through synergistic low-temperature enzymolysis of multiple enzymes such as pectinase and cellulase, then non-saccharomyces cerevisiae and esterifying bacteria are introduced for step-by-step anaerobic fermentation, multi-layer composite aroma components are generated, then freeze drying and three-stage gradient supercritical CO2 extraction are performed, and the multi-layer composite aroma components are obtained. Aroma components with different polarities are enriched in a grading manner; and finally, mixing and stabilizing treatment are performed to obtain an essence finished product. The method constructs a complete process flow from precursor release to structure construction to component extraction, and is suitable for the fields of tobacco flavors, food flavors and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of natural flavor extraction and essence preparation, and particularly relates to a method for preparing essence by low-temperature enzyme fermentation coupled with supercritical CO2 extraction. BACKGROUND

[0002] Tobacco essence is an important component in cigarette products for improving and strengthening aroma, coordinating tobacco aroma, and improving overall flavor quality. Its sources mainly include natural plant extracts, thermal reaction bases, synthetic flavors, and fermentation products. In recent years, with the increasing trend of "natural, green, and safe" consumption, the research and development of tobacco essence prepared from natural plants have continued to heat up. However, there are still a series of technical bottlenecks in the existing preparation process of natural essence, which restricts its wide application in the tobacco industry.

[0003] At present, the main preparation methods of natural tobacco essence include steam distillation, solvent extraction, ultrasonic-assisted extraction, and partial enzyme treatment. The steam distillation method is simple, but high-temperature heating is required, which easily causes the degradation or loss of some heat-sensitive volatile components, resulting in the destruction of aroma spectrum structure and the single flavor of essence. Although the solvent extraction has high extraction efficiency, it often uses organic solvents such as ethanol, ethyl acetate, and petroleum ether, which has problems of solvent residue, environmental pollution, and difficult post-processing, and is difficult to meet the requirements of safety and purity of tobacco additives. Although ultrasonic-assisted or microwave extraction improves the diffusion rate, it still faces problems such as high energy consumption, poor selectivity of aroma substances, and difficult separation of impurities in actual application.

[0004] On the other hand, fermentation technology has been gradually introduced into the field of essence preparation in recent years, which uses microbial metabolism or enzymatic reaction to convert sugar, amino acid, fatty acid, and other precursors in plant raw materials into a series of secondary metabolites with aroma activity. This method has the advantages of low temperature, diverse conversion path, and rich and natural aroma. However, in actual application, due to the influence of factors such as temperature, time, pH, and activity of strains / enzymes, the reaction stability is insufficient, and excessive impurities or off-flavor components are easily generated. Moreover, the concentration of aroma substances in the fermentation broth is low, the extraction and separation are difficult, the processing efficiency is low, and the industrialization ability is limited.

[0005] In the aspect of aroma component purification, supercritical CO2 extraction technology is considered as an ideal solution with high efficiency, greenness and low residue, because it is carried out under lower temperature and anaerobic condition, which can maximize the retention of the activity and integrity of aroma molecules, and has strong selectivity and low energy consumption. However, in practice, supercritical CO2 extraction is suitable for pretreated samples with high enrichment or purification, and when directly acting on original fermentation broth or complex plant mixtures, there are still problems such as low extraction efficiency, unbalanced aroma composition, low yield of some polar components, and the like, which are difficult to fully meet the needs of tobacco flavor for aroma levels and concentration.

[0006] In summary, the existing tobacco flavor preparation technology generally has the following outstanding problems: severe thermal loss of aroma substances during extraction, unstable flavor type, many residual impurities in the preparation system, difficult purification, relatively single aroma level, insufficient naturalness and aroma retention, low preparation process efficiency, harsh process conditions or not conducive to scale-up. Especially for cigarette application, the flavor not only needs to meet the requirements of complex flavor, distinct levels and high fusion with smoke, but also needs to have good thermal stability and safety.

[0007] Therefore, it is urgent to develop a new flavor preparation method which can efficiently generate aroma components under mild conditions and take into account selective extraction and flavor integrity, in order to overcome the above technical bottlenecks and meet the needs of the tobacco industry for high-quality natural flavors. SUMMARY

[0008] The purpose of the present application is to provide a flavor preparation method of low-temperature enzyme fermentation coupled 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 difficult purification in the prior art.

[0009] In order to achieve the above purpose, the present application provides the following technical solutions:

[0010] A flavor preparation method of low-temperature enzyme fermentation coupled with supercritical CO2 extraction, comprising the following steps:

[0011] (1) mixing rose petals, perilla leaves and grapefruit peels, and then crushing, mixing the crushed raw materials with deionized water to obtain a mixture, adding pectinase, cellulase, β-glucosidase and flavor protease to the mixture, and then enzymolysis to obtain an enzymolysis liquid;

[0012] (2) inoculating non-Saccharomyces cerevisiae and esterifying bacteria into the enzymolysis liquid in sequence, and then obtaining a fermentation broth after fermentation;

[0013] (3) centrifuging the fermentation broth to separate out a supernatant, concentrating and freeze-drying the supernatant to obtain a freeze-dried powder;

[0014] (4) The freeze-dried powder is pressed into a tablet (for example, to a thickness of 2-4 mm), placed in a supercritical CO2 extraction kettle, and extracted under three-stage gradient conditions to obtain an extraction liquid;

[0015] (5) The extraction liquids are combined to obtain a composite essence base, and the rosemary extract is added to the composite essence base to obtain an essence.

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

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

[0018] Further, the temperature of the enzymolysis in step (1) is 35-40 DEG C, the pH value is 5.0-5.5, and the time is 2-4 hours.

[0019] The present application has outstanding basic flavor construction advantages in the selection of raw material substrates. The rose petals, perilla leaves and grapefruit peels respectively represent floral, herbal and citrus flavors in the flavor structure, have good complementarity and harmonization potential, and are rich in glycosides, terpenes and aldehyde ketone components, which are important precursors for forming a composite tobacco aroma. The rose is rich in linalool glycoside and geraniol glycoside, which can be converted into typical floral molecules, the perilla provides perilla aldehyde and perilla alcohol as the core skeleton of the herbal flavor, and the grapefruit peel contains naringin and limonene, thereby constructing a top note layer with volatility and penetration. In the selection of enzymes, the pectinase and cellulase act on the complex structure of the plant cell wall to break the cell wall and release the intercellular gap aroma precursors; the beta-glucosidase can hydrolyze the glycosidic bond to release the bound terpenes and phenolic compounds; and the flavor protease further releases flavor peptides and short-chain nitrogen-containing compounds to form a bottom note support. This multi-enzyme synergistic and mild enzymolysis strategy realizes the efficient release of aroma active substances and the structural pre-construction of the substrate flavor under the premise of ensuring the structural integrity, thereby providing highly reactive aroma precursors for subsequent fermentation.

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

[0021] Further, the step (2) is inoculated with non-Saccharomyces after fermentation at 24-28℃ for 24-36 hours, and inoculated with esterification bacteria after fermentation at 20-22℃ for 48-72 hours.

[0022] In the fermentation stage, the first inoculation of non-Saccharomyces (such as Kluyveromyces) has strong ester generation ability and high metabolic activity to polyols and terpenes, which can quickly generate fruit and flower fragrance molecules with top note characteristics such as benzyl acetate and linalool acetate. Subsequently, the esterification bacteria (such as Hansenula debaryana) are introduced to carry out anaerobic fermentation at a lower temperature. The enzyme has strong long-chain fatty acid ester and lactone generation ability, can supplement the middle and rear modulation of roundness and mouthfeel viscosity, and improve the "retention of tobacco essence" and the smoothness of smoke. The two bacteria are operated in cooperation and in stages, avoiding the interference of mutual competition for metabolite substrates or the generation of toxic byproducts. More importantly, this mode naturally converts high-activity precursors into a complex and natural aroma system through microbial enzymatic pathways, ensuring that the aroma source is real, stable, and avoiding the flavor overload and level loss caused by single-bacterium continuous fermentation.

[0023] Further, the temperature of the centrifugal separation in step (3) is 4-10℃.

[0024] Further, the conditions of 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 the entrainer accounts for 3-7% of the total volume of CO2 and the entrainer, extraction time 40-60 minutes;

[0027] Third stage: pressure 30-35 MPa, temperature 48-52℃, 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 the entrainer, extraction time 50-70 minutes;

[0028] Further, the combined extraction liquid in step (5) is as follows: first, the extraction liquids obtained in the second and third stages are subjected to vacuum treatment to remove the entrainer solvent, and the total residual mass of ethanol and water is controlled to be not more than 0.5%, then the extraction liquids of 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 essence base.

[0029] Further, the addition mass of rosemary extract in step (5) is 0.008-0.015% of the composite essence base.

[0030] In the aspect of aroma component extraction, the three-stage gradient supercritical CO2 extraction process is adopted to systematically solve the problems of poor selectivity and aroma imbalance existing in traditional single-stage extraction. In the first stage, non-polar volatile components are extracted under the conditions of low pressure, mild temperature and without entrainer, so as to maximize the retention of headspace substances such as limonene and linalool; in the second stage, low-concentration ethanol is introduced as an entrainer to improve the affinity for medium-polarity components (such as phenols, alcohol esters, etc.); in the third stage, an ethanol-water mixed system is used to further expand the solubility window of polar components, so that components such as coumarin, lactone and esterified components can be efficiently extracted. After the three-stage fractions are proportionally blended, not only the overall extraction of aroma components is realized, but also the aroma gradually changes from fresh to sweet and then to deep woody, which has a higher degree of integration with tobacco aroma and better stability. In addition, CO2 as a green and residue-free solvent not only has good protection for heat-sensitive components, but also does not affect the purity of the essence after volatilization, laying a solid foundation for the production of safe, natural and richly adapted essence for tobacco.

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

[0032] The present application realizes the release and biosynthesis conversion of potential aroma precursors in plant raw materials through the organic integration of a mild enzymatic hydrolysis system with multiple enzymes and a phased and directional fermentation strategy, and then uses a three-stage supercritical CO2 technology to sequentially enrich target aroma substances according to the polarity difference of components. This method not only effectively improves the front note performance and the roundness of middle and rear notes of the essence, but also significantly enhances the level and coordination of the overall aroma. Compared with the unoptimized raw material replacement scheme, enzyme replacement scheme, strain fermentation mode change scheme and single-stage extraction method, the present application shows a more optimal level in terms of headspace release, aroma configuration stability and persistence, etc. This indicates that the synergistic design between the steps is highly targeted and effective, and the technical effect is significant and stable, which is suitable for the development of cigarette and natural essence products with high aroma quality requirements. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present application will be described below in a clear and complete manner. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0034] The raw materials used in the embodiments are ordinary commercially available products unless otherwise specified. The following is an exemplary description:

[0035] Pectinase was purchased from Wuhan Aimeijie Technology, ≥20 U / mg.

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

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

[0038] Flavourzyme was purchased from Lanxu, ≥20 U / mg.

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

[0040] Esterifying bacteria (Debaryomyces hansenii) was purchased from China General Microbiological Culture Collection Center, CGMCC 2.3966.

[0041] Rosemary extract was purchased from Nanjing Daosifu Biotechnology.

[0042] Alpha-amylase was purchased from Guangzhou Kangda Bioproducts Co., Ltd., ≥600 KNU / kg.

[0043] Glucoamylase was purchased from Qingdao Sitan Biotechnology Co., Ltd., 1000 U / g.

[0044] Papain was purchased from Nanning Pangbo Biological Engineering Co., Ltd., 3.5 million U / g.

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

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

[0047] Saccharomyces cerevisiae was purchased from China General Microbiological Culture Collection Center, CGMCC 2.3973.

[0048] Example 1

[0049] The present embodiment provides a method for preparing essence by coupling low-temperature enzyme fermentation with supercritical CO2 extraction, comprising the following steps:

[0050] (1) 500 g of dried rose petals, 200 g of perilla leaves, and 100 g of grapefruit peels were weighed and mixed, and then ground into fine powder below 70 mesh. The obtained powder was mixed with deionized water at a ratio of 1:5 (g:mL), and the pH of the mixture was adjusted to 5.2 using 0.1 mol / L citric acid-sodium citrate buffer. The mixture was heated to 38℃, and enzyme preparations were added to the mixture according to the following proportions: pectinase 80 U / mL, cellulase 100 U / mL, β-glucosidase 20 U / mL, and flavourzyme 25 U / mL. The enzyme hydrolysis was carried out at a constant temperature for 3 hours. After the enzyme hydrolysis was completed, the temperature was raised to 75℃ for 10 minutes to inactivate the enzymes, and then cooled to 26℃ for standby.

[0051] (2) The enzymatic hydrolysate was transferred to a sterilized 10 L fermenter, inoculated with non-Saccharomyces cerevisiae bacteria at a concentration of 1.0 x 10 7 CFU / mL, and anaerobically fermented at 26°C for 30 hours; then inoculated with esterifying bacteria at a concentration of 8.0 x 10 6 CFU / mL, and continued to be anaerobically fermented at 21°C for 60 hours.

[0052] (3) The fermentation broth was placed in a refrigerated centrifuge and centrifuged at 6°C and 4000 rpm for 10 minutes, and the supernatant was collected and transferred to a rotary evaporator and concentrated to 20% of the original volume, followed by freeze-drying to obtain a powder of freeze-dried aroma base powder.

[0053] (4) The freeze-dried powder was pressed into a circular sheet with a thickness of about 3 mm and loaded into a supercritical CO2 extraction kettle, and three-stage extraction was carried out in sequence:

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

[0055] Second stage: pressure 23 MPa, temperature 45°C, entrainer is anhydrous ethanol, the volume of the entrainer accounts for 5% of the total volume of CO2 and the entrainer, extraction time 50 minutes, collect the second fraction;

[0056] Third stage: pressure 33 MPa, temperature 50°C, 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 the entrainer, extraction time 60 minutes, collect the third fraction;

[0057] The second and third fractions were removed from the rotary evaporator to remove the entrainer to a total residual amount of ethanol / water less than 0.5% (w / w), then the three fractions were mixed in a volume ratio of 7:2:1 to obtain a composite aroma base.

[0058] (5) Rosemary extract was added to the composite aroma base at an amount of 0.01% of the mass of the composite aroma base, and after mixing evenly, the aroma was obtained. The aroma was packaged under nitrogen protection and stored at 4°C in the dark.

[0059] Example 2

[0060] The present embodiment provides a method for preparing an aroma by low-temperature enzyme fermentation coupled with supercritical CO2 extraction, comprising the following steps:

[0061] (1) Take 450 g of dried rose petals, 150 g of perilla leaves, and 100 g of grapefruit peels, mix them, and crush them into fine powder below 60 mesh. Mix the obtained powder with deionized water at a ratio of 1:6 (g:mL), slowly add 0.1 mol / L lactic acid solution to adjust the pH of the mixture to 5.0, heat to 37°C, and add enzyme preparations: pectinase 90 U / mL, cellulase 110 U / mL, β-glucosidase 18 U / mL, and flavor protease 22 U / mL to the mixture. Enzymatically hydrolyze at constant temperature for 2.5 hours. After the enzymatic hydrolysis is completed, heat the mixture to 72°C, and keep it at this temperature for 10 minutes for enzyme inactivation. Then, cool it to 25°C for standby.

[0062] (2) Transfer the above-mentioned enzymatic hydrolysate to a sterilized 10 L fermenter. First, inoculate non-Saccharomyces cerevisiae bacteria at a concentration of 1.2 x 10 7 CFU / mL, and anaerobically ferment at 27°C for 36 hours. Then, inoculate esterifying bacteria at a concentration of 6.0 x 10 6 CFU / mL, and continue anaerobic fermentation at 20°C for 72 hours.

[0063] (3) Place the fermentation liquor in a refrigerated centrifuge, centrifuge at 3800 rpm at 4°C for 12 minutes, collect the supernatant, transfer it to a rotary evaporator, concentrate it to 25% of the original volume, and then freeze-dry to obtain a powder-shaped freeze-dried aroma base powder.

[0064] (4) Press the freeze-dried powder into a round tablet with a thickness of 2.5 mm, and load it into a supercritical CO2 extraction kettle. Perform three-stage extraction in sequence:

[0065] First stage: pressure 11 MPa, temperature 39°C, no entrainer, extraction time 35 minutes, collect the first-stage fraction;

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

[0067] Third stage: pressure 32 MPa, temperature 51°C, 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 the CO2 and entrainer, extraction time 55 minutes, collect the third-stage fraction;

[0068] Place the second-stage and third-stage extraction liquids in a rotary evaporator, respectively, to remove the entrainer solvent until the residual amount of ethanol / water is controlled to be below 0.5% (w / w). Then, mix the three fractions at a volume ratio of 6:2.5:1 to obtain a composite aroma base.

[0069] (5) rosemary extract was added into the composite fragrance base, and the addition amount was 0.012% of the mass of the composite fragrance base. After uniform mixing, the fragrance product was obtained. The fragrance was 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), the dried rose petals, perilla leaves and grapefruit peels are replaced by dried loquat, ophiopogon and blueberry, respectively.

[0072] Comparative Example 2

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

[0074] Comparative Example 3

[0075] The difference between this comparative example and Example 1 is that in step (2), the enzyme solution is transferred to a sterilized 10 L fermenter, inoculated with Lactobacillus brevis at an inoculation concentration of 1.0×10 9 CFU / mL, anaerobically fermented at 32°C for 28 hours; then inoculated with Lactobacillus plantarum at an inoculation concentration of 1.0×10 8 CFU / mL, continued anaerobic fermentation at 32°C for 24 hours; then inoculated with Saccharomyces cerevisiae at an inoculation concentration of 1.0×10 7 CFU / mL, anaerobically fermented at 30°C for 48 hours.

[0076] Comparative Example 4

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

[0078] Performance Test

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

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

[0081] 1.00 g of the perfume sample was accurately weighed and placed in a 500 mL round-bottom flask, 200 mL of deionized water was added, and a volatile oil extractor was connected. Heating to boiling and keeping the state of slight boiling for 2 hours, reading the volume of volatile oil after cooling. The collected volatile oil was transferred to a dried and weighed glass weighing bottle (recorded as m0), dried to constant weight in a vacuum drying oven at 40℃, weighed (recorded as m1), and the total volatile matter content was calculated according to the following formula: total volatile matter content (mg / g) = ((m1-m0) x 1000) / sample mass (g).

[0082] The thermal stability was determined according to GB / T 21296-2015, 100 mg of the perfume was placed in a sealed glass tube and heated in an oven at 200℃ for 30 min, and then the total volatile matter content was determined after cooling, and the retention rate (%) = (content after heating / content before heating) x 100%.

[0083] The sensory evaluation was determined according to GB 5606.4-2005, the perfume was uniformly sprayed on blank tobacco (flue-cured tobacco type) at an addition amount of 0.1 wt%, and a cigarette sample was prepared. A 10-person evaluation group evaluated the sample in a temperature (22±1)℃, humidity (60±5)% environment, and the average value was obtained from the scores of aroma characteristics (30 points), irritation (30 points), aftertaste (20 points), and miscellaneous odor (20 points).

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

[0085] Table 1: Perfume performance test results

[0086] Total volatile matter (mg / g) Thermal stability retention rate (%) Sensory score (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 show that the total volatile matter content, thermal stability and sensory score of the perfume prepared in Examples 1-2 are significantly better than those of the comparative examples. The replacement of the raw material in Comparative Example 1 leads to insufficient characteristic aroma precursors, and the total amount of volatile matter decreases significantly. After replacing the enzyme species in Comparative Example 2, the decomposition effect on plant cell walls decreases, and the aroma release decreases significantly. At the same time, the thermal stability decreases significantly. After replacing the strain in Comparative Example 3, lactic acid bacteria fermentation produces acid substances, which promote decomposition at high temperatures, and the stability decreases. Although three-stage extraction is used in Comparative Example 4, due to the unreasonable sequence of extraction parameters, some aroma components are not fully extracted, and the total amount of volatile matter and the sensory score are lower than those of the examples, and the overall flavor performance of the perfume is general.

[0088] The above is the preferred embodiment of the present application, it should be noted that for those skilled in the art, without departing from the principles described in the present application, can also make several improvements and refinements, these improvements and refinements should also be considered as the scope of protection of the present application.

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 compound fragrance base, add rosemary extract to the compound fragrance base to obtain the fragrance.

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 1, characterized in that, The conditions for the three-stage gradient extraction in step (4) are as follows: 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.

9. The method for preparing flavoring according to claim 8, 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.

10. 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.