Fermented bergamot fruit oil as well as preparation method and application thereof

Abstract

The invention relates to fermented bergamot fruit oil as well as a preparation method and application thereof. The preparation method comprises the following steps: inoculating a fermentation culture medium with a fermentation strain, carrying out fermentation culture, carrying out three-phase separation on the obtained fermentation liquid, and collecting an upper-layer oil phase to obtain the fermented bergamot fruit oil, the fermentation culture medium comprises a bergamot peel extract; the bergamot peel extract is obtained by mixing a bergamot peel raw material with an aqueous solution of a compound enzyme, performing ultrasonic-assisted enzymolysis extraction, extracting an extracting solution and collecting an organic phase. Through improvement of an extraction technology and optimization of a fermentation technology, the prepared fermented bergamot fruit oil is high in stability and has excellent antioxidant, anti-inflammatory and repairing effects, and the problems that effective components in bergamot fruit oil obtained through a traditional cold pressing method are unstable and the skin absorption rate is poor are solved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cosmetics, and particularly relates to a fermented bergamot fruit oil as well as a preparation method and application thereof. BACKGROUND

[0002] The bergamot fruit oil is a precious essential oil extracted from the fruit of the plant bergamot of the Rutaceae family, has outstanding application value in the field of skin care, is an important category of natural skin care ingredients, and has multiple physiological functions such as antibacterial and anti-inflammatory, balance of sebum secretion, balance of microecology, antioxidant and repair.

[0003] The bergamot is traditionally extracted by cold pressing, and the essential oil is directly extracted from the fruit of the bergamot through physical pressing by the process. However, the cold pressing method has low oil yield, cannot fully destroy the cell wall structure of the bergamot, and has poor product purity and high impurity content, so that the active ingredients in the bergamot cannot be fully extracted.

[0004] In recent years, the introduction of the fermentation process in the field of natural plant essential oil extraction provides a new direction for solving the pain points of the traditional process. On the one hand, the microbial fermentation product can promote the release of the essential oil components in the cell, greatly improving the oil yield; on the other hand, the fermentation process can selectively convert part of the impurities into small molecule metabolites, reducing the difficulty of subsequent purification, and at the same time retaining or even enhancing the content and stability of the active ingredients in the essential oil. However, there are few reports on the further fermentation of the bergamot fruit peel extract by microorganisms.

[0005] Therefore, developing a fermented bergamot fruit oil, improving the content and stability of the functional ingredients in the fermented bergamot fruit oil, and improving the antioxidant and soothing and repairing effects of the fermented bergamot fruit oil have become one of the technical problems to be solved at present. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application aims to provide a fermented bergamot fruit oil as well as a preparation method and application thereof.

[0007] To achieve the object of the present application, the following technical solutions are adopted:

[0008] In a first aspect, the present application provides a preparation method of a fermented bergamot fruit oil, which comprises the following steps:

[0009] Inoculating the fermentation strain in the fermentation medium, performing fermentation culture, performing three-phase separation on the obtained fermentation liquor, collecting the upper oil phase, and obtaining the fermented bergamot fruit oil.

[0010] The fermentation medium comprises bergamot fruit peel extract.

[0011] The bergamot peel extract is obtained by mixing bergamot peel raw materials with an aqueous solution of complex enzymes, ultrasonic-assisted enzymatic hydrolysis extraction, and collecting the organic phase after extraction.

[0012] The complex enzyme includes pectin lyase, pectin esterase and xylanase.

[0013] The present application improves the extraction technology and optimizes the fermentation process, and the fermented bergamot peel oil prepared by fermenting the bergamot peel extract obtained by ultrasonic-assisted enzymatic hydrolysis extraction and extraction has high stability, excellent antioxidant, anti-inflammatory and repair effects, and solves the problems of unstable effective components and poor skin absorption rate of the bergamot peel oil obtained by traditional cold pressing method.

[0014] The present application uses pectin lyase, pectin esterase and xylanase to hydrolyze the bergamot peel. Among them, pectin lyase acts on highly methylated pectin, can eliminate the alpha-1, 4-glycosidic bond in pectin, and break the gel network in the cell wall of the peel; pectin esterase acts on the methyl ester group in the pectin molecule, reduces the degree of pectin methylation; xylanase acts on the main chain of xylan molecule, breaks the beta-1, 4-xylosidic bond of xylan main chain, destroys the hemicellulose network in the cell wall, and reduces the cross-linking structure. The present application creatively finds that pectin lyase, pectin esterase and xylanase have significant synergistic effect on the release of effective components in the bergamot peel, and the three enzymes can be used together to release the effective components in the bergamot peel.

[0015] Preferably, the solid-liquid ratio of the bergamot peel raw material and the aqueous solution of complex enzymes is 1:8-1:20 g / mL, for example, it can be 1:8 g / mL, 1:10 g / mL, 1:12 g / mL, 1:14 g / mL, 1:16 g / mL, 1:18 g / mL, 1:20 g / mL, etc.

[0016] Preferably, the mass percentage content of complex enzymes in the aqueous solution of complex enzymes is 1-5%, for example, it can be 1%, 2%, 3%, 4%, 5%, etc.

[0017] Preferably, the mass ratio of pectin lyase, pectin esterase and xylanase is (1-5):(1-5):(1-5).

[0018] Specific point values in 1-5 can be selected as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc.

[0019] Preferably, the extraction agent of the extraction includes ethyl acetate. Preferably, the temperature of the ultrasonic-assisted enzymatic hydrolysis extraction is 35-60℃, for example, it can be 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, etc.

[0020] Preferably, the time of the ultrasonic-assisted enzymatic extraction is 60-120 min, for example, it can be 60 min, 70 min, 80 min, 90 min, 100 min, 110 min, 120 min, etc.

[0021] Preferably, the ultrasonic power of the ultrasonic-assisted enzymatic extraction is 400-600 W, and the ultrasonic frequency of the ultrasonic-assisted enzymatic extraction is 30-50 kHz.

[0022] Among them, the specific point value in 400-600 W can be selected as 400 W, 450 W, 500 W, 550 W, 600 W, etc., and the specific point value in 30-50 kHz can be selected as 30 kHz, 35 kHz, 40 kHz, 45 kHz, 50 kHz, etc.

[0023] Preferably, after the extraction, the organic phase is concentrated, dried, and then mixed with cyclodextrin for embedding treatment.

[0024] The application utilizes the cavity structure of cyclodextrin to form inclusion compounds with active ingredients in bergamot peel extract, which can further improve the stability of active ingredients, reduce the damage of volatile components in the extract, and retain the target product precursors.

[0025] Preferably, the cyclodextrin includes β-cyclodextrin.

[0026] Preferably, the mass ratio of the dried product to cyclodextrin is 1:10-3:10, for example, it can be 1:10, 1.5:10, 2:10, 2.5:10, 3:10, etc.

[0027] Preferably, the fermentation strain includes Yarrowia lipolytica, Lactobacillus plantarum, and Aspergillus niger.

[0028] Yarrowia lipolytica is a lipophilic yeast that can efficiently decompose lipid components in peel extract to generate small-molecule volatile aroma components such as terpenol, aldehyde, ester and other metabolites, and its metabolic activity helps to regulate the pH value of the fermentation system and create a microenvironment suitable for the growth of the microbial flora; Lactobacillus plantarum is a gram-positive facultative anaerobe that can degrade macromolecular substances such as pectin and flavonoid glycosides in peel extract to release free flavones and small-molecule sugars, thereby strengthening the anti-inflammatory soothing effect and inhibiting the growth of harmful bacteria; Aspergillus niger is a filamentous fungus that produces a variety of enzymes with high activity, can secrete abundant cellulase, hemicellulase and pectinase, further completely destroys the cell wall structure of the peel to release more encapsulated lipids and active ingredients, and provides more abundant substrates for Yarrowia lipolytica and Lactobacillus plantarum. The present application creatively finds that Yarrowia lipolytica, Lactobacillus plantarum and Aspergillus niger have significant synergistic effect in improving the antioxidant and anti-inflammatory repair effect of the fermentation product.

[0029] Preferably, the ratio of the number of Yarrowia lipolytica, Lactobacillus plantarum and Aspergillus niger is (3-8):(1-5):(1-5).

[0030] Specific point values in 3-8 can be selected as 3, 4, 5, 6, 7, 8, and specific point values in 1-5 can be selected as 1, 2, 3, 4, 5.

[0031] Preferably, the total amount of the inoculated fermentation strains is 7-15% by volume percentage, for example, can be 7%, 9%, 11%, 13%, 15% and the like.

[0032] Preferably, the addition amount of bergamot peel extract in the fermentation medium is 10-40 g / L, for example, can be 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L and the like.

[0033] Preferably, the fermentation medium further comprises geranyl pyrophosphate.

[0034] By adding geranyl pyrophosphate in the fermentation medium, the present application can direct regulate the metabolic pathway of microorganisms, significantly improve the yield of monoterpene and other substances, promote the growth of bacterial cells, and ultimately improve the antioxidant and repair effect of the prepared fermented bergamot fruit oil.

[0035] Preferably, the addition amount of geranyl pyrophosphate in the fermentation medium is 0.05-0.3 g / L.

[0036] Preferably, the fermentation medium comprises a carbon source, a nitrogen source, inorganic salts, bergamot peel extract and geranyl pyrophosphate.

[0037] Preferably, the carbon source comprises glucose and / or glycerol.

[0038] Preferably, the nitrogen source comprises soy peptone and / or yeast extract.

[0039] Preferably, the inorganic salt comprises any one or a combination of at least two of KH2PO4, MgSO4·7H2O, FeCl3.

[0040] Preferably, the fermentation medium comprises glucose, KH2PO4, MgSO4·7H2O, FeCl3, glycerol, soy peptone, yeast extract, cold-pressed olive oil, geranyl pyrophosphate, lemon peel extract.

[0041] Preferably, the fermentation medium comprises glucose 10-20 g / L, KH2PO4 1-2 g / L, MgSO4·7H2O 0.5-1 g / L, FeCl3 0.03-0.08 g / L, glycerol 5-10 g / L, soy peptone 8-13 g / L, yeast extract 3-10 g / L, cold-pressed olive oil 2-5 mL / L, geranyl pyrophosphate 0.05-0.3 g / L, lemon peel extract 10-40 g / L.

[0042] The specific point value in 10-20 g / L can be selected from 10 g / L, 12 g / L, 14 g / L, 16 g / L, 18 g / L, 20 g / L, etc., the specific point value in 1-2 g / L can be selected from 1 g / L, 1.2 g / L, 1.4 g / L, 1.6 g / L, 1.8 g / L, 2 g / L, etc., the specific point value in 0.5-1 g / L can be selected from 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, etc., the specific point value in 0.03-0.08 g / L can be selected from 0.03 g / L, 0.04 g / L, 0.05 g / L, 0.06 g / L, 0.07 g / L, 0.08 g / L, etc., the specific point value in 5-10 g / L can be selected from 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, etc., the specific point value in 8-13 g / L can be selected from 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, etc., the specific point value in 3-10 g / L can be selected from 3 g / L, 5 g / L, 7 g / L, 9 g / L, 10 g / L, etc., the specific point value in 2-5 mL / L can be selected from 2 mL / L, 3 mL / L, 4 mL / L, 5 mL / L, etc., the specific point value in 0.05-0.3 g / L can be selected from 0.05 g / L, 0.1 g / L, 0.15 g / L, 0.2 g / L, 0.25 g / L, 0.3 g / L, etc., the specific point value in 10-40 g / L can be selected from 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, etc.

[0043] Preferably, the fermentation culture comprises a primary fermentation and a secondary fermentation performed in sequence.

[0044] The conditions of the primary fermentation are that the temperature is 26-30℃, the pH value is 5.5-6.5, and the fermentation time is 40-55 h.

[0045] The conditions of the secondary fermentation are that the temperature is 22-25℃, the pH value is 5.5-6.5, and the fermentation time is 60-80 h.

[0046] Specific point values in 26-30℃ can be selected from 26℃, 27℃, 28℃, 29℃, 30℃, etc., specific point values in 22-25℃ can be selected from 22℃, 23℃, 24℃, 25℃, etc., specific point values in 5.5-6.5 can be selected from 5.5, 5.7, 5.9, 6.1, 6.3, 6.5, etc., specific point values in 40-55 h can be selected from 40 h, 45 h, 50 h, 55 h, etc., and specific point values in 60-80 h can be selected from 60 h, 65 h, 70 h, 75 h, 80 h, etc.

[0047] In the present application, the primary fermentation is carried out at 26-30℃, which preferentially promotes the rapid proliferation of the lipolytic yeast Yarrowia lipolytica and occupies the ecological niche advantage, and through the vigorous metabolic activity, it preliminarily decomposes the lipid components in the culture medium, consumes dissolved oxygen, and adjusts the system pH to an acidic environment (5.5-6.5), which creates favorable growth conditions for the subsequent inoculation of Lactobacillus plantarum and Aspergillus niger, which prefer acidic anaerobic environment. The role of the secondary fermentation is to adapt to the normal temperature fermentation characteristics of Aspergillus niger and inhibit its excessive growth, and on the other hand, the lower temperature (22-25℃) is helpful for the retention of volatile aroma components and reduces the escape. In this stage, the three strains enter the stable period of synergistic metabolism, Lactobacillus plantarum continuously carries out biological transformation (such as glycoside hydrolysis), Aspergillus niger secretes a large amount of extracellular enzymes to further enzymatically hydrolyze macromolecular substrates, and Yarrowia lipolytica focuses on the deep conversion of lipids. Extending the time of the secondary fermentation to 60-80 h aims to fully realize this complex multi-strain synergistic conversion process and maximize the accumulation of target functional ingredients.

[0048] Preferably, the primary fermentation comprises inoculating Yarrowia lipolytica in the fermentation medium; after 20-30 h of inoculation of Yarrowia lipolytica, Lactobacillus plantarum and Aspergillus niger are inoculated again.

[0049] Specific point values in 20-30 h can be selected from 20 h, 22 h, 24 h, 26 h, 28 h, 30 h, etc.

[0050] The present application inoculates Yarrowia lipolytica first, and then synchronously adds Lactobacillus plantarum and Aspergillus niger after Yarrowia lipolytica enters the logarithmic growth phase.

[0051] Preferably, the fermentation culture further comprises adding farnesyl pyrophosphate (FPP) and leucine at 32-40 h of the primary fermentation.

[0052] Preferably, the leucine comprises L-leucine.

[0053] Farnesyl pyrophosphate is a 15-carbon sesquiterpene compound generated by isopentenyl pyrophosphate and dimethyl allyl pyrophosphate through the catalysis of farnesyl pyrophosphate synthase, as the core node material of isoprene metabolism, FPP is an important precursor of triterpenoids. In addition, leucine, as a branched-chain amino acid essential for microorganisms, its degradation products can directly enter the mevalonic acid (MVA) pathway to provide precursors for the synthesis of FPP. By exogenous supplementation of leucine and other endogenous synthesis promoters of FPP, the MVA pathway flux is strengthened from the source, and the intracellular level of FPP is targeted to improve, thereby specifically regulating the metabolic flow of microorganisms, significantly promoting the biosynthesis of sesquiterpenes, triterpenoids and other target functional components, and finally realizing the synergistic effect of multi-strain synergistic fermentation.

[0054] The present application creatively finds that farnesyl pyrophosphate and leucine have significant synergistic effect in promoting fermentation and promoting the synthesis of triterpenoids, which can improve the metabolic capacity of strains and promote the generation of high-value functional components.

[0055] Preferably, the total addition amount of farnesyl pyrophosphate and leucine is 1-5 g / L, for example, it can be 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, etc.

[0056] Preferably, the mass ratio of farnesyl pyrophosphate and leucine is 1:1-1:4, for example, it can be 1:1, 1:2, 1:3, 1:4, etc.

[0057] Preferably, the fermentation culture further comprises adding citric acid at the beginning of the secondary fermentation.

[0058] In the present application, citric acid as a characteristic inducer can induce and stimulate metabolic pathways to further promote the synthesis of target active ingredients.

[0059] Preferably, the addition amount of citric acid is 1-5 g / L, for example, it can be 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, etc.

[0060] Preferably, the fermentation culture further comprises supplementing glycerol every 10-14 h to maintain the residual sugar content at 2-3 g / L.

[0061] Specific point values in 10-14 h can be selected as 10 h, 11 h, 12 h, 13 h, 14 h, etc., and specific point values in 2-3 g / L can be selected as 2 g / L, 2.2 g / L, 2.4 g / L, 2.6 g / L, 2.8 g / L, 3 g / L, etc.

[0062] Preferably, after collecting the upper oil phase, the upper oil phase is further subjected to three-stage molecular distillation, and the distillate of secondary distillation and three-stage distillation is collected, and the fermented bergamot fruit oil is obtained after purification.

[0063] Preferably, the temperature of the first-stage distillation in the third-stage molecular distillation is 70-90℃, and the pressure is 8-12 Pa.

[0064] Preferably, the temperature of the second-stage distillation in the third-stage molecular distillation is 50-60℃, and the pressure is 0.5-3 Pa.

[0065] Preferably, the temperature of the third-stage distillation in the third-stage molecular distillation is 40-48℃, and the pressure is 0.01-0.1 Pa.

[0066] Specific point values in 70-90℃ can be selected as 70℃, 75℃, 80℃, 85℃, 90℃, etc., specific point values in 50-60℃ can be selected as 50℃, 52℃, 54℃, 56℃, 58℃, 60℃, etc., specific point values in 40-48℃ can be selected as 40℃, 42℃, 44℃, 46℃, 48℃, etc., specific point values in 8-12 Pa can be selected as 8 Pa, 9 Pa, 10 Pa, 11 Pa, 12 Pa, etc., specific point values in 0.5-3 Pa can be selected as 0.5 Pa, 1 Pa, 1.5 Pa, 2 Pa, 2.5 Pa, 3 Pa, etc., and specific point values in 0.01-0.1 Pa can be selected as 0.01 Pa, 0.03 Pa, 0.05 Pa, 0.07 Pa, 0.1 Pa, etc.

[0067] In a second aspect, the present application provides a fermented bergamot fruit oil prepared by the preparation method of the first aspect.

[0068] In a third aspect, the present application provides use of the fermented bergamot fruit oil of the second aspect in cosmetics.

[0069] Preferably, the cosmetics have any one or a combination of at least two of antioxidant efficacy, anti-inflammatory efficacy, or repair efficacy.

[0070] The numerical ranges described in the present application include not only the point values listed above, but also any point values between the listed point values, limited by the fact that the present application does not exhaustively list all the specific point values included in the ranges.

[0071] Compared with the prior art, the present application has the following beneficial effects:

[0072] The fermented bergamot fruit oil prepared by the improvement of the extraction technology and the optimization of the fermentation process has high stability, excellent antioxidant, anti-inflammatory, and repair efficacy, and solves the problems of unstable efficacy components and poor skin absorption rate of the bergamot fruit oil obtained by the traditional cold pressing method. DETAILED DESCRIPTION

[0073] In order to further clarify the technical means adopted by the present application and its effects, the technical solutions of the present application will be further described below in combination with the preferred embodiments of the present application, but the present application is not limited in the scope of the embodiments.

[0074] The sources of the raw materials in the following specific embodiments are shown in Table 1.

[0075] Table 1

[0076]

[0077] The seed liquid involved in the following specific embodiments is obtained in the following manner:

[0078] (1) Yarrowia lipolytica seed liquid: Yarrowia lipolytica strain (CICC 32187) is inoculated in culture medium I at an inoculation amount of 3% (v / v) and activated under anaerobic conditions at 28°C for 20 h to obtain an activation liquid; the activation liquid is inoculated in culture medium I at an inoculation amount of 3% (v / v) and cultured at 37°C for 24 h to obtain Yarrowia lipolytica liquid, and the concentration of the liquid is adjusted to 1×10 9 CFU / mL.

[0079] (2) Lactobacillus plantarum strain seed liquid: Lactobacillus plantarum strain (CGMCC 1.557) is inoculated in culture medium II at an inoculation amount of 2% (v / v) and activated at 37°C for 20 h to obtain an activation liquid; the activation liquid is inoculated in culture medium II at an inoculation amount of 2% (v / v) and cultured at 37°C for 24 h to obtain Lactobacillus plantarum strain, and the concentration of the liquid is adjusted to 1×10 9 CFU / mL.

[0080] (3) Aspergillus niger seed liquid: First, strain activation and slant culture are performed, PDA culture medium is used, Aspergillus niger strain (CGMCC 3.6189) is streaked on a freshly prepared slant, and the slant is cultured at 30°C for 7 days to produce abundant black spores, then spore harvesting and suspension preparation are performed, sterile water containing 0.1% (v / v) Tween-80 is used as an eluent, 20 mL of the eluent is added to the slant in a clean bench, the mycelium is scraped with a sterile inoculation loop to elute the spores, and finally the mycelium and medium residues are removed by sterile defatted cotton filtration to obtain a pure spore suspension; the spore suspension is inoculated in culture medium III at an inoculation amount of 5%, and the activation liquid is obtained after being activated at 30°C for 48 h; the activation liquid is inoculated in culture medium III at an inoculation amount of 10% (v / v), and the Aspergillus niger seed liquid is obtained after being cultured at 30°C for 36 h, and the concentration of the liquid is adjusted to 1×10 9 CFU / mL.

[0081] The formula of the culture medium involved in the preparation of the seed liquid is as follows:

[0082] (1) The formula of medium I is: glucose 20 g / L, bergamot peel hydrolysate 5.0 g / L, proteose peptone 20 g / L, yeast extract 10 g / L, KH2PO4 2 g / L, MgSO4·7H2O 1 g / L, pH value is 6;

[0083] (2) The formula of medium II is: glucose 20 g / L, bergamot peel hydrolysate 5.0 g / L, proteose peptone 20 g / L, yeast extract 10 g / L, triammonium citrate 2 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.2 g / L, MnSO4·4H2O 0.05 g / L, Tween-80 1 g / L, pH value is 6.5;

[0084] (3) The formula of medium III is: glucose 20 g / L, proteose peptone 10 g / L, yeast extract 5 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.5 g / L, bran leachate 50 mL / L, pH value is 6.0.

[0085] The preparation method of the bergamot peel hydrolysate involved in the above-mentioned culture medium is as follows:

[0086] Fresh bergamot peels are selected, and the moldy and mechanically damaged parts are removed. The peels are washed with water in a circulation mode for 5 min, and then washed with sterile water for 3 times. The peels are dried at 40℃ by hot air until the water content is ≤8%, and then crushed into bergamot peel powder by using an ultrafine grinder. The bergamot peel powder is added into a citric acid-sodium phosphate buffer solution with a pH value of 4.5 at a solid-liquid ratio of 1:10 g / L, and then a compound enzyme (the mass percentage of the compound enzyme in the solution is 3%, and the compound enzyme is a mixture of cellulase and pectinase at a mass ratio of 1:1) is added. The mixture is subjected to enzymatic hydrolysis at 50℃ for 2 h. After enzyme inactivation (90℃ for 10 min), the supernatant is collected by centrifugation at 8000 rpm for 10 min. After freeze-drying, the bergamot peel hydrolysate is obtained.

[0087] The yeast extract involved in the following specific embodiments is prepared by the following method:

[0088] Firstly, the bread yeast cells were collected by centrifugation, washed with sterile normal saline or deionized water for 2-3 times to remove the culture medium residues, then subjected to cell wall disruption, and then adjusted to a mass concentration of 18% and a pH of 7.2; then a multi-stage enzymatic hydrolysis process was adopted, first, alkaline protease and flavor protease were used in a ratio of 1:0.5 for enzymatic hydrolysis at 55°C and a pH of 8.0 for 4 hours (the total amount of enzyme added was 0.3% of the mass of the slurry), then 5'-phosphodiesterase was used at 0.02% of the mass of the slurry for nucleic acid conversion at 60°C and a pH of 6.0 for 2 hours; after the enzymatic hydrolysate was inactivated at 90°C, it was subjected to centrifugation, ceramic membrane filtration and activated carbon adsorption for decolorization and debittering; finally, ultrafiltration-nanofiltration membrane fractionation concentration was adopted, low-temperature evaporation was performed until the solid content reached 40%, and then spray drying was performed to obtain the yeast extract.

[0089] The steps of refining and purifying the secondary fraction and the tertiary fraction, decolorizing and filtering in the following specific embodiments are as follows:

[0090] Firstly, silica gel column chromatography was used for preliminary separation with n-hexane and ethyl acetate (volume ratio of 90:10), then further purification was performed by reverse phase HPLC column (C18) using methanol and water (volume ratio of 85:15) as the mobile phase; next, 1% activated carbon was used as the adsorbent for adsorption of the purified product at 50°C for 30 min for decolorization treatment, and then filtration was performed through a hydrophobic PTFE membrane (pore size of 0.22 μm) under a pressure of 0.2 MPa.

[0091] Example 1

[0092] The present embodiment provides a preparation method of fermented bergamot fruit oil, comprising the following steps:

[0093] (1) Preparation of bergamot fruit peel extract:

[0094] ① Fresh bergamot fruit peels were selected, and the moldy and mechanically damaged parts were removed, washed with water for 5 min, then washed with sterile water for 3 times, dried at 40°C hot air to a moisture content of ≤8%, and crushed into bergamot fruit peel powder with an ultrafine grinder.

[0095] ② The bergamot fruit peel powder was mixed with a composite enzyme aqueous solution (the mass percentage of the composite enzyme in the solution was 3%, and the composite enzyme was pectin lyase, pectin esterase and xylanase at a mass ratio of 1:1:1) at a solid-liquid ratio of 1:12 g / mL, and ultrasonic-assisted enzymatic extraction was carried out at 45°C for 90 min (ultrasonic power was 500 W, and ultrasonic frequency was 40 kHz). The extract was extracted with an equal volume of ethyl acetate, and the organic phase was collected. The organic phase was subjected to high-speed centrifugation, and the supernatant was collected and subjected to cross-flow filtration through a 0.45 μm ceramic membrane (transmembrane pressure difference was 0.3 MPa). After being concentrated under reduced pressure to a solid content of 30%, spray drying was performed. Then, the bergamot fruit peel extract was obtained by embedding treatment in a high-speed shearing mixer for 20 min at a mass ratio of 1:5 with β-cyclodextrin.

[0096] (2) Fermentation process:

[0097] ① The bergamot fruit peel extract was added to the fermentation medium, and at this time, the composition of the medium was as follows: glucose 15 g / L, KH2PO4 1.5 g / L, MgSO4·7H2O 0.8 g / L, FeCl3 0.05 g / L, glycerol 8 g / L, soybean peptone 10 g / L, yeast extract 6 g / L, bergamot fruit peel extract 20 g / L, cold-pressed olive oil 3 mL / L, geranyl pyrophosphate 0.1 g / L, and the pH value was 5.8.

[0098] ② Primary fermentation: 5% of Yarrowia lipolytica seed liquid was inoculated in the fermentation medium, and 3% of Lactobacillus plantarum seed liquid and 3% of Aspergillus niger seed liquid were inoculated after 24 h of culture; farnesyl pyrophosphate and L-leucine (total addition amount was 1.5 g / L, mass ratio was 1:2) were added at the 36th hour, and 50% glycerol aqueous solution was supplemented every 12 h to maintain the residual sugar concentration at 2-3 g / L;

[0099] The temperature of the primary fermentation was controlled at 28°C, the pH value was 6, and the total fermentation time was 48 h;

[0100] ③ Secondary fermentation: 3 g / L of citric acid was added at the beginning of the secondary fermentation;

[0101] The temperature of the secondary fermentation was controlled at 25°C, the pH value was 6, and the fermentation time was 72 h.

[0102] (3) Separation and purification:

[0103] ① The fermentation product is separated by three-phase separation technology. The fermentation broth is centrifuged at a speed of 8000 rpm for 30 min to separate three different components. The upper layer is the crude essential oil containing microbial oil, the middle layer is the microbial residue, and the lower layer is the aqueous solution containing polar metabolites. The upper layer of crude essential oil is introduced into a coalescence water-oil separator, and the flow rate is controlled at 5 L / h and the temperature is controlled at 25℃. The residual water is separated by a glass fiber coalescence filter with a pore size of 1.5 μm.

[0104] ② The upper layer of oil phase after removing the residual water is further separated and purified by three-stage molecular distillation. First, the fatty acid component is removed by primary distillation at 80℃ and 10 Pa. Then, the limonene fraction is collected by secondary distillation at 50℃ and 1 Pa. Finally, the oxygen-containing terpenoid component is obtained by tertiary distillation at 45℃ and 0.05 Pa. The secondary and tertiary fractions are combined.

[0105] ③ The secondary and tertiary fractions are refined and purified by decolorization and filtration, and then vacuum reduced pressure concentration. The concentrated liquid is freeze-dried to obtain the fermented bergamot fruit oil.

[0106] Example 2

[0107] The present embodiment provides a method for preparing a fermented bergamot fruit oil, comprising the following steps:

[0108] (1) Preparation of bergamot fruit peel extract:

[0109] ① Fresh bergamot fruit peels are selected, and the moldy and mechanically damaged parts are removed. The peels are washed with water for 5 min, and then rinsed with sterile water for 3 times. The peels are dried at 40℃ until the water content is ≤8%, and then crushed into bergamot fruit peel powder using an ultrafine grinder.

[0110] ② The bergamot fruit peel powder is mixed with a composite enzyme aqueous solution (the mass percentage of composite enzyme in the solution is 5%, and the composite enzyme is a mixture of pectin lyase, pectin esterase and xylanase with a mass ratio of 1:5:1) at a solid-liquid ratio of 1:8 g / mL. The mixture is subjected to ultrasonic-assisted enzymatic hydrolysis at 35℃ for 60 min (ultrasonic power is 400 W, and ultrasonic frequency is 30 kHz). The extract is extracted with an equal volume of ethyl acetate, and the organic phase is collected. The organic phase is subjected to high-speed centrifugation, and the supernatant is collected. The supernatant is subjected to cross-flow filtration through a 0.45 μm ceramic membrane (transmembrane pressure difference is 0.3 MPa). The filtrate is concentrated under reduced pressure to a solid content of 30%, and then subjected to spray drying. Then, the β-cyclodextrin is added to the filtrate at a mass ratio of 1:10, and the mixture is mixed in a high-speed shearing mixer for 15 min to obtain the bergamot fruit peel extract.

[0111] (2) Fermentation process:

[0112] ①The bergamot fruit peel extract is added to the fermentation medium, and the composition of the medium at this time is: glucose 10 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.5 g / L, FeCl3 0.08 g / L, glycerol 10 g / L, soybean peptone 13 g / L, yeast extract 3 g / L, bergamot fruit peel extract 10 g / L, cold-pressed olive oil 5 mL / L, geranyl pyrophosphate 0.3 g / L, and the pH value is 5.7.

[0113] ②Primary fermentation: inoculate 3% of Yarrowia lipolytica seed liquid in the fermentation medium, inoculate 1% of Lactobacillus plantarum seed liquid and 3% of Aspergillus niger seed liquid after 20 h of culture; add farnesyl pyrophosphate and L-leucine (total addition amount is 1 g / L, mass ratio is 1:1) at the 32nd hour, and supplement 50% glycerol aqueous solution every 10 h to maintain the residual sugar concentration at 2-3 g / L;

[0114] The temperature of the primary fermentation is controlled at 26°C, the pH value is 5.5, and the total fermentation time is 40 h;

[0115] ③Secondary fermentation: 1 g / L of citric acid is added at the beginning of the secondary fermentation;

[0116] The temperature of the secondary fermentation is controlled at 22°C, the pH value is 5.5, and the fermentation time is 60 h.

[0117] (3) Separation and purification:

[0118] ①The fermentation product is separated by three-phase separation technology, and the fermentation liquid is centrifuged at a speed of 8000 rpm for 30 min to separate three different components, the upper layer is the crude essential oil containing microbial oil, the middle layer is the microbial residue, and the lower layer is the aqueous solution containing polar metabolites; the upper layer of crude essential oil is introduced into a coalescence type water-oil separator, the flow rate is controlled at 5 L / h, the temperature is controlled at 25°C, and the residual water is separated by a glass fiber coalescence filter with a pore size of 1-2 μm.

[0119] ②The upper oil phase after removing the residual water is further separated and purified by three-stage molecular distillation: first, the fatty acid component is removed by primary distillation at 70°C and 8 Pa, then the limonene fraction is collected by secondary distillation at 55°C and 0.5 Pa, and finally the oxygen-containing terpenoid component is obtained by tertiary distillation at 40°C and 0.01 Pa; the secondary and tertiary fractions are combined.

[0120] ③The secondary and tertiary fractions are refined and purified, decolorized and filtered, then vacuum reduced pressure concentrated, the concentrated liquid is freeze-dried to obtain the fermented bergamot fruit oil.

[0121] Example 3

[0122] The embodiment provides a preparation method of fermented bergamot fruit oil, and comprises the following steps:

[0123] (1) Preparation of bergamot fruit peel extract:

[0124] ①Fresh bergamot fruit peels are selected, and moldy and mechanically damaged parts are removed. The bergamot fruit peels are washed with water for 5 minutes, and then washed with sterile water for 3 times. The bergamot fruit peels are dried at 40°C until the water content is less than or equal to 8%, and then crushed into bergamot fruit peel powder by using an ultrafine grinder.

[0125] ②The bergamot fruit peel powder is mixed with a composite enzyme aqueous solution (the mass percentage of the composite enzyme in the solution is 1%, and the composite enzyme is composed of pectin lyase, pectin esterase and xylanase at a mass ratio of 5:1:5) at a solid-liquid ratio of 1:20 g / mL, and subjected to ultrasonic-assisted enzymatic hydrolysis extraction at 60°C for 120 minutes (ultrasonic power is 600 W, and ultrasonic frequency is 50 kHz). The extraction liquid is extracted with an equal volume of ethyl acetate, and the organic phase is collected. The organic phase is subjected to high-speed centrifugation, and the supernatant is collected. The supernatant is subjected to cross-flow filtration through a 0.45 μm ceramic membrane (transmembrane pressure difference is 0.3 MPa). After being concentrated under reduced pressure until the solid content is 30%, the supernatant is subjected to spray drying. Then, the supernatant is mixed with β-cyclodextrin in a high-speed shearing mixer at a mass ratio of 3:10 for 30 minutes to perform embedding treatment, so as to obtain the bergamot fruit peel extract.

[0126] (2) Fermentation process:

[0127] ①The bergamot fruit peel extract is added to the fermentation medium, and at this time, the composition of the fermentation medium is as follows: glucose 20 g / L, KH2PO4 2 g / L, MgSO4·7H2O 1 g / L, FeCl3 0.03 g / L, glycerol 5 g / L, soybean peptone 8 g / L, yeast extract 10 g / L, bergamot fruit peel extract 40 g / L, cold-pressed olive oil 2 mL / L, geranyl pyrophosphate 0.05 g / L, and the pH value is 5.9.

[0128] ②Primary fermentation: 8% of the seed liquid of Yarrowia lipolytica is inoculated in the fermentation medium, and 5% of the seed liquid of Lactobacillus plantarum and 2% of the seed liquid of Aspergillus niger are inoculated after 30 hours of culture; at the 40th hour, farnesyl pyrophosphate and L-leucine (total addition amount is 5 g / L, mass ratio is 1:4) are added, and 50% of the aqueous solution of glycerol is supplemented every 14 hours to maintain the residual sugar concentration at 2-3 g / L;

[0129] The temperature of the primary fermentation is controlled at 30°C, the pH value is 6.5, and the total fermentation time is 55 hours;

[0130] ③Secondary fermentation: 5 g / L of citric acid is added at the beginning of the secondary fermentation;

[0131] The secondary fermentation is controlled at a temperature of 25°C, a pH of 6.5, and a fermentation time of 80 h.

[0132] (3) Separation and purification:

[0133] ① The fermentation product is separated by a three-phase separation technique. The fermentation liquid is centrifuged at a speed of 8000 rpm for 30 min to separate three different components. The uppermost layer is a crude essential oil containing microbial oil, the middle layer is microbial residue, and the lowermost layer is an aqueous solution containing polar metabolites. The upper crude essential oil is introduced into a coalescence type water-oil separator, and the flow rate is controlled at 5 L / h and the temperature is controlled at 25°C. The residual water is separated by a glass fiber coalescence filter with a pore size of 1-2 μm.

[0134] ② The upper oil phase after removal of residual water is further separated and purified by three-stage molecular distillation. First, the fatty acid component is removed by primary distillation at 90°C and 12 Pa. Then, the limonene fraction is collected by secondary distillation at 60°C and 3 Pa. Finally, the oxygen-containing terpenoid component is obtained by tertiary distillation at 48°C and 0.1 Pa. The secondary and tertiary fractions are combined.

[0135] ③ The secondary and tertiary fractions are refined and purified by decolorization and filtration, and then vacuum reduced pressure concentration. The concentrated liquid is freeze-dried to obtain the fermented bergamot fruit oil.

[0136] Example 4

[0137] The present example provides a method for preparing a fermented bergamot fruit oil. The difference from Example 1 is that the preparation process of the bergamot fruit peel extract does not involve embedding treatment. The product of ultrasonic-assisted enzymatic extraction is directly obtained after centrifugation, concentration, and drying. The addition amount of the bergamot fruit peel extract in the fermentation medium is adjusted to 3.3 g / L. The remaining steps are consistent with Example 1.

[0138] Example 5

[0139] The present example provides a method for preparing a fermented bergamot fruit oil. The difference from Example 1 is that geranyl pyrophosphate is not added to the fermentation medium. The remaining steps are consistent with Example 1.

[0140] Example 6

[0141] The present example provides a method for preparing a fermented bergamot fruit oil. The difference from Example 1 is that Yarrowia lipolytica is not inoculated in the primary fermentation process. The inoculation amount of Lactobacillus plantarum seed liquid and Aspergillus niger seed liquid is adjusted to 5.5%. The inoculation time and the remaining steps are consistent with Example 1.

[0142] Example 7

[0143] The present example provides a preparation method of fermented bergamot fruit oil, which is only different from example 1 in that, in the primary fermentation process, Lactobacillus plantarum is not inoculated, the inoculation amount of Yarrowia lipolytica seed liquid is adjusted to 6.9%, the inoculation amount of Aspergillus niger seed liquid is adjusted to 4.1%, and the inoculation time and the remaining steps are consistent with example 1.

[0144] Example 8

[0145] The present example provides a preparation method of fermented bergamot fruit oil, which is only different from example 1 in that, in the primary fermentation process, Aspergillus niger is not inoculated, the inoculation amount of Yarrowia lipolytica seed liquid is adjusted to 6.9%, the inoculation amount of Lactobacillus plantarum seed liquid is adjusted to 4.1%, and the inoculation time and the remaining steps are consistent with example 1.

[0146] Example 9

[0147] The present example provides a preparation method of fermented bergamot fruit oil, which is only different from example 1 in that, in the primary fermentation process, L- leucine is not added at 36 h, only 1.5 g / L farnesyl pyrophosphate is added, and the remaining steps are consistent with example 1.

[0148] Example 10

[0149] The present example provides a preparation method of fermented bergamot fruit oil, which is only different from example 1 in that, in the primary fermentation process, farnesyl pyrophosphate is not added at 36 h, only 1.5 g / L L-leucine is added, and the remaining steps are consistent with example 1.

[0150] Example 11

[0151] The present example provides a preparation method of fermented bergamot fruit oil, which is only different from example 1 in that, in the secondary fermentation process, citric acid is not added, and the remaining steps are consistent with example 1.

[0152] Comparative Example 1

[0153] The present comparative example provides a preparation method of fermented bergamot fruit oil, which is only different from example 1 in that, in the preparation process of bergamot fruit peel extract, the total amount of complex enzymes is kept unchanged, the complex enzymes are adjusted to pectinesterase and xylanase with a mass ratio of 1:1, and the remaining steps are consistent with example 1.

[0154] Comparative Example 2

[0155] The comparative example provides a preparation method of fermented bergamot fruit oil, which is only different from example 1 in that, in the preparation process of bergamot fruit peel extract, the total amount of complex enzyme is kept unchanged, the complex enzyme is adjusted to pectin lyase and xylanase with a mass ratio of 1:1, and the remaining steps are consistent with example 1.

[0156] Comparative example 3

[0157] The comparative example provides a preparation method of fermented bergamot fruit oil, which is only different from example 1 in that, in the preparation process of bergamot fruit peel extract, the total amount of complex enzyme is kept unchanged, the complex enzyme is adjusted to pectin lyase and xylanase with a mass ratio of 1:1, and the remaining steps are consistent with example 1.

[0158] Comparative example 4

[0159] The comparative example provides a bergamot fruit oil prepared by a steam distillation method.

[0160] ①Fresh bergamot fruit peels are selected, and moldy and mechanically damaged parts are removed. The bergamot fruit peels are washed with water for 5 minutes, then rinsed with sterile water for 3 times, dried at 40°C until the water content is ≤8%, and then crushed into bergamot fruit peel powder with an ultrafine grinder.

[0161] ②Loading and distillation: the bergamot fruit peel powder obtained above is placed in a distillation kettle of a steam distillation device, and appropriate deionized water is added to ensure that the material is completely immersed. After the device is installed, the heat source is turned on for heating, and water vapor is introduced for distillation.

[0162] ③Condensation and collection: keep boiling, and make the bergamot fruit oil and water vapor evaporate together. After the mixed steam is fully condensed through a condenser tube, it is collected in an oil-water separator.

[0163] ④Separation and drying: separate the layers in the oil-water separator, and after the oil phase and the water phase are completely separated, the lower water phase is separated, and the upper clear oil phase is collected. Anhydrous sodium sulfate is added to the collected crude oil for drying to remove trace amounts of water.

[0164] ⑤Filtration and storage: the oil liquid after drying is filtered through a quantitative filter paper to remove the drying agent, and the obtained filtrate is vacuum reduced pressure concentrated, frozen and dried to obtain the bergamot fruit oil.

[0165] Application example 1

[0166] The application example provides an essence, which comprises, in mass percentage, 5% of the fermented bergamot fruit oil prepared in example 1, 3% of dimethicone, 5% of panthenol, 8% of glycerol, 3% of propylene glycol, 3% of sodium hyaluronate, 3% of Tween-80, and the balance is water.

[0167] The preparation process is:

[0168] (1) Fermented bergamot fruit oil, dimethicone, Tween-80 were stirred and homogenized at 45°C to obtain an oil phase mixture; water, panthenol, glycerol, propylene glycol, sodium hyaluronate were stirred and mixed uniformly at 50°C to obtain an aqueous phase mixture;

[0169] (2) The oil phase mixture was slowly added to the aqueous phase mixture under stirring, and homogenized at 45°C and 500 rpm for 20 min to obtain the serum.

[0170] Application Example 2-11

[0171] Application Example 2-11 each provides a serum, which is only different from Application Example 1 in that the fermented bergamot fruit oil prepared in Example 1 is respectively replaced with the fermented bergamot fruit oil prepared in Example 2-11 in equal quality, and the rest of the formulation and preparation process remain the same as Application Example 1.

[0172] Comparative Application Example 1-4

[0173] Comparative Application Example 1-4 each provides a serum, which is only different from Application Example 1 in that the fermented bergamot fruit oil prepared in Example 1 is respectively replaced with the fermented bergamot fruit oil or bergamot fruit oil prepared in Comparative Example 1-4 in equal quality, and the rest of the formulation and preparation process remain the same as Application Example 1.

[0174] Test Example 1

[0175] Antioxidant capacity test.

[0176] (1) Test principle:

[0177] Excessive production of free radicals is the main cause of natural aging and photoaging of the skin. Therefore, scavenging free radicals can delay skin aging and reduce wrinkles. 1,1-diphenyl-2-trinitrobenzene hydrazine (DPPH) is a stable long-lived free radical, which will weaken the light absorption of DPPH ethanol solution when there is a free radical scavenger due to the pairing of a single electron. The degree of fading of DPPH ethanol solution is linearly related to the number of electrons it receives, so the ability of the test sample to scavenge free radicals, i.e., the size of the antioxidant activity, can be evaluated.

[0178] (2) Test method:

[0179] 1.0 g of the fermented bergamot fruit oil prepared in each of the examples and comparative examples was taken and diluted 5 times with anhydrous ethanol, placed in a 10 mL centrifuge tube, 3.0 mL of DPPH solution (0.008 mmol / L) was added, and the reaction was carried out at room temperature for 30 min in the dark, with anhydrous ethanol as a blank, and the absorbance was measured at 517 nm. The DPPH free radical scavenging rate was calculated according to the following formula.

[0180] DPPH radical scavenging rate = [A0- (As-Ac)] / A0 x 100%;

[0181] In the formula, A0 is the absorbance of 1.0 mL of anhydrous ethanol + 3.0 mL of a DPPH solution, As is the absorbance of 1.0 mL of a sample solution + 3.0 mL of a DPPH solution, and Ac is the absorbance of 1.0 mL of a sample solution + 3.0 mL of anhydrous ethanol.

[0182] The radical scavenging capacity of the fermented bergamot fruit oil or bergamot fruit oil prepared in each example and comparative example is shown in Table 2.

[0183] Table 2

[0184]

[0185] Test Example 2

[0186] Test of soothing and anti-inflammatory effects.

[0187] (1) Test sample: fermented bergamot fruit oil or bergamot fruit oil prepared in Examples 1-11 and Comparative Examples 1-4.

[0188] (2) Test method:

[0189] IL-1α and IL-6 are cell inflammatory factors, and the inflammatory factors are detected by ELISA to evaluate the anti-inflammatory effect of the sample to be tested.

[0190] (2.1) Experimental grouping and treatment method:

[0191] Blank group: HaCat cells without any treatment were cultured in a 12-well plate;

[0192] Sample group: after the HaCat cells were stimulated with 1 μg / mL LPS for 24 h, 100 μL of a sample solution was added to each well of a 12-well plate (wherein the sample solution was prepared by dissolving the bergamot fruit oil in 4 times the mass of DMSO, and then dissolving in deionized water to prepare a sample to be tested with a mass concentration of 1% of the bergamot fruit oil and DMSO mixture) ;

[0193] Model group: after the HaCat cells were stimulated with 1 μg / mL LPS for 24 h, 100 μL of a 1% DMSO aqueous solution was added to each well of a 12-well plate.

[0194] Each experimental group was cultured in a 37°C, 5% CO2 constant temperature incubator for 72 h, and each experimental group had 3 replicate wells. The cell culture supernatant was collected, and the expression levels of IL-1α and IL-6 were detected by an ELISA kit, and the results were averaged.

[0195] The content of each experimental group IL-1α and IL-6 is taken as a reference amount (i.e. 100%), and the relative content results are shown in Table 3.

[0196] Table 3

[0197]

[0198] Test Example 3

[0199] Stability test.

[0200] (1) Test sample: The serum provided in Application Examples 1-11 and Comparative Application Example 1-4.

[0201] (2) Test method:

[0202] The serum of each application example and comparative application example was subjected to a stability test. The test method was as follows: 10 mL of the serum sample was taken and placed in a test tube, which was then sealed and heated in a water bath at 50℃ for 30 days, and the appearance change was observed. Another sample was subjected to irradiation for 24 h under a cold color fluorescent lamp at 25℃, and the appearance change was observed. The light intensity was 2000 Lux.

[0203] The test results are shown in Table 4.

[0204] Table 4

[0205]

[0206] As can be seen from the data in Tables 2-4, the fermented bergamot fruit oil involved in the present application has excellent antioxidant and anti-inflammatory repair effects, and also has excellent stability.

[0207] As can be seen from the data comparison of Example 1 / Application Example 1 and Comparative Example 1-3 / Comparative Application Example 1-3, the pectin lyase and the pectin lyase and xylanase can better hydrolyze the bergamot fruit peel, better release the active ingredients in the bergamot fruit peel, and improve the antioxidant and anti-inflammatory repair effects of the finally prepared fruit oil.

[0208] As can be seen from the data comparison of Example 1 / Application Example 1 and Example 4 / Application Example 4, by embedding the bergamot fruit peel extract with cyclodextrin to form an inclusion compound, the stability of the active ingredients can be further improved, the antioxidant and anti-inflammatory repair effects of the product can be improved, and the storage stability of the product can be improved.

[0209] As can be seen from the data comparison of Example 1 / Application Example 1 and Example 5 / Application Example 5, by adding geranyl pyrophosphate to the fermentation medium, the content of the active ingredients can be significantly improved, and the antioxidant and repair effects of the finally prepared fermented bergamot fruit oil can be improved.

[0210] From the data comparison of Example 1 / Example 1 and Example 6-8 / Example 6-8, it can be seen that in the process of fermentation culture, Yarrowia lipolytica, Lactobacillus plantarum and Aspergillus niger have significant synergistic effect, so that the prepared fermented bergamot fruit oil has excellent antioxidant and anti-inflammatory repair effects.

[0211] From the data comparison of Example 1 / Example 1 and Example 9-10 / Example 9-10, it can be seen that in the process of fermentation, farnesyl pyrophosphate and leucine have significant synergistic effect in promoting fermentation, and promote the generation of high-value functional ingredients.

[0212] From the data comparison of Example 1 / Example 1 and Example 11 / Example 11, it can be seen that in the process of secondary fermentation, citric acid can further promote the synthesis of target active ingredients, and improve the antioxidant and anti-inflammatory effects of the product.

[0213] The applicant declares that the technical scheme of the present application is illustrated by the above examples, but the present application is not limited to the above examples, that is, it does not mean that the present application must rely on the above examples to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes, etc. fall within the protection scope and disclosure scope of the present application.

[0214] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above embodiments, and within the technical concept scope of the present application, various simple modifications can be made to the technical scheme of the present application, and these simple modifications all belong to the protection scope of the present application.

[0215] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combination manners.

Claims

1. A method for preparing fermented bergamot fruit oil, characterized in that, The preparation method includes the following steps: Fermentation culture medium was inoculated with fermentation bacteria and fermented. The fermentation broth was then subjected to three-phase separation, and the upper oil phase was collected to obtain fermented bergamot fruit oil. The fermentation medium includes bergamot peel extract; The bergamot peel extract is obtained by mixing bergamot peel raw material with an aqueous solution of a compound enzyme and then performing ultrasound-assisted enzymatic hydrolysis extraction. The organic phase of the extract is collected after extraction. The complex enzyme includes pectin lyase, pectin esterase, and xylanase.

2. The preparation method according to claim 1, characterized in that, The ratio of the raw material of lemon peel to the aqueous solution of the compound enzyme is 1:8-1:20 g / mL; Preferably, the aqueous solution of the complex enzyme contains 1-5% by mass of the complex enzyme; Preferably, the mass ratio of the pectin lyase, pectin esterase and xylanase is (1-5):(1-5):(1-5).

3. The preparation method according to claim 1 or 2, characterized in that, The temperature for ultrasound-assisted enzymatic hydrolysis extraction is 35-60℃, and the extraction time is 60-120 min. Preferably, the ultrasonic power of the ultrasound-assisted enzymatic hydrolysis extraction is 400-600 W, and the ultrasonic frequency of the ultrasound-assisted enzymatic hydrolysis extraction is 30-50 kHz. Preferably, after extraction, the process further includes filtering, concentrating, and drying the organic phase, then mixing it with cyclodextrin for encapsulation. Preferably, the mass ratio of the dried product to cyclodextrin is 1:10 to 3:

10.

4. The preparation method according to any one of claims 1-3, characterized in that, The fermentation strains include Yersinia lipolytica, Lactobacillus plantarum, and Aspergillus niger. Preferably, the ratio of the number of *Yersinia lipophila*, *Lactobacillus plantarum*, and *Aspergillus niger* is (3-8):(1-5):(1-5); Preferably, the total inoculation amount of the fermentation strain is 7-15% by volume.

5. The preparation method according to any one of claims 1-4, characterized in that, The amount of bergamot peel extract added to the fermentation medium is 10-40 g / L; Preferably, the fermentation medium further includes geranylide pyrophosphate; Preferably, the amount of gerany pyrophosphate added to the fermentation medium is 0.05-0.3 g / L; Preferably, the fermentation medium includes a carbon source, a nitrogen source, inorganic salts, bergamot peel extract, and geraniol pyrophosphate.

6. The preparation method according to any one of claims 1-5, characterized in that, The fermentation culture includes a primary fermentation and a secondary fermentation performed sequentially. The conditions for the primary fermentation are: temperature 26-30℃, pH value 5.5-6.5, and fermentation time 40-55 h; The conditions for the secondary fermentation are: temperature of 22-25℃, pH of 5.5-6.5, and fermentation time of 60-80 h.

7. The preparation method according to claim 6, characterized in that, The primary fermentation includes inoculating *Yersinia lipolytica* into the fermentation medium; 20-30 h after inoculation with *Yersinia lipolytica*, *Lactobacillus plantarum* and *Aspergillus niger* are then inoculated. Preferably, the fermentation culture further includes the addition of farnesyl pyrophosphate and leucine at 32-40 h of the first fermentation; Preferably, the total amount of farnesyl pyrophosphate and leucine added is 1-5 g / L; Preferably, the mass ratio of farnesyl pyrophosphate to leucine is 1:1 to 1:4; Preferably, the fermentation culture further includes adding citric acid at the start of the secondary fermentation; Preferably, the amount of citric acid added is 1-5 g / L.

8. The preparation method according to any one of claims 1-7, characterized in that, The process of collecting the upper oil phase also includes performing a third-stage molecular distillation on the upper oil phase, collecting the fractions from the second-stage and third-stage distillations, and purifying them to obtain fermented bergamot fruit oil. Preferably, the temperature of the first stage of the tertiary molecular distillation is 70-90℃ and the pressure is 8-12 Pa. Preferably, the temperature of the second stage of the tertiary molecular distillation is 50-60℃, and the pressure is 0.5-3 Pa. Preferably, the temperature of the tertiary molecular distillation is 40-48℃ and the pressure is 0.01-0.1 Pa.

9. A fermented bergamot fruit oil prepared by any one of claims 1-8.

10. The use of the fermented bergamot fruit oil as described in claim 9 in cosmetics.

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