A low-oxygen extraction and preparation method for a MITO-type mitochondrial uptake-promoting reduced coenzyme q10 raw material, and use thereof

Abstract

The present invention discloses a low-oxygen extraction and preparation method for a MITO-type mitochondrial uptake-promoting reduced coenzyme Q10 raw material, and use thereof. Coenzyme Q10-containing fermentation biomass is subjected to nitrogen-protected slurry preparation, composite-enzyme-assisted cell disruption, and low-temperature extraction with an ethanol-ethyl acetate mixed solvent. The resulting extract is concentrated, purified with neutral alumina and activated carbon, and subjected to biphasic mild reduction using L-sodium ascorbate to obtain a reduced coenzyme Q10 concentrate. Medium-chain triglycerides, plant-derived phospholipids, phosphatidylserine, ascorbyl palmitate, and mixed tocopherols are added to form a reduced coenzyme Q10 lipid phase. The lipid phase is emulsified with γ-cyclodextrin, gum arabic, and resistant dextrin, followed by low-oxygen spray drying or freeze drying. The method improves extraction yield, reduced-state retention, water dispersibility, storage stability, and mitochondrial-fraction enrichment in an in vitro cell uptake model.

Description

[0001] DESCRIPTION

[0002] A LOW-OXYGEN EXTRACTION AND PREPARATION METHOD FOR A MITO-TYPE MITOCHONDRIAL UPTAKE-PROMOTING REDUCED COENZYME Q10

[0003] RAW MATERIAL, AND USE THEREOF

[0004] TECHNICAL FIELD

[0005] The present invention relates to the technical fields of extraction of coenzyme Q10 raw materials, processing of nutritional supplement raw materials, and stabilization of fat-soluble antioxidant components, and specifically relates to a low-oxygen extraction and preparation method for a MITO-type mitochondrial uptake-promoting reduced coenzyme Q10 raw material, and use thereof.

[0006] BACKGROUND ART

[0007] Coenzyme Q10 is a fat-soluble quinone substance widely present in organisms. Coenzyme Q10 generally includes two forms, namely oxidized coenzyme Q10 and reduced coenzyme Q10, wherein reduced coenzyme Q10 is also known as ubiquinol and belongs to the reduced form of coenzyme Q10. Compared with oxidized coenzyme Q10, reduced coenzyme Q10 has stronger fat-soluble antioxidant characteristics, but it is relatively sensitive to oxygen, light, and high temperature, and is easily re-oxidized into oxidized coenzyme Q10 during extraction, reduction, concentration, crystallization, drying, and storage.

[0008] Existing coenzyme Q10 production technologies mainly include chemical synthesis, biological tissue extraction, and microbial fermentation. Among them, microbial fermentation is widely used because it is suitable for industrial production. For extraction of coenzyme Q10 from fermentation biomass, processes such as saponification, organic solvent extraction, ultrasonic disruption, resin adsorption, silica gel column chromatography, and supercritical extraction have been used. However, conventional processes usually target extraction and purification of oxidized coenzyme Q10, and have problems such as large organic solvent consumption, large amounts of saponification wastewater, relatively high process temperatures,long oxygen exposure time, and insufficient retention rate of reduced coenzyme Q10.

[0009] Reduced coenzyme Q10 stabilization technologies mostly focus on adding antioxidants, controlling crystallization conditions, preparing soft capsules, or preparing common dry powder compositions. Although the above methods can improve the stability of reduced coenzyme Q10 to some extent, there is still a lack of an integrated process solution suitable for development of nutritional supplement raw materials with respect to the synergistic relationship among continuous extraction starting from fermentation biomass, low-oxygen protection, biphasic mild reduction, low-temperature washing and desalting, lipid-phospholipid stabilized dispersion, and promotion of mitochondrial fraction uptake.

[0010] Mitochondria are important organelles for cellular energy metabolism and redox balance. Some mitochondria-enriched antioxidant technologies covalently link antioxidant groups with specific chemical modifying groups so that the antioxidant groups are enriched in mitochondrial regions. However, such compounds are generally novel chemical entities with specific structures and have issues such as raw material compliance, patent licensing, and limitations on application scenarios. For nutritional supplement raw materials, it is more suitable to use reduced coenzyme Q10 having natural or strong nutrient attributes as a core component, and to improve its reduced-state retention rate, water dispersibility, and in vitro mitochondrial fraction enrichment performance through process design.

[0011] The term "MITO type" in the present invention refers to a raw-material technical identifier established around stabilization of reduced coenzyme Q10, improvement of water dispersibility, and evaluation of in vitro mitochondrial fraction enrichment, and does not indicate a mitochondria-enriched compound formed by covalently linking a specific chemical modifying group with coenzyme Q10 or a quinol structure thereof, nor does it indicate mitochondria-specific delivery in a pharmaceutical sense.

[0012] Therefore, it is necessary to develop a MITO-type reduced coenzyme Q10 raw material that uses coenzyme QlO-containing fermentation biomass as a starting material and is prepared by low-oxygen extraction, biphasic mild reduction, adsorptive impurity removal, low-temperaturewashing and desalting, and lipid-phospholipid stabilized dispersion, so that the raw material has a relatively high reduced-state retention rate, good water dispersibility, good storage stability, and in vitro mitochondrial fraction enrichment performance.

[0013] SUMMARY OF THE INVENTION

[0014] In view of the deficiencies of the prior art, the present invention provides a low-oxygen extraction and preparation method for a MITO-type mitochondrial uptake-promoting reduced coenzyme Q10 raw material, and use thereof, thereby solving the technical problems in existing reduced coenzyme Q10 preparation processes, including large oxidation loss, low reduction efficiency, difficulty in removing reducing agent residues, poor water dispersibility, low mitochondrial enrichment level, and insufficient storage stability.

[0015] To achieve the above objectives, the present invention is implemented through the following technical solution: a low-oxygen extraction and preparation method for a MITO-type mitochondrial uptake-promoting reduced coenzyme Q10 raw material, and use thereof, comprising the following steps:

[0016] SI: taking coenzyme QlO-containing fermentation biomass, wherein the fermentation biomass is wet biomass or dry biomass, adding purified water to adjust the solids mass content to 18-35%, and introducing nitrogen for replacement so that the dissolved oxygen of the system is not higher than 1.5 mg / L, thereby obtaining a low-oxygen slurry;

[0017] S2: adding a composite enzyme to the low-oxygen slurry obtained in step SI, treating the same at pH 6.0-7.2 and 42-52°C for 1-3 hours, and then carrying out high-pressure homogenization for cell disruption to obtain a cell-disrupted slurry, wherein the composite enzyme comprises lysozyme, neutral protease, and lipase, and a mass ratio of the three is l:(0.5-2):(0.1-0.8);

[0018] S3: adding an ethanol-ethyl acetate mixed solvent to the cell-disrupted slurry obtained in S2, extracting 2-3 times at 30-45 °C under nitrogen protection, and combining organic phases toobtain a coenzyme QlO-containing extract; wherein a volume ratio of ethanol to ethyl acetate is 35:65-55:45, and a mass ratio of the mixed solvent to the cell-disrupted slurry is 4:1-10:1;

[0019] S4: concentrating the extract obtained in S3 at a temperature not higher than 45°C and under an absolute pressure of 5-20 kPa to obtain a coenzyme Q10 crude extract;

[0020] S5: dissolving the coenzyme Q10 crude extract obtained in S4 in an ethyl acetate-ethanol mixed solvent, adding neutral alumina and activated carbon for adsorptive impurity removal, and filtering to obtain a coenzyme Q10 refined solution;

[0021] S6: under nitrogen protection, adding an aqueous L-sodium ascorbate solution, ascorbyl palmitate, mixed tocopherols, and citric acid to the coenzyme Q10 refined solution obtained in S5 to form a biphasic reduction system composed of an ethyl acetate organic phase and an ethanol-containing aqueous phase, controlling the aqueous phase at a pH of 4.5-5.8 and a temperature of 40-55°C, and reacting for 1-6 hours so that oxidized coenzyme Q10 is converted into reduced coenzyme Q10; after standing for phase separation, taking the organic phase to obtain a reduced coenzyme Q10 organic phase, wherein the ethanol volume fraction in the ethanol-containing aqueous phase is 10-35%, and the volume of the ethanol-containing aqueous phase accounts for 8-25% of the total volume of the biphasic reduction system;

[0022] S7: washing the reduced coenzyme Q10 organic phase obtained in S6 with deoxygenated purified water 1-3 times to remove water-soluble salts and residual reducing agents, washing until the conductivity of the aqueous phase is not higher than 150 pS / cm or until the residue of ascorbic acid substances in the aqueous phase is not higher than 0.5 mg / mL, and then removing the solvent at a temperature not higher than 42°C and under an absolute pressure of 5-20 kPa to obtain a reduced coenzyme Q10 concentrate;

[0023] S8: under nitrogen protection, adding medium-chain triglycerides, plant-derived phospholipids, phosphatidylserine, ascorbyl palmitate, and mixed tocopherols to the reduced coenzyme Q10 concentrate obtained in S7, and stirring and dispersing the same at 40-55°C toobtain a reduced coenzyme Q10 lipid phase, wherein a mass ratio of reduced coenzyme Q10, medium-chain triglycerides, plant-derived phospholipids, and phosphatidylserine is l:(1.0-2.5):(0.5-1.5):(0.05-0.25);

[0024] S9: dissolving or dispersing y-cyclodextrin, gum arabic, and resistant dextrin in deoxygenated purified water, adding the reduced coenzyme Q10 lipid phase obtained in S8, and after high-speed shearing and high-pressure homogenization, carrying out spray drying or freeze drying under low-oxygen conditions to obtain the MITO-type mitochondrial uptake-promoting reduced coenzyme Q10 raw material;

[0025] the MITO type refers to a technical form of a mitochondrial uptake-promoting raw material that is formed with non-covalently modified reduced coenzyme Q10 as a core by low-oxygen extraction, biphasic mild reduction, lipid-phospholipid stabilized dispersion, and low-oxygen microencapsulation, wherein the mitochondrial uptake-promoting effect is achieved by the lipid-phospholipid dispersion system and the low-oxygen stabilization process.

[0026] Preferably, in SI, the coenzyme QlO-containing fermentation biomass is wet biomass or dry biomass obtained by fermentation of Rhodobacter sphaeroides or Paracoccus denitrificans, and in S2, the high-pressure homogenization cell-disruption pressure is 40-80 MPa and the number of homogenization passes is 1-3.

[0027] Preferably, in S5, the addition amount of neutral alumina is 0.5-2.5 times the mass of the coenzyme Q10 crude extract, and the addition amount of activated carbon is 0.02-0.12 times the mass of the coenzyme Q10 crude extract.

[0028] Preferably, in S6, the addition amount of L-sodium ascorbate is 0.8-4.0 times the mass of total coenzyme Q10, the addition amount of ascorbyl palmitate is 0.5-3.0% of the mass of total coenzyme Q10, the addition amount of mixed tocopherols is 0.2-1.5% of the mass of total coenzyme Q10, and the addition amount of citric acid is 0.1-0.8% of the mass of total coenzyme Q10.Preferably, in S8, the plant-derived phospholipid is sunflower seed phospholipid, soybean phospholipid, or a combination thereof, wherein the mass content of phosphatidylcholine is not lower than 40%, and in S9, a mass ratio of the reduced coenzyme Q10 lipid phase, y-cyclodextrin, gum arabic, and resistant dextrin is l:(0.6-1.5):(0.4-1.2):(0.3-1.0).

[0029] Preferably, in S9, the high-speed shearing speed is 8000-12000 r / min, the shearing time is 3-8 minutes, the high-pressure homogenization pressure is 25-45 MPa, the number of homogenization passes is 2-4, an inlet air temperature for spray drying is 105-135°C, an outlet air temperature is 55-70°C, and the oxygen concentration contacted by the material during drying is not higher than 5%.

[0030] Preferably, a MITO-type mitochondrial uptake-promoting reduced coenzyme Q10 raw material prepared by a low-oxygen extraction and preparation method for MITO-type mitochondrial uptake-promoting reduced coenzyme Q10 has a mass proportion of reduced coenzyme Q10 in total coenzyme Q10 of not lower than 85%, a mass content of total coenzyme Q10 of 8-18%, moisture of not higher than 5.0%, and a particle size D50 after water dispersion of 150-400 nm.

[0031] Preferably, after the raw material is sealed, protected from light, and stored at 40°C and 75% relative humidity for 60 days, the mass proportion of reduced coenzyme Q10 in total coenzyme Q10 is still not lower than 78%; in an in vitro cell uptake model, calculated based on the same addition amount of total coenzyme Q10, the relative content of total coenzyme Q10 in mitochondrial fractions isolated from the raw-material treatment group is at least 1.30 times that of a common reduced coenzyme Q10 microcapsule powder treatment group that does not contain plant-derived phospholipids and phosphatidyl serine, and the content of total coenzyme Q10 in the mitochondrial fractions is normalized to the amount of mitochondrial protein.

[0032] Preferably, the reduced coenzyme Q10 in the raw material is non-covalently modified reduced coenzyme Q10.Preferably, the MITO-type mitochondrial uptake-promoting reduced coenzyme Q10 raw material is used in the preparation of a non-therapeutic nutritional supplement in the form of hard capsules, soft capsules, tablets, granules, or powders.

[0033] Preferably, the nutritional supplement is a nutritional supplement containing reduced coenzyme Q10.

[0034] The present invention provides a low-oxygen extraction and preparation method for a MITO-type mitochondrial uptake-promoting reduced coenzyme Q10 raw material, and use thereof, and has the following beneficial effects:

[0035] 1. The present invention carries low-oxygen protection through the processes of slurry preparation, cell disruption, extraction, reduction, washing, solvent removal, and drying, and can reduce oxidation loss of reduced coenzyme Q10 during processing.

[0036] 2. The present invention employs composite enzyme-assisted cell disruption in combination with high-pressure homogenization cell disruption, which is beneficial for improving the release efficiency of coenzyme Q10 from fermentation biomass and reducing side reactions and waste-liquid burden caused by a strong-alkali saponification process.

[0037] 3. The present invention employs low-temperature extraction with an ethanol-ethyl acetate mixed solvent, and combines neutral alumina and activated carbon for adsorptive impurity removal, thereby obtaining a coenzyme Q10 refined solution with relatively high purity.

[0038] 4. The present invention employs a biphasic mild reduction system composed of an ethyl acetate organic phase and an ethanol-containing aqueous phase, so that the water-soluble reducing agent is mainly distributed in the aqueous phase and coenzyme Q10 is mainly distributed in the organic phase. After the reaction is completed, water-soluble salts and residual reducing agents are removed by standing phase separation and low-temperature washing, thereby reducing the impurity burden in subsequent raw materials.

[0039] 5. The present invention constructs a lipid-phospholipid microcapsule system throughmedium-chain triglycerides, plant-derived phospholipids, phosphatidyl serine, y-cyclodextrin, gum arabic, and resistant dextrin. The resulting raw material has good water dispersibility and a relatively high relative enrichment level of coenzyme Q10 in mitochondrial fractions in an in vitro cell uptake model.

[0040] 6. The raw material obtained by the present invention can be used for hard capsules, soft capsules, tablets, granules, or powder nutritional supplements, and has good dosage form adaptability.

[0041] BRIEF DESCRIPTION OF THE DRAWINGS FIG. l is a flow chart of the present invention.

[0042] DETAILED DESCRIPTION OF EMBODIMENTS

[0043] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without creative work shall fall within the scope of protection of the present invention.

[0044] Examples:

[0045] Referring to FIG. 1, an embodiment of the present invention provides a low-oxygen extraction and preparation method for a MITO-type mitochondrial uptake-promoting reduced coenzyme Q10 raw material, and use thereof, comprising the following steps:

[0046] SI: 10 kg of wet biomass obtained by fermentation of Rhodobacter sphaeroides was taken, wherein the solids content of the wet biomass was 28.6%, and the content of total coenzyme Q10 on a dry basis was 5.42%. Purified water was added to the wet biomass to adjust the solids mass content to 24%, and nitrogen was introduced for 30 minutes so that the dissolved oxygen of the system was reduced to 1.1 mg / L, thereby obtaining a low-oxygen slurry:S2: a composite enzyme was added to the low-oxygen slurry. The composite enzyme consisted of lysozyme, neutral protease, and lipase in a mass ratio of 1:1:0.4, and the addition amount of the composite enzyme was 0.45% of the dry-basis mass of the biomass. The pH was adjusted to 6.6, and treatment was performed at 48°C for 2 hours. Subsequently, high-pressure homogenization was performed twice at 60 MPa to obtain a cell-disrupted slurry;

[0047] S3: an ethanol-ethyl acetate mixed solvent was added to the cell-disrupted slurry, wherein the volume ratio of ethanol to ethyl acetate was 45:55, and the mass ratio of the mixed solvent to the cell-disrupted slurry was 6:1. Under nitrogen protection, stirring extraction was performed twice at 38°C, each time for 60 minutes, and the organic phases were combined to obtain a coenzyme QlO-containing extract;

[0048] S4: the extract was concentrated at 42°C and under an absolute pressure of about 10 kPa to obtain a coenzyme Q10 crude extract;

[0049] S5: the coenzyme Q10 crude extract was dissolved in an ethyl acetate-ethanol mixed solvent, wherein the volume ratio of ethyl acetate to ethanol was 70:30. Neutral alumina in an amount equivalent to 1.2 times the mass of the crude extract and activated carbon in an amount equivalent to 0.06 times the mass of the crude extract were added, followed by stirring at 25°C for 40 minutes and filtration to obtain a coenzyme Q10 refined solution;

[0050] S6: under nitrogen protection, an aqueous L-sodium ascorbate solution, ascorbyl palmitate, mixed tocopherols, and citric acid were added to the coenzyme Q10 refined solution to form a biphasic reduction system composed of an ethyl acetate organic phase and an ethanol-containing aqueous phase. The addition amount of L-sodium ascorbate was 2.5 times the mass of total coenzyme Q10, the addition amount of ascorbyl palmitate was 1.5% of the mass of total coenzyme Q10, the addition amount of mixed tocopherols was 0.6% of the mass of total coenzyme Q10, and the addition amount of citric acid was 0.3% of the mass of total coenzyme Q10. The ethanol volume fraction in the ethanol-containing aqueous phase was 25%, the volumeof the ethanol-containing aqueous phase accounted for 15% of the total volume of the biphasic reduction system, the aqueous phase pH was 5.1, the reaction temperature was 50°C, and the reaction time was 3 hours. After completion of the reaction, the system was allowed to stand for phase separation, and the organic phase was taken to obtain a reduced coenzyme Q10 organic phase;

[0051] S7: the reduced coenzyme Q10 organic phase was washed twice with deoxygenated purified water to remove water-soluble salts and residual reducing agents, and the washing endpoint was that the conductivity of the aqueous phase was not higher than 150 pS / cm. Subsequently, solvent was removed at 40°C and under an absolute pressure of about 10 kPa to obtain a reduced coenzyme Q10 concentrate;

[0052] S8: under nitrogen protection, medium-chain triglycerides, sunflower seed phospholipids, phosphatidylserine, ascorbyl palmitate, and mixed tocopherols were added to the reduced coenzyme Q10 concentrate so that the mass ratio of reduced coenzyme Q10, medium-chain triglycerides, sunflower seed phospholipids, and phosphatidyl serine was 1:1.6:0.9:0.12. Stirring and dispersion were performed at 48°C for 40 minutes to obtain a reduced coenzyme Q10 lipid phase;

[0053] S9: y-cyclodextrin, gum arabic, and resistant dextrin were added to deoxygenated purified water and stirred at 45°C for dissolution or dispersion to obtain an aqueous phase. The mass ratio of the reduced coenzyme Q10 lipid phase, y-cyclodextrin, gum arabic, and resistant dextrin was 1:1.0:0.7:0.5. The reduced coenzyme Q10 lipid phase was added to the aqueous phase, sheared at 10000 r / min for 5 minutes, and then subjected to high-pressure homogenization at 35 MPa three times to obtain an emulsion. 0.5% silicon dioxide was added to the emulsion, and spray drying was performed under a condition in which the oxygen concentration contacted by the material was not higher than 5%, with an inlet air temperature of 120°C and an outlet air temperature of 62°C, thereby obtaining a MITO-type mitochondrial uptake-promoting reduced coenzyme Q10raw material.

[0054] Example 2:

[0055] The procedure was substantially the same as that in Example 1, except that:

[0056] in S2, the addition amount of the composite enzyme was 0.60% of the dry-basis mass of the biomass, the enzyme treatment temperature was 46°C, and the treatment time was 2.5 hours; in S3, the volume ratio of ethanol to ethyl acetate was 40:60, the extraction temperature was 35°C, and extraction was performed three times;

[0057] in S6, the addition amount of L-sodium ascorbate was 3.0 times the mass of total coenzyme Q10, the addition amount of ascorbyl palmitate was 2.0% of the mass of total coenzyme Q10, the addition amount of mixed tocopherols was 0.8% of the mass of total coenzyme Q10, the ethanol volume fraction in the ethanol-containing aqueous phase was 20%, the volume of the ethanol-containing aqueous phase accounted for 18% of the total volume of the biphasic system, the aqueous phase pH was 5.0, the reaction temperature was 48°C, and the reaction time was 4 hours;

[0058] in S9, freeze drying was adopted, the pre-freezing temperature was -40°C, the absolute pressure of the drying chamber was about 80 Pa, and the drying time was 28 hours.

[0059] Example 3:

[0060] The procedure was substantially the same as that in Example 1, except that:

[0061] in SI, dry biomass obtained by fermentation of Paracoccus denitrificans was used as the starting material, and the content of total coenzyme Q10 in the dry biomass on a dry basis was 4.86%;

[0062] in S3, the mass ratio of the mixed solvent to the cell-disrupted slurry was 5:1;

[0063] in S6, the addition amount of L-sodium ascorbate was 2.0 times the mass of total coenzyme Q10, the ethanol volume fraction in the ethanol-containing aqueous phase was 30%, the volume of the ethanol-containing aqueous phase accounted for 12% of the total volume of the biphasicsystem, the aqueous phase pH was 5.3, the reaction temperature was 52°C, and the reaction time was 2.5 hours;

[0064] in S8, the mass ratio of reduced coenzyme Q10, medium-chain triglycerides, sunflower seed phospholipids, and phosphatidylserine was 1:1.3:0.7:0.10;

[0065] in S9, the mass ratio of the reduced coenzyme Q10 lipid phase, y-cyclodextrin, gum arabic, and resistant dextrin was 1:0.8:0.6:0.4.

[0066] Comparative Examples:

[0067] Comparative Example 1: compared with Example 1, nitrogen replacement and low-oxygen control were not performed, and the remaining steps were the same;

[0068] Comparative Example 2: compared with Example 1, composite enzyme-assisted cell disruption was not performed, and only high-pressure homogenization cell disruption was adopted, with the remaining steps being the same;

[0069] Comparative Example 3: compared with Example 1, biphasic mild reduction was not performed, and the coenzyme Q10 refined solution was directly subjected to lipid-phospholipid stabilized dispersion and low-oxygen microencapsulation, with the remaining steps being the same;

[0070] Comparative Example 4: compared with Example 1, common ethanol was used as a single extraction solvent, and solvent removal was performed at 60°C, with the remaining steps being the same;

[0071] Comparative Example 5: compared with Example 1, medium-chain triglycerides, sunflower seed phospholipids, and phosphatidylserine were not added, and the reduced coenzyme Q10 concentrate was directly mixed with y-cyclodextrin, gum arabic, and resistant dextrin and then dried, with the remaining steps being the same.

[0072] Detection methods and core calculation formulas:

[0073] Determination of total coenzyme Q10 content and reduced coenzyme Q10 proportion:high-performance liquid chromatography was used to determine the contents of oxidized coenzyme Q10 and reduced coenzyme Q10 in samples. Sample processing was performed under light-shielded and low-oxygen conditions. A C18 reversed-phase chromatographic column was used, the detection wavelength was 275 nm, and the calculation formula for the mass proportion of reduced coenzyme Q10 was as follows:

[0074] ™red =rX 100%

[0075]

[0076] '-'red T Lox

[0077] wherein:

[0078] wred represents the mass proportion of reduced coenzyme Q10 in total coenzyme Q10, %;

[0079] Crecrepresents the mass concentration of reduced coenzyme Q10 in the sample;

[0080] Coxrepresents the mass concentration of oxidized coenzyme Q10 in the sample;

[0081] Based on the total coenzyme Q10 content in the fermentation biomass on a dry basis, the recovery rate of total coenzyme Q10 in the final raw material was calculated, and the calculation formula for the total coenzyme Q10 extraction yield was as follows:

[0082] r = -mV™duct * ™t<>tdl <}W,vrodu« -ioQ%

[0083]

[0084] m biomass dry basis total Q10, biomass

[0085] wherein:

[0086] Y represents the total coenzyme Q10 extraction yield, %;

[0087] Ttlproduct represents the total mass of the finally obtained raw material;

[0088] wtotal Q10, product represents the mass fraction of total coenzyme Q10 in the finished product;

[0089] ^biomass dry basis represents the total dry-basis mass of the starting fermentation biomass;

[0090] total Q10, biomass represents the mass fraction of total coenzyme Q10 in the fermentation biomass on a dry basis.An appropriate amount of the sample was taken, added to purified water at 37°C, gently shaken for dispersion, and then subjected to particle size D50 measurement by dynamic light scattering;

[0091] The total coenzyme Q10 content and surface free coenzyme Q10 content in the sample were measured, and the calculation formula for microcapsule encapsulation efficiency was as follows:

[0092] E=CMalQ10 - CfreeQ10 *lgQ%

[0093]

[0094] C total Q10

[0095] wherein:

[0096] E represents the microcapsule encapsulation efficiency, %;

[0097] C total QW represents the mass content of total coenzyme Q10 in the sample;

[0098] CfreeQIO represents the mass content of surface free coenzyme Q10 in the sample.

[0099] The sample was placed into an aluminum-plastic composite bag, nitrogen-filled and sealed, and stored at 40°C and 75% relative humidity protected from light for 60 days. The reduced coenzyme Q10 proportion and total coenzyme Q10 retention rate were detected on day 0, day 30, and day 60, and the calculation formula for the total coenzyme Q10 retention rate was as follows:

[0100] Wt

[0101] R = — x 100%

[0102] tv0

[0103] wherein:

[0104] R represents the total coenzyme Q10 retention rate, %;

[0105] Wtt represents the mass fraction of total coenzyme Q10 in the sample after storage time t; IVQ represents the mass fraction of total coenzyme Q10 in the initial sample.

[0106] In vitro cell uptake and mitochondrial fraction enrichment test: C2C12 cells were used as an in vitro cell uptake model. The samples of the examples and comparative examples were separately dispersed in a culture medium, and the cells were treated for 4 hours based on the total coenzyme Q10 concentration. After treatment, the cells were collected, and mitochondrialfractions were isolated using a mitochondrial isolation kit. High-performance liquid chromatography was used to detect the total coenzyme Q10 content in whole-cell lysates and mitochondrial fractions. Comparative Example 5 was used as the control group to calculate the relative enrichment fold. The total coenzyme Q10 content in the mitochondrial fractions was normalized to the amount of mitochondrial protein, and the calculation formula for the relative enrichment fold of total coenzyme Q10 in mitochondrial fractions was as follows:

[0107] _ _ Pmitochondria, test sample / ^mitochondria, test sample ^enrichment ~ 77>

[0108] Pmitochondria, control / ^mitochondria, control

[0109] wherein:

[0110] F enrichment represents the relative enrichment fold of total coenzyme Q10 in mitochondrial fractions;

[0111] Pmitochondria, test sample represents the mass concentration of total coenzyme Q10 in the mitochondrial fraction of the test sample group;

[0112] P mitochondria, test sample represents the protein mass concentration of the mitochondrial fraction of the test sample group;

[0113] Pmitochondria, control represents the mass concentration of total coenzyme Q10 in the mitochondrial fraction of the control group;

[0114] F mitochondria, control represents the protein mass concentration of the mitochondrial fraction of the control group.

[0115] Calculation formula for the mitochondria / whole-cell relative distribution index:

[0116] . _ Pmitochondria, test sample I Pwhole cell, test sample 1 distribution ~ 7~

[0117]

[0118] Pmitochondria, control! Pwhole cell, control wherein:

[0119] I distribution represents the mitochondria / whole-cell relative distribution index; Pwhole cell, test sample represents the mass concentration of total coenzyme Q10 in the whole-cell lysate of the test sample group;P whole cell, control represents the mass concentration of total coenzyme Q10 in the whole-cell lysate of the control group.

[0120] The quality of mitochondrial isolation was confirmed using COXIV or TOM20 as a mitochondrial fraction marker protein, and P-actin as a whole-cell protein reference. If obvious cytoplasmic protein contamination occurred in the mitochondrial fraction, the data of that batch of detection were not included in the statistics.

[0121] Evaluation in an in vitro oxidative stress model was performed by establishing an in vitro oxidative stress cell model, and a DCFH-DA fluorescent probe was used to detect the relative level of intracellular reactive oxygen species. The reactive oxygen species level of the model group was set as 100%. This test was used only to evaluate the antioxidant performance of the sample in the in vitro model, and does not represent a therapeutic or preventive effect on human diseases. Unless otherwise specified, the detection results were the average values of three parallel determinations of samples from the same batch.

[0122] Extraction Refined-solution Reduced CoQlO Group

[0123] yield / % CoQlO purity / % after reduction / %

[0124] Example 1 81.9 90.8 89.2

[0125] Example 2 83.4 91.6 90.1

[0126] Example 3 78.8 89.9 88.1

[0127] Comp. Ex. 1 76.5 88.2 72.6

[0128] Comp. Ex. 2 64.8 88.7 86.3

[0129] Comp. Ex. 3 80.1 90.3 58.8

[0130] Comp. Ex. 4 74.6 86.9 66.7

[0131]

[0132] Table 1 Extraction and reduction results

[0133] As can be seen from Table 1, Examples 1-3 had relatively high extraction yields and reduced coenzyme Q10 proportions. In Comparative Example 1, low-oxygen control was not performed, and the reduced coenzyme Q10 proportion was significantly reduced. In Comparative Example 2, composite enzyme-assisted cell disruption was not performed, and the extraction yield decreased. In Comparative Example 3, biphasic mild reduction was not performed, and the reduced coenzyme Q10 proportion was relatively low. In Comparative Example 4, common ethanol was used as a single solvent for extraction and solvent removal was performed at a relatively high temperature, and both the reduced coenzyme Q10 proportion and the extraction yield were lower than those of the examples.

[0134] Total Reduced D50 after water

[0135] Group Moisture / % Encapsulation / % CoQ10 / % CoQ10 / % dispersion / nm

[0136] Example 1 12.1 87.8 3.6 246 90.8

[0137] Example 2 11.9 88.5 3.3 229 91.6

[0138] Example 3 12.6 86.9 3.9 285 89.7

[0139] Comp. Ex. 1 12.0 70.8 3.8 262 89.9

[0140] Comp. Ex. 2 11.3 84.7 3.9 273 88.6

[0141] Comp. Ex. 3 12.3 56.9 3.7 258 90.2

[0142] Comp. Ex. 5 12.5 86.8 4.2 892 71.5

[0143]

[0144] Table 2 Basic indicators of raw material powder

[0145] As can be seen from Table 2, the raw materials obtained in Examples 1-3 had good encapsulation efficiency and water-dispersed particle size. In Comparative Example 5, thelipid-phospholipid stabilized dispersion system was not used, and after water dispersion, the particle size D50 was significantly increased and the encapsulation efficiency decreased;

[0146] Day 0 Day 30 Day 60 Day 60 CoQlO Group

[0147] reduced / % reduced / % reduced / % retention / %

[0148] Example 1 87.8 84.6 80.8 94.6

[0149] Example 2 88.5 85.3 81.5 95.1

[0150] Example 3 86.9 83.5 80.1 93.9

[0151] Comp. Ex. 1 70.8 61.5 52.8 89.7

[0152] Comp. Ex. 3 56.9 48.6 41.2 88.4

[0153] Comp. Ex. 5 86.8 75.6 65.9 86.8

[0154]

[0155] Table 3 Stability test results

[0156] The test conditions for Table 3 were 40°C, 75% relative humidity, nitrogen-filled, protected from light, sealed, and stored for 60 days. As can be seen from Table 3, Examples 1-3 were still able to maintain relatively high reduced coenzyme Q10 proportions under accelerated conditions, indicating that low-oxygen extraction, biphasic mild reduction, low-temperature washing and desalting, and lipid-phospholipid microencapsulation have a synergistic effect on reduced-state retention;

[0157] Whole-cell Mitochondrial Mito / whole-cell Group

[0158] CoQlO (rel.) CoQlO (rel.) index

[0159] Example 1 1.22 1.42 1.16

[0160] Example 2 1.27 1.50 1.18

[0161]

[0162] Whole-cell Mitochondrial Mito / whole-cell Group

[0163] CoQlO (rel.) CoQlO (rel.) index

[0164] Example s 1.18 1.37 1.16

[0165] Comp. Ex. 1 1.01 1.09 1.08

[0166] Comp. Ex. 2 1.13 1.26 1.12

[0167] Comp. Ex. 3 0.84 0.73 0.87

[0168] Comp. Ex. 5 1.00 1.00 1.00

[0169]

[0170] Table 4 In vitro cell uptake and mitochondrial fraction enrichment results The test model in Table 4 was C2C12 cells, the addition concentration of total coenzyme Q10 was 5 pmol / L, and the treatment time was 4 hours, with Comparative Example 5 used as a common reduced coenzyme Q10 microcapsule powder control group. As can be seen from Table 4, under the same addition amount of total coenzyme Q10, the relative content of total coenzyme Q10 in mitochondrial fractions in Examples 1-3 was higher than that in Comparative Example 5, indicating that the lipid-phospholipid stabilized dispersion system adopted by the present invention helps to improve the uptake and mitochondrial fraction enrichment level of reduced coenzyme Q10 in the in vitro cell model;

[0171] Intracellular

[0172] Group Cell viability / %

[0173] ROS / %

[0174] Blank group 44.2 98.3

[0175] Model group 100.0 83.1

[0176] Example 1 65.8 91.6

[0177]

[0178] Intracellular

[0179] Group Cell viability / %

[0180] ROS / %

[0181] Example 2 62.7 92.4

[0182] Example 3 68.4 90.9

[0183] Comp. Ex. 1 78.2 87.9

[0184] Comp. Ex. 2 73.5 88.8

[0185] Comp. Ex. 3 87.6 84.7

[0186] Comp. Ex. 5 82.1 86.3

[0187]

[0188] Table 5 Evaluation results of the in vitro oxidative stress model

[0189] The test model in Table 5 was an EECE-induced in vitro oxidative stress model, and the intracellular reactive oxygen species level of the model group was set as 100%. As can be seen from Table 5, Examples 1-3 were able to reduce the relative intracellular reactive oxygen species level in the in vitro oxidative stress model and maintain a relatively high cell survival rate. This result is consistent with the relatively high reduced coenzyme Q10 proportion and mitochondrial fraction enrichment level thereof.

[0190] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principle and spirit of the present invention, and the scope of the present invention is defined by the appended claims and equivalents thereof.

Claims

CLAIMS1. A low-oxygen extraction and preparation method for a MITO-type mitochondrial uptake-promoting reduced coenzyme Q10 raw material, characterized in that the method comprises the following steps:SI: taking coenzyme QlO-containing fermentation biomass, wherein the fermentation biomass is wet biomass or dry biomass, adding purified water to adjust the solids mass content to 18-35%, and introducing nitrogen for replacement so that the dissolved oxygen of the system is not higher than 1.5 mg / L, thereby obtaining a low-oxygen slurry;S2: adding a composite enzyme to the low-oxygen slurry obtained in step SI, treating the same at pH 6.0-7.2 and 42-52°C for 1-3 hours, and then carrying out high-pressure homogenization for cell disruption to obtain a cell-disrupted slurry, wherein the composite enzyme comprises lysozyme, neutral protease, and lipase, and a mass ratio of the three is l:(0.5-2):(0.1-0.8);S3: adding an ethanol-ethyl acetate mixed solvent to the cell-disrupted slurry obtained in S2, extracting 2-3 times at 30-45 °C under nitrogen protection, and combining organic phases to obtain a coenzyme QlO-containing extract; wherein a volume ratio of ethanol to ethyl acetate is 35:65-55:45, and a mass ratio of the mixed solvent to the cell-disrupted slurry is 4:1-10:1;S4: concentrating the extract obtained in S3 at a temperature not higher than 45°C and under an absolute pressure of 5-20 kPa to obtain a coenzyme Q10 crude extract;S5: dissolving the coenzyme Q10 crude extract obtained in S4 in an ethyl acetate-ethanol mixed solvent, adding neutral alumina and activated carbon for adsorptive impurity removal, and filtering to obtain a coenzyme Q10 refined solution;S6: under nitrogen protection, adding an aqueous L-sodium ascorbate solution, ascorbyl palmitate, mixed tocopherols, and citric acid to the coenzyme Q10 refined solution obtained in S5 to form a biphasic reduction system composed of an ethyl acetate organic phase and anethanol-containing aqueous phase, controlling the aqueous phase at a pH of 4.5-5.8 and a temperature of 40-55°C, and reacting for 1-6 hours so that oxidized coenzyme Q10 is converted into reduced coenzyme Q10; after standing for phase separation, taking the organic phase to obtain a reduced coenzyme Q10 organic phase, wherein the ethanol volume fraction in the ethanol-containing aqueous phase is 10-35%, and the volume of the ethanol-containing aqueous phase accounts for 8-25% of the total volume of the biphasic reduction system;S7: washing the reduced coenzyme Q10 organic phase obtained in S6 with deoxygenated purified water 1-3 times to remove water-soluble salts and residual reducing agents, washing until the conductivity of the aqueous phase is not higher than 150 pS / cm or until the residue of ascorbic acid substances in the aqueous phase is not higher than 0.5 mg / mL, and then removing the solvent at a temperature not higher than 42°C and under an absolute pressure of 5-20 kPa to obtain a reduced coenzyme Q10 concentrate;S8: under nitrogen protection, adding medium-chain triglycerides, plant-derived phospholipids, phosphatidylserine, ascorbyl palmitate, and mixed tocopherols to the reduced coenzyme Q10 concentrate obtained in S7, and stirring and dispersing the same at 40-55°C to obtain a reduced coenzyme Q10 lipid phase, wherein a mass ratio of reduced coenzyme Q10, medium-chain triglycerides, plant-derived phospholipids, and phosphatidylserine is l:(1.0-2.5):(0.5-1.5):(0.05-0.25);S9: dissolving or dispersing y-cyclodextrin, gum arabic, and resistant dextrin in deoxygenated purified water, adding the reduced coenzyme Q10 lipid phase obtained in S8, and after high-speed shearing and high-pressure homogenization, carrying out spray drying or freeze drying under low-oxygen conditions to obtain the MITO-type mitochondrial uptake-promoting reduced coenzyme Q10 raw material;the MITO type refers to a technical form of a mitochondrial uptake-promoting raw material that is formed with non-covalently modified reduced coenzyme Q10 as a core by low-oxygenextraction, biphasic mild reduction, lipid-phospholipid stabilized dispersion, and low-oxygen microencapsulation, wherein the mitochondrial uptake-promoting effect is achieved by the lipid-phospholipid dispersion system and the low-oxygen stabilization process.

2. The low-oxygen extraction and preparation method for a MITO-type mitochondrial uptake-promoting reduced coenzyme Q10 raw material according to claim 1, characterized in that, in SI, the coenzyme QlO-containing fermentation biomass is wet biomass or dry biomass obtained by fermentation of Rhodobacter sphaeroides or Paracoccus denitrificans, and in S2, the high-pressure homogenization cell-disruption pressure is 40-80 MPa and the number of homogenization passes is 1-3.

3. The low-oxygen extraction and preparation method for a MITO-type mitochondrial uptake-promoting reduced coenzyme Q10 raw material according to claim 1, characterized in that, in S5, the addition amount of neutral alumina is 0.5-2.5 times the mass of the coenzyme Q10 crude extract, and the addition amount of activated carbon is 0.02-0.12 times the mass of the coenzyme Q10 crude extract.

4. The low-oxygen extraction and preparation method for a MITO-type mitochondrial uptake-promoting reduced coenzyme Q10 raw material according to claim 1, characterized in that, in S6, the addition amount of L-sodium ascorbate is 0.8-4.0 times the mass of total coenzyme Q10, the addition amount of ascorbyl palmitate is 0.5-3.0% of the mass of total coenzyme Q10, the addition amount of mixed tocopherols is 0.2-1.5% of the mass of total coenzyme Q10, and the addition amount of citric acid is 0.1-0.8% of the mass of total coenzyme Q10.

5. The low-oxygen extraction and preparation method for a MITO-type mitochondrial uptake-promoting reduced coenzyme Q10 raw material according to claim 1, characterized in that, in S8, the plant-derived phospholipid is sunflower seed phospholipid, soybean phospholipid, or a combination thereof, wherein the mass content of phosphatidylcholine is not lower than 40%,and in S9, a mass ratio of the reduced coenzyme Q10 lipid phase, y-cyclodextrin, gum arabic, and resistant dextrin is l:(0.6-1.5):(0.4-1.2):(0.3-1.0).

6. The low-oxygen extraction and preparation method for a MITO-type mitochondrial uptake-promoting reduced coenzyme Q10 raw material according to claim 1, characterized in that, in S9, the high-speed shearing speed is 8000-12000 r / min, the shearing time is 3-8 minutes, the high-pressure homogenization pressure is 25-45 MPa, the number of homogenization passes is 2-4, an inlet air temperature for spray drying is 105-135°C, an outlet air temperature is 55-70°C, and the oxygen concentration contacted by the material during drying is not higher than 5%.

7. A MITO-type mitochondrial uptake-promoting reduced coenzyme Q10 raw material prepared by the low-oxygen extraction and preparation method for MITO-type mitochondrial uptake-promoting reduced coenzyme Q10 according to any one of claims 1-6, characterized in that, in the raw material, a mass proportion of reduced coenzyme Q10 in total coenzyme Q10 is not lower than 85%, a mass content of total coenzyme Q10 is 8-18%, moisture is not higher than 5.0%, and after water dispersion, a particle size D50 is 150-400 nm.

8. The MITO-type mitochondrial uptake-promoting reduced coenzyme Q10 raw material according to claim 7, characterized in that, after the raw material is sealed, protected from light, and stored at 40°C and 75% relative humidity for 60 days, the mass proportion of reduced coenzyme Q10 in total coenzyme Q10 is still not lower than 78%; in an in vitro cell uptake model, calculated based on the same addition amount of total coenzyme Q10, the relative content of total coenzyme Q10 in mitochondrial fractions isolated from the raw-material treatment group is at least 1.30 times that of a common reduced coenzyme Q10 microcapsule powder treatment group that does not contain plant-derived phospholipids and phosphatidyl serine, and the content of total coenzyme Q10 in the mitochondrial fractions is normalized to the amount of mitochondrial protein.

9. The MITO-type mitochondrial uptake-promoting reduced coenzyme Q10 raw material according to claim 7, characterized in that the reduced coenzyme Q10 in the raw material is non-covalently modified reduced coenzyme Q10.

10. Use of the MITO-type mitochondrial uptake-promoting reduced coenzyme Q10 raw material according to any one of claims 7-9 in the preparation of a non-therapeutic nutritional supplement in the form of hard capsules, soft capsules, tablets, granules, or powders.

11. The use according to claim 10, characterized in that the nutritional supplement is a nutritional supplement containing reduced coenzyme Q10.

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