Fermented vegetable oil and preparation method thereof

Abstract

The invention provides fermented vegetable oil and a preparation method thereof.The preparation method of the fermented vegetable oil comprises the following steps that vegetable oil raw materials are smashed and mixed with water, and a raw material solution is obtained; adding biological enzyme into the raw material liquid, performing enzymolysis and enzyme deactivation to obtain an enzymolysis product, then adding zymophyte, and performing fermentation, sterilization and centrifugation to obtain oily liquid; uniformly mixing the oily liquid, 1, 2-dilauroyl phosphatidyl ethanolamine, a self-assembly structure regulator and propylene glycol, adding the mixture into water, and uniformly mixing to obtain a lipidosome suspension of the fermented vegetable oil; 3-thiophenemalonic acid and / or chamalonic acid are / is added into the lipidosome suspension of the fermented vegetable oil, heparan sulfate and calcium chloride are added into the lipidosome suspension, and the fermented vegetable oil encapsulated in the carrier is obtained after reaction. According to the preparation method of the fermented vegetable oil, the obtained fermented vegetable oil is good in long-term stability, and the problem that the fermented vegetable oil is poor in compatibility with a water-based system can be solved.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetic production, in particular to a fermented vegetable oil and a preparation method thereof. Background Art

[0002] In recent years, vegetable oils have been increasingly used in cosmetics and personal care products. Vegetable oils are primarily natural high-molecular compounds formed by the combination of glycerol and fatty acids. However, due to their complex composition, they contain large molecular free groups that cannot be fully absorbed by human skin. Therefore, when vegetable oils are used as matrix raw materials or additives in cosmetics and personal care products, in order to achieve more effective skin protection, microbial fermentation technology is used to convert them into small molecules that are easily absorbed by the skin, which has become one of the important methods.

[0003] Fermented vegetable oil is rich in antioxidants, but its unsaturated fatty acid content is high, and it is easily affected by external oxygen, light, high temperature, and moisture, causing oxidation reactions, resulting in oxidation and deterioration of the oil, degradation or inactivation of active ingredients, and poor long-term stability.

[0004] In addition, due to the strong hydrophobicity of vegetable oils, they have poor compatibility with water-based cosmetics and personal care products, and require a large amount of surfactants to disperse them. This is not only prone to demulsification and stratification, but also easily leads to sticky skin problems after use. Summary of the Invention

[0005] The purpose of the present invention is to provide a fermented vegetable oil and a preparation method thereof, so as to solve the problems of poor long-term stability and poor compatibility of fermented vegetable oil with water-based systems in the prior art.

[0006] The present invention provides the following technical solutions:

[0007] A method for preparing fermented vegetable oil comprises the following steps:

[0008] (1) crushing the vegetable oil raw material and mixing it with water to obtain a raw material liquid;

[0009] (2) adding a biological enzyme to the raw material liquid obtained in step (1), performing enzymatic hydrolysis and inactivating the enzyme to obtain an enzymatic hydrolysis product;

[0010] (3) adding fermentation bacteria to the enzymatic hydrolysis product obtained in step (2), fermenting, sterilizing, and centrifuging to obtain an oily liquid;

[0011] (4) mixing the oily liquid obtained in step (3), 1,2-dilauroylphosphatidylethanolamine, a self-assembly structure regulator, and propylene glycol, and then adding the mixture into water and mixing the mixture to obtain a liposome suspension of fermented vegetable oil;

[0012] The self-assembly structure regulator has the structure shown below:

[0013]

[0014] (5) First, 3-thiophenemalonic acid and / or ferruvic acid are added to the liposome suspension of the fermented vegetable oil obtained in step (4), and then heparan sulfate and calcium chloride are added thereto. After the reaction, the fermented vegetable oil encapsulated in the carrier is obtained.

[0015] Preferably, in step (1), the vegetable oil raw material is crushed to 40-80 mesh; the weight ratio of the vegetable oil raw material to the water is 1:(5-10);

[0016] Optionally, in step (1), the vegetable oil raw material is camellia seeds.

[0017] Preferably, in step (2), the enzymatic hydrolysis is carried out at a temperature of 50-55°C and a pH of 6-7 for 2-4 hours;

[0018] Optionally, the biological enzyme is one or more of cellulase and neutral protease;

[0019] Optionally, the weight ratio of the biological enzyme to the raw material liquid is 1:(0.02-0.06).

[0020] Preferably, in step (3), the fermentation bacteria is one of lactic acid bacteria and yeast;

[0021] The fermentation is carried out for 10-18 hours at a temperature of 30-35° C. and a pH value of 5.5-6.0 according to an inoculum amount of 3-8%.

[0022] Preferably, step (4) specifically includes: first stirring 1,2-dilauroylphosphatidylethanolamine, a self-assembly structure regulator, and propylene glycol at a temperature of 40-45°C, then adding the oily liquid obtained in step (3), stirring evenly, and then adding it to water, stirring at a temperature of 50-55°C for 30-50 minutes, and then ultrasonically treating at a power of 200-300W for 10-20 minutes to obtain a liposome suspension of fermented vegetable oil.

[0023] Preferably, the weight ratio of the oily liquid, 1,2-dilauroylphosphatidylethanolamine, self-assembly structure regulator, propylene glycol, and water is 1: (1-2.5): (0.3-0.5): (0.2-0.4): (8-12).

[0024] Preferably, step (4) further comprises the step of adding methoxy-PEG-N-distearoylphosphatidyl acetamide;

[0025] The weight ratio of the methoxy-PEG-N-distearoylphosphatidylacetamide to the oily liquid is (0.5-0.8):1.

[0026] Preferably, the 3-thiophenemalonic acid and / or ferrous acid is activated 3-thiophenemalonic acid and / or ferrous acid;

[0027] Optionally, the activation is specifically as follows: adding the 3-thiophenemalonic acid and / or ferrugic acid, EDC, and NHS to a PBS solution with a pH value of 6.0-6.5, adding EDC and NHS thereto, and stirring at a temperature of 20-30° C. for 20-40 minutes to obtain an activated 3-thiophenemalonic acid and / or ferrugic acid solution;

[0028] The weight ratio of the 3-thiophenemalonic acid and / or ferruginic acid, EDC, and NHS is 1:(0.5-1.5):(0.5-1.5); and the concentration of the 3-thiophenemalonic acid and / or ferruginic acid in the PBS solution is 1-3 mg / ml.

[0029] Preferably, step (5) specifically includes:

[0030] The activated 3-thiophenemalonic acid and / or ferruvic acid solution is first added to the liposome suspension of the fermented vegetable oil obtained in step (4), and the mixture is stirred at a temperature of 25-35° C. for 30-60 minutes. An aqueous solution of heparan sulfate is then added thereto, and the mixture is stirred for 10-30 minutes. An aqueous solution of calcium chloride is then added dropwise, and the mixture is stirred at a temperature of 25-35° C. and a rotation speed of 100-200 rpm for 20-60 minutes to obtain the fermented vegetable oil encapsulated in the carrier;

[0031] Optionally, the volume ratio of the fermented vegetable oil liposome suspension, the activated 3-thiophenemalonic acid and / or ferruvic acid solution, the heparan sulfate aqueous solution, and the calcium chloride aqueous solution is 1:(0.1-0.2):(0.5-1.5):(0.5-1.5);

[0032] Optionally, the concentration of the aqueous solution of heparan sulfate is 1-5 mg / ml; the concentration of the aqueous solution of calcium chloride is 0.1-0.3 mmol / L.

[0033] The present invention also provides a fermented vegetable oil obtained by the fermented vegetable oil preparation method.

[0034] The above solution of the present invention includes at least the following beneficial effects:

[0035] (1) The method for preparing fermented vegetable oil of the present invention comprises the following steps: crushing a vegetable oil raw material and mixing it with water to obtain a raw material liquid; adding a biological enzyme to the raw material liquid, performing enzymolysis and inactivating the enzyme to obtain an enzymatic hydrolysis product; adding fermentation bacteria to the enzymatic hydrolysis product, performing fermentation, sterilizing, and centrifuging to obtain an oily liquid; uniformly mixing the oily liquid, 1,2-dilauroylphosphatidylethanolamine, a self-assembly structure regulator, and propylene glycol, and then adding the mixture to water and mixing to obtain a liposome suspension of the fermented vegetable oil; first adding 3-thiophenemalonic acid and / or ferrous acid to the liposome suspension of the fermented vegetable oil, and then adding heparan sulfate and calcium chloride thereto, and reacting to obtain the fermented vegetable oil encapsulated in the carrier. The method for preparing fermented vegetable oil of the present invention can obtain a fermented vegetable oil with good long-term stability and can also improve the problem of poor compatibility with water-based systems.

[0036] The oily liquid obtained through fermentation, 1,2-dilauroylphosphatidylethanolamine, a self-assembly structure regulator, and propylene glycol are the oil phase, which are added to the aqueous phase for mixing. The 1,2-dilauroylphosphatidylethanolamine is used as a phospholipid. After the oil phase and the aqueous phase are mixed, the 1,2-dilauroylphosphatidylethanolamine can self-assemble to form liposomes, wrapping the oily liquid inside. The self-assembly structure regulator has a disulfide bond, and the two sides of the disulfide bond are connected in sequence to a carboxyl group, an amide group, and a flexible hydrophobic carbon chain. The carboxyl group and the amide group can combine with the polar group of the 1,2-dilauroylphosphatidylethanolamine through hydrogen bonds, electrostatic interactions, etc., and the flexible hydrophobic carbon chain can be inserted into the phospholipid membrane structure formed by the 1,2-dilauroylphosphatidylethanolamine. The disulfide bond has a certain rigidity, and the sulfur atom has a rotational energy barrier, which allows limited rotation, so that the two flexible hydrophobic carbon chains can be inserted into the phospholipid membrane structure formed by the 1,2-dilauroylphosphatidylethanolamine at a certain angle, limiting its disordered swinging to promote the regularity of the self-assembled structure. It can also undergo reversible deformation when subjected to external force, avoiding membrane rupture and maintaining structural integrity.

[0037] After obtaining a structurally regular liposome suspension, the 3-thiophenemalonic acid and / or ferruginous acid can undergo a nucleophilic substitution reaction with the amino group of the 1,2-dilauroylphosphatidylethanolamine to form an amide bond, allowing it to embed into the phospholipid membrane, reducing membrane fluidity, inhibiting aggregation, and improving its stability. The phenolic component of the ferruginous acid also has antioxidant properties, inhibiting oxidation of the fermented vegetable oil encapsulated within the liposomes.

[0038] After obtaining liposomes containing 3-thiophenemalonic acid and / or ferruginous acid, the heparan sulfate is added to coat the liposomes. Calcium ions initiate ionic crosslinking to form a compact structure, improving the liposome's encapsulation capacity and allowing the fermented vegetable oil to be more stably encapsulated within the liposomes, reducing leakage. Furthermore, the heparan sulfate, as a polysaccharide, enhances the liposome's hydrophilicity.

[0039] (2) The method for preparing fermented vegetable oil of the present invention, in step (4), further comprises the step of adding methoxy-PEG-N-distearoylphosphatidyl acetamide. The methoxy-PEG-N-distearoylphosphatidyl acetamide has distearoylphosphatidyl acetamide, which can form liposomes together with the 1,2-dilauroylphosphatidylethanolamine. At the same time, PEG is a polymer chain, which can be molecularly entangled with the heparan sulfate, so that the heparan sulfate is anchored on the surface of the liposome, thereby increasing the binding strength of the heparan sulfate on the liposome surface and reducing the shedding of the ionic crosslinking layer formed by the heparan sulfate and calcium ions. At the same time, the coating of the ionic crosslinking layer of the heparan sulfate will cause the particle size of the liposome to increase, making the liposome susceptible to external influences and rupture, and the spatial barrier formed by the methoxy group of the methoxy-PEG-N-distearoylphosphatidyl acetamide will reduce the binding sites of the heparan sulfate with the liposome surface, thereby reducing the increase in the particle size of the liposome and weakening the effect of the deterioration of stability caused by the increase in the particle size of the liposome. DETAILED DESCRIPTION

[0040] In the examples of the present invention, if specific conditions are not specified, the experiments were carried out under conventional conditions or the conditions recommended by the manufacturer. Reagents or instruments used without specifying the manufacturer are all commercially available conventional products. Raw materials of different manufacturers and types do not affect the implementation of the technical solutions of the present invention and the achievement of the technical effects.

[0041] In the following examples, the cellulase and neutral protease were purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd., with a product specification of 10,000 u / g;

[0042] The 1,2-dilauroylphosphatidylethanolamine, CAS number is 59752-57-7;

[0043] The 3-thiophenemalonic acid has a CAS number of 21080-92-2;

[0044] The CAS number of the ferrous acid is 67494-15-9;

[0045] The heparan sulfate (HS) is a polyanionic polysaccharide with a CAS number of 9050-30-0;

[0046] The methoxy-PEG-N-distearoylphosphatidyl acetyl, CAS number is 178744-28-0;

[0047] The self-assembly structure regulator is a product in the prior art and can be obtained by commercial purchase or self-preparation.

[0048] The EDC refers to the carboxylic acid activator 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide;

[0049] The NHS refers to N-hydroxysuccinimide, with a CAS number of 6066-82-6.

[0050] Example 1

[0051] The method for preparing the fermented vegetable oil of this embodiment comprises the following steps:

[0052] (1) crushing the vegetable oil raw material into 40 meshes and mixing it with water to obtain a raw material liquid;

[0053] The weight ratio of the vegetable oil raw material to the water is 1:5; the vegetable oil raw material is camellia seeds.

[0054] (2) adding a biological enzyme to the raw material solution obtained in step (1), performing enzymatic hydrolysis for 3 hours at a temperature of 50° C. and a pH value of 6.5, and then inactivating the enzyme to obtain an enzymatic hydrolysis product;

[0055] Wherein, the biological enzyme is cellulase; the weight ratio of the biological enzyme to the raw material liquid is 1:0.02.

[0056] (3) inoculating fermentation bacteria into the enzymatic hydrolysate obtained in step (2) at an inoculum rate of 3%, fermenting at a temperature of 30° C. and a pH value of 6.0 for 10 hours, sterilizing, and centrifuging to obtain an oily liquid;

[0057] Wherein, the fermentation bacteria are lactic acid bacteria. Those skilled in the art can select different lactic acid bacteria for fermentation according to actual conditions. The inoculation amount herein refers to the percentage of the volume of the inoculated bacteria to the total volume of the fermentation substrate (the same below, which will not be repeated hereafter).

[0058] (4) First, 1,2-dilauroylphosphatidylethanolamine, a self-assembly structure regulator, and propylene glycol are stirred at a temperature of 42° C., and then the oily liquid obtained in step (3) is added and stirred evenly, and then added to water, stirred at a temperature of 50° C. for 40 minutes, and then ultrasonically treated at a power of 200 W for 20 minutes to obtain a liposome suspension of fermented vegetable oil;

[0059] The weight ratio of the oily liquid, 1,2-dilauroylphosphatidylethanolamine, self-assembly structure regulator, propylene glycol and water is 1:1:0.4:0.3:10.

[0060] The self-assembly structure regulator has the structure shown below:

[0061]

[0062] (5) adding the activated 3-thiophenemalonic acid solution to the liposome suspension of the fermented vegetable oil obtained in step (4), stirring at 25° C. for 45 minutes, then adding an aqueous solution of heparan sulfate thereto, stirring for 10 minutes, and then adding an aqueous solution of calcium chloride dropwise, stirring at 30° C. and 100 rpm for 40 minutes to obtain the fermented vegetable oil encapsulated in the carrier;

[0063] The volume ratio of the fermented vegetable oil liposome suspension, the activated 3-thiophenemalonic acid solution, the heparan sulfate aqueous solution, and the calcium chloride aqueous solution is 1:0.1:1.5:0.5; the concentration of the heparan sulfate aqueous solution is 3 mg / ml; and the concentration of the calcium chloride aqueous solution is 0.1 mmol / L.

[0064] In this embodiment, activated 3-thiophenemalonic acid is obtained by the following method: adding the 3-thiophenemalonic acid, EDC, and NHS to a PBS solution with a pH value of 6.2, adding EDC and NHS thereto, and stirring at a temperature of 20° C. for 30 minutes to obtain an activated 3-thiophenemalonic acid solution;

[0065] The weight ratio of the 3-thiophenemalonic acid, EDC, and NHS is 1:0.5:0.5; and the concentration of the 3-thiophenemalonic acid in the PBS solution is 3 mg / ml.

[0066] It should be noted that the purpose of steps (1)-(3) is to obtain fermented camellia seed oil from camellia seeds, and steps (4)-(5) are to better encapsulate the obtained camellia seed oil within liposomes. Steps (1)-(3) can be performed using conventional methods in the prior art. Those skilled in the art may also replace them with other conventional methods or add or remove steps, as long as fermented camellia seed oil can be obtained.

[0067] Example 2

[0068] The method for preparing the fermented vegetable oil of this embodiment comprises the following steps:

[0069] (1) crushing the vegetable oil raw material into 80 meshes and mixing it with water to obtain a raw material liquid;

[0070] The weight ratio of the vegetable oil raw material to the water is 1:8; the vegetable oil raw material is camellia seeds.

[0071] (2) adding a biological enzyme to the raw material solution obtained in step (1), performing enzymatic hydrolysis for 4 hours at a temperature of 55° C. and a pH value of 7, and then inactivating the enzyme to obtain an enzymatic hydrolysis product;

[0072] Wherein, the biological enzyme is neutral protease; the weight ratio of the biological enzyme to the raw material liquid is 1:0.06.

[0073] (3) inoculating fermentation bacteria into the enzymatic hydrolysate obtained in step (2) at an inoculum rate of 8%, fermenting at a temperature of 35° C. and a pH value of 5.8 for 18 hours, sterilizing, and centrifuging to obtain an oily liquid;

[0074] Wherein, the fermentation bacteria is yeast.

[0075] (4) First, 1,2-dilauroylphosphatidylethanolamine, a self-assembly structure regulator, and propylene glycol are stirred at a temperature of 45° C., and then the oily liquid obtained in step (3) is added and stirred evenly, and then added to water, stirred at a temperature of 55° C. for 50 minutes, and then ultrasonically treated at a power of 300 W for 15 minutes to obtain a liposome suspension of fermented vegetable oil;

[0076] The weight ratio of the oily liquid, 1,2-dilauroylphosphatidylethanolamine, self-assembly structure regulator, propylene glycol and water is 1:2.5:0.3:0.2:12.

[0077] The self-assembly structure regulator has the structure shown below:

[0078]

[0079] (5) adding the activated ferruvic acid solution to the liposome suspension of the fermented vegetable oil obtained in step (4), stirring at 35° C. for 60 min, then adding an aqueous solution of heparan sulfate thereto, stirring for 20 min, and then adding an aqueous solution of calcium chloride dropwise, stirring and reacting at 35° C. and 200 rpm for 60 min to obtain the fermented vegetable oil encapsulated in the carrier;

[0080] The volume ratio of the fermented vegetable oil liposome suspension, the activated ferrous acid solution, the heparan sulfate aqueous solution, and the calcium chloride aqueous solution is 1:0.2:1.0:1.5; the concentration of the heparan sulfate aqueous solution is 5 mg / ml; and the concentration of the calcium chloride aqueous solution is 0.3 mmol / L.

[0081] In this embodiment, the activated ferruginic acid is obtained by the following method: adding the ferruginic acid, EDC, and NHS to a PBS solution with a pH value of 6.5, adding EDC and NHS thereto, and stirring at a temperature of 30° C. for 40 minutes to obtain an activated ferruginic acid solution;

[0082] The weight ratio of the ferulate acid, EDC, and NHS is 1:1.0:1.5; and the concentration of the ferulate acid in the PBS solution is 1 mg / ml.

[0083] Example 3

[0084] The method for preparing the fermented vegetable oil of this embodiment comprises the following steps:

[0085] (1) crushing the vegetable oil raw material into 60 mesh and mixing it with water to obtain a raw material liquid;

[0086] The weight ratio of the vegetable oil raw material to the water is 1:10; the vegetable oil raw material is camellia seeds.

[0087] (2) adding a biological enzyme to the raw material solution obtained in step (1), performing enzymatic hydrolysis for 2 h at a temperature of 52° C. and a pH value of 6, and then inactivating the enzyme to obtain an enzymatic hydrolysis product;

[0088] The biological enzyme is a mixture of cellulase and neutral protease in a weight ratio of 1:1; the weight ratio of the biological enzyme to the raw material liquid is 1:0.04.

[0089] (3) inoculating fermentation bacteria into the enzymatic hydrolysate obtained in step (2) at an inoculum rate of 5%, fermenting at a temperature of 32° C. and a pH value of 5.5 for 14 hours, sterilizing, and centrifuging to obtain an oily liquid;

[0090] Wherein, the fermentation bacteria is lactic acid bacteria.

[0091] (4) First, 1,2-dilauroylphosphatidylethanolamine, a self-assembly structure regulator, and propylene glycol are stirred at a temperature of 40° C., and then the oily liquid obtained in step (3) is added and stirred evenly, and then added to water, stirred at a temperature of 52° C. for 30 minutes, and then ultrasonically treated at a power of 250 W for 10 minutes to obtain a liposome suspension of fermented vegetable oil;

[0092] The weight ratio of the oily liquid, 1,2-dilauroylphosphatidylethanolamine, self-assembly structure regulator, propylene glycol and water is 1:1.8:0.5:0.4:8.

[0093] The self-assembly structure regulator has the structure shown below:

[0094]

[0095] (5) adding the activated 3-thiophenemalonic acid solution to the liposome suspension of the fermented vegetable oil obtained in step (4), stirring at 30° C. for 30 min, then adding an aqueous solution of heparan sulfate thereto, stirring for 30 min, and then adding an aqueous solution of calcium chloride dropwise, stirring at 25° C. and 150 rpm for 20 min to obtain the fermented vegetable oil encapsulated in the carrier;

[0096] The volume ratio of the fermented vegetable oil liposome suspension, the activated 3-thiophenemalonic acid solution, the heparan sulfate aqueous solution, and the calcium chloride aqueous solution is 1:0.15:0.5:1.0; the concentration of the heparan sulfate aqueous solution is 1 mg / ml; and the concentration of the calcium chloride aqueous solution is 0.2 mmol / L.

[0097] In this embodiment, activated 3-thiophenemalonic acid is obtained by the following method: adding the 3-thiophenemalonic acid, EDC, and NHS to a PBS solution with a pH value of 6.0, adding EDC and NHS thereto, and stirring at a temperature of 25° C. for 20 minutes to obtain an activated 3-thiophenemalonic acid solution;

[0098] The weight ratio of the 3-thiophenemalonic acid, EDC, and NHS is 1:1.5:1.0; and the concentration of the 3-thiophenemalonic acid in the PBS solution is 2 mg / ml.

[0099] Example 4

[0100] The method for preparing the fermented vegetable oil of this embodiment comprises the following steps:

[0101] (1) crushing the vegetable oil raw material into 60 mesh and mixing it with water to obtain a raw material liquid;

[0102] The weight ratio of the vegetable oil raw material to the water is 1:10; the vegetable oil raw material is camellia seeds.

[0103] (2) adding a biological enzyme to the raw material solution obtained in step (1), performing enzymatic hydrolysis for 3 hours at a temperature of 55° C. and a pH value of 6, and then inactivating the enzyme to obtain an enzymatic hydrolysis product;

[0104] The biological enzyme is cellulase and neutral protease mixed in a weight ratio of 2:1; the weight ratio of the biological enzyme to the raw material liquid is 1:0.06.

[0105] (3) inoculating fermentation bacteria into the enzymatic hydrolysate obtained in step (2) at an inoculum rate of 5%, fermenting at a temperature of 32° C. and a pH value of 5.8 for 16 hours, sterilizing, and centrifuging to obtain an oily liquid;

[0106] Wherein, the fermentation bacteria is lactic acid bacteria.

[0107] (4) First, 1,2-dilauroylphosphatidylethanolamine, methoxy-PEG-N-distearoylphosphatidylacetamide, a self-assembly structure regulator, and propylene glycol are stirred at a temperature of 42° C., and then the oily liquid obtained in step (3) is added and stirred evenly, and then added to water, stirred at a temperature of 52° C. for 40 minutes, and then ultrasonically treated at a power of 260 W for 15 minutes to obtain a liposome suspension of fermented vegetable oil;

[0108] The weight ratio of the oily liquid, 1,2-dilauroylphosphatidylethanolamine, self-assembly structure regulator, propylene glycol, and water is 1:1.8:0.4:0.3:10. The weight ratio of the methoxy-PEG-N-distearoylphosphatidylacetamide to the oily liquid is 0.6:1.

[0109] The self-assembly structure regulator has the structure shown below:

[0110]

[0111] (5) adding the activated 3-thiophenemalonic acid and ferruvic acid solution to the liposome suspension of the fermented vegetable oil obtained in step (4), stirring at 30° C. for 40 min, then adding an aqueous solution of heparan sulfate thereto, stirring for 20 min, and then adding an aqueous solution of calcium chloride dropwise, stirring and reacting at 30° C. and 180 rpm for 30 min to obtain the fermented vegetable oil encapsulated in the carrier;

[0112] The volume ratio of the fermented vegetable oil liposome suspension, the activated 3-thiophenemalonic acid and ferruvic acid solution, the heparan sulfate aqueous solution, and the calcium chloride aqueous solution is 1:0.1:1.0:0.8; the concentration of the heparan sulfate aqueous solution is 3 mg / ml; and the concentration of the calcium chloride aqueous solution is 0.2 mmol / L.

[0113] In this embodiment, activated 3-thiophenemalonic acid and ferruvic acid are obtained by the following method: adding the 3-thiophenemalonic acid and ferruvic acid, EDC, and NHS to a PBS solution with a pH value of 6.2, adding EDC and NHS thereto, and stirring at a temperature of 25° C. for 30 minutes to obtain an activated 3-thiophenemalonic acid and ferruvic acid solution;

[0114] The weight ratio of 3-thiophenemalonic acid to ferrous acid, EDC, and NHS is 1:1.2:1.2; the concentration of 3-thiophenemalonic acid and ferrous acid in the PBS solution is 2 mg / ml. The 3-thiophenemalonic acid and ferrous acid are mixed in a weight ratio of 1:2 to form a mixture.

[0115] Example 5

[0116] The method for preparing the fermented vegetable oil in this embodiment is the same as that in Example 4 and uses the same raw materials. The only difference is that in step (4), the weight ratio of the methoxy-PEG-N-distearoylphosphatidylacetamide to the oily liquid is 0.5:1.

[0117] Example 6

[0118] The method for preparing the fermented vegetable oil in this embodiment is the same as that in Example 4 and uses the same raw materials. The only difference is that in step (4), the weight ratio of the methoxy-PEG-N-distearoylphosphatidylacetamide to the oily liquid is 0.8:1.

[0119] Example 7

[0120] The method for preparing the fermented vegetable oil in this embodiment is the same as that in Example 4 and uses the same raw materials, with the only difference being that in step (4), the methoxy-PEG-N-distearoylphosphatidylacetamide is not added.

[0121] Example 8

[0122] The method for preparing the fermented vegetable oil in this embodiment is the same as that in Example 4 and uses the same raw materials. The only difference is that in step (5), the activated 3-thiophenemalonic acid and ferulate solution is replaced with 3-thiophenemalonic acid. That is, instead of using a mixture of 3-thiophenemalonic acid and ferulate acid as the raw material, only an equal amount of 3-thiophenemalonic acid is used for activation and added to step (5).

[0123] Example 9

[0124] The method for preparing the fermented vegetable oil in this example is the same as that in Example 4 and uses the same raw materials. The only difference is that in step (5), the activated 3-thiophenemalonic acid and ferruginic acid solution is replaced with ferruginic acid. That is, instead of using a mixture of 3-thiophenemalonic acid and ferruginic acid as the raw material, only an equal amount of ferruginic acid is used for activation and added to step (5).

[0125] Comparative Example 1

[0126] The preparation method of the fermented vegetable oil in this comparative example is the same as that in Example 4 and uses the same raw materials, with the only difference being that in step (4), the 1,2-dilauroylphosphatidylethanolamine is replaced by lecithin.

[0127] Comparative Example 2

[0128] The preparation method of the fermented vegetable oil in this comparative example is the same as that in Example 4 and uses the same raw materials, with the only difference being that in step (4), the 1,2-dilauroylphosphatidylethanol is not added.

[0129] Comparative Example 3

[0130] The preparation method of the fermented vegetable oil in this comparative example is the same as that in Example 4 and uses the same raw materials, with the only difference being that in step (4), the self-assembly structure regulator is not added.

[0131] Comparative Example 4

[0132] The preparation method of the fermented vegetable oil in this comparative example is the same as that in Example 4 and uses the same raw materials, with the only difference being that in step (4), the self-assembly structure regulator is replaced by sodium N-lauroyl sarcosinate;

[0133] The CAS number of the sodium N-lauroyl sarcosinate is 137-16-6.

[0134] Comparative Example 5

[0135] The preparation method of the fermented vegetable oil in this comparative example is the same as that in Example 4 and uses the same raw materials. The only difference is that in step (5), the activated 3-thiophenemalonic acid and ferruvic acid solution is not added.

[0136] Comparative Example 6

[0137] The preparation method of the fermented vegetable oil in this comparative example is the same as that in Example 4 and uses the same raw materials. The only difference is that in step (5), the activated 3-thiophenemalonic acid and ferrous acid solution is replaced with a PBS solution of 3-thiophenemalonic acid and ferrous acid, that is, unactivated 3-thiophenemalonic acid and ferrous acid.

[0138] Comparative Example 7

[0139] The preparation method of the fermented vegetable oil in this comparative example is the same as that in Example 4 and uses the same raw materials, with the only difference being that in step (5), the aqueous solution of heparan sulfate is not added.

[0140] Comparative Example 8

[0141] The method for preparing the fermented vegetable oil in this comparative example is the same as that in Example 4 and uses the same raw materials, with the only difference being that step (5) is not included.

[0142] Comparative Example 9

[0143] The preparation method of the fermented vegetable oil in this comparative example is the same as that in Example 4 and uses the same raw materials, with the only difference being that the methoxy-PEG-N-distearoylphosphatidylacetamide in step (4) is replaced by distearoylphosphatidylethanolamine-polyethylene glycol-amino, i.e., DSPE-PEG-NH2.

[0144] The CAS number of the distearoyl phosphatidylethanolamine-polyethylene glycol-amino is 474922-26-4.

[0145] Effect Experiment Example

[0146] In order to verify the technical effect of the method for preparing fermented vegetable oil according to the present invention, the following experiments were conducted:

[0147] According to the preparation methods of fermented vegetable oil in Examples 1-9 and Comparative Examples 1-9, fermented vegetable oil encapsulated in a carrier was prepared, and the following experiments were conducted:

[0148] The encapsulation efficiency of the fermented vegetable oil encapsulated in the carrier was detected with reference to the method for detecting the encapsulation efficiency described in Chinese patent document CN111840230B, and the results were recorded.

[0149] The fermented vegetable oils encapsulated in the carriers prepared in Examples 1-9 and Comparative Examples 1-9 were stored at the same room temperature for 1 month and 6 months, respectively, and their acid and peroxide values ​​were measured. Before measuring the acid and peroxide values, the fermented vegetable oils encapsulated in the carriers were subjected to three freeze-thaw cycles (freezing at -20°C and thawing at 37°C) under nitrogen protection, followed by five 2-min ultrasonic treatments at 300W to release the fermented vegetable oil encapsulated in the carriers.

[0150] The fermented vegetable oil encapsulated in the carrier prepared in Examples 1-9 and Comparative Examples 1-9 was centrifuged at 10,000 rpm, the precipitate was collected and washed, and evenly coated on a glass slide and dried into a thin film. Using a contact angle meter, 2 μL of deionized water was added to the glass slide. After stabilization, the water contact angle was measured and photographed. The water contact angle was measured 5 times, and the average value was calculated.

[0151] After testing, the results are as follows:

[0152]

[0153]

[0154] According to the results of Examples 1-9 and Comparative Examples 1-9, the fermented vegetable oil encapsulated in the carrier obtained by the method for preparing fermented vegetable oil of the present invention has a high encapsulation rate and good long-term stability, and can also improve its poor compatibility with water-based systems.

[0155] According to the results of Example 4, Example 8-9, Comparative Examples 5 and 6, the 3-thiophenemalonic acid and halogenated acid also have different degrees of influence on the comprehensive performance of the product. Comparative Example 5, in which no 3-thiophenemalonic acid and halogenated acid solution are added, and Comparative Example 6, in which unactivated 3-thiophenemalonic acid and halogenated acid solution are added, have little change in terms of encapsulation efficiency and water contact angle, but the stability shown by the acid value and peroxide value is slightly better in Comparative Example 6, which may be due to the antioxidant effect of halogenated acid. It can be seen that without activation and participation in the coupling reaction, 3-thiophenemalonic acid and halogenated acid are almost difficult to affect liposomes. Compared with Example 4, in which activated 3-thiophenemalonic acid and halogenated acid are added, Example 8, in which only activated 3-thiophenemalonic acid is added, has little change in terms of encapsulation efficiency and water contact angle, but the stability shown by the acid value and peroxide value decreases. In Example 9, in which only activated halogenated acid is added, the encapsulation efficiency decreases, and the stability shown by the acid value and peroxide value also decreases significantly. This may be due to the fact that cypermethrin is a monoprotic acid containing a rigid multi-ring structure. After being embedded in the phospholipid membrane, it can make the liposomes locally tightly packed, while 3-thiophenemalonic acid is a diprotic acid containing a conjugated thiophene ring. There is a certain flexibility between the dicarboxyl groups, which can form a multi-point connection, adjust the intermolecular force of the phospholipid membrane, and assist in maintaining the stable membrane structure. In addition, the thiophene ring has a certain redox activity, which can inhibit the decomposition of peroxides and play a role in scavenging free radicals to a certain extent. It works in conjunction with the antioxidant effect of the cypermethrin to maintain the long-term stability of the fermented vegetable oil. Therefore, 3-thiophenemalonic acid and cypermethrin can work together to improve the membrane strength of the liposome, maintain membrane stability, improve the encapsulation efficiency, and inhibit the oxidation rate and rancidity of the fermented vegetable oil.

[0156] According to the results of Example 4 and Comparative Examples 1 and 2, the addition of the 1,2-dilauroylphosphatidylethanol and the reaction with the 3-thiophenemalonic acid and the ferrous acid can form liposomes that can achieve a higher encapsulation rate and maintain good stability under long-term storage.

[0157] The results of Example 4 and Comparative Examples 3 and 4 demonstrate that the addition of the self-assembly structure modifier significantly improves the encapsulation efficiency. In particular, the sodium N-lauroyl sarcosinate used in Comparative Example 4, which also has an amide bond and a flexible carbon chain structure, exhibits comparable overall performance compared to Comparative Example 3, which lacks the self-assembly structure modifier. This suggests that the unique structure of the self-assembly structure modifier significantly contributes to the stability of liposomes.

[0158] According to the results of Example 4 and Comparative Examples 7-8, the ion-crosslinked layer of heparan sulfate and calcium chloride has a significant impact on the hydrophilicity of the product. The stability of the liposome suspension (Comparative Example 8) without an ion-crosslinked layer, its acid value and peroxide value, showed a rapid decline over time. However, in Comparative Example 7, which only added the heparan sulfate and did not undergo ion-crosslinking, although the overall performance improved to a certain extent, it was not obvious. This may be because the heparan sulfate without ion-crosslinking cannot be stably coated in the outer layer of the liposome, and is difficult to achieve a good effect.

[0159] According to the result of embodiment 1-3 and embodiment 4-7, comparative example 9, the addition of the methoxyl group-PEG-N-distearoylphosphatidyl acetamide is conducive to the coating of the ion crosslinking layer of the heparan sulfate on the surface of the liposome, and the encapsulation efficiency of the product can be improved, thereby providing a strong guarantee for the long-term stability of the fermented vegetable oil, significantly suppressing the oxidation rate, the degree of sourness of the fermented vegetable oil. Meanwhile, the hydrophilicity of the product is also significantly improved. However, the addition of the distearoylphosphatidylethanolamine-polyethylene glycol-amino group (comparative example 9) can cause the long-term stability of the product to deteriorate, as seen, the spatial shielding effect of the terminal methoxyl group helps to form the ion crosslinking layer of the suitable heparan sulfate, and the ion crosslinking layer of the heparan sulfate is too thick, which can cause the stability of the liposome to decline, and is easily broken in the case of temperature fluctuation, shearing force, etc., causing the long-term stability of the fermented vegetable oil to deteriorate.

[0160] It is understood from common knowledge in the art that the present invention may be implemented by other embodiments that do not depart from its spirit or essential features. Therefore, the embodiments disclosed above are, in all respects, illustrative only and not exclusive. All modifications within the scope of the present invention or equivalent to the scope of the present invention are intended to be encompassed by the present invention.

Claims

1. A method for preparing fermented vegetable oil, characterized in that: The steps include: (1) crushing the vegetable oil raw material and mixing it with water to obtain a raw material liquid; (2) adding a biological enzyme to the raw material liquid obtained in step (1), performing enzymatic hydrolysis and inactivating the enzyme to obtain an enzymatic hydrolysis product; (3) adding fermentation bacteria to the enzymatic hydrolysis product obtained in step (2), fermenting, sterilizing, and centrifuging to obtain an oily liquid; (4) mixing the oily liquid obtained in step (3), 1,2-dilauroylphosphatidylethanolamine, a self-assembly structure regulator, and propylene glycol, and then adding the mixture into water and mixing the mixture to obtain a liposome suspension of fermented vegetable oil; The self-assembly structure regulator has the structure shown below: (5) First, 3-thiophenemalonic acid and / or ferruvic acid are added to the liposome suspension of the fermented vegetable oil obtained in step (4), and then heparan sulfate and calcium chloride are added thereto. After the reaction, the fermented vegetable oil encapsulated in the carrier is obtained.

2. The method for preparing fermented vegetable oil according to claim 1, wherein In step (1), the vegetable oil raw material is crushed to 40-80 mesh; the weight ratio of the vegetable oil raw material to the water is 1: (5-10); Optionally, in step (1), the vegetable oil raw material is camellia seeds.

3. The method for preparing fermented vegetable oil according to claim 1, wherein In step (2), the enzymatic hydrolysis is carried out at a temperature of 50-55° C. and a pH of 6-7 for 2-4 hours; Optionally, the biological enzyme is one or more of cellulase and neutral protease; Optionally, the weight ratio of the biological enzyme to the raw material liquid is 1:(0.02-0.06).

4. The method for preparing fermented vegetable oil according to claim 1, wherein In step (3), the fermentation bacteria is one of lactic acid bacteria and yeast; The fermentation is carried out for 10-18 hours at a temperature of 30-35° C. and a pH value of 5.5-6.0 according to an inoculum amount of 3-8%.

5. The method for preparing fermented vegetable oil according to claim 1, wherein The step (4) specifically comprises: firstly stirring 1,2-dilauroylphosphatidylethanolamine, a self-assembly structure regulator, and propylene glycol at a temperature of 40-45° C., then adding the oily liquid obtained in step (3), stirring evenly, then adding it to water, stirring at a temperature of 50-55° C. for 30-50 minutes, and then ultrasonically treating at a power of 200-300W for 10-20 minutes to obtain a liposome suspension of fermented vegetable oil.

6. The method for preparing fermented vegetable oil according to claim 5, characterized in that: The weight ratio of the oily liquid, 1,2-dilauroylphosphatidylethanolamine, self-assembly structure regulator, propylene glycol and water is 1: (1-2.5): (0.3-0.5): (0.2-0.4): (8-12).

7. The method for preparing fermented vegetable oil according to claim 6, wherein: Step (4) further includes the step of adding methoxy-PEG-N-distearoylphosphatidyl acetamide; The weight ratio of the methoxy-PEG-N-distearoylphosphatidylacetamide to the oily liquid is (0.5-0.8):

1.

8. The method for preparing fermented vegetable oil according to claim 1, wherein In step (5), the 3-thiophenemalonic acid and / or ferulate acid is activated 3-thiophenemalonic acid and / or ferulate acid; Optionally, the activation is specifically as follows: adding the 3-thiophenemalonic acid and / or ferrugic acid, EDC, and NHS to a PBS solution with a pH value of 6.0-6.5, adding EDC and NHS thereto, and stirring at a temperature of 20-30° C. for 20-40 minutes to obtain an activated 3-thiophenemalonic acid and / or ferrugic acid solution; The weight ratio of the 3-thiophenemalonic acid and / or ferruginic acid, EDC, and NHS is 1:(0.5-1.5):(0.5-1.5); and the concentration of the 3-thiophenemalonic acid and / or ferruginic acid in the PBS solution is 1-3 mg / ml.

9. The method for preparing fermented vegetable oil according to claim 8, characterized in that: Step (5) specifically includes: The activated 3-thiophenemalonic acid and / or ferruvic acid solution is first added to the liposome suspension of the fermented vegetable oil obtained in step (4), and the mixture is stirred at a temperature of 25-35° C. for 30-60 minutes. An aqueous solution of heparan sulfate is then added thereto, and the mixture is stirred for 10-30 minutes. An aqueous solution of calcium chloride is then added dropwise, and the mixture is stirred at a temperature of 25-35° C. and a rotation speed of 100-200 rpm for 20-60 minutes to obtain the fermented vegetable oil encapsulated in the carrier; Optionally, the volume ratio of the fermented vegetable oil liposome suspension, the activated 3-thiophenemalonic acid and / or ferruvic acid solution, the heparan sulfate aqueous solution, and the calcium chloride aqueous solution is 1:(0.1-0.2):(0.5-1.5):(0.5-1.5); Optionally, the concentration of the aqueous solution of heparan sulfate is 1-5 mg / ml; the concentration of the aqueous solution of calcium chloride is 0.1-0.3 mmol / L.

10. A fermented vegetable oil obtained by the method for preparing a fermented vegetable oil according to any one of claims 1 to 9.

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