Fermented vegetable oil and method for preparing the same

Abstract

The application provides a fermented vegetable oil and a preparation method thereof. The preparation method comprises the following steps: crushing a vegetable oil raw material and mixing the same with water to obtain a raw material solution; adding a biological enzyme to the raw material solution to perform enzymolysis, and after enzyme inactivation, an enzymolysis product is obtained; then, a fermentation bacteria is added to perform fermentation, sterilization and centrifugation to obtain an oily liquid; the oily liquid, 1,2-dilauroylphosphatidyl ethanolamine, a self-assembled structure regulator and propylene glycol are uniformly mixed, and then added into water to obtain a liposome suspension of the fermented vegetable oil; 3-thiophene malonic acid and / or flower acid are first added into the liposome suspension of the fermented vegetable oil, and then heparan sulfate and calcium chloride are added, and after reaction, the fermented vegetable oil encapsulated in a carrier is obtained. The preparation method of the fermented vegetable oil has good long-term stability, and can also improve the poor compatibility of the fermented vegetable oil with a water-based system.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cosmetic production, in particular to a fermented plant oil and a preparation method thereof. BACKGROUND

[0002] In recent years, plant oils are increasingly widely used in cosmetics and personal care products, and plant oils are mainly natural high molecular compounds formed by glycerol and fatty acid combination, but due to the complex composition, containing macromolecular free radicals that cannot be fully absorbed by human skin, when plant oils are used as matrix raw materials or additives of cosmetics and personal care products, in order to make plant oils achieve more effective skin protection, it is one of the important ways to use microbial fermentation treatment technology to convert them into small molecules that are easily absorbed by the skin.

[0003] The fermented plant oil is rich in antioxidants, but the content of unsaturated fatty acids is high, and it is easy to be affected by external oxygen, light, high temperature and moisture to cause oxidation reaction, resulting in oil oxidation deterioration, degradation or inactivation of active ingredients, and poor long-term stability.

[0004] In addition, due to the strong hydrophobicity of plant oils, the compatibility with water-based system cosmetics and personal care products is poor, and a large amount of surfactant needs to be added for dispersion, which not only easily causes demulsification, delamination and other phenomena, but also easily causes the problem of sticky skin after use. SUMMARY

[0005] The purpose of the present application is to provide a fermented plant oil and a preparation method thereof, in order to solve the problems of poor long-term stability and poor compatibility with water-based system in the prior art fermented plant oil.

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

[0007] A preparation method of a fermented plant oil, comprising the following steps:

[0008] (1) crushing plant oil raw materials and mixing with water to obtain a raw material liquid;

[0009] (2) adding biological enzymes to the raw material liquid obtained in step (1) to perform enzymolysis and enzyme inactivation, and obtaining an enzymolysis product;

[0010] (3) adding fermentation bacteria to the enzymolysis product obtained in step (2) to perform fermentation, sterilization and centrifugation, and obtaining an oily liquid;

[0011] (4) uniformly mixing the oily liquid obtained in step (3), 1,2-dilauroylphosphatidyl ethanolamine, a self-assembled structure regulator and propylene glycol, and then adding them into water and uniformly mixing to obtain a liposome suspension of the fermented plant oil;

[0012] The self-assembled structure regulator has the following structure:

[0013]

[0014] (5) Adding 3-thiophene malonic acid and / or flower acid into the liposome suspension of the fermented vegetable oil obtained in step (4), and then adding heparan sulfate and calcium chloride into the mixture, and obtaining the fermented vegetable oil encapsulated in the carrier after reaction.

[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 seed.

[0017] Preferably, in step (2), the enzymolysis is carried out at a temperature of 50-55℃ and a pH value of 6-7 for 2-4h;

[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 are one of lactic acid bacteria and yeast bacteria;

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

[0022] Preferably, step (4) specifically comprises: firstly, stirring 1,2-dilauroylphosphatidylethanolamine, a self-assembled structure regulator and propylene glycol uniformly at a temperature of 40-45℃, then adding the oily liquid obtained in step (3) and stirring uniformly, and then adding the mixture into water, stirring at a temperature of 50-55℃ for 30-50min, and then treating ultrasonically at a power of 200-300W for 10-20min, to obtain the liposome suspension of the fermented vegetable oil.

[0023] Preferably, the weight ratio of the oily liquid, 1,2-dilauroylphosphatidylethanolamine, the self-assembled 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 a step of adding methoxy-PEG-N-distearylphosphatidylacetyl amide.

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

[0026] Preferably, the 3-thiophene malonic acid and / or berberic acid is activated 3-thiophene malonic acid and / or berberic acid.

[0027] Optionally, the activation is specifically as follows: the 3-thiophene malonic acid and / or berberic acid is added into a PBS solution with a pH value of 6.0-6.5 together with EDC and NHS, and EDC and NHS are added thereto, and stirring is carried out at a temperature of 20-30℃ for 20-40 min to obtain an activated 3-thiophene malonic acid and / or berberic acid solution.

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

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

[0030] The activated 3-thiophene malonic acid and / or berberic acid solution is first added into the liposome suspension of the fermented vegetable oil obtained in step (4), stirring is carried out at a temperature of 25-35℃ for 30-60 min, the aqueous solution of heparan sulfate is then added thereto, stirring is carried out for 10-30 min, and the aqueous solution of calcium chloride is added dropwise, stirring is carried out at a temperature of 25-35℃ and a rotation speed of 100-200 rpm for 20-60 min to obtain the fermented vegetable oil encapsulated in the carrier.

[0031] Optionally, the volume ratio of the liposome suspension of the fermented vegetable oil, the activated 3-thiophene malonic acid and / or berberic acid solution, the aqueous solution of heparan sulfate and the aqueous solution of calcium chloride 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; and the concentration of the aqueous solution of calcium chloride is 0.1-0.3 mmol / L.

[0033] The application further provides a fermented vegetable oil obtained by the preparation method of the fermented vegetable oil.

[0034] The above scheme of the application at least has the following beneficial effects:

[0035] (1) The preparation method of the fermented vegetable oil 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 to perform enzymolysis, and after enzyme inactivation, an enzymolysis product is obtained; adding a fermentation bacteria to the enzymolysis product to perform fermentation, sterilization and centrifugation, and an oily liquid is obtained; the oily liquid, 1,2-dilauroylphosphatidylethanolamine, a self-assembled structure regulator and propylene glycol are uniformly mixed, and then added into water to obtain a liposome suspension of the fermented vegetable oil; 3-thiophene malonic acid and / or flower acid are first added into the liposome suspension of the fermented vegetable oil, and then heparan sulfate and calcium chloride are added, and after reaction, the fermented vegetable oil encapsulated in a carrier is obtained. The preparation method of the fermented vegetable oil has good long-term stability, and can also improve the poor compatibility of the fermented vegetable oil with a water-based system.

[0036] The oily liquid, 1,2-dilauroylphosphatidylethanolamine, a self-assembled structure regulator and propylene glycol obtained by fermentation are oil phases, which are mixed with an aqueous phase, the 1,2-dilauroylphosphatidylethanolamine is a phospholipid, which can self-assemble to form a liposome after mixing of the oil phase and the aqueous phase, and the oily liquid is wrapped in the liposome. The self-assembled structure regulator has a disulfide bond, and carboxyl, amide and flexible hydrophobic carbon chains are sequentially connected on both sides of the disulfide bond. The carboxyl and amide can be combined with the polar groups of the 1,2-dilauroylphosphatidylethanolamine through hydrogen bonding and electrostatic interaction, 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, so it can rotate limitedly, 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, and the disorderly swinging of the two flexible hydrophobic carbon chains is limited, so as to promote the regularity of the self-assembled structure, and the structure can reversibly deform when subjected to external force, so as to avoid membrane rupture and maintain structural integrity.

[0037] After obtaining the liposome suspension with regular structure, the 3-thiophene malonic acid and / or flower acid can undergo nucleophilic substitution reaction with the amino group of the 1,2-dilauroylphosphatidylethanolamine to generate an amide bond, so as to be embedded in the phospholipid membrane, reduce the membrane fluidity, inhibit aggregation and improve the stability. The phenol of the flower acid also has antioxidant properties, which can inhibit the oxidation of the encapsulated fermented vegetable oil in the liposome.

[0038] After obtaining the liposome with 3-thiophene malonic acid and / or flower acid introduced, the heparan sulfate is added to coat the outside of the liposome, and ion crosslinking is induced by calcium ions to form a tight structure, so as to improve the encapsulation capacity of the liposome, make the fermented vegetable oil more stably encapsulated in the liposome, and reduce leakage. At the same time, the heparan sulfate as a polysaccharide can make the liposome have better hydrophilicity.

[0039] (2) The preparation method of the fermented vegetable oil of the present application, in step (4), further comprises the step of adding methoxy-PEG-N-distearyl phosphatidyl acetic amide. The methoxy-PEG-N-distearyl phosphatidyl acetic amide has distearyl phosphatidyl acetic amide, which can form liposomes together with the 1,2-dilauroyl phosphatidyl ethanolamine. Meanwhile, PEG is a high molecular chain, which can entangle with the heparan sulfate to anchor the heparan sulfate on the surface of the liposome, improve the binding strength of the heparan sulfate on the surface of the liposome, and reduce the shedding of the ion cross-linked layer of the heparan sulfate and calcium ions. Meanwhile, the coating of the ion cross-linked layer of the heparan sulfate will cause the particle size of the liposome to increase, so that the liposome is easy to be broken by external influences. The methoxy group of the methoxy-PEG-N-distearyl phosphatidyl acetic amide will reduce the binding sites of the heparan sulfate on the surface of the liposome, so that the particle size increase of the liposome is reduced, and the influence of the poor stability caused by the increase of the particle size of the liposome is weakened. DETAILED DESCRIPTION

[0040] The specific conditions not mentioned in the embodiments of the present application are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not mentioned by the manufacturer are conventional products that can be obtained by market purchase. Different manufacturers and models of raw materials do not affect the implementation and technical effect of the technical solutions of the present application.

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

[0042] The 1,2-dilauroyl phosphatidyl ethanolamine has a CAS number of 59752-57-7;

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

[0044] The flower cypress acid has a CAS number of 67494-15-9;

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

[0046] The methoxy-PEG-N-distearyl phosphatidyl acetic amide has a CAS number of 178744-28-0;

[0047] The self-assembled structure regulator is a product in the prior art, which can be obtained by market 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, CAS No. 6066-82-6.

[0050] Example 1

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

[0052] (1) crushing a vegetable oil raw material to 40 mesh and mixing it with water to obtain a raw material solution;

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

[0054] (2) adding a biological enzyme to the raw material solution obtained in step (1), and carrying out enzymolysis at a temperature of 50°C and a pH value of 6.5 for 3h, and then obtaining an enzymolysis product after enzyme inactivation;

[0055] The biological enzyme is cellulase; the weight ratio of the biological enzyme to the raw material solution is 1:0.02.

[0056] (3) inoculating a fermentation bacteria into the enzymolysis product obtained in step (2) at an inoculation amount of 3%, and carrying out fermentation at a temperature of 30°C and a pH value of 6.0 for 10h, and then obtaining an oily liquid after sterilization and centrifugation;

[0057] The fermentation bacteria is lactic acid bacteria. Those skilled in the art can select different lactic acid bacteria for fermentation according to actual conditions; the inoculation amount 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 described again).

[0058] (4) first stirring 1,2-dilauroylphosphatidylethanolamine, a self-assembly structure regulator, and propylene glycol uniformly at a temperature of 42°C, then adding the oily liquid obtained in step (3), stirring uniformly, and then adding it into water, stirring at a temperature of 50°C for 40min, and then carrying out ultrasonic treatment at a power of 200W for 20min to obtain a liposome suspension of fermented vegetable oil;

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

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

[0061]

[0062] (5) adding the activated 3-thiophene malonic acid solution into the liposome suspension of the fermented vegetable oil obtained in step (4), stirring for 45 min at a temperature of 25°C, then adding the aqueous solution of heparin sulfate into the mixture, stirring for 10 min, and then adding the aqueous solution of calcium chloride dropwise, stirring for 40 min at a temperature of 30°C and a rotating speed of 100 rpm to obtain the fermented vegetable oil encapsulated in the carrier;

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

[0064] In this embodiment, the activated 3-thiophene malonic acid is obtained by adding the 3-thiophene malonic acid, EDC, and NHS into a PBS solution with a pH value of 6.2, and then adding EDC and NHS into the solution, and stirring for 30 min at a temperature of 20°C to obtain the activated 3-thiophene malonic acid solution.

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

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

[0067] Example 2

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

[0069] (1) crushing a vegetable oil raw material to 80 mesh, and mixing the crushed vegetable oil raw material with water to obtain a raw material solution;

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

[0071] (2) adding a biological enzyme into the raw material solution obtained in step (1), and performing enzymatic hydrolysis at a temperature of 55°C and a pH value of 7 for 4 h, and then obtaining an enzymatic hydrolysate after enzyme inactivation;

[0072] The biological enzyme is neutral protease; and the weight ratio of the biological enzyme to the raw material solution is 1:0.06.

[0073] (3) inoculating the enzyme hydrolysate obtained in step (2) with fermentation bacteria at an inoculation amount of 8%, and carrying out fermentation at a temperature of 35°C and a pH value of 5.8 for 18h, sterilizing and centrifuging to obtain an oily liquid;

[0074] The fermentation bacteria are yeast.

[0075] (4) uniformly stirring 1,2-dilauroylphosphatidylethanolamine, a self-assembly structure regulator and propylene glycol at a temperature of 45°C, adding the oily liquid obtained in step (3) and uniformly stirring, then adding the mixture into water, stirring at a temperature of 55°C for 50min, and ultrasonically treating at a power of 300W for 15min to obtain a liposome suspension of fermented vegetable oil;

[0076] The weight ratio of the oily liquid, 1,2-dilauroylphosphatidylethanolamine, the 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 following structure:

[0078]

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

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

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

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

[0083] Example 3

[0084] The preparation method of the fermented vegetable oil of the present embodiment comprises the following steps:

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

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

[0087] (2) adding biological enzymes to the raw material solution obtained in step (1), and carrying out enzymolysis at a temperature of 52℃ and a pH value of 6 for 2h, and then obtaining an enzymolysis product after enzyme inactivation;

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

[0089] (3) inoculating the enzymolysis product obtained in step (2) with fermentation bacteria at an inoculation amount of 5%, and carrying out fermentation at a temperature of 32℃ and a pH value of 5.5 for 14h, and then obtaining an oily liquid after sterilization and centrifugation;

[0090] The fermentation bacteria are lactic acid bacteria.

[0091] (4) first stirring 1,2-dilauroylphosphatidylethanolamine, a self-assembled structure regulator, and propylene glycol uniformly at a temperature of 40℃, then adding the oily liquid obtained in step (3), stirring uniformly, and then adding it into water, stirring at a temperature of 52℃ for 30min, and then ultrasonic treatment at a power of 250W for 10min, to obtain a liposome suspension of fermented vegetable oil;

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

[0093] The self-assembled structure regulator has the following structure:

[0094]

[0095] (5) first adding an activated 3-thiophene malonic acid solution to the liposome suspension of fermented vegetable oil obtained in step (4), stirring at a temperature of 30℃ for 30min, then adding an aqueous solution of heparan sulfate into it, stirring for 30min, and then adding an aqueous solution of calcium chloride dropwise, stirring at a temperature of 25℃ and a rotation speed of 150rpm for 20min, to obtain fermented vegetable oil encapsulated in a carrier;

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

[0097] In the embodiment, the activated 3-thiophene malonic acid is obtained by the following method: the 3-thiophene malonic acid, EDC and NHS are added into a PBS solution with a pH value of 6.0, EDC and NHS are added into the solution, and the solution is stirred at a temperature of 25℃ for 20 min to obtain the activated 3-thiophene malonic acid solution.

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

[0099] Embodiment 4

[0100] The preparation method of the fermented vegetable oil of the embodiment comprises the following steps:

[0101] (1) The vegetable oil raw material is crushed to 60 mesh and mixed with water to obtain a raw material solution;

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

[0103] (2) The biological enzyme is added into the raw material solution obtained in step (1), and the enzyme is hydrolyzed at a temperature of 55℃ and a pH value of 6 for 3 h, and then the enzyme is inactivated to obtain an enzyme hydrolysis product;

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

[0105] (3) The fermentation bacteria are inoculated into the enzyme hydrolysis product obtained in step (2) according to an inoculation amount of 5%, and the fermentation bacteria are fermented at a temperature of 32℃ and a pH value of 5.8 for 16 h, and then the fermentation bacteria are sterilized and centrifuged to obtain an oily liquid;

[0106] The fermentation bacteria are lactic acid bacteria.

[0107] (4) The 1,2-dilauroylphosphatidylethanolamine, the methoxy-PEG-N-distearylphosphatidyl acetyl amide, the self-assembly structure regulator and the propylene glycol are uniformly stirred at a temperature of 42℃, the oily liquid obtained in step (3) is added, and then the mixture is uniformly stirred, and then the mixture is added into water, and the mixture is stirred at a temperature of 52℃ for 40 min, and then the mixture is ultrasonically treated at a power of 260 W for 15 min to obtain the liposome suspension of the 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-distearylphosphatidylacetic amide to the oily liquid is 0.6:1.

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

[0110]

[0111] (5) The activated 3-thiophene malonic acid and ferulic acid solution is first added to the liposome suspension of the fermented vegetable oil obtained in step (4), stirred at a temperature of 30°C for 40 min, then the heparin sulfate aqueous solution is added, stirred for 20 min, and then the calcium chloride aqueous solution is added dropwise, stirred at a temperature of 30°C and a rotation speed of 180 rpm for 30 min, to obtain the fermented vegetable oil encapsulated in the carrier.

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

[0113] In this embodiment, the activated 3-thiophene malonic acid and ferulic acid are obtained by the following method: the 3-thiophene malonic acid and ferulic acid, EDC, and NHS are added to a PBS solution with a pH value of 6.2, and EDC and NHS are added thereto, stirred at a temperature of 25°C for 30 min, to obtain the activated 3-thiophene malonic acid and ferulic acid solution.

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

[0115] Example 5

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

[0117] Example 6

[0118] The method for producing the fermented vegetable oil of this example is the same as that of Example 4, and the same raw materials are used, except that in step (4), the weight ratio of the methoxy-PEG-N-distearylphosphatidylacetamide to the oily liquid is 0.8: 1.

[0119] Example 7

[0120] The method for producing the fermented vegetable oil of this example is the same as that of Example 4, and the same raw materials are used, except that in step (4), the methoxy-PEG-N-distearylphosphatidylacetamide is not added.

[0121] Example 8

[0122] The method for producing the fermented vegetable oil of this example is the same as that of Example 4, and the same raw materials are used, except that in step (5), the activated 3-thiophene malonic acid and flower acid solution is replaced by 3-thiophene malonic acid. That is, instead of using a mixture of 3-thiophene malonic acid and flower acid as a raw material, an equal amount of 3-thiophene malonic acid is activated and added to step (5).

[0123] Example 9

[0124] The method for producing the fermented vegetable oil of this example is the same as that of Example 4, and the same raw materials are used, except that in step (5), the activated 3-thiophene malonic acid and flower acid solution is replaced by flower acid. That is, instead of using a mixture of 3-thiophene malonic acid and flower acid as a raw material, an equal amount of flower acid is activated and added to step (5).

[0125] Comparative Example 1

[0126] The method for producing the fermented vegetable oil of this example is the same as that of Example 4, and the same raw materials are used, except that in step (4), the 1,2-dilauroylphosphatidyl ethanolamine is replaced by lecithin.

[0127] Comparative Example 2

[0128] The method for producing the fermented vegetable oil of this example is the same as that of Example 4, and the same raw materials are used, except that in step (4), the 1,2-dilauroylphosphatidyl ethanolamine is not added.

[0129] Comparative Example 3

[0130] The method for producing the fermented vegetable oil of this example is the same as that of Example 4, and the same raw materials are used, except that in step (4), the self-assembly structure modifier is not added.

[0131] Comparative Example 4

[0132] The preparation method of the fermented vegetable oil of the present comparative example is the same as that of Example 4, and the same raw materials are used, with the only difference being that in step (4), the self-assembled structure modifier is replaced by N-lauroylsarcosine sodium;

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

[0134] Comparative Example 5

[0135] The preparation method of the fermented vegetable oil of the present comparative example is the same as that of Example 4, and the same raw materials are used, with the only difference being that in step (5), no activated 3-thiophene malonic acid and flower acid solution is added.

[0136] Comparative Example 6

[0137] The preparation method of the fermented vegetable oil of the present comparative example is the same as that of Example 4, and the same raw materials are used, with the only difference being that in step (5), the activated 3-thiophene malonic acid and flower acid solution is replaced by a PBS solution of 3-thiophene malonic acid and flower acid, i.e. unactivated 3-thiophene malonic acid and flower acid.

[0138] Comparative Example 7

[0139] The preparation method of the fermented vegetable oil of the present comparative example is the same as that of Example 4, and the same raw materials are used, with the only difference being that in step (5), no aqueous solution of heparan sulfate is added.

[0140] Comparative Example 8

[0141] The preparation method of the fermented vegetable oil of the present comparative example is the same as that of Example 4, and the same raw materials are used, with the only difference being that step (5) is not included.

[0142] Comparative Example 9

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

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

[0145] Effect Experimental Example

[0146] In order to verify the technical effect of the preparation method of the fermented vegetable oil of the present application, the following tests were conducted:

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

[0148] The encapsulation rate of the fermented vegetable oil encapsulated in the carrier was detected by referring to the method for detecting the encapsulation rate described in Chinese Patent Document CN111840230B, and the results were recorded.

[0149] The fermented vegetable oil encapsulated in the carrier prepared in Examples 1-9 and Comparative Examples 1-9 was placed at the same room temperature for 1 month and 6 months, respectively, and the acid value and peroxide value thereof were measured. Before measuring the acid value and peroxide value, the fermented vegetable oil encapsulated in the carrier was subjected to freeze-thaw cycles 3 times under nitrogen protection (-20℃ freezing, 37℃ thawing), and then was placed in 300W ultrasonic treatment for 5 times, 2min each time, to release the fermented vegetable oil encapsulated in the carrier.

[0150] The fermented vegetable oil encapsulated in the carrier prepared in Examples 1-9 and Comparative Examples 1-9 was centrifuged at 10000rpm, the precipitate was collected and washed, and then was uniformly coated on a glass slide and dried into a thin film. A contact angle measuring instrument was used, 2μL of deionized water was added dropwise on the glass slide, and after stabilization, the water contact angle was measured and photographed, and the average value was calculated based on 5 measurements.

[0151] The results of the experiments 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 prepared by the preparation method of the fermented vegetable oil described in the present application has high encapsulation rate, good long-term stability, and can also improve the problem of poor compatibility with water-based systems.

[0155] According to the results of Example 4 and Examples 8-9, Comparative Examples 5, 6, the 3-thiophene malonic acid and the flower acid have different degrees of influence on the comprehensive performance of the product. Comparative Example 5 without adding 3-thiophene malonic acid and flower acid solution and Comparative Example 6 with adding unactivated 3-thiophene malonic acid and flower acid solution have little change in encapsulation rate and water contact angle, but the stability of acid value and peroxide value of Comparative Example 6 is slightly better, which may be due to the antioxidant effect of flower acid. It can be seen that unactivated and involved in the coupling reaction, 3-thiophene malonic acid and flower acid have little effect on the liposome. Compared with Example 4 with adding activated 3-thiophene malonic acid and flower acid, Example 8 with only adding activated 3-thiophene malonic acid has little change in encapsulation rate and water contact angle, but the stability of acid value and peroxide value decreases. Example 9 with only adding activated flower acid has a decrease in encapsulation rate, and the stability of acid value and peroxide value also significantly decreases. This may be due to the fact that flower acid is a monobasic acid containing rigid polycyclic structure, which can make the liposome locally tightly packed after embedding into the phospholipid film, while 3-thiophene malonic acid is a dibasic acid containing conjugated thiophene ring, which has a certain flexibility between the two carboxyl groups and can form multiple point connections to adjust the intermolecular forces of the phospholipid film and help maintain the stability of the membrane structure. In addition, the thiophene ring has a certain redox activity, which can inhibit the decomposition of peroxide and to a certain extent, play a role in removing free radicals, and cooperate with the antioxidant effect of the flower acid to maintain the long-term stability of the fermented vegetable oil. Therefore, 3-thiophene malonic acid and flower acid can synergistically improve the membrane strength of the liposome, maintain the stability of the membrane, improve the encapsulation rate, and inhibit the oxidation rate and rancidity degree of the fermented vegetable oil.

[0156] According to the results of Example 4 and Comparative Examples 1, 2, the addition of 1,2-dilauroyl phosphatidyl ethanol and the reaction of 3-thiophene malonic acid and flower acid can form liposomes with higher encapsulation rate and good stability after long-term storage.

[0157] According to the results of Example 4 and Comparative Examples 3, 4, the addition of the self-assembled structure regulator can significantly improve the encapsulation rate. In particular, N-lauroylsarcosine sodium used in Comparative Example 4 also has structures such as amide bond and flexible carbon chain, but compared with Comparative Example 3 without adding the self-assembled structure regulator, the comprehensive performance is not much different. It can be seen that the special structure of the self-assembled structure regulator has a significant effect on maintaining the stability of the liposome.

[0158] According to the results of the examples 4 and comparative examples 7-8, it is known that the ion cross-linking layer of heparan sulfate and calcium chloride has a significant effect on the hydrophilicity of the product. The acid value and peroxide value of the liposome suspension without the ion cross-linking layer (comparative example 8) show a rapid decline over time, and the comparative example 7, which only adds heparan sulfate without ion cross-linking, has a certain degree of improvement in the comprehensive performance, but it is not obvious. This may be due to the fact that the heparan sulfate without ion cross-linking cannot be stably coated on the outer layer of the liposome, and it is also difficult to play a good role.

[0159] According to the results of the examples 1-3 and examples 4-7, and comparative example 9, it is known that the addition of methoxy-PEG-N-distearylphosphatidylacetamide is conducive to the coating of the ion cross-linking layer of heparan sulfate on the surface of the liposome, which can improve the encapsulation efficiency of the product, thereby providing a strong guarantee for the long-term stability of the fermented vegetable oil, and significantly inhibiting the oxidation rate and rancidity of the fermented vegetable oil. At the same time, the hydrophilicity of the product is also significantly improved. However, the addition of distearylphosphatidyl ethanolamine-polyethylene glycol-amino (comparative example 9) will cause the long-term stability of the product to deteriorate. It can be seen that the steric shielding effect of the terminal methoxy group helps to form a suitable ion cross-linking layer of heparan sulfate. If the ion cross-linking layer of heparan sulfate is too thick, it will cause the stability of the liposome to decrease, and it is easy to rupture under conditions such as temperature fluctuations and shear force, which will cause the long-term stability of the fermented vegetable oil to deteriorate.

[0160] From the technical common sense, the present application can be realized by other embodiments without departing from the spirit or essential characteristics. Therefore, the above disclosed embodiments are only examples, and are not the only ones. All changes within the scope of the present application or within the scope equivalent to the present application are covered by the present application.

Claims

1. A method for the production of a fermented vegetable oil, characterized in that, It comprises the following steps: (1) crushing the plant oil raw material and mixing it with water to obtain a raw material solution; (2) adding a biological enzyme to the raw material solution obtained in step (1) to perform enzymolysis and enzyme inactivation, thereby obtaining an enzymolysis product; The enzymolysis is performed at a temperature of 50-55℃ and a pH value of 6-7 for 2-4h; the biological enzyme is one or more of cellulase and neutral protease; (3) adding a fermentation bacteria to the enzymolysis product obtained in step (2) to perform fermentation, sterilization and centrifugation, thereby obtaining an oily liquid; The fermentation is performed at a temperature of 30-35℃ and a pH value of 5.5-6.0 for 10-18h according to an inoculation amount of 3-8%; the fermentation bacteria is one of lactic acid bacteria and yeast bacteria; (4) uniformly 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 to obtain a liposome suspension of fermented plant oil; The self-assembly structure regulator has the following structure: ； (5) adding 3-thiophene malonic acid and / or flower acid into the liposome suspension of fermented plant oil obtained in step (4) first, and then adding heparan sulfate and calcium chloride into the mixture, and after reaction, a fermented plant oil encapsulated in a carrier is obtained; The 3-thiophene malonic acid and / or flower acid is activated 3-thiophene malonic acid and / or flower acid; the activation is performed as follows: adding the 3-thiophene malonic acid and / or flower acid, EDC and NHS into a PBS solution with a pH value of 6.0-6.5, adding EDC and NHS into the solution, and stirring at a temperature of 20-30℃ for 20-40min to obtain an activated 3-thiophene malonic acid and / or flower acid solution.

2. The process for the preparation of a fermented vegetable oil according to claim 1, characterized in that, In step (1), the plant oil raw material is crushed to 40-80 mesh; the weight ratio of the plant oil raw material to the water is 1:(5-10).

3. The process for the preparation of a fermented vegetable oil according to claim 2, characterized in that, In step (1), the plant oil raw material is camellia seed.

4. The method of producing a fermented vegetable oil according to claim 1, characterized by, In step (2), the weight ratio of the biological enzyme to the raw material solution is 1:(0.02-0.06).

5. The process for the preparation of a fermented vegetable oil according to claim 1, characterized in that, Step (4) specifically comprises the following steps: uniformly stirring 1,2-dilauroylphosphatidylethanolamine, a self-assembly structure regulator and propylene glycol at a temperature of 40-45℃, adding the oily liquid obtained in step (3) into the mixture, stirring uniformly, then adding the mixture into water, stirring at a temperature of 50-55℃ for 30-50min, and then ultrasonic treating for 10-20min at a power of 200-300W to obtain a liposome suspension of fermented plant oil.

6. The process for the preparation of a fermented vegetable oil according to claim 5, characterized in that, The weight ratio of the oily liquid, 1,2-dilauroylphosphatidylethanolamine, a 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 process for the preparation of a fermented vegetable oil according to claim 6, characterized in that, In step (4), a step of adding methoxy-PEG-N-distearylphosphatidyl acetic amide is further included; The weight ratio of the methoxy-PEG-N-distearylphosphatidyl acetic amide to the oily liquid is (0.5-0.8):

1.

8. The process for the preparation of a fermented vegetable oil according to claim 1, characterized in that, In step (5), the weight ratio of 3-thiophene malonic acid and / or berberine acid, EDC and NHS is 1:(0.5-1.5):(0.5-1.5); and the concentration of 3-thiophene malonic acid and / or berberine acid in the PBS solution is 1-3 mg / ml.

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

10. A fermented vegetable oil, which is obtained by the preparation method of the fermented vegetable oil according to any one of claims 1-9.

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