Method for extracting algae oil from phaeodactylum tricornutum and application of algae oil in soothing products

Abstract

The invention relates to a method for extracting algae oil from phaeodactylum tricornutum and application of the method in soothing products in the field of algae oil extraction. The extraction method comprises the following steps: firstly, carrying out fine treatment on algae powder to obtain wall-broken algae powder; extracting wall-broken algae powder by using a mixed solvent formed by absolute ethyl alcohol and ethyl acetate, wherein a microwave-ultrasonic combined reaction system is used for assistance during extraction; performing centrifugal separation on the crude fucoxanthin extract through a centrifugal device to obtain supernate; filtering the supernate through three-stage series membrane filtration equipment to remove impurities, and evaporating by using a rotary evaporator to remove 90% of the solvent, so as to obtain thick dark brown crude extract oil; extracting the crude extract oil by using a methanol aqueous solution and ethyl acetate in sequence to obtain extract liquor; evaporating ethyl acetate in the extract liquor to dryness to obtain a concentrated grease mixture; and finally, refining and separating by using a molecular distillation device to obtain target algal oil, thereby avoiding the degradation of fucoxanthin caused by high temperature. The final peroxide value of the algae oil is easily and effectively reduced.

Description

Technical Field

[0001] The present invention relates to a method for extracting algae oil, and in particular to a method for extracting algae oil from Phaeodactylum tricornutum, which is applied in the field of algae oil extraction, and application of the method in soothing products. Background Art

[0002] Existing algae oil extraction methods mainly include physical, chemical and biotechnology. The mechanical method destroys the algae cell wall through centrifugation, pressing or homogenization to release oil. Among them, the centrifugation method uses density differences to separate oils, which is highly efficient but consumes a lot of energy; the pressing method is suitable for high-oil algae, but the crushing effect is limited. The chemical method often uses organic solvents (such as n-hexane) to extract oils. The operation is simple but there is a risk of solvent residue; the acid-base wall breaking method dissolves the cell wall by adjusting the pH value. It is low-cost but easy to destroy the oil structure. The biological method uses enzymatic hydrolysis or microorganisms to decompose the cell wall. It has the advantages of mild conditions and environmental protection, but the cost of enzyme preparations is relatively high. In recent years, supercritical CO2 extraction technology has attracted attention due to its lack of solvent residue and good selectivity, but its high-pressure equipment investment is relatively large.

[0003] The specification of Chinese invention patent CN111732997B discloses a method for treating microalgae liquid using a membrane bioreactor, including the step of using a membrane bioreactor to demulsify the crushed microalgae fermentation liquid, wherein the pool liquid in the membrane bioreactor contains an immobilized enzyme. In this scheme, the membrane bioreactor is applied to the treatment of the microalgae fermentation liquid, and the immobilized enzyme is used to degrade the protein, polysaccharide and other components in the microalgae liquid, breaking the emulsion to facilitate the separation of microalgae oil. The method has the advantages of continuous use, high processing efficiency, good effect and system stability, and also discloses an algae oil extraction method and apparatus.

[0004] The specification of Chinese invention patent CN118993879B discloses a method for extracting and refining DHA from marine microalgae fermentation broth, which comprises the following steps: enzymatic hydrolysis, extraction, degumming, deacidification, decolorization, and deodorization; enzymatic hydrolysis, mixing Schizochytrium fermentation broth, alkaline protease, and an enzymatic hydrolysis auxiliary agent, adjusting the pH to 8-9, and stirring at 50-60°C for 2.5-3 hours to obtain an enzymatic hydrolysis cell wall-breaking liquid; decolorization, mixing the deacidified DHA crude oil and a decolorizing agent, stirring at room temperature for 30-40 minutes, centrifuging, and taking the supernatant to obtain decolorized DHA crude oil; the invention can improve the cell wall-breaking rate and extraction rate, shorten the enzymatic hydrolysis time, and fully remove the pigment in DHA.

[0005] In the existing extraction process of P. tricornutum algae oil, high temperature or strong chemical conditions often destroy the heat-sensitive active ingredients in the algae oil, such as fucoxanthin. In addition, the extracted algae oil is easily oxidized, resulting in an increase in the peroxide value, affecting its quality and application value. Summary of the Invention

[0006] In response to the above-mentioned existing technologies, the technical problem to be solved by the present invention is that in the existing extraction process of P. tricornutum algae oil, high temperature or strong chemical conditions often destroy the heat-sensitive active ingredients in the algae oil, such as fucoxanthin, and the extracted algae oil is easily oxidized, resulting in an increase in the peroxide value, affecting its quality and application value.

[0007] To solve the above problems, the present invention provides a method for extracting algae oil from Phaeodactylum tricornutum, which specifically comprises the following steps:

[0008] S1, fine processing of algae powder; suspending the algae powder in citric acid-phosphate buffer; adding complex enzyme to the suspension for enzymatic hydrolysis; after enzymatic hydrolysis, heat-treating the algae slurry to inactivate the enzyme, and then cooling it to 10-15 degrees Celsius; breaking the wall of the cooled enzymatically hydrolyzed algae slurry to obtain broken wall algae powder;

[0009] S2, extracting fucoxanthin; using anhydrous ethanol and ethyl acetate mixed in a ratio of 1:1 to form a mixed solvent; transferring the broken algae powder to an extraction device, adding the mixed solvent for extraction; mixing the broken algae powder and the mixed solvent in a solid-liquid ratio of 1:20, and using a microwave-ultrasound combined reaction system to assist in the extraction; and cooling the crude extract in an ice bath to room temperature;

[0010] S3, primary separation of the crude extract; the crude extract is centrifuged for 20 minutes, and the supernatant is collected; the supernatant is passed through a pre-filtration unit, a microfiltration unit, and an ultrafiltration unit in a three-stage tandem membrane filtration device to remove macromolecular colloidal impurities, submicron particles, and biomacromolecular impurities. The filtration process is maintained at a filtration temperature below 15 degrees Celsius and an inert gas is introduced;

[0011] S4, removing impurities and concentrating; using a rotary evaporator to evaporate 90% of the solvent to obtain a thick dark brown crude oil; extracting the crude oil with methanol-water and ethyl acetate in sequence to obtain an extract;

[0012] S5, gentle and efficient concentration; the extract is transferred to a rotary evaporator, and the ethyl acetate is evaporated to obtain a concentrated oil mixture; the concentrated oil mixture is transferred to a molecular distillation device for refined separation to remove thermally unstable impurities and pigment residues to obtain the target algae oil.

[0013] In the above method for extracting algae oil from Phaeodactylum tricornutum, it is easy to effectively reduce the peroxide value of the final algae oil.

[0014] As a further improvement of the present application, the raw material of the triangular kelp used in the production of algae powder is heterotrophically cultured under nitrogen-deficient and high-light conditions, and the algae powder production method is as follows: the algae liquid of the triangular kelp is centrifuged to obtain algae mud, the algae mud is vacuum-freeze-dried to obtain freeze-dried algae mud, and the freeze-dried algae mud is ground to obtain the target algae powder.

[0015] As a further improvement of the present application, the complex enzyme includes: one or more combinations of cellulase, pectinase and β-glucanase, and the dosage of the complex enzyme is 1.5-2.0% of the dry weight of the algae powder; during enzymatic hydrolysis, the cell wall is enzymatically hydrolyzed by gentle shaking at 50 degrees Celsius for 1.5-2.5 hours.

[0016] As a further improvement of the present application, the specific steps of using methanol-water solution and ethyl acetate to extract the crude oil in S4 include: adding n-hexane at 2 times the volume of the crude oil to dissolve it; back-extracting with methanol-water solution, combining the lower methanol-water phase and transferring it into a separatory funnel, adding an equal volume of sodium chloride aqueous solution; extracting the aqueous phase mixture with ethyl acetate 3 times, combining the ethyl acetate-water phase, and obtaining an extract.

[0017] As another improvement of the present application, the working parameters of the microwave-ultrasound combined reaction system for assisted extraction include: microwave power: 200-400W; ultrasonic power is 100-200W; temperature range is controlled at 40±2 degrees Celsius; time is 10-20 minutes.

[0018] As another improved supplement to the present application, the pre-filtration unit includes a stainless steel metal sintered filter element, which is made of 316L stainless steel and has a pore size of 30 μm; the pre-filtration unit is used to remove residual tiny solid particles, flocs and macromolecular colloidal impurities;

[0019] The microfiltration unit includes a tubular ceramic membrane microfiltration component, which is made of alumina ceramic and has a pore size of 0.45 μm. The microfiltration unit is used to efficiently remove submicron particles, partially emulsified oil droplets, bacteria and large-volume colloids.

[0020] The ultrafiltration unit includes an ultrafiltration membrane filter element, the manufacturing material of the ultrafiltration membrane filter element includes polyethersulfone, and the pore size of the ultrafiltration membrane filter element is 0.005-0.02μm; the ultrafiltration unit is used to intercept biological macromolecular impurities with larger molecular weight, while allowing target lipids, fucoxanthin, small molecular pigments and solvents to pass through.

[0021] As another improved supplement to the present application, the pre-filtration unit, the microfiltration unit and the ultrafiltration unit are all installed in a cooling jacket, and an inert gas inlet pipe is passed through the cooling jacket to pass the inert gas into the filter element body.

[0022] As another improvement of the present application, the pre-filtration unit, the microfiltration unit and the ultrafiltration unit all include a filter core body with a protective shell, a partition ring is connected between two adjacent filter core bodies, the input end of the partition ring is installed with an electromagnetic gate valve matching the output end of the filter core body, a gas nozzle connected to the inert gas input pipe is installed in the partition ring 3, and a cooling medium of 5-10 degrees Celsius is introduced into the cooling jacket to maintain the internal temperature of multiple filter core bodies below 15 degrees Celsius.

[0023] As another improvement of the present application, a liquid accumulation detection unit is installed in the partition ring, and the liquid accumulation detection unit includes a pressure detection ring installed in the partition ring. The blockage condition of each filter element body is judged by detecting the pressure change of the pressure detection ring. When the filter element body is blocked, the electromagnetic gate valves on the input and output sides of the filter element body are closed to alarm and inert gas is filled in through the gas nozzle.

[0024] The invention discloses an application of algae oil extracted from Phaeodactylum tricornutum in a soothing product. The algae oil is used in the preparation of the soothing product. The specific operation is as follows: 3% of the algae oil is evenly mixed with 84.5% of water, 5% of propylene glycol, 0.5% of lecithin, 0.5% of tocopherol, 5% of 1.2-hexanediol, 0.5% of p-hydroxyacetophenone and 1% of 1,2-pentanediol according to a weight ratio, and the pH is adjusted to 6.8-7.1 to obtain the soothing product.

[0025] In summary, this scheme, through the synergistic effect of composite enzymatic hydrolysis and low-temperature heat treatment, efficiently destroys the cell wall structure of P. tricornutum under mild conditions, easily improves the dissolution rate of algae oil, and at the same time avoids the degradation of fucoxanthin caused by high temperature; the three-stage membrane filtration combined with the inert gas protection system simultaneously completes the impurity removal and antioxidant treatment in a low-temperature environment, which easily reduces the peroxide value of the final algae oil effectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a process flow chart of the first embodiment of this application;

[0027] Figure 2 A partial three-dimensional diagram of a three-stage series membrane filtration device according to the first and second embodiments of the present application;

[0028] Figure 3 This is a cross-sectional view of a three-stage series membrane filtration device according to the first and second embodiments of the present application;

[0029] Figure 4 for Figure 3 Schematic diagram of the structure at A in the middle;

[0030] Figure 5 This is a schematic diagram of the working state of the three-stage series membrane filtration equipment of the first and second embodiments of this application;

[0031] Figure 6 This is a bar graph of the relative expression levels of the TRPV1 gene in Table 2 in the fourth embodiment of the present application;

[0032] Figure 7 This is a bar graph of the relative expression levels of the HMGB1 gene in Table 4 in the fifth embodiment of the present application;

[0033] Figure 8This is a bar graph of the relative expression levels of the EP2 gene in Table 5 in the fifth embodiment of the present application;

[0034] Figure 9 This is a bar graph of the relative expression levels of the IL-4 gene in Table 6 in the fifth embodiment of the present application.

[0035] Description of the numbers in the figure:

[0036] 1. Cooling jacket; 2. Filter element body; 3. Isolation ring; 31. Solenoid gate valve; 32. Pressure detection ring. DETAILED DESCRIPTION

[0037] The following describes three implementation methods of the present application in detail with reference to the accompanying drawings.

[0038] The first implementation method:

[0039] Figure 1 A method for extracting algae oil from Phaeodactylum tricornutum is shown, which specifically comprises the following steps:

[0040] S1, fine processing of algae powder; suspending the algae powder in citric acid-phosphate buffer; adding a complex enzyme to the suspension for enzymatic hydrolysis; after enzymatic hydrolysis, heat-treating the algae slurry to inactivate the enzymes, and then rapidly cooling it to 10-15 degrees Celsius; the complex enzyme comprises: one or more combinations of cellulase, pectinase, and β-glucanase, with the complex enzyme dosage being 1.5-2.0% of the dry weight of the algae powder; during enzymatic hydrolysis, gently shaking at 50 degrees Celsius for 1.5-2.5 hours to hydrolyze the cell walls;

[0041] The cooled enzymatically hydrolyzed algae slurry is subjected to a wall-breaking treatment to obtain wall-broken algae powder. The raw material of the triangular spherical algae used in the production of the algae powder is heterotrophically cultured under nitrogen-deficient and high-light conditions. The algae powder is produced by centrifuging the triangular spherical algae liquid to obtain algae mud, vacuum freeze-drying the algae mud to obtain freeze-dried algae mud, and grinding the freeze-dried algae mud to obtain the target algae powder.

[0042] S2, extracting fucoxanthin; using anhydrous ethanol and ethyl acetate mixed in a ratio of 1:1 to form a mixed solvent; transferring the broken algae powder to an extraction device, adding the mixed solvent for extraction; mixing the broken algae powder and the mixed solvent in a solid-liquid ratio of 1:20, and using a microwave-ultrasonic combined reaction system to assist in the extraction; the extracted crude extract is cooled to room temperature in an ice bath; the microwave-ultrasonic combined reaction system adopts existing technology, such as: SL-SM series microwave-ultrasonic combined reaction system; microwave power: 200-400W (to maintain low temperature); ultrasonic power: 100-200W (to enhance osmotic mass transfer); temperature range: 40±2 degrees Celsius; time: 10-20 minutes;

[0043] S3, primary separation of the crude extract; the crude extract is centrifuged for 20 minutes, and the upper clear supernatant is collected; the supernatant is filtered and removed by a three-stage tandem membrane filtration device. During filtration, the pre-filtration unit, microfiltration unit, and ultrafiltration unit are sequentially passed through the device to remove macromolecular colloidal impurities, submicron particles, and biomacromolecule impurities. The filtration temperature is maintained below 15 degrees Celsius during the filtration process, and an inert gas is introduced into the three-stage tandem membrane filtration device throughout the filtration process;

[0044] S4, impurity removal and concentration; using a rotary evaporator to evaporate 90% of the solvent to obtain a thick dark brown crude oil; adding n-hexane at a volume twice that of the crude oil to dissolve; back-extraction with methanol-water solution, combining the lower methanol-water phase, transferring it to a separatory funnel, and adding an equal volume of sodium chloride aqueous solution; extracting the aqueous phase mixture three times with ethyl acetate to obtain an extract;

[0045] S5, gentle and efficient concentration; transferring the extract to a rotary evaporator, evaporating the ethyl acetate to obtain a concentrated oil mixture; transferring the concentrated oil mixture to a molecular distillation apparatus for refined separation to remove thermally unstable impurities and residual pigments to obtain the target algae oil;

[0046] Finally, the algae oil is cooled to below 35 degrees Celsius under the protection of inert gas, oxidized by adding natural mixed tocopherols, filtered through a sterilization unit, and then inertly packaged.

[0047] This embodiment realizes the synergistic effect of composite enzymatic hydrolysis and low-temperature heat treatment, efficiently destroys the cell wall structure of P. tricornutum under mild conditions, easily improves the dissolution rate of algae oil, and avoids the degradation of fucoxanthin caused by high temperature; the three-stage membrane filtration is combined with the inert gas protection system to simultaneously complete the impurity removal and antioxidant treatment in a low-temperature environment, which easily makes it possible to effectively reduce the peroxide value of the final algae oil.

[0048] Second implementation method:

[0049] Figure 2-Figure 6 As shown, the pre-filtration unit includes a stainless steel metal sintered filter element, which is made of 316L stainless steel and has a pore size of 30μm; the pre-filtration unit is used to remove residual tiny solid particles, flocs and macromolecular colloidal impurities;

[0050] The microfiltration unit includes a tubular ceramic membrane microfiltration component, which is made of alumina ceramic and has a pore size of 0.45μm. The microfiltration unit is used to efficiently remove submicron particles, partially emulsified oil droplets, bacteria and large-volume colloids.

[0051] The ultrafiltration unit includes an ultrafiltration membrane filter element, which is made of polyethersulfone and has a pore size of 0.005-0.02 μm. The ultrafiltration unit is used to retain large-molecule bio-macromolecule impurities, such as proteins, polysaccharides, and residual cell debris, while allowing target lipids, fucoxanthin, small-molecule pigments, and solvents to pass through.

[0052] The pre-filtration unit, microfiltration unit and ultrafiltration unit are all installed in the cooling jacket 1. The pre-filtration unit, microfiltration unit and ultrafiltration unit all include a filter element filter body 2 with a protective shell; an inert gas input pipe is penetrated in the cooling jacket, and the inert gas input pipe passes the inert gas into the filter element body 2.

[0053] A cooling medium at 5-10 degrees Celsius is passed into the cooling jacket 1 to maintain the internal temperature of multiple filter element bodies 2 below 15 degrees Celsius. The cooling medium includes cooling water. By maintaining the low temperature of the filter element body 2, it is easy to protect the heat-sensitive components in the supernatant and reduce the degradation of heat-sensitive components and the oxidation of oil. The cooling medium passed into the cooling jacket 1 circulates between the inner wall of the cooling jacket 1 and the outer wall of the filter element body 2. The cooling medium is input from the lower part of the cooling jacket 1 and flows out from the upper part. A suitable coolant circulation system in the prior art is used by those skilled in the art to set it up to ensure that the cooling medium of the specified temperature is circulated in the cooling jacket 1.

[0054] A partition ring 3 is connected between two adjacent filter core bodies 2. The input end of the partition ring 3 is equipped with an electromagnetic gate valve 31 that matches the output end of the filter core body 2. A gas nozzle connected to the inert gas input pipe is installed inside the partition ring 3. The gas nozzle is connected to the inert gas input pipe through an electromagnetic three-way valve. The gas nozzle is located on the input side of the next-level filter core body 2; the electromagnetic gate valve 31 can close the output end of the filter core body 2;

[0055] A liquid accumulation detection unit is installed in the partition ring 3, and the liquid accumulation detection unit includes a pressure detection ring 32 installed in the partition ring 3 and the input end of the filter element main body 2. A pressure sensor is installed on the pressure detection ring 32, and the detection end of the pressure detection ring 32 faces the next level. The pressure change detected by the pressure detection ring 32 (when the filter element main body 2 is blocked, liquid accumulates at the input end of the filter element main body 2 and pressurizes the pressure detection ring 32) is used to judge the blockage status of each filter element main body 2. When the filter element main body 2 is blocked, the electromagnetic gate valve 31 on the input and output sides of the filter element main body 2 is closed to alarm and inert gas is filled in through the gas nozzle. After the input and output ends of the filter element main body 2 are closed, inert gas is introduced. At this time, the supernatant is accelerated to be filtered with the assistance of the inert gas flow. After the residual supernatant in the filter element main body 2 is filtered, the electromagnetic gate valve 31 at the output end of the filter element main body 2 is opened to discharge the filtered supernatant. At this time, the technicians can replace or maintain the filter element main body 2.

[0056] This embodiment ensures that the filter element maintenance is carried out after the supernatant is emptied. The outlet gate valve is automatically opened after the filtrate is completely emptied, realizing dry replacement of the filter element, avoiding cross contamination, and ensuring full utilization of the supernatant, which is easy to reduce raw material loss.

[0057] This embodiment achieves efficient and precise impurity removal through three-stage cascade filtration (prefiltration -> microfiltration -> ultrafiltration, low-temperature protection below 15°C, and inert gas protection), improving filtration efficiency and stability, extending membrane life, and ensuring low-temperature protection. Compared to existing membrane tandem filtration devices, this embodiment easily improves filtration efficiency and provides low-temperature protection for the target product during filtration.

[0058] The third implementation method:

[0059] The invention discloses an application of algae oil extracted from brown finger algae in a soothing product. The algae oil is used to prepare a soothing product. The specific operation is as follows: 3% of the algae oil is evenly mixed with 84.5% of water, 5% of propylene glycol, 0.5% of lecithin, 0.5% of tocopherol, 5% of 1.2-hexanediol, 0.5% of p-hydroxyacetophenone and 1% of 1,2-pentanediol according to a weight ratio, and the pH is adjusted to 6.8-7.1 to obtain a soothing product. The soothing product is named Extreme Healing Algae Shield.

[0060] The fourth implementation method:

[0061] Figure 6 Shown, soothing efficacy test-TRPV1 gene relative expression

[0062] Test sample: the sample prepared in the third embodiment (Extremely Effective Algae Shield).

[0063] Experimental methods:

[0064] 1. Immortalized human keratinocytes (HaCaT) were seeded into 6-well plates (3 × 105 cells / well) and incubated at 37°C, 5% CO2 for 24 h. The experimental groups are shown in Table 1:

[0065] Table 1 Experimental grouping table for the fourth implementation method

[0066]

[0067] 2. After incubation, remove the culture medium, wash 1-2 times with D-Hanks, replace with fresh culture medium for the normal control group, replace with fresh culture medium containing the modeling agent (15 μM capsaicin) for the model control group, and replace with fresh culture medium containing the sample and modeling agent (15 μM capsaicin) for the sample group. Continue incubation at 37°C, 5% carbon dioxide for 24 h.

[0068] 3. Total RNA was extracted from each experimental group, cDNA was synthesized, and the gene expression of β-actin and target genes was detected by q-PCR.

[0069] 4. Using β-actin as an internal reference for gene expression, calculate the relative RNA expression of the target gene; the calculation formula is as follows:

[0070] Relative RNA expression = 2 ΔΔC(t) ;

[0071] ΔC(t)=ΔC(t) 模型对照组 -ΔC(t) 样本组 ;

[0072] ΔC(t)=C(t) 目的基因 -C(t) β-acin ;

[0073]

[0074] Where: P: relative expression level of TRPV1 gene.

[0075] Experiment 2: Relative expression of HMGB1, EP2, and IL-4 genes

[0076] The experimental results are shown in Table 2 below.

[0077] Table 2 TRPV1 gene relative expression level comparison table of the third embodiment

[0078]

[0079] The fifth implementation method:

[0080] Figure 7-Figure 9 Shown, soothing efficacy test - relative expression of HMGB1, EP2, and IL-4 genes

[0081] Test sample: the sample prepared in the third embodiment (Extremely Effective Algae Shield).

[0082] Experimental methods:

[0083] 1. Human immortalized keratinocytes were seeded into 6-well plates (3×10 5 cells / well) and incubated at 37° C., 5% CO 2 for 24 h. The experimental groups are shown in Table 3 below.

[0084] Table 3 Experimental grouping table of the fifth embodiment

[0085]

[0086] 2. After incubation, remove the culture medium and gently rinse the cells once or twice with D-Hanks. Replace the culture medium with fresh medium for the normal control group, and then expose the sample group and the model control group to ultraviolet light (80mJ / cm 2 ).

[0087] 3. After modeling, the normal control group and the model control group were replaced with fresh culture medium, and the sample group was added with fresh culture medium containing the sample and incubated at 37°C, 5% carbon dioxide for 24 hours.

[0088] 4. Total RNA was extracted from each experimental group, cDNA was synthesized, and the gene expression of β-actin and target genes was detected by q-PCR.

[0089] 5. Using β-actin as an internal reference for gene expression, calculate the relative RNA expression of the target gene; the calculation formula is as follows:

[0090] Relative RNA expression = 2 ΔΔC(t) ;

[0091] ΔC(t)=ΔC(t) 模型对照组 -ΔC(t) 样本组 ;

[0092] ΔC(t)=C(t) 目的基因 -C(t) β-acin ;

[0093]

[0094] Where: S represents the relative expression level of HMGB1, EP2 or IL-4 gene.

[0095] The relative expression results of the HMGB1 gene in the third embodiment are shown in Table 4 below.

[0096] Table 2 Comparison table of relative expression levels of HMGB1 gene in the fourth embodiment

[0097]

[0098] The relative expression results of the HMGB1 gene in the third embodiment are shown in Table 5 below.

[0099] Table 5 Comparison table of relative expression levels of EP2 gene in the fourth embodiment

[0100]

[0101] The relative expression results of the HMGB1 gene in the third embodiment are shown in Table 6 below.

[0102] Table 6 Comparison table of relative expression levels of IL-4 genes in the third embodiment

[0103]

[0104] Statistical analysis was performed using SPSS 26.0 software, with the independent sample T-test performed between the two groups. A significant difference was determined when α = 0.05 was used as the test threshold, and p < 0.05 (compared with the model control group). Compared with the model control group (NC), the relative expression levels of TRVP1, HMGB1, EP2, and IL-4 genes in the sample group were significantly downregulated, indicating that the sample has a soothing effect.

[0105] The test results showed that the relative expression levels of TRVP1, HMGB1, EP2, and IL-4 genes in the model control group were significantly increased compared to the normal control group, indicating that the stimulation conditions tested were effective. The relative expression levels of TRVP1, HMGB1, EP2, and IL-4 genes in the sample group were significantly decreased compared to the model control group, demonstrating that the third embodiment (Extreme Healing Algae Shield) has a soothing effect.

[0106] In view of current actual needs, the protection scope of the above-mentioned implementation mode adopted in this application is not limited to this. Various changes made within the knowledge scope of technical personnel in this field without departing from the concept of this application still fall within the protection scope of the present invention.

Claims

1. A method for extracting algae oil from Phaeodactylum tricornutum, characterized in that: The specific steps include: S1, fine processing of algae powder; suspending the algae powder in citric acid-phosphate buffer; adding complex enzyme to the suspension for enzymatic hydrolysis; after enzymatic hydrolysis, heat-treating the algae slurry to inactivate the enzyme, and then cooling it to 10-15 degrees Celsius; breaking the wall of the cooled enzymatically hydrolyzed algae slurry to obtain broken wall algae powder; S2, extracting fucoxanthin; using anhydrous ethanol and ethyl acetate mixed in a ratio of 1:1 to form a mixed solvent; transferring the broken algae powder to an extraction device, adding the mixed solvent for extraction; mixing the broken algae powder and the mixed solvent in a solid-liquid ratio of 1:20, and using a microwave-ultrasound combined reaction system to assist in the extraction; and cooling the crude extract in an ice bath to room temperature; S3, primary separation of the crude extract; the crude extract is centrifuged for 20 minutes, and the supernatant is collected; the supernatant is passed through a pre-filtration unit, a microfiltration unit, and an ultrafiltration unit in a three-stage tandem membrane filtration device to remove macromolecular colloidal impurities, submicron particles, and biomacromolecular impurities. The filtration process is maintained at a filtration temperature below 15 degrees Celsius and an inert gas is introduced; S4, removing impurities and concentrating; using a rotary evaporator to evaporate 90% of the solvent to obtain a thick dark brown crude oil; extracting the crude oil with methanol-water and ethyl acetate in sequence to obtain an extract; S5, gentle and efficient concentration; the extract is transferred to a rotary evaporator, and the ethyl acetate is evaporated to obtain a concentrated oil mixture; the concentrated oil mixture is transferred to a molecular distillation device for refined separation to remove thermally unstable impurities and pigment residues to obtain the target algae oil.

2. The method for extracting algae oil from Phaeodactylum tricornutum according to claim 1, wherein: The raw material of the triangular kelp used in the algae powder production is heterotrophically cultured under nitrogen-deficient and high-light conditions. The algae powder production method is as follows: the algae liquid of the triangular kelp is centrifuged to obtain algae mud, the algae mud is vacuum-freeze-dried to obtain freeze-dried algae mud, and the freeze-dried algae mud is ground to obtain the target algae powder.

3. The method for extracting algae oil from Phaeodactylum tricornutum according to claim 1, wherein: The complex enzyme comprises one or more combinations of cellulase, pectinase and beta-glucanase, and the dosage of the complex enzyme is 1.5-2.0% of the dry weight of the algae powder; during enzymatic hydrolysis, the cell wall is enzymatically hydrolyzed by gentle shaking at 50 degrees Celsius for 1.5-2.5 hours.

4. The method for extracting algae oil from Phaeodactylum tricornutum according to claim 1, wherein: The specific steps of extracting the crude oil using methanol-water solution and ethyl acetate in S4 include: adding n-hexane twice the volume of the crude oil to dissolve it; performing back extraction with methanol-water solution, combining the lower methanol-water phase, transferring it into a separatory funnel, and adding an equal volume of sodium chloride aqueous solution; extracting the aqueous phase mixture with ethyl acetate three times, combining the ethyl acetate-water phase, and obtaining an extract.

5. The method for extracting algae oil from Phaeodactylum tricornutum according to claim 1, wherein: The working parameters of the microwave-ultrasound combined reaction system for assisted extraction include: microwave power: 200-400W; ultrasonic power: 100-200W; temperature range controlled at 40±2 degrees Celsius; time: 10-20 minutes.

6. The method for extracting algae oil from Phaeodactylum tricornutum according to claim 1, wherein: The pre-filtration unit includes a stainless steel metal sintered filter element, which is made of 316L stainless steel and has a pore size of 30 μm; the pre-filtration unit is used to remove residual tiny solid particles, flocculent matter and macromolecular colloidal impurities; The microfiltration unit includes a tubular ceramic membrane microfiltration component made of alumina ceramic with a pore size of 0.45 μm. The microfiltration unit is used to efficiently remove submicron particles, partially emulsified oil droplets, bacteria and large-volume colloids; The ultrafiltration unit includes an ultrafiltration membrane filter element, the manufacturing material of the ultrafiltration membrane filter element includes polyethersulfone, and the pore size of the ultrafiltration membrane filter element is 0.005-0.02 μm; the ultrafiltration unit is used to intercept biological macromolecular impurities with larger molecular weights while allowing target lipids, fucoxanthin, small molecular pigments and solvents to pass through.

7. The method for extracting algae oil from Phaeodactylum tricornutum according to claim 1, wherein: The pre-filtration unit, microfiltration unit and ultrafiltration unit are all installed in a cooling jacket (1); the pre-filtration unit, microfiltration unit and ultrafiltration unit all include a filter core body (2) with a protective shell, and multiple inert gas inlet pipes are inserted into the cooling jackets, and the inert gas inlet pipes pass the inert gas into the filter core body (2).

8. The method for extracting algae oil from Phaeodactylum tricornutum according to claim 7, wherein: A partition ring (3) is connected between two adjacent filter core bodies (2), and an electromagnetic gate valve (31) matching the output end of the filter core body (2) is installed at the input end of the partition ring (3). A gas nozzle connected to the inert gas input pipe is installed in the partition ring (3). A cooling medium of 5-10 degrees Celsius is introduced into the cooling jacket (1) to maintain the internal temperature of the multiple filter core bodies (2) below 15 degrees Celsius.

9. The method for extracting algae oil from Phaeodactylum tricornutum according to claim 8, wherein: A liquid accumulation detection unit is installed in the partition ring (3), and the liquid accumulation detection unit includes a pressure detection ring (32) installed in the partition ring (3). The blockage condition of each filter element body (2) is judged by the pressure change detected by the pressure detection ring (32). When the filter element body (2) is blocked, the electromagnetic gate valve (31) on the input and output sides of the filter element body (2) is closed to alarm and inert gas is filled in through the gas nozzle.

10. Use of the algae oil prepared by the method according to any one of claims 1 to 9 in soothing products, characterized in that: It is used in the preparation of a soothing product. The specific operation is: 3% algae oil is evenly mixed with 84.5% water, 5% propylene glycol, 0.5% lecithin, 0.5% tocopherol, 5% 1.2-hexanediol, 0.5% p-hydroxyacetophenone and 1% 1,2-pentanediol according to the weight ratio, and the pH is adjusted to 6.8-7.1 to obtain a soothing product.

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