Preparation method of white tomato exosome freeze-dried powder

Abstract

The invention relates to the technical field of freeze-dried powder preparation, and discloses a white tomato exosome freeze-dried powder preparation method, which comprises: 1, manually picking white tomatoes 3-5 days after a color changing period, carrying out double-frequency ultrasonic cleaning, and pre-cooling in a 4 DEG C phosphate buffer solution containing 0.1% of vitamin C; according to the method, by virtue of an ultrasonic-cellulase synergistic crushing process, cells are mildly and efficiently crushed, so that the damage to an exosome structure is reduced, and the release efficiency and activity of the exosome are improved; the zebra fish embryo melanogenesis can be obviously inhibited, and the whitening and freckle-removing effects are relatively good.

Description

Technical Field

[0001] The present invention relates to the technical field of freeze-dried powder preparation, and specifically to a method for preparing freeze-dried powder of white tomato exosomes. Background Art

[0002] In the fields of cosmetics and biomedicine, whitening and freckle-removing products have always attracted much attention. Traditional whitening and freckle-removing ingredients such as hydroquinone and kojic acid, although having certain whitening effects, have problems such as cytotoxicity and strong irritation. Long-term use may cause damage to human skin and even trigger adverse reactions such as allergies and inflammation. In recent years, natural-source whitening ingredients have gradually become a research hotspot, but some natural ingredients have deficiencies such as weak whitening efficacy and poor stability, making it difficult to meet the market demand for highly efficient and safe whitening products.

[0003] At the same time, for the safety evaluation of products, non-irritation is one of the important indicators. In terms of reducing irritation of existing products, methods such as adding chemically synthesized anti-inflammatory ingredients or adjusting the pH value of the formula are mostly used. However, these methods may introduce new safety risks and cannot guarantee the mildness of the products at the root. Therefore, developing a product that can efficiently whiten and remove freckles has become an urgent problem in the industry. Summary of the Invention

[0004] Aiming at the deficiencies of the existing technology, the present invention provides a method for preparing freeze-dried powder of white tomato exosomes, which has good whitening and freckle-removing functions by optimizing each preparation link.

[0005] To achieve the above object, the present invention provides the following technical solution: A method for preparing freeze-dried powder of white tomato exosomes, comprising the following steps: Step 1: Use white tomatoes, manually pick them 3 - 5 days after the color-changing period, pre-cool them in a 4°C phosphate buffer solution containing 0.1% vitamin C after double-frequency ultrasonic cleaning; Step 2: Adopt an ultrasonic - cellulase synergistic fragmentation process, set the ultrasonic power at 300W / cm 2 , with a pulse mode of a cycle period of 2 seconds of working time and 3 seconds of intermittent time, and at the same time add 0.5% pectinase and 0.3% β-glucanase, and carry out cell wall decomposition for 30 minutes under the protection of inert gas; Step 3: Through a three-stage gradient centrifugation system, first use a gravitational acceleration of 300 times to remove cell debris, second use a gravitational acceleration of 12,000 times to enrich exosomes, and third use a gravitational acceleration of 160,000 times for ultra-high-speed centrifugation purification. Keep the temperature at 4°C throughout the process and cooperate with CO2 for temperature control; Step 4: Adopt a nano-filtration membrane-coated ultrafiltration concentration technology, select a 100kDa tangential flow filtration device, and control the transmembrane pressure ≤ 0.5 bar; Step 5: Prepare a trehalose-mannitol-polyethylene glycol composite lyoprotectant, mix it with the exosome suspension at a volume ratio of 1:3, and then perform a multi-stage freeze-drying process: cool down to -50°C at a rate of 5°C / min in the pre-freezing stage, maintain at -35°C for 8 h in the primary drying stage, and gradually heat up to 25°C in the secondary drying stage.

[0006] Preferably, for the dual-frequency ultrasonic cleaning in Step 1, the two frequencies are 28 kHz and 40 kHz respectively, and the cleaning time is 10 - 15 minutes.

[0007] Preferably, for the phosphate buffer solution in Step 1, its pH value is 7.2 - 7.4, and the pre-cooling treatment time is 20 - 30 minutes.

[0008] Preferably, the inert gas in Step 2 is nitrogen. Before cell wall decomposition, nitrogen is filled into the reaction system to make the nitrogen content in the system reach more than 95%.

[0009] Preferably, for the ultrasonic - cellulase synergistic fragmentation process in Step 2, the addition amount of cellulase is 0.2% - 0.4% of the mass of white tomatoes.

[0010] Preferably, for the three-stage gradient centrifugation system in Step 3, after each centrifugation, the substances in the centrifuge tube are washed with a phosphate buffer solution at 4°C for 2 - 3 times.

[0011] Preferably, for the nano-filtration membrane-coated ultrafiltration concentration technology in Step 4, before ultrafiltration concentration, the nano-filtration membrane is pretreated. The pretreatment method is to rinse it with deionized water for 3 - 5 times, then soak it in a 0.1% sodium hydroxide solution for 1 - 2 hours, and finally rinse it with deionized water until neutral.

[0012] Preferably, for the trehalose-mannitol-polyethylene glycol composite lyoprotectant in Step 5, the mass ratio of trehalose, mannitol, and polyethylene glycol is 2:1:1.

[0013] Preferably, for the gradient heating in the secondary drying in Step 5, the heating rate is 0.5°C / min.

[0014] Compared with the prior art, the present invention provides a method for preparing freeze-dried powder of white tomato exosomes, which has the following beneficial effects: Through the ultrasonic - cellulase synergistic fragmentation process, the present invention gently and efficiently breaks cells, reduces the damage to the exosome structure, improves the release efficiency and activity of exosomes, and can significantly inhibit the melanogenesis of zebrafish embryos, proving its good efficacy in whitening and freckle removal. Detailed implementation mode

[0015] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0016] A preparation method of white tomato exosome freeze-dried powder includes the following steps: Step 1: Use white tomatoes, manually pick them 3 - 5 days after the color-changing period, pre-cool them in a 4°C phosphate buffer solution containing 0.1% vitamin C after double-frequency ultrasonic cleaning; Step 2: Adopt an ultrasonic - cellulase synergistic fragmentation process, set the ultrasonic power at 300W / cm 2 、The pulse mode is a cycle of working time of 2 seconds and intermittent time of 3 seconds. At the same time, add 0.5% pectinase and 0.3% β-glucanase, and carry out cell wall decomposition for 30 minutes under the protection of inert gas; Step 3: Through a three-stage gradient centrifugation system, first use a gravitational acceleration of 300 times to remove cell debris, second use a gravitational acceleration of 12,000 times to enrich exosomes, and third use a gravitational acceleration of 160,000 times for ultra-high speed centrifugation purification. Keep the temperature at 4°C throughout the process and cooperate with CO2 for temperature control; Step 4: Adopt a nano-filtration membrane-coated ultrafiltration concentration technology, select a 100kDa tangential flow filtration device, and control the transmembrane pressure ≤ 0.5 bar; Step 5: Prepare a trehalose - mannitol - polyethylene glycol composite freeze-drying protective agent, mix it with the exosome suspension in a volume ratio of 1:3, and perform a multi-stage freeze-drying procedure: cool down to -50°C at 5°C / min in the pre-freezing stage, maintain at -35°C for 8h in the primary drying stage, and gradually heat up to 25°C in the secondary drying stage.

[0017] For the double-frequency ultrasonic cleaning in Step 1, the two frequencies are 28kHz and 40kHz respectively, the cleaning time is 10 - 15 minutes, and during the cleaning process, the volume ratio of white tomatoes to the cleaning solution is 1:3 - 1:5. The cleaning solution is deionized water, and continuous stirring is carried out during the cleaning process, with the stirring speed being 100 - 150 revolutions per minute. Among them, the 28kHz ultrasonic wave can generate larger cavitation bubbles, which helps to remove larger dirt particles on the surface of white tomatoes; the cavitation bubbles generated by the 40kHz ultrasonic wave are smaller and can penetrate into the tiny pores on the surface of white tomatoes to achieve more thorough cleaning. The role of stirring is to make white tomatoes fully contact with the cleaning solution, improve the cleaning efficiency, and prevent incomplete local cleaning; The phosphate buffer solution in Step 1 has a pH value of 7.2 - 7.4. The phosphate buffer solution is prepared from sodium dihydrogen phosphate and disodium hydrogen phosphate, and the molar ratio of sodium dihydrogen phosphate to disodium hydrogen phosphate is 1:2 - 1:3. The pre-cooling treatment time is 20 - 30 minutes. During the pre-cooling treatment, the phosphate buffer solution is stirred every 5 minutes, and the stirring speed is 50 - 80 revolutions per minute. Maintaining the pH value at 7.2 - 7.4 is to simulate the acid-base environment inside white tomato cells and reduce the structural damage of exosomes during subsequent processing. The specific ratio of sodium dihydrogen phosphate and disodium hydrogen phosphate can ensure that the buffer solution can stably maintain the pH value when affected by external acids and bases. Stirring can cool the white tomatoes evenly and prevent local overheating from affecting the activity of exosomes.

[0018] The inert gas in Step 2 is nitrogen. Before cell wall decomposition, nitrogen is filled into the reaction system to make the nitrogen content in the system reach more than 95%, and the filling rate of nitrogen is 5 - 10 L / min. During cell wall decomposition, the oxygen content in the reaction system is continuously monitored. When the oxygen content exceeds 3%, nitrogen is filled in again. As an inert gas, nitrogen can effectively exclude oxygen in the reaction system, prevent exosomes from being oxidized, and protect the biological activity of exosomes. Setting the nitrogen filling rate and oxygen monitoring mechanism is to ensure that the whole reaction process is in a low-oxygen environment, improving the stability and quality of exosomes.

[0019] In the ultrasonic - cellulase synergistic breaking process in Step 2, the addition amount of cellulase is 0.2% - 0.4% of the mass of white tomatoes. The ultrasonic - cellulase synergistic breaking is carried out in a constant-temperature reaction vessel, and the temperature of the reaction vessel is controlled at 25 - 30 °C. During the breaking process, the reaction system is sampled and detected every 10 minutes to observe the degree of cell breaking. The high-frequency vibration of ultrasonic waves can generate mechanical force to damage the cell wall structure, while cellulase decomposes the cellulose component in the cell wall through specific action. The synergistic action of the two can improve the cell breaking efficiency and reduce the loss of exosomes. Controlling the reaction temperature at 25 - 30 °C is because this temperature range is the optimal reaction temperature for enzymes such as cellulase, which can fully exert the activity of the enzymes. Regular sampling and detection helps to grasp the cell breaking process in real time and ensure that the breaking effect reaches the best.

[0020] In the three-stage gradient centrifugation system in Step 3, after each centrifugation, the substances in the centrifuge tube are washed with phosphate buffer solution at 4°C for 2 - 3 times. Each time during washing, the amount of phosphate buffer solution used is 1 - 1.5 times the volume of the substances in the centrifuge tube. During the washing process, the substances are gently shaken to fully contact the phosphate buffer solution, and the shaking frequency is 50 - 80 times per minute. The purpose of washing is to remove the impurities remaining during centrifugation and the debris generated from broken cells, further purifying the exosomes. Using phosphate buffer solution at 4°C can maintain the activity of exosomes. Gentle shaking can not only ensure the washing effect but also avoid mechanical damage to exosomes. In the three-stage gradient centrifugation system of Step 3, during centrifugation, the centrifugal radius of the centrifuge tube is 10 - 15 cm, and before each centrifugation, the centrifuge tube is balanced, with a balance error not exceeding 0.1 g. An appropriate centrifugal radius can ensure uniform distribution of centrifugal force and improve the centrifugation effect; precise balance treatment can avoid vibration and wear caused by unbalanced centrifuge tubes during centrifugation, ensure the normal operation of the centrifugation equipment, and prevent damage to exosomes due to unbalanced centrifugation.

[0021] In the nano-filter membrane coated ultrafiltration concentration technology in Step 4, before ultrafiltration concentration, the nano-filter membrane is pre-treated. The pre-treatment method is to rinse it with deionized water 3 - 5 times, then soak it in a sodium hydroxide solution with a mass fraction of 0.1% for 1 - 2 hours, and finally rinse it with deionized water until neutral. During ultrafiltration concentration, the circulation flow rate is set at 50 - 100 L / h, and at the same time, the ultrafiltration concentrate is sampled and detected every 30 minutes to monitor the concentration and purity of exosomes. Pre-treatment can remove the impurities and contaminants on the surface of the nano-filter membrane, improving the filtration performance of the filter membrane and the recovery rate of exosomes. Controlling the circulation flow rate can make exosomes evenly distributed on the surface of the filter membrane, avoid local blockage, and improve the concentration efficiency. Regular sampling and detection helps to timely adjust the ultrafiltration concentration conditions to ensure that exosomes reach the required concentration and purity. In the nano-filter membrane coated ultrafiltration concentration technology of Step 4, the selected nano-filter membrane material is polyvinylidene fluoride, and the pore size of the nano-filter membrane is distributed between 10 - 20 nm. Polyvinylidene fluoride has good chemical stability and mechanical strength, and can withstand the pressure and chemical reagents during ultrafiltration; the pore size range of 10 - 20 nm matches the size of exosomes, can effectively retain exosomes, achieve high-efficiency concentration, and at the same time allow small molecule impurities to pass through, improving the purity of exosomes.

[0022] The trehalose-mannitol-polyethylene glycol composite lyoprotectant in step five has a mass ratio of trehalose, mannitol, and polyethylene glycol of 2:1:1. The molecular weight of the polyethylene glycol is 2000 - 4000. When preparing the composite lyoprotectant, trehalose and mannitol are first dissolved in deionized water, and then polyethylene glycol is added and stirred evenly. The stirring speed is 200 - 300 revolutions per minute, and the stirring time is 10 - 15 minutes. Trehalose has excellent vitrification transition characteristics and can form a stable glassy structure during the freeze-drying process to protect the biomembrane structure of exosomes; mannitol can adjust the osmotic pressure of the lyoprotectant to prevent exosomes from rupturing due to osmotic pressure changes during freeze-drying; polyethylene glycol can increase the viscosity of the lyoprotectant and improve the binding force between exosomes and the protectant. Specific stirring speed and time can ensure that the three components are fully mixed evenly to form an effective protection system.

[0023] For the secondary drying in step five with a gradient temperature increase, the heating rate is 0.5 °C / min. During the secondary drying process, when the temperature rises to 10 °C, this temperature is maintained for 2 hours, and then the temperature continues to rise to 25 °C at a rate of 0.5 °C / min. Throughout the freeze-drying process, the vacuum degree in the freeze-dryer is maintained at 10 - 30 Pa. Gradient temperature increase can cause the residual water in exosomes to sublimate slowly, avoiding the destruction of exosome structure due to sudden temperature rise. Maintaining the temperature at 10 °C for 2 hours is to further remove the bound water on the surface and inside of exosomes to ensure the freeze-drying effect. Controlling the vacuum degree at 10 - 30 Pa can provide good conditions for water sublimation, accelerate the freeze-drying speed, and at the same time ensure the quality of exosomes. In the multi-stage freeze-drying procedure of step five, before the pre-freezing stage, the mixed solution of exosome suspension and the composite lyoprotectant is first left standing at 4 °C for 1 - 2 hours to allow the protectant to fully penetrate around the exosomes. The standing process can allow the composite lyoprotectant to fully contact with exosomes, form a stable protection structure, improve the stability of exosomes during the freeze-drying process, and reduce the loss of exosome activity.

[0024] I. Brief description of the laboratory method for the melanin gray value of zebrafish embryos 1. Test principle Melanocytes in human skin can produce tyrosinase, which can convert tyrosine into dopachrome, and dopachrome then forms melanin. Melanin is transferred from melanocytes to surrounding epidermal cells, causing the skin color to darken. When zebrafish embryos develop to 24 h, skin melanin begins to form, and the skin melanin generation mechanism is the same as that of humans. By treating the zebrafish embryos during the skin melanin formation process with the test substance and observing the inhibition of skin melanin using a microscope, it is evaluated whether the test substance has the effect of whitening and freckle removal.

[0025] 2. Materials and methods 2.1 Experimental organisms Zebrafish embryos at the 24th hour of development were selected as experimental organisms.

[0026] 2.2 Reagents and Consumables The main reagents included N-phenylthiourea (positive drug PTU), methylcellulose, MS-222, pronase, calcium chloride, magnesium sulfate, sodium bicarbonate, potassium chloride, etc.; The main consumables included 6-well / 12-well plates, 1.5 mL / 10 mL / 50 mL centrifuge tubes, disposable Pasteur pipettes, and disposable culture dishes.

[0027] 2.3 Instruments and Equipment Nikon upright stereomicroscope, analytical balance (one in ten thousand), pipette, water quality detection equipment (dissolved oxygen meter, salinometer, hardness meter, pH meter), refrigerator, zebrafish breeding system, constant temperature incubator, etc.

[0028] 2.4 Test Procedures The tests were set up with a blank control group, a positive drug group (nine parts per million: 0.009‰), and a sample group (0.1%). Each group had 3 biological replicates, with 8 zebrafish embryos in each replicate. 5 mL of the working solution corresponding to each concentration group was added to each well; the test groups were placed in a constant temperature incubator at 28.5 ± 0.5 °C and incubated for 24 h; the membranes were removed with a 1 mg / mL pronase solution; after fixation with 3% methylcellulose, observations and photographs were taken under a stereomicroscope. When taking pictures, the zebrafish embryos should have their heads facing left, their abdomens facing down, and their bodies horizontal. The photographing results of all zebrafish embryos must be completed under the same instrument and environmental conditions, and the body positions of the zebrafish embryos should be consistent. The melanin gray values of each were analyzed using ImageJ software, and a significant difference test was performed on the data of each group.

[0029] 2.5 Result Calculation Calculate the whitening and freckle-removing effect of the test substance (expressed as the melanin inhibition rate), and the formula is as follows: Melanin production inhibition rate (%) = (S1 - S2) / S1 × 100% (1) In formula (1): S2—the average melanin gray value of zebrafish embryos in the test group; S1—the average melanin gray value of zebrafish embryos in the blank control group; A two-sample T-test was performed on the melanin gray values of the test group fish and the blank control group fish. A p < 0.05 indicates a significant difference.

[0030] II. Detection Results The freeze-dried powder prepared by the present invention was tested for its whitening and freckle-removing efficacy (test for the inhibition rate of zebrafish embryo melanin production). The specific test results are shown in Table 1.

[0031] Compared with the control group, the melanin inhibition rate of the sample on zebrafish embryos at a concentration of 0.1% was 19.66%, showing a significant difference (p < 0.001).

[0032] Table 1 shows the statistical results of the melanin gray values of zebrafish embryos in each group (n = 20). Note: p ≥ 0.05 indicates no significant difference; p < 0.05 indicates a significant difference: "*" indicates 0.01 ≤ p < 0.05; "**" indicates 0.001 < p < 0.01; "***" indicates p < 0.001.

[0033] Under the experimental conditions of this study, the sample "white tomato freeze-dried powder" can significantly inhibit the melanin production of zebrafish embryos (p < 0.001), showing good efficacy in whitening and freckle removal.

[0034] The above are only specific embodiments of the present invention, but the technical features of the present invention are not limited thereto. Any simple changes, equivalent substitutions or modifications made based on the present invention to solve substantially the same technical problems and achieve substantially the same technical effects are all covered by the protection scope of the present invention.

Claims

1. A method for preparing freeze-dried white tomato exosome powder, characterized in that: The following steps are involved: Step 1: White tomatoes were manually picked 3-5 days after the color change period, cleaned by dual-frequency ultrasonic wave, and then precooled in 4°C phosphate buffer containing 0.1% vitamin C; Step 2: Use ultrasonic-cellulase synergistic crushing process and set the ultrasonic power to 300W / cm 2 , the pulse mode is a cycle of 2 seconds of working time and 3 seconds of rest time, and 0.5% pectinase and 0.3% β-glucanase are added at the same time, and the cell wall is decomposed for 30 minutes under the protection of inert gas; Step 3: Through the three-stage gradient centrifugation system, 300 times the gravity acceleration is used to remove cell debris for the first time, 12,000 times the gravity acceleration is used for the second time to enrich exosomes, and 160,000 times the gravity acceleration is used for the third time to purify by ultracentrifugation. The whole process is maintained at 4°C and the temperature is controlled with CO2. Step 4: Use nanofiltration membrane coated ultrafiltration concentration technology, select 100kDa tangential flow filtration device, and control the transmembrane pressure ≤ 0.5bar; Step 5: Prepare a composite freeze-drying protective agent containing trehalose-mannitol-polyethylene glycol, mix it with the exosome suspension at a volume ratio of 1:3, and perform a multi-stage freeze-drying procedure: pre-freezing stage, cooling to -50℃ at 5℃ / min, primary drying at -35℃ for 8h, and secondary drying gradient temperature increase to 25℃.

2. The method for preparing a freeze-dried white tomato exosome powder according to claim 1, characterized in that: The dual-frequency ultrasonic cleaning in step 1 has dual frequencies of 28 kHz and 40 kHz, respectively, and the cleaning time is 10-15 minutes.

3. The method for preparing a freeze-dried white tomato exosome powder according to claim 1, characterized in that: The pH value of the phosphate buffer in step 1 is 7.2-7.4, and the pre-cooling treatment time is 20-30 minutes.

4. The method for preparing a freeze-dried white tomato exosome powder according to claim 1, characterized in that: The inert gas in step 2 is nitrogen. Before cell wall decomposition, nitrogen is charged into the reaction system to make the nitrogen content in the system reach more than 95%.

5. The method for preparing a freeze-dried white tomato exosome powder according to claim 1, characterized in that: In the ultrasonic-cellulase synergistic crushing process in step 2, the amount of cellulase added is 0.2%-0.4% of the mass of the white tomato.

6. The method for preparing a freeze-dried white tomato exosome powder according to claim 1, characterized in that: In the three-stage gradient centrifugation system in step 3, after each centrifugation, the material in the centrifuge tube is washed with 4° C. phosphate buffer for 2-3 times.

7. The method for preparing a freeze-dried white tomato exosome powder according to claim 1, characterized in that: In the nanofiltration membrane coated ultrafiltration concentration technology in step 4, the nanofiltration membrane is pretreated before ultrafiltration concentration. The pretreatment method is to rinse it with deionized water for 3-5 times, then soak it in a sodium hydroxide solution with a mass fraction of 0.1% for 1-2 hours, and finally rinse it with deionized water until it is neutral.

8. The method for preparing freeze-dried white tomato exosome powder according to claim 1, characterized in that: In the trehalose-mannitol-polyethylene glycol composite freeze-drying protective agent in step 5, the mass ratio of trehalose, mannitol and polyethylene glycol is 2:1:

1.

9. The method for preparing freeze-dried white tomato exosome powder according to claim 1, characterized in that: The secondary drying in step 5 is subjected to gradient temperature increase, with a heating rate of 0.5°C / min.

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