Preparation method of high-solubility mussel byssus glandular mucoprotein for thickening skin basal layer

Abstract

The invention discloses a preparation method of high-solubility mussel byssus gland mucoprotein for thickening a skin basal layer, which comprises the following steps: taking out common mussel byssus glands as raw materials, naturally unfreezing, cleaning, keeping the completeness of the byssus glands, carrying out double-enzyme treatment, sequentially carrying out double-enzyme enzymolysis by using neutral protease and pepsase, filtering, washing, and drying to obtain the high-solubility mussel byssus gland mucoprotein for thickening the skin basal layer. After the enzymolysis is finished, heating a reaction system, keeping the activity of the passivating enzyme, and removing an enzymolysis mixed solution; the preparation method comprises the following steps: washing byssus gland subjected to double-enzyme enzymolysis, adding a urea solution for homogenization treatment, removing insoluble impurities through efficient centrifugation, reserving a supernatant as a crude extract of byssus gland mucoprotein, carrying out microfiltration and nanofiltration through a ceramic membrane and a nanofiltration membrane, and carrying out spray drying or freeze drying on the collected liquid to obtain high-solubility mussel byssus gland mucoprotein powder. The high-solubility mussel byssus glandular mucoprotein is obtained through a double-enzyme urea nanofiltration engineering method, and the mussel byssus glandular mucoprotein has molecular uniqueness and high biological activity efficiency and is wide in application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for preparing highly soluble mussel byssus gland mucin for thickening a basal layer of the skin. Background Art

[0002] As the largest organ of the human body, the skin has multiple functions such as protection, temperature regulation, and perception of the external environment. However, with age and the influence of the external environment, the basal layer of the skin gradually becomes thinner, causing the skin to lose elasticity, become dry and loose, and then produce signs of aging. In order to improve skin health and appearance, thickening the basal layer of the skin has become a hot topic in the field of skin care and anti-aging. Thickening the basal layer not only helps to improve the barrier function of the skin, but also promotes the metabolism and regeneration of skin cells.

[0003] At present, common methods for thickening the basal layer of the skin mainly include using various chemical ingredients, hormone drugs or stimulating the skin through physical means. However, these methods often have certain limitations, such as strong skin irritation, obvious side effects, and short-lasting effects. Therefore, the development of a natural, biocompatible and effective ingredient that can effectively and permanently thicken the basal layer of the skin has become a hot topic in the field of skin care and anti-aging.

[0004] Mussel Foot Protein (MFP), as a natural polymer protein, is widely used in the biomedical field due to its unique bioadhesion properties. Perna canaliculus ) is mainly composed of a special protein, which has excellent adhesion, good biodegradability and outstanding biocompatibility, and is used in wound repair, drug delivery, etc. However, the low solubility of mussel foot gland mucin limits its application in skin care and other biomedical fields, especially in promoting the thickening of the basal layer of the skin. Therefore, how to improve the solubility of mucin to enhance its thickening effect and biological activity in the basal layer of the skin has become the key to solving this problem.

[0005] The prior art improves the solubility of mucin by chemical modification or adding excipients. For example, increasing the polar groups of mucin molecules, or increasing their solubility to a certain extent by enzymatic hydrolysis, acid hydrolysis, etc. However, the prior art has failed to effectively solve the problem of how to improve the solubility of mucin in water and promote its effective thickening of the basal layer of the skin. At the same time, the prior art mostly focuses on the moisturizing effect of mucin and its interaction with the surface layer of the skin, and pays less attention to its effect on the thickening of the basal layer of the skin, especially in terms of the thickening mechanism of the basal layer, optimization of the preparation process and improvement of the therapeutic effect. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a method for preparing highly soluble mussel foot gland mucin for thickening the basal layer of the skin. Through the dual enzyme urea nanofiltration engineering method, the high molecular weight protein with low solubility in the outer layer of the foot gland can be removed to obtain the low molecular weight mucin with high solubility in the inner layer of the foot gland. By increasing the solubility, the biological activity of the mussel foot gland mucin is improved.

[0007] To achieve the above object, the present invention adopts the following technical solutions: The present invention provides a method for preparing highly soluble mussel foot gland mucin for thickening the basal layer of the skin, which is prepared by a double enzyme urea nanofiltration engineering method and comprises the following steps: (1) Raw material pretreatment: The foot silk gland of blue mussel is taken out from a -20~-80℃ environment, thawed naturally and then cleaned; (2) Dual enzyme hydrolysis: preheat the reaction vessel in a constant temperature water bath at 35-37°C, and use a magnetic stirrer at a speed of 50-100 rpm to keep the system uniform. First, add the neutral protease to the foot silk gland of the blue mussel pretreated in step (1) for 1.5-3 h. After the neutral protease is completed, adjust the pH of the reaction system to 2.5-3.5, and then add pepsin solution to continue enzymolysis for 2-4 h. (3) Termination of enzymatic hydrolysis: After the double enzyme hydrolysis is completed, the enzymatic hydrolysis solution is drained, the foot silk gland of the blue mussel is washed three times with drinking water, the enzymatic hydrolysis solution and the washing solution are mixed, the enzymatic hydrolysis mixture is heated to 90-95°C and maintained for 10-15 min to completely inactivate the enzyme activity, and the enzymatic hydrolysis mixture is discarded; (4) Preliminary extraction and purification: After double enzyme hydrolysis, the byssin glands were washed and homogenized with 0.25 M-0.75 M urea solution, and insoluble impurities were removed by high-efficiency centrifugation. The supernatant was retained as the crude extract of byssin gland mucin; (5) Microfiltration and nanofiltration: The crude extract is microfiltered through a 150-200 kDa ceramic membrane to remove macromolecular impurities, and then a 10-30 kDa nanofiltration membrane is used to concentrate the protein solution to remove urea and other small molecular impurities, thereby reducing the volume of the protein solution to 1 / 20 to 1 / 10 of the original volume; (6) Post-treatment: The protein solution after microfiltration and nanofiltration is spray-dried or freeze-dried to obtain highly soluble mussel foot gland mucin.

[0008] Preferably, in step (1), ultrasonic treatment is performed at 450-500 W for 40-45 min to remove the attachments on the surface of the foot gland of the blue mussel, and the mixture is washed three times with drinking water until there are no impurities.

[0009] Preferably, in step (2), the neutral protease is added to the foot gland of the blue mussel pretreated in step (1), and the pH is monitored every 20 to 30 minutes during the reaction and adjusted to pH 7.5 to 8.5 to ensure the catalytic efficiency of the enzyme.

[0010] Preferably, in step (2), pepsin solution is added to continue enzymolysis, and the enzymolysis reaction is maintained at 35-37°C and intermittent stirring (for example, stirring for 3-5 min every 15-20 min) is performed to ensure efficient hydrolysis.

[0011] Preferably, in step (2), the concentration of the neutral protease solution is 3000-6000 U / g, the weight ratio of the neutral protease added to the weight of the foot silk gland is 1:3000-6000, and the pH is adjusted to 7.5-8.5.

[0012] Preferably, in step (2), the enzyme activity of the pepsin solution is 1500-3000 U / g, the weight ratio of the added pepsin to the weight of the foot silk gland is 1:1500-3000, and 0.5%-1.0% acetic acid is added to adjust the pH to 2.0-3.0.

[0013] The present invention also provides a product for thickening the basal layer of the skin, which comprises highly soluble mussel byssin, wherein the molecular weight range of the highly soluble mussel byssin is 11-17 kDa, and the average molecular weight is 14.10. The product is prepared by the preparation method of the highly soluble mussel byssin for thickening the basal layer of the skin.

[0014] Compared with the prior art, the beneficial effect of the present invention is that: the method for improving the solubility of the foot gland mucin of blue mussel by using the double enzyme urea nanofiltration engineering technology can remove the high molecular weight protein with low solubility in the outer layer of the foot gland by using the double enzyme urea nanofiltration engineering method, and obtain the low molecular weight mucin with high solubility in the inner part of the foot gland, thereby increasing the solubility and improving the biological activity. The highly soluble mussel foot gland mucin has molecular uniqueness and high biological activity efficiency, and can be widely used in the field of biomedicine, especially in the field of skin medical beauty, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 These are the results of verifying the effect of high-solubility mussel foot gland mucin on thickening of skin basement membrane in the examples.

[0016] Figure 2 The molecular weight of the highly soluble mussel byssin was determined by SDS-PAGE in the examples.

[0017] Figure 3 The ultrastructure of the highly soluble mussel byssin in the example. DETAILED DESCRIPTION

[0018] The present invention will be further described below with reference to examples, but the present invention is not limited to these examples.

[0019] Example 1 In this embodiment, high-solubility mussel byssin powder is prepared, and the steps are as follows: 1. Raw material pretreatment: 4000 g of blue mussel foot glands were taken out from -20℃ environment, thawed naturally, and then cleaned with 480 W ultrasonic for 42 min to remove surface attachments. 40 L of drinking water was used for cleaning three times each time, and the water was drained.

[0020] 2. Double enzyme hydrolysis: Place the cleaned foot silk gland sample in a constant temperature water bath at 35℃, add a neutral protease solution with an enzyme activity of 3500U / g, mix at a solid-liquid ratio of 1:2, add 1.14g of neutral protease, and adjust the pH to 7.5. The stirring paddle speed is 50 rpm, and the pH is tested and adjusted every 20 min. The enzymatic hydrolysis lasts for 2 h. Subsequently, the pH of the sample is adjusted to 3.0, and a pepsin solution with an enzyme activity of 2000 U / g (0.02% acetic acid is added during preparation) is added, mixed at a solid-liquid ratio of 1:2, and the amount of pepsin added is 2.00g. Maintain 35℃ and stir intermittently (stir for 3 min every 20 min), and the enzymatic hydrolysis time is 2.5 h.

[0021] 3. Termination of enzymatic hydrolysis: After the double enzyme hydrolysis is completed, drain the enzymatic hydrolyzate, wash the foot silk gland of the blue mussel three times with 40 L of drinking water, mix the enzymatic hydrolyzate and the washing solution, heat the enzymatic hydrolyzate mixture to 93°C and keep it for 12 min to inactivate the enzyme activity, and discard the enzymatic hydrolyzate mixture (for other uses).

[0022] 4. Preliminary extraction and purification: After double enzyme enzymatic hydrolysis, the foot silk gland was washed and homogenized with 40 L of 0.5 M urea solution and centrifuged, and the supernatant was retained as the crude extract.

[0023] 5. Microfiltration and nanofiltration: The crude extract was microfiltered through a 200 kDa ceramic membrane and then concentrated to 1 / 15 of the original volume using a 10 kDa nanofiltration membrane.

[0024] 6. Post-processing: freeze-dry the protein solution to prepare highly soluble mussel byssus gland mucin powder.

[0025] Example 2 In this embodiment, high-solubility mussel byssin powder is prepared, and the steps are as follows: 1. Raw material pretreatment: 6000 g of blue mussel foot glands were taken out from -80℃ environment, thawed naturally, and then cleaned with 500 W ultrasonic for 40 min to remove surface attachments. 60 L of drinking water was used each time for three times until there were no impurities.

[0026] 2. Double enzyme hydrolysis: Place the cleaned foot silk gland sample in a constant temperature water bath at 37°C, add a neutral protease solution with an enzyme activity of 4500U / g, mix at a solid-liquid ratio of 1:1, add 1.33g of neutral protease, and adjust the pH to 7.8. The stirring speed is 75 rpm, and the pH is tested and adjusted every 30 minutes. The enzymatic hydrolysis lasts for 3 hours. Subsequently, the pH of the sample is adjusted to 2.5, and a pepsin solution with an enzyme activity of 2500U / g (0.05% acetic acid is added during preparation) is added, mixed at a solid-liquid ratio of 1:3, and the amount of pepsin added is 2.40g. Maintain 36°C and stir intermittently (stir for 5 minutes every 15 minutes), and the enzymatic hydrolysis time is 3.5 hours.

[0027] 3. Termination of enzymatic hydrolysis: After the enzymatic hydrolysis is completed, drain the enzymatic hydrolysate, wash the foot silk gland of the blue mussel three times with 40 L of drinking water, mix the enzymatic hydrolysate and the washing solution, heat the enzymatic hydrolysate mixture to 95℃ and keep it for 15 min to inactivate the enzyme activity, and discard the enzymatic hydrolysate mixture (for other uses).

[0028] 4. Preliminary extraction and purification: After double enzyme hydrolysis, the foot silk gland was washed and homogenized with 90 L of 0.25 M urea solution and centrifuged, and the supernatant was retained as the crude extract.

[0029] 5. Microfiltration and nanofiltration: The crude extract was microfiltered through a 150 kDa ceramic membrane and then concentrated to 1 / 10 of the original volume using a 10 kDa nanofiltration membrane.

[0030] 6. Post-processing: The protein solution is spray-dried to prepare highly soluble mussel byssus gland mucin powder.

[0031] Example 3 In this embodiment, high-solubility mussel byssin powder is prepared, and the steps are as follows: 1. Raw material pretreatment: 8000 g of blue mussel foot glands were taken out from -40℃ environment, thawed naturally, and then cleaned with 480 W ultrasonic for 42 min to remove surface attachments. 80 L of drinking water was used each time for three times until there were no impurities.

[0032] 2. Double enzyme hydrolysis: Place the cleaned foot silk gland sample in a constant temperature water bath at 36°C, add a neutral protease solution with an enzyme activity of 6000U / g, mix at a solid-liquid ratio of 1:3, add 1.33g of neutral protease, and adjust the pH to 8.0. The stirring speed is 30 rpm, and the pH is tested and adjusted every 25 minutes. The enzymatic hydrolysis lasts for 1.5 hours. Subsequently, the pH of the sample is adjusted to 2.0, and a pepsin solution with an enzyme activity of 3000U / g (0.01% acetic acid is added during preparation) is added, mixed at a solid-liquid ratio of 1:1, and the amount of pepsin added is 2.67g. Maintain 37°C and stir intermittently (stir for 3 minutes every 20 minutes), and the enzymatic hydrolysis time is 4 hours.

[0033] 3. Termination of enzymatic hydrolysis: After the enzymatic hydrolysis is completed, drain the enzymatic hydrolyzate, wash the foot silk gland of the blue mussel three times with 40 L of drinking water, mix the enzymatic hydrolyzate and the washing solution, heat the enzymatic hydrolyzate mixture to 90℃, keep it for 10 minutes to inactivate the enzyme activity, and discard the enzymatic hydrolyzate mixture (for other uses).

[0034] 4. Preliminary extraction and purification: After double enzyme enzymatic hydrolysis, the foot silk gland was washed and homogenized with 120 L of 0.30 M urea solution and centrifuged, and the supernatant was retained as the crude extract.

[0035] 5. Microfiltration and nanofiltration: The crude extract was microfiltered through a 180 kDa ceramic membrane and then concentrated to 1 / 20 of the original volume using a 10 kDa nanofiltration membrane.

[0036] 6. Post-processing: freeze-dry the protein solution to prepare highly soluble mussel byssus gland mucin powder.

[0037] Effect Example 1 This effect example verifies the effect of the highly soluble mussel foot gland mucin powder prepared in Example 1 on the thickening of the skin basement membrane, as follows: (1) Dissolution preparation of highly soluble mussel byssin. Dissolve the highly soluble mussel byssin powder in sterile distilled water to prepare a highly soluble mussel byssin solution with a concentration of 10-30 mg / mL.

[0038] (2) The drug was administered using a mouse back skin wound healing model. The specific operation was as follows: After creating a standardized wound model with a diameter of 0.8-1.0 cm on the mouse back skin, 80-100 μL of highly soluble mussel foot gland mucin solution was evenly applied each time, and the wound healing status, including the thickness change of the new skin tissue and the speed of wound closure, was regularly observed and recorded.

[0039] (3) Skin basement membrane thickening effect. Figure 1 Compared with the group treated with highly soluble mussel foot gland mucin, the basement membrane of the back skin of mice thickened by 10% to 20% ( Figure 1 right).

[0040] Effect Example 2 The molecular weight and composition of the highly soluble mussel byssin prepared in Example 1 were determined by SDS-PAGE. Figure 2 As shown, the molecular weight spectrum of the highly soluble mussel byssin was 11 ± 1 kDa, 14 ± 1 kDa, 15 ± 1 kDa and 17 ± 1 kDa, with an average molecular weight of 14.10 ± 1 kDa.

[0041] Effect Example 3 The ultrastructure of highly soluble mussel foot gland mucin was observed by scanning electron microscopy. Figure 3 As shown in the figure, the mussel foot gland mucin extracted by the dual enzyme urea nanofiltration engineering method appears as a sheet with a distinct long strip structure under a scanning electron microscope, with a length of 30-50 μm and a width of 5-10 μm. These long strip structures have a uniform shape and a relatively regular size, which may be formed by the aggregation or rearrangement of mucin molecules under enzymatic hydrolysis. Fibrous or ribbon-like structures at the cell or polymer level can be observed on the surface, showing its unique morphological characteristics formed during the processing process.

[0042] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing highly soluble mussel foot gland mucin for thickening the basal layer of the skin, characterized in that: The following steps are involved: (1) Raw material pretreatment: The foot silk gland of blue mussel is taken out from a -20~-80℃ environment, thawed naturally and then cleaned; (2) Dual enzyme hydrolysis: preheat the reaction vessel in a constant temperature water bath at 35-37°C, and use a magnetic stirrer at a speed of 50-100 rpm to keep the system uniform. First, add the neutral protease to the foot silk gland of the blue mussel pretreated in step (1) for 1.5-3 h. After the neutral protease is completed, adjust the pH of the reaction system to 2.5-3.5, and then add pepsin solution to continue enzymolysis for 2-4 h. (3) Termination of enzymatic hydrolysis: After the double enzyme hydrolysis is completed, the enzymatic hydrolysis solution is drained, the foot silk gland of the blue mussel is washed three times with drinking water, the enzymatic hydrolysis solution and the washing solution are mixed, the enzymatic hydrolysis mixture is heated to 90-95°C and maintained for 10-15 min to completely inactivate the enzyme activity, and the enzymatic hydrolysis mixture is discarded; (4) Preliminary extraction and purification: After double enzyme hydrolysis, the byssin glands are washed and homogenized with urea solution of appropriate concentration, and insoluble impurities are removed by high-efficiency centrifugation. The supernatant is retained as the crude extract of byssin gland mucin; (5) Microfiltration and nanofiltration: The crude extract is microfiltered through a 150-200 kDa ceramic membrane to remove macromolecular impurities, and then a 10-30 kDa nanofiltration membrane is used to concentrate the protein solution to remove urea and other small molecular impurities, thereby reducing the volume of the protein solution to 1 / 20 to 1 / 10 of the original volume; (6) Post-treatment: The protein solution after microfiltration and nanofiltration is spray-dried or freeze-dried to obtain highly soluble mussel foot gland mucin.

2. The method for preparing the highly soluble mussel byssin for thickening the basal layer of the skin according to claim 1, characterized in that: In step (1), 450-500 W ultrasonic treatment is used for 40-45 min to remove the attachments on the surface of the foot gland of the blue mussel, and then rinsed with drinking water for 3 times until there are no impurities.

3. The method for preparing the highly soluble mussel byssin for thickening the basal layer of the skin according to claim 1, characterized in that: In step (2), the neutral protease is added to the foot silk gland of the blue mussel pretreated in step (1). During the reaction, the pH is monitored every 20 to 30 minutes and adjusted to pH 7.5 to 8.5 to ensure the catalytic efficiency of the enzyme.

4. The method for preparing the highly soluble mussel byssin for thickening the basal layer of the skin according to claim 1, characterized in that: In step (2), pepsin solution is added to continue enzymolysis. The enzymolysis reaction process is maintained at 35-37°C and intermittent stirring is performed to ensure efficient hydrolysis.

5. The method for preparing the highly soluble mussel byssin for thickening the basal layer of the skin according to claim 1, characterized in that: In step (2), the concentration of the neutral protease solution is 3000-6000 U / g, the ratio of the added weight of the neutral protease to the weight of the foot silk gland is 1:3000-6000, and the pH is adjusted to 7.5-8.

5.

6. The method for preparing the highly soluble mussel byssus gland mucin for thickening the basal layer of the skin according to claim 1, characterized in that: In step (2), the enzyme activity of the pepsin solution is 1500-3000 U / g, the weight ratio of the added pepsin to the solution is 1:1500-3000, and 0.5%-1.0% acetic acid is added to adjust the pH to 2.0-3.

0.

7. A product for thickening the basal layer of the skin, characterized in that: The invention comprises highly soluble mussel foot gland mucin, wherein the molecular weight of the highly soluble mussel foot gland mucin is 14.10±5 kDa, and the mussel foot gland mucin is prepared by the preparation method of the highly soluble mussel foot gland mucin for thickening the basal layer of the skin as claimed in any one of claims 1 to 6.

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