A mussel foot gland mucin subunit and its preparation method and application

Abstract

The present invention discloses a mussel foot gland mucin subunit and its preparation method and application, including: using mussel foot gland as raw material, first removing the outer layer of the foot gland by hydrolysis with neutral protease, then removing the secondary outer layer of the foot gland by hydrolysis with pepsin to obtain the foot gland core, adding urea solution to the foot gland core for homogenization, centrifuging to remove insoluble impurities, adding dithiothreitol to the supernatant to obtain a crude extract of foot gland mucin, microfiltration to remove biological impurities and nanofiltration to obtain foot gland mucin subunit liquid, and spray drying or freeze drying the collected liquid to obtain a highly soluble mussel foot gland mucin subunit powder. The present invention obtains highly soluble mussel foot gland mucin subunits by a dual-enzyme urea nanofiltration engineering reduction method, which has the effects of thickening the basal layer of the skin, promoting hair growth, inducing skin regeneration, etc., and is applied to the field of skin medical beauty and the field of biomedical devices.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a mussel byssal gland mucin subunit and a preparation method and application thereof. Background Art

[0002] Mussel foot protein (Mfp) is a naturally occurring high-molecular-weight protein rich in DOPA from marine organisms. It exhibits excellent biocompatibility, biofunctionality, and material degradability, and is used in tissue repair and functional materials. To date, Mfp has been prepared using molecular sieve chromatography, a process that suffers from low efficiency, time consumption, and yield. Furthermore, Mfp prepared using chromatography also suffers from key application challenges, such as low solubility, which limits its preparation and application.

[0003] The mussel gland, a secretory gland of mussel foot silk protein, is rich in mussel foot silk protein subunits, which are structurally conserved, have precise activity, and significant functions. Compared with adhesive byssal threads or edible parts, the mucin from the byssal gland contains fewer impurities and a higher content of the active ingredient DOPA, making it an excellent raw material for the production of mussel foot silk protein subunits.

[0004] The bioactive mussel protein, mussel byssal protein or mussel byssal gland mucin obtained from the edible parts of mussels, mussel byssal threads or mussel byssal glands using existing molecular sieve chromatography technology have the disadvantages of weak biological activity, poor water solubility and low stability, which limit their use and can only be used in the field of ordinary cosmetics.

[0005] In view of the defects of the existing technology for preparing products, combined with the advantages of mussel foot gland mucin, which is rich in DOPA active ingredients, it is urgent to establish a new method for producing highly soluble mucin subunits using mussel foot gland as raw material. 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 mussel foot gland mucin subunit, which has molecular uniqueness and high biological activity, and can be used in products for thickening the basal layer of the skin, promoting hair growth, and inducing skin regeneration.

[0007] Another object of the present invention is to provide a method for preparing the mussel byssal gland mucin subunit.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] The present invention provides a method for preparing mussel byssal gland mucin subunits, which is prepared by a double-enzyme urea nanofiltration engineering reduction method, comprising the following steps:

[0010] Raw material pretreatment: Use mussel foot gland as raw material, clean it and set aside;

[0011] (2) Double enzyme hydrolysis: add the pre-treated mussel foot gland in step (1) at 35-37°C, add neutral protease solution under stirring, and the hydrolysis time is 1.5-3 h; then adjust the pH of the reaction system to 2.5-3.5, and add pepsin solution to continue the hydrolysis for 2-4 h; drain the hydrolyzed solution, and wash the intact inner core layer of the mussel foot gland with clean water 2-4 times for later use;

[0012] The invention adopts a double-enzyme enzymatic hydrolysis method to completely enzymatically hydrolyze and remove the outer layer and the sub-outer layer of the mussel foot gland macromolecular mussel foot gland mucin layer by layer.

[0013] When neutral protease is added, the outermost layer of the mussel foot gland can be enzymatically hydrolyzed, removing the outermost macromolecular mussel foot gland mucin Mfp-1, with a molecular weight of ~110 kDa.

[0014] Then, pepsin was added to enzymatically hydrolyze the sub-outer layer of the mussel foot gland, removing the sub-outer macromolecular mussel foot gland mucins Mfp-2 and Mfp-4, with a molecular weight of 40-80 kDa.

[0015] Through the above enzymatic hydrolysis process, the outer layer and sub-outer layer of the mussel foot gland macromolecular mussel foot gland mucin can be removed, making it easier to subsequently extract the complete main components of the inner core layer of mussel foot gland mucin subunits with a molecular weight of 9-13kDa, namely Mfp-3 and Mfp-5.

[0016] (3) Preliminary extraction and purification: adding urea solution to the core of the mussel byssal gland in step (2) for homogenization, centrifuging, retaining the supernatant and adding dithiothreitol at a weight ratio (G / G) of 1.5% to 2.5% (mass concentration) to the urea solution to form a crude extract of byssal gland mucin;

[0017] The present invention uses urea of ​​a specific concentration for treatment to remove impurities such as biomacromolecules. Dithiothreitol is used as a reducing agent to ensure that the final product is a subunit of the foot gland mucin, otherwise multi-subunit molecules will appear.

[0018] (4) Microfiltration and nanofiltration: The crude extract is microfiltered through a 100-150 kDa ceramic membrane to remove large molecular impurities. The protein solution is then concentrated using a 5-15 kDa nanofiltration membrane to remove urea and other small molecular impurities, reducing the volume of the protein solution to 1 / 20 to 1 / 10 of its original volume, forming a foot gland mucin subunit solution.

[0019] (5) Post-processing: The byssal gland mucin subunit liquid is spray-dried or freeze-dried to obtain a highly soluble mussel byssal gland mucin subunit powder.

[0020] Preferably, in step (1), ultrasonic treatment is performed at 450-500 W for 40-45 minutes to remove the attachments on the surface of the mussel's foot gland; and then the mussel is washed with clean water for 2-4 times until there is no impurity. The clean water can be drinking water, purified water, distilled water, etc.

[0021] Preferably, the stirring speed in step (2) is 50-100 rpm.

[0022] Preferably, in step (2), the pH of the neutral protease solution is monitored every 20 to 30 minutes during the enzymatic hydrolysis reaction and adjusted to pH 7.5 to 8.5 to ensure the catalytic efficiency of the enzyme.

[0023] Preferably, the enzyme activity of the pepsin solution in step (2) is 1500-3000 U / g, and the weight ratio of pepsin to mussel foot gland is 1:1500-3000;

[0024] The pH of the enzymatic hydrolysate after adding pepsin was maintained at 2.0-3.0. The pH was adjusted using 0.5%-1.0% acetic acid solution.

[0025] Preferably, the concentration of the urea solution in step (3) is 0.25-0.5 mol / L, the gap between the rotor and the stator is 5-10 μm, and insoluble impurities are removed by high-efficiency centrifugation.

[0026] The molecular weight of the mussel byssal gland mucin subunits obtained by the above preparation method ranges from 9 to 13 kDa, with an average molecular weight of 11.10 ± 2 kDa.

[0027] The above-mentioned mussel foot gland mucin subunit can be used to prepare products for thickening the basal layer of the skin, promoting hair growth, and inducing skin regeneration, with significant efficacy.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The mussel byssal gland mucin subunit prepared by the present invention has high solubility, molecular uniqueness, and high biological activity, and is widely used in the fields of skin medical beauty and biomedical devices.

[0030] The present invention utilizes dual-enzyme urea nanofiltration engineering reduction technology to improve the solubility of mussel byssin, which can remove high-molecular-weight proteins with low solubility in the outer and sub-outer layers of the byssin gland, and obtain low-molecular-weight mucin subunits with high solubility in the core of the byssin gland. By increasing the solubility, the bioactivity of the mussel is improved, and the scope of application is expanded. The mussel can be used in products for thickening the basal layer of the skin, promoting hair growth, and inducing skin regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a diagram showing the HE results of using the mussel foot gland mucin subunit prepared in Example 1 to thicken the basal layer of the skin.

[0032] Figure 2 This is the SDS-PAGE result of the molecular weight of the mussel foot gland mucin subunit prepared in Example 1.

[0033] Figure 3 This is a scanning electron microscope ultrastructure image of the freeze-dried powder of mussel foot gland mucin subunit prepared in Example 1.

[0034] Figure 4 This is the SDS-PAGE result of the molecular weight of the mussel foot gland mucin subunit in Comparative Example 1.

[0035] Figure 5 The SDS-PAGE result of the molecular weight of the mussel foot gland mucin subunit in Comparative Example 2 is shown in FIG. DETAILED DESCRIPTION

[0036] The present invention will be further described below with reference to examples, but the present invention is not limited to these examples.

[0037] The commercially available mussel foot gland mucin used in the following experiments has a CAS number of 84195-52-8.

[0038] Example 1

[0039] In this example, mussel byssal gland mucin subunit powder was prepared by the following steps:

[0040] 1. Raw material pretreatment: 4000 g of blue mussel byssus gland was taken out of -20℃ environment, thawed naturally, and then cleaned with 480W ultrasonic for 42 min to remove surface attachments. The byssus gland was cleaned three times with 40L of drinking water each time and the water was drained.

[0041] 2. Dual-Enzyme Hydrolysis: Place the cleaned byssal silk gland sample in a 35°C constant-temperature water bath. Add 1.14 g of a neutral protease solution with an enzyme activity of 3500 U / g, mixing at a solid-liquid ratio of 1:2. The pH is adjusted to 7.5. The stirring speed is 50 rpm, and the pH is checked and adjusted every 20 minutes. The enzymatic hydrolysis is continued for 2 hours. Subsequently, the pH of the sample is adjusted to 3.0, and a pepsin solution with an enzyme activity of 2000 U / g (prepared with 0.02% acetic acid) is added. The solution is mixed at a solid-liquid ratio of 1:2, with a pepsin amount of 2.00 g. The temperature is maintained at 35°C with intermittent stirring (3 minutes every 20 minutes). The enzymatic hydrolysis is continued for 2.5 hours.

[0042] 3. End of enzymatic hydrolysis: After the double enzyme hydrolysis is completed, drain the enzymatic hydrolyzate and wash the mussel foot glands three times with 40 L of drinking water and set aside.

[0043] 4. Preliminary extraction and purification: The mytilus foot gland in step (3) was homogenized with 40 L of 0.5 M urea solution and centrifuged. 616 g of dithiothreitol was added to the supernatant, and the mixture was mixed and retained as the crude extract.

[0044] 5. Microfiltration and nanofiltration: The crude extract was microfiltered through a 150 kDa ceramic membrane and then concentrated to 1 / 15 of its original volume using a 5 kDa nanofiltration membrane.

[0045] 6. Post-processing: freeze-dry the mussel foot gland mucin subunit liquid to prepare highly soluble mussel foot gland mucin subunit powder.

[0046] The mussel byssal gland mucin subunit prepared in this embodiment has high solubility, and the specific monitoring data are shown in Table 1 below:

[0047] Table 1 Solubility of mussel byssal gland mucin subunits

[0048]

[0049] Note: The commercially available mussel foot gland mucin is a white to off-white powder with a molecular weight of <20 kDa but no obvious bands.

[0050] It can be seen from the table that the solubility of the mussel byssal gland mucin subunit prepared by the present invention in water and dilute acid is much higher than that of the mussel byssal gland mucin on the market, and the mussel byssal gland mucin subunit prepared by the present invention has less impurities and better light transmittance.

[0051] The mussel byssal gland mucin obtained by the preparation method has a protein content of more than 5%, a constituent sugar content of more than 93%, and a protein to constituent sugar ratio of 1:18.6.

[0052] Example 2

[0053] In this example, mussel byssal gland mucin subunit powder was prepared by the following steps:

[0054] 1. Raw material pretreatment: 6000 g of blue mussel byssus gland was taken out from -80℃ environment, thawed naturally, and then cleaned with 500 W ultrasonic wave for 40 min to remove surface attachments. 60 L of drinking water was used each time for three times until there was no impurity.

[0055] 2. Dual-Enzyme Hydrolysis: Place the cleaned byssal gland sample in a 37°C water bath and add 1.33 g of a neutral protease solution with an enzyme activity of 4500 U / g. Mix the solution in a 1:1 ratio. The pH is adjusted to 7.8. The stirring speed is 75 rpm. The pH is checked and adjusted every 30 minutes. The enzymatic hydrolysis is continued for 3 hours. Subsequently, the pH of the sample is adjusted to 2.5. A pepsin solution with an enzyme activity of 2500 U / g (prepared with 0.05% acetic acid) is added. The solution is mixed in a 1:3 ratio. The amount of pepsin added is 2.40 g. The solution is maintained at 36°C with intermittent stirring (5 minutes every 15 minutes). The enzymatic hydrolysis is continued for 3.5 hours.

[0056] 3. End of enzymatic hydrolysis: After the enzymatic hydrolysis is completed, drain the enzymatic hydrolyzate and wash the blue mussel foot gland three times with 40 L of drinking water.

[0057] 4. Preliminary extraction and purification: The mytilus foot gland in step (3) was homogenized with 90 L of 0.25 M urea solution and centrifuged. 1663 g of dithiothreitol was added to the supernatant, and the mixture was mixed and retained as the crude extract.

[0058] 5. Microfiltration and nanofiltration: The crude extract was microfiltered through a 100 kDa ceramic membrane and then concentrated to 1 / 10 of its original volume using a 5 kDa nanofiltration membrane.

[0059] 6. Post-processing: The mussel foot gland mucin subunit liquid is spray-dried to prepare a highly soluble mussel foot gland mucin subunit powder.

[0060] Example 3

[0061] In this example, mussel byssal gland mucin subunit powder was prepared by the following steps:

[0062] 1. Raw material pretreatment: 8000 g of blue mussel byssus gland was taken out from -40℃ environment, thawed naturally, and then cleaned with 480 W ultrasonic wave for 42 min to remove surface attachments. 80 L of drinking water was used each time for three times until there was no impurity.

[0063] 2. Dual-Enzyme Hydrolysis: Place the cleaned byssal silk gland sample in a 36°C constant-temperature water bath. Add 1.33 g of a neutral protease solution with an enzyme activity of 6000 U / g at a 1:3 material-to-liquid ratio. The pH is adjusted to 8.0. The stirring speed is 30 rpm. The pH is checked and adjusted every 25 minutes. The enzymatic hydrolysis is continued for 1.5 hours. Subsequently, the pH of the sample is adjusted to 2.0. Add 2.67 g of pepsin solution with an enzyme activity of 3000 U / g (prepared with 0.01% acetic acid) at a 1:1 material-to-liquid ratio. The temperature is maintained at 37°C with intermittent stirring (3 minutes every 20 minutes). The enzymatic hydrolysis is continued for 4 hours.

[0064] 3. End of enzymatic hydrolysis: After the enzymatic hydrolysis is completed, drain the enzymatic hydrolyzate and wash the blue mussel foot gland three times with 40 L of drinking water.

[0065] 4. Preliminary extraction and purification: The mytilus foot gland in step (3) was homogenized with 120 L of 0.30 M urea solution and centrifuged. 2772 g of dithiothreitol was added to the supernatant, and the mixture was mixed and retained as the crude extract.

[0066] 5. Microfiltration and nanofiltration: The crude extract was microfiltered through a 120 kDa ceramic membrane and then concentrated to 1 / 20 of its original volume using a 5 kDa nanofiltration membrane.

[0067] 6. Post-processing: freeze-dry the mussel foot gland mucin subunit liquid to prepare highly soluble mussel foot gland mucin subunit powder.

[0068] Effect Example 1

[0069] This effect example verifies the effect of the mussel foot gland mucin subunit powder prepared in Example 1 on thickening the skin basement membrane. The specific process is as follows:

[0070] (1) The mussel foot gland mucin subunit obtained in Example 1 was dissolved and prepared.

[0071] Dissolve the mussel byssal gland mucin subunit powder in sterile distilled water to prepare a solution with a concentration of 10-100 mg / mL.

[0072] (2) Drug administration was performed using a rat back exposed skin model.

[0073] A rat bare skin model was used for drug administration. Anesthetized female rats weighing 150-200 g, which had undergone one week of adaptive feeding, had their hair removed using a commercially available depilatory cream, exposing the skin to a 0.8-1.0 cm diameter area on their backs, midway between the forelimbs and hindlimbs, 0.5 cm from the spine. A rat bare skin model was created. 80-100 μL of a solution of mussel byssal gland mucin subunits and other patented mussel byssal proteins were evenly applied daily. The basement membrane thickness of the skin was observed microscopically using hematoxylin and eosin staining on days 3, 7, 14, and 21 of treatment.

[0074] Thickening effect of skin basement membrane. Figure 1 A) Compared with the blank group, the back skin of rats coated with the mussel foot gland mucin subunit prepared in Example 1 had a 10% to 20% thickening of the basement membrane ( Figure 1 B). The group receiving commercial mussel foot gland mucin ( Figure 1 C) Compared with the control group, there was no significant effect of skin basement membrane thickening.

[0075] Table 2 Mussel foot gland mucin subunits promote skin basement membrane thickening

[0076]

[0077] Effect Example 2

[0078] The molecular weight of the mussel foot gland mucin subunit prepared in Example 1 was determined by SDS-PAGE. Figure 2 As shown in the figure, the molecular weight of mussel byssal gland mucin subunits ranges from 9 to 13 kDa, with an average molecular weight of 11.10 ± 2 kDa.

[0079] The hair growth-promoting effect of a highly soluble mussel foot gland mucin subunit was evaluated in a female rat dorsal hair removal model. After one week of acclimatization, anesthetized female rats weighing 150-200 g had their dorsal hair shaved, 0.5 cm from the spine, midway between the forelimbs and hindlimbs. A commercially available depilatory cream was evenly applied to the dorsal area. After a short while, the hair was removed and the depilated area was rinsed with clean water. Daily application of the cream was performed, with 100 μL of cream applied to the depilated area on the rat's back. The cream was applied evenly until absorbed and dried on the rat's back, and the rat was returned to its home cage. On days 7 and 14 of treatment, the dorsal hair of rats treated with 10-100 mg / mL mussel foot gland mucin subunit grew denser, more covered, and faster than that of the positive control group. However, the commercial mussel foot gland mucin group showed no significant hair growth-promoting effect.

[0080] Table 3 Mucilagenous subunits of mussel foot gland promote hair growth

[0081]

[0082] Effect Example 3

[0083] The ultrastructure of the freeze-dried powder of mussel foot gland mucin subunit prepared in Example 1 was observed by scanning electron microscopy. Figure 3 As shown in the figure, mussel foot gland mucin extracted by the dual-enzyme urea nanofiltration reduction method appears as distinct elongated sheets under a scanning electron microscope, measuring 30-50 μm in length and 5-10 μm in width. These elongated structures have a uniform shape and relatively regular size, likely formed by the aggregation or rearrangement of mucin molecules under enzymatic degradation. Fibrous or ribbon-like structures at the cellular or polymeric level can be observed on the surface, demonstrating unique morphological characteristics formed during processing.

[0084] The efficacy of mussel foot gland mucin subunit in inducing skin regeneration was evaluated using a dorsal skin defect model in female rats. Anesthetized female rats weighing 150-200 g, which had been acclimated for one week, had skin removed from the back midway between the forelimb and hindlimb, 0.5 cm from the spine, using surgical instruments. This created a 0.8-1.0 cm diameter skin injury model. 80-100 μL of a highly soluble mussel foot gland mucin subunit solution was evenly applied daily. On days 3, 7, and 14 of treatment, skin regeneration in the rats treated with 10-100 mg / mL mussel foot gland mucin subunit was faster than that in the positive control rats. While commercially available mussel foot gland mucin promoted wound healing, the effect was weaker than that of the mussel foot gland mucin subunit obtained in this invention.

[0085]

[0086] Comparative Example 1

[0087] This comparative example is the preparation process in Control Example 1. Other processes are the same, except that the urea treatment is not performed. The finally prepared mussel foot gland mucin subunit is subjected to SDS-PAGE test. The SDS-PAGE results show that if urea treatment is not used, it will contain many macromolecular impurities.

[0088] Figure 4 This is the SDS-PAGE result of the mussel foot gland mucin subunit prepared in this comparative example.

[0089] Comparative Example 2

[0090] This comparative example is the preparation process in Control Example 1. Other processes are the same, except that dithiothreitol is not added. The mussel byssal gland mucin powder obtained is subjected to SDS-PAGE. The SDS-PAGE results show that if dithiothreitol is not used for treatment, it will contain a certain amount of multi-subunit molecules.

[0091] Figure 5 This is the SDS-PAGE result of the mussel foot gland mucin subunit prepared in this comparative example.

[0092] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for preparing mussel byssal gland mucin subunits, characterized in that: The following steps are involved: (1) Raw material pretreatment: Use mussel foot gland as raw material, clean it and set aside; (2) Double enzyme hydrolysis: add the pre-treated mussel foot gland in step (1) at 35-37 °C, add neutral protease solution under stirring, and the hydrolysis time is 1.5-3 h; then adjust the pH of the reaction system to 2.5-3.5, and add pepsin solution to continue the hydrolysis for 2-4 h; drain the hydrolyzed solution, and wash the intact inner core layer of the mussel foot gland with clean water 2-4 times after the hydrolysis, and set aside; The enzymatic activity of the neutral protease is 3000-6000 U / g, and the weight ratio of the neutral protease to the mussel foot gland is 1:3000-6000; The enzymatic activity of the pepsin is 1500-3000 U / g, the weight ratio of pepsin to mussel foot gland is 1:1500-3000, and the pH value of the enzymatic hydrolyzate after adding pepsin is maintained at 2.0-3.0; (3) Preliminary extraction and purification: urea solution was added to the core of the mussel byssal gland in step (2), and the mixture was homogenized using a 5-10 μm slit colloid mill. The mixture was centrifuged, and the supernatant was retained and 100-150 mM dithiothreitol was added to form a crude extract of byssal gland mucin. The concentration of the urea solution is 0.25-0.5 mol / L; (4) Microfiltration and nanofiltration: The crude extract is microfiltered through a 100-150 kDa ceramic membrane, and then the protein solution is concentrated using a 5-15 kDa nanofiltration membrane to reduce the volume of the protein solution to 1 / 20 to 1 / 10 of the original volume, forming a foot gland mucin subunit solution; (5) Post-processing: The byssal gland mucin subunit liquid is spray-dried or freeze-dried to obtain a highly soluble mussel byssal gland mucin subunit powder.

2. The method for preparing the mussel byssal gland mucin subunit according to claim 1, wherein The cleaning process in step (1) is as follows: the mussel foot gland is first treated with 450-500 W ultrasound for 40-45 minutes, and then rinsed with clean water until there is no impurity.

3. The method for preparing the mussel byssal gland mucin subunit according to claim 1, wherein In the step (2), the pH value of the reaction system during enzymatic hydrolysis with the neutral protease solution is 7.5-8.

5.

4. A mussel foot gland mucin subunit, characterized in that Obtained by the preparation method according to any one of claims 1 to 3, The molecular weight of the mussel byssal gland mucin subunit ranges from 9 to 13 kDa, with an average molecular weight of 11.10 ± 2 kDa.

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