Astaxanthin binding protein derived from euphausia superba and use thereof

Abstract

The present application relates to a kind of astaxanthin binding protein derived from Antarctic krill and its application, belong to the field of biotechnology, the astaxanthin binding protein amino acid sequence as shown in SEQ ID NO.1, the present application also provides the application of the astaxanthin binding protein in stabilizing astaxanthin, astaxanthin binding protein is added in astaxanthin solution, astaxanthin binding protein can be combined with astaxanthin and form stable complex, can accurately protect the unsaturated structure of astaxanthin, significantly improve its stability to light, heat and other environmental factors, help to improve its bioavailability in functional food or pharmaceutical field.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for stabilizing astaxanthin based on endogenous proteins from Antarctic krill and its application. Background Technology

[0002] Astaxanthin, also known as astaxanthin, is an oxygen-containing carotenoid derivative with a chemical structure similar to β-carotene. It is found in microorganisms and marine animals. Astaxanthin's unique molecular structure endows it with a variety of biological functions, making it widely used in food, medicine, feed, and health products, with a promising market prospect. Studies have confirmed that in addition to its strong antioxidant properties, astaxanthin also has anti-tumor effects, helps prevent cardiovascular and cerebrovascular diseases, protects against ultraviolet radiation, enhances immunity, slows aging, reduces inflammation, and improves vision.

[0003] Antarctic krill is a small crustacean zooplankton living in the Southern Ocean and is one of the largest single-species biological resources on Earth. Research has found that Antarctic krill is not only rich in nutrients such as protein and fat, but also contains abundant astaxanthin and other bioactive substances. Currently, researchers have conducted systematic identification of astaxanthin in Antarctic krill and its biological products. Studies have shown that Antarctic krill oil contains up to 340.93 mg / kg of astaxanthin, whose molecules are mainly composed of astaxanthin diesters, monoesters, and a small amount of free astaxanthin. The esterified form makes it more stable than the free astaxanthin. Furthermore, researchers have used modern analytical techniques to clarify the composition of three optical isomers and revealed the dynamic changes of astaxanthin molecules during on-site processing. The accumulation of this fundamental data has greatly promoted the efficient utilization and targeted development of Antarctic krill astaxanthin.

[0004] However, due to its highly unsaturated molecular structure, astaxanthin is highly susceptible to oxidative degradation during processing and storage due to factors such as light, heat, and oxygen, which has become a technical bottleneck limiting its widespread application. To address this challenge, researchers have developed various stabilization techniques. For example, using maltodextrin and hydroxypropyl-β-cyclodextrin as wall materials to prepare microcapsules via spray drying can significantly improve the retention rate of astaxanthin under high temperature, light, and aerobic conditions. Furthermore, encapsulation with exogenous proteins such as bovine serum albumin and ovalbumin is also an effective way to improve its stability. However, research on how to utilize proteins derived from Antarctic krill to preserve astaxanthin for efficient utilization remains lacking. Summary of the Invention

[0005] This invention provides an astaxanthin-binding protein derived from Antarctic krill and its application. This technology enables the efficient stabilization of astaxanthin, realizing its application value.

[0006] To achieve the above-mentioned objectives, the present invention employs the following technical solution:

[0007] One of the objectives of this invention is to provide an astaxanthin-binding protein derived from Antarctic krill, wherein the protein is hemocyanin and its amino acid sequence is shown in SEQ ID NO.1.

[0008] A biological agent for preventing astaxanthin oxidation, said biological agent containing astaxanthin-binding protein.

[0009] The present invention also provides the application of the astaxanthin binding protein in stabilizing astaxanthin, wherein the astaxanthin binding protein is added to the astaxanthin solution, and the astaxanthin binding protein can bind with astaxanthin to form a stable complex.

[0010] Furthermore, a method for preparing a solution containing the astaxanthin-binding protein:

[0011] Step 1: Grind the Antarctic krill meat sample and add salt solution, stirring until fully mixed;

[0012] Step 2, protein solution fractionation and extraction: After the solution system prepared in Step 1 is allowed to stand, it is repeatedly centrifuged at low temperature, and the supernatant is retained. The supernatant is the solution containing the astaxanthin binding protein.

[0013] Furthermore, in step one, tissue grinding includes, but is not limited to, liquid nitrogen grinding, high-throughput tissue grinder grinding, etc.; the salt solution includes salt solution (1) and salt solution (2), wherein the salt solution (2) is a 150 mM NaCl solution, and the salt solution (2) is a solution composed of 25 mM NaCl and 5 mM histidine with a pH of 7.4.

[0014] Furthermore, in step one, the ratio of Antarctic krill meat sample to salt solution (1) and salt solution (2) is 1:10 (m:v) and 1:20 (m:v), respectively, and the mixing method includes, but is not limited to, stirring, vortexing, and sonication;

[0015] Furthermore, in step two, the settling time is 10 minutes, the ambient temperature is 4°C, and the centrifugation is performed at 8000-10000 g for 10 minutes;

[0016] Furthermore, after adding astaxanthin solution to the solution containing the astaxanthin binding protein, a reaction mixture is formed. In 1 mL of the reaction mixture, the final concentrations of protein, sodium cholate, and astaxanthin are 0.70-0.75 mg / mL, 200 mM, and 8.5 µM, respectively.

[0017] The present invention also provides the application of the biological agent in stabilizing astaxanthin.

[0018] The advantages of this invention compared to existing technologies are as follows: First, Antarctic krill is rich in high-quality protein resources, enabling the high-value utilization of krill protein resources; second, this strategy utilizes the natural affinity between homologous proteins and astaxanthin to achieve precise protection, avoiding immune rejection that may be caused by heterologous proteins; third, this binding method can precisely protect the unsaturated structure of astaxanthin, significantly improving its stability to environmental factors such as light and heat; finally, this strategy provides a green and simple solution for the efficient delivery of astaxanthin, helping to improve its bioavailability in functional foods or pharmaceuticals. Attached Figure Description

[0019] Figure 1 Assessment of the ability of Antarctic krill proteins to bind astaxanthin;

[0020] Figure 2 Identification of the astaxanthin binding site of Antarctic krill protein. Detailed Implementation

[0021] The technical solution of the present invention will be further explained below through embodiments, but the scope of protection of the present invention is not limited in any way by the embodiments.

[0022] Example 1: Preparation and fractionation of protein solution

[0023] Weigh 2 grams of Antarctic krill meat and grind it for 5 minutes at 4°C using a high-throughput tissue homogenizer. Add 10 mL of 150 mM NaCl solution (1) and homogenize the tissue using a vortex mixer for 1 minute. Centrifuge at 10,000 g for 20 minutes at 4°C and collect the supernatant as muscle protein solution A. Subsequently, at 4°C, extract the particles three times with 20 times the volume of 25 mM NaCl, 5 mM histidine, and pH 7.4 solution (2) for 10 minutes each time. Collect the supernatant and name them muscle protein solution B, muscle protein solution C, and muscle protein solution D, respectively.

[0024] Example 2 Astaxanthin Binding Capacity Assessment

[0025] Centrifuge at 5000 rpm for 10 min using a 5K Millipore ultrafiltration centrifuge tube to adjust the protein concentration in the muscle protein solution to between 1.4 and 1.5 mg / mL. Mix the dissolved muscle protein solution and an astaxanthin solution containing 400 mM sodium cholate at a 1:1 ratio in a brown glass bottle, with a total volume of 1 mL, to achieve final concentrations of 0.70–0.75 mg / mL for protein, 200 mM for sodium cholate, and 8.5 µM for astaxanthin. Vortex briefly and incubate at room temperature for 30 min. Centrifuge at 3500 g for 45 seconds through a 30 kDa Millipore filter, discard the initial filtrate, and centrifuge again for 7 min to separate protein-bound and unbound astaxanthin. Transfer the sample and eluent to a microquartz cuvette, measure the absorbance at 485 nm using a spectrophotometer, and calculate the amount of astaxanthin bound per gram of protein. The results showed that the astaxanthin binding capacity of different batches of muscle protein solutions varied, with values ​​of 1.17 mg·g⁻¹ protein and 0.94 mg·g⁻¹ protein, respectively. -1 Protein, 0.85 mg / g -1 Protein and 1.07 mg / g -1 protein( Figure 1 ).

[0026] Example 3 Protein Identification and Site Recognition

[0027] Take 30 μL of sample and 10 μL of 4× protein loading buffer, mix thoroughly, and incubate in a boiling water bath for 10 min. After incubation, remove the sample, cool to room temperature, add 5 μL of protein marker and 15 μL of sample to the wells of a gradient-like PAGE gel, and perform electrophoresis at a constant voltage of 200 V. Electrophoresis is terminated when the protein migrates to the appropriate position in the gel and forms a clear band. Subsequently, the gel band containing the target protein is accurately cut and sent to Shanghai Meiji Biotechnology Co., Ltd. for proteomics identification. The results show that all four protein solutions are composed of Hemocyanin protein. Based on the obtained Hemocyanin amino acid sequence SEQ ID NO.1, the protein crystal structure is predicted using the Chai discovery online tool. The 3D structure of astaxanthin is downloaded from the PubChem database. Molecular docking is performed using AutoDock Vina 1.2.3 software, and the docking results are visualized using PyMol 2.5.2. The results show that astaxanthin and Antarctic krill protein communicate through a network dominated by an extensive hydrophobic network (key residue LEU). 578 GLU 449 and LEU 556 ), and is assisted by a single high-quality hydrogen bond (key residue SER) 468 The interaction mode of ) forms a stable complex ( Figure 2 ).

[0028] Astaxanthin-binding protein (SEQ ID NO.1)

[0029] MKGPSFLLGCLLAGALASQTQASDPKNQQDINTLLWKVYEPLRKAKMGHPPTDGFSPVGDLAMYSDGGKAAQNLEADVQDGRLLKQKNYFSLFNPRHRQEALMLFETLMNCKDFSPCVSDNAAYFRDIMNEGVFIYALYVSVIHHPIAEGLVLPPLYEVTPHMFTNSEVINQAYTAKMTATPGKFTMGYTGSQKNPEQRVAYFGEDIGMNVHHVSWHMDFPFWWKDSYGYNLDRKGELFFWVHHQLTARFDAERLSNNLNMVDELYWDQPIYEGFAPHTTYKYGGEFPARPDNMKFEDVEGVARVRDLRVWEDRIRDAIAHGYVTGKEGTIIDILNDRGVDIIGDIIESSEYSPNPVYYGALHNTAHIVLGRQGDPKGKFKQPPGVMEHFETATRDPSFFRLHKYMNNIFKEFKDRLPPYTYDELNFEGVSIESVSIEGSLETFFEDYEFSLTNAVDDTAELEDVAISAEVKRLNHKPFSFILNINNNNAAAVTASARIYLCPRRDNNDVPFHPNFGRWGCIEMDKFWADLAPGANTIVRKSSESAVTVPDSPSFLEMIERTDKAAASGAASSGLEELSRTCGIPDRLFLPKGKEEGLEMVLMSFVSDGATDHTDTFQVGGHYGGTHAHCGIHGQKYPDKRPMGFPIDRQITDFRMTAQVTNFKNTLVYVYHKKSA。

Claims

1. An astaxanthin-binding protein derived from Antarctic krill, characterized in that, The amino acid sequence of the astaxanthin-binding protein is shown in SEQ ID NO.

1.

2. The application of the astaxanthin-binding protein according to claim 1 in stabilizing astaxanthin, characterized in that, When the astaxanthin-binding protein is added to the astaxanthin solution, the astaxanthin-binding protein can bind with astaxanthin to form a stable complex.

3. The application according to claim 2, characterized in that, Method for preparing a solution containing the astaxanthin-binding protein: Step 1: Grind the Antarctic krill meat sample and add salt solution, stirring until fully mixed; Step 2, protein solution fractionation and extraction: After the solution system prepared in Step 1 is allowed to stand, it is repeatedly centrifuged at low temperature, and the supernatant is retained. The supernatant is the solution containing the astaxanthin binding protein.

4. The application according to claim 3, characterized in that, Step one, tissue grinding includes, but is not limited to, liquid nitrogen grinding or high-throughput tissue grinder grinding; the salt solution includes salt solution (1) and salt solution (2), wherein the salt solution (1) is a 150 mM NaCl solution and the salt solution (2) is a solution composed of 25 mM NaCl and 5 mM histidine with a pH of 7.

4.

5. The application according to claim 4, characterized in that, In step one, the mass-to-volume ratio of Antarctic krill meat sample to salt solution (1) and salt solution (2) is 1:10 and 1:20, respectively, and the mixing methods include, but are not limited to, stirring, vortexing and ultrasound.

6. The application according to claim 5, characterized in that, In step two, the settling time is 10 minutes, the ambient temperature is 4℃, and the centrifugation is performed at 8000-10000 g for 10 minutes.

7. The application according to claim 6, characterized in that, After adding astaxanthin solution to the solution containing the astaxanthin binding protein, a reaction mixture is formed. In 1 mL of the reaction mixture, the final concentrations of protein, sodium cholate, and astaxanthin are 0.70-0.75 mg / mL, 200 mM, and 8.5 µM, respectively.

8. A biological agent for preventing astaxanthin oxidation, characterized in that, The biological agent contains the astaxanthin-binding protein as described in claim 1.

9. The use of the biological agent according to claim 8 in stabilizing astaxanthin.

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