Method for extracting carotenoid-protein compound from ampullaria gigas eggs and application of carotenoid-protein compound
The carotenoid-protein complex in golden apple snail eggs was extracted by gradient centrifugation, agarose gel electrophoresis, and freeze-thaw cycles. This method solves the problems of complex extraction methods and high costs in existing technologies, and achieves high purity and high recovery rate extraction results, which are applicable to the fields of medicine and cosmetics.
Patent Information
- Application Number
- CN202511600527.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-04
- Publication Date
- 2026-01-23
AI Technical Summary
Existing methods for extracting carotenoids from golden apple snail eggs are complex, costly, and produce low purity, which limits their large-scale production and application.
Carotenoid-protein complexes were extracted from golden apple snail eggs using gradient centrifugation, agarose gel electrophoresis, and freeze-thaw cycles. Extraction conditions were optimized to improve purity and recovery rate.
It achieves a carotenoid purity of 92.3% and a recovery rate of 85.7%, simplifies the extraction process, reduces costs, and provides a green and environmentally friendly extraction method suitable for high-quality fields such as pharmaceuticals and cosmetics.
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Figure CN121370960A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biotechnology, in particular to a method for extracting carotenoid-protein complex in Pomacea canaliculata and application thereof. BACKGROUND
[0002] Pomacea canaliculata belongs to Mollusca, Gastropoda, Mesogastropoda, Ampullariidae and Pomacea, and is originally from the Amazon River Basin in South America. It is the only kind of freshwater snail listed in the global 100 kinds of malignant alien species. Due to its ecological characteristics such as fast growth, high reproduction, large food intake, mixed diet and strong stress resistance, Pomacea canaliculata not only causes serious harm to crop production, water ecological system and biodiversity in the invaded area, but also spreads Fuke and Guangzhou Guanwan, threatening human life and health. Therefore, it is urgent to prevent and control Pomacea canaliculata disaster.
[0003] Carotenoids are fat-soluble natural pigments, which exist universally in microorganisms, plants, animals and human bodies, have important biological functions such as antioxidant, antitumor and immune enhancement, and are widely used in medical health, food health and other fields. It is found that Pomacea canaliculata eggs and perivitelline fluid contain rich carotenoids, 99% of which are astaxanthin and its ester. Carotenoids in Pomacea canaliculata eggs can be obtained by extraction with organic solvents such as methanol, anhydrous ethanol and acetone. The extraction rate is not only related to the type of organic solvent, but also affected by extraction process, light, temperature, volume ratio and other factors, which limits the large-scale production and application of carotenoids. In addition, there are reports in the prior art that astaxanthin is extracted from Pomacea canaliculata eggs and ovaries by using solvent ultrasonic extraction, enzymatic hydrolysis and silica gel column purification, but the extraction process is complex, the cost is high, and the purity is low. SUMMARY
[0004] The purpose of the present application is to provide a method for extracting carotenoid-protein complex in Pomacea canaliculata eggs and application thereof, so as to solve the problems existing in the prior art. The total carotenoid content in the carotenoid-protein complex extracted from Pomacea canaliculata eggs by the method of the present application can reach 45.6 mg / 100g fresh egg block, the carotenoid purity is 92.3%, and the recovery rate is 85.7%. The present application provides a green technical approach for resource utilization and harmless treatment of Pomacea canaliculata eggs, and provides a new idea for biological control and sustainable management of Pomacea canaliculata.
[0005] To achieve the above purpose, the present application provides the following solutions.
[0006] The application provides a method for extracting carotenoid-protein complex in apple snail eggs, comprising the following steps:
[0007] The apple snail egg pieces are added into distilled water for homogenization and gradient centrifugation, and the supernatant is collected to obtain egg perivitelline fluid;
[0008] The egg perivitelline fluid is subjected to agarose gel electrophoresis, and the pink band appearing in the gel is taken out and heated to melt to obtain a mixture;
[0009] The mixture is incubated on ice, and after freezing, thawing and centrifugation, the supernatant is collected, which is the carotenoid-protein complex.
[0010] Preferably, the weight / volume ratio of the apple snail egg pieces to the distilled water is 1g:3mL.
[0011] Preferably, the gradient centrifugation is carried out at 10℃, 10,000xg for 20min, and then at 10℃, 100,000xg for 50min.
[0012] Preferably, the agarose gel concentration of the agarose gel electrophoresis is 2.5%, the voltage is 100V, and the time is 1h.
[0013] Preferably, the incubation time on ice is 30min.
[0014] Preferably, the freezing condition is freezing at-20℃ overnight or at-70℃ for 2h.
[0015] Preferably, the centrifugation condition is centrifugation at 4℃, 16,000xg for 20min.
[0016] The application also provides the use of the carotenoid-protein complex prepared by the method in the preparation of medicines, foods or health products.
[0017] The application discloses the following technical effects:
[0018] The application aims at the problems in the prior art, and explores a non-toxic and harmless extraction method to further reduce the extraction cost, shorten the extraction period and improve the product purity, so as to be applied to the medicine and cosmetic industries with higher quality requirements. The carotenoid-protein complex in apple snail eggs is extracted by the method, the total carotenoid content can reach 45.6mg / 100g of fresh egg pieces, the carotenoid purity is 92.3%, and the recovery rate is 85.7%.
[0019] The carotenoid-protein complex in the apple snail eggs is extracted, a new way for the resource utilization and harmless treatment of the apple snail is provided, the combination of prevention and utilization is realized, the harm is changed into the treasure, the utilization promotes the prevention, and the prevention and utilization are win-win. Moreover, the toxic and harmful organic solvents are avoided, the extraction method is simple, fast, green, environmental protection, low in cost and high in efficiency. In a word, the green technical way for the resource utilization and harmless treatment of the apple snail eggs is provided, and the new idea for the biological prevention and sustainable management of the apple snail is provided. BRIEF DESCRIPTION OF DRAWINGS
[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.
[0021] Figure 1 The flow chart of the method for extracting the carotenoid-protein complex in the apple snail eggs is shown in the figure.
[0022] Figure 2 The HPLC detection chart of the carotenoid in the product extracted from Example 1 and Comparative Examples 1-8 is shown in the figure. Standard: Example 1, Control 1- Control 8: Comparative Examples 1-8.
[0023] Figure 3 The purity and recovery rate statistics chart of the carotenoid in the product extracted from Example 1 and Comparative Examples 1-8 is shown in the figure. Standard: Example 1, Control 1- Control 8: Comparative Examples 1-8. DETAILED DESCRIPTION
[0024] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of some aspects, characteristics and embodiments of the present application.
[0025] It should be understood that the terms described in the present application are only for describing the specific embodiments, and are not used to limit the present application. In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value in the stated range, and any other stated value or intermediate value in the stated range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.
[0026] All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains, unless indicated otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference is not an admission that it is prior art with respect to the present application. All literature and similar materials cited in this application, including but not limited to, patents, genetic code, scientific and / or technical articles, and / or treatises, are expressly incorporated by reference.
[0027] Many modifications and variations of this application of the application can be made without departing from its spirit or scope, which will be apparent to those skilled in the art. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only. It is to be understood that the application is not limited in scope by the
[0028] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms "comprises", "comprising", "includes", "including" and the like are open-ended terms that are intended to denote the presence of stated features, items, actions, components or the like, but do not exclude the presence of one or more other features, items, actions, components or the like.
[0029] The flow chart of the method for extracting carotenoid-protein complex in Pomacea canaliculata eggs provided by the present application is shown in Figure 1 The specific embodiments are shown in the following examples.
[0030] Example 1: A method for extracting carotenoid-protein complex in Pomacea canaliculata eggs
[0031] S1: Collect Pomacea canaliculata egg pieces, and after weighing, add distilled water at a weight-to-volume ratio of 1:3 (g:mL) for homogenization.
[0032] S2: Perform gradient centrifugation under low-temperature conditions, specifically 10℃, 10,000 x g for 20 min, and then 10℃, 100,000 x g for 50 min, and collect the supernatant to obtain egg perivitelline fluid.
[0033] S3: Take 160 μL of the egg perivitelline fluid, mix it with 40 μL of TBE loading buffer, and add it to a 2.5% agarose gel, and perform non-denaturing electrophoresis at a voltage of 100 V for 1 h.
[0034] S4: Cut out the pink band in the gel and place it in a 1.5 mL microcentrifuge tube. Estimate the volume of the gel piece by weight, and add extraction buffer (50 mM Tris-HCl and 1 mM EDTA, pH 8.0) to make the final concentration of agarose 0.5%. Then, melt the gel piece by heating to the melting temperature of the agarose used.
[0035] S5: The mixture was incubated on ice for 30 min, then frozen at -20°C overnight (alternatively, it can be frozen at -70°C for 2 h). After that, the mixture was thawed on ice and centrifuged at 4°C at 16,000 x g for 20 min, and the supernatant containing the carotenoid-protein complex was collected. Note: If the amount of protein in the recovered carotenoid-protein complex is very low, it can be concentrated using a microcentrifugal filter unit with an upper molecular weight cut-off of 100,000.
[0036] The supernatant containing the carotenoid-protein complex obtained above was subjected to routine methods for total carotenoid content, purity, protein content, UV-Vis λmax, HPLC retention time, and mass spectrometry detection, and the results are shown in Table 1. Among them, the specific detection methods of HPLC-DAD and ESI-MS are as follows.
[0037] HPLC-DAD: Column: C18 reversed-phase column (250 mm x 4.6 mm, 5 μm); mobile phase: acetonitrile (A) and water (B), gradient elution program: 0-5 min: 90% A, 10% B; 5-10 min: 95% A, 5% B; 10-15 min: 100% A; flow rate: 1.0 mL / min; detection wavelength: 472 nm; injection volume: 20 μL; column temperature: 25°C.
[0038] ESI-MS: Ion source: electrospray ionization (ESI), positive ion mode; scan range: m / z 100-1000; capillary voltage: 3.5 kV; dry gas flow rate: 10 L / min; dry gas temperature: 300°C; nebulizing gas pressure: 30 psi.
[0039] Table 1
[0040] Analysis item Value / characteristic Method Total carotenoid content 45.6 mg / 100 g fresh egg mass UV-Vis quantification (ε472=125000 L / mol / cm) Purity 92.3% HPLC-DAD (λ=472 nm) Protein content 548 mg / 100 g fresh egg mass BCA method Carotenoid: protein molar ratio 1:3 Calculated from the above quantitative data UV-Vis λmax 472 nm Ultra-violet-visible HPLC retention time 5.42 min (consistent with astaxanthin standard) Acetonitrile: water gradient elution Mass spectrum m / z 597.5 (astaxanthin) ESI-MS
[0041] Comparative Example 1
[0042] The same as Example 1, except that the weight to volume ratio in step S1 was 1:2 (g:mL).
[0043] Comparative Example 2
[0044] The same as Example 1, except that the weight to volume ratio in step S1 was 1:4 (g:mL).
[0045] Comparative Example 3
[0046] The same as Example 1, except that the centrifugation operation in step S2 was at 10°C, 10,000 x g for 60 min.
[0047] Comparative Example 4
[0048] The same as example 1, the only difference is that the centrifugation operation in step S2 is 10℃, 50,000xg centrifugation for 30min, then 10℃, 100,000xg centrifugation for 30min.
[0049] Comparative Example 5
[0050] The same as example 1, the only difference is that in step S3, non-denaturing electrophoresis is performed using 2.0% agarose gel.
[0051] Comparative Example 6
[0052] The same as example 1, the only difference is that in step S3, denaturing electrophoresis is performed using 12% polyacrylamide gel.
[0053] Comparative Example 7
[0054] Methanol ultrasonic extraction is used, and the specific steps are as follows:
[0055] The apple snail egg mass is collected, weighed, and then 1:3 (g:mL) of methanol is added.
[0056] The mixture is placed in an ultrasonic cleaner, and ultrasonic extraction is performed for 30 min, with an ultrasonic frequency of 40 kHz and a power of 200 W. The temperature is maintained below 25℃ during the extraction process.
[0057] After the extraction is completed, centrifugation is performed at 10℃ and 10,000xg for 20 minutes, and the supernatant is collected.
[0058] The extraction is repeated twice, and the supernatants are combined.
[0059] The supernatant is removed by a rotary evaporator to obtain the extract.
[0060] Ethanol ultrasonic extraction is used, and the specific steps are as follows:
[0061] The apple snail egg mass is collected, weighed, and then 1:3 (g:mL) of anhydrous ethanol is added.
[0062] The mixture is placed in an ultrasonic cleaner, and ultrasonic extraction is performed for 40 min, with an ultrasonic frequency of 40 kHz and a power of 250 W. The temperature is maintained below 30℃ during the extraction process.
[0063] After the extraction is completed, centrifugation is performed at 10℃ and 10,000xg for 20 minutes, and the supernatant is collected.
[0064] The extraction is repeated twice, and the supernatants are combined.
[0065] The supernatant is removed by a rotary evaporator to obtain the extract.
[0066] The total carotenoid yield, purity and recovery rate of the extracted product of Example 1 and Comparative Examples 1-8 were detected, and the results are shown in Table 2, Figure 2 - Figure 3 .
[0067] Table 2
[0068] Group Total carotenoid yield (mg / 100 g fresh egg mass) Purity (%) Recovery (%) Remarks Example 1 45.6 92.3 85.7 Best Comparative Example 1 30.2 80.1 56.8 Increased solubility and loading viscosity Comparative Example 2 28.5 78.4 53.6 Over-dilution, increased centrifugation loss Comparative Example 3 25.7 75.2 48.2 High-speed protein not fully separated Comparative Example 4 35.8 82.5 67.4 Complex medium-speed step sample Comparative Example 5 40.1 88.7 75.4 Slightly poor band resolution Comparative Example 6 20.3 70.4 38.1 Protein denaturation, carotenoid loss Comparative Example 7 80.5 60.2 78.3 High impurity content, high purification cost Comparative Example 8 74.2 58.9 72.1 High impurity content, high purification cost
[0069] Note: The recovery rate is 100% relative to the total amount that can be measured in Comparative Example 7.
[0070] As can be seen from Table 2, the purity of carotenoids extracted by the method of the present application is as high as 92.3%, and the recovery rate is also the highest, which is 85.7%. Although the total carotenoid yield of Comparative Example 7 and Comparative Example 8 is higher than that of Example 1, the purity is significantly lower than that of Example 1 and the extracted product of Comparative Examples 7-8 contains many impurities, which has high purification cost; Comparative Example 1 increases the amount of Pomacea canaliculata egg mass, resulting in increased solubility and sample viscosity; Comparative Example 2 reduces the amount of Pomacea canaliculata egg mass, resulting in excessive dilution and increased centrifugal loss; Comparative Examples 3-4 change the centrifugal conditions, resulting in insufficient separation of high-speed proteins or tedious problems of medium-speed step sample retention; Comparative Example 5 uses 2.0% agarose gel for non-denaturing electrophoresis, and the resolution of the obtained band is slightly poor; Comparative Example 6 uses other electrophoresis forms for electrophoresis, resulting in protein denaturation and loss of carotenoids. Only the carotenoids obtained according to Example 1 are the best.
[0071] The above-described examples only describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art shall fall within the protection scope determined by the claims of the present application.
Claims
1. A method for extracting carotenoid-protein complexes from golden apple snail eggs, characterized in that, Includes the following steps: Take golden apple snail egg masses, add distilled water, homogenize, and centrifuge in a gradient. Collect the supernatant to obtain peri-egg fluid. The periovarian fluid was subjected to agarose gel electrophoresis. The pink bands that appeared in the gel were removed and heated to melt them, resulting in a mixture. The mixture was incubated on ice, then frozen, thawed, and centrifuged. The supernatant was collected, which is the carotenoid-protein complex.
2. The method as described in claim 1, characterized in that, The weight-to-volume ratio of the golden apple snail egg mass to the distilled water is 1g:3mL.
3. The method as described in claim 1, characterized in that, The gradient centrifugation conditions are: centrifugation at 10℃ and 10,000×g for 20 min, followed by centrifugation at 10℃ and 100,000×g for 50 min.
4. The method as described in claim 1, characterized in that, The agarose gel concentration for the agarose gel electrophoresis was 2.5%, the voltage was 100 V, and the time was 1 h.
5. The method as described in claim 1, characterized in that, The incubation time on ice is 30 minutes.
6. The method as described in claim 1, characterized in that, The freezing conditions are -20°C overnight or -70°C for 2 hours.
7. The method as described in claim 1, characterized in that, The centrifugation conditions were 4°C, 16,000 × g for 20 min.
8. The use of the method according to any one of claims 1-7 in the preparation of carotenoid-protein complexes.
9. The use of the carotenoid-protein complex prepared by the method according to any one of claims 1-7 in the preparation of pharmaceuticals, food or health products.