Extraction method of carotenoid-rich chromosome substructure
By using frozen extraction buffer and density gradient centrifugation combined with sonication in wolfberry, substructures were isolated from the non-colors, solving the problems of carotenoid extraction and stability, and achieving high content and high bioaccessibility carotenoid extraction effect.
Patent Information
- Application Number
- CN202510179388.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-18
- Publication Date
- 2025-05-23
AI Technical Summary
The prior art is difficult to effectively extract and stabilize carotenoids, especially due to their hydrophobicity and easy degradation problems, resulting in a decrease in biological activity.
The content and bioaccessibility of carotenoids were improved by adding frozen extraction buffer to the wolfberry and then homogenized by density gradient centrifugation and sonication.
It has achieved efficient extraction of carotenoid-rich substructures from wolfberry. It has a smaller size and higher content, which may be better absorbed and utilized by the human body, and is easier to disperse evenly in the food matrix, improving the texture and taste of the food.
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Figure CN120021768A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of extracting pigments from natural raw materials, and specifically relates to a method for extracting chromoplast substructures and the obtained chromoplast substructures. Background Art
[0002] As people's living standards improve, consumers' health awareness has significantly increased, and the market demand for dietary supplements has continued to rise. Lutein (also known as zeaxanthin) is a carotenoid with multiple functions, including antioxidant, antiparasitic, anti-osteoporosis, anti-inflammatory and anti-cancer effects, as well as maintaining the integrity of biological membrane structure and function (Bouyahya et al., 2021; Demmig-Adams et al., 2020), an important class of dietary supplements. Many studies have also found that zeaxanthin has a potential role in maintaining eye health and preventing ophthalmic diseases, especially reducing the risk of age-related macular degeneration (Johra, FT, Bepari, AK, Bristy, AT, & Reza, HM (2020). A mechanistic review of β-carotene, lutein, and zeaxanthin in eye health and disease. Antioxidants, 9(11), Article 11. https: / / doi.org / 10.3390 / antiox9111046). Zeaxanthin is also abundant in brain tissue, especially in areas closely related to learning, memory, and neurotransmission. Therefore, they are thought to play an important role in improving and maintaining cognitive function throughout life. Carotenoids have received considerable attention due to their potential positive effects on health, but their hydrophobicity and interactions with other components of the human dietary matrix have adverse effects on their bioaccessibility. In addition, carotenoids are easily degraded by external environmental factors such as light, heat, and oxygen, resulting in reduced bioactivity (Meléndez-Martínez, AJ, Esquivel, P., & Rodriguez-Amaya, DB (2023). Comprehensive review on carotenoid composition: Transformations during processing and storage of foods. Food Research International, 169, 112773. https: / / doi.org / 10.1016 / j.foodres.2023.112773).
[0003] Carotenoids can be synthesized in various plastids in plants and accumulated in chromoplasts in plant roots, fruits and petals. Chromoplasts originate from chloroplasts or other non-green plastids and store a large amount of carotenoids by generating and accumulating carotenoid-lipoprotein substructures. According to the morphological characteristics of these storage substructures, chromoplast substructures can be divided into spherical, crystalline, membranous, fibrous, tubular and other types. Furthermore, these different deposition patterns within the substructures (or sub-structures) have intrinsic effects on the properties, stability, and bioaccessibility of carotenoids (Schweiggert, RM, Mezger, D., Schimpf, F., Steingass, CB, & Carle, R. (2012). Influence of chromoplast morphology on carotenoid bioaccessibility of carrot, mango, papaya, and tomato. Food Chemistry, 135(4), 2736–2742. https: / / doi.org / 10.1016 / j.foodchem.2012.07.035).
[0004] Extracting chromoplast substructures from plant resources by improving extraction technology is a beneficial approach to obtain more stable and better bioaccessible carotenoids. Summary of the invention
[0005] In order to solve the above problems existing in the prior art, the present invention provides a method for extracting chromoplast substructures rich in carotenoids.
[0006] A second object of the present invention is to propose the products obtained by said process.
[0007] The technical solution for achieving the above-mentioned purpose of the present invention is:
[0008] A method for extracting chromoplast substructures rich in carotenoids comprises the steps of:
[0009] 1) adding frozen extraction buffer to wolfberry and homogenizing with a stirrer; the ratio of wolfberry to frozen extraction buffer is 1 g: 8-15 mL, and the frozen extraction buffer contains HEPES (4-hydroxyethylpiperazineethanesulfonic acid), sorbitol, EDTA and DTT (dithiothreitol) to obtain a suspension;
[0010] 2) Filter the suspension through four layers of Miracloth and centrifuge the filtrate at 1000-5000g for 15-25 minutes. The precipitate obtained by centrifugation is the crude chromoplast;
[0011] 3) Gently resuspend the crude chromoplasts in an extraction buffer containing 5-15% (w / v in g / mL, e.g. 5% means 5 g of Nycodenz in 100 mL) Nycodenz, overlaying the extraction buffer with a discontinuous gradient density, separating the mixed solution into layers by centrifugation, and separating the layers of liquid obtained by centrifugation;
[0012] 4) The separated liquid layers are diluted with the frozen extraction buffer, and Nycodenz is removed by centrifugation;
[0013] 5) Gently resuspend the intermediate product obtained in step 4) with 15-20% (w / v) Nycodenz buffer, and then perform ultrasonic treatment; the ultrasonically treated samples are sequentially covered with extraction buffer containing 5% to 12.5% (w / v) gradient concentrations of Nycodenz, the mixed solution is separated by centrifugation, the substructure layers of the Nycodenz gradient between the layers are collected, and then centrifuged to remove Nycodenz (iohexol).
[0014] Wolfberry (Lycium barbarum L.) is a rich source of zeaxanthin, up to 38 mg / 100 g FW (fresh weight), mainly in the esterified form of zeaxanthin dipalmitate (ZDP). It is speculated that the carotenoids in wolfberry exist in a liquid crystal state in nanoscale tubular substructures at the fully mature stage. At present, related research is relatively limited. This study isolated tubular substructures from fresh mature wolfberry through density gradient separation, providing technical and theoretical support for the effective integration of new carotenoid carrier systems in the field of food processing.
[0015] In step 1), it is preferred to select mature, bright red, well-shaped wolfberries and rinse them. About 250 mL of frozen extraction buffer can be added for every 25 g of wolfberries.
[0016] Furthermore, in step 1), the frozen extraction buffer contains 50 mM HEPES, 330 mM sorbitol, 2 mM EDTA, 5 mM DTT, pH 7 to 8. The pH value of the frozen extraction buffer can be adjusted with 0.1 to 2 M sodium hydroxide solution or a pH adjuster known in the art.
[0017] Preferably, in step 1), homogenization is performed at 300 W with a pre-cooled stirrer for 3 to 5 s (pre-cooled to a temperature of 0 to 4° C.).
[0018] In order to further purify the chromoplasts, in step 3), the crude chromoplasts are gently resuspended in an extraction buffer having a Nycodenz content of 5%, 7.5%, 10%, 12.5% and 15%, w / v gradient concentrations, and after centrifugation, the intermediate suspension solutions of each layer between 0% and 15% are collected.
[0019] It is preferred to collect the solution suspended in the middle of each layer between 5% and 15% (including all layers between 5% and 15%, but excluding the interface layer between 0% and 5% and the layer below 15%).
[0020] The operation of sequential covering is: the solution with high density is at the bottom, and the solutions with low density are added in order. The resuspension of crude chromoplasts is covered on the 5% extract, the 5% extract is covered on the 7.5%, and so on.
[0021] Further preferably, in step 3), the centrifugation conditions are: centrifugation at 5000-6000g for 50-80min at 4°C; collecting the obtained substructures, diluting with the frozen extraction buffer, and then centrifuging at 1000-5000g for 15-25min at 0-4°C to remove Nycodenz.
[0022] In step 4), after removing Nycodenz by centrifugation, the resulting precipitate is quickly frozen in liquid nitrogen, placed at 80° C. for 1.5 h, and then thawed on ice. The freeze-thaw process can promote the fragmentation of chromosomes and assist in the release of substructures by ultrasound.
[0023] Step 5) Using 15-20% (w / v) Nycodenz buffer has no effect on the extraction effect. The last layer can be set to any gradient greater than or equal to 15%. At the beginning of the experiment, the bottom layer uses 20%, and then preferably uses 15% (w / v) Nycodenz buffer to save raw materials.
[0024] Wherein, in step 5), the output power of the ultrasonic treatment is 60-100 W, and the ultrasonic treatment is performed for 3-8 min. More preferably, the output power of the ultrasonic treatment is 100 W, and the ultrasonic treatment is performed for 5 min.
[0025] Another preferred technical solution of the present invention is that in step 5), the ultrasonically treated sample is sequentially covered with extraction buffer having a concentration of 5%, 7.5%, 10%, and 12.5% (w / v) Nycodenz, and the mixture is stratified by centrifugation at 5800g for 60 min at 4°C, and the suspended solution in the middle of each layer between 7.5% and 12.5% is collected, and the collected solution is diluted with the frozen extraction buffer, and then centrifuged at 1000-5000g for 15-25 min at 0-4°C to remove Nycodenz.
[0026] Referring to the existing literature on organelle extraction, the extraction should be performed at low temperature to avoid damaging the effective components. The operations of steps 1) to 5) are all performed at a temperature of 0 to 4°C.
[0027] The chromoplast substructures obtained by the extraction method of the present invention.
[0028] Compared with the prior art, the present invention has the following beneficial effects:
[0029] Zeaxanthin is a naturally occurring hydrophobic carotenoid with multiple biologically active functions; chromoplast substructures are subcellular organelles / ultrastructures where plants accumulate abundant carotenoids. The present invention proposes a method for extracting carotenoid-rich chromoplast substructures, which separates substructures from wolfberry chromoplasts by density gradient centrifugation and ultrasonic treatment. Substructures are smaller in size than chromoplasts and have higher carotenoid content, and may be better absorbed and utilized by the human body; they are more easily evenly dispersed in food matrices, improving the texture and taste of food, thereby enhancing the biologically active functions of carotenoids such as zeaxanthin and broadening their application range in the food industry. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 Physical images of the extracted substructures for different gradient conditions.
[0031] Figure 2 Transmission electron microscopy images of the extracted substructures for different sonication conditions.
[0032] Figure 3 Light microscopy (LM) images of free carotenoids (A), chromoplasts (B) and substructures (C); and transmission electron microscopy (TEM) images of free carotenoids (D, G), chromoplasts (E, H) and substructures (F, I).
[0033] Figure 4 Figure 2 is the particle size distribution diagram of chromoplasts and substructures. DETAILED DESCRIPTION
[0034] The present invention will be further described below in conjunction with specific examples. The following examples are only used to illustrate the present invention in detail rather than to limit the present invention.
[0035] Fresh mature wolfberry (Lycium barbarum L. NingQi No. 10) used in the examples was purchased from Yinchuan Qijindian Food Trading Company. Nycodenz (chemically pure) was purchased from Bairdi Biotechnology Co., Ltd. (Beijing, China). Other chemicals used in this study were of analytical grade and were obtained from Beijing Sola Biotechnology Co., Ltd. (Beijing, China).
[0036] Unless otherwise specified, the technical means used in the specification are all known in the art. All raw materials used can be purchased commercially.
[0037] Preliminary experiment 1
[0038] 1) Select mature, bright red, well-shaped wolfberries, rinse, and remove the remaining stems. Use about 250 mL of chilled extraction buffer (50 mM HEPES, 330 mM sorbitol, 2 mM EDTA, 5 mM DTT, pH 7.5, pH adjusted with 1 M sodium hydroxide solution) per 25 g of wolfberries and homogenize them at 300 W using a pre-cooled stirrer (JYL-CO20E, Jiuyang, Beijing, China).
[0039] 2) The suspension was filtered through four layers of Miracloth (Millipore, Massachusetts, USA), and the mixed filtrate was centrifuged at 3000 g for 20 min at 4° C. The precipitate obtained by centrifugation is the crude chromoplast.
[0040] 3) Gently resuspend the crude chromoplasts in extraction buffer, overlay on a discontinuous density gradient (5%, 7.5%, 10%, 12.5% and 15% Nycodenz in extraction buffer, w / v), and centrifuge at 5800 g for 60 min at 4°C. Carefully collect the chromoplasts between the layers.
[0041] 4) Dilute the collected chromoplasts with the freezing extraction buffer, centrifuge at 3000 g for 20 min at 4°C to remove Nycodenz. Then, snap-freeze the obtained sample in liquid nitrogen, place at 80°C for 1.5 h, and then thaw on ice.
[0042] 5) Gently resuspend the intermediate product obtained in step 4) with 20% (w / v) Nycodenz buffer, with an output power of 80w for 5min. The ultrasonically treated samples were sequentially covered with extraction buffers with concentrations of 5%, 7.5%, 10%, 12.5% and 15% (w / v) Nycodenz, and centrifuged at 5800g for 60min at 4°C. Carefully collect the substructure layers of the Nycodenz gradient between each layer, and repeat the same steps to remove Nycodenz. The last washing step was repeated twice.
[0043] The purified chromoplasts and substructures were recovered for identification and immediately diluted with PBS (0.01 M, pH 7.2-7.4) to a specific content of about 50 nmol / mL of total carotenoids for further evaluation.
[0044] The core purpose of the crushing in step 1) is to crush the wolfberry fruit so that the chromoplasts in the wolfberry plant cells can be released. Appropriate homogenization time is the key to ensuring sufficient cell crushing. In preliminary experiments, it was found that if the homogenization time is too short (less than 4s), the cell crushing is incomplete, and a large number of chromoplasts are still wrapped in intact cells and cannot be effectively separated, resulting in a reduced extraction rate. If the homogenization time is too long (greater than 4s), the integrity of the chromoplasts will be damaged, thereby affecting the extraction of subsequent substructures.
[0045] Therefore, the homogenization time of the mixer during the extraction of crude chromoplasts was set to 4 s.
[0046] Preliminary Experiment 2
[0047] The basic operation is the same as that in Preliminary Experiment 1.
[0048] Lycium barbarum chromoplasts have a complex phospholipid bilayer. Freeze-thaw combined with ultrasonic treatment can effectively break up the chromoplasts, release the substructures therein, and make them evenly dispersed. The ice crystals formed during the freezing stage exert mechanical pressure on the membrane structure and internal tissue of the chromoplasts. During the thawing stage, the ice crystals melt, and the pressure and osmotic pressure inside the chromoplasts change, further destroying the integrity of their structure. In the preliminary experiments, microscopic observation of the chromoplasts after freeze-thaw revealed that their structure was basically destroyed, and a large number of substructures and chromoplast matrix substances aggregated into clumps. The high-frequency vibration and cavitation effect generated by the subsequent ultrasonic treatment can more easily act on the broken chromoplasts, making them more thoroughly broken and forming a good and evenly dispersed state.
[0049] Therefore, the preferred experimental conditions are as follows: after removing Nycodenz by centrifugation in step 4), the obtained chromoplast sample is quickly frozen in liquid nitrogen, placed at 80° C. for 1.5 h, and then thawed on ice.
[0050] Example 1
[0051] This embodiment provides a method for extracting chromoplast substructures rich in carotenoids, comprising the steps of:
[0052] 1) Select mature, bright red, well-shaped wolfberries, rinse, and remove residual stems. Each 25 g of wolfberries was homogenized with about 250 mL of chilled extraction buffer (50 mM HEPES, 330 mM sorbitol, 2 mM EDTA, 5 mM DTT, pH 7.5, pH adjusted with 1 M sodium hydroxide solution) at 300 W using a cold stirrer (JYL-CO20E, Jiuyang, Beijing, China) for 4 s.
[0053] 2) Filter the suspension through four layers of Miracloth (Millipore, Massachusetts, USA), and centrifuge the mixed filtrate at 3000g for 20 min at 4°C. The precipitate obtained by centrifugation is the crude chromoplast;
[0054] 3) Gently resuspend the crude chromoplasts in extraction buffer, overlay on a discontinuous density gradient (5%, 7.5%, 10%, 12.5% and 15% Nycodenz in extraction buffer, w / v), and centrifuge at 5800g for 60 min at 4°C. Carefully collect the chromoplasts between the layers;
[0055] 4) Dilute the collected chromoplasts with extraction buffer and centrifuge at 3000 g for 20 min at 4°C to remove Nycodenz. Then snap-freeze the obtained sample in liquid nitrogen, place at 80°C for 1.5 h, and then thaw on ice.
[0056] 5) Gently resuspend the intermediate product obtained in step 4) with 15% (w / v) Nycodenz extraction buffer, with an ultrasonic output power of 80w and an ultrasonic treatment time of 5min. The ultrasonically treated samples were sequentially covered with extraction buffers with concentrations of 5%, 7.5%, 10%, and 12.5% (w / v) Nycodenz, and centrifuged at 5800g for 60min at 4°C. Carefully collect the substructure layers of the Nycodenz gradient between each layer, and repeat the same steps to remove Nycodenz. The last washing step was repeated twice,
[0057] The purified chromoplasts and substructures were recovered for identification and immediately diluted with PBS (0.01 M, pH 7.2-7.4) to a specific content of about 50 nmol / mL of total carotenoids for further evaluation.
[0058] All the above preparation steps were carried out at 4°C or on ice.
[0059] right Figure 1 The analysis shows that after ultrasonic crushing (80w) of the chromoplasts collected at different gradients, the pigment components in the obtained substructures are basically distributed between 5% and 15% (0% or 15% refers to the interface layer, and the collected target substances are between the two interfaces), especially between 7.5% and 12.5. The chromoplasts obtained at different gradients were ultrasonically crushed and then subjected to gradient density centrifugation again. The gradients where the pigments were mainly enriched were all smaller than the gradients where the original chromoplasts were enriched, indicating that the density of the substructure was smaller than that of the chromoplasts. In subsequent experiments, 5-15% of the chromoplast layer was taken for ultrasonic optimization to obtain more pigment components, and more than 15% of the chromoplast layer was removed.
[0060] Example 2
[0061] This embodiment adopts the same operation as that of embodiment 1, wherein in step 5), 0-15% of the chromogen layer is taken for ultrasonic optimization, the output power of the ultrasonic treatment is 60, 80, 100w, and the ultrasonic treatment is performed for 5min.
[0062] The purified chromoplasts and substructures were recovered and identified.
[0063] Transmission electron microscopy (TEM) analysis: For negative staining sample preparation, substructures and free carotenoids were diluted to appropriate concentrations with PBS (0.01 M, pH 7.2-7.4). 3 μL of sample solution was deposited on a carbon-coated grid copper grid (mesh size 230) treated with glow discharge hydrophilicity. After incubation for 60 seconds, the residual sample solution was removed from the edge of the grid with a piece of filter paper. 3 μL of 2% uranyl acetate solution was quickly added and the stain was quickly removed with a filter paper. After repeating once, staining was performed with 3 μL of 2% uranyl acetate solution for 60 seconds and the staining solution was removed with a filter paper. The grid was allowed to dry in air and placed in a grid storage box. The grid was observed using a transmission electron microscope (ThermoFisher Tecnai Spirit, 120 kV) equipped with an EMSIS Veleta camera (2K*2K).
[0064] like Figure 2 As shown in the figure, the substructures obtained by the 100w and 120w conditions are evenly dispersed, and there are no obvious chromoplasts and their agglomerated structures in the field of view. The chromoplasts are more effectively broken, so the 100w ultrasonic condition is used to extract the substructure and characterize it.
[0065] Example 3
[0066] This embodiment provides a method for extracting chromoplast substructures rich in carotenoids, comprising the steps of:
[0067] 1) Select mature, bright red, well-shaped wolfberries, rinse, and remove any remaining stalks or calyx. About 25 g of wolfberries were homogenized with 250 mL of chilled extraction buffer (50 mM HEPES, 330 mM sorbitol, 2 mM EDTA, 5 mM DTT, pH 7.5) at 300 W using a cold stirrer (JYL-CO20E, Jiuyang, Beijing, China) for 4 s.
[0068] 2) The suspension was filtered through four layers of Miracloth (Millipore, Massachusetts, USA), and the mixed filtrate was centrifuged at 3000 g for 20 min at 4° C. The precipitate obtained by centrifugation is the crude chromoplast.
[0069] 3) Gently resuspend the crude chromoplasts in extraction buffer, overlay on a discontinuous density gradient (5%, 7.5%, 10%, 12.5% and 15% Nycodenz in extraction buffer, w / v), and centrifuge at 5800g for 60 min at 4°C. Carefully collect the 5-15% chromoplast layer.
[0070] 4) Dilute with extraction buffer and centrifuge at 3000 g for 20 min at 4°C to remove Nycodenz.
[0071] The resulting samples were snap-frozen in liquid nitrogen, placed at 80°C for 1.5 h, and then thawed on ice.
[0072] 5) Gently resuspend the chromoplasts with 15% (w / v) Nycodenz extraction buffer, with an ultrasonic output power of 100w and an ultrasonic treatment time of 5min. The ultrasonically treated samples were sequentially covered with extraction buffers with concentrations of 5%, 7.5%, 10%, and 12.5% (w / v) Nycodenz, and centrifuged at 5800g for 60min at 4°C. Carefully collect the 7.5%-12.5% substructure layer and repeat the same steps to remove Nycodenz. The last washing step was repeated twice.
[0073] All the above preparation steps were carried out at 4°C or on ice.
[0074] Comparative Example 1 Extraction of free carotenoids
[0075] Select mature, bright red, well-shaped wolfberries, rinse, separate the remaining stems, cut appropriately and freeze-dry. The freeze-dried powder was sieved to remove the wolfberry seeds. Take 200 mg of wolfberry freeze-dried powder, add 5 mL of extraction solution, 1 mL of PBS solution, where the extraction solution is methanol, ethyl acetate, petroleum ether (1:1:1, v / v / v) and 0.1 g / L BHA (butylated hydroxyanisole) and BHT (2,6-di-tert-butyl-p-cresol). The sample is mixed with the solvent and then sonicated at 300 W for 30 seconds using a probe sonicator (Scientz-ⅡD, Ningbo, China). After centrifugation at 3000g for 10 minutes, the upper organic phase was collected and the residue was re-extracted with the extraction solvent 3-4 times until colorless. All combined organic solvents were evaporated to dryness under a gentle nitrogen stream to obtain free carotenoids.
[0076] Comparative Example 2: Extraction of Chromoplasts
[0077] The method is the same as steps 1) to 4) of Example 3, wherein in step 4), the precipitate after centrifugation to remove Nycodenz is collected without freeze-thawing to obtain chromoplasts.
[0078] Microstructural observation of substructures
[0079] The free carotenoids extracted from wolfberries were dispersed in PBS (not dissolved), and then LM and TEM images were obtained( Figure 3 (A), (D), (G)). In natural wolfberries, carotenoids are orderly aggregated in a liquid crystal state in numerous nanoscale tubules. These tubules are then enclosed in the double membranes of chromoplasts, as Figure 3 (B) shows. As Figure 3 E and (F) show, the tubules are the main substructures of chromoplasts, with a small number of microspheres around them. As Figure 3 C shows, the substructures (extracted in Example 3) are randomly aggregated and the overall morphology is irregular. Limited by the resolution of LM, it is difficult to distinguish the shape and contour of individual structures. However, in the high-resolution TEM images after negative staining( Figure 3 F and I), the substructure samples show a clear tubule, 200 - 800 nm in length and 30 - 60 nm in width. Corresponding to the tubular ultrastructure of the spherical chromoplasts in Figure 3 H, it proves that the intact substructures have been isolated from wolfberries by this method. The free carotenoids extracted from wolfberries show a structured wedge-shaped morphology under LM( Figure 3 A), with a diameter range of 1 - 10 μm. As a natural form extracted from wolfberries, the substructures can play an important role in loading carotenoids in vitro and have the advantage of small size.
[0080] The particle size distributions of chromoplasts (extracted in Comparative Example 2) and substructures (extracted in Example 3) were measured at 25 °C using a particle size analyzer (Mastersizer 3000, Malvern, Beijing, China). Deionized water was used as the dispersant.
[0081] From Figure 4 the position of the first peak shown and combined with the observation according to Figure 3 (I), the individual substructures have the advantage of smaller particle size compared to chromoplasts and are comparable to the particle size of nanoparticles loaded with ZDP. Perhaps because substances such as proteins released from broken chromoplasts may act as "bridges" to promote the adhesion of multiple substructures to each other, another peak appears at around 10 μm for the substructures. In addition, the "substructure clusters" after aggregation are also smaller than the existing liposomes encapsulating mixed carotenoids. In contrast, the substructures save the traditional extraction and encapsulation steps of carotenoids and show the potential to protect and sustain the release of carotenoids in food hydrophilic systems as an alternative to nano- or micro-systems.
[0082] Carotenoid extraction and HPLC analysis
[0083] The extraction solvents were methanol, ethyl acetate, petroleum ether (1:1:1, v / v / v) and 0.1 g / L BHA and BHT. The samples were mixed with the extraction solvent and then ultrasonicated using a probe ultrasonicator (Scientz-
[0084] ⅡD, Ningbo, China) was ultrasonicated at 300W for 30s. After centrifugation at 3000g for 10min, the upper organic phase was collected and the residue was re-extracted with the extraction solvent 3-4 times until colorless. All combined organic solvents were evaporated to dryness under a gentle nitrogen stream. The dried extract was dissolved in a mixture of tert-butyl methyl ether and methanol (1:1, v / v) and filtered through a membrane (0.45μm, Junlibo, Beijing, China). Carotenoids were identified and quantified by HPLC (LC-20A, Shimadzu, Japan). The chromatographic column was a C30 reverse phase column (250mm×4.6mm, particle size 3μm; YMC (Japan) was protected by a YMC C30 column of the same material. The mobile phase was methanol / tBME / water (80:18:2, v / v / v) as eluent A, methanol
[0085] / tBME / water (8:90:2, v / v / v) was used as eluent b, and the standard curve constructed by the corresponding standards was used to determine ZEA (zeaxanthin) and ZDP (zeaxanthin dipalmitate). Among them, zeaxanthin monopalmitate (ZMP) was quantitatively analyzed using the ZEA standard curve
[0086] As shown in Table 1, the total amount of the three carotenoids in the chromoplasts and substructures was 178.50 mg / g DW (dry weight) and 277.80 mg / g DW, respectively. It is worth noting that these values are much higher than the 4.95 mg / g DW found in wolfberry. In all samples, ZDP accounted for more than 90% of the total content of the three, while ZEA (zeaxanthin) accounted for only about 1%. This shows that the extracted chromoplasts and substructures are similar in terms of the composition of these three carotenoid components, while the total carotenoid content of the substructure is about 1.6 times that of the chromoplasts.
[0087] Table 1 Potential and main carotenoid contents in wolfberry and its substructure
[0088]
[0089] In this extraction method, density gradient centrifugation and ultrasonic treatment are used to separate substructures from wolfberry chromoplasts. Substructures are smaller in size than chromoplasts and have higher carotenoid content. They can be better absorbed and utilized by the human body and are easier to be evenly dispersed in the food matrix, thereby improving the texture and taste of the food.
[0090] Although the present invention has been described above through embodiments, those skilled in the art should understand that without departing from the spirit and substance of the present invention, all improvements and modifications made to the present invention should fall within the protection scope of the present invention.
Claims
1. A method for extracting chromoplast substructures rich in carotenoids, characterized in that: Includes steps: 1) adding frozen extraction buffer to wolfberry and homogenizing with a stirrer; the ratio of wolfberry to frozen extraction buffer is 1 g: 8-15 mL, and the frozen extraction buffer contains HEPES, sorbitol, EDTA and DTT to obtain a suspension; 2) Filter the suspension through four layers of Miracloth, centrifuge the filtrate at 1000-5000g for 15-25 minutes, and the precipitate obtained by centrifugation is the crude chromoplast; 3) Gently resuspending the crude chromoplasts in an extraction buffer containing 5-15% Nycodenz by mass volume; covering the extraction buffer with a discontinuous gradient density, separating the mixed solution by centrifugation, and separating the liquid layers obtained by centrifugation; 4) The separated liquid layers are diluted with the frozen extraction buffer, and Nycodenz is removed by centrifugation; 5) resuspending the intermediate product obtained in step 4) with 15-20% (w / v) Nycodenz buffer, and then subjecting the sample to ultrasonic treatment; sequentially covering the sample with Nycodenz extraction buffer with gradient concentrations ranging from 5% to 12.5%, separating the mixed solution by centrifugation, collecting the substructure layers of the Nycodenz gradient between the layers, and centrifuging again to remove the Nycodenz.
2. The extraction method according to claim 1, characterized in that In step 1), the freezing extraction buffer contains 50 mM HEPES, 330 mM sorbitol, 2 mM EDTA, 5 mM DTT, and the pH is 7-8.
3. The extraction method according to claim 1, characterized in that In step 1), homogenize with a pre-cooled stirrer at 300 W for 3 to 5 seconds.
4. The extraction method according to claim 1, characterized in that In step 3), the crude chromoplasts are gently resuspended in an extraction buffer having a gradient concentration of 5%, 7.5%, 10%, 12.5% and 15% Nycodenz, w / v, and after centrifugation, the intermediate suspension solutions of each layer between 0% and 15% are collected.
5. The extraction method according to claim 1, characterized in that In step 3), the centrifugation conditions are: centrifugation at 5000-6000g for 50-80min at 4°C; the obtained substructures are collected, diluted with the frozen extraction buffer, and then centrifuged at 1000-5000g for 15-25min at 0-4°C to remove Nycodenz.
6. The extraction method according to claim 1, characterized in that Step 4) After removing Nycodenz by centrifugation, the resulting precipitate was quickly frozen in liquid nitrogen, placed at 80°C for 1-2 hours, and then thawed on ice.
7. The extraction method according to claim 1, characterized in that In step 5), the output power of the ultrasonic treatment is 60 to 150 W, and the ultrasonic treatment is performed for 3 to 8 minutes.
8. The extraction method according to claim 7, characterized in that In step 5), the output power of the ultrasonic treatment is 100 W, and the ultrasonic treatment is performed for 5 min.
9. The extraction method according to any one of claims 1 to 8, characterized in that In step 5), the ultrasonically treated sample is sequentially covered with extraction buffer having a concentration of 5%, 7.5%, 10%, and 12.5% (w / v) Nycodenz, and the mixture is separated by centrifugation at 5800 g for 60 min at 4°C, and the suspended solution in the middle of each layer between 7.5% and 12.5% is collected, and the collected solution is diluted with the frozen extraction buffer, and then centrifuged at 1000-5000 g for 15-25 min at 0-4°C to remove Nycodenz.
10. The chromoplast substructure obtained by the extraction method according to any one of claims 1 to 9.