Green extraction method for chromoplast, resulting product and use

Abstract

Disclosed are a green extraction method for a chromoplast, a resulting product and a use. The green extraction method comprises: using plant tissues as a raw material, and adding an extraction solvent and homogenizing; filtering, and then collecting a filtrate; carrying out centrifugal separation, and discarding a supernatant to obtain a crude extract; and carrying out sucrose solution gradient separation, aspirating an enrichment layer, adding an extraction solution, carrying out centrifugal separation, and discarding a supernatant to obtain a pure chromoplast extract. The chromoplast extract obtained using the green extraction technique in the present invention has no significant difference in lipid solubility compared to carotenoids extracted using organic reagents, but has significantly better water solubility than the carotenoids extracted using the organic reagents, and also has a significant advantage in rehydration rate.

Description

A green extraction method for chromoplasts, obtained products and applications Technical Field

[0001] The invention belongs to the technical field of natural organic chromoplast extraction, and particularly relates to a method for extracting chromoplasts from various plant tissues, the extracted chromoplast products and applications thereof. Background Art

[0002] Carotenoids are the most abundant natural pigments in nature besides chlorophyll. They are tetraterpenoids that are yellow, orange or red. There are many types of carotenoids, with a total of more than 750 species, which are widely found in animals, plants, microorganisms and algae. As one of the pigment components, carotenoids can not only play a key role in evaluating the appearance and nutritional quality of food, but also promote human health and reduce the risk of disease. They have active functions such as provitamin A, antioxidant, immunomodulatory, and prevention of cardiovascular disease. They are a class of substances with important nutritional value and functional activity for the visual and nervous systems. For example, zeaxanthin (ZEA), as one of the most common carotenoids in food, is an indispensable component of the human retinal macular pigment. Studies have shown that regular intake of foods rich in zeaxanthin can prevent eye diseases and improve human cognitive function. [1-2] Therefore, natural carotenoids are expected to become promising colorants in the food industry due to their coloring properties and high nutritional value for human health.

[0003] Chromosomes are organelles with a phospholipid bilayer membrane that biosynthesize and accumulate carotenoids in plants. Based on the substructure of the carotenoids they store, chromoplasts can be divided into four main types: globular, tubular, membranous, and crystalline. [3-5] , in which carotenoids may exist in the form of fat-soluble or liquid crystal aggregates [6-7] Different aggregation states of carotenoid molecules will cause changes in the visible light absorption spectrum [8] Compared with free monomers, chromoplast substructures may cause visual color changes of carotenoids. In addition, chromoplasts have a phospholipid bilayer membrane similar to liposomes, which has certain advantages in improving the water solubility and stability of carotenoids. A recent study found that under different storage conditions, tomato chromoplasts are more stable than free carotenoid extracts from tomatoes in terms of color properties and carotenoid preservation. [9] Therefore, chromoplasts show great potential as natural water-soluble colorants and food additives to replace carotenoids due to their natural structural advantages.

[0004] During solvent- or ultrasound-assisted extraction, natural carotenoids are released from the chromoplasts and subsequently dissolved in the solvent. Due to their lipophilic nature, carotenoids extracted with organic solvents have very poor water solubility, resulting in low solubility in water, often to the point of being virtually insoluble. Furthermore, during storage, high temperatures, ultraviolet light, and oxygen free radicals can lead to instability (e.g., isomerization, oxidation, and degradation), thus limiting the application of carotenoid extracts. Researchers have developed delivery systems, particularly microcapsules and nanocapsules, to improve the water dispersibility, stability, and even bioavailability of carotenoids. However, the preparation of microcapsules and nanocapsules is complex, costly, and has poor biocompatibility. Extracting natural encapsulation systems directly from plants using environmentally friendly solvents is a more cost-effective approach.

[0005] References

[0006] [1] Izumi-Nagai, K., Nagai, N. & Ohgami, K. (2007). Macular pigment lutein is anti-inflammatory in preventing. Arteriosclerosis, Thrombosis, and Vascular Biology, 27, 2555-2562. DOI:10.1161 / ATVBAHA.107.151431.

[0007] [2] Lopresti, AL, Smith, SJ & Drummond, PD (2022). The effects of lutein and zeaxanthin supplementation on cognitive function in adults with self-reported mild cognitive complaints: A randomized, double-blind, placebo-controlled study. Frontiers in Nutrition, 9, 843512. DOI:10.3389 / fnut.2022.843512.

[0008] [3] SITTE P, FALK H, LIEDVOGEL B. Chromoplasts[M]. Stuttgart: G Fischer, 1980: 117-148.

[0009] [4] WEN Xin, HELLER A, WANG Kunli, et al. Carotenogenesis and chromoplast development during ripening of yellow, orange and red colored Physalis fruit[J]. Planta, 2020, 251(5): 95. DOI:10.1007 / s00425-020-03383-5.

[0010] [5] RUBAN A V, HORTON P, YOUNG A J. Aggregation of higher plant xanthophylls: Differences in absorption spectra and in the dependency on solvent polarity[J]. Journal of Photochemistry and Photobiology B-Biology, 1993, 21(2), 229-234. DOI:10.1016 / 1011-1344(93)80188-F.

[0011] [6] SCHWEIGGERT M R, MEZGER D, SCHIMPF F, et al. Influence of chromoplast morphology on carotenoid bioaccessibility of carrot, mango, papaya, and tomato[J]. Food Chemistry, 2012, 135(4): 2736-2742. DOI: 10.1016 / j.foodchem.2012.07.035.

[0012] [7] KÖHN S, KOLBE H, KORGER M, et al. Carotenoids: Volume 4: Natural Functions[M]. Basel: Birkhäuser Basel, 2008: 53-98.

[0013] [8] CLAUDIO L, ILARIA B, MARCO E. Novel food colorants from tomatoes: Stability of carotenoid-containing chromoplasts under different storage conditions[J]. LWT-Food Science and Technology, 2021, 140(11): 110725. DOI: 10.1016 / j.lwt.2020.110725.

[0014] [9] WANG Yongqiang, YANG Yong, FEI Zhangjun, et al. Proteomic analysis of chromoplasts from six crop species reveals insights into chromoplast function and development[J]. Journal of Experimental Botany, 2013, 64(4): 949-61. DOI:10.1093 / jxb / ers375. Technical issues

[0015] In view of the shortcomings of the existing technology, the first purpose of the present invention is to provide a green extraction method for chromoplasts, which can be applied to the extraction of chromoplasts in any structural form.

[0016] The second purpose of the present invention is to provide a product obtained by the green extraction method.

[0017] The third object of the present invention is to provide applications of the extracted chromoplasts. Technical Solutions

[0018] The technical solution for achieving the above-mentioned purpose of the present invention is:

[0019] A green extraction method for chromoplasts comprises the following steps:

[0020] 1) Using plant tissue as a raw material, adding an extraction solvent, and homogenizing using a spiral juicer, wherein the extraction solvent is selected from one or more of purified water, a sucrose solution, a PBS buffer, and a sorbitol extraction buffer; the concentrations of the sucrose solution and the sorbitol extraction buffer are independently 0.3-0.4 mol / L; the sorbitol extraction buffer contains 0.001-0.002 mol / L EDTA-Na2 and 10-100 mL / L HEPES buffer; the plant tissue is a carotenoid-rich tissue, such as one or more selected from plant tissues such as wolfberry, red sour berry, yellow sour berry, marigold, sweet potato, daffodil, papaya, watermelon, corn, pepper, citrus, pumpkin, tomato, carrot, mango, and palm fruit;

[0021] 2) After step 1), the stirred material is filtered through 1 to 3 layers of filter cloth, and the filtrate is collected;

[0022] 3) Centrifuge the filtrate at 3-8°C, discard the supernatant, and obtain the crude extract.

[0023] Wherein, in step 1), the wolfberry is fresh or dried plant tissue; and / or

[0024] The material-liquid ratio of the extraction solvent to the raw material is 1:5-25 (v / w, mL / g), preferably 1:10 (v / w, mL / g).

[0025] In step 1), a spiral juicer is used to homogenize and stir the juice at a power of 200-500 W, with intermittent stirring 2-5 times, each time for 1 s.

[0026] Wherein, in step 2), the filter cloth is one or a combination of two of ordinary filter cloth, 80 mesh cheese cloth, 120 mesh cheese cloth, 200 mesh cheese cloth, and Miracloth filter cloth.

[0027] A preferred technical solution of the present invention is that it also includes step 4): freeze-drying the obtained crude extract or separating it with a sucrose solution; wherein the freeze-drying conditions are: freeze-drying at -30~-50°C using a freeze dryer; and the separation is separation using a sucrose solution with a mass concentration of 5~50%.

[0028] Another preferred technical solution of the present invention comprises the steps of:

[0029] 1) Using plant tissue as raw material, adding an extraction solvent, and homogenizing using a spiral juicer, wherein the extraction solvent is PBS buffer, the concentration of the PBS buffer is 0.01M, the pH value is 7.2-7.4, and the material-liquid ratio is 1:10-25;

[0030] 2) After filtering through 2 layers of ordinary filter cloth, collect the filtrate,

[0031] 3) Centrifuge at 3-5°C and discard the supernatant to obtain the crude chromoplast extract; centrifuge at 3000-6000g for 10-30 minutes.

[0032] 4) Separate with a sucrose solution with a mass concentration of 5-50%.

[0033] Among them, the more preferred centrifugation condition in step 3) is centrifugation at 3000 g for 15-20 min.

[0034] Further preferably, the separation using sucrose solution in step 4) involves resuspending the crude chromoplast extract in sorbitol extraction buffer or PBS buffer, collecting the resuspension, adding sucrose solution to the resuspension at a volume ratio of 1:(2-3) and centrifuging horizontally at 4000g-5800g for 25-50 minutes at 3-5°C, aspirating the chromoplast-enriched layer, diluting it with sorbitol extraction buffer or PBS buffer, and centrifuging it at 3000-6000g for 10-30 minutes at 3-5°C, and discarding the supernatant. The precipitate at the bottom is the pure chromoplast extract. In this step, the centrifugation conditions and the number of washes have little effect on the separation results; the sucrose removal step can be repeated 1-3 times. The dilution factor can be 5-20 times.

[0035] In step 4), the operation of adding the sucrose solution is as follows: first adding a sucrose solution with a mass concentration of 50%, and then adding a sucrose solution with a concentration of 5 to 20%, and the volume ratio of the two sucrose solutions to the resuspension is independently (1 to 1.5):1.

[0036] More preferably, when adding the sucrose solution for the second time, a sucrose concentration of 20% is used.

[0037] The chromoplasts are obtained by extracting using the green extraction method described in any one of the present inventions.

[0038] The chromoplasts of the present invention are used in the preparation of cosmetics, Chinese cakes, candies and snacks, beverages, baked products, and meat products. Beneficial effects

[0039] The advantages and beneficial effects of the present invention are as follows:

[0040] The present invention provides a method for preparing an aqueous carotenoid ingredient, comprising: 1) obtaining a crude chromoplast extract from plant tissue; 2) separating the crude chromoplast extract using a sucrose gradient to obtain a pure chromoplast extract having approximately uniform size, shape, and density; and 3) dissolving the pure chromoplast extract in water to prepare the aqueous carotenoid ingredient.

[0041] The chromoplast extracts obtained using the green extraction technology of the present invention have no significant difference in fat solubility compared to carotenoids extracted using organic reagents, but their water solubility is significantly better than that of carotenoids extracted using organic reagents, and their rehydration rate is also significantly superior. Therefore, the chromoplasts obtained using the green extraction technology not only retain their chromoplast properties but also exhibit greater advantages in water solubility, which not only helps to improve their applicability in various applications but also is more environmentally friendly.

[0042] The present application uses gradient sucrose extraction, and the obtained chromoplasts are more complete and pure; and the aqueous solution system prepared from the chromoplasts obtained by the optimized gradient sucrose extraction method is more stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0044] Figure 1: Comparison of the purity and extraction rate of wolfberry chromoplasts obtained with different parameters: (A) shows the comparison of the extraction rate of wolfberry chromoplasts obtained with different extraction solvents; (B) shows the comparison of the purity of wolfberry chromoplasts obtained with different extraction solvents; (C) shows the comparison of the extraction rate of wolfberry chromoplasts obtained with different material-liquid ratios; (D) shows the comparison of the purity of wolfberry chromoplasts obtained with different material-liquid ratios; (E) shows the comparison of the extraction rate of wolfberry chromoplasts obtained with different filter cloths; (F) shows the comparison of the purity of wolfberry chromoplasts obtained with different filter cloths;

[0045] Figure 2: Comparison of extraction rate and purity of Example 3;

[0046] Figure 3: Comparison of rehydration rates of wolfberry chromoplasts from different separation treatments;

[0047] Figure 4: Optical microscope images of wolfberry chromoplast extracts separated by different sucrose gradients; in Figure 4, a is an optical microscope image of a wolfberry chromoplast extract without sucrose gradient separation; b is an optical microscope image of a wolfberry chromoplast extract separated by a 30% sucrose solution; c is an optical microscope image of a wolfberry chromoplast extract separated by a 40% sucrose solution; d is an optical microscope image of a wolfberry chromoplast extract separated by a 50% sucrose solution;

[0048] Figure 5: Comparison of the solubility of wolfberry chromoplasts in water;

[0049] Figure 6: Comparison of the solubility of wolfberry chromoplasts in petroleum ether;

[0050] Figure 7: Optical microscope images of wolfberry chromoplast extracts separated by 5% to 20% / 50% sucrose gradient in Example 6. In the figure, A is a 5% / 50% sucrose gradient; B is a 7.5% / 50% sucrose gradient; C is a 10% / 50% sucrose gradient; D is a 12% / 50% sucrose gradient; E is a 15% / 50% sucrose gradient; and F is a 20% / 50% sucrose gradient.

[0051] Figure 8: Characterization of the wolfberry chromoplasts isolated in Example 7. Polarized light micrographs of the chromoplasts under brightfield (A) and polarized light mode (B); Scale bar = 25 μm. Transmission electron micrograph of the chromoplasts (C); Scale bar = 500 nm. Arrows mark the chromoplast bilayer membrane, and arrowheads mark the tubular substructures within the chromoplasts. Particle size distribution of the chromoplast dispersion (D).

[0052] Figure 9: Light microscopic images of chromoplasts in isolated red physalis, carrot, and tomato cells. a. Red physalis; b. Carrot; c. Tomato. Arrows without straight lines point to chromoplasts.

[0053] Figure 10: Transmission electron microscopy image of the ultrastructure of isolated chromoplasts from red physalis. Arrows point to crystals; g plastid globules, m mitochondria, st starch granules, t tubular structures, and th thylakoids.

[0054] Figure 11: Characterization of chromoplast morphology in isolated yellow berries.

[0055] Figure 12: Transmission electron microscopy image of the ultrastructure of isolated yellow berry chromoplasts.

[0056] Figure 13: Biscuits containing water-soluble carotenoid ingredients.

[0057] Figure 14: Bread with water-soluble carotenoid topping.

[0058] Figure 15: Soft candies containing water-soluble carotenoid ingredients.

[0059] Figure 16: Beverages containing water-soluble carotenoid ingredients.

[0060] Figure 17: Mayonnaise with water-soluble carotenoid ingredients.

[0061] Figure 18: Jelly containing water-soluble carotenoid ingredients (left: without apple juice concentrate; right: with additional apple juice concentrate).

[0062] Figure 19: Mooncakes containing water-soluble carotenoid ingredients. Best Mode for Carrying Out the Invention

[0063] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0064] Unless otherwise specified, the technical means used in the specification are all known in the art, and the raw materials used are all commercially available.

[0065] The HPLC quantitative analysis method used in the examples was as follows: A centrifuge tube containing the lyophilized crude color body extract was weighed. Approximately 10 mL of extraction solution (methanol / ethyl acetate / petroleum ether, 1:1:1, v / v / v, each containing 0.1 g / L BHA and BHT) was added to the lyophilized crude color body extract. The extract was then ultrasonically extracted for 30 seconds. The sample was placed in an ice bath. After ultrasonic extraction, the extract was centrifuged (3000 g, 5 min), and the supernatant was pipetted into a brown bottle. The extraction was repeated until the sample became colorless. The supernatants were combined and nitrogen-purged to dryness. The dried extract was dissolved in a mixture of methanol and methyl tert-butyl ether (1:1, v / v), filtered through a 0.45 μm PTFE membrane, and transferred to a brown vial for analysis by HPLC. Liquid chromatography separation was performed using a YMC C30 reversed-phase column (250 mm × 4.6 mm, 3 μm) with a YMC C30 guard column of the same material. The analytical conditions were: 40 °C, mobile phase rate 1 mL / min, mobile phase A consisting of methanol / tBME / water (80:18:2, v / v / v); mobile phase B consisting of methanol / tBME / water (8:90:2, v / v / v). The elution gradient was as follows: mobile phase B concentration increased from 0% to 30% over 5 minutes, from 30% to 86% over 30 minutes, from 86% to 100% over 2 minutes, and then decreased from 100% to 0% over 4 minutes, with a dwell time of 4 minutes at 0%, for a total of 45 minutes. (all-E)-zeaxanthin (ZEA), zeaxanthin monopalmitate (ZMP), and zeaxanthin dipalmitate (ZDP) were used to quantify the chromoplasts in the extract.

[0066] Data processing and analysis: All experiments were repeated three times. The purity and extraction rate of wolfberry chromoplasts were calculated using Microsoft Excel 2019 software. The calculation formula is:

[0067]

[0068]

[0069] SPSS data analysis software was used to perform one-way analysis of variance (ANOVA), and Duncan's method was used to analyze the significant differences of the experimental data, with a significance level of p 0.05.

[0070] Example 1

[0071] This example optimizes the process conditions through single-factor experiments.

[0072] Fresh, stem-removed and cleaned wolfberry samples were weighed and the extraction solvent was added at a specific volume-to-liquid ratio (v / w, mL / g). The mixture was homogenized and stirred using a screw extractor (300 W power), with four 1-second intervals. The filtrate was filtered through two layers of filter cloth and transferred to a centrifuge tube. The tube was weighed and labeled before transfer. The extract was centrifuged at 3000 g at 4°C for 20 min, and the supernatant was discarded. The crude wolfberry chromoplast extract was stored at -80°C for 10 h, then removed and freeze-dried in a freeze dryer for 48 h. The resulting wolfberry chromoplasts were quantified and the optimal extraction conditions were selected. The factor levels for the single-factor experiment are shown in Table 1.

[0073] Table 1 Single factor experimental factors

[0074]

[0075] Selection of extraction solvents: Four solvents were selected, namely purified water, sucrose solution, PBS buffer (0.01M, pH 7.2-7.4) and sorbitol extraction buffer (containing 0.0018 mol / L EDTA-Na2, 50 mL / L HEPES buffer). To ensure that the chromoplasts that store a large amount of carotenoids in wolfberry are not destroyed, the concentrations of sucrose solution and sorbitol solution are both 0.33 mol / L.

[0076] Weigh 4 portions of 10 g fresh wolfberry, add 100 mL of each of the four extraction solvents at a material-liquid ratio of 1:10, use a spiral juicer (power 300 W) to homogenize and stir, intermittently stirring 4 times for 1 s each time, filter through two layers of Miracloth, collect the filtrate, transfer the filtrate of each extraction system to a weighed and marked centrifuge tube, discard the supernatant after centrifugation, and store the crude wolfberry chromoplast extract in a -80 ℃ refrigerator for 10 h and then freeze-dry it in a freeze dryer for 48 h.

[0077] Selection of material-liquid ratio: Weigh 2 portions of 20 g fresh wolfberry and 3 portions of 10 g fresh wolfberry, add 100 mL, 200 mL, 150 mL, 200 mL, and 250 mL of PBS buffer (0.01 M) at a material-liquid ratio of 1:5, 1:10, 1:15, 1:20, and 1:25, respectively. Use a spiral juicer (power 300 W) for homogenization and stirring, with intermittent agitation 4 times for 1 s each time. After filtering through two layers of Miracloth, collect the filtrate, centrifuge, and discard the supernatant. Place the crude wolfberry chromoplast extract in a -80°C refrigerator for 10 h and then freeze-dry it in a freeze dryer for 48 h.

[0078] Selection of filter cloth: Weigh 5 portions of 10 g fresh wolfberry and add 100 mL of PBS buffer (0.01 M) to each portion at a solid-liquid ratio of 1:10. Use a spiral juicer (power 300 W) to beat the juice and then pass it through 2 layers of ordinary filter cloth, 2 layers of 80-mesh cheese cloth, 2 layers of 120-mesh cheese cloth, 2 layers of 200-mesh cheese cloth, and 2 layers of Miracloth. Collect the filtrate and transfer it to weighed and marked centrifuge tubes respectively. After centrifugation, discard the supernatant. Place the crude wolfberry chromoplast extract in a -80°C refrigerator for 10 h and then freeze-dry it in a freeze dryer for 48 h.

[0079] The freeze-dried crude wolfberry chromoplast extracts obtained from the single-factor experiment were quantitatively analyzed by HPLC. The purity and extraction efficiency are shown in Figure 1. Figures 1 (A) and (B) show that under the same conditions, the purity and extraction efficiency of wolfberry chromoplasts extracted with PBS buffer were significantly higher than those of the other groups, indicating that PBS buffer (0.01M, pH 7.2-7.4) is the most effective for extracting wolfberry chromoplasts. Fresh wolfberries were added to PBS buffer (0.01M, pH 7.2-7.4) at a material-to-liquid ratio of 1:5, 1:10, 1:15, 1:20, and 1:25, respectively. The purity and extraction efficiency of the chromoplasts in the crude wolfberry chromoplast extracts are shown in Figures 1 (C) and (D). As can be seen from the figure, under the same conditions, the purity of wolfberry chromoplasts extracted by PBS buffer at a solid-liquid ratio of 1:10 was significantly higher than that of other groups. There was no significant difference in the extraction rate of wolfberry chromoplasts at solid-liquid ratios of 1:10, 1:15, 1:20, and 1:25, which were all significantly higher than the extraction rate at a solid-liquid ratio of 1:5. From the perspective of saving solvent, 1:10 should be used as the solid-liquid ratio for wolfberry chromoplast extraction.

[0080] The purity and extraction rate of chromoplasts in the crude extracts of wolfberry chromoplasts obtained by selecting different filter cloths are shown in Figure 1 (E) and (F). As can be seen from the figure, under the same conditions, the purity and extraction rate of wolfberry chromoplasts obtained by filtering with ordinary filter cloth were significantly higher than those of other groups.

[0081] In this example, a single-factor experiment was conducted to determine the process conditions for the green extraction of natural wolfberry chromoplasts. The results showed that the best extraction effect of wolfberry chromoplasts was achieved when the solvent was PBS (0.01M, pH 7.2-7.4), the solid-liquid ratio was 1:10, and the filter cloth was ordinary.

[0082] Example 2

[0083] This example uses fresh wolfberry as raw material, and the extraction steps are as follows:

[0084] 1) Using fresh wolfberry as raw material, adding an extraction solvent, and homogenizing using a spiral juicer (conditions are the same as in Example 1), the extraction solvent is PBS buffer, the concentration of PBS buffer is 0.01M, the pH value is 7.2-7.4, and the solid-liquid ratio is 1:10;

[0085] 2) After the homogenized product is filtered through 2 layers of ordinary filter cloth, the filtrate is collected.

[0086] 3) Centrifuge the filtrate at 4°C and discard the supernatant to obtain the crude extract of wolfberry chromoplasts; the centrifugation conditions are 3000g for 20 min.

[0087] 4) Freeze-drying: The freeze-drying conditions are the same as those in Example 1.

[0088] The extraction effect of this example: the purity of wolfberry chromoplasts is 5.7%, and the extraction rate is 69.3% Example 3

[0089] In this example, dried wolfberry was used as the raw material, and the extraction steps were basically the same as in Example 2. In step 3), the centrifugal separation stage, the centrifugal force was set to 1000, 3000, 4500, and 5800 g. Figure 2 compares the extraction rate and purity of the product under different centrifugal conditions.

[0090] At 5800g, the extraction rate reached 28.99 ± 2.30%, the highest extraction rate, but the purity was only 3.61 ± 0.69%; at 1000g, the purity reached 5.02 ± 0.64%, the highest purity, but the extraction rate was only 16.00 ± 1.78%; the greater the centrifugal force, the more plant fibers and some dead cells will settle, affecting the purity; but at the same time, the chromoplasts or broken parts containing carotenoids will also settle, and the extraction rate will increase.

[0091] Comparing the extraction in Example 1 and Example 2, it can be seen that fresh wolfberry has higher purity and extraction rate than dry wolfberry, so the subsequent separation experiments were carried out with fresh wolfberry. Example 4

[0092] This embodiment provides a green extraction method for natural wolfberry chromoplasts, comprising the steps of:

[0093] 1) Using fresh wolfberry as the raw material, an extraction solvent was added, and the mixture was homogenized using a spiral juicer at a power of 300 W, with intermittent stirring four times for 1 s each time; the extraction solvent was PBS buffer (0.01 M, pH 7.2-7.4), and the solid-liquid ratio was 1:10;

[0094] 2) The homogenized material is filtered through 2 layers of ordinary filter cloth and the filtrate is collected.

[0095] 3) Centrifuge the filtrate at 3-5°C and discard the supernatant to obtain a crude extract of wolfberry chromoplasts (a paste-like product). Centrifuge at 3000 g for 15-20 min.

[0096] 4) In this step, the crude wolfberry chromoplast extract was freeze-dried and separated using sucrose solutions of varying concentrations. The freeze-drying conditions involved storing the crude wolfberry chromoplast extract at -80°C for 10 hours, removing it from the freeze-dryer, and freeze-drying it for 48 hours. (Freeze-drying is a storage method; the rehydration rate was measured after freeze-drying and was found to be very good.)

[0097] The operation of sucrose solution separation is as follows: Step 3) After discarding the supernatant, the crude wolfberry chromoplast extract in the centrifuge bottle is resuspended with PBS buffer, and the resuspended liquid is collected.

[0098] Prepare 30%, 40%, and 50% sucrose solutions and divide them into 50 mL centrifuge tubes, 10 mL per tube. Take 5 mL of the resuspension and add it to the sucrose solution. Centrifuge horizontally at 5000-5800 g for 30-45 min at 4°C. Carefully pipette the chromoplast-enriched layer into a beaker with a pipette and divide it into weighed and labeled centrifuge tubes. Dilute with PBS buffer and centrifuge at 3000 g for 15-20 min at 4°C. Repeat twice. Discard the supernatant. The precipitate at the bottom is the wolfberry chromoplast extract.

[0099] Another group of wolfberry chromoplast crude extracts were also not freeze-dried, and were resuspended in PBS buffer, and were marked as wolfberry chromoplast extracts not separated by sucrose solution.

[0100] The water solubility and fat solubility tests were conducted on the wolfberry chromoplast extracts that were not separated by sucrose solution, the wolfberry chromoplast extracts that were separated by 30%, 40%, and 50% sucrose solutions, and the wolfberry chromoplasts extracted by organic reagents. Purified water was used as the water-soluble reagent, and petroleum ether was used as the fat-soluble reagent.

[0101] Experimental results and discussion

[0102] Optical microscopic observation of wolfberry chromoplasts: Microscopic observation of wolfberry chromoplast extracts before sucrose solution separation and after 30%, 40%, and 50% sucrose solution separation was performed, and the results are shown in Figure 4. It can be seen that after sucrose solution separation, the orange area decreased, indicating that the content of wolfberry chromoplasts decreased, but the integrity of the chromoplasts improved to varying degrees.

[0103] Observation of the solubility of wolfberry chromoplasts: A certain amount of purified water and petroleum ether were added to the wolfberry chromoplast extracts that had not been separated with sucrose solution, the wolfberry chromoplast extracts that had been separated with 30%, 40%, and 50% sucrose solutions, and the wolfberry chromoplasts extracted with organic reagents, respectively, to ensure that the carotenoid concentration in each sample was approximately 24.9 nmol / mL. The solubility was observed. The results are shown in Figures 5 and 6.

[0104] As can be seen from Figure 5, the wolfberry carotenoids extracted by organic solvents are yellow in color with precipitation when dissolved in pure water. The wolfberry chromoplast extracts that have not been separated with sucrose solution are light orange-red in color when dissolved in water, with a small amount of insoluble matter suspended therein. The wolfberry chromoplast extracts that have been separated with sucrose solution have no precipitation and are orange-red in color when dissolved in water.

[0105] As can be seen from Figure 6, carotenoids dissolved in petroleum ether are yellow and have no precipitation, and the wolfberry chromoplast extract that has not been separated by sucrose solution is yellow and has no precipitation when dissolved in petroleum ether. However, the wolfberry chromoplast extract that has been separated by sucrose solution has a certain degree of insoluble matter precipitated at the bottom after being soaked in petroleum ether, which is yellow.

[0106] Determination of the rehydration rate of wolfberry chromoplasts: As shown in Figure 3, the rehydration rate of wolfberry chromoplast extracts separated with sucrose solution was significantly higher than that of extracts not separated with sucrose solution. Combined with the optical microscopy results, this is because the chromoplasts in the wolfberry chromoplast extract separated with sucrose solution are more intact, better protecting them from damage during subsequent operations, resulting in a more hydrophilic nature.

[0107] Example 5

[0108] This embodiment provides a green extraction method for natural wolfberry chromoplasts, wherein steps 1) to 4) are substantially the same as those in Example 4, wherein the concentration of the sucrose solution in step 4) is 30%.

[0109] Taking into account the application in the food industry, the national standard allows food-grade solvents to be sucrose and sorbitol. Therefore, sorbitol and sucrose were used as extraction solvents in subsequent experiments, and there was no significant difference in the extraction effects of the two.

[0110] Chromoplasts have a phospholipid bilayer membrane similar to that of liposomes, which is more advantageous in improving the water solubility and stability of chromoplasts. Based on the above extraction experiments, the present inventors further optimized the extraction method of chromoplasts.

[0111] 1) Weigh clean carotenoid-rich plant tissue samples, including but not limited to wolfberry, red and yellow berries, marigold, sweet potato, daffodil, papaya, watermelon, corn, pepper, citrus, pumpkin, tomato, carrot, mango, and palm fruit. Add an extraction solvent (v / w, mL / g) at a 1:10 ratio of material to liquid to each sample. This extraction solvent is sorbitol at a concentration of 0.33 mol / L (containing 0.0018 mol / L EDTA-Na2 in 50 mL / L HEPES buffer). Use a spiral juicer (300 W) for rapid homogenization, stirring four times for 1 second each. In this example, fresh, stemmed and cleaned wolfberry samples were used.

[0112] 2) After filtering through two layers of filter cloth, collect the filtrate. Ordinary filter cloth is used for filtering, and a total of 2 layers are used.

[0113] 3) Transfer the filtrate to a centrifuge tube. Weigh and label the tube before transfer. Centrifuge at 3000 g for 20 min at 4°C. Discard the supernatant to obtain the crude chromoplast extract.

[0114] 4) Add extraction solvent to the crude chromoplast extract and resuspend (v / w, mL / g), and further separate and purify the chromoplast extract using a sucrose gradient. The extraction solvent in this step is sorbitol extraction buffer.

[0115] Step 4) Separation using a sucrose gradient is as follows: resuspend the crude chromoplast extract in sorbitol extraction buffer and collect the resuspension; add sucrose solution to the resuspension at a volume ratio of 1:(2-3) and centrifuge horizontally at 4000g-5800g for 25-50 minutes at 3-5°C; aspirate the chromoplast-enriched layer into a beaker, dilute it with sorbitol extraction buffer (pH 7.5) (a dilution factor of 1:10), and then centrifuge to wash away the sucrose; centrifuge at 3000-6000g for 10-30 minutes at 3-5°C; repeat the sucrose washing step one or two times, discard the supernatant, and the precipitate at the bottom is the pure extract of wolfberry chromoplasts (hereinafter referred to as the pure extract). The sucrose solution concentration is in two gradients: 5% to 20% and 50%. The sucrose solution was added as follows: first, a 50% sucrose solution was added, followed by a second sucrose solution with a gradient of 5%, 7.5%, 12%, 15%, and 20%. The volume ratio of the two sucrose solutions to the resuspension solution was 1:1.

[0116] In step 4), the first centrifugation is performed at 5800 g for 30 min and horizontal centrifugation at 4°C; the second centrifugation is performed at 3000 g for 20 min; the precipitate at the bottom is the pure extract of wolfberry chromoplasts, and this is repeated 1 to 2 times.

[0117] An optical microscopic image of the isolated wolfberry chromoplast extract is shown in Figure 7. As can be seen, the higher the sucrose gradient, the more chromoplasts are present. Furthermore, compared to the 12% and 15% sucrose gradients, the 20% sucrose gradient produced a cleaner chromoplast extract, free of cell debris and impurities. Therefore, 50% and 20% sucrose gradients were selected for subsequent experiments.

[0118] The basic operation was the same as in Example 6, except that in step 4) separation using a sucrose gradient, the sucrose solution had two concentration gradients of 20% and 50%, and the volume ratio of the sucrose solution to the resuspension in both gradients was 1:1.

[0119] The morphology of the chromoplasts obtained in this example was observed under an optical microscope. As shown in Figure 8a, the chromoplasts of wolfberry fruit appear orange under bright field conditions and are spherical or elliptical in shape, with a diameter of 4-6 μm. Under polarized light, a distinctive colored, filamentous texture was observed (Figure 8b). Because the nematic carotenoid molecules within the tubular structure of the chromoplasts can produce optical anisotropy and bitropy, they typically exhibit a fibrous or striped optical texture under a polarized light microscope. Therefore, it can be inferred that the carotenoid molecules within the chromoplast tubes are likely deposited in a nematic liquid crystal state. Compared to Figure 4, Figure 4 shows more fragmented chromoplasts and a higher degree of aggregation; Figure 8 demonstrates greater integrity and cleanliness, lacking aggregation.

[0120] TEM observation of isolated wolfberry chromoplasts revealed abundant tubular ultrastructures (arrows, Figure 8c). As lipophilic molecules, carotenoids were assembled into nanoscale tubular structures and encapsulated within the intact hydrophilic bilayer membrane of the chromoplasts (arrows, Figure 8c). The chromoplast dispersion exhibited a unimodal particle size distribution within the range of 1-10 μm (Figure 8d). Particle size analysis calculated the average diameter of the chromoplasts to be 3.53 ± 0.11 μm (D 4,3 ), which is consistent with the microscopic observation results.

[0121] The basic operation is the same as that of Example 7, wherein the extraction solvent in step 1) is a sucrose solution with a concentration of 0.33 mol / L.

[0122] Among plant tissues, the red physalis (P. alkekengi var. franchetii (Mast.) Makino), commonly known as the Red Lady, is a common herb in my country. The yellow physalis, also known as the hairy physalis (P. pubescens), is native to the Americas. Both red and yellow physalis produce edible fruits that are either reddish-orange or light yellow, respectively.

[0123] The extraction operation in this example is the same as that in Example 6, wherein the plant tissue used in step 1) is red sour berry fruit.

[0124] The extraction operation in this example is the same as that in Example 6, wherein the plant tissue used in step 1) is the fruit of the yellow sour berry.

[0125] The extraction process in this example is the same as that in Example 6, wherein the plant tissues used in step 1) are carrot and tomato, respectively. For ease of operation, the carrot or tomato is cut into pieces before being put into the spiral juicer.

[0126] The chromoplast structures of the extracts from the above examples were observed using both optical microscopy and transmission electron microscopy. Figure 9 shows the results of optical microscopic observation of the chromoplast structure of red physalis berries. It can be seen that the chromoplasts in red physalis berries appear red (Figure 9a). Compared to the typical needle-shaped crystal structures of carotenoid deposits in carrot (Figure 9b) and tomato (Figure 9c) extracts, the chromoplasts in red physalis berries (Figure 9a) do not have a typical crystalline structure, but may be deposited in spherical or tubular structures. The detailed ultrastructure requires transmission electron microscopy, as shown below.

[0127] The ultrastructure of the chromoplasts extracted from the red tartary berries was further observed using transmission electron microscopy, as shown in Figure 10. The chromoplasts were filled with small crystalline structures, numerous plastoglobules, and a small number of tubular structures. Therefore, plastoglobules, crystalline structures, and tubular structures represent the deposited forms of carotenoids in the red tartary berries.

[0128] Moreover, it is worth noting that the crystal size in the chromoplasts of red sour berries is much smaller than the larger needle-shaped crystals in carotene and tomatoes (Figure 9b and c), and no visible crystal structure was observed under an optical microscope (Figure 9a). This crystal structure is called submicroscopic crystals, which is a carotenoid deposition structure that is different from the classic crystal structure.

[0129] Figure 11 shows the results of optical microscopic observation of the chromoplasts in the extracted yellow berry. The image shows the presence of some crystals in the chromoplasts. However, the size of the crystals in the yellow berry is significantly smaller than those in carrots (Figure 9b) and tomatoes (Figure 9c).

[0130] Figure 12 shows the ultrastructural observation of chromoplasts in yellow tart berries using transmission electron microscopy. The results show that, in addition to large plastid globules, the chromoplasts in yellow tart berries also exhibit classic crystalline structures, generally located on both sides of the wavy inner membrane, as indicated by the arrows in Figure 12. Modes for Carrying Out the Invention

[0131] Application Examples

[0132] The purified chromoplast extract obtained in Example 7 is a paste, which is dissolved in water to produce a water-soluble carotenoid ingredient. Chromoplasts rich in different carotenoids can be used to produce natural water-soluble carotenoid ingredients of different colors. For example, chromoplasts rich in lycopene can be used to produce a red natural water-soluble carotenoid ingredient, while chromoplasts rich in lutein or zeaxanthin can be used to produce a yellow natural water-soluble carotenoid ingredient. Furthermore, for functional products, the amount of water added is adjusted based on the desired concentration. For colored products, the amount of water added is adjusted based on the desired color, provided that the product meets the "GB 2760-2014 Food Additives Usage Standard."

[0133] ①Application in baked products

[0134] For baked goods, this method can be applied to products such as biscuits, bread, mooncakes, macrones, and egg rolls. For biscuits, for example, the recipe involves 75g butter, 50g sugar, 15g egg liquid, 4mg chromoplast extract, and 115g wheat flour. The mixture is frozen for 1 hour, sliced ​​thinly, and baked at 165°C for 20 minutes. See Figure 13 for a specific product.

[0135] Taking bread as an example, the ingredients are: 120 g of ice milk, 50 g of eggs, 3 g of yeast, 30 g of sugar, 250 g of high-gluten flour, 2 g of salt, 20 g of butter, and 10 mg of a chromoplast pure extract (prepared into a solution with a concentration of 20 mg / kg, i.e., 20 mg of the chromoplast pure extract prepared in Example 7 is added to 1 kg of ingredients, and the remainder is water).

[0136] Steps: Add ice milk, egg liquid, yeast, sugar, and high-gluten flour to the bread machine and start kneading. Add salt and butter halfway through and continue kneading until you can pull out a thin film.

[0137] Place the kneaded dough in a greased bowl, cover with plastic wrap, and let it rise at 26-32°C for 1.5 hours. Once risen, press to release the air. Divide the dough into 8 equal portions, place them on a baking sheet, and cover with plastic wrap for a second rise. Let rise for 20-30 minutes. Brush with egg wash after rising. Bake in the oven at 170°C for 20 minutes. The resulting product is shown in Figure 14.

[0138] Currently, carotenoids are mixed with oil in bread making to facilitate their dissolution. However, the addition of oil results in a high calorie content and reduced solubility of the carotenoids. Other technologies use carotenoids as an encapsulation delivery system and add them to bread, but these systems are chemically synthesized externally and have poor biocompatibility. The chromoplasts used in the present invention are natural carotenoid encapsulation systems, which are healthier and more conducive to carotenoid absorption.

[0139] ②Application in candy products

[0140] Gelatin: 45 g gelatin powder + 70 g water, stir and let it soak;

[0141] To make sugar: heat 250 g apple juice concentrate with 20 g water to 110-120°C, then stop heating.

[0142] Preparation: Add 5 g of lemon juice concentrate and 10 mg of chromoplast pure extract;

[0143] Bottling and pouring: The syrup is poured into squeeze bottles, poured into the mold, cooled, and then demolded. The resulting product is shown in Figure 15.

[0144] ③Application in beverage products

[0145] For beverages, this method can be applied to yogurt, dairy drinks, and fruit and vegetable beverages. For a fruit and vegetable beverage, for example, 2 mg of pure chromoplast extract is added to a mixture of water, an appropriate amount of white sugar, 1.5% citric acid, and 0.1% xanthan gum solution. The mixture is then uniformly mixed and bottled in a 50 mL transparent glass bottle. See Figure 16 for the specific product.

[0146] In another application involving juice, chromoplast extracts or water-soluble carotenoid ingredients can be added to fruit and vegetable juices, such as apple juice, carrot juice, and tomato juice. Adding small amounts of chromoplast extracts, such as less than 0.05 g / kg, can brighten the juice color and help maintain a rich appearance.

[0147] ④Application in meat products

[0148] Adding a small amount of pure color body extract, for example less than 0.02 g / kg, can be used to brighten or color emulsified sausage or red sausage.

[0149] ⑤Application in margarine, mayonnaise and other sauces

[0150] Water-soluble carotenoid ingredients can also be used to brighten or color butter (with less than 1.0 g / kg of pure chromoplast extract) and mayonnaise (with less than 2.0 g / kg of water-soluble carotenoid ingredients). Butter, mayonnaise, and other sauces can be used in sweetcorn, bread, cake mixes, or bread making.

[0151] Place two egg yolks in a bowl, add 30 mg of the pure chromoplast extract, and 15 g of sugar. Whisk until combined. Add 10 mL of olive oil and 1 teaspoon of white vinegar, and continue stirring. Repeat this process until you have a mayonnaise of the desired consistency. See Figure 17 for the resulting product.

[0152] ⑥Application on jelly

[0153] Pour 150g of milk and 15g of sugar into a pot and heat until bubbles form. Add 2mg of the pure chromoplast extract, softened gelatin sheets, and granulated sugar. Turn off the heat, pour into a mold, and refrigerate for 4 hours before removing from the mold. The resulting product is shown in Figure 18.

[0154] ⑦Application in Chinese pastries

[0155] Ingredients: high-gluten flour 28%, low-gluten flour 28%, syrup 30%, oil 13%, alkaline water 1%, 20 mg / kg of pure chromoplast extract;

[0156] Production steps: Mix lye, oil, syrup, and water-soluble carotenoids evenly, knead into a dough, roll into a ball, flatten, wrap with filling, place in a mold, and bake at 180 degrees Celsius for 20 minutes. The resulting product is shown in Figure 19.

[0157] ⑧ Application in cosmetics

[0158] In this embodiment, the chromoplast extract is formulated as a dermatologically acceptable natural water-soluble colorant. According to the usage requirements of the "Catalogue of Used Cosmetic Raw Materials (2021 Edition)", 1.8% of a lutein-rich chromoplast extract, 3% of a astaxanthin-rich chromoplast extract, or a lycopene- or β-carotene-rich chromoplast extract (no specific dosage requirement) can be added to skin coloring products such as lipstick, eye shadow, and blush, or sunscreen and moisturizing products.

[0159] ⑨ Application in functional products

[0160] In this embodiment, the chromoplasts pure extract rich in lutein or zeaxanthin can be prepared into lutein or zeaxanthin soft capsules or tablets for preventing eye diseases or as a dietary supplement; the chromoplasts pure extract rich in astaxanthin can be prepared into oral liquids or capsules for enhancing the body's sun protection mechanism or antioxidant capacity. Functional products rich in chromoplasts pure extracts can be consumed regularly and in appropriate amounts according to the Chinese Dietary Reference Intake of Nutrients.

[0161] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications. Industrial Applicability

[0162] The present invention discloses a green extraction method for chromoplasts, the resulting products, and their applications. The green extraction method comprises the following steps: 1) using plant tissue as the raw material, adding an extraction solvent selected from one or more of purified water, sucrose solution, PBS buffer, and sorbitol extraction buffer, and homogenizing and stirring the resulting product using a spiral juicer; 2) filtering through one to three layers of filter cloth and collecting the filtrate; 3) centrifuging at 3 to 8°C and discarding the supernatant to obtain a crude extract; and 4) separating the resulting product using a 5%-50% sucrose gradient, aspirating the enriched layer, resuspending it in the extraction solution, centrifuging, and discarding the supernatant to obtain a pure chromoplast extract. The chromoplast extracts obtained using the green extraction technique of the present invention exhibit no significant difference in fat solubility compared to carotenoids extracted using organic reagents, but exhibit significantly better water solubility than carotenoids extracted using organic reagents. Therefore, the chromoplasts obtained using the green extraction technique not only retain their chromoplast properties but also exhibit greater water solubility, which not only improves their applicability in various applications but is also more environmentally friendly.

Claims

1. A green extraction method for chromoplasts, characterized in that: Includes steps: 1) Using plant tissue as raw material, adding extraction solvent, and homogenizing with a spiral juicer, wherein the extraction solvent is selected from one or more of pure water, sucrose solution, PBS buffer and sorbitol extraction buffer; the concentrations of the sucrose solution and the sorbitol extraction buffer are independently 0.3-0.4 mol / L; the sorbitol extraction buffer contains 0.001-0.002 mol / L EDTA-Na2, 10-100 mL / L HEPES buffer; the plant tissue is a carotenoid-rich tissue, selected from one or more of wolfberry, red sour berry, yellow sour berry, marigold, sweet potato, daffodil, papaya, watermelon, corn, pepper, citrus, pumpkin, tomato, carrot, mango, and palm fruit; 2) Step 1) The stirred material is filtered through 1 to 3 layers of filter cloth, and the filtrate is collected; 3) Centrifuge the filtrate at 3-8°C, discard the supernatant, and obtain the crude extract.

2. The green extraction method according to claim 1, characterized in that: In step 1), the wolfberry is fresh or dried plant tissue; and / or The material-liquid ratio of the extraction solvent to the raw material is 1:5-25 (v / w, mL / g).

3. The green extraction method according to claim 1, characterized in that: In step 1), use a spiral juicer to homogenize and stir at a power of 200-500 W, stirring intermittently 2-5 times, each time for 1 s.

4. The green extraction method according to claim 1, characterized in that: In step 2), the filter cloth is one or a combination of two of ordinary filter cloth, 80 mesh cheese cloth, 120 mesh cheese cloth, 200 mesh cheese cloth, and Miracloth filter cloth.

5. The green extraction method according to claim 1, characterized in that: The method also includes step 4): freeze-drying the obtained crude extract or separating it with a sucrose solution; wherein the freeze-drying conditions are: freeze-drying at -30 to -50°C using a freeze dryer; and the separation is separation using a sucrose solution with a mass concentration of 5 to 50%.

6. The green extraction method according to any one of claims 1 to 5, characterized in that: Includes steps: 1) Using plant tissue as raw material, adding extraction solvent, and using a spiral juicer to homogenize, the extraction solvent is PBS buffer, the concentration of PBS buffer is 0.01M, the pH value is 7.2-7.4, and the solid-liquid ratio is 1:10-25; 2) After filtering through 2 layers of ordinary filter cloth, collect the filtrate, 3) Centrifuge at 3-5°C, discard the supernatant, and obtain the crude extract of chromoplasts; the centrifugation condition is 3000-6000g for 10-30min; 4) Separate using a sucrose solution with a mass concentration of 5-50%.

7. The green extraction method according to claim 6, characterized in that: Step 4) The operation of separation with sucrose solution is as follows: resuspend the crude chromoplast extract obtained by centrifugation with sorbitol extraction buffer or PBS buffer, and collect the resuspended liquid; add sucrose solution to the resuspended liquid at a volume ratio of 1:(2-3), and centrifuge horizontally at a temperature of 3-5°C and 4000g-5800g for 25-50min; then aspirate the chromoplast enriched layer, add sorbitol extraction buffer or PBS buffer for dilution, and centrifuge at a temperature of 3-5°C and 3000-6000g for 10-30min; discard the supernatant, and the bottom precipitate is the pure chromoplast extract.

8. The green extraction method according to claim 7, characterized in that: In step 4), the operation of adding the sucrose solution is: first add a sucrose solution with a mass concentration of 50%, and then add a sucrose solution with a mass concentration of 5-20%, and the volume ratio of the two sucrose solutions to the resuspension solution is independently (1-1.5):

1.

9. The chromoplast obtained by the green extraction method according to any one of claims 1 to 8.

10. The use of the chromoplast according to claim 9, characterized in that: Used in the preparation of cosmetics, Chinese pastries, candy snacks, beverages, baked products, and meat products.

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