A green extraction method of natural goji berry pigment and the obtained product

Abstract

The present invention provides a green extraction method for natural wolfberry pigment, comprising the following steps: 1) using wolfberry as a raw material, adding an extraction solvent, and stirring using a spiral juicer, wherein the extraction solvent is selected from one or more of purified water, sucrose solution, PBS buffer, and sorbitol extraction buffer; 2) filtering through one to three layers of filter cloth and collecting the filtrate; 3) centrifuging at a temperature of 3 to 8°C and discarding the supernatant to obtain a crude wolfberry pigment extract. The crude wolfberry pigment extract is further separated using a sucrose solution. The wolfberry pigment extract obtained by the green extraction technology of the present invention maintains the properties of the pigment while being more environmentally friendly. After further separation using a sucrose solution, the pigment extract's advantages in solubility and rehydration rate become apparent.

Description

Technical Field

[0001] The invention belongs to the technical field of natural organic pigment extraction, and particularly relates to a method for extracting pigment from wolfberry, and the obtained wolfberry pigment. Background Art

[0002] Wolfberry (Lycium barbarum) is a climbing shrub of the genus Lycium in the Solanaceae family. It is a dual-purpose plant for medicine and food. Its roots, flowers, leaves and fruits can all be used as medicine. In addition, wolfberry pigments also have many physiologically active functions, making it an ideal natural pigment resource.

[0003] Lycium barbarum pigment is one of the main physiologically active ingredients of Lycium barbarum, mainly composed of water-soluble flavonoid pigments and fat-soluble carotenoids and their esters (Zhang Chongjian et al. Study on the extraction and purification process of Ningxia Lycium barbarum pigment [J]. Food Science and Technology, 2015, 40(5): 247-251). It is a safe natural colorant and food nutritional enhancer. As a natural pigment, Lycium barbarum pigment is non-toxic and safe. It can provide certain nutrients to the human body and has important biological functions such as improving eyesight, anti-oxidation, anti-fatigue, anti-inflammatory, tumor prevention and treatment of cardiovascular diseases. It also has rich nutritional value and medicinal health effects, so it has good application and development prospects. The main components of Lycium barbarum pigment are carotenoids and carotenoid esters. Carotenoids are an important class of natural pigments that are widely distributed in animals, plants, microorganisms and algae. The most important carotenoid is lutein and its esters. Due to the presence of 11 conjugated double bonds and terminal hydrocarbon groups in the zeaxanthin molecule, it has strong antioxidant activity (London VU, Beta T. Distribution of carotenoids in endosperm, germ, and aleurone fractions of cereal grain kernels[J]. Food Chemistry, 2013, 139(1): 663-671).

[0004] Currently, several extraction techniques for wolfberry pigments are available, including organic solvent extraction, ultrasonic-assisted extraction, supercritical CO₂ extraction, and microwave-assisted extraction. Organic solvent extraction is the most commonly used method in related research. However, it is time-consuming, and the widely used organic solvents are highly toxic and environmentally polluting. Ultrasonic extraction is less time-consuming, energy-efficient, and less likely to destroy the active ingredients. However, the ultrasonic generator is noisy, and this method still cannot avoid the environmental hazards and human health risks associated with the use of organic solvents. Supercritical CO₂ extraction is limited by its requirement for critical conditions, strict operational control, and high temperatures, which can easily lead to carotenoid losses during extraction. Microwave-assisted extraction offers advantages such as reduced extraction time, high efficiency, ease of operation, solvent savings, low energy consumption, high selectivity, and environmental safety. However, current research on microwave-assisted extraction is insufficient to provide sufficient technical support for its industrial application, and the scale-up of microwave extraction remains to be addressed.

[0005] In recent years, to meet the environmental protection concept and trend of green development, researchers have proposed replacing organic solvents with green solvents and developing new directions for green extraction technology. Green extraction originates from the classic concept of "green chemistry," whose principles include reducing hazardous compounds, using safe solvents and reaction conditions, preventing waste, and reducing hazardous chemical syntheses. There are two overarching principles for solvents used in green extraction: 1) using solvents derived from renewable resources and 2) using solvents that are harmless to the human body (Anastas PT, Warner JC. Green chemistry: theory and practice [M]. Oxford University Press, 1998). Water is the preferred green solvent, but is limited by the solubility of the substance in it; ionic liquids are also good green solvents. However, no research has yet used green solvents to extract large amounts of wolfberry pigments. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the purpose of the present invention is to propose a green extraction method for natural wolfberry pigment with high extraction efficiency, short extraction time and low cost based on the principle of using "green solvent".

[0007] The second object of the present invention is to provide the product obtained by the green extraction method.

[0008] The technical solution for achieving the above-mentioned purpose of the present invention is:

[0009] A green extraction method for natural wolfberry pigment comprises the following steps:

[0010] 1) Using wolfberry as a raw material, adding an extraction solvent, and stirring using a screw juicer, 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 solution are independently 0.3-0.4 mol / L; the sorbitol extraction buffer contains 0.001-0.002 mol / L of EDTA-Na2 and 10-100 mL / L of HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) buffer;

[0011] 2) After filtering through 1 to 3 layers of filter cloth, collect the filtrate.

[0012] 3) Centrifuge at 3-8°C and discard the supernatant to obtain a crude wolfberry pigment extract.

[0013] The sucrose solution and the sorbitol solution are selected to have an isotonic concentration with the chromoplasts, that is, the concentrations thereof are independently 0.3 to 0.4 mol / L, more preferably 0.33 mol / L.

[0014] Wherein, in step 1), the raw material is fresh wolfberry or dried wolfberry; and / or

[0015] The material-liquid ratio of the extractant to the raw material is 1:5-25 (v / w, mL / g).

[0016] The sucrose solution and the sorbitol solution are selected to have an isotonic concentration with respect to the chromoplasts, that is, their concentrations are independently 0.3 to 0.4 mol / L.

[0017] One of the preferred technical solutions of the present invention is that in step 1), a spiral juicer is used to homogenize and stir the juice at a power of 200 to 500 W, with intermittent stirring 2 to 5 times, each time for 1 second.

[0018] For example, use a spiral juicer to homogenize and stir at a power of 300W, intermittently stirring 4 times, each time for 1 second, that is, the total stirring time is 4 seconds, 1 second is the stirring time each time, stop immediately after 1 second, and then stir for another 1 second, for 4 consecutive times.

[0019] Wherein, in step 2), the filter cloth is one or a combination of two of ordinary filter cloth (i.e., absorbent gauze), 80 mesh cheese cloth, 120 mesh cheese cloth, 200 mesh cheese cloth, and Miracloth filter cloth.

[0020] Wherein, in step 3), the centrifugation condition is 3000-6000g for 15-30 minutes.

[0021] Furthermore, the green extraction method further includes step 4): freeze-drying the obtained wolfberry pigment 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 20 to 50%.

[0022] The freeze-drying conditions are as follows: the crude wolfberry pigment extract is placed in a -80°C refrigerator for 10 hours, then taken out and placed in a freeze dryer for freeze-drying for 48 hours.

[0023] A further preferred technical solution of the present invention comprises the steps of:

[0024] 1) using fresh wolfberry as raw material, adding an extraction solvent, and stirring 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;

[0025] 2) After filtering through 2 layers of ordinary filter cloth, collect the filtrate,

[0026] 3) Centrifuge at 3-5° C., discard the supernatant, and obtain a crude wolfberry pigment extract; the centrifugation condition is 3000 g for 15-20 minutes.

[0027] 4) Separate with a sucrose solution having a mass concentration of 30-40%.

[0028] The operation of separating with sucrose solution in step 4) is as follows: resuspending the centrifuged wolfberry pigment crude extract using the PBS buffer and collecting the resuspension; adding sucrose solution (resuspension 1, sucrose solution 2-3) to the resuspension in a volume ratio of 1:(2-3), horizontally centrifuging, aspirating the chromoplast-enriched layer into a beaker (i.e., the liquid layer with a dark red color), then adding PBS buffer to dilute and then centrifuging, discarding the supernatant, and the precipitate at the bottom is the wolfberry pigment extract.

[0029] More preferably, in step 4), the first centrifugation operation is: horizontal centrifugation at 5800g, 30min, 4°C, and a ramp rate of 2; the second centrifugation operation is: centrifugation at 3000g, 20min, 4°C, and a ramp rate of 9.

[0030] The wolfberry pigment is extracted by the green extraction method of the present invention.

[0031] The beneficial effects of the present invention are:

[0032] The wolfberry pigment extract extracted using the green extraction technology of the present invention has no significant difference in water solubility and fat solubility from wolfberry carotenoids extracted using organic reagents. However, after separation with a sucrose solution, the chromoplasts in the pigment extract are more complete, their water solubility is significantly improved, and they are significantly better than wolfberry carotenoids extracted using organic reagents. The rehydration rate is also significantly improved. Therefore, the wolfberry pigment extract obtained by the green extraction technology has an advantage in environmental friendliness while maintaining the pigment properties. After further separation with a sucrose solution, the advantages of the pigment extract in solubility and rehydration rate become apparent.

[0033] The wolfberry pigment extract extracted by the present invention also has better stability at pH=9 or Ca 2+ The pigment precipitation rate was the slowest in the system with a concentration of 500 mM, and it was still not completely precipitated after standing for 1 week. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Comparison of the purity of wolfberry pigment obtained from different extraction solvents;

[0035] Figure 2 Comparison of the extraction rate of wolfberry pigment obtained by different extraction solvents;

[0036] Figure 3 The purity of wolfberry pigment obtained with different material-liquid ratios;

[0037] Figure 4 Comparison of wolfberry pigment extraction rates obtained with different material-liquid ratios;

[0038] Figure 5 Comparison of the purity of wolfberry pigment obtained from different filter cloths;

[0039] Figure 6 is the extraction rate of wolfberry pigment obtained by different filter cloths;

[0040] Figure 7 This is a comparison chart of the extraction rates of Example 3;

[0041] Figure 8 This is a comparison chart of the purity of Example 3;

[0042] Figure 9 To compare the rehydration rate of wolfberry pigments with different separation treatments;

[0043] Figure 10 This is an optical microscope image of the wolfberry pigment extract without sucrose gradient separation;

[0044] Figure 11 This is an optical microscope image of the wolfberry pigment extract separated by 30% sucrose solution;

[0045] Figure 12This is an optical microscope image of the wolfberry pigment extract separated by 40% sucrose solution;

[0046] Figure 13 This is an optical microscope image of the wolfberry pigment extract separated by 50% sucrose solution;

[0047] Figure 14 This is a comparison chart of the solubility of wolfberry pigment in water;

[0048] Figure 15 This is a comparison chart of the solubility of wolfberry pigment in petroleum ether;

[0049] Figure 16 The chromoplast solutions were kept at different pH conditions for different periods of time;

[0050] Figure 17 The microscopic observation pictures of chromoplasts under different pH conditions;

[0051] Figure 18 The final value (a) and change curve (b) of the chromoplast clarification index under different pH conditions;

[0052] Figure 19 For different Na + Color body solutions were left standing for different time periods at different concentrations;

[0053] Figure 20 For different Ca 2+ Color body solutions were left standing for different time periods at different concentrations;

[0054] Figure 21 For different Na + Microscopic observation of chromoplasts at different concentrations (0, 5, 10, 50 mM);

[0055] Figure 22 For different Na + Microscopic observation of chromoplasts at different concentrations (100, 200, and 500 mM);

[0056] Figure 23 For different Ca 2+ Microscopic observation of chromoplasts at different concentrations (0, 5, 10, 50 mM);

[0057] Figure 24 For different Ca 2+ Microscopic observation of chromoplasts at different concentrations (100, 200, and 500 mM);

[0058] Figure 25 For different Na + Final value (a) and change curve (b) of chromogenic body clarification index under different concentrations;

[0059] Figure 26 For different Ca2+ Final value (a) and change curve (b) of the chromogenic body clarity index at different concentrations. DETAILED DESCRIPTION

[0060] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0061] 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.

[0062] The HPLC quantitative analysis method used in the embodiment is as follows: take a centrifuge tube containing a crude extract of wolfberry pigment after freeze-drying and weigh it. About 10mL of extract (methanol / ethyl acetate / petroleum ether, 1:1:1, v / v / v, both containing 0.1g / L BHA and BHT) was added to the freeze-dried crude extract of wolfberry pigment, extracted using an ultrasonic crusher for 30s, the sample was ice-bathed, and after ultrasonic extraction, centrifuged (3000g, 5min), and the supernatant was pipetted into a brown bottle. Repeat the extraction until the sample is colorless, combine the supernatants, blow nitrogen to dryness, and dissolve the dried extract in a mixture of methanol and methyl tert-butyl ether (1:1, v / v), filter through a 0.45μm PTFE membrane into a brown vial, and analyze using HPLC. A YMC C30 reverse phase column (250mm×4.6mm, 3μm) with a YMCC30 guard column of the same material was used for liquid chromatography separation. The analytical conditions were: temperature 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 wolfberry pigments in the extract.

[0063] Data processing and analysis: All experiments were repeated three times. The purity and extraction rate of wolfberry pigment were calculated using Microsoft Excel 2019 software. The calculation formula is:

[0064]

[0065]

[0066] SPSS data analysis software was used to analyze the experimental data using one-way analysis of variance (ANOVA) and Duncan's method for multiple comparisons. The significance level was set at 0.05. The data in this chapter are expressed as mean ± standard deviation. Different letters indicate significant differences between the groups (p < 0.05).

[0067] Example 1

[0068] This example optimizes the process conditions through single-factor experiments.

[0069] Fresh wolfberry samples (Ningqi No. 9 fresh wolfberries) were weighed and washed after removing the stems. The extraction solvent was added at a specific material-to-liquid ratio (v / w, mL / g). The mixture was homogenized using a 300W screw extractor, with four 1-second intervals. The filtrate was filtered through two layers of filter cloth, collected, and transferred to a centrifuge tube. The tube was weighed and labeled before transfer. The extract was centrifuged at 3000g at 4°C for 20 minutes. The supernatant was discarded, and the crude wolfberry pigment extract was stored in a -80°C refrigerator for 10 hours. The extract was then freeze-dried in a freezer for 48 hours. The resulting wolfberry pigment was quantified and the optimal extraction conditions were selected. The factor levels for the single-factor experiment are shown in Table 1.

[0070] Table 1 Single factor experimental factors

[0071]

[0072] Selection of extraction solvents: Four solvents were selected, namely purified water, sucrose solution, PBS buffer (0.01M, pH7.2-7.4) and sorbitol extraction buffer (containing 0.0018mol / L EDTA-Na2, 50mL / L HEPES buffer). To ensure that the chromoplasts in the area where wolfberry stores a large amount of pigments are not destroyed, the concentrations of sucrose solution and sorbitol solution were both 0.33mol / L.

[0073] Weigh 4 portions of 10g fresh wolfberry, add 100mL of each of the four extraction solvents according to the material-liquid ratio of 1:10, use a spiral juicer (power 300W) to homogenize and stir, intermittently stir 4 times, each time for 1s, 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, store the crude wolfberry pigment extract in a -80℃ refrigerator for 10h, and then place it in a freeze dryer for freeze-drying for 48h.

[0074] Selection of material-liquid ratio: Weigh 2 parts of 20g fresh wolfberry and 3 parts of 10g fresh wolfberry, add 100mL, 200mL, 150mL, 200mL, 250mL of PBS buffer (0.01M) at material-liquid ratios of 1:5, 1:10, 1:15, 1:20, 1:25, respectively, use a spiral juicer (power 300W) to homogenize and stir, intermittently point 4 times, each time for 1s, filter through two layers of Miracloth, collect the filtrate, transfer the filtrate obtained for each material-liquid ratio to a weighed and marked centrifuge tube, discard the supernatant after centrifugation, store the crude wolfberry pigment extract in a -80℃ refrigerator for 10h, and then place it in a freeze dryer for freeze-drying for 48h.

[0075] Selection of filter cloth: Weigh 5 portions of 10g fresh wolfberry, add 100mL of PBS buffer (0.01M) to each portion at a material-liquid ratio of 1:10, use a spiral juicer (power 300W) to beat the pulp, 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, respectively. Collect the filtrate and transfer it to weighed and marked centrifuge tubes respectively. After centrifugation, discard the supernatant, store the crude wolfberry pigment extract in a -80℃ refrigerator for 10 hours, and then place it in a freeze dryer for freeze-drying for 48 hours.

[0076] The freeze-dried crude extract of wolfberry pigment obtained from the above single factor experiment was quantitatively analyzed by HPLC, and the purity and extraction rate were as follows: Figure 1 、 Figure 2 As shown. Figure 1 It can be seen that under the same conditions, the purity and extraction rate of wolfberry pigment extracted by PBS buffer were significantly higher than those of other groups, indicating that PBS buffer (0.01M, pH 7.2-7.4) has the best extraction effect on wolfberry pigment.

[0077] Fresh wolfberry was added with PBS buffer (0.01M, pH 7.2-7.4) according to the material-liquid ratio of 1:5, 1:10, 1:15, 1:20, and 1:25, respectively. The pigment purity and extraction rate of the obtained wolfberry pigment crude extract were as follows: Figure 3 、 Figure 4 As shown in the figure, under the same conditions, the purity of wolfberry pigment 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 pigment at solid-liquid ratios of 1:10, 1:15, 1:20, and 1:25, all of which were significantly higher than the extraction rate at a solid-liquid ratio of 1:5. From the perspective of saving solvent, 1:10 should be selected as the solid-liquid ratio for wolfberry pigment extraction.

[0078] The pigment purity and extraction rate of the crude extract of wolfberry pigment obtained by selecting different filter cloths are as follows: Figure 5 、 Figure 6As shown in the figure, under the same conditions, the purity and extraction rate of wolfberry pigment obtained by filtration through ordinary filter cloth are significantly higher than those of other groups.

[0079] This example adopts a single factor experiment to determine the process conditions of the green extraction technology of natural wolfberry pigment. The final conclusion is: when the solvent is PBS (0.01M, pH 7.2-7.4), the solid-liquid ratio is 1:10, and the filter cloth is ordinary filter cloth, the extraction effect of wolfberry pigment is the best.

[0080] Example 2

[0081] This example uses fresh wolfberry as raw material, and the extraction steps are as follows:

[0082] 1) Using fresh wolfberry as raw material, adding an extraction solvent, and stirring 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 material-liquid ratio is 1:10;

[0083] 2) After filtering through 2 layers of ordinary filter cloth, collect the filtrate,

[0084] 3) Centrifuge at 4° C. and discard the supernatant to obtain a crude wolfberry pigment extract; the centrifugation condition is 3000 g for 20 min.

[0085] 4) Freeze-drying: The freeze-drying conditions are the same as those in Example 1.

[0086] The extraction effect of this example: the purity of wolfberry pigment is 5.7%, and the extraction rate is 69.3%

[0087] Example 3

[0088] In this embodiment, dried wolfberry is used as raw material, and the extraction steps are basically the same as those in Example 2. In step 3), the centrifugal separation stage is performed at centrifugal forces of 1000, 3000, 4500, and 5800 g. Figure 7 and Figure 8 The extraction rate and purity of the products under different centrifugation conditions were compared.

[0089] Under 5800g conditions, the extraction rate reached 28.99±2.30%, the highest extraction rate, but the purity was only 3.61±0.69%; under 1000g conditions, 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.

[0090] 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.

[0091] Example 4

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

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

[0094] 2) After filtering through 2 layers of ordinary filter cloth, collect the filtrate,

[0095] 3) Centrifuging at 3-5° C. and discarding the supernatant to obtain a crude wolfberry pigment extract (a paste-like product); the centrifugation condition is 3000 g for 20 min.

[0096] 4) In this step, the obtained wolfberry pigment crude extract is freeze-dried and separated with sucrose solutions of different concentrations; the freeze-drying conditions are as follows: the wolfberry pigment crude extract is placed in a -80°C refrigerator for 10 hours, then taken out and placed in a freeze dryer for freeze-drying for 48 hours.

[0097] The operation of sucrose solution separation is as follows: discarding the supernatant, resuspending the wolfberry pigment crude extract in the centrifuge bottle with PBS buffer, and collecting the resuspended liquid.

[0098] Prepare 20%, 30%, 40%, and 50% sucrose solutions, divide them into 50mL centrifuge tubes, 10mL per tube, take 5mL of the resuspension and add it to the sucrose solution, centrifuge horizontally at 5800g, 30min, 4℃, and a ramp rate of 2, carefully pipette the chromoplast-enriched layer into a beaker, divide it into weighed and marked centrifuge tubes, add PBS buffer to dilute, centrifuge at 3000g, 20min, 4℃, and a ramp rate of 9, discard the supernatant, and the precipitate at the bottom is the wolfberry pigment extract.

[0099] Another group of wolfberry pigment crude extracts was not freeze-dried and resuspended in PBS buffer, which was recorded as the wolfberry pigment extract without sucrose separation. Microscopic observation was performed on the wolfberry pigment extract without sucrose separation and the wolfberry pigment extracts separated by 30%, 40%, and 50% sucrose solutions.

[0100] Solubility Observation and Color Comparison of Wolfberry Pigment

[0101] Water solubility and fat solubility tests were performed on the wolfberry pigment extracts that were not separated by sucrose solution, the wolfberry pigment extracts that were separated by 30%, 40%, and 50% sucrose solutions, and the wolfberry pigments extracted by organic reagents. Purified water was used as the water-soluble reagent, and petroleum ether was used as the fat-soluble reagent.

[0102] Experimental results and discussion

[0103] 20% sucrose solution could not separate the wolfberry pigment extract, while 30%, 40% and 50% sucrose solutions had no significant difference in the separation effect on the wolfberry pigment extract.

[0104] Optical microscopic observation of wolfberry pigment: The wolfberry pigment extracts without sucrose solution separation and the wolfberry pigment extracts separated by 30%, 40%, and 50% sucrose solution were observed under a microscope. Figures 10 to 13 As shown in the figure, it can be seen that after separation with sucrose solution, the orange area is reduced, that is, the content of wolfberry pigment is reduced, but the integrity of the chromoplasts is improved to varying degrees.

[0105] Observation of the solubility of wolfberry pigment: A certain amount of purified water and petroleum ether were added to the wolfberry pigment extract that had not been separated by sucrose solution, the wolfberry pigment extract that had been separated by 30%, 40%, and 50% sucrose solution, and the wolfberry pigment extracted by organic reagent, respectively, to ensure that the carotenoid concentration in each sample was about 24.9nmol / mL, and their solubility was observed. The results are as follows: Figure 14 、 Figure 15 shown.

[0106] Depend on Figure 14 It can be observed that the wolfberry carotenoids extracted by organic solvents are yellow in color and have precipitates when dissolved in pure water. The wolfberry pigment extracts that have not been separated by sucrose solution are light orange-red in color when dissolved in water, with a small amount of insoluble matter suspended in it. The wolfberry pigment extracts that have been separated by sucrose solution have no precipitates when dissolved in water and are orange-red.

[0107] Depend on Figure 15 It can be observed that carotenoids are yellow and have no precipitation when dissolved in petroleum ether, and the wolfberry pigment extract that has not been separated by sucrose solution is yellow and has no precipitation when dissolved in petroleum ether. However, the wolfberry pigment 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.

[0108] Determination of the rehydration rate of wolfberry pigment: The rehydration rate of wolfberry pigment extract in each treatment group was determined. Figure 9As shown in the figure, the rehydration rate of the wolfberry pigment extract separated by sucrose solution was significantly higher than that of the wolfberry pigment extract not separated by sucrose solution. Combined with the optical microscopy results, this is because the chromoplasts in the wolfberry pigment extract separated by sucrose solution are more intact, which better protects the wolfberry pigment from being destroyed during subsequent operations, resulting in a more hydrophilic nature.

[0109] Stability test of wolfberry pigment: Considering environmental protection and resource conservation, the wolfberry pigment extract extracted from 30% sucrose solution was selected for stability analysis among 30%, 40%, and 50% sucrose solutions, which showed no significant difference in the separation effect of wolfberry pigment extract. The specific operation is as follows:

[0110] First, the wolfberry pigment extract was diluted with PBS buffer at pH values of 3, 5, 7, and 9, respectively, to a carotenoid content of 30 nmol / mL in each sample. Four mL of the sample at each pH condition was added to a transparent photography bottle and stored in a dark refrigerator at 4°C for 7 days. The sample's appearance was regularly observed for changes, and photographed and microscopically observed in a photography box. The stability of the wolfberry pigment extract samples at different pH conditions was evaluated using a LUMISizer full-function stability analyzer. The experimental parameters were as follows: temperature 25°C, rotation speed 2000 rpm, acquisition interval 10 seconds, and acquisition number 400 times.

[0111] Second, the wolfberry pigment extract was diluted with PBS buffer at pH 7 to make the carotenoid content in each sample reach 30 nmol / mL. + , Ca 2+ The ion concentrations of the two metal ions were set to 0, 5, 10, 50, 100, 200, and 500 mM. The metal ion concentrations in the colored body weight suspension were adjusted to the set concentrations using deionized water, 2 M NaCl solution, or 2 M CaCl2 solution. The stability was tested using the same method as "1".

[0112] pH stability test: the results are as follows Figure 16-18 As shown. Figure 16 It can be observed that after extraction, the pigment solutions under pH = 3, 5, 7, and 9 conditions all showed a uniform and stable orange-yellow color. After standing for 1 day, the wolfberry pigment under pH = 3 conditions almost completely precipitated, and the precipitate color was bright orange-red. After standing for 1 day, the pigment under pH = 5 and 7 conditions showed a light orange color on the upper solution, and it was slightly uneven. Some pigment precipitated, but the amount of precipitation was less than that of pH = 3. After standing for 2 days, the pigment under pH = 5 conditions was almost completely precipitated, while a light orange color could still be observed in the solution of pH = 7. Compared with the first three pH conditions, the pigment under pH = 9 conditions had the slowest precipitation rate. After standing for 1 week, it was still not completely precipitated, and the solution system could still be observed to be orange.

[0113] Depend on Figure 17 Under the 40x objective lens of a polarizing optical microscope, it can be observed that the wolfberry pigment is orange-yellow in color and irregularly shaped. Under the microscopic field of view, it can be found that the wolfberry pigment exhibits different aggregation forms under different pH conditions: at pH = 3, the pigment aggregation is high and the range is very large; at pH = 5 and 7, the pigment shows dispersed aggregation, and no flake aggregation similar to the pH = 3 condition is observed; at pH = 9, the pigment only has a small amount of aggregation and is generally dispersed.

[0114] Depend on Figure 18 It can be observed that in the rapid stability analysis, the greater the rate of change of the clarity index over time and the steeper the curve, the faster the sample settles and the poorer the stability. The results show that the clarity index first increases over time and then remains stable. The lower the pH value, the faster the clarity index changes and the higher the final value, and all these differences are significant. The curve at pH = 3 is the steepest, indicating that the pigment settles fastest and has the poorest stability under this condition. The curve at pH = 9 changes the slowest, indicating that the pigment settles the slowest under this condition, and the clarity index at the final stabilization is the lowest of the four conditions, indicating that the sample is the most stable compared to the other three conditions. This is consistent with the pigment sedimentation results observed in the statically photographed bottles.

[0115] Metal ions (Na + , Ca 2+ ) Stability test: the results are as follows Figure 19-26 As shown. Figure 19 It can be observed that the cells containing different concentrations (0, 5, 10, 50, 100, 200, 500 mM) of Na + The wolfberry pigment samples were placed in transparent photography bottles and left to stand at 4 ° C. It was found that all Na + The pigment solutions at different concentrations all showed a uniform and stable orange-red color. After standing for 1 day, Na + The solutions with concentrations of 50, 100, and 200 mM began to show pigmentation. After standing for 3 days, Na + Orange-red precipitates were observed in samples with concentrations of 5 and 10 mM, and the solution state was similar to that without Na addition. + The color of the sample is lighter than that of the sample containing 5mM Na + The color of the sample with 10mM is darker than that of the sample with 10mM. Compared with the sample after standing for 1 day, the amount of pigment precipitated in the samples with 50, 100 and 200mM is more. +The state of the samples at different concentrations was not much different from that after standing for 3 days. The samples at 5 and 10 mM still maintained orange-yellow color, and the color of the 5 mM sample was darker than that of the 10 mM sample. The pigments in the three samples at 50, 100, and 200 mM were almost completely precipitated, and the upper solution was colorless and transparent. + The pigment precipitation in the samples with concentrations of 0 and 500 mM was the least. + Pigment samples with concentrations of 50, 100, and 200 mM were the most unstable, while samples with concentrations of 5 and 10 mM were relatively stable. + The stability of the sample with a concentration of 500mM was relatively the best.

[0116] Depend on Figure 20 It can be observed that the cells contain different concentrations (0, 5, 10, 50, 100, 200, 500 mM) of Ca 2+ The wolfberry pigment samples were placed in transparent photography bottles and left to stand at 4 ° C. It was found that on the day of extraction and sample preparation, all Ca 2+ The pigment solutions at different concentrations all showed a uniform and stable orange-red color, which was consistent with Na + After standing for 1 day, Ca 2+ In samples with concentrations of 5, 10, 50, and 100 mM, a large amount of pigment precipitated, and only a very small amount of fine orange particles were observed in the solution, which was almost clear and transparent overall; 2+ A small amount of pigmentation was observed in the sample with a concentration of 200 mM, and the solution color was brighter than that without the introduction of Ca. 2+ After standing for 3 days, the 200mM sample solution became clearer and the orange color became lighter. 2+ The sample with a concentration of 0mM also precipitated, but the degree was not high. There was no significant difference between the state of each sample after standing for one week and that after standing for 3 days. 2+ Only very little precipitation was observed in the sample with a concentration of 500 mM. 2+ The pigment samples with concentrations of 5, 10, 50, and 100 mM were the most unstable, and no Ca was added. 2+ The samples with a concentration of 200mM were relatively stable, and the sample with a concentration of 500mM had the best stability.

[0117] Depend on Figure 21 It can be observed that in the 40x field of view of polarized optical microscope, Na + When the concentration is 0mM, the pigments are relatively complete, in the form of orange irregular round particles, with no obvious aggregation. Therefore, when observed statically, it can be found that the ion-free pigment sample still maintains a uniform and stable orange color after 7 days of static observation. + The introduction of will cause pigments to aggregate to varying degrees.

[0118] Depend on Figure 22 It can be observed that when Na + When the concentration is 100mM, the above-mentioned flake aggregation can also be observed, and the range of each aggregation is small but the number is large; however, some small granular pigments are also observed, which are orange in color and high in brightness, and some areas have light orange "spots", which shows that at this concentration, the pigment is largely destroyed. + When the concentration is 200mM, individual free pigments are almost invisible, and the pigments are still aggregated in small pieces. Careful observation of each aggregate reveals that the fragments are broken but with relatively clear outlines. + At a concentration of 500 mM, large orange-yellow marks were observed in the microscopic field of view, and small orange particles were seen in the marks arranged in a relatively regular and orderly manner.

[0119] Depend on Figure 23 It can be observed that Ca 2+ The introduction of Ca will also cause pigments to aggregate to varying degrees. 2+ At concentrations of 0 and 5 mM, the pigment appears as an irregular (but clearly defined) circle, evenly distributed with no apparent aggregation. At concentrations of 10 and 50 mM, the pigment appears aggregated, slightly fragmented, and with unclear outlines. At concentrations of 100 and 200 mM, some of the pigment appears fragmented and aggregated, while others appear as clearly visible round particles. Compared to the background at lower concentrations, the background appears slightly messy, with light orange "stains." Figure 24 It can be observed that when Ca 2+ When the concentration increased to 500mM, a few pigments were scattered, but the outlines were not very clear. Most of them were aggregated, and large orange-yellow marks were observed. This phenomenon is similar to that of Na + The results of static observation were analyzed, and Na + , Ca 2+ After standing for 7 days, the pigment sample with a concentration of 500 mM only underwent a small amount of sedimentation, and the upper solution showed a uniform and stable orange-yellow color.

[0120] Depend on Figure 25 It can be observed that the final values of the clarification index are significantly different between all concentrations except for 10mM and 200mM. The clarification index of the sample with 0mM is the smallest, which is 0.1202, indicating that the sample does not contain Na + The samples with Na+ concentrations of 10, 50, 100, and 200 mM were the most stable; the clarification index of the samples with Na+ concentrations of 10, 50, 100, and 200 mM were significantly higher than that of 0 mM, indicating that the samples were unstable; compared with 10-200 mM, Na + The clarification index change curves of samples with concentrations of 5 and 500 mM were significantly slower, and the final values of the clarification index were lower, with significant differences, indicating that the stability was relatively good.

[0121] Depend on Figure 26It can be observed that when Ca 2+ When the concentration was 5-200 mM, the final value of the clarification index of the pigment sample was significantly higher than that of the Ca-free sample. 2+ For samples with Ca, the clarification index change curve is extremely steep, indicating that the internal pigment precipitation is extremely fast and the system is unstable. 2+ The clarification index changes most slowly when the concentration is 500 mM, and the final value is the same as that in the absence of Ca 2+ There is no significant difference between them, both are less than 0.2, which shows that the two samples are relatively stable.

[0122] Example 5

[0123] This embodiment provides a green extraction method for natural wolfberry pigment, wherein steps 1) to 4) are the same as those in Example 4, wherein the concentration of the sucrose solution in step 4) is 30%.

[0124] Although the present invention has been described above through the embodiments, those skilled in the art should understand that any improvements and modifications made to the present invention without departing from the spirit and essence of the present invention should fall within the scope of protection of the present invention.

Claims

1. A green extraction method of natural wolfberry pigment, characterized in that: Including steps: 1) Using fresh wolfberry as raw material, adding an extraction solvent, and stirring using a spiral juicer, 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 (v / w, mL / g); 2) After filtering through 2 layers of ordinary filter cloth, collect the filtrate; 3) Centrifuge at 3-5°C and discard the supernatant to obtain a crude extract of wolfberry pigment; centrifuge at 3000 g for 15-20 minutes; 4) Separate with a sucrose solution with a mass concentration of 30~40%.

2. 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 for 1 s each time.

3. The green extraction method according to claim 1, characterized in that Step 4) separation with sucrose solution is as follows: resuspending the centrifuged wolfberry pigment crude extract with the PBS buffer and collecting the resuspension; adding sucrose solution to the resuspension at a volume ratio of 1:(2-3), horizontally centrifuging, aspirating the chromoplast-enriched layer into a beaker, diluting with PBS buffer and then centrifuging, discarding the supernatant, and the precipitate at the bottom is the wolfberry pigment extract.

4. The green extraction method according to claim 3, characterized in that In step 4), the first centrifugation was performed at 5800 g for 30 min, 4°C, and a ramp rate of 2; the second centrifugation was performed at 3000 g for 20 min, 4°C, and a ramp rate of 9.

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