Composite green extraction method of low-methoxyl pectin, hesperidin and synephrine in young citrus fruits

Abstract

The invention relates to the technical field of citrus processing, in particular to a composite green extraction technology for low-ester pectin, hesperidin and synephrine in young citrus fruits, which comprises the following steps: (1) crushing the dried young citrus fruits, putting the young fruit powder into an ultrasonic extraction tank, adding distilled water according to the material-liquid ratio of 1: 5-1: 30 g / mL, performing ultrasonic extraction for 20-60 minutes at the ultrasonic frequency of 20-80 kHz, and filtering to obtain filtrate; the temperature is 50-90 DEG C, centrifuging after the treatment is completed, taking supernate, and using residual filter residues for extracting hesperidin and synephrine. According to the technical scheme provided by the invention, composite extraction of low-ester pectin, hesperidin and synephrine in the young citrus fruits can be realized, and the extraction efficiency of functional active substances in the young citrus fruits is effectively improved; and the used extraction solvent has small pollution, is more environment-friendly compared with dichloromethane, ethyl acetate, methanol, acetone and other organic solvents used in the prior art, realizes reutilization of the extraction solvent, reduces environmental pollution, and reduces the extraction cost.

Description

Technical Field

[0001] The invention relates to the technical field of citrus processing, and in particular to a composite green extraction technology of low-ester pectin, hesperidin and synephrine in young citrus fruits. Background Art

[0002] my country's citrus planting volume ranks among the highest in the world, and countless fruit drops are produced every year. However, due to factors such as the short fruit drop time and small size that make collection difficult, people have not paid much attention to it, resulting in a large number of fruit drops rotting in the ground, which not only increases the environmental burden, but also causes a waste of resources. In fact, young citrus fruits are rich in a variety of functional active ingredients, especially in the first physiological fruit drop from April to May each year. At present, the only methods for extracting active ingredients from young citrus fruits are mostly limited to traditional solvent extraction methods, which can only extract a single functional active ingredient, and the organic solvents used, such as dichloromethane, ethyl acetate, methanol, acetone, etc., are highly volatile, and the extraction process consumes a lot of energy. Application in industrial production causes serious pollution, which does not conform to the green and sustainable development strategy currently advocated by my country. Summary of the invention

[0003] In order to solve the problems of serious organic solvent pollution, complex extraction process, high energy consumption, etc. in the prior art, the present invention provides a green and simple composite extraction process, which can simultaneously extract and separate three active ingredients, hesperidin, synephrine and pectin, from young citrus fruits.

[0004] The present invention is achieved through the following technical solutions:

[0005] Step 1: Extraction and separation of pectin from young citrus fruits. Crush the dried young citrus fruits, place the young fruit powder in an ultrasonic extraction tank, add distilled water at a solid-liquid ratio of 1:5-1:30 g / mL, the ultrasonic frequency is 20-80 kHz, the extraction time is 20-60 min, the temperature is 50-90 ° C, centrifuge after the treatment is completed, take the supernatant, and the remaining filter residue is used for the extraction of hesperidin and synephrine. Then add anhydrous ethanol according to 2-5 times the volume of the supernatant, let it stand for 30 minutes, centrifuge after the pectin precipitates, the precipitate is the pectin product, take it out and dry it to obtain pectin.

[0006] Step 2, pretreatment of macroporous resin. AB-8 macroporous resin is selected for adsorption. Since some industrial impurities will remain in the macroporous resin during the production process, it needs to be pretreated before use. First, immerse the macroporous resin in 95% ethanol to fully swell the resin filler. During this period, the ethanol is replaced every 2 hours. After soaking for 24 hours, it is repeatedly washed with deionized water until there is no alcohol smell. Then soak it in a 4% NaOH solution with a volume fraction for 4 hours, and wash it with deionized water until neutral; then soak it in a 4% HCL solution with a volume fraction for 4 hours, rinse it with deionized water until neutral, and scan the eluent in the wavelength range of 200-400nm with a UV spectrophotometer. If no absorption peak is detected, it can be soaked in deionized water for use.

[0007] Step 3, preparation of a low eutectic solvent. A low eutectic solvent is prepared by a heating method, choline chloride and acetic acid are placed in a mixing tank at a molar ratio of 1:2, and then heated to 80° C. and stirred at a constant temperature for 2 hours until a uniform, clear, transparent liquid is formed.

[0008] Step 4, preparation of choline chloride / acetic acid citrus young fruit residue extract. The residue prepared in step 1 is placed in an ultrasonic extraction tank, a low eutectic solvent is added at a ratio of 1:10-1:50, and then fully stirred and ultrasonically extracted. The extraction conditions are: ultrasonic frequency 20-90kHz, temperature 40-90°C, time 10-60min. After the end of the ultrasound, high-speed centrifugation is performed for 10-30min. The supernatant is used for the extraction of hesperidin, and the residue is used for the extraction of synephrine.

[0009] Step 5: Enrichment and separation of hesperidin in the citrus young fruit residue extract by macroporous adsorption resin. The macroporous resin treated in step 2 is wet loaded into a glass chromatography column, and the low eutectic solvent extract of the citrus young fruit residue is mixed with deionized water in a ratio of 1:1-1:5, and slowly poured into the chromatography column. After the macroporous resin is fully adsorbed, it is repeatedly rinsed with deionized water to remove the low eutectic solvent. After the macroporous resin is fully adsorbed, the hesperidin component adsorbed by the macroporous resin is desorbed with 95% ethanol solution until the outflowing solution is nearly colorless. The enriched hesperidin crude product is vacuum dried to obtain brown-yellow hesperidin.

[0010] Step 6: Reuse of low eutectic solvent. As a new type of green extraction solvent, the low eutectic solvent is easy to recycle, which is one of its excellent characteristics. The adsorption saturated macroporous resin infusion is filtered, and ethanol is added to the macroporous resin for desorption. The low eutectic solvent is recycled and used for the next extraction of hesperidin from young citrus fruits.

[0011] Step 7, enhanced regeneration of macroporous resin. Add 2% to 3% HCL solution 10cm above the resin layer into the container, soak for 2 to 4 hours, then pass the column with 3 times the volume of HCL solution of the resin, and wash with clean water until it is close to neutral; then soak with 5% NaOH solution for 2 to 4 hours, and pass the column with 3 times the volume of the resin with the same concentration of sodium hydroxide solution, and finally wash with clean water until the pH is neutral. Finally, wash the column with 2 to 3 times the volume of the resin with ethanol, wash off the ethanol with pure water, and put it into use.

[0012] Step 8, extraction of synephrine from young citrus fruits. The filter residue prepared in step 4 is dried and placed in an extraction tank, and then a composite enzyme buffer is added at a solid-liquid ratio of 1:10-1:50 g / mL, wherein the ratio of pectinase to cellulase is 1:1-1:5, and the pH is adjusted to 2-5 with a citric acid solution, and a constant temperature heating magnetic stirrer is placed, and the temperature is adjusted to 30-70°C, the speed is 100-300 rpm, and the temperature is adjusted to 70-95°C after enzymolysis for 1-5 hours, and the enzyme is inactivated for 5-30 minutes. After centrifugation, the supernatant is taken for use. The treated 001-7 type cation exchange resin is wet loaded onto the column, and then the synephrine extract is slowly poured into it, and the deionized water is repeatedly washed for about 4 to 5 column volumes at a flow rate of 1-5BV / h. After the exchange resin is fully adsorbed, the flow rate is adjusted to 5BV / h, and the synephrine component adsorbed by the exchange resin is desorbed with a 30-80% ethanol solution until the outflowing solution is nearly colorless, and then vacuum dried to obtain a milky white synephrine product.

[0013] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0014] (1) The technical solution provided by the present invention can realize the composite extraction of low-ester pectin, hesperidin and synephrine in young citrus fruits, effectively improving the extraction efficiency of functional active substances in young citrus fruits.

[0015] (2) The extraction solvent used in the technical solution provided by the present invention has low pollution and is more environmentally friendly than organic solvents such as dichloromethane, ethyl acetate, methanol, acetone, etc. used in the prior art. The extraction solvent can be reused, which reduces environmental pollution and reduces extraction costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments of the present invention. In the drawings:

[0017] Figure 1 This is a flow chart of the extraction process of the present invention;

[0018] Figure 2The infrared spectra of citrus young fruit pectin are as follows: (a) citrus young fruit pectin; (b) commercial low-ester pectin; (c) citrus commercial pectin;

[0019] Figure 3 The ion chromatograms of citrus young fruit pectin are as follows: (a) mixed standard sample; (b) citrus young fruit pectin;

[0020] Figure 4 The microscopic surface morphology of pectin, (a) commercial pectin; (b) freeze-dried pectin; (c) oven-dried pectin;

[0021] Figure 5 This is the static adsorption kinetic curve of hesperidin by AB-8 macroporous resin;

[0022] Figure 6 This is the static desorption kinetic curve of hesperidin by AB-8 macroporous resin;

[0023] Figure 7 This is the infrared spectrum of hesperidin in young citrus fruit, (a) hesperidin standard; (b) young citrus fruit;

[0024] Figure 8 This is the UV spectrum of hesperidin in young citrus fruit;

[0025] Fig. 9 This is the HPLC chromatogram of hesperidin in young citrus fruit. Note: (a) Hesperidin standard; (b) Hesperidin in young citrus fruit

[0026] Fig.10 This is the static adsorption kinetic curve of synephrine by 001×7 type cation exchange resin;

[0027] Fig.11 This is the static desorption kinetic curve of synephrine by 001×7 type cation exchange resin;

[0028] Fig.12 This is the infrared spectrum of synephrine in young citrus fruit, (a) synephrine standard; (b) young citrus fruit;

[0029] Fig.13 This is the UV spectrum of synephrine;

[0030] Fig.14 This is the HPLC chromatogram of synephrine in young citrus fruit. Note: (a) synephrine standard; (b) young citrus fruit. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments and drawings.

[0032] Embodiment 1:

[0033] (1) The dried young citrus fruits were crushed, and the young fruit powder was placed in an ultrasonic extraction tank. Distilled water was added at a solid-liquid ratio of 1:5-1:30 g / mL, the ultrasonic frequency was 20-80 kHz, the extraction time was 20-60 min, and the temperature was 50-90 ° C. After the treatment was completed, centrifugation was performed to take the supernatant, and the remaining filter residue was used to extract hesperidin and synephrine. Anhydrous ethanol was then added according to 2-5 times the volume of the supernatant, and the mixture was allowed to stand for 30 min. After the pectin was precipitated, the mixture was centrifuged. The precipitate was the pectin product, which was taken out and dried to obtain pectin. The infrared spectra, monosaccharide composition, microscopic morphology, moisture content, ash content, yield, degree of esterification, galacturonic acid (GalA) content, pectin relative molecular weight and other indicators of pectin in young citrus fruits were measured respectively.

[0034] The crude citrus pectin extracted from young fruits, commercial low-ester pectin and commercial citrus pectin were analyzed by Fourier transform infrared spectroscopy. Figure 2 As shown. The broad peak at 3500-3000cm-1 is the representative peak of the OH group, which is caused by the stretching vibration of the hydrogen bonds within and between molecules. Since hydroxyl groups exist both within and between molecules, the absorption peak is very broad; the weaker peak at 2930cm-1 is caused by the stretching vibration of the CH bond. Citrus commercial pectin has absorption peaks at around 1750 and 1625cm-1, while citrus young fruit pectin and commercial low-ester pectin only have absorption peaks at 1625. The above two peaks are characteristic peaks for judging pectin. The peak at 1750cm-1 is the absorption peak formed by the stretching vibration of the esterified carboxyl group (-COOR), which can be used to distinguish low-ester pectin from high-ester pectin. The absorption peak at 1625cm-1 is formed by the asymmetric stretching vibration of the free carboxylate (-COO) in pectin. In addition, the carboxylate has another absorption band located at around 1400cm-1, which is formed by weaker symmetric vibration. The spectra of citrus young fruit pectin are highly consistent with those of commercial low-ester pectin, which shows that the type of pectin extracted from citrus young fruit is low-ester pectin, and the calculation of esterification degree can also explain this. Based on the characteristics of the above absorption peaks, it can be inferred that the extract is pectin.

[0035] The results of the determination of the monosaccharide composition of citrus young fruit pectin and commercial pectin are shown in Table 1. Gal is the most abundant monosaccharide in citrus young fruit pectin, with a content of 51.4%, which is greater than commercial pectin. It is followed by Ara, GlcA, Glc and Rha, with contents of 28.3%, 7.2%, 7% and 4.6% respectively. The contents of GalA and Man are very small.

[0036] Table 1 Analysis of monosaccharide composition of pectin in young citrus fruits

[0037]

[0038] The pectin yield of young citrus fruits was calculated to be about 5.2%, and the DE value was 38%, which is less than 50%, so it can be classified as low methoxyl pectin. According to the regulations of the European Union and the Food and Agriculture Organization of the United Nations, the galacturonic acid content in pectin should be greater than 65%. Substituting the standard curve into the measured GalA content in young citrus fruit pectin was 68.27%, which meets the current market requirements for pectin purity. The above test results show that the physical and chemical parameters of young citrus fruit pectin meet the national standards.

[0039] Table 2 Determination results of physicochemical parameters of pectin in young citrus fruits

[0040]

[0041] The weight average molecular weight (MW), number average molecular weight (MN), and polydispersity index MW / MN (PDI) of citrus pectin can be seen to be different from commercial pectin. From the data in Table 3, it can be seen that the MW of citrus pectin is 583.66 kDa, which is a high molecular weight pectin (19.9-2000 kDa), and the value is slightly lower than that of commercial pectin. The advantages of high molecular weight pectin are strong gelling and high viscosity, which are beneficial to improve the emulsification of pectin and are widely used in food. The PDI value of citrus pectin is very close to that of commercial pectin.

[0042] Table 3 Relative molecular weight of pectin from young citrus fruits

[0043]

[0044] Depend on Figure 4 It can be seen that under the same magnification of 20k, the microscopic morphology of the three pectins is different, indicating that different drying methods have different effects on the microstructure of pectin. The surface of commercial pectin is rough, blocky, tight and pore-free, with almost no cracks; the surface of freeze-dried pectin is smooth and continuous, with relatively uniform pores and a regular and dense honeycomb structure; the pores of dried pectin are unevenly distributed, the structure is disorderly arranged, and the surface is rough.

[0045] Table 4 Physical and chemical parameters of pectin in young citrus fruits

[0046]

[0047] As shown in Table 4, the pectin yield of young citrus fruit is calculated to be about 5.2%, and the DE value is 38%, which is less than 50%, so it can be classified as low methoxyl pectin. According to the regulations of the European Union and the Food and Agriculture Organization of the United Nations, the galacturonic acid content in pectin should be greater than 65%. Substituting the standard curve into the GalA content in young citrus fruit pectin is 68.27%, which meets the current market requirements for pectin purity. The above test results show that the physical and chemical parameters of young citrus fruit pectin meet the national standards.

[0048] Table 5 Relative molecular weight of pectin from young citrus fruits

[0049]

[0050]

[0051] As shown in Table 5, the weight average molecular weight (MW), number average molecular weight (MN), and polydispersity index MW / MN (PDI) of citrus pectin can be seen to be different from commercial pectin. From the data in the above table, it can be seen that the MW of citrus pectin is 583.66 kDa, which is a high molecular weight pectin (19.9-2000 kDa), and the value is slightly lower than that of commercial pectin. The advantages of high molecular weight pectin are strong gelling and high viscosity, which are conducive to improving the emulsification of pectin. The PDI value of citrus pectin is very close to that of commercial pectin.

[0052] (2) Pretreatment of macroporous resin. AB-8 macroporous resin was selected for adsorption. Since some industrial impurities will remain in the macroporous resin during the production process, it needs to be pretreated before use. First, immerse the macroporous resin in 95% ethanol to fully swell the resin filler. During this period, the ethanol is replaced every 2 hours. After soaking for 24 hours, it is repeatedly washed with deionized water until there is no alcohol smell. Then, soak it in a 4% NaOH solution by volume for 4 hours, and wash it with deionized water until it is neutral; then soak it in a 4% HCL solution by volume for 4 hours, and rinse it with deionized water until it is neutral. Scan the eluent in the wavelength range of 200-400nm on a UV spectrophotometer. If no absorption peak is detected, it can be soaked in deionized water for use.

[0053] Depend on Figure 5 It can be seen that after the macroporous resin is adsorbed, the concentration of hesperidin in the remaining sample solution in the conical flask gradually decreases. Within 0-6 hours, as the adsorption rate of the macroporous resin gradually increases, the hesperidin content drops sharply. After 6 hours, the concentration changes tend to be constant, indicating that the adsorption is close to saturation and the adsorption rate no longer increases. It can be seen that the static adsorption equilibrium time of hesperidin on AB-8 macroporous resin is 6 hours.

[0054] Depend on Figure 6 It can be seen that in the initial 0-4 hours, the concentration of hesperidin in the desorption solution increased rapidly and then gradually stabilized. When the desorption time reached 4 hours, the concentration of hesperidin basically stopped increasing, indicating that the desorption had reached equilibrium at this time. It can be seen that the static desorption equilibrium time of hesperidin on AB-8 macroporous resin is 4 hours.

[0055] The calculation results show that the adsorption and desorption rates of hesperidin in citrus young fruit residue by AB-8 macroporous resin are 78.57% and 82.15% respectively, which are good indicators and can be used as a filler for hesperidin extraction.

[0056] (3) Preparation of low eutectic solvent. The low eutectic solvent was prepared by heating method. Choline chloride and acetic acid were placed in a mixing tank in a molar ratio of 1:2, and then heated to 80°C and stirred for 2 hours until a uniform, clear and transparent liquid was formed.

[0057] (4) Preparation of choline chloride / acetic acid citrus young fruit residue extract. The residue prepared in step 1 was placed in an ultrasonic extraction tank, and a low eutectic solvent was added at a ratio of 1:10-1:50, and then fully stirred and ultrasonically extracted. The extraction conditions were: ultrasonic frequency 20-90kHz, temperature 40-90°C, and time 10-60min. After the end of the ultrasound, high-speed centrifugation was performed for 10-30min. The supernatant was used for the extraction of hesperidin, and the residue was used for the extraction of synephrine.

[0058] (5) Enrichment and separation of hesperidin in the citrus young fruit residue extract by macroporous adsorption resin. The macroporous resin treated in step 2 is wet loaded into a glass chromatography column, and the low eutectic solvent extract of the citrus young fruit residue is mixed with deionized water in a ratio of 1:1-1:5, and slowly poured into the chromatography column. After the macroporous resin is fully adsorbed, it is repeatedly rinsed with deionized water to remove the low eutectic solvent. After the macroporous resin is fully adsorbed, the hesperidin component adsorbed by the macroporous resin is desorbed with 95% ethanol solution until the outflowing solution is nearly colorless. The enriched hesperidin crude product is vacuum dried to obtain brown-yellow hesperidin.

[0059] according to Figure 7 The results show that the structure of crude hesperidin extracted from young citrus fruits is highly consistent with that of standard hesperidin. The broad peak at 3300-3400cm-1 is the stretching vibration peak of the OH group; the peak in the range of 2850-3000cm-1 is the stretching vibration peak of the CH group; the typical absorption peaks of the hesperidin structure appear at 1636cm-1, 1603cm-1 and between 1000-1100cm-1. The above results are consistent with the literature records and their molecular structure, and it can be inferred that the extract is hesperidin.

[0060] The crude hesperidin obtained by vacuum concentration was added to methanol solution for re-dissolution and then placed in a UV spectrophotometer for measurement. It was found that the maximum wavelength appeared around 283 nm ( Figure 8 ), which is consistent with the characteristics of hesperidin.

[0061] Depend on Fig. 9 It can be seen that the retention time of the crude hesperidin product extracted from the citrus young fruit residue is basically consistent with that of the hesperidin standard, both around 13.9 minutes, and the peak shape is symmetrical with extremely high separation.

[0062] The results of HPLC showed that the hesperidin content in the eluate after adsorption and desorption by macroporous resin using low eutectic solvent as the extraction agent was 726.37 μg / ml, which is almost the same as the extraction amount using methanol as the extraction solvent. The difference is that low eutectic solvent is more environmentally friendly.

[0063] At the same time, no synephrine was detected in the eluate, indicating that the low eutectic solvent composed of choline chloride / acetic acid had a poor extraction effect on synephrine, and a large amount of synephrine still remained in the remaining filter residue, creating conditions for the next step of extracting synephrine.

[0064] (6) Reuse of low eutectic solvents. As a new type of green extraction solvent, the low eutectic solvent is easy to recycle, which is one of its excellent characteristics. The adsorption saturated macroporous resin extract is filtered, and the macroporous resin is desorbed by adding ethanol. The low eutectic solvent is recycled and used for the next extraction of hesperidin from young citrus fruit.

[0065] (7) Enhanced regeneration of macroporous resin. Add 2% to 3% HCL solution to a height of 10 cm above the resin layer in the container, soak for 2 to 4 hours, then pass HCL solution 3 times the volume of the resin through the column, and wash with clean water until it is nearly neutral; then soak with 5% NaOH solution for 2 to 4 hours, and pass the column with sodium hydroxide solution 3 times the volume of the resin in the same way, and finally wash with clean water until the pH is neutral. Finally, wash the column with ethanol 2 to 3 times the volume of the resin, wash away the ethanol with pure water, and then put it into use.

[0066] (8) Extraction of synephrine from young citrus fruits. The filter residue prepared in step 4 is dried and placed in an extraction tank. Then, a composite enzyme buffer is added at a solid-liquid ratio of 1:10-1:50 g / mL, wherein the ratio of pectinase to cellulase is 1:1-1:5. The pH is adjusted to 2-5 with a citric acid solution. The mixture is placed in a constant temperature heating magnetic stirrer, the temperature is adjusted to 30-70°C, the speed is 100-300 rpm, and the temperature is adjusted to 70-95°C after enzymolysis for 1-5 hours. The enzyme is inactivated for 5-30 minutes. After centrifugation, the supernatant is taken for use. The treated 001-7 type cation exchange resin is wet loaded onto the column, and then the synephrine extract is slowly poured into it, and the deionized water is repeatedly washed for about 4 to 5 column volumes at a flow rate of 1-5BV / h. After the exchange resin is fully adsorbed, the flow rate is adjusted to 5BV / h, and the synephrine component adsorbed by the exchange resin is desorbed with a 30-80% ethanol solution until the outflowing solution is nearly colorless, and then vacuum dried to obtain a milky white synephrine product.

[0067] Depend on Fig.10It can be seen that after the exchange resin is adsorbed, the concentration of synephrine in the remaining sample solution in the conical flask gradually decreases. Within 0-4 hours, as the adsorption rate of the exchange resin gradually increases, the synephrine content drops sharply. After 4 hours, the concentration changes tend to be constant, indicating that the adsorption is close to saturation and the adsorption rate no longer increases. It can be seen that the static adsorption equilibrium time of synephrine on 001×7 type cation exchange resin is 4 hours.

[0068] Depend on Fig.11 It can be seen that within the first 0-2 hours, the concentration of synephrine in the desorption solution increased rapidly and then gradually stabilized. When the desorption time reached 2 hours, the concentration of synephrine basically stopped increasing, indicating that the desorption had reached equilibrium at this time. Therefore, it can be seen that the static desorption equilibrium time of synephrine on the 001×7 type cation exchange resin is 2 hours. It was calculated that the adsorption rate and desorption rate of synephrine in citrus young fruit residue by 001×7 type cation exchange resin were 65.78% and 76.13% respectively, with good indicators, proving that it can be used as a filler for synephrine extraction.

[0069] like Fig.12 As shown, the characteristic peak at 3340cm-1 is the stretching vibration peak of OH, the characteristic peaks at 2950 and 2870cm-1 are the stretching vibration peaks of CH3 and CH2, the range of 1000-1600cm-1 is the stretching vibration region of single and double bonds, among which 1248, 1160 and 1058cm-1 are the stretching vibrations of CO, which are consistent with the characteristic structure of synephrine. It can be inferred that the extract is synephrine.

[0070] The crude synephrine obtained by vacuum concentration and drying was added to methanol solution for re-dissolution and then placed in a UV spectrophotometer for measurement. It was found that the maximum wavelength appeared around 275nm ( Fig.13 ), which is consistent with the characteristics of synephrine.

[0071] Depend on Fig.14 It can be seen that the retention time of the crude synephrine product extracted from the citrus young fruit residue is basically consistent with that of the synephrine standard, both of which are about 5.9 minutes, and the peak shape is symmetrical, with extremely high separation. The results of HPLC determination show that the synephrine content in the eluate after adsorption and desorption by 001×7 type cation exchange resin using the composite enzyme extract is 187.28μg / ml, which is about 2 / 3 of the extraction amount of the methanol control group.

[0072] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A composite green extraction method for low-ester pectin, hesperidin and synephrine in young citrus fruits, characterized by: The method comprises the following steps: step 1, extracting and separating pectin from young citrus fruits; Step 2, pretreatment of macroporous resin; Step 3, preparation of a low eutectic solvent; Step 4, preparation of choline chloride / acetic acid citrus young fruit residue extract; Step 5, enriching and separating hesperidin in the citrus young fruit residue extract by using a macroporous adsorption resin; Step 6, reusing the low eutectic solvent; Step 7, enhanced regeneration of macroporous resin; Step 8: Extraction of synephrine from young citrus fruits.

2. The composite green extraction method of low-ester pectin, hesperidin and synephrine in young citrus fruits according to claim 1, characterized in that: The dried young citrus fruits were crushed and the young fruit powder was placed in an ultrasonic extraction tank. Distilled water was added at a solid-liquid ratio of 1:5-1:30 g / mL. The ultrasonic frequency was 20-80 kHz, the extraction time was 20-60 min, and the temperature was 50-90°C. After the treatment, the mixture was centrifuged and the supernatant was taken. The remaining filter residue was used for the extraction of hesperidin and synephrine. Anhydrous ethanol was added according to 2-5 times the volume of the supernatant and the mixture was allowed to stand for 30 min. After the pectin was precipitated, the mixture was centrifuged and the precipitate was the pectin product. The pectin was obtained by taking it out and drying it.

3. The composite green extraction method of low-ester pectin, hesperidin and synephrine in young citrus fruits according to claim 2, characterized in that: AB-8 macroporous resin was selected for adsorption. Since some industrial impurities will remain in the macroporous resin during the production process, it needs to be pretreated before use. First, the macroporous resin was immersed in 95% ethanol to fully swell the resin filler. The ethanol was replaced every 2 hours. After soaking for 24 hours, it was repeatedly washed with deionized water until there was no alcohol smell. Then it was soaked in a 4% NaOH solution with a volume fraction for 4 hours and washed with deionized water until neutral; then it was soaked in a 4% HCL solution with a volume fraction for 4 hours and rinsed with deionized water until neutral. The eluent was scanned in the wavelength range of 200-400 nm by an ultraviolet spectrophotometer. If no absorption peak was detected, it could be soaked in deionized water for standby use.

4. The composite green extraction method of low-ester pectin, hesperidin and synephrine in young citrus fruits according to claim 3, characterized in that: The low eutectic solvent was prepared by a heating method. Choline chloride and acetic acid were placed in a mixing tank in a molar ratio of 1:2, and then heated to 80°C and stirred at a constant temperature for 2 h until a uniform, clear and transparent liquid was formed.

5. The composite green extraction method of low-ester pectin, hesperidin and synephrine in young citrus fruits according to claim 4, characterized in that: The filter residue prepared in step 1 is placed in an ultrasonic extraction tank, and a low eutectic solvent is added at a ratio of 1:10-1:50, and then fully stirred and ultrasonically extracted. The extraction conditions are: ultrasonic frequency 20-90 kHz, temperature 40-90°C, time 10-60 min, and high-speed centrifugation for 10-30 min after the end of ultrasound. The supernatant is used for the extraction of hesperidin, and the filter residue is used for the extraction of synephrine.

6. The composite green extraction method of low-ester pectin, hesperidin and synephrine in young citrus fruits according to claim 5, characterized in that: The macroporous resin treated in step 2 is wet loaded into a glass chromatography column, and the low eutectic solvent extract of the citrus young fruit residue is mixed with deionized water in a ratio of 1:1-1:5, and slowly poured into the chromatography column. After the macroporous resin is fully adsorbed, it is repeatedly rinsed with deionized water to remove the low eutectic solvent. After the macroporous resin is fully adsorbed, the hesperidin component adsorbed by the macroporous resin is desorbed with 95% ethanol solution until the outflowing solution is nearly colorless, and the enriched hesperidin crude product is vacuum dried to obtain brown-yellow hesperidin.

7. The composite green extraction method of low-ester pectin, hesperidin and synephrine in young citrus fruits according to claim 6, characterized in that: As a new type of green extraction solvent, low eutectic solvent has the advantage of being easy to recycle. The adsorption-saturated macroporous resin extract is filtered, ethanol is added to the macroporous resin for desorption, and the low eutectic solvent is recycled for the next extraction of hesperidin from young citrus fruit.

8. The composite green extraction method of low-ester pectin, hesperidin and synephrine in young citrus fruits according to claim 7, characterized in that: Add 2%~3% HCL solution 10 cm above the resin layer in the container, soak for 2~4 hours, then pass the column with HCL solution 3 times the volume of the resin, and wash with clean water until it is close to neutral; then soak with 5% NaOH solution for 2~4 hours, and pass the column with sodium hydroxide solution of the same concentration 3 times the volume of the resin as above, finally wash with clean water until the pH is neutral, finally wash the column with ethanol 2~3 times the volume of the resin, wash away the ethanol with pure water, and put it into use.

9. The composite green extraction method of low-ester pectin, hesperidin and synephrine in young citrus fruits according to claim 8, characterized in that: The filter residue prepared in step 4 is dried and placed in an extraction tank, and then a composite enzyme buffer is added at a solid-liquid ratio of 1:10-1:50 g / mL, wherein the ratio of pectinase to cellulase is 1:1-1:5, and the pH is adjusted to 2-5 with a citric acid solution, and a constant temperature heating magnetic stirrer is placed, and the temperature is adjusted to 30-70°C and the rotation speed is 100-300 rpm. After enzymatic hydrolysis for 1-5 hours, the temperature is adjusted to 70-95°C, and the enzyme is inactivated for 5-30 minutes. After centrifugation, the supernatant is taken for standby use, and the treated 001-7 type cation exchange resin is wet-column-loaded, and then the synephrine extract is slowly poured into it, and the synephrine extract is repeatedly rinsed with deionized water at a flow rate of 1-5 BV / h for about 4 to 5 column volumes. After the exchange resin is fully adsorbed, the flow rate is adjusted to 5 BV / h, and the synephrine component adsorbed on the exchange resin is desorbed with a 30-80% ethanol solution until the outflowing solution is nearly colorless, and then vacuum dried to obtain a milky white synephrine product.

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