Method for extracting zeaxanthine extract from Chinese wolfberry fruits

Abstract

The invention belongs to the technical field of agricultural product processing and natural product extraction, and discloses a method for extracting zeaxanthine extract from fresh wolfberry fruits. The method comprises the following steps: ultrasonically soaking fresh Chinese wolfberry fruits in a 1%-5% Na2CO3 solution for 20-40 seconds, spraying a compound system of 0.2%-0.5% pectinase and / or hemicellulase and 0.2%-1.0% lactic acid bacteria on the surfaces of the Chinese wolfberry fruits, vacuumizing, fermenting at 25-35 DEG C for 4-5 days, vacuumizing, crushing the fruits, and continuously fermenting for 1 day; and drying the fermentation product at 55 + / -5 DEG C, crushing step by step, extracting in a subcritical extraction kettle, extracting most of air, and desolventizing under reduced pressure to obtain the zeaxanthine extract. Through pretreatment, the structure of wolfberry is destroyed, the sugar content is reduced, the content of zeaxanthine in the extract is more than 15% and is increased by more than 3 times compared with the prior art, the process is simple and environment-friendly, and large-scale production is easy to realize.

Description

Technical Field

[0001] This invention belongs to the field of agricultural product processing and natural product extraction technology, specifically relating to a method for extracting zeaxanthin from wolfberry. Background Technology

[0002] Zeaxanthin is a characteristic biomarker and core functional factor in goji berries. It is the main pigment responsible for the bright red color of goji berries and is also one of the key active ingredients responsible for their antioxidant, vision-protecting, immune-boosting, and chronic disease-preventing physiological functions. Zeaxanthin is a key pigment in the macular region of the human retina, playing a vital role in protecting visual health along with lutein. With increasing health awareness, the demand for natural, healthy, and effective active ingredients is growing, resulting in significant potential market demand for zeaxanthin. Therefore, efficient and high-yield extraction of zeaxanthin from goji berries has significant commercial value and practical significance for developing high-value-added raw materials for health foods, pharmaceuticals, and cosmetics. It can also improve the level and competitiveness of goji berry deep processing and promote industrial upgrading.

[0003] Currently, technologies for extracting zeaxanthin from wolfberry are constantly evolving. For example, organic / green solvents and auxiliary technologies (such as ultrasound / microwave) are used to extract zeaxanthin from wolfberry, but large-scale production faces bottlenecks. In addition, there is supercritical extraction, but the equipment operation requirements are high and the industrial production cost is high. In existing reports on subcritical extraction technology, the obtained zeaxanthin content is low and the target substance is not completely extracted.

[0004] The closest existing technology is patent ZL201610768879.3 (a method for extracting zeaxanthin and its derivatives from wolfberries). This technology uses a method of soaking dried wolfberries in water to reduce the sugar content of the raw material, and then performs a second drying to obtain wolfberry raw material for zeaxanthin extraction. However, this method requires a second drying, which significantly increases the drying cost.

[0005] Therefore, there is an urgent need in this field to develop a novel extraction technology specifically for zeaxanthin from wolfberry, which can maximize the extraction rate of zeaxanthin esters and be efficient, green, with mild process conditions, simple process, controllable cost, and easy to achieve large-scale production. Summary of the Invention

[0006] This invention provides a method for extracting zeaxanthin from wolfberry. The method involves uniformly spraying a composite system consisting of 0.2%–0.5% pectinase and / or hemicellulase and 0.2%–1.0% lactic acid bacteria onto the surface of fresh wolfberry fruit. A stepwise fermentation process is then employed: first, fermentation is carried out in a vacuum environment at 25℃–35℃ for 4–5 days; then, after vacuum treatment and fruit crushing, fermentation continues for 1 day. Through the synergistic effect of this stepwise fermentation process, the cell wall structure of wolfberry is disrupted, and zeaxanthin is released and accumulated, ultimately significantly increasing the extraction yield of zeaxanthin.

[0007] This invention provides a method for extracting zeaxanthin from wolfberry, comprising the following steps: S1. Na2CO3 ultrasonic impregnation: Fresh wolfberry fruits are ultrasonically impregnated with a 1%~5% Na2CO3 solution, and then the Na2CO3 solution is drained off. S2. Compound fermentation: The compound system is evenly sprayed onto the surface of whole wolfberry fruits after ultrasonic impregnation with Na2CO3. The compound system contains 0.2%~0.5% pectinase and / or hemicellulase, and 0.2%~1.0% lactic acid bacteria. Vacuum fermentation is carried out for 5~6 days. S3. Drying and grinding: Dry the fermented wolfberries at 55℃±5℃ and grind them to a particle size of 16~22 mesh; S4. Subcritical extraction: The dried and pulverized wolfberries from step S3 are placed into a subcritical extraction vessel for extraction. S5. Post-processing: After extraction, desolventize under reduced pressure to obtain wolfberry zeaxanthin extract.

[0008] Furthermore, the concentration of the Na2CO3 solution in step S1 above is 1% to 5%.

[0009] Furthermore, the ultrasonic impregnation time in step S1 above is 20s~40s.

[0010] Furthermore, the ultrasonic power in step S1 is 240~480 W and the frequency is 9~12 Hz.

[0011] Furthermore, the vacuum fermentation mentioned in step S2 above refers to placing the wolfberry fruit with the composite system evenly sprayed on its surface into a sealed bag, vacuuming the sealed bag, and fermenting it at 25℃~35℃ for 4~5 days; then vacuuming it again, crushing the fruit, and continuing to ferment at 25℃~35℃ for 1 day.

[0012] Furthermore, in step S4 above, the extractant is liquefied butane, the extraction temperature is 40~50℃, the material-to-liquid ratio is 1:2~1:3, the extraction time is 40 min / time, and the number of extractions is 4~5 times.

[0013] Compared with the prior art, the present invention has the following advantages: (1) The present invention uses Na2CO3 ultrasonic impregnation and pectinase and / or hemicellulase treatment to synergistically destroy the waxy layer and cell wall structure of fresh wolfberry epidermis and promote the release of zeaxanthin.

[0014] (2) This invention uses lactic acid bacteria fermentation simultaneously with enzymatic hydrolysis. On the one hand, this reduces the sugar content of the fruit and decreases material adhesion during extraction. On the other hand, the fermentation of lactic acid bacteria increases the zeaxanthin content in the wolfberry raw material. This may be because the fermentation of lactic acid bacteria promotes the conversion and accumulation of zeaxanthin. Combined with a specific fermentation process, the zeaxanthin content in the extract is >15%. Compared with extraction methods without lactic acid bacteria fermentation, the zeaxanthin content in the extract is increased by 1.66 to 1.73 times.

[0015] (3) In this invention, fresh whole wolfberry fruit is directly subjected to Na2CO3 ultrasonic impregnation, enzymatic hydrolysis and fermentation. Compared with dried wolfberry fruit, the zeaxanthin content in the extract can be increased by more than 115%. Compared with wolfberry pulp, the subsequent drying treatment before extraction is more efficient, which can increase the yield of zeaxanthin in the extract by 21%. Detailed Implementation

[0016] The lactic acid bacteria used in the following examples and comparative examples are yogurt starter powders purchased from Beijing Chuanxiu Technology Co., Ltd.

[0017] Example 1 A method for extracting zeaxanthin from wolfberry includes the following steps: (1) Raw material screening: Remove moldy, rotten, and damaged wolfberry fruits from fresh wolfberry; (2) Ultrasonic impregnation: Impregnate the fruits with 5% Na2CO3 solution using ultrasonic power 360 W, ultrasonic frequency 10 Hz, and impregnation time 40 s, and then drain the Na2CO3 solution; (3) Compound fermentation: Spray a compound system evenly onto the surface of the fruit, the compound system containing 0.15% pectinase + 0.15% hemicellulase and 1% lactic acid bacteria; after vacuuming, ferment at 35℃ for 5 days; then vacuum again, crush the fruit, and continue fermenting at 35℃ for 1 day; (4) Drying, crushing, and sieving: Dry the fermented wolfberry with hot air at 55℃, and sieve for 16~22 seconds. (5) Subcritical extraction: The combined raw materials are loaded into the subcritical extraction vessel, most of the air in the extractor is extracted after the feeder is closed, liquefied butane is injected, the extraction temperature is set to 40~50℃, the material-liquid ratio is 1:2~1:3, the extraction time is 40 min / time, and the number of extractions is 4 times; (6) Post-treatment: After extraction, the solvent is removed under reduced pressure and the wolfberry zeaxanthin extract is obtained.

[0018] After testing, the zeaxanthin content in the wolfberry raw material was 144.31 mg / 100g after the completion of the compound fermentation step (3), and the zeaxanthin content in the post-processed extract was 15.70%, with a yield of 2.17%.

[0019] Example 2 A method for extracting zeaxanthin from wolfberry, the specific steps are the same as in Example 1, except that: the concentration of Na2CO3 solution in step (2) is 3%; and the 0.15% pectinase + 0.15% hemicellulase in step (3) is replaced with 0.3% hemicellulase.

[0020] After testing, the zeaxanthin content in the wolfberry raw material was 126.42 mg / 100g after the completion of the compound fermentation step (3), and the zeaxanthin content in the post-processed extract was 15.10%, with a yield of 2.41%.

[0021] Example 3 A method for extracting zeaxanthin from wolfberry, the specific steps are the same as in Example 1, except that: the concentration of Na2CO3 solution in step (2) is 1%; and the concentration of lactic acid bacteria in step (3) is 0.2%.

[0022] After testing, the zeaxanthin content in the wolfberry raw material was 118.29 mg / 100g after the completion of the compound fermentation step (3), and the zeaxanthin content in the post-processed extract was 15.19%, with a yield of 2.70%.

[0023] Example 4 A method for extracting zeaxanthin from wolfberry, the specific steps are the same as in Example 1, except that: in step (3), the 0.15% pectinase + 0.15% hemicellulase is replaced with 0.3% pectinase.

[0024] After testing, the zeaxanthin content in the wolfberry raw material was 107.72 mg / 100g after the completion of the compound fermentation step (3), and the zeaxanthin content in the post-processed extract was 15.24%, with a yield of 2.41%.

[0025] Comparative Example 1 A method for extracting zeaxanthin from wolfberry, the specific steps are the same as in Example 1, except that the pectinase in step (3) is replaced with 0.3% lipase.

[0026] After testing, the zeaxanthin content in the wolfberry raw material was 62.91 mg / 100g after the compound fermentation step was completed, and the zeaxanthin content in the post-processed extract was 7.04%, with a yield of 1.63%.

[0027] Comparative Example 2 A method for extracting zeaxanthin from wolfberry, the specific steps are the same as in Example 1, except that the 5% Na2CO3 solution in step (2) is replaced with pure water.

[0028] After testing, the zeaxanthin content in the wolfberry raw material was 70.94 mg / 100g after the compound fermentation step was completed, and the zeaxanthin content in the post-processed extract was 8.11%, with a yield of 1.31%.

[0029] Comparative Example 3 A method for extracting zeaxanthin from wolfberry, the specific steps are the same as in Example 1, except that the composite system in step (3) contains only 0.3% pectinase and no lactic acid bacteria.

[0030] After testing, the zeaxanthin content in the wolfberry raw material was 54.16 mg / 100g after the compound fermentation step was completed, and the zeaxanthin content in the post-processed extract was 9.07%, with a yield of 1.37%.

[0031] Comparative Example 4 A method for extracting zeaxanthin from wolfberry is described, with the same steps as in Example 1, except that the compound fermentation process in step (3) involves vacuuming and fermenting at 35°C for 5 days; then vacuuming again, crushing the fruit, and continuing fermentation at 35°C for 2 days.

[0032] After testing, the zeaxanthin content in the wolfberry raw material was 73.96 mg / 100g after the compound fermentation step was completed, and the zeaxanthin content in the post-processed extract was 6.01%, with a yield of 1.23%.

[0033] Comparative Example 5 A method for extracting zeaxanthin from wolfberry, the specific steps are the same as in Example 1, the difference being that: the compound fermentation process in step (3) is to be vacuumed and fermented at 35°C for 6 days; then the drying process in step (4) is carried out directly. After testing, the zeaxanthin content in the wolfberry raw material was 75.06 mg / 100g after the compound fermentation step was completed, and the zeaxanthin content in the post-processed extract was 6.13%, with a yield of 1.30%.

[0034] Comparative Example 6 A method for extracting zeaxanthin from wolfberry, the specific steps are the same as in Example 1, except that: in step (1), dried wolfberry fruit is used for screening; After testing, the zeaxanthin content in the wolfberry raw material was 79.69 mg / 100g after the compound fermentation step was completed, and the zeaxanthin content in the post-processed extract was 4.76%, with a yield of 1.29%.

[0035] Comparative Example 7 A method for extracting zeaxanthin from wolfberry is described, with the same steps as in Example 1, except that: in step (1), fresh wolfberry fruit is pulped after removing moldy, rotten, or damaged fruit; step (2) ultrasonic impregnation process is omitted; in step (3), 0.15% pectinase + 0.15% hemicellulase is directly added to the wolfberry pulp for fermentation during compound fermentation.

[0036] After testing, the zeaxanthin content in the wolfberry raw material was 77.72 mg / 100g after the compound fermentation step was completed, and the zeaxanthin content in the post-processed extract was 7.11%, with a yield of 1.13%.

[0037] Comparative Example 8 A method for extracting zeaxanthin from wolfberry, the specific steps are the same as in Example 1, except that: in step (5) subcritical extraction, the extraction solvent used is liquefied propane.

[0038] Comparative Example 9 A method for extracting zeaxanthin from wolfberry, the specific steps are the same as in Example 1, except that: in step (5) subcritical extraction, the extraction solvent used is tetrafluoroethane.

[0039] Test Example 1. Determination of zeaxanthin content in raw materials Weigh 1.000 g of wolfberry raw material, add 0.01 g of butylated hydroxytoluene (BHT), and grind rapidly with liquid nitrogen until powdered. Transfer the powder to a stoppered conical flask, add 10 mL of tetrahydrofuran, and place in a constant temperature water bath ultrasonic instrument. Set the ultrasonic power to 200 W and the temperature to 25℃, and extract ultrasonically for 10 min, shaking once every 2 min during extraction. Filter with qualitative filter paper and collect the filtrate in a 50 mL stoppered conical flask. Add 10 mL of tetrahydrofuran back to the filter residue, and repeat the extraction and filtration process as described above until the extract is colorless (absorbance at 460 nm < 0.010). Combine all filtrates. Transfer the combined filtrate to a rotary evaporator flask, concentrate under reduced pressure at 35℃ to near dryness, dissolve the residue in portions with a small amount of dichloromethane, transfer to a 25 mL volumetric flask, sonicate for 5 min, cool to room temperature, and dilute to the mark with dichloromethane. Shake well to obtain the test sample mother liquor. Accurately measure an appropriate amount of the test sample stock solution and dilute it with dichloromethane to an absorbance value within the range of 0.2–0.8 to prepare the test solution. Using dichloromethane as a blank control, measure the absorbance value at a wavelength of 460 nm using a UV spectrophotometer. Accurately weigh an appropriate amount of zeaxanthin standard, dissolve and dilute it with dichloromethane to prepare a series of standard solutions with concentrations of 0.001, 0.002, 0.004, 0.006, and 0.008 mg / L. Measure the absorbance value of each concentration using the same method. Plot a standard curve with the standard concentration as the x-axis and the absorbance value as the y-axis, and calculate the regression equation (r ≥ 0.999 is required). The formula for calculating the zeaxanthin content in the raw material is: Zeaxanthin content in raw materials (mg / g) = (Zeaxanthin concentration in the test solution (mg / L) × Volume of the test sample mother liquor (0.025 L) × Dilution factor of the test sample mother liquor) ÷ Mass of wolfberry raw materials 2. Determination of zeaxanthin content in extracts Accurately weigh 0.2000 g of wolfberry zeaxanthin extract and dissolve it in dichloromethane to a final volume of 5 mL. Accurately measure an appropriate amount of the test sample stock solution and dilute it with dichloromethane to an absorbance value within the range of 0.2–0.8 to obtain the test solution. Using dichloromethane as a blank control, measure the absorbance value at a wavelength of 460 nm using a UV spectrophotometer.

[0040] Accurately weigh an appropriate amount of zeaxanthin standard, dissolve and dilute it in dichloromethane to prepare a series of standard solutions with concentrations of 0.001, 0.002, 0.004, 0.006, and 0.008 mg / L. Measure the absorbance values ​​at each concentration using the same method. Plot a standard curve with the standard concentration on the x-axis and the absorbance value on the y-axis, and calculate the regression equation (r ≥ 0.999). The formula for calculating the zeaxanthin content in the extract is: Zeaxanthin content in the extract (mg / g) = (Zeaxanthin concentration in the test solution (mg / L) × Volume of the test sample stock solution (0.005 L) × Dilution factor of the test sample stock solution) ÷ Sample amount of wolfberry zeaxanthin extract (g) 3. Determination of zeaxanthin yield

[0041] The formula for calculating zeaxanthin yield is: Zeaxanthin yield (%) = Mass of wolfberry zeaxanthin extract (g) / Amount of wolfberry extract raw material used (g) The test results are shown in Table 1.

[0042] Table 1. Data on Zeaxanthin Extraction

[0043] The test results of the above examples and comparative examples show that the "Na2CO3 ultrasonic impregnation + enzyme-lactic acid bacteria compound fermentation" pretreatment of fresh wolfberry fruit of the present invention can significantly improve the dissolution and extraction efficiency of zeaxanthin. Without any key pretreatment step or after replacing the core reagent, it is impossible to obtain a high concentration of zeaxanthin extract.

[0044] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for extracting zeaxanthin from wolfberry, characterized in that, Includes the following steps: S1. Na2CO3 ultrasonic impregnation: Fresh wolfberry fruits are ultrasonically impregnated with a 1%~5% Na2CO3 solution, and then the Na2CO3 solution is drained off. S2. Compound fermentation: The compound system is evenly sprayed onto the surface of whole wolfberry fruits after ultrasonic impregnation with Na2CO3. The compound system contains 0.2%~0.5% pectinase and / or hemicellulase, and 0.2%~1.0% lactic acid bacteria. Vacuum fermentation is carried out for 5~6 days. S3. Drying and grinding: Dry the fermented wolfberries at 55℃±5℃ and grind them to a particle size of 16~22 mesh; S4. Subcritical extraction: The dried and pulverized wolfberries from step S3 are placed into a subcritical extraction vessel for extraction. S5. Post-processing: After extraction, desolventize under reduced pressure to obtain wolfberry zeaxanthin extract.

2. The method according to claim 1, characterized in that, The concentration of the Na2CO3 solution in step S1 is 5%.

3. The method according to claim 1, characterized in that, The ultrasonic immersion time in step S1 is 20s~40s, the ultrasonic power is 240~480 W, and the ultrasonic frequency is 9~12 Hz.

4. The method according to claim 1, characterized in that, The vacuum fermentation mentioned in step S2 refers to placing the wolfberry fruits with the composite system evenly sprayed on their surface into a sealed bag, vacuuming the bag, and fermenting at 25℃~35℃ for 4~5 days; then vacuuming again, crushing the fruits, and continuing to ferment at 25℃~35℃ for 1 day.

5. The method according to claim 1, characterized in that, The extractant used in step S4 is liquefied butane, the extraction temperature is 40~50℃, the material-to-liquid ratio is 1:2~1:3, the extraction time is 40 min / time, and the number of extractions is 4~5 times.

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