Process for efficiently extracting high-activity black aronia melanocarpa cyanidin

Abstract

The present application belongs to the technical field of plant extracts, and particularly relates to a process for efficiently extracting high-activity blackflower akebia anthocyanidins. The present application uses a new lactic acid / xylose alcohol DES as an extraction solvent and cooperates with a deep cold cell wall breaking technology to efficiently extract high-activity blackflower akebia anthocyanidins through centrifugal high-speed stirring. The extraction process significantly improves the extraction rate of active substances, shortens the extraction time, maximally retains the activity of anthocyanidins, and is green, safe, and low in cost.

Description

Technical Field

[0001] This invention belongs to the field of plant extract technology, specifically relating to a process for efficiently extracting highly active anthocyanins from black chokeberry. Background Technology

[0002] Black-fruited glandular-ribbed chokeberry ( Aronia melanocarpa Sorbus adenophora, also known as wild cherry or ageless berry, is a shrub belonging to the genus Sorbus in the family Rosaceae. It is currently cultivated in many provinces in my country, including Shanxi, Liaoning, Jiangsu, Zhejiang, Hebei, and Jilin. The fruit of Sorbus adenophora is spherical, and the fresh fruit has a bitter taste, rarely eaten directly. Its product development mainly focuses on juice, concentrated juice, fruit wine, fruit pulp, silken tofu, enzyme drinks, and noodles. Sorbus adenophora contains various active ingredients, such as proanthocyanidins, anthocyanins, anthocyanins, phenolic acids, flavanols, and flavonols. Anthocyanins mainly exist in the form of anthocyanins, accounting for 25% of the total phenols.

[0003] Anthocyanins are water-soluble natural plant pigments found in plants such as blueberries, black rice, grapes, and black goji berries. Belonging to the flavonoid family, they possess antioxidant, immune-boosting, anti-tumor, antibacterial, glucose and lipid metabolism-regulating, and anti-cancer properties. Currently, common methods for extracting anthocyanins include direct extraction and assisted extraction. Assisted extraction methods mainly include microwave-assisted extraction, ultrasonic-assisted extraction, and ultra-high pressure-assisted extraction. While ultrasonic-assisted and microwave-assisted extraction offer advantages such as short extraction times, simple operation, and easy purification, the ozone generated during ultrasonic-assisted extraction and the high temperatures during microwave-assisted extraction can destroy anthocyanin activity. Although ultra-high pressure-assisted extraction can overcome the disadvantage of low activity of active substances, its operating conditions are harsh and the equipment investment is high. Conventional extraction agents mainly include acidic ethanol, aqueous two-phase systems, and supercritical fluids, but their extraction efficiency is generally low, and the extraction process requires large amounts of organic solvents, posing multiple drawbacks such as flammability, explosiveness, volatility, environmental pollution, and harm to human health.

[0004] Deep eutectic solvents (DES) possess advantages such as simple preparation, low toxicity, biodegradability, biocompatibility, and recyclability, and have wide applications in metal processing and electrochemistry, biomass conversion, extraction of bioactive components, gas adsorption, pharmaceuticals, and biomolecules. As a novel extraction agent, DES has been reported to be used to extract active ingredients such as plant and animal lactones, polyphenols, astaxanthin, and polysaccharides. The discovery of deep eutectic solvents has opened up new avenues for the extraction of active substances. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention provides a process for efficiently extracting highly active anthocyanins from black chokeberry using a novel DES as the extraction solvent and cryogenic cell disruption technology via centrifugation and high-speed stirring. This process significantly improves the extraction rate of active substances while maximizing the preservation of anthocyanin activity. Furthermore, the extraction process is green, safe, and low-cost.

[0006] To achieve the above objectives, the present invention provides a process for efficiently extracting highly active anthocyanins from black chokeberry, the process comprising the following steps:

[0007] (1) Black chokeberry was pulverized by cryogenic cell wall breaking to obtain ultrafine powder;

[0008] (2) The above ultrafine powder is mixed with lactic acid / xylitol eutectic solvent, and centrifuged and stirred at high speed at room temperature. The supernatant is then taken to obtain the crude anthocyanin extract.

[0009] (3) Evaporate the crude anthocyanin extract until no more liquid evaporates, cool it, and then freeze-dry it under vacuum to obtain anthocyanin extract freeze-dried powder.

[0010] This invention utilizes cryogenic cell disruption technology to fully release anthocyanins from the cells. Then, lactic acid / xylitol is used as a novel DES extraction agent. Under the condition of ensuring that the pH is below 4, high-speed centrifugation and stirring are used to enhance mass transfer, resulting in high extraction efficiency. Furthermore, the low temperature is maintained throughout the extraction process, which better preserves the bioactivity of anthocyanins.

[0011] Furthermore, the eutectic solvent is prepared by stirring lactic acid and xylitol at 80-110°C for 1-2 hours until a uniform and transparent liquid is produced, which is then stored for later use.

[0012] In the eutectic solvent of this invention, the molar ratio of lactic acid to xylitol is 1:(2-6), and the water content of the eutectic solvent is 30-50 wt%. Xylitol acts as a hydrogen bond acceptor, and lactic acid acts as a hydrogen bond donor. Both lactic acid and xylitol are edible. Xylitol, as a sweetener, can improve the sour taste of anthocyanins and has certain health benefits. It will not cause an increase in blood sugar in diabetic patients.

[0013] Further, the specific process of cryogenic cell wall breaking in step (1) is as follows: black chokeberry is placed in a liquid nitrogen environment at -60~-80℃ for 4~6 minutes, and then ultra-finely pulverized at -80~-120℃ using a cryogenic cell wall breaking machine for 20~40 minutes to obtain ultra-fine powder with a particle size of 1~10µm.

[0014] Furthermore, in step (2), the mixing ratio of ultrafine powder to lactic acid / xylitol eutectic solvent is 1g:(20-40)mL, the rotation speed is 3000-5000r / min, the number of stirring times is 3-5 times, and the stirring time is 1-2min / time.

[0015] Furthermore, in step (3), the evaporation method is vacuum rotary evaporation, the evaporation temperature is 30-50℃, and the rotation speed is 99-139r / min.

[0016] Furthermore, the vacuum freeze-drying temperature is -60~-80℃.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) This invention uses cryogenic cell disruption technology instead of conventional ultrasonic cell disruptors, which improves the cell disruption rate and significantly increases the extraction rate of active substances while maximizing the preservation of anthocyanin activity; among them, the extraction rate of anthocyanins from black chokeberry can reach 28.69 mg / g, the DPPH free radical scavenging rate can reach 99.22%, the hydroxyl free radical scavenging rate can reach 90.73%, and the O2 free radical scavenging rate can reach 90.73%. - The free radical scavenging rate reached 73.73%, and the ABTS free radical scavenging rate reached 89.58%.

[0019] (2) A novel eutectic solvent of lactic acid / xylitol is used as the extraction solvent. Both lactic acid and xylitol are edible, the extraction process is green and safe, and the extraction rate is high.

[0020] (3) The present invention uses centrifugal high-speed stirring instead of conventional stirring, which can greatly enhance the interaction between solvent and extract at room temperature, significantly improve the extraction rate, shorten the extraction time, and maximize the preservation of anthocyanin activity; in addition, the cryogenic cell wall breaking technology combined with centrifugal high-speed stirring simplifies the anthocyanin extraction process and is also conducive to process scale-up and industrial production. Attached Figure Description

[0021] Figure 1 This is a comparison chart of the extraction rates of anthocyanins using different eutectic solvents. DES-1 is citric acid: mannitol; DES-2 is citric acid: 1,2-propanediol; DES-3 is malic acid: xylitol; DES-4 is citric acid: xylitol; DES-5 is citric acid: sorbitol; DES-6 is lactic acid: 1,2-propanediol; DES-7 is malic acid: sorbitol; DES-8 is malic acid: mannitol; and DES-9 is lactic acid: sorbitol. Detailed Implementation

[0022] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0023] Example 1

[0024] S1. Black chokeberry was kept in a liquid nitrogen environment at -70℃ for 5 minutes, and then ultra-finely pulverized at -120℃ using a cryogenic wall-breaking machine for 20 minutes to obtain ultra-fine powder with a particle size of 1-10µm.

[0025] S2 is weighed into a beaker at a molar ratio of lactic acid to xylitol of 1:2, and magnetically stirred at 90°C for 1 hour until a uniform and transparent liquid is formed. This yields a lactic acid / xylitol eutectic solvent with a water content of 30 wt%.

[0026] S3 Take 2g of the ultrafine powder obtained in step S1 and mix it with 60mL of the lactic acid / xylitol eutectic solvent prepared in step S2. Centrifuge and stir at high speed at room temperature. The speed is 3000r / min, the stirring is repeated 5 times, and the stirring time is 1min each time. Take the supernatant to obtain the crude anthocyanin extract.

[0027] S4 The crude anthocyanin extract was vacuum rotary evaporated at 40℃ and 130r / min until no more liquid evaporated. After cooling, it was freeze-dried at -70℃ to obtain anthocyanin extract freeze-dried powder.

[0028] Example 2

[0029] S1. Black chokeberry was kept in a liquid nitrogen environment at -80℃ for 4 minutes, and then ultra-finely pulverized at -100℃ using a cryogenic blender for 30 minutes to obtain ultra-fine powder with a particle size of 1-10µm.

[0030] S2 is weighed into a beaker at a molar ratio of lactic acid to xylitol of 1:3, and magnetically stirred at 90°C for 1 hour until a uniform and transparent liquid is formed, thus obtaining the lactic acid / xylitol eutectic solvent with a water content of 40 wt%.

[0031] S3 Take 2g of the ultrafine powder obtained in step S1 and mix it with 40mL of the lactic acid / xylitol eutectic solvent prepared in step S2. Centrifuge and stir at high speed at room temperature. The speed is 4000r / min, the stirring is repeated 5 times, and the stirring time is 1min each time. Take the supernatant to obtain the crude anthocyanin extract.

[0032] S4 The crude anthocyanin extract was vacuum rotary evaporated at 40℃ and 130r / min until no more liquid evaporated. After cooling, it was freeze-dried at -70℃ to obtain anthocyanin extract freeze-dried powder.

[0033] Example 3

[0034] S1. Black chokeberry was kept in a liquid nitrogen environment at -60℃ for 6 minutes, and then ultra-finely pulverized at -80℃ using a cryogenic blender for 40 minutes to obtain ultra-fine powder with a particle size of 1-10µm.

[0035] S2 is weighed into a beaker at a molar ratio of lactic acid to xylitol of 1:4, and magnetically stirred at 90°C for 1 hour until a uniform and transparent liquid is formed. This yields a lactic acid / xylitol eutectic solvent with a water content of 50 wt%.

[0036] S3 Take 2g of the ultrafine powder obtained in step S1 and mix it with 80mL of the lactic acid / xylitol eutectic solvent prepared in step S2. Centrifuge and stir at high speed at room temperature. The speed is 5000r / min, the stirring is repeated 5 times, and the stirring time is 1min each time. Take the supernatant to obtain the crude anthocyanin extract.

[0037] S4 The crude anthocyanin extract was vacuum rotary evaporated at 40℃ and 130r / min until no more liquid evaporated. After cooling, it was freeze-dried at -70℃ to obtain anthocyanin extract freeze-dried powder.

[0038] Comparative Example 1

[0039] The synthesis process is the same as in Example 1, except that the cell wall breaking process uses an ultrasonic cell disruptor (model LD-150T). Specifically, the cell wall is broken up by ultrasonic treatment at 40°C and 90W for 40 minutes to obtain ultrafine powder with a particle size of 1-10µm.

[0040] Comparative Example 2

[0041] The synthesis process is the same as in Example 1, except that the stirring method is a normal manual simulated mechanical stirring with a speed of 400 r / min.

[0042] Comparative Example 3

[0043] The synthesis process is the same as in Example 1, except that different types of eutectic solvents are used, specifically citric acid / mannitol (DES-1), citric acid / 1,2-propanediol (DES-2), malic acid / xylitol (DES-3), citric acid / xylitol (DES-4), citric acid / sorbitol (DES-5), lactic acid / 1,2-propanediol (DES-6), malic acid / sorbitol (DES-7), malic acid / mannitol (DES-8), and lactic acid / sorbitol (DES-9). The extraction rate comparison is shown in the figure below. Figure 1 As shown (Note: The optimal values ​​corresponding to different eutectic solvents were selected for the extraction effect determination in the comparative examples).

[0044] Comparative Example 4

[0045] The synthesis process of Example 1 was the same as that of Example 1, with the same molar ratio of lactic acid to xylitol and water content. The only difference was that lactic acid and xylitol did not form a DES system.

[0046] [Performance Testing]

[0047] The properties of the materials prepared in Examples 1-3 and Comparative Examples 1-4 were measured.

[0048] The specific testing methods are as follows:

[0049] I. Determination of Anthocyanin Content

[0050] Take two 1 mL aliquots of anthocyanin solution. Dilute one aliquot with a KCl-HCl buffer solution (pH 1.0) to 10 mL. Adjust the pH of the other aliquot to 4.5 using a 2 mol / L NaOH solution and a HAc-NaAc buffer solution (pH 4.5) and dilute to 10 mL. Mix well and equilibrate at room temperature in the dark for 30 min. Then centrifuge at 8000 r / min for 5 min. Measure the absorbance of the supernatant at 510 nm and 700 nm. Calculate the anthocyanin extraction rate using the following formula, expressed as the cyanidin-3-glucoside equivalent (mg Cy-3-glu / g DW) contained in the black chokeberry powder.

[0051] Anthocyanin extraction rate (mg / g) = (ΔA×Mω×n×V) / (ε×m)

[0052] ΔA=(A 510 -A 700 ) pH=1.0 -(A 510 -A 700 ) pH=4.5

[0053] In the formula, Mω is the relative molecular mass of cyanidin-3-glucoside, 449.2 g / mol; n is the dilution factor; V is the total volume of the extract, mL; ε is the extinction coefficient of cyanidin-3-glucoside, 26900; and m is the mass of black chokeberry powder, g.

[0054] II. Anthocyanin Activity Assay

[0055] 1. Determination of hydroxyl radical scavenging rate

[0056] The reagents were added sequentially according to Table 1 below to obtain the test samples for the sample group, control group, and blank group:

[0057] Table 1. Sample synthesis for hydroxyl radical scavenging rate determination

[0058]

[0059] The above test samples were placed in a water bath at 37°C for 1 hour, then removed and cooled to room temperature. The zero point was adjusted with distilled water, and the absorbance A was measured at a wavelength of 510 nm.

[0060] Hydroxyl radical scavenging rate (%) = {[A 空白 -(A 样品 -A 对照 )] / A 空白}×100%

[0061] 2. DPPH free radical scavenging rate determination

[0062] Dissolve 2.56 mg of DPPH standard in 95% ethanol and dilute to 100 mL to obtain a DPPH solution. Prepare anthocyanin solution with a mass concentration of 60 µg / mL, and add the reagents in order according to Table 2 below to obtain the test samples for the sample group, control group, and blank group:

[0063] Table 2. DPPH free radical scavenging rate determination sample synthesis

[0064]

[0065] React for 30 min under light-protected conditions, adjust the zero point with 0.5 mL of distilled water and 2.5 mL of 95% ethanol, and measure the absorbance A at a wavelength of 515 nm.

[0066] DPPH free radical scavenging rate (%) = {[A 空白 -(A 样品 -A 对照 )] / A 空白}×100%

[0067] 3. O2 - Free radical scavenging rate determination

[0068] Dilute 40 mL of 0.1 mol / L Tris-HCl aqueous solution to 80 mL, then adjust the pH to 8.2 with 0.1 mol / L HCl solution to obtain a 50 mmol / L Tris-HCl buffer. Take 100 μL of 0.1 mol / L HCl solution and bring the volume to 10 mL to obtain a 1 mmol / L HCl solution. Add 73 mg of pyrogallol, sonicate for 1 min, and then add distilled water to bring the volume to 100 mL again. Shake well to obtain a 60 mmol / L pyrogallol solution. Take 2.25 mL of preheated Tris-HCl buffer (pH 8.2, concentration 50 mmol / L) at 25 °C and place it in a 10 mL centrifuge tube. Add 2 mL of 60 µg / mL anthocyanin solution and 0.05 mL of preheated 60 mmol / L pyrogallol solution at 25 °C, mix rapidly, and react for 4 min. Then add 0.02 mL of 10 mol / L HCl solution to terminate the reaction. Measure the absorbance at 325 nm and record it as A1. Replace the sample solution with 2 mL of 30% ethanol solution and follow the same procedure; record the absorbance as A2. Replace the pyrogallol solution with 0.05 mL of 1 mmol / L HCl solution and follow the same procedure; record the absorbance as A3.

[0069] O2 - Free radical scavenging rate (%) = {[A2-(A1-A3)] / A2} × 100%

[0070] 4. Determination of ABTS free radical scavenging rate

[0071] A 7.4 mM ABTS solution and a 2.6 mM potassium persulfate solution were mixed at a volume ratio of 1:1 and reacted in the dark for 12 h to prepare an ABTS stock solution. The ABTS stock solution was then mixed with anhydrous ethanol at a ratio of 1:70 (v / v), and the absorbance of this mixture at 734 nm was adjusted to 0.700 ± 0.02. The mixture was then preheated in a 30°C water bath for later use. A 60 µg / mL anthocyanin solution was prepared, and reagents were added sequentially according to Table 3 below to obtain the sample group, control group, and blank group test samples.

[0072] Table 3. Sample synthesis for ABTS free radical scavenging rate determination

[0073]

[0074] The reaction was carried out at room temperature and in the dark for 15 minutes, and the absorbance A at a wavelength of 734 nm was measured.

[0075] ABTS free radical scavenging rate (%) = {[A 空白 -(A 样品 -A 对照 )] / A空白}×100%

[0076]

Experimental Results

[0077] Comparison of anthocyanin extraction data between the examples and comparative examples is shown in Table 4 and Figure 1 As shown.

[0078] Table 4 Comparison of anthocyanin extraction data

[0079]

[0080] Note: Experimental results are expressed as mean ± standard deviation (X±S). The significance test was performed using SPSS 15.0 statistical software. p<0.05 was considered significant, and p<0.01 was considered highly significant. Compared with Example 1, ▲ indicates p<0.05, and ▲▲ indicates p<0.01.

[0081] The technical solution of this invention significantly improves the extraction rate of active substances while maximizing the preservation of anthocyanin activity. In Example 1, the anthocyanin extraction rate from *Sorbus nigra* was 28.69 mg / g, the DPPH free radical scavenging rate was 99.22%, the hydroxyl free radical scavenging rate was 90.73%, and the O2... - The free radical scavenging rate was 73.73%, and the ABTS free radical scavenging rate was 89.58%. According to... Figure 1 It can be seen that the novel eutectic solvent of lactic acid / xylitol has the highest extraction rate of anthocyanins compared with other eutectic solvents; and the centrifugal stirring method in this invention can greatly shorten the extraction time compared with the manual stirring method. That is, under the same extraction conditions, only the stirring method is changed, and the extraction time is shorter when centrifugal stirring is used to obtain the same extraction amount, which also improves the extraction efficiency of anthocyanins to a certain extent. Table 4 shows that the anthocyanin activity obtained by cryogenic cell disruption technology is significantly or extremely significantly higher than that extracted by ultrasonic cell disruption technology. Furthermore, the anthocyanin extraction rates obtained by the two methods also differ significantly. The extraction rate using the cryogenic cell disruption technology of this invention reaches as high as 28.69 mg / g, while the extraction rate using ultrasonic cell disruption technology is only 20.26 mg / g. For anthocyanin extraction rate, the centrifugal stirring method is extremely significantly higher than the manual stirring method, with the extraction rates differing by more than double. The stable DES system and the system without DES formation also have a very significant impact on the anthocyanin extraction rate, with the extraction rates differing by more than double. Moreover, the anthocyanin activity extracted using the stable DES system is significantly higher than that extracted using the system without DES formation.

[0082] Therefore, this invention utilizes a novel lactic acid / xylitol DES to extract anthocyanins, and employs cryogenic cell disruption technology combined with high-speed centrifugal stirring. This not only improves the cell disruption rate and significantly increases the extraction rate of active substances, but also maximizes the preservation of anthocyanin activity, simplifies the anthocyanin extraction process, and facilitates process scale-up and industrial production.

[0083] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A process for efficiently extracting highly active anthocyanins from black chokeberry, characterized in that, Includes the following steps: (1) Black chokeberry was pulverized by cryogenic cell wall breaking to obtain ultrafine powder; (2) The above ultrafine powder is mixed with lactic acid / xylitol eutectic solvent, and centrifuged at high speed at room temperature. The supernatant is taken to obtain the crude anthocyanin extract. The centrifugation speed is 3000-5000 r / min. (3) Evaporate the crude anthocyanin extract until no more liquid evaporates, cool it, and then freeze-dry it under vacuum to obtain anthocyanin extract freeze-dried powder.

2. The process according to claim 1, characterized in that: The lactic acid / xylitol eutectic solvent is prepared by stirring lactic acid and xylitol at 80-110°C for 1-2 hours until a uniform and transparent liquid is produced, which is then stored for later use.

3. The process according to claim 1, characterized in that: The molar ratio of lactic acid to xylitol in the lactic acid / xylitol eutectic solvent is 1:(2-6), and the water content of the eutectic solvent is 30-50wt%.

4. The process according to claim 1, characterized in that: The specific process of cryogenic cell wall breaking in step (1) is as follows: place black chokeberry in a liquid nitrogen environment at -60~-80℃ for 4~6 minutes, and use a cryogenic cell wall breaking machine to perform ultra-fine pulverization at -80~-120℃ for 20~40 minutes to obtain ultra-fine powder with a particle size of 1~10µm.

5. The process according to claim 1, characterized in that: In step (2), the mixing ratio of ultrafine powder to lactic acid / xylitol eutectic solvent is 1g:(20-40)mL.

6. The process according to claim 1, characterized in that: In step (2), the stirring is performed 3-5 times, and the stirring time is 1-2 minutes per stirring.

7. The process according to claim 1, characterized in that: In step (3), the evaporation method is vacuum rotary evaporation, the evaporation temperature is 30-50℃, and the rotation speed is 99-139r / min.

8. The process according to claim 1, characterized in that: The vacuum freeze-drying temperature is -60~-80℃.

Images