CN110711404A - Method for extracting anthocyanin from raspberry - Google Patents

Abstract

The invention discloses a method for extracting anthocyanin from raspberry. Meanwhile, the Box-Behnken design of a Response Surface Method (RSM) is adopted to research and optimize main experimental parameters influencing the extraction process. A UPLC-Triple-TOF/MS system is adopted to study and compare the component composition of raspberry anthocyanin extracted by subcritical water, hot water and methanol and the anthocyanin yield. The research shows that under the optimal extraction conditions (extraction pressure of 7MPa, extraction time of 90min and extraction temperature of 130 ℃), subcritical water is utilized to successfully extract 6 kinds of anthocyanin from fresh raspberry fruits, and the yield of the obtained anthocyanin is higher than that of the anthocyanin obtained by the traditional extraction method.

Description

A method for extracting anthocyanins from raspberries

technical field

The invention relates to the field of extraction methods for raspberry active substances, in particular to a method for extracting anthocyanins from raspberries.

Background technique

Anthocyanins are important natural flavonoids that are widely present in fruits, flowers and leaves of various colors, making them appear in different colors from red, purple to blue. Structural diversity endows anthocyanins with a wide range of physiological activities, such as antioxidant, cardiovascular disease prevention and anticancer effects. In addition, anthocyanins also have proliferative lutein, anti-inflammatory, inhibit lipid peroxidation and platelet aggregation, prevent Diabetes, weight loss, vision protection and other functions. Anthocyanins act as effective antioxidants by providing hydrogen atoms to highly reactive free radicals thereby breaking free radical chain reactions. It is well known that many degenerative diseases are currently associated with oxidative damage, such as atherosclerosis, aging, and cancer in humans. Nutritionists suggest that, in view of the broad-spectrum biological activity of anthocyanins, adding a certain amount of anthocyanin-rich fruits and vegetables to the daily diet will benefit people's health.

The raspberry fruit is rich in anthocyanins. Research has confirmed that raspberry is an important source of anthocyanins, and anthocyanins are its main functional components. The existing extraction methods of anthocyanins in raspberries include solvent extraction and enzymatic extraction. , fermentation extraction, etc., there are problems such as complex process and low extraction yield.

SUMMARY OF THE INVENTION

The main technical problem solved by the present invention is to provide a method for extracting anthocyanins from raspberries, which can efficiently and quickly extract anthocyanins in raspberries.

In order to solve the above-mentioned technical problems, a technical scheme adopted in the present invention is:

Provided is a method for extracting anthocyanins from raspberries, comprising the following content: extracting anthocyanins from raspberry fruits through subcritical water extraction.

In the existing technology for extracting anthocyanins, due to the strong polarity of anthocyanins, solvent extraction methods are mostly used, and there is no relevant report on extracting anthocyanins in raspberries by the method of subcritical water extraction.

Further, the described method for extracting anthocyanins from raspberry comprises the following steps:

The raspberry is subjected to subcritical water extraction with an extraction agent under the conditions of an extraction temperature of 100-160° C. and an extraction pressure of 6-8 MPa to obtain a raspberry anthocyanin extract.

Further, the method also includes the following steps: drying the raspberry anthocyanin extract to obtain the raspberry anthocyanin extract.

Further, the raspberries are fresh raspberries.

Further, the dosage ratio of the fresh raspberry fruit and the extractant is 1g:60-120mL; further, the dosage ratio of the fresh raspberry fruit and the extractant is 1g:90mL;

In a specific embodiment of the present invention, the extraction agent is ultra-clean water.

Further, the extraction time is 60-120 min; further selected from 90 min.

Further, the extraction temperature is 130°C.

Further, the extraction pressure is 7MPa.

Further, the anthocyanins are selected from cyanidin-3-sophoroside-5-glucoside, cyanidin-3-sophoroside, cyanidin-3-glucoside, pelargonidin-3-sophoroside One or more of glycosides, cyanidin-3-(6"-citric acid)-sophoroside and pelargonidin-3-glucoside.

The present invention also provides a raspberry anthocyanin extract, comprising 75-85 parts of cyanidin-3-sophoroside, 75-85 parts of cyanidin-3-glucoside and pelargonidin-3-glucoside 8 to 11 servings.

It is found through detection that the raspberry anthocyanin extract prepared by the present invention mainly includes six anthocyanins: cyanidin 3-sophoroside-5-glucoside, cyanidin 3-sophoroside-5-glucoside, cyanidin -3-sophoroside (cyanidin 3-sophoroside), cyanidin-3-glucoside (cyanidin-3-O-glucoside), pelargonidin 3-sophoroside (pelargonidin 3-sophoroside), cyanidin- 3-(6"-citric acid)-sophoroside [cyanidin-3-(6"-citryl)-sophoroside] and pelargonidin 3-O-glucoside. Among them, the three components with higher content are cyanidin-3-sophoroside, cyanidin-3-glucoside, and pelargonidin-3-glucoside. And it is measured by quantitative test that the anthocyanins in raspberries are extracted by the present invention, and both the total yield and the yield of a single anthocyanin monomer are higher than the prior art.

The beneficial effects of the present invention are:

(1) the present invention adopts the method of subcritical water extraction for the first time to extract the anthocyanins in the raspberry, the extraction efficiency is higher than the prior art, the process is simple, the extraction effect is good, and the operation is convenient, which provides for the development and utilization of the raspberry anthocyanins. more efficient strategies.

(2) The present invention adopts the response surface method to optimize the conditions of subcritical water extraction of anthocyanins in raspberries, which can greatly reduce the number of experiments. According to the regression models of different response values, multi-objective co-optimization is realized and an optimal factor combination is established. , scientific and reliable, practical.

Description of drawings

Fig. 1 is the three-dimensional response surface of the effect of the interaction of two factors of the present invention on the extraction rate of raspberry anthocyanins: (a) extraction pressure and extraction time; (b) extraction pressure and extraction temperature; (c) extraction time and extraction temperature; Fig. The middle ordinate is the anthocyanin yield;

Fig. 2 is the HPLC chromatogram of raspberry anthocyanins obtained by comparing three kinds of extraction methods: (a) hot water extraction sample, (b) methanol extraction sample, (c) subcritical water extraction sample;

Fig. 3 is the primary and secondary mass spectrum of compound 1 (negative and positive);

Fig. 4 is the primary secondary mass spectrum of compound 2 (negative and positive);

Fig. 5 is the primary secondary mass spectrum of compound 3 (negative and positive);

Fig. 6 is the primary secondary mass spectrum of compound 4 (negative and positive);

Fig. 7 is the primary secondary mass spectrum of compound 5 (negative and positive);

Figure 8 is the primary and secondary mass spectra of compound 6 (negative and positive).

Detailed ways

The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Example 1

1. Instruments and Reagents

Material: Raspberry, provided by Qinghai Yaochi Biotechnology Co., Ltd., trade name Meek.

Instrument: UPLC-Triple-TOF/MS system: AcquityTM ultra high performance liquid chromatograph (Waters, USA), Triple TOF 5600+ time-of-flight mass spectrometer, equipped with electrospray ion source (AB SCIEX, USA); Eppendorf minispan centrifuge machine (Eppendorf, Germany). The water used for chromatography was Watsons purified water. Chromatographic grade acetonitrile was purchased from Shanghai Chemical Reagent Co., Ltd. All other reagents used were analytical grade reagents.

2. Optimization of subcritical water extraction conditions for raspberry anthocyanin extract by response surface methodology

Three factors and three levels Box-Behnken design method were used to optimize the three key influencing factors in the extraction process of extraction pressure (A), extraction time (B) and extraction temperature (C), in order to obtain subcritical water extraction of raspberry anthocyanins. optimal extraction conditions.

The operation method is as follows: Precisely weigh 20g of fresh raspberry fruit and place it in a subcritical water extraction kettle. When all parts of the system reach 100 to 160 °C, pump the extraction agent (ultra-pure water) into the extraction system, and the pressure of the extraction kettle is increased. To 6-8MPa, the collector collects the extract, dynamically extracts it for 60-120min, the extract is recovered, and the content of anthocyanins in the raspberry anthocyanin extract is determined by pH differential method.

The pH differential method is as follows: the raspberry anthocyanin extract is dissolved in 0.025mol/L potassium chloride buffer solution, and the pH value is adjusted to 1.0 with sodium acetate buffer solution. Accurately weigh 1.64 g of sodium acetate, dilute to 100 mL with distilled water, and adjust the pH to 4.5 with 0.2 mol/L salt solution. Accurately weigh 0.1 g of raspberry anthocyanin extract, diluted 10 times with distilled water as a blank control, and measure the sample solution diluted with potassium chloride buffer solution at 520 nm and 700 nm, respectively. In the same way, the samples diluted with sodium acetate buffer solution were measured in two places. Note that the absorbance of the samples should be measured within 20 to 50 minutes. The concentration of anthocyanin pigment is calculated by using bromine pigment-3,5-diglucoside, and the formula is as follows:

Wherein: A=(A _520nm -A _700nm )pH 1.0-(A _520nm -A _700nm )pH 4.5;

MW (molecular weight) = the molar mass of malvide-3,5-diglucoside 655.2 g/mol;

DF is the dilution factor 10; 10 ³ is the conversion of the unit g to mg;

ε is the extinction coefficient of malvide-3,5-diglucoside, which is 20500L/mol/cm;

l is the thickness of the cuvette, in cm.

The Box-Benhnken experimental design and results are shown in Table 1.

Table 1 Experimental design and data of subcritical water extraction of raspberry anthocyanins (n=3)

Use Design Expert (Trial Version 7.1.6) software to perform regression fitting on the experimental data in Table 1 to obtain the regression equation:

Y=+7.99-0.077A+0.036B-0.014C-0.0025AB-0.22AC ⁺ 0.075BC ^{- 0.80A2-0.86B2-0.97C2} .

The multivariate quadratic response surface regression model of raspberry anthocyanins subcritical water extraction was analyzed by variance. The impact is very significant. The above variance analysis results show that the model fits well with the actual experiment, and can better reflect the relationship between the independent variables and the influencing factors. The model can be used to predict the optimal subcritical water extraction of raspberry anthocyanins. condition. In order to further study the interaction between the influencing factors and determine the optimal conditions for subcritical water extraction of raspberry anthocyanins, this experiment used Design Expert (Trial Version 7.1.6) software to draw the extraction pressure, extraction time and extraction temperature. The surface plot of the interaction between factors on the subcritical water extraction yield of raspberry anthocyanins is shown in Figure 1. Figure 1(a) reflects the effect of extraction pressure and extraction time on the extraction yield of raspberry anthocyanins in subcritical water; Figure 1(b) reflects the effect of extraction pressure and extraction temperature on the extraction yield of raspberry anthocyanins in subcritical water. Figure 1(c) reflects the effect of extraction time and extraction temperature on the subcritical water extraction yield of raspberry anthocyanins.

Combined with the results of the optimization model and considering the convenience of practical operation, the optimal conditions for subcritical water extraction of raspberry anthocyanins are: extraction pressure 7MPa, extraction time 90min, extraction temperature 130℃.

In order to verify the reliability of the model, under the above optimal conditions, three parallel experiments were repeated, and the average anthocyanin content obtained was close to the total anthocyanin content predicted by the model, indicating that the results obtained by using the Box-Behnken experimental design The model can be used to predict the relationship between the subcritical water extraction of raspberry anthocyanins and the factors of extraction pressure, extraction time and extraction temperature.

By comparing with the raspberry anthocyanin extract obtained by hot water extraction and methanol extraction in the prior art, and analyzing and comparing by high performance liquid chromatography, mass spectrometry and other analytical methods, the excellent technical effects of the present invention are reflected.

Comparative Example 1

1. Hot water extraction and methanol extraction

Accurately weigh 20 g of homogenized raspberry fruit into a brown reagent bottle, and add 150 mL of deionized water. The ultrasonic power is 150W, the frequency is 20kHz, and the ultrasonic wave is 90min at 70℃. To prevent the degradation of anthocyanins, phosphoric acid solution was added to stabilize the pH of the solvent at pH 1.95.

The methanol extraction conditions were the same as the hot water extraction process, converting deionized water to 50% methanol. After hot water extraction and methanol extraction, the extract was filtered and stored for further analysis.

2. High performance liquid chromatography and mass spectrometry

Liquid phase conditions are: mobile phase: A: 0.1% formic acid aqueous solution, B: 0.1% formic acid acetonitrile; flow rate: 0.8 mL/min; detection wavelength: 520 nm; chromatographic column: Agilent Zorbax-SB C ₁₈ (100 mm×4.6 mm id, 1.8 μm); injection volume: 5 μL; column oven: 30° C.; gradient elution program: 9-13% B, 0-35 min; 13-9% B, 35-37 min; 9% B, 37-40 min.

The analytical results of HPLC have good repeatability and resolution, and the HPLC chromatogram is shown in Figure 2. With a unique anthocyanin fingerprint, raspberries can be distinguished from other species. Figure 2(a) shows the liquid phase spectrum of anthocyanins obtained by hot water extraction. Figure 2(b) shows the liquid phase spectrum of anthocyanins obtained by methanol extraction. Figure 2(c) depicts the liquid chromatogram of raspberry anthocyanins extracted by subcritical water. A total of six anthocyanin monomers were obtained, and the total amount of anthocyanins was significantly higher than that obtained by the other two conventional extraction methods. In conclusion, compared with the conventional method, the subcritical water extraction technology of the present invention can significantly improve the extraction efficiency of raspberry anthocyanins.

The structure of anthocyanins is similar to that of flavonoids, and it is easy to be confused in routine mass spectrometry analysis. Therefore, the present invention adopts positive and negative ion detection, using negative ions [M-2H] ^- , [M-2H+H ₂ O] ^- and positive ions [M] ⁺ to detect The first-order mass spectrometry rule was used to determine the presence of anthocyanins.

Mass spectrometry conditions: UPLC-Triple-TOF 5600 ⁺ time-of-flight LC/MS: positive and negative ion scanning mode; scanning range: 100～1500m/z; nebulizer gas (GS1): 50psi, nebulizer gas (GS2): 50psi, Curtain gas (CUR): 35psi; ion source temperature (TEM): 550°C (negative) 600°C (positive); ion source voltage (IS): -4500V (negative) 5500V (positive); primary scan: declustering voltage (DP): 100V; Focusing voltage (CE): 10V; Secondary scan: MS data was collected using TOF MS-Product Ion-IDA mode with CID energies of -20, -40 and -60V, CDS pump before injection Do mass axis correction to make the mass axis error less than 2ppm.

Composition 1: [M-2H] ^- is m/z 771.1997, [M-2H+H ₂ O] ^- is m/z 789.2094 [M+H] ⁺ is m/z 773.2150, according to the results of high-resolution mass spectrometry The combined molecular formula is C ₃₃ H ₄₁ O ₂₁ . According to secondary mass spectrometry, the parent nucleus of the compound is 287, which is cyanidin, and there are three 6-carbon sugar structures in the structure. According to Scifinder and Reaxy database retrieval and It is speculated that the compound is cyanidin3-sophoroside-5-glucoside. The primary and secondary mass spectra (negative and positive) of this compound are shown in Figure 3. The possible structural formula is as follows:

Composition 2: [M-2H] ^- is m/z 609.1457, [M-2H+H ₂ O] ^- is m/z 627.1564 [M+H] ⁺ is m/z 611.1601, according to high-resolution mass spectrometry results The fitted molecular formula is C ₂₇ H ₃₁ O ₁₆ . According to the secondary mass spectrometry, the parent nucleus of the compound is 287, which is cyanidin. There are two 6-carbon sugar structures in the structure. Search according to Scifinder and Reaxy databases And it is speculated that the compound is cyanidin-3-sophoroside (cyanidin 3-sophoroside). The primary and secondary mass spectra (negative and positive) of this compound are shown in Figure 4, and the possible structural formula is as follows:

Composition 3: [M-2H] ^- is m/z 447.0928, [M-2H+H ₂ O] ^- is m/z 465.1032 [M+H] ⁺ is m/z 449.1081, according to high-resolution mass spectrometry results The fitted molecular formula is C ₂₁ H ₂₁ O ₁₁ . According to secondary mass spectrometry, the parent nucleus of the compound is 287, which is cyanidin, and there is a 6-carbon sugar structure in the structure, which is searched according to Scifinder and Reaxy databases And it is speculated that the compound is cyanidin-3-O-glucoside. The primary and secondary mass spectra (negative and positive) of this compound are shown in Figure 5, and the possible structural formula is as follows:

Composition 4: [M-2H] ^- is m/z 593.1509, [M-2H+H ₂ O] ^- is m/z 611.1609 [M+H] ⁺ is m/z 595.1674, according to high-resolution mass spectrometry results The fitted molecular formula is C ₂₇ H ₃₁ O ₁₅ . According to secondary mass spectrometry, the parent nucleus of the compound is 271, which is pelargonidin. There are two 6-carbon sugar structures in the structure. According to Scifinder and Reaxy database search and inference The compound is pelargonidin 3-sophoroside, the primary and secondary mass spectra (negative and positive) of this compound are shown in Figure 6, and the possible structural formula is as follows:

Composition 5: [M-2H] ^- is m/z 593.1509, [M-2H+H ₂ O] ^- is m/z 611.1609 [M+H] ⁺ is m/z 595.1674, according to high-resolution mass spectrometry results The fitted molecular formula is C ₃₃ H ₃₇ O ₂₂ . According to secondary mass spectrometry, the parent nucleus of the compound is 287, which is cyanidin. There are two 6-carbon sugars and a citrate monoacyl structure in the structure. According to Scifinder and Reaxy database search and speculation, the compound is cyanidin-3-(6"-citric acid)-sophoroside (cyanidin-3-(6"-citryl)-sophoroside), the primary and secondary level of this compound The mass spectrum (negative and positive) is shown in Figure 7, and the possible structural formula is as follows:

Composition 6: [M-2H] ^- is m/z 431.0980, [M-2H+H ₂ O] ^- is m/z 449.1090 [M+H] ⁺ is m/z 433.1121, according to high-resolution mass spectrometry results The fitted molecular formula is C ₂₁ H ₂₁ O ₁₀ . According to secondary mass spectrometry, the parent nucleus of the compound is 271, which is pelargonidin. There is a 6-carbon sugar structure in the structure, which is searched and speculated according to Scifinder and Reaxy databases The compound is pelargonidin 3-O-glucoside, the primary and secondary mass spectra (negative and positive) of this compound are shown in Figure 8, and the possible structural formula is as follows:

3. Quantitative analysis of anthocyanins in raspberries

For the main anthocyanin components in the raspberry anthocyanin extract obtained by hot water extraction, methanol extraction and subcritical water extraction of the present invention (optimal extraction conditions: extraction pressure 7MPa, extraction time 90min, extraction temperature 130°C). That is, components 2, 3, 5) and the total amount of anthocyanins were quantitatively analyzed.

Using malvain-3,5-diglucoside as a semi-quantitative standard, the content of each anthocyanin monomer in the extract was calculated by the standard curve. The standard curve is:

Y=513.27X+0.2937(r=0.9995)

Wherein, Y is the peak area, and X is the content of the equivalent of malvain-3,5-diglucoside.

The amount of each anthocyanin monomer extracted by each method is expressed as the mass of the equivalent of malvain-3,5-diglucoside extracted per 100 g of fresh raspberry fruit, all samples were repeated three times, and the average was calculated value. The total amount of extracted anthocyanins is obtained by adding up the measured amounts of all anthocyanin monomers.

The analysis results are shown in Table 3.

Table 3 Composition and quantitative analysis of main anthocyanins

As can be seen from the data in Table 3, using the subcritical water extraction method of the present invention to extract raspberry anthocyanins, about 81.52 mg of the anthocyanin monomer of component 2, about 6.39 mg of anthocyanins can be extracted from every 100 g of raspberry fresh fruit. The total amount of anthocyanins extracted from each 100g of fresh raspberry fruit is as high as 98.91mg, which can extract more anthocyanins, the total amount and The extraction efficiency of each anthocyanin monomer is significantly improved, which is obviously higher than the extraction efficiency of the prior art.

Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, and substitutions can be made in these embodiments without departing from the principle and spirit of the invention and modifications, the scope of the present invention is defined by the appended claims and their equivalents.

Claims (10)

1. A method for extracting anthocyanin from raspberry is characterized by comprising the following steps: extracting from raspberry fruit by subcritical water extraction.

2. The method of claim 1, comprising: performing subcritical water extraction on the raspberry by an extracting agent under the conditions that the extraction temperature is 100-160 ℃ and the extraction pressure is 6-8 MPa to obtain raspberry anthocyanin extract.

3. The method of claim 2, further comprising the steps of: drying the raspberry anthocyanin extract to obtain the raspberry anthocyanin extract.

4. The method of claim 2, wherein the raspberry is a raspberry fresh fruit.

5. The method as claimed in claim 4, wherein the dosage ratio of the fresh raspberry fruit to the extractant is 1 g: 60-120 mL; further, the dosage ratio of the fresh raspberry fruits to the extracting agent is 1 g: 90 mL; further, the extracting agent is ultra-pure water.

6. The method according to claim 2, wherein the extraction time is 60-120 min; further selected from 90 min.

7. The method of claim 2, wherein the extraction temperature is 130 ℃.

8. The process according to claim 2, characterized in that the extraction pressure is 7 MPa.

9. The method according to any one of claims 1 to 8, wherein the anthocyanin is selected from one or more of cyanidin-3-sophoroside-5-glucoside, cyanidin-3-sophoroside, cyanidin-3-glucoside, pelargonidin-3-sophoroside, cyanidin-3- (6' -citric acid) -sophoroside and pelargonidin-3-glucoside.

10. A raspberry anthocyanin extract is characterized by comprising 75-85 parts of cyanidin-3-sophoroside, 75-85 parts of cyanidin-3-glucoside and 8-11 parts of pelargonidin-3-glucoside.

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