Process for extracting pyracantha fortuneana fruit polyphenol by enzyme-assisted method

Abstract

The invention discloses a process for extracting pyracantha fortuneana fruit polyphenol by an enzyme-assisted method, which comprises the following steps: S1, raw material pretreatment: drying, crushing and sieving pyracantha fortuneana fruits to obtain pyracantha fortuneana fruit powder; s2, enzyme-assisted extraction: mixing the pyracantha fortuneana fruit powder with cellulase and purified water, carrying out an enzymolysis reaction, adding an ethanol solution after enzymolysis is completed, carrying out extraction, and carrying out centrifugal separation after extraction to obtain a pyracantha fortuneana fruit polyphenol extract solution. The efficient extraction of the pyracantha fortuneana fruit polyphenol is realized through a process for extracting the pyracantha fortuneana fruit polyphenol by an enzyme-assisted method.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polyphenol extraction, and particularly relates to an enzyme-assisted extraction process of Pyracantha fortuneana polyphenols. BACKGROUND

[0002] Pyracantha fortuneana is a wild evergreen plant of the Rosaceae-Pyreae-Pyracantha genus, which is widely distributed in the southwest of China. Its roots, stems, leaves, flowers and fruits have medicinal value, and it has strong growth ability in the wild, has low requirements for the environment and soil, and has high yield. It is a typical medicinal and edible plant. Pyracantha fortuneana is red or orange-red after maturity, and contains rich nutritional ingredients. According to the Record of Materia Medica, it has the effects of invigorating the spleen, promoting digestion, clearing heat and detoxifying, and promoting blood circulation. Modern research further confirms that Pyracantha fortuneana has the effects of whitening, antioxidant, anti-tumor, antibacterial, free radical scavenging, etc. Pyracantha fortuneana polyphenols, as an important active ingredient in Pyracantha fortuneana, have high safety, can significantly reduce the weight and fat of obese mice caused by high-fat food, repair the intestinal barrier, and have antioxidant and anti-acid hydrolysis effects on oil, and the concentration and inhibition effect are positively correlated. It has broad application prospects in the fields of functional food and cosmetics.

[0003] The pharmacological effects of Pyracantha fortuneana are closely related to the chemical components contained therein, such as alcohol, fat, aldehyde, ketone, quercetin, etc. In recent years, there have been many studies on the components such as pigments, vitamins, flavonoids and polysaccharides in Pyracantha fortuneana, but relatively few studies on Pyracantha fortuneana polyphenols with important physiological activity. At present, the polyphenol extraction process mainly includes high-pressure pulse electric field extraction, organic solvent extraction, macroporous resin separation, ultrasonic-assisted extraction, etc. The high-pressure pulse electric field extraction process requires a special high-voltage pulse generator, and the cost of equipment manufacturing and maintenance is high, which is difficult to realize large-scale industrial production. The organic solvent extraction process usually uses organic solvents such as methanol and ethanol for extraction, which is relatively simple to operate, but has the problem of organic solvent residue. The macroporous resin separation process is mainly used for the separation and purification of total polyphenols in Pyracantha fortuneana, not for direct extraction, and the crude extract still needs to be combined with other extraction methods in the early stage, which is complicated. The ultrasonic-assisted extraction process uses the cavitation effect and mechanical vibration of ultrasonic waves to improve the extraction rate, but the range of action of ultrasonic waves is limited, and it is difficult to achieve uniform action in large-scale extraction equipment, resulting in unstable extraction effect.

[0004] In summary, the existing Pyracantha fortuneana polyphenol extraction process has obvious deficiencies in equipment requirements, production cost, extraction efficiency, product safety, environmental friendliness, etc., and it is difficult to meet the needs of industrial production and practical application. Therefore, developing a Pyracantha fortuneana polyphenol extraction process with mild reaction conditions, simple operation, low equipment requirements, high extraction rate, good product safety and meeting the green production concept has become a problem to be solved in the field of Pyracantha fortuneana resource development and utilization. SUMMARY

[0005] The application aims to provide an enzyme-assisted extraction process of Pyracantha fortuneana fruit polyphenols.

[0006] The enzyme-assisted extraction process of Pyracantha fortuneana fruit polyphenols comprises the following steps: S1, raw material pretreatment: drying, crushing and sieving Pyracantha fortuneana fruit to obtain Pyracantha fortuneana fruit powder; S2, enzyme-assisted extraction: mixing the Pyracantha fortuneana fruit powder with cellulase and pure water, performing enzymolysis reaction, adding ethanol solution for leaching after the enzymolysis is completed, and centrifugally separating to obtain Pyracantha fortuneana fruit polyphenol extract solution.

[0007] As a preferred, in the Pyracantha fortuneana fruit pretreatment step, the drying temperature is 40-50 DEG C, the drying time is 10-14h, and the crushed product is sieved through a 50-70 mesh sieve.

[0008] As a preferred, the mass ratio of Pyracantha fortuneana fruit powder to cellulase is 20-40, the amount of pure water added is 18-22 mL per gram of Pyracantha fortuneana fruit powder, the enzymolysis temperature is 55-65 DEG C, the enzymolysis time is 80-120 min, the volume fraction of ethanol solution is 40%-55%, the amount of ethanol solution added is 8-12 mL per gram of Pyracantha fortuneana fruit powder, the leaching time is 25-35 min, and the centrifugal speed is 4500-5500 r / min, and the centrifugal time is 8-12 min.

[0009] As a preferred, the enzymolysis time is 95-99 min, the enzyme dosage is 0.038-0.042 g per gram of Pyracantha fortuneana fruit powder, and the ethanol volume fraction is 45%-49%.

[0010] As a preferred, the method further comprises a total Pyracantha fortuneana fruit polyphenol content determination step, a standard curve is drawn by using Folin-phenol colorimetry with gallic acid as a standard, and the content and extraction rate of Pyracantha fortuneana fruit polyphenols are calculated.

[0011] The application of Pyracantha fortuneana fruit polyphenols in functional food, cosmetics and medicine.

[0012] The working principle and beneficial effects of the application are as follows: The cell wall of Pyracantha fortuneana fruit is mainly composed of polysaccharide substances such as cellulose and hemicellulose, which hinders the release of active ingredients such as Pyracantha fortuneana fruit polyphenols in the cell. The cellulase added in the process can specifically act on the cellulose in the cell wall of Pyracantha fortuneana fruit, decompose it into small molecules such as glucose, destroy the structural integrity of the cell wall, form porous channels, and make the Pyracantha fortuneana fruit polyphenols in the cell more fully contact with the extraction solvent, thereby promoting its release into the extraction system.

[0013] The application can reach 45.9% of the actual extraction rate of the pyracantha fortuneana fruit polyphenol under the optimal process condition, which is significantly higher than the extraction efficiency of the existing part of the extraction process, and can more fully utilize the pyracantha fortuneana fruit resources. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 is the experimental process flow chart; Figure 2 is the influence of enzymolysis time on the extraction rate of pyracantha fortuneana fruit polyphenol; Figure 3 is the influence of enzymolysis temperature on the extraction rate of pyracantha fortuneana fruit polyphenol; Figure 4 is the influence of enzymolysis pH on the extraction rate of pyracantha fortuneana fruit polyphenol; Figure 5 is the influence of enzyme dosage on the extraction rate of pyracantha fortuneana fruit polyphenol; Figure 6 is the influence of ethanol volume fraction on the extraction rate of pyracantha fortuneana fruit polyphenol. DETAILED DESCRIPTION

[0015] The following is further described in detail through specific embodiments: The wild pyracantha fortuneana fruit picked in Sinan County of Tongren City in Guizhou Province is dried and sealed for dry storage. After the pyracantha fortuneana fruit is cleaned, it is dried in a 45℃ drying box for 12h to constant weight, then crushed for 1min by a universal crusher and sieved through a 60 mesh sieve to obtain pyracantha fortuneana fruit powder. The pyracantha fortuneana fruit powder is sealed and dry stored for standby use.

[0016] 1g of pyracantha fortuneana fruit powder and 0.01g of cellulase are placed in a beaker, 20mL of pure water is added to the beaker, mixed, and then enzymolysis is carried out in a 60℃ water bath for 60min. After taking out, 10mL of ethanol with a volume fraction of 80% is added, and then extraction is continued for 30min. The extracted sample is transferred into a centrifuge tube and centrifuged at 5000r / min for 10min to separate the pyracantha fortuneana fruit polyphenol crude extract solution, which is sealed and stored at 0-4℃ for standby use.

[0017] 1. Pyracantha fortuneana fruit total polyphenol content determination The Folin-phenol colorimetric method was used to draw the gallic acid standard curve. 2.0 mg of gallic acid standard was accurately weighed, and distilled water was added to make the concentration of the gallic acid mother liquor 0.1 mg / mL. 0.0 mL, 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL and 0.5 mL of the gallic acid mother liquor were precisely taken into 6 test tubes, and distilled water was added to make the volume 1 mL to obtain a series of gallic acid standard solutions. Then 0.5 mL of Folin-phenol reagent was added, shaken well, and reacted for 5 min in the dark, and then 2 mL of 20% anhydrous sodium carbonate solution was added, and finally distilled water was added to make the volume 10 mL, and reacted for 20 min in a water bath at 40°C. The absorbance was measured at 765 nm. The gallic acid standard curve was drawn with the gallic acid mass concentration (mg / mL) as the abscissa and the absorbance as the ordinate, and the gallic acid standard curve was y=0.1041x-0.0347, R2=0.9993, which had a good linear relationship in the range of 0-0.005 mg / mL.

[0018] 2. Pre-experiment of firethorn fruit polyphenol extraction 1 g of firethorn powder and 0.01 g of cellulase were weighed into a beaker, 20 mL of pure water was added to the beaker, mixed well, and enzyme hydrolysis was carried out in a 60°C water bath for 60 min, then removed, 10 mL of 80% ethanol was added, and extraction was continued for 30 min. The extracted sample was transferred to a centrifuge tube and centrifuged at 5000 r / min for 10 min.

[0019] 3. Single factor experiment The sieved firethorn fruit powder was used as raw material, and the enzyme hydrolysis time, enzyme hydrolysis temperature, enzyme hydrolysis pH, enzyme dosage and ethanol volume fraction were investigated under the same experimental conditions. The yield of firethorn fruit powder polyphenol was used to judge the degree of influence.

[0020] (1) Effect of enzyme hydrolysis time on polyphenol yield 1 g of firethorn fruit powder and 0.01 g of cellulase were accurately weighed into a beaker, numbered 1-5, 20 mL of pure water was added to the beaker, mixed well, and enzyme hydrolysis was carried out at 60°C for 60 min, 80 min, 100 min, 120 min and 140 min, respectively, then removed, 10 mL of 80% ethanol was added, and extraction was continued for 30 min. The extracted sample was transferred to a centrifuge tube and centrifuged at 5000 r / min for 10 min, and the supernatant was determined by Folin-phenol method to calculate the yield of firethorn fruit polyphenol.

[0021] (2) Effect of enzyme hydrolysis temperature on polyphenol yield Accurately take 1 g of fire jiaoshu fruit powder, 0.01 g of cellulase in a beaker, number 1-5, add 20 mL of pure water into the beaker, mix well, and hydrolyze at 50℃, 60℃, 70℃, 80℃, 90℃ water temperature for 60 min, take out, add 10 mL of 80% ethanol, continue to extract for 30 min, and move the extracted sample into a centrifuge tube and centrifuge at 5000 r / min for 10 min, take the supernatant, and measure the concentration of gallic acid by Folin-phenol method to calculate the yield of fire jiaoshu fruit polyphenol.

[0022] (3) Effect of enzyme hydrolysis pH on polyphenol yield Accurately take 1 g of fire jiaoshu fruit powder, 0.01 g of cellulase in a beaker, number 1-5, add 20 mL of pure water into the beaker, mix well, and adjust the pH to 3, 4, 5, 6, 7, and hydrolyze at 60℃ water temperature for 60 min, take out, add 10 mL of 80% ethanol, continue to extract for 30 min, and move the extracted sample into a centrifuge tube and centrifuge at 5000 r / min for 10 min, take the supernatant, and measure the concentration of gallic acid by Folin-phenol method to calculate the yield of fire jiaoshu fruit polyphenol.

[0023] (4) Effect of enzyme dosage on polyphenol yield Accurately take 1 g of fire jiaoshu fruit powder, 0.01 g, 0.02 g, 0.03 g, 0.04 g, 0.05 g of cellulase in a beaker, number 1-5, add 20 mL of pure water into the beaker, mix well, and hydrolyze at 60℃ water temperature for 60 min, take out, add 10 mL of 80% ethanol, continue to extract for 30 min, and move the extracted sample into a centrifuge tube and centrifuge at 5000 r / min for 10 min, take the supernatant, and measure the concentration of gallic acid by Folin-phenol method to calculate the yield of fire jiaoshu fruit polyphenol.

[0024] (5) Effect of ethanol volume fraction on polyphenol yield Accurately take 1 g of fire jiaoshu fruit powder, 0.01 g of cellulase in a beaker, number 1-5, add 20 mL of pure water into the beaker, mix well, and hydrolyze at 60℃ water temperature for 60 min, take out, add 10 mL of 80% ethanol, continue to extract for 30 min, and move the extracted sample into a centrifuge tube and centrifuge at 5000 r / min for 10 min, take the supernatant, and measure the concentration of gallic acid by Folin-phenol method to calculate the yield of fire jiaoshu fruit polyphenol.

[0025] 4. Response surface optimization test design Based on the results of single-factor analysis, the yield of polyphenols from Pyracantha fortuneana was investigated. Enzymatic hydrolysis time (min), enzyme dosage (g), and ethanol volume fraction (%) were selected as the factors to be investigated. The central composite design of Box-Behnken was used in the experimental analysis using DesignExpert11 software. The experimental design is shown in Table 1, and the data were analyzed.

[0026] Table 1 Response Surface Experimental Design

[0027] The following conclusions were drawn: 1. Univariate Results and Analysis (1) Effect of enzymatic hydrolysis time on the extraction rate of polyphenols from Pyracantha fruit The trend of polyphenol extraction rate from Pyracantha fortuneana at different enzymatic hydrolysis times is shown in the figure. Figure 2 ,Depend on Figure 2 It was found that within the enzymatic hydrolysis time range of 60 to 140 minutes, the yield of Pyracantha fortuneana polyphenols initially increased and then decreased with increasing hydrolysis time. The highest polyphenol yield (53.24%) was achieved at a hydrolysis time of 100 minutes. When the hydrolysis time exceeded 100 minutes, other impurities dissolved and formed complexes with the polyphenols, leading to a decrease in the polyphenol yield. To improve experimental efficiency, the optimal hydrolysis time was determined to be 100 minutes.

[0028] (2) Effect of enzymatic hydrolysis temperature on the extraction rate of polyphenols from Pyracantha fruit The trend of polyphenol extraction rate from Pyracantha fortuneana at different enzymatic hydrolysis temperatures is shown in the figure. Figure 3 .Depend on Figure 3 It was found that within the enzymatic hydrolysis temperature range of 50℃ to 90℃, the yield of polyphenols from Pyracantha fortuneana fruit initially increased and then decreased with increasing hydrolysis temperature. The highest polyphenol yield (40.89%) was achieved at 80℃. Above 80℃, excessively high temperatures caused enzyme inactivation and the dissolution of other substances, affecting polyphenol solubility and thus decreasing the polyphenol yield. To improve experimental efficiency, the optimal hydrolysis temperature was determined to be 80℃.

[0029] (3) Effect of enzymatic hydrolysis pH on the extraction rate of polyphenols from Pyracantha fruit The trend of polyphenol extraction rate from Pyracantha fortuneana at different enzymatic hydrolysis pH values ​​is shown in the figure. Figure 4 .Depend on Figure 4It was found that within the enzymatic hydrolysis pH range of 3 to 7, the yield of Pyracantha fortuneana polyphenols initially increased and then decreased with increasing pH. The highest polyphenol yield (34.62%) was achieved at pH 6. When the pH exceeded 6, excessively high pH levels caused protein denaturation, leading to enzyme inactivity and the dissolution of other substances, thus affecting polyphenol solubility and resulting in a decreased polyphenol yield. To improve experimental efficiency, the optimal enzymatic hydrolysis pH was determined to be 6.

[0030] (4) Effect of enzyme dosage on the extraction rate of polyphenols from Pyracantha fruit The trend of polyphenol extraction rate from Pyracantha fruit with different enzyme dosages is shown in the figure. Figure 5 .Depend on Figure 5 It was found that within the enzyme dosage range of 0.01 to 0.05 g, the yield of Pyracantha fortuneana polyphenols initially increased and then decreased with increasing enzyme dosage. The highest polyphenol yield (50.84%) was achieved with an enzyme dosage of 0.04 g. At this dosage, the enzyme mass fraction may have reached saturation, inhibiting enzyme activity and causing it to bind with the Pyracantha fortuneana polyphenols to form precipitates, thus reducing the polyphenol yield. To improve experimental efficiency, the optimal enzyme dosage was determined to be 0.04 g.

[0031] (5) Effect of ethanol volume fraction on the extraction rate of polyphenols from Pyracantha fruit The trend of polyphenol extraction rate from Pyracantha fruit under different ethanol volume fractions is shown in the figure. Figure 6 ,Depend on Figure 6 It was found that within the ethanol volume fraction range of 50% to 90%, the yield of Pyracantha fortuneana fruit polyphenols initially increased and then decreased with increasing ethanol volume fraction. The highest polyphenol yield (42.63%) was achieved at an ethanol volume fraction of 60%. However, at this 60% volume fraction, further increases in ethanol concentration led to protein denaturation, affecting enzyme activity and causing impurities to dissolve, thus decreasing the polyphenol yield. To improve experimental efficiency, the optimal ethanol volume fraction was determined to be 60%.

[0032] 2. Response surface optimization experimental design Analysis of the experimental data showed that the optimal extraction conditions for enzyme-assisted extraction of polyphenols from Pyracantha fortuneana fruit were: enzymatic hydrolysis time of 97.034 min, enzyme dosage of 0.041 g, and ethanol volume fraction of 47.165%. The theoretical predicted extraction rate of polyphenols was 45.654%.

[0033] It should be noted that, for those skilled in the art, without departing from the technical solutions of the present application, a number of modifications and improvements can be made, which should also be considered as the protection scope of the present application, which will not affect the effect of the present application and the practicality of the patent. The protection scope claimed in the present application should be subject to the content of its claims, and the specific implementation mode and the like recorded in the specification can be used to explain the content of the claims.

Claims

1. A process for the extraction of polyphenols from Pyracantha fortuneana fruit by enzyme-assisted method, characterized by, It comprises the following steps: S1, raw material pretreatment: drying, crushing, and sieving the Pyracantha fortuneana fruit to obtain Pyracantha fortuneana fruit powder; S2, enzyme-assisted extraction: mixing the Pyracantha fortuneana fruit powder with cellulase and purified water, performing enzymatic reaction, adding ethanol solution for extraction after the enzymatic reaction is completed, and centrifugally separating the Pyracantha fortuneana fruit polyphenol extract solution obtained after the extraction.

2. The process for enzyme-assisted extraction of polyphenols from Pyracantha fortuneana fruits according to claim 1, characterized in that, In the Pyracantha fortuneana fruit pretreatment step, the drying temperature is 40-50℃, the drying time is 10-14h, and the crushed Pyracantha fortuneana fruit is sieved through a 50-70 mesh sieve.

3. The process for enzyme-assisted extraction of polyphenols from Pyracantha fortuneana fruits according to claim 2, characterized in that, The mass ratio of the Pyracantha fortuneana fruit powder to cellulase is 20-40, the amount of purified water added is 18-22mL per gram of Pyracantha fortuneana fruit powder, the enzymatic reaction temperature is 55-65℃, the enzymatic reaction time is 80-120min, the volume fraction of the ethanol solution is 40%-55%, the amount of ethanol solution added is 8-12mL per gram of Pyracantha fortuneana fruit powder, the extraction time is 25-35min, the centrifugal speed is 4500-5500r / min, and the centrifugal time is 8-12min.

4. The process for enzyme-assisted extraction of polyphenols from Pyracantha fortuneana fruits according to claim 3, characterized in that, The enzymatic reaction time is 95-99min, the cellulase amount is 0.038-0.042g per gram of Pyracantha fortuneana fruit powder, and the volume fraction of the ethanol solution is 45%-49%.

5. The process for enzyme-assisted extraction of polyphenols from Pyracantha fortuneana fruits according to claim 4, characterized in that, It further comprises a Pyracantha fortuneana total polyphenol content determination step, a standard curve is drawn using gallic acid as a standard, and the content and extraction rate of the Pyracantha fortuneana polyphenols are calculated using the Folin-phenol colorimetric method.

6. The Pyracantha fortuneana fruit polyphenols extracted according to any one of claims 1-5 are used in functional food, cosmetics, and medicines.

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