KR102053941B1 - Method for extracting phenolic compounds comprising resveratrol from peanut sprouts - Google Patents

Abstract

The present invention relates to a method for extracting a phenolic compound including resveratrol from peanut sprouts, and more specifically, to a method for extracting a phenolic compound including resveratrol from peanut sprouts, which comprises the steps of: making enzyme reaction of peanut sprouts with celluclast, viscozyme, amyloglucosidase, promozyme, maltogenase, dextrozyme, termamyl, papain, protamex, neutrase and flavourzyme to yield a reaction product; and performing hot water extraction on the reaction product. According to the present invention, without using an organic solvent such as ethanol, a phenolic compound including resveratrol can be efficiently extracted from peanut sprouts by means of methods such as enzyme treatment and hot water extraction, which are safe to human bodies. According to the method where effective ingredients are extracted from peanut sprouts for application to foods and human bodies, wasting of peanut sprouts, or a material can be reduced to save raw material costs. In addition, a product obtained from processing of peanut sprouts can be directly utilized as a material for foods in a safe manner, without a separate process for removing a solvent.

Description

Method for extracting phenolic compounds comprising resveratrol from peanut sprouts}

The present invention relates to a method for extracting phenolic compounds including resveratrol from peanut sprouts, specifically peanut sprouts (celluclast), biscozyme (amycolucosidase), amyloglucosidase, promozyme ( enzymatic reaction with promozyme, maltogenase, dextrozyme, termamyl, papain, protamex, neutrase and flavorzyme To obtain a reaction product; It relates to a method for extracting phenolic compounds, including resveratrol from peanut sprouts comprising; and hot water extraction from the reaction product.

Peanuts are annual herbaceous plants belonging to the legumes and are high in fat and protein. It is consumed in various forms, such as immediate eating, butter, margarine and cooking oil (Lee et al., 2004). Peanut is a representative plant that contains resveratrol, a natural polyphenol compound. Germinated peanut sprouts have abundant functional nutrition, high water content, good taste, and a wide range of use as food materials (Ikeda). et al., 1984). During sprouting of peanuts, fat and calories are lowered and the content of resveratrol, a natural polyphenol compound present in trace amounts in peanuts, is increased (Lee et al., 2003; Kang et al., 2010; Kim et al. , 2010). These peanut sprouts are known to be effective in preventing cancer, diabetes, heart disease, antioxidant activity, inhibiting inflammation, preventing aging, hardening of arteries, lowering blood cholesterol and improving memory. Pharmacological effects are reported (Kim, 2013).

Recently, by decomposing plant cell walls with enzymes, attempts have been made to increase the production efficiency of plant useful substances or to improve process and product quality. Apples, pears, carrots, cabbages, mangoes, bananas, and other plants are being studied, but peanut sprouts have not been studied. In addition, rather than improving the production efficiency of useful substances through one enzyme, the various types of enzymes will be used to break down the cell wall more quickly and increase the amount of useful substances and production efficiency. Although peanut sprouts are increasing in availability as food materials and demand is increasing in the international market, the production method of peanut sprouts has been inefficient (Kang et al., 2011). Korean Peanut and Peanut Sprout Research has been conducted for breeding to improve productivity (Pae et al., 2004), cultivation improvement (Cheong et al., 2001), useful ingredients (Wang et al., 2005; Lee et al., 2003) There have been studies on the back, but studies related to extraction efficiency have been insufficient.

Therefore, the present inventors have attempted to develop a method for efficiently extracting useful components such as resveratrol and phenolic compounds from peanut sprouts, and in particular, by using a method safe for the human body without using an organic solvent such as ethanol. It was intended to increase the usability by allowing it to be used directly in food without a process.

J. Agric Food Chem. 47: 3963-3966. Kor Soc Crop Sci. 46: 206-270. Cereal Chem. 61: 236-240. J. Kor Soc Food Sci Nutr. 39: 941-946. Kor J. Horti Sci Tech. 29: 202-203. Food Chem. 81: 321-326. Kor J. Food Tech. 35: 764-768. Kor J. Crop Sci. 51: 259-263. J. Kor Academia-Industrial cooperation Soc. 14: 4100-4105. J. Kor Soc Food Sci Nutr. 39: 406-413. J. Env Sci intern. 24: 763-767. Kor J. Crop Sci. 48: 429-433. J. Kor Soc Food Sci Nutr. 33: 941-945. Kor J. Food Sci Technol. 38: 571-576. Kor J. Food Cookery Sci. 28: 51-55. Kor J. Breed. 36: 375-376. J. Agric Food Chem. 53: 242-246.

Therefore, the main object of the present invention is to provide a method for efficiently extracting useful components such as resveratrol and phenolic compounds from peanut sprouts in a safe manner to the human body without using an organic solvent such as ethanol.

According to one aspect of the present invention, the present invention is a peanut sprout cellulast (celluclast), biscozyme (amycolucosidase), amyloglucosidase (promozyme), maltogenase (dextozyme), dex Enzymatic reaction with dextrozyme, termamyl, papain, papain, protamex, neutrase and flavorzyme to obtain a reaction product; It provides a method for extracting phenolic compounds, including resveratrol from peanut sprouts comprising; and extracting hot water from the reaction product.

In the method of the present invention, the peanut sprout is preferably hot air dried.

In the method of the present invention, the enzyme reaction is preferably carried out for 10 to 20 hours at pH conditions of pH 5.3 to 5.7 and temperature conditions of 50 to 60 ℃.

In the method of the present invention, it is preferable to perform the hot water extraction for 30 minutes to 2 hours at 90 to 100 ℃.

According to the present invention, it is possible to efficiently extract phenolic compounds including resveratrol from peanut sprouts in a human-safe method such as enzyme treatment and hot water extraction without using an organic solvent such as ethanol. Therefore, the method of the present invention has the advantage of reducing raw material costs by reducing the waste of peanut sprouts as a material in extracting the active ingredient from peanut sprouts for the purpose of applying to humans such as food. In addition, the peanut sprout processed product obtained by the method of the present invention has the advantage that it can be safely used as a material such as food immediately without a separate process for removing the solvent.

1 is a result of analyzing the total phenolic compound content of the hot water extract, ethanol extract and homoenzyme reaction product of peanut sprout. If the alphabet at the top of the graph is different, it means that there is a statistically significant difference ( P <0.05).
Figure 2 shows the results of analyzing the total phenolic compound content of the complex enzyme reaction product of peanut sprout. If the alphabet at the top of the graph is different, it means that there is a statistically significant difference ( P <0.05).
Figure 3 shows the results of analyzing the total phenolic compound content of the hot water extract after the complex enzyme reaction of peanut sprout. If the alphabet at the top of the graph is different, it means that there is a statistically significant difference ( P <0.05).

The method of the present invention comprises peanut sprouts (celluclast), biscozyme (amycolucosidase), amyloglucosidase (promozyme), maltogenase (maltogenase), dextrozyme (dextrozyme), Enzymatic reaction with termamyl, papain, protamex, neutrase and flavorzyme to obtain a reaction product; And extracting hot water from the reaction product.

Peanut sprouts mean that the sprouts are sprouted by germinating peanuts for about a week, and generally can be grown only with water, such as bean sprouts. The method of the present invention can efficiently extract phenolic compounds including resveratrol from these peanut sprouts. The peanut sprout in the present invention is preferably used in a hot air dry state.

The enzymes used in the present invention are industrially produced and sold enzymes that can be utilized in foods, etc., and can be easily purchased on the market.

Cellulast is an enzyme produced from the fungus Trichoderma resei , which is known to break down cellulose into glucose.

Biscozyme is a multi-enzyme complex produced from Aspergillus and contains carbohydrates including arabanase, cellulase, beta-glucanase, hemicellulase and xylanase It is known to contain hydrolases.

Amyloglucosidase is an enzyme produced from Aspergillus niger and is known to hydrolyze starch's 1,4- and 1,6-alpha bonds.

Promozyme is a pullulanase produced from Bacillus acidopullulyticus that breaks down 1,6-alpha bonds in amylopectin partially degraded by pullulan or alpha-amylase. Known.

Maltogenase is a low-molecular-weight oligosaccharide containing 1,4 starch and maltotriose partially decomposed with high-maltogenic amylase produced from Bacillus subtilisamyloliquefaciens containing Bacillus stearothermophilus gene. It is known to hydrolyze alpha glycoside bonds to produce maltose from non-reducing ends.

Dextrozyme is a mixed enzyme of glucoamylase produced from a genetically modified strain of Aspergillus niger and pullulanase produced from a genetically modified strain of Bacillus and is a 1,4- and 1,6-starch of starch. It is known to hydrolyze alpha bonds.

Thermamil is a thermostable alpha amylase produced from a genetically modified strain of Bacillus licheniformis and is known to hydrolyze the 1,4-alpha glycoside bonds of amylose and amylopectin.

Papain is a cysteine protease belonging to the peptidase C1 family produced from Carica papaya and is known to hydrolyze proteins, peptides, amides, and esters of amino acids and peptides.

Protamex is known as protease produced from batch fermentation from the response surface methodology from Bacillus .

Neutraase is known as an endoprotease produced from Bacillus amyloliquefaciens .

Flavorzyme is a protease produced from Aspergillus oryzae and is known to exhibit endo- and exo-protease activity.

In the present invention, the enzymatic reaction may be achieved through a conventional method of enzymatically reacting plant material, but it is preferable to use a method of keeping the peanut sprout and the enzyme in contact with each other in an aqueous solution. For example, a method of adding peanut sprouts and the enzymes to water and maintaining them at a pH and temperature condition within a range in which the enzymes can act may be used.

According to the present invention, the pH conditions of the enzyme reaction to pH 5.3 ~ 5.7 and the temperature conditions to 50 ~ 60 ℃ to perform the enzyme reaction for 10 to 20 hours to increase the extraction efficiency of phenolic compounds, including resveratrol desirable. More preferably, the enzyme reaction is performed at pH 5.4 to 5.6 and 53 to 57 ° C. for 14 to 18 hours.

In the present invention, the hot water extraction may be achieved through a conventional method for extracting useful components from plant materials by using water as a solvent and heating, but the extraction temperature is increased to 90 to increase the extraction efficiency of phenolic compounds, including resveratrol It is preferable to extract for 30 minutes-2 hours as ~ 100 degreeC. More preferably it is extracted for 40 minutes to 80 minutes at 93 ~ 97 ℃. The hot water extraction may be carried out by performing an enzymatic reaction in an aqueous solution and then heating without adding water separately.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

EXAMPLE

1. How to

1-1. material

Peanut sprouts cultivated directly from domestic peanuts were hot-air dried at 50 ° C. for 24 hours, and cut into 3 to 5 cm and used as samples for enzyme treatment or extraction.

1-2. Hot water extraction and enzyme treatment

Hot water extraction, ethanol extraction, single enzyme treatment, complex enzyme treatment, complex enzyme treatment + hot water extraction were performed in five ways.

1-2-1. Hot water or ethanol extraction

Distilled water was added to the peanut sprout sample of 1-1 in a weight ratio of 1:50, and hot water was extracted by varying the extraction time at 95 ± 5 ° C. for 1, 2 and 4 hours.

Ethanol extraction was added to the same ratio instead of distilled water and extracted with reflux cooling for 2 hours.

1-2-2. Single enzyme treatment

Distilled water was added to the peanut sprout sample of 1-1 in a weight ratio of 1:50, and any enzyme selected in Table 1 was added (added to be the final 2% by weight in the enzyme reaction solution) according to each pH and temperature conditions. Enzyme reaction was carried out for 8 hours or 16 hours while stirring in a shaker water bath, and then heat-treated at 105 ° C. for 15 minutes to inhibit enzyme activity.

enzyme Optimal condition pH ℃ Biscozyme 4.5 50 Cellulast 4.5 50 AMG 4.5 60 Termamil 6.0 60 Promozyme 4.5 60 Maltogenase 4.5 50 Dextrozyme 4.5 50 Neutase 6.0 50 Flaborzyme 7.0 50 Alcalase 8.0 50 Papain 6.0 50 Protamex 6.0 40

1-2-3. Complex enzyme treatment

Distilled water was added to the peanut sprout sample of 1-1 in a weight ratio of 1:50, and enzymes were added for each enzyme composition according to each experimental group as shown in Table 2 (each enzyme added per experimental group in the same weight ratio, and the enzyme reaction The total weight of each enzyme in the solution was added to the final 2% by weight), followed by enzymatic reaction for 16 hours with stirring in a shaker water bath according to each pH and temperature conditions, and then at 105 ° C to inhibit enzymatic activity. Heat treatment was performed for 15 minutes.

In the case of Experimental Groups 7 and 8, the mixture was heat-treated at 105 ° C. for 15 minutes in order to inhibit enzymatic activity after the complex enzyme treatment, followed by extraction by heating at 95 ° C. for 1 hour or 2 hours.

Enzyme composition pH ℃ Enzyme Treatment Time /
Hot water extraction time Experimental Group 1 Cellulast, Biscozyme, AMG, Promozyme 4.5 55 16 hours / 0 hours Experimental Group 2 Cellulast, Biscozyme, AMG, Promozyme, Maltogenase, Dextrozyme 4.5 55 16 hours / 0 hours Experimental Group 3 Papain, Protamex, Neutrase, Flavorzyme 6 55 16 hours / 0 hours Experimental Group 4 Cellulast, Biscozyme, AMG, Promozyme, Maltogenase, Dextromezyme, Thermamil, Papain, Protamex, Neutase, Flavorzyme 4.5 55 16 hours / 0 hours Experimental Group 5 Cellulast, Biscozyme, AMG, Promozyme, Maltogenase, Dextromezyme, Thermamil, Papain, Protamex, Neutase, Flavorzyme 5 55 16 hours / 0 hours Experimental Group 6 Cellulast, Biscozyme, AMG, Promozyme, Maltogenase, Dextromezyme, Thermamil, Papain, Protamex, Neutase, Flavorzyme 5.5 55 16 hours / 0 hours Experimental Group 7 Cellulast, Biscozyme, AMG, Promozyme, Maltogenase, Dextromezyme, Thermamil, Papain, Protamex, Neutase, Flavorzyme 5.5 55 16 hours / 1 hour Experimental Group 8 Cellulast, Biscozyme, AMG, Promozyme, Maltogenase, Dextromezyme, Thermamil, Papain, Protamex, Neutase, Flavorzyme 5.5 55 16 hours / 2 hours

1-3. Total Phenolic Compound Analysis

In 1 to 2, 1 ml of the solution obtained by hot water extraction, ethanol extraction, single enzyme treatment, complex enzyme treatment, complex enzyme treatment + hot water extraction and 9 ml of tertiary distilled water were mixed, 1 ml of Ciocalteu's phenol reagent) was added and mixed for 5 minutes at room temperature. Thereafter, 10 ml of a 7% Na ₂ CO ₃ solution was added to the reaction solution, followed by 25 ml of distilled water. The mixed solution was allowed to stand at 23 ° C. for 2 hours, and then absorbance was measured at 760 nm with a UV-spectrophotometer (UV-1201, Shimadzu, Tokyo, Japan). The total phenolic compound content was calculated by applying absorbance measured to the calibration curve prepared using gallic acid (Kim et al., 2003).

1-4. Resveratrol Quantitative Analysis

The solution obtained by hot water extraction, ethanol extraction, complex enzyme treatment, complex enzyme treatment + hot water extraction in 1-2 was subjected to filtration and concentration, respectively, and lyophilized to be used as a sample while being frozen and stored.

Lyophilized resveratrol obtained by the enzymatic treatment or hydrothermal extraction of the above 1-2 using an HPLC shimadzu LC-20AT pump, SPD-M20A photodiode array detector (shimadzu, Tokyo, Japan). Quantitative analysis.

As a column, a C18 column (250 mm × 4.6 mm, 5.0 μm, Simpek, shimadzu, Tokyo, Japan) was used, and the mobile phase was A: acetonitrile + 0.1% formic acid, B: Water + 0.1% formic acid was used. Mobile phases A: B were detected from 40 to 100: 0 to 100 over time. In this case, the mobile phase velocity was 1.2 ml / min, the sample injection amount was 20 µl, and the wavelength was analyzed at 310 nm. Standard resveratrol was used by dissolving in methanol (Kang et al., 2010).

1-5. Statistical processing

All experiments were repeated three times and expressed as mean and standard deviation (mean ± SD), and each mean value was tested by Duncan's multiple test (Duncan's) using SAS® version 9.11 (SAS Institute, Cary, NC, USA). multiple range test).

2. Results

2-1. Phenolic Compound Content

The results of analyzing the total phenolic compound content of hot water extraction, ethanol extraction, single enzyme treatment, complex enzyme treatment, complex enzyme treatment + hot water extraction are as shown in Figs.

The extraction time was about 17.5mg GAE / g for 1 hour hot water extraction, 18.75mg GAE / g for 2 hours hot water extraction and about 20.25mg GAE / g for 4 hours hot water extraction. It was found that the content increased. Ethanol 2-hour extraction showed 25 mg GAE / g. In the single enzyme treatment, two random enzymes were selected and used for enzyme treatment one by one. When the enzyme treatment was carried out for 8 hours, it was 22.75 and 24 mg GAE / g, respectively. When the enzyme treatment was carried out for 16 hours, 25.25 and 24.5 mg. The total phenolic compound content was increased when the enzyme was treated with GAE / g for 16 hours rather than 8 hours. These results show that the enzyme treatment can increase the content of phenolic compounds rather than hot water extraction, and can increase the content of phenolic compounds to a degree similar to ethanol extraction, which is an organic solvent.

The complex enzyme treatment used a mixture of four and six types of glycolytic enzymes, four kinds of proteolytic enzymes, and 11 types of glycolytic enzymes and proteolytic enzymes. The combinations are shown in Table 2. When the enzyme treatment time is 16 hours, the content analysis result of the total phenolic compound is shown in FIG. 2. Experimental group 6 showed the highest total phenolic compound content in the complex enzyme treatment, and the content was 24.25mg GAE / g, and the next highest content was experimental group 2, experimental group 3, and the contents were 23.25, 22, respectively. Mg GAE / g. It is believed that the content of phenolic compounds can be increased in the complex enzyme composed of a mixture of glycoprotein and proteolytic enzyme rather than the complex enzyme composed of sugar or protease alone.

The result of measuring the total phenolic content of the hydrothermally extracted sample after the complex enzyme treatment is shown in FIG. 3. The complex enzyme used here was experimental group 6, and the enzyme treatment was carried out for 16 hours, and then the reaction was terminated, 1 hour hydrothermal extraction, and 2 hours hydrothermal extraction were conducted. The result of measuring total phenolic compound content was about 38% higher than 33.5mg GAE / g when hot water extraction was performed for 1 hour than 24.25mg GAE / g, and 28.4mg GAE / g when hot water extraction was performed for 2 hours. It was higher than when the reaction was terminated immediately with g, but showed a lower content than when hot water was extracted for 1 hour.

2-2. Resveratrol content

Table 3 shows the results of resveratrol content of hot water extraction, ethanol extraction, complex enzyme treatment, complex enzyme treatment + hot water extraction.

The ethanol extraction method showed the highest content of 112.94 ㎍ / g, followed by 91.57 ㎍ / g in Experimental Group 7. The resveratrol content was relatively low in the hydrothermal extraction and the rest of the experimental groups.

These results suggest that resveratrol can be efficiently extracted from peanut sprouts in the same degree as ethanol extract when hot water extraction is performed for 1 hour after complex enzyme treatment as in Experimental Group 7.

Way Resveratrol content (㎍ / g) Ethanol Extract 112.94 ± 12.82 ^a Hydrothermal extraction 28.50 ± 5.89 ^f Experimental group 1 31.03 ± 5.71 ^f Experiment group 2 33.12 ± 12.79 ^ef Experiment group 3 42.28 ± 2.86 ^d Experimental Group 4 40.06 ± 3.05 ^de Experimental group 5 27.99 ± 1.62 ^f Experimental Group 6 40.29 ± 8.29 ^de Experimental group 7 91.57 ± 4.52 ^b Experimental Group 8 49.57 ± 9.52 ^c

Claims (4)

Peanut sprouts are made from celluclast, biscozyme, amyloglucosidase, promozyme, maltogenase, dextrozyme, termamyl Enzymatic reaction with papain, protamex, neutrase and flavorzyme to obtain a reaction product; And
And extracting hot water from the reaction product.
The enzyme reaction is carried out for 10 to 20 hours at a pH of pH 5.3 to 5.7 and a temperature of 50 to 60 ℃,
The hot water extraction is characterized in that for 30 minutes to 2 hours at 90 to 100 ℃, extracting phenolic compounds, including resveratrol from peanut sprouts. The method of claim 1,
The peanut sprout is characterized in that the hot air dried. delete delete

Images