KR102586529B1 - Method for producing FA-1 from Prunus mume using microwave enzyme catalysis, high temperature and high pressure treatment - Google Patents

Abstract

The present invention relates to a method of producing 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal from plums, which involves irradiating plums with microwaves to hydrolyze them and then subjecting them to high temperature and high pressure treatment. Therefore, FA-1 production can be controlled easily and with high efficiency in a short period of time compared to conventional methods.

Description

Method for producing FA-1 from Prunus mume using microwave enzyme catalysis, high temperature and high pressure treatment}

The present invention relates to a method of producing FA-1 from plums by irradiating microwaves and high-temperature and high-pressure treatment.

Plum ( Japanese apricot , scientific name: Prunus mume ) has long been popular as a health food among Koreans, Japanese, and Chinese. Japanese apricot extract (JAE) has several beneficial biological effects, such as reducing oxidative stress and inflammation, and strengthening innate immunity.

Meanwhile, microwaves refer to electric waves. They are electromagnetic waves with a frequency of 1 GHZ-300 GHZ and a wavelength of 1 mm to 1 m, and are widely applied in communications, remote sensing, navigation, spectroscopy, etc. On the other hand, when microwaves are applied to a material, the material itself can be affected, so it can be used for heating or chemical reactions. A representative example is the micro oven used at home, which utilizes the phenomenon that when food containing water is heated, the food itself is heated and warmed. This heating phenomenon is also used in drying, hardening reactions, and synthesis.

In the present invention, an attempt was made to develop a method for producing useful compounds from plums with beneficial biological activity by irradiating microwaves.

Republic of Korea Patent Publication No. 10-2013-0017163

The present inventors confirmed that the sugar content increased in the hydrolyzate obtained by mixing sugar hydrolyzing enzymes with plums and irradiating them with microwaves. In addition, it was confirmed that FA-1 was produced when the hydrolyzate was treated at high temperature and high pressure, unlike samples that were not hydrolyzed or treated at high temperature and high pressure, and the present invention was completed.

The purpose of the present invention is to provide a method for producing 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal from plum.

In one aspect for achieving the above object, the present invention includes the steps of generating a hydrolyzate of plum by irradiating a mixture containing plum and sugar hydrolyzing enzyme with microwaves; And treating the hydrolyzate at a high temperature of 100 to 200°C and a high pressure of 0.005 to 0.3 MPA for more than 1 minute. 5-Hydroxymethyl-2-furaldehyde bis(5-formylfur) in plum Provides a method for producing furyl)acetal.

The method of producing 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal from plum of the present invention involves irradiating a mixture containing plum and sugar hydrolyzing enzyme with microwaves to produce hydrolyzate of plum. Includes creation steps.

In the present invention, the term "plum", whose scientific name is Prunus mume , is the fruit of the plum tree and is used as a health supplement or medicine. Plums are known to be sour and non-toxic, control the liver and phlegm, strengthen cells, normalize blood circulation, sterilize, relieve fatigue, eliminate inflammation, have excellent detoxification properties, eliminate indigestion and gastrointestinal disorders, and are effective in improving physical constitution.

In the present invention, the plum may be a freeze-dried powder of plum with the seeds removed. Specifically, it may be a powder obtained by removing the seeds from plum fruits, pulverizing them, and freeze-drying them, or it may be a powder obtained by removing the seeds, freeze-drying, and then pulverizing them.

In the present invention, the term "FA-1" refers to 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl) acetal.

The FA-1 has the structural formula of Formula 1 below.

The FA-1 can be isolated from plum. Through previous research, the present inventor isolated the FA-1 compound from the chloroform fraction obtained by adding chloroform to the plum extract extracted by adding sugar and heating the plum extract aged at 4°C for more than a week at a low temperature for more than a week. In addition, it was confirmed that FA-1 is effective in chronic obstructive pulmonary disease and protects skin cells.

The FA-1 is a precursor of hydroxymethylfurfural (HMF), and FA-1 is produced from hydroxymethylfurfural. Additionally, xylose is a precursor of hydroxymethylfurfural, and xylose is converted to hydroxymethylfurfural under conditions of approximately pH 5.0. In the case of plums, the pH is approximately 5.0, and when xylose is produced within the plums, xylose is easily converted to hydroxymethylfurfural (HMF), and FA-1 is converted from the produced hydroxymethylfurfural (HMF). can be created.

In the present invention, the sugar hydrolyzing enzyme is from the group consisting of Celluclast, Viscozyme, Amylogucosidase (AMG), Termamyl, and Ultraflo. It may be selected, and specifically may be Viscozyme.

In the present invention, the sugar hydrolyzing enzyme mixed in the mixture may have a pH of 4.5 to 5.5. The sugar hydrolyzing enzyme can be dissolved in water and mixed with plums in the form of a solution with a pH of 4.5 to 5.5.

The plum and sugar hydrolyzing enzyme may be mixed at a weight ratio (w/w) of 5:1 to 5000:1. Alternatively, when the sugar hydrolyzing enzyme is mixed with plum in the form of a solution dissolved in water, it can be mixed at a weight ratio (w/v) of 5:1 to 5000:1 to form a mixture. Specifically, they are mixed at a weight ratio (w/v, w/w) of 5:1 to 200:1, or more specifically, they are mixed at a weight ratio (w/v, w/w) of 10:1 to 100:1 to form a mixture. can do.

The microwave may be irradiated with a power of 10 to 800 W for 5 to 1440 minutes. More specifically, it can be irradiated with a power of 200 to 600 W for 30 to 120 minutes.

The microwaves can be generated using MAREDS (microwave assistant rapid enzyme digest system), and any machine that can generate microwaves can be used.

The MAREDS means that there is a constant temperature water bath in which a tube, etc. can be placed in a chamber where microwaves are radiated, so that the composition or enzyme in the tube is activated by microwaves, thereby reducing the effect of the enzyme on the composition (e.g. , hydrolysis, etc.).

In one embodiment of the present invention, plum and sugar hydrolyzing enzyme are mixed at a weight ratio of 10:1, and the hydrolyzate produced by irradiating a microwave with a power of 400 to 600 W for 60 to 120 minutes has a high yield and a high sugar content. confirmed.

The sugar content in the hydrolyzate may be increased, and specifically, the monosaccharide or polysaccharide content may be increased. Additionally, the sugar may be xylose.

In one embodiment of the present invention, it was confirmed that the polysaccharide concentration significantly increased when hydrolysis was performed for 30 to 120 minutes with microwave irradiation compared to the case where hydrolysis was performed for 24 to 48 hours without microwave irradiation. Additionally, compared to proteolytic enzyme (alkalase), when hydrolyzate of plum was prepared by mixing sugar hydrolytic enzyme with plum and irradiating it with microwaves, the polysaccharide content produced in the hydrolyzate increased.

In addition, among sugar hydrolysates, when hydrolysis was performed by adding Viscozyme and irradiating microwaves, the polysaccharide content and degree of hydrolysis were the highest, and the xylose content produced was also high.

The method for producing 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal from plum of the present invention is to heat the hydrolyzate at a high temperature of 100 to 200°C and a high pressure of 0.005 to 0.3 MPA for 1 minute. It includes the step of high temperature and high pressure treatment.

In the present invention, the high temperature and high pressure treatment step may be performed at a high temperature of 100 to 200 ℃ and a high pressure of 0.005 to 0.3 MPA for 1 minute or more, specifically, 1 minute at a high temperature of 100 to 200 ℃ and high pressure of 0.005 to 0.3 MPA. It may be carried out for from 160 minutes, and more specifically, it may be carried out for 15 to 60 minutes at a high temperature of 120°C and high pressure of 0.01 MPA.

In addition, the high-temperature and high-pressure treatment step may be performed by repeating the high-temperature and high-pressure treatment 2 to 6 times at a high temperature of 100 to 200 ° C. and a high pressure of 0.005 to 0.3 MPA. Specifically, the step of high-temperature and high-pressure treatment at a high temperature of 100 to 200°C and a high pressure of 0.005 to 0.3 MPA for 10 to 20 minutes may be repeated 2 to 6 times. More specifically, the step of high-temperature and high-pressure treatment at a high temperature of 120°C and a high pressure of 0.01 MPA for 15 minutes may be repeated three times.

In one embodiment of the present invention, through high-performance liquid chromatography and LC-MS analysis, the plum extract was extracted using the existing plum extract processing method (adding sugar and heating the extract aged at 4°C for more than a week at low temperature for more than a week). In the same way as FA-1 isolated from the chloroform fraction, which is the main fraction containing FA-1 component in plum extract, FA-1 was obtained by treating the hydrolyzate using Viscozyme and microwaves at high temperature and high pressure. Creation was confirmed.

Additionally, in one embodiment of the present invention, when the high-temperature and high-pressure treatment time at a high temperature of 120°C and a high pressure of 0.01 MPA is increased to 60 minutes, or the number of high-temperature and high-pressure treatments is increased from 1 to 3, the amount of FA-1 produced is It was confirmed that it was increasing.

However, samples of plum freeze-dried powder (PM) and plum freeze-dried powder hydrolyzed with a sugar hydrolase (PMDV) were treated without hydrolysis at a high temperature of 120°C and high pressure of 0.01 MPA for 15 minutes. In the case of the sample (PM HTHP15), it was confirmed through high-performance liquid chromatography that FA-1 was not produced.

Therefore, when the hydrolyzate obtained by irradiating plums with sugar hydrolyzing enzymes and microwaves is treated at high temperature and high pressure, it is easy and takes a short time to separate FA-1 from the chloroform fraction of the plum extract obtained by the conventional plum extract processing method. FA-1 production can be controlled simply and with high efficiency.

The present invention relates to a method of producing 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal from plums, which involves irradiating plums with microwaves to hydrolyze them and then subjecting them to high temperature and high pressure treatment. This has the advantage of being able to easily and highly efficiently control FA-1 production in a short period of time compared to conventional methods.

Figure 1 shows a method of hydrolysis of freeze-dried plum powder.
Figure 2 compares the yield of hydrolyzates using four types of proteolytic enzymes with samples hydrolyzed without proteolytic enzymes.
Figure 3 shows the results of comparing the sugar content after hydrolysis for each proteolytic enzyme.
Figure 4 compares the yield of hydrolyzates using five types of sugar hydrolyzing enzymes with samples hydrolyzed without sugar hydrolyzing enzymes.
Figure 5 shows the results of comparing the sugar content after hydrolysis for each sugar hydrolyzing enzyme.
Figure 6 shows a method of hydrolysis of freeze-dried plum powder using microwaves.
Figure 7 shows the polysaccharide concentration (sugar content) of the hydrolyzate hydrolyzed by applying microwaves to freeze-dried plum powder without hydrolytic enzyme treatment by adding only water.
Figure 8 shows the polysaccharide concentration (sugar content) after adding alcalase, a proteolytic enzyme, to freeze-dried plum powder and hydrolyzing it by varying the microwave power and substrate (freeze-dried plum powder) and enzyme ratio. It represents.
Figure 9 shows the polysaccharide concentration after adding amylogucosidase (AMG), a sugar hydrolyzing enzyme, to freeze-dried plum powder and hydrolyzing it by varying the microwave power and substrate (freeze-dried plum powder) and enzyme ratio. (sugar content).
Figure 10 shows the polysaccharide concentration (sugar content) after adding Viscozyme, a sugar hydrolyzing enzyme, to freeze-dried plum powder and hydrolyzing it by varying the microwave power and substrate (freeze-dried plum powder) and enzyme ratio. It is shown.
Figure 11 shows polysaccharide concentration (sugar content) after adding Celluclast, a sugar hydrolyzing enzyme, to freeze-dried plum powder and hydrolyzing it by varying the microwave power and substrate (freeze-dried plum powder) and enzyme ratio. It represents.
Figure 12 shows the addition of Amylogucosidase (AMG), Viscozyme, and Celluclast, microwave power of 400W, substrate (lyophilized plum powder), and glycolytic enzyme ratio of 10:1 ( The polysaccharide concentration (sugar content) in the case of weight ratio (w/w) is compared and shown in one graph.
Figure 13 shows the addition of Amylogucosidase (AMG), Viscozyme, and Celluclast, microwave power of 400W, substrate (lyophilized plum powder), and glycolytic enzyme ratio of 100:1 (weight ratio). , w/w), the polysaccharide concentration (sugar content) is compared in one graph.
Figure 14 shows the polysaccharide concentration (sugar content) when hydrolyzed according to the general conditions shown in Figure 1 without using microwaves.
Figure 15 shows the polysaccharide concentration after adding Viscozyme, a sugar hydrolyzing enzyme, and hydrolyzing with different ratios of the substrate (freeze-dried plum powder) and the sugar degrading enzyme under microwave power conditions of 400 W, and after hydrolyzing under general conditions. Polysaccharide concentration is compared in one graph.
Figure 16 shows the polysaccharide concentration and general conditions after addition of amylogucosidase (AMG), a sugar hydrolyzing enzyme, and hydrolysis by varying the ratio of the substrate (lyophilized plum powder) and the glycolytic enzyme under microwave power conditions of 400W. The polysaccharide concentration after hydrolysis is compared in one graph.
Figure 17 compares the degree of hydrolysis of amylogucosidase (AMG) and Viscozyme.
Figure 18 shows the addition of Viscozyme and amylogucosidase (AMG) under conditions of a substrate (lyophilized plum powder) and glycolytic enzyme ratio of 10:1 (weight ratio, w/w) in a microwave of 400 W. After hydrolysis, the xylose concentration (μM) was measured.
The top of Figure 19 shows the results of high-performance liquid chromatography (HPLC) using the chloroform fraction of plum extract extracted by the existing plum extract processing method.
The bottom of Figure 19 shows the hydrolyzate hydrolyzed by adding Viscozyme and irradiating 400 W of microwaves under the conditions of a substrate (lyophilized plum powder) and sugar decomposing enzyme ratio of 10:1 (weight ratio, w/w). This is the result of high-performance liquid chromatography (HPLC) after high-temperature and high-pressure treatment for 15 minutes at a high temperature of ℃ and high pressure of 0.01 MPA.
Figure 20 shows the presence or absence of high-temperature and high-pressure treatment after adding Viscozyme and hydrolyzing 400W of microwaves under the condition of a substrate (lyophilized plum powder) and glycolytic enzyme ratio of 10:1 (weight ratio, w/w). This is a comparison of FA-1 production.
Figure 21 confirms the production of FA-1 using LC-MS.
Figure 22 shows the high-temperature and high-pressure treatment time after adding Viscozyme and hydrolyzing irradiation with 400 W of microwaves under the condition of substrate (lyophilized plum powder) and glycolytic enzyme ratio of 10:1 (weight ratio, w/w). FA-1 production was compared using high-performance liquid chromatography at different times of 15, 30, and 60 minutes.

Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings so that those skilled in the art can easily implement the present invention. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein.

Experimental Example 1: Preparation of plum extract

Plum ( Prunus mume ) freeze-dried powder was prepared by grinding and freeze-drying, excluding the seeds.

Experimental Example 2: Hydrolysis using protein hydrolytic enzyme

2-1. Proteolytic enzyme preparation

A proteolytic enzyme was prepared to hydrolyze freeze-dried plum powder. The proteolytic enzymes used were flavourzyme, neutrase, protamex, and alcalase, and their application temperature and pH are shown in Table 1 below.

Types Enzymes Temperature
(℃) Applied Temperature
(℃) pH Applied pH Protease Flavorzyme 50 40 5.0-7.0 7 Neutrase 45 6 Protamex 30 - 65 6.0 - 9.0 Alcalase 60 60 6.5 - 8.5

2-2. Production of hydrolysates using protein hydrolytic enzymes

The four types of proteolytic enzymes listed in Table 1 were added to the freeze-dried powder of plum to prepare plum hydrolyzate. The hydrolysis method is as shown in Figure 1.

Figure 1 shows a method of hydrolysis of freeze-dried plum powder. First, 30 ml of DW was added to 100 mg of plum freeze-dried powder, and then 0.1 M NaOH was used to adjust the application pH of each proteolytic enzyme listed in Table 1. Afterwards, 1 mg of enzyme was added, and the mixture was shaken in an incubator for 1 hour, 3 hours, 6 hours, 12 hours, and 24 hours while reacting at the temperature at which each hydrolytic enzyme reacts. Flavozyme, neutrase, and protamex were reacted at a hydrolysis application temperature of 40°C, and alcalase was reacted at 60°C. Afterwards, the enzyme reaction was inactivated at 100°C for 30 minutes to stop hydrolysis. After centrifugation, the supernatant excluding the residue was filtered using filter paper to obtain four types of hydrolysates, which were freeze-dried and powdered.

2-3. Comparison of hydrolyzate yield using proteolytic enzymes

Figure 2 compares the yield of hydrolyzates using four types of proteolytic enzymes with samples hydrolyzed without proteolytic enzymes. Samples were hydrolyzed at 40°C by adding water (DW) and flavourzyme, neutrase, and protamex to the freeze-dried plum powder, respectively, and water (DW) to the freeze-dried plum powder. Samples in which DW) and alcalase were added and hydrolyzed at 60 ℃, and samples in which water (DW) was added to plum freeze-dried powder without hydrolyzing enzyme and hydrolysis was performed at 40 ℃ or 60 ℃. represents.

As a result, as shown in Figure 2, the yield of the hydrolyzate using protein hydrolysis was similar to that of the sample hydrolyzed only with water (DW) without treatment with proteolytic enzyme.

2-4. Sugar content measurement

Figure 3 shows the results of comparing the sugar content after hydrolysis for each proteolytic enzyme. Sugar content was analyzed using the phenol-sulfuric acid colorimetric method of Dubois et al. (Anal. Chem, 1954, 28(3): 350-356.). As shown in Figure 3, among proteolytic enzymes, when hydrolyzed with alcalase, compared to samples hydrolyzed with flavourzyme, neutrase, and protamex. The polysaccharide concentration (sugar content) increased after 10 to 12 hours, and the polysaccharide concentration (sugar content) also increased compared to the sample hydrolyzed with water only without hydrolytic enzyme.

Experimental Example 3: Comparison of hydrolysis yield and sugar content using sugar hydrolyzing enzyme

3-1. Sugar hydrolytic enzyme preparation

A sugar hydrolytic enzyme was prepared to hydrolyze freeze-dried plum powder. The sugar hydrolyzing enzymes used were Celluclast, Viscozyme, Amylogucosidase (AMG), Termamyl, and Ultraflo, and their application temperature and The pH is shown in Table 2 below.

Types Enzymes Temperature
(℃) Applied Temperature
(℃) pH Applied pH Glucosidase Celluclast 40 40 4.8 5 Viscozyme 40-50 3.3 - 3.5 Amylogucosidase (AMG) 60 60 4.3 Termamyl 60 5.6 Ultraflo 45-70 7 7

3-2. Production of hydrolysates using sugar hydrolyzing enzymes

Hydrolysis was performed in the same manner as in Experimental Example 2-2 using the five types of sugar hydrolyzing enzymes listed in Table 2, and five types of sugar hydrolysates were obtained.

Celluclast and Viscozyme were reacted at a hydrolysis application temperature of 40°C, and for Amylogucosidase (AMG), Termamyl, and Ultraflo, the reaction was performed at 60°C. The reaction was carried out at ℃. Afterwards, the enzyme reaction was inactivated at 100°C for 30 minutes to stop hydrolysis. After centrifugation, the supernatant excluding the residue was filtered using filter paper to obtain five types of hydrolysates, which were freeze-dried and powdered.

3-3. Comparison of hydrolyzate yield using sugar hydrolyzing enzymes

Figure 4 compares the yield of hydrolyzates using five types of sugar hydrolyzing enzymes with samples hydrolyzed without sugar hydrolyzing enzymes. Samples were hydrolyzed at 40°C by adding water (DW), Celluclast, and Viscozyme to freeze-dried plum powder, respectively, and water (DW) and amyloglucosid to freeze-dried plum powder. Amylogucosidase (AMG), Termamyl, and Ultraflo were added to the sample and hydrolyzed at 60°C, and water (DW) was added to the freeze-dried plum powder without hydrolyzing enzyme and heated to 40°C or 60°C. shows a sample that underwent hydrolysis reaction.

As a result, as shown in Figure 4, the yield of the hydrolyzate using the sugar hydrolytic enzyme was similar to that of the sample hydrolyzed only with water (DW) without treatment with the sugar hydrolytic enzyme, at approximately 80%.

3-4. Sugar content measurement

Figure 5 shows the results of comparing the sugar content after hydrolysis for each sugar hydrolyzing enzyme. As shown in Figure 5, compared to the sample hydrolyzed only with water (DW) without treatment with a sugar hydrolytic enzyme, when hydrolyzed with a sugar hydrolytic enzyme as a whole, the sugar content does not change significantly until 12 hours, but then decreases after 12 hours. It was confirmed that there was a significant increase in ~24 hours.

Experimental Example 4: Optimization of hydrolysis conditions for each enzyme using microwaves

4-1: Preparation of sugar and proteolytic enzymes

As the proteolytic enzyme, alcalase was adopted as the proteolytic enzyme with increased polysaccharide content in Figure 3. Sugar hydrolyzing enzymes Celluclast, Viscozyme, and Amylogucosidase (AMG) were prepared, and their application temperature and pH are shown in Table 3 below.

Hydrolysis was performed by setting the substrate (freeze-dried plum powder) and enzyme ratios to 10:1 and 100:1, the microwave power (W) to 200, 400, 600 W, and the time to 10, 30, 60, and 120 minutes. proceeded.

Enzyme Temperature(℃) pH Protease Alcalase 60 7 Glucosidase AMG 60 5 Viscozyme 40 Cellularst

4-2. Production of hydrolysates using protein hydrolytic enzymes

Specifically, plum hydrolyzate was prepared using the proteolytic enzyme alcalase listed in Table 3 on freeze-dried plum powder. The hydrolysis method is shown in Figure 6. Figure 6 shows a method of hydrolysis of freeze-dried plum powder using microwaves.

First, 2 ml of water (enzyme solution) dissolved in enzymes adjusted to the pH at which each enzyme acts as shown in Table 3 was added to 20 mg of freeze-dried plum powder. Afterwards, the reaction was performed by irradiating microwave power of 200, 400, and 600W for 10, 30, 60, and 120 minutes using a Microwave Assistant Rapid Enzyme Digestion System (MAREDS SPM0201000) equipment that can irradiate microwaves. After centrifugation for 3 minutes, the supernatant excluding the residue was filtered using filter paper, and the enzyme reaction was inactivated at 100°C for 30 minutes to stop hydrolysis.

Figure 7 shows the polysaccharide concentration (sugar content) of the hydrolyzate hydrolyzed by adding only water to freeze-dried plum powder and irradiating (applying) microwaves without hydrolytic enzyme treatment. As a result, as shown in Figure 7, when hydrolysis was performed by applying microwaves without treatment with hydrolytic enzymes, there was no significant difference in polysaccharide concentration depending on temperature and microwave power.

Figure 8 shows the polysaccharide concentration (sugar content) after adding alcalase, a proteolytic enzyme, to freeze-dried plum powder and hydrolyzing it by varying the microwave power and substrate (freeze-dried plum powder) and enzyme ratio. It represents. As a result, compared to hydrolysis using only water without a hydrolytic enzyme (see Figure 7), alcalase enzyme had no effect on the increase in polysaccharide content even when hydrolysis was performed by irradiating microwave power. .

4-3. Production of hydrolysates using sugar hydrolyzing enzymes

Plum hydrolyzate was prepared by adding three types of sugar hydrolyzing enzymes listed in Table 3 to freeze-dried plum powder. The hydrolysis method was performed using MAREDS SPM0201000 equipment to irradiate microwaves in the same manner as in Experimental Example 4-2, according to FIG. 6.

Figure 9 shows the polysaccharide concentration after adding amylogucosidase (AMG), a sugar hydrolyzing enzyme, to freeze-dried plum powder and hydrolyzing it by varying the microwave power and substrate (freeze-dried plum powder) and enzyme ratio. (sugar content).

Figure 10 shows the polysaccharide concentration (sugar content) after adding Viscozyme, a sugar hydrolyzing enzyme, to freeze-dried plum powder and hydrolyzing it by varying the microwave power and substrate (freeze-dried plum powder) and enzyme ratio. It is shown.

Figure 11 shows polysaccharide concentration (sugar content) after adding Celluclast, a sugar hydrolyzing enzyme, to freeze-dried plum powder and hydrolyzing it by varying the microwave power and substrate (freeze-dried plum powder) and enzyme ratio. It represents.

As a result, as shown in Figures 9 to 11, when hydrolyzed for 60 to 120 minutes at a microwave power of 400W at a ratio of substrate (freeze-dried plum powder) and glycolytic enzyme of 10:1 (weight ratio, w/w), Polysaccharide concentration (sugar content) was the highest.

Figure 12 shows the addition of Amylogucosidase (AMG), Viscozyme, and Celluclast, microwave power of 400W, substrate (lyophilized plum powder), and glycolytic enzyme ratio of 10:1 ( The polysaccharide concentration (sugar content) in the case of weight ratio (w/w) is compared and shown in one graph.

As a result, the polysaccharide concentration was the lowest when hydrolysis was performed with only water (DW) without sugar hydrolyzing enzyme, and Viscozyme was added, microwave power of 400W, substrate (freeze-dried plum powder), and glycolytic enzyme were added. When hydrolyzed for 60 minutes at a ratio of 10:1 (weight ratio, w/w), the polysaccharide concentration (sugar content) was highest at 3062.73 μg/mL.

Figure 13 shows the addition of Amylogucosidase (AMG), Viscozyme, and Celluclast, microwave power of 400W, substrate (lyophilized plum powder), and glycolytic enzyme ratio of 100:1 (weight ratio). , w/w), the polysaccharide concentration (sugar content) is compared in one graph.

As a result, the polysaccharide concentration was the lowest when hydrolysis was performed with only water (DW) without sugar hydrolyzing enzyme, and using Viscozyme, microwave power of 400W, substrate (lyophilized plum powder) and enzyme ratio of 100 :1 (weight ratio, w/w), the highest polysaccharide concentration (sugar content) was 1784.77 μg/mL when hydrolyzed for 60 minutes and 1973.18 μg/mL when hydrolyzed for 120 minutes. However, compared to Figure 12, hydrolysis was performed using Viscozyme under the conditions of 400W of microwave power and a substrate (freeze-dried plum powder) and glycolytic enzyme ratio of 10:1 (weight ratio, w/w). In this case, the sugar content was the highest.

9 to 13, polysaccharide concentration (sugar content) under the conditions of adding Viscozyme, microwave power of 400W, and substrate (freeze-dried plum powder) and glycolytic enzyme ratio of 10:1 (weight ratio, w/w) ) was confirmed to be the highest.

4-4. Production of hydrolyzate using sugar hydrolyzing enzyme under general conditions (comparative experiment)

Figure 14 shows the polysaccharide concentration (sugar content) when hydrolyzed according to the general conditions shown in Figure 1 without using microwaves.

As a result, as a result of hydrolysis for 48 hours using Amylogucosidase (AMG), Viscozyme, and Celluclast, the polysaccharide concentration (sugar content) was about 1500-2000 μg/ It was mL. Therefore, in the case of hydrolysis using microwaves, the polysaccharide content was high and the optimal hydrolysis efficiency was shortened to less than 2 hours, showing that using microwaves was an efficient hydrolysis method.

Figure 15 shows the polysaccharide concentration after adding Viscozyme, a sugar hydrolyzing enzyme, and hydrolyzing with different ratios of the substrate (freeze-dried plum powder) and the sugar degrading enzyme under microwave power conditions of 400 W, and after hydrolyzing under general conditions. Polysaccharide concentration is compared in one graph.

As a result, when Viscozyme was added and hydrolyzed for 60 minutes under the conditions of microwave power of 400W and substrate (freeze-dried plum powder) and enzyme ratio of 10:1 (weight ratio, w/w), the substrate was 3062.73 μg/mL. The polysaccharide concentration was significantly higher than in the case of a per-enzyme ratio of 100:1 (weight ratio, w/w), and also significantly higher than in the case of hydrolysis for 48 hours under general conditions without using microwaves.

Figure 16 shows the polysaccharide concentration and general conditions after addition of amylogucosidase (AMG), a sugar hydrolyzing enzyme, and hydrolysis by varying the ratio of the substrate (lyophilized plum powder) and the glycolytic enzyme under microwave power conditions of 400W. The polysaccharide concentration after hydrolysis is compared in one graph.

As a result, when amylogucosidase (AMG) was added and hydrolyzed for 120 minutes under conditions of 400 W of microwave power and a substrate (freeze-dried plum powder) and enzyme ratio of 10:1 (weight ratio, w/w), the substrate The polysaccharide concentration was significantly higher compared to the condition of (freeze-dried plum powder) and enzyme ratio of 100:1 (weight ratio, w/w), and also, compared to the case of hydrolysis for 48 hours under general conditions without using microwaves, the polysaccharide concentration was significantly higher. It was significantly high.

Experimental Example 5: Comparison of hydrolysis degree of hydrolytic enzymes using microwaves

Next, the sugar content was measured using amylogucosidase (AMG) and Viscozyme to compare the degree of hydrolysis. Figure 17 compares the degree of hydrolysis of amylogucosidase (AMG) and Viscozyme. In order to measure the degree of hydrolysis, the inactivated hydrolyzate was additionally subjected to ultrafiltration as shown in FIG. 6 (A ₁ ⁾ , and the degree of hydrolysis (DH) was measured.

The degree of hydrolysis was compared using the formula below.

A ₁ = Carbohydrates contents in sample after ultra-filtration (30,000 MWCO) of REDS

A ₀ = Carbohydrates contents in sample after ultra-filtration (30,000 MWCO) of no REDS

B = carbohydrate content of sample after REDS

DH(%) = A ₁ - A ₀ /B * 100

As a result, as shown in Figure 17, the degree of hydrolysis was significantly higher than when hydrolyzed using amylogucosidase (AMG) and compared to when hydrolyzed using Viscozyme.

Figure 18 shows the addition of Viscozyme and amylogucosidase (AMG) under conditions of a substrate (lyophilized plum powder) and glycolytic enzyme ratio of 10:1 (weight ratio, w/w) in a microwave of 400 W. After hydrolysis, the xylose concentration (μM) was measured.

As a result, the content of xylose produced when hydrolyzed using Viscozyme was significantly higher than when hydrolyzed with amylogucosidase (AMG). Therefore, from the above results, when Viscozyme is added and hydrolyzed at a microwave power of 400W under conditions of a substrate (freeze-dried plum powder) and a glycolytic enzyme ratio of 10:1 (weight ratio, w/w), It was found that maximum loss (μM) could be generated.

In the case of xylose, it is a precursor of hydroxymethylfurfural (HMF), and at approximately pH 5.0, xylose is converted to hydroxymethylfurfural. In addition, 5-hydroxymethylfurfural is 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl) acetal (5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl) acetal, hereinafter referred to as “FA- It is a precursor of "1"). In the case of plums, the pH is approximately 5.0 and xylose is automatically converted to hydroxymethylfurfural. Additionally, as more hydroxymethylfurfural is produced, the production of FA-1 also increases.

Hereinafter, we attempted to develop a method to convert the sugar containing xylose produced by hydrolysis by adding viscozyme and applying microwaves to hydroxymethylfurfural, and ultimately to produce a large amount of FA-1.

Experimental Example 6: FA-1 production according to high temperature and high pressure treatment

Previously, the present inventors added n-hexane to the plum extract extracted by the existing plum extract processing method (adding sugar and heating the extract aged at a low temperature of 4°C for more than a week) to separate the hexane fraction. After adding chloroform, 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal (“FA-1”) was separated from the chloroform fraction (PMC, Prunus mume chloroform fraction) obtained.

The top of Figure 19 shows the results of high-performance liquid chromatography (HPLC) using the chloroform fraction of plum extract extracted by the existing plum extract processing method. As a result, as confirmed at the top of Figure 19, a peak in which FA-1 was produced was confirmed in the chloroform fraction of the plum (PMC, Prunus mume chloroform fraction).

The HPLC reaction conditions are as follows.

The bottom of Figure 19 shows the hydrolyzate hydrolyzed by adding Viscozyme and irradiating 400 W of microwaves under the conditions of a substrate (lyophilized plum powder) and sugar decomposing enzyme ratio of 10:1 (weight ratio, w/w). This is the result of high-temperature and high-pressure treatment for 15 minutes at a high temperature of ℃ and high pressure of 0.01 MPA, and then analyzed by high-performance liquid chromatography (HPLC).

As a result, it was confirmed that at the bottom of Figure 19, the same peak corresponding to FA-1 as confirmed at the top of Figure 19 appeared. Therefore, through the above results, it was found that FA-1 can be produced by treating the hydrolyzate hydrolyzed using Viscozyme and microwaves at high temperature and pressure.

Experimental Example 7: Verification of FA-1 production according to presence/number of high-temperature and high-pressure treatments

Next, the presence or absence of FA-1 was compared depending on the presence or absence of high temperature and high pressure treatment. Figure 20 shows the presence or absence of high-temperature and high-pressure treatment after adding Viscozyme and hydrolyzing 400W of microwaves under the condition of a substrate (lyophilized plum powder) and glycolytic enzyme ratio of 10:1 (weight ratio, w/w). This is a comparison of FA-1 production. As a result, in the case of a sample (PMDV HTHP0) that was hydrolyzed by applying microwaves and did not undergo high temperature and high pressure treatment, FA-1 was not produced from the hydrolyzate of Viscozyme. However, in the case of a sample (PMDV HTHP1) that was subjected to high temperature and high pressure treatment once at a high temperature of 120°C and high pressure of 0.01 MPA for 15 minutes after hydrolysis, FA-1 was produced. In addition, in the case of a sample (PMDV HTHP2) that was subjected to high temperature and high pressure treatment twice for 15 minutes at a high temperature of 120°C and a high pressure of 0.01 MPA, it was confirmed through high performance liquid chromatography that the amount of FA-1 produced was significantly increased.

Next, through LC-MS analysis, it was confirmed whether the peak generated in Figure 20 corresponds to FA-1. Figure 21 shows the results of confirming the production of FA-1 using LC-MS. According to the inventor's previous research results, it was confirmed that FA-1 has a molecular formula of C ₁₈ H ₁₆ O ₈ and a molecular weight of approximately 378.1182. As a result of LC-MS analysis, as confirmed in Figure 21, when a mixture of plum and sugar hydrolase is hydrolyzed by irradiating microwaves and then treated at high temperature and pressure, FA-1 with a molecular weight of about 378.1182 is produced. It was confirmed that it was done.

Experimental Example 8: Verification of FA-1 production according to high temperature and high pressure treatment time

Next, the amount of FA-1 produced at different high temperature and high pressure treatment times was compared using HPLC.

The HPLC reaction conditions are as follows.

Figure 22 shows the high-temperature and high-pressure treatment time after adding Viscozyme and hydrolyzing irradiation with 400 W of microwaves under the condition of substrate (lyophilized plum powder) and glycolytic enzyme ratio of 10:1 (weight ratio, w/w). FA-1 production was compared using high-performance liquid chromatography at different times of 15, 30, and 60 minutes.

As a result, as shown in Figure 22, it was confirmed that FA-1 was produced in the case of a sample (PMDV HTHP15) treated at high temperature and high pressure for 15 minutes at a high temperature of 120°C and high pressure of 0.01 MPA after hydrolysis. In addition, it was confirmed that the amount of FA-1 produced in the sample (PMDV HTHP30) in which the high-temperature and high-pressure treatment time was increased to 30 minutes was increased compared to the case of high-temperature and high-pressure treatment for 15 minutes. In addition, it was confirmed that the production of FA-1 increased the most in the sample (PMDV HTHP60) in which the high temperature and pressure treatment time was increased to 60 minutes.

However, plum freeze-dried powder (PM), a sample in which plum freeze-dried powder was hydrolyzed using Viscozyme only (PMDV), was not hydrolyzed using Viscozyme, and was treated at a high temperature of 120°C and high pressure of 0.01 MPA. In the case of the sample (PM HTHP15) that was only processed, it was confirmed through high-performance liquid chromatography that FA-1 was not produced.

In this way, it was found that FA-1 was produced when a mixture of plum and sugar hydrolyzing enzyme was hydrolyzed using microwaves and subjected to high temperature and high pressure treatment.

Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims are also possible. falls within the scope of rights.

Claims (10)

Generating a hydrolyzate of plum by irradiating a mixture containing plum and sugar hydrolyzing enzyme with microwaves; and
5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl) in plums, comprising the step of treating the hydrolyzate at a high temperature of 100 to 200 ° C. and a high pressure of 0.005 to 0.3 MPA for more than 1 minute )How to produce acetal. According to paragraph 1,
The sugar hydrolyzing enzyme is selected from the group consisting of Celluclast, Viscozyme, Amylogucosidase (AMG), Termamyl, and Ultraflo. , Method for producing 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal from plum. According to paragraph 1,
A method of producing 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal from plum, wherein the sugar hydrolyzing enzyme has a pH of 4.5 to 5.5. According to paragraph 1,
A method for producing 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal from plum, wherein the plum and sugar hydrolyzing enzyme are mixed at a weight ratio of 5:1 to 5000:1. According to paragraph 1,
A method of producing 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal from plum, wherein the plum is freeze-dried powder of plum with the seeds removed. According to paragraph 1,
A method for producing 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal from plum, wherein the microwave is irradiated with a power of 10 to 800 W for 5 to 1440 minutes. According to paragraph 1,
The high-temperature and high-pressure treatment step is performed at a high temperature of 100 to 200 ° C. and a high pressure of 0.005 to 0.3 MPA for 1 to 160 minutes. 5-Hydroxymethyl-2-furaldehyde bis (5-formyl) in plums Method for producing furfuryl)acetal. According to paragraph 1,
The high-temperature and high-pressure treatment step is repeated 2 to 6 times at a high temperature of 100 to 200 ° C. and a high pressure of 0.005 to 0.3 MPA. 5-Hydroxymethyl-2-furaldehyde bis (5-formyl) in plums Method for producing furfuryl)acetal. According to paragraph 1,
A method for producing 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal from plum, wherein the sugar content in the hydrolyzate increases. According to paragraph 1,
A method for producing 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal from plum, wherein the sugar is xylose.