마이로시네이즈 활성에 따른 십자화과 채소의 설포라판 함량 변화와 기능적 특성에 관한 연구

Abstract

Commercially available myrosinase refining enzyme (thioglucosidase, CAT number T4528) costs more than 4,000 Korean won, which makes it impossible to apply the method to producing vegetable juices, beverages, etc. (the definition of 1 unit: the amount of enzyme produced by the decomposition of 1.0 u㏖ from sinigrin under a condition of pH 6.0 at 25 ℃). Therefore, the method to refine the myrosinase needs developing in an economical way. In the cruciferous plant such as broccoli, kohlabi, red beet, and cabbage, there is a glucoraphanin (GR) of the glucosinolate group in the form of a glycoside synthesized with sugar. It is hydrolyzed into sugar and non-sugar and is produced with SFN of the group of isothiocyanate, which is reported to have a possibility of possessing functional properties such as antioxidant and anti-cancer. However, when SFN is produced by the myrosinase enzyme, it brings about taking a negative effect in obtaining SFN by the way in which nucleophile (most proteins, peptides or amino acids) in the plant forms the sulforaphane conjugate with SFN. In order to address the problem, and develop a method to obtain sulforaphane from a cruciferous plant with high yield. In order to prove the function of sulforaphane extracted at a high rate of efficiency, DPPH assay was conducted to evaluate the antioxidant activity which eliminates free radicals that cause aging. In addition, by evaluating live cells, the MTT assay, a test for evaluating changes in cell proliferation or apoptosis was conducted and oral health tests, which aim to inhibit the growth of oral microbes causing bad breath, were conducted and effects on anti-cancer, antioxidant and anti tooth decay were validated. Studies comprised experiments for three topics and the results obtained in the study are summarized as follows:

Experiment 1. The Optimization of extracting myrosinase deriving from the cruciferous plants

This study is mainly concerned with a method to refine myrosinase enzyme from a cruciferous plant; more in detail, the research was conducted to economically refine myrosinase by taking advantage of then ammonium sulfate (AS) method from a cruciferous plant. The results obtained in the study are summarized as follows:

1) A method to refine myrosinase by using the ammonium sulfate method (AS), not column type high refinement, has been developed from broccoli, a cruciferous vegetable. After precipitating and removing the protein by treating 50∼60 % (w/v) AS of cruciferous vegetables, crude protein was precipitated and fractionated including myrosinase. When myrosinase enzyme activity was measured after obtaining the crude protein fraction, and when crude protein from Sri Lanka mustard seed was applied with glucoraphanin (GR) of 66.6 ㎎ per ℓ, 44.02 % of GR was converted to sulforaphane (SFN) and the result showed about higher activity of 2.6 % compared with myrosinase purchased from Sigma-Aldrich (the column type refinement method).

2) Compared with the column type refinement method, the enzyme titer of myrosinase was proved to be excellent. In order to confirm the advantage the two-tier AS refinement method has over one-tier refinement, refinement yields of the control group of 55-80 %, which used 55 % of AS in the first step, and in the second stage, used 80 % of AS were compared with those of 80 % of AS in its counterpart. SFN content of broccoli treated with 55-80 % compared with that of broccoli treated with 80 % of AS was shown to be twice higher. It was found that from this result, the two-tier AS processing method was much higher than one-tier AS treatment method.

3) The refinement and activation of myrosinase was measured in accordance with the inactivation of ESP (epithiospecifier proteins). First, by treating 55 % (w/v) of AS on pulverized mustard seeds, and precipitating and removing the protein, AS of 80 % (w/v) was treated and crude protein containing myrosinase was obtained by precipitating crude protein. In the following process, the crude protein fraction containing myrosinase obtained to inactivate ESP protein was dissolved by the buffer and heat-treated at 60 ℃ for 10 minutes (the control group). On the control group, at this stage, heat was not treated to inactivate ESP protein. When undergoing inactivation the produced content of SFN was 4,200 ㎎/dry ㎏, which showed a higher rate of 55 % compared with the SFN content of 2,700 ㎎/dry kg of the content in the subject group. From this result it draws a conclusion that the activation can enhance the enzyme activity.

4) Myrosinase refinement and activity were measured from oiled mustard seeds. Based on the judgment that in the course refining myrosinase into a water-soluble substance, removing fat soluble ingredients in the refinement process would obtain a highly dense concentration of myrosinase, it was refined after oiling mustard seeds by using the oil extractor (the oil presso oil extractor manufactured by Liquid), the enzymatic assay confirmed enzymatic activation. The result showed that while the use of the oiled mustard seeds in 30 minutes of measurement showed the activation of 39.63 units/㎎, the use of the un-oiled mustard seeds in 30 minutes of measurement showed the activation of 29.33 units/㎎. Therefore, it is estimated that oiling the seeds produces the higher activation of enzymes.

5) Change in the activation of myrosianse enzymes depending on the treatment of cell wall degrading enzymes was confirmed. The mustard seeds treated with Viscozyme® L (Cellulase, multi enzyme), Pectinex® Ultra Pulp (Pectin lyase), Fungamyl® 800L (Fungal alpha amylase) produced by Novozyme company by 0.5 % for each were used. In the case of refined mustard seeds without adding cell wall disintegrating enzymes, the activation of the enzymes was measured 87.67 units/㎎ after measuring for thirty minutes. In contrast, in the case of the sample of refined mustard seeds after adding cell disintegrating enzymes, the activation of the enzymes was 103.92 units/㎎ after thirty minutes of measurement. From these results more myrosinase with a higher degree of enzyme activation when using refined mustard seeds after adding cell wall disintegrating enzymes than not using them at all.

Experiment 2. The Optimization of the enzyme treatment condition for the extraction of high content sulforaphane

This research is concerned with the method of producing a high content sulforaphane (SFN); more specifically, it was conducted on the method of producing a high yield of SFN from a cruciferous plant by preventing SFN from being compounded by treating phenyl isothiocyanate (PITC), the results are as follow:

1) In order to establish the concentration conditions of the enzyme myrosinase, myrosinases extracted from four mustard seeds (from USA, India, Sri Lank and Canada) on the same condition were mixed by the same ratio and were treated with broccoli crushing solution depending on concentrations. For two hours the treatment of the enzyme was conducted at 60 ℃ after myrosinase mixed with broccoli grinding solution was added at a sequence of 1 %, 5 %, 10 % and then myrosinase manufactured by Sigma-Aldrich Company was added 5 %. Less SFN content was detected. Despite adding myrosinase from Sigma-Aldrich, the fact that it has less SFN than the negative control group showed the result in which there are a variety of proteins and as higher the concentration of refined myrosinase is, the more the protein content increased and it decreased by mixing with SFN.

2) A broccoli sprout known to have a higher SFN content than broccoli was obtained. It was crushed under the same conditions (broccoli: water=1:4), all of which showed substantial difference from broccoli. The broccoli sprout with the highest content was 3,800 mg/kg, which was 63 times higher than broccoli. When myrosinase extracted from mustard seeds and broccoli was added, the higher result was shown, and when adding mustard seeds, the content of SFN increased by 3.2 times. In addition, it showed that myrosinase derived from mustard seeds and broccoli can be activated and myrosinase derived from mustard seeds in comparison with that from broccoli was more effective. As mentioned earlier, ESP (epithiospecifier protein) prevents the action of myrosinase and converts GR into sulforaphane nitrile. After heat treatment of broccoli sprouts at 100 ℃ for 20 minutes, in order to remove ESP the treatment of heat was conducted at 60 ℃ for ten minutes. An experiment was conducted to reduce the action of ESP and maximize the content of SFN. The treatment of heat inactivated ESP, which led to an increase in the size of content but did not show much difference; therefore, PITC treatment was conducted.

3) In order to conduct the experiment, after treating PITC of 100 g/ℓ in the pulverized solution obtained by crushing broccoli heat treated at 100 ℃ for 20 minutes, and after centrifugation was conducted for two hours at a rate of 10,000 rpm, a supernatant was obtained. After treating myrosinase with 5 % of the obtained supernatant, the reaction was induced at 40 ℃ for two hours, and the amount of sulforaphane present in the reaction solution sample was measured. Meanwhile, in the case of the control group, after myrosinase was treated by 5 % at the broccoli crushed liquid, the sample treated at 40 ℃ for two hours, after treating PITC at the broccoli crushed liquid, was used. As a result of the experiment to compare the amount of production depending on the treatment of PITC, it was confirmed that the content of SFN improved by 5.7 % compared with that of the control sample, which did not treat PITC.

4) By treating PITC and eliminating unnecessary proteins pure SFN was obtained and experiments were conducted to establish the temperature of enzyme treatment and the pH condition. Enzyme is the main component of protein and the rate of activity and reaction vary depending on the concentration of hydrogen ions. In order to establish the optimal pH condition, pH was adjusted with acetic acid and NaOH. As a result of checking the safety depending on the temperature of SFN and pH, it showed a peak of activity at 40 ℃ and pH was best at pH 4-5; however, the acidity was too low and in the following experiments, the supernatant treated with PITC was used at pH 5.5.

Experiment 3. Experiment on functionality and evaluation of broccoli enzyme treatment solution and fermentation liquid

This study crushed traditional fermented foods of Jeju Province, smeared them on MRS solid medium in which lactic acid bacteria and gram positive bacteria were selectively cultivated, and cultivated separated 1000 individuals; then, each was fermented individually and DPPH analysis, a test method for antioxidant and MTT analysis, and a test method for cancer cell death were conducted. Microorganisms which exhibited a radical scavenging activity (antioxidant capacity) with an absorbance rate of 0.15 or less were selected based on the criteria for native fermentation microorganisms and among them, the fermentation of broccoli pulverized liquid was conducted to the top 25 microorganisms. The results obtained in the study are summarized as follows:

1) As a result of measuring the antioxidant capacity, it was shown that the antioxidant capacity was about 3 times higher than negative control group, which indicated that a large number and a large amount of antioxidant active substance exist in fermented broccoli solution. The measurement of the antioxidant capacity of the enzyme solution and the fermented broth showed the negative control group showed an antioxidant capacity of 4.4 %, when diluted with untreated broccoli solution. The enzyme solution. compared with negative control group, showed 17.6 %, four times more, and the fermentation broth was 20.5 %, which amounted to 4.7 times higher antioxidant capacity.

2) The test was conducted by using macrophage RAW 264.7 cells, and in the case of the macrophage cells, which are related to immunity, the test was conducted to select strains toxic to the cells through the macrophage cells. It was confirmed that when MTT assay was conducted by diluting unfermented broccoli four times, other microorganisms were shown to increase more than 3 times other than Weissella cibaria, and in the case of enzyme treated solution, it was shown that after diluted four times, it showed more than potent toxity against more than 95 % of macrophages. In addition, all broccoli treated with enzymes showed the inhibition of the growth of the macrophage cells more than 10 times more. The results of liver toxicity test of broccoli enzyme solution showed 27.6 % of cell growth inhibition when diluted 4 times with broccoli solution. Compared with the negative control group, the enzyme solution was 63.9 %, which amounted to 2.3 times higher and the fermentation broth was 70.7 %, which amounted to 2.6 times higher.

3) Based on the judgment that adding 1/20, 1/50, 1/100 of fermentation broth in order to check oral health was thought too low in its concentration, the rate of concentration increased to 1/2, 1/4. Though positive results were obtained about the dilution of bacteria, more tests were conducted to dilute it further based on the result of 108. Cavity bacteria are Streptococcus mutans and the results of the inhibition capacity of cavity bacteria by broccoli fermentation broth showed that the negative control group showed 1.3×108 number of the bacteria with untreated broccoli solution and when they were dispensed into the medium of broccoli fermentation broth, the number decreased by 100 %. It was confirmed that adding fermentation solution has a much higher inhibition capacity than the unfermented broccoli solution of the negative control group.