Method for preparing anthocyanin oligomers by using coenzyme derived from <i>Aspergillus </i>sp. strain

Abstract

The present invention relates to a method of preparing an anthocyanin oligomer using a coenzyme derived from an Aspergillus sp. strain, and more particularly to a method of preparing an anthocyanin oligomer by fermenting an anthocyanin monomer with a coenzyme of Aspergillus niger, which is a kind of Aspergillus sp. strain. According to the present invention, in order to overcome contamination problems during the culturing process using Aspergillus niger, a coenzyme of Aspergillus niger is extracted and the fermentation process is performed using the same, whereby an anthocyanin oligomer characterized by reduced concern of contamination and superior radical-scavenging effects, compared to existing anthocyanin monomers, can be produced. Also, an anthocyanin oligomer, obtained through fermentation using glucosidase as an enzyme contained in the coenzyme, can exhibit excellent fermentation efficiency and radical-scavenging ability, and polymerization of the anthocyanin oligomer can be confirmed even upon the fermentation of the enzyme including glucosidase.

Claims

1. A method of preparing an anthocyanin oligomer, comprising: (i) mixing an anthocyanin monomer and distilled water at a mass ratio of 1:8 to 1:15 to prepare an anthocyanin monomer solution; (ii) mixing the anthocyanin monomer solution and a multi-enzyme complex comprising arabanase, cellulase, -glucanase, hemicellulase, and xylanase, the multi-enzyme complex being available under the trade name Viscozyme L, at a mass ratio of 500:1; (iii) fermenting the solution prepared in step (ii) at a temperature of 15 C. to 30 C. for 5 days to 10 days; and (iv) filtrating the fermented solution using filter paper to obtain the anthocyanin oligomer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a process of preparing an anthocyanin oligomer by fermenting an anthocyanin monomer with an Aspergillus niger culture media in Example 1 of the present invention;

(2) FIG. 2 is a graph showing the results of ESI mass spectrometry of an anthocyanin monomer serving as a control in Example 1 of the present invention;

(3) FIG. 3 is a graph showing whether synthesis of an anthocyanin oligomer occurred in Example 1 of the present invention through ESI mass spectrometry;

(4) FIG. 4 is a graph showing the hydroxyl radical-scavenging activity of the anthocyanin oligomer synthesized by fermenting the anthocyanin monomer with an Aspergillus niger culture media in Example 1 of the present invention, depending on the concentration;

(5) FIG. 5 shows a process of obtaining a coenzyme from an Aspergillus niger culture media in Example 2 of the present invention;

(6) FIG. 6 shows a process of preparing an anthocyanin oligomer by fermenting an anthocyanin monomer with the coenzyme obtained from Aspergillus niger in Example 2 of the present invention;

(7) FIG. 7 is a graph showing the results of ESI mass spectrometry of an anthocyanin monomer serving as a control in Example 2 of the present invention;

(8) FIG. 8 is a graph showing whether synthesis of an anthocyanin oligomer occurred in Example 2 of the present invention through ESI mass spectrometry;

(9) FIG. 9 is an SDS-PAGE image showing the expression of an Aspergillus niger coenzyme protein depending on the culturing period in Example 2 of the present invention;

(10) FIG. 10 is an SDS-PAGE image for analysis of the enzyme contained in the Aspergillus niger coenzyme in Example 2 of the present invention;

(11) FIG. 11 shows a process of preparing an anthocyanin oligomer by fermenting an anthocyanin monomer with glucosidase, which is an enzyme obtained from Aspergillus niger, and with the multi-enzyme complex containing a wide range of carbohydrases (Viscozyme L) in Example 3 of the present invention;

(12) FIG. 12 shows the results of ESI mass spectrometry of an anthocyanin monomer serving as a control in Example 3 of the present invention;

(13) FIG. 13 is a graph showing whether synthesis of an anthocyanin oligomer occurred when using glucosidase as an enzyme in Example 3 of the present invention through ESI mass spectrometry;

(14) FIG. 14 is a graph showing whether synthesis of an anthocyanin oligomer occurred when using the multi-enzyme complex containing a wide range of carbohydrases (Viscozyme L) as an enzyme in Example 3 of the present invention through ESI mass spectrometry; and

(15) FIG. 15 is a graph showing the hydroxyl radical-scavenging activity of two kinds of anthocyanin oligomers prepared through fermentation with glucosidase, which is an enzyme obtained from Aspergillus niger, and with the multi-enzyme complex containing a wide range of carbohydrases (Viscozyme L) in Example 3 of the present invention, depending on the concentration.

DETAILED DESCRIPTION

(16) Hereinafter, a detailed description will be given of the present invention.

(17) The present invention pertains to a method of preparing an anthocyanin oligomer, comprising (1) isolating a water-soluble coenzyme from a culture media of an Aspergillus sp. strain and (2) fermenting an anthocyanin monomer with the coenzyme isolated in step (1).

(18) The Aspergillus sp. strain in step (1) is preferably Aspergillus niger.

(19) The strain in step (1) is preferably cultured at a temperature of 15 to 30 C. for 4 to 8 days, and more preferably at 25 C. for 5 days.

(20) The coenzyme in step (1) is preferably isolated as an enzyme by adding the culture media with an organic solvent to give a precipitate and dissolving the precipitate in distilled water.

(21) The fermenting in step (2) is preferably includes mixing the anthocyanin monomer and distilled water at a mass ratio of 1:8 to 1:15 to prepare an anthocyanin monomer solution, after which mixing the anthocyanin monomer solution and the coenzyme isolated in step (1) at a substrate-to-enzyme mass ratio of 40:1 to 60:1. The substrate is an anthocyanin monomer. More preferably, the anthocyanin monomer and distilled water are mixed at a mass ratio of 1:10 to prepare an anthocyanin monomer solution, after which the anthocyanin monomer solution and the coenzyme isolated in step (1) are mixed at a mass ratio of 50:1.

(22) The fermenting in step (2) is preferably at a temperature of 15 to 30 C. for 5 to 10 days, and more preferably at 25 C. for 7 days. The amount of the anthocyanin oligomer that is synthesized is increased up to 7 days, but does not change further even over time under conditions after 8 days.

(23) The coenzyme isolated in step (1) contains glucosidase as an active ingredient.

(24) In addition, the present invention pertains to a method of preparing an anthocyanin oligomer, comprising fermenting an anthocyanin monomer by adding the anthocyanin monomer with a coenzyme, which is present in a culture media of an Aspergillus sp. strain and contains glucosidase as an active ingredient, and with the multi-enzyme complex containing a wide range of carbohydrases (Viscozyme L).

(25) A better understanding of the present invention will be given through the following examples, which are merely set forth to illustrate the present invention but are not to be construed as limiting the scope of the present invention, as will be apparent to those skilled in the art.

Example 1. Evaluation of Ability of Aspergillus Sp. Strain Culture Media to Synthesize Oligomer

(26) As summarized in FIG. 1, an anthocyanin monomer and distilled water were mixed at a mass ratio of 1:10 to give an anthocyanin monomer solution. Then, the anthocyanin monomer solution and an Aspergillus niger strain culture media were mixed at a mass ratio of 95:5 and fermented at 25 C. for 5 days. The strain culture media was made by culturing an Aspergillus niger strain in 1 L of a medium solution at 25 C. for 5 days.

(27) After the fermentation, a filtration process was performed using filter paper, whereby materials other than anthocyanin, such as the strain and the like, were filtered and thus the anthocyanin oligomer was isolated and lyophilized, thereby obtaining an anthocyanin oligomer. In order to purify the anthocyanin oligomer, filtration is preferably conducted using a tubular, capillary, coiled spiral, or plane membrane.

(28) An anthocyanin monomer serving as a control was subjected to peak observation through ESI mass spectrometry. As shown in FIG. 2, the peak near the molecular weight of 300 was very high. However, based on the results of peak observation of the obtained anthocyanin oligomer through ESI mass spectrometry, as shown in FIG. 3, a high peak was observed near a molecular weight of 600, and peaks were also observed near 900 and 1200. This means that the anthocyanin monomer was fermented and thus converted into an anthocyanin oligomer, such as a dimer, a trimer, a tetramer, etc., from which the anthocyanin oligomer can be found to be synthesized.

(29) In order to compare the efficacy of the anthocyanin monomer with that of the anthocyanin oligomer, as shown in FIG. 4, hydroxyl radical-scavenging activity was tested using the monomer and the oligomer at different concentrations. Based on the test results, the inhibitory concentration (IC.sub.50) of the oligomer was only about half that of the monomer, from which the anthocyanin oligomer can be found to exhibit radical-scavenging activity even at a low concentration.

Example 2. Evaluation of Ability of Coenzyme Obtained from Aspergillus sp. Strain Culture Media to Synthesize Oligomer

(30) As summarized in FIG. 5, in order to obtain a coenzyme from an Aspergillus niger strain culture media, an Aspergillus niger strain was cultured in 2 L of a medium solution at 25 C. for 5 days, thus affording a culture media, after which the strain was removed through filtration using filter paper and precipitation was performed at 4 C. for 8 to 12 hr by the addition of acetone. Thereafter, centrifugation was performed at 3000 rpm for 20 min to give a culture precipitate, and the enzyme of the culture precipitate, which was dissolved in deionized water, was isolated and lyophilized, whereby the coenzyme was prepared.

(31) Next, as summarized in FIG. 6, the anthocyanin monomer and distilled water were mixed at a mass ratio of 1:10 to give an anthocyanin monomer solution. Furthermore, the anthocyanin monomer solution and the coenzyme were mixed at a mass ratio of 500:1 and fermented at 25 C. for 5 days.

(32) After the fermentation, a filtration process was performed using filter paper, whereby materials other than anthocyanin were filtered and thus the anthocyanin oligomer was isolated and lyophilized, thereby obtaining an anthocyanin oligomer. In order to purify the anthocyanin oligomer, filtration is preferably conducted using a tubular, capillary, coiled spiral, or plane membrane.

(33) An anthocyanin monomer serving as a control was subjected to peak observation through ESI mass spectrometry. The results are shown in FIG. 7. Based on the results of peak observation of the obtained anthocyanin oligomer through ESI mass spectrometry, as shown in FIG. 8, high peaks were observed near the molecular weights of 600, 900 and 1200 compared to the results shown in FIG. 7. This means that the anthocyanin monomer was fermented and thus converted into an anthocyanin oligomer, such as a dimer, a trimer, a tetramer, etc., from which the anthocyanin oligomer can be found to be synthesized.

(34) In order to investigate the properties of the isolated coenzyme for synthesizing an anthocyanin oligomer and the culture conditions thereof, SDS-PAGE was performed. The results are shown in FIG. 9. Based on the results of SDS-PAGE for the amount of extracted coenzyme upon culturing for 4 to 8 days, the amount of the coenzyme that was extracted was similar even over time under conditions of 6 to 8 days. Thus, in order to prepare the coenzyme necessary to synthesize an anthocyanin oligomer, culturing Aspergillus niger for 5 days was found to be optimal.

(35) As indicated by the rectangle in FIG. 10, nine thin fragments were obtained, digested with trypsin protease, and analyzed by LC-MS/MS in a Q-STAR Pulsar ESI-hybrid Q-TOF instrument. As results thereof, tens of proteins were validated and MS/MS spectrum peaks thereof were analyzed with Analyst QS (v1.1, Applied Biosystems) to identify proteins. The identified proteins are shown in the following Tables. Aspergillus niger is currently receiving attention as industrially useful model fungi, and these fungi are known to secrete hydrolytic proteins which are very suitable for the production of various food additives, pharmaceuticals and industrial enzymes. Among the identified proteins of Tables 1 and 2 below, some proteins expected to be involved in anthocyanin oligomer metabolism were selected and represented as italic types.

(36) TABLE-US-00001 TABLE 1 Molecular Gene I.D. Protein name Probability Weight gi|224027 glucoamylase G1 627 65448 gi|134081727 unnamed protein product [Aspergillus niger] 274 75190 gi|765328 acid phosphatase, orthophosphoric monoester 265 64211 phosphohydrolase, APase {EC 3.1.3.2} [Aspergillus ficuum, NRRL 3135, Peptide, 583 aa] gi|257187 alpha-glucosidase P2 subunit, ANP P2 subunit {EC 181 79656 3.2.1.20} [Aspergillus niger, Peptide, 719 aa] gi|2344 preproglucoamylase G2 [Aspergillus niger] 531 56695 gi|145242978 hypothetical protein ANI_l_1546094 [Aspergillus 351 59208 niger CBS 513.88] gi|145231236 phospholipase C PLC-C [Aspergillus niger CBS 410 49652 513.88] gi|145235505 serine carboxypeptidase [Aspergillus niger CBS 297 62560 513.88] gi|145252338 phosphatidylglycerol specific phospholipase 261 53895 [Aspergillus niger CBS 513.88] gi|4185610 phytase [Aspergillus niger] 218 50997 gi|145241119 3-phytase B [Aspergillus niger CBS 513.88] 256 52453 gi|145241490 1,3-beta-glucanosyltransferase gel3 [Aspergillus 161 56721 niger CBS 513.88] gi|83655609 acid phosphatase [Aspergillus niger] 142 52725 gi|145242970 hypothetical protein ANl_l_1540094 [Aspergillus 128 45753 niger CBS 513.88] gi|145256696 protein ecm33 [Aspergillus niger CBS 513.88] 125 41026 gi|317026828 serine-type carboxypeptidase F [Aspergillus niger 118 57756 CBS 513.88] gi|145248273 polyamine oxidase [Aspergillus niger CBS 513.88] 110 58728 gi|145248205 aspartic-type endopeptidase opsB [Aspergillus 104 50958 niger CBS 513.88] gi|145234270 glutaminase GtaA [Aspergillus niger CBS 513.88] 99 75470 gi|350633205 hypothetical protein ASPNIDRAFT_55058 87 22487 [Aspergillus niger ATCC 1015] gi|350631594 hypothetical protein ASPNIDRAFT_53033 63 57162 [Aspergillus niger ATCC 1015] gi|145235707 FAD binding domain protein [Aspergillus niger 59 61292 CBS 513.88] gi|145233743 alpha-galactosidase B [Aspergillus niger CBS 392 48796 513.88] gi|317031802 histidine acid phosphatase [Aspergillus niger CBS 153 53047 513.88]

(37) TABLE-US-00002 TABLE 2 gi|317025164 aspartic endopeptidase (AP1) [Aspergillus niger 483 46701 CBS 513.88] gi|145242664 sulphydryl oxidase [Aspergillus niger CBS 513.88] 264 43471 gi|74626383 RecName: Full = Probable alpha-galactosidase B; 175 48753 AltName: Full = Melibiase B; Flags: Precursor gi|134083538 unnamed protein product [Aspergillus niger] 173 45226 gi|400801 RecName: Full = Pectin lyase A; Short = PLA; 135 39830 AltName: Full = Pectin lyase II; Short = PLII; Flags: Precursor gi|145235303 hypothetical protein ANI_1_496034 [Aspergillus 103 52301 niger CBS 513.88) gi|134055991 unnamed protein product [Aspergillus niger] 85 41620 gi|134076313 unnamed protein product [Aspergillus niger] 85 45581 gi|145251519 phosphoglycerate mutase family protein 79 19282 [Aspergillus niger CBS 513.88] gi|350633205 hypothetical protein ASPNIDRAFT_55058 73 22487 [Aspergillus niger ATCC 1015] gi|145232359 endopolygalacturonase C [Aspergillus niger CBS 241 37796 513.88] gi|145235523 glucan endo-1,3-beta-glucosidase eglC [Aspergillus 129 46778 niger CBS 513.88] gi|145230419 glycosidase crf1 [Aspergillus niger CBS 513.88] 107 39862 gi|129935 RecName: Full-Endopolygalacturonase II; 89 37489 Short = EPG-II; AltName: Full = Pectinase 2; AltName: Full = Polygalacturonase II; Short = PG-II; AltName: Full = Polygalacturonase X2; Flags: Precursor gi|133176 RecName: Full = Ribonuclease M; Short = RNase M 89 26590 gi|134055750 unnamed protein product [Aspergillus niger] 84 27072 gi|145229151 endo-1,3(4)-beta-glucanase [Aspergillus niger CBS 83 46311 513.88] gi|134075575 hypothetical protein An07g00170 [Aspergillus 69 90993 niger] gi|134083538 unnamed protein product [Aspergillus niger] 67 45226 gi|145252266 GPI anchored cell wall protein [Aspergillus niger 64 19022 CBS 513.88] gi|83638302 xylanase [Aspergillus phoenicis] 117 10944 gi|350633205 hypothetical protein ASPNIDRAFT_55058 92 22487 [Aspergillus nigerATCC 1015]

Example 3. Evaluation of Ability of Glucosidase in Coenzyme Obtained from Aspergillus sp. Strain Culture Media and of the Multi-Enzyme Complex Containing a Wide Range of Carbohydrases (Viscozyme L) to Synthesize Oligomer

(38) As summarized in FIG. 11, an anthocyanin monomer and distilled water were mixed at a mass ratio of 1:10 to give an anthocyanin monomer solution. Then, the anthocyanin monomer solution was mixed with each of glucosidase, which is an enzyme isolated from the Aspergillus niger strain coenzyme, and the multi-enzyme complex containing a wide range of carbohydrases (Viscozyme L), at a mass ratio of 1000:1 and 500:1, respectively (100:1 of FIG. 11 is the mass ratio of the anthocyanin monomer itself, rather than the solution, to the enzyme, and the multi-enzyme complex containing a wide range of carbohydrases (Viscozyme L) was used in double the amount in order to ensure similar effects due to the high enzyme efficiency of glucosidase), and fermented at 25 C. for 5 days.

(39) The multi-enzyme complex containing a wide range of carbohydrases (Viscozyme L) is an enzyme that includes glucosidase and is commercially available.

(40) After the fermentation, a filtration process was performed using filter paper, whereby materials other than anthocyanin were filtered and thus the anthocyanin oligomer was isolated and lyophilized, thereby obtaining an anthocyanin oligomer. In order to purify the anthocyanin oligomer, filtration is preferably conducted using a tubular, capillary, coiled spiral, or plane membrane.

(41) An anthocyanin monomer serving as a control was subjected to peak observation through ESI mass spectrometry. The results are shown in FIG. 12. However, based on the results of peak observation through ESI mass spectrometry of the anthocyanin oligomers obtained using glucosidase, which is an enzyme isolated from the Aspergillus niger strain coenzyme, and the multi-enzyme complex containing a wide range of carbohydrases (Viscozyme L), as shown in FIGS. 13 and 14, high peaks were observed near molecular weights of 600, 900 and 1200 compared to the results shown in FIG. 12. This means that the anthocyanin monomer was fermented and thus converted into an anthocyanin oligomer, such as a dimer, a trimer, a tetramer, etc., from which the anthocyanin oligomer can be found to be synthesized. Moreover, the amount of the synthesized oligomer was high when using glucosidase as the enzyme (FIG. 13) compared to when using the multi-enzyme complex containing a wide range of carbohydrases (Viscozyme L) (FIG. 14).

(42) In order to compare the efficacies of the anthocyanin oligomers obtained using individual enzymes, as shown in FIG. 15, the oligomer obtained using glucosidase and the oligomer obtained using the multi-enzyme complex containing a wide range of carbohydrases (Viscozyme L) were set to different concentrations and tested for hydroxyl radical-scavenging activity. Based on the test results, the oligomer obtained using glucosidase exhibited an inhibitory concentration (IC.sub.50) of 0.217 mg/ml, which is much lower than 0.278 mg/ml, which is the inhibitory concentration (IC.sub.50) of the oligomer obtained using the multi-enzyme complex containing a wide range of carbohydrases (Viscozyme L), and was thus concluded to exhibit radical-scavenging activity even at a low concentration.

(43) Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. Accordingly, the actual scope of the present invention will be defined by the appended claims and equivalents thereof.