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Biosynthesis of Oligomeric Anthocyanins from Grape Skin Extracts

We synthesized oligomeric anthocyanins from grape skin-derived monomeric anthocyanins such as anthocyanidin and proanthocyanidin by a fermentation technique using Aspergillus niger, crude enzymes and glucosidase. The biosyntheses of the oligomeric anthocyanins carried out by the conventional method using Aspergillus niger and crude enzymes were confirmed by ESI-MS. The molecular weight of the synthesized anthocyanin oligomers was determined using MALDI-MS. The yield of anthocyanin oligomers using crude enzymes was higher than that of the synthesis using Aspergillus fermentation. Several studies have been demonstrated that oligomeric anthocyanins have higher antioxidant activity than monomeric anthocyanins. Fermentation-based synthesis of oligomeric anthocyanins is an alternative way of producing useful anthocyanins that could support the food industry.


Introduction
Anthocyanins are naturally occurring water-soluble plant pigments belonging to the group of phytochemicals known as flavonoids [1]. Anthocyanins are present in many plants which display colorful flowers, and different kinds of fruits and vegetables [2][3][4]. The quality and nutritional value of fruits and their products is commonly associated with the color that is derived from anthocyanins [5,6]. Anthocyanins are very useful for the food industry, due to their good water solubility and safety. They have been recognized internationally for their applications, including the replacement of synthetic colorants [7,8]. Anthocyanins have antioxidant activity which contributes to many biological activities such as anticancer, cardiovascular protection, ocular protection and protection against some other chronic diseases [9][10][11][12]. Several studies have been demonstrated that the oligomeric derivatives of anthocyanin have higher activity than the monomeric versions. For example, the anthocyanin oligomers derived from bilberry fruit such as small anthocyanidin glycoside polymers, particularly in the form of dimers, trimers, tetramers and pentamers have higher antioxidant activity than the monomers. These compounds are highly hydro-and liposoluble in nature and are not known to accumulate in the human body [13].
The biosynthesis of oligomeric anthocyanins is the best alternative to overcome the problem of deficiency. At present, studies on the synthesis of anthocyanin oligomers are scarce, and only one related paper is available [13]. Aspergillus species such as Aspergillus niger, A. sojae and A. oryzae have long been used for the production of traditional fermented foods such as doenjang, cheonggukjang, soy sauce and sake in Asian countries [14,15]. Fungi are rich sources of citric acid [16], C 8 volatiles [14] and many enzymes such as xylanase, cellulose [17], amyloglucosidase and exopolygalacturonase [18]. However, in the industrial applications of these fungi overcoming their contamination is a big challenge. The present study focuses on the synthesis of oligomeric anthocyanins by fermentation of monomeric anthocyanins with Aspergillus niger, as well as with crude enzymes derived from the fungus.

Results and Discussion
The oligomeric anthocyanins were successfully synthesized by fermentation using Aspergillus niger ( Figure 1) as well as crude enzyme ( Figure 2) as confirmed by ESI-MS (Figures 3 and 4). The oligomeric anthocyanins showed higher peak values and higher molecular weight than the monomeric anthocyanins. The higher peak value might be attributed to the presence of higher amount of oligomeric anthocyanins under similar experimental conditions [19]. It was confirmed that the yield of oligomeric anthocyanins derived from the fermentation by crude enzyme was better than that derived from the fermentation with Aspergillus niger (Table 1). We have previously reported the synthesis and characterization of anthocyanin oligomers produced by A. niger fermentation using anthocyanin monomers as substrate [13]. The molecular weight of the anthocyanin oligomers was determined using Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) [20]. In this study, the biosynthesis of oligomeric anthocyanins was detected using the relative absorbance values of compounds estimated by ESI-MS. ESI-MS is an important technique to detect femtomole quantities of sample, including non-volatile and thermally labile biomolecules that are difficult to analysis by other conventional techniques [21]. Liu et al. [22] detected the monomers, dimers, tetramers and hexamers of purified oligomeric proanthocyanins using ESI-MS. Therefore, the present study also used ESI-MS to analyze the various structures of the oligomeric proanthocyanins.
The monomeric anthocyanins such as anthocyanidin and proanthocyanidin give peaks at 288 m/z ( Figure 3A) and 381 m/z ( Figure 4A), respectively. The oligomeric anthocyanins synthesized from anthocyanidin monomers showed the peak values of m/z 905, 1193 ( Figure 3B). Similarly, the oligomeric anthocyanins synthesized from the other monomer (proanthocyanidin) showed the following highest peak values: m/z 903, 1191, 1479 ( Figure 4B). Therefore, the differentiation of peak values between before and after fermentation confirmed the synthesis of oligomeric anthocyanins using crude enzyme as well as fermentation with Aspergillus niger. The amount of oligomeric anthocyanin synthesized from fermented crude enzyme was higher than that synthesized from fermentation with Aspergillus niger.       Table 2). According our literature search, some of the carbohydrate hydrolases mentioned in Table 2 were found to produce condensation reactions [23]. After the synthesis (Figure 8), the content of anthocyanin was determined based on the presence of glucosidase in the product ( Figure 9). The results demonstrate the similarity of pattern between Figures 3 and 4. Figure 9 indicates a difference in molecular weight of m/z 288 for each oligomeric anthocyanin peak, which corresponds to the molecular weight of cyanidin. Consequently, the structure of anthocyanins was presumed to be as shown in Figure 10.
Comparing the yield of the oligomeric anthocyanins, it was seen that the highest yield was obtained using crude enzyme (Table 1), but crude enzymes are difficult to obtain. Therefore, it was thought that it might be more economical to use a commercially available glucosidase. In addition, as seen in Figures 4B and 9B, the oligomeric anthocyanins synthesized with glucosidase showed higher oligomer content than oligomeric anthocyanin synthesized with crude enzyme.   Table 2). According our literature search, some of the carbohydrate hydrolases mentioned in Table 2 were found to produce condensation reactions [23]. After the synthesis (Figure 8), the content of anthocyanin was determined based on the presence of glucosidase in the product ( Figure 9). The results demonstrate the similarity of pattern between Figures 3 and 4. Figure 9 indicates a difference in molecular weight of m/z 288 for each oligomeric anthocyanin peak, which corresponds to the molecular weight of cyanidin. Consequently, the structure of anthocyanins was presumed to be as shown in Figure 10.
Comparing the yield of the oligomeric anthocyanins, it was seen that the highest yield was obtained using crude enzyme (Table 1), but crude enzymes are difficult to obtain. Therefore, it was thought that it might be more economical to use a commercially available glucosidase. In addition, as seen in Figures 4B and 9B, the oligomeric anthocyanins synthesized with glucosidase showed higher oligomer content than oligomeric anthocyanin synthesized with crude enzyme.  Table 2). According our literature search, some of the carbohydrate hydrolases mentioned in Table 2 were found to produce condensation reactions [23]. After the synthesis (Figure 8), the content of anthocyanin was determined based on the presence of glucosidase in the product (Figure 9). The results demonstrate the similarity of pattern between Figures 3 and 4. Figure 9 indicates a difference in molecular weight of m/z 288 for each oligomeric anthocyanin peak, which corresponds to the molecular weight of cyanidin. Consequently, the structure of anthocyanins was presumed to be as shown in Figure 10.
Comparing the yield of the oligomeric anthocyanins, it was seen that the highest yield was obtained using crude enzyme (Table 1), but crude enzymes are difficult to obtain. Therefore, it was thought that it might be more economical to use a commercially available glucosidase. In addition, as seen in Figures 4B and 9B, the oligomeric anthocyanins synthesized with glucosidase showed higher oligomer content than oligomeric anthocyanin synthesized with crude enzyme.           The comparison of monomeric and oligomeric anthocyanins using HPLC with UV detection confirmed that the fractions have different patterns (Figure 11). Different patterns were fractionated and analyzed by LC/MS. In the second fraction, a single substance showing a molecular weight of m/z 429, 871 was identified at 7 min ( Figure 12). It was assumed that the compound with molecular weight m/z 871 was the dimer and that with m/z 429 was the monomer. This assumption was correlated to the results obtained by ESI-MS for the m/z 871 peak ( Figure 13A). Based on the results of Figure 13, the m/z 429 compound consisted of anthocyanidin (m/z 310) and glucose. The m/z 871 peak is the dimeric form of the m/z 429 species. An NMR study is needed for better understanding of these molecular structures. The comparison of monomeric and oligomeric anthocyanins using HPLC with UV detection confirmed that the fractions have different patterns (Figure 11). Different patterns were fractionated and analyzed by LC/MS. In the second fraction, a single substance showing a molecular weight of m/z 429, 871 was identified at 7 min ( Figure 12). It was assumed that the compound with molecular weight m/z 871 was the dimer and that with m/z 429 was the monomer. This assumption was correlated to the results obtained by ESI-MS for the m/z 871 peak ( Figure 13A). Based on the results of Figure 13, the m/z 429 compound consisted of anthocyanidin (m/z 310) and glucose. The m/z 871 peak is the dimeric form of the m/z 429 species. An NMR study is needed for better understanding of these molecular structures. The comparison of monomeric and oligomeric anthocyanins using HPLC with UV detection confirmed that the fractions have different patterns (Figure 11). Different patterns were fractionated and analyzed by LC/MS. In the second fraction, a single substance showing a molecular weight of m/z 429, 871 was identified at 7 min ( Figure 12). It was assumed that the compound with molecular weight m/z 871 was the dimer and that with m/z 429 was the monomer. This assumption was correlated to the results obtained by ESI-MS for the m/z 871 peak ( Figure 13A). Based on the results of Figure 13, the m/z 429 compound consisted of anthocyanidin (m/z 310) and glucose. The m/z 871 peak is the dimeric form of the m/z 429 species. An NMR study is needed for better understanding of these molecular structures.    In summary, the biosynthesis of oligomeric anthocyanins using fermentation is an alternative approach to overcome the problem of their natural scarcity to avoid the overexploitation of natural resources.

Materials
5-Dimethyl-1-pyrroline-N-oxide (DMPO), FeSO4, and H2O2 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). KH2PO4, KCl and NaCl were purchased from Junsei (Tokyo, Japan). Saccharose, dextrose, urea, MgSO4, MnSO4, and ZnSO4 were purchased from Deajung (Siheung, Korea). Peptone In summary, the biosynthesis of oligomeric anthocyanins using fermentation is an alternative approach to overcome the problem of their natural scarcity to avoid the overexploitation of natural resources.  In summary, the biosynthesis of oligomeric anthocyanins using fermentation is an alternative approach to overcome the problem of their natural scarcity to avoid the overexploitation of natural resources. Peptone G was purchased from Acumedia (Lansing, MI, USA). The grape skin-derived anthocyanins were purchased from Kitolife Co. Ltd. (Pyeongtaek, Korea).

Synthesis of Oligomeric Anthocyanin by Fermentation Using Aspergillus niger
Synthesis of oligomeric anthocyanins using Aspergillus niger was described by Lee et al. [13]. In this study, monomeric anthocyanins such as anthocyanindins and proanthocyanidins were used to synthesize oligomeric anthocyanins. The monomeric anthocyanin powders were fermented with Aspergillus niger at 25 • C in a shaking incubator for 5 days. The fermented cultures were centrifuged at 3000 rpm, 4 • C, for 20 min. The supernatants were filtered with Whatman No. 41 filter paper and the filtrate was freeze-dried by a freeze drier system (SFDSM06, Samwon, Busan, Korea) in order to obtain the synthesized oligomeric anthocyanins. The concentrations of fermented oligomeric anthocyanin produced by fermentation were estimated using Electrospray Ionization-Mass Spectrometry (ESI-MS) at the Korea Basic Science Institute (KBSI, Ochang, Korea). The molecular mass values of the compounds were analyzed by a Synapt G2 HDMS quadrupole time-of-flight (TOF) mass spectrometer equipped with an electrospray ion source (Waters, Milford, MA, UK) in positive ion mode at a spray voltage of 2.5 kV. MS spectra were obtained with the capillary heated to 150 • C. The instrument was calibrated using NaF solution. The sample was dissolved in 100% MeOH and introduced by direct infusion at a flow rate of 20 µL/min into the ion source operating in positive mode. All spectra were acquired at a range of 50 to 2500 m/z. Leucine enkephalin was used as the lock mass for the exact mass measurement correction.

Separation of Fermented Crude Enzyme from the Culture of Aspergillus niger
The fungal strain Aspergillus niger was cultured in 100 mL saccharose medium or potato dextrose agar (PDA) medium and incubated at 25 • C, for 7 days in an shaking incubator. The Aspergillus niger culture medium was centrifuged at 3000 rpm, 4 • C, for 20 min. The supernatant was precipitated with an equal volume of acetone at 4 • C, overnight (10-12 h) and this mixture was then centrifuged at 3000 rpm, 4 • C for 20 min. After removal of the supernatant, the pellet was dissolved using 5 mL of distilled water and further centrifuged at 13,000 rpm, 4 • C, for 5 min. The supernatant (crude enzyme) was freeze-dried in order to synthesize the oligomeric anthocyanin.

Synthesis of Oligomeric Anthocyanin Using Crude Enzyme
The anthocyanin powder was fermented with crude enzyme at 25 • C in a shaking incubator for 7 days. The fermented stuff was centrifuged at 4 • C, 3000 rpm for 20 min. The supernatant was filtered with Whatman No. 41 filter paper and the filtrate was freeze-dried in a SFDSM06 freeze drier system in order to obtain the synthesized oligomeric anthocyanins. The concentration of oligomeric anthocyanin was examined by ESI-MS at KBSI.
3.6. Analysis of Crude Enzyme from the Culture of Aspergillus niger SDS-PAGE gel slicing was used for LC-MS/MS analysis of the secretory proteins from Aspergillus niger. The soluble proteins in urea lysis buffer containing 8 M urea and 4% CHAPS after the acetone precipitation of the secretory fraction from Aspergillus niger was subjected to 6-15% SDS-PAGE and stained with colloidal Coomassie solution. The lanes, which are cut as nine slices, were excised from the gel in three of the protein lanes for the mass spectrometry experiments by destaining and in-gel digestion followed by peptide extraction. The tryptic peptides obtained from each gel slice were analyzed by LC-MS/MS running on the Q-STAR Pulsar ESI-hybrid Q-TOF instrument.

Synthesis of Oligomeric Anthocyanin Using Various Enzymes
The anthocyanin powder was fermented with various enzymes at 25 • C in a shaking incubator for 7 days. The fermented product was centrifuged at 4 • C, 3000 rpm for 20 min. The supernatant was filtered with Whatman No. 41 filter paper and the filtrate was freeze-dried by a freeze dry system (SFDSM06) in order to obtain the synthesized oligomeric anthocyanins The concentration of oligomeric anthocyanin was examined by ESI-MS at KBSI.

Isolation of Oligomeric Anthocyanin and Analysis
The monomeric and oligomeric anthocyanins were further isolated using reversed-phase HPLC (RP-HPLC) on a C 18 column (4.0 × 250 mm) with a linear gradient of MeOH (0-60%) at a flow rate of 1.0 mL/min. The eluted peaks were detected at 272 nm. The collected samples were pooled and concentrated using a rotary evaporator, then lyophilized for 3 days. The lyophilized sample was further analyzed by LC/MS followed by ESI-MS at KBSI.

Statistical Analysis
The statistical analyses was carried out by the paired t-test (p < 0.05) and comparisons made between monomeric anthocyanins and oligomeric anthocyanins. The data are presented as mean ± SD. All analyses were performed using the SPSS software (SPSS Institute, Chicago, IL, USA).

Conclusions
Our results indicate that the synthesis of oligomeric anthocyanins using glucosidase from A. niger is better than that possible with fermentation of A. niger. Synthesis of oligomeric anthocyanins was confirmed by ESI-MS and HPLC analysis. The present study successfully overcome the problem of fungal contamination during synthesis of oligomeric anthocyanins. Further studies are however required to assess the biological activities of the produced oligomeric anthocyanins.