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Microbial Production of Novel Pigments from Black Rice Anthocyanin
2014 Volume 20 Issue 5 Pages 1013-1016
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2014 Volume 20 Issue 5 Pages 1013-1016
We screened soil microorganisms with the potential to modify the structure of black rice anthocyanin. After culturing the microorganisms on PDA medium containing black rice anthocyanin, caffeic acid, p-hydroxybenzoic acid, ferulic acid, and malonic acid for 4 days at 30°C, one strain that changed the red medium to reddish purple was isolated. Anthocyanins present in the color-changed medium were extracted using 15% (w/v) acetic acid and their absorption spectra were then determined. The analysis revealed that the peak wavelength of the extracted anthocyanins was shifted to a shorter wavelength (500 nm) from that of black rice anthocyanin (520 nm). Further HPLC separation of the anthocyanins confirmed that four new species were present in the medium. Of the four pigments, two showed an absorption maximum at 500 nm. Analysis of their basic structure by acid hydrolysis showed that the two pigments had clearly different retention times from cyanidin, which is the main anthocyanidin in black rice, and were not malvidin or peonidin. A 16S rDNA homology search indicated that the isolated microorganism with the ability to modify the structure of black rice anthocyanin was an Enterobacter aerogenes strain.
Anthocyanins are a class of flavonoid-based compounds that appear as various shades of blue, purple, and red in the flowers and fruits of higher plants. Anthocyanins undergo structural changes in response to pH, temperature, and oxygen, among other factors, resulting in color changes. As a consequence of this property, various plant colors have arisen due to differences in the components and environmental factors, which lead to subtle changes in the color and tone of anthocyanins. Anthocyanins are derived from anthocyanidins, which comprise the basic structure, by the addition of an organic acid and sugar. Six types of anthocyanidins (cyanidin, delphinidin, malvidin, pelargonidin, petunidin and peonidin) are most commonly found in nature. Sugars such as glucose, galactose, and rhamnose are often bound to anthocyanidins through a β-glycosidic bond. Anthocyanins that have ester bonds with organic acids are called acylated anthocyanins and display increased stability. In contrast, anthocyanidins have low stability and rapidly fade as a result of degradation. Organic acids that bind to anthocyanins are classified as either aliphatic or aromatic. Among aliphatic organic acids, acetic acid and malonic acid are the most common, whereas the most frequently encountered aromatic acids are p-coumaric acid, ferulic acid, and caffeic acid. Although anthocyanins are easily discolored and susceptible to fading, their safety is higher than that of compounds used for artificial coloring. For this reason, anthocyanins are often used for the coloring of processed foods. Recently, anthocyanins were found to exhibit antioxidant activity (Tsuda, 2012) in addition to coloring activity. The anthocyanin of purple sweet potato was demonstrated to have antioxidant activity (Kano et al., 2005) and anti-mutagenic properties (Yoshimoto et al., 1999). In addition, Matsui et al. (2004) reported that caffeoylsophorose, a novel natural α-glucosidase inhibitor, was found in red vinegar produced from fermented purple sweet potato.
From the viewpoint of safety and security, anthocyanins are attractive compounds for use in food coloring. However, to obtain natural plant pigments, several steps including crop production, extraction, filtration, and purification are required, and the resulting yields vary depending on weather conditions during crop cultivation. For this reason, in vitro approaches for anthocyanin production have been investigated. For example, Nawa et al. (1993) reported the production of anthocyanin using cultured cells of rabbiteye blueberry. Using film culture vessels with Euphorbia millii cells, Hamade et al. (1994) produced anthocyanin, while more recently, Asano et al. (2002) reported the production of anthocyanin by habituated Nyoho strawberry cell cultures. However, despite these advances in in vitro anthocyanin production, the yields are too low for commercial applications.
To overcome this limitation, we speculated that if microorganisms capable of modifying anthocyanins could be identified, it may be possible to rapidly and inexpensively produce novel anthocyanins for use in food products. If we could produce novel anthocyanins in a short period by the reaction of black rice anthocyanin, microbial enzymes and ingredients for structural modification, a stable supply would be ensured independent of weather conditions. Moreover, we anticipate that the development of anthocyanins with higher antioxidant activity and thermal stability would lead to increasing demand for their use as a food additive.
In the present study, we screened soil samples for microorganisms capable of modifying the structure of anthocyanins from black rice. From the viewpoint of safety, we aimed for production using a microbial enzyme, specifically an extracellular enzyme. The aim of this study is to isolate novel anthocyanins that can be used in food applications.
Materials Caffeic acid, p-hydroxybenzoic acid, ferulic acid, and malonic acid were purchased from Nacalai Tesque Co., Ltd. (Kyoto, Japan). PDA (Potato Dextrose Agar) medium was purchased from Nissui Co., Ltd. (Tokyo, Japan). Anthocyanidins (cyanidin, peonidin and malvidin) were obtained from Funakosi Co., Ltd. (Tokyo, Japan). All other reagents were of the highest available grade. Black rice (Oryza sativa var. Indica cv. Shiun) was purchased from Kajiwara Beikoku Co., Ltd. (Kyoto, Japan).
Screening of microorganisms We screened soil microorganisms for strains capable of producing new types of anthocyanins. For this experiment, we used a medium containing phenolic compounds and anthocyanins extracted from black rice. To separate the bran fraction, unpolished black rice was ground in a rice milling machine (MK Seiko Co., Ltd., Nagano, Japan). To extract anthocyanins, 10.0 g of the bran fraction was added to 200 mL of 10% (w/v) acetic acid and the resulting mixture was allowed to sit at 15°C for 12 h in the dark. After filtration through gauze, the mixture was centrifuged (10 min, 3,000 rpm, 4°C), and the supernatant was filtered using No. 2 and No. 5 filter paper (ADVANTEC Toyo Co., Ltd., Tokyo, Japan).
The obtained filtrate (black rice anthocyanin) contained the following anthocyanins: cyanidin-3,5-diglucoside, cyanidin-3-sophoroside, cyanidin-3-glucoside, cyanidin-3-rutinoside, peonidin-3-glucoside, peonidin-3-rutinoside, and others at 7.1, 3.8, 55.8, 1.5, 16.4, 0.7, and 14.7%, respectively (Terahara et al., 1994). The obtained filtrate was added to the PDA medium, which was prepared by mixing 100 mL black rice anthocyanin, 3.9 g PDA medium, 0.01 g caffeic acid, 0.01 g malonic acid, 0.01 g ferulic acid, and 0.01 g p-hydroxybenzoic acid in an Erlenmeyer flask, then immersed in boiling water for 5 min. To maintain the structure of anthocyanins, sterilization of the medium by autoclaving was avoided. After cooling the prepared medium, it was poured into a petri dish on a clean bench. As a control, medium without added phenolic compounds was also prepared as above. Soil samples were collected from the soil of an athletic field at Sojo University. Next, the soil samples were suspended in deionized water and inoculated onto PDA medium containing black rice anthocyanin extract and four phenolic compounds. After incubation at 30°C, colonies were isolated from media exhibiting color changes. The isolated colonies were inoculated onto fresh PDA medium containing the black rice anthocyanin extract and four phenolic compounds. After incubation at 30°C, color changes were observed.
Absorption spectra The medium showing color changes was collected and added to a 15% (w/v) acetic acid solution. Pigment was extracted from the medium for 12 h at 5°C. After filtration of the extracted solution through filter paper (No. 5C; ADVANTEC Toyo), the obtained extract was analyzed using a U-3010 spectrophotometer (Hitachi, Tokyo, Japan).
Anthocyanin analysis Anthocyanins were analyzed using a high-performance liquid chromatograph (HPLC) (Shimadzu, Kyoto, Japan) equipped with a CTO-10AC column oven and SPD-10AV UV-VIS detector. Analytical HPLC was performed using an ODS-3 column (4.6 mm i.d. × 250 mm; GL Sciences, Inc., Tokyo, Japan) with solvents A (H3PO4 : H2O = 15 : 985) and B (H3PO4 : AcOH : CH3CN : H2O = 15 : 200 : 250 : 535) under gradient conditions at a flow rate of 1 mL min−1. Anthocyanin analysis was carried out using the following gradient conditions: 0 − 100 min, linear gradient from 35% to 55% of solvent B in solvent A; 100 − 140 min, linear gradient from 55% to 65% of solvent B in solvent A. Detection was performed at 520 nm. Column temperature was 35°C. Anthocyanin extract was diluted two-fold with solvent (solvent A : B = 65 : 35) and filtered through a disposable membrane filter unit DISMIC-13HP (0.2 µm; ADVANTEC, Tokyo, Japan).
Anthocyanidin analysis Anthocyanidin analysis of anthocyanins was performed according to the standard method (Terahara, 1993). Briefly, anthocyanin extracts were first hydrolyzed with 2 N hydrochloric acid for 45 min in a boiling water bath. A small amount of iso-amyl alcohol was then added and the reaction mixture was then shaken. The upper (iso-amyl alcohol) layer was then diluted two-fold with solvent (solvent A : B = 65 : 35), filtered through a disposable membrane filter unit DISMIC-13HP (0.2 µm; ADVANTEC), and then directly applied for anthocyanidin identification by HPLC.
Identification of microorganisms Identification of microorganisms was performed by Techno Suruga Laboratory Co., Ltd. (Shizuoka, Japan).
Screening of microorganisms During the screening of soil microorganisms, one plate changed to reddish purple from red. One strain from this plate was isolated and inoculated on medium containing black rice anthocyanin with and without phenolic components, respectively. After further culture, the strain that changed the red color of the medium supplemented with phenolic components to a reddish purple was isolated (Fig. 1). Because the medium changed to reddish purple, it was thought to contain novel pigments that were microbially formed from the black rice anthocyanin.
Effects of microorganisms on the color of black rice anthocyanin. PDA medium containing phenolic compounds changed from red to reddish purple (right side in left photo; +Phenolic compounds, +Microorganism). The color did not change in PDA medium without phenolic compounds (−Phenolic compounds, +Microorganism).
Analysis of pigments The reddish purple pigments were extracted from the medium using 15% (w/v) acetic acid. The absorption spectra of the extracted pigments revealed that the peak wavelength (500 nm) was shifted to a shorter wavelength from that of black rice anthocyanin (520 nm) (Fig. 2). The extracted pigments were further separated by HPLC, demonstrating that four new compounds were microbially formed from the black rice anthocyanin in the medium (Fig. 3). Of the four separated pigments, two pigments, corresponding to peaks 1 and 4, showed an absorption maximum at 500 nm (data not shown). On the other hand, peaks 2 and 3 showed an absorption maximum at 520 nm (data not shown). To analyze the basic structure of these two pigments (peaks 1 and 4), the separated pigments were hydrolyzed with hydrochloric acid. The retention times of the hydrolyzed pigments of peaks 1 and 4 were 7.20 and 12.52 min, respectively, which clearly differed from that of cyanidin (3.22 min), which is the main anthocyanidin found in black rice, as well as from malvidin (2.54 min) and peonidin (4.22 min). In this study, we did not analyze the basic structure of peaks 2 and 3. However, we presumed that the two pigments are novel anthocyanins produced from black rice pigments. The role of phenolic compounds on new anthocyanin production remains unknown. Further, it is also unclear why the absorption maximum of black rice anthocyanin changed to 500 nm from 520 nm. In this study, we aimed to improve the antioxidant activity and thermal stability of black rice anthocyanin by acylation with microbial enzymes. Microbiallyinduced changes in plate color did not occur without phenolic compounds. Although phenolic compounds in the black rice anthocyanin solution were not analyzed, the importance of the four phenolic compounds in pigment modification is suggested. Also, it was determined that the basic structure of the novel pigments differed from that of the black rice anthocyanin, cyanidin.
Absorption spectra of pigments extracted from PDA medium. The spectrum shown by the solid line in the left figure is from a compound extracted from PDA medium +Phenolic compounds, +Microorganism, corresponding to the right side of the left image of Figure 1. The dotted line in the left figure shows the spectrum of a compound extracted from PDA medium +Phenolic compounds, −Microorganism, corresponding to the left side of the left image of Figure 1. The solid line in the right figure shows the spectrum of a compound extracted from PDA medium −Phenolic compounds, +Microorganism, corresponding to the right side of the right image of Figure 1. The dotted line in the right figure shows the spectrum of a compound extracted from PDA medium −Phenolic compounds, −Microorganism, corresponding to the left side of the right image of Figure 1.
HPLC chromatograms of black rice anthocyanin (left) and black rice anthocyanin exposed to microorganisms (right). Four new peaks were detected as shown on the right (peaks 1–4). Two pigments, corresponding to peaks 1 and 4, showed an absorption maximum at 500 nm. Detection, 520 nm.
Identification of microorganisms The isolated microorganism was entrusted to Techno Suruga Lab for identification based on 16S rDNA sequencing. Results of homology searches revealed that the strain had the highest homology (99.8%) to Enterobacter aerogenes strains RW9516 and RW7M1 stored at the NITE Biological Resource Center (NBRC), Department of Biotechnology National Institute of Technology and Evaluation (Chiba, Japan).
In this study, we found that a microbial strain with high homology to E. aerogenes shifted the absorption peak and the basic structure of black rice anthocyanin. Based on this result, we speculate that the novel pigments were produced by microbial enzymes. It may be possible to rapidly and cost-effectively produce novel anthocyanins for use in various food applications.
To date, there have been no reports on the structural modification of anthocyanins by microorganisms. In order to improve the antioxidant activity and thermal stability of anthocyanins, we attempted to rapidly produce acylated anthocyanins by microbial enzymes. In this study, four novel pigments not included in black rice were confirmed. In two of these pigments, the structure of the aglycone differed from the black rice pigment, and exhibited a maximum absorption at 500 nm. In future, it is necessary to examine the antioxidant activity and thermal stability of novel pigments. However, the ability to structurally modify anthocyanins from black rice using the microorganism “E. aerogenes” is noteworthy.
