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LWT 41 (2008) 14121416 www.elsevier.com/locate/lwt

Composition and colour stability of anthocyanins extracted from fermented purple sweet potato culture
Gongjian Fan, Yongbin Han, Zhenxin Gu, Feirong Gu
College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, Peoples Republic of China Received 28 May 2007; received in revised form 2 September 2007; accepted 6 September 2007

Abstract The components and colour stability of anthocyanins in purple sweet potato were investigated. Anthocyanins were extracted from fermented purple sweet potato culture fermented by Suzhou wine starter (Rhizopus 3.851, 3.866 and Saccharomyces cerevisiae). In these purple sweet potato anthocyanins (PSPAs), ve major anthocyanins were detected by high performance liquid chromatography (HPLC). Cyanidin and peonidin were found to be the major anthocyanidins in PSPAs by acid hydrolysis of anthocyanins. PSPAs were more stable under the acid conditions (pH 2.04.0) than the subacid conditions (pH 5.06.0) as per UVVis absorption spectra and CIELAB colour coordinates. r 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
Keywords: Purple sweet potato; Anthocyanin; Fermentation; Composition; Colour stability

1. Introduction Public concern about the safety of synthetic colourants in food has led to increasing attention to natural colourants as alternatives. Anthocyanins, as a group of phenolic compounds widely existing in the plant kingdom, present a spectrum from orange to blue in colour in the natural world, satisfying consumers demand for food colours. Therefore, anthocyanins have been be used instead of synthetic pigments (Mazza & Miniati, 1993). They also possess known pharmacological properties and are used for therapeutic purpose (Rice-Evans & Packer, 1998; Smith, Marley, Seigler, Singletary, & Meline, 2000; Wang, Wang, Lin, Chu, Chou, & Tseng, 2000). Anthocyanins are normally obtained by extraction from plants. The extraction methods currently employed are to use methanol, ethanol, acetone, water or mixtures as solvents. However, the stability of these anthocyanins are easily affected by structural modications with hydroxyl, methoxyl, glycosyl, and especially acyl groups and by
Corresponding author. Tel.: +86 025 84396293; fax: +86 025 84396293. E-mail address: guzx@njau.edu.cn (Z. Gu).

environmental factors such as temperature and light (Francis, 1989; Wrolstad, 2000). Therefore, hydrochloric acid or formic acid is often added to the solvents to prevent degradation of the anthocyanins (Brigita, Mirko, & n, Jose , & Ana, 2006; Gonnet Alenka, 2005; Glenda, Julia & Fenet, 2000; Kjell & yvind, 2005). But in these extracts, there are more impurities such as amylose and proteins. These extraction methods also require relatively high cost due to use of various solvents and need for purifying the anthocyanins. A less expensive way to directly extract purer anthocyanins is therefore needed. The extraction of anthocyanins from fermented purple sweet potato culture was investigated by Japanese scientists (Kousuke, Hiroaki, Kinnosuke, & Teruyo, 1990). The purple sweet potato anthocyanins (PSPAs) were extracted from purple sweet potato mixed with rice with the help of rhizopus and yeast. The anthocyanins thus extracted are purer than those extracted by the chemical methods. However, the components of these anthocyanins and their stability were not mentioned in patent le. And there has been no report in this regard to our knowledge. Furthermore, the yeast during fermentation produces not only ethanol and CO2 from the sugar but also secondary metabolites such as higher alcohols, ethyl ethers, fatty acids

0023-6438/$34.00 r 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2007.09.003

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and acetates. These secondary metabolites might combine anthocyanins and form pyroanthocyanins which are more stable than anthocyanins (Pretorius, 2000). However, there was no mention of pyroanthocyanins detected in PSPAs fermented by the Japanese scientists (Kousuke et al., 1990), though it is reported that pyroanthocyanins could be detected in red wine (Morate, mez-Cordove s, Caldero n, & Sua rez, 2006). Purple sweet Go potato has enough starch and reducing sugars for rhizopus and yeast. We wondered if we can obtain pyroanthocyanins in the fermented PSPAs and if we could ferment only purple sweet potato with rhizopus and yeast to obtain PSPAs. This would be a more economical and more ideal extraction of PSPAs. In this research, PSPAs were extracted from fermented purple sweet potato culture which was fermented by Suzhou wine starter (Rhizopus 3.851, 3.866 and Saccharomyces cerevisiae). The components of the PSPAs were determined by reverse-phase HPLC. The stability of the PSPAs was examined at six different pH values between 2.0 and 7.0 and was measured both by UVvis absorption spectra and CIELAB colour coordinates. The objective of this study was to nd a useful way to extract purer PSPAs, and to determine their components and colour stability for their potential application as a natural food colourant. 2. Materials and methods 2.1. Materials Purple sweet potatoes (Ipomoea batatas cv. ZiA0) were harvested in Xuzhou, Jiangsu Province in 2005. These sweet potatoes were cleaned with tap water and stored at 20 1C before experiment. Cyanidin and peonidin were purchased from Sigma Chemical Co. Suzhou wine starter (Rhizopus 3.851, 3.866 and S. cerevisiae) was purchased from Suzhou Rice Factory, Jiangsu Province. 2.2. Extraction of PSPAs One kilogram of frozen purple sweet potatoes was steamed and mashed with 1 L of citric acid solution (10 g/ L). The purple sweet potato mash was inoculated with 4 g of Suzhou wine starter and was fermented at 2872 1C for 72 h. The fermented culture was centrifuged at 1776 g for 15 min. The supernatant was concentrated to 50 mL by the rotatory evaporator (45 1C) and stored at 4 1C in a refrigerator for later analysis. Another 1 kg of frozen purple sweet potatoes was steamed and mashed with 1 L of acid-ethanol (0.05N, HCl; 95% ethanol, the ratio 15:85, v/v), and then put in thermostatic water at 50 1C for 5 h. After centrifugation at 1766 g for 15 min, the supernatant was concentrated to 50 mL by the rotatory evaporator (45 1C) and stored at 4 1C in a refrigerator for later analysis

2.3. Acid hydrolysis of PSPAs One millilitre of concentrated PSPAs solution was dissolved in 10 mL of hydrochloric acid (1.0 mol/L) in a screw-cap test tube. The solution was hydrolyzed at 98 1C for 1 h, cooled in an ice bath (Luigia & Giuseppe, 2006)Please check the unlinked reference Luigia and Giuseppe (2006).4 and stored at 4 1C. 2.4. Quantitative analysis The anthocyanins were quantied following the spectrophotometric method proposed by Francis (1989). The concentration of anthocyanins was determined applying the LambertBeer law. The spectra recorded in a UV-2802 diode array spectrophotometer (UNIC, USA) were measured at 25 1C and 530 nm, against the solvent. For that purpose 10 mm quartz cells were used. Anthocyanins yield (g/mL) was calculated using the following equation: Total anthocyanins TA A530 dilution factor . 98:2

2.5. HPLC analysis of PSPAs PSPAs were separated by reverse-phase high performance liquid chromatography (HPLC) using Agilent 1100 (Agilent, USA) with a Prodigy C18 reversed-phase column (5 mm), 4.6 250 mm i.d. (Agilent, USA), and were detected at 520 nm using UVvis diode-array absorbance detection (DAD). The HPLC temperature was at 30 1C, the ow rate was 1 mL/min, and the injection volume was 20 mL. The mobile phase A consisted of 100% HPLC-grade acetonitrile, whereas mobile phase B was a mixture of 10% (v/v) acetic acid in distilled water. Separation of anthocyanins was carried out for 25 min. The elution prole was a linear gradient elution with 1025% solvent A from 0 to 15 min, 2530% from 15 to 25 min. Separation of anthocyanidins was carried out for 30 min. The elution prole was a linear gradient elution with 1040% solvent A from 0 to 30 min. 2.6. Analysis of PSPAs stability at different pH values Buffers at six different pH values (2.0, 3.0, 4.0, 5.0, 6.0, and 7.0) were prepared with Na2HPO3 (0.2 mol/L) and citric acid (0.2 mol/L). The pH values of the buffersolutions were measured by a Thermo Orion 868 pH-meter (Orion, USA) equipped with an Orion CHN 060 pH electrode (Orion, USA). Six concentrated PSPAs samples (1 mL each) were dissolved in different buffers in separate volumetric asks (10 mL) and made up to 10 mL. These solutions were then centrifuged at 1776 g for 15 min in order to obtain supernatant. Spectroscopic analyses were carried out by a UNIC-2802 spectrophotometer (Unic, USA), and the data were recorded by UNIC SB Spectroscopy Software (Version

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1.0). Colour intensity (CI520) index was obtained from the visible spectra as A520 (Real Decree No. 2107/1996). The colour quality of PSPAs was evaluated by colour density, colour tonality and degradation index (Bol var & Luis, zquez, Salinas, & Alonso, 2004; Prodanov, Dom nguez, Bla 2005). Colour density A420 A520 A620 , A420 , Colour tonality A530 A420 . Degradation index Almax Tristimulus parameters (L*, C* and H) were calculated using CR-S4w software (CR400, Konica Minolta, Japan), based on CIELAB (CIE, 1976) colour space, and with reference to standard observation angle of 101 visual eld and a standard illuminator D65 at Dl of 5 nm (Montes, Vicario, Raymundo, Fett, & Heredia, 2005). 2.7. Statistics All trials were carried out in triplicate and data were reported as mean7standard deviation (S.D., n 3). The statistical signicance was evaluated using Students t-test and set at po0.05. 3. Results and discussion 3.1. Quality and quantity of extracted purple sweet potato anthocyanins Two extractions were compared in the study (Table 1). Each 1 mL concentrated PSPAs was dissolved in separate volumetric asks (10 mL) and made up to 10 mL with acidethanol. The higher colour intensity was found in the chemistry extraction, but the lower degradation index was found in the fermentation extraction. Chemically, anthocyanin extracts are an intricate mixture of glycosylated polyhydroxy and polymethoxy avilium salts. However, some water-soluble composition such as organic acids, colourless polyphenol, amylose and soluble protein were also extracted in these extracts. This anthocyanin extract needs purifying until we can use it in food industry. PSPAs extracted by fermentation had a lower degradation index, which indicated that these extracts have little impurities and were more pure. In our research, we compared the anthocyanins yield of the two extractions. The results indicated that spectrophotometric method could not

represent the actual yield of anthocyanins extracts because of the impurities. 3.2. HPLC characterization of purple sweet potato anthocyanins Fig. 1 shows that 18 peaks appeared in the chromatograms of PSPAs, which were detected at 520 nm. From the chromatographic and UVvisible spectral features, peaks 1, 2, 3, 4 and 5 were accounting for 8.3%, 7.3%, 22.9%, 22.6% and 19.6%, respectively, of the total amount of all the anthocyanins, and they were eluted after 9.3, 12.1, 14.3, 14.5 and 15.6 min, respectively. An absorption peak appeared at 330 nm of the UVvis characteristic of the ve major anthocyanins, which indicates that these anthocyanins are acylated anthocyanins. This result agreed with the research of Suda, Oki, Masuda, Kobayashi, Nishiba, & Furuta (2003). They have indicated that the anthocyanins in purple sweet potato are mono- or diacylated forms. Calculation from the chromatograms showed that more than 80.7% of the PSPAs were acylated anthocyanins. This suggests a high stability of the PSPAs and conrms the potential use of PSPAs as a source of natural colourants for the food industry (Fernando & Cisneros-Zevallos, 2007). Otherwise, we did not nd pyroanthocyanins in the fermented purple sweet potato as Morate et al. (2006) did in red wine. Goda et al. (1997) and Bridle and Timberlake (1997) have indicated that the anthocyanins in purple sweet potato are mono- or di-acylated forms of cyanidin and peonidin. Fig. 2 was the chromatograms of PSPAs after acid hydrolysis, and peaks 1 and 2 were detected compared with the standard samples. They accounted for about 48% of the total anthocyanidins. Cyanidin accounted for about 30% of the anthocyanidins and was eluted after 19.3 min. Peonidin accounted for about 18% and was eluted after 23.2 min. Further studies would be needed to determine those anthocyanidins in PSPAs.

4 5 400 300 mAU 200 100 1 2 3

Table 1 Quality and quantity of PSPAs in acidethanol Extraction method Chemistry Fermentation CI 0.58170.001 0.46370.004 DI 0.32570.005 0.19670.002 Yield (mg/mL) 0.07470.005 0.05170.003

0 0 5 10 min
Fig. 1. HPLC prole of purple sweet potato anthocyanins.

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3.3. Colour stability in various pH values The stability of PSPAs in this research was determined by reference to the legal requirements in Spain (Real Decree No. 2107/1996), which was used as a standard of colour intensity. CD and CT indicate the relationship between yellow-brownish colour and red colour (Prodanov et al., 2005). With pH value being raised from 2.0 to 7.0, CI and CD decreased from 0.424 to 0.099 and from 0.511 to 0.187, respectively, while CT increased from 0.221 to 0.717 (Table 2). These indicated that PSPAs degraded as the pH value increased and the colour of PSPAs solution changed from red to purple. The colour stability of anthocyanins depends on the structural changes between avylium cation, quinonoidal bases, colourless carbinol pseudobases and yellow chalcone due to different pH values or protonation or hydration reactions (Jacinto, Beatriz, Marta, Rosaa, Carlos & Roger, 2002; Mazza & Brouillard, 1987). In this research, the lmax shifted from 527 to 548 nm long as the pH value increased from 2.0 to 7.0, which was visually conrmed by differences in the colour of the solution. The increase in pH values brought about browning at 420 nm which then led to an increase in absorbance of the PSPAs, and the formation of less carbinol bases at 520 nm which led to a decrease in the absorbance. These reactions suggest that
1 30 25 mAU 20 15 10 50 0 0 5 10 min
Fig. 2. HPLC prole of purple sweet potato anthocyanins after acid hydrolysis.

the PSPAs were more stable at lower pH values than at higher pH values (Table 2). The colour stabilities of PSPAs at different pH values could be conrmed by the uniform CIELAB colour space parameters of L* (lightness), C* (chroma), H (hue) (Table 3). The C* represents distance away from the grey tone (i.e., degree of colour saturation) in a scale from 0 to 100. Results showed that C* increased when pH values decreased, and the highest C* was found at pH 2.0, while the lowest C* was around pH 7.0. The low C* values indicate that the coloured material in the model solution decreased. The anthocyanins changed from red to colourless at the subacid condition. The H values indicate colour varieties at different pH values. Our results showed that H decreased from 0.621 to 15.161. The anthocyanins solution was red at pH 2.0 and gradually turned to more red-lilac by raising pH to 7.0. It is believed that at low pH, anthocyanin exists as avylium cation which is the most stable form. This bright red form transforms into blue quinonoidal bases or colourless carbinol pseudobases as the pH value increases. In the alkaline condition, the anthocyanin exists as yellow chalcone species (Brouillard, 1982). The L* had no relationship with the alteration of pH values. 4. Conclusions To sum up, anthocyanins extracted from fermented purple sweet potato were more pure than chemistry extraction. More than 80.7% of the PSPAs are acylated anthocyanins. Cyanidin and peonidin were the major anthocyanidins in PSPAs. The determination of pyr-

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Table 3 CIELAB values of PSPAs at different pH values pH values CIELAB L* 2.0 3.0 4.0 5.0 6.0 7.0 18.7070.01 18.8370.07 18.7570.09 18.5370.08 18.9870.04 19.2370.08 C* 3.0270.05 2.8070.19 2.6970.02 2.3570.17 2.3170.10 2.3170.02 H 0.6270.00 1.0470.01 2.5670.02 3.5170.01 12.9670.01 15.1670.01

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Table 2 Colour characterization of PSPAs at different pH values pH value 2.0 3.0 4.0 5.0 6.0 7.0 CI 0.42470.002 0.35170.000 0.25370.003 0.15670.000 0.11070.000 0.09970.001 DI 0.21470.000 0.23070.001 0.27970.002 0.37970.000 0.49470.005 0.63170.000 CD 0.51170.002 0.42670.000 0.32470.005 0.22270.000 0.17870.001 0.18770.002 CT 0.22170.000 0.23870.001 0.29370.002 0.40570.000 0.54170.004 0.71770.001 lmax 527.070.0 530.070.0 531.570.5 535.570.5 545.070.0 548.071.0

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oanthocyanins requires further research. PSPAs extracted from fermented purple sweet potato were found to be more stable at low pH values. The research result has a positive implication for using PSPAs as natural colourants in food industry. References
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