Anthocyanins from Mulberry (Morus Rubra) Fruits As Potential Natural Colour Additives in Yoghurt

Vol. 8(12), pp. 182-190, December, 2014 DOI: 10.5897/AJPAC2014.0594 Article Number: 124600249302 African Journal of Pure and Applied ISSN 1996 - 0840 Copyright © 2014 Chemistry Author(s) retain the copyright of this article http://www.academicjournals.org/AJPAC Full Length Research Paper Anthocyanins from mulberry (Morus rubra) fruits as potential natural colour additives in yoghurt Robert Byamukama1*, Moses Andima1,2, Angella Mbabazi1 and Bernard T. Kiremire1 1Department of Chemistry, Makerere University, P. O. Box 7062, Kampala, Uganda. 2Department of Chemistry, Busitema University, P. O. Box 236, Tororo, Uganda. Received 27 September, 2014; Accepted 16 December, 2014 Colouring potential of anthocyanins from whole fruit juice of mulberry (Morus rubra) was studied in yoghurt. Whole fruit juice from M. rubra rich in non-acylated anthocyanins was incorporated into plain yoghurt (100 g) at increasing concentration levels of the juice; 10, 20, 25, 30, 40 and 50 mg cyanidin-3- glucopyranoside equivalents (cy-3-glu eqv) and stored under refrigerated condition (< 8°C) for two weeks. Colour properties, pigment and colour stability and degradation kinetics were studied using a UV-Vis spectrophotometer (UV-1700 CE Shimadzu, Japan).Yoghurt coloured with mulberry anthocyanins between 25 to 40 mg cy-3-glu eqv concentration levels of anthocyanins produced a colour which was very much comparable to commercial brand strawberry yoghurt coloured with 20 mg FD & C red No. 3 in 100 g of yoghurt. Pigment and colour stabilities of the anthocyanins increased with increasing concentration of anthocyanins added to yoghurt. The tendency to polymerise decreased with increasing concentration of the pigments added to yoghurt. The degradation of the anthocyanins was fitted to first-order reaction kinetics. Moderate concentration levels (25 to 40 mg cy-3-glu eqv in 100 g of yoghurt) of mulberry anthocyanins were found to be ideal to colour yoghurt. Key words: Mulberries, anthocyanins, cyanidin-3-glucopyranoside (cy-3-glu), natural colourants, colour stability, yoghurt. INTRODUCTION One of the vital constituents of some foods and colours is diminishing drastically due to associated health beverages is colour. In addition to other functions (Mendi problems and legislative action against some of them et al., 2000), colour plays a very important role in the (Cevallos-Casals and Cisneros-Zevallos, 2004; Markakis, acceptability of some foods by many consumers (Giusti 1982). and Wrolstad, 2003). In practice, most manufacturers Anthocyanins have been of great interest as tend to colour products which have dull colours and look alternatives to synthetic colourants due to their bright unappealing to most consumers. Synthetic colourants colours and associated health benefits (Cevallos-Casals have often been used in attempts to colour some foods and Cisneros-Zevallos, 2004; Fan et al., 2008). They are and beverages (Delgados-Vargas and Paredes-López, considered to be safe because they have been 2003). However, the demand for foods with synthetic consumed for centuries in fruits, and vegetables without *Corresponding author. E-mail: [email protected], Tel: +256392548585. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Byamukama et al. 183 any health risks (Bridle and Timberlake, 1997). The Xu, 2007). The fruits were then blended in a laboratory blender application of anthocyanins as food colourants faces (Waring 700S 1 L, Lab depot, Inc.) and the juice was filtered some limitations such as low stability to several through a clean piece of nylon clothe. The juice was centrifuged at 4000 rpm and the supernatant was concentrated in a vacuum rotary processing, formulation and storage conditions. Despite evaporator at 35°C to 15% brix. The concentrate was kept below the drawback, highly acylated anthocyanins possess high 4°C in a freezer until analysis. colour stability in some food matrices (Giusti and Wrolstad, 2003; Kirca et al., 2006). Nevertheless, Wallace and Giusti (2008) reported that whole fruit High-performance liquid chromatography (HPLC) analysis extracts containing non-acylated anthocyanins from To confirm the anthocyanins in M. rubra, high performance liquid Berberis boliviana L. showed improved colour and chromatography (Shimadzu, Columbia, Md.,U.S.A) was performed pigment stability when incorporated in yoghurt. This on a Hypercil goldTM reverse phase 250 x 4.6 mm symmetry, 5 μm information led us to believe that whole fruit juice extracts size C18 column (Thermo Fisher Scientific Inc.) equipped with a from mulberries (Morus rubra) could serve as an photo array detector (PAD). Separation of the anthocyanins was appropriate colourant and nutraceautical in yoghurt. achieved using a linear gradient. Solvent A was HCOOH:H2O; 1:19, Solvent B was HCOOH:H2O:MeOH; 1:9:10. The elution profile was Mulberries (M. rubra) are a rich source of anthocyanins, 0 to 23 min, 10 to 100% B, 0 to 5 min 10% B, 5 to 23 min, 100% B, with fully ripe fruits yielding between 2.53 to 8.30 mg cy- 23 to 28 min, 100% B 28 to 29 min, 10% B at a flow rate of 0.75 ml 3-glu/gfresh weight of anthocyanins (Özgen et al., 2009). min-1 and the injection volume was 15 μl. UV-Vis spectral data was Other studies reported the content of anthocyanins to obtained from 250 to 560 nm (Wallace and Giusti, 2008). vary from 0.59 mg/ g to 10.90 mg/ g fresh weight infrom different cultivars of mulberries (Liu et al., 2004).The Colouring yoghurt with the juice extract fruits possess many health benefits such asantithrombotic, antioxidant, antimicrobial, anti- 100 g portions of plain yoghurt at the point of adding colour were inflammatory, and neuroprotective properties(Aramwit et coloured with the whole fruit juice extract of mulberries at increasing al., 2010). In recent years, mulberry anthocyanins have in concentration levels of the juice (10, 20, 25, 30, 40, and 50 mg Cy- many occasions been applied in baked foods, and 3-glu eqv) in 100 g of yoghurt. The amount of the juice extract for each portion of yoghurt was chosen in order to study the effect of confectioneries (Wu et al., 2011). In Turkey, they are increasing pigment concentration on the colour of yoghurt and the used to make special important traditional products such stability of the pigment in yoghurt (Wallace and Giusti, 2008). Plain as mulberry pekmez, mulberry pestil, and mulberry kome yoghurt was manually mixed gently and thoroughly with the juice (Ercisli and Orchan, 2007). The anthocyanins are stable concentrate to ensure uniformity and avoid syneresis of the set at low temperatures and in the dark (Aramwit et al., yoghurt. Yoghurt samples were prepared in triplicate at each 2010). In this study, we decided to investigate whether colouring level. The coloured yoghurt samples were packed in McCartney bottles (50 ml, 144 bottles in total, 3 at each colouring anthocyanins from mulberries (M. rubra) could be used level) and stored in a laboratory refrigerator (< 8°C)for two weeks as potential colour additives in yoghurt since yoghurt has during which, colour properties, pigment concentration and pH were a low pH and it is stored under refrigerated conditions. measured at regular time intervals (Wallace and Giusti, 2008). MATERIALS AND METHODS Extraction of anthocyanins from the yoghurt matrix Plant materials and yoghurt Anthocyanins were recovered from the coloured yoghurt matrix using the method adopted by (Wallace and Giusti, 2008). Coloured Ripe fruits of mulberries (M. rubra) were collected from Masaka yoghurt samples (100 g) at each concentration level, were District in Uganda and stored in a laboratory freezer before use. thoroughly mixed with methanol acidified with hydrochloric acid Plain yoghurt (1.5% fat) at the point of adding colours and (99.9%, 0.1%, 30 ml) for 5 min and centrifuged at 4000 rpm for 15 commercial brand strawberry flavoured yoghurt were kindly min. The extraction procedure was carried out twice on each provided by fresh dairy factory (Sameer Agriculture and Livestock sample to ensure maximum recovery of anthocyanins from the (U) ltd, Plot 49-53/55 fifth street, Industrial Area, Kampala, Uganda). yoghurt matrix. The supernatant in each case was carefully filtered and concentrated to a known volume at 35°C with a rotary evaporator. The concentrates were used for spectrophotometric analysis in Chemicals, reagents and equipment triplicate. To determine the recovery rates, juice containing anthocyanins (20 mg cy-3-glu eqv) were added to plain yoghurt Analytical grade methanol, potassium chloride, sodium acetate, and (100 g) and extracted similarly with acidified methanol. The recovery potassium metabisulphite used in this study were purchased from rate was expressed as a percentage of the recovered amount in the BDH laboratory suppliers (U) ltd. A UV-Vis spectrophotometer (UV- total initial amount added to yoghurt Equation (1). This procedure 1700 CE Shimadzu) was used for spectrophotometric was repeated six times to determine the extraction efficiency. measurements using 1 cm path length disposable cuvette tubes. (1) Preparation of whole-fruit juice concentrate Frozen fruits of mulberry were allowed to defrost and the fruits were Determination of anthocyanin content pasteurised immediately to denature enzymes such as polyphenol oxidase (PPO) which causes browning of anthocyanins (Wang and The content of total anthocyanins in the coloured samples 184 Afr. J. Pure Appl. Chem. mAU Extract-518nm4nm (1.00)/smth 1 2 3 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 Min Figure 1. HPLC-PAD chromatogram of anthocyanins in M. rubra fruit juice extract recorded at 520 nm. Cyanidin-3-O- glucopyranoside (peak 1), cyanidin-3-O-rutinoside (peak 2), pelagornidin 3-O-glucopyranoside (peak 3). remaining at any time was determined using the pH-differential calculated from Equation (5) (Kirca et al., 2006; Wallace and Giusti, method described by Giusti and Wrolstad (2001).

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