Abstract: The anthocyanin pigments was extracted from the four Malus varieties such as M. domestica cv. red starking,M. domestica cv. red delicious, M. domestica cv. jonathan and M. domestica cv. abbasi mashhad using the soaking in Ethanol (1% acidified).The extracted anthocyanin pigments then were exposed to number of environmental factors which could destabilize the anthocyanin molecules. These environmental factors were included three different pH (1, .5 and 4), four various temperatures (5, 0, 0 and 40°C) and presence or absence of light.The results showed that increasing in pH, temperature or exposure to light is able to destruction the anthocyanin pigments. Another factor affecting the tolerance of anthocyanin towards the environmental condition is the role of different varieties. Among the various Malus varieties anthocyanin pigment in M. domestica cv. red starking, showed the greatest resistance to destruction by environmental conditions, other varieties followed: M. domestica cv. red delicious M. domestica cv. jonathan and M. domestica cv. abbasi mashhad consecutively.
INTRODUCTION
The anthocyanins comprises the largest group of water soluble pigments in the plant kingdom and are especially characteristic of the angiosperms or flowering plants, which themselves provide our major source of food crops in food plants, anthocyanins are widespread occurring in at least 27 families, 73 genera and a multitude of species and can be classified chemically as both flavonoid (related to flavone/isoflavone, C15H10O2) and phenolic related to phenol, C6H5OH) (Malien-Aubert et al., 2001). Anthocyanins are synthesized in the cytoplasm of plant cells and actively transported across the tonoplast into the vacuole, where they accumulate (Leng et al., 2000). They are responsible for the red, pink, mauve, purple and blue colours found in the flowers fruits and leaves of Angiosperms, as well as some Gymnosperms and some Bryophytes (Yamazaki and Saito, 2002). There has been a great increase in utilization of natural plant pigments in the food industries, replacing the implementation of artificial coloring agents, in order to provide a healthier food for consumers. However, in comparison with the natural coloring agents, the artificial coloring agents show greater resistance and stability when exposed to oxidation changes in temperature, pH and other factors (Navidra et al., 2002). Although anthocyanins are less stable in various environmental condition, they include varieties of colors such as orange, red, maroon and blue which make them an attractive alternative as coloring agents in food industries (Mazza and Minitiati, 1993). The intensity and stability of the anthocyanin pigments is dependent on various factors including structure and concentration of the pigments, pH, temperature, light intensity, quality and presence of other pigments together, metal ions, enzymes, oxygen, ascorbic acid, sugar and sugar metabolites, sulfur oxide etc. (Lee, 2002). Anthocyanins have four different structures, which are in equilibrium and include flavylium cation, quinoidalbase, carbinolpseudobase and chalcone. The relative amounts of these structures in equilibrium are varied and depend on the pH and anthocyanin structure (Spayd et al., 2002). Some anthocyanins are more stable than other depends on their molecular structure. The example of this is the Malvidin glycosides, the major anthocyanin in grape, which due to dimethyloxylation of t he molecules are more stable than other anthocyanins (Bridle and Timberlake, 1997). Moreover hydroxylation of organic acids results in more stable molecules in most cases (Cooper-Driver, 2001). Anthocyanins have applications in chemotaxonomical and ecological studies. They have bright attractive colours. Another factor in increasing the stability of the anthocyanin is the co-pigmentationv (Cordenunsi et al., 2003). The skin of Malus of red fruit is also among the plants, which contains large amount of anthocyanin. Transformation of these pigments to other forms by enzymes, oxidation, light, temperature, etc., during storage cause color change from red to brown in Malus fruits, which has a negative impact on appearance of the product.
There are also more stable color pigments exist in the fruits and vegetables which their phyto-chemical structure and anthocyanin properties need to be investigated with regard to importance of anthocyanin in different industries in the current research, effects of some environmental factors were evaluated on the stability of anthocyanin in four Malus varieties in uromieh such as: M. domestica cv. jonathan M. domestica cv. red starking, M. domestica cv. red delicious and M. domestica cv. abbasi mashhad.
MATERIALS AND METHODS
Sample preparation: Samples of Malus varieties such as M. domestica cv. red starking, M. domestica cv. red delicious, M. domestica cv. jonathan and M. domestica cv. abbasi mashhad gathered in urmia city area of Ghasemlo in provence of West Azarbayjan in Iran. Then fruits were washed with distilled water and dried and kept frozen at -8°C till use.
Methods: We used the methods of Chiriboga and Francis (1970) using ethanol, acidified with 0.1% hydrochloric acid 1% for extraction of anthocyanin. Samples were taken out of the freezer, left at room temperature for 30 min to defrost. Three hundred grams skin of each Malus variety were put in a mixer, solvent added and a mixed for 10 min. The products then filtered in vacuum using Bochner funnel and wattman filter (grade 1), the remain of the mixture on the filter paper was washed again with the solvent and filter again to get a clear liquid. The filtered product then placed in a balloon container in a vacuum evaporator at 35°C to separate the ethanol-acid solvent. The balloon container was separated from the vacuum evaporator and distilled water was added to dissolve the powder which was formed at the bottom of the balloon container. The product then transferred to a 500 mL container and brought the volume to 500 mL using distilled water. The product then centrifuged at 8000 rpm, the supernatant was separated and kept for further analysis. The anthocyanin contents of the extracts was measured by Spectrophotometer (UV/Visible). The following two buffers were used: a) 0.13 M HCl-0.05 M KCl, pH = 1.0 and (b) 0.05 M HCl -0.5M CH3COONa, pH = 5. The mixture of buffer and samples were equilibrated in darkness for 1 h and their absorbance was measured at 520 nm by spectrophotometer.
RESULTS AND DISCUSSION
The role of Malus varieties on the stability f anthocyanin: There are great inter-genus and inter-species variations between the level of phenolic compounds in fruits. Depends on the Malus varieties the stability of anthocyanin varies. Stability of anthocyanin extracted from four different Malus varieties in a fixed temperature of 25°C and pH = 2 measured. The results show that M. domestica cv. red starking contains the most stable form of anthocyanin and M. domestica cv. abbasi mashhad has the least stable form of anthocyanin. The level of anthocyanin destruction after 90 days in Malus varieties was 51.92, 64.67, 68.77 and 94.49% for M. domestica cv. red starking, M. domestica cv. red delicious, M. domestica cv. jonathan and M. domestica cv. abbasi mashhad consecutively (Fig. 1 and Table 1).
The effect of pH on the stability of anthocyanin: Another factor which affects the stability of anthocyanin is the pH (1, 2.5 and 4). All the experiments were performed in a fixed temperature of 25°C in 90 days period and total of 5 separate measurements for each variety. Present results show that increasing pH cause greater destruction of anthocyanin in samples (Fig. 2 and Table 2). The degree of anthocyanin destruction between pH = 2.5 and 4 is considerably greater than that of pH = 1 to pH = 2.5. Flavylium salts are stable only in highly acidic conditions. These salts loose the proton in higher pH and transform into quinoidal base, which is an unstable pigment and immediately bound to water and form colourless compound called chromenol. Delpech (2000) have reported that in warm agricultural areas, high pH of the grapes at the time of harvest could cause problem for the juice making industry. Higher pH in grapes can cause fading the colour and decrease in stability of the products. Little (1997) studied the combination of the colour pigments in strawberry, jam and packaged strawberries at the 37.7°C during time. Also recorded the data for pH of 2, 3 and <1 and showed that destruction of anthocyanin pigments increases with increase in pH.
The effect of temperature on the destruction of anthocyanin: Temperature is also another factor, which has a role in destabilising the anthocyanin molecular structure; with increase in temperature we see a greater degree of destruction in anthocyanin. In a fixed pH of 2, the effect of four different temperatures of 5, 20, 30 and 40°C on level of anthocyanin extracted from four different Malus varieties during 90 days period were measured in 5 separate instances (Fig. 3).
| Table 1: | Comparison effect of varieties on the percentage of destruction
of anthocyanin in four Malus varieties |
| Fig. 1: | Comparison of Anthocyanin in four Malus varieties |
| Fig. 2: | pH effects on the stability of Anthocyanin in four Malus varieties |
| Table 2: | The percentage of anthocyanin destruction in pH of 1, 2.5 and 4 in varieties of Malus |
| Table 3: | The percentage of anthocyanin destruction in temperatures of 5, 20, 30 and 40°C in varieties of Malus |
| Fig. 3: | The effects of temperature on the stability of Anthocyanin in four Malus varieties |
The results show that percentage of destruction of anthocyanin in 5, 20, 30 and 40°C which are presented in the Table 3. We suggest that the speedy destruction of anthocyanin in higher temperatures could be due to hydrolisation of 3-Glycoside structure, which has a protective effect in unstable anthocyanin.
| Table 4: | Effect of presence or absence of light the percentage of destruction of anthocyanin in four Malus varieties |
| Fig. 4: | The effects of Light on the stability of Anthocyanin in four Malus varieties |
The other suggestion is that the hydrolisation of the pyrilium ring resulted in production of chalcone, which are responsible for brown colour developed in food containing anthocyanin (Giusti and Wrolstad, 2000). Palamidis and Markakis (1975) has studied the effect of temperature on the stability of anthocyanin in soft drinks and have shown that increase in the storage temperature greatly accelerate the destruction of pigments in soft drinks. Spayd et al. (2002) found that the increase in temperature accelerates the destruction of anthocyanins.
The effect of presence or absence of light on the stability of anthocyanin: Light is another factor, which affects the stability of anthocyanin. pH in all samples was 2 and temperature was kept at 25°C (Fig. 4). The period of experiments was 90 days and data was recorded in 5 separate instances. In the presence or absence of light (400 Lux) the percentage of destruction of anthocyanin is shown in Table 4 for four Malus varieties. Similar results for grapes anthocyanin had been reported by Palamidis and Markakis (1975). They exposed the pigments of the light for 135 days at 20°C and observed 50% of pigments destruction, at the same time in dark the level of destruction was 30%. Timberlake (1989) suggested that light increase the flavylium cation construction, but in the absence of light the amount of chalcone in the extract containing anthocyanin was higher than its flavylium cation.