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Antioxidant activity of Agaricus heterocystis hot water extract and their effect on apple browning were investigated. Antioxidant activities were assayed spectrophotometrically against ABTS and DPPH radicals. The mushroom extract showed effective antioxidant activity against both DPPH and ABTS radicals, with the EC50 value of 0.202Â±0.55 mg/mL and 6.42Â±0.261 mg/mL respectively. The extract inhibited the browning of fresh apple, analysed by immersing sliced apple into the mushroom extract with appropriate control prevented effectively the browning development. These observations suggested that 'Agaricus heterocystis' contained certain antioxidant substance or compounds which inhibited the oxidation of endogenous compounds present in the apple.
Oxidation is essential to living organisms for the production of energy to fuel biological processes. However, oxygen-centered free radicals (oxidation) produced continuously in vivo, results in cell death and tissue damage. Oxidative damage in fruits and vegetables during handling, processing and storage after harvest leads to oxidative deterioration or due to enzymatic oxidation of endogenous phenolic compounds catalysed by polyphenol oxidase (PPO) or tyrosinase inherent in biological tissues (Mayer and Harel, 1981; Macheix et al., 1991; Nicolas et al., 1994) leads to browning of tissue. The brown pigment formed in food and food stuffs, looks in unpleasant appearance and concomitant generally result in loss of nutritional and market values (Chen et al., 1991)
Synthetic antioxidants have been used in stabilization of foods. The most commonly used synthetic antioxidants was bisulfite (Sayavedra-Soto and Montgomery, 1986), ascorbic acid and its analogs (Hsu et al., 1988) as well as cysteine (Richard-Forget et al., 1992) for the prevention of PPO-catalysed browning in food. However, the bisulfite used, would found to create lot of side effects in human health, especially in asthmatic patients (Taylor and Bush, 1986). Hence, there was an increasing demand of consumers for substituting synthetic compounds with natural substances as food ingredients. Therefore, the compounds inherent in natural origins are widely accepted by consumers in the market. Based on this notion, there have been a lot of reports on the PPO inhibitors occurring in natural resources as well as numerous species of mushrooms, including Agaricus bisporus (Espín et al., 1999). However, no reports have been found on the scavenging effect of free radicals from the wild edible mushroom 'Agaricus heterocystis' that could prevent the oxidative damage that increase the shelf life of food stuffs. Therefore, the aim of the present study is to evaluate the antioxidant potential of hot water extract of A. heterocystis and their inhibitory effects on browning of apple an important topic from the standpoint of food science and technology (Murata et al., 1995).
Material and methods
The wild edible mushroom Agaricus heterocystis, indigenously collected and identified, was cultivated under laboratory condition to produce fruitbody using compost. The mushroom cultivated and harvested was shade dried at room temperature. Dried mushroom samples (50g) was extracted by stirring with 500 mL of boiling water for 3h and filtered through Whatman No. 4 filter paper. The filtered extracts was then subjected in rotor evaporator to dryness and stored at 4 °C for further use.
ABTS radical scavenging activity
The Trolox equivalent antioxidant capacity (TEAC) was estimated using the Feryl Myoglobulin/ABTS method for total antioxidant activity (Arnao, Cano and Asota, 2001). In this assay, ABTS is oxidized by peroxyl radicals or other oxidants to its radical cation, ABTS.+, which is intensely colored, and AOC (Antioxidant capacity) is measured as the ability of test compounds to decrease the color reacting directly with the ABTS.+ radical. The stock solution included 7.4 mM ABTS solution and 140 mM Potassium per-sulphate solution. The working solution was prepared by mixing the two stock solutions in equal quantities and allowing them to react for 12 h at room temperature in dark. The solution was then diluted by mixing 1 ml ABTS solution with ethanol to obtain an absorbance of 0.7 Â± 0.02 units at 734 nm using UV-1601 spectrophotometer (Shimadzu, Kyoto, Japan). The antioxidant property was determined by reduction in the O.D compared with the standard Vitamin E. Inhibition of free radical by ABTS.+ in percent (I%) was calculated in following way:
I% = (A blank - A sample/A blank) X 100
Where Ablank is the absorbance of the control reaction (containing all reagents except the test compound), and Asample is the absorbance of the test compound. Extract concentration providing 50% inhibition (EC50) was calculated from the graph, plotted in percentage against extract concentration. Tests were carried out in triplicate.
The hydrogen atom or electron donation abilities of the corresponding extracts and some pure compounds were measured from the bleaching of the purple-coloured methanol solution of 2, 2 diphenylpicrylhydrazyl (DPPH). DPPH the stable free radical was used in this spectrophotometric assay (Shimada, Fujikawa, Yahara, & Nakamura, 1992). A stock solution of 1.3 mg/mL in methanol was prepared from that 75 Î¼l of it was added in 3 mL methanol and gave an initial absorbance of 0.9. Decrease in absorbance in the presence of sample extract at different concentration was noted after 15 min. Inhibition of free radical by DPPH in percent (I %) was calculated in following way:
I% = (A blank - Asample/A blank) X 100
Where Ablank is the absorbance of the control reaction (containing all reagents except the test compound) and Asample is the absorbance of the test compound. Extract concentration providing 50% inhibition (EC50) was calculated from the graph, plotted in percentage against extract concentration. Tests were carried out in triplicate.
Treatment of Apple
A 100 mg of Agaricus heterocystis hot water extract was dissolved in 100mL of distilled water. In which two apple slices were immersed and kept for 15 min at ambient temperature. The sliced apples were taken out from the immersing solution and changes in color were observed in the air at room temperature (28Â±2Â°C). The apple slices immersed in normal distilled water and without any immersing treatment were served as control.
Results and Discussion
The antioxidant properties assayed herein were summarized in Table 1 and the results were normalized and expressed as EC50 values (effective concentration scavenging 50% of free radicals) for comparison. The hot water extract against ABTS was found to be effective and their EC50 value was found to be 0.78 Â± 0.05mg/mL. A similar result was also inferred in Agaricus bisporus (Jagadish et al., 2009). The EC50 value of 7.03 mg/mL in Agaricus heterocystis hot water extract was lesser with regard to the other mushrooms Lactarius deliciosus (8.52mg/mL) and Tricholoma protentosum (22.9mg/mL) (Ferreira et al., 2007). Similarly, a study from Barros et al., (2008) on antioxidant property of Agaricus sp. against DPPH radical and their EC50 values varied from 5.37 -15.85 mg/ml in A.silvaticus, A.silvicola, A.bisporus and A.arvensis respectively. The difference in the EC50 value with the previous results may be due to the discrepancy in the experimental protocol.
Table 1 EC50 values obtained from the hot water extract of Agaricus heterocystis against the free radicals
Each value is expressed as mean Â± SD value (n=3).
Inhibitory effect on the browning of sliced apple
The inhibitory effect on apple browning by Agaricus heterocystis hot water extract is shown in Fig. 1. A remarkable browning phenomenon occurred in the controls without mushroom extract. On the contrary, no browning took place in the distilled water added with mushroom extract. These results suggested that Agaricus heterocystis extract contained certain effective inhibitors against browning of apple. The inhibition of browning in apple has been reported by several researchers (Hsu et al., 1988; Santerre et al., 1988; Friedman and Molnar-Perl, 1990). Browning has also been studied in other fruits, including pear and banana (Galeazzi et al., 1981; Dong et al., 2000). The discoloration correlated with the PPO an enzymatic reaction was usually accomplished by the addition of ascorbic acid, citric acid, benzoic acid and glutathione were an antioxidant compounds. Santerre et al. (1988) showed that a combination of ascorbic acid, erythorbic acid and citric acid was efficient in preventing browning of sliced apples. Similar to that the Agaricus heterocystis extract inhibited the browning of apple, also found to have an antioxidant property that may be due to the presence of antioxidant compounds like ascorbic acid and glutathione enriched in the hot water extract. Nevertheless, there have been no reports on the effect of wild edible mushroom Agaricus heterocystis on antioxidant and inhibitory effect on apple browning so far.
Fig. 1 Inhibitory effect of immersing slice apples in 'Agaricus heterocystis' hot water extract on the browning. a)Fresh apple slices without any treatment; b) slices were immersed in distilled water and c) 'Agaricus heterocystis' hot water acetone extract (T) for 15 min.
In conclusion, though a number of other antibrowning treatments, including reducing agents, acidulants, chelating agents, polyphenol oxidase inhibitors, inorganic salts and enzymes have been investigated but are not applied in commercial use (Sapers et al., 1989). Utilization of this wild edible mushroom extract will be promised as a natural food additive for the prevention of color browning caused by PPO or tyrosinase. Further research will be required for the identification of the effective compound.