Food Sci. Technol. Res., 15 (5), 547 – 552, 2009
Note Effects of Different Drying Methods and Extraction Condition on Antioxidant Properties of Shiitake (Lentinus edodes) Zuofa Zhang, Guoying Lv, Huijuan Pan, Yongzhi Wu and Leifa Fan* Institute of Horticulture, Zhejiang Academy of Agricultural Science, Hangzhou 310021, China
Received November 6, 2008; Accepted May 25, 2009 This work studies the effect of four drying methods, microwave-, oven-, freeze- and sun-drying on the parameters of antioxidant properties for Shiitake (Lentinus edodes): total phenolic content, total antioxidant capacity and DPPH scavenging activity. Freeze-drying Shiitake showed the highest activities in above three parameters. In addition, the optimum extraction condition (90℃, 40% ethanol and 1 h) of Shiitake was determined with orthogonal array design matrix using the total phenolic content as an indication. The results may be an economically interesting phytochemical source for the nutraceutical and functional food market. Keywords: Lentinus edodes, antioxidant activity, phenolics, DPPH, freeze-drying
Introduction Oxidative damages caused by free radicals to living cells mediate the pathogenesis of many chronic diseases, such as atherosclerosis, Parkinson’s desease, Alzheimer’s disease, stroke, arthritis, chronic inflammatory diseases, cancers and other degenerative diseases (Halliwell and Grootveld, 1987). Antioxidants are not only needed by our body to combat reactive oxygen species (ROS) but are also important as food additives. Many antioxidant compounds, naturally℃curring from plant sources, have been identified as a free radical or active oxygen scavengers (Zheng and Wang, 2001). Recently, interest has increased considerably in finding naturally℃ curring antioxidants for use in foods or medicinal materials to replace synthetic antioxidants, which are being restricted due to their side effects (Ito et al., 1983). In addition, natural antioxidants have the capacity to improve food quality and stability and can also act as nutraceuticals to terminate free radical chain reactions in biological systems, and thus may provide additional health benefits to consumers. Mushrooms have been used for traditional foods and medicines in Asia (Chang, 1996). Generally, mushrooms are rich in dietary fiber, minerals, vitamins and low in fat (Manzi, 2001; Mattila et al., 2001). Moreover, mushrooms contain *To whom correspondence should be addressed. E-mail:
[email protected]
various polyphenolic compounds recognized as an excellent antioxidant (Ishikawa and Morimoto, 1984). Especially, Shiitake (Lentinus edodes) is the second most popular and the third widely cultivated edible mushroom in the world. Several important compounds including bioactive polysaccharides (lentinan), dietary fiber, ergosterol, vitamins B1, B2 and C, and minerals have been isolated from the fruiting body, mycelia, and culture medium of this mushroom. Resent numerous studies have shown its medicinal attributes including anti-tumor, antimicrobial, liver function improving and cholesterol lowering activity (Fukushoma et al., 2001; Mizuno et al., 1995). Some mushrooms are perishable in their fresh state and may deteriouate within a few days after harvest. One way to preserve this products is to dry them in order to conserve their desirable qualities, reduce storage volume and to extend their shelf life. Drying functions to inactivate the enzymes polyphenol oxidases and leads to significant changes in the composition of phytochemicals (Capecka et al., 2005). Extraction is the first step in the isolation of phenolic compounds from plant materials. Extraction is influenced by the chemical nature of the compounds (simple and complex phenolics), the extraction method employed, the storage time and conditions, and the presence of interfering substances (Naczk and Shahidi, 2004). Solvent extraction is widely used for phenolic extraction from various vegetable materials
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(Lapornik et al., 2005; Liyana-Pathirana and Shahidi, 2005; Liu et al., 2000; Li et al., 2006). Many factors contribute to the efficiency of the solvent extraction steps, ratio solvent/ solid, and particle size of the solid matrix. The present study was conducted to analysis the effects of different drying methods and determinate the optimal extraction conditions on the antioxidant properties of Shiitake with an orthogonal array design matrix. To the best of our knowledge, it was never investigated. Materials and Methods Materials Folin-Ciocalteu reagent, gallic acid, 1,1-diphenyl-2-picrylhydrazyl (DPPH) were purchased from Sigma Chemical Co. (St. Louis, MO, USA) and all other reagents were of analytical grade. Raw Shiitake was purchased form local market in China on September, 2008. The raw Shiitakes was sliced and divided into several groups (accurately 2 g each, weighted as it is). Drying processes Shiitake were subject to four different drying methods, i.e., microwave-, oven-, sun-, and freezedrying. For each drying method, 2 g of fresh Shiitake was used. In microwave-drying, Shiitake were dried in a microwave oven (Sharp R-248e; 800W) for 4 min. Oven-drying involved drying for 8 h in an oven at 50℃. Shiitake were sun-dried in the greenhouse for three days with about 36 h of daylight. Mid-day temperature in the greenhouse can reach 26℃. For each of the above drying methods, the materials were spread out evenly on a Petri dish. In freeze-drying, the samples were lyophilized overnight in a vacuum flask at 0.125 Pa and -50℃ in a freeze-dryer (Alpha 1-4, Martin Christ, Germany). Sample extraction Ethanol was selected as extraction solvent due to its wide use in different processes of phenolic extraction from various diverse plant materials. Fresh (2 g) and dried (2 g fresh Shiitake equivalent) Shiitake were extracted with 30 mL 80% ethanol in an orbital shaker (Heidolph, Schawabach, Germany) at 50 rpm under darkness for 2 h at room temperature (22℃), respectively. The mixtures were flushed with nitrogen to prevent phenolic oxidation during extraction. The mixtures were centrifuged at 2000 × g for 10min at 4℃ and the supernatants were collected. The supernatants of each extraction were stored at -20℃ until analysis. Analysis of extracts was done in triplicate. Measuerment of total phenolic content (TPC) Total phenolics in all samples were determined using FolinCiocalteu reagent according to the method of Singleton and Rossi (1965) using gallic acid as a standard. 0.3 mL each extract was added into test tubes followed by 1.5 mL of FolinCiocalteau reagent (diluted 10 times) and 1.2 mL of sodium carbonate (7.5 g/100 mL). The contents of the tubes were
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mixed thoroughly and stored in the dark for 30 min before the absorbance was measured at 765 nm. Total phenolic content was expressed as gallic acid equivalent (GAE) in mg per 100 g fresh Shiitake. Measurement of total antioxidant capability (TAC) The total antioxidant activity of the extracts was evaluated by the phosphomolybdenum method according to the procedure of Prieto et al. (1999). 0.3 mL each extract was combined with 3 mL of the reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes containing the reaction of solution were incubated at 95℃ for 90 min. Then the absorbance of the solution was measured at 695 nm after cooling to room temperature. The antioxidant activity was expressed as asorbic acid equivalent (AAE) in mg per 100 g fresh Shiitake. Measurement of DPPH scavenging activity (DSA) The DPPH scavenging activity of the extracts was measured by the method of Shimada et al. (1992). 1 mL of each extract was added to 2 mL of 0.15 mM of DPPH. The mixture was allowed to stand for 30 min before measuring the absorbance at 517 nm. The antioxidant activity was expressed as a percentage of scavenging activity on DPPH radical: Scavenging activity % = (1 – A1 / A0) × 100
(1)
where A0 is the absorbance of the control, and A1 is the absorbance of the samples. The control contains all reagents except the sample. Optimization of the extraction condition with orthogonal array design matrix Extractions were conducted in conical flask (100 mL). On the basis of our previous study, temperature, time and the concentration of ethanol were three important factors affecting the extraction yields. The parameters considered in optimization of the extraction conditions are temperature (30, 60 and 90℃), time (1, 2 and 3 h) and ethanol concentration (40%, 60% and 80%). About 0.3 g of lyophilized Shiitake powder and 30 mL ethanol with different concentration were packed into the flask and subjected to the above procedures. The supernatants of each extraction were stored at -20℃ until analysis for determinating TPC. Analysis of extracts was done in triplicate. Results and Discussion The four different drying methods resulted in drastic weight loss due to loss of water (Table 1). Drying except freeze-drying method also resulted in considerable shrinkage of the materials and caused them to become in nature, thus making them easier to grin during extraction. Total phenolic content The Folin-Ciocalteu phenol method is not an antioxidant test but an alternative assay for the quantitation of phenolic compounds (Wangensteen et
Effects of Different Drying Methods and Extraction Condition on Antioxidant Properties of Shiitake (Lentinus edodes)
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Table 1. Effect of drying method on total phenolic content (TPC), total antioxidant capability (TAC) and DPPH scavenging activities (DSA) of Shiitake.
Drying method
Water loss (%)
TPC (mg GAE/100 g fresh Shiitake)
TAC (mg AAE/100 g fresh Shiitake)
DSA (%)
Fresh
----
149.6 ± 4.7
167.3 ± 13.2
67.4 ± 5.8
Microwave-drying
78.2 ± 1.5
106.7 ± 6.1
98.0 ± 9.1
68.3 ± 4.5
Sun-drying
79.6 ± 2.1
93.4 ± 3.9
109.5 ± 5.4
43.2 ± 6.6
Oven-drying
82.3 ± 0.8
132.1 ± 8.6
131.9 ± 6.6
58.5 ± 3.8
Freeze-drying
84.2 ± 0.3
160.1 ± 10.5
192.6 ± 3.7
81.6 ± 7.2
Data are mean values ± SD of 3 determinations.
al., 2004). Phenolic compounds undergo a complex redox reaction with phosphotungstic and phosphomolybdic acids present in the reagent. Table 1 presents the results of TPC of fresh and dried Shiitake. Different drying treatments affected the TPC, freeze-drying method showed the highest TPC, which followed by fresh, oven-drying, microwave-drying and sun-drying. Total antioxidant capacity The phosphomolybdenum method was based on the reduction of Mo (VI) to Mo (V) by the antioxidant compound and the formation of a green phosphate Mo (V) complex with the maximal absorption at 695 nm. The total antioxidant capacity are given in Table 1, the antioxidant activity of the Shiitake with different drying method are in the order: freeze-drying > fresh > oven-drying > sun-drying > microwave-drying. DPPH scavenging activity The model of scavenging the stable DPPH radical is a widely used method to evaluate antioxidant activities in a relatively short time compared with other methods. The addition of the extracts to the DPPH solution caused a rapid decrease in the optical density at 517 nm. The degrees of discoloration indicate the scavenging capacity of the sample. From Table 1, we observed that the Shiitake was treated with different drying methods exhibiting different DPPH scavenging activity. There could be few explanations for the decrease in both TPC and antioxidant activity of the samples. For sun-drying method, loss of TPC may be caused by enzymatic processes that℃curred during sun drying. Sun drying did not immediately deactivate degradative enzymes such as phenolic
oxidases, which are able to degrade phenolic compounds before the Shiitake are completely dry. Okuda et al. (1989) have mentioned that rosmarinic acid was degraded when it is dried under direct sunlight, and in oven at 60 and 80℃ and Mueller-Harvey (2001) has reported that some phenolic compounds decompose rapidly in direct sunlight. So solar radiation may also cause some degradation of the compounds. Oven heating at 50℃ was shown to rapidly inactive polyphenol oxidase present in plant materials, however, some of their initial activities may have occurred earlier and caused some polyphenols to be degraded. Microwave heating, brought about by absorption of microwave energy by water molecules, is more energy efficient than conventional heating and it could inactivate degradative enzymes very much faster than oven heating and yet drastic loss of TPC was observed, the degradetion of phytochemicals may be the main reason. Interestingly, in the present study, freeze-drying of Shiitake showed higher TPC, TAC and DSA than fresh Shiitake. For fresh Shiitake, the cause of depletion of antioxidants could be due to operations such as cutting and slicing as they induce a rapid enzymatic oxidation of natural antioxidants (Lim and Murtijaya., 2007). For freeze-drying, there is no thermal degradation and neither dose the process allow degradative enzymes to function (Chan et al., 2008). Furthermore, freeze-drying is known to have high extraction efficiency because ice crystals formed within the sample matrix can rupture cell structure, which allows exit of cellular components and access of solvent, and consequently better extraction (Asami and Hong, 2003). The effect of
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antioxidants on DPPH radical scavenging was thought to result from their hydrogen donating ability (Baumann and Bruchlarusen, 2002). In the present study, the high DPPH scavenging activity of microwave-drying can be explained that high temperature might disrupt the cell wall and liberate antioxdant compounds from insoluble portion of this mushroom or the formation of novel compound having powerful donating ability (Peleg et al., 1991; Choi et al., 2006). Optimization of the extraction condition with orthogonal array design matrix An orthogonal array design, L9 (33), was performed to optimize the extract temperature, time and ethanol concentration. Table 2 shows the results of total phenol yield obtained under the experimental conditions tested. Analyses on the orthogonal array design indicated that ethanol concentration had the highest R value of 41.9 and exerted the largest effect on the extraction of total phenolic. The extent of the impact of variables on extraction yields followed the order: ethanol concentration > extraction temperature > extraction time. We also concluded that extraction tempera-
ture and ethanol were the two major factors affecting extraction yield of total phenolic. The optimum extraction yield was provided at 90℃, 40% ethanol and 1 h. Extraction yields of total phenolic increased with the increasing temperaure, which was supported by the results of Eggers (1996) and Oghaki et al. (2002). The explanation of this result could be that the intense heat was able to release cell wall phenolics or bound phenolics due to the breakdown of cellualr constituents, thus causing more polyphenols to be extracted (Toor and Savage, 2006). Moreover, the high temperature also increases the solubility of phenols (Amin et al., 2006). However, in the present study, the extraction yields of total phenolic was decreased with the increased ethanol concentration, which may be determined by the composition of the phenolics in Shiitake. It is interesting to find that the extraction time have little effect on the yield of total phenol, so in the present study, we choose 1 h as our optimum extraction time.
Table 2. Results obtained under the experimental conditions using L9 (33) orthogonal array design.
Run
Extraction temperature (℃)
Extraction time (h)
Ethanol concentration (%)
TPC (mg GAE/100 g fresh Shiitake)
1
30
1
40
122.9 ± 3.5
2
30
2
60
147.7 ± 1.4
3
30
3
80
87.7 ± 2.7
4
60
1
60
133.4 ± 4.4
5
60
2
80
96.3 ± 1.8
6
60
3
40
174.3 ± 3.6
7
90
1
80
129.6 ± 0.9
8
90
2
40
142.0 ± 1.5
9
90
3
60
134.4 ± 2.1
R value
15.9
3.5
41.9
Data are mean values ± SD of 3 determinations.
Effects of Different Drying Methods and Extraction Condition on Antioxidant Properties of Shiitake (Lentinus edodes)
Conclusion Results showed that freeze-drying was superior to other drying methods in preserving the antioxidant properties of Shiitake. Thermal dry (microwave-, oven, and sun-drying) resulted in significant declines in TPC, TAC, and DSA. Freeze-drying Shiitake even showed higher antioxidant properties than fresh Shiitake. In the present study, we optimized the extraction conditions with the indication of TPC using orthogonal array design matrix. These best extracts are bearing a high antioxidant properties and therefore be an economically interesting phytochemical source for the nutritional and functional food market.
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