JFS and Safety Antioxidant Capaity, Phenoli Content, and Polysaharide Content of Lentinus edodes Grown in Whey Permeate-Based Submerged Culture X.J. WU ANDC. HANSEN ABSTRACT: Total antioxidant apaity (TAC), phenoli ontent, and rude water-soluble polysaharide (WSP) were determined for Lentinus edodes myelia grown on both whey permeate (WP)-based medium with latose ontent of 4.5% and ontrolled medium, and harvested after 5, 10, 15, or 20 d of fermentation at 25 C. Both methanol extrats and water extrats of L. edodes in this study were found to exhibit high free radial savenging apaity. The harvesting time was found to ontribute to most of the variability in the free radial savenging apaity. High levels of antioxidant apaities (0.28 ± 0.03 and 0.29 ± 0.06 mmol TAE/g dry weight for methanol and water extrats, respetively) were observed in myelia grown on whey permeate and harvested on day 10. Harvesting time and the type of media an interat to alter the hemial ontent of myelia. Myelia grown in whey permeate had greater (P < 0.05) WSP than myelia grown in the syntheti media. High levels of WSP (4.1 10 2 ± 71 mg polysaharide/g dried myelia) were found in myelia grown in whey permeate and harvested on day 10. Whey permeate grown myelia had phenoli ompounds ranging from 4.2 ± 0.1 to 8.0 ± 0.8 mg GAE/g dried myelia. The overall means of total phenoli ontents of myelia grown in whey permeate were 5.9 ± 0.5 and 6.2 ± 0.6 mg GAE/g dried myelia for methanol and water extrats, respetively. Keywords: antioxidant apaity, Lentinus edodes, myelia, phenoli ompound, polysaharides, whey permeate Introdution Free radials released during oxidative stress pose the major endogenous damage in the biologial system (Cheung and others 2003). This type of damage is often assoiated with various degenerative diseases and disorders suh as aner, ardiovasular disease, immunofuntion deline, and aging (Kaur and Kapoor 2001; Cheung and others 2003). Free radials are highly reative moleules having unpaired eletrons (Kaur and Kapoor 2001). They an be produed by radiation or as by-produts of the metaboli proess (Cheung and others 2003; Kang and others 2003). To gain stability, free radials apture eletrons quikly from other ompounds. The attaked ompound beomes a free radial itself, whih ontinues to attak other ompounds and leads to a hain reation. This results in the disintegration of ell membranes and ell ompounds, inluding lipid, protein, and nulei aids (Kaur and Kapoor 2001). Besides damage to living ells, free radials are the major ause of food deterioration through lipid oxidation, whih ultimately affets the organolepti properties and edibility of foods (Cheung and others 2003). Hene, intervention of an antioxidant may provide therapeuti funtions (Jose and others 2002) and maintain the freshness of food produts (Kaur and Kapoor 2001). Reent researh suggested that syntheti antioxidants ould promote tumor formation as well as provide antiarinogeni properties (Cheung and others 2003). Due to these ontraditory properties, the appliation and exploration of natural antioxidants has reeived more attention. The onsumption of fruits and vegetables MS 20070263 Submitted 4/11/2007, Aepted 9/27/2007. Authors are with Dept. of Nutrition and Food Siene, 750 North 1200 East, Utah State Univ., Logan, UT 84322-8700, U.S.A. Diret inquiries to author Hansen (E-mail: hansen@.usu.edu). has been proven to provide protetion against various degenerative diseases (Kaur and Kapoor 2001; Cheung and others 2003). Dietary antioxidants from fruits and vegetables have been shown to inrease plasma antioxidant apaities (Kaur and Kapoor 2001). Other researh has also shown that the onsumption of plants is assoiated with lower inidenes of aner (Kaur and Kapoor 2001). A great number of naturally ourring substanes have been reognized to have antioxidant abilities. Flavonoids and other phenoli ompounds are among the major ontributors to suh ativity (Kang and others 2003). Other ative ompounds inlude fiber, onjugated isomers of linolei aid, some vitamins, alium, uri aid, glutathinone, and protease inhibitors. These ompounds may work independently or synergistially (Kaur and Kapoor 2001). Mushrooms have been reognized as a nutritious food and an important soure of bioative ompounds. Studies in antioxidant apaity of mushrooms have begun to take the spotlight in reent researh (Mattila and others 2001; Mau and others 2002, 2004; Badalyan 2003a, 2003b; Cheung and others 2003). Antioxidant ativities have been disovered in some mushrooms at different growth stages (Badalyan 2003a, 2003b). However, data from most studies often fous on ertain antioxidant apaity of fruiting bodies. Studies onerning antioxidant apaity of mushroom myelia using radial savenging apaity as measurement are rare. There have been no data on the antioxidant ativity for myelia grown in whey permeate ulture and its hanges ourring during growth of myelia. Our previous researh (Wu and Hansen 2006) suggests that whey permeate an be utilized as an alternative medium for the growth of Lentinus edodes. However, the interation between the type of media and harvesting time hanges the proximate omposition of the myelia. C 2007 Institute of Food Tehnologists Vol. 73, Nr. 1, 2008 JOURNAL OF FOOD SCIENCE M1 doi: 10.1111/j.1750-3841.2007.00595.x Further reprodution without permission is prohibited
The objetive of this study was to evaluate the effets of whey permeate-based medium and harvesting time on the antioxidant apaity measured by radial savenging apaity, phenoli ontent, and rude water-soluble polysaharide (WSP) of L. edodes myelia. Materials and Methods Culture, subulture, and storage ondition L. edodes ulture was obtained from the Amerian Type Culture Colletion (ATCC, Manassas, Va., U.S.A.) and maintained in a potato dextrose agar (PDA) (Beton Dikinson, Sparks, MD., U.S.A.) slant at 4 C. The seed ulture was transferred to a Petri dish ontaining PDA medium and inubated at 25 C for 8 d. Miroorganisms were subultured at regular intervals (50 d) to maintain viability (Mukhopadhyay and others 1999). Media preparation The fungi were grown on both whey permeate and ontrolled medium (syntheti medium). The whey permeate was supplied by Gooding Cheese and Whey Plant (Glanbia In., Gooding, Idaho, U.S.A.). The latose onentration of the whey permeate-based medium was adjusted to 4.5% (Mukhopadhyay and others 2002). The ph of whey permeate was adjusted to ph 5.5. Sterilization of the whey permeate media was ahieved by filtration through a 0.2-μm membrane filter (Fisherbrand, Pittsburgh, Pa., U.S.A.). The ontrol medium onsisted of 10 g/l yeast extrat (Difo Laboratories, Detroit, Mih., U.S.A.), 10 g/l gluose (Mallinkrodt Chemials, Phillipsburg, N.J., U.S.A.) and 10 g/l peptone (Beton Dikinson,) (Song and others 1987). The ph of the ontrol medium was also adjusted to ph 5.5 prior to autolaving. Inoulation and fermentation ondition Myelial agar diss from the margin of 8-d-old olonies were isolated from the PDA ulture by using inoulating loops. The myelia agar pellets were transferred to potato dextrose broth (PDB) (Beton Dikinson,) enrihment media (5 diss/ 100 ml media) and ultivated at 25 ± 1 C, ph 5.5 for 8 d. Fermentation was arried out in 2000 ml baffled flasks. Eah flask ontained 600 ml media. About 60 ml of the same PDB ulture were inoulated in eah 600 ml media. The flasks were inubated at 25 ± 1 C on a rotary shaker (New Brunswik Sientifi Co. In., Series 25 Inubator Shaker, Edison, N.J., U.S.A.) at 120 rpm until the assigned harvesting days (Mukhopadhyay and others 1999; Wasser and others 2003; Wu and Hansen 2006). Harvesting The fermentation was terminated at 4 different intervals (5, 10, 15, 20 d). At the end of eah fermentation proess, the myelia were harvested and separated from the media by filtration through Whatman Nr 1 filter paper. About 20 ml of distilled water were used to wash the filtered myelia 3 times (Elisashvili and others 2004). The growth of myelia was measured as dried myelial mass. The filtered myelia were dried by lyophilization for 40 h. Dried myelia were kept at 4 C until use (Mukhopadhyay and others 1999). Preparation of myelial extrats The freeze-dried myelia (about 0.02 g) were extrated with 80% (v/v) methanol (1 ml) or distilled water (1 ml) at 80 C for 30 min. The extrats were entrifuged at 6000 rpm for 5 min, and the supernatant was olleted. Extrats from 80% methanol were vaporized under vauum at 40 C for 3 h. All extrats were stored at 20 C. The powders of methanol extrats were reonstituted with 1 ml assay buffer from the antioxidant assay kit (Cayman Chemial Co., Ann Harbor, Mih., U.S.A.) prior to analyses. Trolox equivalent antioxidant apaity (TEAC) assay The total antioxidant apaity (TAC) was measured as desribed by Miller and others (1993, 1995) and Miller and Rie-Evans (1997). The antioxidant assay kit was purhased from Cayman Chemial Co. Absorbane was monitored at 750 nm for 5 min. Changes in absorbane were alulated and plotted with respet to onentrations of the standards and samples. The final TEAC value was expressed as millimoles of Trolox antioxidant equivalent (TAE) per gram of dried myelia. Higher TEAC values suggested a stronger free radial savenging apaity. Crude WSP and protein ontents The proedure used in determining the total protein ontent was adapted from the AOAC Offiial Method of Analysis (1990). The Kjeldahl method was used to determine the total nitrogen. The fator of 6.25 was used to alulate the rude protein ontent (Wang and others 2001). The rude water WSP were measured by the phenol-sulfuri aid method as desribed by Dubois and others (1956). The dried myelia were mixed with distilled water at a ratio of 1:4. Mixture was boiled in a water bath at 100 C for 12 h. The extrats were then entrifuged (Bekman Coulter TM, Allegra TM X-22 Centrifuge, Germany) at 9000 rpm for 15 min. The supernatant was olleted for the assay with the phenol-sulfuri aid (Basbitskaya and others 2003). Absorbane was measured at 490 nm with standard gluose solutions from 0 to 100 mg/l spetrophotometrially (Perkin Elmer, Lambda 3B, Norwalk, Conn., U.S.A.). Determination of total phenoli ontent The total phenoli ontent was determined using the Folin Cioalteau method (Singleton and Rossi 1965). About 100 μlofthe extrats, 625 μl of Folin Cioalteau reagent, 4 ml distilled water, and 4 ml of 10% sodium arbonate were added and mixed ompletely. After 2-h inubation in a 35 C water bath, the absorbane of the solution at 765 nm was measured with a spetrophotometer (Perkin Elmer). Quantifiation was based on the standard urve for galli aid (0 to 0.5 mg/ml). The results were expressed in milligrams of galli aid equivalents (GAE) per gram of dried myelia. Statistial analysis The results were expressed as means ± the standard error of means (SEM) of the tripliate experimental results. Statistial analysis was performed using SAS version 9.1 (SAS Inst. In., Cary, N.C., U.S.A.). Data sets were evaluated by analysis of variane (ANOVA). Statistially signifiant differene was identified at the 95% onfidene level. Post-ho means omparison were made based on P value (α = 0.05) using the Ryan Einot Gabriel Welsh method. Correlation oeffiient (r) was used to evaluate the relationship of myelia omposition and TEAC values. A 2-way fatorial model with tripliates was used for analyzing the hemial results. The type of media (whey permeate, ontrol media) and harvesting times (5, 10, 15, 20 d) were set as fixed fators. Dependent variables were analyzed using a natural log transformation to satisfy the assumption of normality and homosedastiity of the data. Untransformed data were used in tables and figures for the ease of interpretation. M2 JOURNAL OF FOOD SCIENCE Vol. 73, Nr. 1, 2008
Results and Disussion Water-soluble polysaharides Data in Figure 1 show that whey permeate always resulted in a higher onentration of WSPs in the myelia, as ompared to the ontrolled media, at eah level of harvesting time. The interation term between harvesting time and type of media was found to be statistially signifiant (P = 0.0258, Table 1). The fator of harvesting time was nonsignifiant (P = 0.0916, Table 1). The type of media was found to ontribute to most of the variability. Highly statistially signifiant differenes (P < 0.0001) were found between the pooled means of myelia grown on whey permeate (2.6 10 2 ± 36 mg polysaharide/g dried myelia) and the pooled means of myelia grown on the ontrolled media (72 ± 4mgpolysaharide/g dried myelia). This indiated that the prodution of polysaharide greatly depended on the ompound presented in the medium. As shown in our previous study (Wu and Hansen 2006), whey permeate was more favorable than the ontrolled media in the prodution of WSPs for L. edodes. The highest mean value of WSP was found in whey-permeate grown myelia harvested on day 10 (4.1 10 2 ± 71 mg polysaharide/g dried myelia; Figure 1). The WSP/g of myelia inreased from day 5 to day 10. However, a redution in the WSP/g of myelia ourred from day 10 to day 20. By day 20, WSP/g of myelia was statistially signifiantly lower than the myelia harvested on day 10. WSP ontent of the myelia (mg WSP/g dried myelia) 600 500 400 300 200 100 0 a,b a 4 6 8 10 12 14 16 18 20 22 Harvesting Time (d) a,b Total phenoli ontent Cheung and others (2003) said that most soluble omponents in mushroom fruiting bodies have high polarity. The antioxidant ativities of mushrooms seemed to rely highly on the polarity of the solvent used in extration. Extrats using polar solvents suh as methanol and water had higher total phenoli ontent (TPC) than those extrated with hexane (Cheung and others 2003; Kang and others 2003). Total phenoli ontent varied in myelia harvested at different times and grown with different media. Whey permeate grown myelia had less phenoli ompounds, ranging from 4.2 ± 0.1 to 8.0 ± 0.8 mg GAE/g dried myelia, while myelia grown on ontrolled media were omposed of phenoli ompounds ranging from 9 ± 1to14± 1 mg GAE/g dried myelia (Figure 2 and 3). Methanol extrats. There was no statistially signifiant interation between the harvesting time and the type of media on the variability of total phenoli ontent in myelia, if they were extrated with 80% methanol. Harvesting time and type of media were found to ontribute to most of the variability independently (P = 0.0002 or 0.0001, respetively; Table 1). As shown in Figure 2, myelia harvested on day 5 or day 15 onsisted of signifiantly higher phenoli ontent than myelia harvested on day 10 or day 20, regardless of the type of media used for growth. When grown in whey permeate, myelia had a lower overall mean value of total phenoli (5.9 ± 0.5 mg GAE/g dried myelia), in omparison to myelia grown in ontrolled media (11 ± 1 mg GAE/g dried myelia). However, in our study, in the methanol extrats, myelia Whey permeate Controlled media b, Figure 1 --- Water-soluble polysaharides ontent of the myelia due to the interation of the types of media and the harvesting time. Means sharing letter are not different at P < 0.05. Table 1 --- Summary of signifiane as determined by the ANOVA. WSP a PRO b PCME PCWE d TEAC-ME e TEAC-WE f Media NS NS Harvesting time NS Media harvesting time NS NS = signifiant at P < 0.05; = signifiant at P < 0.01; NS = not signifiant at P < 0.05. a Water-soluble polysaharides (WSP). b Protein ontent (PRO). Phenol ontent in methanol extrats (PCME). d Phenol ontent in water extrats (PCWE). e TEAC in methanol extrats (PCME). f TEAC in water extrats (PCWE). Vol. 73, Nr. 1, 2008 JOURNAL OF FOOD SCIENCE M3
grown in whey permeate was found to produe phenoli ompounds with a value lose to or even higher than the value of L. edodes fruiting bodies reported by Cheung and others (2003), whih was about 4.79 ± 1.2 mg GAE/g dried fruiting bodies weight. Water extrats. There was a statistially signifiant interation (P < 0.05) between the harvesting time and the type of media on the variability of total phenoli ontent in myelia (Table 1), if they were extrated with water. Water extrats from myelia grown on whey permeate exhibited peak values of total phenoli ontent at day 5, while extrats from myelia grown on the ontrolled media exhibited peak values at day 10 (Figure 3). Phenoli ontent of myelia in water extrats evaluated in this study was muh higher (Figure 3) than phenoli ontent of the fruiting bodies of L. edodes (1.33 ± 0.04 mg GAE/g dry weight) reported by Cheung and others (2003). When grown in whey permeate, myelia had a lower overall mean value of total phenoli (6.2 ± 0.6 mg GAE/g dried myelia), in omparison to myelia grown in ontrolled media (17 ± 1 mg GAE/g dried myelia). Water extrats showed different patterns in phenoli ontent if different media were used for ultivation. When ontrolled media was used, the highest phenoli ontent was observed on day 10; and then a redution of phenoli ontent was seen (Figure 3). When whey permeate was used, the pattern of phenoli ontent in water extrat was similar to the pattern for methanol extrats. Myelia 16 14 Whey permeate Controlled media Figure 2 --- Total phenoli ontent of Lentinus edodes grown on whey permeate and ontrolled media extrated by 80% (v/v) methanol. Phenoli ontent (mg GAE/g dry wt) 12 10 8 6 4 2 0 0 5 10 15 20 25 Harvesting time (d) Phenoli ontent (mg GAE/g dry wt) 25 20 15 10 5 Whey permeate Controlled media Figure 3 --- Total phenoli ontent of Lentinus edodes grown on whey permeate and ontrolled media extrated by water. 0 0 5 10 15 20 25 Harvesting time (d) M4 JOURNAL OF FOOD SCIENCE Vol. 73, Nr. 1, 2008
Protein ontent of the myelia ( w/w %) 50 40 30 20 10 a a,b a a b, a,b b, Whey permeate Controlled media Figure 4 --- Protein ontent of the myelia due to the interation of the types of media and the harvesting time. Means sharing letter are not different at P < 0.05. 0 4 6 8 10 12 14 16 18 20 22 Harvesting Time (d) harvested on day 5 or day 15 onsisted of higher phenoli ontent than myelia harvested on day 10 or day 20. A slightly higher phenoli ontent was found in water extrats than in methanol extrats. This suggested that the inrease of polarity ould ontribute to more effetive extration of phenoli ompound in the myelia of L. edodes. Protein ontent The rude protein ontent of myelia was found to be statistially signifiantly different due to the effet of the type of media (Figure 4). Myelia grown on the whey permeate media had an overall mean value of 19 ± 2% in rude protein ontent, whih was about 50% of the overall mean of myelia grown on ontrolled media (40 ± 3%). Total antioxidant apaity The antioxidant ativities of myelia extrats were evaluated by means of Trolox equivalent antioxidant apaity (TEAC) assay. Miller and others (1993) introdued the TEAC assay by the measurement of the olor in the solution with ABTS + radial ation. In the presene of antioxidative substanes, the olorization proess an be delayed due to the radial savenging ability of antioxidants. Miller and others (1995) in their later study further demonstrated that the assay ould be applied to pure solutions as well as mixtures of substanes. It provided reproduible and pratial ways to estimate antioxidant ativity, espeially for food matrixes. Both methanol extrats and water extrats of L. edodes were found to exhibit free radial savenging apaity (Table 2). Methanol extrats. No interation between harvesting time and type of media was found in the ANOVA for the TEAC of the methanol extrats. The harvesting time was found to ontribute to most of the variability (P < 0.0001) (Table 1). There was no signifiant differene between methanol extrats from myelia grown on either medium. This indiated that the antioxidative ability of methanol extrats from myelia was not affeted by substituting ontrolled medium with whey permeate. For methanol extrats, highly statistially signifiant differenes were found among the TEAC of myelia harvested at different time intervals. Methanol extrats of myelia harvested on day 10 showed Table 2 --- Antioxidant apaity (AOA) of the methanol and water extrats of L. edodes myelia grown on whey permeat (WP) or ontrolled media (CM) expressed as millimoles of Trolox antioxidant equivalent (TAE) per gram of dried myelia (mmol Trolox/g dry weight). Methanol extrat Water extrat Harvest time (d) WP grown CM grown WP grown CM grown 5 0.06 ± 0.02 b 0.11 ± 0.02 b 0.12 ± 0.03 b 0.13 ± 0.03 b 10 0.28 ± 0.03 a 0.29 ± 0.06 a 0.29 ± 0.03 a 0.16 ± 0.04 a,b 15 0.11 ± 0.02 b 0.12 ± 0.01 b 0.07 ± 0.01 b 0.10 ± 0.02 b 20 0.06 ± 0.02 b 0.12 ± 0.03 b 0.22 ± 0.02 a 0.20 ± 0.02 a Means from the same type of extrat olumns and sharing the same letter are not signifiantly different (P < 0.05). signifiantly higher ability in inhibiting free radials. When expressed as millimoles of Trolox antioxidant equivalent per gram of dried myelia (mmol TAE/g dry weight), TEAC of myelia grown on whey permeate and ontrolled media were 0.28 ± 0.03 and 0.29 ± 0.06, respetively for day 10 harvest. These TEAC values were at least 2 times higher than extrats from the myelia harvested on other intervals. As myelia grew, the TEAC inreased until it reahed a peak value at day 10; and this was followed by a redution of the TEAC value (Table 2). Badalyan s study (2003b) suggested that the strength of antioxidant ativities in ertain types of mushroom ould depend on the length of its growth time. In their study, studying another mediinal mushroom Flammulina velutips, they found no notable antioxidant ativity until the growth at week 3. This suggested that the timing of myelia harvesting ould ontribute to the variability of their nutrieutial properties. Water extrats. The interation term between the type of media and harvesting time was found to be statistially signifiant (P = 0.045), whih suggested a ontribution of that interation to variability in the TEAC of harvested myelia (Table 1). Unlike methanol extrats, water extrats of the myelia exhibited peak values of TEAC at both days 10 and 20 (Table 2). The TEAC of water extrats from myelia grown on whey permeate was slightly higher than one grown on ontrolled media, even though no signifiant differene was found between the types of media. Harvesting time Vol. 73, Nr. 1, 2008 JOURNAL OF FOOD SCIENCE M5
was found to ontribute to most of the variability in TEAC (P < 0.001), as shown in Table 1. Water extrats of myelia harvested on the 10th and 20th day showed signifiantly higher ability in the inhibition of free radials. The TEAC values of myelia grown on whey permeate and ontrolled media were 0.29 ± 0.03 and 0.16 ± 0.04 mmol TAE/g dry weight, respetively, if the harvesting was onduted on day 10. Pellegrini and others (2003) provided detailed data on the antioxidant ativity of vegetables and fruits by the TEAC assay. Spinah and pepper in the vegetable ategory were found to have the highest antioxidant apaity. Among the 30 different types of fruits, high antioxidant apaity was found in berries, suh as blakberry, strawberry, and raspberry. Compared to the data provided by Pellegrini and others (2003) (Table 3), the TEAC values of myelia grown on whey permeate and harvested on day 10 in our study were muh higher, regardless of the type of extrat solvent used. Correlation of myelia omposition and TEAC value High TEAC values and rude WSP ontent were deteted in mushroom myelia grown in whey permeate and harvested on day 10. In spite of a lower level of phenoli ontent in the whey permeate grown myelia when ompared to myelia from ontrolled media, similar or higher levels of total antioxidant apaity were observed on day 10. However, neither total phenoli ontent nor rude WSP ontent in this study was found to be signifiantly orrelated with the TEAC values of either methanol or water extrats diretly. Rather than a single type of omponent, it is possible that TAC of mushroom extrats is the result of a sum of various antioxidative omponents in extrats, inluding WSP and various phenoli ompounds. Mau and others (2005) reported in their researh on Ganoderma tsugae, a different type of mushroom, that total phenol ontent in methanol extrats did not orrelate with the disrepany in antioxidant ativities. In our study, when water was used as solvent for extration, the TEAC value of the extrat was found to be loosely but positively orrelated to the ratio of WSP to protein (r = 0.46, P = 0.023) as well as the ratio of WSP to total phenoli in water extrats (r = 0.45, P = 0.026). The low value of orrelation oeffiient suggested that in spite of a signifiant orrelation between the TEAC value and the 2 ratio values, the relationship was nonlinear. Liu and others (1997) suggested in their researh that the free radial savenging ativity of polysaharide ould depend on the ratio of WSP to protein. More speifially, the ratio of bound protein in the polysaharide protein omplexes was onsidered to be more essential to the savenging ativity. A loose orrelation was also found between the TEAC values of methanol extrats and water extrats (r = 0.60, P = 0.039). The types of media were found to affet the orrelations of various omponents in myelia with TAC of mushroom. When myelia grew in the ontrolled media, no signifiant orrelation was found between the hemial parameters and the TEAC values. However, when whey permeate was used to grow L. edodes, the ratio of WSP to total phenoli in extrats was loosely but positively orrelated to the TEAC values of mushrooms (in water extrats: r = 0.58, P = 0.048; in methanol extrats: r = 0.64, P = 0.026). Again, the low values of orrelation oeffiient suggested that in spite of a signifiant orrelation between the TEAC value and the 2 ratio values, the relationship was nonlinear. For methanol extrats from WP grown myelia, there was a lose to linear relationship between the TEAC value and the ratio of WSP to protein (r = 0.82, P = 0.011). The TEAC value inreased along with the inrease of the ratio value. The TEAC values of the methanol extrats were also found to be loosely but negatively orrelated to total protein ontent of myelia (r = 0.67, P = 0.017). Due to the onern of potential toxi and arinogeni effets in some syntheti antioxidants, studies in the effetiveness of natural antioxidants have gained inreased interest sine the 1990s (Kaur and Kapoor 2001). A number of plant extrats and plant byproduts have been identified with antioxidative properties. Brooli, abbage, auliflower, spinah, and beet have been reported to have high levels of antioxidant apaity. The ative omponents being studied inlude fiber, polyphenol, flavonoids, vitamins, and other phytohemials (Kaur and Kapoor 2001). It is believed that the antioxidant properties of phenolis are a result of their ability to at as reduing agents, hydrogen donors, and free radial quenhers (Rie-Evans and others 1997; Kaur and Kapoor 2001). Depending on the struture of the moleules, phenolis an also at as metal helators whih prevent the atalyti funtion of metal in the proess of initiating radials (Salah and others 1995; Kaur and Kapoor 2001). There are few reports on the antioxidant apaity of polysaharides. The mehanism of polysaharides as antioxidative reagents is still not fully understood (Liu and others 1997; Jiang and others 2005). Chen and others (2005) found that in lowgrade green tea, whih ontained low levels of phenoli ontent, Table 3 --- Comparison of TEAC values of various vegetables, fruits, and L. edodes myelia grown on whey permeate. Extrat solvent Growth length (d) TEAC (mmol/g dry weight) Myelia a L. edodes from WP Methanol 10 0.28 ± 0.03 Water 10 0.29 ± 0.03 Moisture b (%) TEAC (mmol/kg FW) TEAC d (mmol/g dry weight) Vegetable Red pepper 92.21 8.40 0.108 Spinah 91.40 8.49 0.099 Asparagus 93.22 3.92 0.058 Zuhini 94.64 2.86 0.053 Radish 95.27 2.22 0.047 Beet 87.58 5.21 0.042 Fruit Blakberry 85 20.24 0.135 Strawberry 90 10.94 0.109 Raspberry 84 16.79 0.105 Pineapple 85 9.91 0.066 Orange 86 8.74 0.062 a Data of myelia were obtained from this study. b Moisture ontents of vegetables and fruits were obtained from USDA (1981, 2006), respetively. TEAC values, based on fresh weight, were initially reported by Pellegrini and others (2003). d For the ease of omparison, TEAC values of fresh weight were onverted to values based on dry weight, aording to data from USDA. M6 JOURNAL OF FOOD SCIENCE Vol. 73, Nr. 1, 2008
polysaharides ontributed to the antioxidant apaity. Polysaharides from green tea exerted inhibitory effets on both hydroxyl and superoxide radials. Jiang and others (2005) reported that WSPs extrated from Isaria farinose myelia had Fe 2+ helating ativity. This minimizes the amount of atalyti metal available for the Fenton Reation, whih ultimately inhibits the generation of free radials (Jiang and others 2005). When myelia were grown in whey permeate and harvested on day 10 in our study, rude WSP made up almost 40% of the dried weight. It was found that gluan and its derivatives, whih were identified to be the major omponents of the water soluble fration of polysaharides in mushroom fruiting bodies (Mizuno 1995), ould exhibit various degrees of free radial savenging ativities in Tsiapali and others study (2001). The antioxidant ativities appeared to be partially due to the monosaharide onstituents. And the polymeri struture in polysaharides seems to enhane the free radial savenging apaity, when ompared to monomeri units, suh as dextrose (Pathen and others 1987; Tsiapali and others 2001). In Pathen and others study (1987), the ability to savenge radiation indued free radials was ompared between D-gluose and its polymer gluan. The gluan onentration required for exhibiting the savenging ability was 10 times lower than the gluose onentration. Tsiapali and others (2001) proposed that this enhanement may be due to the ease of abstration of an anomeri hydrogen from one of the monosaharide units on the bakbone rather than from the reduing ends. Conlusions This study provided data on total antioxidant apaity (TAC), phenoli ontent, and rude WSP of myelia grown on WPbased media at different growth stages in omparison with the ontrol media. In this study, both methanol extrats and water extrats of L. edodes were found to exhibit free radial savenging apaity, whih was used to measure the total antioxidant apaity. Harvesting time played an important role in the variability of the free radial savenging apaity. Methanol and water extrats of myelia harvested on day 10 showed signifiantly higher ability to inhibit free radials. In methanol extrats, the antioxidative ability of myelia was not affeted by the substitution of ontrolled medium with whey permeate. In water extrats, the antioxidative ability of myelia was affeted by the interation between the types of media and harvesting time. The proportion of different hemials in the myelia hanged due to the impat of the type of media and harvesting time. When grown in whey permeate and extrated with methanol, myelia had a lower overall mean value of total phenoli (5.9 ± 0.5 mg GAE/g dried myelia) in omparison to myelia grown in ontrolled media (11 ± 1 mg GAE/g dried myelia). Similar to methanol extrats, water extrat of whey permeate grown myelia had a lower overall mean value of total phenoli (6.2 ± 0.6 mg GAE/g dried myelia) in omparison to myelia grown in ontrolled media (17 ± 1 mg GAE/g dried myelia). But ompared to other reported phenoli ontents of the fruiting bodies of L. edodes, phenoli ontent of myelia evaluated in our study was muh higher. Growing myelia in whey permeate was more favorable for the prodution of WSP in this study in omparison to the syntheti medium. The maximum prodution of WSP (4.1 10 2 ± 71 mg polysaharide/g dried myelia) in this study was found in the whey-permeate grown myelia harvested on the 10th day. It is possible that TAC of mushroom extrats an be the result of a sum of various antioxidative omponents suh as WSP and phenoli ompounds. Aknowledgments Appreiation is expressed to Dairy Management In., Rosemont, Ill., Utah Agriultural Experiment Station, Logan, Utah, and Glanbia Foods, Gooding, Idaho. 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