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Prevention of Agaricus bisporus postharvest browning with tyrosinase inhibitors
Postharvest Biology and Technology 39 (2006) 272–277
Prevention of Agaricus bisporus postharvest browning with tyrosinase inhibitors Ohad Nerya a,b , Ruth Ben-Arie b , Tal Luzzatto a , Ramadan Musa a , Soliman Khativ a , Jacob Vaya a,c,∗ a
c
Laboratory of Natural Compounds for Medicinal Use, Migal, Galilee Technological Center, P.O. Box 831, Kiryat Shmona 11016, Israel b Fruit Storage Research Laboratory, Kiryat Shmona 10200, Israel Department of Biotechnology and Environmental Science, Tel-Hai Academic College, Israel Received 6 March 2005; accepted 30 October 2005
Abstract Postharvest browning of Agaricus bisporus mushrooms is a severe problem that reduces the shelf life of harvested mushrooms. Mushroom browning occurs mainly as a result of tyrosinase activity, an enzyme belonging to the polyphenol oxidase (PPO) family and known to be a key enzyme in melanin biosynthesis. An ethanolic extract from licorice roots (Glycyrrhiza glabra) and [3-(2,4-dihydroxyphenyl propionic acid)] (DPPacid) isolated from fig leaves and fruit have been shown to inhibit tyrosinase activity. Adding these inhibitors to sliced mushrooms had a very strong inhibitory effect on browning, but pre-storage immersion of intact mushroom in the licorice extract did not prevent browning after 8 days storage at 4 ◦ C. By contrast, treatment with DPPacid at 1 g/mL reduced browning by half. Measurement of inhibitor uptake by mass spectra (MS) and assay of tyrosinase activity indicated that penetration into the mushroom tissue was inadequate for tyrosinase inhibition. Moreover, DPPacid was found to be unstable in the mushroom tissue and within a short time it was, presumably, metabolised. © 2005 Elsevier B.V. All rights reserved. Keywords: PPO; Licorice root extract; Browning; DPPacid
1. Introduction Agaricus bisporus mushrooms have a short shelf life compared to most fruit and vegetables. The intact mushrooms lose their commercial value within a few days, due to senescence, water loss, microbial attack and browning. Mushroom browning occurs as a result of two distinct mechanisms of phenol oxidation: (a) activation of tyrosinase, an enzyme belonging to the polyphenoloxidase (PPO) family; (b) and/or spontaneous oxidation (Jolivet et al., 1998). The PPO family includes catechol oxidase and laccase, both of which oxidize diphenols to the corresponding quinones (Kertesz and Zito, 1962), but the former can also oxidize some monophenols into o-diphenols (Mayer, 1987).
∗
Corresponding author. Tel.: +972 4 6953512; fax: +972 4 6944980. E-mail address: [email protected] (J. Vaya).
0925-5214/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2005.11.001
This activity is also referred to as ‘phenolase’ or ‘tyrosinase’, which has become a trivial name for catechol oxidase. The monophenolase activity of tyrosinase is usually much lower than the diphenolase activity, and this ratio, which can vary from 1:40 to 1:1, (Vamos-Vigyazo, 1981) is regarded as the key step in browning biosynthesis. Several authors have described tyrosinase activity in the fruit body of A. bisporus during its development and there is a consensus that a latent form of tyrosinase occurs, the extent of which is not yet clear (Yamaguchi et al., 1970; Flurkey and Ingebrigtsen, 1989). Latent tyrosinase can become active after contact with bacteria or a toxin such as tolaasin (Soler-Rivas et al., 1997). The problem of postharvest browning of mushrooms has been tackled from several aspects. Addition of calcium chloride during irrigation enhanced the white color at harvest and decreased postharvest browning (Philippoussis et al., 2001); modified atmosphere packaging reduced browning rate, due to reduced oxygen levels (Kuyper et al., 1993). Browning of
O. Nerya et al. / Postharvest Biology and Technology 39 (2006) 272–277
fresh sliced mushrooms was inhibited by immersion in citric acid (4%) or hydrogen peroxide (5%) (Brennan et al., 2000). In this case, however, the authors suggested that shelf life extension was the result of antibacterial activity rather than tyrosinase inhibition. Chemical control of enzymatic browning includes chelation of the copper present at the active site of the enzyme (Martinez and Whitaker, 1995) and reduction of the diquinone to its uncolored form (Kubo and Kinst-Hori, 1998). In previous work (Nerya et al., 2003), we extracted three new tyrosinase inhibitors from licorice roots (Glycyrrhiza glabra): glabridin, a non-competitive inhibitor eliciting 50% inhibition at a concentration (IC50 ) of 0.09 M, glabrene and isoliquiritigenin—both competitive inhibitors with IC50 values of 3.5 and 8.1 M, respectively. The molecular structures of these three inhibitors are given in Fig. 1. McEvilly et al. (1992) isolated several inhibitors of tyrosinase activity from fig latex (Ficus carica), one of them identified as DPPacid (Fig. 1). Shimizu et al. (2001) examined the biochemical parameters of tyrosinase inhibition by DPPacid and concluded that it is a competitive inhibitor with an IC50 of 3.2 M. We have isolated this compound from both fig leaves and fruit (unpublished data). The aim of the present study was to inhibit the postharvest browning of A. bisporus mushrooms (strain U1) by applying new safe, edible tyrosinase inhibitors from licorice roots and fig fruit. During the study we observed differences in the efficiency of browning inhibition between intact and sliced mushrooms. We have, therefore, also addressed the question whether the difference is due to limited permeability of the intact mushroom to the inhibitors.
2. Materials and methods 2.1. Tyrosinase inhibition Tyrosinase (EC1.14.18.1, Sigma product T7755) activity was determined using 0.03 mL tyrosinase (333 units/mL) in
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PBS (phosphate buffer solution) (50 mM, 0.07 mL) at pH 6.5 and 2 L of the tested inhibitor compounds (0.18–18 g/mL) dissolved in absolute ethanol in 96 well plates. After 5 min incubation at room temperature, 0.1 mL l-tyrosine (2 mM) was added and optical density at 492 nm measured every 5 min for 20 min (Elisa SLT Labinstruments co. A-5082). 2.2. Inhibition of sliced mushroom browning A. bisporus mushrooms (strain U1—second harvest) were harvested at a commercial farm. For IC50 determination, the mushroom caps were sliced and 20 L aliquots of the inhibitors (0.18–0.91 g/mL) were pipetted onto the cut surface. After 24 h incubation at 20 ◦ C and 97% relative humidity, the browning of the treated area was monitored by reading the L, a, b parameters with a CR-200 Minolta chromameter. 2.3. Inhibition of intact mushroom browning The inhibition of browning during storage was examined using mushrooms immersed in the tested inhibitors or in water (control) for 10 min. After drying, the mushrooms were packed in high-density polyethylene bags and stored at 4 ◦ C for 10 days. Storage changes in mushroom cap color (L) were monitored on days 1, 3, 5 and 8. 2.4. Inhibition of tyrosinase activity in vivo The influence of the inhibitors on mushroom tyrosinase activity was tested using cut mushroom caps that had been immersed in the tested inhibitors (0.6 and 1.35 g/mL for DPPacid and licorice extract, respectively), or in water (control) for 10 min. After drying, the mushroom caps were cut and frozen in PBS (50 mM at pH 6.5). For enzyme extraction, the frozen caps were ground in the buffer and centrifuged at 4000 rpm for 20 min. An aliquot (0.3 mL) of the supernatant was diluted in 0.34 mL PBS and 0.36 mL tyrosine 2 mM was added. The rate of DOPA-quinone formation was monitored at 475 nm, after 10 min incubation at room temperature
Fig. 1. Molecular structure of tyrosinase inhibitors extracted from fig leaf (A) DPPacid and from licorice root: (B) glabridin, (C) isoliquiritigenin and (D) glabrene (Vaya et al., 1997).
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using a Shimadzu A-187 spectrophotometer. Protein content was measured using Coomassie blue (Bradford, 1976), and the tyrosinase activity was calculated. The specific effect of fresh inhibitors on the extracted enzyme activity in vitro was assayed by adding 2 L of the test compounds to the reaction mixture described above. Two different experiments were conducted to compare the kinetics of tyrosinase activity in vitro and in vivo. The in vitro test was performed with a commercially purified mushroom tyrosinase and the in vivo test was conducted with an enzyme preparation from peeled mushrooms. The in vitro reaction was started after incubation periods of the enzyme with the inhibitors for 10, 70 and 250 min. In vivo, the peeled caps were immersed in the inhibitor solution for 10 min and the enzyme was extracted after 10, 70 and 250 min incubation. 2.5. Mushroom cap permeability to inhibitors Peeled and intact mushrooms were immersed in 10 g/mL solutions of the test inhibitors for 10 min. After drying, the mushrooms were peeled and longitudinal slices 2 mm thick were cut, using a vegetable peeler. Extraction for tyrosinase assay activity was as described above. In addition, the amount of DPPacid absorbed in the mushroom tissue was extracted by grinding 5–93 mg cut tissue in 2 mL double distilled water (ddw) pH 2. Ethyl acetate (4 mL) was added and after strong vortex mixing, the extract was collected by centrifugation. Extraction with ethyl acetate was repeated twice. After evaporation of the solvent, the dry residue was dissolved in 0.5 mL acetonitrile. The DPPacid content in the tissue was assayed by MS, as described below. 2.6. Quantification of DPPacid by MS/MS The MS/MS (Micromass Quattro Ultima MS, UK) analysis of the oxidized products was performed using electrospray negative ions (ES− ) mode. The source temperature of the mass spectrometer was set at 150 ◦ C, with a cone gas flow of 124 L/h and a desolvation gas flow of 525 L/h. Collision-induced dissociation MS of the DPPacid using 15-collision energy and 3 (kV) capillary voltage revealed with m/z 181 (M−1 ) and its fragmentation at m/z 137 (M-1CO2). Multiple-reaction-monitoring (MRM) was performed using the same conditions and analyzed by the m/z 137 ion fragmentation.
Table 1 Inhibitory effects of a licorice root extract and DPPacid on mushroom tyrosinase activity in vitro and on mushroom slice browning in vivoa AA
IC50 (g/mL) DPPacid
Licorice extract
In vitro tyrosinase activity In vivo browning
0.27 ± 0.05 0.18 ± 0.07
0.01 ± 0.0005 0.11 ± 0.05
a
Data are averages ± S.E., n = 3–8 replicates.
the in vivo test there was very little difference between their degrees of browning inhibition (Table 1). With the information that these products have not only a strong inhibitory effect on the purified enzyme, but also a strong potential as browning inhibitors of the cut surface of mushroom caps, a trial was conducted to inhibit browning of intact mushrooms in storage. A pre-storage mushroom immersion in the licorice extract did not prevent browning after 8 days storage at 4 ◦ C, and although immersion in DPPacid at a concentration of 1 g/mL reduced browning by half, compared to the untreated control, the variance was too great for the difference to be significant (Fig. 2). When the kinetics of tyrosinase activity and inhibition were compared in vivo and in vitro two different patterns of tyrosinase activity and inhibition evolved. With the commercial enzyme, a very strong inhibition of tyrosinase activity that was observed after 10 min, gradually dissipated after longer incubations, with the enzyme loosing 75% of its activity (Fig. 3A). Inhibition by the licorice extract and DPPacid decreased from 99% and 86%, respectively, after 10 min incubation, to 83% for both, after 70 min. After 250 min neither had any inhibitory effect on commercial tyrosinae activity. With the mushroom extract, there was a gradual increase in enzyme activity with time, even in the untreated control, and the effects of both inhibitors were not significant even after 10 min (Fig. 3B).
3. Results The tyrosinase inhibitory potentials of DPPacid and licorice extract were compared, in vitro on a commercial enzyme preparation from mushrooms, and in vivo, using a chromameter, on the assumption that browning of disks cut from mushroom caps resulted from tyrosinase activity. The licorice extract showed a very strong inhibition of tyrosinase activity compared to DPPacid in the in vitro test, whereas in
Fig. 2. The effect of licorice extract and DPPacid on intact mushroom cap browning, after storage for 8 days. (Initial average L value was 92.3 ± 2.5).
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Fig. 4. The amount of DPPacid in three layers of the peeled mushroom caps, after 10 min immersion in a 10 g/mL solution. Layer 1 = beneath the skin–2 mm; layer 2 = 2–4 mm and layer 3 = 4–6 mm.
Fig. 3. Change in the inhibitory effect of licorice extract and DPPacid after different incubation times prior to reaction: (A) with the commercial enzyme ((a and b) treatments with different letters at a specific incubation time are significantly different at p ≤ 0.05, according to Duncan’s multiple range test; (A and B) activity bars with different letters at various incubation times are significantly different at p ≤ 0.05 for each treatment, according to Duncan’s multiple range test); (B) with the peeled mushroom extract ((A and B) average enzyme activities at different incubation times, designated by different letters at p = 0.0169).
Possible explanations for the lack of any significant effect of either inhibitor on intact mushrooms are: (1) the occurrence of a structural change of the inhibitors, due to enzymatic or chemical reactions on the mushroom surface or (2) lack of penetration into the outer layer of the mushroom cap. The amount of DPPacid remaining after 5 h incubation with the commercial enzyme fell from 10 ng/mL at the beginning of the test to less than 0.1 ng/mL (below the detection limits) at the end of the incubation period. With the extract from the treated mushrooms the amount of DPPacid detected decreased almost by half from 5.5 ng/mg tissue after 10 min to 2.9 ng/mg tissue after 250 min. In order to define the reasons for the difference in the efficiency of the product to reduce browning of sliced and intact mushrooms, the permeability of the skin was examined. After 10 min immersion, the weight of peeled mush-
rooms increased by 13.1%, whereas that of intact mushrooms increased by only 1.6%. The low efficiency of the inhibitors in preventing browning of the intact mushroom could well be due to their limited penetration. This was demonstrated by monitoring the amount of DPPacid observed in three layers of the mushroom’s cap: skin level and 2 and 4 mm beneath the skin, comparing intact and peeled mushrooms. Tyrosinase inhibition by the licorice extract and DPPacid was also tested in each layer. In the intact mushrooms, there was no detected sign of DPPacid in the second and third layers, whereas the amount of the DPPacid in the peeled mushrooms did not decrease significantly (p = 0.1655) and was still detectable in the third layer (Fig. 4). Moreover, the inhibitory effect of DPPacid (but not of the licorice extract) was strong in all three layers of the peeled mushrooms (Fig. 5). DPPacid inhibited enzyme
Fig. 5. Tyrosinase activity in three layers of peeled mushroom caps, immersed for 10 min in DPPacid or licorice extract (10 g/mL). Layer 1 = beneath the skin–2 mm; layer 2 = 2–4 mm and layer 3 = 4–6 mm. (a and b) Treatments with different letters in each layer are significantly different at p ≤ 0.05, according to Duncan’s multiple range test. (A and B) Layers with different letters are significantly different at p ≤ 0.05, according to Duncan’s multiple range test.
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activity by 68%, 79% and 87% in the first, second and third layers respectively. In the samples treated with licorice extract inhibition rates of 53%, 16% and 44% were observed in the first, second and third layers, respectively.
4. Discussion The advantage of adding natural inhibitors to a product to prevent postharvest enzymatic browning, as is described in the present paper, is that they are derived from edible plant organs—the fig fruit or licorice roots. Glabridin, an extremely potent tyrosinase inhibitor, extracted from licorice root (Nerya et al., 2003), is also known for it strong antioxidant activity (Vaya et al., 1997; Belinky et al., 1998), and therefore we expected it to provide an effective solution for the postharvest browning of sliced and intact mushrooms. Both DPPacid, extracted from fig leaves and fruit, and the licorice root extract demonstrated strong tyrosinase inhibitory effects in vitro (Nerya et al., 2003), and in vivo, prevented browning of sliced mushrooms but had very little effect on the browning of intact mushroom caps during 8 days under cold storage. A possible explanation for this difference could be the lack of contact between the inhibitors and the enzyme in situ. The cap of A. bisporus is covered with a polysaccharide skin to protect it from the external environment (NovaesLedieu and Garcia-Mendoza, 1981; Bernardo et al., 1999). Thus, the tissue does not absorb raindrops or applied sprays. Kuyper et al. (1993) found that after spraying intact mushrooms with 0.4 g/L calcium hypochlorite, only 1 g of the active compound remained on 200 g of mushrooms. In our work, when the mushroom skin was removed by peeling the cap, both inhibitors were able to penetrate the tissue and the uptake increased from 1.6% to 13.1% of the initial weight, due to peeling. This should have enabled the inhibitors to come into close contact with the cells of the interior layers of the peeled mushroom and the amount of DPPacid in the same layers ranged from 3.8 to 5.5 ng/mg according to the depth of the layer. However, with the intact mushrooms the small amount of uptake appeared to be insufficient for enzyme inactivation. Tyrosinase activity has been shown to be highest in the skin and the layer just beneath it (Van Leeuwen and Whichers, 1999) and to be significantly correlated (R2 = 0.72–0.74) with the change in cap color. We have also shown that tyrosinase activity declined by 42% from the first layer beneath the skin to the more interior layers. MS analysis revealed that the amount of DPPacid was less than 0.1 ng/mL in the interior layers, whereas the IC50 concentration in vivo was 0.18 g/mL. This means that for the intact mushroom, in order to increase the inhibitory level at the enzyme location, at least a 1000-fold higher concentration of the inhibitors should be applied, or alternatively a better way to improve the penetration to the target layers needs to be developed. Water movement in the intact mushroom is naturally from stipe to cap with transpira-
tion from the gill-tissue. Application of the inhibitors by employing this mode of water movement may improve their efficacy. With sliced mushrooms there is no penetration problem to the intercellular space, but a large sized inhibitor or hydrophilic molecule may not penetrate through the cell membrane barrier to the cytoplasm. Kojic acid, a wellrecognized tyrosinase inhibitor, has limited use because of its instability and limited permeation into animal skin. Kojyl APPA is a kojic acid derivative that does not inhibit the enzyme in vitro, but the added group improved the stability of the molecule and increased its penetration into mammalian cells eight-fold. Within the cell the molecule is converted to kojic acid which inhibits tyrosinase activity and melanin formation (Kim et al., 2003). Shimizu et al. (2001) synthesized a derivative of DPPacid by adding the active group of Vitamin E to the carboxylic group of the molecule. The IC50 of the new molecule (TM4R) is 10-fold higher than DPPacid and also has a good antioxidant activity. In a depigmentation trial with guinea pigs’ skins, the new derivate decreased skin darkness. Accordingly, modification of the active molecule without changing the active resorcinol group may well improve the molecule’s activity, as was noticed in a similar study with compounds of the chalcone family (Khatib et al., 2005). However, the intra-cellular compartmentation of tyrosinase, which keeps it separate from its substrate, probably also isolates it from the inhibitors, unless the tissue is wounded. During the mushroom peeling process, a layer of cells is presumably wounded, thus releasing cellular components into the intercellular spaces and enhancing the browning process. Moreover, tyrosinase activity in the peeled mushroom was found to be higher than in the intact mushroom (data not shown) and it also increased by 150% after 70 min incubation. On the other hand, the inhibitory effect on tyrosinase activity declined with increasing incubation time, both in vitro and in vivo. MS analysis revealed that the amount of DPPacid had also dramatically declined after 5 h incubation with the enzyme below (<0.1 ng/mL). Possible changes in the original DPPacid structure could not be detected using HPLC and MS methods. The possibility of irreversible binding to the enzyme also has to be rejected, as the inhibitory effect gradually dissipated. However, the possible binding to another protein cannot be ruled out. In conclusion, in spite of the potent anti-browning effect of the tyrosinase inhibitors studied on sliced mushrooms, their inability to penetrate and remain available in sufficient amounts in the relevant tissues of the intact mushroom, eliminates them for application to prevent postharvest browning of stored intact mushrooms.
Acknowledgment This study was supported by Grant No. 01-18-00314/2, from the Ministry of Science and Technology, Israel.
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References Belinky, P.A., Aviram, M., Fuhrman, B., Rosenblat, M., Vaya, J., 1998. The antioxidative effects of the isoflavan glabridin on endogenous constituents of LDL during its oxidation. Atherosclerosis 137, 49–61. Bernardo, D., Mendoza, C.G., Calonje, M., Novaes-Ledieu, M., 1999. Chemical analysis of the lamella walls of Agaricus bisporus fruit bodies. Curr. Microbiol. 38, 364–367. Bradford, M., 1976. A rapid and sensitive method for the quantition of microgram quantities of protein utilizing the principle of dye-binding. Anal. Biochem. 72, 248–254. Brennan, M., Le Port, G., Gormley, R., 2000. Post-harvest treatment with citric acid or hydrogen peroxide to extend the shelf life of fresh sliced mushrooms. Lebensm.-Wiss. u.-Technol. 33, 285–289. Flurkey, W.H., Ingebrigtsen, J., 1989. Polyphenoloxidase activity and enzymatic browning in mushrooms. In: Jen, J.J. (Ed.), Quality Factors of Fruits and Vegetables. Chemistry and Technology. ACS American Chemical Society, Washington, DC, pp. 45–54. Jolivet, S., Arpin, N., Wichers, H.J., Pellon, G., 1998. Agaricus bisporus browning: a review. Mycol. Res. 102, 1459–1483. Kertesz, D., Zito, R., 1962. Kinetic studies of the polyphenoloxidase action; kinetics in the presence of reducing agents. The indirect oxidation of reduced cytochrome c by polyphenol oxidase. Biochim. Biophys. Acta 64, 153–167. Khatib, S., Nerya, O., Musa, R., Shmuel, M., Tamir, S., Vaya, J., 2005. Chalcones as potent tyrosinase inhibitors: the importance of a 2,4substituted resorcinol moiety. Bioorg. Med. Chem. 13, 433–441. Kim, H.D., Hwang, J.S., Baek, H.S., Kim, K.J., Lee, B.G., Chang, I., Kang, H.H., Lee, O.S., 2003. Development of 5-[(3aminopropyl)phosphinooxyl]-2-(hydroxymethyl)-4H-pyran-4-one as a novel whitening agent. Chem. Pharm. Bull. 51, 113–116. Kubo, I., Kinst-Hori, I., 1998. Tyrosinase inhibitors from anis oil. J. Agric. Food Chem. 46, 1268–1271. Kuyper, L., Weinert, I.A.G., McGill, A.E.J., 1993. The effect of modified atmosphere packaging and addition of calcium hypochlorite on the atmosphere composition, color and microbial quality of mushrooms. Lebensm.-Wiss. u.-Technol. 26, 14–20.
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Martinez, M.V., Whitaker, J.R., 1995. The biochemistry and control of enzymatic browning. Trends Food Sci. Technol. 6, 195–200. Mayer, A.M., 1987. Polyphenol oxidases in plants—recent progress. Phytochemistry 26, 11–20. McEvilly, A.J., Iyengar, R., Gross, A.T., 1992. Inhibition of polyphenol oxidase by phenolic compounds. In: Phenolic Compounds in Food and Their Effects on Health 1. Am. Chem. Soc. Symp. Ser. 506. Cambridge, MA, USA, pp. 318–325. Nerya, O., Vaya, J., Musa, R., Izrael, S., Ben-Arie, R., Tamir, S., 2003. Glabrene and isoliquiritigenin as tyrosinase inhibitors from licorice roots. J. Agric. Food Chem. 51, 1201–1207. Novaes-Ledieu, M., Garcia-Mendoza, C., 1981. The cell walls of Agaricus bisporus and Agaricus campestris fruiting body hyphae. Can. J. Microbiol. 27, 779–787. Philippoussis, A., Diamantopoulou, P., Zervakis, G., 2001. Calcium chloride irrigation influence on yield, calcium content, qualitynd shelf-life of the white mushroom Agaricus bisporus. J. Sci. Food Agric. 81, 1447–1454. Shimizu, K., Kondo, R., Sakai, K., Takeda, N., Nagahata, T., Oniki, T., 2001. Novel vitamin E derivative with 4-substituted resorcinol moiety has both antioxidant and tyrosinase inhibitory properties. Lipids 36, 1321–1326. Soler-Rivas, C., Arpin, N., Olivier, J.M., Wichers, H.J., 1997. Activation of tyrosinase in Agaricus bisporus strains following infection by Pseudomonas tolaasii with tolaasin-containing preparation. Mycol. Res. 101, 375–382. Vamos-Vigyazo, L., 1981. Polyphenol oxidase and peroxidase in fruits and vegetables. Crit. Rev. Food Sci. Nutr. 15, 49–127. Van Leeuwen, J., Whichers, H.J., 1999. Tyrosinase activity and isoform composition in separate tissues during development of Agaricus bisporus fruit bodies. Mycol. Res. 103, 413–418. Vaya, J., Belinky, P.A., Aviram, M., 1997. Antioxidant constituents from licorice roots: isolation, structure elucidation and antioxidative capacity toward LDL oxidation. Free Radic. Biol. Med. 23, 302–313. Yamaguchi, M., Hwang, P.M., Campbell, J.D., 1970. Latent o-diphenol oxidase in mushrooms (Agaricus bisporus). Can. J. Biochem. 48, 198–202.
Prevention of Agaricus bisporus postharvest browning with tyrosinase inhibitors Ohad Nerya a,b , Ruth Ben-Arie b , Tal Luzzatto a , Ramadan Musa a , Soliman Khativ a , Jacob Vaya a,c,∗ a
c
Laboratory of Natural Compounds for Medicinal Use, Migal, Galilee Technological Center, P.O. Box 831, Kiryat Shmona 11016, Israel b Fruit Storage Research Laboratory, Kiryat Shmona 10200, Israel Department of Biotechnology and Environmental Science, Tel-Hai Academic College, Israel Received 6 March 2005; accepted 30 October 2005
Abstract Postharvest browning of Agaricus bisporus mushrooms is a severe problem that reduces the shelf life of harvested mushrooms. Mushroom browning occurs mainly as a result of tyrosinase activity, an enzyme belonging to the polyphenol oxidase (PPO) family and known to be a key enzyme in melanin biosynthesis. An ethanolic extract from licorice roots (Glycyrrhiza glabra) and [3-(2,4-dihydroxyphenyl propionic acid)] (DPPacid) isolated from fig leaves and fruit have been shown to inhibit tyrosinase activity. Adding these inhibitors to sliced mushrooms had a very strong inhibitory effect on browning, but pre-storage immersion of intact mushroom in the licorice extract did not prevent browning after 8 days storage at 4 ◦ C. By contrast, treatment with DPPacid at 1 g/mL reduced browning by half. Measurement of inhibitor uptake by mass spectra (MS) and assay of tyrosinase activity indicated that penetration into the mushroom tissue was inadequate for tyrosinase inhibition. Moreover, DPPacid was found to be unstable in the mushroom tissue and within a short time it was, presumably, metabolised. © 2005 Elsevier B.V. All rights reserved. Keywords: PPO; Licorice root extract; Browning; DPPacid
1. Introduction Agaricus bisporus mushrooms have a short shelf life compared to most fruit and vegetables. The intact mushrooms lose their commercial value within a few days, due to senescence, water loss, microbial attack and browning. Mushroom browning occurs as a result of two distinct mechanisms of phenol oxidation: (a) activation of tyrosinase, an enzyme belonging to the polyphenoloxidase (PPO) family; (b) and/or spontaneous oxidation (Jolivet et al., 1998). The PPO family includes catechol oxidase and laccase, both of which oxidize diphenols to the corresponding quinones (Kertesz and Zito, 1962), but the former can also oxidize some monophenols into o-diphenols (Mayer, 1987).
∗
Corresponding author. Tel.: +972 4 6953512; fax: +972 4 6944980. E-mail address: [email protected] (J. Vaya).
0925-5214/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2005.11.001
This activity is also referred to as ‘phenolase’ or ‘tyrosinase’, which has become a trivial name for catechol oxidase. The monophenolase activity of tyrosinase is usually much lower than the diphenolase activity, and this ratio, which can vary from 1:40 to 1:1, (Vamos-Vigyazo, 1981) is regarded as the key step in browning biosynthesis. Several authors have described tyrosinase activity in the fruit body of A. bisporus during its development and there is a consensus that a latent form of tyrosinase occurs, the extent of which is not yet clear (Yamaguchi et al., 1970; Flurkey and Ingebrigtsen, 1989). Latent tyrosinase can become active after contact with bacteria or a toxin such as tolaasin (Soler-Rivas et al., 1997). The problem of postharvest browning of mushrooms has been tackled from several aspects. Addition of calcium chloride during irrigation enhanced the white color at harvest and decreased postharvest browning (Philippoussis et al., 2001); modified atmosphere packaging reduced browning rate, due to reduced oxygen levels (Kuyper et al., 1993). Browning of
O. Nerya et al. / Postharvest Biology and Technology 39 (2006) 272–277
fresh sliced mushrooms was inhibited by immersion in citric acid (4%) or hydrogen peroxide (5%) (Brennan et al., 2000). In this case, however, the authors suggested that shelf life extension was the result of antibacterial activity rather than tyrosinase inhibition. Chemical control of enzymatic browning includes chelation of the copper present at the active site of the enzyme (Martinez and Whitaker, 1995) and reduction of the diquinone to its uncolored form (Kubo and Kinst-Hori, 1998). In previous work (Nerya et al., 2003), we extracted three new tyrosinase inhibitors from licorice roots (Glycyrrhiza glabra): glabridin, a non-competitive inhibitor eliciting 50% inhibition at a concentration (IC50 ) of 0.09 M, glabrene and isoliquiritigenin—both competitive inhibitors with IC50 values of 3.5 and 8.1 M, respectively. The molecular structures of these three inhibitors are given in Fig. 1. McEvilly et al. (1992) isolated several inhibitors of tyrosinase activity from fig latex (Ficus carica), one of them identified as DPPacid (Fig. 1). Shimizu et al. (2001) examined the biochemical parameters of tyrosinase inhibition by DPPacid and concluded that it is a competitive inhibitor with an IC50 of 3.2 M. We have isolated this compound from both fig leaves and fruit (unpublished data). The aim of the present study was to inhibit the postharvest browning of A. bisporus mushrooms (strain U1) by applying new safe, edible tyrosinase inhibitors from licorice roots and fig fruit. During the study we observed differences in the efficiency of browning inhibition between intact and sliced mushrooms. We have, therefore, also addressed the question whether the difference is due to limited permeability of the intact mushroom to the inhibitors.
2. Materials and methods 2.1. Tyrosinase inhibition Tyrosinase (EC1.14.18.1, Sigma product T7755) activity was determined using 0.03 mL tyrosinase (333 units/mL) in
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PBS (phosphate buffer solution) (50 mM, 0.07 mL) at pH 6.5 and 2 L of the tested inhibitor compounds (0.18–18 g/mL) dissolved in absolute ethanol in 96 well plates. After 5 min incubation at room temperature, 0.1 mL l-tyrosine (2 mM) was added and optical density at 492 nm measured every 5 min for 20 min (Elisa SLT Labinstruments co. A-5082). 2.2. Inhibition of sliced mushroom browning A. bisporus mushrooms (strain U1—second harvest) were harvested at a commercial farm. For IC50 determination, the mushroom caps were sliced and 20 L aliquots of the inhibitors (0.18–0.91 g/mL) were pipetted onto the cut surface. After 24 h incubation at 20 ◦ C and 97% relative humidity, the browning of the treated area was monitored by reading the L, a, b parameters with a CR-200 Minolta chromameter. 2.3. Inhibition of intact mushroom browning The inhibition of browning during storage was examined using mushrooms immersed in the tested inhibitors or in water (control) for 10 min. After drying, the mushrooms were packed in high-density polyethylene bags and stored at 4 ◦ C for 10 days. Storage changes in mushroom cap color (L) were monitored on days 1, 3, 5 and 8. 2.4. Inhibition of tyrosinase activity in vivo The influence of the inhibitors on mushroom tyrosinase activity was tested using cut mushroom caps that had been immersed in the tested inhibitors (0.6 and 1.35 g/mL for DPPacid and licorice extract, respectively), or in water (control) for 10 min. After drying, the mushroom caps were cut and frozen in PBS (50 mM at pH 6.5). For enzyme extraction, the frozen caps were ground in the buffer and centrifuged at 4000 rpm for 20 min. An aliquot (0.3 mL) of the supernatant was diluted in 0.34 mL PBS and 0.36 mL tyrosine 2 mM was added. The rate of DOPA-quinone formation was monitored at 475 nm, after 10 min incubation at room temperature
Fig. 1. Molecular structure of tyrosinase inhibitors extracted from fig leaf (A) DPPacid and from licorice root: (B) glabridin, (C) isoliquiritigenin and (D) glabrene (Vaya et al., 1997).
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using a Shimadzu A-187 spectrophotometer. Protein content was measured using Coomassie blue (Bradford, 1976), and the tyrosinase activity was calculated. The specific effect of fresh inhibitors on the extracted enzyme activity in vitro was assayed by adding 2 L of the test compounds to the reaction mixture described above. Two different experiments were conducted to compare the kinetics of tyrosinase activity in vitro and in vivo. The in vitro test was performed with a commercially purified mushroom tyrosinase and the in vivo test was conducted with an enzyme preparation from peeled mushrooms. The in vitro reaction was started after incubation periods of the enzyme with the inhibitors for 10, 70 and 250 min. In vivo, the peeled caps were immersed in the inhibitor solution for 10 min and the enzyme was extracted after 10, 70 and 250 min incubation. 2.5. Mushroom cap permeability to inhibitors Peeled and intact mushrooms were immersed in 10 g/mL solutions of the test inhibitors for 10 min. After drying, the mushrooms were peeled and longitudinal slices 2 mm thick were cut, using a vegetable peeler. Extraction for tyrosinase assay activity was as described above. In addition, the amount of DPPacid absorbed in the mushroom tissue was extracted by grinding 5–93 mg cut tissue in 2 mL double distilled water (ddw) pH 2. Ethyl acetate (4 mL) was added and after strong vortex mixing, the extract was collected by centrifugation. Extraction with ethyl acetate was repeated twice. After evaporation of the solvent, the dry residue was dissolved in 0.5 mL acetonitrile. The DPPacid content in the tissue was assayed by MS, as described below. 2.6. Quantification of DPPacid by MS/MS The MS/MS (Micromass Quattro Ultima MS, UK) analysis of the oxidized products was performed using electrospray negative ions (ES− ) mode. The source temperature of the mass spectrometer was set at 150 ◦ C, with a cone gas flow of 124 L/h and a desolvation gas flow of 525 L/h. Collision-induced dissociation MS of the DPPacid using 15-collision energy and 3 (kV) capillary voltage revealed with m/z 181 (M−1 ) and its fragmentation at m/z 137 (M-1CO2). Multiple-reaction-monitoring (MRM) was performed using the same conditions and analyzed by the m/z 137 ion fragmentation.
Table 1 Inhibitory effects of a licorice root extract and DPPacid on mushroom tyrosinase activity in vitro and on mushroom slice browning in vivoa AA
IC50 (g/mL) DPPacid
Licorice extract
In vitro tyrosinase activity In vivo browning
0.27 ± 0.05 0.18 ± 0.07
0.01 ± 0.0005 0.11 ± 0.05
a
Data are averages ± S.E., n = 3–8 replicates.
the in vivo test there was very little difference between their degrees of browning inhibition (Table 1). With the information that these products have not only a strong inhibitory effect on the purified enzyme, but also a strong potential as browning inhibitors of the cut surface of mushroom caps, a trial was conducted to inhibit browning of intact mushrooms in storage. A pre-storage mushroom immersion in the licorice extract did not prevent browning after 8 days storage at 4 ◦ C, and although immersion in DPPacid at a concentration of 1 g/mL reduced browning by half, compared to the untreated control, the variance was too great for the difference to be significant (Fig. 2). When the kinetics of tyrosinase activity and inhibition were compared in vivo and in vitro two different patterns of tyrosinase activity and inhibition evolved. With the commercial enzyme, a very strong inhibition of tyrosinase activity that was observed after 10 min, gradually dissipated after longer incubations, with the enzyme loosing 75% of its activity (Fig. 3A). Inhibition by the licorice extract and DPPacid decreased from 99% and 86%, respectively, after 10 min incubation, to 83% for both, after 70 min. After 250 min neither had any inhibitory effect on commercial tyrosinae activity. With the mushroom extract, there was a gradual increase in enzyme activity with time, even in the untreated control, and the effects of both inhibitors were not significant even after 10 min (Fig. 3B).
3. Results The tyrosinase inhibitory potentials of DPPacid and licorice extract were compared, in vitro on a commercial enzyme preparation from mushrooms, and in vivo, using a chromameter, on the assumption that browning of disks cut from mushroom caps resulted from tyrosinase activity. The licorice extract showed a very strong inhibition of tyrosinase activity compared to DPPacid in the in vitro test, whereas in
Fig. 2. The effect of licorice extract and DPPacid on intact mushroom cap browning, after storage for 8 days. (Initial average L value was 92.3 ± 2.5).
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Fig. 4. The amount of DPPacid in three layers of the peeled mushroom caps, after 10 min immersion in a 10 g/mL solution. Layer 1 = beneath the skin–2 mm; layer 2 = 2–4 mm and layer 3 = 4–6 mm.
Fig. 3. Change in the inhibitory effect of licorice extract and DPPacid after different incubation times prior to reaction: (A) with the commercial enzyme ((a and b) treatments with different letters at a specific incubation time are significantly different at p ≤ 0.05, according to Duncan’s multiple range test; (A and B) activity bars with different letters at various incubation times are significantly different at p ≤ 0.05 for each treatment, according to Duncan’s multiple range test); (B) with the peeled mushroom extract ((A and B) average enzyme activities at different incubation times, designated by different letters at p = 0.0169).
Possible explanations for the lack of any significant effect of either inhibitor on intact mushrooms are: (1) the occurrence of a structural change of the inhibitors, due to enzymatic or chemical reactions on the mushroom surface or (2) lack of penetration into the outer layer of the mushroom cap. The amount of DPPacid remaining after 5 h incubation with the commercial enzyme fell from 10 ng/mL at the beginning of the test to less than 0.1 ng/mL (below the detection limits) at the end of the incubation period. With the extract from the treated mushrooms the amount of DPPacid detected decreased almost by half from 5.5 ng/mg tissue after 10 min to 2.9 ng/mg tissue after 250 min. In order to define the reasons for the difference in the efficiency of the product to reduce browning of sliced and intact mushrooms, the permeability of the skin was examined. After 10 min immersion, the weight of peeled mush-
rooms increased by 13.1%, whereas that of intact mushrooms increased by only 1.6%. The low efficiency of the inhibitors in preventing browning of the intact mushroom could well be due to their limited penetration. This was demonstrated by monitoring the amount of DPPacid observed in three layers of the mushroom’s cap: skin level and 2 and 4 mm beneath the skin, comparing intact and peeled mushrooms. Tyrosinase inhibition by the licorice extract and DPPacid was also tested in each layer. In the intact mushrooms, there was no detected sign of DPPacid in the second and third layers, whereas the amount of the DPPacid in the peeled mushrooms did not decrease significantly (p = 0.1655) and was still detectable in the third layer (Fig. 4). Moreover, the inhibitory effect of DPPacid (but not of the licorice extract) was strong in all three layers of the peeled mushrooms (Fig. 5). DPPacid inhibited enzyme
Fig. 5. Tyrosinase activity in three layers of peeled mushroom caps, immersed for 10 min in DPPacid or licorice extract (10 g/mL). Layer 1 = beneath the skin–2 mm; layer 2 = 2–4 mm and layer 3 = 4–6 mm. (a and b) Treatments with different letters in each layer are significantly different at p ≤ 0.05, according to Duncan’s multiple range test. (A and B) Layers with different letters are significantly different at p ≤ 0.05, according to Duncan’s multiple range test.
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activity by 68%, 79% and 87% in the first, second and third layers respectively. In the samples treated with licorice extract inhibition rates of 53%, 16% and 44% were observed in the first, second and third layers, respectively.
4. Discussion The advantage of adding natural inhibitors to a product to prevent postharvest enzymatic browning, as is described in the present paper, is that they are derived from edible plant organs—the fig fruit or licorice roots. Glabridin, an extremely potent tyrosinase inhibitor, extracted from licorice root (Nerya et al., 2003), is also known for it strong antioxidant activity (Vaya et al., 1997; Belinky et al., 1998), and therefore we expected it to provide an effective solution for the postharvest browning of sliced and intact mushrooms. Both DPPacid, extracted from fig leaves and fruit, and the licorice root extract demonstrated strong tyrosinase inhibitory effects in vitro (Nerya et al., 2003), and in vivo, prevented browning of sliced mushrooms but had very little effect on the browning of intact mushroom caps during 8 days under cold storage. A possible explanation for this difference could be the lack of contact between the inhibitors and the enzyme in situ. The cap of A. bisporus is covered with a polysaccharide skin to protect it from the external environment (NovaesLedieu and Garcia-Mendoza, 1981; Bernardo et al., 1999). Thus, the tissue does not absorb raindrops or applied sprays. Kuyper et al. (1993) found that after spraying intact mushrooms with 0.4 g/L calcium hypochlorite, only 1 g of the active compound remained on 200 g of mushrooms. In our work, when the mushroom skin was removed by peeling the cap, both inhibitors were able to penetrate the tissue and the uptake increased from 1.6% to 13.1% of the initial weight, due to peeling. This should have enabled the inhibitors to come into close contact with the cells of the interior layers of the peeled mushroom and the amount of DPPacid in the same layers ranged from 3.8 to 5.5 ng/mg according to the depth of the layer. However, with the intact mushrooms the small amount of uptake appeared to be insufficient for enzyme inactivation. Tyrosinase activity has been shown to be highest in the skin and the layer just beneath it (Van Leeuwen and Whichers, 1999) and to be significantly correlated (R2 = 0.72–0.74) with the change in cap color. We have also shown that tyrosinase activity declined by 42% from the first layer beneath the skin to the more interior layers. MS analysis revealed that the amount of DPPacid was less than 0.1 ng/mL in the interior layers, whereas the IC50 concentration in vivo was 0.18 g/mL. This means that for the intact mushroom, in order to increase the inhibitory level at the enzyme location, at least a 1000-fold higher concentration of the inhibitors should be applied, or alternatively a better way to improve the penetration to the target layers needs to be developed. Water movement in the intact mushroom is naturally from stipe to cap with transpira-
tion from the gill-tissue. Application of the inhibitors by employing this mode of water movement may improve their efficacy. With sliced mushrooms there is no penetration problem to the intercellular space, but a large sized inhibitor or hydrophilic molecule may not penetrate through the cell membrane barrier to the cytoplasm. Kojic acid, a wellrecognized tyrosinase inhibitor, has limited use because of its instability and limited permeation into animal skin. Kojyl APPA is a kojic acid derivative that does not inhibit the enzyme in vitro, but the added group improved the stability of the molecule and increased its penetration into mammalian cells eight-fold. Within the cell the molecule is converted to kojic acid which inhibits tyrosinase activity and melanin formation (Kim et al., 2003). Shimizu et al. (2001) synthesized a derivative of DPPacid by adding the active group of Vitamin E to the carboxylic group of the molecule. The IC50 of the new molecule (TM4R) is 10-fold higher than DPPacid and also has a good antioxidant activity. In a depigmentation trial with guinea pigs’ skins, the new derivate decreased skin darkness. Accordingly, modification of the active molecule without changing the active resorcinol group may well improve the molecule’s activity, as was noticed in a similar study with compounds of the chalcone family (Khatib et al., 2005). However, the intra-cellular compartmentation of tyrosinase, which keeps it separate from its substrate, probably also isolates it from the inhibitors, unless the tissue is wounded. During the mushroom peeling process, a layer of cells is presumably wounded, thus releasing cellular components into the intercellular spaces and enhancing the browning process. Moreover, tyrosinase activity in the peeled mushroom was found to be higher than in the intact mushroom (data not shown) and it also increased by 150% after 70 min incubation. On the other hand, the inhibitory effect on tyrosinase activity declined with increasing incubation time, both in vitro and in vivo. MS analysis revealed that the amount of DPPacid had also dramatically declined after 5 h incubation with the enzyme below (<0.1 ng/mL). Possible changes in the original DPPacid structure could not be detected using HPLC and MS methods. The possibility of irreversible binding to the enzyme also has to be rejected, as the inhibitory effect gradually dissipated. However, the possible binding to another protein cannot be ruled out. In conclusion, in spite of the potent anti-browning effect of the tyrosinase inhibitors studied on sliced mushrooms, their inability to penetrate and remain available in sufficient amounts in the relevant tissues of the intact mushroom, eliminates them for application to prevent postharvest browning of stored intact mushrooms.
Acknowledgment This study was supported by Grant No. 01-18-00314/2, from the Ministry of Science and Technology, Israel.
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References Belinky, P.A., Aviram, M., Fuhrman, B., Rosenblat, M., Vaya, J., 1998. The antioxidative effects of the isoflavan glabridin on endogenous constituents of LDL during its oxidation. Atherosclerosis 137, 49–61. Bernardo, D., Mendoza, C.G., Calonje, M., Novaes-Ledieu, M., 1999. Chemical analysis of the lamella walls of Agaricus bisporus fruit bodies. Curr. Microbiol. 38, 364–367. Bradford, M., 1976. A rapid and sensitive method for the quantition of microgram quantities of protein utilizing the principle of dye-binding. Anal. Biochem. 72, 248–254. Brennan, M., Le Port, G., Gormley, R., 2000. Post-harvest treatment with citric acid or hydrogen peroxide to extend the shelf life of fresh sliced mushrooms. Lebensm.-Wiss. u.-Technol. 33, 285–289. Flurkey, W.H., Ingebrigtsen, J., 1989. Polyphenoloxidase activity and enzymatic browning in mushrooms. In: Jen, J.J. (Ed.), Quality Factors of Fruits and Vegetables. Chemistry and Technology. ACS American Chemical Society, Washington, DC, pp. 45–54. Jolivet, S., Arpin, N., Wichers, H.J., Pellon, G., 1998. Agaricus bisporus browning: a review. Mycol. Res. 102, 1459–1483. Kertesz, D., Zito, R., 1962. Kinetic studies of the polyphenoloxidase action; kinetics in the presence of reducing agents. The indirect oxidation of reduced cytochrome c by polyphenol oxidase. Biochim. Biophys. Acta 64, 153–167. Khatib, S., Nerya, O., Musa, R., Shmuel, M., Tamir, S., Vaya, J., 2005. Chalcones as potent tyrosinase inhibitors: the importance of a 2,4substituted resorcinol moiety. Bioorg. Med. Chem. 13, 433–441. Kim, H.D., Hwang, J.S., Baek, H.S., Kim, K.J., Lee, B.G., Chang, I., Kang, H.H., Lee, O.S., 2003. Development of 5-[(3aminopropyl)phosphinooxyl]-2-(hydroxymethyl)-4H-pyran-4-one as a novel whitening agent. Chem. Pharm. Bull. 51, 113–116. Kubo, I., Kinst-Hori, I., 1998. Tyrosinase inhibitors from anis oil. J. Agric. Food Chem. 46, 1268–1271. Kuyper, L., Weinert, I.A.G., McGill, A.E.J., 1993. The effect of modified atmosphere packaging and addition of calcium hypochlorite on the atmosphere composition, color and microbial quality of mushrooms. Lebensm.-Wiss. u.-Technol. 26, 14–20.
277
Martinez, M.V., Whitaker, J.R., 1995. The biochemistry and control of enzymatic browning. Trends Food Sci. Technol. 6, 195–200. Mayer, A.M., 1987. Polyphenol oxidases in plants—recent progress. Phytochemistry 26, 11–20. McEvilly, A.J., Iyengar, R., Gross, A.T., 1992. Inhibition of polyphenol oxidase by phenolic compounds. In: Phenolic Compounds in Food and Their Effects on Health 1. Am. Chem. Soc. Symp. Ser. 506. Cambridge, MA, USA, pp. 318–325. Nerya, O., Vaya, J., Musa, R., Izrael, S., Ben-Arie, R., Tamir, S., 2003. Glabrene and isoliquiritigenin as tyrosinase inhibitors from licorice roots. J. Agric. Food Chem. 51, 1201–1207. Novaes-Ledieu, M., Garcia-Mendoza, C., 1981. The cell walls of Agaricus bisporus and Agaricus campestris fruiting body hyphae. Can. J. Microbiol. 27, 779–787. Philippoussis, A., Diamantopoulou, P., Zervakis, G., 2001. Calcium chloride irrigation influence on yield, calcium content, qualitynd shelf-life of the white mushroom Agaricus bisporus. J. Sci. Food Agric. 81, 1447–1454. Shimizu, K., Kondo, R., Sakai, K., Takeda, N., Nagahata, T., Oniki, T., 2001. Novel vitamin E derivative with 4-substituted resorcinol moiety has both antioxidant and tyrosinase inhibitory properties. Lipids 36, 1321–1326. Soler-Rivas, C., Arpin, N., Olivier, J.M., Wichers, H.J., 1997. Activation of tyrosinase in Agaricus bisporus strains following infection by Pseudomonas tolaasii with tolaasin-containing preparation. Mycol. Res. 101, 375–382. Vamos-Vigyazo, L., 1981. Polyphenol oxidase and peroxidase in fruits and vegetables. Crit. Rev. Food Sci. Nutr. 15, 49–127. Van Leeuwen, J., Whichers, H.J., 1999. Tyrosinase activity and isoform composition in separate tissues during development of Agaricus bisporus fruit bodies. Mycol. Res. 103, 413–418. Vaya, J., Belinky, P.A., Aviram, M., 1997. Antioxidant constituents from licorice roots: isolation, structure elucidation and antioxidative capacity toward LDL oxidation. Free Radic. Biol. Med. 23, 302–313. Yamaguchi, M., Hwang, P.M., Campbell, J.D., 1970. Latent o-diphenol oxidase in mushrooms (Agaricus bisporus). Can. J. Biochem. 48, 198–202.