Antioxidant Compounds for the Inhibition of Enzymatic Browning by Polyphenol Oxidases in the Fruiting Body Extract of the Edible Mushroom Hericium erinaceus

Abstract

Mushrooms are attractive resources for novel enzymes and bioactive compounds. Nevertheless, mushrooms spontaneously form brown pigments during food processing as well as extraction procedures for functional compounds. In this study, the dark browning pigment in the extract derived from the edible mushroom Hericium erinaceus was determined to be caused by the oxidation of endogenous polyphenol compounds by the polyphenol oxidase (PPO) enzyme family. These oxidized pigment compounds were measured quantitatively using a fluorospectrophotometer and, through chelation deactivation and heat inactivation, were confirmed to be enzymatic browning products of reactions by a metalloprotein tyrosinase in the PPO family. Furthermore, a transcript analysis of the identified putative PPO-coding genes in the different growth phases showed that tyrosinase and laccase isoenzymes were highly expressed in the mushroom fruiting body, and these could be potential PPOs involved in the enzymatic browning reaction. A metabolite profiling analysis of two different growth phases also revealed a number of potential enzymatic browning substances that were grouped into amino acids and their derivatives, phenolic compounds, and purine and pyrimidine nucleobases. In addition, these analyses also demonstrated that the mushroom contained a relatively high amount of natural antioxidant compounds that can effectively decrease the browning reaction via PPO-inhibitory mechanisms that inhibit tyrosinase and scavenge free radicals in the fruiting body. Altogether, these results contribute to an understanding of the metabolites and PPO enzymes responsible for the enzymatic browning reaction of H. erinaceus.

Keywords: Hericium erinaceus; antioxidant compounds; enzymatic browning; laccase; mushroom metabolites; natural inhibitor; polyphenol oxidase; tyrosinase.

Figure 1

The fruiting body extract fractions (F1–F12) eluted from an ion-exchange column, DEAE-Sepharose, in the edible mushroom Hericium erinaceus, with dark brown pigments in F3 and F4.

Figure 2

Fluorescence emission spectra of the dark brown pigment in the protein fraction. (a) Emission spectrum of the dark pigment fraction scanned by a fluorospectrophotometer with an excitation wavelength of 380 nm. (b) Fluorescence spectra of the pigment fractions for relative quantification of the brown pigment, which increased in a time-dependent manner under the same conditions. The fluorescence intensity of every fraction was measured with the following time points: initial 0 h, purple; 48 h, blue; 72 h, green; 96 h, yellow; 120 h, orange; 144 h, red.

Figure 3

Measurement of the browning pigment substances and tyrosinase activities in the fraction pools collected in the DEAE-Sepharose column. (a) The protein fraction pools eluted from the DEAE-Sepharose column: 1, crude extract fraction pool (CL); 2, flow-through fraction pool (FT); 2, wash fraction pool (W); 4–8, elution fraction pools with 50 mM (E50), 250 mM (E250), 500 mM (E500), 1000 mM (E1000), and 5000 mM NaCl (E5000), respectively, in 50 mM Tris-HCl buffer. (b) Fluorescence intensities (Arbitrary Unit, A.U.) of these fraction pools for the measurement of brown pigment under the optimum conditions with λ_{Ex380 nm}~λ_Em460nm. (c) Tyrosinase-specific activities of these fraction pools under the enzyme assay conditions described in the material and method section.

Figure 4

Measurement of fluorescence intensities and tyrosinase activities in the mushroom lysate treated with EDTA or heat. (a) Fluorescence intensities of the EDTA-treated lysates at the initial time (black circles) and 14 days later (white circles). (b) Total tyrosinase activities of EDTA-treated lysates. (c) Fluorescence intensities of the heat-treated lysates at the initial time (black squares) and 14 days later (white squares). (d) Total enzyme activities of the heat-treated lysates. Fluorescence intensities (Arbitrary Unit, A.U.) and tyrosinase activities of all fractions were measured under the optimum conditions with λE × 380 nm~λEm 460 nm and the enzyme assay conditions described, respectively.

Figure 5

Transcription levels of tyrosinase- and laccase-coding genes involved in the enzymatic browning reaction in the fruiting body (dark gray) and the mycelium (gray-white) of H. erinaceus. (a) Log scale for transcription levels expressed as fragments per kilobase of transcript per million (FPKM). The log (FPKM) values of lac2c and lac2d were less than −0.1. (b) Normalized FPKM values relative to a suite of endogenous actin genes in each growth phase.