Eur Food Res Technol (2014) 238:727–739 DOI 10.1007/s00217-013-2114-7

ORIGINAL PAPER

Polymerization of catalyzed by polyphenol oxidase from lotus seedpod

Xiao-ru Liu · Ru-peng Xie · Ya-wei Fan · Jiang-ning Hu · Ting Luo · Hong-yan Li · Ze-yuan Deng

Received: 28 March 2013 / Revised: 9 October 2013 / Accepted: 14 October 2013 / Published online: 7 January 2014 © Springer-Verlag Berlin Heidelberg 2014

Abstract This study aimed to investigate the profiles MWS Model wine system change in proanthocyanidins (PAs) catalyzed by polyphenol CD Circular dichroism spectroscopy oxidase (PPO) from lotus seedpod in a model wine system FT-IR Fourier transform infrared spectroscopy (MWS). Results showed that PAs from lotus seedpod con- SDS-PAGE Sodium dodecyl sulfate–polyacrylamide gel sisted of dimer (74.00 %) and trimer (22.75 %). PPO could electrophoresis tolerate ethanol concentrations below 20 % (v/v). The opti- TPC Total other phenolic compounds (excluding mum temperature of PPO activity was 80 °C, and the optimal proanthocyanidins) pH was 9.0. Its molecular weight was approximately 31 kDa, and its secondary structures were α-helix (59.0 %), β-sheet (4.3 %), turns (14.1 %), and random coils (22.6 %). In the Introduction MWS, the trimers gradually increased from 22.55 % at 0 h (control) to 100 % at 10 h incubation, while the dimers Lotus (Nelumbo nucifera, Gaertn) is a perennial aquatic herb decreased from 74.33 % (control) to 0 % at 10 h incubation. with creeping rhizome throughout the world. All parts of lotus Moreover, the composition of the precipitate formed at dif- can be used for culinary and medicinal purposes. Its seedpod is ferent incubation time points was approximately 16.54 % of usually discarded after harvest of lotus seeds. In recent years, monomers, 21.03 % of dimers, and 62.43 % of trimers at 2 h polyphenols, especially proanthocyanidins (PAs) in lotus incubation with PPO. The results from this study have pro- seedpod, have been found to exhibit many health benefits such vided in vitro evidence for a possible application of PPO in as anti-cancer, anti-oxidation, anti-myocardial ischemia, and red wine aging. anti-radiative effects [1–4]. Furthermore, their activities against melanoma, cognitive dysfunction, and Alzheimer’s Keywords · Polyphenol oxidase · disease have also been reported recently [5–7]. Lotus seedpod · Polymerization Polyphenols are considered as crucial components in terms of the sensory characteristics and nutritional benefits Abbreviations of red wine. Polyphenols in red wine can usually be clas- PPO Polyphenol oxidase sified into two types: compounds of low molecular weight PAs Proanthocyanidins such as phenolic acids, stilbenes, flavanols, flavonols and anthocyanins and compounds of high molecular weight such as PAs [8]. All these compounds are released from the Xiao-ru Liu and Ru-peng Xie have contributed equally to this work; they are co-first authors. grape seeds and skins into the wine during fermentation and maceration. The crucial factors affecting the phenolic X. Liu · R. Xie · Y. Fan · J. Hu · T. Luo · H. Li · Z. Deng (&) profiles of wines are the making process itself and the State Key Laboratory of Food Science and Technology, reactions during aging [9, 10]. Profiles analyses revealed Institute for Advanced Study, Nanchang University, that the polymerization of low molecular weight phe- NO. 235, Nanjing East Road, 330047 Nanchang, Jiangxi, People’s Republic of China nols progressively occurred throughout the wine-making e-mail: [email protected] process [11–15]. However, the red wine aging is a time- 123 728 Eur Food Res Technol (2014) 238:727–739 consuming and inefficient process by some conventional Germany). All other reagents used in this study were of techniques. Enzyme-assisted autolysis is a common analytical grade. method to ameliorate the problem. β-glucanase and pec- tinase were reported to accelerate the release of Preparation and characterization of PAs from lotus anthocyanins and saccharides before or during the fer- seedpod mentation[9, 16]. But these methods could not shorten the aging time for that red wine aging is mostly reflected in PAs preparation phenolic compounds polymerization and color stabiliza- tion. However, to our knowledge, little related research has The powder (100 g) of lotus seedpods was first extracted by been carried on so far. 200 ml of 70 % acetone (v/v) containing 1 g/L of Polyphenol oxidase (PPO) may be a highly efficient cata- L-ascorbic acid to avoid oxidation for 12 h. The filtrate was lyst of phenolic polymerization. It is known that PPO exists in vacuum-dried to 50 ml at 45 °C. Then, 100 ml of trichlo- numerous plants and seems always harmful in food industry romethane, 100 ml of diethyl ether and 100 ml of ethyl because of its contribution to enzymatic browning in vegeta- acetate were successively added to remove the lipids, bles and fruits. The catalytic process is composed of two steps: flavonoids and chlorophylls, respectively. The aqueous first, the monophenols changed into O-diphenols by hydrox- layer was vacuum-dried to remove the remnant organic ylation, and then, the O-diphenols are oxidized and solvent and freeze-dried to powder, and 0.5 g of powder accumulated into O-quinones [17–19]. The excess presence was dissolved in 10 ml of H O for following purification. and activity of the PPO are undesirable when exposed to 2 AB-8 resin column (Φ 1.5 cm 9 35 cm) was used for oxygen in red wine. PPO may cause browning in wines under preliminary purification, with 200 ml of water and 200 ml aerobic condition; however, under anoxic condition, trans- of 50 % ethanol (v/v) as eluents, respectively. The ethanol formation from phenols to O-quinones might be the first step eluent was collected and vacuum-dried to 10 ml and further of polymerization in red wine. The formed quinones will react purified by Sephadex LH-20 column (Φ 3.2 cm 9 40 cm). with other phenols with an electron-rich ring. When the Then, 400 ml of water, 600 ml of 25 % methanol (v/v), reaction occurs, a new bond is formed between the two phe- 200 ml of 50 % methanol (v/v) and 300 ml of 70 % acetone nolic compounds resulting in the generation of a PA [20, 21]. (v/v) were successively added to elute the column. The Based on these studies, we think about whether this system acetone and methanol eluents were collected, concentrated could be used to improve the quality of young red wine. and freeze-dried to powder as described above. The objectives of the present study were mainly to examine the characteristics of the PAs and PPO extracted from lotus seedpod, respectively. The changes in profiles of Determination of PAs by HPLC–QTOF–MS total phenolic acids and PAs catalyzed by the PPO in a model wine system were investigated. The results from this The PAs powder (0.1 mg) was dissolved in methanol study might provide not only a valuable way to utilize the (1 ml), and concentration of PAs was determined by an lotus seedpod, but also may put forward a new efficient Agilent 1200 N HPLC system (Agilent Technologies, PPO-assisted method for red wine aging. Shanghai, China) using an ODS C18 column (250 9 4.6 mm, 5 μm) at 25 °C with a flow rate of 0.5 ml/ min and injection volume of 10 μl. Mobile phases consisted Materials and methods of 0.5 % formic acid in water (A) and acetonitrile (B). The diode array detector (DAD) monitored at 278 nm, and Plant materials and chemical reagents linear gradient was carried out in 50 min under the fol- lowing conditions: 0–20 min, A 3.0–8.0 %; 20–40 min, A Lotus seedpods were obtained from Guangchang County, 8.0–13.0 %; 40–41 min, A 13.0–3.0 %; 41–50 min A Jiangxi Province, China. The undamaged ripe lotus seed- 3.0 %. The HPLC system was coupled to a hybrid quad- pods without seeds were stored at 2 °C within 12 h after rupole time-of-flight (QTOF) mass spectrometer (ABSciex, harvest to minimize oxidation and microbial contamina- USA). The MS was performed in negative ionization mode, tion. The lotus seedpods were divided into two groups. One and data of full scans were collected between a m/z range group was dried to constant weight. The other was stored at of 50–1,300. Mass parameters were as follows: ion spray −20 °C for PPO extraction. Each group was in triplicate. voltage, −4,200 V; declustering potential, −60 V; focusing The chromatographic polyphenolic reference standards potential, −190 V; declustering potential 2, −15 V; ion of HPLC grade, including ferulic acid, chlorogenic acid, release delay, 6 V; ion release width, 5 V; temperature, caffeic acid, vanillic acid, trans-cinnamic acid and quer- 400 °C with curtain gas (N2), 50 a.u. (arbitrary units); cetin, were purchased from Sigma Company (Steinheim, auxiliary gas, 50 a.u. and nebulizer gas (N2), 50 a.u. IDA 123 Eur Food Res Technol (2014) 238:727–739 729 was performed using the criteria: ions that exceeded 5 A Jasco MOS-450 spectropolarimeter circular dichroism counts, ion tolerance 50 mDa, collision energy fixed at (CD) chromatograph was used to determine the secondary −30 V, dynamic background substract activated. Quantifi- structure of PPO. Sample solution (1 ‰, m/v) was prepared cation was measured by the area of each peak through the in PBS (0.1 M, pH 6.8) and filtered by 0.02 μm membrane HPLC diagram using (+)- as external standard via prior to CD measurements with the working parameters: calibration curve method. 0.1 cm of optical distance, 2 mdeg/cm of sensibility and 0.01 mdeg of distinguish ability. Scanning wavelength range of CD was from 190 to 250 nm with 3.3 nm/s of Preparation and characterization of PPO scanning velocity at 25 °C. CD spectra were corrected for solvent contributions and were expressed in terms of spe- PPO preparation cific ellipticities versus wavelength. Estimation of secondary structure composition was performed using an The PPO was extracted from lotus seedpods by the fol- online server DICHROWEB, located at http://dichroweb. lowing method of Aydemir with minor modification [22]. cryst.bbk.ac.uk/html/home.shtml [24]. The information and Briefly, lotus seedpods (80 g) were homogenized and operation were according to Whitmore [24, 25] extracted with 100 ml of phosphate buffer solution (PBS) (0.2 M, pH 7.2) at 25 °C for 30 min. Then, the paste was Determination of the optimal pH and temperature, ethanol filtered, the supernatant was treated with (NH ) SO to 4 2 4 resistance capacity of PPO 30 % saturation, and centrifuged, successive supernatant was adjusted to 80 % of (NH ) SO saturation to precipi- 4 2 4 The optimal pH of PPO was determined by measuring its tate PPO. The crude PPO extract was further purified activity in different buffers of different pH values (citrate by DEAE-Sephadex A-25 ion-exchange column buffer of pH 2.0–3.0, acetate buffer of pH 4.0–5.0, phosphate (Φ 3.2 cm 9 40 cm) and Sephacryl S-200 column buffer of pH 6.0–7.0, Tris–HCl buffer of pH 8.0–9.0 and (Φ 1.5 cm 9 35 cm) chromatography with a linear gradient NH Cl–NH OH buffer for pH 9.5–10.5) for 10 min at 30 °C of NaCl (0.2–1.0 M) as the eluent. 4 4 [19]. The temperature stability was carried out by deter- Polyphenol oxidase activity was assayed by a modified mining PPO activities at different temperatures ranging from method based on previous reports [19, 23]. The sample 10 to 100 °C in PBS (0.2 M, pH 6.8). The ethanol resistance cuvette contained 2.90 ml of 20 mM (+)-catechin solution in capacity of PPO was determined by the variance in its 0.1 M PBS (pH 6.0) and 0.10 ml of the enzyme solution. The activity at different concentrations of ethanol (5, 10, 15, 20 blank sample (the control) contained only 3 ml of substrate and 50 %, v/v) at 30 °C for 10 min. PPO activity was mea- solution. (+)-catechin was chosen as the substrate because it sured as the method mentioned above except for buffers of was the best substrate for lotus seedpod PPO among five different pH values, temperatures and concentrations of tested phenols such as resorcinol, (+)-catchin, hydroquinone, ethanol, respectively. L-tyrosine and pyrogallol, whereas its Km and Vmax of PPO with (+)-catechin were 0.19 mol/L and 1.01 OD/min, Detection of PPO catalytic reaction in MWS respectively. A Jasco spectrophotometer was employed to monitor the reaction of the enzyme catalysis at 276 nm and Foundation of MWS and PAs polymerization by PPO 430 nm. The enzyme activity was expressed in a Q-product. ÀÁ The MWS was designed according to the method of Lee with Q product ¼ A276ÀÁtestedsample A276control some modification [11]. Briefly, 200 mg of PAs powder was A430testedsample A430control dissolved in the MWS (total 1,000 ml), consisting of ethanol Determination of molecular weight and secondary structure (150 ml), acetaldehyde (100 μl) and pyruvic acid (100 μl) in of PPO 850 ml of H2O, adjusted to pH 4.5 with formic acid. Acet- aldehyde and pyruvic acid were replaced by H2O in control The apparent molecular weight of the purified PPO was esti- group. In order to expediently separate the PPO and MWS, mated by sodium dodecyl sulfate–polyacrylamide gel 2.0 mg of purified PPO sample was packaged in a dialysis electrophoresis (SDS-PAGE). The procedures were per- bag (20 cm 9 10 cm) and dissolved in 50 ml of PBS (0.2 M, formed according to Laemmli’s method, using a Bio-Rad pH 6.0). The dialysis bags were immersed in the MWS, and Mini Gel system. The samples were fully denatured by heating the whole simulation system is the inadequate supply of at 100 °C for 6 min. Commassie Brilliant Blue was used as oxygen by nitrogen passed through, when the limited oxygen colorant. Molecular weights were estimated by comparison was used up, it change into anaerobic condition. The samples with the protein markers (14, 24, 30, 40, 62 and 100 kDa). were incubated at 40 °C for 0, 0.5, 1, 2, 5 and 10 h, respec- Purity was measured by the number of the protein bands. tively, in a shaker. As soon as the incubation completed, the 123 730 Eur Food Res Technol (2014) 238:727–739

MWS was shifted to −20 °C to prevent further reactions. for 40 min, centrifuged at 2,500 g for 10 min; then, the Meanwhile, the dialysis bag was taken out and then put in supernatant was collected and combined after washing with 500 ml of PBS (0.2 M, pH 6.0) at 4 °C for 12 h, then freeze- 5 ml of butanol twice [26]. The determination and calcu- dried to powder. lation were the same as described above.

Determination of phenolic acids in MWS by HPLC–ESI–MS Statistical analysis

The HPLC–ESI–MS method was used to monitor the Results were expressed as mean ± SD. One-way analysis change in other phenolic compounds (excluding proanth- of variance (ANOVA) was performed to compare the ocyanidins) in the MWS. The LC/MS system (Model 6430, means. Differences were considered significant at p\0.05 TRIPLE QUAD LC/MS, Agilent Technologies, Shanghai, and highly significant at p\0.01. Statistical analyses were

China) was equipped with a DAD. An ODS C18 column performed using Statistix for Windows version 9.0 (Ana- (250 9 4.6 mm, 5 μm) was used for separation of other lytical Software, Tallahassee, FL, USA). phenolic compounds. About 10 μl of sample was injected into the column by an auto-sampler, thermostatically con- trolled at 30 °C, and the flow rate was set at 0.8 ml/min. Results and discussion The DAD was monitored and analyzed for 25 min at 327 and 280 nm. The mobile phases consisted of acetic acid Profiles of PAs and phenolic acids

(0.5 %) in H2O (A) and acetonitrile (B). A linear gradient was applied as 0–20 min, 95 % A; 20–25 min, 70 % A. Profiles of the purified PA (purity of 98.67 %) from lotus Level of total phenolic acids was determined by the total seedpod were detected by HPLC–QTOF–MS according to the area of peaks through the HPLC diagram using chlorogenic previous method [27]. From the result of HPLC–QTOF–MS acid as an external standard. (Fig. 1;Table1), the proanthocyanidins were identified. M/z values of 289.0892 [(+)-catechin or (−)-epicatechin], 305.0718 UV–Vis scanning and FTIR measurement of PAs in MWS [(+)-gallocatechin or (−)-epigallocatechin], 577.1408 (pro- anthocyanidin B1 or B4), 573.5289 (proanthocyanidin A1 or The reaction solution of MWS (incubated with PPO for 0, A2), 591.1199 [(+)-catechin or (−)-epicatechin linked to a (+)- 0.5, 1, 2, 5 and 10 h) was filtrated by 0.45 μm membrane gallocatechin or (−)-epicatechin through an ether bond], and was directly scanned by a 2450-Series UV–Vis spec- 593.1366 (proanthocyanidin B2 or B3), 609.1297 [(+)-gallo- trophotometer (Shimadzu, Japan). The scanning catechin or (−)-epigallocatechin dimer], 865.2076 wavelength was selected from 600 to 200 nm after cor- [proanthocyanidinB1orB4linkedtoa(+)-catechinor(−)- rection by solvent contributions. epicatechin unit], 881.1087 [proanthocyanidin B2 or B3 linked The incubation MWS was freeze-dried to powder for the to a (+)-catechin or (−)-epicatechin unit], 897.1934 [proanth- FT-IR measurement. About 1 % of the sample (w/w, ocyanidin B2 or B3 linked to a (+)-gallocatechin or (−)- sample powder/KBr) was used for the Fourier transform epigallocatechin unit], 913.1878 [(+)-gallocatechin or (−)-epi- infrared (FTIR) spectroscopy analysis. A Bruker IFS 48 gallocatechin tripolymer through two ether bonds].The PAs FT-IR spectrophotometer (Bruker, Germany) over the fraction in the purified extract was only comprised of mono- wave range of 4,000–500 cm−1 (2.5–25 μm) with the res- mers (3.25 %), dimers (74.00 %) and trimer (22.75 %). olution of 2 cm−1 was performed. Compared with the known data on their dissociation pathways and retention time matched, the monomers consisted of Determination of polymerization degree of PAs in MWS 13.04 % (+)-catechin/(−)-epicatechin and (+)-gallocatechin/ (−)-epigallocatechin monomers, which were the basic units of The analyses of polymerization degree for the soluble PAs other PAs in lotus seedpod. There were both A-type and B-type in MWS (incubated with PPO for 0, 0.5, 1, 2, 5 and 10 h) of PAs, the greatest amount fraction was B1 or B4 were based on the results of HPLC–QTOF–MS as descri- (56.97 %) and the smallest amount of fraction was procyanidin bed above. Total contents of monomer, dimer and trimer A1/A2 (0.81 %). were calculated by the total peaks area according to the MS There were some other phenolic compounds in the identification data using (+)-catechin as the external stan- purified PAs extract, which were analyzed by HPLC–ESI– dard via a standard curve method. Moreover, full scanning MS. Ferulic acid, chlorogenic acid, caffeic acid, quercetin of m/z from 150 to 1,350 was applied to monitor the derivative, vanillic acid derivative and trans-cinnamic acid polymerization degree of PAs in the MWS. The insoluble derivative were identified by the retention time in HPLC PAs (precipitate) in the MWS were dissolved in 100 ml of chromatograms and m/z values from ESI–MS. The other butanol/HCl (97.5:2.5 v/v) with 7.0 g of FeCl3 at 100 °C phenols such as hydroxybenzoic acid derivative were 123 Eur Food Res Technol (2014) 238:727–739 731

Fig. 1 Q-TOF HPLC–MS analysis result of the oligomeric proanth- epigallocatechin, D (+)-afzelechin, E (+)-catechin, F (+)-gallocate- ocyanidins from lotus receptacle. The letters indicate the base units of chin, G Proanthocyanidin B1, H Proanthocyanidin B2, I Proantho- proanthocyanidins. A (−)-epiafzelechin, B (−)-epicatechin, C (−)- cyanidin B3, J Proanthocyanidin B4 identified only by comparison with published MS data purification with both Sephadex A-25 column (Lane B) and [28–32]. The level of total phenolic compounds was Sephacryl S-200 column chromatography (Lane C), a measured by area of total peaks in the HPLC diagram using much higher purity of PPO was obtained, as shown by the chlorogenic acid as an external standard. Among the SDS-PAGE analysis. The molecular weight of PPO was identified phenolic compounds, chlorogenic acid had the approximately 31 kDa compared to the markers. highest content (1.06 mg/L) and trans-cinnamic acid The CD chromatography of PPO was shown in Fig. 3a. The derivative the second highest (0.57 mg/L). CD spectrum of PPO in the far-UV showed a negative mini- mum at approximately 208 nm, indicating formation of α- Characteristics of polyphenol oxidase (PPO) from lotus helix, and a positive band of comparable magnitude was also seedpod observed at 195 nm, suggesting β-sheet formation. PPO consists of α-helix (59 %), β-sheet (4.3 %), turns (14.1 %) and Molecule weight and secondary structure of PPO random coil (22.6 %) at the secondary structure, which is considered as highly ordered and stable conformation. Such As shown in Fig. 2, the precipitation obtained from 80 % conformation probably provides evidence for its ability to

(NH4)2SO4 saturation was observed to have more than 15 withstand extreme environments to some degree such as high- bands (Lane A), suggesting it was an impure PPO. After level alcohol, high temperature and a wide pH range [19, 22].

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Table 1 Profiles of phenolic compounds from PA extract of lotus seedpod Identified compounds Ret. time (min) m/z value Equivalent Percentage (M–H)+ content (mg/L) content (%)

Phenolic acids and Ferulic acida 17.85 175.1 – – flavonoids [29–32] Chlorogenic acida 10.65 353.1 – – Caffeic acida 12.51 179.1 – – Vanillic acid derivativea 15.52 167.1 – – Trans-cinnamic acid 14.21 145.1 – – derivativesa Quercetin derivativesa 13.17 300.2 – – Hydroxybenzoic acid 3.82 136.1 – – deriviative Total Phenol – – 2.66 ± 0.18 – PAs [9, 13, 32] EC/C 36.19; 47.72 289.0892 5.20 ± 0.37 12.03 ± 1.33 EGC/GC 6.83 305.0718 1.30 ± 0.12 1.01 ± 0.15 B2/B3 isomerides 17.26; 21.13; 22.65; 23.72 593.1366 25.27 ± 2.45 7.38 ± 1.23 B1/B4 isomerides 28.50;30.12;31.89;39.53;42.35;46.08 577.1408 109.66 ± 10.58 49.59 ± 5.30 A1/A2 16.65 573.5289 1.63 ± 0.29 0.81 ± 0.14 (EC/C)–O–(EGC/GC) 21.13; 49.70 591.1199 4.18 ± 0.48 2.09 ± 0.24 (B1/B4)–(EC/C) 15.38; 33.52; 44.71 865.2076 19.42 ± 2.48 10.70 ± 1.44 (EGC/GC)–(EGC/GC) 14.61 609.1297 7.28 ± 0.16 1.81 ± 0.08 (B2/B3)–(EGC/GC) 5.76; 9.67; 18.80 897.1934 12.39 ± 2.35 7.18 ± 1.18 isomerides (B2/B3)–(EC/C) 10.96; 12.21; 13.13; 26.50 881.1087 10.36 ± 1.16 5.18 ± 0.71 isomerides (EGC/GC)–O–(EGC/ 5.48; 7.52 913.1878 3.33 ± 0.36 2.22 ± 0.18 GC)–O–(EGC/GC) Total monomer – – 6.50 ± 0.49 3.25 ± 0.25 Total dimer – – 148.01 ± 13.97 74.00 ± 6.98 Total trimer – – 45.49 ± 6.35 22.75 ± 3.18 Total PAs – – 202.36 ± 20.81 – Quantifications were measured by the area of each peak through the HPLC diagram using catechin (for PAs) and chlorogenic acid (for phenolic acids) as external standards via standard curve (R2 = 0.9995). The method had acceptable accuracy (90–110 % of true value) and precision (the intraday and interday coefficient variations\15 %) over the concentration range (0.10–100 μM). The recovery of samples was between 85 and 115 %. The limit of quantitation (LOQ) defined as minimum concentration which could be determined with acceptable accuracy and precision was 0.5 mg/L for PAs and 0.1 mg/L for phenolic acids and other flavonoids PA, proanthocyanidin; EC, (−)-epicatechin; C, (+)-catechin; EGC, (−)-epigallocatechin; GC, (+)-gallocatechin; B1, ; B2, ; B3, ; B4, ; A1, procyanidin A1; A2, ; –O–, ether bond a Commercial standards

Stability of PPO making. Excessive drastic reaction rate of polymerization in red wine is not desirable. The activities of purified PPO were measured using (+)- The effect of temperature on PPO activity was examined catechin as substrate at different pH values from 4.0 to in the range of 0–100 °C. The enzyme kept higher activity 10.5. The result showed that optimum pH of PPO was 9.0 between 60 and 100 °C with an optimum temperature of (Fig. 4a). This result was partially in agreement with pre- 80 °C using (+)-catechin as the substrate (Fig. 4b). Com- vious reports in which PPOs were from bitter or astringent pared with the highest activity of PPO at the optimum tissues, such as leaves of Cleome gynandra L. (Aydemir, temperature of 80 °C and the optimum pH of 9, its activity 2004) and green bean of Pouteria sapota [22, 33]. At the at 50–60 °C and pH of 4–5 was much lower, in which the pH of 4–5, the PPO presented much lower activity. How- enzyme could keep appropriate reaction rates when used in ever, the reaction rates could be controlled in much lower red wine aging after fermentation for that the reaction was level under pH of 4–5, which was preferable for red wine easily controlled.

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making process of red wine and it would cause a loss of wine color. The condensation between anthocyanins and tannins produced ethanal, and the cycle addition between anthocyanins and ethanal or pyruvic acid is one of the most important ways for precipitates formation. In this study, however, the polymerization and condensation of PAs could be one of the main reactions forming the precipitates due to the low content of anthocyanins in the MWS. There was no further change in the MWS after 5 h incubation. These results indicated that the formation of precipitates may be catalyzed by PPO, and its addition may strongly affect the phenolic profiles in MWS with its gradual loss of enzyme activity [35]. The alteration of the total phenolic compounds (excluding PAs) (TPC) and total PAs in MWS after incu- bation with PPO at different time points was shown in Fig. 5b. TPC concentration increased significantly at 0.5 h incubation with PPO compared to the control (9.76 vs. 0.56 mg, p \ 0.01). However, it kept a relative constant level in the subsequent periods from 1 to 5 h. A slight decrease in PAs content occurred in 1 h incubation com- Fig. 2 SDS-PAGE analysis of polyphenol oxidase purified from lotus pared to the control (186.10 vs 193.77 mg, p [ 0.05), but seedpod. a Crude enzyme extract after ammonium sulfate (80 % significant decrease in PAs content was observed in 2 h saturation) precipitation. b Further purified enzyme by Sephadex A-25 column gel filtration. c Final purified polyphenol oxidase after incubation (145.32 mg). The increased phenolic com- Sephacryl S-200 column gel filtration. d Protein markers pounds in MWS may result from broken PAs or catechin units by hydrolysis during 0.5 h incubation. The acidic Ethanol tolerance was assayed with a gradient concen- condition, arisen from the addition of pyruvic acid and tration ranging from 5 to 50 % (v/v, pH 7.0). The relative formic acid, probably contributed to a series of hydrolysis enzyme activities were 90.3 and 85.1 % at alcoholicity of reactions. A slight decrease in PA content occurring in 1 h 10 and 15 %, respectively (Fig. 4c), suggesting that PPO provided strong evidence (Fig. 5b). may be active in red wine with 11–20 % alcoholicity, Profiles of the soluble PAs (Fig. 5d) showed that poly- particularly the dry red wine [16]. meric PAs formed gradually in the MWS. The proportion of total monomer significantly increased at 0.5 h (22.47 %) Profiles change in phenolic compounds and PAs in and 1 h (24.23 %) incubation compared to the control MWS (3.12 %), then slightly decreased at 2 h (20.24 %), dropped to 16.35 % at 5 h and 0 % at 10 h incubation. The dimer When the MWS was incubated with PPO at 40 °C for 0, proportion in MWS was 74.33 % in 0 h incubation, 0.5, 1, 2, 5 and 10 h, respectively, the supernatant, pre- decreased slightly to 70.40 % in 0.5 h incubation, signifi- cipitate and PPO were analyzed, respectively. In the treated cantly to 48.51 % in 2 h incubation, 22.00 % in 5 h groups, acetaldehyde and pyruvic acid addition presented incubation and 0 % in 10 h incubation with PPO. The little difference on profile composition of PAs compared trimer proportion of PAs in MWS decreased at 0.5 h with control group, suggesting that acetaldehyde and incubation (7.13 %) compared to the control (22.55 %), pyruvic acid were not the major factors affecting the con- then began to increase in 1 h incubation (11.34 %), sig- densation of flavanols in the MWS. The color of the MWS nificantly increased to 31.25 % in 2 h incubation, to became continuously darken and muddy during 2 h incu- 61.65 % in 5 h incubation, and to 100 % in 10 h incubation bation. After 2 h, the color slowly transformed to clear with with PPO. The results of full scanning of m/z from 150 to the formation of visible precipitate (Fig. 5a), suggesting 1,350 in the control group and 5 h groups (Fig. 6) also that content of soluble PAs decreased in the MWS. A small revealed that polymerization gradually took place during amount of light brown precipitate was found in the dialysis incubation with PPO, because the low molecular weight bags after 2 h incubation (no precipitate was observed (\ m/z 500) of PAs (or flavanols) at high level in the before 1 h) and increased significantly with the incubation control group decreased gradually, the PAs with the time prolonging from 5 to 10 h (p \ 0.01). According to molecular weight between m/z 500 and 865.2 became main Sacchi [34], precipitation commonly occurred in the components at 2 h incubation with PPO, the PAs with 123 734 Eur Food Res Technol (2014) 238:727–739

Fig. 3 CD spectrum and secondary structure composition of the purified PPO. a PPO before incubation in MWS, b PPO after 10 h incubation in MWS

Fig. 4 StabilityÀÁ of PPO in different pH values (a), temperatures (b), and concentrations of ethanol (c). (+)-catechin was used as a substrate (Q product ¼ A276testedsample A276control ðA430testedsample A430control) molecular weight between m/z 865.2 and 1,280 were rare in 0 % at 10 h incubation with PPO, in a reverse gradual the control, and were up to high level at 5 h incubation. increase in trimers to 100 % at 10 h, was observed, sug- The initial increase in monomers was likely due to the gesting that the monomers and the dimers transformed to hydrolysis of the dimers and the trimers in acidic solution. trimers as the only soluble PAs and to tetramers or other The monomers ( and gallocatechins) and dimmers oligomers (degree of polymerization [ 4) as the precipi- might play a precursor role in the polymerization catalyzed tate. In the initial periods (0–0.5 h), the significant decrease by PPO. A gradual decrease in monomers and dimers to in the trimers might be partially due to the formation of

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Fig. 5 Change in PAs and other phenolic compounds with PPO different adjacent letters in each set indicate significance at p \ 0.05. incubation in the MWS. a Change in precipitate content with PPO Values marked with different nonadjacent letters in each set indicate incubation in different groups; b change in total PAs and total other significance at p \ 0.01. The method had acceptable accuracy (90– phenolic compounds with PPO incubation in different groups; c 110 % of true value) and precision (the intraday and interday monomer, dimer and trimer composition of precipitate with PPO coefficient variations \ 15 %) over the concentration range (0.10– incubation in different groups; d monomer, dimer and trimer 100 μM). The recovery of samples was between 85 and 115 %. The proportion in the supernatant with PPO incubation in different limit of quantitation (LOQ) defined as minimum concentration which groups. Total PAs total proanthocyanidins; TPC total other phenolic could be determined with acceptable accuracy and precision was compounds (excluding PAs). Data are expressed as the mean fold 0.5 mg/L for PAs and 0.1 mg/L for phenolic acids and other changes in three individual experiments ± SD. Values marked with flavonoids tetramers and other oligomers from trimers, which could polymerization of soluble PAs in which the soluble PAs not be detected by HPLC [36]. The addition of PPO such as monomer, dimmer and trimer of PAs might be obviously boosted the polymerization in the MWS, which combined together or with other phenolic compounds to was in accordance with the profiles change in PAs during form some insoluble high molecular weight compounds aging of red wine making. However, the optimal amount of such as the tetramer and higher polymers. PPO and incubation condition remains to be determined. The spectra of the PAs of lotus seedpod incubated with Go´mez-Plaza [37] reported that the phenolic compounds PPO at different time points were shown in Fig. 7a (FTIR) directly reacted with each other to form polymeric com- and b (UV–vis). Ling [38] has reported a strong O–H pounds. As the molecular weight was getting higher absorption band from 3,000 to 3,750 nm and the benzene enough, it became insoluble. On the other hand, the PAs ring absorption band from 1,300 to 1,700 nm. There was were combined with the phenolic compounds or other PAs little difference between O–H band and benzene ring band, to form insoluble compounds. In this study, the latter was but ketonic bond or ether bond from 800 to 1,200 nm in the probably the main reaction because the PAs had a much PAs incubated with PPO at different time points appeared, higher proportion (98.67 %) in the MWS than other phe- with no band in the control. It indicated that a series of nolic compounds. The significant decrease in PAs was oxidation reactions catalyzed by PPO may occur in PAs. probably due to the formation of precipitate from the This result was in accordant with the profile variance in 123 736 Eur Food Res Technol (2014) 238:727–739

Fig. 6 Full scanning mass spectra of the MWS incubated with PPO at different time points

PAs and precipitate formation found in the study (Fig. 5). A butanol/HCl/FeCl3 acidolysis method was used to C–O–C form is much stable than C = O form during the analyze the insoluble precipitate. A similar PA composition oxidative processes. C = O form mostly appears in the in the precipitates formed in MWS incubated with PPO at processes as the intermediate product and will be auto- 2, 5 and 10 h was observed, which were 16.54 % of oxidized into more stable form (C–O–C). The FTIR fin- monomers, 21.03 % of dimers and 62.43 % of trimers (2 h) gerprints of C = O and C–O–C are different, in which C– (Fig. 5c). The insoluble polymeric PAs were in an invari- O–C presents a stronger trough. Compared with the other able polymeric form in some degree, and their molecular time points of treated groups, the FTIR band of 1 h showed composition was probably alike in different incubation a different behavior (Fig. 7a). The results reflected that the time points. formation of C = O bond and C–O–C bond was a dynamic Polymerization in the system was constricted and lim- process during the time period from 0.5 to 1 h. In the initial ited by the enzyme activity, pH value, and balance of stage of the process, oxidative reaction took place in a reaction potential between different phenols [20]. The smaller scale under the oxygen-lacked environment. formation of carbocation intermediate was regarded as a Minority phenolic compounds were involved in the reac- most important step in this biochemical process. The car- tion, and the end products containing C–O–C bond were bocation intermediate, a hyperactive electrophile, existed therefore generated. However, other phenolic compounds in the solution system for a very short time and would be with low potential would compete for limited oxygen. New combined with another flavan-3-ol unit, the dimer was O-quinones (containing C = O) therefore formed again. therefore formed. The chain reaction continued; trimer and The reaction did not stop until the system had an equilib- higher polymers were synthesized and produced the pre- rium potential. cipitate [20, 39]. 123 Eur Food Res Technol (2014) 238:727–739 737

Fig. 7 FTIR (a) and UV–Vis (b) spectra of the MWS incubated with PPO at different time points

Change in PPO conformation in the MWS 1990 s [41]. Some replacement methods of sulfur dioxide have been reported and applied to improve red wine As shown in Fig. 3b, the conformation of PPO after incu- making [42–44]. In the MWS, high concentrations of bation in MWS appeared to be different from that of PPO ethanol and phenols, as well as low pH value, were the before incubation. The α-helix content decreased from 59.0 main inhibiting factors in the activity of PPO. These to 39.1 %. Meanwhile, β-sheet content increased from 4.30 characteristics and polymerizing reaction with PA by PPO to 9.28 %, turns content from 14.1 to 23.7 %, and random may have potential application in improving the quality of coil content from 22.6 to 27.9 %, respectively. These red wine (Fig. 6). results indicate that the PPO after incubation may be more In summary, the oligomeric PAs could combine with the irregular and instable in conformation than before incuba- PPO which catalyzed them to form polymeric PA with tion, which leads to a possible change in its bioactivity another phenolic compound unit in the MWS, such as (−)- because the irregular and instable conformation could be epicatechin in the chain reaction. As the polymerization more readily to be combined with other compounds [19]. level was higher enough, the precipitate would be formed. The activity change in the PPO may be mainly caused At the same time, the various kinds of phenols would by two steps. Firstly, as the incubation time increased, the weaken the enzyme activity of PPO and the conformation conformation of PPO became more irregular and instable. of PPO was changed. Finally, the PPO combined with the And the irregular and instable conformation could be polymeric phenols to form the insoluble compounds and more readily to be combined with other compounds, lose its catalytic activity. Our results have shown that PPO especially the phenolic compounds; the combination may may be used to accelerate the polymerization of PAs in the lead to a decrease in the enzyme activity. Secondly, as model red wine system. the polymerization of the PAs increased over tetramer or pentamer in the MWS, astringency of PAs enhanced, the Acknowledgments This research was supported by the National polymers more likely combined with proteins by the Natural Science Foundation of China (Project No. 31301578); the complexation of hydrophobic bonds and hydroxyl bonds Natural Science Foundation of Jiangxi Province (Project No. to form the precipitate and lose its catalytic activity. 20132BAB214002); the Science Foundation of Jiangxi provincial According to a previous study, all these reactions department of education (Project No. GJJ13024); the Free Explora- tion and the Research Program of State Key Laboratory of Food occurred in a hydrophobic pocket formed from protein Science and Technology, Nanchang University (Project No. SKLF- molecules [40]. TS-200921 and SKLF-MB-201003). All the authors would like to There are sulfur dioxide/sulfites addition in the con- thank for these financial support. Also, the authors would like to thank ventional processing of red wine, and it will inactivate Dr Deming Gong for editing the manuscript. PPO. However, sulfur dioxide residues in traditional red Conflict of interest None. wine processing have aroused extensive attention nowa- days. Its harmful effects, especially on asthma, allergy Compliance with Ethics Requirements This article does not and toxic headache, were continually reported from contain any studies with human or animal subjects.

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