Preparation of modified citrus pectin (MCP) using an advanced oxidation process with hydroxyl radicals generated by UV-H2O2

Food Hydrocolloids 102 (2020) 105587

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Preparation of modified citrus pectin (MCP) using an advanced oxidation process with hydroxyl radicals generated by UV-H2O2 Jing Cao a, Jian Yang b, Kaiting Yue a, Zhaomei Wang a, * a b

School of Food Science & Engineering, South China University of Technology, Guangzhou, Guangdong Province, 510640, PR China College of Pharmacy and Nutrition, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK S7N 5E5, Canada

A R T I C L E I N F O

A B S T R A C T

Keywords: Modified citrus pectin UV-H2O2 Advanced oxidation process Degradation mechanism

An advanced UV-H2O2 oxidation system was developed to prepare modified citrus pectin (MCP) from citrus pectin. A rhamnogalacturonan I (RG-I) enriched MCP fraction with the weight-average molecular weight (Mw) of 44.9 kDa and a MCP recovery rate of 91.2% was obtained under a condition of 30 mM H2O2 and UV irradiation for 5 h. The kinetic study implied that UV-H2O2 oxidation process is more efficient in reducing the Mw than the most commonly used approaches. By comparison of the chemical compositions of MCP and relevant in­ termediates during continuous UV irradiation, we proposed a functional mechanism of the UV-H2O2 oxidation process. Pectin depolymerization started with an initial random scission, followed by main chain scission through selective oxidative degradation of galacturonic acid linkages, and ended with a chain-end scission. In a minor extent, β-elimination and de-esterification occurred simultaneously throughout the process.

1. Introduction Modified citrus pectin (MCP) is normally produced from degradation of citrus pectin (CP). During this process, pectin is cleaved and deesterified, generating linear homogalacturonan (HG), rhamnogalactur­ onan I (RG-I) and rhamnogalacturonan II (RG-II) (Maxwell, Belshaw, Waldron, & Morris, 2012). Compared to unmodified CP, MCP is easier to be digested in the small intestine and absorbed into the bloodstream due to the lower molecular weight. This confers MCP a wide range of ap­ plications. It is generally regarded safe by the U.S. Food and Drug Administration (FDA) and commercially distributed as a dietary sup­ plement since it possesses many important health-promoting functions, such as antioxidative activity, hypocholesterolemic effect and removal of heavy metals (Brouns et al., 2012; Ramachandran, Wilk, Melnick, & Eliaz, 2017; Zhao et al., 2008). Recently, MCP was reported to possess immunomodulatory effects via increasing the expression of pro-inflammatory cytokines (Merheb, Abdel-Massih, & Karam, 2019). Furthermore, MCP was shown to inhibit proliferation and metastasis and induce apoptosis in cancer cells (Ai et al., 2018; Maxwell et al., 2016; Morris, Belshaw, Waldron, & Maxwell, 2013). Two commercial MCPs, the pH-modified pectin GCS-100 and the enzymatically modified pectin Pectasol-C, have fulfilled phase II clinical trials for chronic kidney dis­ ease and the latter is under phase III clinical trial in treating patients

with prostate cancer (Ruvolo et al., 2016). So far, MCP has shown a promising potential in the treatment of cancers. To prepare MCP, various degradation methods have been developed, including chemical treatment, enzymatic hydrolysis and physical pro­ cess. Chemical treatment is widely used due to the low cost and imme­ diate availability. However, chemical degradation by either acid or alkaline requires high quality anti-corrosion equipment and might cause serious environmental pollution (Zhang, Hu, Wang, Liu, & Pan, 2018). Enzymatic degradation has been emerging as an environment-friendly alternative to chemical treatment. However, it requires different types of enzymes, leading to increased costs of the depolymerization process (Ma, Wang et al., 2018). Physical process, including photocatalysis and ultrasonic approaches, has also been considered as a green technique to prepare MCP. For instance, UV-TiO2 photolytic process can decrease the Mw of CP from 400 kDa to 200 kDa in 6 h (Burana-osot, Soon­ thornchareonnon, Hosoyama, Linhardt, & Toida, 2010), and an ultra­ sound process can degrade CP to a final Mw around 84 kDa ~ oz-Almagro, Montilla, Moreno, & Villamiel, 2017). Apparently, (Mun physical method alone is not sufficient in producing MCP with desired Mw (10–60 kDa). An advanced oxidation process, which is based on the cleavage re­ action by hydrogen peroxide (H2O2)-generated free radicals, has been evidenced as a promising way for depolymerization of macromolecules

* Corresponding author. E-mail address: [email protected] (Z. Wang). https://doi.org/10.1016/j.foodhyd.2019.105587 Received 23 March 2019; Received in revised form 3 December 2019; Accepted 9 December 2019 Available online 9 December 2019 0268-005X/© 2019 Elsevier Ltd. All rights reserved.

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and found several applications in polysaccharide degradation (Ma, Wu et al., 2018; Wu et al., 2018; Zhang et al., 2018; Zhang, Wang, Zhao, & Qi, 2014). Fenton-type reactions are among the most-widely-accepted H2O2-participted oxidative degradation, which utilize the powerful oxidative radical �OH generated from H2O2 under inorganic particle mediated catalysis. Up to now, a large quantity of iron or its oxidized derivatives such as nano Fe3O4, CuO and ZnO have been applied in Fenton oxidation systems. However, these particles are difficult to be separated after the treatment and might be harmful in the final products. Apart from the inorganic catalysts, some physical technologies were reported to catalyze the generation of �OH in a H2O2 system. For instance, solution plasma process (SPP) was combined with H2O2 to prepare low-molecular-weight polysaccharide from Auricularia auricula (Ma, Wu et al., 2018). Li et al. set up an ultrasonically accelerated metal-free Fenton reaction to fast-prepare RG-I enriched low Mw pectin (Li et al., 2019). Both studies show the potential application of the H2O2-related oxidation process in pectin modification. Ultraviolet light catalytic hydrogen peroxide (UV–H2O2) oxidation system is a very efficient �OH generator, which significantly accelerates the disassociation of �OH from H2O2 by UV radiation. With the increased capability of producing high concentrations of �OH over very short period of time, UV-H2O2 oxidative technique has been widely used to degrade organic pollutants rapidly and efficiently in industrial waste water treatment (Zhang, Xiao, Zhang, Chang, & Lim, 2017). Wang et al. first applied the UV-H2O2 technique in polysaccharide degradation and reduced the viscosity of chitosan through a synergetic degradation effect of ultraviolet light and H2O2 (Wang, Huang, & Wang, 2005). Since then, little had been conducted in this research area until our recent study showing that pectin in sisal cell wall can be efficiently destructed by UV-H2O2 (Yang et al., 2018b). Therefore, we proposed that the UV-H2O2 technique would be an efficient alternative to prepare bioactive MCP. Despite the evident efficacy of the advanced oxidative process on polysaccharides degradation, the underlying mechanism is poorly un­ derstood. The oxidative degradation of polysaccharides was roughly viewed as a cleavage of backbone glycoside in a random pattern (Bur­ ana-osot et al., 2010; Wu et al., 2018). In recent years, several fast oxidation techniques have been developed in pectin degradation and the possible degradation mechanism was assumed as a depolymerization by backbone oxidation cleavage preferentially at the acid glycosidic bonds between GalAs in HG domain (Li et al., 2019; Zhi et al., 2017). However, an important question is what would happen when the HG part was reduced too short to be the main chain and lost the easy availability for reaction at later reaction period. Apparently, along with the proceeding of the reaction, secondary degradation will happen on the already degraded intermediates and the degradation patterns may vary signifi­ cantly depending on the distinct structural characteristics of the in­ termediates. Take acid degradation of pectin as an example, it firstly occurred on the more susceptible linkages between neutral sugar resi­ dues, followed by a slow cleavage of the linkages within GalA residues (Thibault, Renard, Axelos, Roger, & Cr� epeau, 1993). As for enzymatic degradation of pectin, it has been shown to involve multiple degradation pathways, including a random cleavage pattern at the beginning and adopts an exo-mode of cleavage at a later stage (Combo, Aguedo, Goffin, Wathelet, & Paquot, 2012). Pectin degradation under alkaline condition was realized by two pH-modulated competitive reactions, β-elimination degradation and demethylation by saponification (Kravtchenko, Arnould, Voragen, & Pilnik, 1992). Therefore, it is essential to identify the degradation stages and study the degradation mechanism of each stage independently based on the variation of the substrates produced from the previous stage. In the current study, we evaluated the efficacy of the UV-H2O2 advanced oxidation process in producing low molecular weight MCP and investigated the underlying mechanism using product release ki­ netic analysis. To our knowledge, this is the first study on MCP prepa­ ration using a UV-H2O2 advanced oxidation process.

2. Materials and methods 2.1. Chemicals Citrus pectin material type P9135 (Mw: ~640 kDa; degree of ester­ ification: 62%; GalA: > 74%) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Monosaccharide standards, including fucose (Fuc), glucose (Glc), arabinose (Ara), xylose (Xyl), galactose (Gal), rhamnose (Rha) and GalA, were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). H2O2 (30% wt) was supplied by Tianjin Damao Chemical Reagent Co., Ltd (Tianjin, P. R. of China). Pectinase S10007 (enzyme activity: 500 U/mg) from Aspergillus Niger was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, P.R. of China). The pectinase solution was prepared by dis­ solving 1.0 g pectinase powder in 100 mL 0.2 M sodium acetate buffer (pH 4.0). 2.2. Photochemical reaction apparatus The photochemical reaction device (CEL-LAB500), which was pre­ viously described by Yang et al., 2018a, was provided by Beijing Aulight Co., Ltd. (Beijing, P.R. of China). Briefly, the main section of the device consists of a quartz glass sleeve, a magnetic stirrer, a reaction tank with six reaction tubes (25 cm, 50 mL) and a high-pressure mercury lamp CEL-LAM500 (500 W, 200 mm). The UV light source has a 365 nm cutoff filter to assure the presence of only UV light. The apparatus was exter­ nally connected to a water circulating system in order to maintain the temperature below 25 � C. 2.3. Preparation of MCP by UV-H2O2 The process for MCP preparation was shown in Fig. 1. Briefly, various amount of CP (0.1, 0.3, 0.5, 0.7 or 1.0 g) was dispersed in 100 mL distilled water and an ultrasonic homogenizer was applied to assist in pectin dispersing. The solution pH was adjusted to 1, 4, 7, 10 or 12 using HCl (0.1 M) or NaOH (2.5 M) to examine the effect of pH on CP degradation at varied irradiation time. Aliquots (10 mL) of the dispersed solutions were transferred to 50 mL-screw-capped test tubes with sub­ sequent addition of H2O2 (5, 10, 20, 30, 40, 50 and 60 μL, respectively, per 100 mg CP). The sample tubes with loosely screwed caps were then placed into the photochemical reactor and exposed to UV light irradia­ tion (500 W, 365 nm). All degradation experiments were carried out under UV irradiation for 1, 3, 5, 7 and 9 h, respectively, at room tem­ perature (~25 � C) under a constant magnetic stirring (~1000 rpm) before the reaction being terminated by turning off the UV lamp. The solution was neutralized with 1 M HCl or 1M NaOH, and subsequently poured into anhydrous ethanol. The ratio of anhydrous ethanol to re­ action solution is 4:1 (v/v). After gently stirring for 2 min using a glass

Fig. 1. The scheme for citrus pectin degradation method by UV-H2O2. 2

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rod, the mixture was rested in a fridge (4 � C) for 24 h. Then, the mixture was centrifuged at 4000 rpm for 10 min, and the precipitate was ob­ tained, which was washed for three times with anhydrous ethanol and then vacuum-dried to constant at 60 � C to give crude MCP powder. The MCP powder (1 g) was dissolved in 20 mL deionized water via rapid stirring and then dialyzed against deionized water for 3 days with fresh water exchange twice daily. The Mw cutoff of the dialyzing membrane is 1000 Da. Finally, the dialyzed solution was concentrated to 1/5 of its original volume under reduced pressure by rotary evaporator and vacuum-dried at 60 � C, resulting in the pure degraded citrus pectin (MCP). The MCP recovery rate (%) was calculated using Eq. (1). Recovery ​ rate ​ ð%Þ ¼ MMCP =MCP � 100%

2.6. Quantitative detection of the chemical composition of MCP and reaction intermediates Through time-course of the UV-H2O2 treatment, aliquots of the CP reaction solution was extracted and divided into solid and liquid frac­ tions through ethanol precipitation. The solid fraction was vacuumdried to obtain the final product MCPs, which were subjected to chemical composition analysis. The left supernatant, i.e. liquid fraction, was concentrated and vacuum-dried to constant weight at 60 � C to give the intermediate powder, which was also subjected to chemical composition analysis. Concisely, component determination for both MCP and the intermediate is described below.

(1)

2.6.1. GalA and neutral sugars in CP, MCP and the intermediate Content of GalA was measured using the m-hydroxydiphenyl method (Blumenkrantz & Asboe-Hansen, 1973). The monosaccharide composition of neutral sugars was determined using a modified protocol (Guo, Guo, Yu, & Kong, 2018). Briefly, a re­ action sample (~10 mg) was first decomposed into separated mono­ saccharides in 2 mL pectinase solution at 45 � C for 12 h, followed by acid hydrolysis with 2 mL 0.4 M trifluoroacetic acid at 110 � C for 2 h. Before analysis, the hydrolysate was diluted with distilled water and the final pH was adjusted to 8–10 using ammonium hydroxide. Then, the hy­ drolysate was filtered through a 0.45 μm membrane and the obtained supernatant (25 μL) was subjected to high performance anion-exchange chromatography analysis and detected by a pulsed amperometric de­ tector (ICS-5000, Dionex, USA). The instrument was equipped with a CarboPac PA1 analytical column (250 � 4 mm) and a CarboPac PA1 guard column (50 � 4 mm). Neutral monosaccharides were separated in a single run using a mobile phase (1.0 mL/min) of 10 mM NaOH for 23 min at 30 � C, followed by a 170 mM CH3COONa in 100 mM NaOH for 12 min. The total neutral sugar (NS) content was calculated as the sum of Fuc, Rha, Gal, Ara, Xyl and Glc. The contents of GalA, each mono­ saccharide and total NS in the intermediate were expressed as weight percentage, based on the milligrams per 100 mg of MCP or CP. The contents of these sugars in MCP and CP were then transferred into molars in order to compare the numbers among specific sugars.

where MMCP and MCP denote the weight of MCP (g) and the weight of CP (g), respectively. 2.4. Molecular weight analysis The molecular weight of CP was examined before and after modifi­ cation by gel permeation chromatograph (GPC) using a modified pro­ tocol by Yang, wang et al. (2018). The MCP samples (4–5 mg) were prepared in 2 mL 20 mM KH2PO4 buffer (pH 4) and filtered with 0.45 μm drainage membrane before being injected into two consecutive TSK-GEL columns (G5000 PWXL, 7.8 � 300 mm and G3000 PWXL, 7.8 � 300 mm). The analysis was performed at 35 � C using a GPC connected with a Waters 2414 refractive index detector (Bio-rad, Richmond, CA, USA). The elution solvent was 20 mM KH2PO4 buffer solution (pH 4) and the flow rate was 0.6 mL/min. The dextran standards with the peak mo­ lecular weight (Mp) of 4.4 k, 9.9 k, 21.4 k, 43.5 k, 124 k, 196 k, 277 k, 401 kDa were used to obtain the calibration curves. A linear equation, Log Mp ¼ 0.1987*X þ 9.6453, where X ¼ retention time, was estab­ lished for the molecular weight estimation (R2 ¼ 0.9965). The molecular weight of MCP or CP was determined using the Breeze software asso­ ciated with Waters GPC system. 2.5. Kinetic analysis

2.6.2. Degree of esterification in CP or MCP The degree of esterification (DE) in the CP samples before and after modification was determined using the titrimetric method (Yang, wang et al., 2018).

Two simplified kinetic models were explored to quantitatively describe the degradation process of CP by UV-H2O2 method based on the assumption that the degradation occurs predominantly on main-chain linkage. Assuming that the degradation reaction obeys first-order kinetics for random chain scission mechanism, a random scission model was set up using the reciprocal Mw versus degradation time relationship according to Eq. (2), which was proposed by Tanford (1961, pp. 611–618) and applied by a recent research on Auricularia auricula polysaccharide degradation by combined H2O2-SPP (Wu et al., 2018). 1 = MwðtÞ

1=Mwð0Þ ¼ kt=m ¼ k’ t

2.6.3. Oligosaccharide, reducing sugar and double bond in the intermediate In order to quantify the content of oligosaccharides released in the reaction solution, the intermediate was subjected to conversion of oli­ gosaccharides into monosaccharides by a post-hydrolysis process using the same method as for the monosaccharide composition determination described in 2.8.1. The oligosaccharide content was calculated based on the difference of the NS content before and after post-hydrolysis as below (Chen, Li, Sun, Cao, & Sun, 2018).

(2)

where Mw(t) and Mw (0) (Da) represent the Mw of pectin at time t and 0, respectively; k (min 1) and k’ (mol g 1 min 1) represent the degrada­ tion rate constant; t is the reaction time; and m is the monomeric mo­ lecular weight of CP of ~194 g/mol. The main-chain scission model was adopted to evaluate the effect of H2O2 concentration on average number of chain scission (N) using Eq. (3), which was previously applied in glycosaminoglycan depolymer­ ization by H2O2–Cu (II) method (Wu, Xu, Zhao, Kang, & Ding, 2010). N ¼ Mwð0Þ=MwðtÞ

1

Oligosaccharides ​ ðmg = 100 mgÞ ¼ NSah

NSbh

(4)

where NSbh and NSah denote the total neutral sugar contents in the su­ pernatant before and after post-hydrolysis, respectively. The reducing sugar generated during the degradation of CP was determined by the dinitrosalicylic acid method (Miller, 1959). The double bond content was determined by measuring the absor­ bance of the diluted samples at 235 nm using a UV–vis spectropho­ tometer (UV-5200, Unico, Shanghai, China). The concentration of 4, 5unsaturated uronides was calculated using an extinction coefficient of 5412 L mol 1 cm 1.

(3)

The average number of chain scission (N) is defined as (Mw (0) - Mw (t))/Mw(t), where Mw (0) is weight average molecular weight of CP sample at time t ¼ 0, and Mw(t) is weight average molecular weight of the fragment at the end of reaction time t. 3

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2.7. Statistical analysis

3.3. Kinetics during molecular weight reduction

All the experiments were carried out in triplicate. Statistical analysis was performed with one-way ANOVA with Tukey’s multiple comparison tests using SPSS 22.0 software (SPSS Inc. Chicago, IL, USA). Statistical significance was set at p < 0.05.

3.3.1. Effect of UV irradiation time The chromatographic profiles of CP exposed to UV light irradiation for 0–9 h in the presence of 15 μL/100 mg H2O2 were shown in Fig. 2.

3. Results and discussion 3.1. Effect of pectin concentration Effect of pectin concentration on the molecular weight and recovery rate of MCP was summarized in Table 1. The Mw of MCP decreased significantly from 74k to 15 kDa, associating with the polydispersity (P) index (Mw/Mn) being lowered from 2.6 to 1.9, when the pectin con­ centration was reduced. This phenomenon is likely due to the better dispersity of pectin solution at low concentration, which, in turn, would provide a better contact with H2O2 molecules and improve the degree of oxidative degradation. However, the recovery rate of MCP decreased from 94.7 � 1.2% to 54.3 � 2.3% upon reducing pectin concentration from 10 mg/mL to 3 mg/mL. Based on the limit of CP dispersion in water and in consideration of the recovery rate of MCP, we decided to proceed subsequent studies at CP concentration of 10 mg/mL. 3.2. Effect of pH Effect of pH on the molecular weight and recovery rate of MCP was also summarized in Table 1. As pH of the pectin solution increased, the recovery rate of MCP was only marginally changed (between 94.3 � 2.3 and 91.2 � 2.1%). However, the P index was reduced from 2.8 at pH 1 to 2.1 at pH 12, implicating that MCP fractions became homogenized, which may be attributed to the different degradation mechanisms involved at acid or basic environment. The Mw of CP was reduced from 641 kDa to the minimum of 45 kDa at pH 4, being consistent with the observation that H2O2 is more stable and effective in weak acidic solu­ tions (Gogate & Prajapat, 2015). It is notable that the Mw of CP was significantly decreased to 50 kDa at pH 12, equally as efficient as what occurred at pH 4, despite the fact that H2O2 is prone to self-decompose at alkaline pH and leads to loss of the actual available H2O2 during the reaction. We propose that there are other degradation ways happened in alkaline environment other than the advanced oxidation. For instance, de-esterification of high methoxyl pectin becomes more prominent at alkaline environment according to Jiang’s report (Jiang, Liu, Wu, Chang, & Chang, 2005). In addition, depolymerization by β-elimination is expected to occur at relatively high pHs.

Fig. 2. (A) Molecular weight distribution curves of CP degradation for different time. (■) refer to the right vertical axis. (B) The degradation kinetics curves of 10 mg/mL of CP solution exposure to the UV light in the presence of 15 μL/100 mg H2O2 at pH 4.

Table 1 Molecular weight and recovery rate of MCP prepared at different pectin concentration and pH.a Factors

Treatments

Pectin concentration (mg/mL)

1 3 5 7 10 1 4 7 10 12

Molecular weight

Polydispersity (Mw/Mn)

Mw (kDa)

pH

15.4 � 38.7 � 52.3 � 58.9 � 74.2 � 82.4 � 44.9 � 53.6 � 58.4 � 49.6 �

Recovery rate (%)

Mn (kDa)

1.0d 3.3c 5.3b 5.0b 7.2a 7.8a 4.2c 4.3cb 4.4b 3.2cb

8.1 � 0.6d 19.1 � 1.3c 22.0 � 1.3cb 23.5 � 1.2b 28.6 � 2.8a 30.0 � 2.0a 19.4 � 1.3c 22.0 � 1.4cb 23.7 � 1.2b 23.6 � 1.4b

1.90 � 2.03 � 2.38 � 2.51 � 2.59 � 2.75 � 2.32 � 2.43 � 2.46 � 2.10 �

0.02d 0.04c 0.08b 0.07a 0.01a 0.04a 0.04c 0.06b 0.05b 0.03d

–b 54.3 � 87.2 � 92.8 � 94.7 � 94.3 � 91.2 � 93.5 � 92.9 � 91.6 �

2.3c 2.5b 1.3a 1.2a 2.3a 2.1a 2.2a 2.4a 2.6a

Values with different small case superscript letters (a-d) in the same column within each factor indicate significant differences as estimated by Duncan’s multiple range test (P < 0.05). a H2O2 dosage 30 μl/100 mg CP, UV irradiation for 5 h. b – means the recovery rate is too low to be detected. 4

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Unmodified CP appeared to be the earliest in the elution profile, and displacement of the peaks gradually shifted to the right with prolonga­ tion of photolysis time. This implicates that pectin was degraded to lower Mw fragments. Based on the commonly accepted random scission model for polysaccharide degradation (Wu et al., 2018; Li, Li, Geng, Song, & Wu, 2017), the relationship between the reciprocal of Mw and degradation time was evaluated using Eq. (2). As shown in Fig. 2(B), a linear relationship (R2 ¼ 0.9787) was observed during the degradation process, indicating that the adopted random scission model was appli­ cable in explaining pectin degradation by UV-H2O2. The random scission implied that photochemical degradation might occur through random breakage of the glycosidic bonds and releases a random series of het­ erogeneous oligomers. We also noticed that data point at 9 h deviated from the linear relationship, which might be attributed to the change of degradation mechanism from random scission to chain-end scission, a process occurred right after random scission with the increasing amount of reduced ends released from random scission (Thibault et al., 1993). Similar phenomenon was also observed in chitosan degradation by H2O2–Fe (II) (Chang, Tai, & Cheng, 2001). The rate constant k was determined to be (6.93 � 0.44) � 10 6 min 1 (i.e. (4.16 � 0.26) � 10 4 h 1) which was equivalent to k’ of (3.57 � 0.23) � 10 8 mol g 1 min 1, during pectin degradation by UV-H2O2. It is 2.6 times higher than that for the CP degradation by UV-TiO2 at pH 4 (1.59 � 10 4 h 1) (Bur­ ana-osot et al., 2010). Furthermore, the rate was one order of magnitude higher than those (4.2 � 10 7 and 4.3 � 10 7 min 1, respectively) for acid hydrolysis (pH 2) of two kinds of carrageenan (κ-carrageenan and τ-carrageenan) at 35 � C (Karlsson & Singh, 1999). This suggests that UV-H2O2 is a highly efficient system in producing MCP. Fast degradation of pectin was also reported in a recent study with a combined ultrasound-Fenton oxidative reaction (Zhi et al., 2017). The high effi­ ciency of UV-H2O2 process might be due to the multiple degradation mechanisms, which are discussed in the following sections. 3.3.2. Effect of H2O2 concentration Clear difference in molecular weight distribution patterns was observed for pectin treated at different H2O2 concentrations (Fig. 3 (A)). With increased H2O2 concentration, the retention time of pectin was delayed, which corresponds to the decrease of the molecular weight of CP, and the peaks were gradually becoming narrower, which suggested a decreased polydispersity (Mw/Mn). Consequently, homogeneity in the Mw distribution was increased. On the other hand, continuing degra­ dation happened on the degraded fraction at high H2O2 concentration, which led to an excessive degradation and brought low recovery of MCP. Therefore, the optimal H2O2 loading for a desired final Mw of 45 kDa was 30 μL/100 mg (~30 mM). This is much lower than 833 mM by H2O2 oxidation treatment at 80 � C, 714 mM by SPP with H2O2 degradation, 200 mM by H2O2 oxidation method at 120 � C, and 176 mM by ultrasound-assistant Fenton method (Liang, Liao, Ma, Li, & Wang, 2017; Wu et al., 2018; Wu & Yu, 2015; Zhi et al., 2017). In Fig. 3 (B), we evaluated the effect of H2O2 concentration on average number of chain scission (N) using Eq. (3). The curve of average number of chain scission versus H2O2 concentration was close to linear (R2 ¼ 0.9836) in the range of 0–60 μL/100 mg, implicating main chain scission was random. This process of stochastic chain cleavage was first reported by Jellinek in 1955 (Jellinek, 1955). It is assumed that polymers can be degraded randomly and any chain connection has an equal chance of rupture. In the current study, an increase in average number of chain scission (N) was observed upon elevating the H2O2 concentration, suggesting that main chain scission is predominant. The reason lies in the fact that there are a larger number of bonds in the main chains to degrade than in the side chains, and thus giving a larger probability of chain cleavage in the main chain.

Fig. 3. (A) Molecular weight distribution curves of CP degradation by different concentration of H2O2 (0–60 μL/100 mg). (●) refer to the right vertical axis. (B) Average number of main chain scission, N, as a function of concentration of H2O2. In the process of degradation, 10 mg/mL of CP solution exposure to the UV light for 5 h at pH 4.

MCPs were shown in Table 2, and the structural variation of MCP with irradiation time was analyzed. There was a fast decrease of Mw from 641 to 191 kDa within the first 1 h, followed by a slow stage from 1 to 9 h with final Mw of 54 kDa. The distinct decrease of Mw in the early stage might be largely attributed to de-aggregation effect on the CP cluster in aqueous solution, which was also observed in polysaccharide degrada­ tion by other techniques (Li et al., 2019; Zhi et al., 2017). Along with the decrease of Mw, the polydispersity (Mw/Mn) reduced significantly (P < 0.05), suggesting that MCP became smaller and more homogeneous with prolonged UV-H2O2 treatment. Decrease of the DE (representing the total degree of methoxylation and acetylation in CP) suggested that part of these two small groups was broken into even smaller molecules. In this study, DE of MCP declined (P < 0.05) with prolonged irradiation, however, it remained at a high content of more than 60%, indicating that MCP belonged to a high DE pectin like CP. For the monosaccharide composition, a significant loss of GalA was observed, from original GalA content of 77.59% down to 44.89% in the 9h-treated MCP. In regard to neutral sugars, molar percentage of most monosaccharides, such as Gal, Ara, Glc and Xyl, had a significant in­ crease (P < 0.05) upon UV-H2O2 treatment. Meanwhile, molar per­ centage of total neutral saccharides (NS) exhibited a progressive increase from 22.41% at the beginning to 55.11% after irradiation for 9

3.4. Changes in chemical composition of MCP with UV irradiation time Molecular weight and chemical composition of the final product 5

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3.5. Analysis of the intermediate products generated during UV-H2O2 process

Table 2 Molecular weight and chemical compositions of MCP prepared at varied irra­ diation time.a

During the CP degradation process by UV-H2O2, some intermediates were released and solubilized into the reaction solution, which were subsequently separated and collected from the final products MCP by ethanol precipitation for further analysis. As shown in Table 3, there was a constant presence of double bond in the solution in spite of a mild decrease at longer irradiation time. This might be due to double bond destruction by UV-H2O2. The double bond we examined here was the 4, 5-unsaturated double bond, which is generally produced through β-elimination reaction by removing of the H atom at C-5, leading to formation a double bond between C-4 and C-5 in the sugar ring of GalA unit by losing the C–O linkage in the β-position (BeMiller & Kumari, 1972). Hence, the progressive increase of double bond suggested that β-elimination reaction occurred throughout the UV-H2O2 process. However, we cannot confirm the β-elimination was due to UV-H2O2 oxidation or acid hydrolysis, since β-elimination has been noticed at pH-values as low as 3.8 (Krall & Mcfeeters, 1998). For sugars liberated during the degradation, neutral and acid sugars, mono- and oligo-saccharides, and reducing sugar were examined. Dur­ ing the course of the reaction, reducing sugar increased steadily, cor­ responding to progressive cleavage of the glycosidic linkages connected to neutral sugars, such as the bonds between the GalA and Rha residues in the RG-I region and its side-chains and the linkages between Xyl and GalA in the XGA area. Furthermore, we examined content changes of specific monosaccharides in the supernatant. Untreated CP supernatant has a high content of mono- and oligo-saccharides than those in the treatment groups. This is possibly due to the large quantity of smaller molecule weight fractions in CP, most of which, however, disappeared after 1 h of treatment and would not have any effect on sugar content

UV irradiation time (h) 0

1

191.0 641.2 � � 13.1b 52.4a Polydispersity 4.25 � 2.70 � (Mw/Mn) 0.08a 0.06b b DE (%) 67.52 66.31 � � 0.31a 0.34b Monosaccharides composition (mol %) 77.59 70.76 GalAc � � a 1.13 2.15b Fuc 0.38 � 0.19 � 0.05b 0.12b Rha 3.43 � 4.18 � 0.15c 0.09c Ara 1.63 � 2.00 � 0.11e 0.17d Gal 12.67 16.38 � 0.17f � 0.13e Glc 2.29 � 3.34 � 0.08e 0.02d Xyl 2.02 � 3.16 � 0.06d 0.10c d NS 22.41 29.24 � 0.36f � 0.41e Molar ratios of monosaccharides GalA/NS 3.46 � 2.42 � 0.15a 0.05b Rha/GalA 0.04 � 0.06 � 0.00c 0.01c (Gal þ Ara)/ 4.17 � 4.40 � Rha 0.04d 0.10c Mw (kDa)

3

5

7

9

99.3 � 8.7c

72.9 � 9.4dc

57.3 � 3.9dc

54.1 � 4.2d

2.53 � 0.03c 65.42 � 0.56c

2.51 � 0.07c 61.34 � 0.47d

2.36 � 0.08d 60.93 � 0.26d

2.31 � 0.04d 60.71 � 0.28d

52.81 � 1.86c

51.96 � 1.67c

0.32 � 0.10b 8.12 � 1.12a 3.04 � 0.30c 26.35 � 0.07d 4.70 � 0.05c 4.65 � 0.04b 47.19 � 0.47d

0.35 � 0.06b 7.08 � 0.85a 3.35 � 0.21c 27.30 � 0.11c

47.31 � 1.02d 0.61 � 0.14a 5.77 � 0.48b 3.91 � 0.04b 31.44 � 0.06b 6.31 � 0.07a 4.66 � 0.13b 52.69 � 0.29b

44.89 � 1.42d 0.35 � 0.16b 5.64 � 0.19b 4.35 � 0.26a 33.14 � 0.03a 6.32 � 0.06a 5.30 � 0.04a 55.11 � 0.38a

1.12 � 0.08c 0.15 � 0.02a 3.62 � 0.18e

1.08 � 0.03c 0.14 � 0.02ba 4.33 � 0.08dc

0.90 � 0.02d 0.12 � 0.01b 6.13 � 0.03b

0.81 � 0.03d 0.13 � 0.01ba 6.65 � 0.06a

5.40 � 0.03b 4.56 � 0.07b 48.04 � 0.43c

Table 3 The content of the intermediates released into in the reaction solution during CP degradation of by UV-H2O2 process.a

Values with different small case superscript letters (a-f) in the same row indicate significant differences as estimated by Duncan’s multiple range test (P < 0.05). a Preparation conditions fixed: CP concentration 10 mg/mL, H2O2 dosage 30 μl/100 mg CP, pH 4. b DE: degree of de-esterification. c GlaA: galacturonic acid. d NS: neutral sugars.

UV irradiation time (h)

h. We elucidated the structural difference between MCP and CP by comparing the molar ratio of GalA to specific NS (GalA/NS). GalA/NS, which represents the linearity of pectin, showed a significant decrease from 3.46 to 0.81, suggesting that the degradation process changed CP from linear-dominant structure into a branched one. Moreover, the molar ratio of Rha to GalA (Rha/GalA), which reflects the contribution of RG-I to pectin population, increased significantly from 0.04 to 0.15 (P < 0.05) when UV-H2O2 treatment was more than 3 h. This implies that content of the RG-I region was elevated in MCP, which was consistent with the observation of decreased GalA/NS. These results implicated that UV-H2O2 had more degradation effect on HG region than the RG-I part of CP, compared with the thermal and acid degradations (Khalikov & Mukhiddinov, 2004; Zhang, Zhang, Liu, Ding, & Ye, 2015). Another index, (Gal þ Ara)/Rha, standing for the length of side chains attached to RG-I, increased gradually during the degradation process, again indicating an increase of RG-I content in MCP. In summary, the degra­ dation of CP was mainly caused by destroying the major chemical composition GalA, resulting in a highly branched RG-I type MCP fragment.

0

1

3

5

7

9

Double bond (mM) Reducing Sugar (%)

1.30 � 0.01e 5.60 � 0.21e

1.50 � 0.01c 7.83 � 0.30d

GalAb

16.84 � 1.02e 0.11 � 0.02a 0.05 � 0.00a 2.69 � 0.04a 0.16 � 0.01d 1.97 � 0.04a 0.73 � 0.03a 5.71 � 0.03a 13.76 � 0.33a

16.36 � 1.26e 0.02 � 0.01d 0.01 � 0.00b 0.18 � 0.01e 0.07 � 0.01e 0.61 � 0.02e 0.19 � 0.01d 1.07 � 0.01f 3.86 � 0.17d

1.58 � 0.01a 13.95 � 0.31c 19.74 � 1.14d 0.06 � 0.00b 0.06 � 0.01a 1.03 � 0.02c 0.20 � 0.03c 1.63 � 0.02c 0.41 � 0.02c 3.39 � 0.02d 3.96 � 0.21d

1.52 � 0.01b 14.63 � 0.23b 30.35 � 1.10c 0.07 � 0.00b 0.06 � 0.00a 1.36 � 0.02b 0.23 � 0.02cb 1.75 � 0.01b 0.40 � 0.03c 3.87 � 0.02c 5.05 � 0.34c

1.49 � 0.01c 16.30 � 0.12a 35.48 � 1.03b 0.07 � 0.01b 0.05 � 0.01a 1.34 � 0.03b 0.29 � 0.01a 1.95 � 0.01a 0.52 � 0.02b 4.22 � 0.01b 7.32 � 0.16b

1.42 � 0.01d 16.70 � 0.22a 39.17 � 1.38a 0.04 � 0.01c 0.06 � 0.00a 0.90 � 0.01d 0.24 � 0.02b 1.35 � 0.03d 0.47 � 0.02c 3.05 � 0.02e 7.55 � 0.18b

Fuc Rha Ara Gal Glc Xyl NSc Oligosaccharides

Values with different small case superscript letters (a-f) in the same row indicate significant differences as estimated by Duncan’s multiple range test (P < 0.05). a The content of monosaccharides and oligosaccharides the reaction solution (mg/100 mg) was refer to the monosaccharides and oligosaccharides content in the supernatant before hydrolysis. b GlaA: galacturonic acid. c NS: neutral sugars. 6

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variation over irradiation time. Among the intermediate compounds released in the reaction solution, GalA was predominant, especially when irradiation time was above 3 h. This is consistent with the serious loss of GalA in MCP. Due to the degradation on GalA, the missing GalA, which was not perceptible, was washed away after ethanol precipita­ tion. Meanwhile, the amount of NS released into the reaction solution was much less than GalA. Nevertheless, the relative content of NS increased over the reaction, suggesting that some linkages within neutral sugars or between neutral and acid sugars were attacked and freed neutral sugars were produced. This was consistent with the result obtained from the variation of reducing sugar. Oligosaccharides, which are another type of intermediates in CP degradation, were present in a higher concentration in reaction solution than NS but showed similar trend to NS in the concentration-time course. In term of specific monosaccharides, Rha was minor in the reaction solution with only slight increase during the 1–3 h period and main­ tained constant afterwards (3–9 h). As the second largest neutral sugar in CP, Rha was expected to be in a higher amount in the solution should the degradation be nonselective. Thus, it implies that preferential cleavage of the linkage between GalA and Rha occurred at around 1–3 h, liberating RG-I with Rha as the reducing end and producing GalA as intermediates, since Rha is an important unit in RG-I to separate RG-I from HG. Linkage between Rha and GalA in the RG-I was not cleaved, likely due to steric hindrance of the Rha side branches. As the main components of RG-I side chain, three monosaccharides Gal, Ara and Glc were released little in the reaction solution, consistent with our obser­ vation that NS was highly branched in MCP. Moreover, all of the three neutral sugars were produced during 1–3 h treatment, which partially contributed to the rapid increase of reducing sugar within 1–3 h. Similar to Rha, Xyl was liberated fairly low in the solution and appeared during the 1–3 h period, producing XGA fragment and leaving GalA in the so­ lution. Based on the chemical composition of the final product MCP and the percentage of intermediates formed over the degradation process, we deduced the susceptibility of glycosidic linkages in CP during UVH2O2 degradation. The linkage between GalA residues was predominant in CP and readily cleaved during the whole process, producing GalA, oligo-GalA or HG fraction. Preferential attack of free radicals on GalA backbone in the HG region has also be previously reported (Li et al., 2019). The GalA-neutral sugar linkages, including GalA-Rha and GalA-Xyl, in the backbone of CP appeared to be stronger than the GalA-GalA bond as they started to be disrupted only after 1 h treatment, leading to the appearance of products RG-I and XGA rich in neutral sugars. As a result, more cleavage points became exposure and saccha­ rides with reducing ends were created. The rest linkages were those between neutral sugars, which were relatively stable during UV-H2O2 treatment. Only a small amount of monosaccharides was released slowly during the 1–3 h period. In addition to the oxidation degradation, acid hydrolysis at pH 4 could lead to hydrolysis of side-chains and the acid-labile linkages between GalA and Rha residues in the RG-I region (Khalikov & Mukhiddinov, 2004).

Fig. 4. The shear schematic diagram of pectin degradation under UV-H2O2 process. In the process of degradation, 10 mg/mL of CP solution exposure to the UV light for 5 h at pH 4.

by reduced DE. Therefore, the initial stage of CP degradation can be summarized as a random scission process through different possible reactions with equal opportunities, resulting in a random series of MCP fragments, heterogeneous oligomers and small amount of low molecular weight intermediates. In most cases, random scission was proposed as a general pathway for the whole process based on the kinetic analysis (Li et al., 2017; Wu et al., 2018). Our current study showed that random scission mainly occurred at the beginning phase of degradation, which was consistent with previous reports on polysaccharide degradation (Narrainen & Lovell, 2010; Wu et al., 2010). In the second stage (1–9 h), a slower degradation proceeded with a quick onset within 1–3 h and continued steadily until the end of the reaction (3–9 h). Following the fast random scission stage, a large quantity of long and un-hindered linear structure of CP backbone was exposed to attack by �OH radicals, leading to the main chain scission mode. During this stage, the main chain scission, preferentially occurred at the GalA linkages, producing lower Mw HG fractions and RG-I enriched portions. It is well-known that the main chain scission is a prime cleavage mode in most of the pectin degradation processes due to the large portion of main chains. Particularly for pectin as a diverse heteropolysaccharide, the main chain is composed of varied glycosidic linkages. The susceptibility of different chemical bonds on pectin backbone varies with the degradation methods. For instance, acid hy­ drolysis selectively split the glycosidic linkage at C-1 of neutral sugars, whereas glycosidic bonds between GalAs were highly resistant to acidic conditions (Thibault et al., 1993). In contrast, CP degradation by UV-H2O2 occurred preferentially at the linkages between GalAs, while linkages between neutral sugars remained untouched, which was consistent with the literature reports on pectin degradation with other oxidative degradation process (Hu, Chen, Wu, Zheng, & Ye, 2019; Li et al., 2019; Zhi et al., 2017). In addition to the easy accessibility of the GalA linkages original from the high content of galacturonic acid, the relatively lower energy barrier of a-1, 4-glycoside bond in GalA, calcu­ lated using density functional theory (Wu, Liu, Xu, Li, & Li, 2019), also contributed to favored cleavage on GalAs linkages. With the vast release of terminal linkages, chain-end scission occurred simultaneously with main chain scission at the later stage (7–9 h). It proceeded by breaking down the linkage end of HG, RG-I or their neutral sugar branches to slightly reduce the Mw. Chain-end scission is a well-accepted degradation mode for polysaccharides hydrolysis under acid condition because the hydrolysis rate of terminal bonds was higher than that of internal bonds (BeMiller, 1967; Chang et al., 2001; Thibault et al., 1993). In theory, terminal linkages, both reducing and non-reducing, are acid-hydrolyzed easier because they have more

3.6. Proposed mechanism of degradation of CP by UV-H2O2 Based on analysis of the degradation kinetics and comparison of the chemical composition of MCP and the intermediates varied with irra­ diation time, we herein propose a putative model to explain the CP degradation mechanism under UV-H2O2 oxidative reaction (Fig. 4). In view of the rate of Mw decrease, the whole degradation process can be divided into three stages. The first stage (0–1 h) led to a remarkable decrease of Mw via random scission, which included several degradation modes. Firstly, aggregation of CP was disrupted because the inter- or inner-molecular H-bonds are weak and easily susceptible to �OH attack (Wang, Cheung, Leung, & Wu, 2010). Secondly, the acidic glycosidic linkage was broken down, decreasing GalA content in MCP and releasing reducing sugar in the reaction solution. Finally, break­ down of small groups such as methoxyl and acetyl groups was evidenced 7

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Food Hydrocolloids 102 (2020) 105587

tendency to form a conjugate acid (BeMiller, 1967). In addition, the β-elimination reaction and de-esterification occurred throughout the CP degradation process by UV-H2O2 to a lesser extent because the reaction condition, either low pH (<5) or low temperature, was not favorable for β-elimination reaction (Fraeye et al., 2007; Kravtchenko et al., 1992). In summary, CP degradation by UV-H2O2 process is a reaction con­ current with an initial random chain cleavage, dominated chain-end scissions, and minor β-elimination and de-esterification, generating a mixture of MCP fragments with varied Mw and DE.

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