Effects of ultrasound pretreatment on the enzymolysis of pectin: Kinetic study, structural characteristics and anti-cancer activity of the hydrolysates

Accepted Manuscript Effects of ultrasound pretreatment on the enzymolysis of pectin: Kinetic study, structural characteristics and anti-cancer activity of the hydrolysates Xiaobin Ma, Danli Wang, Weijun Chen, Balarabe Bilyaminu Ismail, Wenjun Wang, Ruiling Lv, Tian Ding, Xingqian Ye, Donghong Liu PII:

S0268-005X(17)31749-6

DOI:

10.1016/j.foodhyd.2017.12.008

Reference:

FOOHYD 4184

To appear in:

Food Hydrocolloids

Received Date: 16 October 2017 Revised Date:

7 December 2017

Accepted Date: 7 December 2017

Please cite this article as: Ma, X., Wang, D., Chen, W., Ismail, B.B., Wang, W., Lv, R., Ding, T., Ye, X., Liu, D., Effects of ultrasound pretreatment on the enzymolysis of pectin: Kinetic study, structural characteristics and anti-cancer activity of the hydrolysates, Food Hydrocolloids (2018), doi: 10.1016/ j.foodhyd.2017.12.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Effects of ultrasound pretreatment on the enzymolysis of pectin:

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kinetic study, structural characteristics and anti-cancer activity of

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the hydrolysates

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Xiaobin Ma a, Danli Wang a, Weijun Chen a, Balarabe Bilyaminu Ismail

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Wang a, Ruiling Lv a, Tian Ding a, Xingqian Ye a,b, Donghong Liu a,b*

, Wenjun

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a,c

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a

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Engineering Laboratory of Intelligent Food Technology and Equipment, Zhejiang Key

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Laboratory for Agro-Food Processing, Zhejiang R & D Center for Food Technology

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and Equipment, Zhejiang University, Hangzhou 310058

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b

Fuli Institute of Food Science, Zhejiang University, Hangzhou 310058, China

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Department of Food Science and Technology, Bayero University Kano, PMB 3011,

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Nigeria

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College of Biosystems Engineering and Food Science, National-Local Joint

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* Corresponding author

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Address: College of Biosystems Engineering and Food Science, Zhejiang University,

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866 Yuhangtang Rd., Hangzhou 310058, China.

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Tel.: +86 057188982169; Fax: +86 057188982144; E-mail: [email protected]

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Abstract In this study, the effects of ultrasound pretreatment on the enzymolysis of pectin

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were investigated. Ultrasound at an intensity of 18.0 W mL-1 for 30 min significantly

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decreased pectin molecular weight by 50.50%, whereas it increased the degree of

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hydrolysis (DH) for enzymatic reactions by 20.22%. After ultrasound treatment, the

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maximum velocity of the enzymatic reaction (Vmax) increased but the Michaelis

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constant (Km) decreased, indicating an improved enzymolysis efficiency and stronger

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affinity between pectin and pectinase. Investigations into pectin structures

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demonstrated that, ultrasound effectively decreased the degree of methoxylation (DM)

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resulting

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homogalacturonan (HG) regions of the pretreated pectin were more completely

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degraded during enzymolysis compared with the control. According to the results of

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FT-IR and NMR analysis, both ultrasound and pectinase had no effect on pectin

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primary structures. However, ultrasound pretreatment could induce higher content of

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galactose in pectin hydrolysates, which contributed to an improved inhibitory activity

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against HT-29 colon cancer cells as shown by MTT assay.

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Keywords: ultrasound; pectin; enzymolysis; degradation; structure; anti-cancer

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activity

more

favorable

substrates

for

enzymatic

attack;

thus,

the

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1.

Introduction Found mostly in the cell walls of the fruits, vegetables, and plants we encounter

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daily, pectin is a colloidal acidic heteropolysaccharide that has many industrial

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applications and beyond. Despite its availability in ubiquitous plant processing wastes,

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pectin from citrus peel accounts for more than 85% of its commercial sources (Chan,

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Choo, Young & Loh, 2017). Pectin has a backbone of galacturonic acid (GalUA)

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residues linked by α-1,4-linkages, which is called the homogalacturonan (HG) region.

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The carboxyl group can be partly methyl esterified or acetylated; different degrees of

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methoxylation (DM) and acetylation (DAc) allow it to generate multiple functional

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properties (Ma et al., 2016). The HG region is known as the smooth region of pectin,

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versus

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rhamnogalacturonan II (RG-II) and xylogalacturonan (XG), known as hairy regions.

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In plant cell walls, pectin is mainly in charge of tissue sustainability and cell cohesion

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(Banerjee, Vijayaraghavan, Arora, MacFarlane & Patti, 2016). Due to this property, it

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is often used as a gelling agent, texturizer, stabilizer or emulsifier (Liu, Guo, Liang,

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Liu & Chen, 2017). But another role pectin has, being used as a nutraceutical or

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pharmaceutical for cancer therapy, is in the induction of apoptosis and inhibition of

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galectin-3 (Gal-3), a special chimeric protein with the ability to promote cell adhesion

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and metastasis (Maxwell, Belshaw, Waldron & Morris, 2012). This accredits the

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importance of pectin in some food supplements and therapeutic processes. However,

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the structure-bioactivity relationships of pectin are still under debate due to its

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extremely complicated structures and diverse sources.

other

parts,

including

the

rhamnogalacturonan

I

(RG-I),

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ACCEPTED MANUSCRIPT While pectin has revealed promising effects on preventing the generation and

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development of a series of cancers, its large molecular weight and poor solubility

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impede its absorption into human digestive systems, and thus limit its applications in

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the medical field (Maxwell et al., 2012). This is why the “modified pectin (MP)” has

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become so prevalent. Modifying pectin is commonly conducted using chemicals,

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enzymes, or through physical methods, to remove the structure regions irrelevant to

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its bioactivity, while still maintaining its functional regions. This kind of modification

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will also result in smaller fragments with lower molecular mass and increased

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solubility, making it possible to enter the digestive system for effective functions.

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Enzymatic modification is an environmentally-friendly method for MP production

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with high efficiency and specificity. However, the large amounts of structure barriers

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that exist in pectin molecules generally obstruct the action sites for enzymatic attack,

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leading to the decreased output and increased production cost (Chen et al., 2015). In

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this case, it is necessary for pectin to go through some pretreatment procedures before

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enzymatic hydrolysis.

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In recent years, ultrasound is found versatile in improving the quality of diverse

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food products and accelerating various processes in the food industry (Chemat et al.,

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2017). A number of researchers have adopted ultrasound as a pretreatment method for

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substrate degradation before enzymatic reactions, to obtain products with increased

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hydrolysis rate and better functionality (Abadia-Garcia et al., 2016; Abdualrahman et

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al., 2017; Luo, Fang & Jr. Smith, 2014; SriBala, Chennuru, Mahapatra & Vinu, 2016;

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Yang et al., 2017; Zhang et al., 2016). When ultrasound is delivered into a liquid

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ACCEPTED MANUSCRIPT system, the vigorous shear force generated from the transient cavitation effect could

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randomly cleavage the polymer, which reduces the resistance of enzymes to diffuse in;

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meanwhile, free radicals produced through the sonolysis of water molecules can

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further change the structure of the substrates and make them more accessible for

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enzymatic attack, thus resulting in the improved enzymolysis process (Weiss,

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Kristbergsson & Kjartansson, 2011). In a study by Abdualrahman et al. (2017), the

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degree of hydrolysis (DH) of sodium caseinate protein was significantly increased

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under an ultrasonic field at a frequency of 28 kHz and an intensity of 450 W L-1. The

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authors have hypothesized this situation happening and accredited it to the increased

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enzymes/proteins contact after ultrasound pretreatment. In addition to the enhanced

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hydrolysis extent, the enzymolysis products with ultrasound pretreatment also

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demonstrate better bioactivity. As supported by Yang et al. (2017), the

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angiotensin-I-converting enzyme (ACE) inhibitory activity of the wheat germ protein

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pretreated with dual-fixed frequency ultrasound (at an intensity of 60 W L-1 for 70

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min) has increased by 62.30% compared to the control; results are in accordance with

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studies on combined ultrasound/enzyme treatments on whey protein (Abadia-Garcia

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et al., 2016) and wheat gluten (Zhang et al., 2016).

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Despite the potential of ultrasound pretreatment to accelerate the enzymatic

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process and generate hydrolysates with more desirable bioactivity, few studies were

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undertaken on the enzymolysis of ultrasound-treated pectin. Also, there is a paucity of

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information on the effects of ultrasound pretreatment on the structures and bioactivity

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of pectin hydrolysates. In the present study, pectin was treated with ultrasound before 5

ACCEPTED MANUSCRIPT enzymatic hydrolysis. Effects of pretreatment conditions on pectin molecular weight

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and its polydispersity, DH and enzymatic kinetics were studied. Furthermore,

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structural characteristics (molecular weight parameters, DM, DAc, monosaccharide

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component, primary structures, etc.) and anti-cancer activity of pectin hydrolysates

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with and without ultrasound pretreatment were also investigated to clarify the

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structure-bioactivity relationship of MP. Results of this study will provide an

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innovative green and efficient method for MP production, and pave the way for the

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application of ultrasound in enzymatic reactions.

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2.

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2.1. Materials

Materials and Methods

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Pectinase from Aspergillus niger (EC No. 3.2.1.15), pectin from a citrus peel, standard

dextran,

standard

monosaccharides,

and

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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased

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from Sigma–Aldrich (Shanghai, China). HT-29 colon cancer cells were obtained from

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the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences

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(Shanghai, China). RPMI 1640 medium (RPMI), penicillin/streptomycin (100 U mL-1)

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and fetal bovine serum (FBS) were purchased from Ji Nuo Biotechnology Co., Ltd.

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(Zhejiang,

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1-phenyl-3-methyl-5-pyrazolone (PMP) were of HPLC-grade; all other chemicals,

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including 3,5-dinitrosalicylic acid (DNS), trifluoroacetic acid, dimethylsulfoxide

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(DMSO), etc., were of analytical grade and were used without further purification.

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2.2. Ultrasound pretreatment of citrus pectin (CP)

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China).

Methyl

alcohol,

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acetonitrile,

isopropanol

and

ACCEPTED MANUSCRIPT The commercial CP powder was dissolved in 1.0 mol L-1 citric acid–phosphate

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buffer at a pH of 4.0 with a final concentration of 5.0 mg mL-1. Twenty milliliters of

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CP solution were added to a glass tube (with an inner diameter of 2.77 cm) and then

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sonicated with a probe ultrasonic homogenizer (JY92-IIDN, frequency: 22 kHz,

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nominal power: 900 W, diameter of the probe tip: 1 cm; Ningbo Scientz

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Biotechnology Co., Ningbo, China) at different intensities (9.0, 13.5, 18.0, 22.5 and

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27.0 W mL-1; calculated according to our previous study (Ma et al., 2015)) and

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different times (10, 20, 30, 40, 50 and 60 min). The temperature of CP solution during

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sonication was controlled at 20 °C with a low-temperature thermostatic water bath

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(DC-1006, Safe Corporation, Ningbo, China) and monitored by a temperature probe

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connected to the ultrasound controller. The ultrasound-pretreated pectin sample was

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named as “UCP”.

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2.3. Enzymatic hydrolysis of pectin

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The pectinase powder was dissolved in 1.0 mol L-1 citric acid-phosphate buffer

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(pH 4.0) with a final concentration of 1.0 mg mL-1. Pectinase solution (50 µL) was

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added to 950 µL of pectin samples with and without ultrasound treatment (i.e. UCP

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and CP samples) in a 10 mL colorimetric tube. Enzymatic hydrolysis was conducted

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at 50 °C for 30 min. After hydrolysis, the mixture was immediately put into a boiling

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water-bath for 3 min to inactivate the enzyme. The DH of pectin samples was

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calculated as follows:

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=

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where

× 100 (%)

is the GalUA concentration at an enzymolysis time of 30 min (µM), 7

(1) is

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the ultimate GalUA concentration (µM) after complete hydrolysis by concentrated

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H2SO4. The hydrolysates of CP and UCP were named as “ECP” and “UECP”,

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respectively.

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2.4. Enzymatic kinetics

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The CP and UCP samples with a series of initial concentrations (0.95–9.52 mg

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mL-1) were incubated with the prepared pectinase solution at 30 °C for 10 min. The

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reaction rate at each substrate concentration was measured to draw the Lineweaver–

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Burk plots and calculate the kinetics parameters (Vmax and Km).

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2.5. Structural characteristics of pectin samples

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2.5.1. Molecular weight and its distributions

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The weight-average molecular weight (Mw) and its polydispersity index (ratio of

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Mw to number-average molecular weight) were measured using SEC-HPLC (Ma et

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al., 2016). Pectin samples were dialyzed in deionized water for 48 h, then filtered

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through a 0.45 µm membrane and preserved at 4 °C before injection. Standard

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dextrans with different Mw of 670, 270, 150, 50, 25 and 12 kDa were dissolved in

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deionized water with a concentration of 2.0 mg mL-1. The standard samples were then

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filtered and preserved at -80 °C.

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HPLC analysis: HPLC instrument: Waters 1525 HPLC system (Waters, US);

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column: TSK-GEL mixed-bed column (G4000PWXL, 300 × 7.8 mm, 10 µm; Tosoh

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Bioscience, Tokyo, Japan); column temperature: 40 °C; detector: refractive index

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detector (Waters 2414, US); injection volume: 50 µL; mobile phase: 0.2 M NaCl; flow 8

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rate: 0.5 mL/min; elution time: 30 min. Mw and its distributions were analyzed using

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Breeze 2 GPC.

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2.5.2. DM and DAc DM and DAc of pectin samples were measured by HPLC (Ma et al., 2016). The

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freeze-dried pectin samples were saponified at 4 °C for 30 min and were then

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centrifuged. The supernatant was adjusted to pH 2.0 and was filtered through a 0.45

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µm membrane prior to HPLC analysis (using isopropanol as an inner standard). The

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standard mixtures were comprised of methyl alcohol, glacial acetic acid and

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isopropanol.

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HPLC analysis: HPLC instrument: Waters 1525 HPLC system (Waters, US);

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column: C18 column (SinoChrom ODS-BP, 250 × 4.6 mm, 5 µm; Elite Corp., Dalian,

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China); column temperature: 25 °C; detector: refractive index detector (Waters 2414,

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US); injection volume: 20 µL; mobile phase: 0.4 mM H2SO4 solution; flow rate: 0.8

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mL min-1; elution time: 20 min.

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2.5.3. Monosaccharide component

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The monosaccharide component was measured using HPLC (Ma et al., 2016).

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The freeze-dried samples were hydrolyzed by trifluoroacetic acid at 110 °C for 8 h.

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The acidolysis products were dried and neutralized. Then, 450 µL of 0.3 M NaOH

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solution, 450 µL of 0.5 M methanol solution of PMP and 50 µL of 2.0 mM lactose

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solution (internal standard) were mixed with 400 µL of acidolysis samples. The

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mixture was incubated at 70 °C for 30 min and then neutralized. The resultant

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solution was then extracted by chloroform and the aqueous layer was filtered through

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ACCEPTED MANUSCRIPT a 0.22 µm membrane prior to injection. Standard monosaccharides, including

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mannose (Man), rhamnose (Rha), glucuronic acid, galacturonic acid (GalUA), lactose,

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glucose (Glu), galactose (Gal), xylose (Xyl), arabinose (Ara) and fucose (Fuc), were

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dissolved in deionized water to obtain standard mixtures with a concentration of 2.0

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mM for each monosaccharide. The standard mixtures were derivatized as described

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above.

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HPLC analysis: HPLC instrument: Waters 2695 HPLC system (Waters, US);

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column: C18 column (Zorbax Aclips XDB, 250 × 4.6 mm, 5 µm; Agilent Technologies

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Inc., CA, US); column temperature: 25 °C; detector: PDA 2996 detector (wavelength:

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250 nm; Waters, US); injection volume: 20 µL; mobile phase: prepared using

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acetonitrile and KH2PO4-NaOH buffer (0.05 M, pH 6.9), phase A: acetonitrile

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contents of 15% (v/v), phase B: acetonitrile contents of 40% (v/v); gradient time

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pattern: 0 min→10 min→30 min→35 min→40 min, the concentration gradient

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pattern of phase B: 0→15%→25%→25%→0; elution time: 45 min.

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2.5.4. Fourier transform infrared spectroscopy (FT-IR) analysis

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The freeze-dried pectin samples were mixed with KBr and pressed into KBr

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pellets. FT-IR spectra were recorded using an IR spectrometer (Nicolet 5700; Thermo

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Fisher Scientific, MA, US) at the absorbance mode, with a frequency range of 4000–

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400 cm-1 and a resolution of 4 cm-1. The data were exported from Omnic 9.0 (Nicolet,

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US).

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2.5.5. Nuclear magnetic resonance (NMR) analysis

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The freeze-dried pectin samples were deuterium-exchanged twice and then 10

ACCEPTED MANUSCRIPT dispersed in deuterium oxide with final concentrations of 3–5%. NMR spectra were

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collected by a 600 MHz NMR spectrometer (DD2-600; Agilent Technologies Inc., CA,

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US) at 25 °C. The spectra were processed using the MestReNova 6.1.1 (MestreLab

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Research, Santiago de Compostela, Spain)

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2.6. Cytotoxicity assay

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The anti-cancer activity of pectin samples was studied with their inhibitory

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effects on HT-29 colon cancer cells. The HT-29 cells were incubated in the

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RPMI-1640 medium with 10% FBS in a 96-well plate (5 × 103 cells/well). The

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culture plate was placed in a humidified atmosphere containing 5% CO2 at 37 °C.

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Cytotoxicity of pectin samples was measured by MTT assays. HT-29 cells were

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incubated with 20 µL of pectin solution at different concentrations (0.2, 0.4 and 1.0

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mg mL-1) and cells cultured without pectin samples were designated as the control.

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After 72 h, the pectin samples were removed and MTT was added. After another

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incubation period of 4 h, the supernatants were removed and 200 µL of DMSO was

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added. The mixture was shaken for 10 min and then measured at 570 nm. Each

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experiment was performed in sextuplicate as technical replicates. The inhibitory rate

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of cells can be calculated according to Eqn. 2:

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ℎ

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= (1 −

!"#$

) × 100 (%)

(2)

%&' (&#

2.7. Statistical analysis and figure plotting

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All the experiments were performed in triplicate, and the data was represented as

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a mean ± standard deviation (SD). Statistical analysis was conducted by ANOVA

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() < 0.05) and Duncan’s multiple range tests with the SPSS 17.0 (SPSS Inc., Chicago, 11

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IL, US). Figures were exported from Origin Software 8.5 (OriginLab Corp., MA,

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US).

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3.

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3.1. Effects of ultrasound conditions on the Mw and its polydispersity of pectin

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3.1.1. Effect of ultrasound intensity on the Mw and its polydispersity of pectin

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Results and discussions

Fig. 1 (a) depicts the Mw and its polydispersity of pectin with ultrasound

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treatment at intensities of 0–27.0 W mL-1 for 30 min. The molecular weight of pectin

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decreased with an increase in ultrasound intensity, and the whole degradation process

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can be divided into two stages: firstly, the increase in ultrasound intensity from 0 to

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18.0 W mL-1 significantly reduced pectin Mw from 485.10 kDa to 240.11 kDa. This

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suggested that elevating ultrasound intensity could significantly enhance the

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degradation efficiency of pectin. While at the second stage, pectin Mw decreased

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from 240.11 kDa to 230.80 kDa as the intensity increased from 18.0 W mL-1 to 27.0

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W mL-1, indicating that further energy input did not lead to an effective degradation

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after exceeding a certain level of ultrasound irradiation. Similarly, the polydispersity

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index of pectin Mw significantly decreased from 3.64 to 2.70 as the intensity

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increased from 0 to 18.0 W mL-1, and tended to be stable afterwards.

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In recent years, power ultrasound (20–100 kHz) has proven to be a very efficient

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method in degrading diverse polymers, such as starch (Lima & Andrade, 2010;

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Brenner, Kiessler, Radosta & Arndt, 2016), cellulose (Barbash et al., 2016; Nagula &

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Pandit, 2016), chitosan (Gomes et al., 2016; Taghizadeh & Bahadori, 2014), pectin

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(Zhang, Zhang, Liu, Ding & Ye, 2015; Zhang et al., 2013a, b), etc. The rapid collapse 12

ACCEPTED MANUSCRIPT of cavitation bubbles is usually accompanied by strong mechanical shear forces and

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reactive free radicals, which could break down the long chains of polymers into

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shorter segments. But it is noteworthy that there is a threshold, or a minimum value,

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that the ultrasound intensity must achieve to allow the cavitation phenomenon to

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occur (Czechowska-Biskup, Rokita, Lotfy, Ulanski & Rosiak, 2005). However, there

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is also another threshold, beyond which the further increase in ultrasound intensity

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would lead a “cushioning effect” to take place (Ugarte-Romero, Feng, Martin,

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Cadwallader & Robinson, 2006). Under this situation, the strong vertical circulation

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of the liquid system would entrain lots of air into the reactor, resulting in the lowered

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cavitational activity (Ugarte-Romero, Feng & Martin, 2007). Therefore in Fig. 1 (a),

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pectin degradation was firstly promoted by the enhanced cavitation effect at elevated

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ultrasound intensity; nevertheless, after achieving the “upper threshold”, further

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increase in the output power induced massive air-entrainment and the subsequent

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cushioning effect, thus leading to the reduced degradation extent. Results are in

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agreement with the earlier reported studies on ultrasound degradation processes for

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β-carotene (Sun, Ma, Ye, Kakuda & Meng, 2010), chitosan (Czechowska-Biskup,

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Rokita, Lotfy, Ulanski & Rosiak, 2005) and apple pectin (Zhang et al., 2013a). Taking

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both productivity and energy efficiency into consideration, the ultrasound intensity for

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pectin degradation was selected at 18.0 W mL-1.

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3.1.2. Effect of ultrasound time on the Mw and its polydispersity of pectin

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The Mw and its polydispersity of pectin with ultrasound treatment at an intensity

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of 18.0 W mL-1 for 0–60 min are shown in Fig. 1 (b). Similar to Fig. 1 (a), the 13

ACCEPTED MANUSCRIPT degradation process also consisted of a rapid decline stage at 0 to 30 min, and a steady

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stage at 30 to 60 min. Polymer degradation induced by power ultrasound is mainly

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attributed to the shear forces generated from fluid current motion (Koda, Taguchi &

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Futamura, 2011); thus, variations in pectin Mw and its polydispersity were closely

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related to variations in sono-mechanical effects. When pectin solution was introduced

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to ultrasound, energy was gradually accumulated in the liquid system with the

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prolonged treatment time, resulting in the steady increase in cavitational activity.

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Furthermore, shear forces tend to act on the midpoint of a polymer chain (Koda,

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Taguchi & Futamura, 2011), which also contributed to the rapid decline in pectin Mw

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at the early stage of ultrasound irradiation. While mechanical breakage is effective, it

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only occurs to long-chain polymers with Mw greater than a certain limit, otherwise

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this effect will disappear (Portenlanger & Heusinger, 1997). In a study by

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Portenlanger & Heusinger (1997), dextran molecules with Mw below 40 kDa were

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found so small that they could just follow the high velocity gradients without being

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cracked mechanically. As shown in Fig. 1 (b), very little changes in Mw and its

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polydispersity were observed after 30 min, which could be partly attributed to the

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diminished sono-mechanical effect on smaller pectin molecules. Therefore, the best

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ultrasound duration for pectin pretreatment is 30 min.

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3.2. Effects of ultrasound conditions on the DH of pectin samples

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Fig. 2 shows the DH of pectin with ultrasound pretreatment at different

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intensities (a) and different times (b). Ultrasound pretreatment effectively improved

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the enzymolysis process for pectin. For example, the DH for pectin with ultrasound 14

ACCEPTED MANUSCRIPT treatment at 18.0 W mL-1 intensity for 30 min was significantly increased by 20.22%

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compared to the DH of original CP. Furthermore, it can be seen that the variation

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trend for DH under different ultrasound conditions is substantially in line with that for

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pectin Mw and its polydispersity as described in Fig. 1. That is, with the increase in

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ultrasound intensity and treatment time, there is a gradual improvement in the DH of

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pectin hydrolysates, followed by a stable phase thereafter. After moderate ultrasound

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irradiation, DH increases because the pectin structure is modified enough to prepare

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more readily available substrate, and this is closely related to variations in ultrasound

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mechanical effects as described in Section 3.1. Overall, preparation of UCP would be

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optimally conducted under an ultrasonic field at 18.0 W mL-1 intensity for 30 min.

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3.3. Effects of ultrasound pretreatment on enzymatic kinetics

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The Lineweaver-Burk plots based on Michaelis-Mentent equation for CP and

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UCP are illustrated in Fig. 3. The corresponding coefficients for CP and UCP were

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0.9929 and 0.9910, respectively. Values of Vmax, Km and Vmax/Km are listed in Table 1.

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Ultrasound pretreatment is observed to have a positive effect on enzymatic kinetics by

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increasing the Vmax and Vmax/Km for UCP by 29.41% and 41.95%, respectively, as

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well as decreasing the Km from 3.28 mg mL-1 to 2.99 mg mL-1, compared with the

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control. This meant an improved enzymolysis efficiency and stronger affinity between

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pectin and pectinase were achieved after ultrasound irradiation. Similar findings were

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reported by Zhang et al. (2015), where the Km for ultrasound-pretreated wheat gluten

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significantly decreased by 15.83%.

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As stated before, ultrasound cavitation effects induced pectin degradation 15

ACCEPTED MANUSCRIPT resulting in smaller molecules and simpler structures, allowing the pectinase to easily

329

reach the action sites during enzymatic reactions. Furthermore, the main composition

330

of pectinase enzymes used in this study is a polygalacturonase, which only functions

331

on the α-1,4-glycosidic bond between non-esterified GalUAs, but has no effect on

332

bonds between esterified ones. Therefore in addition to the reduced stereo hindrance

333

of the substrate, the de-esterification effect of ultrasound (Zhang et al., 2013a) also

334

has a hand in improving the enzymatic efficiency and the enzyme-substrate affinity by

335

providing more readily accessible substrates.

336

3.4. Effects of ultrasound pretreatment on the structural characteristics of pectin hydrolysates

338

3.4.1. Mw and its distributions

M AN U

337

SC

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328

As shown in Table 2, the Mw of pectin was decreased by 50.50% after

340

ultrasound treatment, distributing within a narrower range. In comparing the Mw of

341

ECP and UECP (the enzymatic hydrolysates of CP and UCP, respectively), both

342

efficiently decreased with enzymatic hydrolysis, but UECP had a Mw even 58.69%

343

lower

344

ultrasound-pretreated pectin. This is consistent with variations in the DH of pectin as

345

mentioned in Section 3.2, which is ascribed to the more favorable pectin structures

346

formed under an ultrasonic field. Although there is no clear evidence that pectin with

347

low molecular weight possessing higher bioactivity, but large-Mw pectin usually can

348

not get into cells to effectively play its role in cancer therapy (Maxwell, Belshaw,

349

Waldron & Morris, 2012). Therefore, decreasing the molecular weight of pectin is a

ECP,

demonstrating

the

enhanced

enzymolysis

efficiency of

AC C

than

EP

TE D

339

16

ACCEPTED MANUSCRIPT prerequisite to achieving desirable bioactivity. On the other hand, degradation of ECP

351

(18.64 kDa) is found to be much more complete compared with UCP (240.11 kDa) as

352

it has a significantly lower Mw. Attributes of ultrasound mechanical forces shearing

353

just large-Mw polymer limit pectin degradation extent, suggesting that ultrasound is

354

still more suitable to be a pretreatment method rather than being used individually for

355

pectin degradation.

356

3.4.2. DM and DAc

SC

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350

Table 2 indicated that CP was converted from a high-methoxyl pectin (HM) to a

358

low-methoxyl pectin (LM) under ultrasound irradiation, with the DM remarkably

359

decreasing from 54.63% to 36.66%. When ultrasound is introduced to pectin samples,

360

shear forces produced by transient cavitation mechanically break down the C-O bond

361

causing the DM to decrease. The ultrasound also prompts formation of free radicals

362

from water sonolysis, which can react with methoxy group resulting in bond rupture

363

(Zhang et al., 2013b). These two effects work together to effectively transform HM

364

into LM. Wang et al. (2016) found that pectin extracted using ultrasound-assisted

365

method had an overall lower DM compared to that extracted by conventional heating

366

method. Following that, in Ogutu and Mu’s literature (2017) it was seen that

367

ultrasound treatment at a power of 400 W for 20 min on sweet potato pectin

368

significantly decreased its DM from 12% to 5.25%. On the other hand, ECP showed a

369

similar DM compared to CP and was still a HM, because there was no pectin

370

methylesterase (PME) in the applied pectinase enzymes. Therefore, the sharp decrease

371

in the DM of UECP was attributed to ultrasound functions rather than enzymatic

AC C

EP

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M AN U

357

17

ACCEPTED MANUSCRIPT hydrolysis. In comparing CP and ECP, or UCP and UECP, the latter even had a

373

slightly higher DM than the former, which was due to dialysis of degradation products

374

causing small fragments to diffuse out while maintaining complex esterified

375

structures.

RI PT

372

It is well known that DM determines the gel-formation property of pectin, but

377

Jackson et al. (2007) found it may also play a role in cancer therapy. In their study, the

378

apoptosis inducing activity of heat-modified pectin on human prostate cancer cells

379

was destroyed after mild base treatment, suggesting the importance of ester-based (or

380

related) cross-link in apoptosis induction. However, there are also some studies that

381

reported DM to be irrelevant to pectin’s bioactivity (Bergman, Djaldetti, Salman &

382

Bessler, 2010; Maxwell et al., 2016) . Effects of DM on the anti-cancer activity of

383

pectin will be further discussed in Section 3.5.

TE D

M AN U

SC

376

Existence of acetyl groups can enhance the steric-hindrance effect in pectin

385

structures and affect pectin’s hydrophobic nature (Maxwell et al., 2016). Thus,

386

decrease in DAc may promote the exposure of more functional sites buried in pectin

387

structures, resulting in an improved bioactivity. However, ultrasound has been

388

reported to have no influence on DAc (Lima & Andrade, 2010; Ma et al., 2016),

389

which is in line with the results in this study. DAc of CP was only 1.56% and

390

remained unchanged during ultrasound or enzymatic treatments. In this case, effects

391

of DAc can be ignored in the subsequent structure-bioactivity analysis.

392

3.4.3. Monosaccharide component

393

AC C

EP

384

Monosaccharide components of pectin samples are listed in Table 2. All four 18

ACCEPTED MANUSCRIPT samples mainly consist of 6 monosaccharides, including Rha, GalUA, Gal, Xyl, Ara

395

and Fuc. Small amounts of Glu presented in pectin samples might have been derived

396

from the extraction process and are not a composition of pectin (Zhang et al., 2013a).

397

Ultrasound and pectinase did not change the monosaccharide type in pectin samples

398

but caused alterations in the monosaccharide contents. GalUA is the principal

399

composition in all pectin samples, accounting for 68.19%, 67.27%, 58.53% and 54.37%

400

in CP, UCP, ECP and UECP, respectively. The Rha/GalUA ratio shows the proportion

401

of RG-I backbone in pectin main chain and thus is an indication of degradation

402

conditions of the main chain. As can be seen, there were little differences in

403

Rha/GalUA for CP and UCP, while the ratio for ECP and UECP significantly

404

increased from 0.13 to 0.19 and 0.21 respectively. On the other hand, the (Gal +

405

Ara)/Rha ratio is used to demonstrate the amount of neutral sugar residues within the

406

RG-I region. According to Table 2, ECP and UECP had higher (Gal + Ara)/Rha ratios

407

compared to CP and UCP. The aforementioned results indicated that ultrasound had

408

similar influences on pectin main chains and side chains, and thus both ratios nearly

409

remained unchanged. Pectinase randomly hydrolyzed α-1,4-glycosidic bonds and

410

caused severe degradation in HG regions resulting in a marked increase in both ratios.

411

In comparing ECP and UECP, both had increased from CP, but UECP had even more

412

increase than ECP, suggesting a higher hydrolysis rate with ultrasound pretreatment.

413

The reasoning for this is due to the beneficial structural alterations in pectin substrate

414

under an ultrasonic field as mentioned before.

415

AC C

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394

Gal and Ara are compositions of RG-I side chains and are thought to be related 19

ACCEPTED MANUSCRIPT to pectin anti-cancer activity (Maxwell, Belshaw, Waldron & Morris, 2012). Gunning

417

et al. (2009) removed Ara from RG-I via enzymolysis and found this significantly

418

improved the combinations of pectin and Gal-3, thus inferring that the anti-cancer

419

activity is mainly contributed by Gal. Looking at Table 2, Gal contents of CP, UCP,

420

ECP and UECP are 12.44%, 14.09%, 16.94% and 19.45%, respectively, which means

421

both ultrasound and pectinase could increase the Gal content resulting in possibly

422

higher bioactivity. Amongst the four samples, UECP has the highest Gal content and

423

thus has the greatest potential in apoptosis induction, indicating that the combining

424

ultrasound-pectinase treatment could possibly produce MP products with satisfying

425

anti-cancer activity.

426

3.4.4. FT-IR

M AN U

SC

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416

Fig. 4 illustrates the FT-IR spectra for pectin samples. The wide absorption bands

428

at 3000 to 3700 cm-1 are related to O–H stretching of hydroxyl groups. Two weak

429

bands near 2923 cm-1 and 2933 cm-1 are caused by antisymmetric and symmetric C–H

430

vibrations, respectively (Sinitsya, Copikova, Prutyanov, Skoblya & Machovic, 2000).

431

All pectin samples had two important bands at 1744 cm-1 and 1614 cm-1 assigned to

432

the methylesterified carbonyl groups (C=O) and the ionic carboxyl groups (COO–),

433

respectively. Thus, the DM of pectin can be measured based on these two absorptions

434

by calculating the ratio of the peak area at 1744 cm-1 over the sum of the areas at 1744

435

cm-1 and 1614 cm-1. Compared to CP, UCP and UECP had greater absorption at 1614

436

cm-1 and less at 1744 cm-1, while ECP had almost same absorptions at both peaks. In

437

accordance with HPLC, this indicated that the DM of pectin was significantly

AC C

EP

TE D

427

20

ACCEPTED MANUSCRIPT decreased by ultrasound but kept unchanged under enzymolysis reactions. The bands

439

at 1010–1150 cm-1 represent all the pectin samples contain pyranose (Wang et al.,

440

2016). Absorbance regions at 1146 cm-1, 1103 cm-1 and 1016 cm-1 display that

441

samples are rich in uronic acid (Coimbra, Barros, Barros, Rutledge & Delgadillo,

442

1998). As Fig. 4 shows, IR characteristic absorptions of three MP samples (UCP, ECP

443

and UECP) are in good match with the spectra of CP, indicating that both ultrasound

444

and pectinase did not change the primary structure of pectin, except for decreasing its

445

polymeric level and DM. Many physical degradation methods, including high

446

pressure homogenization (Hu, Nie & Xie, 2013), microwave (Bezakova et al., 2008)

447

and high speed shearing (Chen et al., 2014), have proven to be able to effectively

448

degrade polysaccharides without altering their primary structures.

449

3.4.5.

H and 13C NMR

TE D

1

M AN U

SC

RI PT

438

During NMR analysis, each pectin sample was saturated in deuterium oxide to

451

strengthen the signals of sugar residues and give sharper spectra. As solubility of

452

pectin samples is negatively correlated to their Mw, concentrations of samples

453

followed the following sequence: CP < UCP < ECP < UECP. Therefore in this study,

454

NMR spectra were simply used to clarify primary structures of pectin samples rather

455

than quantitative analysis. Fig. 5 shows the 1H NMR spectra of pectin samples and

456

Table A. 1 lists the chemical shifts of major signals. Five major signals in pectin were

457

attributed to protons of esterified GalUA (H-1, 5.03; H-2, 3.67; H-3, 3.95; H-4, 4.12;

458

H-5, 4.40) (Zhang, Zhang, Liu, Ding & Ye, 2015), suggesting that GalUA is the main

459

composition in all samples. The α-glycoside linkage of GalUA did not change with

AC C

EP

450

21

ACCEPTED MANUSCRIPT ultrasound or pectinase treatment as all the chemical shifts of H-1 were beyond 4.95

461

ppm. Other structural information will be analyzed taking CP as an example. In the

462

anomeric region, peaks at 5.09 ppm and 5.17 ppm are attributed to the H-1 of Ara and

463

Rha, respectively (Zhi et al., 2017; Zhang et al., 2013a). A strong signal at 3.67 ppm is

464

derived from –CH3 protons attached to carboxyl groups of esterified GalUA residues.

465

Signals at 2.12 ppm and 2.01 ppm are assigned to acetyl groups binding at 2- and

466

3-O-GalUA, respectively. All the pectin samples showed very weak signals around

467

these areas due to the low DAc as demonstrated by HPLC analysis. Peaks at 1.20 and

468

1.26 ppm are derived from –CH3 protons of O-2 and O-2,4 linked L-Rha,

469

respectively.

M AN U

SC

RI PT

460

The 13C NMR spectra are illustrated in Fig. 6 and the chemical shifts of major

471

signals are listed in Table A. 2. Compared to CP and UCP, ECP and UECP had better

472

signal-to-noise ratio and shaper spectra due to their lower Mw and higher solubility.

473

Six major signals of pectin samples were all derived from D-GalUA (C-1, 104.78; C-2,

474

70.48; C-3, 73.25; C-4, 81.33; C-5, 76.00; C-6, 173.36/176.88) (Tamaki, Konishi,

475

Fukuta & Tako, 2008). Signals at 173 and 177 ppm are derived from –COOH and –

476

COOCH3, respectively. Peaks at 55.37 ppm are assigned to –CH3 carbon binding to

477

the GalUA carboxyl groups. According to the spectra, CP, UCP, ECP and UECP were

478

all partly methoxylated. In conclusion, the 1H and

479

CP and modified pectin samples (UCP, ECP and UECP) showed overall similar

480

patterns, proving that both modification processes had little influence on the

481

configurations and glycosidic linkages of pectin. In similar findings by Zhang (2013a)

AC C

EP

TE D

470

22

13

C NMR spectra for commercial

ACCEPTED MANUSCRIPT 482

and Iida (2008), it was found that ultrasound treatment significantly decreased the

483

molecular weight of polysaccharides but reserved their primary structures.

484

3.5. Effects of ultrasound pretreatment on the anti-cancer activity of pectin hydrolysates

RI PT

485

Fig. 7 depicts the effects of CP, UCP, ECP and UECP on HT-29 cell proliferation

487

after incubation for 72 h. All the pectin samples can be seen to have an inhibitory

488

effect on this cell line in a concentration-dependent manner, i.e., the inhibition rate of

489

HT-29 cells increased with an increase in sample concentrations. Looking at Fig. 7,

490

CP with a concentration of 0.2 mg mL-1 even promoted the proliferation process,

491

possibly by being used as a nutrient during cell growth. Modification via ultrasound

492

or pectinase positively improved the anti-cancer activity of pectin, so UCP, ECP and

493

UECP had obviously higher inhibition rates against HT-29 cells when compared with

494

CP. The inhibitory activity was observed to be positively correlated to Gal content.

495

For example, the inhibition rates of CP (12.44% Gal), UCP (14.09% Gal), ECP

496

(16.94% Gal) and UECP (19.45% Gal) at a same concentration of 1 mg mL-1 were

497

4.59%, 13.77%, 20.60% and 30.10%, respectively. In a study by Maxwell et al.

498

(2016), the alkali-modified sugar beet pectin (1.0 mg mL-1) was seen to reduce HT-29

499

cell proliferation by 20.7% after 48 h. Under a further investigation of

500

monosaccharide compositions, they found that modified pectin had a higher Gal

501

content of 18.5%, whereas the untreated pectin only had a content of 12.4%. In this

502

case, the improvement of anti-cancer activity has been ascribed to the elevated Gal

503

content. Results can be seen parallel to our findings.

AC C

EP

TE D

M AN U

SC

486

23

ACCEPTED MANUSCRIPT As presented in Table 2 and Fig. 7, compared to ECP, UECP with a lower DM

505

had a stronger inhibitory effect on cancer cells, suggesting that the DM was not a

506

decisive factor for anti-cancer activity. On the other hand, although low Mw per se is

507

inadequate for anti-cancer activity, yet it is indispensable to guarantee the entry of MP

508

into cells. In this study, ultrasound pretreatment effectively removed structural

509

barriers against enzymatic attack leading to a minimized Mw and higher Gal content,

510

thus dramatically improving the anti-cancer activity of UECP. Results demonstrated

511

that ultrasound pretreatment played an important role in bioactivity improvement.

512

4.

M AN U

SC

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504

Conclusions

In this study, a commercial CP was subjected to ultrasound before being

514

hydrolyzed by pectinase. An ultrasonic field at 18.0 W mL-1 intensity for 30 min was

515

selected as the pretreatment conditions given both productivity and energy efficiency.

516

Ultrasound was observed to have a positive effect on enzymatic kinetics by increasing

517

the Vmax and Vmax/Km while decreasing the Km. Structures of pectin samples with

518

different treatments were also studied. Results showed that ultrasound could

519

effectively decrease the Mw and DM of pectin and induce degradation in both HG

520

and RG-I regions, while enzymatic hydrolysis only occurred at the HG regions

521

without affecting its DM. Both ultrasound and pectinase had no effect on pectin

522

primary structures. The possible degradation paths of pectin with different treatments

523

are summarized in Fig. 8. Furthermore, the combining ultrasound-pectinase treatment

524

improved the Gal contents in pectin samples resulting in higher anti-cancer activity.

525

Results of this study suggested an innovative green and efficient method for MP

AC C

EP

TE D

513

24

ACCEPTED MANUSCRIPT 526

production.

527

Appendices

528

Table A. 1; Table A. 2 Acknowledgement

530

This work was financially supported by National Natural Science Foundation of

531

China (Project 31371872) and Key Research and Development Project of Zhejiang

532

Province, China (2015C02036).

SC

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529

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ultrasound pretreatment under low power density on the enzymolysis and the

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structure characterization of defatted wheat germ protein. Ultrasonics

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nanostructure of citrus pectin. Journal of the Science of Food and Agriculture,

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Wheat Gluten. Food Biophysics, 10(4), 385–395.

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S., & Ye, X. (2017). Fast preparation of RG-I enriched ultra-low molecular

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Figure captions

Fig. 1. Effects of (a) ultrasound intensity and (b) ultrasound duration on the Mw and

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its polydispersity of pectin. Fig. 2. Effects of (a) ultrasound intensity and (b) ultrasound duration on the DH of pectin hydrolysates.

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samples with and without ultrasound pretreatment.

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Fig. 3. Plots of the enzymatic kinetics using Lineweaver–linearization for pectin

Fig. 4. The FT-IR spectra of pectin samples. (a) CP; (b) UCP; (c) ECP; (d) UECP Fig. 5. The 1H NMR spectra of pectin samples. (a) CP; (b) UCP; (c) ECP; (d) UECP Fig. 6. The 13C NMR spectra of pectin samples. (a) CP; (b) UCP; (c) ECP; (d) UECP

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Fig. 7. Effects of pectin samples on HT-29 cell proliferation. Different letters (a–f) indicate significant differences as estimated by Duncan’s multiple range test (P < 0.05).

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Fig. 8. The schematic diagram of pectin degradation paths under different treatments.

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Tables

Table 1 Effect of ultrasound pretreatment on the enzymatic kinetics parameters. / -1

-1

(mg mL )

0.34 ± 0.01 0.44 ± 0.00

3.28 ± 0.21 2.99 ± 0.02

Control With ultrasound

× 10

(min-1)

102.52 ± 0.46 145.53 ± 1.43

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(mg mL min )

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Table 2 Structural characteristics of pectin samples with different treatments CP

Mw (kDa)

485.10 ± 5.61 a

Polydispersity

3.64 ± 0.03 a

DM (%)

54.63 ± 1.01 a

DAc (%)

1.56 ± 0.17 a

Monosaccharide component (%) 8.85 ± 0.06 b 68.19 ± 0.31 a 4.34 ± 0.06 c 12.44 ± 0.08 d 1.38 ± 0.05 a 3.30 ± 0.04 c 1.50 ± 0.09 a

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ECP

UECP

240.11 ± 6.55 b

18.64 ± 0.13 c

7.70 ± 0.04 c

2.70 ± 0.08 b

2.44 ± 0.03 c

2.30 ± 0.01 c

36.66 ± 0.68 b

56.98 ± 2.80 a

39.60 ± 1.23 b

1.56 ± 0.07 a

1.58 ± 0.08 a

1.56 ± 0.03 a

9.31 ± 0.28 b 67.27 ± 0.40 a 4.39 ± 0.26 c 14.09 ± 0.34 c 1.41 ± 0.09 a 2.05 ± 0.13 d 1.47 ± 0.04 a

11.20 ± 0.45 a 58.53 ± 0.25 b 5.44 ± 0.24 b 16.94 ± 0.66 b 1.60 ± 0.06 a 4.76 ± 0.10 a 1.53 ± 0.31 a

11.67 ± 0.34 a 54.37 ± 0.30 c 6.70 ± 0.08 a 19.45 ± 0.26 a 1.77 ± 0.22 a 4.12 ± 0.09 b 1.91 ± 0.36 a

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Rha GalA Glu Gal Xyl Ara Fuc

UCP

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Structural properties

Rha / GalA

0.13 ± 0.00 c

0.14 ± 0.00 c

0.19 ± 0.01 b

0.21 ± 0.00 a

(Gal + Ara) / Rha

1.78 ± 0.02 a ,b

1.74 ± 0.07 b

1.94 ± 0.11 a, b

2.02 ± 0.06 a

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Note: Rha: GalA and (Gal + Ara): Rha represent molar ratios; values with different italic superscript letters

(a–d) in the same column within each pectin indicate significant differences as estimated by Duncan’s multiple range test (P < 0.05).

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4. 5.

Ultrasound pretreatment improved the enzymatic degradation of pectin. The Vmax increased but the Km decreased after ultrasound pretreatment. The degree of methylation (DM) of pectin significantly decreased under ultrasound. Hydrolysates of the pretreated pectin had higher galactose content. Hydrolysates of the pretreated pectin had higher anti-cancer activity.

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1. 2. 3.