Characterizations of a pectin extracted from Premna microphylla turcz and its cold gelation with whey protein concentrate at different pHs

Journal Pre-proof Characterizations of a pectin extracted from Premna microphylla turcz and its cold gelation with whey protein concentrate at different pHs

Ming-Kai Pan, Fang-Fang Zhou, Rong-Hua Shi, Yong Liu, Qiang Zhang, Jun-Hui Wang PII:

S0141-8130(19)34109-1

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.08.074

Reference:

BIOMAC 13051

To appear in:

International Journal of Biological Macromolecules

Received date:

2 June 2019

Revised date:

31 July 2019

Accepted date:

7 August 2019

Please cite this article as: M.-K. Pan, F.-F. Zhou, R.-H. Shi, et al., Characterizations of a pectin extracted from Premna microphylla turcz and its cold gelation with whey protein concentrate at different pHs, International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/j.ijbiomac.2019.08.074

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© 2018 Published by Elsevier.

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Characterizations of a pectin extracted from Premna microphylla turcz and its cold gelation with whey protein concentrate at different pHs

Ming-Kai Pan a, b, Fang-Fang Zhou

a, b

, Rong-Hua Shi c, Yong Liu

a, b,

*, Qiang Zhang

d

, Jun-Hui Wang a, b, * Engineering Research Center of Bio-process, Ministry of Education, Hefei

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f

a

University of Technology, Hefei, 230009, People’s Republic of China

230009, People’s Republic of China

School of Life Sciences, University of Science & Technology of China, Hefei,

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c

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230027, China

Anhui Province Key Laboratory of Functional Compound Seasoning, Jieshou City

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236500, Anhui, China

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d

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School of Food and Biological Engineering, Hefei University of Technology, Hefei,

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b

------------------------------------------------------------------------------------------------------* Correspondence Author: Jun-Hui Wang & Yong Liu E-mail: [email protected] (J.-H. Wang), [email protected] (Y. Liu)

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Abstract In this study, the physicochemical properties of an ammonium oxalate extraction pectin (AOP) from Premna microphylla turcz was investigated. Moreover, its cold gelation with undenatured whey protein concentrate (WPC) was studied at room temperature and different pHs. Characterizations of AOP demonstrated that AOP was

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a linear low-methoxyl pectin rich in homogalacturonan with low branching degree of

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RG-I, leading to its good gelling properties. Gelation between AOP and WPC was

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mainly investigated by turbidity measurement, FTIR, CLSM and ITC. The results

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showed that an optimal complex ratio for gelation was observed at 1:5 at pH 6.0.

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Moreover, AOP was the backbone of the composite gel and WPC might act as crosslinking agents through electrostatic or hydrophobic interaction at different pHs.

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When pH was around the pHΦ of the complex, composite gel was mainly constructed

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by electrostatic interaction. With the increase of pH, the electrostatic interaction

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between AOP and WPC gradually weakened, while the hydrophobic interaction constantly increased. When pH was higher than the pH c of the complex, composite gel was mainly formed by hydrophobic interaction. The results of this study are conducive to further utilization of Premna microphylla turcz pectin to develop related food products.

Key words: Premna microphylla turcz; Low-methoxyl pectin; Cold gelation

1. Introduction 2

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For a long time, people in China have mixed the juice of Premna microphylla turcz leaves (Fig. 1A) with plant ashes to prepare a green tofu-like food [1], commonly known as “Guanyin tofu”, underlining that the pectin in Premna microphylla turcz features good thickening and gelling properties. Chen et al. [2] studied the pectin obtained from Premna microphylla turcz leaves by sequential

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extraction. They found that the main pectin in Premna microphylla turcz was

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calcium-bound pectin (oxalate soluble pectin), which exhibited the best gelling

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properties and high apparent viscosity because of its high galacturonic acid content,

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high molecular weight, low neutral sugar content and low degree of esterification.

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Therefore, the oxalate soluble pectin in Premna microphylla turcz can be used as a commercial thickener or gelling agent in the food production. Whey protein

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concentrate (WPC) is obtained from whey by a series of purification and

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concentration processes. It is characterized by good solubility, water absorption,

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gelling and foaming properties and provides many nutritional advantages for food products [3]. According to Ikeda et al., WPC can form viscoelastic gels during heating which can immobilize large amount of water and food components [4]. Oboroceanu et al. found that fibrillized whey proteins are efficient foaming agents even at low content (1-3% w/w) [5]. Moreover, WPC can be used in the processing of meat, aquatic products and cakes to improve the taste of food [6,7]. Due to the low cost and good functional properties of WPC [8], it is widely used in food industry. In recent years, researchers have paid much attention on the cold gelation of protein-hydrocolloid gels. Generally speaking, cold gelation is a two-step process [9]. 3

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In the first step, protein solution is heated to obtain protein aggregates. The second step is lowering pH to the isoelectric point of protein aggregates to form gel [10]. With addition of another biopolymer, pectin, the process of cold gelation will be changed and the gel properties will be improved [11]. As to these composite gels, cold gelation is promoted by the electrostatic interaction between protein and pectin when

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two biopolymers carry opposite charges during acidification [12]. The composite gels

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result in a more homogeneous gel network and improved water holding capacity [11,

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13]. Previous studies have reported the cold gelation between whey protein and

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pectins of different types. Low-methoxyl pectin behaves very different from

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high-methoxyl pectin in electrostatic interaction with whey protein [14]. The interaction between whey protein and high-methoxyl pectin leads to the coarsening of

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gel network with higher phase separation [15]. While the interaction between whey

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protein and low-methoxyl pectin can overcome the incompatibility between them to

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form composite gel with homogeneous microstructure and no phase separation [11]. Due to the good gelling properties and high commercial value of WPC and the oxalate soluble pectin in Premna microphylla turcz, we chose to study the cold gelation between them. In this study, the physicochemical and structural properties of ammonium oxalate extraction pectin AOP from Premna microphylla turcz were characterized. Moreover, different pHs of AOP-WPC complexes were prepared in order to study the cold gelation between AOP and undenatured WPC at constant pH and room temperature. Through visual observation, physicochemical tests, microstructure characterization and thermodynamics study, the properties of the 4

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composite gels formed at different pHs were investigated. Meanwhile, the process of cold gelation at different pHs were inferred according to these results. Because no heating, enzymatic or any other denaturation treatment is used, the gels prepared in this study may have positive effects on enhancing food stability and protecting micronutrients. In addition, they also possess potential application value in the

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pharmaceutical industry for transferring and protecting drugs or active molecules.

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2. Materials and methods

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2.1. Materials and reagents

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Premna microphylla turcz powders (Fig. 1B) were purchased from Huangshan Senlinzhibao Biotechnology Co., Ltd. Whey protein concentrate was purchased from

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Hilmar Ingredients, Inc. The product contained 82% w/w of protein on dry basis, 5%

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w/w of lactose, 4.5% w/w of moisture and 325 mg calcium /100 g WPC according to

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the manufacturer. The water used in the experiment was prepared by the ultra-pure water purification system (Hefei Corning Water Treatment Equipment Co., Ltd.). Ammonium oxalate monohydrate (99.8% purity) was purchased from Shanghai Macklin Biochemical Co., Ltd (Shanghai, China). Bovine serum albumin (≥98% purity) was obtained from Guangzhou Saiguo Biotech Co., Ltd (Guangzhou, China). D-(+)-galacturonic acid monohydrate (≥97% purity), ethanol (≥95% purity), trifluoroacetic acid (≥99% purity) and phenolphthalein indicator solution (10 g/L) were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Rhodamine B (dye content: 95%) was obtained from Beijing Solarbio Science & 5

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Technology Co., Ltd (Beijing, China). Unless stated otherwise, all the other chemical reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).

2.2. Extraction of AOP

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100 g of dried Premna microphylla turcz powders were weighed and

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preprocessed by Soxhlet method to remove pigments and lipids. After that, the

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powders were stirred vigorously in 95% v/v ethanol (powders: ethanal 1:30 w/v) for

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1 h at room temperature. Then, the suspension was put in a fridge at 4 ℃ for 24 h to obtain alcohol insoluble solids. The suspension was treated by suction filtration upon

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quantitative filter paper (medium speed, Φ=9 cm, 10-20 μm of pore size) for 30 min

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at 25 ℃. After that, 0.1 M 600 mL ammonium oxalate monohydrate (pH=6.4)

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solution was added to the residues at the ratio of 1:30 w/v, and the extraction was

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conducted at 70 ℃ for 30 minutes. The extracts were filtered by four layers of gauze (110 meshes, Hualu Sanitary Material Co., Ltd, Shandong, China) and the supernatant was then centrifuged (11000 rpm, 10 min, 25 ℃) to remove the residues. Subsequently, the supernatant was added to 95% v/v ethanol (supernatant: ethanal 1:30 v/v) and placed for 24 h at 4 ℃. After suction filtration and drying, the solids were dissolved in ultra-pure water for 48 h of dialysis (molecular weight cut-off 14000 Da) and freeze-dried to obtain the ammonium oxalate soluble pectin AOP (Fig. 1C).

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2.3. Characterizations of AOP The galacturonic acid content of AOP was analyzed by m-hydroxyldiphenyl method with D-(+)-galacturonic acid monohydrate as the standard [16]. The protein content of AOP was measured by Coomassie brilliant blue method with bovine serum albumin as the standard [17]. The neutral sugar content was determined by HPAEC-PAD system. 5 mg of AOP

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sample was decomposed by 3 mL 2 M trifluoroacetic acid (TFA) for 2 h at 121 ℃.

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Then, the TFA in the sample was removed by nitrogen blow. 0.1 mg/mL of the sample

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solution was prepared by dissolving the dried sample to ultra-pure water. After being

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filtered through a 0.22 μm membrane, 25 μL of the sample solution was injected into HPAEC system (ICS-5000, Dionex Corp., USA) with a CarboPac PA20 column

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(3×150 mm). The neutral monosaccharides and uronic acids were respectively eluted

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with NaOH solution (200 mM) and NaAc/NaOH solution (500 mM/200 mM) at the

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flow rate of 0.5 mL/min.

The molecular weight and polydispersity of AOP were measured by the combination of size-exclusion chromatography (SEC) and multi-angle laser light scattering (MALLS). The SEC-MALLS system mainly consisted of a HPLC pump (1525, Waters, USA), Ultrahydrogel 2000 (7.8×300 mm) and Ultrahydrogel 500 (7.8×300 mm) in series, a refractive detector (RI-2410, Waters, USA) and a multi-angle laser light scattering detector (DAWN HELEOS Ⅱ, Wyatt, USA). 0.1 mol/L of NaNO3 was used as eluent. The sample solution was prepared to 2 mg/mL by dissolving AOP in the eluent. After being filtered through a 0.45 μm 7

Journal Pre-proof membrane, 200 μL of the sample solution was injected into the SEC-MALLS system at the flow rate of 0.5 mL/min and the experiment was conducted at 25 ℃. The refractive index increment (dn/dc) of AOP was taken as 0.135 mL/g [18]. The experimental data was collected and analyzed by ASTRA 6.0 software. Degree of esterification (DE) of AOP was analyzed by the titration method. 2 mL

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ethanol was used to wet 500 mg of AOP sample, and the sample was then dissolved in

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100 mL ultra-pure water. After AOP was fully dissolved, the solution was titrated by

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0.1 M NaOH and phenolphthalein indicator solution was used as indicator. The initial

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titer volume (V1) was recorded. Subsequently, 20 mL of 0.5 M NaOH was added to

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the solution and the solution got stirred vigorously for 20 min. Then, 20 mL of 0.5 M HCl was added to the solution and the solution was stirred until the pink color

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disappeared. Finally, the solution was titrated with 0.1 M NaOH and the titer volume

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of NaOH (V2) was recorded. The DE was calculated according to the Eq:

2.4. Preparation of AOP-WPC mixtures According to the method described by Wang et al. [19], WPC (5%, w/w) and AOP (1%, w/w) stock solutions with different pHs were prepared by dissolving dry powders in ultra-pure water (pH 6.0) and 5 mM buffer solutions (pH 3.5 citric acid-sodium citrate buffer; pH 4.5 disodium hydrogen phosphate-citric acid buffer; pH 7.0 phosphate buffer; pH 9.0 sodium bicarbonate-sodium carbonate buffer) respectively, with stirring (600 rpm) at room temperature for 1 h. AOP (0, 0.1%, 0.2%, 8

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0.4%, 0.5% w/w) - WPC (0, 0.25%, 0.5%, 1%, 2%, 2.5% w/w) mixtures were prepared by mixing AOP and WPC stock solutions (dissolved in ultra-pure water) with proper amount of ultra-pure water added in. In addition, AOP (0.2% w/w) - WPC (1% w/w) mixtures with different pHs (pH 3.5, 4.5, 7.0, 9.0) were prepared by mixing AOP and WPC stock solutions of different pHs with proper amount of corresponding

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buffer added in. The mixtures were fiercely stirred for 30 s and placed at room

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temperature for 24 h. After centrifugation (3000 rpm, 5 min), the visual appearance

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was obtained by photographing. Then, different pHs of the composite gels (AOP 0.2%

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w/w, WPC 1% w/w) were freeze-dried for the subsequent analysis.

2.5 Gel strength analysis

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Gel hardness of the composite gels (containing WPC 1% w/w) with different

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complex ratios (1:10, 1:5, 1:2.5 and 1:2) were measured by TA-XT Plus (Stable

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Micro Systems, Surrey, UK). The gel samples were respectively placed in 10 mL glass tubes and the parameters of the test were set as follows: GMIA Gelatine mode, P/0.5 probe, 1.5 mm/s pre-test speed, 1.0 mm/s test speed, 1.0 mm/s post-test speed, auto trigger type, 5.0 g trigger force and 4 mm distance.

2.6. Turbidity measurement and critical transition pHs The turbidity of the AOP-WPC mixed solution (composite ratio 1:5) was determined over a pH range of 3.0-9.0 by an SP-752 UV-VIS spectrophotometer (Jinghua, Shanghai, China) with quartz colorimeters at 600 nm [20]. Critical transition 9

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pHs (pHc, pHΦ, pHmax) were obtained according to the methods reported by Mattison, Dubin and Brittain [21]. AOP and WPC solutions were used as controls.

2.7. ζ-potential measurement According to the method of Anvari et al. [22], AOP and WPC were respectively prepared to 1 mg/mL solutions with buffers of different pHs (3.0, 4.0, 5.0, 6.0, 7.0, 8.0,

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9.0). The ζ-potential was measured at 25 ℃ by Zetasizer Nano ZS (Malvern

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Instruments Ltd).

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2.8. Confocal laser scanning microscopy (CLSM)

The microstructures of the composite gels (AOP 0.2% w/w, WPC 1% w/w)

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formed at different pHs (3.5, 4.5, 7.0, 9.0) were observed by Zeiss LSM710 (Carl

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Zeiss, Jena, Germany) confocal laser scanning microscope at room temperature.

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Before observation, 1 mL of the newly prepared samples were mixed with 20 μL of 0.4% w/w Rhodamine B solution to stain the protein rich phases. Then, 30 μL of the sample was placed on a microslide, covered with a coverslip. The excitation wavelength was 488 nm and 40 × objective lens was used for observation. Digital image files were acquired in 1024 × 1024 pixels resolution. For each sample, 6 pictures were taken at various parts of it. Only repeatedly confirmed trends were reported and representative pictures were shown.

2.9. Fourier transform infrared (FTIR) spectroscopy 10

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Fourier transform infrared (FTIR) spectroscopy of AOP, WPC and the gel powders (AOP 0.2% w/w, WPC 1% w/w) formed at different pHs were obtained by a Perkin Elmer spectrophotometer (Spectrum 100, Perkin Elmer, Waltham, MA, USA) in the wavelength range of 4000-650 cm-1. Ominic 8.0 software was used to analyze

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2.10. Isothermal calorimetric titration (ITC)

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the experimental data.

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The thermodynamics properties of the interaction between AOP and WPC was

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analyzed by isothermal calorimetric titration (Microcal ITC 200, GE Healthcare, USA)

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to infer the interaction forces between the two components at different pHs. 2 mg/mL of WPC solution with different pHs (4.5, 7.0, 9.0) was loaded in the ITC reaction cell

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(200 μL of cell volume), equilibrated at 25 ℃. 5 mg/mL of the AOP solution with

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corresponding pH was loaded in the injector cell of ITC. During the experiment, AOP

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solution would be titrated into WPC solution by adding 1 μL initial injection and 19 successive 2 μL injections with continuously stirring at 1000 rpm. Each injection lasted 10 s and there was an interval of 120 s between two successive injections. The experimental data were analyzed by Origin 7.0.

2.11. Data Analysis All the experiments were replicated. Statistical significance analysis (p < 0.05) was analyzed by the SPSS 21.0 statistical analysis program. The data showed in corresponding figures were expressed in the form of mean values ± standard 11

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deviation.

3. Result and discussion 3.1. Physicochemical and structural properties of AOP Basic information about the physicochemical properties of AOP were shown in

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Table 1. The yield of AOP was 17.63% which further confirmed that the main pectin

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in Premna microphylla turcz was calcium-bound pectin. The protein and galacturonic

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acid content of AOP were 0.29% and 73.5% respectively. Fig. 2 also verified that

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AOP contained high content of galacturonic acid. These results showed that AOP was

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mainly composed of galacturonic acid with very little protein. The DE of AOP was 13.69 %, indicating that the AOP extracted in this experiment was a low-methoxyl

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pectin (LMP). In addition, the neutral sugar of AOP was mainly composed of

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rhamnose, arabinose, galactose and glucose. According to Al-Amoudi et al. [23], the

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values of GalA/(Rha+Ara+Gal) and Rha/GalA could reflect the linearity of pectin structure. Moreover, (Ara+Gal)/Rha and HG/RG-I could reflect the branching degree of RG-I and the main composition of pectin backbone. The values of GalA/(Rha+Ara+Gal), Rha/GalA, (Ara+Gal)/Rha and HG/RG-I of AOP were 7.88, 0.02, 4.52 and 6.51 respectively, indicating that AOP was a linear low-methoxyl pectin rich in homogalacturonan with low branching degree of RG-I. The molecular weight and polydispersity of AOP were 128.60 kg/mol and 1.56, reflecting the high homogeneity of AOP. The pectin extracted in this study was similar to Chen et al. [2], except for the molecular weight. The molecular weight of oxalate soluble pectin in 12

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their study was 980.67 kg/mol. The distinction of molecular weight might result from the differences in ingredient sources and extraction method. Meanwhile, the oxalate soluble pectin in their study was also an LMP rich in homogalacturonan. It contained 76.15% of galacturonic acid, and its DE was 14.90%.

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3.2. Inspection of gel formation

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The visual appearance provides further information on the status of AOP-WPC

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mixtures at different complex ratios, concentrations and pHs. Fig. 3A-C showed the

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visual appearance of the mixtures prepared by ultra-pure water with different complex

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ratios and concentrations. According to Fig. 3A-C, it was showed that when the content of WPC was below 0.5% w/w, the mixtures were solution. When the content

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of WPC was above 1% w/w, gelation occurred. While the volume of the centrifuged

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water increased with the increase of WPC. These phenomena might result from the

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role of AOP as the backbone of the composite gel and WPC as the crosslinking agent [24]. According to the results of characterizations of AOP, it was a linear low-methoxyl pectin rich in homogalacturonan with low branching degree of RG-I. These properties contributed to the good gelling properties of AOP, and AOP would be easily crosslinked by crosslinking agents to form gels [2]. However, gel network couldn’t be formed when there was not enough crosslinking agent. Conversely, in the case of excessive crosslinking agent, a small amount of added pectin would form gels, while the water retention of the gel would decrease. Wang et al. [25] studied the effect of crosslinking agents on polysaccharide-protein mixed gels and found that high 13

Journal Pre-proof dosage of crosslinking agents (Ca2+ concentration > 0.015 M) could lead to the decrease in water retention of gels. In addition, there might be an optimal complex ratio for the interaction between AOP and WPC at constant pH, enabling the mixture to form a solid composite gel [24]. According to Fig. 3B, when the WPC content was 1% w/w and the content of

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AOP was higher than 0.4% w/w, the composite gels slid from top to bottom in the

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inverted photograph, indicating the weakening of the composite gel strength. Fig. 4

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showed that the gel strength of the composite gel containing 1% w/w WPC increased

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to the maximum at the complex ratio of 1:5 and then decreased with the increasing

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complex ratio. Hence, the complex ratio of 1:5 might be the optimal complex ratio for gelation at pH 6.0. The change in gel strength of the composite gel was similar to the

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results of Le et al. [24]. They found that the optimal ratio for the interaction between

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β-lactoglobulin (βlg) and xanthan gum (XG) was at XG:βlg=1:3.5. In the light of Le

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et al. [24] and Wang et al. [19], it could be deduced that the excessive WPC might promote the conjunction of WPC with individual AOP molecules to form individual complexes when AOP was at low concentration (AOP:WPC<1:5), decreasing the junction zones of composite gel network and reducing its gel strength. When AOP was at high concentration (AOP:WPC>1:5), the excessive AOP might not be crosslinked by WPC due to the limited positively-charged patches on WPC molecules, leading to the decrease in the gel strength of composite gel. According to Fig. 3A-C and Fig. 4, we chose the concentration and complex ratio at AOP (0.2% w/w) – WPC (1% w/w) to further study the effect of pH on the cold gelation between AOP and 14

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WPC. The visual appearance of the composite gels formed at AOP (0.2% w/w) - WPC (1% w/w) with different pHs were shown in Fig. 3D. According to Fig. 3D, AOP and WPC aggregated to coacervates at pH 3.5 likely due to the strong electrostatic interaction between AOP and WPC [26]. Gelation occurred from pH 4.5 to 9.0. The

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composite gel of pH 4.5 was relatively turbid (OD600=0.214 in Fig. 5A) while the

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composite gels of pH 7.0 (OD600=0.041 in Fig. 5A) and pH 9.0 (OD600=0.022 in Fig.

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5A) were relatively transparent. These phenomena could be attributed to the insoluble

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complexes formed between AOP and WPC by electrostatic interaction at pH 4.5. With

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the increase of pH, the electrostatic repulsion between two components was strengthened. Thus, less or no insoluble complex was formed, leading to the

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transparency of the composite gels formed at pH 7.0 and pH 9.0. These results were

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slightly different from those reported by Wijaya et al. [13]. They studied the cold

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gelation between whey protein and low-methoxyl apple pectin (DE: 33%-38%) at four different pHs (5.5, 3.5, 2.5, 2.0) and reported that soluble complexes with low turbidity was formed at pH 5.5, while insoluble complexes with high turbidity was formed at pH 3.5 and 2.5. However, gels were not formed at pH 5.5, 3.5 and 2.5. Gelation only occurred at pH 2.0. The difference in gelation behavior might result from the structural and physicochemical distinctions between AOP and low-methoxyl apple pectin.

3.3. Critical transition pHs 15

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The electrostatic complex status of AOP and WPC at different pHs can be deduced from the critical transition pHs on the turbidity curve of the mixed solution. The pH where soluble complexes are formed is labeled as pH c. At this pH, the electrostatic interaction between the two components is weak. The pH where insoluble complexes are formed is labeled as pHΦ, indicating the strengthening of

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electrostatic interaction between the two components. The maximum turbidity of the

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mixture is labeled as pHmax. The electrostatic interaction between two components is

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the strongest at this pH. Fig. 5A demonstrated the turbidity curve and critical

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transition pHs of AOP-WPC mixed solution. The pH c of the mixed solution was

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present at pH 7.5, indicating that the electrostatic interaction between AOP and WPC began at pH 7.5. As shown in Fig. 5B, the isoelectric point of WPC was around pH

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4.8. The formation of soluble complexes at a pH above the isoelectric point of WPC

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was due to the heterogeneity of the charges on protein surface. At pH c, some moieties

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of protein molecules still carried positive charges and these positively charged patches could interact with negatively charged polysaccharides to form complexes [27]. The pHΦ of the mixed solution was at pH 4.5 where AOP and WPC carried opposite charges according to Fig. 5B. From this pH, insoluble complexes began to form in the mixed solution, and phase separation began to occur. The formation of insoluble complexes made the turbidity of the mixed solution increase significantly with the decrease of pH until it reached to pHmax (pH 3.5). According to the Wang et al. [19], insoluble complexes begin to form when pH reaches the point where two biopolymers carry opposite charges, and associative phase separation occurred by strong 16

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electrostatic interaction. So, pHΦ should be lower than the isoelectric point of protein.

3.4. CLSM Fig. 6A-D shows the microstructures of the composite gel (AOP: WPC= 1: 5, 1% w/w WPC) formed at pH 3.5, 4.5, 7.0 and 9.0. At pH 3.5, the composite gel

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exhibited a significant phase separation microstructure, which might result from the

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coacervation of AOP and WPC. The strong electrostatic interaction between the two

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components would lead to coacervates at pH 3.5 according to turbidity measurement

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and visual inspection. At pH 4.5, AOP and WPC produced coarsening of

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microstructure, due to the phase separation caused by formation of insoluble complexes. However, the electrostatic repulsion between the two components

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increased at pH 4.5, so aggregation was restricted and no coacervate was formed. At

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pH 7.0, the composite gel showed homogenous microstructure with some small

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protein aggregates. The homogenous microstructure was resulted from the interaction between WPC and high charged AOP, which could overcome the incompatibility of them [11]. The small protein aggregates might attribute to the formation of soluble complexes by weak electrostatic interaction at pH 7.0. At pH 9.0, both WPC and AOP carried strong negative charges according to Fig. 5B. The composite gel presented a very uniform microstructure that no obvious concentrated particle was observed. Since pH 9.0 was higher than pHc, no soluble complex would be formed due to the strong electrostatic repulsion between AOP and WPC.

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3.5. FTIR As to the infrared spectra of AOP in Fig. 7A, the broad absorption peaks at 3198 cm-1 corresponding to O-H stretching vibration, attributed to the hydrogen bonds of water. The absorption peaks at 2927 cm-1 and 1328 cm-1 were caused by C-H stretching and bending vibration. The peaks at 1736 cm-1 was derived from the stretching vibration of C=O in -COOCH3 and the undissociated carboxyl acid (COOH)

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[28]. The weak absorption of peak 1736 cm-1 indicated the low esterification of AOP,

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which was consistent with the result of DE analysis. The absorption peaks at 1583

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cm-1 and 1411 cm-1 were caused by C=O asymmetric and symmetric stretching vibration of -COO-, respectively. The peaks at 1094 cm-1 and 1014 cm-1 resulted from

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C-O-H and C-O-C stretching vibration of sugar ring. The peaks at 951 cm-1, 889 cm-1

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and 852 cm-1 were the absorption peaks of glucose, galactose and arabinose, caused

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by C-H bending vibration of β-D-pyranose.

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As to the infrared spectrum of WPC in Fig. 7A, the broad absorption peak at 3274 cm-1 was caused by O-H stretching vibration, attributed to the hydrogen bonds of water. The absorption peak of 2930 cm-1 was caused by C-H stretching vibration. The absorption peaks at 1633 cm-1, 1539 cm-1, 1248 cm-1 corresponded to Amide I (C=O stretching vibration), Amide II (N-H bending and C-N stretching vibration) and Amide III (C-N stretching vibration), respectively [29]. The peak at 1397 cm-1 was caused by the symmetrical stretching vibration of -COO-. The peaks at 1067 cm-1 and 1032 cm-1 represented C-O-H and C-O-C stretching vibration, derived from the lactose in WPC [30]. 18

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The infrared spectra of the complex gels (AOP: WPC= 1: 5, 1% w/w WPC) at pH from 3.5 to 9.0 were shown in Fig. 7B. At pH 3.5, Amide I and Amide II exhibited large peak absorption at 1633 cm-1 and 1540 cm-1, while the AOP sugar ring presented weak peak absorption at 1026 cm-1. The strengthened Amide I and Amide II peaks and the weakened sugar ring peak might result from the strong aggregation between

f

WPC and AOP. The structure of AOP was thus significantly affected by WPC, leading

oo

to the weakened peak absorption. With the increase of pH, the peak absorption of

pr

Amide I and Amide II decreased, while the peak absorption of AOP sugar ring

e-

increased significantly. The electrostatic binding between the two components would

Pr

become weaker and finally disappeared with the increasing pH. Accordingly, the characteristic peaks of AOP sugar ring would become obvious and their peak

al

absorption would enhance.

rn

The change of the Amide III peak could also reflect the degree of electrostatic

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interaction between two components. With the decrease of pH from 9.0 to 3.5, the Amide III peak shifted from 1248 cm-1 to 1231 cm-1 and the peak absorption gradually increased. The shift of the Amide III peak indicated that the electrostatic interaction between AOP and WPC became stronger, due to the strengthening of electrostatic attraction between the two components. Timilsena et al. [31] found that the Amide III peak, which previously appeared at 1228 cm-1 in chia seed protein isolate (CPI), shifted to 1224 cm-1 in the complex coacervation with chia seed gum and the peak intensity was also substantially increased. This shifting of Amide III peak indicated the conformational change of WPC from random coil and α-helix type 19

Journal Pre-proof to more organized β-sheet structure [32]. At pH 3.5, the absorption peak of C-H bending vibration (around 1330 cm-1) almost disappeared, which might be attributed to the strong electrostatic binding between AOP and WPC, restricting the C-H bending vibration of AOP. With the increase of pH, the absorption of C-H bending vibration peak increased at 1336 cm-1 due to the increase of electrostatic repulsion

oo

f

between AOP and WPC.

pr

3.6. ITC

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The thermodynamic properties of the interaction between AOP and WPC at

Pr

different pHs were studied by ITC. Fig. 8A-C reflected the ITC curves of their interaction at pH 4.5, pH 7.0 and pH 9.0, respectively. It was showed that the

al

thermodynamic properties of the interaction gradually changed from exothermic to

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endothermic with the increase of pH.

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At pH 4.5, the exothermic binding enthalpy increased continuously until the complex ratio reached to 0.25. Then, the binding enthalpy began to decrease with the increase of complex ratio. Li et al. [33] pointed out that the complexation of proteins and polysaccharides was an enthalpy driven process caused by electrostatic forces when polysaccharides and proteins exhibited a two-step binding behavior with a "v" type ITC curve. Accordingly, it could be inferred that the gelation between AOP and WPC was an enthalpy driven process caused by electrostatic forces at pH 4.5, which was consistent with the results above.

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At pH 9.0, the ITC image showed an endothermic curve, which might result from the hydrophobic interaction between AOP and WPC [34]. According to Benjamin, Lassé, Silcock, and Everett [35], the structural of β-lactoglobulin (the main component of WPC) would change from being compact and rigid to a more open and flexible structure at pH above 6.0. Moreover, it would aggregate at pH 9.0 due to alkaline denature. These changes would lead to the significant increase in the

oo

f

hydrophobicity of β-lactoglobulin. Besides, the hydrophobic interaction will be

pr

dominant in polymer-polymer interaction when the electrostatic repulsion between the

e-

components is strong [36]. Therefore, it could be inferred that the complexation of

Pr

AOP and WPC was an entropy driven process caused by hydrophobic forces at pH 9.0.

al

The ITC curve showed very little heat release at pH 7.0. The electrostatic

rn

interaction between AOP and WPC became continuously weaker with the increase of

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pH, while the hydrophobic interaction increased. At pH 7.0, the combination of enthalpy and entropy driven processes led to micro heat release in the complexation of AOP and WPC, resulting from weak electrostatic forces and hydrophobic forces. These results of ITC were similar to those obtained from de Jong et al. [15]. They suggested that pectin and protein mainly interacted by electrostatic interaction at low pH, while they mainly interacted by entropy force at high pH. Another point which must be taken into account is that AOP is very sensitive to calcium. Meanwhile, WPC contains little amount of calcium (325 mg/100 g WPC). Thus, the calcium in WPC might also contribute to the gelation. 21

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4. Conclusions The pectin extracted from Premna microphylla turcz was a linear low-methoxyl pectin rich in homogalacturonan with low branching degree of RG-I, leading to the good gelling properties of AOP. The optimal complex ratio between AOP and WPC

f

was at 1:5 (AOP 0.2% w/w, WPC 1% w/w) under pH 6.0. In addition, the structure of

oo

the composite gel was mainly based on AOP, whereas WPC acted as a crosslinking

pr

agent through electrostatic or hydrophobic interaction. The extend of interaction was

e-

affected by different pHs. When pH was around the pHΦ of the AOP-WPC complex,

Pr

gelation was an enthalpy driven process. When pH was around pHc, both enthalpy and entropy driven interactions led to gelation. When pH was higher than pH c, gelation

al

occurred by entropy driven interaction. The results of this study underline the

rn

potential for further utilization of the resources of Premna microphylla turcz to

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develop jelly-like food products low in sugar, fat and salt, which are good for health. In addition, the gels prepared in this study can be used to encapsulate micronutrients or active molecules over a wide range of pH.

Acknowledgments This work was supported by grants from National Natural Science Foundation of China （No. 21571095）and the Anhui Province Science & Technology Major Projects (No. 16030701081).

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[33] X. F. Li, Y. F. Hua, Y. M. Chen, X. C. Kong, C. M. Zhang, Two-step complex behavior between bowman–birk protease inhibitor and ι-carrageenan: effect of

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protein concentration, ionic strength and temperature. Food Hydrocoll. 62

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[34] W. Y. Chen, Z. C. Liu, P. H. Lin, C. I. Fang, S. Yamamoto, The hydrophobic

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interactions of the ion-exchanger resin ligands with proteins at high salt concentrations by adsorption isotherms and isothermal titration calorimetry.

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Sep. Purif. Technol. 54 (2007) 212-219.

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[35] O. Benjamin, M. Lassé, P. Silcock, D. W. Everett, Effect of pectin adsorption on

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the hydrophobic binding sites of β-lactoglobulin in solution and in emulsion systems. Int. Dairy J. 26 (2012) 36-40.

[36] P. Gilsenan, R. Richardson, E. Morris, Thermally reversible acid-induced gelation of low-methoxy pectin. Carbohydr. Polym. 41 (2000) 339-349.

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Figures captions Fig. 1. (A) Premna microphylla turcz leaves; (B) Dried powders of Premna microphylla turcz; (C) Ammonium oxalate extraction pectin AOP from Premna microphylla turcz. Fig. 2. HPAEC-PAD chromatograms of AOP and standard monosaccharides. Fig. 3. (A) Upright and (B) inverted photographs of AOP-WPC mixtures prepared by ultra-pure water at different complex ratios and concentrations; (C) abstract image of the status of AOP-WPC mixtures.

f

◒:

composite gel with obvious

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○: solution, ●: composite gel, ⊕ : weak composite gel and

pr

centrifuged water; (D) photographs of AOP-WPC mixtures (AOP 0.2% w/w, WPC 1% w/w) prepared

e-

at different pHs (3.5, 4.5, 7.0, 9.0).

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Fig. 4. Gel hardness of AOP-WPC composite gels (WPC 1% w/w) at different complex ratios (1:10 1:2). a-c represented a significant difference in gel hardness of the composite gels with different

al

complex ratios (p < 0.05).

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Fig. 5. (A) Turbidity curves (OD600) for AOP, WPC and AOP-WPC mixed solution (composite ratio

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1:5), and the critical transition pH of the given solution. (B) ζ-potential of AOP and WPC. Fig. 6. Microstructures of AOP-WPC composite gels. A-D: CLSM images of AOP-WPC (AOP 0.2% w/w, WPC 1% w/w) composite gels prepared at different pHs (3.5, 4.5, 7.0, 9.0). Fig. 7. FTIR spectra of (A) AOP and WPC, (B) AOP-WPC (AOP 0.2% w/w, WPC 1% w/w) composite gels prepared at different pHs. Fig. 8. ITC titration curves of AOP-WPC at (A) pH 4.5, (B) pH 7.0 and (C) pH 9.0. Temperature: 25 °C. WPC solution (2 mg/mL) was loaded in the reaction cell and AOP solution (5 mg/mL) was loaded in the syringe.

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Table 1 Yield, chemical composition, monosaccharide molar ratios, weight average molar mass and degree of esterification (DE) of AOP. Composition

AOP

Yield (%)

17.63 ± 1.32

Protein (%)

0.29 ± 0.04

Galacturonic acids (GalA, %)

73.50 ± 3.16

Total neutral sugars (%)

12.34 1.43 ± 0.18

Arabinose (Ara)

2.44 ± 0.13

oo

f

Rhamnose (Rha)

Galactose (Gal)

4.16 ± 0.32 3.64 ± 0.25

pr

Glucose (Glc) Fucose (Fuc)

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Mannose (Man) Molar ratio of GalA:Rha:Ara:Gal:Glc

HG/ RG-I Mw (kg/mol) Mn (kg/mol) Mw/Mn DE (%)

Pr

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RG-I (mol%)

b

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HG (mol%) a

al

Molar ratio of (Ara+Gal)/Rha

0.44± 0.03

84.73:1.95:3.64:5.17:4.52

Molar ratio of GalA/(Rha+Ara+Gal) Molar ratio of Rha/GalA

0.23 ± 0.02

a

HG (mol%) = GalA (mol%) - Rha(mol%)

b

RG-I (mol%) = 2×Rha (mol%) + Ara (mol%) + Gal (mol%)

7.88 0.02 4.52 82.78 12.71 6.51 128.60± 2.57 82.53± 1.65 1.56± 0.05 13.69 ± 1.43

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Fig. 1. (A) Premna microphylla turcz leaves; (B) Dried powders of Premna microphylla turcz; (C)

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rn

al

Pr

e-

pr

oo

f

Ammonium oxalate extraction pectin AOP from Premna microphylla turcz.

31

Pr

e-

pr

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f

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rn

al

Fig. 2. HPAEC-PAD chromatograms of AOP and standard monosaccharides.

32

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rn

al

Pr

e-

pr

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f

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Fig. 3. (A) Upright and (B) inverted photographs of AOP-WPC mixtures prepared by ultra-pure water at different complex ratios and concentrations; (C) abstract image of the status of AOP-WPC mixtures. ○: solution, ●: composite gel, ⊕ : weak composite gel and

◒:

composite gel with obvious

centrifuged water; (D) photographs of AOP-WPC mixtures (AOP 0.2% w/w, WPC 1% w/w) prepared at different pHs (3.5, 4.5, 7.0, 9.0).

33

e-

pr

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f

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Pr

Fig. 4. Gel hardness of AOP-WPC composite gels (WPC 1% w/w) at different complex ratios (1:10 -

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rn

complex ratios (p < 0.05).

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1:2). a-c represented a significant difference in gel hardness of the composite gels with different

34

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rn

al

Pr

e-

pr

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f

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Fig. 5. (A) Turbidity curves (OD600) for AOP, WPC and AOP-WPC mixed solution (composite ratio 1:5), and the critical transition pH of the given solution. (B) ζ-potential of AOP and WPC.

35

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rn

al

Pr

e-

pr

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Fig. 6. Microstructures of AOP-WPC composite gels. A-D: CLSM images of AOP-WPC (AOP 0.2% w/w, WPC 1% w/w) composite gels prepared at different pHs (3.5, 4.5, 7.0, 9.0).

36

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rn

al

Pr

e-

pr

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Fig. 7. FTIR spectra of (A) AOP and WPC, (B) AOP-WPC (AOP 0.2% w/w, WPC 1% w/w) composite gels prepared at different pHs.

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f

Fig. 8. ITC titration curves of AOP-WPC at (A) pH 4.5, (B) pH 7.0 and (C) pH 9.0. Temperature:

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25 °C. WPC solution (2 mg/mL) was loaded in the reaction cell and AOP solution (5 mg/mL) was

pr

loaded in the syringe.

A pectin with good gelling properties in Premna microphylla turcz was

Pr

•

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Highlights

Low content of AOP (0.2% w/w) and native WPC (1% w/w) could gelate at

rn

•

al

extracted and characterized.

pH from 4.5 to 9.0.

No heating, denaturing or acidification treatment was used to induce gelation.

•

Gelation of AOP (0.2% w/w) with WPC (1% w/w) were studied at pH 4.5, 7.0

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•

and 9.0.

38

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8