Emulsifying properties of pectic polysaccharides obtained by sequential extraction from black tomato pomace

Journal Pre-proof Emulsifying properties of pectic polysaccharides obtained by sequential extraction from black tomato pomace

Wei Zhang, Xie Fan, Xinzhe Gu, Shengxiang Gong, Jinhong Wu, Zhengwu Wang, Qiang Wang, Shaoyun Wang PII:

S0268-005X(19)30429-1

DOI:

https://doi.org/10.1016/j.foodhyd.2019.105454

Reference:

FOOHYD 105454

To appear in:

Food Hydrocolloids

Received Date:

24 February 2019

Accepted Date:

17 October 2019

Please cite this article as: Wei Zhang, Xie Fan, Xinzhe Gu, Shengxiang Gong, Jinhong Wu, Zhengwu Wang, Qiang Wang, Shaoyun Wang, Emulsifying properties of pectic polysaccharides obtained by sequential extraction from black tomato pomace, Food Hydrocolloids (2019), https://doi. org/10.1016/j.foodhyd.2019.105454

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Journal Pre-proof Emulsifying properties of pectic polysaccharides obtained by sequential extraction from black tomato pomace Wei Zhang1, Xie Fan1, Xinzhe Gu1, Shengxiang Gong1, Jinhong Wu*,1, Zhengwu Wang*,1, Qiang Wang2, Shaoyun Wang3

1School

of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240,

China 2Key

Laboratory of Agro-Products Processing, Ministry of Agriculture and Rural

Affairs/Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences 3Institute

of Food and Marine Bioresources, College of Biological Science and

Technology, Fuzhou University, Fuzhou 350108, China

*Corresponding

author:

Zhengwu Wang, Shanghai Jiao Tong University, NO. 800, Dongchuan Road, Shanghai, 200240, People's Republic of China; Phone： +86-21-34206613; Fax: +86-2134205748; E-mail: [email protected]. Jinhong Wu, Shanghai Jiao Tong University, NO. 800, Dongchuan Road, Shanghai, 200240, People's Republic of China; Phone： +86-21-34206613; Fax: +86-2134205748; E-mail: [email protected].

Journal Pre-proof Abstract: The polysaccharides in black tomato pomace (BTP) were extracted by water, cyclohexane-trans-1,2diamine tetraacetic acid, Na2CO3 and KOH to obtain hemicellulose fraction (HF) and pectic fractions including water-soluble pectin (WEP), chelator-extractable pectin (CEP), sodium carbonate extractable pectin (NEP). Structural characterization indicated that WEP, CEP and NEP were high-methylated (68.02%, 61.58% and 59.70%, respectively). Percentage content of the major domain, linear homogalacturonan (HG), in WEP, CEP and NEP were 70.44%, 84.07% and 60.08%, respectively, while the percentage content of branched rhamnogalacturonan (RG-I) were 10.97%, 5.32% and 29.08%, respectively. The results were also confirmed by atomic force microscope (AFM) images. DSC results showed that the Tm of CEP and NEP was higher than WEP. Evaluation of emulsifying property suggested that the pectic fractions could stabilize emulsion containing 50% oil phase at a concentration as high as 1.5% (w/v) and pH 4.0-8.0. Pectin’s ability to lower surface tension and increase viscosity, along with the repulsion in pectin molecules, facilitated emulsion formation. Protein residue (WEP) and structure difference (CEP and NEP) might be responsible to the distinction of emulsifying capacity. Keywords: Black tomato pomace, Pectin, HG, RG-I, Emulsifying properties.

Journal Pre-proof 1. Introduction The oil-water (O/W) emulsion is an important delivery system for lipophilic bioactive compounds and also wide applicated in modulating sensory properties, digestibility and spreadability in certain food materials (Capron, Costeux, & Djabourov, 2001; Frank, Köhler, & Schuchmann, 2011; Yang et al., 2018). Yet, emulsions are thermodynamically unstable colloidal systems that tend to eventually result in two immiscible phases (McClements, 2015). Therefore, emulsifiers are often employed to either reduce the interfacial tension between the oil and water phase or to generate repulsive interactions in order to stabilize the emulsion system (Kralova & Sjöblom, 2009). Recently, the demand for food safety and the development of emulsions with enhanced functionality have propelled the exploration of new emulsifiers from natural sources. Proteins (e.g., whey protein, soy proteins, rice glutelin and bean proteins) (Gumus, Decker, & McClements, 2017; Wan et al., 2014; Xu et al., 2016) and polysaccharides (e.g., gum arabic, modified starches and pectin) (Estrada-Fernández et al., 2018; Verkempinck et al., 2018; Zhao et al., 2018) are the most widely used natural food emulsifiers. An effective emulsifying agent consists of water-soluble domains (hydrophilic) and water-insoluble domains (hydrophobic). Although pectin, traditionally used as gelling and thickening reagent, usually exhibits less hydrophobicity and higher molarity than proteins, the emulsifying capacity of pectin is generally weaker than proteins (Li & Nie, 2016). Protein emulsifiers that are absorbed in the oil-water interface are usually in unfolded conformations and share similar molecular characteristic (Żmudziński et al., 2014). In addition, emulsions that are stabilized by proteins usually lack diversity (e.g. in terms of droplet size and rheology) (Li & Nie, 2016). On the other hand, pectin, as an emulsifying agent, exhibits varied emulsifying functionalities attributing to its tunable structure (Kpodo et al., 2018). Furthermore, as the main components in cell walls, rich sources of pectin are abundant in the food and vegetable processing industry. Recently, reports of the remarkable emulsifying ability of pectin have attracted much attention, with sugar beet pectin (Karnik & Wicker, 2018; Zhang et al., 2015) and pomegranate peel pectin (Yang et al., 2018) as examples. Pectin is a complex galacturonic acid-rich heteropolysaccharide. The most abundant blocks of pectin are HG and RG-I, which account for 65% and 20% to 35% of total pectin, respectively (Ngouemazong et al., 2015). HG is a linear and smooth segment composed of 1→4 linked α-Dgalacturonic acid (GalpA), which could be methyl esterified at C-6 and/or possibly substituted by acetyl groups at O-2 and O-3. According to the degree of methylation (DM), pectin could be divided into high

Journal Pre-proof methoxyl pectin (HMP, DM>50%) and low methoxyl pectin (LMP, DM<50%) (Yapo, 2011). RG-I is a “hairy” part with a backbone composed of [α-(1→2)-D-GalpA-α-(1→4)-L-Rhap]n (n>100) and sidechains rhamnosyl residues substituted by neutral sugars, such as arabinans, galactans and arabinogalactans, at O-4 (Koubala et al., 2014). Proteins may also be found to link with side chains of RG-I, which make the structure of pectin very complicated (Funami et al., 2011). Other moieties, such as rhamnogalacturonan (RG-II), xylogalacturonan (XGA) and apiogalacturonan (AGA) may be found depending on the pectin source and extraction method (Alba & Kontogiorgos, 2017). Commercial pectin is obtained under strong acidic condition and usually contains high level of DM and large proportion of HG regions. Some examples of such pectin are citrus pectin (DM>55% and ratio of HG/RGI >15.6) and apple pectin (DM varied from 54.5 to 67.1%, HG/RGI varied from 5.6 to 16.3)(Harris & Smith, 2006; Kaya et al., 2014; Cho et al., 2019), which are regarded as effective gelling agents rather than as emulsifier. In general, the emulsifying property of pectin is associated with the tunable HG and RG-I blocks, especially the hydrophobic groups and neutral sugar side chains, which provide pectin the ability to anchor oil-water interface (Yang, Mu, & Ma, 2018; Yang et al., 2018). Extensive studies focusing on the effect of extraction conditions on yield and structure of pectin have been conducted. For example, methods relying on ammonium oxalated, HCl or water have been employed to obtain tomato pectin, which has been confirmed to be a high methoxyl pectin with DMs of 76.3%, 87.8% and 84.51%, respectively (Alancay, Lobo, Quinzio, & Iturriaga, 2017; Grassino et al., 2016). Ammonium oxalate and sulfuric acid have also been reported to allow sequential extraction of sugar beet pectin, however, the fractions obtained using ammonium oxalate were found to exhibit poor emulsifying stability (Liu et al., 2019). To the best of our knowledge, little literature has reported on the effects of extraction method on the domain structure of pectin and its emulsifying properties. Tomato (Solanum lycopersicum) is one of the most important vegetables in the world and the major source of lycopene, β-carotene and vitamins (Burton‐Freeman et al., 2012). The black tomato has a noticeable blackish-red skin with higher lycopene and β-carotene contents than typical red tomatoes (Park, Sangwanangkul, & Baek, 2018). Besides carotenes, protein, chlorophyll, organic acid (e.g. chlorogenic acid, neochlorogenic acid, citric acid and amino acid), phenolic compound (e.g. naringenin and naringenin chalcone) were found in black tomato, the extraction contained phenolic and pigmented compounds from black tomato was also reported to be with ability of antioxidant and growth inhibition of cancer cells (Choi et al., 2014; Erdinc et al., 2018). However, the published work on black tomato has

Journal Pre-proof largely focus on chemical composition and nutritional functions related, the knowledge of polysaccharide in black tomato is lack. The industrial transformation of tomato usually leads to large quantities of tomato pomace, a mixture of tomato peels, seeds and pulp, which has been reckoned as a good source of pectin recently (Schieber, Stintzing, & Carle, 2001; Grassino et al., 2016; Zhang et al., 2019). The availability of black tomato is usually lower than red tomato for the thickness and compactness of peel. The objectives of this study are to obtain the polysaccharide in black tomato pomace by sequential extraction, to investigate the physicochemical character and emulsifying properties of polysaccharide fractions. Moreover, the emulsifying mechanisms has also been discussed. The results are particularly useful for efficient utilization of black tomato pomace. 2. Materials and methods 2.1. Materials Black tomatoes were cultivated and harvested from the farmland of Nanjing Lehehe Agricultural Technology Co., Ltd (Nanjing, China). BTP was collected by sieving the tomato pulp immediately after the cold break during ketchup production using black tomato, then freeze-dried and stored at -20C until prior to experiments. L-Rhamnose (Rha), L-(+)-Arabinose (Ara), D-(+)-Galactose (Gal), L-(-)-Fucose (Fuc), D-(+)-Xylose (Xyl), D-(+)-Mannose (Man), D-(+)-Glucose (Glc) and D-(+)-Galacturonic acid

(GalA) monohydrate standards were provided by Sigma-Aldrich (Bornem, Belgium). Soy protein isolate (SPI) was purchased from Ourchem (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China). All chemical reagents were purchased from Macklin (Shanghai, China) and were of analytical grade. 2.2. Polysaccharide fractions Polysaccharides were prepared as described in literatures with slight modifications (Meersman et al., 2017; Sila et al., 2006). In brief, BTP was homogenized with 95% ethanol at a solid/liquid ratio of 1:20 by ultra-turrax T18 (IKA, Staufen, Germany), followed by filtration (Grade GF10 Φ 80 mm, Whatman, Maidstone, England). The procedure was repeated with acetone and the residual was vacuum dried at 40C. The obtained alcohol-insoluble residue (AIR) was ground and stored in a desiccator. The polysaccharides in the AIR were fractionated into WEP, CEP, NEP and HF by sequential extraction. The AIR (500 mg) was dispersed in 40 mL of boiling demineralized water and continued boiling for 5 min. The mixture was cooled and filtered. WEP was obtained by adjusting the filtrate to 50 mL with demineralized water, supposedly consisting of high methyl esterified parts weakly bound to cell material. The residue was then dispersed in 0.05 M cyclohexane-trans-1,2-diamine tetraacetic acid in 0.1

Journal Pre-proof M potassium acetate (40 mL, pH was adjusted to 6.5 with 0.1 M KOH) and incubated for 6 h at 28C. CEP was collected by adjusting the filtrate to 50 mL. The residue was redispersed in 0.05 M Na2CO3 containing 0.02 M NaBH4 (40 mL) and incubated for 16 h at 4C, then for 6 h at 28C. The suspension was filtrated and adjusted to 50 mL to extract the NEP. The residue was then dispersed in 40 mL aqueous solutions containing 4 M KOH, 20 mM NaBH4 and 3.5% borate, and stirred for 22 h at room temperature. The suspension was filtered, diluted to 50 mL and adjusted to pH 5 with 0.1 M HCl to obtain the HF. All the fractions were frozen at -40C. 2.3. Polysaccharide characterization 2.3.1 Uronic acid content Uronic acid (UA) contents of AIR and polysaccharide fractions were determined by the mhydroxydiphenyl method introduced by Blumenkrantz & Asboe-Hansen (1973), using galacturonic acid as standard. The AIR solutions were prepared by hydrolyzation with sulfuric acid prior to the determination according the method described by Ahmed & Labavitch (1978). 2.3.2 Monosaccharide composition For the determination of neutral sugars, pH of the polysaccharide fractions was adjusted to 6.0, dialyzed (with a 3.0 kDa molecular weight cut off) against ultrapure water for 48 h and then lyophilized. Before acetylation, the polysaccharide fractions (2 mg) were hydrolyzed with 2 M TFA (0.5 mL) at 100C for 6 h (Colodel et al., 2017). After cooling, the samples were dried by nitrogen blowing at 50C and neutralized with NH2OH·HCl (10 mg), followed by an acetylation step with pyridine-acetic anhydride (1:1 v/v, 1.0 mL) at 90C for 30 min. The resulting alditol acetates were extracted by chloroform (5 mL) for further analysis. The samples (1 μL) were analyzed in a GC system equipped with quadrupole MS (7890A-

5975C, Agilent, CA, USA). The fitted HP-5 column was subjected to the following procedure: 160 to 210C at 2C/min, 210-240C at 5C/min, then held for 10 min at 240C (Liang et al., 2012). For identification and quantification, mixtures of neutral sugar standards were used for calibration. The sugar molar ratios were calculated based on content in weight according to the formulas: R1=GalA/(Fuc+Rha+Ara+Gal+Xyl), R2=Rha/GalA, R3=(Ara+Gal)/Rha, R4=Gal/Rha, HG = (GalARha)/(GalA+ total sugar), RG-I=2Rha+Ara+Gal/(GalA+ total sugar) (Kpodo et al., 2017). 2.3.3 DM

Journal Pre-proof DM was determined by the method introduced by Ogutu et al., (2017). In brief, pectic fractions (100 mg) was wetted with ethanol (5 mL) in a 250 mL conical flask. Sodium chloride (1g), deionized water (100 mL) and six drops of phenolphthalein (0.1% in ethanol) were added. The mixture was stirred until pectic sample was fully dissolved, and titrated with 0.1 M NaOH until the colour changed to light pink (Titration A). Then, 0.25 M NaOH (25 mL) was added and mixed thoroughly. Next, 0.25 M HCI (25 mL) was added after standing for 30 minutes. The mixture was titrated again with 0.1M NaOH (Titration B). DM (%) =

𝑚𝑒𝑞 𝑇𝑖𝑡𝑟𝑎𝑡𝑖𝑜𝑛 B100 meq Titration A + meq Titration B

(1)

2.3.4 Degree of acetylation (DAc) DAc, defined as the molar ratio of acetyl groups to uronic acid, was determined by a Magazyme kit (K-ACETRM, Ireland) as descripted by Njoroge et al (2014). Prior to acetic acid determination, pectic fractions were hydrolyzed with 2 M NaOH (500 μL) at 25oC for 1h, and the hydrolyzation was stoped by adding 2 M HCI and incubating at 25oC for 15 min. Hydrolyses of the pectic samples were performed in triplicate. 2.3.5 Fourier transform-infrared (FT-IR) spectroscopy An FT-IR (Nicolet 6700, Thermo Fisher Scientific, Watltham, USA) was employed to characterize the spectra of the polysaccharide fractions. In brief, polysaccharide samples (1 mg) were pressed into KBr-sample (100 mg) discs and scanned at a resolution of 4 cm-1 from 64 scans and the range of 4000 to 400 cm-1. The spectroscopy was recorded by Omnic (Version 8.2, Thermo Fisher Scientific Watltham, USA). 2.3.6 Thermal analysis The thermal properties of the polysaccharide fractions were investigated by differential scanning calorimetry (DSC204F1, Netzsch, Selb, Germany) according to described method (Wang, Chen, & Lu, 2014). Dried sample (3 mg) was sealed in a standard aluminum crucible, then heated from 40C to 250C at a rate of 10C/min in dynamic nitrogen atmosphere. An empty standard aluminum crucible was used as reference. 2.3.7 Nanostructure analysis The nanostructure of polysaccharide fractions was analyzed by atomic force microscopy AFM (Nanonavi E-Sweep, HITACHI, Tokyo, Japan). About 10 μL of polysaccharides (0.1 μg/mL) was

Journal Pre-proof dropped onto to a stripped mica sheet surface and dried by nitrogen purging at room temperature. A Si3N4 probe was used for scanning at response frequency of 0.1-150 Hz, vertical distances of 0.1 nm and horizontal distances of 5 nm (Zhang et al., 2017). 2.4. Emulsifying properties Emulsifying capacity (EC) and emulsifying stability (ES) were evaluated according to the methods described in Liang et al., (2015). In brief, polysaccharide fractions were dissolved into deionized water to achieve a concentration gradient (0.3%, 0.6%, 0.9% and 1.20%, w/v). Then, the pectin solution (4 mL) was mixed with olive oil (4 mL) and subjected to homogenization at 20000 rpm for 90s in a rotor homogenizer (T18 digital ultra-turax, IKA, Germany) to prepare emulsions. The volume of the prepared emulsion was recorded as Wv. The emulsions were subjected to centrifugation at 1315  g for 5 min and the volume of emulsified layer was recorded as ELv. Then, the emulsions were kept at 80C for 1 h, followed by centrifuged at 1315  g for 5 min, and the volume of emulsified layer was recorded as ELSv. The emulsifying capacity (EC) and emulsion stability (ES) are calculated according to the following equations (Schmidt, Schutz, & Schuchmann, 2017): EC (%) = (ELv/Wv)  100

(2)

ES (%) = (ELSv/ELv)  100

(3)

Deionized water was used as the control. SPI, a commercial emulsifier from a natural source, was used for comparison. The emulsifying properties of polysaccharide fractions at a concentration of 0.3% to 1.2% (w/v) and pectic fractions at pH value of 2.0 to 10.0 were also investigated. 2.5. Surface tension measurement The ability of polysaccharide fractions to lower surface tension was investigated by Mobile Surface Analyzer (DSA 100, KRüSS, Hamburg, Germany). In brief, the polysaccharide fractions were dispersed in distilled water at a concentration of 0.6% (w/v), pendant drop method was employed to determine the surface tension. 2.6. Rheological properties To further understand the emulsifying mechanism of polysaccharide fractions, the rheological properties of emulsions prepared with polysaccharide fractions were investigated according to the method reported by Yang et al., (2018) with some modifications. Dynamic oscillatory measurements were performed by a rheometer (MCR 302, Anton Paar, Austria) equipped with parallel plate (40 mm and 0.5 mm of gap). In brief, 1.5 mL of sample was loaded onto the sample plate and adjusted to the

Journal Pre-proof designated value. Frequency sweep (0.1-10 Hz) were recorded with a strain 1% at 25C (which was confirmed by strain sweep within the linear viscoelastic region). Temperature ramp was also performed within the temperature range of 25 to 80C at a rate of 1C/min and at a shear rate of 1 s-1. 2.7. Microscopic morphology of the emulsions The size of microscopic droplet was used to characterize the microscopic morphology of emulsions. The droplet size was obtained by a Laser Partical Analyzer (S3500, Mircrotrac Inc., USA) with wet measurement (Ye et al., 2018). The refractive index of water and olive oil were set as 1.333 and 1.467, respectively. The volume-average droplet size (D4,3) was calculated based on Mie theory (Chen et al.,

2016). 2.8. Statistical analysis Statistical analyzes were performed by SPSS (19.0, SPSS Inc, Chicago, IL, USA). Results were expressed as mean ± standard deviation of at least 3 repeated experiments. Significant differences were determined by One-way analysis of variance (ANOVA) conducted by Tukey test (p< 0.05). 3. Results and discussion 3.1. Characterization of polysaccharide fractions Commercial pectin of complex polysaccharides of uniform quality is usually obtained from food waste by single extraction. In order to take full advantage of tomato pomace as a rich source of pectin that could be used as emulsifier, four fractions of polysaccharides in the extracted pectin were assessed. First, polysaccharides from BTP were separated as alcohol-insoluble residue (AIR) and further isolated as pectin fractions (WEP, CEP, NEP) and HF. The yield of AIR from BTP was 50.57% (w/w) with a water content of 21.98% (w/w). Results showed that WEP and NEP were most abundant with relative weight contributions to total AIR at 24.02% and 22.01%, respectively, while a small fraction of CEP was observed (Table 1). In addition, a high weight percent yeild of HF (16.76%, w/w) was obtained. The total weight-based pectin fractionation yields from AIR and BTP were 54.25% and 27.4%, respectively. The yield based on BTP corresponded with the previously reported values in red tomato pomace (15.2-36.0%) (Grassino et al., 2016). The UA content, neutral sugar concentrations, DM and protein content were reported in Table 1. As expected, UA was the dominate component in the pectic fractions while no UA was detected in the HF. The weight-based content of GalA in pectin fractions (w/w) followed the order of: CEP (86.28%) > WEP (73.85%)> NEP (73.23%). Since HG chains can be cross-linked by divalent ions and the uronic acids are

Journal Pre-proof prone to migrate to the water-soluble fraction, the pectin extracted by chelating agents (CEP) and water (WEP) usually has high uronic acid (Colodel & de Oliveira Petkowicz, 2019; Gawkowska, Cybulska, & Zdunek, 2018). These results were similar to the pectin obtained by nitric acid from passion fruit peel (82.3%) and cacao pod husks (72.0%) (Gawkowska, Cybulska, & Zdunek, 2018; Seixas et al., 2014), but higher than pectin obtained by water from citrus (52.33-68.88%) and apple (20.67-44.37%) expressed in GalA (Wang, Chen, & Lu, 2014). The UA in pectin is mainly GalA and minor amounts of glucuronic acid, and the UA content is commonly representative for GalA content in pectin (Kermani et al., 2015). As the requirement of commercial pectin (at least 65% GalA) according to JECFA (2009), the BTP will be a potential commercial pectin source. As for neutral sugar composition, arabinose and galactose were the predominant neutral sugars in WEP, indicating the contribution of RG-I in the WEP fraction. In addition, large amount of glucose was observed, which suggested that WEP might bind to non-pectin polysaccharides such as hemicellulose, cellulose (Meersman, et al., 2017). Arabinose and rhamnose were the main neutral sugars in the NEP fraction, which could be related to the high degree of branching of RG-I from the linear structure. To better interpret the structural characteristic, molar ratios (R1-R4) and domains (HG, RG-I) based on sugar content were calculated (Houben et al., 2011; Kpodo et al., 2017). R1 represented the ratio of backbone sugar GalA to side chains sugars (Rha, Ara and Gal) and indicated the linearity of pectin. R2 revealed the contribution of RG to total pectin branching. R3 and R4 represented the ratio of side-chain sugars to RG branch sugar Rha and were reckoned as an estimate of the length of RG-I and RG branching, respectively (Denman & Morris, 2015). High values represented highly branched structures, which usually exhibited higher degree of esterification and weaker bonding with cell wall materials. Results showed that the CEP fraction contained the most linear pectin and had the least RG-I branching, while the NEP fraction had the highest proportion of RG-I but the shortest RG-I branches as reflected by R4. Both WEP and CEP exhibited low contribution of RG as indicated by R2, which was consistent with similar results reported in coca pulp (Meersman, et al., 2017). Our observations also aligned with the results on HG and RG-I. Furthermore, the large proportions of Glc and Ara indicated possible existence of arabinoxylan and cellulose residue in HF. FT-IR spectra of polysaccharide fractions were shown in Fig.1. The peak of 3426 cm-1 corresponded to the O-H stretch attached to the GalA backbone or that of other neural sugars. The peak of 2935 cm-1 represented the C-H vibrations caused by CH, CH2 and CH3 (Monsoor, Kalapathy, & Proctor, 2001). The

Journal Pre-proof most significantly absorption peaks located at 1731 cm-1 and 1631 cm-1 were also observed, which could be supposed to be caused by C=O stretch of methyl esterified group and COOH groups, respectively (Kpodo et al., 2017). The low-resolution of the peak at 1731 cm-1 and the acromion observed at 1631cm-1 were similar with the spectra pectin from red tomato pomace (Grassino et al., 2016). That might be attributed to the protein, which typically occur at around 1670 cm-1 (amide I) and 1588 cm-1 (amide II) (Wang, Chen, & Lü, 2014). The results were in agreement with the results of protein (Table 1). With protein in WEP, HF and NEP fraction, the peaks at 1631 cm-1 were with poor shape. The peak at 1403 cm-1 might be related to bending of CH2OH and -CH3CO stretching (Kpodo et al., 2017). In the pectin fingerprint region (1200-900 cm-1), the peaks at 1105 and 1012 cm-1 were assigned to vibrations of glycosidic bonds (-C-O-C-) and pyranoid rings (C-C). It has to be noted that spectral peaks in the region 1105-1012 cm-1 are commonly attributed to the vibrations of monosaccharide. The result suggested similarities in monosaccharide composition between WEP, CEP and NEP, while the spectra were significantly different with HF. The peaks at 831,704 and 619 cm-1 were related to skeletal C-C bond deformation vibrations and were similar in the pectic fractions and HF. Since the peak at 1045 cm-1 was found in spectrum of the HF fraction, which is identified as characteristic of hemicellulose, and usually signs of xylan, glucan, mannan, corresponding to the characteristic absorption of C–O deformation in primary alcohol (Du et al., 2016; Wang, Yuan, Ji, & Li, 2013), the HF was a different type of polysaccharides in BTP. The DM of pectic fractions from BTP were in the range of 59.70% - 68.02%, suggested that WE, CEP and NEP were HMP. It is known that pectin could be demethoxylated via acid and alkaline treatments and are sensitive to thermal degradation (Einhorn-Stoll et al., 2019). The DM of NEP was slight lower than those of WEP and CEP, potentially due to extraction condition of NEP at room temperature in a mild alkaline solution. DAc are reported to act as an effective anchor in emulsions and a positive role in formation of emulsifying droplets (Ngouemazong et al., 2015). WEP (14.07) and NEP (19.56%) could be considered as high acetylated pectin (DAc > 8%) and the DAc was also higher than that of apple pectin (5%) and commercial citrus pectin (1.4%-1.6%) (Chan & Choo, 2013; Yang, Mu, & Ma, 2018). However, the CEP had a low DAc (2.37%), the strength of complexation might be specific to the smooth part of pectin, which could be lowly acetylated (Willats et al., 2001). In general, the sequential extraction of BTP successfully generated polysaccharide fractions that contained different chemical structures.

Journal Pre-proof 3.2. Thermal properties The thermodynamic properties of polysaccharide fractions were examined by DSC from 40oC to 300C (Fig. 2). Obvious endothermic and exothermic peak appeared in the thermograms of all fractions. The melting temperature (Tm), degradation temperature (Td), melting enthalpy (ΔHm) and degradation enthalpy (ΔHd) were listed in Table 2. At the endothermic stage, the transition of GalA is an essential step. As the conformation of GalA transform from a stable 4C1 chair into the inverse 1C4 chair, the exposure of hydroxyl groups is reduced, and the hydrogen bonds among GalA units or water is also destroyed. To complete the transition, the water in pectin sample has to be removed. Thus, higher Tm and ΔHm suggests the amount of energy required to remove water from pectin fractions is more (EinhornStoll & Kunzek, 2009; Li et al., 2019). Tm of pectin fractions ranged from 180.04 to 196.44C following the order of: NEP>CEP>WEP. Tm of pectin fractions from BTP was significantly higher than that of pectin from apple (86.46C-136.36C) and citrus (105C to 137.18C), with similar results also observed regarding △Hm (Wang, Chen, & Lu, 2014). The results indicated that the energy needed to break hydrogen bonds among GalA units or water in pectin from BTP is higher than that from apple and citrus. Considering the pectic fraction were HMP, higher Tm of CEP compared to that of WEP could potentially be attributed to the higher proportion of HG in CEP, which could be ascribed to the higher galacturonic acid proportions (83.78 and 70.99%, respectively). The higher content of GalA in pectin means that more energy is needed to complete the transitions. The even higher Tm of NEP might be attributed to the neutral sugar attached to the RG-I regions and the hydrogen bond among the chains. The exothermic peak was resulted from the degradation of pectin during heat processing (Godeck, Kunzek, & Kabbert, 2001). As shown in Fig. 2, although WEP showed the highest △Hd (270.97C), the △Hd of WEP was significantly lower than those of CEP and NEP. Accordingly, Td of the pectin is much affected by the DM and hydrogen bonds, the pyrolyzation starts as a random breakdown of the glycoside bond, followed by the decomposition of main chains (Einhorn-Stoll & Kunzek, 2009; Mudgil, Barak, & Khatkar, 2012). The results observed in the WEP fractions could be attributed to the low GalA content and the protein content in the fraction, whose NH2 groups provided more hydrogen bond acceptors. The thermal properties of HF fraction were significant different from the pectin fractions, Tm was much lower while the Td was higher, indicating that HF exhibited poor hydration ability but good thermal stability. 3.3. Nanostructure properties of polysaccharide fractions

Journal Pre-proof The AFM results of polysaccharide fractions from BTP were shown in Fig. 3. The morphologies of the polysaccharide fractions were significantly different. The light features represented polymers which are usually highly present in three-dimensional image and can be attributed to glucose in neutral sugars (Li et al., 2018). Pectin chains were found to have self-assembled network and clumps in the WEP solution, including short chains of length 2 nm to 10 nm and polymers of width 42 nm to 90 nm that were sporadically distributed. In the CEP fraction, more short and straight chains were founded which coincided with the linearity of pectin as reflected by the sugar composition. In addition, polymer content in CEP was much higher than those of all other fractions. In the scan of NEP, linear strands and branched structures were found, however, polymers and the associated self-assembly and aggregation phenomenon were absent, which further highlighted the difference between NEP and the CEP and WEP fractions. Furthermore, only clumps and polymers were found in the HF fraction. Taken together, the polysaccharide fractions exhibited different nanostructure morphologies, observations from images of the pectic fractions were in agreement with the structural analyses based on neutral sugar ratios, demonstrating that the extraction process was efficient in separating pectin of different properties. 3.4. Emulsifying ability of pectin The effects of concentrations and pH on the emulsifying properties of pectic fractions were investigated and the results were shown in Fig. 4. As the pectin solutions were viscous and hard to disperse, the emulsions were prepared at the highest concentration of 1.20% (w/v). The EC of pectic fractions ranged from 53.17% to 82.46%, which was much higher than that of commercial citrus pectin (44.87%), apple pectin (45.34%), and potato pectin extracted by acid (44.97%-47.71%), while being lower than the EC of pomegranate pectin (96.70%) (Yang, Mu, & Ma, 2018; Yang et al., 2018; Yapo et al., 2007). The EC of WEP, CEP and NEP rose with increasing concentration. The best EC of 79.38% was observed at concentration of 0.90% for WEP, 75.71% at concentration of 1.20% for CEP and 82.46% at concentration of 0.90% for NEP. ECs of all the pectic fractions at optimal concentrations were comparable to that of SPI. Based on our results, we deduced that higher concentration of pectin could afford more droplet surface to facilitate the formation of emulsify droplet. Interestingly, the emulsifying capacity of the HF fraction was independent from the concentrations of HP and was relative lower than those of other pectin fractions. The hydrophobic groups in pectin chains are known to be important to the emulsifying capacity, with higher content of hydrophobic groups usually indicating better emulsifying capacity (Chen, Fu, & Luo, 2016). Moreover, the stability of the prepared emulsion, as

Journal Pre-proof defined by the ratio of layer volume after incubation at 80C, was also evaluated. As shown in Fig. 4b, ES of WEP and NEP fractions increased significantly as pectin concentration increased from 0.3% to 0.9% and followed by a slight decrease, while the ES of CEP and HF exhibited a relatively stable trend despite being much lower than the WEP and CEP fraction, as pectin concentration increased from 0.3% to 1.2%. Since polysaccharides consist of aldonic acids, pectin solution is vulnerable to changes in pH. Besides the dispersing morphology, certain reactions of pectin occurred as pH varies, such as degradation and β-elimination, which could affect the emulsifying properties of pectin (Einhorn-Stoll et al., 2019). As shown in Fig. 4c, the emulsifying capacity of CEP and NEP increased as the pH increased from 2.0 to 8.0 and subsequently decreased as the pH increased to 10.0. However, the emulsifying capacity of WEP reached 74.86% at pH 6.0 then decreased sharply as the pH increased to 10.0. Results of WEP were consistent with those reported by Yang et al., (2018), which suggested a similar same trend of emulsifying capacity as pH increases. The emulsions prepared by WEP, CEP and NEP exhibited obvious phase separation after storing at room temperature for 1 h at pH 12.0, which could be attributed to the conformational change and molecular interactions induced by pH. Pectin is an anionic polysaccharide, whose pKa is usually higher than that of galacturonic acid, most abundant units of pectin, alone (3.5), (Chang, McLandsborough, & McClements, 2011). As the pH gets closer to the pKa, conformation of molecules is mostly relaxed and is largely dependent on the concentration and steric hindrance within the solution. At pH lower than the pKa, pectin becomes less negatively charged, which indicates that intramolecular repulsions of pectin are weaker, resulting in a more compact conformation of the anchored structures. In addition, the formation of hydrogen bond with water is also stronger, which could explain the poor emulsifying capacity of pectin fraction at pH of 2.0. At pH higher than the pKa, the electrostatic repulsion provided by charged groups would prevent the formation of intermolecular hydrogen bond, pectin in chain form exposed more anchored groups and tail to coat the oil droplets. The electrostatic repulsion also prevented the coalescence and flocculation of coated oil droplets (Ngouemazong, et al., 2015). As the pH values deviate farther from the pKa, the repulsion force becomes too strong for anchoring and de-polymerization of pectin, which ultimately results in poor emulsifying capacity of pectin faction at pH 10.0. The differences in pH values at which pectin fractions demonstrated the best capacity emulsifying could be attributed to their different pKa values. The WEP fraction could have a lower pKa than CEP and NEP. As the pH reached 6.0, the status of pectin was at the suitable conformation

Journal Pre-proof for emulsion to occur. Unlike the emulsifying capacity, emulsify stability of pectin was relatively stable (Fig. 4d) except for that of WEP. 3.5. Emulsifying mechanism In order to better understand the emulsifying mechanism of pectin, the surface tension of pectin at the oil-water interface, the droplets size and the rheological properties of emulsion were investigated. The emulsions were prepared by dissolving pectin fractions in distilled water at the concentration of 0.6% (w/v). As shown in Table 3, the surface tension was reduced by polysaccharides fractions following the order of: WEP=HF>NEP>CEP. Nevertheless, the results were higher than that of pomegranate peel pectin (68.63 to 59.14 mN/m) but similar to that of pumpkin pectin (62.5 to 52.5 mN/m) (Cui & Chang, 2014; Yang, et al., 2018). Although low surface tension facilitated droplets formation, it was not the only element that could affect the emulsifying activity of pectin. The role of protein remained in pectin could not be ignored. Reports had shown that deproteinized polysaccharides, such as fenugreek gum and Acacia gum, exhibit higher surface tension (Brummer, Cui, & Wang, 2003; Dickinson, 2003). Considering the selectivity of the extraction process and that many of the proteins in tomato pomace are water-soluble (Alancay, Lobo, Quinzio, & Iturriaga, 2017), the residual proteinaceous matters in WEP and HF (Table 1) may play an important role in lowing surface tension. Results of emulsifying capacity for CEP and NEP further confirmed the surface tension lowering capacity of these pectins, and the higher content of RG-I segments with longer chains in NEP might be responsible. The emulsions stabilized by polysaccharide fractions were relatively homogeneous as reflected by particle size distribution (Fig. 5). The D4,3 of emulsions were significantly smaller than the control group (Table 3). The results indicated that no aggregation of polysaccharide fractions occurred at the concentration and the emulsions were more stable against oil droplet coalescence compared to the control group. Of note, the emulsion prepared by CEP exhibited the smallest droplet size, which might be related to the short chains and little branching of RG-I, that are usually associated with small molecular weight and domains for oil droplets. The watersoluble protein and high proportion of RG-I could be responsible for the bigger droplet size in emulsions prepared by WEP and CEP, respectively, while the small droplet size of emulsions prepared by HF might be related to the surface tension lowering ability. The rheological properties of emulsion prepared by polysaccharide fractions were also investigated (Fig. 6). The storage modulus (G') and loss modulus (G'') as a function of sweep frequency were shown in Fig. 6a and Fig. 6b. For emulsions prepared by CEP, NEP, HF and control group, the G'> G'' indicated

Journal Pre-proof that the emulsions were elasticity-dominated, which was similar to other pectin solutions (Yuliarti & Othman, 2018). The G''/ G' of emulsions followed the order of: WEP>CEP>NEP>control>HF. These results suggested that there was no rearrangement of the droplets between the control group and the emulsions prepared by the CEP, NEP and WEP. In addition, the decrease in emulsions droplets size, static repulsion and steric hindrance might be the dominant force for the formation of emulsion, a phenomenon that was very different from emulsions prepared by proteins, which showed that the G'> G'' caused primarily by the covalent and hydrogen bonds among droplets (Xi et al., 2019). Furthermore, the stability of emulsion versus temperature was also studied. As shown in Fig. 6c, the viscosity of all emulsions decreased as the temperature increased from 20C to 80C, and the viscosity of emulsions prepared by pectic fractions was higher than that of the control group. Interestingly, the viscosity of emulsion prepared by WEP increased at temperatures ranged from 25C to 37.5C, which might be attributed to the intermolecular interactions between WEP and proteins. The viscosity of emulsion prepared by NEP decreased sharply despite being higher than those prepared by WEP and CEP, indicating that emulsions prepared by NEP were more sensitive to temperature and exhibited better emulsion stability versus temperature. However, as the temperature increased to 80C, the viscosity of emulsions showed little influence on stability. Furthermore, the difference among emulsions might be attributed to structural differences. 4. Conclusions In the present study, pectin in black tomato pomace was subsequently extracted using water, chelator, sodium carbonate and alkali, and then separated into WEP, CEP, NEP and HF fractions. The pectic fractions demonstrated emulsifying capacity and emulsifying stability, which possible could rely upon the presence of protein, acetyl groups and affected by the HG and RG-I segments. The emulsions fabricated by pectic fraction exhibited different stability at various pectin concentrations and pH values. The WEP, CEP and NEP fractions resulted in stable emulsions at the highest concentration of 1.2% (w/v). The emulsion capacity of WEP, CEP and NEP increased as the concentration increased. The NEP fraction showed the best emulsion stability, while the emulsion stability of CEP was independent from concentration. The pectin fractions exhibited good emulsion capacity at pH values range of 4.0-8.0. The different results of CEP and NEP might attribute to the structure traits (e.g. DM, DAc and HG/RG-I segments), while the good emulsifying properties of WEP might be related to the proteins remained in the fraction. Furthermore, the pectic fractions of BTP exhibited good surface tension lowering properties

Journal Pre-proof and formatted small droplets size of emulsions. Finally, increased viscosity by pectin fractions played an important role in enhancing the emulsifying properties. Results from this study is of special significance for the development of pectin from BTP as an effective emulsifier.

Journal Pre-proof Acknowledge This work was financially supported by funding sponsored by National Key R&D Program of China (No. 2016YFD0400206), Shanghai Innovation Action Plan (No. 18DZ2200500) and the project from the Key Laboratory of Synthetic and Biological Colloids, Ministry of Education, Jiangnan University (No. JDSJ2017-09). The authors were grateful for the equipments supported by the Instrumental Analysis Center of Shanghai Jiao Tong University.

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Journal Pre-proof Food Hydrocolloids Conflict of Interest Statement

Titile:Emulsifying properties of pectic polysaccharides obtained by sequential extraction from black tomato pomace Authors: Wei Zhang, Xie Fan, Xinzhe Gu1, Shengxiang Gong, Jinhong Wu, Zhengwu Wang, Qiang Wang, Shaoyun Wang The authors declare that: 1) All authors of this manuscript have directly participated in planning, execution, or analysis of this study. 2) The contents of this manuscript have not been copyrighted or published previously. 3) There are no directly related manuscripts or abstracts, published or unpublished, by any authors of this manuscript. 4) The contents of this manuscript are not now under consideration for publication elsewhere. 5) The contents of this manuscript will not be copyrighted, submitted, or published elsewhere while the manuscript is under consideration by food hydrocolloids. 6) All the financial support was provided in the manuscript.

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Fig. 1. FI-IR spectra of polysaccharide fractions

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Fig. 2. DSC thermograms of polysaccharide fractions.

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(a)

(b)

(c)

(d)

Fig. 3. The nanostructured morphologies of polysaccharide fractions from BTP. (a) WEP (b) CEP (c) NEP (d) HF. Scan area: 1.001.00 μm2.

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Fig. 4. Emulsifying properties of polysaccharide fractions from BTP at various concentrations and pH. a: EC at different concentrations (0.2% to 1.2%); b: ES at different concentrations (0.2% to 1.2%); c: EC at different pH values (2.0 to 10.0); d: ES at different pH values (2.0 to 10.0).

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Fig. 5. Distribution of particle size of freshly prepared emulsions stabilized by polysaccharide fractions.

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Fig. 6. The rheological behaviors of emulsions prepared by different pectic fractions. a: storage modulus of emulsions at frequency range of 0.1 to 10 Hz. b: loss modulus of emulsions at frequency range of 0.1 to 10 Hz. c: apparent viscosity of pectic fractions at the temperature range of 25C to 80C.

Journal Pre-proof Highlights:



Four polysaccharide fractions were separated from black tomato pomace and their emulsifying properties were investigated.



WEP and NEP exhibited good emulsifying properties at the concentration of 1.2% and a pH range of 4.0-8.0.



Protein in WEP was responsible for the good emulsifying capacity.



Structural difference was responsible for emulsifying properties distinction of CEP and NEP.

Journal Pre-proof Table 1 Average weight-based fractionation yields, sugar composition and structure character reflected by sugar molar ratios of WEP, CEP, NEP and HF from AIR. WEP Yield in AIR (%) Neutral sugar (mg/g AIR) Fuc Ara Rha Gal Glc Xyl Man Total pectin related sugar (mg/g AIR) UA (mg/g AIR) Linearity R1* Contribution of RG R2* R3* Branching of RG R4* HG (%) RG-I (%) DM (%) DAc (%) Protein (mg/g AIR) a-d:

24.02 ±

1.47a

CEP 8.22 ±

0.43c

NEP 22.01 ±

HF

0.96a

16.76 ± 0.85b

6.12 ± 0.49a

2.44 ± 0.22c

4.06 ± 0.07b

0.35 ± 0.01d

11.80 ± 0.26a

1.44 ± 0.13c

13.65 ± 1.21a

3.80 ± 0.10b

1.64 ± 0.00b

0.36 ± 0.03c

17.63 ± 1.38a

ND

6.60 ± 0.32a

1.56 ± 0.14c

3.78 ± 0.68b

1.81 ± 0.08c

24.83 ± 0.55a

4.00 ± 0.26d

7.44 ± 1.34c

12.43 ± 0.57b

0.03b

0.03c

0.11a

0.46 ± 0.02c

1.80 ±

0.71 ±

5.93 ±

7.60 ± 0.14a

0.60 ± 0.01c

2.92 ± 0.05b

2.93 ± 0.13b

60.39 ± 4.26a

11.11 ± 0.69c

55.40 ± 2.33b

NC

8.26a

2.17c

9.23b

NC

170.54 ±

69.87 ±

151.87 ±

5.03

8.99

2.76

NC

0.01

0.01

0.14

NC

0.05

0.03

0.16

NC

3.67

3.95

0.20

NC

70.44

84.07

60.08

NC

10.97

5.32

29.08

NC

68.02 ±

2.32a

14.07 ±

1.54b

11.52 ±

1.07a

61.45 ± 1.87b 2.37 ± 0.75c ND

59.70 ±

1.19c

NC

19.56 ±

1.46a

NC

0.00c

9.46 ± 0.85b

2.78 ±

Different letters indicated significant deference (p< 0.05) at the same row. NC: Not calculated.

Journal Pre-proof Table 2 Thermal properties of polysaccharides fractions determined by DSC. Fractions

Tm (oC)

△Hm (J/g)

Td (oC)

△Hd (J/g)

WEP

180.04

300.71

270.97

2.15

CEP

190.23

348.07

244.12

94.37

NEP

196.44

314.46

251.92

116.54

HF

115.32

186.78

257.66

124.36

Journal Pre-proof Table 3 The surface tension and droplets size of emulsions prepared by polysaccharides fractions Groups Control WEP CEP NEP HF *:

D4,3 (μm) * 112.6 ±

Surface tension (mN/m)

3.43a

68.50 ± 1.87a

93.91 ± 1.88b

46.30 ± 0.93d

86.98 ± 0.85d

55.84 ± 1.62b

93.74 ± 1.11b

50.93 ± 1.57c

90.10 ± 0.79c

47.83 ± 1.45d

Mean volume-weighted diameter.

a-d:

Results were expressed as mean value ± standard deviation. Values were compared with significant

difference within the same columns (p<0.05).