Emulsion stability of sugar beet pectin increased by genipin crosslinking

Food Hydrocolloids 101 (2020) 105459

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Emulsion stability of sugar beet pectin increased by genipin crosslinking Jiawei Lin a, Shujuan Yu a, b, *, Chao Ai a, Tao Zhang a, Xiaoming Guo a, ** a b

School of Food Sciences and Engineering, South China University of Technology, Guangzhou, 510640, China Overseas Expertise Introduction Center for Discipline Innovation of Food Nutrition and Human Health (111 Center), Guangzhou, 510640, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Sugar beet pectin Genipin Crosslinking Emulsion Emulsifying stability

This work aimed to enhance the emulsifying properties of sugar beet pectin (SBP) by adopting a crosslinking strategy that covalently bridged the amino groups (–NH2) of lysine residues of the proteinaceous moiety using genipin (GP) as the crosslinker. Compared to control-SBP (C-SBP), GP crosslinked SBP (G-SBP) was larger in molecular weight (from 3.04 � 105 g/mol to 1.31 � 106 g/mol) and mean radius of gyration (from 34.8 nm to 39.2 nm), and showed a more compact conformation, as supported by analysis of high-performance size exclusion chromatography coupled with multiple angle light scattering (SEC-MALLS) and atomic force micro­ scopy (AFM). G-SBP was featured by a new absorbance peak at ~595 nm, resulting in a distinct blue color of the aqueous solution. G-SBP suffered a slight decrease in interfacial properties and emulsifying activities in com­ parison to C-SBP, leading to a lower specific surface area and a larger volume-weighted mean diameter (d4,3) for the fresh emulsion. Nevertheless, the G-SBP stabilized emulsion was more stable than the C-SBP stabilized emulsion during storage for 21 d with d4,3 increasing from 0.541 μm and 0.425 μm to 0.625 μm and 1.66 μm for G-SBP and C-SBP, respectively. The improved emulsifying stability of G-SBP can be ascribed to the combination of the following two aspects: 1) G-SBP exerted a lager viscosity-enhancing effect to slow down droplet collision and flocculation; 2) the carbohydrate moiety of G-SBP formed a dense and compact hydrated layer providing strong steric stabilization for the oil droplets to resist flocculation and coalescence. Altogether, this work dem­ onstrates that GP is a favorable agent to crosslink the side chains of SBP and modify its emulsification performance.

1. Introduction

upper gastrointestinal tract (such as mouth and stomach) and di­ gestibility by microorganism in colon, pectin (e.g. from sugar beet and okra) stabilized emulsions are a type of vehicles for delivering acid sensitive bioactive compounds and probiotics to target sites of interest (Alba, Lmc, & Kontogiorgos, 2016; Ghori, Alba, Smith, Conway, & Kontogiorgos, 2014). However, SBP faces the drawback of insufficient emulsifying stability within 30 d or even shorter (Jung & Wicker, 2012; Williams, Sayers, Viebke, Senan, & Boulenguer, 2005), which severely restricts its application. Structural alteration seems necessary for SBP to overcome its intrinsic limitation of undesirable emulsifying properties to cater mar­ ket’s requirement, particularly the emulsifying stability. For this pur­ pose, several methods based on polymerization or polysaccharideprotein conjugation have been developed, such as oxidative cross­ linking of side chains of SBP via its feruloylated groups (Jung & Wicker, 2012; Zhang et al., 2015), covalent conjugation of endogenous proteins to galacturonate moieties of SBP at the C1 position of the terminal end

Extensive studies on the unique emulsifying capacity of sugar beet pectin (SBP) have demonstrated that its emulsifying activity is pre­ dominantly related to the hydrophobic proteinaceous moiety and ferulic acid which may act as anchors facilitating the adsorptivity of SBP to oily phase (Siew & Williams, 2008), whilst the long-term emulsifying sta­ bility is maintained by hydrophilic polysaccharide chains protruding into the continuous phase for steric stabilization and increasing viscosity (Funami et al., 2011). SBP has the advantages in stabilizing an oil-in-water emulsion at a relatively lower dosage than other poly­ saccharide emulsifiers such as gum arabic and soybean soluble poly­ saccharide (Nakauma et al., 2008). In addition, SBP shows better emulsifying properties in acidic emulsions around the protein isoelectric point as compared with protein emulsifier, which is mainly stabilized by electrostatic repulsion (Mcclements, Bai, & Chung, 2017; Surh, Decker, & Mcclements, 2006). Owing to the resistance against enzymolysis in

* Corresponding author. 381 Wushan, Guangzhou, China. ** Corresponding author. 381 Wushan, Guangzhou, China. E-mail addresses: [email protected] (S. Yu), [email protected] (X. Guo). https://doi.org/10.1016/j.foodhyd.2019.105459 Received 6 July 2019; Received in revised form 8 October 2019; Accepted 21 October 2019 Available online 24 October 2019 0268-005X/© 2019 Elsevier Ltd. All rights reserved.

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(Funami et al., 2011; Funami et al., 2008), coupling of whey protein isolate or bovine serum album to SBP via laccase and Maillard reaction (Hao et al., 2018; Qi, Xiao, & Wickham, 2017), in which SBP provided the end carbonyl groups or phenolic compounds like ferulic acid, tyro­ sine, cysteine and tryptophan as reactive sites. These methods endow modified SBP with stronger steric hindrance to prevent oil droplet from aggregation and flocculation. Additionally, non-covalent complexation induced by electrostatic and hydrophobic interactions is another method for modifying emulsi­ fying properties of SBP. For instance, by forming an electrostatic com­ plex with either bovine serum albumin or sodium caseinate, the proportion of SBP adsorbing on oil-water interface was increased significantly, concomitantly with improvement in emulsifying stability (Li, Fang, Al-Assaf, Phillips, & Jiang, 2012; Li, Fang, Phillips, & Al-Assaf, 2013). Genipin (GP), a hydrolyzed product of gardenoside isolated from Gardenia jasminoides, is a natural covalent crosslinking agent that crosslinks the primary amine groups (–NH2, from such as casein, gelatin and chitosan) to form a larger-sized polymer (Neri-Numa, Pessoa, Pau­ lino, & Pastore, 2017) with improved functionality like gelation and emulsification (Muzzarelli, 2009; Nickerson, Patel, Heyd, Rousseau, & Paulson, 2006; Wang et al., 2018). Chemically, GP spontaneously reacts with –NH2 at room temperature (Fujikawa et al., 1987) through two stages: 1) a fast reaction of nucleophilic attack by an –NH2 to C3 of GP, which results in the opening of the dihydropyran ring and subsequently the attack on aldehyde group by the preformed secondary amine; 2) at a slower reaction rate, a nucleophilic substitution of the ester group of GP to form an amide (Butler, Ng, & Pudney, 2003). In either case the crosslinking between GP and –NH2 is time-, temperature- and concentration-dependent. Normally, polymers crosslinked by GP show brilliant blue color and are advantageous for lower cytotoxicity and higher biocompatibility (Semenova, 2016; Sung, Huang, Huang, & Tsai, 1999), thereby having the potential to be used as a food colorant (Ramos-de-la-Pena, Renard, Montanez, Reyes-Vega, & Contreras-Esquivel, 2016). Although there are extensive studies on crosslinking between GP and hydrocolloids such as chitosan, protein (Mi, Sung, & Shyu, 2015; Wang et al., 2018), to our best of knowledge, the crosslinking between GP and SBP is still to be undertaken. Studies have shown that pectin of different origins usually contains small amounts of amino acids that have free primary amine groups, such as lysine, arginine and glutamine residues in SBP (Liu, Pi, Guo, Guo, & Yu, 2019; Siew, Williams, Cui, & Wang, 2008). Based on these findings, the reactivity of GP with –NH2 located in pectin chains provides a chemical basis for the reaction between GP and SBP. We speculated that the GP-crosslinked SBP might feature novel structures and emulsifying properties. For these purposes, the present work aimed to produce a new type of SBP crosslinked by GP with enhanced emulsification properties and to characterize the physicochemical properties of the resulting crosslinked SBPs. Also, relationships of structure-emulsifying perfor­ mance were discussed for G-SBP.

2.2. Crosslinking reaction of the SBP and GP In our preliminary experiments, reaction conditions including reac­ tion time, pH, amount of GP, and concentration of SBP were optimized (see Fig. S1). A 3.5% w/w SBP solution was prepared by dispersing the SBP into phosphate buffer (10 mM, pH 7.5) under magnetic stirring at room temperature overnight, and by adjusting the pH to 7.5 until the SBP was fully hydrated. A weighted GP powder was added to the SBP solutions, to reach a predesignated concentration of 10 mM, after which the resulting solution was stirred for 0–48 h at 25 � C. The reaction was terminated by adding 3 vol of 95% v/v ethanol to the solution. After standing for 3 h, precipitated pectins were separated by centrifugation (10,000�g for 10 min), washed with 75% v/v ethanol and 95% v/v ethanol, and dried at 45 � C for 12 h. SBP crosslinked by GP was named G-SBP. For a fair comparison, a control sample, named C-SBP, was prepared by treating SBP with the aforementioned treatments except GP crosslinking. Residual GP in the obtained G-SBP was determined ac­ cording to the method described in the literature (Bell� e et al., 2018) and the results showed nearly no GP was detectable (<0.1 μg/g G-SBP). 2.3. Ultraviolet spectroscopy UV spectrum of 0.1% w/w pectin solutions was recorded using a spectrophotometer in the wavelength range from 800 to 250 nm. The blue color was indicative for the reaction between GP and –NH2, which was responsible for the new peak with maximal absorbance at 595 nm (OD 595) (Fig. 1b). Taking advantages of the dependence of absorbance on reaction time (Park, Lee, Kim, Hahn, & Paik, 2002; Zhang et al., 2015), OD 595 was used to monitor the kinetic process of the cross­ linking reaction (Lee, Lim, Bhoo, Paik, & Hahn, 2003; Silva, Arnaud, Carvalho, & Fr�ed�eric, 2014). 2.4. Chromatography The amino acid composition of C- and G-SBPs was determined by the HPLC method described in our previous study (Liu et al., 2019). Weight-average molecular weight (Mw), z-average root mean square radius of gyration (Rg) and mass recovery (MR) were analyzed by size exclusion chromatography (SEC) coupled with 2998 photodiode array detector (PAD) (Waters Corp., Milford, USA), multi-angle light scat­ tering (MALLS) detector (DAWN HELEOS, Wyatt Corp., USA), 2414 refractive index (RI) detector (Waters Corp., Milford, USA). Prior to analysis, normalization of light scattering and alignment of all detectors were carried with bovine serum albumin (BSA) obtained from SigmaAldrich (St. Louis, USA). After filtering through 0.45 μm filters, 100 μL 0.1% w/w pectin solution (prepared using the eluent as the solvent) was passed through three columns, an Ultrahydrogel Guard (40 mm � 6 mm), an Ultrahydrogel 2000 (300 mm � 7.5 mm) and an Ultrahydrogel 1000 Column (300 mm � 7.5 mm) (Waters Corp., Mil­ ford, USA), at a flow rate of 0.6 mL/min, using 100 mM NaNO3 solution as eluent with 0.05% Na3N as preservative. Calculation was carried out by ASTRA software (version 6.1.1.17, Wyatt Corp., USA) and a dn/dc of 0.135 mL/g was taken from the literature (Williams et al., 2005).

2. Materials and methods 2.1. Materials Sugar beet pectin (SBP) is homemade using heat-acid extraction previously described (Guo et al., 2016), and its chemical features were shown in Table S1 (refer to supplemental materials). GP (purity > 98%) was purchased from Linchuan Zhixin Biotechnology Co., Ltd. (Jiangxi, China). Medium-chain triglyceride (MCT) was a product of Britz Net­ works Sdn. Bhd (Melaka, Malaysia). 4-Chloro-3,5-dinitro tri­ fluoromethylbenzene (CNBF) was obtained from Sigma-Aldrich (St. Louis, USA).

2.5. Atomic force microscopy (AFM) Molecular morphologies of C- and G-SBP were imaged under tapping mode using a Multimode 8 AFM instrument (Bruker, USA). Each pectin sample was dissolved in Mill-Q water to set a concentration of 3 μg/mL. An aliquot of pectin solution (3 μL) was drop-deposited onto freshly cleaved mica sheet and air dried at room temperature for 20 min. Captured images were processed by the Nanoscope analysis software (Version 1.7, Bruker). 2

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Fig. 1. Visual observation of the solutions made of C-SBP and G-SBP (a). UV–Vis spectra of C-SBP and GSBP (b). Dependence of OD 595 on crosslinking time (c). Changes in the lysine content for C-SBP and GSBP as a function of crosslinking time (d). In panel a, the concentration of pectin was 3.5% w/w and the pH was maintained at 7.5 using a 10 mM phosphate buffer. In panel b–c, the concentration of each tested solution was set at 1 mg/mL and the pH maintained at 3.5 with 10 mM citrate buffer. In panel b, the GSBP was obtained by crosslinking with GP for 20 h.

2.6. Emulsion preparation

where parameters ks represents the consistency coefficient (Pa⋅s); n represents the flow behavior index (dimensionless); γs is shear viscosity (Pa⋅s) and η (∞) stands for asymptotic viscosity at high shear rate.

Emulsion formula consisted of 1% w/w pectin as emulsifier, 15% w/ w as oil phase, 0.1% sodium benzoate as preservative. One gram pectin and 0.1 g sodium benzoate were dissolved in 80 g citrate buffer (50 mM, pH 3.5) with stirring for 12 h at room temperature. Next, 15 g of MCT was added to the as-prepared solution, after which an adequate amount of citrate buffer was added to make the total weight 100 g. The twophase was pre-homogenized by high-speed shear mixer (Slientcrusher M, Heidolph Corp., Germany) at 20,000 rpm for 2 min to obtain a coarse emulsion, then passed through the high-pressure homogenizer (NANO, ATS Engineer Inc., China) two times at 50 MPa to obtain a fine emulsion. Unless otherwise noted, the concentrations of SBP solution covered in the following tests were all 1% and the pH was buffered at 3.5. For a fair comparison, commercial gum arabic (GA) was chosen as a reference emulsifier and specifically used at a concentration of 18% w/w with the purpose of obtaining an optimum emulsification effect.

2.9. Interfacial behavior 2.9.1. Interfacial tension The interfacial tension of the MCT-water interface was measured using an optical drop shape tensiometer (OCA-20, DataPhysics, Ger­ many). Each pectin solution was prepared by the same method for emulsion preparation as described in Section 2.6 and the citrate buffer was used as a reference. A pendant drop (25 μL) of sample solution was formed by a syringe needle and immersed in MCT. The shape of the pendant drop was recorded by a CCD camera and the interfacial tension was calculated by SCA 20 software (DataPhysics, Germany) according to the Young-Laplace equation. 2.9.2. SBP adsorption on emulsion droplet Adsorbed concentration (C, mg/mL emulsion) of pectin on the emulsion droplet surface was inferred from the polysaccharide remaining in the serum phase after the freshly prepared emulsion was ultracentrifuged (800,000�g for 30 min) by Optima XE-100 (Beckman Coulter, Inc., USA), quantified by SEC-MALLS method (Section 2.4). The interfacial concentration (Г , mg/m2) of pectin at the surface of oil droplet and specific surface area (Sv, m2/mL emulsion) of the emul­ sion was calculated (Puppo et al., 2005).

2.7. Droplet size measurement Measurements of the droplet size distribution (DSD) and volumeweight mean diameter (d4,3) was performed using a Mastersizer 3000 (Malvern Instruments Ltd., UK). The refractive indexes were 1.45 and 1.33 for MCT and water, respectively. For freshly prepared emulsions, d4,3 was determined within 2 h after preparation. For stored emulsions, d4,3 was determined at days 2, 5, 10, 15, and 21 after being treated at 35 � C for destabilization.

Г ¼ C / Sv

(2)

2.8. Rheological determination

Sv ¼ 6Φ/ d3,2

(3)

Rheological measurements of emulsion were determined by a rota­ tional rheometer (MCR301, Anton Paar, Graz, Austria) equipped with plate geometry CP-50 at a temperature of 25 � C (shear rate increasing from 0.1 to 100 s 1). The obtained data were fitted to Sisko’s power law model equation (Sisko, 1958):

where Φ represents the volume fraction of the dispersed phase; d3,2 is the surface-volume mean diameter (d3,2) measured in section 2.7.

η ¼ η (∞) þ ks � γs (n

1)

2.9.3. Layer thickness of adsorbed SBP onto latex surface A 1% w/w stock SBP solution was prepared as described in Section 2.6 and then diluted into a series of concentration ranging from 0.01% to 0.5% with citrate buffer (10 mM, pH 3.5). Prior to dynamic light

(1) 3

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scattering measurement (Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK), 2440 μL SBP solution was mixed with 60 μL monodisperse polystyrene latex beads (2.5% w/v, DAE Scientific Co., Ltd, Tianjin, China) and lasting for 8 h to achieve adsorption equilib­ rium. The particle size of polystyrene latex beads provided by the manufacturer was 200 nm and was measured as 199.4 � 2.2 nm with Zetasizer. The thickness of the adsorbed layer was calculated from the difference in particle size of adsorbed and naked polystyrene latex beads.

G-SBP (Fig. 1b), which was attributable to crosslinking between –NH2 of SBP and GP. Similar findings were reported by Butler et al. (2003) who studied the reaction between GP and chitosan. The intensity at 595 nm increased in a time-dependent manner and eventually leveled off at 20 h (Fig. 1c), suggesting that the extent of crosslinking reaction between SBP and GP had reached the maximum. Since the –NH2 residues in SBP only existed in the proteinaceous moiety, changes in contents of amino acids can reveal their consumption by GP during reaction, thus providing useful information for kinetic properties of the crosslinking reaction. As the peptide linkage of protein is formed by α-NH2 and α-carboxy groups, only non-α-NH2 is available for crosslinking with GP. For this reason, we further measured the contents of non-α-NH2 containing amino acids for C-SBP (Table S2), i.e., glutamine, arginine, lysine, asparagine, and histidine. The results showed the contents of glutamine, arginine, and lysine were 2.77, 2.15, and 6.14 mg/g, respectively. In contrast, asparagine and histidine were not detectable. These results were consistent with those reported for SBP extracted by chelators and acids (Liu et al., 2019). Previously, Lee et al. (2003) reported that these three amino acids reacted with GP to produce a blue-colored product. However, in present work, only lysine residues were consumed as a function of crosslinking time (Fig. 1d) and the contents of other amino acids were almost constant (Table S2). The lysine content of G-SBP significantly decreased from 6.14 mg/g to 0.53 mg/g after reaction with GP for 20 h, thereafter it held steady. These results agreed with the trend of OD 595 (Fig. 1c). Through investigation of crosslinking reaction between casein and GP, Silva, Arnaud, Carvalho, De, and Fr� ed�eric (2014) reported that GP preferred to react with lysine residues rather than arginine residues, and the author attributed the selectivity of GP toward lysine to its lower pKa of –NH2. Other factors affecting the reaction between GP and casein might include pH, accessibility of free –NH2, and some degree of reversibility of the reaction, but they played minor roles. For glutamine and arginine, the highly branched side chain structure of SBP may create a steric hindrance for their free –NH2 groups to be crosslinked by GP. Another explanation might be the different molecular structures of amino acid. The non-α-NH2 of lysine is attached to a saturated carbon atom, whereas non-α-NH2 of glutamine or arginine is attached with an unsaturated carbon atom, presumably, the latter being chemically inactive in the crosslinking by GP. To support this speculation, an experiment was performed separately by incubating GP with acrylamide (one –NH2 attached with carbonyl) and guanidine hydrochloride (one –NH2 in guanidine group) in solution at pH ¼ 7 and 45 � C for 24 h, but no crosslinking action was developed.

(4)

dT ¼ (Ra - Ri) / 2

where dT is the thickness of the adsorbed layer; Ra and Ri represent the particle size of polystyrene latex beads with and without pectin adsorption, respectively. The total latex surface area of particles per milliliter (M) was calculated as follow. (5)

M ¼ N � S � π � R2i 10

where N is the number of latex particle, which is 22,748 � 10 per gram of polystyrene latex beads provided by the manufacturer; S is the con­ centration of particle solids (2.5% w/v). This gives a total latex surface area of 0.7146 m2/mL. 2.10. Statistical analysis Unless otherwise stated, all experiments were performed in tripli­ cate. Statistical evaluation was performed by SPSS software (IBM Corp., USA) using one-way analysis of variance. Means were considered significantly different at p-value < 0.05. Date fitting of rheological de­ terminations was carried by Origin software (OriginLab Corp., USA). 3. Results and discussion 3.1. Crosslinking between SBP and GP C-SBP solutions were initially clear and slightly yellow. Upon crosslinking between SBP chains by GP, the color first changed to cyan and then blue, which showed that SBP could react with GP (Fig. 1a). Interestingly, no gel or precipitate could be formed even after complete reaction between SBP and GP for 48 h, as indicated by the high degree of transparency (data not shown). The UV spectra of C- and G-SBP are presented in Fig. 1b. The spec­ trum of C-SBP had two peaks at 280 nm and 325 nm, as has been re­ ported for an SBP (Williams et al., 2005). Normally, the absorption peak at 280 nm in pectin can be assigned to the aromatic residues from tryptophan, tyrosine, and phenylalanine, and the absorption peak at 325 nm is attributed to ferulic acid covalent bonding with SBP (Fishman, Chau, Qi, Jr, & Yadav, 2013; Qi, Chau, Fishman, Wickham, & Hotchkiss, 2014). As expected, amino acid analysis showed that C-SBP contained small amounts of tyrosine (3.16 mg/g) and phenylalanine (1.86 mg/g) (Table S2). Tryptophan was undetectable due to destruction during acid hydrolysis of samples. At the same concentration of 1 mg/mL, the in­ tensity of peaks at 280 nm of the spectrum of G-SBP was notably higher than C-SBP. A possible reason could be attributed to the formation of heterocyclic amino compound in the reaction of –NH2 and GP (Butler et al., 2003; Mi, Shyu, & Peng, 2004). Since the crosslinking of GP with –NH2 of protein may expose the buried tryptophan, tyrosine, and phenylalanine, such alteration in protein structure may also contribute to an increase of OD 280. However, given the above three amino acids only presented in very small amounts and the total protein content of SBP was 6.48% (Table S1), their influence in increasing OD 280 may be limited as compared to the contribution of newly formed heterocyclic amine compound. As such, the impact of protein structural changes exposed by crosslinking with GP on OD 280 was not discussed further. It was noted that a new peak centered at 595 nm occurred in the case of

3.2. Macromolecular characteristics Superimposed elution profiles of SBPs treated by GP for 1, 4, 10, 20, 48 h and control sample are shown in Fig. 2. It can be seen from the RI profiles (Fig. 2a) that all of the samples were polydispersed, containing at least three sub-fractions. Notably, the RI elution profile of C-SBP consisted of three peaks centered at ~25 min, ~29 min, and ~32 min, respectively. This elution pattern was similar to that of an SBP oxida­ tively crosslinked by horseradish peroxidase in the presence of H2O2 (Zhang et al., 2015). When crosslinked by GP, a new elution peak appeared at 21–24 min, whose intensity gradually increased with pro­ longing crosslinking time and eventually plateaued at the maximum at 20 h or even longer. Hence, this result again reflected that the reaction between SBP and GP had reached an equilibrium state at 20 h. However, the intensity of the three peaks eluted between 25 and 32 min, in turn, decreased, suggesting that the three sub-populations might be partially crosslinked into the newly occurred sub-population with higher Mw. Table 1 shows the Mw, Rg, and MR of SBP as a function of cross­ linking time. As can be seen, the MR of any tested sample was higher than 82.4%, indicating the good solubility of G-SBPs. This MR was much higher than that of SBP crosslinked by laccase (Jung & Wicker, 2012) or horseradish peroxidase (Zhang et al., 2015). According to Zaidel, 4

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Table 1 Weight-average molecular weight (Mw), mass recovery (MR), z-average root mean square radius of gyration (Rg), hydrodynamic radius (Rh), and ζ-potential of the untreated control and crosslinked SBPs by GP for different times. Samples

Mw a (g/ mol)

Rg a (nm)

MR a (%)

Rh (nm)

C-SBP G-SBP-1h G-SBP-4h G-SBP10 h G-SBP20 h G-SBP48 h

3.04 � 105 6.09 � 105 8.41 � 105 1.22 � 106

34.8 36.6 37.8 38.6

82.4 83.5 87.7 85.7

330�6a

1.28 � 106

38.8

86.4

384�2b

1.31 � 106

39.2

89.4

ζ-potential (mV) b

b

c

23�1a

25�0b

a

Single replication. Determined using a Malvern Zetasizer Nano ZS with pectin concentration and pH being 1 mg/mL and 3.5, respectively. c Date are presented as mean � standard deviation (n ¼ 3). Values in the same column followed by different letters (a and b) indicate statistically significant (p < 0.05) differences among samples. b

Similar results were reported by Zhang, Shi, et al. (2015) and Zhang, Wang, et al. (2015), who reported that SBP, once oxidatively coupled by horseradish peroxidase, had greater values of Mw and Rg than the un­ treated SBP. Fig. 2c shows the UV GPC elution profiles recorded at 280 nm at which variation of the protein (associated with SBP) distribution during treatment by GP. The UV profile of C-SBP consisted of three elution peaks (at ~25 min, ~29 min, and ~33 min), hence, this UV elution pattern resembled the according RI profile pattern (Fig. 2a). When the crosslinking of GP to SBP was initiated, the intensity of peak at ~25 min started to increase in a time-dependent manner. Meanwhile, the maximum absorbance shifted from ~25 min to ~23 min remarkably, leaving the intensity at 29 min nearly unchanged and overlapped by the 33-min peak. Since GP crosslinked SBP chains via the –NH2 of lysine residues, differences in peak intensity are possibly due to variations in both structure and amount of GP-lysine complex in each SBP fraction. It is likely that GP and –NH2 may form a heterocyclic amine structure (Butler et al., 2003). Further evidence is the gradual increase in absor­ bance at 595 nm with prolonging crosslinking time, as can be seen in the GPC elution profiles (Fig. 2d), in comparison to no measurable absor­ bance for C-SBP. By relating the absorbance at 595 nm to the elution time which is inversely proportional to molecular sizes of a polymer, it is suggested that the reaction between GP with SBP may contribute more to the large-sized components of G-SBP eluted at 21–24 min. Note that absorbance intensity at 280 nm for the 33-min peak was unaffected when the crosslinking reaction prolonged, revealing that considerable amount of lower Mw fraction carrying with aromatic residues not be affected even crosslinking for over 20 h. Comparison of RI elution pattern between G-SBP-20 h and G-SBP48 h (Fig. 2a) clearly revealed that the reaction between SBP and GP reached a plateau at crosslinking up to 20 h, which was in agreement with the results of Mw measurement, amino acid analysis, and UV spectrum. Therefore, the crosslinking time of 20 h was defined as the end point of the crosslinking reaction. For this reason, G-SBP-20 h was thereby used as a representative sample (below, the SBP sample cross­ linked by GP for 20 h was referred to as G-SBP for short) for both atomic force microscopy (AFM) observation and assessment of emulsifying properties.

Fig. 2. GPC elution profiles of control and crosslinked SBP samples constructed using signals from refractive index (RI) (a), light scattering (LS) at 90� angle (b), absorbance at 280 nm (c), and 595 nm (d).

Chronakis, and Meyer (2012), the reduction in MR associated with enzymatically crosslinked SBP was probably due to the loss of one part of SBP in the form of gel. Previously, gelation of chitosan (Delmar & Bianco-Peled, 2015) or gelatin (Nickerson et al., 2006) were induced by considerable crosslinking between GP and free –NH2 groups. In this work, however, no gelation of SBP could be formed even crosslinking by GP for 48 h. In fact, the MR slightly increased from 82.4% to 89.4% as the crosslinking reaction went on. As SBP contained much lower reactive –NH2 than chitosan and gelatin, only a small number of sites (6.15 mg/g of lysine in this work) available for crosslinking by genipin, which might not enough for gelation to take place. The Mw of SBP increased dramatically from 3.04 � 105 g/mol to the highest Mw of 1.31 � 106 g/mol after 48 h of crosslinking and the Rg from 34.8 to 39.2 nm, confirming the intermolecular crosslinking of SBP molecules.

3.3. AFM Fig. 3 depicts molecular morphologies of pectin samples imaged by AFM. Under AFM observation, the C- and G-SBP molecules distributed on the freshly cleaved mica sheet fairly evenly, thus individual mole­ cules were clearly imaged. C-SBP molecules turned out to be linear and 5

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Fig. 3. Atomic force microscopy (AFM) images captured from the solutions made of C-SBP (a) and G-SBP (b) at a concentration of 3 μg/mL.

branched strands, which assigned to polysaccharide chains, as well as tadpole-looking structure, which assigned to protein-polysaccharide complexes (Fig. 3a), as has been reported for SBP previously (Kirby, Macdougall, & Morris, 2006). It is worth noting that some larger sized spherical structures (Fig. 3a) were attached to multiple divergent stands, resulting in an irregularly shaped aggregate (labeled as ‘A’). In this case, such aggregate structure possibly resulted from the aggregation of SBP molecules during air drying (Kirby, Macdougall, & Morris, 2008). For G-SBP, most molecules showed spherical-like structures of different sizes, and a few displayed either linear or tadpole structures. Some of the spherical structures in G-SBP (labeled as ‘B’), bearing no connection to divergent lines, were obviously larger than that of C-SBP (labeled as ‘A’). These results revealing that proteinaceous moiety of side chains cross­ linked by GP made G-SBP a more compact conformation. 3.4. Emulsifying properties Fig. 4 depicts changes in droplet size distribution (DSD) of emulsions upon storage at 35 � C for 21 d. It could be seen from Fig. 4a that the percentages of larger droplets of sizes of 2–10 μm gradually increased with prolonging storage time, probably compensated by coalescence of fine droplets of sizes 0.05–1 μm. Since d4,3 is more sensitive to show the occurrence of aggregated or flocculated droplets (Mcclements, 2007), the emulsion stability was evaluated by changes in d4,3 (Fig. 5). Fresh emulsions stabilized by C-SBP and G-SBP had d4,3 of 0.425 μm and 0.541 μm, respectively, and both of them showed nearly gaussian DSD (Fig. 4a–b), indicating their good emulsifying activity. Compared to the C-SBP stabilized emulsion, the fresh G-SBP stabilized emulsion was larger in d4,3 value, which reflected the higher emulsifying activity of C-SBP. Basically, the crosslinking reaction between GP and SBP did not decrease the total protein content, but did change the structure of pro­ teinaceous moieties by bridging amino acids. Hence, the decreased emulsifying activity is primarily correlated to alteration in the hydro­ phobicity of the proteinaceous moiety. A possible explanation could be that the amount of protein accessible to the droplet surface decreases in G-SBP, leading to decreased emulsification activity of G-SBP. Another possible reason may lay in the accessibility of proteinaceous moiety based on that the crosslinking of GP between two lysine residues from two constituent peptide chains will make the proteinaceous moiety less flexible. As a consequence, G-SBP was more difficult to absorb onto the newly form oil-water interface. Previous studies showed that the emulsifying activity of pectin was also affected by Mw. Akhtar, Dick­ inson, Mazoyer, and Langendorff (2002) demonstrated that depoly­ merized pectin of Mw ranging from 5.62–6.97 � 104 g/mol showed better emulsifying performance. Jung & Wicker (2012) speculated that negative carboxyl groups in SBP might be buried due to the crosslinking of side chains via ferulic acid by laccase, making the ζ-potential of laccase-modified SBP less negative than control SBP. Considering the results obtained in this work, G-SBP was larger in hydrodynamic radius (Rh) and Rg, and was more negative in ζ-potential than C-SBP (Table 1)

Fig. 4. Droplet size distributions measured at different storage times at 35 � C for the emulsions stabilized by C-SBP (a), G-SBP (b), and GA (c), respectively. All the emulsions were prepared with 15% w/w MCT and 1% w/w polymer at pH 3.5 except that the concentration of GA was 18% w/w.

Fig. 5. Changes in d4,3 values of the emulsions stored at 35 � C for 21 d. All the emulsions were prepared with 15% w/w MCT and 1% w/w polymer at pH 3.5 except that the concentration of GA was 18% w/w.

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despite its side chains were partly crosslinked by GP, indicating that it was not the negatively charged groups but the positively charged amino groups being consumed or even buried after crosslinking reaction. The amphiphilic properties of C- and G-SBP were assessed through measurement of the MCT-water interfacial tension. As shown in Table 2, the measured values ranged from 11.84 to 13.82 mN/m, which were in agreement with the results measured with citrus pectin (~13 mN/m) (Schmidt et al., 2014). However, the measured interfacial tension of SBP was much lower than that (~19 mN/m) reported previously (Funami et al., 2007; Leroux, Langendorff, Schick, Vaishnav, & Mazoyer, 2003). Discrepancy may be ascribed to some deviations in determined condi­ tions like oil source, pectin concentration, pH. Comparison in interfacial tension among sample solutions revealed that both C- and G-SBP possessed interfacial activities, but the interfacial tension of G-SBP (13.82 mN/m) was significantly higher (Table 2). This result again supported the lower emulsifying activity of G-SBP as compared to C-SBP. Slight decreases in the emulsion stability of the G-SBP emulsion were observed in the colloidal test. The DSD measured with the G-SBP emulsion shifted to larger droplet sizes continuously but very slightly (Fig. 4b). Besides, a small proportion of large-sized droplets was formed at day 5 and then this part of oily droplets became larger in quantity and size with increasing storage time (the inserted panel in Fig. 4b). Despite these changes in tested G-SBP emulsions, a comparison of DSD profiles of the G-SBP emulsion pattern showed no sign of emulsion breakdown. Oppositely, the DSD of the C-SBP emulsion at day 5 not only signifi­ cantly decreased in the main peak across 0.5–1 μm but also gave a new peak across 2–10 μm (Fig. 4a). This bimodal DSD for the stored C-SBP emulsion clearly indicated breakdown in emulsion stabilization. When reflected in mean droplet diameter, the d4,3 increased dramatically up to 0.622 μm (Fig. 5). GA, recognized emulsifier, was used for a fair com­ parison of evaluation on the emulsifying stability of G-SBP. As expected, GA resulted in a fine emulsion with a monodisperse DSD. The d4,3 of GAstabilized emulsion increased from 0.303 μm at day 0 to 0.378 μm at day 21. Meanwhile, the DSD was changed to be bimodal by the formation of droplets larger than 2 μm (Fig. 4c). Comparison of DSD between GA and G-SBP (Fig. 4b–c) showed that G-SBP had higher emulsifying stability because it experienced relatively less change in DSD. Enhancement in the emulsifying stability of G-SBP was also shown by a slightly upward trend of d4,3 in opposition to a steep increasing trend for C-SBP (Fig. 5). Fig. 6 presents changes in droplet shape and size for the emulsions stabilized by G-SBP, C-SBP, GA, respectively. For the GSBP emulsion, droplet flocculation and coalescence were evident by the presence of droplets of sizes ca. 4 μm at day 21 (Fig. 6). This is possible that some droplets may aggregate into large flocs greater than 6 μm and subsequently recorded by laser particle sizer (Fig. 4b). Flocs in the G-SBP stabilized emulsion could be attributed to the Ca-bridging flocculation since SBP had a high content of uronic acids with extended molecular configuration (Nakauma et al., 2008). In the case of C-SBP, the emulsion stability was rapidly broken during storage as indicated by the formation of droplets larger than 10 μm (Fig. 6). The GA-coated droplets were more resistant to flocculation, which was probably because of the strong steric stabilization provided by GA and also the low affinity of GA to elec­ trostatically bind divalent ions like Ca2þ (Braccini, Grasso, & Prez, 1999;

Chanamai & McClements, 2002). Based on the above results, the emulsion stability of SBP was significantly enhanced after crosslinking of the lysine residues and GP. It has been shown that reinforcement in the emulsifying stabilities of SBP could be achieved by adopting a similar modification method based on crosslinking of side (Jung & Wicker, 2012; Zhang et al., 2015) or nonspecific chains of SBP (Funami et al., 2011). The mechanism by which G-SBP stabilized the 15% MCT emulsion may be attributable to both the viscosity-enhancing effect induced by an increase in Mw and steric stabilization from the cross­ linked pectin chains. First, G-SBP is able to increase the viscosity of the aqueous phase effectively due to its remarkably large Mw, which may reduce the degree of droplet collision and subsequently droplet floccu­ lation and coalescence. Secondly, G-SBP, when adsorbed onto oil droplets, is expected to form a dense layer for protecting the droplets stable, taking advantage of its crosslinked chains and favorable compact conformation. 3.5. Apparent viscosity of emulsions The flow curves of apparent viscosity (η) against shear rate (γs) for the emulsions stabilized by C-SBP, G-SBP, and GA are presented in Fig. 7. All curves were well fitted to Sisko’s model (R2 > 0.99). As shown in Table 3, the flow behavior index n was 0.56, 0.39, and 0.66 (n < 1) for the C-SBP, G-SBP, and GA emulsions, respectively, showing their pseu­ doplasticity behavior of non-Newtonian fluid. Similar pseudoplasticity behaviors have been reported for brea gum or GA stabilized emulsions (Castel, Rubiolo, & Carrara, 2017) and also for aqueous dispersions of �ndez, Acacia cochliacantha or Acacia farnesiana gums (Sibaja-Herna �n-Guerrero, Sepúlveda-Jim� Roma enez, & Rodríguez-Monroy, 2015). Within the measured shear rate range (0.1–100 s 1), the G-SBP emulsion had a higher η and a greater consistency coefficient (ks) than C-SBP emulsion (Table 3) because of the higher Mw of G-SBP. At low shear rates (0.1–1 s 1), the difference in η between G- and C-SBP was much larger than those recorded at high shear rates (10–100 s 1), indicating the stronger intermolecular forces of G-SBP under static state. Since all of the three emulsions received no further stirring during storage, the greatest viscosity-enhancing effect of G-SBP allowed it to more effi­ ciently retard the movement of droplets and reduce the probability of contact between droplets. 3.6. Interfacial behavior Specific surface area (Sv), interfacial concentration (Г ), and SBP fraction adsorbed onto the emulsion droplet surface are presented in Table 4. The C-SBP stabilized emulsion (3.08 m2/mL) had a higher Sv than the G-SBP stabilized emulsion (2.59 m2/mL), indicating more droplet surfaces were generated with C-SBP as the emulsifier. This result correlated well with the interfacial tensions (Table 2) and again indi­ cated the slightly lower surface activity of G-SBP. By subtracting the non-adsorbed fractions, it was calculated that 24.51% of C-SBP and 24.14% of G-SBP adsorbed onto droplet surfaces during emulsification and there were no significant differences between them. These values were in line with the result reported by Akhtar et al. (2002), who demonstrated that the proportion of depolymerized citrus pectin adsorbed onto rapeseed oil droplet surface was ca. 25%, independent of the concentration of emulsifier used. On the basis of Sv and the amount of fractions on the total droplet surfaces, the calculated interfacial concentration of G-SBP was 1.07 mg/m2, which was significantly higher than that of C-SBP (0.90 mg/m2). These two values were close to 1 mg/m2, the typical value of monolayer coverage for many polymer/­ particle systems that adsorbed onto a surface (Siew & Williams, 2008), and they also fell into the range from 0.610 to 1.571 mg/m2 for an SBP with MCT as oily phase (Funami et al., 2011). To evaluate the steric hindrance generated by C- and G-SBP, the adsorbed hydrated layer thickness surrounding monodisperse poly­ styrene latex was determined, and the results are shown in Fig. 8. The

Table 2 Interfacial tension of the sample dispersions prepared using C- and G-SBP at the MCT oil-water interface a. Interfacial tension (mN/m)

Buffer

C-SBP

G-SBP

18.9 � 0.2a b

11.8 � 0.4b

13.8 � 0.3c

a

The concentration of pectin was 1% w/w and the pH was buffered at 3.5 (50 mM citrate buffer). The G-SBP was obtained by crosslinking with GP for 20 h. b Data are presented as mean � standard deviation (n ¼ 3). Values in the same row followed by different letters (a,b and c) indicate statistically significant (p < 0.05) differences among samples. 7

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Food Hydrocolloids 101 (2020) 105459

Fig. 6. Light micrograph of the emulsions made by 1% C-SBP, G-SBP, and GA after storage at 35 � C for 0, 5, and 21 d. All the emulsions were prepared with 15% w/w MCT and 1% w/w polymer at pH 3.5 except that the concentration of GA was 18% w/w. Table 4 Specific surface area Sv (m2/mL), surface coverage Γ (mg/m2) and adsorbed amount of C-SBP and G-SBP at the surface stabilized using them at a concen­ tration of 1% w/w a. Samples

Sv (m2/mL emulsion)

Γ (mg/m2)

Fraction adsorbed onto emulsion droplet surface b (%)

C-SBP G-SBP

3.08 � 0.04a 2.59 � 0.06b

0.90 � 0.03a 1.07 � 0.01b

24.51 � 0.75a 24.14 � 0.31a

a Data are presented as mean � standard deviation (n ¼ 3). Values in the same column followed by different letters (a and b) indicate statistically significant (p < 0.05) differences among samples. b Weight percentage of fraction adsorbed onto emulsion as compared to whole SBP fraction before adsorption.

Fig. 7. Flow curves of emulsions stabilized by C-SBP, G-SBP, and GA. All the emulsions were prepared with 15% w/w MCT and 1% w/w polymer at pH 3.5 except that the concentration of GA was 18% w/w. Table 3 Sisko model parameters of emulsions stabilized by C-SBP, G-SBP, and GA a. 3

Sample ID

η∞ ( � 10

C-SBP G-SBP GA

8.67 � 0.27 9.15 � 0.52 53.9 � 1.2

Pa s)

ks ( � 10

3

Pa s)

6.75 � 0.35 17.6 � 0.7 31.2 � 1.4

n

R2

0.56 � 0.02 0.39 � 0.02 0.66 � 0.02

0.99 0.99 0.99

a All the emulsions were prepared with 15% w/w MCT and 1% w/w polymer at pH 3.5 except that the concentration of GA was 18% w/w.

thickness of the hydrated layer increased with the increasing amount of beet pectin added but not tended to plateau, the rate of increase in layer thickness was more rapidly at low surface coverages (amount of SBP

Fig. 8. Layer thickness of C- and G-SBP adsorbed onto the polystyrene (PS) surface as a function of pectin dosage. 8

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Food Hydrocolloids 101 (2020) 105459

added < 10 mg per m2 PS particles), as has been reported for SBP by Siew & Williams (2008). In contrast to the consensus viewpoint that a larger size of emulsifier will result in a thicker hydrated layer onto the covered droplet, the thickness value of G-SBP, however, was lower than that of C-SBP irrespective of its larger Mw. This phenomenon could be explained by the aspects of molecular conformation. Studies relevant to SBP modification showed that enhancement of the emulsifying proper­ ties was achieved by adopting a dry-heating method to induce cova­ lently bonding between endogenous proteins and galacturonate moieties (Funami et al., 2011; Funami et al., 2008). Interestingly, the enhanced SBP not only adopted a very extended and bulky branched configuration under AFM observation but also experienced a significant increase in Rg (from 51.2 nm to 76.5 nm) (Funami et al., 2011; Funami et al., 2008). However, in this work, GP mediated crosslinking of SBP via primary amine groups, induced local aggregation of molecular chains as the substantial increase in Mw accompanied with a slight and unpro­ portional increase in Rg (from 34.8 nm to 39.2 nm). Hence, these results suggested a more compact conformation of G-SBP when compared to C-SBP, as confirmed by AFM observation (Fig. 3). Since pectin inher­ ently adopts a semi-flexible structure conformation, it is possible that the SBP may undergo conformational transition upon adsorbing onto oily surfaces. In fact, polysaccharide chains are unfolding and bending so as to expose more hydrophobic proteinaceous moieties and facilitate their anchoring process (Ngou� emazong, Christiaens, Shpigelman, Loey, & Hendrickx, 2015). When adopting an extended conformation, SBP anchoring onto the droplet surface will allow the polysaccharide chains to protrude inside the aqueous phase; when adopting a less extended and compact conformation, polysaccharide chains of G-SBP adsorbed may tend to tangle within a limited space. The pH-regulated adsorption behavior onto oil droplets also proposed that SBP chains can be arranged in a more compact manner at low pHs (below pKa of galacturonic acid) than at high pHs, providing effective steric hindrance and inhibiting desorption of pectin (Alba & Kontogiorgos, 2017; Alba et al., 2016). Based on the higher interfacial concentration of G-SBP (Table 4), we hypothesis that G-SBP may form a denser and more compact but thinner hydrated layer surrounding droplets, providing stronger steric stabili­ zation for oil droplets and avoiding permeation of oil phase.

Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.foodhyd.2019.105459. References Akhtar, M., Dickinson, E., Mazoyer, J., & Langendorff, V. (2002). Emulsion stabilizing properties of depolymerized pectin. Food Hydrocolloids, 16(3), 249–256. Alba, K., & Kontogiorgos, V. (2017). Pectin at the oil-water interface: Relationship of molecular composition and structure to functionality. Food Hydrocolloids, 68, 211–218. Alba, K., Lmc, S., & Kontogiorgos, V. (2016). Engineering of acidic O/W emulsions with pectin. Colloids and Surfaces B: Biointerfaces, 145, 301–308. Bell� e, A. S., Hackenhaar, C. R., Spolidoro, L. S., Rodrigues, E., Klein, M. P., & Hertz, P. F. (2018). Efficient enzyme-assisted extraction of genipin from genipap (Genipa americana L.) and its application as a crosslinker for chitosan gels. Food Chemistry, 246, 266–274. Braccini, I., Grasso, R. P., & Prez, S. (1999). Conformational and configurational features of acidic polysaccharides and their interactions with calcium ions: A molecular modeling investigation. Carbohydrate Research, 317(1–4), 119–130. Butler, M. F., Ng, Y. F., & Pudney, P. D. A. (2003). Mechanism and kinetics of crosslinking reaction between biopolymers containing primary amine groups and genipin. Journal of Polymer Science Part A Polymer Chemistry, 41(24), 3941–3953. Castel, V., Rubiolo, A. C., & Carrara, C. R. (2017). Droplet size distribution, rheological behavior and stability of corn oil emulsions stabilized by a novel hydrocolloid (Brea gum) compared with gum Arabic. Food Hydrocolloids, 63, 170–177. Chanamai, R., & McClements, D. (2002). Comparison of gum Arabic, modified starch, and whey protein isolate as emulsifiers: Influence of pH, CaCl2 and temperature. Journal of Food Science, 67(1), 120–125. Delmar, K., & Bianco-Peled, H. (2015). The dramatic effect of small pH changes on the properties of chitosan hydrogels crosslinked with genipin. Carbohydrate Polymers, 127, 28–37. Fishman, M. L., Chau, H. K., Qi, P. X., Jr, A. T. H., & Yadav, M. P. (2013). Physicochemical characterization of protein-associated polysaccharides extracted from sugar beet pulp ☆. Carbohydrate Polymers, 92(2), 2257–2266. Fujikawa, S., Fukui, Y., Koga, K., Iwashita, T., Komura, H., & Nomoto, K. (1987). Structure of genipocyanin G1, a spontaneous reaction product between genipin and glycine. Tetrahedron Letters, 28(40), 4699–4700. Funami, T., Nakauma, M., Ishihara, S., Tanaka, R., Inoue, T., & Phillips, G. O. (2011). Structural modifications of sugar beet pectin and the relationship of structure to functionality. Food Hydrocolloids, 25(2), 221–229. Funami, T., Nakauma, M., Noda, S., Ishihara, S., Al-Assaf, S., & Phillips, G. O. (2008). Enhancement of the performance of sugar beet pectin as an emulsifier. Foods food ingredients J. Jpn., 213(4), 347–356. Funami, T., Zhang, G., Hiroe, M., Noda, S., Nakauma, M., Asai, I., et al. (2007). Effects of the proteinaceous moiety on the emulsifying properties of sugar beet pectin. Food Hydrocolloids, 21(8), 1319–1329. Ghori, M. U., Alba, K., Smith, A. M., Conway, B. R., & Kontogiorgos, V. (2014). Okra extracts in pharmaceutical and food applications. Food Hydrocolloids, 42, 342–347. Guo, X., Meng, H., Tang, Q., Pan, R., Zhu, S., & Yu, S. (2016). Effects of the precipitation pH on the ethanolic precipitation of sugar beet pectins. Food Hydrocolloids, 52, 431–437. Hao, C., Ji, A., Shuang, Q., Yan, L., Zhu, Q., & Yin, L. (2018). Covalent conjugation of bovine serum album and sugar beet pectin through Maillard reaction/laccase catalysis to improve the emulsifying properties. Food Hydrocolloids, 76, 173–183. Jung, J., & Wicker, L. (2012). Laccase mediated conjugation of sugar beet pectin and the effect on emulsion stability. Food Hydrocolloids, 28(1), 168–173. Kirby, A. R., Macdougall, A. J., & Morris, V. J. (2006). Sugar beet pectin–protein complexes. Food Biophysics, 1(1), 51–56. Kirby, A. R., Macdougall, A. J., & Morris, V. J. (2008). Atomic force microscopy of tomato and sugar beet pectin molecules. Carbohydrate Polymers, 71(4), 640–647. Lee, S.-W., Lim, J.-M., Bhoo, S.-H., Paik, Y.-S., & Hahn, T.-R. (2003). Colorimetric determination of amino acids using genipin from Gardenia jasminoides. Analytica Chimica Acta, 480(2), 267–274. Leroux, J., Langendorff, V., Schick, G., Vaishnav, V., & Mazoyer, J. (2003). Emulsion stabilizing properties of pectin. Food Hydrocolloids, 17(4), 455–462. Li, X., Fang, Y., Al-Assaf, S., Phillips, G. O., & Jiang, F. (2012). Complexation of bovine serum albumin and sugar beet pectin: Stabilising oil-in-water emulsions. Journal of Colloid and Interface Science, 388(1), 103–111. Li, X., Fang, Y., Phillips, G. O., & Al-Assaf, S. (2013). Improved sugar beet pectinstabilized emulsions through complexation with sodium caseinate. Journal of Agricultural and Food Chemistry, 61(6), 1388–1396. Liu, Z., Pi, F., Guo, X., Guo, X., & Yu, S. (2019). Characterization of the structural and emulsifying properties of sugar beet pectins obtained by sequential extraction. Food Hydrocolloids, 88, 31–42. Mcclements, D. J. (2007). Critical review of techniques and methodologies for characterization of emulsion stability. Critical Reviews in Food Science and Nutrition, 47(7), 611–649. Mcclements, D. J., Bai, L., & Chung, C. (2017). Recent advances in the utilization of natural emulsifiers to form and stabilize emulsions. Annu. Rev. Food Sci. Technol., 8 (1), 205–236. Mi, F. L., Shyu, S. S., & Peng, C. K. (2004). Characterization of ring-opening polymerization of genipin and pH-dependent cross-linking reactions between

4. Conclusion SBP was successfully crosslinked by GP via –NH2 of its lysine residues under controlled conditions. The covalent crosslinking between lysine residues and GP was corroborated by amino acid analysis and macro­ molecular characterization. Compared to the control SBP, G-SBPs had larger molecular sizes and more compact conformation. These molecu­ lar features endowed G-SBP with superior interfacial and emulsifying properties. When modified by GP, the emulsifying activity of SBP was slightly decreased, but the emulsifying stability was significantly improved. Enhancement in emulsifying stability by the employed GP crosslinking strategy can be attributed to strengthening effects of emulsion viscosity and stronger steric hindrance provided by the unique physiochemical properties of G-SBP. Altogether, the results obtained in the present work suggest that the emulsification properties of SBP can be tailored by using GP as the crosslinker. Declaration of competing interest The authors declare no competing financial interest. Acknowledgment This work was supported by grants from the National Natural Science Foundation of China (No. 31601423 and No.31771931) and the China Postdoctoral Science Foundation (No. 2019M653279).

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Food Hydrocolloids 101 (2020) 105459 Semenova, M. (2016). Advances in molecular design of biopolymer-based delivery micro/nanovehicles for essential fatty acids. Food Hydrocolloids, 68. Sibaja-Hern� andez, R., Rom� an-Guerrero, A., Sepúlveda-Jim�enez, G., & RodríguezMonroy, M. (2015). Physicochemical, shear flow behaviour and emulsifying properties of Acacia cochliacantha and Acacia farnesiana gums. Industrial Crops and Products, 67, 161–168. Siew, C. K., & Williams, P. A. (2008). Role of protein and ferulic acid in the emulsification properties of sugar beet pectin. Journal of Agricultural and Food Chemistry, 56(11), 4164–4171. Siew, C. K., Williams, P. A., Cui, S. W., & Wang, Q. (2008). Characterization of the surface-active components of sugar beet pectin and the hydrodynamic thickness of the adsorbed pectin layer. Journal of Agricultural and Food Chemistry, 56(17), 8111–8120. Silva, N. F. N., Arnaud, S. J., Carvalho, A. N. F., De, & Fr� ed�eric, G. (2014). Development of casein microgels from cross-linking of casein micelles by genipin. Langmuir, 30 (34), 10167–10175. Sisko, A. (1958). The flow of lubricating greases. Ind. Eng. Chem., 50(12), 1789–1792. Sung, H. W., Huang, R. N., Huang, L. L., & Tsai, C. C. (1999). In vitro evaluation of cytotoxicity of a naturally occurring cross-linking reagent for biological tissue fixation. Journal of Biomaterials Science, Polymer Edition, 10(1), 63–78. Surh, J., Decker, E. A., & Mcclements, D. J. (2006). Influence of pH and pectin type on properties and stability of sodium-caseinate stabilized oil-in-water emulsions. Food Hydrocolloids, 20(5), 607–618. Wang, P., Chen, C., Guo, H., Zhang, H., Yang, Z., & Ren, F. (2018). Casein gel particles as novel soft pickering stabilizers: The emulsifying property and packing behaviour at the oil-water interface. Food Hydrocolloids, 77, 689–698. Williams, P. A., Sayers, C., Viebke, C., Senan, C., M, J., et al. (2005). Elucidation of the emulsification properties of sugar beet pectin. Journal of Agricultural and Food Chemistry, 53(9), 3592–3597. Zaidel, D. N. A., Chronakis, I. S., & Meyer, A. S. (2012). Enzyme catalyzed oxidative gelation of sugar beet pectin: Kinetics and rheology. Food Hydrocolloids, 28(1), 130–140. Zhang, L., Shi, Z., Shangguan, W., Fang, Y., Nishinari, K., Phillips, G. O., et al. (2015). Emulsification properties of sugar beet pectin after modification with horseradish peroxidase. Food Hydrocolloids, 43(3), 107–113. Zhang, Q., Wang, X., Mu, Q., Liu, P., Jia, S., Chen, L., et al. (2015). Genipin-cross-linked silk sericin/poly(N-isopropylacrylamide) IPN hydrogels: Color reaction between silk sericin and genipin, pore shape and thermo-responsibility. Materials Chemistry and Physics, 166, 133–143.

chitosan and genipin. Journal of Polymer Science Part A Polymer Chemistry, 43(10), 1985–2000. Mi, F. L., Sung, H. W., & Shyu, S. S. (2015). Synthesis and characterization of a novel chitosan-based network prepared using naturally occurring crosslinker. Journal of Polymer Science Part A Polymer Chemistry, 38(15), 2804–2814. Muzzarelli, R. A. A. (2009). Genipin-crosslinked chitosan hydrogels as biomedical and pharmaceutical aids. Carbohydrate Polymers, 77(1), 1–9. Nakauma, M., Funami, T., Noda, S., Ishihara, S., Al-Assaf, S., Nishinari, K., et al. (2008). Comparison of sugar beet pectin, soybean soluble polysaccharide, and gum Arabic as food emulsifiers. 1. Effect of concentration, pH, and salts on the emulsifying properties. Food Hydrocolloids, 22(7), 1254–1267. Neri-Numa, I. A., Pessoa, M. G., Paulino, B. N., & Pastore, G. M. (2017). Genipin: A natural blue pigment for food and health purposes. Trends in Food Science & Technology, 67, 271–279. Ngou�emazong, E. D., Christiaens, S., Shpigelman, A., Loey, A. V., & Hendrickx, M. (2015). The emulsifying and emulsion-stabilizing properties of pectin: A review. Comprehensive Reviews in Food Science and Food Safety, 14(6), 705–718. Nickerson, M. T., Patel, J., Heyd, D. V., Rousseau, D., & Paulson, A. T. (2006). Kinetic and mechanistic considerations in the gelation of genipin-crosslinked gelatin. International Journal of Biological Macromolecules, 39(4), 298–302. Park, J.-E., Lee, J.-Y., Kim, H.-G., Hahn, T.-R., & Paik, Y.-S. (2002). Isolation and characterization of water-soluble intermediates of blue pigments transformed from geniposide of Gardenia jasminoides. Journal of Agricultural and Food Chemistry, 50 (22), 6511–6514. �n, M. C., & Anton, M. Puppo, M. C., Speroni, F., Chapleau, N., Lamballerie, M. D., A~ no (2005). Effect of high-pressure treatment on emulsifying properties of soybean proteins. Food Hydrocolloids, 19(2), 289–296. Qi, P. X., Chau, H. K., Fishman, M. L., Wickham, E. D., & Hotchkiss, A. T. (2014). Investigation of molecular interactions between β-lactoglobulin and sugar beet pectin by multi-detection HPSEC. Carbohydrate Polymers, 107(1), 198–208. Qi, P. X., Xiao, Y., & Wickham, E. D. (2017). Changes in physical, chemical and functional properties of whey protein isolate (WPI) and sugar beet pectin (SBP) conjugates formed by controlled dry-heating. Food Hydrocolloids, 69, 86–96. Ramos-de-la-Pena, A. M., Renard, C. M. G. C., Montanez, J., Reyes-Vega, M. D., & Contreras-Esquivel, J. C. (2016). A review through recovery, purification and identification of genipin. Phytochemistry Reviews, 15(1), 37–49. Schmidt, U. S., Koch, L., Rentschler, C., Kurz, T., Endreß, H. U., & Schuchmann, H. P. (2014). Effect of molecular weight reduction, acetylation and esterification on the emulsification properties of citrus pectin. Food Biophysics, 10(2), 1–11.

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