Physicochemical properties and bioactivity of whey protein isolate-inulin conjugates obtained by Maillard reaction

Journal Pre-proof Physicochemical properties and bioactivity of whey protein isolate-inulin conjugates obtained by Maillard reaction

Wen-Duo Wang, Chao Li, Zhang Bin, Qiang Huang, Li-Jun You, Chun Chen, Xiong Fu, Rui Hai Liu PII:

S0141-8130(19)40730-7

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.02.086

Reference:

BIOMAC 14703

To appear in:

International Journal of Biological Macromolecules

Received date:

30 December 2019

Revised date:

1 February 2020

Accepted date:

9 February 2020

Please cite this article as: W.-D. Wang, C. Li, Z. Bin, et al., Physicochemical properties and bioactivity of whey protein isolate-inulin conjugates obtained by Maillard reaction, International Journal of Biological Macromolecules(2020), https://doi.org/10.1016/ j.ijbiomac.2020.02.086

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© 2020 Published by Elsevier.

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Physicochemical properties and bioactivity of whey protein isolate-inulin conjugates obtained by Maillard reaction Wen-Duo Wanga, Chao Li a,b, Zhang Bin a,b, Qiang Huang a,c, Li-Jun You a,c, Chun Chen a, c, d *, Xiong Fu a, c, e **, Rui Hai Liua, f

a

School of Food Science and Engineering, South China University of Technology, 381 Wushan

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Road, Guangzhou 510640, China SCUT-Zhuhai Institute of Modern Industrial Innovation, Zhuhai 519715, China

c

Guangzhou Institute of Modern Industrial Technology, Nansha, 511458, China Guangdong Province Key Laboratory for Green Processing of Natural Products and Product

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d

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b

e

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Safety, Guangzhou 510640, China

Overseas Expertise Introduction Center for Discipline Innovation of Food Nutrition and Human

Department of Food Science, Stocking Hall, Cornell University, Ithaca, NY, 14853, USA

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f

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Health (111 Center), Guangzhou 510640, China

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Co-corresponding authors:

*Chun Chen, Tel.: +86-20-87112894, Fax: +86-20-87112894. E-mail: [email protected]

** Xiong Fu, Tel.: +86-20-87112894, Fax: +86-20-87112894. E-mail: [email protected]

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Journal Pre-proof Abstract The functional properties and physiological functions of whey protein isolate (WPI) decreased near its isoelectric point (PI). The Maillard reaction covalently binding polysaccharides to proteins is an effective method to improve the functional activities of proteins. WPI-inulin conjugates were prepared by wet-heating method at 70 oC for 2h,4h and 6h, respectively. New bonds at higher molecular zone appearing

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at SDS-PAGE, decreased free amino acid content and new formed C-N bonds in

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FT-IR of conjugates compared with WPI confirmed the formation of the covalent bonds between WPI and inulin. As the increase of the reaction time, both the brown

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intensity and fluorescence intensity of WPI-inulin conjugates became higher. Amino

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acid contents, Circular dichroism analysis and SEM analysis presented the primary

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structure, secondary structure and surface structure change of protein after covalent with inulin. Emulsion properties of emulsion activity (EAI) and emulsion stability (ES)

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of WPI-inulin conjugates were assessed and both showed significantly enhanced

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compared with WPI at range of pH3 to pH7. AAPH+ scavenging test and ORAC measurement also revealed that covalent binding with inulin enhanced the antioxidant activities of WPI. This work presented the conjugation with inulin successfully enhanced the functional properties of WPI. Key words: Whey protein isolate; Inulin; Glycation; Emulsibility; Antioxidant

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Journal Pre-proof 1. Introduction As one natural food ingredient, whey protein isolate (WPI) has rich physiological activities and functional properties [1–3]. Thus, the WPI was a potential alternative in protein beverages, salad dressings, baking food, ice-cream and candy products as thickener, stabilizer and emulsifier. However, the poor solubility, emulsibility and gel property of WPI in the system with the pH near its Isoionic point (PI) limited its industrialized application. In addition, there are few applications in the oil/water (O/W)

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system for their poor oxidative stability[1,3–6]. Therefore, it is necessary to improve

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the functional properties of WPI for a wider range of use in the food industry.

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Glycation with polysaccharides through Maillard reaction, a non-enzymatic

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reaction, which formed a covalent bond between protein and polysaccharide has been reported as a excellent modification manner for protein to enhance its functional

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properties [2,3,7–10]. Qu et al. has successfully improved the application range of

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rapeseed protein isolate in the food industry by enhancing its emulsifying property via grafted with dextran[11,12]. Furthermore, accumulating evidences have demonstrated

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that Maillard reaction products were good natural emulsifiers in the O/W emulsion-containing foods for they possessed strong antioxidant activity and could prevent the degradation of active compounds or oil oxidation [13,14]. The natural polysaccharides obtained from plants have shown good antioxidant activity [15–18], and many kinds of

polysaccharides like dextran, pectin, carboxymethyl cellulose

have been applied to manufacture protein-polysaccharides conjugates [8,19–22]. Among these polysaccharides, inulin as one nature polysaccharide is linear type fructans, which is mainly made up of D-fructose molecules connected with β-(2, 1)-glycoside bonds and usually ends with one molecule of α-D-glucose residue. Due to there is no digestive enzyme in human digestive system can decompose β-(2, 1) 3

Journal Pre-proof bonds, inulin can reach intestine and consumed by the flora colonized in the large intestine to act as the prebiotics and produce biomass, short chain fatty acids (acetate, butyrate and propionate et al) [23–29]. However, there is limited information about WPI-inulin conjugates. In this study, WPI-inulin conjugates were manufactured by Maillard reaction under wet-heating conditions with different reaction time. Meanwhile, the role of glycation with inulin on the structure and functional properties change of WPI has

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been investigated.

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2. Materials and methods

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2.1. Materials

WPI was purchased from Hilmar Ingredients International Inc. (Hilmar, CA 95324,

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USA) with protein content of 91.38% (on dry basis), fat content of 1.06%, ash content

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of 2.325% and pH value of 6.54. Inulin (Shanghai, China) was obtained from Yuanye biotechnology Co., LTD. The corn oil (Shanghai, China) was supplied by Yihai Kerry

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group (Shanghai). Protein Marker with molecular weight range from 10 to 250 kDa for SDS-PAGE was purchased from Bio-Rad Inc. (California, USA). All reagents used in this work were of analytical grade. 2.2. Preparation of WPI-inulin conjugates The WPI-inulin conjugates was synthesized by the wet-heating method [21]. WPI solution (5 mg/mL) was made by deionized water and stirred for 2 h at 25 ºC. The WPI solution was mixed with inulin with weight ratio of 1:1 and stirred thoroughly for 2 h, followed by adjusted to the pH 9.0 by 0.1 M HCl and 0.1M NaOH. The WPI/inulin mixtures were incubated in water bath of 70 ºC for 2 h, 4 h and 6 h, to get the different glycation degree conjugates respectively. The reaction was stopped by 4

Journal Pre-proof put reaction vessel into ice-water bath. Then, after dialysis for 36 h at 4 ºC to remove unreacted inulin and other small molecules, WPI-inulin conjugates were lyophilized and stored under -20 ºC for use. 2.3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE analysis was operated by the method of previous report by Laemmli with slight modification [30]. After mixing with 3 times of loading buffer, the

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WPI-inulin conjugates were heated at 90 ºC for 5min then for electrophoresis. 10 µL

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volume of each sample solutions were loaded onto the gel and the working voltage

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was set at 180 V. When electrophoresis finished, gels were stained with Coomassie brilliant blue R-250 and destained by a solution constitute of 10% methanol and 10%

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2.4. UV spectroscopy

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acetic acid.

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UV absorption of of samples were scaled at 294 nm (characteristic absorption wavelengths for intermediate materials generation) and 420 nm (characteristic

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absorption wavelengths for brown intensity generation) [21] using a microplate reader (Molecular Device, CA , USA).

2.5. Determination of graft degree (GD) The covalent grafting degree of WPI-inulin conjugates were measured according to report of Guo et al [31]. Briefly, 1 mL TNBS (0.1% w/v) solution, 1 mL NaHCO3 solution (pH 8.5, 4%) and 1 mL of SDS solution (0.1%) were well mixed well with 1 mL sample solution (0.5 mg/mL) and incubated at 40 ºC for 2 h in dark place. 2 mL of HCl solution (0.1 mol/L) was used to finish the reaction. Absorbance at 340 nm was measured by a UV spectrophotometer for the solution. The grafting degree was calculated as following: 5

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GD (%) =

（1）

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Where, A1 means the absorbance value of control WPI solution, and A2 means that of glycated WPI solution. 2.6. Intrinsic fluorescence spectroscopy The intrinsic fluorescence spectra of WPI-inulin conjugates and WPI samples

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(1mg/mL) were measured by a fluorescence spectrophotometer (F-7000, Hitachi,

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Japan). For fluorescence excitation spectra, excitation wavelength was set from 250

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nm to 400 nm and the emission wavelength at 420 nm. For the fluorescence emission spectra, the excitation wavelength was set at 280 nm and emission wavelength range

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2.7. Amino acid content analysis

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from 300 to 600 nm with slit at 5 nm[31,32].

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Amino acid content of WPI-inulin and WPI samples were measured by the method of previous reports with some adjustment[11,21]. Protein samples were added into 15mL

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6 M HCl and hydrolyzed under nitrogen at 110±1℃ for 22h. After hydrolysis, the sample solutions were cooled down, and diluted using the 50mL volumetric flask. 1 mL of sample dilution was evaporated 2 times for dryness and redissolved with 1 mL pH 2.2 buffer solution. After samples filtered through the filter membrane of 0.22 um, the amino acid content of samples was analyzed with the automatic amino acid analyzer (A300 advanced, MembraPure, Germany). 2.8. FT-IR spectroscopy FT-IR spectra of WPI-inulin and WPI were analyzed by KBr-disk method [11,33]. Samples were well mixed with KBr and grinded to fine powder (2~3 µm), finally 6

Journal Pre-proof pressed into sheet. The spectra were gathered by a Vector 33 FT-IR Spectrometer (Burker, Germany) in the frequency domain of 4000- 400 cm-1 at a resolution of 2 cm-1. 2.9. Circular dichroism (CD) spectroscopy The second structure of WPI-inulin conjugates and WPI samples were collected by a CD spectrometer (J-810, JASCO, Japan). The sample was prepared at protein

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concentration of 1mg/mL using 10 mM sodium phosphate buffer (pH 7.0) at 25 ºC.

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The CD spectra were obtained from 190 to 260 nm with scanning rate of 100 nm/minutes under nitrogen. Data was revealed as the mean residue ellipticity (θ) in

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unit of deg cm2 dmol-1 [34]. The secondary structure compositions were generated

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using the CDNN program of the CD Pro software.

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2.10. Scanning electron microscopy (SEM) analysis

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Surface structure of WPI-inulin conjugates and WPI were obtained by SEM analysis [35]. Samples were fixed on the sample table with conductive double-sided

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adhesive tape, the excess powder was blown off, and gold was sprayed over the surface of samples under vacuum environment. Then appearance of samples was observed using the scanning electron microscope (JSM-7500F, JEOL, Japan). 2.11. ζ-Potential measurement ζ-Potential of WPI-inulin conjugates and WPI were determined using a Zetasizer Nano Series (Malvern Instruments, UK). Samples were diluted to 0.05% (w/v) at protein concentration with Milli-Q water before measurement [36]. 2.12. Surface hydrophobicity measurement (H0) Determination of H0 was according to the method of previous reports using 7

Journal Pre-proof 1-anilino-8-naphthale-nesulfonate (ANS) as the hydrophobic fluorescent probe [37]. Sample solutions were attenuated to a different protein concentration of 0.02-0.1% (w/v) with sodium phosphate-buffer solution (10Mm, pH7.0). Samples of 4 mL volume were reacted with 20uL ANS solution (1mM) for 15min, and then the fluorescence intensity of samples were acquired at excitation wavelength and emission wavelength of 390nm and 470 nm, respectively. H0 was determined by the

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initial slope of FI versus the sample concentration.

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2.13. Emulsifying activity (EAI) and emulsion stability (ES)

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Emulsifying activity (EAI) and emulsion stability (ES) of samples were measured by the turbidimetric method with slight modification [11,34]. In brief, corn oil of 2

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mL volume was added to 8 mL volume of 0.25% (w/v) protein solution with pH range

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of pH3.0-pH7.0. After a homogenization at 20000 rpm for 1min with a mechanical homogenizer (IKA ULTRA-RURRAX), emulsion from the bottom of the container

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was pipetted 50µL at 0 and 10 min respectively and diluted with 5 mL 0.1% (w/v)

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SDS solution. Absorbance of the diluted emulsion was measured at 500 nm. And EAI and ES indices were calculated as following: EAI (m2/g) = (

(2)

ES (min) =

(3)

Where N is the dilution factor (N=101)，ρ is the protein solution concentration(g/mL), φ is optical path ( φ=0.01 m), θ is the fraction of oil phase (θ=0.20), A0 is the absorbance of sample at 0 min, and At is the absorbance of sample at t min(t =10). 2.14. Assay for antioxidant scavenging activity

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Journal Pre-proof 2.14.1. ABTS radical scavenging assay The ABTS radical scavenging activity was conducted as Zeng et al. [38]. By reference, 7 mM ABTS was reacted with potassium persulfate of 2.5 mM for 16 h keep in dark place at 25 ºC. The working solution was diluted using pure water to get the absorbance of 0.70±0.02 at the wavelength of 734 nm. Working solution of 3 mL was stirred with 0.4 mL sample solutions, and the mixture was incubated at 25 ºC in

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dark for 30 min. The absorbance of samples at 734 nm was recorded by MAX i3

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microplate reader. The value of ABTS radical scavenging activity of samples was =

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assessed as following:

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(4)

water.

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Where A1 means the absorbance of samples, and A0 means the absorbance of pure

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2.14.2. Oxygen radical absorbance capacity (ORAC)

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The total antioxidant activity of the sample was determined by ORAC assay [39] . Trolox solution with a gradient concentration (6.25, 12.5, 25, 50 µM) was used for the standard curve. Both sample solution and Trolox solution were added into each well of black-walled 96-well plates for 20 µL, subsequently, 200 µL fluorescein sodium salt (0.0956 µM) was added for each well. The plate was put into the microplate reader and automatically shaken 10 s. After incubation, AAPH solutions (119.4 mM) of 20 µL volume were added. The fluorescence intensity was measured by microplate reader (Molecular Device, CA, USA) with excitation and emission wavelength of 485 nm and 538 nm respectively for 38 cycles every 3.5 min. All tests were repeated three times and the ORAC indices of samples were expressed as mean µM Trolox equivalent (TE) per gram of dry protein sample weight (DW). 9

Journal Pre-proof 2.15. Statistical analysis All experiments were measured in triplicate. And the SPSS 16.0 statistical analysis system (SPSS Inc., Chicago, IL) was used to conduct analysis of variance (ANOVA) with significant difference defined at p<0.05 by Tukey’s test. 3. Results and Discussion

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3.1. SDS-PAGE SDS-PAGE is an important method to analysis the protein molecule composition,

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from which different components were separated by molecular weight [30]. Fig. 1

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represented the electrophoretic result of WPI , WPI/inulin mixture, WPI heated alone

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and WPI-inulin conjugates (with reaction time of 2h, 4h, and 6h), respectively. By

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reference to the marker (lane M), the native WPI (lane 1) showed two major bands at around 13.9, 16.0 kDa ascribed to α-lactalbumin and β-lactoglobulin monomer

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repectively, which was consistent with previous study [1,2,19]. The band patterns of WPI heated alone (lane 6) and WPI-inulin mixture (lane 2) were similar to that of

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WPI (lane 1) and didn’t appear new bands making clear that physical mixture with inulin or heated alone didn’t form the new substances with high molecular weight. For the different time of WPI and inulin common heat-treated samples, new bands at the top zone were observed of the gel lanes (lane 3, 4, and 5) compared with WPI, which revealed new substances with higher molecular weight as protein-polysaccharides conjugates were generated. Obviously, much more new bands at the top zone were observed as the increase of the reaction time and the characteristic protein bands at around 13.9, 16.0 kDa were much paler correspondingly. The results suggested that the WPI-inulin conjugates with higher molecular weight were formed by wet-heating reaction might by the α-lactalbumin and β-lactoglobulin monomers of WPI, and much 10

Journal Pre-proof more protein -polysaccharides conjugates generated increasing with reaction time( 2h to 6h range). This results were in consistence with the report of previous reports [11,31]. 3.2. The degree of browning When in early Maillard reaction stage, intermediate compounds were formed. Among these compounds, early stage intermediate matter like amadori compounds

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which has characteristic absorption wavelength at 294nm and advanced stage

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products like melanoidins which presents brown color and can be monitored at

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wavelength at 420nm [21,40]. As shown in Fig. 2(A), in comparison to the absorbance of WPI heated alone didn’t significantly increase, the absorbance of

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WPI-inulin conjugates samples at 294 nm increased significantly over reaction time

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(2h to 6h), suggesting more amount of Amadori compounds formed between WPI and inulin over reaction time [14,21,41]. Pictures of the samples that WPI-inulin

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conjugates (2 h, 4 h and 6 h) all had brown color, and became darker as time

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extended also indicated that more intermediate matters generated over reaction time, which is consistent with the absorbance data. However, no significant changes of color change for WPI alone were observed during incubation, which indicated that WPI heated alone cann’t success formation of WPI-polysaccharide conjugates. The absorbance of WPI-inulin conjugates at 420nm didn’t significant higher than that of WPI heated alone, showing that wet-heat method is a good way to produce WPI-inulin conjugates without much brown color compounds formed. 3.3. Degree of glycation In the process of Maillard reaction, WPI and inulin were covalently linked between the carbonyl groups of the reducing end of inulin and the free amino groups 11

Journal Pre-proof of WPI. Thus, the degree of glycation of WPI could be measured by demining the decrese of free amino group content of WPI using TNBS method [20]. From fig. 2(B), we can see that The glycation degree of WPI when it glycated with inulin of different reaction time (2 h, 4 h, and 6 h, respectively). As the incubation time increased, the DG of WPI-inulin conjugates became larger, which is consistent with the results of SDS-PAGE. In addition, the DG of WPI-inulin conjugates of 4 h and 6 h has no significant difference, this suggested the reaction became gentle when the reaction

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time reached 4h, which was same as the UV absorbance at 420 nm.

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3.4. Fluorescence spectrum

Previous studies showed that there would generate fluorescent substances when

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proteins conjugated with polysaccharides through Maillard reaction [42]. The

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fluorescence of the fluorescent substances generated in Maillard reaction (the

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maximum excitation wavelengths are between 340 nm and 370 nm, and the maximum emission wavelengths are between 420 and 450 nm) is obviously different from that

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of native proteins (the maximum excitation wavelengths and maximum emission wavelengths for which are 290 nm and 336 nm, respectively) [31,42]. From Fig. 3 (A), it can be seen that all the samples have maximum excitation wavelengths at 290 nm, while WPI-inulin conjugates (2 h, 4 h, 6 h) have another maximum excitation wavelength at 344 nm, which indicated the formation of covalent binding between WPI and inulin. From Fig.3 (B), emission maximum wavelength was around 420 nm, which was in accordance with previous study. During the incubation, the emission maximum of the WPI-inulin conjugates shifted toward long wavelength direction. Furthermore, as incubation time extended, the FI of WPI-inulin conjugates became stronger which further confirmed the formation WPI-inulin conjugates. There might have polyreaction between reactive intermediary compounds to form more complex 12

Journal Pre-proof chemical structures during the wet-heat treatment. Guo et al studied WPI - sugar beet pectin conjugates using dry heating treatment presented the same change trend [20]. 3.5. Amino acid analysis The formation protein-polysaccharide conjugates was based on the glycation between amino groups and reducing-end carbonyl groups. Compared with native WPI, after glycation with inulin, the total hydrophobic and hydrophilic amino acids ratio of WPI-inulin didn’t show significant difference (shown in Table 1). However, compared

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to native WPI, the levels of lysine and arginine were both slightly decreased, which

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amino acids taking part in Maillard reaction.

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was in accordance with the fact that lysine and arginine of proteins were the main

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3.6. FT-IR spectrum

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The Fourier Transmittance Infrared Spectrum (FT-IR) was used to reflect the structure changes of proteins based on the hydrogen bonding force [39]. The protein

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has three important and typical absorption bands including 1700-1600 cm-1 band of

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amide I group corresponding to N-H bending, 1500-1550 cm-1 amide II group of protein structure and 1300-1200 cm-1 band of amide III group corresponding to N-H bending and C-N stretching vibrations [21]. As shown in Fig. 4, the absorption bands at 1636.30, 1540.85 and 1243.38 cm-1 were related to amide I, amide II and amide III group of protein respectively. And the intensity of these bands decreased after glycated with inulin. This was attributed to the loss of special groups such as –NH2 groups of the lysine in protein through the Maillard reaction, which has been proven by other studies [11,43]. Furthermore, at wavenumber of 1036.07 cm-1, absorption intensities of all WPI-inulin conjugates were distinctively higher than that of native WPI, which revealed newly formed C-N covalent bond has been generated in WPI-inulin conjugates was consist with previous study between rapeseed protein 13

Journal Pre-proof isolate (RPI) and dextran [11] and that between WPI and dextran [19]. 3.7. CD spectrum Circular dichroism (CD) was used to indicate the conformation change of proteins at the molecular level. Far-UV CD spectroscopy at the wavelength of 190-260 nm can especially offer the information about protein’s secondary structure conformation. The secondary structure conformation of WPI and WPI-inulin

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conjugates was shown in Fig.6 that noticeably peak position and height changed in the

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wavelength range of 190-250 nm, which displayed the effect of glycation with inulin

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on the secondary structure of WPI.

The secondary structure compositions change of WPI and WPI-inulin conjugates

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by CD spectra were shown in Fig. 5. Obviously, β-sheet and random coil were the

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major secondary structures in WPI (38.46% and 32.63%, respectively), which was consistent with the previous report [1]. After glycation with inulin, the β-sheet

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contents decreased and the content of random coil increased. The decrease of a-helix

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and β-sheet content and increase of random coil content were mainly due to the protein’s unfold and transform into random coil during the glycation of inulin. Previous study about the peanut protein isolate-polysaccharide conjugates also reported a same trend [7]. Qu et al pointed out that both glycation with polysaccharides and extending the glycation degree had an effect on the secondary structure conformation of RPI [11]. CD spectrum result indicated that glycation with inulin through wet-heat treatment affected secondary structure of WPI, which also consisted with above data of FT-IR analysis. 3.8. Scanning electron microscopy (SEM) In comparison to native WPI exhibited spherical surface conformation, WPI-inulin 14

Journal Pre-proof conjugates showed more sheet surface conformation with more incompact and porous (shown in Fig. 6). The surface structure of conjugates loosened much more as the incubation time increased. This may be due to two reasons , on one hand inulin attached to the surface of WPI let the conformation change from polysaccharides accumulated, on the other hand, the wet-heat treatment also made protein structure became unfold [32], which was consist with the results of FT-IR and CD analyses.

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3.9. ζ-Potential

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ζ-potential of emulsifier is one of the key factors that affect the stability of

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emulsions, as it has effect on electrostatic repulsive forces between emulsion droplets and the biomacromolecule conformation in the aqueous phase. Table 2 showed that

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the native WPI had ζ-Potential of -29.8 mV , while WPI-inulin conjugates had ζ

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-Potential range from -37.64 to -39.5 mV. This might because glycation with inulin

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changed the surface charge distribution of WPI, which was in consistent with researches of Safoura Pirestani et al. [36]. WPI-inulin conjugates had much largerζ

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-Potential value than native WPI, might have better ability to stabilize emusion . Larissa Consoli et al also reported thatζ-Potential of sodium caseinate after glycated with corn starch hydrolysates rangeed from -35 to -40 mV , during which could be deemed as appropriately steady on oil droplet surface in emulsion [44,45]. 3.10. Effect of glycosylation on surface hydrophobicity (H0) Fig. 7 showed that H0 index of WPI-inulin conjugates was higher than that of WPI (517). And the H0 value was increased from 1218 to 2500 as the extension of reaction time from 2 h to 6 h. This might due to glycation with inulin attached to the protein and wet-heat treatment let WPI conformation unfolding and expose more hydrophobic groups initially buried in the interior of protein molecules, which was in agreement 15

Journal Pre-proof with the conformational variation as described in Chen et al. [15] and Li et al. [46]. 3.11. Emulsifying property The EAI and ES indices are always used to measure the emulsifying property[11,34]. The EAI values of WPI and WPI-inulin conjugates with different glycation degree at pH range of 3.0-7.0 are shown in Fig. 8 (A). The EAI values of conjugates (17.50-21.77 m2/g) were significantly higher than WPI (5.22-18.48 m2/g)

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under selected pH values (3.0-7.0). This indicated that the emulsifying activity of WPI

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in the range from pH 3.0 to 7.0 was significantly improved by covalent binding with

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inulin, especially at the pH 4.0 and pH5.0, which are near the isoelectric point of WPI. This was mainly because the chain conformation extended and surface structure

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became looser of WPI-inulin conjugates through the glycation reaction, which

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promoted the biomolecule adsorption on the oil-water interface more quickly to

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improve the emulsifying activity[8,47].

The ES values of native WPI and WPI-inulin conjugates samples at pH 3.0 to pH

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7.0 are shown in Fig.8 (B). ES values of WPI-inulin conjugates (17.44-45.26 min) were much higher than that of WPI (11.69-17.01 min) under various pH values (pH3.0-7.0). This indicated that the emulsifying stability of WPI-inulin conjugates in the range of (pH3.0-7.0) was significantly improved by covalent reaction. Especially, at pH 4.0, near the PI of WPI, WPI showed the lowest ES of 11.69 min, while all the conjugates were from 18.78 to 20.08 min, significantly higher than that of WPI. This results showed that WPI-inulin conjugates had better emulsifying stability than WPI, which was consistent with many previous studies [11,34]. The secondary structure shown in FT-IR and CD, looser surface structure revealed by SEM of WPI-inulin conjugates promoted the enhancement of emulsifying properties after glycation with 16

Journal Pre-proof inulin. These structure change was beneficial for the conjugates to adsorb quickly and firmly on the oil-water interface, which also could improve the emulsifying properties[8,47]. In addition, WPI-inulin conjugates have stronger electrostatic repulsive forces than native WPI due to their relatively high interfacial charge (-37.7 to 39.5 mV). This property made WPI-inulin conjugates more stable to spread and adsorb on the emulsion droplets surface than native WPI and slow down the rate of

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droplet flocculation during emulsion formation [44,45].

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3.12. Antioxidant activities

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To prevent the oxidation in the emulsion system, one of the efficient pathways is to scavenging the free radicals [48,49]. As shown in Fig. 9(A), it can be seen that the

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ABTS radical scavenging activity of WPI-inulin conjugates were significantly higher

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than WPI and inulin within the concentration from 0.25 mg/mL to 1 mg/mL and showed concentration dependent manner. Especially at concentration of 1 mg/mL, the

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ABTS radical scavenging activity of WPI-inulin conjugates of 2 h, 4 h and 6 h were

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2.38-fold, 2.54-fold and 2.64-fold higher than that of WPI and 4.38-fold, 4.68-fold and 4.86-fold higher than that of inulin, respectively. The Oxygen radical absorbance capacity (ORAC) is another important evaluation for antioxidants which overall consideration the degree of inhibition of reactive oxygen species over time [39,49]. Fig.9 (B).revealed The TEAC values of WPI, conjugates of 2 h, 4 h, and 6 h, and inulin were 65.41±13.21, 302.63±62.10, 305.42±52.49, 312.26±66.98 and 6.16±2.51 µ TE per g, respectively. Obviously, the WPI-inulin conjugates had significant stronger ORAC than WPI and inulin, which was consistent with the ABTS result. Previous studies gave the explanation that during glycation with polysaccharides through Maillard reaction, some advanced 17

Journal Pre-proof stage components were generated, which had antioxidant activities and showed brown color because these compounds can serve as hydrogen donors in the process of scavenging free radicals [14,50]. The scavenging activity between conjugates also became stronger along over reaction time, which was in consistence with the results of WPI-inulin conjugates’ UV absorbance at 294nm and 420nm. 4. Conclusion

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In this study, WPI-inulin conjugates generation in aqueous solution system was

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subjected to wet-heat treatment at 70 ºC for 2 h, 4 h and 6 h respectively. SDS-PAGE,

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glycation degree, browning degree, fluorescence intensity and FT-IR spectrum were used to verify the formation of WPI-inulin conjugates, which mainly linked by C-N

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covalent binding. After glycation reaction, the secondary structure changed showing

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that α-helix, β-sheet contents decreased slightly, while random coil contents increased slightly. SEM also showed that surface structure became looser and more porous after

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glycation with inulin. These changes improved the both the functional characters

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including the EA, ES and physiological activities including ABTS radical scavenging and oxygen radical scavenging activity of WPI-inulin conjugates, which presented that wet-heat treatment of Maillard reaction was an efficient method to produce highly functional WPI-inulin conjugates in food system. Conflict of interest There are no conflicts to declare.

Acknowledgements Financial and moral assistances supported by the National Natural Science Foundation of China (31972022), Guangdong Basic and Applied Basic Research Foundation

(2019A1515011996),

China

Postdoctoral

Science

Foundation

(2018M643092), National Natural Science Foundation of China(31972011), the 18

Journal Pre-proof Guangzhou Science and Technology Program (201907010035), the Natural Science Foundation of Guangdong Province (2019A1515011670 ） , National Key R&D Program (2017YFD0400703), Guangzhou Science Technology and Innovation Commission (201803050001), Science & Technology Planning Project of Nansha, Guangzhou (2016GJ001), and 111 Project (B17018) to conduct the project are gratefully acknowledged.

G. Liu, Q. Zhong, Thermal aggregation properties of whey protein glycated with

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Table 1. Amino acid contents of native WPI and WPI-inulin conjugates with different reaction time Amino acid

Content(%) WPI

WPI-I (2 h)

WPI-I (4 h)

WPI-I (6 h)

3.97

3.28

3.29

2.45

Thr

7.89

7.99

7.1

7.69

Pro

0.37

0.44

0.54

0.23

Ala

8.36

8.33

8.25

Val

7.34

7.06

of

8.59

7.54

7.39

Ile

6.22

6.46

6.54

6.73

Leu

11.13

11.09

11.31

11.6

Phe

2.08

2.36

2.4

2.37

Total

47.36

46.97

47.05

10.04

9.96

10.42

6.19

5.77

5.99

15.9

16.47

16.21

16.39

2.74

2.93

3.32

3.07

1.76

1.45

1.65

1.59

Tyr

2.12

2.12

2.35

2.04

His

1.7

1.78

1.88

1.93

Lys

10.83

10.34

10.19

10.11

Arg

1.72

1.65

1.69

1.41

Total

52.62

52.97

53.02

52.95

9.8

Ser

6.05

Glu Gly Cys

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Asp

47.01

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Hydrophilic amino acids

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Met

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Hydrophobic amino acids

Note: The values in parenthesis are the change percentage points of amino acid contents in WPI-inulin conjugates compared to WPI. 26

Journal Pre-proof Table 2. ζ-potential of native WPI and WPI-inulin conjugates with different reaction time a ζ-potential (mV)

WPI

-29.87±1.73 b

WPI-I (2 h)

-39.50±1.81 a

WPI-I (2 h)

-38.80±0.28 a

WPI-I (2 h)

-37.67±1.96 a

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Different letters represented statistically significant difference (p < 0.05).

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a

Sample

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Fig.1 SDS-PAGE pattern of molecular weight marker (lane M), WPI (lane 1), WPI/inulin

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h), respectively.

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mixture (lane 2), WPI-I (2 h) (lane 3), WPI-I (4 h) (lane 4), WPI-I (6 h) (lane 5) and WPI (6

28

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Fig. 2 (A) Changes in absorbance at 294 nm and 420 nm of WPI, WPI-I (2 h), WPI-I (4 h), WPI-I (6 h), respectively. Different letters (A. B, C) means significantly different (P<0.05) for absorbance of 420 nm; Different letters (a. b, c, d, e, f) means significantly different (P<0.05) for absorbance of 294 nm. (B) Degree of glycation of WPI/inulin mixture samples with heated for different time( 0h, 2h, 4h and 6h respectively). 29

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Fig. 3 (A) Fluorescence excitation spectra and (B) Fluorescence emission spectra of WPI, WPI-I (2 h), WPI-I (4 h) and WPI-I (6 h), respectively.

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Fig. 4 FT-IR spectra of native WPI and WPI-inulin conjugates with different reaction time.

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Fig. 5 CD spectrum and secondary structure composition of native WPI and WPI-inulin

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conjugates with different reaction time.

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WPI-I (6 h).

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Fig. 6 Scanning electron microscopy data of (A) WPI; (B) WPI-I (2 h); (C) WPI-I (4 h); (D)

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2000

1500

1000

of

Surface hydrophobicity

2500

ro

500

0

W-I(2 h)

W-I(4 h)

-p

WPI

W-I(6 h)

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Fig. 7 The surface hydrophobicity of native WPI and WPI-inulin conjugates with different

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reaction time.

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Fig. 8 The emulsifying activity (A) and emulsifying stability (B) of WPI, WPI-I (2 h), WPI-I (4 h) and WPI-I (6 h) at pH (3.0-7.0), respectively.

36

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Fig. 9 The ABTS radical scavenging activity (A) and Oxygen radical absorbance capacity (B) of WPI, inulin, WPI-I (2 h), WPI-I (4 h) and WPI-I (6 h), respectively.

37

Journal Pre-proof Author Statement

Wang Wen-Duo: Conceptualization, Conducting the experiment Li Chao: Methodology Zhang Bin: Data curation Huang Qiang: Writing- Original draft preparation You Li-Jun: Editing Chen Chun: Investigation Fu Xiong: Supervision

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Liu Rui-Hai: Writing- Reviewing

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Highlight · Incubation of whey protein isolate with inulin under wet-heated conditions formed conjugates. · The emulsibility of whey protein isolate was greatly enhanced after grafted with inulin.

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· The whey protein isolate-inulin conjugates exhibited enhanced antioxidant activity.

39