Improved Low pH Emulsification Properties of Glycated Peanut Protein

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Improved low pH emulsification properties of glycated peanut protein isolate by ultrasound Maillard reaction Lin Chen, Jianshe Chen, Kegang Wu, and Lin Yu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00989 • Publication Date (Web): 22 Jun 2016 Downloaded from http://pubs.acs.org on June 28, 2016

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Journal of Agricultural and Food Chemistry

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Improved low pH emulsification properties of glycated peanut protein isolate by

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ultrasound Maillard reaction

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LIN CHEN †, JIANSHE CHEN ‡,§, KEGANG WU † AND LIN YU †,*

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†

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Guangzhou, China, 510006

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‡

College of Chemical Engineering and Light Industry, Guangdong University of Technology,

School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, China,

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310018

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§

School of Food Science and Nutrition, University of Leeds, LS2 9JT, Leeds, UK

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* corresponding author: Lin Yu, Prof.

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Tel/Fax: ＋86 203 9322 203.

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E-mail: [email protected]

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6 Figures

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2 Tables

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Running title: Emulsification properties of pea protein-maltodextrin conjugates

ACS Paragon Plus Environment


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ABSTRACT

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In this work, peanut protein isolate (PPI) was grafted with maltodextrin (MD) through the

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ultrasound-assisted Maillard reaction. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

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(SDS-PAGE) analysis showed link between PPI and MD. The substantially increased accessibility

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of the major subunits (conarachin, acidic subunit of arachin, and basic subunit of arachin) in PPI

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under high-intensity ultrasound treatment led to changes in the degree of graft (DG), zeta-potential,

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protein solubility, and surface hydrophobicity of conjugates. Emulsion systems (20 % v/v oil, 2.0 %

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w/v PPI equivalent, pH 3.8) formed by untreated PPI, PPI-MDC (PPI-MD conjugates obtained with

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wet-heating alone) and UPPI-MDC (PPI-MD conjugates obtained with ultrasound-assisted wet-

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heating) were characterized using light-scatter particle size analyzer and confocal laser scanning

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microscope. Results showed that emulsions of untreated PPI and PPI-MDC were not stable due to

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immediate bridging flocculation and coalescence of droplets, whilst that formed by UPPI-MDC

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with 32.4 % DG was stable with a smaller mean droplet size. It was believed that high-intensity

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ultrasound promoted production of glycated PPI that was soluble and surface active at pH 3.8 and

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thus improved emulsification properties for UPPI-MDC. This study showed that glycated PPI by

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ultrasound-assisted Maillard reaction is an effective emulsifying agent for low pH applications.

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KEYWORDS: peanut protein isolate; maltodextrin; ultrasound treatment; glycation; emulsification

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property

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INTRODUCTION

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Interest has developed in the use of peanut protein isolate (PPI) as emulsifying agent or emulsion

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stabilizer in formulated foods, ranging from sausages to vegetable creams (1–3). PPI is favoured

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because of the surface active properties of its major constitutive proteins: conarachin and arachin,

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which take up nearly 75 % of the total proteins (2). Furthermore, peanut protein is considered as

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good nutritional source for its high content of essential amino acids and has palatable taste (3).

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However, emulsifying behaviour of PPI is poor under certain conditions due to aggregation or

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precipitation of protein and the associated loss of colloidal stability. This instability is most

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pronounced under acidic pH near the isoelectric point (pI, 4.5) of peanut protein (4,5).

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Unfortunately, many emulsion-based foods (e.g., mayonnaise, salad dressings, cream beverages,

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sauces) are normally quite acidic (pH 3.6–4.6).

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One way to improve the solubility and emulsification properties of proteins under unfavourable

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aqueous conditions of low pH, is by direct conjugation with a hydrophilic polymer such as a non-

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ionic polysaccharide (e.g., maltodextrin, dextran) (6–11). The formation of these high-molecular-

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weight protein-polysaccharide conjugates combines the characteristic property of proteins to adsorb

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strongly to the oil-water interface with that of the polysaccharide for quick solvation in aqueous

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medium (12). A safe and practical way to achieve grafting of protein with polysaccharide is by

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using the Maillard reaction carried out under controlled dry-heating or wet-heating conditions (6–

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11). During this complex sequence of reactions, the terminal and side-chain amine groups on a

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protein molecule become covalently linked to the terminal reducing group of the carbonyl of

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polysaccharide (13).

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However, the grafting of protein-polysaccharide through the Maillard reaction is a time-

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consuming process using dry-heating or wet-heating alone, which usually takes several days (6–11).

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Moreover, previous studies found that peanut protein was generally resistant to graft reaction due to

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their compact quaternary and tertiary structures that protect many of the reactive amine groups (9,

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10). Therefore, new emerging technologies, avoiding high temperatures and/or long processing

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times, should be applied to improve the efficiency of graft reaction for peanut protein. Recently,

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several studies have reported that the grafting of protein-polysaccharide through the Maillard

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reaction could be substantially enhanced and accelerated under high-intensity ultrasound treatment

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(10, 14–16). For example, Li et al. (10) reported that after wet-heating at 80 ºC for 24 h, a DG

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(degree of graft) of 35.7 % was obtained by PPI-dextran conjugate; however, with the assistance of

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ultrasound, a significantly higher DG of 45.4 % was reached at a much shorter reaction time (40

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min). Stanic-Vucinic et al. (16) also observed such an sonocatalysis effect during the Maillard

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reaction of β-lactoglobulin with different kinds of saccharide. It has been suggested that local

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translational motions induced by ultrasound cavitation might allow reactive groups to be brought

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into closer proximity, resulting in more steady Maillard reaction (17). In addition, high-intensity

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ultrasound could disrupt the tertiary and quaternary structure of globular proteins, causing the

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exposure of reactive amine groups (i.e., free amine groups) for grfat reaction (10, 14).

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Although ultrasound-assisted Maillard reaction has been used to modify the emulsifying

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properties of food proteins (10, 14, 15), the experimental results reported in the literature seems

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ambiguous. As Li et al. (10) reported, ultrasound treatment caused an increase in both emulsifying

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activity index (EAI) and emulsion stability index (ESI) for PPI-dextran conjugates, while Mu et al.

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(14) observed that such a treatment caused a decrease in ESI for soy protein isolate-gum aracia

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conjugates. It should be noted that emulsifying properties in these studies were mainly characterized

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by EAI and ESI using turbidity meansurement. However, this relatively simple approach should be

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treated with caution, because the differences in turbidity between emulsions may not only reflect

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differences in droplet size but also in the state of the oil droplets and/or polymer aggregation, which

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may give incorrect conclusions about their emulsifying properties (6, 18). Among various

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experimental techniques, multi-angle static light-scattering and confocal laser scanning microscopy

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(CLSM) are usually preferred for determining droplet sizes and visualizing microstructures in fine

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emulsions (18) and are widely used not only for emulsification capability assessment of an

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emulsifying agent, but also for exploring fundamental mechanisms of emulsion formation and

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stabilization (19, 20). So far, however, few such work has been done to investigate the

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emulsification properties of glycated PPI.

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Therefore, the current study is designed to improve emulsification properties of PPI-MD

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conjugates made with ultrasound-assisted Maillard reaction and the possible mechanisms.

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Emulsification properties of different samples were studied by analyzing the droplet size and

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microstructure of freshly made O/W emulsions (20 vol.% oil, 2 % w/v PPI equivalent, pH 3.8) by

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light-sactter particle size analysis and CLSM. Results obtained from this study could provide a new

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possibility to food industry of improved emulsification functionality for low pH applications.

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MATERIALS AND METHODS

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Materials and Chemicals. PPI was prepared from low-temperature defatted peanut meal

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(Tianshen Bioprotein Co. Ltd., Taixing, China) according to the method of Liu et al. (9), with slight

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modifications. The protein content of PPI was 88.6 g/100 g of powder, which was determined by

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micro-Kjeldahl method (N × 6.25). MD sample (Glucidex IT 6) with a dextrose equivalent of 4.2

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was obtained from Roquette (Lestrem, France). According to the manufacturer, it had an average

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molecular weight Mw of 8.1 kDa. O-Phthaldialdehyde (OPA), 6-propionyl-2-(N,N-dimethy-amino)

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naphthalene (PRODAN) and Nile Red were obtained from Sigma Chemicals (St. Louis, MO, USA).

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All other chemicals used in this study were at least of analytical grade. Water purified with a Milli-

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Q filtration unit (Millipore, Bedford, UK) was used for the preparation of solutions. The pH was

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adjusted using 0.1–1.0 M HCl solutions and 0.1–1.0 M NaOH solutions.

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Preparations of PPI-MDC and UPPI-MDC. Based on the previously reported method (11), PPI

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(10.0%, w/v) and MD (10.0%, w/v) were fully dispersed in phosphate buffer solution (0.2 M, pH

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7.0, 0.02% (w/v) sodium azide to prevent bacterial growth) by magnetically stirring at 4 ºC

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overnight. The following day, an ultrasound processor (XingDongLi Ultrasonic Electron Equipment

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Co. Ltd, Guangzhou, China) with a 1.5 cm diameter titanium probe was used to sonicate 150 mL of

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PPI-MD dispersions in 250 mL tall beakers that were immersed in a temperature-controlled shaking

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water bath set at 70 ºC and 10 rpm. Samples were sonicated with a power output of 250 W at a

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frequency of 20 kHz and an amplitude of 95 % (wave amplitude of 121 µm at 100 % amplitude) for

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different time (10, 20, 40, 60, 80, 100 min). Pulsed sonication (2 s on and 2 s off) was used to

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minimize the intrinsic heating effect of ultrasound and ensured that samples remained within ± 5 ºC

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of the ultrasonic bath temperature. After ultrasound-assisted wet-heating, the PPI-MD dispersions

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were cooled to room temperature and dialyzed at 4 ºC for 24 h. Finally, the resulting products were

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lyophilized, finely ground and kept in a desiccator for further use. The same PPI-MD dispersion

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was treated by wet-heating alone at 70 ºC for different times (1, 4, 12, 24, 32, 48 h), and the

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subsequent procedure was the same to ultrasound treatment. As controls, PPI was treated under

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glycation conditions in the absence of MD as described above. Samples were designated

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subsequently according to the grafting methods used and the DG obtained. For example, UPPI-

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MDC-32.4% means PPI-MD conjugate was prepared with ultrasound-assisted wet-heating and had

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a DG of 32.4 %.

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Degree of Graft (DG) Measurements. Free amine groups were measured using the o-

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phthaldialdehyde (OPA) assay according to the method of Vigo et al. (21), with slight modifications.

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OPA of 40 mg was dissolved in 1 mL methanol and mixed with 25 mL of 10 mM sodium

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tetraborate, 100 µL of β-mercaptoethanol, and 2.5 mL of 20% (w/v) sodium dodecyl sulphate (SDS),

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and was then diluted to a final volume of 50 mL with deionized water to form the OPA reagent. 4

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mL of OPA reagent was added into 200 µL of sample solution (10 mg/mL). After incubated at 35

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°C for 2 min, the absorbance at 340 nm was measured immediately. A calibration curve was made

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using 0.25–2 mM L-Lysine. DG was calculated as follow: DG = (Ac – At)/ A0 × 100%, where Ac is

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the level of free amine groups in controls; At is the level of free amine groups in conjugates; A0 is

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the level of free amine groups in untreated PPI.

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Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). SDS-PAGE

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was carried out using a precast 4–15 % gradient polyacrylamide gel (Bio-Rad Laboratories,

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Hercules, CA) according to the method of Liu et al (7), with slight modifications. Samples were

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dissolved in a SDS-PAGE sample buffer (catalog no. 161-0737, Bio-Rad Laboratories) to a

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concentration of 1 µg/µL. The mixture was shaken for 10 s and heated at 95 °C for 5 min. After

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centrifuging at 12,000 × g for 10 min, fifteen microliters of each sample were applied to the gel

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lanes for electrophoresis running in a Mini-protean IV system (Bio-Rad Laboratories). Afterward,

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the gel sheets were stained for protein with 0.2 % Coomassie Brilliant Blue G-250 and were stained

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for carbohydrate with 0.5% periodic acid fuchsin (PAS).

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Protein Solubility (PS) Measurements. The PS refers to the percentage of soluble protein

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against original protein presented in samples, and is measured as described by Petruccelli et al. (22).

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Zeta Potential (ζ-potential) Measurements. The ζ-potential of protein samples was measured

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using a Zetasizer Nano ZS (Malvern Instruments, Worcs, UK). Sample dispersions (0.2 %, w/v)

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were adjusted to pH 7.0 or 3.8, and then were magnetically stirred at room temperature for 2 h. The

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resulting dispersions were filtered through a 0.45 µm HA Millipore membrane prior to analysis.

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Based on an analysis of particle electrophoretic mobility measurements, the ζ-potential of samples

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was automatically calculated by the instrument.

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Surface Hydrophobicity (H0) Measurements. An unchanged fluorescent probe, PRODAN, was

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used to measure the H0 of different samples at pH 7.0 or 3.8 according to the method of Haskard et

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al. (23), with slight modifications. For buffers at pH 7.0 and 3.8, a mixture of 0.1 M citric acid and

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0.2 M Na2HPO4 were used in the following proportions (v/v), respectively: 3.5/16.5, 12.9/7.1.

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Sample dispersions were serially diluted with appropriate buffers at pH 7.0 or 3.8 to a final

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concentration of 0.004–0.02% (w/v), and then were centrifuged (12,000 × g, 20min) to obtain the

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supernatants. Ten microliters of PRODAN solution (1.5 mmol/L in the same buffer) was mixed to 4

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mL aliquots of diluted samples. After 15 min in the dark, the fluorescence intensity (FI) was

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measured at 465 nm (emission) using an excitation at 365 nm. A plot of initial slope of the FI versus

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protein concentration (mg/mL) plot was used as an index of surface hydrophobicity (H0).

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Emulsion Preparation and Characterization. Dispersions of different samples were adjusted to

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pH 3.8, and were magnetically stirred at room temperature for 2 h to ensure complete hydration,

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with 0.02% (w/v) sodium azide added to retard microbial growth. The subsequently quoted pH

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referred to that of sample dispersions. The dispersions contained 2.0 % (w/v) PPI equivalent for

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samples of pure protein or Maillard conjugates. O/W emulsions containing 20 vol.% sunflower seed

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oil were made using a high-pressure jet homogenizer operating at a pressure of 300 bar.

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Droplet size distributions (DSD) of freshly made emulsions were determined using a Mastersizer

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2000 particle size analyzer (Malvern Instruments). Refractive indices of water and sunflower seed

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oil were taken as 1.330 and 1.462, respectively. The average droplet size was reported as volume

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mean diameter d43 = ∑nidi4/∑nidi3, where ni is the number of droplets of diameter di.

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Microstructure of freshly made emulsions was visualized using an upright Leica TCS SP2

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confocal laser scanning microscope (CLSM) (Leica, Heidelberg, Germany) operating in

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fluorescence mode. A 63× water-immersion objective (NA 1.20) was used to scan the samples. Nile

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Red dye (Twenty microliters of 0.01% w/v dye in polyethylene glycol mixed with 5 mL of emulsion)

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was used to stained the emulsion oil phase, with fluorescence excited with an Argon laser line (488

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nm). Because only oily substances in emulsions were stained by Nile Red, the oil phase appeared as

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bright blobs, whilst the water/protein phase appeared dark in all the CLSM images.

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Statistical Analysis. All the tests were performed in triplicate, and results were expressed as

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mean ± standard deviation. Duncan’s multiple-range test was applied to identify significant

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differences between results (p < 0.05).

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RESULTS AND DISCUSSION

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Effects of Ultrasound Treatment on the Graft Reaction between PPI and MD. In order to

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analyze the effect of ultrasound treatment on the graft reaction between PPI and MD, the degree of

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graft (DG) and SDS-PAGE profiles of conjugates prepared with and without ultrasound treatment

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were investigated. Table 1 shows the DG values of PPI-MDC and UPPI-MDC prepared at different

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reaction times. A DG of 26.8 % was obtained by PPI-MDC prepared with wet-heating alone for 48h.

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In contrast, with the assistance of ultrasound, a significantly (p < 0.05) higher DG of 35.6 % was

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obtained by UPPI-MDC at a much shorter reaction time of 100 min. These results were similar with

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previous studies and confirmed that ultrasound treatment was an highly effective way to accelerate

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and enhance the graft reaction between proteins and polysaccharides (10, 14–16). It was reported

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that ultrasound posed a sonocatalysis effect during the protein-polysaccharide Maillard reaction,

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which made the occurence of protein glycation more readily (16). In addition, high-intensity

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ultrasound could disrupt the tertiary and quaternary structure of globular proteins, causing the

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exposure of more reactive amine groups (i.e., free amine groups) for the Maillard reaction (10, 14,

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15).

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SDS-PAGE has been proved to be a reliable method to establish the covalent link between

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proteins and polysaccharides during the Maillard reaction (9, 10). Fig. 1 shows the SDS-PAGE

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profiles of untreated PPI-MD mixture, PPI-MDC and UPPI-MDC prepared at different reaction

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times. Protein components stained by Coomassie blue appeared as blue and polysaccharide

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components stained by PAS appeared as pink. We can see that for both PPI-MDC and UPPI-MDC,

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with the increase of reaction time, the polysaccharide components was increasingly attached to the

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protein components, forming characteristic new polydisperse bands, suggesting that the PPI was

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indeed complexed with MD to form conjugates with high molecular weight. The positions of

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protein bands in Fig. 1A are in accordance with polysaccharide bands in Fig. 1B provides further

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evidence for strong linkage between PPI and the MD.

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On the other hand, as shown in Fig. 1A, there were five major bands presented in the SDS-

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PAGE profile of untreated PPI (lane 1), referring to S1−S5, respectively. Band S1 at around 64 kDa

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was identified as the subunit of conarachin. Bands S2−S4 (approximately 40, 39, and 37 kDa) were

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identified as the acidic subunits of arachin (AS-arachin), and band S5 (around 25 kDa) was

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identified as the basic subunit of arachin (BS-arachin) (24). Under wet-heating condition, different

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subunits in PPI showed different grafting accessibilities: a gradual disappearance in the

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characteristic band for the conarachin (S1) could be observed as the graft reaction proceeded from

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60 min to 24 h; AS-arachin (S2−S4) and BS-arachin (S5) appeared highly resistant to graft reaction,

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and were still identifiable after incubation for 24 h. In contrast, under ultrasound-assisted wet-

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heating condition, conarachin, AS-arachin and BS-arachin turned out to be grafted with MD more

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readily: after incubated for 60 min, conarachin and AS-arachin disappeared almost completely; the

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band intensity of BS-arachin in UPPI-MDC was noticably weaker than that in PPI-MDC. It can be

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inferred from these observations that the accessibility of the major subunits in PPI to graft with MD

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were substantially increased under ultrasound treatment, which made UPPI-MDC obtain higher DG

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at a much shorter reaction time as compared with PPI-MDC.

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Zeta-Potential of PPI-MDC and UPPI-MDC. In order to investigate the changes in

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electrostatic interactions of PPI after different treatments, we measured the ζ-potentials of

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dispersions of untreated PPI, PPI-MDC and UPPI-MDC at pH 7.0 and 3.8. As shown in Table 2 the

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absolute magnitude of the ζ-potential was significantly (p < 0.05) lower at pH 3.8 (+21.3 mV) than

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that at pH 7.0 (-35.2 mV) for untreated PPI dispersion. This indicated that peanut protein carried

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less net charge at acidity near its pI (pH 4.5), which might weaken the intra- and inter- molecular

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electrostatic repulsions, promote the folding of protein, enhance the protein-protein aggregation and

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decrease the protein-water interaction (25). In contrast, after 20 min of ultrasound treatment, UPPI-

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20min dispersion showed a higher negative ζ-potential (-43.7 mV) at pH 7.0, which might suggest

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that ultrasound treatment induced the exposure of charged groups previously hidden in protein

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structures, as also reported by Gülseren et al. (26). However, at pH 3.8, the ζ-potential of UPPI-

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20min dispersion was not significantly (p > 0.05) different from that of untreated PPI dispersion.

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After glycation, the magnitude of the ζ-potential of PPI-MDC and UPPI-MDC dispersions both

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decreased gradually with increasing DG (Fig. 2). This may be because the ζ-potentials were

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measured at the slip plane of the PPI-MD conjugates, which is the edge of the MD layer. But the

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charge was on the protein which was several nanometers away from the slip plane. As the MD layer

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got denser, more charge on the protein would be shielded, thus reducing the measured protein

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charge (8). It can be seen that UPPI-MDC dispersions showed a relatively lower ζ-potential than

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PPI-MDC dispersions at high DG, which may suggest that more peanut protein in UPPI were

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grafted with MD, as also observed in SDS-PAGE profiles.

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Protein Solubility of PPI-MDC and UPPI-MDC. Protein solubility is probably the most

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important influencing factor on its functionality. Poor solubility is often accompanied with poor

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functionality. As shown in Table 2, The PS of untreated PPI was 79.6 % at pH 7.0, and decreased to

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43.5 % at pH 3.8, which may be due to the aggregation of peanut protein near its pI (pH 4.5). UPPI-

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20min showed a PS of 45.4 % at pH 3.8, which suggested that ultrasound treatment had a limited

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effect on improving the PS of PPI at acidity near its pI.

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The PS of PPI-MDC and UPPI-MDC as a function of DG at pH 3.8 is shown in Fig. 3. After

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glycation, with the increase of DG, the PS of both PPI-MDC and UPPI-MDC increased gradually,

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and then decreased slightly at high DG. The higher PS of protein-polysaccharide conjugates than

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the original protein around pI could be attributed to the steric effects provided by the glycated MD,

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as suggested by Liu et al. (7). The nonionic MD used in this study kept almost completely soluble at

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pH 3.8, and provided steric repulsion as a dominant mechanism account for the improved PS of

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glycated PPI around its pI. However, it is noteworthy that UPPI-MDC showed higher PS than PPI-

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MDC, even though they had similar DG values. For example, UPPI-MDC-21.7% showed a PS of

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60.3 % at pH 3.8, significantly higher than that of PPI-MDC-22.5% (PS = 52.3 %). These results

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suggest that ultrasound-assisted Maillard reaction exerted a marked effect on improving the PS of

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glycated PPI at acidity around its pI. This finding may be due to the fact the grafting accessibility of

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the major subunits in PPI (conarachin, AS-arachin and BS-arachin) were increased under ultrasound

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treatment, which made more peanut protein in UPPI can be grafted readily with MD and keep

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soluble around its pI. However, both PPI-MDC and UPPI-MDC showed a slight decrease in PS at

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high DG. This finding was similar with some previous studies that denaturation and aggregation of

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peanut proteins may occur at long incubation periods during the Maillard reactoin, resulting in the

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decrease of PS (6, 9).

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Surface Hydrophobicity of PPI-MDC and UPPI-MDC. In order to accurately predict protein

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functionality, quantitative analysis of protein surface hydrophobicity is essential. Table 2 shows that

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the H0 of untreated PPI was 537.6 at pH 7.0, and decreased to 423.2 at pH 3.8. It has been reported

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that compared to a more hydrophobic and flexible molecule at neutral pH, peanut protein underwent

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specific structural changes characterized by a tighter conformation at acidic pH (25). This is to be

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expected since the intra- and inter- molecular electrostatic repulsions decreased near the pI of

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peanut protein as demonstrated before, which could lead to the folding/aggregation of protein

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molecules and therefore the loss of hydrophobic groups on protein surface. In contrast, UPPI-20min

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showed significantly higher H0 at both pH 7.0 (581.3) and 3.8 (457.5), which might be due to the

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exposure of previously buried hydrophobic groups after ultrasound treatment, as suggested

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previously (27, 28). In addition, this finding may imply that the compacted structure of peanut

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protein could be effectively unfolded by high-intensity ultrasound, causing the exposure of

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hydrophobic/hydrophilic groups as well as the reactive amine groups previously buried. The H0 of

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of PPI-MDC and UPPI-MDC as a function of DG at pH 3.8 is shown in Fig. 4. After glycation, the

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H0 of PPI-MDC decreased markedly with increasing DG. The reduced H0 after glycation is also

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reported in the literature, which might be due to (1) the shielding effect of the polysaccharide chain

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bound to proteins, (2) burial of effective hydrophobic regions due to protein aggregation during the

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glycation, and (3) formation of advanced glycation products or melanoidins with little surface

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hydrophobicity (7, 9, 29). As a consequence, PPI-MDC exhibited very low H0 at high DG, although

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increased PS at pH 3.8. On contrast, it is noteworthy that when the glycation was conducted under

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ultrasound treatment, the H0 of UPPI-MDC increased at initial stage, and then decreased relatively

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moderately with a further increase in DG. The reason for this may be due to the fact that the

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ultrasound treatment unfolded globular peanut protein and/or dissociated protein aggregates,

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causing the exposure of hydrophobic groups previously buried. More significantly, with the

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assistance of ultrasound, more peanut protein could be grafted readily with MD and kept stable at

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acidic pH as demonstrated before, resulting in the preservation of more hydrophobic groups on

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protein surface. We can see that compared with the untreated PPI, some UPPI-MDC (DG = 18.7–

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32.4 %) had much increased PS at pH 3.8, but still maintained comparably high H0.

297

Emulsification Properties of PPI-MDC and UPPI-MDC. An effective emulsifying agent

298

should rapidly lower the interfacial tension at the freshly created oil-water interface during

299

emulsification, thus facilitating droplet fromation, and quickly form an interfacial membrane at the

300

droplet surface, effectively protecting the newly created droplets against immediate bridging

301

flocculation and coalescence. Therefore, the emulsification property of an emulsifying agent refers

302

to its ability to generate and stabilize fine droplets during emulsification (18). In this study, the

16


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303

emulsification properties of different samples were assessed according to average droplet size d43

304

and microstructure of freshly made O/W emulsions (20 vol.% oil, 2 % w/v PPI equivalent, pH 3.8)

305

as measured by light-sacttering particle size analysis and CLSM. The d43 of emulsions formed by

306

PPI-MDC and UPPI-MDC at pH 3.8 is plotted as a function of DG in Fig. 5, and CLSM images of

307

some selected sample emulsions with their DSD superimposed are shown in Fig.6. Emulsions

308

formed by untreated PPI and UPPI-20min both showed a relatively big d43 of ca. 21 µm at pH 3.8.

309

Based on CLSM observation, these emulsions were found to be highly flocculated, containing some

310

big flocs/droplets (see Fig. 6A and Fig. 6D). These findings suggest that PPI alone exhibited a poor

311

emulsification property at acidity near the pI of peanut protein, which was consistent with previous

312

studies (4, 5). This may be mainly attributed to the aggregation of peanut protein induced by

313

inadequate electrostatic repulsion under conditions close to pI, resulting in the loss of protein

314

solubility and the formation of insoluble protein particles. It should be noted that under the turbulent

315

flow conditions of homogenization, protein particles could also adsorb onto the surface of newly

316

created droplets (30). However, compared to soluble proteins, protein particles are less effective in

317

covering oil droplets due to their much higher saturation surface load and much slower diffusion.

318

Consequently, emulsions tend to have bridging flocculation (the sharing of adsorbed particles or

319

macromolecules amongst adjacent droplets) and coalescence during and immediately after the

320

homogenization (31, 32), as illustrated here in the emulsion formed by PPI alone at pH 3.8.

17



321

However, d43 of emulsions formed by PPI-MDC and UPPI-MDC showed gradual decrease

322

gradually at initial stage, and then sharply increased at high DG. This finding is in accordance with

323

some previous research and suggests that controlled glycation by the Maillard reaction improved

324

the emulsification property of PPI, but excessive glycation was detrimental (6, 14, 33). Emulsions

325

formed by PPI-MDC showed a minimum d43 of 14.6 µm at DG 18.3 %. Microstructure observation

326

showed that flocculation still occurred in the emulsion formed by PPI-MDC-18.3% (see Fig. 6B),

327

although at a less extent as compared with that in untreated PPI emulsion. In addition, an increase in

328

droplets with small size (ranging from 0.1 to 1 µm) was observed. It seems that PPI-MDC-18.3%

329

contained some conjugates that are soluble and surface active at pH 3.8, enabling the formation and

330

stabilization of fine droplets. However, with only 53.5% PS for PPI-MDC-18.3% at pH 3.8, it is

331

obvious that emulsifying agent is not sufficiently available for full surface coverage of newly

332

formed droplets. Therefore, it is reasonable to conclude that wet-heating of PPI with MD could only

333

induce a limited improvement in emulsification property under acidic pH.

334

In contrast, emulsions formed by UPPI-MDC showed a minimum d43 of ca. 6.0 µm at DG of

335

18.7–32.4 %. Microstructure observation showed that the emulsion formed by UPPI-MDC-32.4%

336

was homogeneous with no sign of flocculation (Fig. 6E). Compared with PPI-MDC-18.3%

337

emulsion, UPPI-MDC-32.4% emulsion contained many more fine droplets ranged between 0.1 and

338

10 µm. These results clearly showed that glycation of PPI with MD using ultrasound-assisted wet-

339

heating was more effective in improving the emulsification property of PPI at acidic pH than that

18


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340

using wet-heating alone. As has been shown that under ultrasound treatment, the grafting

341

accessibility of the major subunits (conaracin, AS-arachin and BS-arachin) in PPI was substantially

342

increased, so more peanut protein in PPI could be readily grafted with MD and remained soluble

343

and surface active around its pI. As a result, compared with untreated PPI and PPI-MDC-18.3%,

344

UPPI-MDC-32.4% showed a much increased PS (65.3 %) and a comparably high H0 (409.5) at pH

345

3.8, and might contain a lot of more conjugates that were soluble and surface-active at pH 3.8, a

346

highly feasible explanation for its much improved emulsification property under acidic pH.

347

However, it was observed that the d43 of emulsions formed by PPI-MDC and UPPI-MDC both

348

increased strongly at high DG. In addition, both CLSM images and DSD measurements showed the

349

appearance of many big droplets in emulsions formed by PPI-MDC-26.8% (Fig. 6C) and UPPI-

350

MDC-35.6% (Fig. 6F). This phenomenon might arise from the strong decrease in H0 at high DG for

351

both PPI-MDC and UPPI-MDC, resulting in the loss of surface activity for the conjugates. It has

352

been reported that some techniques (e.g. static high pressure, microwave heating) used to accelerate

353

the the Maillard reaction between proteins and polysaccharides tended to promote the formation of

354

advanced glycation products or melanoidins, which had shown to possess poor surface activity (34–

355

36). By contrast, it seems that high-intensity ultrasound is a more appropriate technique to promote

356

the Maillard reaction for the functional modifications of globular proteins, since the extent of the

357

Maillard reaction could be well controlled in terms of DG as demonstrated in this study.

19



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358

In summary, this study demonstrated that with the assistance of ultrasound, the grafting between

359

PPI and MD through the Maillard reaction was markedly accelerated and enhanced in terms of DG.

360

Under ultrasound-assisted wet-heating condition, the major graft-resistant subunits of conarachin,

361

AS-arachin and BS-arachin in PPI, became more readily grafted with MD through the Maillard

362

reaction. Compared with PPI-MDC, UPPI-MDC showed a stronger increase in PS and a more

363

moderate change in H0 at pH 3.8 during the Maillard reaction, resulting in the production of some

364

UPPI-MDC with much increased PS and comparably high H0. Fresh emulsions formed by untreated

365

PPI and PPI-MDC were unstable due to the bridging flocculation and coalescence of droplets

366

during homogenization, whilst that formed by UPPI-MDC with 32.4 % DG was stable and had a

367

smaller average droplet size d43, suggesting its remarkably improved emulsification properties at

368

acidity near the pI of peanut protein. In conclusion, glycated PPI could be an effective emulsifying

369

agent for acidic emulsion-based foods. High-intensity ultrasound is an efficient technique to

370

promote the Maillard reaction for the functional modifications of globular proteins. But how could

371

ultrasound treatment make PPI readily graft with MD is still not fully clear, and future work on this

372

topic is needed.

373

374 375

376

ACKNOWLEDGEMENTS This work was supported by National Research Fund for Doctoral Program of China (No. 20134420120008)

and

Guangdong

Provincial

Science

and

20


Technology

Project

(No.

Page 21 of 36

377


2013B020311015). We thank Professors E. Dickinson and B. Murray for useful discussions.

378

379

LITERATURE CITED

380

(1) Zhao, X. Y.; Chen, J.; Du, F. L. Potential use of peanut by-products in food processing: a

381

review. Int. J. Food Sci. Tch. 2012, 49, 521–529.

382

(2) Neucere, N. J.; Conkerton, E. J. Some physicochemical properties of peanut protein isolates. J.

383

Agric. Food Chem. 1978, 26, 683–686.

384

(3) Ghatak, S. K.; Sen, K. Peanut proteins: Applications, ailments and possible remediation. J. Ind.

385

Eng. Chem. 2013, 19, 369–374.

386

(4) Wu, H.; Wang, Q.; Ma, T.; Ren, J. Comparative studies on the functional properties of various

387

protein concentrate preparations of peanut protein. Food Res. Int. 2009, 42, 343–348.

388

(5) Liang, H. N.; Tang, C. H. pH-dependent emulsifying properties of pea [Pisum sativum (L.)]

389

proteins. Food Hydrocolloids 2013, 33, 309–319.

390

(6) Diftis, N.; Kiosseoglou, V. Physicochemical properties of dry-heated soy protein isolate-dextran

391

mixtures. Food Chem. 2006, 96, 228–233.

392

(7) Liu, G.; Zhong, Q. X. Glycation of whey protein to provide steric hindrance against thermal

393

aggregation. J. Agric. Food Chem. 2012, 60, 9754–9762.

394

(8) Wooster, T. J.; Augustin, M. A. β-Lactoglobulin-dextran Maillard conjugates: Their effect on

395

interfacial thickness and emulsion stability. J. Colloid Interf. Sci. 2006, 303, 564–572.

21



396

(9) Liu, Y.; Zhao, G. L.; Zhao, M. M.; Ren, J. Y.; Yang, B. Improvement of functional properties of

397

peanut protein isolate by conjugation with dextran through Maillard reaction. Food Chem. 2012,

398

131, 901–906.

399

(10) Li, C.; Xue, H. R.; Chen, Z. Y.; Ding, Q.; Wang, X. G. Comparative studies on the

400

physicochemical properties of peanut protein isolate-polysaccharide conjugates prepared by

401

ultrasonic treatment or classical heating. Food Res. Int. 2014, 57, 1–7.

402

(11) Dan, Z.; Srinivasan, D.; Lucey, J. A. Physicochemical and emulsifying properties of whey

403

protein isolate (WPI)-dextran conjugates produced in aqueous solution. J. Agric. Food Chem. 2010,

404

58, 2988–2994.

405

(12) Evans, M.; Ratcliffe, I.; Williams, P. A. Emulsion stabilisation using polysaccharide-protein

406

complexes. Curr. Opin. Colloid Interface Sci. 2013, 18, 272–282.

407

(13) Oliver, C. M.; Melton, L. D.; Stanley, R. A. Creating proteins with novel functionality via the

408

Maillard reaction: A review. Crit. Rev. Food Sci. Nui. 2006, 46, 337–350.

409

(14) Mu, L. X.; Zhao, M. M.; Yang, B.; Zhao, H. F.; Cui, C.; Zhao, Q. Z. Effect of ultrasounic

410

treatment on the graft reaction between soy protein isolate and gum acacia and on the

411

physicochemical properties of conjugates. J. Agric. Food Chem. 2010, 58, 4494–4499.

412

(15) Zhang, B.; Chi, Y. J. J.; Li, B. Effect of ultrasound treatment on the wet heating Maillard

413

reaction between β-conglycinin and maltodextrin and on the emulsifying properties of conjugates.

414

Eur. Food Res. Technol. 2014, 238, 129–138.

22


Page 22 of 36

Page 23 of 36


415

(16) Stanic-Vucinic, D.; Prodic, I.; Apostolovic, D.; Nikolic, M.; Velickovic, T. C. Structure and

416

antioxidant activity of β-lactoglobulin-glycoconjugates obtained by high-intensity-ultrasound-

417

induced Maillard reaction in aqueous model systems under neutral conditions. Food Chem. 2013,

418

138, 590–599.

419

(17) Gogate, P. R. Cavitational reactors for process intensification of chemical processing

420

applications: a critical review. Chem. Eng. Process 2008, 47, 515–527.

421

(18) McClements, D. J. Critical review of techniques and methodologies for characterization of

422

emulsion stability. Crit. Rev. Food Sci. Nutr. 2007, 47, 611–649.

423

(19) Dickinson, E. Food colloids research: historical perspective and out look. Adv. Colloid Interf.

424

Sci. 2011, 165, 7–11.

425

(20) Moschakis, T., Murray, B. S., Dickinson, E. Particle tracking using confocal microscopy to

426

probe the microrheology in a phase-separating emulsion containing nonadsorbing polysaccharide.

427

Langmuir 2006, 22, 4710-4719.

428

(21) Vigo, M. S.; Malec, L. S.; Gomez, R. G.; Llosa, R. A. Spectrophotometric assay using o-

429

phthaldialdehyde for determination of reactive lysine in dairy products. Food Chem. 1992, 44, 363–

430

365.

431

(22) Petruccelli, S.; Añón, M. C. Partial reduction of soy proteins isolate dissufide bonds. J. Agric.

432

Food Chem. 1995, 43, 2001–2006.

433

(23) Haskard, C. A.; Li-Chan, E. C. Y. Hydrophobicity of bovine serum albumin and ovalbumin

23



434

determined using uncharged (PRODAN) and anionic (ANS-) fluorescent probes. J. Agric. Food

435

Chem. 1998, 46, 2671–2677.

436

(24) Chiou, R. Y. Y. Effects of heat treatment on peanut arachin and conarachin. J. Food Biochem.

437

1990, 14, 219–232.

438

(25) Gharsallaoui, A.; Cases, E.; Chambin, O.; Saurel, R. Interfacial and emulsifying characteristics

439

of acid-treated pea protein. Food Biophys. 2009, 4, 273–280.

440

(26) Gülseren, Đ.; Güzey, D.; Bruce, B. D.; Weiss, J. Structural and functional changes in

441

ultrasonicated bovine serum albumin solutions. Ultrason. Sonochem. 2007, 14, 173–183.

442

(27) O’sullivan, J.; Murray, B.; Flynn, C.; Norton, I. The effect of ultrasound treatment on the

443

structural, physical and emulsifying properties of animal and vegetable proteins. Food

444

Hydrocolloids 2016, 53, 141–154.

445

(28) Zhang, Q. T.; Tu, Z. C.; Xiao, H.; Wang, H.; Huang, X. Q.; Liu, G. X., et al. Influence of

446

ultrasonic treatment on the structure and emulsifying properties of peanut protein isolate. Food

447

Bioprod. Process. 2014, 92, 30–37.

448

(29) de Oliveira, F. C.; Coimbra, J. S. R.; de Oliveira, E. B.; Zuñiga, A. D. G.; Rojas, E. E. G. Food

449

protein-polysaccharide conjugates obtained via the Maillard reaction: a review. Crit. Rev. Food Sci.

450

Nutr. 2016, 56, 1108–1125.

451

(30) Dickinson, E. (2010). Food emulsions and foams: Stabilization by particles. Curr. Opin.

452

Colloid Interface Sci. 2010, 15, 40–49.

24


Page 24 of 36

Page 25 of 36


453

(31) Dickinson, E. Flocculation of protein-stabilized oil-in-water emulsions. Colloids Surf. B. 2010,

454

81, 130–140.

455

(32) Tcholakova, S.; Denkov, N. D.; Lips, A. Comparison of solid particles, globular proteins and

456

surfactants as emulsifier. Phys. Chem. Chem. Phys. 2008, 10, 1608–1627.

457

(33) Martinez-Alvarenga, M. S.; Martinez-Rodriguez, E. Y.; Garcia-Amezquita, L. E.; Olivas, G. J.;

458

Zamudio-Flores, P. B.; Acosta-Muniz, C. H., et al. Effect of Maillard reaction conditions on the

459

degree of glycation and functional properties of whey protein isolate-Maltodextrin conjugates. Food

460

Hydrocolloids 2014, 38, 110–118.

461

(34) Xu, C. H.; Yu, S. J.; Yang, X. Q.; Qi, J. R.; Lin, H.; Zhao, Z. G. Emulsifying properties and

462

structural characteristics of β-conglycinin and dextran conjugates synthesised in a pressurised liquid

463

system. Int. J. Food Sci. Tech. 2010, 45, 995–1001.

464

(35) Niu, P. P. Meissner, K.; Erbersdobler, H. F. Maillard reaction in microwave cooking:

465

comparison of early Maillard products in conventionally and microwave-heated milk. J. Sci. Food

466

Agr. 1996, 70, 307–310.

467

(36) Tu, Z. C.; Hu, Y. M.; Wang, H.; Huang, X. Q.; Xia, S. Q. Microwave heating enhances

468

antioxidant and emulsifying activities of ovalbumin glycated with glucose in solid-state. J. Food

469

Sci. Technol. 2015, 52, 1453–1461.

470

471

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472

FIGURE CAPTIONS

473

Fig. 1. Changes of SDS-PAGE profiles of PPI-MDC and UPPI-MDC prepared at different reaction

474

times. S1: conarachin; S2–S4: acidic subunits of arachin; S5: basic subunits of arachin; M,

475

molecular weight markers.

476

Fig. 2. Effects of DG on zeta-potentials (ζ-potential) of PPI-MDC and UPPI-MDC at pH 3.8.

477

Fig. 3. Effects of DG on protein solubility (PS) of PPI-MDC and UPPI-MDC at pH 3.8.

478

Fig. 4. Effects of DG on surface hydrophobicity (H0) of PPI-MDC and UPPI-MDC at pH 3.8.

479

Fig. 5. Effects of DG on average droplet size (d43) of fresh emulsions (20 vol.% oil, 2.0 % w/v PPI

480

equivalent) formed by PPI-MDC and UPPI-MDC at pH 3.8.

481

Fig. 6. Microstructure of fresh emulsions (20% vol.% oil, 2 % w/v PPI equivalent) formed by PPI-

482

MDC and UPPI-MDC with selected DG values at pH 3.8: (A) untreated PPI; (B) PPI-MDC-18.3%;

483

(C) PPI-MDC-26.8%; (D) UPPI-20min: (E) UPPI-MDC-32.4%; (F) UPPI-MDC-35.6%. DSD is

484

superimposed on the microimages, with horizontal scale showing particle size (µm).

26


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485


Table 1. DG values of PPI-MDC and UPPI-MDC prepared at different reaction times* PPI-MDC

486

UPPI-MDC

Time (h)

DG (%)

Time (min)

DG (%)

1

9.5 (±0.5) i

10

3.2 (±0.4) j

4

12.7 (±0.4) h

20

9.3 (±0.7) i

12

18.3 (±0.7) g

40

21.7 (±0.5) f

24

22.5 (±0.6) f

60

32.4 (±0.6) c

32

24.6 (±0.4) e

80

34.1 (±0.3) b

48

26.8 (±0.3) d

100

35.6 (±0.4) a

* Results having different letters are significantly different (p < 0.05).

27



Page 28 of 36

487

Table 2. Protein solubility (PS), surface hydrophobicity (H0) and zeta-potential (ζ-potential) of

488

different samples* Samples

489

PS (%)

H0

ζ-potential (mV)

PPI (pH 7.0)

79.6 (±0.3) b

537.6 (±4.7) b

-35.2 (±0.3) b

PPI (pH 3.8)

43.8 (±0.4) f

423.2 (±5.2) d

+21.3 (±0.4) c

UPPI-20 min (pH 7.0)

83.6 (±0.5) a

581.3 (±6.4) a

-43.7 (±0.4) a

UPPI-20 min (pH 3.8)

45.4 (±0.6) e

457.5 (±6.6) c

+21.6 (±0.5) c

PPI-MDC-18.3% (pH 3.8)

53.5 (±0.4) d

366.2 (±4.5) f

+13.4 (±0.5) d

UPPI-MDC-32.4% (pH 3.8)

65.3 (±0.6) c

409.5 (±4.3) e

+9.1 (±0.4) e

* Results within a column having different letters are significantly different (p

Improved Low pH Emulsification Properties of Glycated Peanut Protein

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