Gum Arabic mediated synthesis of glyco-pea protein hydrolysate via

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Food and Beverage Chemistry/Biochemistry

Gum Arabic mediated synthesis of glyco-pea protein hydrolysate via Maillard reaction improves solubility, flavor profile, and functionality of plant protein fengchao zhao, Zhongyu Yang, Jiajia Rao, and Bingcan Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04099 • Publication Date (Web): 22 Aug 2019 Downloaded from pubs.acs.org on August 26, 2019

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Journal of Agricultural and Food Chemistry

1 2

Gum Arabic mediated synthesis of glyco-pea protein hydrolysate

3

via Maillard reaction improves solubility, flavor profile, and

4

functionality of plant protein

5

Fengchao Zha1, Zhongyu Yang2*, Jiajia Rao1, Bingcan Chen1* 1 Department

6 7

2

of Plant Sciences, North Dakota State University, Fargo, ND 58108, USA

Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND 58108,

8

United States

9 10 11 12 13 14 15 16 17 18 19

*

To

whom

correspondence

should

be

addressed.

[email protected]; [email protected]

ACS Paragon Plus Environment

Tel.

(701)

231-9450,

e-mail:


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20

ABSTRACT

21

Pea protein hydrolysate (PPH) is successfully conjugated with gum arabic (GA)

22

through Maillard-driven chemistry. The effect of cross-linking conjugation on the

23

structure, solubility, volatile substances, emulsification, and antioxidative activity of

24

glyco-PPH is investigated, and found to improve all properties. The formation of

25

glyco-PPH

26

electrophoresis (SDS-PAGE), Fourier-transform infrared (FTIR), and scanning

27

electron microscopy (SEM). Size exclusion chromatography-multi angle light

28

scattering (SEC-MALS) unveils that the maximum molecular mass of glyco-PPH

29

occurs after 1 day of conjugation and approximately 1.2 mole of gum arabic

30

conjugates on one mole of PPH. Headspace solid-phase microextraction gas

31

chromatography-mass spectrometry (HS-SPME-GC-MS) reveals the odor changes of

32

glycoprotein before and after cross-linking. We have also prepared oil-in-water

33

emulsions using glyco-PPH which have enhanced physical stability against pH

34

changes and chemical stability against lipid oxidation. The mechanism proposed

35

involves Maillard-driven synthesis of the cross-linked PPH-GA conjugates which

36

increase the surface hydrophilicity and steric hindrance of glyco-PPH. These findings

37

could provide a rational foundation for tailoring the physicochemical properties and

38

functionalities of plant-based protein, which are attractive for food and functional

39

materials applications.

40

KEYWORDS: glycoprotein, pea protein hydrolysate, Maillard-driven, conjugation,

41

volatile substance, emulsification, antioxidant

is

confirmed

by

sodium

dodecyl


sulfate-polyacrylamide

gel

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42

INTRODUCTION

43

Owning to the combined characteristics of low allergens and lipids, as well as

44

high versatility and abundance, plant-based pea protein has been breaking into the

45

mainstream as a critical functional protein contender to supplant animal protein 1.

46

Since the presence of certain anti-nutritional factors and high level of fibrous material

47

in pea proteins can somehow be digestive discomfort 2,3, especially for people with

48

sensitivity or poor tolerance to intact proteins, pea proteins are often enzymatic

49

hydrolyzed commercially to break down the intact proteins into peptides to enhance

50

its digestibility and absorbability 4. The resulting pea protein hydrolysate (PPH) is

51

able to deliver the optimal performance and allow for nutritional and simultaneously

52

functional contribution to food, pharmaceutical, and cosmetic industry

53

plant proteins, several major obstacles restrict consumer acceptability of PPH and the

54

derived products. Off-flavors of plant proteins caused by the oxidative degradation of

55

unsaturated fatty acid in protein-lipid complexes during the storage and processing of

56

pea imposes restrictions on their utilization

57

followed by acid (isoelectric) precipitation is performed commercially to manufacture

58

plant proteins; this extraction process, however, could lower the solubility of final

59

products 8. Other functionalities including emulsification, foaming, and gelation

60

properties are impaired accordingly as they are highly associated with protein

61

solubility

62

high in solubility and low in off-flavors.

63

8,9.

4,7.

5,6.

Like other

In addition, alkaline extraction

Consequently, there is a tremendous demand for plant protein that are

Conjugation of polysaccharide with plant proteins through Maillard-driven



64

reaction, a so-called cooking chemistry, has become a promising green chemistry to

65

ameliorate the general functionality of proteins

66

conjugates via Maillard-driven reaction involves the Amadori rearrangement of Schiff

67

base adducts of carbonyl-containing polysaccharides with protein -amino groups in

68

early stage

69

Maillard-driven reactions were conducted on animal-based proteins focusing

70

primarily on characterizing the functionality of the conjugates; only a few studies

71

have been done on the formation mechanisms. Our recent research has shown that

72

Maillard-driven reaction allows plant-based protein, pea proteins concentrate (PPC) or

73

isolate (PPI) with low initial solubility, to be covalently linked with Gum arabic (GA)

74

in a dry state, thus enhancing their emulsification and solubility particularly around

75

the isoelectric point (IEP) of the protein. Equally important, conformational changes

76

of pea proteins after conjugation could potentially impact the level of off-flavors, as

77

well as the formation of some pleasant aroma-active volatiles derived from the

78

Amadori rearrangement and Strecker degradation 15,16.

13,14.

10–12.

The formation of glycoprotein

Unfortunately, most protein-polysaccharide conjugation studies via

79

Leveraging on the exceptional functionality (e.g., hydrophilicity, steric hindrance,

80

and viscosity) offered by polysaccharides, and coupling the unique volatile aroma of

81

glycoprotein mediated by polysaccharides could provide enhanced functionality and

82

flavor profile of plant-based proteins. However, the use of protein hydrolysate for

83

glycoprotein synthesis via Maillard-driven reaction is not well explored. In this work,

84

PPH, for the first time, was selected to conjugate with gum arabic via Maillard-driven

85

chemistry. Comparing to animal proteins pea protein has the advantages of


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17,

86

environmental sustainability, cultural acceptability, and low-cost accessibility

87

while comparing to PPC or PPI, PPH has the advantages of greater nutritional

88

efficacy (digestibility and absorbability). Gum arabic is selected because it is a natural

89

polysaccharide with high biocompatibility and biodegradability comparing to

90

synthetic organic compounds. Therefore, we are improving plant protein functionality

91

as functional resources via a mild Maillard-driven green chemistry. Our goals were to

92

systematically understand the cross-linking mechanisms and the relationship between

93

glyco-PPH structure and functionality. Gum arabic mediated synthesis of glyco-PPH

94

was controlled by conjugation time. Thus, the structural characteristics of glyco-PPH

95

was fully characterized and compared with the raw PPH. Additionally the

96

functionality

97

physicochemical stability of emulsion it prepared was evaluated. We found that upon

98

mild conjugation, not only the solubility and flavor issues were alleviated but also the

99

functionality, including emulsification and antioxidant activity of PPH are improved.

100

These findings are useful for the rational modification and generation of food proteins

101

as well as protein-based functional biomaterials.

102

MATERIALS AND METHODS

103

Materials

of

glyco-PPH,

including

volatile

substances,

solubility,

and

104

Both PURIS pea protein 870 (PPI) and PURIS pea protein 870H (PPH, the

105

hydrolysates of PPI; protein ~80%, moisture ~6%, ash ~5%, carbohydrate ~6%, lipid

106

~ 8%) were obtained from Cargill. TIC Pretested® gum arabic Spray Dry Powder

107

(moisture content ~6.7%, GA content ~90%, protein ~3.2%, and minerals ~ 0.2%)



108

was kindly offered by TIC Gums (Belcamp, MD). Mazola® Corn oil (saturated fatty

109

acid ~14.3%, monounsaturated fatty acid ~28.6%, polyunsaturated fatty acid ~57.1%)

110

was purchased locally. All other chemicals were of analytical grade. All bulk samples

111

were used as received.

112

Gum arabic mediated synthesis of glyco-pea protein hydrolysate

113

The glycoprotein was synthesized as described by Zha and co-workers with a 16,18.

114

slight modification

115

mass ratio of 1:4, followed by the hydration in deionized water (1:2, w/v) for 24 h on

116

a stir plate (300 rpm) at room temperature (22 °C). The pH of the hydrated mixture

117

was adjusted to 7.0 which was lyophilized to dryness (Lyophilizer, SP scientific,

118

Gardiner, New York). Five grams of lyophilized mixture was transferred in a VWR

119

clear glass straight-sided jar (60 mL). The jar was uncovered and set on a perforated

120

porcelain plate in a desiccator. The relative humidity and temperature of the

121

desiccator was maintained at 79% by saturated KBr solution and 60 °C by a

122

pre-heated incubator (Heratherm IMH180, Thermo Fisher Scientific, Inc., USA),

123

respectively. Maillard-driven conjugation was performed with variable time (0, 1, 3,

124

and 5 days) to prepare glyco-PPH with different structure.

125

Characterization of structure and degree of conjugation

126

Briefly, PPH and Gum arabic (GA) were firstly mixed at a

The structure of PPH-GA and degree of conjugation were characterized following 16,18.

127

our previous studies

Briefly, Amadori compounds and melanoidins formation,

128

free amino groups in PPH were recorded using a Shimadzu UV-1100

129

spectrophotometer (Shimadzu Corp., Kyoto, Japan). Color development (L*, a*, b*)


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130

was measured with a Minolta CR-310 Chroma Meter (Osaka, Japan). Sodium dodecyl

131

sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with a

132

Bio-Rad Mini-Protein apparatus III (Bio-Rad Laboratories Inc., Richmond, CA).

133

Fourier Transform Infrared Spectroscopy-Attenuated Total Reflection (FTIR-ATR)

134

was applied on a Varian 600-IR series spectrometer (Varian, Palo Alto, CA), and

135

scanning electron microscopy (SEM) of glyco-PPH was characterized with a

136

Cressington 108 auto sputter (Ted Pella Inc., Redding, CA) coupled with a JEOL

137

JSM-6490LV scanning electron microscope (JEOL USA, Peabody, MA)

138

Determination of Molecular Weight by Size Exclusion Chromatography with

139

Multiangle Laser Light Scattering (SEC-MALLS)

140

Accurately weighed glyco-PPH obtained (0.10 g) was dissolved in 10 mL PBS

141

(10 mM, pH 7.0). The sample solution was hydrated for 2 h, followed by a

142

centrifugation at 2,000 g for 30 min. The supernatant was then filtered through a 0.45

143

μm nylon filter to remove any insoluble precipitation or dust. Glyco-PPH solution

144

was separated by an Agilent 1200 HPLC using a tandem array of a polySep-GFC-P

145

(35×7.8mm) and polySep-GFC-P linear (300×7.8mm) columns (Phenomenex,

146

Torrance, CA, USA). One hundred microliter of sample was injected, and eluted

147

using PBS buffer (10 mM, pH7.0) at a flow rate of 0.3 mL/min. Elution from columns

148

was monitored sequentially with a DAD detector (280 nm), a refractive index detector

149

(Agilent 1362 A), and a DAWN HELEOS II multiangle laser light scattering detector

150

(Wyatt Technology, Santa Barbara, CA) equipped with a helium-neon laser (λ = 661

151

nm).



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152

The gum arabic refractive index increment (dn/dc) was set at 0.141 mL/g

153

according to the previous study 19. A known dn/dc (0.174 mL/g) of NaCl was used to

154

calibrate the refractive index detector (RI). Data accumulated by the UV, RI, and

155

MALLS detectors were analyzed by the ASTRA 7.1.2.5 software (Wyatt Technology).

156

The SEC-MALLS measurement was carried out at room temperature (22°C). The number of Gum arabic molecules (N) attached to each PPH molecule was

157 158

calculated using the following formula 20;

159

N = (Mw2 - Mw1)/ Mw3

160

Mwi = [∑ (Fi Mi)/ ∑Fi]

——

——

——

(1)

——

——

——

(2) ——

161

Where Mw1, Mw2, and Mw3 are the average molecular masses of monomeric

162

PPH, glyco-PPH, and Gum arabic, respectively; Fi is the proportion of fraction, and

163

Mi is the molecular mass of the fraction.

164

Volatile substances in glyco-pea protein hydrolysate

165

The volatile substances in glyco-PPH were sampled by headspace solid-phase

166

microextraction (CTC Analytics, Zwingen, Switzerland), separated by an Agilent

167

7890B gas chromatography, and identified by an Agilent 5977A mass spectrometry

168

on the basis of the NIST database. The detailed parameters can be found in our

169

previous work 16.

170

Relative solubility of glyco-pea protein hydrolysate

171

Protein solubility was determined according to our previous work without any

172

modification

173

Bradford

21.

16,

and protein concentration was determined following the method of

The solubility was expressed as the percentage of the initial PPH


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174

concentration.

175

Corn oil-in-water emulsion prepared by glyco-pea protein hydrolysate

176

A course corn oil-in-water emulsion was prepared by mixing 2 wt% corn oil with

177

98 wt% emulsifier solution (0.20 wt% glyco-PPH in 10 mM, pH 7.0 PBS buffer)

178

using a high-speed blender (M133/128-0, Biospec Products, Inc., ESGC, Switzerland)

179

for 2 min. A fine emulsion with reduced particle size was prepared by passing the

180

course emulsion through a two-stage high-pressure valve homogenizer (LAB 2000,

181

APV-Gaulin, Wilmington, MA) at first and second stage pressure of 5,000 and 500

182

psi, respectively, for three times. In order to prevent microbial growth during

183

emulsion storage, 0.04% of sodium azide was added to the final emulsions. The

184

emulsions prepared by the same amount of PPH or the mixture of PPH and GA were

185

used as controls.

186

Physical stability of emulsions against pH changes

187

The stability of emulsions against pH (2.0–8.0) changes was determined by

188

measuring the particle size and ζ-potential of emulsions after 30 min storage at room

189

temperature (22°C). The particle size was directly determined using a Mastersizer

190

3000 from Malvern (Malvern Instruments Ltd., U.K.) and reported as the

191

volume-weight mean diameter (d43 = ∑nidi4/∑nidi3), where ni was the number of

192

droplets of diameter di. The ζ-potential (mV) of droplets was measured using a

193

Malvern Nano-ZS (Malvern Instruments Ltd., U.K).

194

Lipid oxidation kinetics of emulsions

195

Primary oxidation marker lipid hydroperoxides were quantified using a method



196

adapted from Chen, McClements and Decker without any modofication 22. Secondary

197

oxidation product marker hexanal was determined using the methods described by

198

Zhao and coworkers without any modification

199

quantified using a calibration curve prepared from an authentic standard (LOD: 7.89

200

ng/mL). The lag phase is defined as the time at which a sudden increase of hexanal

201

formation is observed.

202

Statistical analysis

18.

The concentration of hexanal was

203

At least two independent experiments was conducted to prepare the fresh samples.

204

All measurements were performed with triplicate samples. The values reported herein

205

were means ± standard deviation (SD) of triplicates from fresh samples. The data were

206

analyzed using SAS version 9.4 (SAS institute Inc. Cary, NC). One-way analysis of

207

variance (ANOVA) was conducted and significant difference among the treatment

208

was defined at p < 0.05 by Tukey’s test.

209

RESULTS AND DISCUSSION

210

Structure characterization of glyco-pea protein hydrolysate

211

To confirm the formation of glyco-PPH, SDS-PAGE, FTIR and SEM (Fig.1 A, B

212

& C) were employed to determine molecular weight and structure changes of the

213

conjugates at different conjugation time.

214

Figure 1 inserted

215

One of the characteristic bands presenting in raw PPI is 2S albumin (Fig. 1A lane

216

1) that constitutes a light (~4.5 kDa) and a heavy (~10 kDa) polypeptide chains 9.

217

Additionally, the monomer of 11S legumin consisting of one subunit of convicilin


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218

(~72.4 kDa), one (~28.7 kDa) subunit of 7S vicilin, as well as acidic (~40 kDa)

219

subunit was identified in lane 1

220

globulin and albumin are the primary constituents in pea protein that involved in the

221

conjugation with GA

222

proteins were cleaved into small peptides ( 1-octen-3-ol (1.10 ppm) > 1-octen-3-one (1.07 ppm) > 3-methyl-1-butanol

445

(0.45 ppm) > acetophenone (0.40 ppm) > 2-pentylfuran (0.19 ppm). Based on the

446

thresholds of these volatiles (40-164 ppb)

447

(OAV) of these volatiles was above 1, and hence were considered as main

448

flavor-active components. Upon covalent cross-linking between PPH and GA, the

449

concentration of the beany flavor markers in glyco-PPH reduced significantly, and

450

more than two-fold less than in PPH even after 1 day of conjugation. Extending the

451

conjugation time could greatly reduce the beany flavors in glyco-PPH. The reduction

452

of beany flavor compounds in glyco-PPH might be related to structural reorientation

453

and conformational change of PPH (SEM results) upon conjugation resulting in the

454

release of hydrophobic beany flavors compounds.

455

Solubility and emulsification properties of glyco-pea protein hydrolysate

44

were quantified (Table 2). The eluting profiles of selected

45,

it is concluded that odor activity value

456

As aforementioned, low solubility of pea protein is the biggest challenge to

457

incooperate them into liquid food. The solubility of PPH and glyco-PPH with

458

different conjugation time was measured (Fig. 5A).

459

Figure 5 inserted



460

The solubility of PPH-GA mixture was similar to that of PPH (p>0.05) which

461

meant the absence of physicochemical interactions between PPH and GA at a neutral

462

pH. The solubility of PPH significantly improved from 19.4 % to 26.2 % after 1 day

463

of conjugation with GA. A sudden decline occurred as conjugation time exceeded to 3

464

days. In terms of the solubility of PPH alone, a consistently downward trend was

465

exhibited as PPH subjected to the same conjugation time. Clearly, overreaction of

466

crosslinking that occurs during Maillard reaction under prolonged time is responsible

467

for the decrease of its solubility. Thus, modulating conjugation time is of great

468

importance to synthesize glyco-PPH with desirable solubility.

469

The emulsification property of glyco-PPH produced with different conjugation

470

time (0, 1, 3, and 5 days) was compared by measuring the particle size of corn

471

oil-in-water emulsion it stabilized (Fig. 5B). A U shape pattern for the changes of

472

particle size in emulsion stabilized by glyco-PPH was observed. The particle size (d43)

473

of PPH stabilized corn oil-in-water emulsions was 20.7 μm (Fig. 5B), which was far

474

bigger than other particle size in emulsions stabilized either by PPH-GA mixture or

475

glyco-PPH. A layer of creaming was observed in PPH stabilized emulsions compared

476

to others (Fig. 5B inserted image). The mixture of PPH-GA was able to reduce the

477

particle size of emulsion to 5.08 μm, presumably because of the inherent

478

emulsification property of GA. The emulsion droplet size (d43) stabilized by

479

glyco-PPH with 1 day of conjugation was significantly reduced to 0.75 μm, which

480

suggested that emulsification property of PPH can be substantially enhanced upon

481

covalent cross-linking with GA via Maillard reaction. However, such enhancement


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482

was vanished in emulsion stabilized by the glyco-PPH with 3 or 5 days of conjugation,

483

which resulted in the increased particle size of emulsions. In general, the emulsion stabilized by pea proteins is unstable against pH changes

484 485

46.

486

various pHs (2–8), glyco-PPH synthesized after 1 day of conjugation was used to

487

prepare corn oil-in-water emulsion as it can form the smallest droplets (0.75 μm). The

488

impact of pH on the physical stability of the emulsions (PPH and mixture as controls)

489

was investigated by measuring the particle size (d43) and ζ-potential (Fig. 5C). As can

490

be seen, serious phase separation was visualized in PPH stabilized emulsions across at

491

pH 2–5; whereas macroscopic stable emulsions was observed in both PPH-GA

492

mixture and glyco-PPH stabilized emulsions at pH 2–8 (Fig. 5C). In addition, the

493

particle size of PPH stabilized emulsion was significantly larger than those prepared

494

by glyco-PPH or PPH-GA mixture at a same pH. A similar particle size of the

495

emulsions prepared by glyco-PPH or mixture was detected at pH 5–8 (p > 0.05). The

496

particle size (d43) of emulsions stabilized by PPH-GA mixture was considerably

497

increased at pH 3 or below, corresponding to a reduced emulsification effect of GA at

498

an acidic condition 47. By contract, particle size of emulsions prepared by glyco-PPH

499

remained steady across a broad pH range (2–8) suggesting that the presence of GA to

500

be covalently cross-linked with PPH can effectively enhance the physical stability of

501

emulsions against pH.

To examine if glyco-PPH could improve the physical stability of emulsion under

502

In order to elucidate the mechanism of the enhanced physical stability of

503

emulsion stabilized by glyco-PPH, we compared the ζ-potential of the emulsions



504

under different pH (Fig. 5C). PPH stabilized emulsions exhibited a high ζ-potential

505

(–36.5 mV) at pH 6 indicating a strong electrostatic repulsion between emulsion

506

droplets; this, however, did not warrant a greater physical stability, again denoting the

507

poor emulsification property of PPH. The IEP of PPH was ~3.5 and the conjugation

508

with GA lowered it to 2.5 (Fig. 5C). The modification of available amino groups in

509

protein might be responsible for the lowered IEP of glyco-PPH 48. The ζ-potential of

510

the emulsion stabilized by glyco-PPH or PPH-GA mixture had no significant

511

difference across the entire pH range (p > 0.05) (Fig. 5C). Consequently, the

512

similarity in electrostatic interactions cannot interpret the difference of physical

513

stability between glyco-PPH and PPH-GA mixture based emulsions at acidic pH (2 &

514

3). Alternatively, the attachment of GA on PPH could improve the steric hindrance in

515

emulsion droplets covered by glyco-PPH. The enhanced steric repulsion could

516

prevent emulsion droplets from flocculation and phase separation by counteracting the

517

van der Waals force between emulsion droplets at pH close to the IEP.

518 519

Oxidative stability of corn oil-in-water emulsions stabilized by glyco-pea protein hydrolysate

520

The iron binding capacity of anionic polysaccharides may exert preventive effect

521

against emulsion oxidation. However, when they are coated on emulsion droplets,

522

negative surface charge of droplets has the potency to bring transition metals (e.g.,

523

Fe2+) into close proximity to hydroperoxides (LOOH), thus accelerating emulsion

524

oxidation by decomposing LOOH and producing rancid flavors. This was a particular

525

concern in glyco-PPH stabilized emulsions as it had the highest negative surface


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526

charge at pH 6–8. And, hence, the oxidative stability of emulsions stabilized by

527

glyco-PPH after 1 day of conjugation was examined by measuring the formation of

528

LOOH and hexanal in emulsion during storage at 37°C (Fig. 6 A & B). Fig. 6 inserted

529 530

LOOH in PPH stabilized emulsion increased slightly after 3 days of storage, so

531

did the generation of hexanal (Fig. 6 A&B). Similarly, the development of LOOH in

532

emulsion prepared by PPH-GA mixture was boosted after 3 days of storage; however,

533

a significant increase in hexanal occurred after 7 days of storage. This results

534

suggested that the presence of GA in PPH-GA mixture stabilized emulsion extended

535

the lag phase of emulsion in terms of hexanal formation. In terms of glyco-PPH based

536

emulsion, the level of LOOH retained constant after 5 days of storage while a

537

considerable increase tendency appeared after 6 days of storage. That indicated

538

glyco-PPH synthesized by 1 day of conjugation could delay the development of

539

LOOH in emulsion it stabilized. Surprisingly, the concentration of hexanal in

540

glyco-PPH stabilized emulsion was still lower than LOD even after 9 days of storage.

541

This result implied that glyco-PPH can considerably prevent the formation of hexanal.

542

We attributed the improved oxidative stability of emulsion to the stronger steric

543

hindrance derived from the thicker layer of glyco-PPH on the emulsion droplet

544

surface which hinders the transition metals getting into close proximity to the core

545

lipids.

546

CONCLUSION

547

In this study, the successful covalent cross-linking of gum arabic to pea protein



548

hydrolysates via a mild Maillard reaction at 60°C and 79% relative humidity was

549

confirmed by SDS-PAGE, FTIR-ATR, and SEM characterizations. SEC-MALLS

550

indicated that approximately 1.2 mole of GA were covalently linked to 1 mole of PPH

551

after 1 day of conjugation. The degree of conjugation between GA and PPH can be

552

predicted by measuring the development of non-specific Maillard reaction marker

553

(Ab420

554

Strecker degradation products, i.e. aldehyde and pyrazines aromatic components

555

associated with Maillard reaction, were identified in glyco-PPH via SPME-GC-MS. A

556

remarkable beany flavor mitigation effect appeared in glyco-PPH with 1 day of

557

conjugation. Extending conjugation time greatly diminished the formation of beany

558

flavor markers. The solubility and emulsification properties of glyco-PPH were

559

sufficiently improved by controlling conjugation time to 1 day. The physical stability

560

of corn oil-in-water emulsions stabilized by glyco-PPH with 1 day of conjugation

561

were improved, particularly at pHs close to IEP. Emulsions stabilized by glyco-PPH

562

with 1 day of conjugation also exhibited superior chemical stability against lipid

563

oxidation. The improved physicochemical stability of emulsion stabilized by was

564

attributed to the increased steric hindrance of emulsion droplet surface. The

565

remarkable functionality and antioxidant activity of glyco-PPH with 1 day of

566

conjugation give it great potential for use as a natural plant protein based functional

567

material. These findings may provide valuable information for tailoring the properties

568

of plant protein hydrolysate via controlled Maillard-driven cross-linking with

569

polysaccharides.

nm)

and the available free amino groups in glyco-PPH. The characteristic


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570 571

Supporting Information: SPME-GC-MS profiled volatile compounds in pea protein

572

hydrolysate-arabic gum conjugates with different conjugation time (Supplementary

573

Table 1)

574 575

Conflict of interest

576

The authors declare no conflict of interest.

577

Acknowledgements

578

Mr. Fengchao Zha would like to thank China Scholarship Council (CSC) for the

579

financial support.

580 581

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731 732 733 734 735 736 737 738


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739 740

Table 1. Molecular mass parameters of various glyco-PPH determined by SEC-MALLS. Samples PPI

PPH

GA PPH-GA-0 glyco-PPH1 glyco-PPH3 glyco-PPH5 741 742 743 744

——

1 2

——

Fraction (n)

Mass fraction (%)

1 2 3 1 2 3 1 2 1 2 1

48.7 44.6 6.70 58.9 34.2 6.90 80.1 19.9 31.5 68.5 47.4

Mw (g/mol) 3.59×105 ± 12.6% 1.72×105 ± 8.42% 6.01×104 ± 3.41% 2.81×105 ± 5.20% 1.52×105 ± 0.82% 5.61×104 ± 1.11% 2.17×106 ± 0.48% 2.64×105 ± 5.49% 2.48×106 ± 0.39% 3.23×105 ± 1.34% 4.17×106 ± 0.38%

2 1

52.6 39.9

6.22×105 ± 5.37% 3.66×106 ± 1.19%

2 1

60.1 36.9

2

63.1 ——

——

——

——

——

Mass recovery (%)

R g (nm) 34.0 ± 0.81% 28.6 ± 1.82% 22.4 ± 0.90% 32.8 ± 0.10% 22.6 ± 1.80% 20.4 ± 1.93% 30.5 ± 0.15% 27.7 ± 0.72% 36.8 ± 1.19% 26.3 ± 1.52% 38.8 ± 0.18%

I =Mw /Mn 2.76 ± 14.2% 2.89 ± 2.14% 2.36 ± 3.07% 3.43 ± 8.37% 3.69 ± 28.8% 2.83 ± 7.36% 1.26 ± 0.64% 1.08 ± 6.70% 1.03 ± 0.54% 6.34 ± 1.56% 1.45 ± 0.52%

98.2 11 7.1 94.1 34 12.1 86.3 19.3 92.5 89.5 99.1

1.31×105 ± 1.39% 3.27×106 ± 0.39%

34.2 ± 0.21% 32.2 ± 0.18%

4.75 ± 5.55% 1.14 ± 0.61%

90.1 99.4

7.75×105 ± 7.29% 3.36×106 ± 0.38%

3.97×105 ± 6.67% 3.33×106 ± 0.35%

28.5 ± 0.33% 36.7 ± 0.14%

1.95 ± 9.88% 1.01 ± 0.52%

68.7 98.1

7.96×105 ± 6.64%

4.68×105 ± 5.33%

26.8 ± 0.25%

1.70 ± 8.52%

78.6

Mn (g/mol) 1.29×105 ± 6.41% 5.95×104 ± 8.60% 2.54×104 ± 4.11% 8.19×104 ± 6.60% 4.16×104 ± 8.60% 1.98×104 ± 4.12% 1.72×106 ± 0.43% 2.43×105 ± 3.84% 2.41×106 ± 0.38% 5.18×104 ± 0.80% 2.87×106 ± 0.36%

——

Mw, weight-average molecular weight; Mn, number-average molecular weight; R g, radius of gyration; I, polydispersity PPI, pea protein isolate; PPH, pea protein hydrolysate; GA, Gum arabic;



Page 36 of 45

745 746

Table 2. The level of selected beany flavor volatiles in various glyco-PPH conjugates (n = 3)

747

Identification method qualitative ion quantitative (m/z) ion (m/z)

no. compound 1 2 3 4 5 6

hexanal 3-methyl-1-butanol 2-pentyl furan 1-octen-3-one 1-octen-3-ol acetophenone

St St St St St St

56, 44, 41 55, 70, 42 81, 138, 53 55, 70, 27 57, 72, 43 105, 120, 77

56 55 81 55 57 105

sample PPH

GA

LOD PPH-GA-0 glyco-PPH-1 glyco-PPH-3 glyco-PPH-5 (mg·L-1)

3.54±0.04a 0.45±0.02a 0.19±0.04a 1.07±0.01a 1.10±0.03a 0.40±0.07a

nd nd nd nd nd nd

3.17±0.06a 0.36±0.01b 0.16±0.02a 0.82±0.02a 0.95±0.02a 0.45±0.03a

1.28±0.07b 0.12±0.01c 0.05±0.01b 0.33±0.01b 0.34±0.02b 0.17±0.04b

0.78±0.02c 0.05±0.01c 0.02±0.01c 0.09±0.01c 0.19±0.01c 0.13±0.01b

0.42±0.04d 0.03±0.01c 0.01±0.00c 0.04±0.02d 0.11±0.01d 0.07±0.01c

0.025 0.023 0.008 0.015 0.016 0.012

Recovery %

RSD%

92.59 96.40 91.83 97.97 95.86 94.83

4.68 1.64 2.61 7.12 0.12 6.14

748 749 750 751 752 753 754 755 756

1

Units of milligrams per gram of dry weight; Number (no.) corresponds to the elution order by GC-MS analysis in Figure 4-C; 3 St, standard; nd, not detected; PPH, pea protein hydrolysate; GA, Gum arabic; 0, 1, 3, and 5, different conjugation time (day) at 60°C and 79% relative humidity; LOD, limit of quantitation; RSD, relative standard deviation; 4 Different lowercases represent significant difference at p < 0.05. 2


Page 37 of 45


757 758 759

Figure captions

760

Fig. 1. (A) SDS-PAGE patterns for different glyco-PPH: lane M for protein markers;

761

lanes 1-8 for pea protein isolate (PPI), PPH-0 day, PPH–1 day, PPH–3 day, PPH–5

762

day, glyco-PPH-1 day, glyco-PPH-3 day, glyco-PPH-5 day, respectively. The + &–

763

mean include and exclude, respectively; (B) The characteristic structure of glyco-PPH

764

(1 day) by Fourier transform infrared spectroscopy-attenuated total reflection

765

(FTIR-ATR); (C) SEM for surface characters profiles of glyco-PPH: 1-3 for PPH, GA,

766

mixture of PPH and Gum arabic, 4-6 for glyco-PPH with different times of 1, 3, 5 day,

767

respectively. Magnification 1000×; scale bar =10 μm. PPH and GA represent pea

768

protein hydrolysate and Gum arabic, respectively.

769

Fig. 2. (A-H) A range of selected samples were characterized by a size-exclusion

770

chromatography with multiangle laser light scattering (SEC-MALLS). Molar mass,

771

UV and differential refractive index (dRI) as a function of retention time of various

772

glyco-PPH. (H) Comparison of the elution profiles monitored by UV at 280 nm for

773

different glyco-PPH

774

Fig. 3. (A) Changes in absorbance at 304 nm and 420 nm in the mixture of PPH and

775

GA reacted at 60°C and 79% relative humility for 0-5 day; (B) Changes of free amino

776

groups as a function of reaction time during cross-linking of PPH and GA at 60°C and

777

79% relative humidity; (C) Color development in samples reacted at 0, 1, 3, 5 day,

778

respectively. The size of bubble is related to redness (a*) value; PPH and GA

779

represent pea protein hydrolysate and Gum arabic, respectively.

780

Fig. 4. Principal component analysis (PCA) (A) loading plot (B) score plot of



781

identified flavor compositions in different glyco-PPH, and (C) chromatograms of

782

beany flavor markers; Note: PPH-GA-0 represented the mixture of pea protein

783

hydrolysate (PPH) and Gum arabic (GA); glyco-PPH-1, glyco-PPH-3 and

784

glyco-PPH-5 represented glyco-PPH cross-linked between PPH and GA for 1, 3, 5

785

day, respectively.

786

Fig.5. (A) Relative protein solubility of different glyco-PPH cross-linked at 1, 3, 5

787

day, respectively, at pH 7.0; (B) changes of particle size (d43) for emulsions stabilized

788

with PPH alone, mixture of PPH and Gum arabic, and various glyco-PPH at pH 7.0;

789

(C) changes of particle size (d43) and -potential for emulsions stabilized with PPH

790

alone, mixture of PPH with GA, and glyco-PPH with 1 day reaction against different

791

pHs; PPH and GA represent pea protein hydrolysate and Gum arabic, respectively.

792

Note: for (C), the lowercase is for comparison among groups at the same pH values;

793

the uppercase is for comparison in groups at the different pH values. Different letters

794

indicated significant at p < 0.05

795

Fig. 6. The formation of (A) lipid hydroperoxides and (B) hexanal in corn oil-in-water

796

emulsions (pH 7.0) stabilized by PPH, PPH-GA mixture, and glyco-PPH cross-linked

797

for 1 day during storage at 37 °C

798 799 800 801 802 803 804 805 806 807


Page 38 of 45

Page 39 of 45

808 809 810 811 812 813


Figure 1.

814 815 816 817 818 819 820 821 822



823 824 825

Figure 2.

826 827 828


Page 40 of 45

Page 41 of 45


829 830 831 832 833 834

Figure 3.

835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862



863 864 865 866 867 868 869 870

Figure 4.

871 872 873 874 875 876 877 878 879 880 881 882 883 884


Page 42 of 45

Page 43 of 45


885 886 887 888 889 890 891 892

Figure 5.

893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919



920 921 922 923 924 925 926 927

Figure 6.

928 929


Page 44 of 45

Page 45 of 45


930

Graphic abstract

931

The proposed mechanism of gum arabic mediated synthesis of glyco-pea protein hydrolysate via Maillard reaction

932 933


Gum Arabic mediated synthesis of glyco-pea protein hydrolysate via

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