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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
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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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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
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gel
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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
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reaction, a so-called cooking chemistry, has become a promising green chemistry to
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ameliorate the general functionality of proteins
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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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environmental sustainability, cultural acceptability, and low-cost accessibility
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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.
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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%)
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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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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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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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concentration.
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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
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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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(~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
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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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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
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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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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
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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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Supporting Information: SPME-GC-MS profiled volatile compounds in pea protein
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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
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Mr. Fengchao Zha would like to thank China Scholarship Council (CSC) for the
579
financial support.
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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;
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Table 2. The level of selected beany flavor volatiles in various glyco-PPH conjugates (n = 3)
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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
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Figure captions
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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
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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
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The proposed mechanism of gum arabic mediated synthesis of glyco-pea protein hydrolysate via Maillard reaction
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