Comparative Study on Breadmaking Quality of Normoxia- and

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Functional Structure/Activity Relationships

Comparative Study on Breadmaking Quality of Normoxia- and Hypoxia-germinated Wheat: Evolution of #-Aminobutyric Acid, Starch Gelatinization and Gluten Polymerization during Steamed Bread Making Pei Wang, Kexin Liu, Runqiang Yang, Zhenxin Gu, Qin Zhou, and Dong Jiang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00200 • Publication Date (Web): 28 Feb 2019 Downloaded from http://pubs.acs.org on March 2, 2019

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Journal of Agricultural and Food Chemistry

1

Comparative

Study

on

Breadmaking

Quality

of

Normoxia-

and

2

Hypoxia-germinated Wheat: Evolution of γ-Aminobutyric Acid, Starch

3

Gelatinization and Gluten Polymerization during Steamed Bread Making Pei Wangab, Kexin Liua, Runqiang Yanga, Zhenxin Gua*, Qin Zhoub, Dong Jiangb

4 5

a College

6

Jiangsu 210095, People's Republic of China

7

b

8

Laboratory of Crop Physiology, Ecology and Management, Ministry of Agriculture/

9

National Engineering and Technology Center for Information Agriculture, Nanjing

10

Agricultural University, Nanjing, Jiangsu 210095, People's Republic of China

of Food Science and Technology, Nanjing Agricultural University, Nanjing,

National Technique Innovation Center for Regional Wheat Production/Key

1

ACS Paragon Plus Environment


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11

ABSTRACT

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To explore breadmaking characteristic of germinated wheat flour, current study

13

focused on the componential evolution throughout steamed bread making process.

14

Hypoxia-germinated wheat (HGW) dough produced the maximum GABA due to high

15

glutamic

16

normoxia-germinated wheat (NGW) and sound wheat (SW). HGW was superior to

17

NGW in terms of rheological properties, and restored the organoleptic characteristics

18

as SW bread. Blocking of α-amylase activity and protein polymerization

19

demonstrated that the decline in pasting and gelation properties was not caused by

20

changes in intrinsic starch and protein properties. Polymerization of α- and γ-gliadin

21

to glutenin was facilitated in germinated-wheat bread, while the crosslinking degree

22

of glutenin-gliadin was suppressed. Compared with NGW bread, more high molecular

23

weight glutenin subunits but less α-gliadin fractions polymerized upon steaming of

24

HGW dough. Results demonstrate that HGW has the great potential to be exploited as

25

a nutritious functional ingredient for wheat-based food.

26

KEYWORDS: GABA; hypoxia-germinated wheat; steamed bread; componential

27

variation

acid

decarboxylase

activity

during

fermentation

2


compared

with

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INTRODUCTION

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Wheat is one of the most important cereal grains and usually consumed as flour.

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Many nutritious components such as dietary fiber, minerals, vitamins, are eliminated

31

in the refined flour with the developed milling process. Though the sensory quality of

32

whole wheat products declines, consuming whole wheat meals can effectively reduce

33

the morbidity of colon cancer and cardiovascular disease. Thus they are recommended

34

to partially replace the refined grain meals.1 Moreover, germinated grains have

35

attracted more and more attention nowadays, due to their potential to enrich the

36

bioactive compounds such as phenolic compounds, folates and γ-aminobutyric acid

37

(GABA) under the controlled germination condition.2-4

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Among the bioactive substances, GABA functionalizes as the main

39

neurotransmitter in brain, and has the positive effects on lowering the blood pressure

40

and regulating the hormone secretion.5 In advanced plants, GABA is synthesized by

41

L-glutamate through glutamic acid decarboxylase (GAD). It can be a signal substance

42

produced by plants under the high stress environment, and the acidification of the

43

cytoplasm caused by hypoxia can lead to the optimum reaction pH of GAD (5.5 to

44

6.0). Meanwhile, GABA can be significantly accumulated during the germination of

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various plant seeds, e.g. legume, barley, wheat and millet.6 The induced accumulation

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of GABA can be realized under the condition of adversity such as salt stress, low

47

temperature and hypoxia.7 On the other hand, wheat belongs to the anoxia-intolerant

48

cereals, the protein and carbohydrates will be less hydrolyzed due to the suppressed

49

germination under hypoxia, which is beneficial for the technofunctionality of

50

germinated wheat flour.8

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Germinated wheat is considered as the inferior ingredient in flour processing

52

industry, due to the deteriorated rheological, pasting and gelation properties. The

3



53

activated hydrolytic enzymes such as α-amylase and protease results in the hydrolysis

54

of starch and protein in germinated wheat, and further leads to the deterioration of

55

flour quality.9 During the further mixing, fermentation and baking stage, the degraded

56

gluten network deterioration significantly contributed to the degraded quality of final

57

products.2 Nevertheless, there is a general consensus that the technofunctionality of

58

germinated what flour can be preserved with the controlled-germination protocol. It

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was even found that wheat germination at low degree could improve the baking

60

quality.10 In the study of Marti et al.,11 a substitution of 1.5% sprouted wheat flour in

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whole wheat flour increased the specific volume and reduced the firmness of baked

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bread. Exploring controlled-germination techniques have been an updated attentive

63

study, since the nutritional quality as well as the technofunctionality are considerable

64

important for the exploitation and promotion of the new functional product.

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In our previous study, we have successfully enriched GABA and alleviated the

66

componential degradation in whole wheat flour using the hypoxia strategy.12

67

However, little is known about the end-use quality of flour, as well as the

68

transformation of GABA evoked by the endogenous GAD during the subsequent

69

breadmaking process. Consequently, the dynamic changes of GABA, GAD activity,

70

rheological properties, starch and protein characteristics and the attributes of Chinese

71

steamed bread (CSB) made by sound, normoxia and hypoxia-germinated wheat flour

72

were comparatively investigated. The aim of this study is to comprehensively evaluate

73

the GABA and technofunctional components of wheat flour during CSB making, and

74

the resluts may further provide theoretical guidances for the exploitation of a new

75

GABA-enriched CSB product.

76

MATERIALS AND METHODS

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Materials

4


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Wheat (Triticum aestivum L.) variety Huaimai was cultivated in Jiangsu

79

province of China and harvested in 2017. The grains were stored at -20 °C before use.

80

Water was purified by a Millipore system (Waters, Mississauga, ON, USA). All the

81

reagents were of analytical grade unless otherwise specified.

82

Preparation of Germinated Wheat Flour

83

The germinated wheat flour was prepared according to Wang et al.12 Briefly,

84

wheat seeds were sterilized by 1% sodium hypochlorite, steeped in purified water and

85

placed in the automatic sprout machine (BX-801, Beixin Hardware Electrical Factory,

86

Zhejiang, China). The normoxia-germinated wheat (NGW) was obtained in darkness

87

at 25 °C, and water was sprayed for 1 min automatically every 1 h. For the

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hypoxia-germinated wheat (HGW), the wheat seeds were incubated using the

89

cultivated pots with lids (φ 6.0×9.0 cm) at 25 °C in a dark condition with deionized

90

water, and was aerated by a pump at flow rate at 0.9 L/min (final dissolved oxygen

91

concentration was 9.6 mg/L). The germinated wheat seeds were collected after 12 h

92

and washed with purified water, freeze-dried and milled. The obtained whole wheat

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flour was stored at -20 °C before further analysis. The sound wheat (SW) was used as

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the control sample.

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Approximate Composition of Wheat Flour and the Steamed Bread Quality

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The approximate composition of flour samples were measured according to the

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official methods of the AACCI Approved Method 44-15.02, 46-13.01, 76-13.01,

98

30-10.01, 08-01.01, 32-05.01, 38-12.02.13 Chinese steamed bread (CSB) were

99

prepared according to the method described by Wang et al.14 with modifications.

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Flour sample (100 g) was mixed with dry yeast (1 g) in a flour mixer, the mixing time

101

and added water amount were adjusted based on the farinograph test. Then the dough

102

was transferred into a refrigerator at 4 °C to rest for 10 min. The dough was divided

5



103

into 80 g, shaped and fermented at 35 °C and 80% relative humidity to reach the

104

optimum height. Afterwards, the fermented dough was steamed above the boiling

105

water for 30 min. The bread was cooled to room temperature and analyzed within 12

106

h. The bread was weighed and the volume was determined by rapeseed displacement.

107

Firmness of steamed bread was tested using the TA-XT2i texture analyzer (Scarsdale,

108

Stable MicroSystems, UK) using a 40 mm cylindrical acrylic probe. A single 30×30

109

mm field of view in the center of each slice was captured for each image analysis. The

110

cell parameters (cell to total area ratio, cell density and cell mean area) were analyzed

111

by Image J software v. 1.49 (NIH, Bethesda, USA). The color of bread crumb was

112

measured by a CR-400 color meter (Konica Minolta Industries, Tokyo, Japan).

113

GABA, Glutamic Acid (Glu) Content and Glutamate Decarboxylase (GAD)

114

Activity

115

Wheat flour (1.00 g) was mixed with 5 mL of 7% (v/v) acetic acid. After

116

centrifugation (10,000×g, 20 min), the supernatant was mixed with 5 mL of ethanol.

117

After 2 h precipitation at 4 °C, the slurry was centrifuged as above. The supernatant

118

was evaporated at 90 °C to volatilize the acetic acid and ethanol. The residues were

119

dissolved with 1 mL of 1 M NaHCO3 (pH 9), centrifuged at 10,000×g for 10 min and

120

the supernatant was used for the determination of free GABA and Glu content. To

121

determine the total GABA and Glu content, another batch of flour samples was

122

hydrolyzed with 1.0 mL of 6.0 M HCl at 110 °C for 24 h under the nitrogen

123

atomosphere. After hydrolysis, samples were subsequently evaporated at 110 °C for

124

180 min, and resuspended in 5 mL of water. GABA and Glu content were determined

125

by an Agilent 1200 Series HPLC (Agilent Technologies, Santa Clara, CA, USA) with

126

a ZORBAX Eclipse AAA reversed-phase column. The amino acid solution (1 mL, pH

127

9.0) was mixed with 1 mL of dabsyl chloride (4 mg/mL, in acetone) and reacted at 67

6


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128

°C for 10 min. The reaction was stopped by an ice bath and detected at 425 nm using

129

UV-vis diode-array detector.15 The bound GABA and Glu content were calculated as

130

the difference between the total and free content of GABA and Glu, respectively.

131

The determination of GAD activity was conducted following the method of Bai et

132

al.16 with some modifications. Fresh samples (1 g) were homogenizated and mixed

133

with 6 mL of potassium phosphate buffer (66.66 mM, pH 5.8), which contained 2 mM

134

β-mercaptoethanol, 2mM Eyhylene diamine tetraacetic acid (EDTA) and 0.2 mM

135

pyridoxal phpsphate (PLP). The homogenate was centrifuged at 10,000×g for 20 min.

136

The reaction solution containing 200 μL of crude enzyme solution and 100 μL of

137

substrate (1% Glu, pH 5.8) was incubated at 40 °C for 2 h and then terminated at 90

138

°C for 10 min. The GABA content was determined following the method as

139

mentioned above. One unit of enzyme activity was defined as the release of 1 μmol of

140

GABA produced per 1 h at 40 °C.

141

Mixolab Test

142

Mixing and pasting behavior of the wheat flour dough was studied using the

143

Mixolab (Chopin, Tripette et Renaud, Paris, France). Wheat flour (50 g) with known

144

moisture content was placed into the analyzer bowl, the water required for optimum

145

consistency (1.1±0.1 Nm) was added after tempering the solids. The temperature

146

setting in the test were initially maintaining at 30 °C for 4 min, with the temperature

147

increase of 4 °C/min until the mixture reached to 90 °C, then holding at 90 °C for 11

148

min, followed by a temperature decrease of 4 °C/min until the mixture reached to 50

149

°C, and holding at 50 °C for 5 min. Five distinct phases (C1-C5) can be distinguished

150

on the Mixolab curves.17

151

Viscoelastic Property

152

Dynamic rheological measurement was performed by an Anton Paar Physica

7



153

MCR 301 rheometer (Anton Paar GmbH, Graz, Austria) according to Wang et al.18

154

with modifications. A circular parallel-plate geometry was used (40 mm plate), and

155

the gap between the two plates was set to 1 mm for all samples. The edge of the

156

sample was covered with paraffin oil to avoid moisture loss. Dynamic rheological

157

measurements were conducted at 25 ºC over the frequency range of 0.1 to 10 Hz, with

158

the deformation of 0.2% within the linear viscoelastic region. Dynamic temperature

159

sweep measurements were conducted at a deformation of 0.2% within the linear

160

viscoelastic region, and at a frequency of 1 Hz. The temperature of the plate was set to

161

increase from 25 to 95 °C at a heating rate of 4 °C/min, then holding at the

162

temperature at 95 °C for 15 min.

163

Rapid Visco Analyzer (RVA)

164

Pasting properties were measured by a RVA-4 series (Newport Scientific, NSW,

165

Australia). The flour samples (4.0 g, 14% moisture) were suspended and

166

homogenized in 25 mL of water, or equal amount of 1 mM silver nitrate (AgNO3)

167

solution, or 3.4 μM dithiothreitol (DTT), or 4.5 μM N-Ethylmaleimide (NEMI),

168

respectively. The suspension was kept at 50 °C for 1 min, and raised to 95 °C in 6.0

169

min. Then the slurry was held at 95 °C for 8 min, cooled to 50 °C in 9 min, and kept

170

for 10 min.19

171

Thermal Properties of Dough

172

Thermal properties of dough were analyzed by differential scanning calorimeter

173

(DSC, Q100, TA Instrument, New Castle, DE, USA). The measurement was

174

performed according to the method of Wang et al.20 with slight modifications. Dough

175

sample (10-15 mg) was accurately weighed into aluminum sample pans. An empty

176

pan was used as a reference. The pans were sealed, and the sample was kept at 25 °C

177

for 10 min and then heated from 25 to 110 °C at 10 °C/min. The onset, peak, conclude

8


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temperature and enthalpy (ΔH) were recorded from the corresponding thermograms

179

by the Universal Analysis version 2000 software (TA Instruments, New Castle, DE,

180

USA).

181

Molecular Weight Distribution of Proteins

182

Size-exclusion high performance liquid chromatography (SE-HPLC) was

183

conducted on an Agilent 1200 Series HPLC system (Agilent Technologies, Santa

184

Clara, CA, USA). Freeze-dried samples (40 mg) was dispersed in 4 mL sodium

185

phosphate buffer (0.05 M, pH 6.8) containing 2.0% sodium dodecylsulphate (SDS),

186

and the soluble protein was extracted with a magnetic stirrer at room temperature for

187

1 h. All the reduced samples were extracted with SDS buffer in the presence of 1.0%

188

dithiothreitol (DTT). After centrifugation (10,000×g, 10 min), 20 μL of the

189

supernatant was loaded on a Shodex Protein KW-804 column (Showa, Kyoto, Japan).

190

The elution was achieved with sodium phosphate buffer (0.05 M, pH 6.8) containing

191

0.2% SDS at a flow rate of 0.7 mL/min. The thermostat was set at 30 °C and the

192

elution was detected at 214 nm. All the extractions were performed at least in

193

triplicate. The areas of SDS-soluble polymers (SDS-P), mononmers (SDS-M) and

194

insoluble proteins (SDS-I) content were normalized to corresponding peak area of the

195

SW flour.21

196

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

197

SDS-PAGE analysis was performed with 4% stacking gels and 12% separating

198

gels. Samples (50 mg) was extracted with 1.5 mL of non-reduced sample buffer

199

solution of pH 6.8 containing 0.125 M Tris-HCl, 2% (w/v) SDS, 10% (v/v) glycerol

200

and 0.01% w/v bromophenol blue, for 3 h at 25 ºC. Then the dispersions were heated

201

in boiling water for 3 min, after centrifugation at 10,000×g for 20 min at 4 ºC, the

202

supernatant (10 µL for each lane) was loaded for SDS-PAGE analysis, protein

9



203

separation was conducted on a VE-180 vertical slab of gel with a thickness of 1 mm

204

(Puyang Institute of Scientific Instruments, Nanjing China). For reduced proteins,

205

sample buffer contained 5% (v/v) β-mercaptoethanol. Gels were stained with

206

Coomassie brilliant blue G-250 and then scanned with Image Scanner III (GE

207

Healthcare Biosciences, Uppsala, Sweden).

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Determination of Free Sulfhydryl (SH) Groups

209

Total free SH content were determined according the method of Beveridge et al.22

210

with some modifications. Samples (40 mg) were mixed with 4 mL of reaction solution

211

consisting of 86 mM Tris, 92 mM glycine and 4.1 mM EDTA (Tris-glycine-EDTA,

212

TGE) and 2.5% SDS (pH 8.0), and then incubated at 25 °C for 30 min. The mixed

213

solution was oscillated every 10 min. Afterwards, Ellman's reagent (40 μL)

214

[5,5-dithiobis-2-nitrobenzoic acid (DTNB) in TGE (4 mg/mL)] was added and the

215

tubes were wrapped with aluminum foil, and incubated at 25 °C for 30 min. The

216

absorbance of the supernatant was measured at 412 nm against the blank (without

217

Ellman’s reagent and the sample).

218

Distribution of Different Protein Subunits

219

Freeze-dried samples (50 mg) were extracted three times with 1.5 mL of 60%

220

(v/v) ethanol and the residues were again extracted three times with 1.5 mL of 0.05 M

221

Tris-HCl (pH 7.5) containing 50% (v/v) 1-propanol, 2.0 M urea and 1% DTT (w/v) at

222

room temperature. Both of the ethanol soluble and insoluble extracts were diluted to 5

223

mL with the respective extraction solvent. The extracts (20 μL) were separated by a

224

Nucleosil 300-5 C8 column (Machery-Nagel, Duren, Germany). The elution system

225

consisted of deionized water (A) and acetonitrile (B), both containing 0.1% (v/v)

226

trifluoroacetic acid. Proteins were eluted with a linear gradient from 24% B to 56% B

227

in 50 min at a flow rate of 1.0 mL/min and detected at 214 nm.

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228


Solubility of Proteins in Different Solvents

229

Selective buffers prepared in phosphate buffer (0.05 M, pH 7.0) were used to

230

solubilize proteins as follows: 0.05 M NaCl (S1), 0.6 M NaCl (S2), 0.6 M NaCl and

231

1.5 M urea (S3), 0.6 M NaCl and 8 M urea (S4). Samples (300 mg) were extracted

232

with 10 mL solvents by magnetic stirrer at 25 °C for 1 h, and centrifuged at 10,000×g

233

for 20 min. The supernatant was determined using the Kjeldahl method and expressed

234

as the percentage of the total protein content.

235

Statistical Analysis

236

All data were expressed as mean ± standard deviation (SD) of at least three

237

replicates. Significant differences of evaluated parameters among different samples

238

were analyzed by SPSS statistical software (version 19.0 for Windows, SPSS Inc.,

239

Chicago, IL). The probability value of p

Comparative Study on Breadmaking Quality of Normoxia- and

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