Inactivation of Soybean Bowman-Birk Inhibitor by Stevioside

Subscriber access provided by TULANE UNIVERSITY

Food and Beverage Chemistry/Biochemistry

Inactivation of Soybean Bowman-Birk Inhibitor by Stevioside: Interaction Studies and Application to Soymilk Chun Liu, Lijuan Luo, Ying Wu, Xiaoquan Yang, Jie Dong, Feijun Luo, Yuan Zou, Ying-Bin Shen, and Qinlu Lin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05609 • Publication Date (Web): 07 Feb 2019 Downloaded from http://pubs.acs.org on February 7, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

Journal of Agricultural and Food Chemistry

1

Inactivation of Soybean Bowman-Birk Inhibitor by Stevioside: Interaction

2

Studies and Application to Soymilk

3 4

Chun Liu a, Lijuan Luo a, Ying Wu a, Xiaoquan Yang b, Jie Dong a, c, Feijun Luo a, Yuan Zou d,

5

Yingbin Shen e, Qinlu Lin * a

6 7

a

8

of Processed Food For Special Medical Purpose, School of Food Science and Engineering, Center

9

South University of Forestry and Technology, Changsha 410004, China

National Engineering Laboratory for Rice and By-product Deep Processing, Hunan Key Laboratory

10

b

11

South China University of Technology, Guangzhou 510640, China

12

c

Xiangya School of Pharmaceutical Sciences, Central South University, Changsha 410013, China

13

d

School of Food Science, South China Agricultural University, Guangzhou 510642, China

14

e

Department of Food Science and Engineering, School of Science and Engineering, Jinan University,

15

Guangzhou 510632, China

Research and Development Center of Food Proteins, School of Food Science and Engineering,

16 17

*Corresponding author: Qinlu Lin

18

Tel: (+86) 0731-85623241

19

Fax: (+86) 0731-85623241

20

E-mail addresses: [email protected]

21 22 1

ACS Paragon Plus Environment


Page 2 of 32

23

ABSTRACT:

24

In this work, interaction of soybean Bowman-Birk inhibitor (BBI) with stevioside (STE) was studied

25

by stopped-flow/fluorescence and molecular docking. STE inactivating protease inhibitor activities

26

in soymilk and the influence of STE addition on the sensory character of soymilk were also

27

evaluated. The results indicate that STE binds BBI with a binding constant (Ka) of 3.38 ×105 L mol−1

28

to form a 1:1 complex. Docking study reveals that two hydrogen bonds are formed between the

29

side-chain of Lys16 (reactive site 1) of BBI and the hydroxyl groups of the glucose-ring of STE,

30

which may block BBI from contacting with trypsin and thus deactivate the trypsin inhibitor activity

31

(TIA) of BBI. Moreover, the residual TIA in soymilk could also be inactivated by STE. A mixture of

32

159 mg/L of STE and 60 g/L of sucrose could be used for sweetening soymilk without affecting the

33

sensory characteristics from a reference product sweetened with 9% sucrose.

34 35

KEYWORDS: soybean Bowman-Birk inhibitor, stevioside, inactivation, interaction, soymilk

36 37 38 39 40 41 42 43 44 2


Page 3 of 32


45

■ INTRODUCTION

46

Numerous Asian people have an affection for starting a day with a cup of soymilk for its great

47

significance in health. Recently, soymilk has also become an integral beverage on the tables of many

48

families in western countries.1 However, There are some antinutritional factors in soy products, for

49

instance, both Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI) was identified to

50

decrease the protein digestibility as well as lead to pancreatic hypertrophy via binding to trypsin.1

51

According to Xu et al.,1 by heating, KTI was easily to be integrated in protein aggregates with

52

non-covalent bonds and/or disulfide bonds and thus losing its trypsin inhibitor activity (TIA), while

53

the free KTI could lose its TIA via changing the conformation. As BBI was more likely to keep its

54

natural conformation, the residual TIA could derive from BBI.

55

Heating is an important step in soymilk processing as it not only ensures microbial safety but

56

also improves flavor and nutritional quality. As is reported by Yuan et al., only 13% of TIA retained

57

after the soymilk was heated at 100 °C for 20 min.2 Xu et al. found that more than 80% residual TIA

58

gathered in the supernatant, about 89% of the original chymotrypsin inhibitor activity (CIA) could

59

remain after being heated at 100 °C for 15 min, and > 90% residual CIA was congregated in the

60

supernatant.1 It is showed in Tricine-SDS-PAGE that soymilk BBI primarily exists in supernatant

61

rather than in precipitate with such heating treatment.1

62

Complexing of soybean trypsin inhibitors with some natural ingredients of plants, like

63

polyphenols, has been confirmed the inactivation action for soybean trypsin inhibitors.3 Tea

64

polyphenols (TPs) were found to inactivate both KTI and BBI in soy products, and KTI was affected

65

more greatly by TPs than BBI.4 Additionally, although TPs are recognized to be beneficial to health

66

for their distinguished physiological functions, the taste of polyphenols is always bitter, which does 3



67

Page 4 of 32

not suit the palate of most of the people.

68

To make the taste more acceptable, sugar is generally added to soymilk. However, sugar readily

69

induces a glycemic response when ingested. Hence, as an alternative for sucrose, natural sweeteners

70

have garnered much attention for its benefits of reducing obesity and diabetes. Stevioside (STE)

71

(Figure 1), a high-profile natural sweetener, is the most abundant component of steviol glycosides

72

and 250 to 300 times sweeter than sucrose.5 It is both heat-stable and pH-stable. And it does not

73

ferment.6 Furthermore, being ingested, STE will not cause a glycemic response. As a result, it is

74

suitable for diabetics and people who have a preference for carbohydrate-controlled diets.5

75

Interestingly, the amphiphilic structure of STE molecules is found to be similar to triterpenoid

76

saponins, and may determine their potential capacity as polyphenols to interact with proteins, which

77

may lead to changes in the structure as well as functional and nutritional properties of proteins.

78

Structurally, soybean-derived BBI is a double-headed inhibitor with independent activity

79

against both trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1). The Lys16-Ser17 and

80

Leu43-Ser44 bonds were assigned to be the anti-tryptic and anti-chymotryptic sites, respectively.7

81

The three-dimensional structure of soybean BBI has been determined (Figure 2),8, 9 which confirmed

82

that the protein was characterized by two distinct binding domains for serine protease. The crystal

83

structure of soybean BBI complex with bovine trypsin has also been determined.10 The

84

triple-stranded β-hairpin unique to the BBI-family and the surface loops surrounding the active site

85

of the enzyme formed a protein-protein-interface far extended beyond the primary contact region.

86

The buried salt-bridge responsible for trypsin specificity was stabilized in a polar, and destabilized in

87

a hydrophobic environment, which suggests that any factors affecting the polar residues, hydrophilic

88

bridges and hydrophobic contacts can change the inhibitory activity of BBI. 4


Page 5 of 32


89

Our exploration in the study can be sum up in the following aspects: to start with, the effect of

90

STE on the TIA and CIA of BBI against trypsin and chymotrypsin were determined, respectively,

91

and the effect of STE on trypsin and chymotrypsin activity was also measured; in addition, a

92

stopped-flow-based fast mixing technique, steady-state fluorescence, and molecular docking were

93

used to explore the way BBI interacted with STE; finally, the effect of BBI inactivated by STE

94

addition on the sensory profile of soymilk was explored.

95

■ MATERIALS AND METHODS

96

Materials. Soybean Bowman-Birk inhibitor (BBI) (product number: T9777, > 95% pure),

97

soybean Kunitz trypsin inhibitor (KTI) (product number: T2327, > 95% pure), porcine pancreatic

98

trypsin (product number: T7409, ~ 1500 U/mg, 1496 ± 23 U/mg solid was detected in this work),

99

bovine pancreatic α-chymotrypsin (product number: C4129, ≥ 40 U/mg, 49 ± 2.1 U/mg solid was

100

detected in this work), Nα-benzoyl-DL-arginine 4-nitroanilide hydrochloride (BAPNA) (product

101

number: B4875, > 98% purity), and N-benzoyl-L-tyrosine p-nitroanilide (BTpNA) (product number:

102

B6760, > 98% purity) were purchased from Sigma-Aldrich (Shanghai, China). Stevioside (> 95%

103

purity) was purchased from Jining Aoxing Stevia Products Co., Ltd. (Jining, China). The soybean

104

cultivar ‘ Nannongdahuangdou ’ was used in this study. Sucrose was purchased from the local

105

supermarket. All other chemicals were of analytical grade.

106

Inactivation of BBI and KTI Inhibitory Activity Assays. The TIA of BBI, KTI and STE against

107

trypsin were determined according to Smith et al.11 The CIA of BBI and STE against

108

α-chymotrypsin were determined according to Tan et al.12 BBI or KTI (0.1 mg/mL) and STE (1

109

mg/mL) solution were prepared in deionized water and then diluted to different concentrations in

110

deionized water. All these operations were carried out in deionized water, because BAPNA or 5



Page 6 of 32

111

BTpNA would precipitate in a phosphate buffer solution. The residual activities of trypsin or

112

chymotrypsin were measured according Huang et al.13 and Tan et al.12

113

Interaction of STE with Proteins (BBI, KTI, Trypsin and Chymotrypsin). Stopped-Flow

114

Kinetic Experiments and Data Analysis. The kinetic measurements were carried out by using a

115

stopped-flow apparatus with an MOS-450 light chamber and an SFM-20 device (Bio-Logic, France)

116

according to our previous work.3 The kinetic data [kobs = kon [STE] + koff (eq1), KD = koff/kon] were

117

analyzed according to Patrick and Turchi.14

118

Steady-State Fluorescence. Fluorometric experiment was carried out on a F7000 fluorescence

119

spectrophotometer (Hitachi Co., Japan) at 25 oC according to our previous work.3 Solutions used for

120

fluorescence spectra contained a fixed protein (BBI or KTI or Trypsin or Chymotrypsin)

121

concentration (0.4 mg/mL) and the concentration of STE ranging from 0 to 3 mg/mL. 1.0 mL of BBI

122

stock solution (2 mg/mL) and different volumes of STE stock solutions (10 mg /mL) were mixed and

123

then diluted to 5 mL with 10 mM phosphate buffer (pH 7.4) to obtain final STE concentration. The

124

data on the tyrosine (Tyr in BBI) or tryptophan (Trp in KTI, trypsin or chymotrypsin) fluorescence

125

quenching were analyzed according to our previous work.3

126

Docking Studies. Docking of STE with BBI, KTI, trypsin or chymotrypsin protein was performed

127

with AUTODOCK4.1.15 The three-dimensional structure of proteins was obtained from Protein Data

128

Bank (PDB IDs of BBI, KTI, trypsin and chymotrypsin are 1BBI, 1AVU, 1V2O and 1YPH,

129

respectively) as templates and the three-dimensional structure of STE was obtained from PubChem

130

Compound (PubChem CID: 24721412). The binding affinity of the STE to the locations of the

131

proteins was studied by using simulated annealing procedure of Accelrys Discovery Studio 3.5,

132

respectively. 2D of protein-STE complexes was described according to Clark and Labute.16 6


Page 7 of 32


133

Soymilk Preparation. Soymilk was prepared according to the method of Chao.17 Then the

134

soymilk were analyzed immediately or freeze-dried and stored at 4 oC. The protein content of the

135

freeze-dried soymilk was measured by the micro-Kjeldahl method.

136

The Effect of STE on the TIA and CIA in Soymilk. The soymilk was prepared by dispersing the

137

freeze-dried soymilk into distilled water (the protein concentration was about 3%). The TIA and CIA

138

in soymilk with different concentrations of STE (from 50 mg/L to 300 mg/L with interval of 50

139

mg/L) was determined according to the methods as mentioned above. To verify whether sucrose also

140

affects the TIA and CIA in soymilk, the TIA and CIA in soymilk formulations sweetened with STE

141

and sucrose (Table 1 below) are determined.

142

Preparation of Soymilk Formulation Sweetened with STE and/or Sucrose. The concentrated

143

soymilk was prepared by dispersing the freeze-dried soymilk into distilled water (the protein

144

concentration was about 6%). Formulations for further analyses were prepared by mixing the

145

concentrated soymilk (always 50% of final volume) with the sweetener solution(s) (the following

146

stock solution were used: STE 2 g/L; Sucrose 300 g/L) and water to the final volume of 100 mL.

147

Details on the final concentration of sweetener(s) of the formulations are reported in Table 1. After

148

their preparation, the formulations were stored overnight at 4 oC in PET bottles and warmed at room

149

temperature (25 oC) before use.

150

Sensory Evaluation of Soymilk Formulations. Many sweeteners have a non-sweet aftertaste,

151

this problem limit their use at high concentration. Consequently, a low concentration of sucrose (3%)

152

(R1) was selected to assess the STE concentration with sweetness equivalent to 3% sucrose (SEC),

153

namely, the quantity of STE that in a binary mixture with 6% sucrose (samples F1-F5) gives the same

154

sweetness as a 9% sucrose reference (R2), since this concentration (R2) usually present in 7



Page 8 of 32

155

commercial soft drinks.18 Paired preference test on the two final formulations (PTJ) was also carried

156

out. Sensory evaluation of soymilk formulations was carried out in light of the work of Parpinello et

157

al.18 Twenty-one subjects were recruited from the students of School of Food Science and

158

Engineering, Center South University of Forestry and Technology (Changsha, China). The subjects

159

were asked to refrain from eating, smoking or drinking anything (except water) 30 min prior to each

160

testing assay. The sensory tests were carried out in a standard gray individual booth in a sensory

161

evaluation facility. In these tests the formulations (Table 1) were served in transparent glasses and

162

coded with numbers. For SEC experiments, a paired comparison, constant stimulus, forced-choice

163

method was used according to Amerine et al.19 with 21 subjects indicating the sweetest sample. The

164

equisweetness was identified according to Parpinello et al.18 Each test was carried out in triplicate.

165

Statistical Analysis. Results were expressed as mean values ± standard deviations. Sample

166

comparison, by multivariate analysis of variance (ANOVA) followed by Duncan’s comparison test,

167

was used to assess the differences.

168

■ RESULTS

169

Inactivation of BBI by STE. Figure 3A shows the TIA and CIA of BBI when BBI was

170

complexed with STE in various ratios. The curves show that the TIA of BBI could be inactivated

171

prominently by such complexation, while the CIA of BBI could only be inactivated slightly at the

172

same situation. In the absence of STE, the TIA was 1297 U/mg, revealing the effective inhibition of

173

trypsin activity by BBI. When BBI (0.02 mg/mL) of the same concentrations was complexed with

174

STE of different concentrations (0-1.0 mg/mL) in deionized water, the TIA decreased with the

175

increase of the STE concentration. It meant that the TIA of BBI was reduced by complexing with

176

STE. The inhibition rate of the TIA of BBI achieved a maximal value (81.63%) when the STE/BBI 8


Page 9 of 32


177

ratio (w/w) reached to 20, while the maximal inhibition rate of the CIA of BBI was only 33.50% at

178

the same STE/BBI ratio (w/w) (20). When the STE/BBI ratio was over 20, the TIA or CIA showed

179

an increasing trend, which likely resulted from the interaction of the surplus STE with trypsin or

180

chymotrypsin as it lowered their activities.

181

To verify whether KTI can be inactivated by STE, the effect of STE on TIA of KTI was studied.

182

The result indicated that the TIA of KTI could not be inactivated by STE (Figure 3B). Therefore, the

183

decrease in TIA was arised from the interaction between BBI and STE, although the commercial BBI

184

(T9777, http://www.sigmaaldrich.com/catalog/ product/sigma/t2327) contains a little residual KTI.

185

To verify the capacity of STE to inactive trypsin and chymotrypsin, the experiment for the

186

effect of STE on trypsin or chymotrypsin activity was performed. Figure 3B reveals that both trypsin

187

and chymotrypsin could also be inactivated by STE especially at relatively high concentration (> 0.4

188

mg/mL). However, the inhibition effect on trypsin or chymotrypsin was not always increased with

189

the increasing of STE concentration. The maximal inhibition effect on trypsin (about 26.35%) or

190

chymotrypsin (about 31.83%) appeared at the STE concentration of 1.0 mg/mL, when continuing to

191

increase the STE concentration till it is above 1.0 mg/mL, the inhibition effect on trypsin or

192

chymotrypsin had no evident increase. These results verified that the TIA of BBI could be

193

inactivated exactly by STE, whereas the CIA of BBI could not be inactivated obviously by STE, the

194

inactivation effect of STE on the CIA of BBI was likely to result from the interaction between the

195

STE and chymotrypsin.

196

Interaction of STE with BBI, KTI, Trypsin and Chymotrypsin. Stopped-Flow Fluorescence.

197

The stopped-flow-based fast mixing technique has been extensively utilized to investigate the folding

198

and unfolding mechanisms of proteins in solution.20,

21

The tyrosine fluorescence of BBI, as an

9



Page 10 of 32

199

indicator to follow the binding-induced structural changes of BBI by this technique, is presented in

200

Figure 4. As shown in Figure 4A, the fluorescence of BBI declined dramatically upon mixing with

201

STE, indicating the quick structural change in BBI upon binding. The amplitude of the overall

202

fluorescence change was directly related to the content of STE added to BBI solution. The observed

203

rate constant, kobs, varies from ~0.38 to ~3.00 s-1 depending on the STE concentration (Figure 4B).

204

Fitting the relationship of kobs with STE concentration using eq 1 yields kon = 6.46 mM-1s-1 and koff =

205

0.07 s-1. Consequently, the dissociation constant of STE to BBI, KD, is 0.01 mM. These data reveal

206

that STE tends to associate with BBI rather than to dissociate from BBI, especially at a higher

207

concentration of STE.

208

In the case of binding interactions between STE and KTI, a similar result was obtained (KD =

209

0.018 mM) (Figure 4C and 4D), which indicate that there is a strong interaction between STE and

210

KTI. In the case of binding interactions between STE and trypsin, the kobs, varies from ~0.099 to

211

~0.272 s-1 depending on the STE concentration (Figure 4E and 4F), the dissociation constant of STE

212

to trypsin, KD = 0.19 mM. These data indicate that STE has weak interaction with trypsin at

213

concentration of STE (0.1 ~ 0.5 mg/mL). In the case of binding interactions between STE and

214

chymotrypsin, similar results were found (KD = 0.20 mM) (Figure 4G, 4H).

215

Steady-State Fluorescence. Figure 5A shows the effect of STE on the Tyr intrinsic fluorescence

216

emission spectrum of BBI. With increasing concentration of STE (0.5 mg/mL to 3 mg/mL), the

217

fluorescence intensity of BBI decreased markedly, revealing that the interaction between STE and

218

BBI occurred. Fitting the data to Stern-Volmer plots can obtain the Stern-Volmer quenching constant

219

(Figure 5B). The value of Kq (3.29 × 1013 M-1 s-1) indicated that the fluorescence quenching induced

220

by STE was static quenching, since it was much higher than the maximal scatter collision quenching 10


Page 11 of 32


221

constant of various quenchers (2.0 × 1010 M-1 s-1).22 This result suggests that STE could act as a

222

direct Tyr fluorescence quencher and the structure of BBI has been changed. For the BBI molecule,

223

there are two Tyr residues: Tyr45 is located in solvent exposed molecular surface, and Tyr59 is

224

buried in the relatively hydrophobic environment of the folded protein molecule. The binding

225

interactions of STE with BBI likely lead to partially unfolding of the protein, thus Tyr59 residue is

226

exposed to the aqueous medium, which is in accordance with the result of stopped-flow fluorescence

227

measurement (Figure 4).

228

As shown in Figure 5C, the binding constants (Ka) and the number of binding sites per BBI

229

molecule (n) for STE with BBI were 3.38 × 105 L·M-1 and 1.01, respectively. The great magnitude of

230

Ka suggested a strong binding force between BBI and STE. The value of n was 1.01, demonstrating

231

about one association site on BBI for STE.

232

In the case of interaction between STE and KTI, a similar result was obtained (Kq = 3.48 × 1013

233

M-1 s-1, Ka = 1.99 × 107 L·M-1) (Figure 5D, 5E and 5F). The results suggested that there was a strong

234

interaction between STE and KTI. In the case of interactions between STE and trypsin or

235

chymotrypsin, a strong binding force between STE and trypsin (Kq = 3.97 × 1013 M-1 s-1, Ka = 2.13 ×

236

107 L·M-1) (Figure 5G, 5H and 5I) or chymotrypsin (Kq = 5.58 × 1013 M-1 s-1, Ka = 4.36 × 107 L·M-1)

237

(Figure 5J, 5K and 5L) at a concentration of STE (0.5 ~ 3 mg/mL) was observed.

238

Docking Studies. The representative conformation derived from the best pose with the minimal

239

binding energy is shown in Figure 6A. A pose of the binding of STE within a region near the reactive

240

site (Lys16-Ser17) of BBI was obtained (Figure 6A). As can be seen from Figure 6A, STE is in the

241

vicinity of Ala13, Cys14, Thr15, Lys16, Gln21, Arg23, Ser38, Val52, Asp53 and Ile54. Van der

242

Waals, hydrogen bonds and electrostatic interactions were observed between STE and these amino 11



Page 12 of 32

243

acid residues that stabilize STE-BBI complexes (Figure 6B). Moreover, hydrophobic interactions

244

between the triterpenoid ring of STE and non-polar amino acids in the binding location of BBI

245

existed to some extent. Among these interactions, it is noteworthy that two hydrogen bonds were

246

formed between the side-chain of Lys16 of BBI and the hydroxyl groups of the glucose-ring of STE

247

(Figure 6A). According to the previous work,10 in the crystal structure of BBI which is in ternary

248

complex with bovine trypsin, the NH atoms of Lys16 were in contact with the carbonyl group

249

oxygen atom of Ser214 of trypsin and the P1 side-chains of Lys16 was fully embedded into the S1

250

pocket of trypsin. Therefore, the interaction between STE and Lys16 may change the reactive loop

251

conformation and consequently change the TIA of BBI. The binding interaction of STE with the

252

surface of BBI indicates that STE locates at relatively hydrophobic region (Figure 6C).

253

In the case of interaction between STE and KTI, docking study revealed that STE could bind to a

254

pocket on KTI molecular surface, but the binding region of STE was not near the reactive site

255

(Arg63-Ile64) on KTI (Figure 6C, 6D). In the case of interactions between STE and trypsin or

256

chymotrypsin, Docking study indicated that the binding of STE within a region near the reactive site

257

(His57, Asp 102 and Ser 195) on trypsin (Figure 6E and 6F) or chymotrypsin (Figure 6G and 6H).

258

The Effect of STE on the TIA and CIA in Soymilk. The TIA and CIA in raw soymilk were

259

3128 ± 159 U/mL and 682 ± 35 U/mL, respectively. After heating treatment, the residual TIA and

260

CIA in soymilk reduced to 489 ± 29 U/mL (about 15.63% of that in raw soymilk) and 298 ± 14

261

U/mL (about 43.70% of that in raw soymilk), respectively. Figure 7 shows that the TIA in soymilk

262

decreased from 417 ± 21 U/mL to 171 ± 8 U/mL with an increase in the concentration of STE from

263

50 mg/L to 300 mg/L, while the CIA in soymilk only slightly decreased from 283 ± 14 U/mL to 247

264

± 14 U/mL with an increase in the concentration of STE from 50 mg/L to 300 mg/L. The inhibition 12


Page 13 of 32


265

rate of the TIA in soymilk was maximum (65.03%) when the concentration of STE reached to 300

266

mg/L, while the maximal inhibition rate of the CIA in soymilk was only 12.72% at 300 mg/L of STE.

267

When the concentration of STE was over 300 mg/L, the TIA or CIA showed an increasing trend,

268

which probably resulted from the interaction between the surplus STE and trypsin or chymotrypsin.

269

This phenomenon is consistent with the result in the section of “Inactivation of BBI by STE” in the

270

preceding part of the text.

271

Sensory Profile and TIA and CIA of Soymilk Formulations. The linear regression analysis of

272

the SEC experiment (y = 0.456x – 22.6, R2 = 0.9899) indicates that a concentration of 159 mg/L of

273

STE is found to be equisweet to 30 g/L sucrose on soymilk (Figure 8A). To confirm the nonsweet

274

aftertaste perceived at 159 mg/L of STE, paired preference test (PTJ) on the two final formulations

275

was carried out. As a result, there was no significant difference between the reference (R2) and the

276

formulation (F6). The TIA and CIA in these soymilk formulations were also determined (Figure 8B),

277

and decreasing tendency of the TIA and CIA in these formulations is similar with the effect of STE

278

on the TIA and CIA in soymilk described above, indicating that sucrose has no effect on protease

279

inhibitors in soymilk.

280

■ DISCUSSION

281

Soymilk, which resembles with dairy milk, can be used as a vital and cheaper substitute for milk. It

282

is lactose-free, and is of low starch content, and hence qualifies for utilization in ‘specialty’ foods.23

283

Generally, KTI is heat labile, while the BBI is unusually heat stable. Therefore, it is considered that

284

BBI contributes a lot to the protease inhibitory activity in processed soybean products such as

285

soymilk.24

286

In the present study, an alternative approach to inactivating TIA of BBI was successfully 13



Page 14 of 32

287

developed by employing STE as an inhibitor (Figure 3). This is mainly because the location that STE

288

binds was near to P1 reactive site (Lys16-Ser17) (Figure 6A). The binding interactions between STE

289

and BBI in this site may change the polar residues, hydrophilic bridges, and weak hydrophobic

290

contacts that interacting specifically with trypsin, which may prevent BBI from making contact with

291

trypsin and thereby inactivating the TIA of BBI. However, the CIA of BBI could not be inactivated

292

by STE (Figure 3). This occurs due to the location that STE bind to was not near the P2 reactive site

293

(Leu43-Ser44). In addition, the results of fluorescence analysis (stopped-flow kinetic and

294

steady-state fluorescence) revealed that STE has weak interaction with chymotrypsin (KD = 0.20 mM)

295

at low concentration of STE (0.1 ~ 0.5 mg/mL) (Figure 4E and 4F), while a strong binding force (Ka

296

= 4.36 × 107 L·M-1) between chymotrypsin and STE at a higher concentration of STE (0.5 ~ 3

297

mg/mL) (Figure 5G, 5H and 5I). Docking study showed that the binding of STE within a region near

298

the reactive site (His57, Asp 102 and Ser 195) of chymotrypsin (Figure 6E and 6F). These data

299

confirmed that the interaction between the STE and chymotrypsin results to the inactivation effect of

300

STE on the CIA of BBI. Moreover, the order of STE binding to BBI as compared with binding to

301

trypsin and chymotrypsin can be differed by stopped-flow kinetic experiments (KD) (in the low

302

concentration of STE). KD of STE binding to BBI, trypsin and chymotrypsin is 0.01, 0.19 and 0.20

303

mM, respectively. These data indicate that STE tends to associate with BBI, while it tends to

304

dissociate from trypsin and chymotrypsin in the low concentration of STE.

305

The phenomenon of STE inactivating residual TIA in soymilk was observed (Figure 7). The

306

effect of adding STE on the sensory profile of soymilk was also studied (Figure 8A). High-intensity

307

sweeteners may substitute sucrose in soft drinks but they modify the flavor due to their organoleptic

308

properties or interactions with the aromatic compounds.25 In addition, the non-sweet aftertaste 14


Page 15 of 32


309

associated with many sweeteners including STE which limits their use at high concentration.

310

Therefore, STE was used for partial substitution of sucrose in soymilk in the present study (Table 1).

311

Although the inhibition rate of the TIA in soymilk achieved a maximal value when the concentration

312

of STE reached to 300 mg/L, a mixture of 159 mg/L of STE and 60000 mg/L of sucrose is suitable

313

for soymilk formulation. By this means, the typical bitter taste of stevioside was eliminated, and the

314

residual TIA in soymilk could be inactivated to some extent (49.90%) by STE (Figure 8B).

315

For soymilk, it contained KTI and BBI together, the content of KTI was high than that of BBI,

316

therefore, the decrease in TIA may be arised from the interaction between KTI and STE, not only

317

BBI and STE. In order to confirm the fact that inactivation of BBI by STE, the interaction of STE

318

with KTI has been studied by using stopped-flow kinetic experiments, steady-state fluorescence and

319

molecular docking. The results of fluorescence analysis indicated that there was a strong interaction

320

between STE and KTI (KD = 0.018 mM, Ka = 1.99 × 107 L·M-1) (Figure 4C and 4D; Figure 5D, 5E

321

and 5F). However, docking study revealed that the binding region of STE was not near the reactive

322

site (Arg63-Ile64) of KTI (Figure 6C, 6D). Therefore, the decrease in TIA may be not arised from

323

the interaction between KTI and STE. In order to further confirm whether KTI can be inactivated by

324

STE, the effect of STE on TIA of KTI was studied (Figure 3B). The result indicated that STE could

325

not inactivate the TIA of KTI.

326

Besides BBI and KTI, there are various proteins in soymilk. 7S and 11S globulin are the two

327

major storage protein fractions in soymilk, thus a pertinent question arises here: why BBI still could

328

be inactivated by STE rather than preferential binding to other proteins especially to 7S and 11S

329

proteins? In our previous work,26 the interactions between soybean protein isolate (SPI, mainly

330

consists of 7S and 11S) and STE in aqueous phase have been studied, and the value of Kq (6.68 ×108 15



Page 16 of 32

331

M-1 s-1) is much lower than the value of Kq (3.29 × 1013 M-1 s-1) (Figure 5B) derived from interaction

332

between STE and BBI. Furthermore, we found that no obvious changes in the fluorescence intensity

333

of SPI were observed for STE concentrations below 0.8 mg/mL, indicating that there were no or

334

weak interaction of SPI with STE. As the concentration of STE was increased from 2 to 20 mg/mL,

335

the addition of STE led to a progressive quenching of the fluorescence of SPI, revealing that there

336

was interaction between SPI and STE.26 In this study, the distinct changes in the fluorescence

337

intensity of BBI were observed when STE concentration was 0.5 mg/mL. Moreover, a progressive

338

quenching of the fluorescence of BBI was found when STE concentration was 3 mg/mL (Figure 5).

339

These data revealed that STE was preferentially complexed with BBI and residual KTI relative to

340

SPI in soymilk. Although further studies are needed to evaluate the effect of STE on in vitro and in

341

vivo digestibility of soymilk proteins, it is suggested that STE could be used to improve nutritional

342

value and sweet taste of soymilk.

343 344

Funding

345

This research was supported by grants from the National Natural Science Foundation of China (No.

346

31571874), the program for Science & Technology Innovation Talents of Hunan Province (No.

347

2017TP1021 kc1704007) and Natural Science Foundation of Hunan Province (No. 2017JJ3528;

348

2018JJ2672).

349

Notes

350

The authors declare no competing financial interest.

351

Acknowledgements

352

We thank Dr. Ruipu Xin (National University of Singapore) for discussion of the results of 16


Page 17 of 32

353


molecular docking.

354 355

■ REFERENCES

356

(1)Xu, Z.; Chen, Y.; Zhang, C.; Kong, X.; Hua, Y. The heat-induced protein aggregate correlated

357

with trypsin inhibitor inactivation in soymilk processing. J. Agric. Food Chem. 2012, 60 (32),

358

8012-8019.

359

(2)Yuan, S.; Chang, S. K. C.; Liu, Z.; Xu, B. Elimination of trypsin inhibitor activity and beany

360

flavor in soy milk by consecutive blanching and ultrahigh-temperature (UHT) processing. J. Agric.

361

Food Chem. 2008, 56 (17), 7957-7963.

362

(3)Liu, C.; Cheng, F.; Yang, X. Inactivation of soybean trypsin inhibitor by epigallocatechin gallate:

363

stopped-flow/fluorescence, thermodynamics, and docking studies. J. Agric. Food Chem. 2017, 65 (4),

364

921-929.

365

(4)Huang, H.; Kwok, K. C.; Liang, H. Effects of tea polyphenols on the activities of soybean

366

trypsin inhibitors and trypsin. J. Sci. Food Agr. 2004, 84 (2), 121-126.

367

(5)Geuns, J. M. Stevioside. Phytochemistry 2003, 64 (5), 913-921.

368

(6)Brandle, J. E.; Starratt, A. N.; Gijzen, M. Stevia rebaudiana: its agricultural, biological, and

369 370 371 372 373 374

chemical properties. Can. J. Plant Sci. 1998, 78 (4), 527-536. (7)Terada, S.; Sato, K.; Kato, T.; Izumiya, N. Inhibitory properties of nonapeptide loop structures related to reactive sites of soybean Bowman-Birk inhibitor. FEBS Lett. 1978, 90 (1), 89-92. (8)Werner, M. H.; Wemmer, D. E. Three-dimensional structure of soybean trypsin/chymotrypsin Bowman-Birk inhibitor in solution. Biochemistry 1992, 31 (4), 999-1010. (9)Chen, P.; Rose, J.; Love, R.; Wei, C. H.; Wang, B. C. Reactive sites of an anticarcinogenic 17



Page 18 of 32

375

Bowman-Birk proteinase inhibitor are similar to other trypsin inhibitors. J. Biol. Chem. 1992, 267 (3),

376

1990-1994.

377

(10)

Koepke, J.; Ermler, U. E.; Wenzl, G.; Flecker, P. Crystal structure of cancer

378

chemopreventive Bowman-Birk inhibitor in ternary complex with bovine trypsin at 2.3 å resolution.

379

Structural basis of Janus-faced serine protease inhibitor specificity. J. Mol. Biol. 2000, 298 (3),

380

477-491.

381 382 383 384 385 386 387

(11)

Smith, C.; Megen, W. V.; Twaalfhoven, L.; Hitchcock, C. The determination of trypsin

inhibitor levels in foodstuffs. J. Sci. Food Agr. 1980, 31 (4), 341-350. (12)

Tan, N. H.; Zha, R.; Khor, H. T.; Wong, K. C. Chymotrypsin inhibitor activity in winged

beans (Psophocarpus tetragonolobus). J. Agric. Food Chem. 1984, 32 (1), 163-166. (13)

Huang, H.; Zhao, M. Changes of trypsin in activity and secondary structure induced by

complex with trypsin inhibitors and tea polyphenol. Eur. Food Res. Technol. 2008, 227 (2), 361-365. (14)

Patrick, S. M.; Turchi, J. J. Stopped-flow kinetic analysis of replication protein a-binding

388

damage recognition and affinity for single-stranded DNA reveal differential contributions of kon and

389

koff rate constants. J. Biol. Chem. 2001, 276 (25), 22630-22637.

390

(15)

Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.; Olson,

391

A. J. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy

392

function. J. Comput. Chem. 1998, 19 (14), 1639-1662.

393 394 395 396

(16)

Clark, A. M.; Labute, P. 2D depiction of protein-ligand complexes. J. Chem. Inf. Model.

2007, 47, 1933-1944. (17)

Chao, W. X.; Wood, C. M.; Robertson, P.; Gilani, G. S. Protease inhibitor activities and

isoflavone content in commercial soymilks and soy-based infant formulas sold in Ottawa, Canada. J. 18


Page 19 of 32

397 398 399 400 401 402


Food Compos. Anal. 2012, 25 (2), 130-136. (18)

Parpinello, G. P.; Versari, A.; Castellari, M.; Galassi, S. Stevioside as a replacement of

sucrose in peach juice: sensory evaluation. J. Sens. Stud. 2010, 16 (5), 471-484. (19)

Amerine, M. A.; Pangborn, R. M.; Roessler, E. B. Principles of Sensory Evaluation of Food.

1965, 159-176. Academic Press, New York. (20)

Wensley, B. G.; Batey, S.; Bone, F. A.; Chan, Z. M.; Tumelty, N. R.; Steward, A.; Kwa, L.

403

G.; Borgia, A.; Clarke, J. Experimental evidence for a frustrated energy landscape in a

404

three-helix-bundle protein family. Nature 2010, 463 (7281), 685-688.

405 406 407 408 409

(21)

Pan, H.; Qin, M.; Meng, W.; Cao, Y.; Wang, W. How do proteins unfold upon adsorption

on nanoparticle surfaces? Langmuir 2012, 28 (28), 12779-12787. (22)

Lakowicz, J. R.; Masters, B. R. Principles of fluorescence spectroscopy, third edition. J.

Biomed. Opt. 2008, 13 (2), 029901. (23)

Iwuoha, C. I.; Umunnakwe, K. E. Chemical, physical and sensory characteristics of soymilk

410

as affected by processing method, temperature and duration of storage. Food Chem. 1997, 59,

411

373-379.

412

(24)

Rouhana, A.; Adler-Nissen, J.; Cogan, U.; Frøkiær, H. Heat inactivation kinetics of trypsin

413

inhibitors during high temperature-short time processing of soymilk. J. Food Sci. 1996, 61 (2),

414

265-269.

415 416 417 418

(25)

Nahon, D. F.; Roozen, J. P.; Graaf, C. D. Sweetness flavor interaction in soft drinks. Food

Chem. 1996, 56 (3), 283-289. (26)

Wan, Z. L.; Wang, J. M.; Wang, L. Y.; Yang, X. Q.; Yuan, Y. Enhanced physical and

oxidative stabilities of soy protein-based emulsions by incorporation of a water-soluble 19



419

stevioside-resveratrol complex. J. Agric. Food Chem. 2013, 61 (18), 4433-4440.

420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 20


Page 20 of 32

Page 21 of 32


444

Figure captions

445

Figure 1. Chemical structures of stevioside (PubChem CID: 24721412).

446 447

Figure 2. Pymol representation of the structure of soybean BBI. The three-dimensional structure of

448

BBI in solution (PDB ID: 1BBI) was used to generate this diagram. Two reactive site loops, interact

449

at Lys16-Ser17 (subdomain 1) with trypsin and at Leu43-Ser44 (subdomain 2) with chymotrypsin,

450

are shown in a red stick and a purple stick representation respectively.

451 452

Figure 3. (A) Inactivation of BBI by STE. The concentration of BBI is 0.02 mg/mL. (B) Inactivation

453

of KTI by STE. The concentration of KTI is 0.02 mg/mL. (C) Inactivation of trypsin and

454

chymotrypsin activity by STE, respectively. The concentrations of trypsin and chymotrypsin are 0.02

455

mg/mL.

456 457

Figure 4. Stopped-flow analysis of STE binding proteins. (A), (C), (E) and (G): Kinetic traces for

458

STE (at 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 mg/mL) binding to BBI, KTI, trypsin and chymotrypsin in 10

459

mM PBS buffer at pH 7.4, respectively. Each trace is the average of 8-12 independent measurements

460

and is fitted with a single-exponential function (

461

constants (kobs) plotted vs STE concentration provided a linear fit for BBI, KTI, trypsin and

462

chymotrypsin, respectively. Each point represents the average of three individual experiments.

). (B), (D), (F) and (H): The observed rate

463 464

Figure 5. Steady-state fluorescence analysis of STE binding proteins. (A), (D), (G) and (J): Effect of

465

STE on the intrinsic fluorescence of BBI, KTI, trypsin and chymotrypsin, respectively. (B), (E), (H) 21



Page 22 of 32

466

and (K): The Stern-Volmer plots describing fluorescence quenching of BBI, KTI, trypsin and

467

chymotrypsin in the presence of STE, respectively. (C), (F), (I) and (L): The double-logarithmic

468

Stern-Volmer plot describing static quenching of BBI, KTI, trypsin and chymotrypsin in the

469

presence of STE, respectively.

470 471

Figure 6. Molecular docking studies. (A), (C), (E) and (G): The binding interactions of STE with the

472

molecular surface of BBI, KTI, trypsin and chymotrypsin, respectively. (B), (D), (F) and (H): Type

473

of residue interaction at the binding site. Amino acid residues in the vicinity of the STE molecule are

474

labeled, and hydrogen bonds are shown as arrows.

475 476

Figure 7. The effect of STE on the TIA and CIA in soymilk.

477 478

Figure 8. (A) Determination of equisweet between STE and sucrose in soymilk. (B) TIA and CIA in

479

different formulations (F0-F5 in Table 1), Inset: TIA and CIA in two final formulations (F6 and R2 in

480

Table 1).

22


Page 23 of 32


Tables Table 1. Experimental Design of the Soymilk Formulations Sweetened with STE and/or Sucrose SEC

PTJ

Sample F0 STE (final conc. mg/L)

0

Sucrose (final conc. g/L) 60

F1

F2

R1

F6

R2

100 125 150 175 200

0

159

0

60

90

60

90

60

F3

60

F4

60

F5

60

Formulations (F0-F6). Reference (R1-R2). SEC: sweetness equivalency between 3% sucrose (reference R1) and STE at different concentrations (samples F0-F5); PTJ: paired preference test on the two final formulations.

23



Figure 1

24


Page 24 of 32

Page 25 of 32


Figure 2 Anti-chymotryptic site Anti-tryptic site

25



Page 26 of 32

Figure 3

560

1400 TIA retained CIA retained

TIA retained (U/mg)

1200

520

1000

480

800

440

600

400

400

360

200

CIA retained (U/mg)

A

320 0.0

0.2

0.4

0.6

0.8

1.0

0.8

1.0

STE (mg/mL)

TIA retained (U/mg)

B

1600

1200

800

400

0 0.0

0.2

0.4

0.6

STE (mg/ml) 1600 50

Trypsin activity (U/mg)

1500 Trypsin activity Chymotrypsin activity

1400

45

1300

40

1200 35

1100

30

1000 0.0

0.2

0.4

0.6

0.8

1.0

STE (mg/mL)

26


1.2

Chymotrypsin activity (U/mg)

C

Page 27 of 32


Figure 4

A

7.8

B

3.5 3.0 2.5

0.1

2.0

-1

7.4

0.05

kobs (s )

Intensity (a.u.)

7.6

0.2

7.2

0.3

0.5

0.5

6.8 0

1

2

3

1.5 1.0

0.4

7.0

y = 6.4643x + 0.0708 2 R = 0.9951

0.0 0.0

4

0.1

Time (s)

0.3

0.4

0.5

0.4

0.5

0.4

0.5

0.4

0.5

STE (mg/mL)

7.8

3.0

D

7.6

2.5

0.05

7.4

2.0

0.1 0.2 0.3

7.0

0.4

-1

7.2

kobs (s )

Intensity (a.u.)

C

0.2

y = 5.037x + 0.091 2 R = 0.9959

1.5 1.0 0.5

6.8 0.5

0.0 0.0

6.6 0

1

2

3

4

0.1

7.8

F 0.05

0.3

0.30

0.25

0.1 0.2

7.4

0.3

7.2

-1

Intensity (a.u.)

7.6

kobs (s )

E

0.2

STE (mg/mL)

Time (s)

y = 0.3838x + 0.075 2 R = 0.9921

0.20

0.15

0.4

0.10

0.5

7.0 0

1

2

3

4

0.0

0.1

Time (s)

G

0.3

STE (mg/mL)

7.6 0.05

H

0.10

7.5 0.1

-1

0.2

y = 0.1313x + 0.0266 2 R = 0.9946

)

7.4

0.08

7.3

0.3

7.2

kobs (s

Intensity (a.u.)

0.2

0.06

0.4

0.04

7.1 0.5

7.0 0

1

2

3

4

0.0

0.1

Time (s)

0.2

0.3

STE (mg/mL)

27



Page 28 of 32

Figure 5 1400

B

0 mg/mL STE

1200

2.4 2.2

1000

600 400

3 mg/mL STE

1.8

5

Ka = 3.38 × 10 0.0

13

Kq = 3.29 × 10

-0.2

lg[(F0-F)/F]

800

0.2

C

5

Ksv = 3.29 × 10

2.0

F0/F

Fluorescence intensity

A

y = 0.4082x + 0.9596 2 R = 0.9781

1.6

n = 1.01 y = 1.0073x + 5.5293 2 R = 0.9903

-0.4

1.4

-0.6

200

1.2 -0.8

0 320

340

360

380

400

420

0.5

440

1.0

2000

E

0 mg/mL STE

1500

1000

F0/F

2.2

F

5

320

340

360

380

400

420

Kq = 3.48 × 10

1.8

y = 0.4335x + 0.8472 2 R = 0.9890

1.6

-0.2 -0.4

-5.6

-5.4

-5.6

-5.4

-5.6

-5.4

n = 1.327 y = 1.327x + 7.299 2 R = 0.9823

-0.6 -0.8

0.5

H

0 mg/mL STE

1.0

1.5

2.0

2.5

-1.0

3.0

-6.2

I

5

2.4

Ksv = 3.97 × 10

2.2

Kq = 3.97 × 10

13

3 mg/mL STE

1.8

0.4 7

0.2

y = 0.4938x + 0.9058 2 R = 0.9889

-0.2

1.6

380

400

420

-0.6 -0.8 0.5

440

1.0

1.5

2.0

2.5

K

0 mg/mL STE

1500

L

5

Ksv = 5.58 × 10

2.0

y = 0.6937x + 0.6393 2 R = 0.9791

0.0 -0.2

Wavelength (nm)

420

440

y = 1.3643x + 7.6408 2 R = 0.9862

-0.4 -0.6 -0.8

1.0

400

7

Ka = 4.36 × 10 n = 1.3643

Kq = 5.58 × 10

3 mg/mL STE

380

-5.8

0.4 0.2

13

2.5

1.5

360

-6.0

lg[c(STE)]

3.0

F0/F

1000

340

-6.2

3.0

STE (mg/mL)

Wavelength (nm)

320

y = 1.3069x + 7.3286 2 R = 0.9838

-0.4

1.0

360

Ka = 2.13 × 10 n = 1.3069

1.2

340

-5.8

0.0

1.4

320

-6.0

lg[c(STE)]

2.0

500

0 300

-5.4

Ka = 1.99 × 10

STE (mg/mL)

1000

500

-5.6

7

0.0

2.0

1.0

440

1500

2000

-5.8

0.2

13

lg[(F0-F)/F]

2000

-6.0

lg[c(STE)]

1.2

F0/F


-6.2

3 mg/mL STE

0 300


3.0

Ksv = 3.48 × 10

Wavelength (nm)

J

2.5

1.4

500

0 300

G

2.0

lg[(F0-F)/F]


D

1.5

STE (mg/mL)

Wavelength (nm)

lg[(F0-F)/F]

300

0.5

1.0

1.5

2.0

2.5

3.0

STE (mg/mL)

28


-6.2

-6.0

-5.8

lg[c(STE)]

Page 29 of 32


Figure 6

A

B

C

D

F

E

G

H

29



Page 30 of 32

500

300

400

250

300

200

150

TIA CIA

200

100

100 0

100

200

300

STE (mg/L)

30


400

CIA (U/mL)

TIA (U/mL)

Figure 7

Page 31 of 32


Figure 8

TIA CIA

500

y = 0.456x - 22.6 2 R = 0.9899

60

250

400

40 30 20

300

200

200 Protease inhibitor activities (U/mL)

50

500

120

140

160

180

200

TIA CIA

400

150

300

200

100 F6

100 100

300

100

R2

Formulations

F0

F1

F2

F3

Formulations

Stevioside (mg/L)

31


F4

F5

CIA (U/mL)

B

70

TIA (U/mL)

Stevioside sweetner than sucrose (response %)

A


Page 32 of 32

TOC Graphic

Stevioside Trypsin

+

×

Reactive site

Soymilk

STE

Trypsin-BBI complex BBI-STE complex

32


Inactivation of Soybean Bowman-Birk Inhibitor by Stevioside

Recommend Documents