1 Supplementary materials

2 Assessment of soy genotype and processing method on trypsin inhibitors, urease and lectins

3 content of tofu

5 Sladjana P. Stanojević*, Aleksandar Ž. Kostić, Danijel D. Milinčić and Mirjana B. Pešić

6 University of Belgrade, Faculty of Agriculture, Department of Chemistry and Biochemistry,

7 Belgrade, Serbia

8 *Corresponding author: Sladjana P. Stanojević, sladjas@agrif.bg.ac.rs

9 Abstract

10 Tofu has a high nutritional value, but it may also contain components that may have an

11 antinutritional effect, such as trypsin inhibitors (TI), lectins and ureases. The aim of this study

12 was to investigate the influence of the hydrothermal-cooking process of soybean in combination

13 with commercial chymosin-pepsin rennet on the content and activity of TI, urease and lectins in

14 tofu. High total TI content was found in tofu (5.00-16.87%). In addition, Kunitz (KTI=3.52-

15 4.32%) and Bowman-Birk (BBI=5.00-12.53%) TI were registered, and BBI was detected in

16 polymeric (1.38-2.71%) and monomeric (3.42-9.82%) forms. TI activity of tofu was very low

17 (5.86-9.34%), corresponding to the very low activity of urease (0.51-3.07%). The percentage of

18 lectin (2.62-4.63%) and urease (0.03-0.12%) in tofu was low. The results showed that the applied

19 tofu production process is very effective in reducing the content and activity of TI, urease and

20 lectin and provides the values without nutritional harmful effect.

21

22 Keywords: hydrothermal-cooking process, chymosin-pepsin rennet, Kunitz-type trypsin

23 inhibitor; Bowman-Birk- type trypsin inhibitor

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24 Experimental

25 Materials

26 For the preparation of tofu, six commercial soybean genotypes were used: Novosadjanka,

27 Balkan, Krajina (by the Institute of Field and Vegetable Crops, Novi Sad, Serbia) and ZPS-015,

28 Nena and Lana (by the Maize Research Institute Zemun Polje, Belgrade). The soybean

29 genotypes used were characterized by: total trypsin inhibitor content of 3.10-12.17% (Nena

30 7.50%; Krajina 12.17%; Novosadjanka 6.76%; Balkan 7.13%: ZPS-015 9.48% and Lana 3.10%),

31 trypsin inhibitory activity of 95.61-197.68 TUI/mg(Nena 197.68 TUI/mg; Krajina 181.69

32 TUI/mg; Novosadjanka 197.50 TUI/mg; Balkan 195.36 TUI/mg; ZPS-015 184.86 TUI/mg and

33 Lana 95.61 TUI/mg), urease index activity of 0.02-0.08 (Nena 0.04; Krajina 0.08; Novosadjanka

34 0.04; Balkan 0.06: ZPS-015 0.03 and Lana 0.02) and urease activity of 1.03-3.81% (Nena

35 1.75%; Krajina 3.81%; Novosadjanka 1.77%; Balkan 2.70%: ZPS-015 1.39% and Lana 1.03%)

36 (Stanojević et al., 2013).

37 Preparation of tofu

38 Tofu was made from soy milk obtained using the hydrothermal cooking process (high

39 pressure/high temperature/short time), and then coagulation of soy milk with proteolytic

40 enzymes (commercial chymosin-pepsin rennet) according to Stanojevic et al. (2011). Briefly,

41 soybeans were soaked at 5-7 °C, in water (soybeans:water = 1:5) at 5-7 °C, for 14 hours. Soaked

42 seeds were simultaneously ground and cooked by steam injection system (soybeans:water = 1:6)

43 at 1.8 bar and 110 °C, for 8 min (SoyaCow VS 30/40, model SM-30, Russia). Soy milk was

44 obtained after filtering the cooked porridge and separating the okara. After cooling the soy milk,

45 coagulant (10 ml coagulant/L soy milk) was added with manual mixing, and then the mixture

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46 was allowed to stabilize. The obtained curd was pressed, in order to separate the tofu curd and

47 obtain tofu. Tofu samples were stored at 4°C before further analysis.

48 Sodiumdodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and native-

49 polyacrylamide gel electrophoresis (PAGE)

50 For electrophoretic analyses, tofu proteins extracted was prepared by mixing for 1 h at room

51 temperature with 0.055 M Tris-HCl-glycerin buffer (pH 6.80), which contained 0.64 M β-

52 mercaptoethanol (buffer:sample=1:20). Then, the mixture was centrifuged at 7558g, for 15 min

53 at room temperature (Stanojevic et al. 2011). With sample buffer, pH 6.80 [5% (v/v) β-

54 mercaptoethanol, 0.055 M Tris-HCl, 0.0025% (w/v) bromophenol blue, 7% (v/v) glycerin, 2%

55 (w/v) SDS] the supernatant was diluted (to a concentration of 2 mg/mL). 25 μL of each sample

56 was used for electrophoretic analysis.

57 The tofu protein extracts were analyzed in dissociating and reducing conditions by SDS-

58 PAGE by Fling and Gregerson (1986), detailed by Stanojevic et al. (2011). Polyacrylamide gels

59 for SDS-PAGE consisted of 5% (m/v) (pH 6.80) stacking gel and 12.5% (m/v) (pH 8.85) running

60 gel. The gels were run in a buffer solution of pH 8.30 [0.19 M glycine, and 0.1% (w/v) SDS,

61 0.05 M Tris] for 6 h at 80 mA/gel. Then, the gels were fixed and stained for 50 min, with 0.23%

62 (m/v) Coomassie-brilliant-blue-R-250 and destained with the mixture of 188% (v/v) acetic acid

63 and % ethanol. For detection and estimation of molecular weight of polypeptides low molecular

64 weight calibration kit (Pharmacia, Uppsala, Sweden) was used (Stanojevic et al. 2011).

65 The tofu protein extracts were analyzed by PAG electrophoresis by Davis (1964) detailed

66 by Stanojevic et al. (2010). Polyacrylamide gels comprising of 5% (m/v) (pH 6.70) stacking gel

67 and 7% (m/v) running gel (pH 8.90) were used for PAGE analysis under conditions 90 mA/gel,

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68 for 4.30 h. Following the same procedure as after SDS-PAG electrophoresis, the gels were fixed

69 stained and destained (Stanojevic et al. 2010).

70 SDS-PAGE and PAG electrophoresis was performed in two repetitions in electrophoresis

71 unit LKB-2001-100 with power supply LKB-Macrodrive 5 and LKB-Multitemp as a cooling unit

72 (Pharmacia, Sweden). Densitometric analysis of both scanned decolorized gels was performed

73 on SigmaGel software version 1.1 (JandelScientific, San Rafael, CA, USA). Quantity of each

74 identified protein subunit was calculated as the percentage of the respective area with respect to

75 total area of the densitogram and presented as a percentage of extractable proteins (Stanojevic et

76 al. 2010, 2011).

77 Trypsin inhibitor activity

78 Trypsin inhibitory activity in tested tofu samples was evaluated according to Liu and Markakis

79 (1989) detailed by Stanojevic et al. (2013). As the substrate crystalline bovine trypsin (Sigma,

80 USA) and α-N-benzoyl-DL-argininep-nitroanilide hydrochloride (BAPA; Sigma, USA) was

81 using. With distilled water (sample/water =1:100) the tofu samples were extracted, for 30 min,

82 and then filtered (Whatman-paper No. 4). The filtrates were diluted with 0.05 M Tris-HCl buffer

83 (pH 8.2) (extract/buffer = 1:1) and then filtered. The filtrates were diluted with distilled water so

84 that 1 mL of filtrate inhibited 30−70% of the enzyme. A 1 mL of the diluted filtrates was

85 incubated for 10 min at 37 °C with enzyme solution (16 μg/mL in 0.001 MHCl) and 0.92 mM

86 BAPA (1 mL), and then the reaction was stopped with 30% (v/v) acetic acid. Absorbance was

87 measured at 410 nm. The analysis was done in three repetitions Stanojevic et al. (2013). The

88 inhibitor activity was expressed in percent as residual trypsin inhibitor activity relative to

89 respective defatted soybean flour (rTIA) and also in trypsin units inhibited per milligram of the

90 dry sample (TUI/mg). In addition, the degree of reduction of TIA in tofu in relation to seed was

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 91 calculated as a percentage of the reduction of the TUI/mg value for tofu in relation to the

92 TUI/mg value for soy seeds. Values for TUI/mg soy seed were previously published in our study

93 (Stanojević et al., 2013).

94 Urease activity

95 Urease activity was determined according to Caskey and Knapp (1944), detailed by Stanojevic et

96 al. (2013). Over the years, many protocols have been developed that allow the measurement of

97 urease activity. They are based on the quantification of liberated ammonia (Caskey and Knapp

98 1944). To determine urease activity, the investigated tofu samples were extracted with a buffered

99 solution of urea at pH 7.00 (sample/urea = 1:50) and incubated at 30 °C, for 30 min, with

100 intensively shaken every 5 min. From tofu samples according to the same procedure, but with

101 phosphate buffer pH 7.00 (sample/buffer = 1:50) control samples were prepared. The incubation

102 of control samples was started exactly 5 min after the incubation of test samples. After

103 incubation, the pH value of all samples was measured. Urease activity was defined as the amount

104 of urease required to release nitrogen per minute from 1 g of urea. It was found to increase of pH

105 values as compared to the control. The analysis was done in three repetitions Stanojevic et al.

106 (2013). Urease activity was expressed as urease index of activity (pH) by calculating the

107 difference between the pH values of tofu samples and control samples and it was also expressed

108 in percent as urease activity (UA) relative to respective defatted soy seeds. Values for pH and

109 UA of soy seeds were previously published in our study (Stanojević et al., 2013).

110 Statistical analysis

111 For statistical analysis Statistic Software Version 8.0 (StatSoft Co., Tulsa, Oklahoma, USA) was

112 applied. Data are expressed as mean and pooled standard deviation (Pooled std). Pearson's

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113 correlation coefficients and Tukey's test were applied in the statistical analysis of the results at

114 the probability level of p<0.05.

115 Acknowledgments

116 The authors thank the Institute of Field and Vegetable Crops, Novi Sad, Serbia and the Maize

117 Research Institute, Zemun Polje, Serbia, for providing genotypes of soybean.

118 References

119 Caskey CD, Knapp FC. 1944. Method for detecting inadequately heated soy‐ bean oil meal. Ind

120 Eng Chem. 16(10):640-641.

121 Davis J. 1964. Disc electrophoresis-II. Method and application to human serum proteins. Ann the

122 NY Acad Sci. 121:404-427.

123 Fling PS, Gregerson SD. 1986. Peptide and protein molecular weight determination by

124 electrophoresis using a high-molarity tris-buffer system without urea. Anal Biochem. 155:83-

125 88.

126 Liu F, Markakis P. 1989. An improved colorimetric method for determining antitryptic activity

127 in soybean products. Cereal Chem. 66:415-422.

128 Stanojevic PS, Barac BM, Pesic BM, Milovanovic MM, Vucelic-Radovic VB. 2010. Protein

129 composition in tofu of corrected quality. APTEFF. 41:77-86.

130 Stanojevic PS, Barac BM, Pesic BM, Vucelic-Radovic VB. 2011. Assessment of soy genotype

131 and processing method on quality of soybean tofu. J Agr Food Chem. 59(13):7368-7376.

132 Stanojevic S, Barac M, Pesic M, Jankovic V, Vucelic-Radovic B. 2013. Bioactive proteins and

133 energy value of okara as a byproduct in hydrothermal processing of soymilk. J Agr Food

134 Chem. 61:9210-9219.

135

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137 Table S1. Correlation coefficients between properties of tofu
Relationship r* Relationship r*
TI - KTI 0.94 TUI/mg - UA 0.97
TI - BBI 0.98 rTIA - UA 0.92
TI - BBIp 0.99 TUI/mg - % reduction of TIA from seed -0.90
TI - BBIm 0.96 rTIA - % reduction of TIA from seed -0.99
TI - rTIA -0.26 UA - TI -0.44
KTI - BBI 0.86 UA - % reduction of TIA from seed -0.91
KTI - BBIp 0.94 TUI/mg - rTIA 0.92
KTI - BBIm 0.83 TUI/mg - UA 0.97
KTI - KTI/BBI -0.71 TUI/mg - % reduction of TIA from seed -0.90
BBI - KTI/BBI -0.94 rTIA - % reduction of TIA from seed -0.99
BBI - BBIp 0.97 rTIA - UA 0.92
BBI - BBIm 0.99 UA - % reduction of TIA from seed -0.91
BBI - BBIm 0.94 UA - ΔpH 0.99
SBA - TI -0.79 UA - urease 0.92
SBA - KTI -0.55 ΔpH - urease 0.89
SBA - BBI -0.88 ΔpH - TUI/mg 0.97
SBA - KTI/BBI -0.87 ΔpH - % reduction of TIA from seed -0.94
SBA - urease -0.08 ΔpH - rTIA 0.95
urease - TI -0.48 TUI/mg soybean - TUI/mg tofu -0.006
urease - KTI -0.66 TUI/mg soybean - rTIA -0.39
urease - BBI -0.29 TUI/mg soybean – TI tofu 0.11
TUI/mg - rTIA 0.92 TUI/mg soybean - % reduction of TIA from seed 0.43
*
138 Significant at P < 0.05. TI-total content of trypsin inhibitors; KTI-Kunitz trypsin inhibitor; BBI-
139 Bowman-Birk trypsin inhibitor; BBIm-monomeric forms of BBI; BBIp-polymeric forms of BBI;
140 rTIA-residual trypsin inhibitor activity. TIU/ mg-trypsin inhibitor units inhibited per milligram
141 of the dry sample; ΔpH- index of urease activity; UA-urease activity
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184 Figue S1. SDS-PAGE analysis of tofu proteins from investigated genotypes; Lanes: 1-Nena, 2-

185 ZPS-015, 3-Lana, 4-Balkan, 5-Krajina, 6-Novosadjanka, mws molecular weight standards. KTI-

186 unitz trypsin inhibitor; SBA-lectins.

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207 Figue S2. PAGE analysis of tofu proteins from investigated genotypes; Lanes: 1-Lana; 2-ZPS-

208 015; 3-Balkan; 4-Nena; 5-Krajina; 6-Novosadjanka; mws-molecular weight standards. BBIp-

209 polymeric forms of Bowman-Birk-type trypsin inhibitor; BBIm-monomeric forms of Bowman-

210 Birk-type trypsin inhibitor.

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225 Highlights
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227  Relatively high total trypsin inhibitor content was found in the extractable proteins of
228 tofu.
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230  The trypsin inhibitor activity of tofu was very low.
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232  Low trypsin inhibitor activity of tofu corresponded to very low urease activity.
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234  Low trypsin inhibitor activity of tofu corresponded to very low lectins activity.
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236  Low levels of lectins and urease were observed.
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238  The trypsin inhibitors, urease and lectin have no potential antinutritional effect on human
239 health.
240
241  The applied tofu processing method had a positive effect on the content/activity of
242 trypsin inhibitors, urea and lectins.
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