Barać Acta Alimentaria

Acta Alimentaria, Vol. 34 (1), pp. 23–31 (2005)

0139-3006/$ 20.00 2005 Akadémiai Kiadó, Budapest

THE EFFECT OF MICROWAVE ROASTING ON SOYBEANPROTEIN COMPOSITION AND COMPONENTS

WITH TRYPSIN INHIBITOR ACTIVITYM. BARAĆ* and S. STANOJEVIĆ

Faculty of Agriculture, Institute for Food Technology and Biochemistry, Nemanjina 6. Belgrade.Serbia and Montenegro

(Received: 15 April 2003; accepted: 25 September 2004)

The effect of microwave roasting on protease inhibitor activity and soluble protein content andcomposition in cracked soybeans was investigated in relation to the duration of treatment. Soybeans ofHodgson var. were cracked to 1/6–1/8 of the size of whole bean, dehulled and were exposed tomicrowaves at a frequency of 2.450 MHz. Soluble protein content of hexane defatted samples weredetermined and PAGE, SDS-PAGE and densitometric analyses were used to determine the change ofmajor soybean protein subunits as a function of roasting time. Residual protease inhibitor activities andisoinhibitor composition were also determined.

Microwave treatment is an effective way for inactivation of protease inhibitor activity in crackedsoybeans. Roasting for only two minutes reduced the trypsin inhibitor activity to 13.33% of the initialvalue. Both types of inhibitors, Kunitz (KTI) and Bowman-Birk (BBI) were responsible for residualinhibitor activity. The duration of microwave roasting had strong influence on soluble protein contentand polypeptide composition. Microwave treated samples were characterized by dominant content ofglycinin, and high stability of acidic (-A1,2,3-, -A5-) and basic (-B1,2,3,4-) glycinin subunits wereestablished.

Keywords: cracked soybean, microwave roasting, protease inhibitor activity, glycinin, β-conglycinin

Soybean seeds are the most important vegetable source of edible oils and high qualityproteins, but they contain various components with potential antinutritive effects, suchas protease inhibitors and lectins. However, the latest investigations point to the healthbenefits of these components. Therefore, different methods including thermal andchemical modification have been used to balance them and improve nutritive value ofsoybeans. Heat treatments such as live steam treatment, dry roasting (JOHNSON et al.,1980), hydrothermal cooking (WANG & JOHNSON, 2001) are frequently used methodsfor that purpose. The effect of treatment is determined by the level of residual proteaseinhibitor activity and the change of major storage protein content, composition andstructures.

The principal storage proteins are glycinin (11S) and β-conglycinin (7S). Theircontent, ratio and dynamic of biosynthesis vary with soybean varieties and environment * To whom correspondence should be addressed.E-mail: [email protected]

24 BARAĆ & STANOJEVIĆ: EFFECT OF MICROWAVE ROASTING ON SOYBEAN PROTEINS

Acta Alimentaria 34, 2005

(BLANUŠA et al., 2000, PEŠIĆ et al., 2003). Glycinin (Mw∼360 kDa) is a protein withcompact quaternary structure stabilized via disulfide, electrostatic and hydrophobicinteractions. It is made up of six A-SS-B subunits. Each subunit is composed of an acid(Mw∼38 kDa) and basic polypeptide (Mw∼20 kDa) (STASWICK et al., 1981) linked by asingle disulfide bond, except for the acid polypeptide -A4- (STASWICK et al., 1984).Subunits are packed into two hexagons placed one over the other to form a hollowoblate cylinder. According to MARCONE and co-workers (1998) and LAKEMOND and co-workers (2000), basic polypeptides are placed in the interior of the glycinin molecule.

Beta-conglycinin is a major protein of 7S fraction with molecular weight of 150–180 kDa (THAN & SHIBASAKI, 1976). It is composed of three subunits, -α’-, -α- and -β-which interact to produce seven isomers (B0–B6).

The influence of heat on soybean protein structure in solutions is wellcharacterized. Many workers such as HERMANSON (1978; 1986), KINSELLA (1979),GERMAN and co-workers (1982), DAMODORAN & KINSELLA (1982), have studied theeffects of heat treatment on soybean protein solutions under different conditions(temperature, time, protein concentration, pH, ionic strength). Generally, heatingsoybean proteins at temperatures higher than 70 °C dissociates their quaternarystructures, denatures subunits and promotes the formation of protein aggregates viaelectrostatic, hydrophobic and disulfide interchange bond mechanism (MORR, 1990).Several workers (FRIEDMAN et al., 1991; VELIČKOVIĆ et al., 1992; 1994) reported abouthigh thermal stability of soy flour proteins.

Microwave treatment is an important means of heating, which is readilycommercially available, and is forecast to be utilized much more extensively in thefuture (MUDGETT, 1989). Microwave heating is considered to be an interaction of polarmolecules with the electric component of the magnetic field, heat being generated byfriction as the molecules attempt to orient themselves within the oscillating field(YOSHIDA et al., 1997). The effect of microwave energy on lipase and lipoxygenaseactivity (ESSAKA et al., 1987; VETRIMANI et al., 1992) as well as inhibitor activity(YOSHIDA & KAJIMOTO, 1988) was reported. However, the influence of microwaveheating on storage soybean protein composition and structures are not wellcharacterized.

The aim of this work was to investigate the influence of microwave roasting onprotease inhibitor activity as well as soluble protein content and protein subunits incracked soybeans in relation to the duration of treatment.

1. Materials and methodsSoybean seeds of Hodgson var. were cracked to 1/6–1/8 of the size of whole bean anddehulled. One hundred grams of cracked soybeans (moisture content 6.70%) werespread in single layer in a flat, glass vessel and treated for 1.0, 1.5 and 2.0 min. at620W-2450 MHz (EP-2000-DR Ei, Yugoslavia). As soon as they were taken out of theoven, the temperatures were measured with a temperature probe. The temperatures were

BARAĆ & STANOJEVIĆ: EFFECT OF MICROWAVE ROASTING ON SOYBEAN PROTEINS 25


88.5 °C, 97.4 °C and 111.4 °C, respectively. Depending on duration of treatment, theinitial moisture content decreased to 6.62–6.30%. The roasted samples were allowed tocool to ambient temperature prior to further analysis.

The raw and treated samples were ground and defatted with hexane. Trypsininhibitor activity of untreated and microwave heated samples was assayed as describedby LIU & MARKAKIS (1989) using α-N-benzoyl-DL-arginine-p-nitroanilidehydrochloride (BAPA) as substrate and the solution of bovine trypsin (Sigma, USA).The samples were extracted with distilled water (1:100, flour/water, w/v) for 30 min ona mechanical shaker. The extract was filtered through No 4 Whatman paper. Ten cm3 ofthe extract was diluted with 0.05 M Tris/HCl buffer pH 8.2 (1:1, extract/buffer) andfiltered. The filtrate was then further diluted with distilled water (1:5, filtrate/water).One cm3 of the diluted filtrate was incubated with 1 ml of 0.92 mM BAPA and enzymesolution (16 µg cm–3 in the 0.001 M HCl) at 37 °C for 10 min. The reaction wasstopped by the addition of 30% (v/v) acetic acid. The reaction was also run in theabsence of inhibitors by replacing the sample with 1 cm3 of water. The absorbance wasmeasured at 410 nm (Unicam, England). Distilled water was used as a blank. Onetrypsin unit was defined as 0.01 increase in absorbance at 410 nm under the conditionsof the assay, and the inhibitor activity was expressed in trypsin units inhibited (TUI) permilligram of dry sample.

Soluble proteins were extracted according to the method of THAN & SHIBASAKI(1976) and detected by the method of LOWRY (1951), using bovine serum albumin(Sigma, USA) as standard.

Protease inhibitors of the samples were detected by polyacrilamide gelelectrophoresis (PAGE) performed according to DAVIS (1964) on 14.5×15.5 cm×1.5-mm slab gel. Stacking gel was 5% and running gel was 7%. Gel was stained with 0.1%solution of Coomassie Blue R-250 for 45 min, destained for two days (7% methanoland 10% acetic acid). Prior to electrophoresis, the protein extracts were diluted to2 mg cm–3 with sample buffer (pH 6.8). Protease inhibitors were identified according tothe relative mobility by the use of KTI and BBI standards (Sigma, USA). Their contentswere determined by densitometric analysis of the gel and expressed as % of solubleproteins.

The change in polypeptide composition of soluble proteins was detected by SDS-polyacrylamide gel electrophoresis according to FLING & GREGERSON (1986) on 12.5%slab gel, and quantified by densitometric analysis. Vertical electrophoresis unit LKB-2001-100 was used in conjunction with power supply Macrodrive 5 and Multitemp II(LKB, Sweden). Samples (0.025 cm3) were run at 50 mA for 6 h, the gel was stainedwith 0.23% solution of Coomassie blue R-250 for 90 min, and destained inmethanol/acetic acid water solution (5% methanol, 7% acetic acid). Destained gels werescanned using Scanexpress 12000SP (Mustac, Germany) and analysed with SigmaGelfor Windows version 1.1 software (Jandel Scientific Corporation, USA). Detectedsubunits were identified with low molecular weight kit (LKB-Pharmacia, Sweden) andprotein fractions were separated according to THAN & SHIBASAKI (1976).

Each reported value is the mean of three determinations from three replicates.



2. Results and discussionThe degree of microwave induced denaturation in protein subunits and proteaseisoinhibitors was determined by the measurement of residual activity as a function oftreating time (Table 1).

Table 1. Protein solubility and protease inhibitor activity of raw and treated soybeanSample Soluble proteina Trypsin inhibitora Soluble inhibitor (%)b

(mg g–1) activity KTI BBI(TUI/mg) A B

Raw soybean 379.80±0.45 178.50±0.71 5.94 0.71 0.581.0 min MW treated soy 125.70±0.49 95.00±1.41 6.48 1.43 0.961.5 min MW treated soy 151.20±0.10 48.55±0.07 5.49 0.73 0.942.0 min MW treated soy 145.50±0.46 23.80±0.03 4.89 1.83 0.50

a The values presented are means ± standard deviation.b Means of three densitometric analyses of soluble protein extracts by PAGE.

Microwave treatment is an effective way to inactivate trypsin inhibitors in crackedsoybeans. Roasting for 1–2 min reduced protease inhibitor activity to 95.00±1.41–23.80±0.03 TUI/mg. Residual inhibitor activity registered in samples treated for twominutes (13.33%) have no antinutritive effect. Microwave energy seems to be moreefficient than traditional heat treatments. According to RAJKO and co-workers (1997),this is the result of great penetration depth and homogenous effect in whole volume ofthe substance.

The residual protease inhibitors of microwave treated samples were detected byPAGE (Fig. 1).

Both types of protease inhibitors, KTI and BBI were detected in raw soybeanextracts as well as in treated samples. According to our results, two bands correspond toBBI inhibitor (indicated with arrows, Fig. 1). This may be due to the presence ofisoinhibitors or may be result of their self-aggregation under non-dissociatingconditions (SESSA & BIETZ, 1986).

KTI and BBI represent 5.94% and 1.29% (the sum of A and B values) of rawsoybean soluble proteins, respectively (Table 1). After treatment, cracked soybeanextracts had 1.67–2.33% BBI and 4.89–6.48% KTI content, respectively. These resultsindicate that both types of inhibitors are responsible for residual activity of microwavetreated samples. Relatively high content of soluble inhibitors and low residual activityof two-minute treated samples (13.33%) suggest that most of these components exist inpartially disrupted native conformation and inactive form. The presence of inactiveform of BBI inhibitors is very important because of their high sulfur amino acidscontent and their potential role in cancer prevention (WANG & WIXON, 1999; WAN etal., 1999; FRIEDMAN & BRANDON, 2001).



Fig. 1. PAGE electrophoretic analysis of microwave treated cracked soybeans. 1. Raw soybean; 2. 3. 4.samples treated for 1.0, 1.5, and 2.0 min, respectively; 5. standard of Bowman-Birk inhibitor;

6. standard of Kunitz inhibitor

The relatively high ratio of protease inhibitors indicates that microwaves haveselective influence on soybean storage proteins. Thus, solubility decrease from 37.98% to15.12–12.57% (Table 1) is due to higher thermal degradation of major storage proteins.

Major storage proteins have complex and heterogeneous structure andcharacteristics. Treatment mode essentially determined the degree of their changes(VELIČKOVIĆ et al., 2000). Microwave heating caused partial degradation of dominantpolypeptides and their aggregation into soluble high molecular weight (128. 800 Da)fractions (Fig. 2).

The formation of soluble aggregates is promoted in the first 1.5 min of treatment.These components represent 3.71–3.91% of 1.0–1.5 min treated sample extracts.Longer microwave treatment caused higher degradation degree of denaturedpolypeptides and higher decrease of soluble aggregates content to 1.31%. In addition,longer treatments induce the formation of low molecular weight polypeptides(<20 000 Da). Low molecular polypeptides made up 27.39% of the detected fractions.In this zone three, new dominant bands were detected (Mw 19 400 Da, 15 200 Da,12 400 Da). These fractions represented 11.53% of soluble protein subunits. Whensoluble fractions of microwave treated samples (Fig. 2) are compared with native ones,decrease could be observed not only in the α-, α’- and -β- 7S- subunits content but also11S-polypeptides. Thus, low molecular weight fractions could be a result of partialdegradation of both major protein subunits.



Fig. 2. Polypeptide composition (determined by the SDS-PAGE) of microwave treated cracked soybeans. 1.Raw soybean; 2. microwave treated soybean, 1.0 min; 3. microwave treated soybean, 1.5 min; 4. microwave

treated soybean, 2.0 min; 5. molecular weight standard; 6. 11S fraction of raw soybean

Table 2. The change of dominant polypeptide composition of soybean proteinas a function of microwave treatment

Protein polypeptide w/w Content (%)a sampleRaw soybean 1.0 min 1.5 min 2.0 min

Lipoxygenase 1 93.900 1.18 2.70 3.22 3.66α’- 80.900 5.04 4.59 4.91 2.89α- 73.800 6.07 5.15 5.41 5.10

β-conglycinin

β- 49.500 6.79 4.92 5.18 4.76γ-conglycinin 1 65.100 4.35 4.58 4.26 4.66β-amylase 1 57.100 0.86 4.04 3.28 2.63

SBA 1 33.700 5.84 3.57 3.24 3.91A3- 39.700 3.93 4.59 4.75 3.29

A 1.2.4- 36.300 16.05 12.47 10.13 8.32A5- 14.930 7.39 6.91 6.58 5.84

A 7.6- 32.100 2.45 4.19 5.06 5.43B3- 25.500 3.00 2.04 2.74 4.29

Glycinin

B 1.2.4- 22.750 15.87 12.35 12.30 9.18a Means of three densitometric analyses of soluble protein extracts by SDS-PAGE

Major soybean proteins, glycinin and β-conglycinin expressed relatively highstability against microwave heating. Due to the high content and lower stability ofβ-conglycinin, soluble protein extracts were mainly characterized by glycinin subunits.



Depending on the duration of heating, these polypeptides make up 43.13–36.35% ofextracted proteins. Acid –A1,2,4- and basic –B1,2,4- subunits are dominant. Thesesubunits represent 8.32–12.47% and 9.18–12.35% of soluble polypeptides, respectively.

It is known that glycinin subunits have different thermal stability in solution.Insoluble aggregates of glycinin formed in solutions are the results of exclusivereassociation of denatured basic subunits. SDS-PAGE analysis showed that microwavetreated samples had high ratio of basic subunits (13.47–15.04%). These results suggesta different type of changes in glycinin subunits as a result of microwave treatment incomparison with heat treatment in solution. Similar behavior of these subunits wasobserved by VELIČKOVIĆ and co-workers (1994) in the case of live steam treatment ofcracked soybean.

Comparing the SDS-electrophoretic protein profiles (Fig. 2) it can be stated thatthe duration of treatment determined the soluble β-conglycinin subunit composition.The content of -α-, -α’- subunits decreased from 11.11% to 9.74%–7.13, while β-subunits decreased from 6.79% to 5.18–4.76% according to treatment time. Longertreatment caused high denaturation degree of -α’- subunits, which represent only 2.89%of soluble polypeptides.

3. ConclusionMicrowave roasting is an effective way to inactivate protease inhibitors in crackedsoybeans. Treating for two minutes reduced inhibitor activity to the level, which haveno antinutritive effect. Both types of inhibitors are responsible for residual activity ofmicrowave treated samples. Because of their amino acid composition and theirpreventive effect against cancer, the presence of Bowman-Birk inhibitors is desirable(GONZALES DE MEJLA et al., 2003).

Particularly, microwave roasting is useful when it is necessary to obtain goodsolubility of major soybean proteins, such as in processing soy flour and isolate. Themajor storage proteins expressed high thermal stability. The influence of microwavetreatment on cracked soybean proteins is characterized by the formation of soluble highmolecular weight aggregate and their partial degradation to low molecular weightproducts. The degree of protein denaturation is determined by the duration ofmicrowave treatment. Furthermore, high content of basic subunits of glycinin and -α-,-β- subunits of 7S- globulin indicates different types of alteration in relation to theheating of soybean protein solutions.

Abbreviations: PAGE-polyacrylamide gel electrophoresis, SDS-PAGE-sodiumdodecylsulphate- polyacrilamide gel electrophoresis, TI-trypsin inhibitor, KTI-Kunitztrypsin inhibitor, BBI- Bowman-Birk inhibitor.



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