Elution Behavior Analysis of Starch Degrading Enzymes During Rice Cooking with Specific Antibodies

Abstract

Rice grains contain starch degrading enzymes, including α-glucosidases, α-amylases, β-amylases, pullulanase and isoamylases. To investigate the elution behavior of these enzymes from rice grains into cooking water during rice cooking, we separated cooking water from rice grains after soaking (20℃) and when the temperature reached 40℃ and 60℃ during cooking. Immunological detection of these enzymes in rice grains and cooking water was carried out with SDS-PAGE and immunoblot. Bands corresponding to pullulanase, α-glucosidase, isoamylase 1, and α-amylase E were detected in both rice grains and cooking water at each temperature. Bands corresponding to α-amylases A + B and H were not detected in rice grains, but were detected in the cooking water at each temperature. The β-amylase band was detected in rice grains but was not detected in cooking water. These results suggest that the amount of enzyme eluted into cooking water depends on enzyme localization and quantity in rice grains.Keywords: rice, starch degrading enzymes, immunoblot, rice cooking, elution Abbreviations: PBSD medium, sodium phosphate buffer (pH 7.0) containing DTT and NaCl; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis *To whom correspondence should be addressed. E-mail: ookura@affrc.go.jp IntroductionRice (Oryza sativa) is a major food in Asian countries, including Japan. Extensive studies have been carried out to determine how a preferable taste can be achieved in rice cooking. It was found that physical properties such as stickiness and hardness (Maruyama, 1991;Kainuma, 1992), and chemical components such as sugars and amino acids contribute to the taste of cooked rice (Matsuzaki et al., 1992;Tajima et al., 1992;Ikeda, 2001). The amounts of sugars (reducing sugars and oligosaccharides) and amino acids correlate with the activities of starch-degrading enzymes and proteases, which are activated during cooking (Maruyama et al., 1981;Kasai et al., 2000;Maruyama, 2002).The enzyme isoforms present in dormant rice, which degrade starch granules during germination, include: α-amylases (EC 3.2.1.1) (Mitsui et al., 1996;Yu et al., 1996), β-amylases (EC 3.2.1.2) (Matsui et al., 1975;Yamaguchi et al., 1999), isoamylases (EC 3.2.1.68) (Fujita et al., 1999), pullulanase (EC 3.2.1.41) (Takeuchi et al., 1999), and α-glucosidases (EC 3.2.1.20) (Takahashi et al., 1971;Matsui et al., 1988). Two studies demonstrated that rice grains contain more than 10 types of α-amylase isoforms (Daussant et al., 1983;Mitsui et al., 1996). These isoforms are classified into two groups on the basis of their optimum temperature: α-amylase I (70℃) and α-amylase II (37℃) (Mitsui et al., 1996). These α-amylase isoforms are remarkably induced during germination (Alpi and Beevers, 1983; Perata et chi (Hokkaido University, Japan). The polyclonal antibody against isoamylase 1 was kindly provided by Dr. Nakamura (Akita Prefectural University, Japan).Rice Sample Brown rice (Oryza sativa L., cv. Koshihikari) was purchased from Uonuma in Niigata in 2008 and stored at 4℃ u...

Distribution of Starch-degrading Enzymes in Rice Grains of Different Cultivars and Elution Behavior during Cooking

“…In our previous studies, we revealed the distribution of starch-degrading enzymes in rice grains and their elution behavior during cooking with a specific antibody against each enzyme, including α-glucosidase, α-amylases, β-amylase, pullulanase and isoamylase, in Koshihikari cultivars (Tsuyukubo et al, 2010;Tsuyukubo et al, 2012). We also showed that starch hydrolytic activities and reducing sugar production differ with cooking temperature among Koshihikari, Nipponbare, Habutaemochi, Yumetoiro and Milkyqueen cultivars .…”

“…For this analysis, crude enzyme extracts from each fraction of raw rice grains, and those from cooking water were loaded as described previously (Tsuyukubo et al, 2012).…”

“…Transferred membranes were incubated in 20 mM Tris buffered saline (TBS, pH 7.5) containing 0.1% Tween-20 and 5% skim milk for blocking. Transferred membranes were reacted with specific antibodies: anti-rice α-glucosidases (1:10,000 dilution) (Tsuyukubo et al, 2012), anti-pullulanase (1:10,000 dilution) (Tsuyukubo et al, 2012) anti-isoamylase 1 (ISA1) (1:500 dilution) , anti-α-amylase isoform A+B (1:300 dilution), isoform H (1:500 dilution) and isoform E (1:500 dilution) (Mitsui et al, 1996), and anti-β-amylase (1:100 dilution) . Horseradish peroxidase-labeled anti-rabbit or anti-mouse IgG antibody and chemiluminescence reagent were used to detect immunoreactive bands with a LAS-4000 molecular imager (Fujifilm, Tokyo, Japan).…”

“…We here discuss the results for these two cultivars in comparison with the previous results for Koshihikari. To describe Koshihikari, we quoted part of the results of the distribution and enzyme behavior in Koshihikari from previous reports (Tsuyukubo et al, 2010;Tsuyukubo et al, 2012) (Fig. 6).…”

Characteristics of Opaque and Translucent Parts of High Temperature Stressed Grains of Rice

“…However, it was shown that RNAi-mediated suppression of α-amylase genes in ripening seeds resulted in fewer chalky grains under high-temperature conditions, and the extent of the decrease in the ratio of chalky grains was highly correlated to decreases in the expression of Amy1A, Amy1C, Amy3A and Amy3B. 32) Furthermore, Tsuyukubo et al (2010Tsuyukubo et al ( , 2012 36,37) have demonstrated that AmyI-1 (Amy1A) and AmyII-4 (Amy3D) proteins existed in the outer layers (100 to 80% fractions) of rice grain (cv. Koshihikari), while α-glucosidase and AmyII-3 (Amy3E) were mainly detected in the inner layers (80 to 0% fractions) by immunoblotting with the specific antibodies.…”