Function of Carbohydrate-Related Endogenous Enzymes in Milled Japonica-type Non-glutinous Rice and Their Behavior During Cooking

Abstract

This review provides a detailed overview of rice carbohydrate-related endogenous enzymes and their roles in milled japonica-type non-glutinous rice. Rice contains various endogenous enzymes that act during cooking and have important effects on the properties of cooked rice. Although endogenous enzyme activities differ among raw rice varieties, the enzymic reaction can be optimized by controlling the cooking conditions, including the heating temperature, heating time, and the amount of water during rice cooking to obtain cooked rice with desirable properties. Added enzymes allow for reactions that are not possible with the endogenous enzymes in the rice during cooking and change its physical properties. Understanding the localization of carbohydrate-related endogenous enzymes and their function in the ‘Takiboshi’ method used to cook japonica-type non-glutinous rice provides insight into the control of starch structure and its function.

1. Introduction

Rice is an important cereal in agriculture. It cannot be eaten raw but is made edible by cooking in water. Starch is the main component of the rice and when starch is heated in water, it begins to gelatinize at around 60 °C. Gelatinized starch is sticky and readily degraded by enzymes. By contrast, raw starch is insoluble and hard to digest and not sticky. Rice cooking methods are diverse and complex and have varied throughout history. There are also regional differences in cooking methods that are linked to the availability of rice varieties and compatibility with side dishes [1, 2]. The most common rice in East Asia, including Japan, China, South Korea, and Taiwan, is japonica-type non-glutinous rice, which has short grains. This rice is cooked using the ‘Takiboshi’ method. In the Takiboshi method, the lid is not removed during cooking and the added water is absorbed into the rice grains [2, 3, 4]. The most common rice in Southeast Asia, including Malaysia, the Philippines, and Thailand, and in South Asia (e.g., India) is indica-type non-glutinous rice, which has long grains. This rice is cooked using the ‘Yutori’ method. In the Yutori method, a large amount of water is added to the rice and brought to a boil, after which the lid is removed. Any liquid remaining after cooking is discarded and the rice is steamed over low heat [2 , 4, 5]. In both methods, the rice absorbs water and swells, which causes the starch in the rice to gelatinize. In the Takiboshi method, low-molecular-weight starch is released from the rice into the cooking water during heating and then returns to the rice grain. This creates a thin membrane of starch (‘oneba’ in Japanese), on the surface of the rice grain, which makes it sticky. In the Yutori method, the oneba is removed together with the boiled water, and the resulting cooked rice is not sticky [2]. The cooking method is thus related to the properties of the cooked rice, with rice cooked by the Takiboshi method being sticky and that cooked by the Yutori method being less sticky. Various studies have been conducted on how the properties of cooked rice change depending on the cooking method [6, 7].

The Takiboshi method begins with weighing the raw rice, which is followed by washing, soaking in water, heating the water containing the rice to boil, and continuing the boiling process. After heating, the pan lid is kept shut to maintain the temperature. Then, after a certain period of time, the lid is removed and the cooked rice is stirred briefly to separate the grains. For short-grain sticky japonica rice, the weight of water added for cooking is 1.3–1.5 times the weight of the rice. Rice cooked with less than 1.3 times the weight of water is hard and not sticky, which results in poor properties and noticeable retrogradation [8]. Studies have shown that the long-grain variety of indica rice requires more water when cooking by the Takiboshi method, with a water volume twice that of the rice [9]. The soaking step after washing is an important process in the Takiboshi method [3]. This process allows water to fully penetrate the starch in the center of the rice grains, which increases the mass of the raw rice and promotes starch gelatinization, which affects the softness and stickiness of cooked rice [10].

The properties of the cooked rice are evaluated in terms of firmness, stickiness, taste, aroma, and appearance using established sensory evaluation methods [11]. Cooked rice’s properties are largely influenced by the physical properties of firmness and stickiness but are also determined by chemical properties of aroma and taste (such as sweetness and umami), which are perceived when the rice is chewed. In terms of appearance, rice that is white and shiny is preferred. These properties are affected by both the cooking method and the variety, origin, growing conditions, degree of milling, cooking conditions, and storage conditions of the raw rice [12]. Reduced sugars [13], oligosaccharides [14] and glutamic and aspartic acids [13] are positively correlated with the properties of rice, whereas the proteins content [15] is negatively correlated with the properties of rice. The sugar content in cooked rice increases during cooking because of the action of endogenous starch-degrading enzymes in the rice grains [13]. In raw rice, the enzymes are less likely to be active, and heating during cooking induces enzymatic activity.

Recently, genomic information has become available and rice enzymes have been studied from a molecular biology perspective. However, most studies have focused on starch biosynthesis, and enzymes involved in this have been isolated from seeds during germination [16, 17]. Few studies have investigated carbohydrate-related endogenous enzymes in milled rice. Endogenous enzymes in milled rice are the main cause of changes in rice components during the cooking process. It is important to understand composition changes caused by endogenous enzymes, the structural changes that occur during cooking of milled rice, and the mechanisms.

This review focuses on the effects of carbohydrate-related endogenous enzymes in japonica-type non-glutinous rice during cooking by the Takiboshi method. The information presented here provides an understanding of whether endogenous enzymes vary with the rice variety and degree of milling, and where the endogenous enzymes are located in the rice grain and when they act during cooking. Research on the function of carbohydrate-related endogenous enzymes during the cooking process by the Takiboshi method of japonica-type non-glutinous rice is often written in Japanese; however, publications in English are cited where possible.

2. Overview of carbohydrate-related endogenous enzymes in milled rice grains

2.1 Carbohydrate-related endogenous enzymes in milled rice

Starch-degrading enzymes in japonica-type non-glutinous rice include α-glucosidase, α-amylase, β-amylase, pullulanase, and isoamylase. The cell wall polysaccharide-degrading enzymes include β-glucanase, α-galactosidase, β-galactosidase, α-mannosidase, β-xylanase, and polygalacturonase. Aspartic acid protease and carboxypeptidase are proteases that contribute to the generation of amino acids and peptides. [18, 19, 20]. Enzymes can be active at lower temperatures but are more likely to be active at higher temperatures, and each enzyme has its own optimum temperature. Generally, these enzymes are inactivated at temperatures above 80 °C.

2.2 Differences in endogenous starch-degrading enzymes among different rice varieties and degrees of milling

It has been reported that the activities of endogenous starch-degrading enzymes differ among rice varieties [21, 22]. The activity of α-glucosidase, which plays an important role in the properties of rice differs among varieties. Iwata et al. [23] studied 24 varieties of rice and found that the activity of α-glucosidase was higher in varieties considered to have good properties. The endogenous α-glucosidase activity is high in the rice cultivars Koshihikari (Japonica-type non-glutinous rice) and Akitakomachi (Japonica-type non-glutinous rice) and low in Tsushima (Japonica-type non-glutinous red rice) and Hoshiyutaka (Japonica-type non-glutinous long-grain rice). α-Amylase activity is high in Tanegashima (Japonica-type non-glutinous red rice) and low in Koshihikari (Japonica-type non-glutinous rice) [23]. α-glucosidase activity is negatively correlated with the swelling volume and amylose content of the rice and positively correlated with the maximum viscosity. These activities of these endogenous enzymes are closely related to the properties of the cooked rice [23].

In a study by Tsujii et al. [24] of rice endosperm enzyme activity in 17 rice varieties, Hanaemaki, Asatsuyu, Milky Queen, Soft 158 (Japonica-type non-glutinous rice with a low amylose content) and Yumetoiro (Japonica-type non-glutinous rice with a high amylose content) had lower α-glucosidase contents than Koshihikari (Japonica-type non-glutinous rice).Within a rice variety, Nishimoto et al. [25] reported that enzyme activity of the starch-degrading enzymes α-amylase, α-glucosidase, β-amylase, and pullulanase varied between harvest years and the activities of these enzymes were negatively correlated with rice firmness. The activity bands of enzymes from three short-grain japonica rice varieties, two medium-grain japonica rice varieties, and one long-grain indica rice variety were detected using zymography. α-Amylase bands were observed at approximately 44 kDa and were thinner, showing lower activity, in the long-grain indica rice than in the japonica rice. α-Amylase activity is positively correlated with the content of maltooligosaccharides that enter the cooking liquid and the stickiness of cooked rice [26]. There are several isoforms of α-amylase that have different optimum temperatures [27], and the correlation with texture may differ between the isoforms.

Mabashi et al. [28] studied the effect of rice endogenous enzymes in rice with different degrees of milling (percentage of the grain remaining after outer removal) by milling the rice. Amylases activity (activity of all carbohydrate hydrolyzing enzymes except α-glucosidase) at approximately 60 °C during cooking did not change with the degree of milling but was higher at approximately 30 °C for 100% milled rice, followed by 95%, 90%, and 85% milled rice. It is thought that amylases with optimum temperatures of approximately 30 °C are present in the outer layer of the rice grain. Tran et al. [29] reported that samples with a lower degree of milling had higher α-amylase, β-amylase, and protease activities and samples with a higher degree of milling had higher α-glucosidase activity.

2.3 Localization of endogenous starch-degrading enzymes in rice grains

Tsuyukubo [30] investigated where endogenous enzymes were located in rice grains and whether they leached into cooking liquid. Rice grains were milled into four fractions with 100% outside and 0% center, 100%–90%, 90%–80%, 80%–70%, and 70%–0%. A crude enzyme solution was prepared for each milling fraction and analyzed by SDS-PAGE with Coomassie brilliant blue staining and western blotting using antibodies that specifically reacted with various starch-degrading enzymes. SDS-PAGE showed that the proteins in the whole brown rice were distributed below approximately 100 kDa, with particularly high concentrations of proteins at approximately 80–100 kDa, and approximately 55 kDa and below. Proteins below approximately 55 kDa were abundant in the 100%–90% and 90%–80% rice fractions, while the 80–100 kDa proteins (including α-glucosidase) were abundant in the 70%–0% rice fraction [30]. α-Glucosidase (95 kDa) was distributed throughout the rice grain but was particularly abundant in the inner layers (< 80% fractions). α-Amylase (42–44 kDa) was abundant in the outer layers (100%–80% fraction) but was also distributed in the inner layers. β-amylase (53 kDa), pullulanase (100 kDa), and isoamylase (83 kDa) were distributed throughout the rice grain, with pullulanase abundant in the inner layer [30]. β-Amylase was distributed throughout the rice grain in Koshihikari, but was not detected in Nipponbare, which showed that there were varietal differences for the enzymes [31].

Tsuyukubo et al. [32] conducted western blotting analysis of crude enzyme solutions extracted from rice grains and rice cooking liquid. The rice was soaked at 20 °C for 1 h in an electric rice cooker before turning it on. When the temperature reached 40 °C or 60 °C, the mixture in the rice cooker was separated into rice grains and cooking water to investigate whether enzymes in the rice grains leached out during cooking [32]. Enzyme transfer into the cooking liquid was observed after soaking at 20 °C, but the differences in enzyme transfer between cooking at 40 °C or 60 °C were small [32]. α-Glucosidase is abundant in the inner layer of the rice grain and is less likely to transfer into the cooking liquid, while α-amylase is abundant in the outer layer of the rice grain and is more likely to transfer into the cooking liquid. β-Amylase is distributed throughout the rice grain and does not transfer into the cooking liquid. This suggests that β-amylase is strongly bound to the starch grains. Pullulanase and isoamylase, which are distributed throughout the rice grains, transfer into the cooking liquid but mostly remain inside the rice grains. Enzymes on the outside of the rice grains transfer into the cooking liquid, whereas enzymes in the inner layer of the rice grains are less likely to move out of the rice grains. This trend is observed for different varieties of rice, with no apparent differences between varieties [33].

Eleven isoforms of α-amylase have been detected and can be stained using different antibodies [31, 32]. The optimum temperature for starch degradation activity by α-amylase varies according to the isoform. Class I α-amylases are recognized by anti-α-amylase A + B antibodies, class II-3 α-amylases are recognized by anti-α-amylase E, and class II-4 α-amylases are recognized by anti-α-amylase H. Class II-3 α-amylase are present throughout the rice grain and do not readily transfer into the cooking liquid. Class I and II-4 α-amylase are more abundant in the outer layers and transfer from the rice grains into the cooking liquid.

Tsuyukubo et al. [34] investigated the localization of pullulanase and α-glucosidase in frozen sections of Koshihikari, Nipponbare, and Milky Queen rice. Immunofluorescence staining using antibodies that bound specifically to each enzyme showed that pullulanase, which is present inside the rice grain, was mainly detected around the starch grains of the amyloplast [34]. α-Glucosidase was mainly detected along the endosperm cell walls [34]. These features were common to the three rice varieties and indicate that the endogenous enzymes are localized in different areas within the rice grains. α-Glucosidase in brown rice is localized in the endosperm and the aleurone layer. In milled rice with a milling yield of 90%, α-glucosidase is present inside the rice grain, whereas in brown unmilled rice, it is also present in the aleurone layer [34]. The layer between the bran and the endosperm is called the aleurone layer and is rich in endogenous enzymes [35]. Consumption of rice milled using a technique that leaves the aleurone layer intact is reportedly beneficial for health-related factors such as blood pressure and hemoglobin A1C [36]. Consequently, this method of milling rice has attracted attention.

3. Behavior of endogenous enzymes and changes in carbohydrates during cooking

3.1 Increase in sugar content during cooking

Sugars are produced by starch degradation. Heating is necessary for the starch in the hard tissue of the rice grain to become gelatinized by cooking. Kasai et al. [13] found that the amount of glucose, which is a reducing sugar, did not change with washing of the rice, but increased markedly with heating. The amount of other reducing sugars (fructose and maltose) did not change with either washing or heating from 40 °C to 60 °C [13]. Mabashi et al. [37] investigated the effect of varying the time from room temperature to boiling. They found that the total sugar and reducing sugar contents of rice cooking liquid increased with a slower rate of temperature increase, and the temperature protocol used for heating affected the formation of chemical components in the rice. The ratio of total sugar to reducing sugar tended to decrease with a slower rate of temperature increase [37]. These results indicate that the average chain length of the sugar decreases and sugar degradation increases under endogenous enzymes action. Mabashi et al. [37] also found that the amounts of fructose and sucrose did not change greatly with changes in the rate of temperature increase. However, the amount of glucose in the rice greatly increased with a slower rate of temperature increase, and glucose was produced by rice endogenous glycolytic enzymes during the temperature increase phase. In this study, the amounts of maltose and maltotriose produced by starch degradation and the amount of free amino acids increased with cooking but did not change with changes in the rate of temperature increase [37]. The rate of temperature increase during cooking mainly affects sugars. Tsujii et al. [38] found that starch and cell wall polysaccharides were broken down during the cooking process, with approximately 5 g of sugars leaching from 100 g of milled rice into the cooking liquid during the cooking process. Maltooligosaccharides with a glucose degree of polymerization of 9–15, which were not present in milled rice, were detected in the cooking liquid, while the content of sugars with a degree of polymerization of 5–8 in the cooking liquid was approximately 25 times that in the raw rice [38].

In a study where cooked rice was held at 40 °C, 60 °C or 80 °C for 15 min during the temperature increase period in the usual rice cooking process, the total sugar and reducing sugar contents in the cooking liquid increased the most at 60 °C [21]. The temperature of rice after soaking is 20 °C and it takes approximately 8 min of heating to get the rice from this temperature to 80 °C (the temperature at which enzyme inactivation occurs) and approximately 10 min of heating to get the rice to boiling. When the rice is heated from 20 °C to 80 °C, starch-degrading enzymes within the rice grains act and increase the reducing sugars content to four times that in the raw rice. Glucose accounts for 80% of the total content of reducing sugars. This sugar production takes place within the rice grains. Sugars leach from the rice grains into the cooking liquid but are also absorbed back into the rice grains. In the Takiboshi method, some of the cooking liquid remains as a thin film (oneba) on the rice grains and this affects their texture (i.e., stickiness) [13].

3.2 Starch hydrolysis by endogenous enzymes in rice during cooking

Changes in the composition of rice during cooking are attributed to the action of rice endogenous enzymes. Rice starch-degrading endogenous enzymes that produce sugars are α-glucosidase, α-amylase, β-amylase, isoamylase, and pullulanase [26, 31, 38, 39, 40, 41, 42, 43, 44, 45]. Starch-degrading enzymes are classified as endo-type enzymes if they hydrolyze randomly and exo-type enzymes if they hydrolyze sequentially from the end of the chain. α-Glucosidase is an exo-type enzyme that hydrolyzes the α-1,4 bonds of starch from the non-reducing end to produce glucose [41]. α-Amylase is an endo-type enzyme that randomly hydrolyzes α-1,4 bonds to produce oligosaccharides. Amylases can be classified as type I or II according to their optimum temperature for function [42]. β-Amylases are exoenzymes that hydrolyze the α-1,4 bonds of starch from the non-reducing end in disaccharide units [43]. Pullulanase and isoamylase catalyze the hydrolysis of α-1,6 glycosidic branch chains in amylopectin. Pullulanase requires the presence of at least two α-1,4-linked glucose units on two sugar chains linked by α-1,6 bonds, while isoamylase requires at least three α-1,4-linked glucose units [44, 46]. The functions, optimum temperatures, and sizes of starch-degrading endogenous enzymes are listed in Table 1.

Table 1: Functions, optimum temperatures, and sizes of starch-degrading endogenous enzymes

Enzyme	EC number	Function	Optimum temperature (°C)	Size (kDa)	References
α-Amylase	3.2.1.1	Hydrolyzes α-1,4 bonds Endo-type enzyme	II-3 (isoform E): 26 II-4 (isoform H): 37 I (isoform A + B): 70	44	[18] [31] [40] [42]
β-Amylase	3.2.1.2	Hydrolyzes α-1,4 bonds in disaccharide units Exo-type enzyme	37	53	[18] [31] [40][43]
α-Glucosidase	3.2.1.20	Hydrolyzes α-1,4 bonds Exo-type enzyme	60	95	[18] [31] [40] [41]
Pullulanase	3.2.1.41	Hydrolyze s α-1,6 bonds Requires the presence of at least two α-1,4-linked glucose	40–55	100	[18] [32] [44]
Isoamylase	3.2.1.68	Hydrolyze s α-1,6 bonds Requires the presence of at least three α-1,4-linked glucose	30	83	[18] [32] [45]

In a study examining the pH and the level of starch-degrading enzyme activity, the enzyme activity at a cooking pH of 7 was lower than that at the optimum pH of the rice plant (pH 5) [26]. Reportedly, both starch hydrolysis activity and proteolytic activity are higher at pH 5 than at pH 7. A cooking method has been proposed where raw rice is soaked in a pH 5 solution for 16 h before heating, and this results in accumulation of glucose and amino acids in the rice grains because of the action of starch degrading enzymes and protease [47].

During the cooking process, several hydrolysis reactions proceed simultaneously, including the degradation of starch by amylases such as α-amylase, β-amylase, and isoamylase, and the release of glucose from the non-reducing end by α-glucosidase [39]. The amounts of reducing sugars, including glucose, produced by the degradation of soluble starch are maximized at 60 °C and the highest sum of the activities of the various starch-degrading enzymes also occurs at approximately 60 °C [48]. α-Amylase produces oligosaccharides at lower temperatures in the early stages of cooking rice. Maruyama [39] found that α-amylase produced maltotetraose to maltoheptaose in the initial stage of cooking; β-amylase, pullulanase, and isoamylase increased the contents of low-molecular-weight oligosaccharides below maltotetraose in the middle stage of cooking; and α-glucosidase degraded low-molecular-weight oligosaccharides to glucose in the final stage of cooking. Tsujii et al. [26] stated that the activity levels of α-amylase and isoamylase in the rice endosperm were positively correlated with the oligosaccharides leached during cooking.

Awazuhara et al. [40] reported that the optimum temperatures for reducing sugar-producing enzymes differed between the outer layer of milled rice (outer 13% of the grain) and the inner layer of the endosperm (inner 87% of the grain). The optimum temperature for starch hydrolysis endogenous enzymes in the outer layer was approximately 40 °C and that in the inner layer was approximately 60 °C. The main enzymes were α-amylase in the outer layer and α-glucosidase in the inner layer [40]. These results show that different parts of the rice grain contain different enzymes with different heat-dependent properties for the degradation of starch. Kishio et al. [49] also reported that glucose production at 60 °C mainly occurred in the center of the rice endosperm for various rice varieties, and that α-glucosidase, which was highly active, was present in the inner layer of the rice. Nakai et al. [50] found that α-glucosidase bound directly to starch grains and degraded starch into glucose, with the binding site located in the C-terminus of the enzyme.

The activity of the endogenous enzyme α-glucosidase is temperature dependent in crude enzyme solutions. When the substrate is soluble starch or gelatinized starch, maximum sugar production occurs at 60 °C, while sugar production at 50 °C is 70%–80% of the maximum. By contrast, when the substrate is raw rice flour, sugar production at 50 °C is only 20%–30% of the maximum production at 60 °C [48]. This suggests that when considering the temperature dependence of starch degrading enzymes in the actual rice cooking process, it is necessary to consider both the temperature dependence of the enzyme and starch gelatinized state. Starch starts to gelatinize at approximately 60 °C. According to Kasai et al. [51], this temperature is both the optimum temperature for the enzyme and the temperature at which the substrate starch becomes gelatinized and susceptible to degradation (digestion) reactions by endogenous enzymes. Therefore, it is the key temperature for sugar production.

Mabashi et al. [52] clarified the relationship between the activities of endogenous enzymes in milled rice and the accumulation of chemical components in the rice grains during cooking in five genetically distinct rice varieties (Koshihikari, Nipponbare, Habutaemochi, Yumetoiro, and jasmine rice). The sugar content of rice greatly increased with cooking in all varieties. During the temperature increase, a process was inserted to maintain the temperature at 60 °C for 15 min, which was effective for increasing the sugar content [52]. The combined activity of the various sugar hydrolyzing enzymes was highest at 60 °C; however, the rate of increase in sugar content changed with the rice variety. In jasmine rice, compared with Koshihikari, the amounts of reducing sugars and glucose in raw rice extracts were lower but the rate of increase in glucose with cooking was higher [52]. Differences in the sugar content between raw rice varieties were reduced by cooking the rice, and the sugar content of the cooked rice varied more with the cooking method than with varietal differences in raw rice components [52].

The localization of starch-degrading endogenous enzymes in rice grains and their behavior during rice cooking, the degradation of starch to sugar by endogenous enzymes, and the increase in sugar in the center and outer edge of the endosperm at various temperatures are summarized in Figure 1 [18, 31, 39, 49]. In the cooking process, isoamylase, pullulanase, β-amylase, α-amylase II-3, and α-amylase II-4 act on the whole rice grain from the start of heating. During this process, some of the enzymes transfer into the cooking liquid. At approximately 60 °C, α-glucosidase hydrolyzes the already degraded starch from the rice grains to produce glucose. α-Amylase I, which has a higher optimum temperature than α-glucosidase, acts on the outer layer after the α-glucosidase reaction. The sugar content in rice grains reportedly increases at 60 °C [13]. α-Glucosidase has an optimum temperature of 60 °C and the action of α-glucosidase is largely responsible for the increase in the sugar content in cooked rice.

Figure 1: (a) Activation of endogenous starch-degrading endogenous enzymes during rice cooking and their location in the rice grains (where 100% is the outside of the grain and 0% is the center). (b) Degradation pathway of starch to sugar. (c) Increase in the sugar content in the center and on the outer edge of the endosperm with changes in the temperature

3.3 Changes in the cell walls in the endosperm in rice during cooking

The presence of cell walls in the endosperm controls over-swelling of the starch during cooking, which maintains the shape of the rice grain. The cell walls in the endosperm are mainly composed of pectin, hemicellulose, cellulose, and small amounts of protein. Cooking rice causes the cell wall polysaccharides to decompose, which softens the rice. Tsujii et al. [19] studied the degradation of cell wall polysaccharides during rice cooking and reported that differences in the cell wall shape were observed between raw milled rice and cooked rice. For the pectin fraction, the fibrous tissue was densely and regularly arranged in raw milled rice, but this structure collapsed in the cooked rice. For the hemicellulose fraction, the raw milled rice’s cell wall was flat, but this flat shape was degraded in the cooked rice and the fibrous tissues were intertwined with each other. In cooked rice, the cellulose fraction did not show any large changes in shape that could lead to disintegration. The molecular weight distribution in gel filtration chromatography showed that both the pectin and hemicellulose fractions had low molecular weights because of cooking, which suggested that the cell walls degraded during cooking. The properties of cooked rice are affected by starch and structural changes in the cell wall. Rice proteins reportedly interfere with the starch gelatinizing process [53], and it is necessary to ascertain changes in the cell wall and starch during cooking.

Proteins and lipids are abundant in the outer layer of milled rice, and the protein and lipid contents of rice decrease when the rice is milled. The protease activities of crude enzyme solutions measured using casein as a substrate are higher for less milled rice (milling yields: 95% and 100%) than for milled rice (milling yields: 85% and 90%) [28]. There is a clear difference in the activity for rice with milling yields below 90% and above 95%, which indicates that the protease is localized in the outer layers of the rice and its localization range is narrower than that of starch hydrolyzing enzymes [28]. Controlling the abundance of proteins and proteolytic enzymes on the outside of the rice grain could potentially alter the penetration of water during cooking.

3.4 Enzyme penetration into the rice

To investigate whether enzymes penetrated rice during soaking, Sano et al. [54] mixed barley and Nipponbare (Japonica-type non-glutinous rice) and soaked them in water for 1 h. They then used immunoblotting to investigate the presence of barley β-amylase in the barley, soaking water, and rice. Nipponbare is a variety with very low β-amylase activity. Sano et al. [54] reported that barley β-amylase was released into the cooking liquid during soaking and transferred into the rice grains via the soaking water. Conversely, transfer of rice α-glucosidase into barley via the soaking water was reported by Hamamori et al. [55]. Sano et al. [56] also reported that in mixed cooked barley and rice, the amounts of reducing sugars and glucose were much higher than the values calculated for cooked rice without barley. This indicated that enzymes from both barley and rice contributed to starch degradation, with each enzyme acting on the other and the barley starch-degrading enzyme also acting on rice starch. β-Amylase from barley produced maltose in the rice grains, and glucose formation from maltose was thought to occur because of the action of α-glucosidase from rice [56]. The behaviors of various sugars in mixtures of glutinous barley and non-glutinous rice at different ratios and with different cooking temperatures were investigated by Hamamori et al. [57]. The pH of the cooked rice liquid decreased with increases in the barley content in the mixture. Sucrose, maltose, and glucose levels were higher in mixtures than in plain rice, and the rice grains were softer and stickier in the mixtures. Furthermore, retrogradation associated with refrigeration storage decreased in mixtures compared with in plain rice [57]. These results suggest that enzymes added during soaking may penetrate the rice grains.

When raw rice is placed in water, the rice grains absorb water, which increases the mass. Mezaki et al. [58] visualized water absorption in polished rice using liquid nitrogen as a refrigerant to stop the movement of permeated water. The surface of polished rice has several aleurone layers, and the walls of the cells in these layers inhibited water absorption into the rice grain. Inside the aleurone layer is the starch endosperm. The boundary between the endosperm and starch grains provided a possible pathway for transfer of water. Proteins were distributed inside the cell wall in the endosperm, and granular proteins were also present between starch grains in the cells and around the internal starch. Water penetrated primarily through the compound starch granules and secondarily through the intragranular single starch granules [58].

4. Effects of added enzymes on the properties of the cooked rice

Carbohydrate-related enzymes can be added to the cooking water with raw rice to change the taste and improve the quality of cooked rice. These enzymes include the hydrolytic enzymes α-amylase (EC 3.2.1.1), β-amylase (EC 3.2.1.2), α-glucosidase (EC 3.2.1.20), maltotriohydrolase (EC 3.2.1.116), maltogenic α-amylase (EC 3.2. 1.133), and neopullulanase (EC 3.2.1.135); branching enzyme (EC 2.4.1.18); lipase (EC 3.1.1.3); the cell wall degrading enzyme hemicellulase (EC 3.2.1.8); and the proteolytic enzymes papain (EC 3.4.22.2) and protease (EC 3.4.23.6). The EC 3.2.1.x enzymes hydrolyze two or more glycosidic bonds.

Hayashi [59] reported that addition of α-amylase, hemicellulase, and papain softened cooked rice in the Takiboshi method to make it easy to eat and digest. The addition of α-amylase is effective for softening rice even in the Yutori method. Interestingly, in the Yutori method, instead of adding the enzyme during cooking, the gelatinized rice is immersed in the enzyme solution after discarding the hot water [60]. Miyazaki [61] reported that addition of α-glucosidase in the Takiboshi method improved the softness and stickiness of cooked rice and the taste of freshly cooked rice. Sato et al. [62] added neo-pullulanase to a solution that contained soluble solids and was obtained from cooked rice with poor taste. They detected high molecular weight components in this solution because the starch was degraded by the neo-pullulanase, which indicated that it improved the properties of the cooked rice. Addition of neopullulanase may improve the properties of rice with a poor taste by increasing polymerization. Cooking rice with maitake mushrooms reportedly produces cooked rice that is very sticky because of the action of protease from the mushrooms on the rice protein [63]. Wu et al. reported that hydrolysis of corn starch with glucan 1,4-alpha-maltotriohydrolase produced maltotriose and changed the starch digestibility [64].

When cooked rice is stored in the refrigerator, retrogradation of rice starch will occur and the texture will become less sticky and harder. The degree of retrogradation increases the longer the rice is kept at a low temperature, and this results in undesirable properties. Adding enzymes complements the action of rice’s intrinsic carbohydrate-related enzymes to reduce the starch chain length and increase the branched structure of the starch compared with not adding enzymes. These changes slow the rate at which the rice becomes hard and degrades in quality over time after cooking. Addition of the highly thermostable exoenzyme maltogenic α-amylase, which has an optimum temperature of 95 °C –100 °C, reportedly makes the starch surface more porous [65], shortens the starch chains, and inhibits starch retrogradation [66]. Addition of α-glucosidase and branching enzyme also reportedly inhibits retrogradation of cooked rice [67]. Lipase may alter the swelling characteristics of rice starch during cooking and inhibit retrogradation of rice [68]. These studies show that added enzymes induce reactions in cooked rice and affect its physical properties, functionality, and shelf life.

5. Conclusions

A particular focus of Japanese food is the taste of Japanese-type non-glutinous rice cooked by the Takiboshi method. There have been several studies in this area. The function of endogenous starch-degrading enzymes in milled rice influences the texture, stability, and nutritional value of cooked rice. Endogenous enzymes for the carbohydrates can be classified as starch-degrading enzymes and cell wall polysaccharide-degrading enzymes, which act during the short cooking period when raw rice is converted to cooked rice. The main change in the chemical composition of cooked rice is the reducing sugar produced by starch-degrading enzymes. Hydrolysis of starch by starch-degrading enzymes produces monosaccharides and oligosaccharides. Sugar production occurs within the rice grains and sugar also leaches into the cooking liquid. In the Takiboshi method the sugars leached into the cooking water are absorbed back into the rice grains in the end, which leaves small amount on the surface of the rice grains as oneba to make the cooked rice sticky. The highest activity of the various starch-degrading enzymes occurs at approximately 60 °C, when the starch begins to gelatinize. The optimum temperature ranges from room temperature to 80 °C during rice cooking, with different enzymes acting at different times and in different locations within the rice grains. Furthermore, there are varietal differences in the activities and localization of the enzymes. In particular, the activity of endogenous α-glucosidase, which is present inside the rice grains, is associated with good taste in the cooked rice. Although there are differences in the enzyme activity between raw rice varieties, major changes in the firmness and sugar content of cooked rice are caused by cooking. The effects of differences in the endogenous enzyme activity are small compared with the changes caused by cooking.

The cell wall structure of raw rice is changed by cooking, which makes the cooked rice soft. The condition of cooked rice is affected by both the starch and structural changes in the cell wall. It is possible that enzymes contained in the water used to soak the rice are transferred to the rice, and the combination of endogenous and added enzymes changes the properties of the cooked rice. Varying the degree of milling of the rice grains can change the penetration of water into the rice grains during cooking and change the swelling characteristics of the starch.

An understanding of the function of carbohydrate-related endogenous enzymes can be used to produce desirable characteristics, such as reduced retrogradation and increased digestibility of cooked rice. It is important to make the most of enzymic reactions by controlling the cooking conditions, for example, using a temperature protocol for rice cooking to obtain desirable properties in the cooked rice. Furthermore, other enzymes can be added during the cooking process to introduce other reactions to the rice cooking process. Additive enzymes can penetrate into the rice grains during cooking and change the starch properties. These changes help with controlling the taste and quality of the cooked rice.

Acknowledgement

We thank Gabrielle David, PhD, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

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