Palatability Evaluation, DNA Analyses, and Functional Processing of Rice

Quality evaluations of rice are performed by sensory testing and physico-chemical measurements. The former is a basic method that requires large amounts of samples and several panelists. The latter is an indirect method that estimates the eating quality based on the chemical composition, cooking quality, gelatinization properties, and physical properties of cooked rice. Many novel methods to evaluate the rice quality using various kinds of apparatus, such as Tensipresser, RVA, NIR, and spectrophotometer were developed. Simple, rapid, and accurate method to evaluate the quality of rice grains is very valuable. As the cultivars of rice affect markedly eating quality, processing suitability and price, identification or differentiation of rice cultivar is very important. The present authors developed suitable STS (sequence-tagged-site) primers for PCR (Polymerase Chain Reaction) and it became possible to identify rice cultivar using template DNA extracted and purified from rice grains. Two kinds of multiplex kit for identification of “Koshihikari”, dominant cultivar Abstract Rice is one of the most important crops in the world. It is useful not only for the supply of energy, but also for giving palatability and good health to the people. The rice quality depends on various aspects such as consumer safety, nutrition, appearance, price level, and palatability. Sensory test and physico-chemical measurements are important measures for quality assay of rice grains. Sensory test is a fundamental method that requires large amounts of samples, many panel members, and long hours. Physico-chemical measurements estimate the eating quality based on the results of various kinds of measurements, such as chemical composition analyses, cooking quality test, gelatinization property test, and the measurements of physical properties of cooked rice. Novel method to evaluate the quality of the cooked rice is necessary to breed high-quality rice cultivars and to select the suitable rice for each consumer and each utilization purpose. It is necessary to develop the novel method to evaluate the rice quality using various kinds of novel measurements, such as Texture Analyzer, Tensipresser, RVA, NIR, and spectrophotometer. Simple, rapid, and accurate method to assay the quality of rice grains would be very valuable. Abstract As the cultivars of rice affect markedly eating quality, processing suit-ability and price, identification or differentiation of rice cultivar is very important. The present authors developed suitable STS (sequence-tagged-site) primers for PCR (Polymerase Chainm Reaction) and it became possible to identify rice cultivar using template DNA extracted and purified from rice grains. Multiplex primer set was shown to be useful to differentiate effectively rice cultivars produced in various countries by PCR. Two kinds of multiplex kit for identification of “Koshihikari”, dominant cultivar in Japan, have been developed. Application of cultivar identification method by PCR method to processed rice products was investigated. The present authors developed “enzyme treatment method”, in which the gelatinized starch is decomposed by the heat stable alpha-amylase at 80 °C , followed by hydrolysis of proteins by proteinase K with SDS and purification of extracted DNAs by PCI (Phenol/Chloroform/iso-amyl alcohol). It became possible to identify the material rice cultivars of the Abstract Diabetes and dementia are life-style related diseases, and patients have been increased all over the world. We tried to prevent diabetes using the high-amylose rice cultivars. It was shown that the postprandial BGL of high-amylose rice was lower than that of low-amylose rice. “Super hard rice”, of which starch contains long-chain amylopectin, was developed in Kyushu University, Japan, and we clarified its characteristics by the cooperative re-search. It was very hard after cooking, and rich in resistant starch. “Super hard rice” lowered postprandial BGL not only as cooked rice grains but also as rice noodle and rice bread. We prepared super-hard rice bread blended with 5% of black rice bran (SRBBB), which contained much amylose and showed inhibitory activity against β secretase after heating. Aged mice, which were fed SRBBB diet for 4 weeks, showed lower amyloid β 40 in the blood than the control. We investigated the effects of the blend of ordinary brown rice and black rice cooked after a high-pressure treatment (HPTKO). A randomized, single-blind, crossover-designed study was conducted using 15 subjects, and BGLs at 90 and 120 min after ingesting the cooked HPTKO were significantly lower than that for cooked ordinary polished rice. Furthermore, postprandial blood amyloid β 40 did not increased markedly compared with the control.


Introduction
Rice, wheat, and maize are the most important crops in the world. Rice consumers need not only enough amount of rice but also high-quality rice grains. The many rice cultivars grown in the world vary markedly in their cooking, sensory, and processing quality [1]. Consumers are heterogeneous with respect to their perceived differentiation of rice quality among regions, countries, cities, and urbanization levels [2] The rice quality depends on various aspects such as consumer safety, nutrition, appearance, price level, and palatability. Because rice is eaten every day, it is indispensable that it should be safe to eat and highly nutritious. Furthermore, because rice provides income for farmers and is prepared, milled, and sold by wholesalers and retailers, the rice yield and price are extremely important to the farmers. Consumers have been asking for more palatable rice because they have recently become more affluent.

Calingacion et al engaged local experts across the world and they
showed that there are at least 18 different quality types of rice that are favored around the world and this complexity immediately reveals the extent of the specificity of consumer preference [3].
As described by Juliano, rice quality evaluation includes eating quality assays for sensory evaluation and aroma testing, as well as physical property measurements using Instron, Texturometer, or Tensipresser etc., which were highlighted as indirect methods, in addition to amylose content, starch gelatinization temperature, gel consistency, amylography, and protein content determination [4].
Ohtsubo et al. reported on the quality assay of rice using traditional and novel tools, such as the measurements of amylose, physical properties and pasting properties [5].
Apparent amylose content (AAC) is measured by the iodine colorimetric method [6] and high-AAC rice becomes harder and non-sticky on cooking [1,6,7]. Lian et al. investigated on the identification of the main retrogradation-related properties of rice starch and reported that retrogradation rates of different rice starch showed significant positive correlation with proportion of the chains (DP>10) in amy-lopectin [8]. Protein content is another extremely important factor that determines rice quality [1]. Protein contents are measured by Kieldahl method, combustion method or NIR method. As Juliano [8] and Bhattacharya [9] pointed out, the physical properties of cooked rice grains are important determinants of the eating quality of rice. The pasting properties of rice are useful indicators in the quality assay of cooked rice, rice cake, rice bread, rice extrudate, etc. In several countries, such as spectrometer, are utilized to estimate the chemical components and to develop the estimation formulae for palatability. "Taste Analyzer" is an example of the NIR system as a non-destructive estimation apparatus for palatability of rice grains. Examples of sensory test and physico-chemical evaluation of rice palatability are shown in Table 1.

Amylose Content and Amylopectin Molecular Structure:
Amylose content is the most important factor that determines rice quality because starch shares approximately 85% (w/w, dry basis) of the milled rice grains. Because North-east Asian consumers prefer soft and sticky cooked rice, low amylose rice is the dominant rice sold in the rice market. Amylose content is generally measured by the iodine colorimetric method of Juliano [6]. Li, et al. [10] reported that stickiness of cooked rice is affected not only by AAC but also the proportion of short amylopectin chain. They found that the autoclave cooking, which is used to produce sticky "convenience rice", effects on sensory properties and increase leached amylopectin [11].
Protein Content: Protein content is another extremely important factor that determines the eating quality of rice. Cooked rice with high protein content tends to be hard and non-sticky.
Protein content is measured by the Kieldahl or the combustion method. It can be measured by NIR method (Near Infrared Reflectance) easily and non-destructively. Recently, not only protein content, but also molecular compositions of protein, such as glutelin or prolamin, is reported to affect the quality of rice [12,13]. for simulation and monitoring of processes, and on enzymatic reactions [14]. Comejo and Rosell used RVA in the research on the influence of germination time of brown rice in relation to flour and gluten free bread quality [15]. Although the Brabender Viscoamylograph has been used to assess rice pasting properties [1], Blakeney, et al. [16] and Champagne, et al. [17] showed that the RVA is useful in determining the "degree of cook" after processing of rice into precooked and extruded products. Yao et al. reported that the high temperature during ripening of rice decreased setback and trough viscosities and increased breakdown, implying that the pasting properties were slightly better [18]. Zhu et al. investigated the effect of soaking and cooking on structure formation of cooked rice through thermal properties, dynamic viscoelasticity, and enzyme activity, in which they used RVA for the measurements of enzyme activity [19].   [20], enabling evaluation of the starch properties and processing suitability of material rice flours. Furthermore, we developed novel estimation formulae for oleic and linoleic acid contents based on the pasting properties of brown rice flours using an RVA, making it possible to predict easily and rapidly the nutritive and bio-functional characteristics of material rice [21]. To evaluate the pasting properties, the behavior of rice starch at temperatures higher than 100 ‹C should be examined to elucidate the changes in rice quality after cooking and processing, such as extrusion cooking [22] or super-heated moisture treatment [23]. Recently, Nakamura et al. reported about the comparison of the three different programs and estimating the hardness and retrogradation of cooked rice based on its pasting properties using a novel high-temperaturetype programs [24]. They have made improvements to analyzing pasting properties in a new program using RVA 4800 ( Figure 2), and novel estimation formulae were developed in this paper for estimates of the retrograded hardness of cooked rice H1(R) and the degree of retrogradation H1(RD), which has led to an easy and rapid evaluation of the cooking qualities of various rice samples.
The following are the main features;   Texturometer [27]. Low-compression and high-compression test using a Tensipresser [28] is shown in Figure 3. Recently, Texture Analyser has been used for measuring the physical properties of the cooked rice [29] or the parboiled rice [30]. And dynamic viscoelasticity gives valuable information for the elucidation of the relationship between the texture and the molecular structure of starch, which showed that gconsistency coefficient h affected hardness and tanƒÂ affected stickiness of cooked rice grains [31]. assay, and iodine blue value measurements [32]. The formula showed a high correlation between the results of a sensory test (R = 0.84) and the rice samples that will be harvested next year.

Application of Near Infra-Red Spectroscopy to Evaluate Rice
Quality: Near Infra-red spectroscopy is a rapid, non-destructive, and extremely promising technique to evaluate qualities of the rice grains. In Japan, NIR spectroscopy was initially used to determine the protein and moisture content of the rice flours. Satake Co. Ltd subsequently combined NIR and palatability estimation formula and developed a new system called "Taste Analyser".  [38]. There were 8 Japanese 2 companies that developed the characteristic systems to evaluate the palatability of rice 3 grains based on the spectroscopic technique. More than 1000 agricultural cooperatives, 4 wholesalers, and retailers introduced these NIR systems to evaluate the quality of their rice 5 grains ( Figure 4). In 1996, the Japanese Food Agency conducted a survey regarding the palatability evaluation system, and the results showed that 67% users were satisfied with the performance of the system. The protein and moisture data were extremely reliable, but 8 some users requested an improvement in the taste score and amylase accuracy.

Proposal as Novel Physicochemical Measurement, Iodine Colorimetric Analysis, for Rice Palatability Evaluation [39]
Introduction: As described in section 3, physicochemical test is very important because it is time-saving and labor-saving objective test for rice palatability. Main component of rice grains is starch, therefore, amylose content affects eating quality [6]. Low-amylose rice generally be-comes soft and sticky after cooking, whereas high-amylose rice be-comes hard with fluffy separated grains [40]. The most widely used method for amylose determination is a colorimetric assay where io-dine binds with amylose to produce a blue-purple color, which is measured spectrophotometrically at a single wavelength (620nm) [6]. Nakamura   property, resistant starch content, texture of cooked rice grains, and SDS PAGE of proteins [42]. Based on these quality evaluations, we can conclude that Chinese rice cultivars are characterized by their high protein content. The hardness of the surface layer (H1) and overall layer (H2) were higher in the Chinese rice cultivars than the Japanese rice cultivars, whereas the stickiness of the surface layer, and the balance degree of the surface layer were lower in the Chinese rice cultivars than the Japanese rice cultivars, although the stickiness of the overall layer |H2 was higher in the Chinese rice cultivars than the Japanese rice cultivars. In addition, rate of staling of cooked rice was faster for Chinese rice than Japanese one.
In a previous study, they developed a novel formula for estimating AAC based on the iodine absorption curve [39]. The validation test showed a determination coefficient of 0.996 for estimating AAC of these Chinese rice cultivars as unknown samples as shown in Figure   6. Therefore, it became possible to estimate AAC, using iodine scanning colorimetric method [39], or the RVA method [20], easily and rapidly not only for Japanese rice but also various japonica rice cultivars produced in all over the world.

Summary of Evaluation of Rice Palatability
There are various kinds of rice cultivars of which qualities are diversified, such as hard Indica rice and soft Japonica rice in the world. Consumers in the Southern Asia prefer to hard rice grains and people in the North-eastern Asia like soft and sticky rice grains.
Novel method to evaluate the quality of the cooked rice is necessary to breed high-quality rice cultivars and to select the suitable rice for each consumer and each purpose. Many researchers are trying to develop the novel method to evaluate the rice quality using various kinds of apparatus, such as Tensipresser, RVA, NIR, and spectrophotometer, etc. Simple, rapid, and accurate method to evaluate the quality of rice grains is very valuable.

Abstract
As the cultivars of rice affect markedly eating quality, processing suit-ability and price, identification or differentiation of rice cultivar is very important. The present authors developed suitable STS (sequence-tagged-site) primers for PCR (Polymerase Chainm Reaction) and it became possible to identify rice cultivar using template DNA extracted and purified from rice grains.
Multiplex primer set was shown to be useful to differentiate effectively rice cultivars produced in various countries by PCR.
Two kinds of multiplex kit for identification of "Koshihikari", dominant cultivar in Japan, have been developed. Application of cultivar identification method by PCR method to processed rice products was investigated. The present authors developed "enzyme treatment method", in which the gelatinized starch is decomposed by the heat stable alpha-amylase at 80°C, followed by hydrolysis of proteins by proteinase K with SDS and purification of extracted DNAs by PCI (Phenol/Chloroform/iso-amyl alcohol). It became possible to identify the material rice cultivars of the processed rice products, such as cooked rice and rice cake, by PCR method using template DNA prepared by the "enzyme treatment method" [43].

Introduction
Rice grains of the famous cultivars are traded or distributed at higher price as "Premium rice" than the ordinary rice because of their high palatability, processing suitability or special aroma, etc.
As those premium rice grains sell at high price, some dishonest rice wholesalers or retailers blend low quality cheap rice to high quality premium rice and mislabel to be "high-quality premium rice".
Recently, rice consumers are tend to demand more information, such as cultivar name, location of production, year of production, etc., about rice which they purchase. For example, it is obliged to display the name of rice cultivar on its package under the JAS

LAW (Law Concerning Standardization and Proper Labeling of
Agricultural and Forestry Products in Japan). Therefore, technology to identify rice cultivar is very important for breeders, farmers, inspectors, wholesalers, retailers, food industries and consumers.
Rice cultivar has been identified based on morphological characteristics of rice plant or grains, ripening ratio after crossing, or the isozyme patterns, etc. Cultivated rice (Oryza sativa L.) is one of the most polymorphic crop species in the world.
On the contrary, many high-quality rice or premium rice is closely related because inbred lines are often used for their development.
Therefore, it is necessary to develop a time-saving technology to differentiate rice cultivars clearly and precisely. DNA fingerprinting was developed in 1985 [44] and is used for criminal investigation and trial at court. Recently, novel cultivar identification method based on DNA polymorphism has been developed accompanied with the progress in molecular biology. DNA-based markers have the obvious advantage of sampling the genome directly and RFLP analysis has been widely used for assessing variation of plants [45,46].

RFLP analysis has been used to distinguish between species
of Oryza [47] and particularly between indica and japonica type of Oryza sativa [48]. Recently, PCR-based marker system has been developed by Williams et al [49]. In this RAPD (Random Amplified Polymorphic DNA) method, short oligo-nucleotides of arbitrary sequence are used to support the amplification of regions of the test plant genome and amplified DNAs are separated by gel electrophoresis. There are some reports on RAPD analysis of rice germplasm including indica and japonica types, to identify suitable parents for linkage map construction, and for gene tagging for drought resistance [50,51]. RAPD analysis was revealed to be reproducible and amenable for identification of each single plant line of F1 hybrid rice in China [52] and Australian rice cultivars [53]. Diversified rice cultivars were classified into separate groups by PCR using RAPD markers, but many primers were needed to resolve closely related japonica cultivars [54][55][56]. In case of RAPD markers, many other DNA bands than the target DNA for cultivar differentiation appear in the electrophoregram after PCR.
Therefore, it is recommended to develop the STS (sequence tagged site) markers or SCAR (sequence characterized amplified region) markers based on RAPD markers. In addition to RAPD markers, microsatellites or simple sequence repeats (SSRs), which are DNA sequences with repeat lengths of a few base pairs and variation in the number of nucleotide repeats, can be detected with PCR by selecting the conserved DNA sequences flanking the SSR as primers.
SSR markers are useful not only characterize the relationship between heterosis and marker genotype heterozygosity but also to identify chromosome segments that may have significant effects on yield and its component traits in rice [59]. Genetic diversity among Indian elite rice varieties was evaluated using three different types of DNA markers and parentage analysis [60]. SSLP was reported to be more reliable than AFLP for identify rice cultivars [61]. RAPD, RFLP, nuclear SSLP and chloroplast SSLP analyses were carried out to clarify the phylogenetic relationships among A-genome species of rice [62]. SSR was reported to provide useful genetic information on weedy rice [63]. Development of genome-wide DNA polymorphism database for map-based cloning of rice genes was investigated using SNP markers [64]. And SNPs were used for the discussion on the sequence variations between the rice cultivars [65]. Although SSR and SNPs are very useful DNA markers, it is impossible to use several PCR primers simultaneously, which is "multiplex PCR", to simplify the PCR and electrophoresis and save time.
Furthermore, it was not reported to identify or differentiate the material rice cultivars by PCR method using the processed rice products, such as boiled rice or the rice cake. The present authors have investigated about the cultivar identification of rice by PCR method [66][67][68][69][70]. In the present study, application of PCR technology to cultivar identification of rice grains was investigated.
SCAR markers and multiplex primer sets were developed based on RAPD marker analyses to differentiate closely related rice cultivars clearly and efficiently by PCR. The efficient method to prepare the template DNA for PCR from the processed rice products, such as cooked rice or rice cake, was investigated. The identification or the differentiation of material rice cultivars by the multiplex PCR was carried out using the processed rice products as samples.   Polymerase Chain Reaction (PCR): DNAs were proliferated by the PCR method using 600 commercial random primers (10 mers or 12 mers) as primers [56]. Taq-DNA polymerase (Takarabio Inc., Otsu, Japan) was used for amplification of DNAs. Each DNA was denatured for 1 min at 94°C, annealed for 1 min at 62°C and elongated for 2 min at 72°C. This procedure was repeated for 40

Materials
times. As a PCR apparatus, Thermal Cycler MP (Takara-bio, Otsu, Japan) was adopted.

Development of SCAR Markers and Multiplex Primer Sets:
In the present study, SCAR markers were developed based on RAPD analysis to differentiate rice cultivars by PCR (25)(26)(27)(28). DNAs were extracted from the agarose gel after the electrophoresis of the PCR products using "Easy trapＴＭ" (Takara-bio, Otsu, Japan).
DNA cloning was carried out using "TOPO XL PCR Cloning KitＴ   Table 2. The sequence of the differential DNA, which was extracted from the electrophoresis agarose gel after PCR of RAPD method, was determined and the suitable forward and reverse primers were designated as shown in Table 2 [59,60].

Results and Discussion
As shown in Figure 7, SCAR markers are more useful than RAPD markers [59,60,66,67] because it is clearer in electrophoregram.
Furthermore, it is possible to use several SCAR markers simultaneously in PCR, which leads to saving of time and labor. As shown in Table 2, several kinds of SCAR markers were developed.
The length of markers was adjusted so that the transition temperatures of the markers are around same temperature, 62°C.
Therefore, several SCAR markers can be used simultaneously in "multiplex PCR." Properties of Rice Samples: Various rice samples were collected from different district in the world and their chemical and physical properties were measured [60]. The results are shown in Table 3. As shown in Table 3, Indica subspecies, such as IR2061 and Nanjing 11, showed high amylose content and less adhesiveness, on the contrary, Japonica sub-species, such as Hinohikari and Akitakomachi, revealed low amylose content and more adhesiveness. In case of trade con-tract, inspection or grading or the survey of traceability of rice grains, PCR method must be applied using rice grains as samples. Shoot or leaves are not available as sample specimen. Therefore, milled rice flour, single kernel of polished rice or even boiled rice is used as materials for PCR in the inspection carried out in post-harvest inspection.
Rice is markedly diversified from the viewpoint of genetics, morphology and properties. On the contrary, high-quality rice is closely related by inbred breeding to attain high-palatability, high processing suitability or characteristic aroma, etc. Rice grains of the famous cultivars are traded or distributed at higher price as "Premium rice" because of their high palatability, processing suitability or special aroma, etc. As those premium rice grains sell at high price [1]. In the present study, various rice samples were collected from different district in the world and their chemical and physical properties were measured. As shown in Table 3, the grain properties between Indica subspecies, such as IR2061, and Japonica sub-species, such as Hinohikari, were revealed to be very different, but those among the same sub-species are not different. Note: Adhesion was measured by a Tensipresser and the unit is mm.

Application of PCR to Various Rice Samples: CTAB method
can be applied to milled rice flour for extraction and purification of template DNA for PCR [72,73]. The result of PCR using 15 rice cultivars from various countries, such as USA, India, Thailand or Japan, as samples was successful. The primer set used for PCR was our commercial "Koshihikari Positive Kit" (Takara-bio). Four DNA bands appeared clearly in case of Hitomebore and three ones appeared clearly in case of Koshihikari, which were revealed to be useful to differentiate various rice cultivars by a single time PCR and electrophoresis. As shown in Figure 8, the multiplex primer set for PCR, "Koshihikari Positive Kit" is very useful to differentiate various rice cultivars from the different district in the world. It was shown that RAPD analysis is a useful tool in determining the genetic relationships among rice cultivars [51][52][53][54][55][56].  Primer Set for "Koshihikari" Identification: In Japan, leading variety Koshihikari shares more than one thirds of total cultivation area of rice because it is palatable and traded at higher price than other rice cultivars. It is obliged, in Japan, that the name of rice cultivar, location of cultivation, and year of rice production are labeled on the package of rice by the Japan Agricultural Standard (JAS) Act. Therefore, it was necessary to develop the technology to identify Koshihikari cultivar by DNA analysis. As shown in Figure   9A, Koshihikari can be discriminated from any other cultivars using this "primer set for identification" showing three specific DNA bands [69]. There is no other cultivar rice which shows the same three band pattern with Koshihikari among 50 dominant non-glutinous rice cultivars in Japan. All the Koshihikari from the different districts showed the same pattern of three DNA bands, which correspond to the same grouping under Japanese Seeds and Seedling Law based on representative characteristics of rice plant ( Figure 9B). On the contrary, no cultivars other than Koshihikari showed the same DNA patterns with Koshihikari even Hitomebore or Hinohiari, which are descendant cultivars of Koshihikari as shown in Figure 9A [69].

Primer Set for Differentiation of Other Rice with Koshihikari:
As the premium rice Koshihikari sells at high price, mislabeling "Koshihikari 100%" occurs by the dishonest rice retailers in Japan.
For the purpose of detection of rice grains of other cultivars which were dishonestly blended into Koshihikari, "Detection-kit" was developed. In case of detection-kit, no DNA is proliferated in case of all Koshihikari rice grains of various prefectures ( Figure 10A), and more than one DNA bands never fail to in-crease by PCR in case of rice grains of other cultivars ( Figure 10B). This "primer set for detection" can be used to detect the illegal blending of other rice to Koshihikari labeled as "Koshihikari 100% premium rice." [69] SSR markers are useful not only characterize the relationship between heterosis and marker genotype heterozygosity but also to identify chromosome segments that may have significant effects on yield and its component traits in rice [57][58][59][60][61][62]. And SNPs were used for the discussion on the sequence variations between the rice cultivars [64,65]. Nevertheless, "multiplex primer sets" using SCAR markers were revealed to be more useful and practical for efficient and precise cultivar differentiation. Comparison of template DNAs prepared by the three methods, commercial kit method, CTAB method and "enzyme treatment method" was carried out [67]. "Enzyme treatment method" was shown to be profitable from the viewpoint of quality and quantity.  [67].
In case of CTAB method, it is possible to prepare purified DNAs, but it was inferior to "enzyme treatment method" in terms of quantity of DNAs. Lysozyme is sometimes used for DNA preparation [58]. But lysozyme decomposes cell wall mainly, and it is difficult to digest the gelatinized starch and denatured proteins. It became possible to proliferate specific DNAs by PCR using template DNA prepared by the "enzyme treatment method" and the multiplex primer set is very useful to differentiate various rice cultivars by a single PCR and electrophoresis as shown in Figure 11 [67]. In case of boiled white rice grain, as shown in Figure 11, it became possible to differentiate Hitomebore or Hinohikari from Koshihikari, their parental cultivar by PCR using the template DNA prepared from each single grain of boiled rice by the "enzyme treatment method", which was de-scribed in Materials and Methods [67].  Figure 11: Differentiation of Japanese rice cultivars by PCR using template DNA extracted from a single boiled rice grain by the "enzyme treatment method"

PCR:
The results of PCR using template DNAs prepared from each rice cake by "enzyme treatment method" were same with those using template DNAs from Multiplex PCR for identification of material rice of rice cake. "Ezymetreatment method" is also useful for preparation of template DNAs from rice cake. Solubility of rice cake in boiling water and expansion on heating varies depending on cultivar and producing area of the material glutinous rice [69].
Therefore, identification of cultivars of the material glutinous rice using rice cake as a sample is very important. The results of PCR using template DNAs prepared from each rice cake by "enzyme treatment method" were same with those using template DNAs from material raw rice flour directly by CTAB method as shown in Figure   12. It became possible to differentiate each material waxy rice cultivar by the single PCR and electrophoresis by the development of primer set for "multiplex PCR" to identify waxy rice cultivars [70].  prohibit the DNase during the protein digestion [67]. Single boiled rice grain or rice cake can be used as a sample for identification or differentiation of material rice cultivars by PCR method [67,68].

Abstract
Diabetes and dementia are life-style related diseases, and patients have been increased all over the world. We tried to prevent diabetes using the high-amylose rice cultivars. It was shown that the postprandial BGL of high-amylose rice was lower than that of low-amylose rice. "Super hard rice", of which starch contains longchain amylopectin, was developed in Kyushu University, Japan, and we clarified its characteristics by the cooperative re-search. It was very hard after cooking, and rich in resistant starch. "Super hard rice" lowered postprandial BGL not only as cooked rice grains but also as rice noodle and rice bread. We prepared super-hard rice bread blended with 5% of black rice bran (SRBBB), which contained much amylose and showed inhibitory activity against βsecretase after heating. Aged mice, which were fed SRBBB diet for 4 weeks, showed lower amyloid β40 in the blood than the control.
We investigated the effects of the blend of ordinary brown rice and black rice cooked after a high-pressure treatment (HPTKO). A randomized, single-blind, crossover-designed study was conducted using 15 subjects, and BGLs at 90 and 120 min after ingesting the cooked HPTKO were significantly lower than that for cooked ordinary polished rice. Furthermore, postprandial blood amyloid β40 did not increased markedly compared with the control.

Introduction
According to WHO report, globally, an estimated 422 million adults were living with diabetes in 2014, and diabetes of all types can lead to complications in many parts of the body and increase the overall risk of dying prematurely [79]. Therefore, there is an urgent need to implement population-based interventions that prevent diabetes, enhance its early detection, and use lifestyle and pharmacological interventions to prevent or delay its progression to complications [80]. The WHO and Food and Agriculture Organization of the United Nations recommend foods with low glycemic index (GI) to prevent diabetes [81]. The concept of GI was introduced by Jenkins, et al. [82]. as a ranking system for carbohydrates based on their immediate impact on blood glucose levels, and low GI and glycemic load diets have more recently been widely recommended for the prevention of chronic diseases including diabetes, obesity, cancer and heart disease [83], because the re-sulting glycemic index classification of foods provided a numeric physiologic classification of relevant carbohydrate foods in the prevention and treatment of diseases such as diabetes [84].
Alzheimer's disease affects more than 25 million people worldwide and is the most common form of dementia [85]. β-amyloid precursor protein (APP) is processed to generate β-amyloid (Aβ) by β-and γ-secretase, in a highly regulated process. Many drugs have been approved for the treatment of Alzheimer's disease, at different stages of the disease, although they all have limited efficacy. Recent epidemiological studies have suggested a link between Alzheimer's disease and type 2 diabetes mellitus associated with insulin resistance [86][87][88].
Diabetes is a lifestyle disease, and its prevention and treatment are extremely important. Low glycemic index (GI) foods inhibit rapid increases in blood glucose and insulin secretion after meals.
Because working memory is impaired by AD, the disease has spawned dynamic research investigating the influence of gammaaminobutyric acid (GABA) on working memory performance in AD sufferers [89]. It is well-recognized that the prevalence of dementia is higher in diabetic patients than in non-diabetic subjects [90], and Tokutake, et al. [91] showed that there is a molecular link between AD and insulin signaling. Cereal grains are indispensable for the people all over the world. Intake of calories, Several studies have reported the development of highly resistant starch rice [92,93] as well as high-amylose and high-dietary fiber rice [94] via physical or chemical mutation. The hydrolysis of starch is a key factor in controlling the GI of foods. Functional foods that have α-glucosidase inhibi-tory activities have proven effective in controlling blood sugar levels in people at risk of developing diabetes [95]. Furthermore, epidemiological studies suggest that the low incidences of certain chronic diseases in rice-consuming regions of the world may be attributable to the antioxidant compounds found in rice. The molecules with antioxidant activity contained in rice include phenolic acids, flavonoids, anthocyanins, proanthocyanidins, tocopherols, tocotrienols, γ-oryzanol, and phytic acid. Rice bran also contains various functional substances, such as γ-oryzanol, ferulic acid, sterol, wax, ceramide, phytin, inositol, and protein [96].
Rice bran oil, the only domestic edible vegetable oil made from the rice bran produced in Japan, is known to have high oxidative stability and serum cholesterol-lowering activity [97,98] inhibited Aβ production in neuroblastoma cells without causing cytotoxicity, suggesting such foods may prevent Alzheimer's disease [103]. Pigmented rice contains naturally occurring colored substances that be-long to the flavonoid group called anthocyanins.
Positive health effects of these pigments present in the bran layer of rice have been reported [104].
Ling et al. showed that red and black rice decreases atherosclerotic plaque formation and increase antioxidant status in rabbits due to their enhanced serum high-density lipoprotein (HDL) cholesterol and apolipoprotein A1 concentrations [105].
Recently, food technology to prevent the decrease of those biofunctional components during the cooking and increase their functions by the germination, high-pressure treatment, or the coextrusion cooking have been reported. High-pressure treatment (HPT), a technological process that limits the negative effects of food preparation on hydro-soluble vitamins, is recognized as being very useful in preserving nutritional quality in foods [106,107].
Yamakura, et al. [108] showed that subjecting rice to HPT before cooking results in more free amino acids and stickier cooked rice.
The consumption of a western diet, which is characterized by a high in-take of red meats and high-fat dairy products, may contribute to obesity and metabolic syndrome, as well as increase the risk of developing type 2 diabetes and cardiovascular disease. In contrast, the traditional Asian diet, which is rich in soy and fish but low in animal protein and fat, may help reduce the frequency of severe chronic diseases [109]. There is also a significant association between a Mediterranean diet and reduced risk of major chronic de-generative diseases, including Alzheimer's disease. The optimal diet for the prevention of cardiovascular and other major chronic diseases has rapidly evolved [110].

Development of Specialty Rice Cultivars (High-Amylose Rice, Black Rice)
Super rice research project of Japan started in 1989. It was supported by Ministry of Agriculture, Forestry and Fisheries, Japan, to enhance rice consumption. Low-amylose, high-amylose, giant embryo, aromatic, pigmented and high-prolamin rice cultivars were bred and utilized ( Figure 14). We prepared wheat/rice bread blending three kinds of specialty rice, high-amylose, sugary, and purple rice cultivars. The bread showed good taste and maintained softness even after 4 days [111].

Utilization of High-Amylose Rice (Prevention of Diabetes)
Diabetes patients are more than 8.2 million and 18.7 million including those to be diabetes in near future in Japan. It is indispensable to prevent diabetes for the reduction of the increase of the medical costs. It has been reported by the large-scale medical tests that the inhibition of drastic increase of the blood glucose after meals reduce the rate of diabetes initiation [81][82][83][84]93].
Cristiana reviewed about the rice compounds with impact on diabetic control [112]. They pointed out that rice macro nutrients, such as starch, proteins, lipids, and dietary fiber, and rice bran compounds, such as gam-ma-oryzanol, phytic acid, ferulic acid, and gamma-aminobutyric acid (GABA)and vitamin E, and elucidated the metabolic mechanisms and bioactive towards diabetes [112].
We aimed to evaluate the palatability of the high-amylose rice of which eating qualities are inferior to ordinary Japonica rice.
Another objective of this research was to clarify the mechanism to pre-vent diabetes initiation by the high-amylose rice by the feeding test of rats and diet test by the human beings. Proximate components and gelatinization properties of the high-amylose rice were clarified and texture and eating qualities of the cooked rice from high-amylose rice were reported [113]. In the case of highamylose rice, drastic increase of blood glucose and insulin after meals were inhibited ( Figure 15) [113]. By serving the retrograded rice samples (2hrs after cooking), increase of blood insulin was more inhibited for high-amylose rice than for low-amylose one in animal feeding test [113].  (Figure 16) [114]. Its starch is characteristic because its apparent amylose content is markedly high because its amylopectin has much amount of middle and long glucan chains [114]. Although its cooked rice is unpalatable due to hard texture, it contains high amount of resistant starch [115][116][117][118]. Therefore, super-hard rice is promising as a material to prevent obesity and diabetes. Nakamura and Ohtsubo [119], and Maeda, et al. [120] tried to improve the texture of cooked rice from super-hard rice (CRSH), and they showed that the high-pressure treatments improve the texture of CRSH and maintained the effect to inhibit abrupt increase of postprandial blood glucose level.

Bio-Chemical Test for Prevention of Life-Style Diseases
Type-2 diabetes and Alzheimer's disease are very serious disease and the former has been suggested to be one of the causes of the latter [86][87][88]. Low glycemic index foods inhibit rapid increases in blood glucose and insulin secretion after meals [93,94]. Antioxidative capacity of brown rice and rice oil may be useful for the prevention of dementia [96,98,103]. In this study, we investigated the palatability of boiled rice and inhibition of an abrupt increase in blood glucose level (BGL) and amyloid β peptide production after eating blend of ordinary brown rice, "Koshihikari" and anthocyaninrich black-rice, "Okunomurasaki" unpolished rice cooked after a high-pressure treatment (HPT KO). "Okunomurasaki" showed a high antioxidant capacity and high inhibitory activity against β-secretase even after HPT and cooking [121].

Animal Feeding Test
Multiple prevention of type-2 diabetes and Alzheimer's disease by the rice products would be very important objective for us [86,88,93,94]. Therefore, we prepared super-hard rice bread blended with black rice bran (SRBBB), which showed strong inhibitory activities against β-secretase and acetyl-cholinesterase and contained high amount of resistant starch even after heating.
Black rice bran showed greater β-secretase inhibitory activity (3.6fold) than Koshihikari rice [121]. The bran contained more oleic acid and anthocyanin, meaning that it is potentially a bio-functional food with a high antioxidant capacity. Furthermore, aged mice, which were fed a SRBBB diet for 4 weeks, showed lower amyloid β 40 peptide in the blood than mice fed a commercial diet (p < 0.01) [121] ( Figure 17B). Additionally, their initial blood glucose levels after 12 weeks of being fed SRBBB were significantly lower than those in the control group ( Figure 17A). Taken together, our results indicate that SRBBB seems to be promising for inhibiting not only amyloid β production but also abrupt in-creases in post prandial BGL.

Human Intervention Test (Single-Dose Test)
It seemed to be necessary to conduct a human intervention test of the promising rice product in terms of inhibitory activity against the increase of BGL and amyloid β peptide production to prove the probability to prevent type-2 diabetes and Alzheimer's disease. Therefore, we prepared heat moisture treatment cooked brown rice (HMT), and high-pressure treatment cooked brown rice (HPT), which contained higher amounts of resistant starch, antioxidant capacity, and β-secretase inhibitory activity than cooked ordinary white rice (Table 4) [122]. A randomized, single-blind, crossoverdesigned study was conducted using 15 subjects with a normal BGL.
It was reported that GI varies depending on the subjects (e.g., young men and women), so we divided the 15 subjects into two groups; a high BGL subclass (seven subjects) and a low BGL subclass (eight subjects). BGL at 90, and 120 min after eating cooked HPT KO unpolished rice for 15 subjects showed a significantly lower BGL compared with that after eating cooked Koshihikari polished rice (p < 0.05). BGL at 90 min and 120 min after eating cooked HPT KO unpolished rice for high BGL subjects (n = 7) showed a significantly lower BGL compared with that after eating cooked Koshihikari polished rice (p < 0.05). AUC was significantly lower compared with cooked Koshihikari white rice 120 min after eating (p < 0.05) [122].
Furthermore, the increase in the amyloid β40 peptide in the blood 120 min after eating HPT KO (unpolished rice, blend of Koshihikari, ordinary rice, and Okunomurasaki, black rice (ratio was 6:4)) tended to be lower than that of cooked Koshihikari polished rice in human intervention test (single-dose test) [123].

Long-Term Human Intervention Test (12-Week Test)
Furthermore, we conducted a long-term human intervention test using the HPSHRB (high-pressure treated super-hard rice (40%) blended with black rice (40%)) in terms of inhibitory activity against the abrupt increase of BGL ( Figure 18) and amyloid β peptide production for the probability to prevent type-2 diabetes and Alzheimer's disease. Tokutake et al. pointed out that amyloidβ secretion and hyperphosphorylation of tau are closely related [91], and we had ascertained it by the cell-culture test. By the long-term in-tervention test, we aimed to confirm the effects to inhibit both of abrupt in-crease of post-prandial blood glucose and amyloid β peptide production. And we could ascertain the safety and acceptability of our multi-preventive rice product by this long-term test. Test was performed under the permission of ethical committee for the human test. As a result, significant reduction of abrupt increase of blood glucose ( Figure 18) and inhibition of decrease in amyloid β ratio (amyloid β42/amyloid β40, Figure 19), bio-marker of Alz-heimer's disease [124], were shown after the 12-week human test [125]. And all 12 subjects accepted the rice sample and finished test with no adverse effects.

Summary
Diabetes and dementia are life-style related diseases, and patients have been increased all over the world. Rice is a staple food for Japanese and they eat rice at least once or twice every day. Rice does not only supply calories and give tasty dishes but also maintains healthy life for consumers and prevents some diseases. Since 1989, specialty ricecultivars, such as hard rice, soft rice, colored rice, aromatic rice, giant-embryo rice, etc. have been developed in Japan. We tried to prevent diabetes using the highamylose rice cultivars. It was shown that the postprandial BGL of high-amylose rice was lower than that of ordinary rice. It was shown that not only high-amylose rice but also pre-germinated giantembryo rice have the effects to lower the postprandial BGL and to lower the bloodpressure compared with the ordinary rice. "Super hard rice", of which starch contains long-chain amylopectin, was developed in Kyushu University and we showed its characteristics by the cooperative research. It was very hard after cooking, and rich in resistant starch. "Super hard rice" lowered postprandial BGL not only as cooked rice grains but also as rice noodle and rice bread.
High pressure treatment was introduced to food processing by Hayashi in 1980's. We showed that the super rice becomes softer and sticker by the high-pressure treatment preserving high amount of resistant starch.
We prepared super-hard rice bread blended with 5% of black rice bran (SRBBB), which contained a high amount of resistant starch that showed strong inhibitory activities against β-secretase after heating. Aged mice, which were fed SRBBB diet for 4 weeks, showed lower amyloid β 40 in the blood than the control. We investigated the effects of the blend of ordinary brown rice, super-hard rice, and black rice (2:4:4, w/w) cooked after a high-pressure treatment (HPT KO). A randomized,single-blind, crossover-designed study was conducted using 15 subjects with a normal BGL, and BGLs at 90 and 120 min after ingesting the cooked HPT KO were significantly lower than that for cooked ordinary polished rice (p < 0.05). Furthermore, we conducted a long-term test using the HPSHRB in terms of inhibitory activity against the abrupt increase of BGL and amyloid β peptide production for the probability to prevent type-2 diabetes and Alzheimer's disease. As a result, significant reduction of abrupt increase of blood glucose and change in amyloid β ratio were shown after the 12-week human test. And all 12 subjects accepted HPSHRB and finished test with no adverse effects. Furthermore, the ratio ofamyloid β42 to amyloid β40, an indicator for the proceeding of Altzheimer disease, did not increase markedly compared with the control.