JP6187062B2 - Evaluation method of rice starch properties by iodometric colorimetry - Google Patents

Description

  The present invention simply and quickly evaluates starch properties such as resistant starch content and sugar chain fraction content closely related to resistant starch based on iodine colorimetric spectral characteristics of rice starch. Regarding technology.

  In recent years, Japan has become a society with a declining birthrate and an aging society, and it is necessary to promote health maintenance and disease prevention with a diet rich in functional ingredients.

  Rice with a high amylose content of starch and rice with a long amylopectin chain are indigestible, have a slow increase in blood sugar after meals, and are expected to prevent the onset of diabetes (Non-Patent Documents 1 and 2). It is extremely important to develop a technology that can easily and quickly measure the content of resistant starch and related sugar chain fractions in various rice.

  Conventionally, the amylopectin chain length distribution of rice starch has been measured by high performance liquid chromatography after debranching with isoamylase (Non-patent Document 3).

  As a precise analysis method for the molecular weight distribution of amylose, Hanaki et al. Developed a fluorescence-labeled gel filtration method (Non-patent Document 4).

  Igarashi et al. Reported that the amylose molecular weight distribution and amylopectin chain length distribution of rice starch analyzed by the fluorescence-labeled gel filtration method affect the thermal gelatinization characteristics and taste of rice starch (Non-patent Document 5).

  In addition, Igarashi et al. Show that the amylopectin unit chain length distribution of glutinous rice analyzed by a fluorescence-labeled gel filtration method strongly affects the curability of rice bran (Non-patent Document 6) and iodometric color absorption of rice starch from 400 nm to 900 nm. From the curve, the ratio of the two peak areas on the low wavelength side and the high wavelength side with 600 nm as the boundary is obtained, and it has been reported that this ratio is useful as an aging index of rice starch (Non-patent Document 7).

  Although these conventional measurement methods provide accurate measurement values such as amylopectin chain length distribution, they do not measure the resistant starch content that is expected to prevent disease in rice starch. Is a costly method because it requires expensive equipment such as a high performance liquid chromatograph, and it is simple, quick, low cost and highly resistant to starch in the fields of breeding selection and food processing. In order to evaluate the sugar chain fraction content closely related to the content or resistant starch content, a new measurement method was required.

Kenichi Otsubo, Sumiko Nakamura, Kazunori Utsunomiya, Yasunobu Masuda, Keisuke Tsuji: Efforts to prevent and commercialize hard rice and diabetes. Food industry, 53 (14), 46-51, 2010. Ken’ichi Ohtsubo, Sumiko Nakamura, Keisuke Tsuji, Kazunori Utsunomiya, Yasunobu Masuda, Mineo Hasegawa: Possibility of diabetes prevention by high-amylose rice.Rice studies, present and future, Sankyosyuppan Inc., pp.109-115, 2012. Masako Asaoka, Kazutoshi Okuno, Yasumi Sugimoto, Masahiro Yano, Takeshi Omura, and Hidetsugu Fuwa: Structure and properties of endosperm starch and wated soluble polysaccharides from sugary mutant of rice, Starch, 37, 364-366 (11), 1985. Isao Hanashiro and Yasuhito Takeda: Examination of number average degree of polymerization and molar based distribution of amylose by fluorescent labeling with 2-aminopyridine. Carbohydrate Research, 306, 421-426, 1998. Toshinari Igarashi, Isao Hanashiro, and Yasuhito Takeda: Molecular structures and some properties of rice starches from Hokkaido cultivars.J. Applied Glycoscience, 55, 5-12, 2008. Toshinari Igarashi, Masafumi Kinoshita, Hideki Kanda, Tomoko Nakamori, and Toshimi Kusume: Evaluation of hardness of waxy rice cake based on the amylopectin chain-length distribution.J. Applied Glycoscience, 55, 13-19, 2008. Toshinari Igarashi: Molecular structure and properties of Hokkaido rice starch. Hokkaido Agricultural Experiment Station Report, 127, 12-19, 2010.

  It is an object of the present invention to provide a method for simply and quickly evaluating low-cost and high-accuracy rice starch characteristics related to functionality, such as the content of resistant starch, by iodine colorimetric spectrometry.

  As a result of earnest research to solve the above problems, the present inventors added an iodine reagent to a rice starch sample, analyzed the absorbance curve obtained by ultraviolet-visible spectroscopy, and applied the analysis value to the estimation formula. Thus, the present inventors have found that the indigestible starch content or the content of sugar chain fraction deeply related to the indigestible starch content can be estimated simply, rapidly, at low cost and with high accuracy.

That is, the method for evaluating the characteristics of rice starch of the present invention is based on the absorbance (Aλ _max ) at the maximum absorption wavelength in the visible region, which is obtained from the absorbance curve from the ultraviolet region to the visible region of rice starch to which an iodine reagent is added. When the absorbance is measured by scanning the wavelength of the incident light from the long wavelength side into the region, the area under the absorption curve from the wavelength where absorption in the visible region appears to 400 nm (F1 + F2), 400 nm from the maximum absorption wavelength in the visible region It is characterized in that the resistant starch content is evaluated by an estimation formula using any of the areas under the absorption curve (F2) .

Further, the maximum absorption wavelength (λ _max ) in the visible region, the absorbance (Aλ _max ) at the maximum absorption wavelength in the visible region, obtained from the absorbance curve from the ultraviolet region to the visible region of rice starch added with an iodine reagent, from the visible region to the ultraviolet region When the absorbance is measured by scanning the wavelength of the incident light from the long wavelength side into the region, the area under the absorption curve from the wavelength where absorption in the visible region appears to 400 nm (F1 + F2), 400 nm from the maximum absorption wavelength in the visible region Polymerization of glucose by an estimation formula using either the area under the absorption curve (F2) up to 400 nm or the area under the absorbance curve from the absorption peak wavelength that appears next to the maximum absorption wavelength in the visible region (F3) It is characterized in that the content of the sugar chain fraction having a degree of 10 is evaluated.

  Further, the absorbance at the maximum absorption wavelength in the visible region (Aλ) obtained from the absorbance curve from the ultraviolet region to the visible region of rice starch to which an iodine reagent was added. _max ) When the absorbance is measured by scanning the wavelength of incident light from the long wavelength side from the visible region to the ultraviolet region, the area under the absorption curve from the wavelength at which the absorption in the visible region appears to 400 nm (F1 + F2), the visible region The area under the absorption curve (F1) from the wavelength at which absorption occurs to the maximum absorption wavelength in the visible region, the area (F2) under the absorption curve from the maximum absorption wavelength in the visible region to 400 nm, and the maximum absorption in the visible region from 400 nm The content of the sugar chain fraction having a polymerization degree of glucose of 22 is evaluated by an estimation formula using any of the areas (F3) under the absorbance curve up to the wavelength of the absorption peak appearing next to the wavelength.

  Further, the maximum absorption wavelength (λ in the visible region) obtained from the absorbance curve from the ultraviolet region to the visible region of rice starch to which an iodine reagent is added. _max ) Is a method for evaluating the characteristics of rice starch, wherein the content of resistant starch is evaluated by an estimation formula using
The estimation formula for resistant starch content is
Resistant starch content (%) = 21.2 x Amylose content of sample rice (%) / (Maximum absorption wavelength in visible region (λ _max ) (Nm)-Maximum absorption wavelength in the visible region of glutinous rice (nm))-3.50
It is characterized by being.

  According to the present invention, it is necessary to use only an incubator and a spectrophotometer for the content of indigestible starch in rice that is expected to have functions such as prevention of diabetes and prevention of obesity by slowing the increase in postprandial blood glucose in humans. Can be measured easily and quickly at low cost.

  In addition, according to the present invention, it is possible to estimate the content of sugar chain fraction having a polymerization degree of 10 or a polymerization degree of 22 by iodine colorimetric spectroscopy measurement of rice starch, and the possibility of the functionality of the rice sample. Can be estimated.

  Furthermore, in addition to the conventional amylose content, information on the structure of amylopectin can be obtained by simple iodine colorimetric spectroscopy, so that the quality characteristics of the sample rice can be estimated to some extent.

It is a graph which shows the resistant starch content of various sample rice. It is a spectral characteristic in the iodine colorimetric color of starch. It is an iodine colorimetric spectral characteristic of high amylose rice and waxy rice starch. It is an iodine colorimetric spectral characteristic of starch of good taste rice and super hard rice. It is a graph which shows the amylose content of various sample rice. It is explanatory drawing which shows the analysis method of an iodine absorption curve. It is a graph which shows the maximum absorption wavelength ((lambda) max) of the starch iodine colorimetric in various sample rice. It is a graph which shows the light absorbency in the iodine colorimetric maximum absorption wavelength of various sample rice starch. It is a graph which shows the area of F1 in the spectroscopic measurement of various rice starch. It is a graph which shows the area of F2 in the spectroscopic measurement of various rice starch. It is a graph which shows the area of F1 + F2 in the spectroscopic measurement of various rice starch. It is a new index (New λmax) showing a correlation with resistant starch. It is a formula for estimating the sugar chain fraction content based on the spectral characteristics of rice starch. It is an estimation formula of resistant starch content based on spectral characteristics of rice starch. It is an estimation formula for resistant starch using the short-chain sugar chain fraction as a variable.

  The present invention easily and quickly evaluates starch properties such as the resistant starch content and the sugar chain fraction content closely related to the resistant starch based on the iodine colorimetric spectral characteristics of rice starch. Regarding technology.

  The rice starch in the present invention is a product obtained by defatting and deproteinizing a polished rice sample, which is obtained by removing protein with a dilute alkali at a low temperature and subsequently defatted with ethanol and acetone. The protein may be deproteinized by heating, or degreased by hot butanol or hexane.

  The iodo reagent in the present invention is a reagent for developing blue-violet color by binding iodine to a long chain of amylose or amylopectin, and refers to a solution in which 2 g of iodine and 20 g of potassium iodide are dissolved in 1 L of water. .

  The UV-visible spectroscopic measurement in the present invention refers to obtaining an absorbance curve by irradiating the gelatinized starch aqueous solution with light in the visible region (380 to 780 nm) and ultraviolet region (200 to 380 nm), and has a wide wavelength range. The wavelength at which the absorbance in the visible region when the light is continuously incident and the absorbance is measured is referred to as the maximum absorption wavelength (λmax).

  The multiple regression analysis in the present invention is to predict one objective variable with a plurality of explanatory variables, and is a kind of multivariate analysis. Which explanatory variable influences the objective variable to what extent. I can know. For example, when there are three independent variables, the multiple regression equation is as follows. The coefficient applied to each independent variable is called “partial regression coefficient”. The suitability of the model is expressed as a coefficient of determination (the square of the multiple correlation coefficient) and can be tested by analysis of variance.

  The starch characteristic in the present invention refers to a functional characteristic of starch, for example, a physiological characteristic such as digestion and absorption after feeding, and a physical characteristic such as viscosity characteristic of gelatinized starch.

  The indigestible starch in the present invention refers to a starch that is difficult to digest and absorb to the human stomach and small intestine after feeding, and when starch molecules are mutually cross-linked by a covalent bond or chemically modified. In the case of crystallization by hydrogen bonding, when starch molecules are structurally covered with other materials, gelatinized starch is aged, etc. It also refers to starch that has been reported to have an intestinal effect.

  The sugar chain fraction in the present invention refers to a glucose polymer formed by partial decomposition of amylose or amylopectin constituting starch by amylase or isoamylase, and using a technique such as gel filtration chromatography, Separation and fractionation by molecular weight can be performed.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to these Examples at all.

(Measurement of resistant starch content of sample rice)
2 g of the polished rice was suspended in 10 mL of 0.1% sodium hydroxide and deproteinized by shaking at 4 ° C. for 3 hours. After light centrifugation, the precipitate was washed three times with pure water, twice with 99% ethanol, then washed with acetone and air-dried to obtain a sample starch.

  A starch sample having a dry matter weight of 100 mg was taken in a test tube, and the resistant starch content was measured using a resistant starch measurement kit manufactured by Megazyme. The results are as shown in FIG. 1, and EM174, Kaori Koshino, Hokuriku powder 243, etc. showed high values.

(Quantification of sugar chain fractions with different chain lengths)
4 mg of the starch sample prepared in Example 1 was suspended in 1.6 mL of distilled water, the starch was gelatinized at 100 ° C., and 16 μL of 1M acetate buffer pH 3.5 was added to adjust the pH to 3.5. 0.67 μL (0.03 U / mg) was added and reacted at 45 ° C. for 15 hours to debranch the 1,6-glucoside bond of starch. Subsequently, the enzyme was inactivated by heating at 100 ° C. for 10 minutes, cooled in water, and used as a dry sample with a centrifugal concentrator (Tomy CC-105).

  To this sample, 111 μL of dimethyl sulfoxide was added and dissolved by heating. To this, 89 μL of distilled water and 200 μL of 2-aminopyridine were added and mixed well, and incubated at 60 ° C. for 1 hour in the dark. Thereafter, 200 μL of sodium cyanoborohydride was added and incubated for 24 hours to prepare a sample for HPLC. Next, this sample was filtered through a membrane filter (manufactured by Millipore, pore size 0.2 μm), and 20 μL of the filtrate was injected into HPLC manufactured by JASCO Corporation for analysis. The column was connected with SHODEXOHPAKSB-803HQ and SHODEXOHPAKSB-802.5 and detected with a fluorescence detector LP2020. The measurement results of the sugar chain fraction of the rice sample starch are shown in Table 1.

(Iodine colorimetric spectrophotometry of rice starch)
The concentration of the sample starch solution was determined according to Juliano's iodine colorimetric method. First, a 200 to 900 nm scan was performed, and the maximum absorption wavelength λmax in the visible region and the absorbance at this wavelength, Aλmax, 400 nm, shown in FIG. The first maximum absorption wavelength B on the lower wavelength side and the area of 400 nm as F1, the area from 400 nm to λmax as F2, the area from λmax to 900 nm as F3, and the spectral characteristics of 32 types of rice starch Evaluated. As a measurement example, FIG. 3 shows a measurement example of high amylose rice and mochi rice, and FIG. 4 shows a measurement example of good taste rice and amylopectin long-chain ultra-hard rice.

(Analysis of iodine colorimetric spectroscopic measurement results of rice starch)
FIG. 5 shows the amylose content of various rices according to Juliano's method.

  As an analysis of a new iodine absorption curve in FIG. 6, the difference between the iodine absorption curve value of glutinous rice is obtained from the value of the iodine absorption curve of starch, and the ratio of this value and the amount of iodine binding (amylose content) is calculated. This value was defined as New λmax.

  FIG. 7 shows measured values of the maximum absorption wavelength λmax in the visible region of the iodine absorption curves of 32 kinds of sample rice in iodine colorimetric spectrometry, and FIG. 8 shows the absorbance (Aλmax) at the maximum absorption wavelength in the visible region of each sample rice. Shown in In the value of λmax, the high amylose rice has the highest Koshi Kaori, Carnaloli of India-Japan hybrid rice not having a very high amylose content, Taiwanese rice fragrant rice, Chinese rice No. 88 of good taste rice with low amylose content, In addition, the value of Yumepirika from Hokkaido rice was high. As for the value of Aλmax, AE rice except Hokuriku Flour No. 243 shows a very high value, Indian type Yumeji, Hoshiyutaka of India-India hybrid rice, high amylose rice shows a high value, Carnaloli, Mizuhochikara etc. showed a slightly higher value than good-tasting rice.

  Moreover, F1 of each sample rice is shown in FIG. 9, F2 is shown in FIG. 10, and the total of F1 and F2 is shown in FIG.

  Next, Table 2 shows the results of the correlation analysis of the chain length distribution and iodine absorption curve analysis values shown in Table 1 and the resistant starch content. The content of the sugar chain fraction from DP7 to DP12 (polymerization degree 7 to polymerization degree 12) is F2, and the content of the sugar chain fraction from DP12 to DP16 (polymerization degree 12 to polymerization degree 16) is Aλmax, The content of the sugar chain fraction from DP17 to DP19 (degree of polymerization 17 to polymerization degree 19) is F1, and the content of the sugar chain fraction from DP20 to DP23 (degree of polymerization 20 to degree of polymerization 23) is F2. The content of the sugar chain fraction of DP24 (polymerization degree 24) or higher showed the highest correlation with Aλmax, and the content of the sugar chain fraction from DP25 to DP30 (polymerization degree 25 to polymerization degree 30) showed the highest correlation with F2. As a result, it can be confirmed that F2 in the iodine absorption curve has a high correlation with the medium chain fraction of amylopectin, and F1 has a high correlation with the medium chain fraction of DP18 to DP23 (degree of polymerization 18 to degree of polymerization 23). It was. Relative to this, λmax has no correlation with the fraction from DP17 to DP23 (polymerization degree 17 to polymerization degree 23), and is a short-chain DP7 to DP15 (polymerization degree 7 to polymerization degree 15). Minutes showed a significant difference with a risk rate of 5%.

  Table 3 shows the relationship between the resistant starch and the maximum absorption wavelength (λmax) in the visible region, Aλmax, F1, F2, and F1 + F2. Aλmax showed the highest correlation with the resistant starch content, and F2 and F1 + F2 showed a significant correlation with a risk rate of 1%.

  FIG. 12 shows the results of measuring New λmax described with reference to FIG. 6 for various sample rice. Normal λmax did not correlate with resistant starch (r = 0.133), but New λmax showed a significant positive correlation with 1% risk (r = 0.5967) From the results of iodine colorimetric spectroscopy, it was considered a promising index for estimating resistant starch.

(Preparation formula for sugar chain fraction content based on iodine colorimetric spectroscopic measurement results)
As shown in FIGS. 13A and 13B, analysis values of iodine absorption curves of 10 varieties are used as explanatory variables. The objective variable is DP10 (degree of polymerization 10) for the sugar chain fraction content in A, and DP22 (degree of polymerization 22 in B). As a result of multiple regression analysis and formulas for estimation, the multiple correlation coefficients of these estimation formulas were 0.9467 for A and 0.9062 for B, creating an estimation formula with a very high correlation coefficient. Became possible.

(Preparation formula for resistant starch content based on iodine colorimetric spectroscopic measurement results)
Based on the results of Example 4, the results of multiple regression analysis with New λmax added to the explanatory variables are shown in FIG. As shown in A and B of the figure, the results of creating an estimation formula by performing multiple regression analysis using the resistant starch content as an objective variable and the analysis values of iodine absorption curves of 12 varieties as explanatory variables are shown in A. Thus, a calibration curve with a coefficient of determination of 0.9332 was obtained. As a result of testing the applicability to unknown samples with 10 varieties different from A, this estimation formula shows a coefficient of determination of 0.9326 as shown in B, and has a very high correlation coefficient and is applicable to unknown samples. It was shown that this is an estimation formula with high characteristics.

  For comparison, Fr.I / Fr.II was obtained by the method of Igarashi et al. Shown in Patent Document 1 and the correlation with the indigestible starch content of this example was found to be r = 0.58. Thus, it has been clarified that the estimation formula of the present invention is remarkably superior to the prior art.

(Preparation formula for resistant starch content-Part 2-)
As shown in FIG. 15, as a result of creating an estimation formula by performing multiple regression analysis using the resistant starch content as an objective variable, and analyzing values of iodine absorption curves of nine varieties as explanatory variables, as shown in the figure, A calibration curve with a coefficient of determination of 0.8523 was obtained.

Claims (4)

Scan the absorbance (Aλ _max ) at the maximum absorption wavelength in the visible region, obtained from the absorbance curve from the ultraviolet region to the visible region of rice starch to which the iodine reagent is added, and the wavelength of incident light from the long wavelength side from the visible region to the ultraviolet region. When the absorbance is measured, either the area under the absorption curve from the wavelength at which absorption in the visible region appears to 400 nm (F1 + F2) or the area under the absorption curve from the maximum absorption wavelength in the visible region to 400 nm (F2) A method for evaluating the characteristics of rice starch, wherein the content of indigestible starch is evaluated by an estimation formula using a rice cake. The maximum absorption wavelength in the visible region (λ _max ), the absorbance at the maximum absorption wavelength in the visible region (Aλ _max ), obtained from the absorbance curve from the ultraviolet region to the visible region of rice starch added with an iodine reagent, from the visible region to the ultraviolet region When the absorbance is measured by scanning the wavelength of the incident light from the long wavelength side, the area under the absorption curve from the wavelength where absorption in the visible region appears to 400 nm (F1 + F2), the maximum absorption wavelength from the visible region to 400 nm The degree of polymerization of glucose by an estimation formula using either the area under the absorption curve (F2), the area under the absorbance curve from 400 nm to the wavelength of the absorption peak appearing next to the maximum absorption wavelength in the visible region (F3) A method for evaluating the characteristics of rice starch, characterized by evaluating the content of a 0 sugar chain fraction. The absorbance at the maximum absorption wavelength in the visible region (Aλ) obtained from the absorbance curve from the ultraviolet region to the visible region of rice starch to which an iodine reagent was added. _max ) When the absorbance is measured by scanning the wavelength of incident light from the long wavelength side from the visible region to the ultraviolet region, the area under the absorption curve from the wavelength at which the absorption in the visible region appears to 400 nm (F1 + F2), the visible region The area under the absorption curve (F1) from the wavelength at which absorption occurs to the maximum absorption wavelength in the visible region, the area (F2) under the absorption curve from the maximum absorption wavelength in the visible region to 400 nm, and the maximum absorption in the visible region from 400 nm Rice characterized by evaluating the content of a sugar chain fraction having a polymerization degree of glucose of 22 by an estimation formula using any of the areas (F3) under the absorbance curve up to the wavelength of the absorption peak appearing next to the wavelength Evaluation method of starch properties. Maximum absorption wavelength in the visible region (λ) obtained from the absorbance curve from the ultraviolet region to the visible region of rice starch with iodine reagent added. _max ) Is a method for evaluating the characteristics of rice starch, wherein the content of resistant starch is evaluated by an estimation formula using
The estimation formula for resistant starch content is
Resistant starch content (%) = 21.2 x Amylose content of sample rice (%) / (Maximum absorption wavelength in visible region (λ _max ) (Nm)-Maximum absorption wavelength in the visible region of glutinous rice (nm))-3.50
The method for evaluating the characteristics of rice starch,