JP2009254265A - Hardly-digestible rice, and hardly-digestible starch - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide new hardly-digestible rice containing hardly-digestible starch, inhibiting abrupt increase in blood glucose caused by digestion of amylose, and allowing an eater to enjoy crispy palate feeling without high-pressure treatment. <P>SOLUTION: The hardly-digestible rice comprising wx/ae rice as mutant rice lack in amylose synthetic enzyme I (GBSSI) and amylopectin branching enzyme II (BEIIb), is obtained by subjecting wild nonglutinous rice to N-methyl-N-nitrosourea treatment or the like. The rice substantially contains no amylose, and contains hardly-digestible starch grains where the peak of the glucose polymerization degree (DP) of an amylopectin lateral chain falls on 13-15, and the amylopectin lateral chain having glucose polymerization of ≥12 accounts for ≥65 wt.% of the whole amylopectin side chain. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to resistant rice grains and resistant starch.

  In recent years, there has been an interest in so-called metabolic syndrome such as diabetes and hyperlipidemia. Daily eating habits are regarded as important as one of the preventive measures for metabolic syndrome. For this reason, various foods called low-calorie foods and foods that suppress a rapid increase in blood sugar after eating, for example, health foods using components that have an effect of suppressing sugar absorption have been developed.

  In addition, deliciousness is important for food. The deciding factor for the deliciousness of rice is the physical taste, in other words, the texture. The texture depends on the structure of the starch. Therefore, by changing the structure of starch in detail, the texture can be changed and more delicious foods can be created.

  Therefore, Japanese Patent Application Laid-Open No. 2006-217813 (Patent Document 1) discloses a processed rice product obtained from mutant rice lacking the amylopectin branching enzyme IIb (BEIIb) involved in the formation of amylopectin side chains. . This processed rice product is indigestible and suppresses blood sugar levels after eating. Moreover, the processed rice product using the expanded rice obtained by applying a high pressure to the mutant rice and rapidly depressurizing it has a good texture.

  In the amylopectin contained in this mutant rice, the ratio of the amylopectin side chain having a glucose polymerization degree of 6 to 12 to the total amylopectin side chain of the amylopectin is 20% or less, which is smaller than that of the wild type. ing.

  On the other hand, in order to further analyze amylopectin obtained from a mutant rice deficient in amylopectin branching enzyme (BEIIb), the present inventors have developed mutant rice (not only BEIIb but also amylose synthase I (GBSSI)). wx / ae rice) and the structure of the amylopectin side chain of amylopectin in the rice grain is reported (Non-patent Document 1).

JP 2006-217813 A Kubo, A., et al., Journal of Cereal Science (2007), doi: 10.1016 / j.jcs.2007.08.005

  However, since amylose is digested by amylase, when a large amount of mutant rice disclosed in Patent Document 1 is ingested, there is a possibility that a rapid increase in blood glucose level may be caused. In addition, rice with a high amylose content is said to be hard to gelatinize, weakly sticky, and prone to aging when cooled. And in order to obtain a crunchy texture from rice deficient only in the amylopectin branching enzyme, it was necessary to perform a puffing treatment applying high pressure, as described in Patent Document 1.

  Under such a background, in rice lacking both BGSSI and BEIIb, a change in the structure of amylopectin in the rice is observed, and starch obtained from this rice is also derived from the rice-derived starch described in Patent Document 1. It was also found to be an indigestible starch, and the above problem was solved.

  The rice grain of the present invention is an indigestible rice grain that does not contain amylose and contains amylopectin in which the distribution peak of the degree of glucose polymerization of the side chain of amylopectin is located at 13-15.

  Moreover, the resistant starch of this invention consists of amylopectin which does not contain amylose and the distribution peak of the glucose polymerization degree of an amylopectin side chain is located in 13-15.

  According to the rice grain of the present invention, it is a rice grain that does not contain amylose, but unlike mochi rice, it has a good texture that is crispy. In particular, processed rice products such as cooked rice and bread having a good texture can be obtained without the need for puffing as described in Patent Document 1. Moreover, since the so-called aging that the pregelatinized starch returns to the pregelatinized starch is fast, processed rice products that are less likely to be altered during storage are provided by aging early in the production process. And since gelatinization occurs at a high temperature, it can be processed at a higher temperature, and a processed rice product that is excellent in preventing bacterial growth and hygienic is provided.

  Moreover, since it is indigestible, the blood glucose raising action after eating becomes moderate, and various foods that are convenient for so-called metabolic measures such as diabetic patients are also provided.

  The indigestible rice cereal of the present invention is an indigestible rice cereal containing amylopectin, which does not contain amylose and has a markedly high amylopectin side chain having a glucose polymerization degree (DP) of 13 to 15. The starch content in this rice grain is at least 60% or more, preferably about 70% or more, based on the brown rice mass excluding moisture-containing rice husks. Therefore, most of the rice grains of the present invention are made of indigestible amylopectin, excluding moisture, and can be said to be indigestible rice grains. This rice cereal is mutant rice (wx / ae rice) lacking amylose synthase I (GBSSI) and amylopectin side chain forming enzyme (BEIIb) of wild glutinous rice, and the presence of starch grains is confirmed. . This mutant rice is treated with, for example, methyl nitrosourea (MNU) treated with methylnitrosourea (MNU) and so on. To obtain a mutation (see Non-Patent Document 1).

  In the present invention, rice grain means rice harvested from rice lacking amylose synthase I (GBSSI) and enzyme (BEIIb) that synthesizes the side chain of amylopectin, which are included in glutinous rice. In addition, it is used to include so-called brown rice in a threshed state, as well as polished rice from which bran has been removed.

  In the present invention, the processed rice product is a processed product using rice grains harvested from the rice as a raw material, cooked rice using the rice, germinated rice, koji, rice cake, etc. Not only rice flour obtained by pulverizing rice, but also processed foods that use the rice flour, such as noodles such as rice noodles, breads using rice flour, rice confectionery such as taro, rice crackers, rice crackers, cakes and cookies, etc. This means all foods that use rice or rice flour as a raw material, such as various confections made from rice flour, such as Japanese confectionery, Japanese confectionery such as buns. The amount of rice grains used is not particularly limited, and all or part of rice or rice flour used as a raw material for the processed rice product, or all or part of flour used as a raw material for processed product, for example, Originally used wheat flour of 10 to 80% by mass, preferably 30 to 50% by mass, can be produced using the rice grains and rice flour of the present invention in place of conventional rice, rice flour and wheat flour. The production method is not limited, and various known methods capable of producing various target foods are used.

  The indigestible starch in the present invention means a starch obtained from the rice cereal of the present invention as a raw material, regardless of the production method thereof, and in the same manner as when producing cereals such as ordinary rice and corn as a raw material. Obtainable. For example, a method of pulverizing the above-mentioned mutant rice and immersing it in water to separate the precipitated precipitate is exemplified.

  The processed food in the present invention is made from the above resistant starch, for example, rice confectionery such as noodles, breads, rice cakes and rice crackers using the starch, as well as protein in the starch. It means various foods using the starch, such as artificially prepared nutritional supplements, Western confectionery, Japanese confectionery, etc. obtained by using oil, fats and oils, sweeteners, acidulants and the like. The amount of starch used is not particularly limited, and all or part of the starch used as a raw material for the processed food can be produced using the starch of the present invention instead of the conventional starch. Although the specific usage-amount changes with processed foods, it is 0.1-99.9 mass% in processed foods.

  Amylose is a kind of polysaccharide constituting starch, and means a linear polymer in which glucose is mainly α-1,4 glycosidic bonded. Amylopectin is also a kind of polysaccharide constituting starch, and means a polymer having a branched main chain branched by α-1,6 glycosidic bonds in a linear main chain in which glucose is α-1,4 glycosidic bonded. To do.

  The amylopectin side chain in the present invention means a branched chain branched from an α-1,4 glycosidic bonded main chain, and is composed of those having various degrees of glucose polymerization. The degree of glucose polymerization (DP), that is, the number of glucose bonds in the side chain of amylopectin, is determined by using an analytical device such as chromatographies after decomposing denven molecules with an enzyme that digests the branched portion of the glucose chain, such as isoamylase. It is obtained by sieving using different molecular weights. In the present invention, the degree of glucose polymerization measured by the method described in Non-Patent Document 1 is used. More specifically, amylopectin is decomposed by isoamylase obtained from a Pseudomonas bacterium, and it is converted into 8-amino-1,3,6-pyrenetrisulfonic acid (8-amino-1,3,6-pyrentrisulfonic acid). The degree of glucose polymerization determined by performing capillary electrophoresis after labeling with acid (APTS) is used.

  The rice grain or resistant starch of the present invention does not contain amylose. Here, the meaning of not containing amylose is intended to be substantially free of amylose. That is, it means that the amylose content is considered to be logically zero, and does not mean that the measurement result is zero. The rice grain of the present invention does not have amylose synthase and should theoretically be zero, but amylose may be detected depending on the measurement method, its detection limit, and contamination. Moreover, if the presence of amylose synthase I is denied by an antigen-antibody reaction against amylose synthase I (GBSSI), it can be said that amylose is not substantially contained. In addition to amylose synthase I (GBSSI), amylose synthase may be detected, but it is considered that BGSSI is the only enzyme involved in amylose synthesis. It can be said that it does not contain amylose.

  Therefore, the rice grain of the present invention is a rice grain substantially containing only amylopectin as a starch component. Further, the resistant starch of the present invention is a starch substantially consisting only of amylopectin.

  The amylopectin contained in the rice grain of the present invention has a distribution peak of glucose polymerization degree (DP) of the side chain of amylopectin in 13-15. That is, the amylopectin side chain has the most glucose polymerization degree of 13 to 15, specifically, around 14 amylopectin side chains, and the amylopectin side chain has a higher glucose polymerization degree than wild type rice. In the amylopectin, the amylopectin side chain having a glucose polymerization degree of 12 or more and having a long glucose chain length accounts for 65% of the entire amylopectin side chain, and 70% or more of the good amylopectin (see FIG. 1). In addition, the ratio with respect to the whole amylopectin side chain is a ratio when the sum total of the amylopectin side chain with a polymerization degree of 3-35 is 100, and is specifically calculated | required by the method as described in an Example.

  The rice grain and starch of the present invention are characterized by being indigestible. That is, when eating, a rapid increase in blood glucose is not observed, but the blood glucose gradually increases after eating (see FIG. 9). In other words, it can be said that it has very favorable characteristics as a food material for diabetic patients (including not only humans but also animals) and their preparatory people and animals.

  The rice grain of the present invention does not contain amylose, but has a crystal-like structure similar to that of starch grains containing amylose (see FIG. 2). In other words, the crystallinity of starch granules can be determined by the presence of birefringence (polarization cross) when observed under a polarizing microscope. As can be seen from FIG. 2, the presence of a clear polarization cross is confirmed. The

  Gelatinization and aging are unique phenomena found in starch, and it is known that gelatinization and aging differ depending on the grain structure of starch, amylose, amylopectin content, chain length distribution, and the like. It is known that starch takes two forms: β-starch in a raw starch state and α-starch in a swollen state by addition of water and heat. Gelatinization is a phenomenon in which it is converted to α-starch and becomes sticky, and aging is a phenomenon in which water is lost from α-starch and returns to β-starch.

  The rice grain of the present invention does not contain amylose as described above, and has a long chain amylopectin side chain, so unlike conventional rice that is wild rice, the endothermic peak when gelatinized is at a high temperature. It will also shift, and it will age faster than traditional rice. In other words, pregelatinization occurs only in a high temperature state, and it is necessary to increase the temperature by adding water in order to obtain α-starch. For this reason, rice grains and starch can be exposed to high temperatures, and sterilization at high temperatures becomes possible. Moreover, pregelatinized starch is easy to return to the original starch, and aging is completed in a short time after being processed into processed foods such as koji and kaki. Therefore, the food containing this rice flour or the like hardly changes, and the quality change in the distribution stage is reduced.

  The rice cereal of the present invention is rice that does not contain amylose, and after cooking rice, it produces stickiness like glutinous rice. In addition, processed foods using rice grains, especially baked confectionery, have a crispy and crispy feeling, and can enjoy an unprecedented texture. In particular, a rice confectionery with a crisp feeling can be produced under the same conditions as those for ordinary confectionery, even without high-temperature and high-pressure treatment.

  Of course, the rice grains of the present invention lack amylose synthase and the enzymes that synthesize the side chain of amylopectin, so there are few proteins that can be allergens, and they are also suitable as foods for hypoallergenic foods.

  The present invention will be further described below based on examples. However, the present invention is not limited to the following examples.

[Sample preparation]
A wx mutant rice EM21 (GBSSI-deficient strain: by the method of Satoh and Omura 1979) obtained by mutating a japonica rice gold-nanbu fertilized egg cell with N-methyl-N-nitrosourea (MNU), ae Mutant rice EM16 (BEIIb-deficient strain) was crossed to obtain AMF18 strain which is a double mutant wx / ae strain. This strain was cultivated in Osaka Prefecture University and harvested as mutant rice (wx / ae mutant rice) 30 days after flowering. AMF18 was provided by Kyushu University Satoh Hikari Laboratory. In addition, wild-type (WT) Kinnan style, EM21 which is a wx mutant, and EM16 which is an ae mutant rice were used as controls.

  These mutant rices were threshed into brown rice. In addition, brown rice was polished with a test rice mill to obtain polished rice from which rice bran had been completely removed. Thereafter, it was pulverized to obtain rice powder. Moreover, the threshed brown rice was pulverized to obtain brown rice flour.

  Further, each polished rice was dipped in water and then pulverized, passed through a sieve (140 mesh), stirred in a 2% SDS solution, and the precipitate was recovered to remove proteins. Water was added to the precipitate from which the protein had been removed, and the precipitate was collected again. This washing with water was repeated 5 times and dried at room temperature to obtain purified starch.

[Measurement of amylopectin side chain]
The glucose polymerization degree of the amylopectin side chain was measured by the following method (see Non-Patent Document 1). In accordance with the method of O'Shea and Morell (1996), the purified starch obtained above was digested with isoamylase obtained from Pseudomonas spp. To produce 8-amino-1,3,6-pyrenetri Labeled with sulfonic acid (8-amino-1,3,6-pyrentrisulfonic acid: APTS). Next, capillary electrophoresis using ABI PRISM 310 Genetic Analyzer (Applied Biosystems) was performed. A POP-4 polymer column (36 cm long) was used. Electrophoresis was performed at 15 kV for 60 minutes using Genetic Analyzer Buffer (Applied Biosystems). Data was collected and analyzed using Gene Scan software (Applied Biosystems). Each peak value of glucose polymerization degree (DP) of each amylopectin side chain was expressed as a peak area ratio (%) with respect to the total area having a polymerization degree of 3 to 35. The result is shown in FIG. FIG. 1B shows the difference between the peak area ratio of mutant rice (wx / ae mutant rice and wx mutant rice) and the peak area ratio of wild species for each degree of glucose polymerization (DP).

[Crystal-like structure of starch granules]
The crystal-like structure of starch granules was investigated. The purified starch was stained with iodine and observed with a polarizing microscope and an optical microscope. The micrograph is shown in FIG.

[Western blot analysis]
The milled rice was pulverized and the protein extracted from the milled rice was separated by SDS-PAGE according to the method of Laemli (1970), and then Western blot analysis was performed. Anti-rice GBSSI antibody and anti-rice BEIIb antibody (both antibodies were provided by Prof. Yasunori Nakamura, Akita Prefectural University) were used as antibodies. Detection was performed using a horseradish peroxidase detection reagent (Biorad). The result is shown in FIG.

[Characterizing characteristics by DSC]
The gelatinization characteristics were investigated using polished rice, brown rice flour, and purified starch. The polished rice was prepared by swelling three grains with 1.2 times the volume (volume ratio) of water. Moreover, brown rice flour and purified starch were prepared by suspending 40 mg of each in 0.16 mL of water. These samples were measured with a differential scanning calorimeter (Micro DSC VII, manufactured by Setaram). The measurement conditions are 20 ° C. (30 minutes hold) → 105 ° C. (20 minutes hold) → 20 ° C. (10 minutes hold), and the temperature rising / falling rate is 0.5 ° C./minute. The result is shown in FIG.

[Aging characteristics by DSC]
Aging characteristics were examined using the purified starch of the present invention. 40 mg of purified starch was suspended in 0.16 mL of water to prepare a sample. The sample was measured with a differential scanning calorimeter (Micro DSC VII, manufactured by Setaram). The measurement conditions are 20 ° C. (holding for 30 minutes) → 105 ° C. (holding for 20 minutes) → 5 ° C. (holding for 24 hours or 72 hours), and the temperature rising / falling rate is 0.5 ° C./minute. The result is shown in FIG.

[Viscosity measurement by RVA]
Using each purified starch, the viscosity was measured with a viscometer (RVA Super3, Newport Scientific). A 10 w / w% purified starch solution was used as a sample. RVA is a device that measures the resistance of starch paste applied to blades in a measurement container by heating, cooling, or holding starch slurry at an arbitrary temperature condition. The measurement conditions are 35 ° C. (1 minute hold) → 95 ° C. (10 minute hold) → 50 ° C. (1 minute hold), temperature rising / falling speed 5 ° C./min, and rotation speed 160 rpm. The result is shown in FIG.

[Digestibility test (in vitro)]
The suspension of purified starch was adjusted to 0.2 w / w%, porcine pancreas-derived α-amylase 110U (manufactured by Sigma) was added, the enzyme reaction was carried out at 37 ° C., and the reaction solution supernatant over time The amount of reducing sugar contained in was determined. Quantification of reducing sugars was performed using the method of Park and Johnson (1949) et al. The result is shown in FIG. In addition, the purified starch solution of 10 w / w% was heated at 100 ° C. for 2 hours and then cooled at 4 ° C. for 12 hours to homogenize the gel, and the reaction solution was adjusted so that the final concentration was 0.2 w / w%. In addition, the amount of reducing sugar was determined over time in the same manner as in the case of purified starch. The result is shown in FIG.

[Digestibility test (in vivo)]
Purified starch and gel were administered once to Wistar male rats (8 months old, body weight 219.2-243.1 g), and blood glucose levels were measured over time. The purified starch was made into a suspension of 7 w / w%, and a volume corresponding to the amount of purified starch of 0.5 g per kg body weight was administered with an intragastric sonde. In the case of gels, a 10 w / w% suspension is heated at 100 ° C. and cooled to form a gel, homogenized and then a 7 w / w% suspension, and 0.5 g of purified starch per kg body weight. The corresponding volume was administered with an intragastric sonde. The blood glucose level was measured using a glucose pilot (manufactured by Aventir Biotech). The result is shown in FIG.

〔Test results〕
From the results of FIG. 1, the amylopectin in the starch of the present invention has an amylopectin side chain having a peak of the degree of glucose polymerization in the vicinity of 14, and the amylopectin side chain having a glucose polymerization degree of 12 or more is 69.1 of all amylopectin side chains. %, At least 65% or almost 70%. Moreover, in the starch of this invention, there are many amylopectin side chains whose glucose polymerization degree is 12 or more. As shown in FIGS. 3 (a) and 3 (b), the rice cereal as a practical product did not show bands for GBSSI protein and BEIIb protein according to Western blotting using anti-GBSSI antibody and anti-BEIIb antibody. Moreover, as a result of the analysis test of brown rice based on the nutrition labeling standard, the sugar was 58.9% with respect to the water content of 18.4%, and 72.2% or more of the dried brown rice was starch.

  Moreover, according to the digestion test (refer FIG. 7) of refined starch (raw starch) and the digestion test of starch gel (refer FIG. 8), although it is recognized that reducing sugar increases gradually, when there is no wild species or amylose It was found that the production of reducing sugar was delayed compared to the sticky rice (wx strain). As a result of the digestion test using mice (see FIG. 9), the rate of blood glucose rise is slow, the blood glucose level at the peak is lower than that of wild-type glutinous rice (WT) and mochi rice (wx strain), and the absorption rate is low. It was very relaxing. From these facts, the starch of the present invention was judged to be indigestible.

  Next, when the heat gelatinization characteristics of the starch of the present invention were examined, a gelatinization endothermic peak was detected at a significantly higher temperature than wild-type rice (WT) and waxy rice (wx strain) (see FIG. 4). . Moreover, the viscosity start temperature also became high by the viscosity measurement by RVA, and the temperature at the time of gelatinization was also higher than other rice. As a result, it is considered that processing at a high temperature becomes possible.

  Cookies were baked using brown rice flour and the texture was confirmed. Two types of samples shown in Table 1 were produced. Twelve people sampled the product according to the present invention and the comparative product, and compared the texture between the product and the comparative product. The results are shown in Table 2.

  Rolled bread was baked with polished rice flour to confirm its texture. Rolls were baked using flour obtained by mixing the refined rice flour of the present invention at a ratio of 10 w / w%, 20 w / w%, and 50 w / w% to the strong flour. Mix 10g of sugar and 1g of salt with 100g of flour. Next, add 30 g of milk, 36 g of water and 15 g of egg and mix well. Add 15g of butter and knead it as it comes together so that it doesn't get on your hands. Incubate at a temperature of about 35 ° C. for primary fermentation until the dough swells 2 to 2.5 times (bench time). Next, the gas is degassed, divided by a scraper, and subjected to secondary fermentation at a temperature of about 35 ° C. until the dough swells 2 to 2.5 times (bench time). Thereafter, the product was degassed, formed into a butter roll shape, the dough was expanded in an oven having an internal temperature of about 35 ° C., and baked at a temperature of about 190 ° C. to obtain a butter roll.

  Among the products of the present invention, 50% blended bread was crunchy and crumbly. The taste was also delicious. Also, the texture after leaving at room temperature for 2 to 3 was almost the same as that at the time of production and was delicious. On the other hand, 10% and 20% blended breads had the same softness and moist feeling as 100% flour breads. The taste was as good as 100% flour bread.

  According to the present invention, a new resistant starch having a high gelatinization temperature and a fast aging and a rice grain containing the starch are provided. By using this starch or rice grain, it is possible to obtain a processed rice product having a sticky texture and a crisp texture. In particular, since the rice grains and starch of the present invention are indigestible, various processed foods with a new texture suitable for so-called metabolic countermeasures are provided because the action of increasing blood sugar is moderate.

(A) is a figure which shows distribution of the glucose polymerization degree of an amylopectin side chain, (b) is a figure which shows the difference of the peak area in each glucose polymerization degree, and the peak area in a wild type. It is a photograph which shows the crystal-like structure of the starch grain in rice grain. (A) is a photograph taken with a polarizing microscope, (b) is a photograph taken with a polarizing microscope through which a filter (530 nm) is applied, and (c) is a photograph taken with an optical microscope of rice grains subjected to iodine staining. (A), (b), and (c) show the wild species, the wx strain, and the wx / ae strain from the top. It is a Western blot figure with respect to the protein in rice grains (milled rice). (A) shows the case where an anti-GBSSI antibody is used, and (b) shows the case where an anti-BEIIb antibody is used. In (a), S represents a soluble protein, and G represents a starch-binding protein. It is a chart which shows the heat gelatinization characteristic by DSC. (A) shows the thermal gelatinization characteristics of polished rice, (b) shows the thermal gelatinization characteristics of brown rice flour, and (c) shows the thermal gelatinization characteristics of purified starch. It is a chart which shows the aging characteristic by DSC. It is a chart which shows the viscosity change by RVA. It is a chart which shows the result of the digestibility test (in vitro) using refined starch. It is a chart which shows the result of the digestibility test (in vitro) using the gel of refined starch. It is a graph which shows the blood glucose change after ingestion of cooked rice in a rat.

Claims (8)

  A non-digestible rice grain containing amylopectin that does not contain amylose and has a distribution peak of the degree of glucose polymerization of the side chain of amylopectin located at 13-15.   The rice grain according to claim 1, wherein the amylopectin side chain having a glucose polymerization degree of 12 or more is 65% or more of the total amylopectin side chain.   The rice grain according to claim 1 or 2, which is wx / ae rice lacking amylose synthase I (GBSSI) and amylopectin branching enzyme IIb (BEIIb) from wild glutinous rice.   The processed rice product obtained from the rice grain of any one of Claims 1-3.   An indigestible starch comprising amylopectin, which does not contain amylose and has a distribution peak of the degree of glucose polymerization of the side chain of amylopectin located at 13-15.   The indigestible starch according to claim 5, wherein the amylopectin side chain having a glucose polymerization degree of 12 or more is 65% or more of the total amylopectin side chain.   7. The indigestible starch according to claim 5 or 6, obtained from wx / ae rice lacking amylose synthase I (GBSSI) and amylopectin branching enzyme IIb (BEIIb) from wild glutinous rice.   Processed food which uses the indigestible starch of any one of Claims 5-7 as a raw material.