Resistant protein-rich sweetener derived from koji raw material and method for producing same

Abstract

[Problem] The purpose of the present invention is to provide a novel form of rice-malt-derived foodstuff that makes it possible to enjoy the healthful effect of sweet sake on a daily basis and enables storage for prolonged periods of time, and to provide a novel manufacturing technique with which it is possible to produce said rice-malt-derived food while suppressing manufacturing costs by using existing equipment without requiring investment in new equipment. [Solution] Provided are: a high-resistant-protein-content sweetener derived from a rice malt raw material, the rice malt raw material comprising 1 part by weight of water and 1.5-2 parts by weight of rice malt, and the resistant protein content of the sweetener being 1000 mg / 100 mL or greater; and a method for producing the high-resistant-protein-content sweetener.

Description

Sweetener with high resistant protein content derived from koji ingredients and its manufacturing method

[0001] The present invention relates to a sweetener with a high content of resistant protein derived from koji raw materials and a method for producing the same.

[0002] Amazake is a traditional Japanese drink with a long history dating back to the Nara period (8th century). The process of making amazake is relatively simple: first, koji mold is added to steamed rice to create rice koji, and then the rice koji is mixed with water and left to saccharify at a certain temperature. During this process, the sugar and amino acids increase, creating amazake's distinctive sweetness.

[0003] In recent years, the demand for amazake has been growing rapidly due to an increase in health-conscious consumers and growing interest in traditional Japanese cuisine and fermented foods. Amazake contains enzymes that aid digestion, which has the effect of promoting the digestion and absorption of food. Amazake also contains lactic acid bacteria, which has the effect of improving the intestinal environment and improving immunity. Furthermore, amazake is rich in nutrients such as B vitamins and minerals, and is attracting attention for its functionality, such as fatigue recovery and skin-beautifying effects.

[0004] Japanese Patent Application Laid-Open No. 2020-000153 discloses amazake (sweet sake) containing prolamins that are contained in the steamed rice and rice koji used as raw materials and remain without being decomposed by enzymes, and in which the residual ratio, which represents the proportion of remaining prolamins relative to the prolamins in the raw materials, is 95% by mass or more (Patent Document 1).

[0005] Japanese Patent Application Laid-Open Publication No. 2011-55749 discloses a method for producing amazake, which comprises a first step of steaming white rice to produce steamed rice, mixing this steamed rice with pumped water, adding rice koji and allowing it to saccharify and ferment for a predetermined period of time, and a second step of adding more rice koji to the steamed rice that has been saccharified and fermented, and allowing it to saccharify and ferment for a predetermined period of time, with the second step being repeated once or twice or more times (Patent Document 2).

[0006] JP 2020-000153 A JP 2011-055749 A

[0007] In order to meet the recent demand for amazake, it is necessary to find further added value for amazake and establish a mass production system. The invention described in Patent Document 1 claims to increase the residual rate of prolamin, also known as resistant protein, and to effectively exert prolamin-based physiological effects such as cholesterol-lowering, intestinal regulation (improving bowel movements), and obesity suppression. However, the amount of prolamin obtained is only 158-173 mg per 100 ml of amazake, compared to 147 mg per 100 ml of amazake in the comparative example, a mere 7.5-17.7% increase. Furthermore, the invention described in Patent Document 2 uses a paddle (a rod-shaped tool used to stir and homogenize liquids or liquid-solid mixtures contained in containers such as tanks) in the amazake production process, which tends to separate water and rice koji, so it can only produce amounts that can be produced manually, making it difficult to adapt to mass production.

[0008] However, when sweeteners are produced using a saccharification tank equipped with a mixer designed for mechanization and automation, the mixture separates into a watery, low-viscosity lower layer and an upper layer of solidified unsaccharified rice koji, and the mixer cannot adequately mix the mixture. Reducing the water content to create a sweetener with a higher concentration of resistant protein results in a porridge-like mixture with a high viscosity, making uniform mixing even more difficult. Therefore, the manufacturing process needed to be revised in order to produce a sweetener with a high concentration of resistant protein.

[0009] Furthermore, to enjoy the health benefits of resistant protein, a certain amount of amazake must be consumed. However, typical rice koji amazake is said to contain approximately 18g of sugar per 100g, making it a high-sugar beverage. Furthermore, the unique texture of amazake, combined with its sweetness, is a hard sell for many people. Therefore, it is difficult to drink large amounts of amazake at one time, multiple times a day, or every day with rice koji amazake. Therefore, there was a need to develop a new form of rice koji amazake that would allow for daily consumption and allow the health benefits of amazake to be enjoyed every day.

[0010] Furthermore, while amazake is rich in nutrients, its shelf life is relatively short, generally about one week when refrigerated and about three months when frozen. However, health-conscious consumers tend to avoid the addition of preservatives, so it is necessary to find ways to extend the shelf life in a natural form without adding additives.

[0011] Therefore, the present invention aims to provide a new type of rice koji-derived food that allows the health benefits of amazake to be enjoyed on a daily basis and can be stored for a long period of time, as well as to provide a new manufacturing technology that enables the production of the rice koji-derived food using existing equipment while reducing manufacturing costs and eliminating the need for new capital investment.

[0012] In order to solve the above problems, the inventors conducted extensive research into new forms of rice koji amazake and methods for producing it. As a result, they discovered that by using a multi-stage saccharification process in which rice koji is added in stages rather than all at once, the saccharification decomposition of rice koji is promoted while suppressing excessive increases in viscosity, making it possible to mass-produce a sweetener that contains a high concentration of resistant protein, a functional protein.

[0013] The present invention was made based on this finding, and provides a sweetener with a high content of resistant protein derived from rice koji raw materials, wherein the rice koji raw materials consist of 1 part by weight of water and 1.5 to 2 parts by weight of rice koji, and the content of the resistant protein is 1000 mg / 100 ml or more.

[0014] The present invention also provides a method for producing a sweetener with a high content of resistant protein derived from a rice koji raw material, the method comprising: a first saccharification step in which the rice koji raw material consists of 1 part by weight of water and 1.5 to 2 parts by weight of rice koji; adding 1 part by weight of water and a portion of the rice koji to a tank and performing saccharification while stirring to obtain a partially saccharified liquid A in which the rice koji has been partially saccharified; a second saccharification step in which, after a predetermined time has elapsed since the addition of the rice koji, another portion of the rice koji is added and performing saccharification while stirring to obtain a partially saccharified liquid B in which the rice koji has been partially saccharified; a third saccharification step in which the second saccharification step is repeated; and a fourth saccharification step in which the addition of the rice koji is stopped, and saccharification is performed while stirring, terminating the saccharification when the saccharified liquid reaches a desired sugar content.

[0015] According to the sweetener with a high content of resistant protein derived from koji ingredients of the present invention, the resistant protein derived from rice koji ingredients is increased in concentration by approximately 13.5 times compared to amazake, allowing users to enjoy the health benefits of resistant protein on a daily basis without consuming large amounts like amazake. Furthermore, the sweetener with a high content of resistant protein derived from koji ingredients of the present invention has a lower moisture content than amazake, allowing it to be stored for a long period of time. When used as a food processing ingredient, it also has the advantage of being able to be baked more quickly due to its lower moisture content compared to amazake. In addition, it can enjoy effects such as suppressing blood sugar level increases, lowering blood sugar levels, and improving the intestinal flora.

[0016] Furthermore, according to the method of the present invention for producing a sweetener with a high content of resistant protein derived from rice koji as a raw material, it is possible to produce a sweetener with a high content of resistant protein derived from rice koji as a raw material, which enables the production of the rice koji-derived food, while keeping production costs down using existing equipment and without requiring new capital investment.

[0017] 1 is a diagram showing the results of comparing the resistant protein content of commercially available amazake and the sweetener of the present invention. 2 is a diagram showing the results of a fasting glucose tolerance test. 3 is a diagram showing the results of intestinal flora analysis. 4 is a diagram showing the proportions of (a) Bifidobacterium bacteria and (b) Lactobacillus bacteria relative to the entire intestinal flora. 5 is a diagram showing the results of examining the effects of rice koji-derived sauces on the expression of glucose metabolism-related hormones. 6 is a diagram showing the results of comparing the resistant protein content of commercially available amazake and the sweetener of the present invention. 7 is a diagram showing the results of a fasting glucose tolerance test. 8 is a diagram showing the results of an intestinal flora analysis. 9 is a diagram showing the proportions of (a) Bifidobacterium bacteria and (b) Lactobacillus bacteria relative to the entire intestinal flora. 10 is a diagram showing the results of examining the effects of rice koji-derived sauces on the expression of glucose metabolism-related hormones.

[0018] First, we will explain a sweetener with a high content of resistant protein derived from koji raw materials according to an embodiment of the present invention. In the sweetener with a high content of resistant protein derived from koji raw materials according to an embodiment of the present invention, the rice koji raw material comprises 1 part by weight of water and 1.5 to 2 parts by weight of rice koji, and the resistant protein content is 1000 mg / 100 ml or more.

[0019] Here, "resistant protein" refers to a food component that is difficult to digest and absorb in the human small intestine and exerts physiological effects that help maintain health via the digestive tract. Resistant protein, specifically rice-derived prolamin, is known to exhibit physiological functions similar to dietary fiber, and has been reported to improve bowel movements, reduce LDL cholesterol, suppress obesity, suppress postprandial blood glucose elevation, improve intestinal flora, and increase keratinocyte moisture (J. Brew. Soc. Japan. Vol. 116, No. 10, pp. 719-723 (2021)).

[0020] The rice koji can be one or more of yellow koji mold (Aspergillus oryzae, Aspergillus sojae), white koji mold (Aspergillus Kawachii), and black koji mold (Aspergillus uchuensis), but yellow koji mold, which has a high enzyme activity of starch-degrading enzymes (α-amylase, glucoamylase), is preferred. By using 1 part by weight of water and 1.5 to 2 parts by weight of rice koji as the rice koji raw material, a sweetener with a high resistant protein content and low water activity can be obtained.

[0021] It is said that 113 mg / day of resistant protein is required to achieve effects such as cholesterol reduction, improved bowel movements, and obesity suppression, while 247 mg / day is required to achieve effects such as improved intestinal flora and increased keratinocyte moisture (Ozeki, Jyokyo, 2022, Vol. 117, No. 9, pp. 627-634). The koji-derived sweetener with a high resistant protein content according to this embodiment can achieve a resistant protein content of 1000 mg / 100 ml or more. For example, 10 ml of the koji-derived sweetener with a high resistant protein content according to this embodiment (134 mg of resistant protein) can be expected to have effects such as cholesterol reduction and obesity suppression, and 20 ml (268 mg of resistant protein) can be expected to have effects such as improved intestinal flora and increased keratinocyte moisture.

[0022] The content of resistant protein can be adjusted as appropriate depending on the blending amounts of rice koji and water, which are the rice koji raw materials. However, to achieve a resistant protein content of 1000 mg / 100 ml or more, the blending amount should at least be rice koji > water.

[0023] The sweetener with a high content of resistant protein derived from koji ingredients according to this embodiment has a lower moisture content than rice koji, making it more shelf-stable than amazake. Furthermore, because of its lower moisture content, it also has the secondary effect of being easier to transport.

[0024] The sweetener with a high resistant protein content derived from koji raw materials according to this embodiment contains a large amount of sugar, and therefore can be used as an ingredient in various foods and beverages as a sweetener to replace conventional sweeteners.

[0025] Food and beverage products refer to foodstuffs or beverages. Specific examples of food and beverage products include, but are not limited to, cereals such as cornflakes, oatmeal, granola, and muesli; confectioneries such as chewing gum, candy, cookies, and chocolate; jams such as fruit jam, marmalade, and vegetable jam; ice creams such as shaved ice, popsicles, and sorbet; ice creams such as ice milk and lacto ice cream; lactic acid bacteria drinks; soft drinks that do not contain alcohol (less than 1% alcohol) but do not contain milk or dairy products; and pickles. Resistant protein can also be made into tablet, capsule, granule, liquid, or powder supplements. Of course, resistant protein can be efficiently ingested simply by sprinkling it on or mixing it with food. Furthermore, it can also be used as an ingredient in pet food and animal feed.

[0026] The sweetener with a high content of resistant protein derived from koji raw materials according to this embodiment can also be used as a raw material for seasonings.

[0027] Examples of seasonings include soups, sauces, stocks, extracts, sauces (pasta sauce, pizza sauce, Worcestershire sauce, ketchup sauce, oyster sauce, salsa sauce, sambal sauce, chili sauce, demi-glace sauce, white sauce, etc.), dressings, vinegar, seasoned vinegar, soy sauce, ponzu seasoning, seasonings for pickles, seasonings for meat, seasonings containing spices, chutney, mustard, mayonnaise, etc.

[0028] Next, a method for producing a sweetener with a high content of resistant protein derived from rice koji raw material according to an embodiment of the present invention will be described. The method for producing a sweetener with a high content of resistant protein derived from rice koji raw material according to an embodiment of the present invention includes the following steps: a first saccharification step in which 1 part by weight of water and a portion of the rice koji are added to a tank and saccharified with stirring to obtain a partially saccharified liquid A in which the rice koji is partially saccharified; a second saccharification step in which, after a predetermined time has elapsed since the addition of the rice koji, another portion of the rice koji is added and saccharified with stirring to obtain a partially saccharified liquid B in which the rice koji is partially saccharified; a third saccharification step in which the second saccharification step is repeated; and a fourth saccharification step in which the addition of the rice koji is stopped, saccharification is performed with stirring, and saccharification is terminated when the saccharified liquid reaches a desired sugar content.

[0029] The first saccharification step is a step in which 1 part by weight of water and a portion of the rice koji are placed in a tank and saccharified while stirring, to obtain a partially saccharified liquid A in which a portion of the rice koji is saccharified.

[0030] There are no particular limitations on the water used, and groundwater or tap water can be used, but it is preferable to use the brewing water used in sake production.

[0031] The tank is preferably a stainless steel tank, and is equipped with a stirring device for stirring the solution and a temperature adjusting device for heating and maintaining the temperature.

[0032] The amount of rice koji added is preferably 35 to 40% by weight of the total weight of the rice koji. By adding the rice koji in stages, rather than all at once, separation into a lower layer with high moisture content and low viscosity and an upper layer of solidified unsaccharified rice koji can be prevented, and uniform stirring can be achieved even with a stirring device for amazake production. However, this embodiment does not exclude the use of manual stirring (stirring with a paddle), so stirring with a paddle may be used as an auxiliary method.

[0033] After adding water and rice koji, the temperature is adjusted to a range of 50 to 70°C to promote saccharification and prevent bacterial contamination.

[0034] Through the above steps, the rice koji in the solution in the tank is partially saccharified, resulting in a partially saccharified solution A.

[0035] The second saccharification step is a step in which, after a predetermined time has elapsed since the rice koji was added, a portion of the rice koji is further added and saccharification is carried out while stirring, to obtain a partially saccharified liquid B in which a portion of the rice koji has been saccharified.

[0036] As saccharification progresses, the koji decomposes and the viscosity of the solution gradually decreases. Therefore, after a predetermined time, for example, 10 to 240 minutes, has passed since the previous rice koji was added, 10 to 15% by weight of the remaining rice koji is added to the tank as additional koji. After the additional koji is added, the temperature is maintained at around 65°C while stirring to promote saccharification.

[0037] Through the above process, the rice koji in the solution in the tank is partially saccharified, resulting in a partially saccharified solution B.

[0038] The third saccharification step is a step in which the second saccharification step is repeated. That is, after a predetermined time, for example, 10 to 240 minutes, has elapsed since the previous rice koji was added, 10 to 15% by weight of the remaining rice koji is added to the tank as additional koji.

[0039] In the fourth saccharification step, the addition of the rice koji is stopped, and saccharification is carried out while stirring, ending when the saccharified liquid reaches the desired sugar content. After all the rice koji has been added, saccharification continues with stirring while maintaining the temperature. The desired sugar content is reached after 5 to 25 hours, so saccharification is ended when the desired sugar content is reached.

[0040] The sugar content of typical amazake is 15 to 21 degrees, but according to the production method of this embodiment, it is possible to produce a sweetener with a high content of resistant protein derived from rice koji ingredients and a sugar content (Brix) of 25 to 60 degrees.

[0041] The method for producing a sweetener with a high content of resistant protein derived from rice koji raw material of this embodiment preferably further includes a grinding step of grinding residual grains in the saccharified solution.

[0042] The pulverization method is not particularly limited, and examples thereof include methods using a mill (mycoloider, masscolloider), ball mill, coffee mill, power mill, pin mill, airflow pulverizer (jet mill), shear friction pulverizer, cutter pulverizer, impact pulverizer (hammer mill, ball mill), roll pulverizer, and homogenizer.

[0043] By undergoing the grinding process, it is possible to prepare a sweetener with a smooth mouthfeel and reduced graininess.

[0044] The method for producing a sweetener with a high resistant protein content derived from rice koji raw material of this embodiment preferably further includes a filtration step of filtering the saccharified solution.

[0045] The filtration method is not particularly limited, but examples thereof include suction filtration using a Buchner filter (Nuchner) equipped with filter paper or filter cloth, and filter press.

[0046] By going through the filtration process, it is possible to prepare a sweetener with a smooth mouthfeel and reduced graininess.

[0047] The resulting sweetener with a high content of resistant protein derived from rice koji ingredients is finally heat sterilized, filled into containers, and cooled to become the final product.

[0048] In addition, for matters common to the invention relating to the sweetener with a high content of resistant protein derived from rice koji ingredients described above, the matters explained for the sweetener with a high content of resistant protein derived from rice koji ingredients apply as appropriate.

[0049] 1. Production of a sweetener with a high resistant protein content derived from rice koji ingredients. A sweetener with a high resistant protein content derived from rice koji ingredients was produced as follows. To produce approximately 218 kg of sweetener with a Brix of 60, 86 kg of water (groundwater) and 150 kg of rice koji were prepared as rice koji ingredients. The saccharification process of the entire production process is shown in Table 1.

[0050]

[0051] (First saccharification step) First, 86 kg of water was charged into a tank (Shinyo Giken Co., Ltd., 240 L saccharification tank, stirrer: Robocup stick blender MP-550U), then 60 kg of rice koji was charged, and the temperature inside the tank was heated to 65°C. Saccharification was then carried out while stirring the solution with the stirrer attached to the tank. Stirring was continued until the end of the saccharification step.

[0052] (Second saccharification step) 15 minutes after the previous addition of rice koji, 15 kg of additional koji was added. The sugar content (Brix) at the time of the additional addition was 22 degrees.

[0053] (Third to fifth saccharification steps) After 15 minutes had passed since the previous rice koji addition, the step of adding 15 kg of additional koji was repeated. The sugar content (Brix) at the time of adding the additional koji is as shown in Table 1.

[0054] (Sixth to Seventh Saccharification Steps) After 60 minutes had passed since the previous rice koji addition, a step of adding 15 kg of additional koji was carried out. The sugar content (Brix) at the time of the additional koji addition is shown in Table 1.

[0055] (Eighth Saccharification Step) After 120 minutes had passed since the previous rice koji addition, a step of adding 15 kg of additional koji was carried out. The sugar content (Brix) at the time of the additional koji addition is shown in Table 1.

[0056] (Ninth Saccharification Step) After the addition of the rice koji was completed, stirring was continued to carry out saccharification. Then, 7 hours after the final addition of the rice koji in the eighth step, the target sugar content was reached, and the saccharification step was terminated.

[0057] (Crushing and filtration processes) After saccharification, the saccharified liquid was completely crushed using a stirrer attached to the saccharification tank until all the rice koji grains were gone.Then, it was filtered through a filter (1.5 mm openings) to obtain a sweetener with a high content of resistant protein derived from rice koji ingredients.

[0058] (Heat Treatment Step) The filtered sweetener with a high content of resistant protein derived from rice koji raw material was heat treated at 85°C for 30 minutes, filled into a container and cooled to obtain a final product.

[0059] 2. Analysis of resistant protein content Using a sweetener with a high resistant protein content (Brix sugar content 50 degrees) derived from rice koji, produced in the same manner as in 1 above, as a sample, the amount of resistant protein was quantified as follows. As a comparative example, a commercially available amazake was also measured in the same manner.

[0060] The sample was separated into a supernatant and a residue by centrifugation (4°C, 8000 rpm, 15 minutes). Next, 25 ml of urea phosphate buffer was added to 1 g of residue, and the mixture was homogenized using a homogenizer. The homogenized sample was heated at 60°C for 2 hours to extract proteins containing resistant proteins. The supernatant was then recovered by centrifugation (15000 rpm, 10 minutes). The resulting supernatant was subjected to SDS-PAGE (polyacrylamide gel electrophoresis), and after electrophoresis, the proteins were stained and the gel was photographed. The photographed gel was analyzed using ImageJ (image analysis software), and the intensity of the stained bands was compared with that of sample bands of known concentration to quantify the amount of resistant protein.

[0061] The results are shown in Figure 1. While the amount of resistant protein contained in commercially available amazake was 99.1 mg / 100 ml, the amount of resistant protein contained in the sweetener with a high resistant protein content derived from rice koji ingredients of the present invention was 1340.0 mg / 100 ml, which was found to contain approximately 13.5 times as much resistant protein as amazake.

[0062] 3. Production of Food Using the Rice Koji-derived Sweetener with a High Resistant Protein Content as an Ingredient Granola was produced in the following manner using the rice koji-derived sweetener with a high resistant protein content obtained in 1 above as an ingredient.

[0063] The granola was made by mixing 350 ml of the rice koji-derived sweetener with a high content of resistant protein and 150 ml of rice oil, emulsifying the mixture, mixing it with oats, and baking it in an oven at 138°C for 26 minutes.

[0064] When the amount of resistant protein was measured as described in 2 above, the granola contained 8.0 mg of resistant protein per gram.

[0065] 4. Evaluation of the effects of administering rice koji-derived sauce at a constant sugar content (1) Experimental animals: Male ddY mice (Japan SLC Co., Ltd.), 6 weeks old and weighing 26-29 g at the start of the experiment, were used in the experiment after a 9-day preliminary breeding period in a room set at 23±1°C with a light cycle of 6:00-18:00. During the experiment, the mice were given free access to solid MF feed (Oriental Yeast Co., Ltd.) and drinking water (tap water). All animal experiments were conducted after review and approval by the Utsunomiya University Animal Care and Use Committee (A24-0003).

[0066] (2) Experimental Materials A sweetener with a high resistant protein content (Brix sugar content: 50 degrees) derived from rice koji, prepared in the same manner as in 1 above, was used as the sample (hereinafter referred to as "rice koji-derived sauce" or "sauce"). This sample was diluted with purified water to 0.3 g / ml and used for oral administration. Sucrose (Wako, 196-00015) was dissolved in purified water to 0.12 g / ml and used for oral administration.

[0067] (3) Experimental Overview After the preliminary breeding period, a nutritional test period was conducted in which mice were orally administered sucrose once daily for 51 days. During the nutritional test period, mice were divided into two groups (n = 10): one group received 133.3 μl / 10 g mouse body weight of sucrose solution (cont) (sugar group), and the other group received 134.4 μl / 10 g mouse body weight of diluted rice koji-derived sauce (sauce group). Sucrose and rice koji-derived sauce were administered in amounts equivalent to 320 ml of a liquid with a sugar content of 2.5 (equivalent to two cups of tea with one sugar cube), calculated as a human equivalent dose.

[0068] (4) Evaluation Items (4-1) Fasting Glucose Tolerance Test The fasting glucose tolerance test was conducted once during the preliminary breeding period and once during the nutritional test period. During the preliminary breeding period, food was removed from the cage after oral administration on the day before the fasting glucose tolerance test, and during the nutritional test period, the mice were fasted overnight. The following day, the mice were weighed and then orally administered 100 μl / 10 g of glucose solution. At 0 (before administration of the glucose solution), 15, 30, 60, and 90 minutes after oral administration, the mice's tails were injured with a scalpel, and blood glucose levels were measured from the tail vein using a glucose meter (Nipro Corporation, Nipro FS Blood Glucose Sensor Light, 30854000). The glucose solution was prepared by dissolving 20 g of D(+)-glucose (Kanto Chemical Co., Ltd., 10017-00) in 100 ml of purified water. Furthermore, on the day of the fasting glucose tolerance test during the nutritional test period, purified water and rice koji-derived sauce were not orally administered.

[0069] (4-2) Gut Microbiota Analysis Mouse feces from the day before and the day of dissection were collected in a 15 ml tube, and sterile water was added to a concentration of 60 mg / 500 μl. After vortexing, the tube was left on ice for 30 minutes. The tube was then vortexed again to prepare a fecal suspension. DNA extraction from the feces was performed using a DNA extraction kit (TaKaRa, NucleSpin DNA Stool, U0472B). Approximately 200 mg of fecal suspension was placed in a tube containing beads, and 500 μl of ST1 was added. The tube was then incubated in a water bath set at 70°C for 5 minutes and vortexed for 10 minutes. After centrifugation (13,000 × g, 3 min, room temperature), 600 μl of the resulting supernatant was transferred to a 2 ml tube. 100 μl of ST2 was added, vortexed for 5 seconds, and then left on ice for 5 minutes. After centrifugation (13,000 × g, 3 min, room temperature), 550 μl of the resulting supernatant was transferred to a red ring column and centrifuged (13,000 × g, 1 min, room temperature). 200 μl of ST3 was added and vortexed for 5 seconds, after which 700 μl was transferred to a green ring column and centrifuged (13,000 × g, 1 min, room temperature). The liquid in the tube was discarded, and 600 μl of ST3 was added to the green ring column and centrifuged (13,000 × g, 1 min, room temperature). The liquid in the tube was discarded, and 550 μl of ST4 was added to the green ring column and centrifuged (13,000 × g, 1 min, room temperature). The liquid in the tube was discarded, and 700 μl of ST5 was added to the green ring column, vortexed for 2 seconds, and centrifuged (13,000 × g, 1 min, room temperature). The liquid in the tube was discarded, and 700 μl of ST5 was added to the green ring column, followed by centrifugation (13,000 × g, 1 min, room temperature). The liquid in the tube was discarded, and the column was centrifuged (13,000 × g, 1 min, room temperature). The green ring column was placed in a 1.5 ml tube, 50 ml of SE was added, and the column was centrifuged (13,000 × g, 1 min, shaking), followed by vortexing for 2 seconds. Finally, the liquid was drained using a small centrifuge and placed on ice. The extracted DNA was subjected to a quality check and then stored at -80°C. Sequencing of the extracted DNA was outsourced to Macrogen Japan Co., Ltd. Intestinal microbiota analysis was performed using the analysis software "mother" based on the sequenced DNA data.

[0070] (4-3) Plasma Insulin Measurement An ELISA kit (Wako, LBI™ Mouse Insulin ELISA Kit (High Sensitivity), 296-89801) was used to measure insulin concentrations. A 2500 pg / ml standard solution was prepared by diluting the standard solution (5000 pg / ml) two-fold with buffer. Similarly, serial dilutions were performed to prepare standard solutions of 1250, 625, 313, 156, 78, and 39 pg / ml. The biotin-conjugated antibody solution was diluted 4000-fold with buffer, the peroxidase-conjugated streptavidin solution was diluted 500-fold with buffer, and the washing solution was diluted 10-fold with purified water. The original liquid in the plate was discarded, 300 μl of washing solution was added, and the plate was shaken by hand for approximately 10 seconds. The washing solution was then discarded and the plate was gently tapped against a paper towel for four wash cycles to remove the washing solution. 45 μl of biotin-conjugated antibody solution was added and shaken three times (1000 rpm, 10 s, room temperature). 5 μl of standard solution and sample were added to each well, gently mixed, and then the plate was sealed and left at room temperature for 2 hours. After washing four times, 50 μl of peroxidase-conjugated streptavidin solution was added, and the plate was shaken three times (1000 rpm, 10 s, room temperature). After a seal was applied, the plate was left at room temperature for 30 minutes. After washing four times, 50 μl of TMB solution was added, and the plate was shaken three times (1000 rpm, 10 s, room temperature). After a seal was applied, the plate was left at room temperature for 30 minutes. 50 ml of stop solution was added, and the plate was shaken three times (1000 rpm, 10 s, room temperature). The absorbance at 450 nm was immediately measured using a spectrophotometer.

[0071] (4-4) Measurement of GIP Concentration in Plasma An ELISA kit (Cosmo Bio Co., Ltd., Mouse GIP (Total) ELISA, YK225) was used to measure GIP concentrations. The standard was dissolved in buffer to prepare an 800 pM standard solution. The 800 pM standard solution was diluted two-fold with buffer to prepare a 400 pM standard solution. Similarly, serial dilutions were performed to prepare 200, 100, 50, 25, 12.5, and 6.3 pM standard solutions. The wash solution was diluted 20-fold with purified water. 350 μl of wash solution was added, and the plate was shaken by hand for approximately 10 seconds. The wash solution was then discarded and gently tapped against a paper towel for three wash cycles. 25 μl of buffer solution, 25 μl of standard solution, and 25 μl of plasma were added to each well, followed by shaking (200 rpm, 2 h, room temperature) with a plate seal. After washing four times, 100 μl of labeled specific antibody was added and the mixture was shaken (200 rpm, 1 hour, room temperature). After washing four times, 100 μl of enzyme substrate solution was added and the mixture was left standing at room temperature in the dark for 30 minutes. 100 μl of reaction stop solution was added, and the absorbance at 450 nm was measured immediately using a spectrophotometer.

[0072] (4-5) Measurement of Plasma GLP-1 Concentration An ELISA kit (Cosmo Bio Co., Ltd., Total GLP-1-HS ELISA, YK161) was used to measure GLP-1 concentrations. A 300 pM standard solution was prepared by dissolving the standard in buffer. A 100 pM standard solution was prepared by diluting the 300 pM standard solution 3-fold with buffer. Similarly, serial dilutions were performed to prepare standard solutions of 33.3, 11.1, 37.0, and 1.24 pM. Plasma was diluted 5-fold with buffer, and the washing solution was diluted 20-fold. 350 μl of washing solution was added, the plate was shaken by hand for approximately 10 seconds, the washing solution was discarded, and the plate was gently tapped against a paper towel to remove the washing solution. This washing procedure was repeated three times. 200 μl of buffer was added to each well, and 30 ml of standard solution and diluted plasma were added. The plate was then sealed and shaken (200 rpm, 18 h, room temperature). After washing three times, 100 μl of labeled specific antibody was added, a plate seal was attached, and the plate was shaken (200 rpm, 1 hour, room temperature). After washing three times, 100 μl of SA-HRP solution was added, a plate seal was attached, and the plate was shaken (200 rpm, 30 minutes, room temperature). After washing four times, 100 μl of enzyme substrate solution was added, and the plate was left to stand for 30 minutes at room temperature in the dark. After adding 100 μl of reaction stop solution, the absorbance at 450 nm was measured immediately using a spectrophotometer.

[0073] (5) Statistical processing All data were expressed as mean ± standard error, and significance tests were performed using t-tests. After each significant difference test, p<0.05 was considered to indicate a significant difference, and p<0.1 was considered to indicate a tendency.

[0074] (6) Results Figure 2 shows the results of the fasting glucose tolerance test. Figure 2(a) shows the change in blood glucose levels during the preliminary breeding period, and (b) shows the change in blood glucose levels during the nutritional test period. (a) The change in blood glucose levels during the preliminary breeding period confirmed that there was no difference in basic glucose metabolic function between the control group (labeled "cont" in the figure) that received sucrose solution and the sauce group (labeled "sauce" in the figure) that received rice koji sauce. Therefore, the period of nutritional testing (the period of administration of sucrose and rice koji sauce) was started.

[0075] (b) Changes in blood glucose levels during the nutritional test showed a tendency for the rise in blood glucose levels to be suppressed in the sauce group (sauce) given rice koji sauce compared to the control group (cont). This suggests that rice koji sauce may have the ability to suppress rises in blood glucose levels and promote blood glucose reduction.

[0076] Figure 3 shows the results of the intestinal flora analysis. Figure 4 shows the proportions of (a) Bifidobacterium bacteria and (b) Lactobacillus bacteria relative to the total intestinal flora. As shown in Figures 3 and 4, the rice koji sauce-fed sauce group (Sauce) showed an increase in lactic acid-producing bacteria, such as Lactobacillus and Bifidobacterium bacteria, compared with the control group (Cont). Therefore, it is possible that the increase in lactic acid-producing bacteria in the intestinal tract is related to the blood sugar suppression function of rice koji-derived sauce.

[0077] Figure 5 shows the results of examining the effect of rice koji-derived sauce on the expression of glucose metabolism-related hormones. As shown in Figure 5, no significant change was observed in the secretion of incretins following administration of rice koji-derived sauce, suggesting that the blood glucose regulation function of rice koji-derived sauce is not mediated by the pancreas. Therefore, it is possible that the consumption of rice koji-derived sauce and the increase in lactic acid-producing bacteria in the intestinal tract somehow regulate insulin sensitivity in skeletal muscle, etc.

Claims

1. A sweetener with a high content of resistant protein derived from rice koji raw materials, wherein the rice koji raw materials consist of 1 part by weight of water and 1.5 to 2 parts by weight of rice koji, and the content of the resistant protein is 1000 mg / 100 ml or more.

2. A food or drink made from the sweetener with a high resistant protein content according to claim 1.

3. A seasoning made from the sweetener with a high resistant protein content according to claim 1.

4. A method for producing a sweetener with a high content of resistant protein derived from rice koji raw materials, wherein the rice koji raw material consists of 1 part by weight of water and 1.5 to 2 parts by weight of rice koji, comprising: a first saccharification step in which 1 part by weight of water and a portion of the rice koji are added to a tank and saccharified with stirring to produce a partially saccharified liquid A in which a portion of the rice koji has been saccharified; a second saccharification step in which, after a predetermined time has passed since the addition of the rice koji, a further portion of the rice koji is added and saccharified with stirring to produce a partially saccharified liquid B in which a portion of the rice koji has been saccharified; a third saccharification step in which the second saccharification step is repeated; and a fourth saccharification step in which the addition of the rice koji is stopped, and saccharification is carried out with stirring, terminating the saccharification when the saccharified liquid reaches a desired sugar content.

5. The method for producing a sweetener with a high resistant protein content according to claim 4, further comprising a grinding step of grinding residual grains in the saccharified solution.

6. The method for producing a sweetener with a high resistant protein content according to claim 4, further comprising a filtration step of filtering the saccharified solution.

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