Method for producing crude or purified. beta. - glucan from barley bran

Abstract

The present invention provides an improvement in the production of crude or purified β - glucan from barley bran.SOLUTION: To provide a method for producing a crude product of β - glucan or its purified product. The objective component is extracted from the bran of barley by hot water extraction, a liquid phase containing the objective component is separated from the residue of the bran, enzyme treatment is carried out to decompose at least one of starch, protein and other impurities contained in the liquid phase, and the objective component is separated from a decomposition product by the enzyme. In another embodiment, the crude or purified product is combined with other ingredients to produce a food product containing or enriched in β - glucan.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing crude or purified β-glucan products using barley bran as a raw material, and more particularly to a method that utilizes extraction and purification from barley bran. [Background technology]

[0002] Patent Document 1 discloses a method for producing water-soluble dietary fiber whose main component is barley bran-derived β-glucan. This method is characterized by hot water extraction of barley bran generated during the barley milling process. In Example 1, 90°C hot water and thermostable α-amylase are added to barley bran, and extraction is carried out at 90°C for 3 hours. After further cooling to 50°C, glucoamylase is added and the mixture is treated at 50°C for 12 hours. This is then centrifuged for solid-liquid separation, clarified by filtration, and concentrated. After removing insoluble matter by centrifugation, the mixture is desalted using an ion exchange resin, ethanol is added, and the resulting precipitate is collected by centrifugation. This is then dried to obtain a dry powder.

[0003] Patent Document 2 discloses an extract of Kumai bamboo grass extracted as a water-soluble component under multiple high-temperature and high-pressure steam treatment, which contains 1,3-β-glucan. Patent Document 3 discloses the extraction of an antioxidant composition from rice bran using subcritical water. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 11-225706 [Patent Document 2] Special Publication No. 2011-505832 [Patent Document 3] Japanese Patent Application Laid-Open No. 2006-160825 [Non-patent literature]

[0005] [Non-Patent Document 1] Naoyuki Ishikawa, Masaru Ohtsuka, Masaharu Kodama and Noriko Kashima, Improvement of the Congo Red Method, a Rapid β-gIucan Quantification Method for the Breeding of High Quality Malting Barley, Bull Tochigi Agr.Exp.stn No.47:57-64(1998) Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention provides an improved method for producing crude or purified β-glucan from barley bran. [Means for solving the problem]

[0007] <1> A method for producing a crude or purified β-glucan product, comprising: The target components are extracted from barley bran using hot water extraction, Separating the liquid phase containing the target component from the bran residue; performing an enzyme treatment to decompose at least one of starch, protein, and other impurities contained in the liquid phase; Separating the target component from enzymatic degradation products; method.

[0008] <2> Maintaining the liquid phase at a temperature above 60°C from the time the target component is extracted until the time the liquid phase is separated from the residue; <1> The method described below.

[0009] <3> The extract containing the target component is filtered to separate the liquid phase from the residue. <2> The method described below.

[0010] <4> filtering the extract containing the target component to separate the liquid phase from the residue; The enzyme treatment is carried out by adding an enzyme directly to the filtered liquid phase; The liquid phase is maintained at a temperature above 60°C during the extraction of the target component, separation of the liquid phase from the residue, and activation of the enzyme. <1> The method described below.

[0011] <5> performing an enzymatic treatment to decompose at least the starch contained in the liquid phase; The enzyme treatment is carried out in at least two stages, First, treating the liquid phase with a thermostable α-amylase; second, cooling the liquid phase and then treating the liquid phase with glucoamylase; <4> The method described below.

[0012] <6> performing an enzymatic treatment to decompose at least one of starch and protein contained in the liquid phase; <1> The method described below.

[0013] <7> The hot water extraction is carried out at a temperature of 70, 80, or 90°C or higher, but lower than 100°C. <1> The method described below.

[0014] <8> The hot water extraction is carried out at a temperature of 100°C, 110°C, or 120°C or higher under a pressure higher than standard atmospheric pressure. <1> The method described below.

[0015] <9> The purity of the crude or purified β-glucan produced is 20% or more and 100% or less. <1> The method described below.

[0016] <10> adding a polar solvent to the enzyme-treated solution to precipitate the target component and separate it from the degradation products; The method further comprises washing the precipitate containing the target component with water at 80°C or less and recovering the insoluble portion, The purity of the crude or purified β-glucan produced is 40% or more and 100% or less. <1> The method described below.

[0017] <11> adding a polar solvent to the enzyme-treated solution to precipitate the target component and separate it from the degradation products; The precipitate containing the target component is mixed with water at a temperature of 4°C or higher and 60°C or lower, The mixture is treated for a predetermined time by at least one of standing and continuous stirring, further comprising separating the insoluble portion from the water; The crude product or its refined product of the produced β-glucan is 40% or more and 100% or less. <1> The method described below.

[0018] <12> <1> from <11> A crude β-glucan product or a purified β-glucan product is produced by any one of the methods described above. The crude product or its refined product is combined with other ingredients to produce a food product containing or having an increased amount of β-glucan. method. [Effects of the Invention]

[0019] The present invention provides an improved method for producing crude or purified β-glucan from barley bran. [Brief explanation of the drawings]

[0020] [Figure 1] Extraction and purification flow chart [Figure 2] Enzyme treatment flow chart [Figure 3] Flow chart of hot water extraction and enzyme treatment according to the embodiment [Figure 4] Flow chart of hot water extraction and enzyme treatment according to the embodiment DETAILED DESCRIPTION OF THE INVENTION

[0021] <Beta-glucan production flow>

[0022] Figure 1 shows the process for extracting and purifying β-glucan from barley bran to produce crude or purified β-glucan. Hereinafter, crude and purified β-glucan products may be collectively referred to as β-glucan.

[0023] The raw material 10 is barley bran. The raw material 10 is mixed with water. Mixing may be performed by stirring the raw material added to a large amount of water. Then, hot water extraction 11 is performed by maintaining an extraction medium containing the raw material 10, such as water, at a predetermined temperature and pressure for a predetermined time. By hot water extraction 11, at least one of the target components, β-glucan, is extracted from the interior of the barley bran toward the extraction medium. This results in an extract containing the target component, β-glucan.

[0024] As shown in Figure 1, a liquid phase 15 containing the target component β-glucan, i.e., the extract, is separated from a solid phase 16 containing the bran residue by solid-liquid separation 13. The bran residue may then be subjected to one or more additional hot water extractions and solid-liquid separations to obtain further liquid phase 15. Solid-liquid separation 13 may be performed by filtration or centrifugation. The target component in liquid phase 15 may be precipitated using a polar solvent, and the precipitate may then be regenerated by dissolving or suspending the precipitate in water or another medium.

[0025] As shown in Figure 1, in enzyme treatment 21, enzymes are introduced into liquid phase 15 and reacted. In hot water extraction 11 prior to enzyme treatment 21, the target component, β-glucan, is extracted from the bran toward the water. Therefore, liquid phase 15 contains substances other than β-glucan, such as polymers other than β-glucan, as impurities. Examples of such impurities include starch, proteins, and other impurities. One example of an enzyme is an enzyme that breaks down starch to produce sugars with smaller molecular weights. Another example is an enzyme that breaks down proteins to produce peptides and amino acids with smaller molecular weights. Another example of an enzyme is an enzyme that breaks down other impurities and breaks them down into smaller molecules. Because the bran residue in liquid phase 15 is reduced by solid-liquid separation 13, starch, proteins, and other impurities can be efficiently broken down.

[0026] 1, a liquid phase 25 containing the target component β-glucan, i.e., the enzyme-treated solution or treated solution, is separated from a solid phase 26 containing impurities not decomposed by the enzyme by solid-liquid separation 23. Solid-liquid separation 23 may be omitted.

[0027] As shown in Figure 1, precipitation 31 precipitates the target component, β-glucan, in liquid phase 25. A solid phase 36 containing the precipitated β-glucan is separated from liquid phase 35 by solid-liquid separation 33. Liquid phase 35 contains degradation products from enzyme treatment 21. The separated solid phase 36 is a form of a crude product. Solid phase 36 may also be used as final product 45.

[0028] As shown in FIG. 1 , the solid phase 36 may be subjected to washing 41 or other purification steps (not shown) to obtain a purified product. The purified product may be the final product 45. The purified final product 45 contains β-glucan of higher purity. The β-glucan purity in the solid phase 36 or the final product 45 may be 20% or greater, or may be 100% or less. The β-glucan purity may be 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In one embodiment, the β-glucan purity is the mass (weight) fraction of β-glucan in the dried solid phase 36 or the final product 45. The amount of β-glucan in the solid phase 36 or the final product 45 may be determined in accordance with AOAC 995.16, as defined by the Association of Official Analytical Chemists, or AACC 32-23.01, as defined by the American Association of Cereal Chemists.

[0029] <Raw materials>

[0030] As shown in Figure 1, raw material 10 is barley bran. Barley bran may contain the aleurone layer and husk. The bran may be considered to be the portion removed from the barley caryopsis when the barley caryopsis is milled to 10, 20, 30, or 40% by weight, starting from 0% by weight, relative to 100% by weight of the barley caryopsis. Raw material 10 may further contain barley germ and endosperm. Raw material 10 may also be whole wheat flour.

[0031] The variety of barley from which the bran is provided is not limited. Barley is classified into hulled barley and naked barley. In hulled barley, the outer husk, also called the husk, is tightly attached to the grain, making it difficult to peel off from the grain. In naked barley, the husk is easily separated from the grain. When using either type of barley, the husk may be removed. Barley is also classified into two-row varieties with large grains and six-row varieties with small grains. Barley is also classified into non-glutinous and waxy varieties. One of the varieties suitable for this embodiment is Viewfiber. Viewfiber is naked barley, a two-row variety, and non-glutinous. Viewfiber contains a higher amount of beta-glucan than conventional varieties.

[0032] The β-glucan contained in the above raw materials is mainly β-1,3-1,4-glucan. The chemical structure of barley β-glucan is different from the chemical structure of β-1,3-1,6-glucan contained in mushrooms and yeast. Barley β-glucan has β-1,4-bonds, but also has β-1,3-bonds, which makes it different from cellulose. Barley β-glucan has β-1,3-bonds, but also has β-1,4-bonds, which makes it different from curdlan.

[0033] <Quality and uses of β-glucan>

[0034] The purity of β-glucan in the crude product or its purified product may be 20% or more, or 100% or less. The purity of β-glucan may be any of 30%, 40%, 50%, 60%, 70%, 80%, and 90%. In one aspect, the purity of β-glucan is the mass (weight) fraction of β-glucan in the crude product or its purified product. The amount of β-glucan in the crude product or its purified product may be determined according to the standard method described above.

[0035] Compositions containing β-glucan are useful for suppressing blood sugar elevation, lowering cholesterol levels, and promoting bowel movements. Foods containing β-glucan may be provided by combining the crude or purified β-glucan produced by the above method with other ingredients that do not contain β-glucan. Foods with an increased β-glucan content may be provided by combining the crude or purified β-glucan with other ingredients that naturally contain β-glucan. Other ingredients include, but are not limited to, water and seasonings. Foods may be provided in various forms, such as powders, tablets, beverages, sweets, bread, and prepared foods.

[0036] <Hot water extraction conditions>

[0037] In Figure 1, hot water extraction 11 is preferably performed at a temperature higher than 60°C, and may be performed at 70°C or higher. Hot water extraction 11 may be performed at a temperature lower than 100°C. The temperature of hot water extraction 11 may be any of 75, 80, 85, 90, 95, 96, 97, 98, and 99°C. The temperature of hot water extraction 11 may be lower than the boiling point of the extraction medium under a standard atmospheric pressure of 101.325 kilopascals. The upper limit of the temperature for hot water extraction 11 using pure water under standard atmospheric pressure is 100°C.

[0038] In Figure 1, when hot water extraction 11 is performed at 100°C or less, the time can be set appropriately. For example, it may be longer than 20 minutes or shorter than 4 hours. The time may be any of 30 minutes, 40 minutes, 1 hour, 1 hour 30 minutes, 2 hours, and 3 hours. The length of time for hot water extraction 11 may or may not include the time from starting heating from room temperature (20°C ± 15°C) (Japanese Industrial Standards JIS) to reaching the target temperature. Heating and temperature maintenance may be performed in a normal pressure kettle or constant temperature bath.

[0039] In Figure 1, hot water extraction 11 may be carried out at a temperature of 100°C or higher under a pressure higher than standard atmospheric pressure. As long as the extraction medium is liquid, the temperature and pressure may be increased as appropriate. When the extraction medium is pure water, the temperature is preferably 374°C or lower and the pressure is preferably 22.1 MPa or lower. In other words, it is preferable that the water does not exceed its critical point and remains in a subcritical state.

[0040] In FIG. 1, the pressure of the hot water extraction 11 may be any of 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 and 300 kPa.

[0041] In FIG. 1 , the temperature of the hot water extraction 11 may be any of 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, and 140°C.

[0042] In FIG. 1, when hot water extraction 11 is performed at a temperature higher than 100°C under a pressure higher than standard atmospheric pressure, the time can be set appropriately. For example, it may be 1 minute or more and 1 hour or less. The time may be any of 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, and 50 minutes. The length of time for hot water extraction 11 may or may not include the time from starting pressurization and heating at standard atmospheric pressure and room temperature of 20°C ± 15°C (Japanese Industrial Standards JIS) until the target pressure and temperature are reached. Pressurization, heating, and maintaining the pressure and temperature may be performed using a pressure cooker or autoclave.

[0043] In FIG. 1, the extraction medium used in the hot water extraction 11 may be pure water or an aqueous solution in which an electrolyte and a non-electrolyte are dissolved. The pure water may be distilled water or ion-exchanged water. In this embodiment, the extraction medium containing an aqueous solution may be simply referred to as water. The aqueous solution used as the extraction medium may be neutral. Using pure water or a neutral aqueous solution as the extraction medium makes it easier to suppress the hydrolysis of β-glucan compared to using an alkaline aqueous solution as the extraction medium.

[0044] In the hot water extraction 11 shown in Figure 1, the pH of the extraction medium may be greater than 3 and less than 10 at room temperature (20°C ± 15°C). The pH may be any of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, and 9.0 at room temperature (20°C ± 15°C). Hot water extraction 11 may be performed using pure water, and the pH of liquid phase 15 may be adjusted after solid-liquid separation 13. It is also possible not to adjust the pH of liquid phase 15 after solid-liquid separation 13.

[0045] <Temperature control from hot water extraction to solid-liquid separation>

[0046] 1, after the target component is extracted by hot water extraction 11, high temperatures may be maintained until liquid phase 15 is separated from solid phase 16 containing bran residue by solid-liquid separation 13. This increases the purity of β-glucan in liquid phase 15 that is brought to enzyme treatment 21. In other words, by precipitating β-glucan in the steps up to that point, the amount of β-glucan carried away by solid phase 16 can be reduced.

[0047] In FIG. 1, the extract liquid before solid-liquid separation 13 and the liquid phase 15 are preferably maintained at a temperature higher than 20°C even after cooling. The lower limit of the maintained temperature may be any of 30, 40, 50, 60, 70, 80, and 90°C. The upper limit of the temperature is not particularly limited. The temperature may be equal to or lower than the boiling point of the liquid phase 15. The temperature may be equal to or lower than 100°C.

[0048] <Temperature control from solid-liquid separation to enzyme treatment>

[0049] 1, a high temperature may be maintained from the time when liquid phase 15 is separated from solid phase 16 containing bran residue in solid-liquid separation 13 until the enzyme is reacted in enzyme treatment 21. Furthermore, if solid-liquid separation 23 is performed after enzyme treatment 21, a high temperature may be maintained until solid-liquid separation 23 is performed. This increases the purity of β-glucan in liquid phase 25 that is carried into precipitation 31. In other words, by precipitating β-glucan in the steps up to that point, the amount of β-glucan carried away by solid phase 26 can be reduced.

[0050] In FIG. 1, it is preferable to maintain the liquid phase 15 or liquid phase 25 at a temperature higher than 20°C even after it cools. The lower limit of the maintained temperature may be any of 30, 40, 50, 60, 70, 80, and 90°C. The upper limit of the temperature is not particularly limited. The temperature may be equal to or lower than the boiling point of the liquid phase 15 or liquid phase 25. The temperature may be equal to or lower than 100°C.

[0051] <Details of enzyme treatment>

[0052] Figure 2 shows an example of the flow of the enzyme treatment 21 shown in Figure 1. In the first treatment 18, α-amylase is introduced into the liquid phase 15 (Figure 1). The α-amylase may be thermostable. A suitable thermostable α-amylase is Clistase T10S (trademark) from Amano Enzyme. Starch is decomposed by the α-amylase reaction to produce oligosaccharides.

[0053] In the first process 18 shown in Figure 2, the reaction temperature is greater than 60°C and may be less than or equal to 100°C. The temperature may be any of 65, 70, 75, 80, 85, 90 and 95°C.

[0054] 2, the reaction time in first process 18 may be greater than 1 minute and less than 24 hours. The reaction time may be 5, 15, or 30 minutes, or 1, 2, 4, 8, or 16 hours. This may or may not include the time it takes to reach the reaction temperature in first process 18.

[0055] In the second treatment 19 shown in Figure 2, glucoamylase is introduced into the liquid phase 15 (Figure 1). Glucoamylase NL4.2 ​​(trademark) available from Amano Enzyme is preferably used as the glucoamylase. The glucoamylase reaction breaks down oligosaccharides to produce glucose.

[0056] In the second treatment 19 shown in Figure 2, an enzyme for cleaving α-1,6 glucoside bonds may be further introduced into the liquid phase 15 (Figure 1). Pullulanase is preferably used as the enzyme for cleaving α-1,6 glucoside bonds. Pullulanase "Amano" 3 (trademark) provided by Amano Enzyme is preferably used as the pullulanase. The reaction of the enzyme for cleaving α-1,6 glucoside bonds decomposes the partial structure of amylopectin.

[0057] In the second treatment 19 shown in Figure 2, a protease may be further introduced into the liquid phase 15 (Figure 1). A cysteine ​​protease is preferably used as the protease. Papain 300 (trademark) provided by Nippon Biocon is preferably used as the cysteine ​​protease. Proteins are decomposed by the reaction of the protease.

[0058] Other enzymes may be added in the second treatment 19 shown in FIG. 2 . Furthermore, α-amylase, glucoamylase, α-1,6-glucosidic bond cleaving enzyme, protease, and other enzymes may have β-glucanase activity as a side activity, i.e., catalysis of other reactions. The lower the side activity, the better. Here, β-glucanase activity refers to the enzyme activity that cleaves β-1,3 and β-1,4 bonds in β-glucan. This side activity reduces the molecular weight of β-glucan. In the extraction and purification of β-glucan in this embodiment, it is preferable that this side activity be low.

[0059] The β-glucanase activity of the enzyme may be 0 to 10%. The β-glucanase activity is measured, for example, by the method described in Patent Document 4. A β-glucan standard is dissolved in pure water to a concentration of 5 mg / ml to prepare a β-glucan solution. The enzyme sample is diluted with pure water to a concentration of 5 mg / ml. The β-glucan solution and the diluted enzyme sample are mixed in a test tube. The sample is incubated in a 50°C thermostatic chamber for 14 hours and then cooled on ice. For example, by the Congo Red method described in Non-Patent Document 1, the β-glucan in the solution is measured using a β-glucan enzyme having a molecular weight of 10 5The above β-glucan is measured. Instead of the enzyme sample, a blank can be prepared by performing the same procedure using pure water. The β-glucanase activity can be calculated using the following formula:

[0060] β-Glucanase activity (%) = (1-B / B0) x 100 where B = [β-glucan concentration in enzyme sample] B0 = [β-glucan concentration in blank]

[0061] The reaction temperature in the second treatment 19 shown in Figure 2 may be lower than the reaction temperature in the first treatment 18. The reaction temperature may be higher than 20°C and lower than 90°C. The temperature may be any of 40, 50, 55, 60, 65, 70, and 80°C. The reaction temperature in the second treatment 19 may not exceed the inactivation temperature of the enzyme used in the second treatment.

[0062] In the second process 19 shown in Figure 2, the reaction time may be longer than 1 hour and shorter than 7 days. The time may be any of 4, 8, 12, 16, and 24 hours, or any of 2, 3, 4, 5, and 6 days. This may or may not include the time it takes to reach the reaction temperature in the second process 19.

[0063] <Precipitation of target components>

[0064] Returning to FIG. 1, in precipitation 31, β-glucan, the target component, is precipitated in order to separate it from liquid phase 25. Precipitation 31 is performed, for example, by adding a polar solvent, such as a polar organic solvent with a lower polarity than water, to the liquid phase. Examples of polar organic solvents include ethanol and isopropanol. Solid phase 36 is separated from liquid phase 35 by solid-liquid separation 33. Solid-liquid separation 33 may be performed, for example, by centrifugal separation to precipitate solid phase 36, followed by removal of the supernatant liquid phase 35. The target component, β-glucan, is obtained in solid phase 36. Solid phase 36 may be dissolved again in water or another solvent and then precipitated. This may provide a washing effect.

[0065] <Cleaning of precipitates>

[0066] Washing 41 shown in FIG. 1 is performed as needed. A purified β-glucan product is obtained by washing 41. In washing 41, the solid phase 36, which is a precipitate, is washed. For example, the solid phase 36 is mixed with washing water, such as pure water, and the portion insoluble in the washing water is collected. The washing water may contain other components to the extent that the washing effect is not impaired. The washing water may be an electrolyte or a buffer solution. In this specification, washing with washing water may sometimes be simply referred to as washing with water.

[0067] In the cleaning 41 shown in FIG. 1, when the cleaning liquid is water, for example, pure water, its temperature may be higher than 0°C and may be lower than 80°C. The higher the water temperature, the greater the cleaning effect tends to be. Also, the lower the water temperature, the less β-glucan is dissolved in the water and lost. The water temperature may be any of 4, 10, 20, 25, 30, 35, 40, 50, 60, and 70°C.

[0068] The duration of the wash 41 shown in Figure 1 may be greater than 1 minute and less than 24 hours, and may be any of 5, 10, 15, 20, 30, and 45 minutes, or any of 1, 2, 4, 8, and 16 hours.

[0069] 1 may be performed by allowing the solid phase 36 to stand in wash water for a period of time greater than 1 minute and less than 24 hours. The period of time may be any of 5, 10, 15, 20, 30, and 45 minutes, or any of 1, 2, 4, 8, and 16 hours.

[0070] Washing 41 shown in FIG. 1 may be performed by continuously stirring the solid phase 36 in wash water. The duration of continuous stirring may be longer than 1 minute and shorter than 24 hours. The duration may be any of 5, 10, 15, 20, 30, and 45 minutes, or any of 1, 2, 4, 8, and 16 hours. Standing for the above period and continuous stirring for the above period may be combined. Standing for the above period may be performed after continuous stirring for the above period.

[0071] 1, the solid phase 36 before washing may be dried to increase the hydrogen bonds between the β-glucan molecules, thereby preventing the β-glucan from dissolving in the washing water in the washing step 41.

[0072] <Production of a composition containing barley bran components>

[0073] In the process shown in Figure 1, or by adding a purification step to the process, bran components other than β-glucan are removed as much as possible. This allows for the provision of highly pure β-glucan. From a different perspective, it is also possible to separate some of the bran components other than β-glucan from the bran together with β-glucan in the process shown in Figure 1. In one embodiment, a composition containing β-glucan and other barley bran components may be produced by extracting and purifying β-glucan from barley bran. In other words, the extraction and purification procedures may provide a useful composition containing the bran component extracted as the target component together with β-glucan. Such a composition may be further purified. [Example]

[0074] <Example 1. Hot water extraction>

[0075] The upper part of Figure 3 shows the process for obtaining a crude product by hot water extraction. 10 g of barley bran was subjected to hot water extraction with 200 ml of purified water at 121°C for 20 minutes. The extract was separated from the residue by centrifugation at 20°C. The crude product was obtained by ethanol precipitation. The crude product was then washed and dried. In the hot water extraction using purified water, the crude product yield was 8.4%, the β-glucan purity was 21.4%, and the β-glucan yield was 65.9%.

[0076] Unless otherwise specified, in this and subsequent examples, purity refers to the weight (mass) fraction (w / w %) of β-glucan in the dry weight (mass) of the product or precipitate. Unless otherwise specified, in these examples, yield refers to the weight (mass) yield (w / w %) of β-glucan in the product or precipitate relative to the dry weight (mass) of the raw barley bran.

[0077] In this and subsequent examples, the weight (mass) of β-glucan in the product is measured as follows: The product is suspended in 20 mM phosphate buffer (pH 6.5) as a measurement sample, hydrated, and then reacted with purified lichenase (licheninase). The β-1,4 bonds of the β-glucan in the sample are cleaved to produce β-glucooligosaccharides. β-Glucosidase is then added to cleave the β-glycosidic bonds to produce D-glucose. Finally, GOPOD reagent (glucose oxidase / peroxidase / 4-aminoantipyrine) is added. The color reaction of the produced quinone is measured at a wavelength of 510 nm. The weight (mass) of β-glucan is converted from the measured weight (mass) of D-glucose. A Megazyme β-glucan measurement kit can be suitably used for this measurement.

[0078] <Example 2. Alkaline extraction>

[0079] In a separate test, hot water extraction using an alkaline solution was performed. For the alkaline extraction, an alkaline solution consisting of 0.2 M Na2CO3 in the same volume as pure water was used. The extraction temperature and time were the same as for hot water extraction using pure water. When the two were compared, the purity and yield of β-glucan were superior in the hot water extraction using pure water.

[0080] <Example 3-1. Enzyme treatment>

[0081] The lower part of Figure 3 shows the flow of enzyme treatment of the crude product. First, a crude product was obtained by hot water extraction as in Example 1. The crude product was resuspended in 0.2 M acetate buffer (pH 5.0) and boiled. The enzyme was added to the resuspended buffer.

[0082] The enzymes used were the protease Papain 300 (Nippon Biocon Co., Ltd.), the heat-stable α-amylase Chrystase T10S (Amano Enzyme Co., Ltd.), the pullulanase "Amano" 3 (Amano Enzyme Co., Ltd.) which acts to cleave α-1,6 glucoside bonds, and the glucoamylase Glucozyme NL4.2 ​​(Amano Enzyme Co., Ltd.). Hereafter, Papain 300 will be referred to as papain, Chrystase 10S as Chrystase, pullulanase "Amano" 3 as pullulanase, and Glucozyme NL4.2 ​​as gluczyme. Papain was adjusted to 5%, and the other enzyme solutions were used undiluted.

[0083] In the enzyme addition / reaction step shown in the lower part of Figure 3, 200 μL of clastase was added and the mixture was incubated at 90°C for 1 hour in a constant temperature air dryer (WFO-500W, Tokyo Rikakikai). After the sample was cooled to 60°C, 200 μL each of 5% papain, gluczyme, and pullulanase was added and the mixture was incubated at 60°C overnight in a constant temperature air dryer (WFO-500W, Tokyo Rikakikai). The next day, the mixture was transferred to a 50 mL glass centrifuge tube and centrifuged at 570 G for 15 minutes in a tabletop centrifuge (LC-200). The supernatant was collected.

[0084] In the ethanol precipitation step shown in the lower part of Figure 3, an equal volume of 100% ethanol was added, stirred for 1 hour using a magnetic stirrer (HS-3B, AS ONE), and then allowed to stand overnight. The next day, the mixture was transferred to a 50 mL glass centrifuge tube and centrifuged at 570 G for 15 minutes using a tabletop centrifuge (LC-200). The supernatant was discarded, and an appropriate amount of 100% ethanol was added to the precipitate. The mixture was then transferred to a 50 mL glass centrifuge tube and centrifuged at 570 G for 15 minutes using a tabletop centrifuge (LC-200). After washing with 100% ethanol, an appropriate amount of acetone was added, transferred to a 50 mL glass centrifuge tube, and centrifuged at 570 G for 15 minutes using a tabletop centrifuge (LC-200). The precipitate was then collected and air-dried overnight in a fume hood. The next day, the mixture was dried under reduced pressure for approximately 30 minutes, weighed, and stored in a vial.

[0085] The analytical results of the precipitate are as follows: In Example 3-1, in which the enzyme treatment was carried out after the hot water extraction, the purity of β-glucan was increased compared to Example 1, in which the enzyme treatment was not carried out.

[0086] [Table 1]

[0087] <Example 3-2. Enzyme treatment while maintaining high temperature after hot water extraction>

[0088] Figure 4 shows the flow of hot water extraction followed by enzyme treatment while maintaining high temperatures. The procedure up to hot water extraction was the same as in Example 3-1. After hot water extraction, the extract was separated from the residue by suction filtration using two layers of Miracloth (Merck).

[0089] In the enzyme addition / reaction step shown in Figure 4, 100 μL of clastase was added to the filtrate and incubated at 90°C for 1 hour in a constant temperature oven (WFO-500W, Tokyo Rikakikai). The mixture was then cooled to 60°C, and 100 μL each of 5% papain, gluczyme, and pullulanase was added. The mixture was then incubated at 60°C overnight in a constant temperature oven (WFO-500W, Tokyo Rikakikai). The next day, the filtrate was collected by vacuum filtration through two layers of Miracloth (Merck).

[0090] In the ethanol precipitation step shown in FIG. 4, an equal volume of 100% ethanol was added to the filtrate, and the mixture was stirred for 1 hour using a magnetic stirrer (HS-3B, AS ONE) and then allowed to stand overnight. The next day, the sample containing the precipitate was transferred to a 250 mL centrifuge tube and centrifuged at 10,200 G for 15 minutes at 20°C in a centrifuge (CT-13, HITACHI). The precipitate was collected, an appropriate amount of 80% ethanol was added, and the sample was centrifuged at 10,200 G for 1 hour at 20°C in a centrifuge (CT-13, HITACHI) for washing. The supernatant was discarded, and the precipitate was added to an appropriate amount of 100% ethanol and centrifuged twice at 10,200 G for 15 minutes at 20°C in a centrifuge (CT-13, HITACHI). The supernatant was discarded after each centrifugation. After washing twice with 100% ethanol, the precipitate was transferred to a 50 mL glass centrifuge tube, an appropriate amount of acetone was added, and the sample was centrifuged twice at 570 G for 15 minutes in a tabletop centrifuge (LC-200). The precipitate was collected and air-dried overnight in a fume hood. The next day, the mixture was dried under reduced pressure for about 30 minutes, weighed, and stored in a vial.

[0091] As described above, in this example, the extract was separated from the residue by suction filtration without cooling, and the extract was directly subjected to enzyme treatment.

[0092] <Example 3-3. Enzyme treatment without filtering the extract>

[0093] Unlike Example 3-2, the bran residue was treated with the enzyme without filtering after the hot extraction of the bran. Other conditions were the same as in Example 3-2.

[0094] <Evaluation of Example 3>

[0095] As shown in Table 1, compared to Example 3-1, in which the crude product was resuspended and then enzymatically treated, the purity of β-glucan increased by approximately 7% when the filtered extract was subjected to enzyme treatment. The β-glucan yield was comparable. In this example, the recovery rate of β-glucan was increased by suppressing the temperature drop after thermal extraction and before enzyme treatment. Furthermore, in Example 3-2, in which the extract was separated by suction filtration, the procedure was simpler than in Example 3-1, which involved precipitation and resuspension.

[0096] As shown in Table 1, the purity of the crude β-glucan was higher in Examples 3-1 and 3-2 than in Example 3-3, in which the enzyme treatment was carried out without separating the extract from the residue. This indicates that separating the extract from the residue allows for efficient decomposition of unnecessary components, including starch.

[0097] <Example 4. Cleaning with water> The dried precipitate containing β-glucan obtained in Example 3-2 shown in Figure 4 was washed to obtain β-glucan of higher purity. 0.30 g of the dried precipitate was suspended in 30 mL of distilled water. The suspension was allowed to stand for 2 hours at the temperature shown in Table 2. The water-insoluble components were collected by centrifugation at 540 G for 15 minutes and then lightly rinsed.

[0098] [Table 2]

[0099] As shown in Table 2, the β-glucan purity exceeded 50% at washing temperatures between 4°C and 60°C, and exceeded 60% at temperatures other than 40°C. In contrast, the β-glucan purity of the dried precipitate containing β-glucan obtained in Example 3-2 was 41.6%. The β-glucan purity increased by 14 to 20% at washing temperatures between 4°C and 60°C. As described above, washing with distilled water increased the β-glucan purity.

[0100] <Example 5. Temperature and pressure of hot water extraction>

[0101] It was confirmed that hot water extraction at a temperature below the boiling point of water is effective. 10 g of barley bran was mixed with 200 mL of distilled water and stirred for 5 minutes. In Examples 5-1 and 5-2, the mixture was left to stand in a constant temperature oven at 70°C and 90°C for 2 hours. In Example 5-3, the mixture was autoclaved at 121°C, 2 atmospheres, and 20 minutes. The extract was separated from the residue by suction filtration using a filter made of two layers of Miracloth.

[0102] 250 μL of clastase was added to the extract, stirred, and then allowed to stand at 90°C for 1 hour. The sample was then cooled slightly, and 250 μL of 5% papain, gluczyme, and pullulanase were added and stirred. The mixture was then allowed to stand overnight at 60°C. The enzyme-treated solution was separated from the insoluble components by suction filtration using a filter made of two layers of Miracloth.

[0103] An equal volume of ethanol was added to the treated solution, and the mixture was stirred for 1 hour and then allowed to stand overnight. The sample was centrifuged (8000 rpm, 15 minutes) to obtain a precipitate. The precipitate was washed once with 80% ethanol, twice with 100% ethanol, and twice with acetone. The collected precipitate was air-dried by leaving it in a fume hood overnight and then further dried in a vacuum dryer for 30 minutes.

[0104] [Table 3]

[0105] Table 3 shows the average of three independent trials for each example. Both the purity and yield of β-glucan increased with increasing temperature and pressure.

[0106] <Example 6. Separating hot water extraction and enzyme treatment>

[0107] It was confirmed whether simultaneous enzyme treatment under hot water extraction conditions at 90°C was effective. In Example 6-2, extraction and purification were carried out in the same manner as in Example 5-2, except for the following points. In Example 6-2, extraction and purification were carried out in the same manner as in Example 5-2.

[0108] In Example 6-1, 250 μL of clastase was added to the water and stirred before hot water extraction at 90°C. The mixture was left to stand at 90°C for 3 hours while reacting with clastase. The liquid phase was separated from the residue using a filter made of two sheets of Miracloth. After cooling slightly, 250 μL each of 5% papain, gluczyme, and pullulanase was added and stirred. The mixture was left to stand overnight at 60°C. The enzyme-treated solution was then purified in the same manner as in Example 5-2.

[0109] [Table 4]

[0110] Table 4 shows the average of three independent trials for each example. The purity of β-glucan was higher in Example 6-2, in which hot water extraction and enzyme treatment were performed separately, than in Example 6-1, in which they were performed simultaneously. The yield in Example 6-1 was higher than that in Example 6-2. However, the yield in Example 6-1 was lower than that in Example 5-3, in which extraction was performed at a temperature higher than the boiling point of water.

[0111] <Example 7-1. Effect of time on cleaning>

[0112] To further investigate the water washing conditions described in Example 4, the effect of washing time was investigated. 0.3 g of dried β-glucan precipitate was mixed with 30 mL of distilled water at 4°C, 30°C, and 70°C. Some samples were left to stand at 4°C for 10 minutes or 1 hour in a refrigerator. Other samples were left to stand at 30°C for 10 minutes or 1 hour in a dry incubator. Other samples were left to stand at 70°C for 10 minutes or 1 hour in a dry incubator. Each sample was centrifuged at 750G for 15 minutes. The collected precipitate was washed once with 100% ethanol and once with acetone. After air drying, the liquid components were removed by vacuum drying. The β-glucan purity measured for each sample is shown in the table below.

[0113] [Table 5]

[0114] Except for when the mixture was left to stand at 70°C for one hour, the purity increased by 6 to 7% compared to the original dried precipitate. The purity tended to be particularly high when the temperature was 30°C. Therefore, it was found that washing at room temperature (20°C ± 15°C), including 30°C, is preferable. Except for when the mixture was left to stand at 70°C, no effect on purity was observed depending on the length of time the mixture was left to stand.

[0115] <Example 7-2. Effect of continuous stirring on cleaning>

[0116] To further investigate the water washing conditions described in Example 4, the effect of continuous stirring during washing was investigated. The temperature throughout the process was kept at room temperature (20°C ± 15°C). 30 mL of distilled water was mixed with 0.3 g of dried β-glucan precipitate. The mixture was stirred continuously for 5 minutes or 1 hour using a stirrer. Each sample was centrifuged at 750 G for 15 minutes. The collected precipitate was washed once with 100% ethanol and once with acetone. After air drying, the liquid components were removed by vacuum drying. The purity of β-glucan measured for each sample is shown in the table below.

[0117] [Table 6]

[0118] As shown in Table 6, purity could be increased even with continuous stirring. No effect on purity was observed depending on the duration of stirring. Note that the purity of the original dried precipitate was slightly lower than that of Example 3-2. Because turbidity was observed in the supernatant after centrifugation, it was determined that there was room for improvement in the recovery of β-glucan, and the following test was carried out.

[0119] <Example 7-3. Effect of a combination of continuous stirring and standing>

[0120] In this example, a settling period was added after continuous stirring. The temperature throughout the process was room temperature (20°C ± 15°C). 30 mL of distilled water was mixed with 0.3 g of dried precipitated β-glucan. One sample was continuously stirred for 5 minutes using a vortex mixer and then settling for 5 minutes. Another sample was continuously stirred for 1 hour using a stirrer and then settling for 1 hour. The vibration of this stirrer was stronger than in Example 7-2. Each sample was centrifuged at 750 G for 15 minutes. The recovered precipitate was washed once with 100% ethanol and once with acetone. After air drying, the liquid components were removed by vacuum drying. The β-glucan purity measured for each sample is shown in the table below. Note that the purity of the original dried precipitate was slightly higher than that of Example 7-1.

[0121] [Table 7]

[0122] By stirring for 1 hour and then leaving to stand for 1 hour, the purity improved by approximately 15% compared to the original dried precipitate. Furthermore, the supernatant after centrifugation was colorless and transparent for all samples. It is expected that the continuous stirring allowed the impurities in the original dried precipitate to dissolve well in water. It is also expected that leaving to stand improved the efficiency of β-glucan recovery after centrifugation. It should be noted that the purity of the original dried precipitate was slightly lower than that of Example 7-2. [Explanation of symbols]

[0123] 10: Raw material, 11: Hot water extraction, 13: Solid-liquid separation, 15: Liquid phase, 16: Solid phase, 18: First treatment, 19: Second treatment, 21: Enzyme treatment, 23: Solid-liquid separation, 25: Liquid phase, 26: Solid phase, 31: Precipitation, 33: Solid-liquid separation, 35: Liquid phase, 36: Solid phase, 41: Washing, 45: Final product

Claims

1. A method for producing a crude or purified β-glucan product, comprising: The target components are extracted from barley bran using hot water extraction, Separating the liquid phase containing the target component from the bran residue; performing an enzyme treatment to decompose at least one of starch, protein, and other impurities contained in the liquid phase; Separating the target component from enzymatic degradation products; method.

2. Maintaining the liquid phase at a temperature above 60°C from the time the target component is extracted until the time the liquid phase is separated from the residue; The method of claim 1.

3. The extract containing the target component is filtered to separate the liquid phase from the residue. The method of claim 2.

4. filtering the extract containing the target component to separate the liquid phase from the residue; The enzyme treatment is carried out by adding an enzyme directly to the filtered liquid phase; The liquid phase is maintained at a temperature above 60°C during the extraction of the target component, separation of the liquid phase from the residue, and activation of the enzyme. The method of claim 1.

5. performing an enzymatic treatment to decompose at least the starch contained in the liquid phase; The enzyme treatment is carried out in at least two stages, First, treating the liquid phase with a thermostable α-amylase; second, cooling the liquid phase and then treating the liquid phase with glucoamylase; The method of claim 4.

6. performing an enzymatic treatment to decompose at least one of starch and protein contained in the liquid phase; The method of claim 1.

7. The hot water extraction is carried out at a temperature of 70, 80, or 90°C or higher, but lower than 100°C. The method of claim 1.

8. The hot water extraction is carried out at a temperature of 100°C, 110°C, or 120°C or higher under a pressure higher than standard atmospheric pressure. The method of claim 1.

9. The purity of the crude β-glucan or its purified product is 20% or more and 100% or less. The method of claim 1.

10. adding a polar solvent to the enzyme-treated solution to precipitate the target component and separate it from the degradation products; The method further comprises washing the precipitate containing the target component with water at 80°C or less and recovering an insoluble portion, The purity of the crude β-glucan or its purified product produced is 40% or more and 100% or less. The method of claim 1.

11. adding a polar solvent to the enzyme-treated solution to precipitate the target component and separate it from the degradation products; The precipitate containing the target component is mixed with water at a temperature of 4°C or higher and 60°C or lower, The mixture is treated for a predetermined time by at least one of standing and continuous stirring, further comprising separating the insoluble portion from the water; The crude β-glucan or its purified product produced is 40% or more and 100% or less. The method of claim 1.

12. A crude β-glucan product or a purified β-glucan product is produced by the method according to any one of claims 1 to 11, The crude product or its refined product is combined with other raw materials to produce a food product containing β-glucan or having an increased β-glucan content. method.

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