JP4964710B2 - Process for producing β-glucan-containing cereal saccharified product - Google Patents
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- JP4964710B2 JP4964710B2 JP2007221921A JP2007221921A JP4964710B2 JP 4964710 B2 JP4964710 B2 JP 4964710B2 JP 2007221921 A JP2007221921 A JP 2007221921A JP 2007221921 A JP2007221921 A JP 2007221921A JP 4964710 B2 JP4964710 B2 JP 4964710B2
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- 229920002498 Beta-glucan Polymers 0.000 title claims description 75
- FYGDTMLNYKFZSV-URKRLVJHSA-N (2s,3r,4s,5s,6r)-2-[(2r,4r,5r,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5r,6s)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1[C@@H](CO)O[C@@H](OC2[C@H](O[C@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-URKRLVJHSA-N 0.000 title claims description 72
- 235000013339 cereals Nutrition 0.000 title claims description 48
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
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- YZIYKJHYYHPJIB-UUPCJSQJSA-N chlorhexidine gluconate Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O.C1=CC(Cl)=CC=C1NC(=N)NC(=N)NCCCCCCNC(=N)NC(=N)NC1=CC=C(Cl)C=C1 YZIYKJHYYHPJIB-UUPCJSQJSA-N 0.000 description 2
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- 240000006394 Sorghum bicolor Species 0.000 description 1
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- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
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- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
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- Coloring Foods And Improving Nutritive Qualities (AREA)
- Cereal-Derived Products (AREA)
Description
本発明は、甘味素材である糖化物に機能性を付与した食品素材の提供、およびその製造法に関するものである。本発明の製造方法により得られる穀物糖化物は、機能性成分であるβ−グルカンを１〜１５質量％含有し、かつヨード発色テストが（−）である糖化物であり、特に血清コレステロール低下作用や免疫調節作用を有する食品素材として、健康食品分野におけるサプリメント用、一般食品分野における飲料、パン、和洋菓子等に幅広く使用することができる。
The present invention relates to the provision of a food material in which functionality is imparted to a saccharified product, which is a sweet material, and a method for producing the same. The cereal saccharified product obtained by the production method of the present invention is a saccharified product containing 1 to 15% by mass of β-glucan, which is a functional component, and having an iodine color development test of (−). As a food material having an immunomodulatory action, it can be widely used for supplements in the health food field, beverages in the general food field, bread, Japanese and Western confectionery, and the like.
近年、β−グルカンの生理機能が見出されている。β−グルカンの構造は、グルコースを構成ユニットとし、これらがβ−１，３結合、β−１，４結合する直鎖上の高分子である。例えば、β−グルカン量が異なる大麦粉を原料とするケーキ、マフィン等を摂取することにより、摂取したβ−グルカン量に依存して総コレステロール値が減少する事例が報告されている（例えば、非特許文献１及び非特許文献２参照。）。また、米国ＦＤＡは、大麦β−グルカンに血清コレステロール値を低下させる効果があり、ヘルスクレームとして冠状心疾患の危険を減らす表示を認めている。 In recent years, physiological functions of β-glucan have been found. The structure of β-glucan is a straight-chain polymer in which glucose is a structural unit and these are β-1,3 bonds and β-1,4 bonds. For example, there have been reports of cases in which total cholesterol levels are reduced depending on the amount of β-glucan consumed by ingesting cakes, muffins and the like made from barley flour having different amounts of β-glucan (for example, non- (See Patent Document 1 and Non-Patent Document 2.) In addition, the US FDA recognizes that barley β-glucan has an effect of lowering serum cholesterol level, and as a health claim, the indication for reducing the risk of coronary heart disease is accepted.
またマウスに大麦β−グルカンを腹腔内投与することにより、腹腔内細胞中の好中球集積が観察され、マクロファージ様細胞数の増加、活性化や細胞培養液中のＩＬ−１２、ＩＦＮ−γの産生増強が観察された報告があり（例えば、非特許文献３参照。）、大麦β−グルカンによる免疫調節作用が謳われている。 Further, when barley β-glucan was intraperitoneally administered to mice, neutrophil accumulation in the intraperitoneal cells was observed, and the number of macrophage-like cells increased, activated, and IL-12, IFN-γ in the cell culture medium. (See, for example, Non-Patent Document 3), and the immunoregulatory action by barley β-glucan is expected.
穀物には、水溶性食物繊維であるβ−グルカンが含まれている。β−グルカンが比較的多く含まれる穀物としては、麦やオーツ麦が知られ、その含有量は６質量％とされる（例えば、非特許文献４参照。）。これら大麦やオーツ麦において、β−グルカンは、デンプン貯蔵細胞の細胞壁成分として存在する。こうした事実に鑑み、様々な大麦等穀物からのβ−グルカン製造法が考えられている。例えば、多ろう質大麦を原料とし、水抽出により製造する方法（例えば、特許文献１参照。）、あるいは、大麦、オーツ麦を原料として、アルカリ抽出、中和、アルコール沈殿により、重量平均分子量１０万〜１００万のβ−グルカンを得る方法（例えば、特許文献２参照。）、搗精歩留まり８２質量％以下の大麦糠類を原料として、８０〜９０℃の熱水にてβ−グルカンを抽出する方法（例えば、特許文献３参照。）等がある。しかし、これらの方法は基本的に大麦等穀物からβ−グルカンを抽出する方法であり、抽出効率を上げるために様々な工夫はされているものの、いずれも抽出効率は不十分である。また、大麦等穀物中のβ−グルカン量は６〜１０質量％程度と澱粉質やタンパク質等他の成分に比べると含有量が著しく低く、抽出後の残渣は製品であるβ−グルカンに比べ、著しく多い。これらは、飼料等付加価値の低い利用、または廃棄物にせざるを得ないため、製品単価が高くならざるを得ず、多くのサプリメント用、一般食品用に使用することが困難であった。 Cereals contain β-glucan, which is a water-soluble dietary fiber. As grains containing a relatively large amount of β-glucan, wheat and oats are known, and the content thereof is 6% by mass (see, for example, Non-Patent Document 4). In these barley and oats, β-glucan exists as a cell wall component of starch storage cells. In view of these facts, methods for producing β-glucan from various grains such as barley have been considered. For example, a method of producing by wax extraction using waxy barley as a raw material (see, for example, Patent Document 1), or using barley or oat as a raw material by alkali extraction, neutralization, alcohol precipitation, and a weight average molecular weight of 10 A method for obtaining 10,000 to 1,000,000 β-glucan (see, for example, Patent Document 2). Extracting β-glucan with hot water at 80 to 90 ° C. using barley koji with a fine yield of 82% by mass or less. There are methods (for example, see Patent Document 3). However, these methods are basically methods for extracting β-glucan from cereal grains such as barley, and although various efforts have been made to increase the extraction efficiency, all of them are insufficient in extraction efficiency. In addition, the amount of β-glucan in grains such as barley is about 6 to 10% by mass, which is significantly lower than other components such as starch and protein, and the residue after extraction is compared to β-glucan as a product, Remarkably many. These have to be used for low value-added feeds or waste, so that the unit price of the product has to be high, and it has been difficult to use for many supplements and general foods.
実際、β−グルカンの生理機能に基づいた食品素材が近年開発されている。例えば、キッコーマン社から「オーツ麦５０ＥＸ」、「オーツ麦ＥＸ」、ＡＤＥＫＡ社から「大麦β−グルカン」等が販売されている。これらは機能的には優れているものの、高価である（例えば、非特許文献５参照。）。 In fact, food materials based on the physiological function of β-glucan have been developed in recent years. For example, “Oat 50EX” and “Oat EX” are sold by Kikkoman, and “Barley β-glucan” is sold by ADEKA. Although these are functionally superior, they are expensive (see, for example, Non-Patent Document 5).
穀物を原料とする糖化物は古くから開発されている。原料としてモチゴメ、ウルチゴメ、砕米、モロコシ、トウモロコシ、アワ、サツマイモデンプン、ジャガイモデンプンが用いられ、これらを液化・糖化した後、金網等でアクを取り、自然濾過、加熱濃縮することにより麦芽飴や米飴が得られる。（例えば、非特許文献６参照。）これらの特徴は、米や麦芽に由来するビタミンやミネラル類等栄養素を含み、かつ香ばしい風味を持つ点であり、栄養強化、風味を増強する甘味料として活用されている。しかし、これらの糖化物は、微量成分としてミネラルや香気成分が含まれているものの、マルトースを主体とする糖類が主成分であり、各種菓子やつくだ煮等に利用されるが、β−グルカン等機能成分を含まず、サプリメントとして利用されることはなかった。 Saccharified products made from grains have been developed for a long time. Mochigome, uruchigome, crushed rice, sorghum, corn, millet, sweet potato starch, and potato starch are used as raw materials. After liquefying and saccharifying these, take acupuncture with a wire mesh, etc., naturally filter, heat concentrate, A cocoon is obtained. (For example, see Non-Patent Document 6.) These features include nutrients such as vitamins and minerals derived from rice and malt, and have a fragrant flavor, and are used as a sweetener to enhance nutrition and enhance flavor. Has been. However, although these saccharified products contain minerals and aroma components as trace components, they are mainly composed of saccharides mainly composed of maltose, and are used for various confectionery, tsukudani, etc. It contains no ingredients and was not used as a supplement.
また、穀物中のタンパク質やミネラルの栄養性に着眼したものが開発されている。米を液化・糖化した後、適当なｐＨに調整しタンパク質分解酵素処理することにより、アミノ酸がリッチな米飴が製造できる（例えば、特許文献４参照。）。また、小麦を原料としたシロップ（例えば、特許文献５参照。）、大麦を原料とした水飴（例えば、特許文献６及び特許文献７参照。）があるが、いずれもタンパク質を分解したアミノ酸やミネラルの一部である亜鉛含量を増やしたものであり、β−グルカン等機能成分を含有するものではなく、やはりサプリメントとして利用されることはなかった。 In addition, products that focus on the nutritional properties of proteins and minerals in grains have been developed. After liquefaction and saccharification of rice, a rice bran rich in amino acids can be produced by adjusting to an appropriate pH and treating with proteolytic enzyme (see, for example, Patent Document 4). In addition, there are syrups made from wheat as a raw material (for example, see Patent Document 5) and varicella made from barley as a raw material (see, for example, Patent Document 6 and Patent Document 7). The content of zinc, which is a part of the above, was increased and did not contain functional components such as β-glucan, and was never used as a supplement.
こうした事実に鑑み、大麦等β−グルカンを含有する穀物を液化反応、糖化反応を行う前にβ−グルカナーゼ活性度が０〜１０％のタンパク質分解酵素によりタンパク質を分解し、次にβ−グルカナーゼ活性度が０〜１０％の液化酵素、およびβ−グルカナーゼ活性度が０〜１０％の糖化酵素により澱粉質を分解することにより、大麦等穀物のβ−グルカンを損傷、損失することなく糖化物中に混合させることができること、本発明は抽出法に比べ、低コストで製造できることを見出し、本発明に至った。
本発明の目的は、大麦等穀物を原料とし、穀物が含むβ−グルカンを効率よく糖化物中に入れ込むことにより、β−グルカンの持つ機能性を活かした食品素材で、かつ低コストで製造可能な食品素材を開発することである。 The object of the present invention is to produce a food material utilizing the functionality of β-glucan at low cost by efficiently incorporating β-glucan contained in the grain into saccharified products using grains such as barley as a raw material. The development of possible food ingredients.
本発明は、β−グルカンを含む穀物を原料とし、液化反応、糖化反応を行う前に、β−グルカナーゼ活性度が０〜１０％のプロテアーゼタンパク質分解酵素を用いてタンパク質分解反応を行い、次にβ−グルカナーゼ活性度が０〜１０％の液化酵素、β−グルカナーゼ活性度が０〜１０％の糖化酵素により澱粉質分解を行い、固液分離、スプレードライ、凍結乾燥法等を行うことにより得られた素材が１〜１５質量％のβ−グルカンを含有し、ヨード発色テストが（−）である穀物糖化物であり、本糖化物が上記目標を達成することを見出し、本発明を完成させたものである。 The present invention uses cereals containing β-glucan as a raw material, and performs a proteolytic reaction using a protease proteolytic enzyme having a β-glucanase activity of 0 to 10% before performing a liquefaction reaction or a saccharification reaction. It is obtained by degrading starch with a liquefying enzyme with a β-glucanase activity of 0-10% and a saccharifying enzyme with a β-glucanase activity of 0-10%, followed by solid-liquid separation, spray drying, freeze drying, etc. The obtained material is a cereal saccharified product containing 1 to 15% by mass of β-glucan and having an iodine color development test of (−), and the saccharified product achieves the above-mentioned target, thereby completing the present invention. It is a thing.
本発明により、β−グルカンが１〜１５質量％を含有し、ヨード発色テストが（−）であることを特徴とする穀物糖化物を得ることができる。本発明品は、β−グルカンの持つ機能性を活かした糖化物であり、健康食品分野におけるサプリメント用、一般食品分野における飲料、パン、和洋菓子等に幅広く使用することができる。 According to the present invention, a saccharified grain product containing 1 to 15% by mass of β-glucan and having an iodine color development test of (−) can be obtained. The product of the present invention is a saccharified product utilizing the functionality of β-glucan, and can be widely used for supplements in the health food field, beverages, breads, Japanese and Western confectionery in the general food field.
以下に本発明を詳細に説明する。原料は、β−グルカンを含む穀物を用いる。澱粉質とβ−グルカンを含む穀物であれば本発明に使用することができるが、β−グルカン含量が高いものほど得られる糖化物中のβ−グルカン含量が高く、商品価値が高い。β−グルカンを含む穀物としては、大麦やオーツ麦があり、本発明に使用することができるが、これ以外の穀物でもβ−グルカンを含めば本発明に使用することができ、特にこれにこだわらない。なお、β−グルカンは低分子化されたものよりも分子量が１０５以上の高分子β−グルカンのほうが商品価値が高い。その理由は、大麦やオーツ麦由来の分子量が１０５以上のβ−グルカンに関しては古くから幅広くその生理機能が調べられ、多くの知見が得られているのに対し、低分子化されたものは新規に上市されたものであり、その生理機能の確認はまだまだ十分でないためである。 The present invention is described in detail below. The raw material is a grain containing β-glucan. Grains containing starch and β-glucan can be used in the present invention, but the higher the β-glucan content, the higher the β-glucan content in the saccharified product, and the higher the commercial value. Cereals containing β-glucan include barley and oats and can be used in the present invention. However, other grains can be used in the present invention if β-glucan is included, and this is particularly particular. Absent. It should be noted that β-glucan has a higher commercial value for high-molecular β-glucan having a molecular weight of 10 5 or more than that obtained by reducing the molecular weight. The reason is that the molecular weight derived from barley and oats are examined extensively its physiology from ancient times with respect to 10 5 or more β- glucans, whereas many findings have been obtained, those low molecular weight is This is because it has been newly launched and its physiological function has not been confirmed.
まず始めに、本糖化物を製造するにあたり、上記穀物を水に分散させる。この際、穀物の濃度として、１〜２０質量％であることが重要である。穀物の仕込み濃度が２０質量％より高い場合、十分な液化反応を行うことができず、糖化物に未分解の澱粉が残ってしまう。未分解の澱粉が残った糖化物は、老化しやすい等の理由により商品価値が著しく低い。未分解の澱粉の有無はヨード発色テストにより確認できる。また、未分解の澱粉が残った糖化液は粘度が高く、固液分離が困難である。 First, in producing the saccharified product, the cereal is dispersed in water. At this time, it is important that the concentration of the grain is 1 to 20% by mass. When the charged concentration of cereal is higher than 20% by mass, sufficient liquefaction reaction cannot be performed, and undegraded starch remains in the saccharified product. A saccharified product in which undegraded starch remains has a remarkably low commercial value for reasons such as easy aging. The presence or absence of undegraded starch can be confirmed by an iodine color development test. Moreover, the saccharified solution in which undegraded starch remains has a high viscosity and is difficult to separate into solid and liquid.
次にタンパク質分解反応を行う。市販の酵素は、主活性のみならず、他の反応を触媒する活性を持つことが多く、これを副活性という。副活性作用であるβ−グルカナーゼ活性のレベルをβ−グルカナーゼ活性度と定義する。なお、β−グルカナーゼ活性とは、β−グルカンのβ−１，３結合、β−１，４結合を切断する酵素活性である。タンパク質分解酵素は、β−グルカナーゼ活性度が０〜１０％のものを使用することが重要である。β−グルカナーゼ活性度が１０％以上のものを使用するとβ−グルカンが分解してしまい、分子量が１０５以上のβ−グルカンが１質量％以下になる。 Next, a proteolytic reaction is performed. Commercially available enzymes often have not only a main activity but also an activity that catalyzes other reactions, which is called a secondary activity. The level of β-glucanase activity, which is a side activity, is defined as β-glucanase activity. The β-glucanase activity is an enzyme activity that cleaves β-1,3 bond and β-1,4 bond of β-glucan. It is important to use a protease having a β-glucanase activity of 0 to 10%. When beta-glucanase activity is to use a more than 10% beta-glucan would decompose a molecular weight of 10 5 or more beta-glucan is less than 1 wt%.
タンパク質分解反応は、液化反応の前に行うことが重要である。β−グルカンはタンパク質と相互作用しているため、タンパク質分解反応を行わなければ、β−グルカンが液部に十分に遊離しない。このため、この順序で反応を行わなければ、固液分離の際に、残渣側にβ−グルカンが残ったままとなり、最終的にβ−グルカンが十分含まれない糖化物しか得られない。 It is important to carry out the proteolytic reaction before the liquefaction reaction. Since β-glucan interacts with protein, β-glucan is not sufficiently released into the liquid part unless a proteolytic reaction is performed. For this reason, if the reaction is not performed in this order, β-glucan remains on the residue side during solid-liquid separation, and finally only a saccharified product that does not sufficiently contain β-glucan is obtained.
実際には上記穀物含有の水溶液の温度をタンパク質分解酵素の反応温度まで上げて、タンパク質分解酵素を添加し、タンパク質を分解する。この際、水の温度を反応温度まで上げた後に、穀物、タンパク質分解酵素を添加してもよい。この際の反応温度とは、穀物の糊化温度よりも低く、かつタンパク質分解酵素が活性をもつ温度のことである。前処理の際の反応温度は穀物の糊化温度以上まで上げてはならない。粘度が上がりすぎ、液化反応が不十分と成るためである。また、タンパク質分解酵素はその温度に応じて活性が異なり、温度が低すぎても反応が進まない。反応温度範囲は、穀物の種類やタンパク質分解酵素の種類により異なるが、通常２０℃から６０℃で行うのが好ましい。タンパク質分解酵素の添加量は特に限定するものではないが、固形分１ｇに対して、５０〜１０００Ｕ添加する。反応ｐＨは、タンパク質分解酵素が活性をもつ範囲ならよく、特に限定する必要はない。また、反応時間も特に限定するものではないが、生産性を考慮すると通常１〜２４時間で行う。例えば、大麦５部に対し水を９５部添加し、固形分１ｇ当たりタンパク質分解酵素を３００Ｕ添加し、５５℃まで温度を上げ、３時間反応させることにより、反応を行うことができる。 In practice, the temperature of the aqueous solution containing grains is raised to the reaction temperature of the proteolytic enzyme, and the proteolytic enzyme is added to decompose the protein. At this time, the cereal and the proteolytic enzyme may be added after raising the temperature of the water to the reaction temperature. The reaction temperature at this time is a temperature that is lower than the gelatinization temperature of the grain and at which the proteolytic enzyme is active. The reaction temperature during pretreatment should not be raised above the gelatinization temperature of the grain. This is because the viscosity increases too much and the liquefaction reaction becomes insufficient. Proteolytic enzymes have different activities depending on the temperature, and the reaction does not proceed even if the temperature is too low. The reaction temperature range varies depending on the type of grain and the type of proteolytic enzyme, but it is usually preferable to carry out the reaction at 20 ° C to 60 ° C. The addition amount of the proteolytic enzyme is not particularly limited, but 50 to 1000 U is added to 1 g of the solid content. The reaction pH is not particularly limited as long as the proteolytic enzyme is active. Moreover, although reaction time is not specifically limited, When productivity is considered, it is normally performed in 1 to 24 hours. For example, the reaction can be carried out by adding 95 parts of water to 5 parts of barley, adding 300 U of proteolytic enzyme per gram of solid content, raising the temperature to 55 ° C., and reacting for 3 hours.
その後、上記反応液に、液化酵素を添加して液化反応を行う。ここで言う液化とは、澱粉質をランダムに切断し、水に可溶化させることである。液化酵素は、α−アミラーゼを用いればよく、植物由来、微生物由来のものがあるが、β−グルカナーゼ活性度が０〜１０％のものであればいずれを用いても良い。β−グルカナーゼ活性度が１０％以上のものを使用すると前処理反応でβ−グルカンが分解してしまい、分子量が１０５以上のβ−グルカンが１質量％以下になる。また、液化酵素添加量は特に限定するものではないが、通常１ｇあたり１０〜１０００Ｕ添加する。液化酵素は、β−グルカナーゼ活性度が０〜１０％であれば特に由来に限定されるものではない。液化酵素添加後、穀物の糊化温度以上まで昇温して、液化する。液化温度は穀物の糊化温度よりも高ければよく、特に限定するものではない。例えば、上記前処理液に、β−グルカナーゼ活性度が０〜１０％の液化酵素を固形分１ｇ当たり５０Ｕ添加し、３０分かけて８０℃まで昇温し、８０℃の状態を３０分保持することにより液化することができる。 Thereafter, a liquefaction enzyme is added to the reaction solution to perform a liquefaction reaction. Liquefaction as used herein refers to randomly cutting starch solubilized in water. As the liquefaction enzyme, α-amylase may be used, and there are those derived from plants and microorganisms, but any may be used as long as β-glucanase activity is 0 to 10%. β- glucanase activity ends up decomposition β- glucan treatment reaction before With more than 10%, a molecular weight of 10 5 or more β- glucan is below 1 wt%. The amount of liquefied enzyme added is not particularly limited, but is usually 10 to 1000 U added per 1 g. The liquefying enzyme is not particularly limited to its origin as long as the β-glucanase activity is 0 to 10%. After the liquefaction enzyme is added, the temperature is raised to a temperature equal to or higher than the gelatinization temperature of the grain and liquefies. The liquefaction temperature is not particularly limited as long as it is higher than the grain gelatinization temperature. For example, 50 U of liquefied enzyme having a β-glucanase activity of 0 to 10% per 1 g of solid content is added to the pretreatment liquid, the temperature is raised to 80 ° C. over 30 minutes, and the state at 80 ° C. is maintained for 30 minutes. Can be liquefied.
さらに、上記液化液に糖化酵素を添加し、糖化反応を行う。ここで言う糖化とは、液化により生じるデキストリン類をさらに分解し、少糖類とすることである。β−グルカナーゼ活性度が０〜１０％のものであることが重要であり、この条件さえ満たせば、糖化酵素は液化液により可溶化されたデキストリンを分解するものであれば、特に限定されるものではない。また、酵素の種類をかえることにより、目的に応じた糖組成とすることができる。なお、糖化反応の際の反応温度は、短時間で失活しない程度に低く、雑菌汚染の恐れがない程度に高い温度であれば特に問題はなく、通常は５０〜７０℃にするが、酵素の至適温度にするのがより好ましい。また、ｐＨは短時間で失活しない程度であれば特に問題はないが、酵素の至適ｐＨにするのがより好ましい。なお、ここで言う至適ｐＨとは酵素活性が最も高くなるｐＨのことであり、至適温度とは酵素活性が最も高くなる温度のことである。 Further, a saccharification enzyme is added to the liquefied liquid to perform a saccharification reaction. The saccharification here means to further decompose dextrins generated by liquefaction into oligosaccharides. It is important that the activity of β-glucanase is 0 to 10%, and as long as this condition is satisfied, the saccharifying enzyme is particularly limited as long as it decomposes dextrin solubilized by the liquefied liquid. is not. Moreover, it can be set as the saccharide | sugar composition according to the objective by changing the kind of enzyme. The reaction temperature during the saccharification reaction is low enough not to be deactivated in a short time and is not particularly problematic as long as it is high enough not to cause contamination with bacteria, and is usually 50 to 70 ° C. It is more preferable to set it to the optimum temperature. Further, there is no particular problem as long as the pH is such that it does not deactivate in a short time, but it is more preferable to adjust the pH to the optimum value for the enzyme. The optimum pH referred to here is the pH at which the enzyme activity is highest, and the optimum temperature is the temperature at which the enzyme activity is highest.
糖化酵素としてβ−アミラーゼを用いれば、マルトースが多く含まれる糖化物を得ることができる。この際、β−アミラーゼとしては、大豆や大麦麦芽など植物由来のものやＢａｃｉｌｌｕｓ属、Ｐｓｅｕｄｏｍｏｎａｓ属など微生物由来のものがあるが、β−グルカナーゼ活性度が０〜１０％のものであればいずれを用いてもよい。マルトースの量は、β−アミラーゼの添加量と反応時間により調整することができるが、更に、枝切酵素を使用することにより増やすことができる。枝切酵素とは、澱粉やデキストリンのα−１，６結合を切断する酵素で、種類としてプルラナーゼやイソアミラーゼがある。β−グルカナーゼ活性度が０〜１０％のものであれば特に由来を限定するものではなく、プルラナーゼ「アマノ」（天野エンザイム、Ｋｌｅｂｓｉｅｌｌａ ｐｎｅｕｍｏｎｉａｅ由来）やプロモザイム（ノボザイムズ、Ｂａｃｉｌｌｕｓ ａｃｉｄｏｐｕｌｌｕｌｙｔｉｃｕｓ由来）等が使用できる。最終的に、糖組成としてマルトース量は、液化の際の分解度にもよるが、３０〜７５質量％まで調整することができる。 If β-amylase is used as a saccharifying enzyme, a saccharified product containing a large amount of maltose can be obtained. In this case, β-amylase includes those derived from plants such as soybeans and barley malt, and those derived from microorganisms such as Bacillus genus and Pseudomonas genus, and any of those having β-glucanase activity of 0 to 10%. It may be used. The amount of maltose can be adjusted by the amount of β-amylase added and the reaction time, but can be further increased by using a debranching enzyme. The debranching enzyme is an enzyme that cleaves α-1,6 bonds of starch and dextrin, and includes pullulanase and isoamylase as types. The origin is not particularly limited as long as the β-glucanase activity is 0 to 10%, and pullulanase “Amano” (derived from Amano Enzyme, Klebsiella pneumoniae), promozyme (derived from Novozymes, Bacillus acidopullulyticus) and the like can be used. Finally, the amount of maltose as the sugar composition can be adjusted to 30 to 75% by mass, depending on the degree of decomposition during liquefaction.
また、糖化酵素としてグルコアミラーゼを用いれば、グルコースが多く含まれる糖化液を得ることができる。この際、グルコアミラーゼとしては、β−グルカナーゼ活性度が０〜１０％のものであれば特に由来にはこだわらない。グルコースの量は、グルコアミラーゼの量と反応時間により調整することができるが、更に、枝切酵素の使用することにより増やすことができる。市販の酵素では、プルラナーゼが配合されたグルコアミラーゼがあり、これらを用いることにより作業性を上げることができる。最終的に、糖組成としてグルコース量は、液化の際の分解度にもよるが、５０質量％以上まで調整することができる。 If glucoamylase is used as the saccharifying enzyme, a saccharified solution containing a large amount of glucose can be obtained. In this case, the origin of glucoamylase is not particularly limited as long as the β-glucanase activity is 0 to 10%. The amount of glucose can be adjusted by the amount of glucoamylase and the reaction time, but can be further increased by using a debranching enzyme. Among commercially available enzymes, there are glucoamylases in which pullulanase is blended. By using these enzymes, workability can be improved. Finally, the amount of glucose as a sugar composition can be adjusted to 50% by mass or more, depending on the degree of decomposition during liquefaction.
この様にして製造した糖化液から遠心分離やフィルタープレスにより固液分離をして不溶部を除くことにより、液部を得る。このまま、本液部を後工程に使用しても良いが、ケイソウ土や活性炭などを助材とするろ過を行うことにより、清澄な液を得ることができる。また、濾過は遠心分離やフィルタープレスを行わない液を直接行うこともできる。固液分離した液や濾過液でも食品素材として利用することができるが、栄養豊富なため、わずかな微生物の混入でも微生物が増殖してしまい、運送するのが困難である。 A liquid part is obtained by carrying out solid-liquid separation from the saccharified liquid manufactured in this way by centrifugation or a filter press and removing an insoluble part. The liquid part may be used in the subsequent step as it is, but a clear liquid can be obtained by performing filtration using diatomaceous earth or activated carbon as an auxiliary material. Filtration can also be performed directly with a liquid that is not subjected to centrifugation or filter press. Although liquids and filtrates that have been separated into solid and liquid can be used as food materials, they are abundant in nutrients, so that even if a slight amount of microorganisms are mixed, microorganisms grow and are difficult to transport.
通常の穀物糖化物は、得られた清澄な濾過液を濃縮することにより、微生物に汚染されにくい糖化物とすることができるが、本発明品は濃縮中固形分濃度を上げるとゲル化してしまうため、微生物が増殖しないレベルまで濃縮することができない。 Ordinary cereal saccharified products can be made into saccharified products that are not easily contaminated by microorganisms by concentrating the clear filtrate obtained, but the product of the present invention gelates when the solid content concentration is increased during concentration. Therefore, it cannot be concentrated to a level at which microorganisms do not grow.
得られた清澄な濾過液を粉体とすることにより、微生物に汚染されにくい運送に適した糖化物に仕上ることができる。粉体化する方法は、特にこだわらないが、例えばスプレードライ法、凍結乾燥法がある。 By making the obtained clear filtrate into powder, it can be finished into a saccharified product suitable for transportation that is not easily contaminated by microorganisms. The method for powdering is not particularly limited, but there are, for example, a spray drying method and a freeze drying method.
以下、実施例により本発明を更に説明するが、本発明はこれら実施例によって限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited by these Examples.
［糖組成測定、ヨード発色テスト、および水分量の測定方法］
本発明において行った一連の分析は次の方法で行った。糖組成はＨＰＬＣにより測定した。ＨＰＬＣの構成は、ポンプが日本ウォーターズ製６００コントローラ、カラムが島津製作所製ＳＣＲ−１０１Ｎ、溶媒が純水、検出器が日本ウォーターズ製示差式屈折率計２４１４である。ヨード発色テストは、澱粉化学実験法（朝倉書店、１９７９年）に基づいて行った。すなわち、１２．７ｇヨウ素、４０ｇヨードカリを純水２５ｍｌに溶解させたものを純水で５倍に希釈した溶液を、固形分１０質量％に希釈した糖化物と混合し、発色の状況を目視で確認する。黄色を（−）、青色を（＋３）とし、その青色程度に応じて、その間を（＋１）、（＋２）とする。水分量は、常圧加熱乾燥法（五訂日本食品標準成分表分析マニュアルの解説、財団法人日本食品分析センター編）による。すなわち、アルミ容器にサンプルを入れ、１０５℃乾燥後の質量変化より求めた。
[Sugar composition measurement, iodine color development test, and water content measurement method]
A series of analyzes performed in the present invention was performed by the following method. The sugar composition was measured by HPLC. The configuration of the HPLC is that the pump is a 600 controller manufactured by Nippon Waters, the column is SCR-101N manufactured by Shimadzu Corporation, the solvent is pure water, and the detector is a differential refractometer 2414 manufactured by Nippon Waters. The iodine color development test was performed based on the starch chemical experiment method (Asakura Shoten, 1979). That is, a solution in which 12.7 g iodine and 40 g iodokari were dissolved in 25 ml of pure water was diluted 5 times with pure water and mixed with a saccharified product diluted to a solid content of 10% by mass. Check. Yellow is (−), blue is (+3), and (+1) and (+2) are set between them according to the degree of blue. The moisture content is determined by the atmospheric pressure heating and drying method (explanation of the 5th edition Japanese Food Standard Composition Table Analysis Manual, edited by the Japan Food Analysis Center). That is, the sample was put in an aluminum container and obtained from the mass change after drying at 105 ° C.
［β−グルカンの定量方法］
β−グルカンの定量は、２通り行うことにより、低分子から高分子までのβ−グルカン全量と分子量が１０５以上の高分子β−グルカンをそれぞれ測定する。低分子から高分子を含むβ−グルカン総量は、メガザイム社のβ−グルカン測定キットを用いて、ＭｃＣｌｅａｒｙ法（酵素法）により行った。すなわち、固形分約１ｇをメスフラスコを用いて１００ｍｌに希釈する。希釈した糖化物５ｍｌを遠心管に入れ、細かく粉砕した硫酸アンモニウム２．５ｇを加え、溶解する。４℃、２０時間静置した後、４℃、３０００ｒｐｍ、１０分遠心し、上清を除去する。残ったペレットに５０質量％エタノール水溶液１ｍＬを加え、激しく攪拌してペレットを懸濁させ、さらに５０質量％エタノール水溶液１０ｍＬ加えて混合する。再び、４℃、３０００ｒｐｍ、５分遠心し、上清を除去する。再度、ペレット懸濁、エタノール添加、遠心の操作を繰り返す。ペレットを２０ｍＭ リン酸ナトリウムバッファー（ｐＨ ６．５）４．８ｍＬに再溶解し、リケナーゼ溶液２００μＬを加え、４０℃、５分インキュベーションする。２５℃、３０００ｒｐｍ、１０分遠心した上清を１００μＬずつエッペンチューブに移す。チューブにβ-グルコシダーゼ溶液１００μＬ加えて４０℃、１５分反応させる。その後、チューブにｇｌｕｃｏｓｅ ｏｘｉｄａｓｅ／ｐｅｒｏｘｉｄａｓｅ（ＧＯＰＯＤ）を３ｍＬずつ加え、４０℃、２０分反応させる。５１０ｎｍの吸光度を測定する。なお、β-グルコシダーゼ溶液のかわりに５０ｍＭ 酢酸バッファー（ｐＨ４．０）１００μＬを加えたものをブランクとする。β−グルカン含有量は、次式により求めた。
β−グルカン（質量％）＝△Ａ×Ｆ×９×Ｄ
ここに、
△Ａ＝サンプルの吸光度−ブランクの吸光度
Ｆ＝１００／グルコース１００μｇの吸光度
Ｄ＝糖化物をメスフラスコで希釈した際の希釈倍率
[Method for quantifying β-glucan]
beta-glucan quantified by performing two ways, beta-glucan total amount and molecular weight of a low molecular until the polymer is measured 10 5 or more polymeric beta-glucan, respectively. The total amount of β-glucan including low to high molecules was measured by McCleary method (enzyme method) using a β-glucan measurement kit manufactured by Megazyme. That is, about 1 g of solid content is diluted to 100 ml using a volumetric flask. Place 5 ml of diluted saccharified product in a centrifuge tube and add 2.5 g of finely pulverized ammonium sulfate to dissolve. After leaving still at 4 ° C. for 20 hours, the supernatant is removed by centrifugation at 4 ° C., 3000 rpm for 10 minutes. Add 1 mL of 50 mass% aqueous ethanol solution to the remaining pellets, vigorously stir to suspend the pellets, and add 10 mL of 50 mass% aqueous ethanol solution and mix. Again, centrifuge at 4 ° C. and 3000 rpm for 5 minutes to remove the supernatant. Repeat the procedure of pellet suspension, ethanol addition, and centrifugation again. Redissolve the pellet in 4.8 mL of 20 mM sodium phosphate buffer (pH 6.5), add 200 μL of the lichenase solution, and incubate at 40 ° C. for 5 minutes. 100 μL of the supernatant obtained by centrifugation at 25 ° C., 3000 rpm, and 10 minutes is transferred to an Eppendorf tube. 100 μL of β-glucosidase solution is added to the tube and reacted at 40 ° C. for 15 minutes. Thereafter, 3 mL of glucooxidase / peroxidase (GOPOD) is added to the tube at 40 ° C. for 20 minutes. The absorbance at 510 nm is measured. A blank obtained by adding 100 μL of 50 mM acetate buffer (pH 4.0) instead of the β-glucosidase solution is used. The β-glucan content was determined by the following formula.
β-glucan (mass%) = ΔA × F × 9 × D
here,
ΔA = absorbance of the sample−absorbance of the blank F = 100 / absorbance of 100 μg of glucose D = dilution ratio when the saccharified product was diluted in the measuring flask
［分子量が１０５以上の高分子β−グルカンの定量］
分子量が１０５以上の高分子β−グルカンはコンゴレッド法（栃木農試研報、Ｎｏ４７、５７−６４、１９９８年）により行った。
[Determination of molecular weight of 10 5 or more polymeric β- glucan]
A molecular weight of 10 5 or more polymeric β- glucan Congo red method (Tochigi agriculture試研report, No47,57-64, 1998 years) was carried out by.
［β−グルカナーゼ活性度の測定方法］
β−グルカナーゼ活性度は、次の方法により測定される。β−グルカンの標準（大麦由来、シグマ社）を５ｍｇ／ｍｌとなるよう純水で溶解して、β−グルカン溶液を作製する。酵素サンプルは純水で５ｍｇ／ｍｌとなるよう希釈する。β−グルカン溶液と希釈した酵素サンプルを試験管内で混ぜて、５０℃恒温槽に１４時間インキュベーションした後、氷冷する。コンゴレッド法により、溶液中の分子量１０５以上のβ−グルカンを測定する。酵素サンプルのかわりに、純水で同様の操作を行ったものをブランクとした。β−グルカナーゼ活性度は次式より求めた。
β−グルカナーゼ活性度（％）＝（１−Ｂ／Ｂ０）×１００
ここに、
Ｂ＝酵素サンプル中のβ−グルカン濃度
Ｂ０＝ブランク中のβ−グルカン濃度
[Method for Measuring β-Glucanase Activity]
β-glucanase activity is measured by the following method. A β-glucan standard (derived from barley, Sigma) is dissolved in pure water so as to be 5 mg / ml to prepare a β-glucan solution. The enzyme sample is diluted with pure water to 5 mg / ml. The β-glucan solution and the diluted enzyme sample are mixed in a test tube, incubated in a thermostat at 50 ° C. for 14 hours, and then cooled on ice. The Congo red method to measure the molecular weight 105 or more β- glucan in solution. A blank obtained by performing the same operation with pure water instead of the enzyme sample was used. The β-glucanase activity was determined from the following equation.
β-glucanase activity (%) = (1−B / B 0 ) × 100
here,
B = β-glucan concentration in enzyme sample B 0 = β-glucan concentration in blank
［β−グルカナーゼ活性度の測定例］
β−グルカナーゼ活性度の測定法に基づいて、タンパク質分解酵素、液化酵素、糖化酵素のβ−グルカナーゼ活性度を測定した。タンパク質分解酵素として、スミチームＦＰ、スミチームＰ（以上、新日本化学社製 「スミチーム」（登録商標））、プロテアーゼＰアマノ３Ｇ（天野エンザイム社製）、サモアーゼＰＣ１０Ｆ（大和化成製 「サモアーゼ」（登録商標））を、液化酵素として、クライスラ−ゼＴ１０Ｓ（大和化成製 「クライスターゼ」（登録商標））、ＢＡＮ（ノボザイムズ製）を、糖化酵素として、βアミラーゼ＃１５００Ｓ（ナガセケムテックス製）、プルラナーゼ「アマノ」３（天野エンザイム製）、ハイマルトシンＧＬ（エイチビィアイ製 「ハイマルトシン」（登録商標））、シルバラーゼ（大和化成製 「シルバラーゼ」（登録商標））をそれぞれ用いた。結果を表１に示す。
[Measurement example of β-glucanase activity]
Based on the method for measuring β-glucanase activity, β-glucanase activity of proteolytic enzymes, liquefaction enzymes, and saccharifying enzymes was measured. As proteolytic enzymes, Sumiteam FP, Sumiteam P (hereinafter, “Sumiteam” (registered trademark) manufactured by Shin Nippon Chemical Co., Ltd.), Protease P Amano 3G (manufactured by Amano Enzyme), Samoaase PC10F (“Samoase” manufactured by Yamato Kasei) (registered trademark) )) As a liquefying enzyme, Chrysalase T10S (“Chrystase” (registered trademark) manufactured by Daiwa Kasei) and BAN (manufactured by Novozymes) as β-amylase # 1500S (manufactured by Nagase ChemteX), pullulanase “ Amano 3 (manufactured by Amano Enzyme), Hymaltocin GL (manufactured by HIBI, “Himaltocin” (registered trademark)), and Silvalase (manufactured by Daiwa Kasei “Sylvalase” (registered trademark)) were used. The results are shown in Table 1.
表１から、タンパク質分解酵素として、スミチームＰ、サモアーゼＰＣ１０Ｆは、β−グルカナーゼ活性度が１０％以下のため、本発明に使用することができるが、スミチームＦＰ、プロテアーゼＰアマノ３Ｇは、１０％以上のβ−グルカナーゼ活性度をもつため、本発明には用いることができない。また、液化酵素として、クライスターゼＴ１０Ｓは、β−グルカナーゼ活性度が１０％以下のため、本発明に使用することができるが、ＢＡＮは、１０％以上のβ−グルカナーゼ活性度をもつため、本発明には用いることができない。さらに、糖化酵素として、βアミラーゼ＃１５００Ｓ、プルラナーゼ「アマノ」３、シルバラーゼは、β−グルカナーゼ活性度が１０％以下のため、本発明に使用することができるが、ハイマルトシンＧＬは、１０％以上のβ−グルカナーゼ活性度をもつため、本発明には用いることができない。 From Table 1, as proteolytic enzymes, Sumiteam P and Samoase PC10F can be used in the present invention because the β-glucanase activity is 10% or less, but Sumiteam FP and Protease P Amano 3G are 10% or more. Therefore, it cannot be used in the present invention. As a liquefying enzyme, Christase T10S has a β-glucanase activity of 10% or less and can be used in the present invention. However, BAN has a β-glucanase activity of 10% or more. It cannot be used in the invention. Further, β-amylase # 1500S, pullulanase “Amano” 3, and sylvalase as saccharifying enzymes can be used in the present invention because β-glucanase activity is 10% or less, but hymaltocin GL is 10% or more. Since it has β-glucanase activity, it cannot be used in the present invention.
［実施例１］
大麦（栽培品種：ＣＤＣファイバー）の粉砕物５０ｇを純水９５０ｇに分散させる。これに苛性ソーダを加えて、ｐＨを６．０に調整する。これに、スミチームＰ（新日本化学工業製、Ｂａｃｉｌｌｕｓ Ｓｕｂｔｉｌｉｓ由来プロテアーゼ）を１５０００Ｕ添加し、５５℃で１時間反応する。この後、クライスターゼＴ１０Ｓ（大和化成製、Ｂａｃｉｌｌｕｓ Ｓｕｂｔｉｌｉｓ由来α−アミラーゼ）を２５００Ｕ添加した後、加熱して１時間かけて９０℃に昇温し、９０℃で１時間反応する。次に、６０℃まで冷却し、ｐＨを変えずに糖化酵素としてβアミラーゼ＃１５００Ｓ（ナガセケムテックス製、大豆由来）を５００Ｕ、プルラナーゼ「アマノ」３（天野エンザイム製、Ｋｌｅｂｓｉｅｌｌａ ｐｎｅｕｍｏｎｉａｌ由来）を５００Ｕ添加し、６０℃で２４時間反応する。反応液を７０℃に加熱し、これをろ紙Ｎｏ．５Ｃ（東洋濾紙製）上に１０ｇの珪藻土＃８００Ｓ（昭和化学工業製）をコートしたヌッチェに通液する。このろ過液を孔径５μのニトロセルロースタイプメンブランフィルター（東洋濾紙製）に通液した後、スプレードライ試験装置Ｌ−８i（大川原化工機製）にかけた。運転条件は、原液温度８０℃、ディスクＭＣ−５０、回転数２５０００ｒｐｍ、入口温度１５０℃で行い、粉体状の糖化物が得られた。結果を表２に示す。
[Example 1]
50 g of pulverized barley (cultivar: CDC fiber) is dispersed in 950 g of pure water. Caustic soda is added to this to adjust the pH to 6.0. To this, 15000 U of Sumiteam P (manufactured by Shinnippon Kagaku Kogyo Co., Ltd., Bacillus Subtilis- derived protease) is added and reacted at 55 ° C. for 1 hour. After this, 2500 U of Christase T10S (manufactured by Daiwa Kasei, Bacillus Subtilis- derived α-amylase) is added, heated to 90 ° C. over 1 hour, and reacted at 90 ° C. for 1 hour. Next, after cooling to 60 ° C., 500 U of β-amylase # 1500S (manufactured by Nagase ChemteX, derived from soybeans) and 500 U of pullulanase “Amano” 3 (manufactured by Amano Enzyme, derived from Klebsiella pneumonial ) are added as saccharifying enzymes without changing the pH. And react at 60 ° C. for 24 hours. The reaction solution was heated to 70 ° C. The solution is passed through Nutsche in which 10 g of diatomaceous earth # 800S (manufactured by Showa Chemical Industry) is coated on 5C (manufactured by Toyo Roshi). This filtrate was passed through a nitrocellulose type membrane filter (manufactured by Toyo Filter Paper) having a pore size of 5 μm, and then applied to a spray dry test apparatus L-8i (manufactured by Okawara Kako). The operating conditions were a stock solution temperature of 80 ° C., a disk MC-50, a rotation speed of 25000 rpm, and an inlet temperature of 150 ° C., and a powdery saccharified product was obtained. The results are shown in Table 2.
［実施例２］
大麦（栽培品種：ＣＤＣファイバー）の粉砕物１００ｇを純水９００ｇに分散させる。これに苛性ソーダを加えて、ｐＨを６．０に調整する。これに、スミチームＰを３００００Ｕ添加し、５５℃で１時間反応する。この後、クライスターゼＴ１０Ｓ（大和化成製、Ｂａｃｉｌｌｕｓ Ｓｕｂｔｉｌｉｓ由来α−アミラーゼ）を５０００Ｕ添加した後、加熱して１時間かけて９０℃に昇温し、９０℃で１時間反応する。次に、６０℃まで冷却し、ｐＨを変えずにβアミラーゼ＃１５００Ｓ（ナガセケムテックス製、大豆由来）を１０００Ｕ、プルラナーゼ「アマノ」３（天野エンザイム製、Ｋｌｅｂｓｉｅｌｌａ ｐｎｅｕｍｏｎｉａｌ由来）を１０００Ｕ添加し、６０℃で２４時間反応する。その後、実施例１と同様の操作を行うことにより糖化物が得られた。結果を表２に示す。
[Example 2]
100 g of pulverized barley (cultivar: CDC fiber) is dispersed in 900 g of pure water. Caustic soda is added to this to adjust the pH to 6.0. To this, 30000 U of Sumiteam P is added and reacted at 55 ° C. for 1 hour. Thereafter, 5000 U of Christase T10S (manufactured by Daiwa Kasei Co., Ltd., Bacillus Subtilis- derived α-amylase) is added, heated to 90 ° C. over 1 hour, and reacted at 90 ° C. for 1 hour. Next, it is cooled to 60 ° C., and 1000 U of β-amylase # 1500S (manufactured by Nagase ChemteX, derived from soybean) and 1000 U of pullulanase “Amano” 3 (manufactured by Amano Enzyme, derived from Klebsiella pneumonial ) are added without changing the pH, and 60 React at 24 ° C. for 24 hours. Thereafter, the same operation as in Example 1 was performed to obtain a saccharified product. The results are shown in Table 2.
［実施例３］
大麦（品種：ミサトゴールデン）の粉砕物を使用し、糖化酵素としてβアミラーゼ＃１５００Ｓ（ナガセケムテックス製、大豆由来）を５００Ｕ添加するのみであることの他は、実施例１と同様の操作を行うことにより糖化物が得られた。結果を表３に示す。
[Example 3]
The same operation as in Example 1 was performed, except that barley (variety: Misato Golden) was ground and only 500 U of β-amylase # 1500S (manufactured by Nagase ChemteX, derived from soybeans) was added as a saccharifying enzyme. The saccharified product was obtained by carrying out. The results are shown in Table 3.
［実施例４］
麦として、オーツ麦（品種：ヒダカ）の粉砕物を使用し、タンパク質分解酵素として、サモアーゼＰＣ１０Ｆ（大和化成製、Ｂａｃｉｌｌｕｓ ｓｔｅａｒｏｔｈｅｒｍｏｐｈｉｌｕｓ由来プロテアーゼ）を使用する他は、実施例１と同様の処理を行うことにより、糖化物が得られた。結果を表２に示す。
[Example 4]
The same treatment as in Example 1 is carried out except that a ground product of oat (variety: Hidaka) is used as the wheat and Samoaase PC10F (manufactured by Daiwa Kasei Co., Ltd., Bacillus stearothermophilus protease) is used as the proteolytic enzyme. As a result, a saccharified product was obtained. The results are shown in Table 2.
［実施例５］
糖化酵素としてβアミラーゼ＃１５００Ｓ、プルラナーゼ「アマノ」３の代わりに、シルバラーゼ（大和化成製、Ｂａｃｉｌｌｕｓ ｓｐ．およびＡｓｐｅｒｇｉｌｕｓ ｎｉｇｅｒ由来）１５００Ｕ使用する他は、実施例１と同様の処理を行うことにより、糖化物が得られた。結果を表２に示す。
[Example 5]
Saccharification is carried out in the same manner as in Example 1 except that 1500 U is used instead of β-amylase # 1500S and pullulanase “Amano” 3 as a saccharification enzyme, except for using 1500 U of Silvalase (from Daiwa Kasei, Bacillus sp. And Aspergillus niger ) . Things were obtained. The results are shown in Table 2.
［比較例１］
大麦（栽培品種：ＣＤＣファイバー）の粉砕物３００ｇを純水７００ｇに分散させる。これに苛性ソーダを加えて、ｐＨを６．０に調整する。これに、スミチームＰを９００００Ｕ添加し、５５℃で１時間反応する。この後、クライスターゼＴ１０Ｓ（大和化成製、Ｂａｃｉｌｌｕｓ Ｓｕｂｔｉｌｉｓ由来α−アミラーゼ）を１５０００Ｕ添加した後、加熱して１時間かけて９０℃に昇温し、９０℃で１時間反応した後、６０℃まで冷却し、ｐＨを変えずにβアミラーゼ＃１５００Ｓ（ナガセケムテックス製、大豆由来）を３０００Ｕ、プルラナーゼ「アマノ」３（天野エンザイム製、Ｋｌｅｂｓｉｅｌｌａ ｐｎｅｕｍｏｎｉａｌ由来）を３０００Ｕ添加し、６０℃で２４時間反応する。その後、実施例１と同様の操作を行うことにより糖化物が得られた。実施例１と同様の操作を行うことにより糖化物が得られた。結果を表２に示す。
[Comparative Example 1]
Disperse 300 g of barley (cultivar: CDC fiber) in 700 g of pure water. Caustic soda is added to this to adjust the pH to 6.0. To this, 90000 U of Sumiteam P is added and reacted at 55 ° C. for 1 hour. After that, after adding 15000 U of Christase T10S (manufactured by Daiwa Kasei, Bacillus Subtilis- derived α-amylase), the mixture was heated to 90 ° C. over 1 hour, reacted at 90 ° C. for 1 hour, and then to 60 ° C. Cool, add 3000 U β-amylase # 1500S (manufactured by Nagase ChemteX, derived from soybean) and 3000 U pullulanase “Amano” 3 (manufactured by Amano Enzyme, Klebsiella pneumonial ) without changing pH, and react at 60 ° C. for 24 hours. . Thereafter, the same operation as in Example 1 was performed to obtain a saccharified product. By performing the same operation as in Example 1, a saccharified product was obtained. The results are shown in Table 2.
［比較例２］
大麦（栽培品種：ＣＤＣファイバー）の粉砕物５０ｇを純水９５０ｇに分散させる。これに苛性ソーダを加えて、ｐＨを６．０に調整する。この後、タンパク質分解酵素を添加せずに、５５℃で１時間反応する。クライスターゼＴ１０Ｓ（大和化成製、Ｂａｃｉｌｌｕｓ Ｓｕｂｔｉｌｉｓ由来α−アミラーゼ）を２５００Ｕ添加した後、加熱して１時間かけて９０℃に昇温し、９０℃で１時間反応した後、実施例１と同様の操作を行うことにより糖化物が得られた。結果を表２に示す。
[Comparative Example 2]
50 g of pulverized barley (cultivar: CDC fiber) is dispersed in 950 g of pure water. Caustic soda is added to this to adjust the pH to 6.0. Then, it reacts at 55 degreeC for 1 hour, without adding a proteolytic enzyme. After adding 2500 U of Christase T10S (manufactured by Daiwa Kasei, Bacillus Subtilis- derived α-amylase), the mixture was heated to 90 ° C. over 1 hour, reacted at 90 ° C. for 1 hour, and then the same as in Example 1 A saccharified product was obtained by performing the operation. The results are shown in Table 2.
［比較例３］
大麦（栽培品種：ＣＤＣファイバー）の粉砕物５０ｇを純水９５０ｇに分散させる。これに苛性ソーダを加えて、ｐＨを６．０に調整する。この後、タンパク質分解酵素としてタンパク質分解酵素アマノＰアマノ３Ｇ（天野エンザイム製、Ａｓｐｅｒｇｉｌｌｕｓ ｍｅｌｌｅｕｓ由来プロテアーゼ）を１００００Ｕ添加して５５℃で１時間反応した後、実施例１と同様の操作を行うことにより糖化物が得られた。結果を表２に示す。
[Comparative Example 3]
50 g of pulverized barley (cultivar: CDC fiber) is dispersed in 950 g of pure water. Caustic soda is added to this to adjust the pH to 6.0. Thereafter, 10000 U of proteolytic enzyme Amano P Amano 3G (manufactured by Amano Enzyme, Aspergillus melleus- derived protease) is added as a proteolytic enzyme, reacted at 55 ° C. for 1 hour, and then subjected to the same operation as in Example 1 for saccharification. Things were obtained. The results are shown in Table 2.
［比較例４］
大麦（栽培品種：ミサトゴールデン）の粉砕物５０ｇを純水９５０ｇに分散させる。スミチームＰを１５０００Ｕ添加し、５５℃で１時間反応する。この後、ＢＡＮ（ノボザイムズ製、Ｂａｃｉｌｌｕｓ Ｓｕｂｔｉｌｉｓ由来α−アミラーゼ）を２０００Ｕ添加した後、実施例１と同様の操作を行うことにより糖化物が得られた。結果を表２に示す。
[Comparative Example 4]
50 g of pulverized barley (cultivar: Misato Golden) is dispersed in 950 g of pure water. Add 15000 U of Sumiteam P and react at 55 ° C. for 1 hour. Thereafter, 2000 U of BAN (manufactured by Novozymes , Bacillus Subtilis- derived α-amylase) was added, and the same operation as in Example 1 was performed to obtain a saccharified product. The results are shown in Table 2.
［比較例５］
大麦（栽培品種：ミサトゴールデン）の粉砕物５０ｇを純水９５０ｇに分散させる。スミチームＰを１５０００２５００Ｕ添加し、５５℃で１時間反応する。この後、クライスターゼＴ１０Ｓ（大和化成製、Ｂａｃｉｌｌｕｓ Ｓｕｂｔｉｌｉｓ由来α−アミラーゼ）を２５００Ｕ添加した後、加熱して１時間かけて９０℃に昇温し、９０℃で１時間反応した後、６０℃まで冷却し、ｐＨを変えずにハイマルトシン（エイチビィアイ製、小麦由来β−アミラーゼ）を５００Ｕ、プルラナーゼ「アマノ」３を５００Ｕ添加した後、実施例１と同様の操作を行うことにより糖化物が得られた。結果を表２に示す。
[Comparative Example 5]
50 g of pulverized barley (cultivar: Misato Golden) is dispersed in 950 g of pure water. Add 150002500 U of Sumiteam P and react at 55 ° C. for 1 hour. After this, 2500 U of Christase T10S (manufactured by Daiwa Kasei, Bacillus Subtilis- derived α-amylase) was added, heated to 90 ° C. over 1 hour, reacted at 90 ° C. for 1 hour, and then to 60 ° C. After cooling and adding 500 U of hymaltocin (produced by HIBI, wheat-derived β-amylase) and 500 U of pullulanase “Amano” 3 without changing the pH, the same operation as in Example 1 was performed to obtain a saccharified product. . The results are shown in Table 2.
表２から、穀物の仕込み濃度が２０重量％より高い場合、もしくは液化反応・糖化反応を行う前にタンパク質分解酵素を用いたタンパク質分解反応を行わない場合は、ヨード発色テストが（−）にならず未分解の澱粉が残ることがわかる。また、製造に使用する液化酵素、糖化酵素、タンパク質分解酵素として１０％以上のβ−グルカナーゼ活性度をもつ酵素を用いた場合、β−グルカンが分解して十分なβ−グルカンが得られないことがわかる。 From Table 2, if the feed concentration of cereal is higher than 20% by weight, or if the proteolytic reaction using a proteolytic enzyme is not performed before the liquefaction reaction or saccharification reaction, the iodine color development test should be (-). It can be seen that undegraded starch remains. In addition, when an enzyme having a β-glucanase activity of 10% or more is used as a liquefying enzyme, saccharifying enzyme, or proteolytic enzyme used for production, β-glucan is degraded and sufficient β-glucan cannot be obtained. I understand.
Claims (7)
前記タンパク質分解酵素、液化酵素及び糖化酵素は、いずれも下記β−グルカナーゼ活性度が０〜１０％であり、The proteolytic enzyme, liquefying enzyme and saccharifying enzyme all have the following β-glucanase activity of 0 to 10%,
前記タンパク質分解酵素が、スミチームＰ（新日本化学社製、「スミチーム」（登録商標））及びサモアーゼＰＣ１０Ｆ（大和化成製、「サモアーゼ」（登録商標））の少なくとも一方であり、The proteolytic enzyme is at least one of Sumiteam P (manufactured by Shin Nippon Chemical Co., Ltd., “Sumiteam” (registered trademark)) and Samoaase PC10F (manufactured by Yamato Kasei, “Samoase” (registered trademark)),
前記液化酵素が、クライスターゼＴ１０Ｓ（大和化成製、「クライスターゼ」（登録商標））であり、The liquefying enzyme is Christase T10S (manufactured by Daiwa Kasei, “Chrytase” (registered trademark)),
前記糖化酵素が、βアミラーゼ＃１５００Ｓ（ナガセケムテックス製）、プルラナーゼ「アマノ」３（天野エンザイム製）及びシルバラーゼ（大和化成製、「シルバラーゼ」（登録商標））からなる群から選ばれる少なくとも１種であり、The saccharifying enzyme is at least one selected from the group consisting of β-amylase # 1500S (manufactured by Nagase ChemteX), pullulanase “Amano” 3 (manufactured by Amano Enzyme) and silvalase (manufactured by Daiwa Kasei, “Sylvalase” (registered trademark)). And
得られる穀物糖化物が、１〜１５質量％のβ−グルカンを含有し、下記ヨード発色テストが（−）であることを特徴とする穀物糖化物の製造方法。A method for producing a saccharified cereal according to claim 1, wherein the saccharified cereal obtained contains 1 to 15% by mass of β-glucan and the following iodine color development test is (-).
（β−グルカナーゼ活性度）(Β-glucanase activity)
β−グルカナーゼ活性度は、以下の方法で求められる値である。The β-glucanase activity is a value determined by the following method.
β−グルカンを５ｍｇ／ｍｌとなるように純水に溶解したβ−グルカン溶液と、酵素サンプルを５ｍｇ／ｍｌとなるように純水で希釈したものとを試験管内で混ぜ、５０℃の恒温槽で１４時間インキュベーションした後に氷冷し、コンゴレッド法（栃木農試研報、Ｎｏ４７、５７−６４、１９９８年）により、溶液中の分子量１０A β-glucan solution obtained by dissolving β-glucan in pure water to 5 mg / ml and an enzyme sample diluted with pure water to 5 mg / ml are mixed in a test tube, and a constant temperature bath at 50 ° C. After 14 hours of incubation in ice, the mixture was ice-cooled, and the molecular weight in the solution was 10 by the Congo Red method (Tochigi Agricultural Research Laboratories, No 47, 57-64, 1998). ５5 以上のβ−グルカンの濃度を測定する。また、前記酵素サンプルの代わりに純水を使用して同様の操作を行ったブランク中の分子量１０The above β-glucan concentration is measured. Moreover, the molecular weight 10 in the blank which performed the same operation using the pure water instead of the said enzyme sample. ５5 以上のβ−グルカンの濃度を同様に測定し、下式によりβ−グルカナーゼ活性度を求める。The above β-glucan concentration is measured in the same manner, and the β-glucanase activity is determined by the following equation.
β−グルカナーゼ活性度（％）＝（１−Ｂ／Ｂβ-glucanase activity (%) = (1−B / B ０0 ）×１００) × 100
ただし、前記式中、Ｂは酵素サンプルを使用した溶液中の分子量１０However, in the said formula, B is molecular weight 10 in the solution which uses an enzyme sample. ５5 以上のβ−グルカン濃度であり、ＢΒ-glucan concentration above, B ０0 はブランク中の分子量１０Is the molecular weight of 10 in the blank ５5 以上のβ−グルカンの濃度である。It is the density | concentration of the above (beta) -glucan.
（ヨード発色テスト）(Iodine coloring test)
１２．７ｇのヨウ素と、４０ｇのヨードカリを純水２５ｍｌに溶解させ、さらに純水で５倍に希釈した溶液を、固形分が１０質量％となるように希釈した穀物糖化物と混合し、発色の状況を目視で確認し、黄色のものを（−）とする。A solution obtained by dissolving 12.7 g of iodine and 40 g of iodokari in 25 ml of pure water and further diluting 5 times with pure water is mixed with cereal saccharified product diluted to a solid content of 10% by mass, and coloring is performed. Confirm the situation of () visually and make the yellow one (-).
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