JP2005075957A - Water-soluble chitosan - Google Patents
Water-soluble chitosan Download PDFInfo
- Publication number
- JP2005075957A JP2005075957A JP2003309224A JP2003309224A JP2005075957A JP 2005075957 A JP2005075957 A JP 2005075957A JP 2003309224 A JP2003309224 A JP 2003309224A JP 2003309224 A JP2003309224 A JP 2003309224A JP 2005075957 A JP2005075957 A JP 2005075957A
- Authority
- JP
- Japan
- Prior art keywords
- chitosan
- molecular weight
- water
- soluble
- taste
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Abstract
Description
本発明は、呈味が改善された中性域ないしアルカリ域でも澄明で安定かつ抗酸化作用および免疫活性を有する水溶性キトサンに関するもので、食品用途であるところの機能性飲料の素材提供に関する。 The present invention relates to a water-soluble chitosan that is clear and stable in neutral to alkaline regions with improved taste and has antioxidant and immunological activities, and relates to the provision of a functional beverage material for food use.
日本の死亡原因の上位は（平成14年）、悪性新生物（31.0％）、心疾患（15.5％）、脳血管疾患（13.2％）である。また、日本は高齢社会であり、総人口に対する65歳以上の高齢者の占める割合も年々増加している（1950年4.9％、1980年9.1％、1990年12.0％、2000年17.3％、・・・、2020年35.7％、非特許文献１）。健康で長生きするためには、生活習慣を改善し、ストレスをなくして、身体の免疫力をつけること、動脈硬化や癌、心臓病、糖尿病等のいわゆる生活習慣病にかからないように予防することが大切である。これら生活習慣病に関わる疾病の多くは、近年の医学、生化学等の分野の研究によると、ストレス、アルコール、過酸化物、薬物、紫外線等によって生体内で生成する活性酸素やフリーラジカル（一重項酸素1Ｏ2、スーパーオキシドＯ2-、水酸化ラジカルＯＨ・、過酸化水素Ｈ2Ｏ2等、以下「活性酸素」と略す）が起因しているとされている。すなわち、活性酸素は、生体の感染等に対する防御機構を受け持つ反面、コントロールできない余剰に生成した活性酸素は、非常に不安定で酸化力を持ち、核酸やタンパク質を変性させ、脂質の過酸化反応等を促進し、生活習慣病や癌、難病等の疾病を引き起こす原因となることがわかっている（非特許文献２）。人体にとって副作用がなく安全で、安定供給でき、各種障害を起こす活性酸素の消去効果に優れた抗酸化作用や免疫活性の有する素材の開発がこれまで以上に望まれている。
キトサンは、カニやエビなどの甲殻類の殻、イカの軟膏、昆虫の骨格、菌体の細胞壁に含まれるキチンを40〜50％濃度の苛性ソーダ中で加熱し得られる天然由来の素材であって、分子量が数十万以上の陽イオン性高分子ポリマーである。キトサンの特徴は、水に不溶、塩酸や硝酸などの無機酸、酢酸や乳酸などの有機酸などに溶ける。生分解性に優れ、毒性は極めて低い（非特許文献３）。キトサンの用途は、高分子凝集剤、食品日保向上剤、シャンプーやリンスの化粧品基材、繊維処理剤、医薬研究材料（ドラッグデリバリーシステム）、農業資材など幅広い。また、その生理機能は、分子量数10万以上のキトサンやオリゴ糖を用いた実験で、生活習慣病に関わるコレステロール低下や脂肪排泄、血圧降下、血糖値上昇抑制、免疫賦活、金属イオン選択吸着などがある（非特許文献４）。しかし、キトサンは、粘性が非常に高く、キトサンのアミノ基に起因する呈味の悪さと、中性域ないしアルカリ域では不溶化し、タンパク質やアミノ酸、核酸、酸性多糖などと凝集沈殿を起こしたりすることもある。
The top causes of death in Japan (2002) are malignant neoplasms (31.0%), heart disease (15.5%), and cerebrovascular disease (13.2%). In addition, Japan is an aging society, and the proportion of elderly people aged 65 and over is increasing year by year (4.9% in 1950, 9.1% in 1980, 12.0% in 1990, 17.3% in 2000, ...・ 2020, 35.7%, non-patent document 1). In order to live a healthy and long life, it is necessary to improve lifestyle, eliminate stress, improve body immunity, and prevent so-called lifestyle-related diseases such as arteriosclerosis, cancer, heart disease, diabetes, etc. It is important. Many of these diseases related to lifestyle-related diseases are based on recent research in the fields of medicine, biochemistry, etc., and active oxygen and free radicals (single-fold) generated in vivo by stress, alcohol, peroxides, drugs, ultraviolet rays, etc. The term oxygen 1 O 2 , superoxide O 2- , hydroxyl radical OH., Hydrogen peroxide H 2 O 2 and the like (hereinafter abbreviated as “active oxygen”) are considered to be caused. In other words, active oxygen is responsible for defense mechanisms against living body infections, etc., but excessively generated active oxygen that cannot be controlled is extremely unstable and has oxidizing power, denatures nucleic acids and proteins, lipid peroxidation, etc. It is known that it causes a disease such as lifestyle-related diseases, cancer, and intractable diseases (Non-patent Document 2). The development of a material having antioxidative action and immunological activity that has no side effects for the human body, is safe, can be stably supplied, and has an excellent scavenging effect on active oxygen causing various disorders has been desired more than ever.
Chitosan is a naturally-occurring material that can be obtained by heating chitin contained in crustacean shells such as crabs and shrimps, squid ointment, insect skeletons, and cell walls of bacterial cells in caustic soda with a concentration of 40-50%. A cationic polymer having a molecular weight of several hundred thousand or more. Chitosan is insoluble in water, soluble in inorganic acids such as hydrochloric acid and nitric acid, and organic acids such as acetic acid and lactic acid. Excellent biodegradability and extremely low toxicity (Non-patent Document 3). Chitosan has a wide range of uses, including polymer flocculants, food day protection agents, cosmetic bases for shampoos and rinses, fiber treatment agents, pharmaceutical research materials (drug delivery systems), and agricultural materials. In addition, its physiological function is based on experiments using chitosan and oligosaccharides with a molecular weight of 100,000 or more, such as cholesterol lowering and fat excretion associated with lifestyle-related diseases, blood pressure lowering, suppression of blood sugar level increase, immune activation, selective adsorption of metal ions, etc. (Non-Patent Document 4). However, chitosan has a very high viscosity, and has a bad taste due to the amino group of chitosan and insolubilizes in neutral or alkaline range, causing aggregation and precipitation with proteins, amino acids, nucleic acids, acidic polysaccharides, etc. Sometimes.
飲料に適用するためには「溶解性」や「呈味」の改善が極めて重要である。溶解性を解決する手段としては、これまでにアミノ基や分子量を制御するなどの検討が行われてはいた。具体的には、均一反応系でキチンを脱アセチル反応したもの（特許文献１）、キトサンを無水酢酸でアセチル反応（合成）したもの（特許文献２）、これらは低カチオン度の30〜60％アセチル体の高分子ポリマーであって、一般のキトサンに比べるとアミノ基含量が少ないのでカチオンとしての機能が十分に発揮できるものではなかった。しかも合成品であれば食品としては利用することはできなかった。また、低分子化による溶解性の改善も多く検討された。その大部分は亜硝酸や臭素酸などの酸化的に分解するもので（特許文献３、４）、中性域ないしアルカリ域で可溶であっても非常に低分子であり、アミノ基含量の減少やアンヒドロマンノースの生成などの理由により食品への利用に際しては安全性への十分な配慮も必要であった。これに対して酵素による方法がある（特許文献５、６）。最近では市販でキトサン分解酵素も手に入り易くなっており、水溶性で溶解性が高く呈味が良好である低級オリゴ糖が工業生産されている。
低分子の生理機能は、主としてグルコサミンやオリゴ糖であって、それ以上の分子量になると分子量１万以上による報告（非特許文献５、６）があるのみであって分子量2,000〜10,000未満の機能は明らかにはされていない。キトサンオリゴ糖の生物活性は、５糖よりは６糖に活性があるとしており、アセチルグルコサミンの６量体の生物活性においてはマウス非経口投与による移植腫瘍細胞の増殖抑制効果やマクロファージ活性化等が報告されている（非特許文献７、８）。オリゴ糖のレベルになると、重合度がある程度あった方が活性に寄与することが言われているが、本願に関わる10糖や15糖以上の活性について詳細なデータは見当たらない。
すなわち、オリゴ糖よりも平均重合度が高く、溶解性や呈味が良好で、しかも生理機能を有した分解組成物に関して詳細な検討はこれまでされていなかった。
また、キトサンは健康食品に利用する場合、カプセルや錠剤の形態でよく使われるが、飲料の形態ではほとんど利用されることはなかった。
Physiological functions of low molecules are mainly glucosamine and oligosaccharides. If the molecular weight is higher than that, there are only reports with molecular weights of 10,000 or more (Non-Patent Documents 5 and 6). It is not clear. The bioactivity of chitosan oligosaccharides is more active on hexasaccharides than on pentasaccharides, and the bioactivity of hexamers of acetylglucosamine includes the effects of inhibiting the growth of transplanted tumor cells and macrophage activation by parenteral administration in mice. It has been reported (Non-Patent Documents 7 and 8). Although it is said that a certain degree of polymerization contributes to the activity at the oligosaccharide level, no detailed data on the activity of 10 sugars or 15 sugars or more related to the present application is found.
That is, a detailed examination has not been made so far regarding a decomposition composition having an average degree of polymerization higher than that of oligosaccharide, good solubility and taste, and having a physiological function.
In addition, chitosan is often used in the form of capsules and tablets when used in health foods, but is rarely used in the form of beverages.
生活習慣病の予防、美容や老化予防といった関心が高まる中で、健康食品素材が注目されている。キトサンの生理機能は、分子量数10万以上のキトサンやオリゴ糖を用いた実験で、生活習慣病に関わるコレステロール低下や脂肪排泄、血圧降下、血糖値上昇抑制、免疫賦活、金属イオン選択吸着などがある。キトサンの期待はこれまで以上に大きく、キトサンの機能性食品を目的とした研究開発が精力的に行われている。健康食品として利用する場合、カプセルや錠剤があるが、飲料であれば、より手軽に摂取できて、ダイレクトに生活習慣病予防、老化予防や美容効果が期待できる。しかし飲料へ利用するには「機能」のほかに「溶解性」や「呈味」の点が極めて重要となる。従来のキトサンでは、高分子のために希酸に溶解した場合に粘性が極めて高く水に溶け難く、中性域ないしアルカリ域では不溶化したり、タンパク質や核酸、酸性多糖と凝集沈殿を起こしたりするため難点があった。さらに、飲料に利用する場合において呈味にも重大な問題があり、効能効果が期待できるほどの量を配合することはこれまでできなかった。 Health food materials are attracting attention as interest in prevention of lifestyle-related diseases, beauty and aging prevention increases. Chitosan's physiological functions are based on experiments using chitosan and oligosaccharides with a molecular weight of 100,000 or more, such as cholesterol lowering and fat excretion, blood pressure lowering, blood glucose lowering suppression, immunostimulation, and metal ion selective adsorption related to lifestyle-related diseases. is there. The expectations of chitosan are greater than ever, and research and development aimed at functional foods of chitosan are being energetically conducted. When used as a health food, there are capsules and tablets, but if it is a beverage, it can be taken more easily, and direct prevention of lifestyle-related diseases, prevention of aging and beauty effects can be expected. However, in addition to “function”, “solubility” and “taste” are extremely important for use in beverages. Conventional chitosan is extremely viscous and difficult to dissolve in water when dissolved in dilute acid due to high molecular weight. It becomes insoluble in neutral or alkaline regions, and causes aggregation and precipitation with proteins, nucleic acids, and acidic polysaccharides. Therefore, there was a difficulty. Furthermore, when used for beverages, there is a serious problem in taste, and it has not been possible to formulate such an amount that an effect can be expected.
本発明の目的は、機能性飲料の素材として、キトサンの基本性質を有し、中性域ないしアルカリ域でも安定で可溶性を示し、しかも生活習慣病予防、老化予防や美容効果が期待でき、呈味が改善されたキトサン素材を提供することにある。 The object of the present invention is to provide the basic properties of chitosan as a functional beverage material, exhibit stability and solubility in neutral to alkaline regions, and can be expected to prevent lifestyle-related diseases, aging prevention and beauty effects. The object is to provide chitosan materials with improved taste.
本発明は、以下の(１)ないし(３)のキトサン素材を要旨としている。
(１) 呈味が改善されたキトサン素材であって、抗酸化作用および免疫活性を有し、重量平均分子量が3,000〜7,000の水溶性キトサンよりなることを特徴とするキトサン素材。
(２) 上記の水溶性キトサンが、分子量2,000以下のオリゴ糖成分を40％以上、分子量2,000〜50,000の低分子キトサンを60％未満含む特定の分子量分布を示す上記(１)のキトサン素材。
(３) 中性域ないしアルカリ域で安定な可溶性を示す上記(１)または(２)のキトサン素材。
The gist of the present invention is the following chitosan materials (1) to (3).
(1) A chitosan material having improved taste, comprising a water-soluble chitosan having an antioxidant action and immunological activity and having a weight average molecular weight of 3,000 to 7,000.
(2) The chitosan material according to (1), wherein the water-soluble chitosan has a specific molecular weight distribution including 40% or more of oligosaccharide components having a molecular weight of 2,000 or less and less than 60% of low-molecular chitosan having a molecular weight of 2,000 to 50,000.
(3) The chitosan material according to (1) or (2), which exhibits stable solubility in a neutral region or an alkaline region.
また、本発明は、以下の(４)の機能性飲料を要旨としている。
(４) 上記(１)、(２)または(３)のキトサン素材を配合したことを特徴とする機能性飲料。
The gist of the present invention is the functional drink (4) below.
(4) A functional beverage comprising the chitosan material of (1), (2) or (3) above.
機能性飲料の素材として、キトサンの基本性質を有し、中性域ないしアルカリ域でも安定で可溶性を示し、しかも抗酸化作用および免疫活性を有し、呈味が改善されたキトサン素材を提供することができる。また、キトサンの需要拡大が期待でき、未利用資源であるカニ殻やエビ殻などの資源の有効活用につながる。 Provided as a functional beverage material is a chitosan material that has the basic properties of chitosan, is stable and soluble in neutral to alkaline regions, has antioxidant activity and immune activity, and has improved taste. be able to. In addition, demand for chitosan can be expected to increase, leading to effective use of resources such as crab shell and shrimp shell, which are unused resources.
そこで本発明者らは、課題を詳細に検討を重ねた結果、酵素または酸でキトサンのβ１−４グリコシド結合を切断する加水分解反応を基本とした低分子化技術を用いることで、中性域ないしアルカリ域でも可溶で、しかも呈味が改善され、抗酸化作用および免疫活性も有することを見出し、本発明を完成するに至った。さらにオリゴ糖をある一定含有していることにより呈味が改善されるばかりか、飲料に配合した場合の白濁沈殿が起こり難いことを見いだした。 Therefore, the present inventors have studied the problem in detail, and as a result, by using a low molecular weight technology based on a hydrolysis reaction that cleaves the β1-4 glycoside bond of chitosan with an enzyme or an acid, It was also found that it is soluble even in the alkaline region, has improved taste, has antioxidant activity and immune activity, and has completed the present invention. Furthermore, it has been found that not only the taste is improved by containing a certain amount of oligosaccharide, but also that white turbidity precipitation hardly occurs when blended in a beverage.
本発明は、キトサンの重量平均分子量が3,000〜7,000まで分解し、特定の分子量分布を持つことにより、中性域ないしアルカリ域でも安定で、しかも抗酸化作用および免疫活性を有し、呈味の良好な飲料適性に優れた水溶性キトサンに関するもので、酵素法叉は酸分解法のいずれかの方法で調製することができる。次ぎに順を追って解説する。 The present invention decomposes chitosan to a weight average molecular weight of 3,000 to 7,000 and has a specific molecular weight distribution, so that it is stable even in a neutral or alkaline range, has an antioxidant action and immunological activity, and has a taste. The present invention relates to water-soluble chitosan having excellent beverage suitability, and can be prepared by either an enzymatic method or an acid decomposition method. Next, I will explain in order.
まずは、酵素法によるキトサンを低分子化の場合には、「食品衛生法の改正と食品添加物の規制／平成８年、厚生省生活衛生局食品化学課監修、日本食品添加物協会発行」の既存添加物名簿リストに収載されている糸状菌（Aspergillus niger、Trichoderma reesei、Trrichoderma viride、Verticillium）や細菌（Aeromonas、Bacillus）が生産するエンド型キトサナーゼあるいはエキソ型キトサナーゼを含む粗製酵素あるいは精製された酵素のいずれかを用いるのが適当であるが、エンド型キトサナーゼの方が単糖を生成せずに目的物を得やすい。
溶解する酸としては、好ましくは乳酸、酢酸、クエン酸、リンゴ酸、グルコン酸などの有機酸、塩酸などの無機酸が挙げられる。酢酸は臭気が強く、機能性飲料に用いる場合には爽やかな酸味を呈する乳酸やクエン酸、塩酸などを溶解剤として用いるのが最も好ましい。
本発明に用いるキトサンは、キチンを40〜50％濃度の苛性ソーダで加熱することで得られるが、キチンの原料は特に限定するものではない。キトサンの脱アセチル化度は、遊離したアミノ基の割合を示すもので、一般にコロイド滴定法により知ることができる。通常75％以上であれば容易に希酸に溶解することができる。従って、酵素法で用いる原料は脱アセチル化度75％以上が好ましいが、原料によって得られる分解物は異なるので、エンド型キトサナーゼを用いる場合には、脱アセチル化度85％〜95％が最も好ましく、目的物が得やすい。キトサンの粘度は100mPa・s以上で高いものでは数千mPa・sにも達する原料が一般的であるが、酵素反応する場合にはこれら高粘度の原料では希薄溶液での反応となり生産性が非常に悪くなるので適当ではない。従って、低分子のキチンからキトサンを調製するか或は別途出願中の特願2001-40758号（不均一な系による低分子キトサンの製造方法）などの方法で適度に低分子化したキトサンを用いることが好ましく、粘度が500mPa・s/5.0％以下であって分子量50万以下の原料を使用することが最も好ましい。酵素反応は、キトサンを溶解しキトサン１ｇに対し３〜10Ｕのキトサナーゼ（１Ｕは１分間当たり１μmolのグルコサミンに相当する還元糖が生成する活性量）を添加して６〜24時間酵素分解し酵素失活させる。なお、酵素反応の終点は、ゲルパーミエーションクロマトグラフィー法（昭和電工製Asahipack GF-7M HQカラム等、溶離液：0.2Ｍ酢酸−0.2Ｍ酢酸ナトリウム緩衝液、ＲＩ検出、標準試料：昭和電工製プルラン、生化学工業製キトサンオリゴ糖）によって知ることができる。さらに簡単な確認法としては、反応液を苛性ソーダで中和し反応中和液の濁度から判断することができる。すなわち、４％濃度の場合、分子量７千以下は澄明、８千〜1.0万は半透明、２万以上では白濁ゲル化する。
First of all, in the case of reducing the molecular weight of chitosan by the enzyme method, the existing “Revision of Food Sanitation Law and Regulation of Food Additives / 1996, Supervision of Food Chemistry Department of Health and Welfare Bureau, Ministry of Health and Welfare, published by Japan Food Additives Association” A crude or purified enzyme containing endo-type chitosanase or exo-type chitosanase produced by filamentous fungi ( Aspergillus niger , Trichoderma reesei , Trrichoderma viride , Verticillium ) and bacteria ( Aeromonas , Bacillus ) on the additive list. Either one is suitable, but endo-type chitosanase is easier to obtain the target product without producing monosaccharides.
The acid to be dissolved is preferably an organic acid such as lactic acid, acetic acid, citric acid, malic acid or gluconic acid, or an inorganic acid such as hydrochloric acid. Acetic acid has a strong odor, and when used in a functional beverage, it is most preferable to use lactic acid, citric acid, hydrochloric acid, or the like that exhibits a refreshing acidity as a solubilizer.
The chitosan used in the present invention can be obtained by heating chitin with 40-50% caustic soda, but the chitin raw material is not particularly limited. The degree of deacetylation of chitosan indicates the ratio of free amino groups and can be generally known by colloid titration. Usually, if it is 75% or more, it can be easily dissolved in dilute acid. Therefore, the raw material used in the enzymatic method preferably has a degree of deacetylation of 75% or more, but the degradation product obtained differs depending on the raw material, so when using endo-type chitosanase, the degree of deacetylation is most preferably 85% to 95%. , Easy to get the object. Chitosan's viscosity is 100mPa · s or higher, and raw materials that reach several thousand mPa · s are common. However, when enzymatic reaction occurs, these high-viscosity raw materials react in dilute solutions and are very productive. It is not appropriate because it gets worse. Therefore, chitosan is prepared from low-molecular chitin, or chitosan moderately reduced in molecular weight by a method such as Japanese Patent Application No. 2001-40758 (a method for producing low-molecular chitosan using a heterogeneous system), which has been filed separately, is used. It is preferable to use a raw material having a viscosity of 500 mPa · s / 5.0% or less and a molecular weight of 500,000 or less. The enzyme reaction is performed by dissolving chitosan and adding 3 to 10 U of chitosanase (1 U is the amount of activity capable of producing reducing sugar equivalent to 1 μmol of glucosamine per minute) per 1 g of chitosan, followed by enzymatic degradation for 6 to 24 hours. Make it live. The end point of the enzyme reaction was gel permeation chromatography (Asahipack GF-7M HQ column, manufactured by Showa Denko, etc., eluent: 0.2 M acetic acid-0.2 M sodium acetate buffer, RI detection, standard sample: Pullulan manufactured by Showa Denko Chitosan oligosaccharide manufactured by Seikagaku Corporation). As a simpler confirmation method, the reaction solution can be neutralized with caustic soda and judged from the turbidity of the reaction neutralization solution. That is, when the concentration is 4%, the molecular weight is 7,000 or less, the molecular weight is 8,000 to 10,000, translucent, and the 20,000 or more is cloudy gel.
また、酸分解法による低分子化の場合には、食品添加物として認められ、食品工業で広く一般的に使われている塩酸を用いるのが適当である。10モル以上の高濃度の塩酸下ではオリゴ糖まで容易に分解してしまうので7.0〜8.0％程度の塩酸、80℃以上の条件で分解するのが好ましい。酵分解に用いるキトサンは、脱アセチル化度75％以上であればよく、粘度は100mPa・s以上で、数千mPa・sにも達する原料であっても高温で加熱するため原料投入と同時に急激に粘度低下が起きるので分解時間が多少長くなるだけで特に問題にはならない。原料の添加量は、生産性の面から塩酸溶媒に対して５％以上投入するのが適当で、好ましくは10％前後である。酸分解の反応終点は、酵素法と同様にゲルパーミエーションクロマトグラフィー法で確認することができる。さらに簡便法として、反応液を中和して濁度から判断することもできる。酸分解後は、水を加えて直ちに冷却し、苛性ソーダでｐＨ５〜７に調整する。 In addition, in the case of reducing the molecular weight by the acid decomposition method, it is appropriate to use hydrochloric acid which is recognized as a food additive and widely used in the food industry. Under high-concentration hydrochloric acid of 10 mol or more, oligosaccharides are easily decomposed. Therefore, it is preferable to decompose under conditions of about 7.0 to 8.0% hydrochloric acid at 80 ° C. or higher. Chitosan used for fermentation decomposition should have a degree of deacetylation of 75% or more, and its viscosity is 100 mPa · s or more, and even raw materials that reach several thousand mPa · s are heated at high temperatures. In this case, a decrease in viscosity occurs, so that the decomposition time is only slightly longer and is not a problem. The addition amount of the raw material is suitably 5% or more with respect to the hydrochloric acid solvent from the viewpoint of productivity, and is preferably around 10%. The end point of the acid decomposition reaction can be confirmed by gel permeation chromatography as in the enzyme method. Further, as a simple method, the reaction solution can be neutralized and judged from turbidity. After acid decomposition, water is added and immediately cooled, and the pH is adjusted to 5 to 7 with caustic soda.
その後の工程は、酵素法も酸分解法も、酸分解法で脱塩処理する以外は同じである。すなわち、限外ろ過膜（例えば公称分画分子量５万等）或は活性炭やろ過助剤を使ってフィルタープレスなどの多段式ろ過器でろ過し、未分解物のキトサンや酵素などが除去される。酸分解法の場合にはここで電気透析（ＥＤ）或は逆浸透膜（ＲＯ）で中和塩を脱塩除去される。次ぎに陰イオン交換樹脂（例えばアンバーライトIRA400等）でｐＨを調整するか、或は溶解に使用した酸でｐＨを調整することで各種の水溶液ができる。それから0.2μm或は0.45μmのカートリッジ式フィルターで除菌するか或は熱交換器に通して加熱滅菌処理される。それから減圧濃縮機などで任意の濃度まで濃縮しそのまま製品とすることもできるが凍結乾燥（フリーズドライ）や噴霧乾燥（スプレードライ）の方法で処理し、水溶性キトサンの乾燥物としても提供することができる。なお、本発明においては、分子量分布の調整を目的としたＵＦ膜やクロマトグラフィーによる精密な分画は必要としない。その理由は、オリゴ糖画分そのものに有効活性があること、オリゴ糖画分が全体の呈味に関与すること、収率面でコスト的に割高になることが挙げられる。 The subsequent steps are the same except that the enzymatic method and the acid decomposition method are desalted by the acid decomposition method. That is, ultrafiltration membranes (for example, nominal molecular weight cut off 50,000) or filtered with a multistage filter such as a filter press using activated carbon or a filter aid to remove undegraded chitosan and enzymes . In the case of the acid decomposition method, the neutralized salt is desalted and removed by electrodialysis (ED) or reverse osmosis membrane (RO). Next, various aqueous solutions can be formed by adjusting the pH with an anion exchange resin (for example, Amberlite IRA400) or adjusting the pH with an acid used for dissolution. It is then sterilized with a 0.2 μm or 0.45 μm cartridge filter or passed through a heat exchanger and heat sterilized. Then, it can be concentrated to any concentration with a vacuum concentrator, etc., but it can be used as it is, but it can be processed by freeze-drying (freeze-drying) or spray-drying (spray-drying) and provided as a dried product of water-soluble chitosan. Can do. In the present invention, precise fractionation by UF membrane or chromatography for the purpose of adjusting the molecular weight distribution is not required. The reason is that the oligosaccharide fraction itself has effective activity, the oligosaccharide fraction is involved in the overall taste, and the yield is expensive in terms of cost.
以上によれば、分子量2,000以下のオリゴ糖成分が40％以上含まれ、分子量2,000〜50,000の低分子キトサン成分が60％未満含む特定の分子量分布を示し、重量平均分子量3,000〜7,000まで分解することにより、中性域ないしアルカリ域でも可溶で、しかも抗酸化作用および免疫活性を有し、呈味が良好な飲料適性に優れた水溶性キトサンが得られることを発見し、本発明に至った。
本発明の水溶性キトサンは、生活習慣病の予防や美容、老化予防を目的とした機能性飲料への利用が期待できる。
According to the above, it shows a specific molecular weight distribution that contains 40% or more of oligosaccharide components with a molecular weight of 2,000 or less and low molecular weight chitosan components with a molecular weight of 2,000 to 50,000, and decomposes to a weight average molecular weight of 3,000 to 7,000. Thus, it was discovered that a water-soluble chitosan that is soluble in a neutral region or an alkaline region, has an antioxidative action and immunological activity, has a good taste, and is excellent in drinkability can be obtained. .
The water-soluble chitosan of the present invention can be expected to be used for functional beverages for the purpose of preventing lifestyle-related diseases, beauty, and aging.
次ぎに水溶性キトサンに関して実施例及び試験例を挙げるが、本発明を何ら制限するものではない。 Next, although an Example and a test example are given regarding water-soluble chitosan, this invention is not restrict | limited at all.
調製例１（酵素法）
10Ｌジャーファーメンター（柴田科学製）に蒸留水７Ｌ、原料キトサン490ｇ（脱アセチル化度91％、粘度10mPa・s／1.0％、灰分0.3％）を入れ、氷酢酸180ｇで溶解し、Bacillus由来エンド型キトサナーゼの標品を入れ（キトサン１gに対して６Ｕ添加した）、ｐＨ5.5、40℃で24時間、酵素処理した。それから80℃で酵素失活、粉末活性炭（二村化学工業製）５ｇを加え、ろ紙（アドバンテック製No5C）でろ過した。ろ液を凍結乾燥装置（東京理化製FD550P）で乾燥物620ｇを得た（黄褐色、水分15.0％、灰分0.4％、重量平均分子量4,800、分子量分布：分子量2,000以下：56.4％；分子量2,000−10,000：27.1％；分子量10,000−20,000：10.5％；分子量20,000−50,000：5.7％；分子量50,000−100,000：0.3％；分子量100,000以上：0.0％）。再溶解後、中性域ないしアルカリ域にしても白濁せずに可溶な水溶性キトサンであった。また、エグ味もなく呈味も良好であった。
Preparation Example 1 (Enzymatic method)
Put 10L jar fermenter (manufactured by Shibata Kagaku) with 7L distilled water and 490g raw chitosan (deacetylation degree 91%, viscosity 10mPa · s / 1.0%, ash content 0.3%), dissolve with 180g glacial acetic acid, and end with Bacillus origin A sample of type chitosanase was added (6 U was added to 1 g of chitosan), and the enzyme treatment was carried out at pH 5.5 and 40 ° C. for 24 hours. Then, the enzyme was inactivated at 80 ° C., 5 g of powdered activated carbon (manufactured by Nimura Chemical Co., Ltd.) was added, and the mixture was filtered with filter paper (No5C manufactured by Advantech). The filtrate was lyophilized (FD550P manufactured by Tokyo Rika Co., Ltd.) to obtain 620 g of a dried product (yellow brown, moisture 15.0%, ash content 0.4%, weight average molecular weight 4,800, molecular weight distribution: molecular weight 2,000 or less: 56.4%; molecular weight 2,000-10,000 : 27.1%; Molecular weight 10,000-20,000: 10.5%; Molecular weight 20,000-50,000: 5.7%; Molecular weight 50,000-100,000: 0.3%; Molecular weight 100,000 or more: 0.0%). After re-dissolution, it was a water-soluble chitosan that was soluble in neutral or alkaline regions without becoming cloudy. Moreover, there was no taste and the taste was good.
調製例２（酵素法）
10Ｌジャーファーメンター（柴田科学製）に蒸留水７Ｌ、原料キトサン490ｇ（脱アセチル化度91％、粘度10mPa・s/1.0％、灰分0.3％）を入れ、90％乳酸237ｇで溶解し、Bacillus由来エンド型キトサナーゼの標品を入れ（キトサン１gに対して６Ｕ添加した）、ｐＨ5.5、40℃で４時間、酵素処理した。それから80℃で酵素失活、粉末活性炭（二村化学工業製）５ｇを加え、ろ紙（アドバンテック製Ｎｏ５Ｃ）でろ過した。ろ液を凍結乾燥装置（東京理化製FD550P）で乾燥物703ｇを得た（黄褐色、水分12.0％、灰分0.4％、重量平均分子量7,000、分子量分布：分子量2,000以下：40.0％；分子量2,000−10,000：34.1％；分子量10,000−20,000：14.8％；分子量20,000−50,000：10.1％；分子量50,000−100,000：1.0％；分子量100,000以上：0.0％）。再溶解後、中性域ないしアルカリ域にしても白濁せずに可溶な水溶性キトサンであった。また、エグ味もなく呈味も良好であった。
Preparation Example 2 (Enzymatic method)
Distilled water 7L, raw material chitosan 490g (deacetylation degree 91%, viscosity 10mPa · s / 1.0%, ash content 0.3%) in 10L jar fermenter (manufactured by Shibata Kagaku), dissolved in 237g of 90% lactic acid, derived from Bacillus An endo-type chitosanase preparation was added (6 U was added to 1 g of chitosan), and the enzyme treatment was performed at pH 5.5 and 40 ° C. for 4 hours. Then, the enzyme was deactivated at 80 ° C., 5 g of powdered activated carbon (manufactured by Nimura Chemical Co., Ltd.) was added, and the mixture was filtered with a filter paper (No5C manufactured by Advantech). The filtrate was lyophilized (FD550P manufactured by Tokyo Rika Co., Ltd.) to obtain 703 g of a dried product (yellow brown, moisture 12.0%, ash content 0.4%, weight average molecular weight 7,000, molecular weight distribution: molecular weight 2,000 or less: 40.0%; molecular weight 2,000-10,000 34.1%; molecular weight 10,000-20,000: 14.8%; molecular weight 20,000-50,000: 10.1%; molecular weight 50,000-100,000: 1.0%; molecular weight 100,000 or more: 0.0%). After re-dissolution, it was a water-soluble chitosan that was soluble in neutral or alkaline regions without becoming cloudy. Moreover, there was no taste and the taste was good.
調製例３（酵素法）
10Ｌジャーファーメンター（柴田科学製）に蒸留水７Ｌ、原料キトサン490ｇ（脱アセチル化度91％、粘度10mPa・s/1.0％、灰分0.3％）を入れ、90％乳酸237ｇで溶解し、Bacillus由来エンド型キトサナーゼの標品を入れ（キトサン１ｇに対して６Ｕ添加した）、ｐＨ5.5、40℃で８時間、酵素処理した。それから80℃で酵素失活、粉末活性炭（二村化学工業製）５ｇを加え、ろ紙（アドバンテック製Ｎｏ５Ｃ）でろ過した。ろ液を凍結乾燥装置（東京理化製FD550P）で乾燥物690ｇを得た（黄褐色、水分14.2％、灰分0.4％、重量平均分子量4,900、分子量分布：分子量2,000以下：56.6％；分子量2,000−10,000：26.1％；分子量10,000−20,000：10.8％；分子量20,000−50,000：6.1％；分子量50,000−100,000：0.4％；分子量100,000以上：0.0％）。再溶解後、中性域ないしアルカリ域にしても白濁せずに可溶な水溶性キトサンであった。また、エグ味もなく呈味も良好であった。
Preparation Example 3 (Enzymatic method)
Distilled water 7L, raw material chitosan 490g (deacetylation degree 91%, viscosity 10mPa · s / 1.0%, ash content 0.3%) in 10L jar fermenter (manufactured by Shibata Kagaku), dissolved in 237g of 90% lactic acid, derived from Bacillus An endo-type chitosanase preparation was added (6 U was added to 1 g of chitosan), and enzyme treatment was performed at pH 5.5 and 40 ° C. for 8 hours. Then, the enzyme was deactivated at 80 ° C., 5 g of powdered activated carbon (manufactured by Nimura Chemical Co., Ltd.) was added, and the mixture was filtered with a filter paper (No5C manufactured by Advantech). The filtrate was lyophilized (Tokyo Rika FD550P) to obtain 690 g of a dried product (yellow brown, moisture 14.2%, ash content 0.4%, weight average molecular weight 4,900, molecular weight distribution: molecular weight 2,000 or less: 56.6%; molecular weight 2,000-10,000 26.1%; molecular weight 10,000-20,000: 10.8%; molecular weight 20,000-50,000: 6.1%; molecular weight 50,000-100,000: 0.4%; molecular weight 100,000 or more: 0.0%). After re-dissolution, it was a water-soluble chitosan that was soluble in neutral or alkaline regions without becoming cloudy. Moreover, there was no taste and the taste was good.
調製例４（酵素法+イオン交換処理）
10Lジャーファーメンター（柴田科学製）に蒸留水4.7L、原料キトサン200g（脱アセチル化度95％、粘度11mPa・s/1.0％、灰分0.3％）を入れ、90%乳酸124gで溶解し、Bacillus由来エンド型キトサナーゼの標品を入れ（キトサン１gに対して６U添加した）、pH5.4、40℃で16時間、酵素処理した。それから80℃で酵素失活、粉末活性炭（二村化学工業製）２gを加え、ろ紙（アドバンテック製No5C）でろ過した。ろ液を陰イオン交換樹脂(オルガノ製IRA410)に通し、ろ液をpH6.2に調整、凍結乾燥装置(東京理化製FD550P)で乾燥物（遊離体）250gを得た（微黄色、灰分0.4%、重量平均分子量3,600、分子量分布：分子量2,000以下：56.2%；分子量2,000−10,000：35.4％；分子量10,000-20,000：6.1％；分子量20,000-50,000：2.3％；分子量50,000-100,000：0.0％；分子量100,000以上：0.0％）。調製例１と同様、再溶解後、中性域ないしアルカリ域にしても白濁せずに可溶な水溶性キトサンであった。また、エグ味もなく呈味も良好であった。
Preparation Example 4 (Enzyme method + ion exchange treatment)
Put 10L jar fermenter (made by Shibata Kagaku) with distilled water 4.7L, raw material chitosan 200g (deacetylation degree 95%, viscosity 11mPa · s / 1.0%, ash content 0.3%), dissolve with 90% lactic acid 124g, Bacillus A sample of derived endo-type chitosanase was added (6 U was added to 1 g of chitosan), and the enzyme treatment was carried out at pH 5.4 and 40 ° C. for 16 hours. Then, the enzyme was deactivated at 80 ° C., 2 g of powdered activated carbon (manufactured by Nimura Chemical Co., Ltd.) was added, and the mixture was filtered with a filter paper (No5C manufactured by Advantech). The filtrate was passed through an anion exchange resin (organo-made IRA410), the filtrate was adjusted to pH 6.2, and 250 g of dried product (free form) was obtained with a freeze-drying device (Tokyo Rika FD550P) (slight yellow, ash content 0.4 %, Weight average molecular weight 3,600, molecular weight distribution: molecular weight 2,000 or less: 56.2%; molecular weight 2,000-10,000: 35.4%; molecular weight 10,000-20,000: 6.1%; molecular weight 20,000-50,000: 2.3%; molecular weight 50,000-100,000: 0.0%; 100,000 or more: 0.0%). As in Preparation Example 1, it was a water-soluble chitosan that was soluble and did not become cloudy even after being redissolved and neutral or alkaline. Moreover, there was no taste and the taste was good.
調製例５（酸分解法＋脱塩処理）
２Ｌ丸底フラスコに高粘度キトサン（脱アセチル化度99％、粘度600mPa・s/0.5％、灰分0.8％）100ｇを入れ、7.0％塩酸１kgを加え、80℃で５時間、酸処理した。蒸留水で分解液を２倍希釈後、１Ｎ苛性ソーダでｐＨ6.0に調整、粉末活性炭（二村化学工業製）２ｇを加え、ろ紙でろ過した。ろ液（アドバンテック製No5C）を卓上型電気透析装置S3（旭化成製）で脱塩、凍結乾燥装置（東京理化製FD-1）で分解物96ｇを得た（淡褐色、灰分0.3％、重量平均分子量6,400、分子量分布：分子量2,000以下：54.2％；分子量2,000−10,000：25.6％；分子量10,000−20,000：10.8％；分子量20,000−50,000：8.2％；分子量50,000−100,000：1.1％；分子量100,000以上：0.1％）。調製例１と同様に、再溶解後、中性域ないしアルカリ域にしても白濁せずに可溶な水溶性キトサンであった。また、エグ味もなくて呈味も良好であった。
Preparation Example 5 (acid decomposition method + desalting treatment)
100 g of high-viscosity chitosan (degree of deacetylation 99%, viscosity 600 mPa · s / 0.5%, ash content 0.8%) was placed in a 2 L round bottom flask, and 1 kg of 7.0% hydrochloric acid was added, followed by acid treatment at 80 ° C. for 5 hours. The decomposition solution was diluted twice with distilled water, adjusted to pH 6.0 with 1N caustic soda, 2 g of powdered activated carbon (manufactured by Nimura Chemical Co., Ltd.) was added, and the mixture was filtered with filter paper. The filtrate (Advantech No5C) was desalted with a desktop electrodialyzer S3 (Asahi Kasei), and 96 g of decomposition product was obtained with a freeze-drying device (Tokyo Rika FD-1) (light brown, ash content 0.3%, weight average) Molecular weight 6,400, molecular weight distribution: molecular weight 2,000 or less: 54.2%; molecular weight 2,000-10,000: 25.6%; molecular weight 10,000-20,000: 10.8%; molecular weight 20,000-50,000: 8.2%; molecular weight 50,000-100,000: 1.1%; molecular weight 100,000 or more: 0.1 %). In the same manner as in Preparation Example 1, it was a water-soluble chitosan that was soluble and did not become cloudy even after being redissolved and neutral or alkaline. Moreover, there was no taste and the taste was good.
キトサンの溶解性と呈味についてより詳細に調べた。
キトサンの溶解性については、吸光度660nmによる濁度をみた。すなわち、各種キトサン分解物を得るため原料キトサン91ｇ（脱アセチル化度92％、粘度71mPa・s/0.5％、分子量81万）を90％乳酸28.2ｇで溶解して１％濃度とし、エンド型キトサナーゼ320Ｕ、ｐＨ5.0、45℃の条件で酵素分解し、重量平均分子量の異なる各種キトサン分解物の１％液を得て、原料キトサンを含むそれらを試験試料とした（表１）。なお、分子量2,000のキトサン分解物は、脱アセチル化度100％のキトサン（粘度30mPa・s/0.5％、分子量36万）を用いて同様に調製した。キトサン各ｐＨ条件下で試料液の清澄（濁り）を見るために50％濃度の苛性ソーダでｐＨ6〜10に調整し、分光光度計（島津製作所製UV1600）で、蒸留水を透過率100％として、660nmにおける試料液の透過率％を測定した。その結果を図１に示す。原料である重量平均分子量800,000のキトサンは、ｐＨ6.0で透過率が30％まで低下し、ｐＨ7.0では透過率が10％以下となり、中性域では澄明性はなく溶解しなかった。重量平均分子量120,000であるキトサン分解物は、ｐＨ6.5で透過率が70％、ｐＨ7.0では透過率が20％と中性域では澄明性はなく溶解しなかった。重量平均分子量48,000であるキトサン分解物は、ｐＨ7.0で透過率70％、ｐＨ7.5では透過率が20％以下と中性域では澄明性はなく溶解しなかった。重量平均分子量12,000であるキトサン分解物は、ｐＨ7.5までは透過率が96％と中性域でも澄明であったが、ｐＨ8.0では透過率が20％まで低下し、アルカリ域では溶解しなかった。これらに対し、重量平均分子量7,000まで分解したキトサンは、オリゴ糖が主成分の重量平均分子量2,000の分解物と同様、ｐＨ10でも透過率が96％以上で、中性域ないしアルカリ域でも澄明性を示し溶解した。
Regarding the solubility of chitosan, the turbidity at an absorbance of 660 nm was observed. That is, in order to obtain various chitosan degradation products, 91 g of raw material chitosan (deacetylation degree 92%, viscosity 71 mPa · s / 0.5%, molecular weight 810,000) was dissolved in 28.2 g of 90% lactic acid to a concentration of 1%, and endo-type chitosanase Enzymatic degradation was performed under conditions of 320 U, pH 5.0, and 45 ° C. to obtain 1% solutions of various chitosan degradation products having different weight average molecular weights, and those containing raw material chitosan were used as test samples (Table 1). A chitosan decomposition product having a molecular weight of 2,000 was prepared in the same manner using chitosan having a deacetylation degree of 100% (viscosity 30 mPa · s / 0.5%, molecular weight 360,000). Chitosan In order to see the clearness (turbidity) of the sample solution under each pH condition, adjust the pH to 6-10 with caustic soda of 50% concentration, with a spectrophotometer (Shimadzu UV1600), with distilled water at 100% transmittance, The% transmittance of the sample solution at 660 nm was measured. The result is shown in FIG. The raw material, chitosan having a weight average molecular weight of 800,000, had a transmittance of 30% at pH 6.0, a transmittance of 10% or less at pH 7.0, and was not clear and did not dissolve in the neutral range. The degradation product of chitosan having a weight average molecular weight of 120,000 had a transmittance of 70% at pH 6.5 and a transmittance of 20% at pH 7.0 and was not clear and did not dissolve in the neutral region. The degradation product of chitosan having a weight average molecular weight of 48,000 had a transmittance of 70% at pH 7.0 and a transmittance of 20% or less at pH 7.5 and was not clear and did not dissolve in the neutral region. The degradation product of chitosan with a weight average molecular weight of 12,000 had a transmittance of 96% up to pH 7.5 and was clear even in the neutral range, but at pH 8.0 the transmittance dropped to 20% and dissolved in the alkaline region. There wasn't. On the other hand, chitosan decomposed to a weight average molecular weight of 7,000 has a transmittance of 96% or more even at pH 10 as well as a degradation product having a weight average molecular weight of 2,000, the main component of which is an oligosaccharide. Shown dissolved.
次ぎに表１の原料キトサンおよびキトサン分解物の呈味について調べた。すなわち、１％濃度のキトサン液10mlを口の中に10秒間含ませた後の呈味をみた。重量平均分子量48,000および800,000をそれぞれ経口摂取した場合、口の中でキトサンが凝固し収斂性を伴ったエグ味を感じた。重量平均分子量48,000以上のキトサンは飲料素材としては適していなかった。重量平均分子量12,000のキトサン分解物を経口摂取した場合、口の中で強く凝固することはないがややエグ味が感じられた。重量平均分子量10,000程度に分解したキトサンは、飲料への適用性としては多少の違和感があったので中程度とし、高濃度に配合した場合の考慮が必要で十分に満足するものではなかった。それに対して、重量平均分子量6,000叉は7,000のキトサン分解物をそれぞれ経口摂取すると、口の中で凝固することは全くなく、比較的重合度の高いキトサンが含まれているにも関わらずエグ味も感じず良好な呈味を有していた。重量平均分子量2,000まで分解するまでもなく、重量平均分子量7,000程度に分解したキトサンは、飲料素材としての適用性が認められた。以上の結果を表２に示した。
(１) 水溶性キトサンの粘性
調製例２で得た重量平均分子量7,000の水溶性キトサンの粘度について回転粘度計（東機産業製RB-80L）で調べた。その結果を図２に示す。重量平均分子量7,000まで分解した水溶性キトサンは、従来の高分子キトサンに比べ、20％濃度でも粘度が10mPa・s程度と非常に低く、高濃度の溶液が調整可能であった。
(1) Viscosity of water-soluble chitosan The viscosity of the water-soluble chitosan having a weight average molecular weight of 7,000 obtained in Preparation Example 2 was examined with a rotational viscometer (RB-80L manufactured by Toki Sangyo). The result is shown in FIG. Water-soluble chitosan decomposed to a weight average molecular weight of 7,000 had a very low viscosity of about 10 mPa · s even at a concentration of 20% compared to conventional polymer chitosan, and a high-concentration solution could be prepared.
(２) 水溶性キトサンの抗酸化作用
調製例３で得た重量平均分子量4,900の水溶性キトサンの添加によるリノール酸の自動酸化について、生成した過酸化物量をロダン鉄法（500nm）によって測定し酸化率を求めた（土屋；北海道女子大学短期大学部研究紀要No.35,p27-32,1998：寺沢ら；日本食品科学工学会誌Vol.48,No.2,p99-104,2001）。なお、比較として、脂溶性のビタミンＥ（dl-α-トコフェロール）、水溶性のビタミンＣ（L(+)-アスコルビン酸）および合成抗酸化剤であるBHA(3 (2)-ｔ-ブチル-４-ヒドロキシアニソール）を用いた。本試験系ではラジカル促進剤としてＡＡＰＨ（2,2-アゾビス(2-アミジノプロパン）２塩酸塩）を使用した。
また、水溶性キトサンの添加によるDPPH（1,1-ジフェニル-2-ピクリヒドラジル）ラジカルの消去能について、520nmの波長でラジカルの減少を測定しラジカル消去率を求めた（篠原ら；食品機能研究法，光琳，p218‐220,2000）。なお、比較として、高分子キトサン（重量平均分子量10万以上を乳酸で溶解）を用いた。さらに水溶性誘導体であるビタミンＥ（Trolox）をラジカル消去能の標準物質とし、Trolox濃度に対する水溶性キトサン量を算出した。その結果、リノール酸自動酸化試験では、水溶性キトサンは既存の合成の抗酸化剤に比べて強くはないが、過酸化物の生成を抑制し、抗酸化作用を示した（図３）。また、DPPHラジカル消去試験では、水溶性キトサンは１nmolのTrolox濃度で276μg量に相当し、高分子キトサンよりラジカル消去能を有し、抗酸化作用を示した（図４）。
(2) Antioxidant action of water-soluble chitosan For the auto-oxidation of linoleic acid by addition of water-soluble chitosan with a weight average molecular weight of 4,900 obtained in Preparation Example 3, the amount of peroxide produced was measured by the rodan iron method (500 nm) and oxidized. The rate was calculated (Tsuchiya; Bulletin of Hokkaido Women's University Junior College No. 35, p27-32, 1998: Terasawa et al .; Journal of Japanese Society for Food Science and Technology Vol. 48, No. 2, p99-104, 2001). For comparison, fat-soluble vitamin E (dl-α-tocopherol), water-soluble vitamin C (L (+)-ascorbic acid) and synthetic antioxidant BHA (3 (2) -t-butyl- 4-hydroxyanisole) was used. In this test system, AAPH (2,2-azobis (2-amidinopropane) dihydrochloride) was used as a radical accelerator.
In addition, the radical scavenging rate was determined by measuring the decrease in radicals at a wavelength of 520 nm for the scavenging ability of DPPH (1,1-diphenyl-2-piclyhydrazyl) radicals by adding water-soluble chitosan (Shinohara et al .; Food Function Research method, Korin, p218-220, 2000). For comparison, polymer chitosan (weight average molecular weight of 100,000 or more dissolved in lactic acid) was used. Furthermore, the amount of water-soluble chitosan relative to the Trolox concentration was calculated using vitamin E (Trolox), which is a water-soluble derivative, as a standard substance for radical scavenging ability. As a result, in the linoleic acid auto-oxidation test, water-soluble chitosan was not as strong as the existing synthetic antioxidants, but inhibited the formation of peroxides and showed an antioxidant action (FIG. 3). In the DPPH radical scavenging test, water-soluble chitosan was equivalent to 276 μg at 1 nmol Trolox concentration, and had radical scavenging ability and higher antioxidant activity than polymeric chitosan (FIG. 4).
（３）水溶性キトサンの免疫活性
調製例３で得た重量平均分子量4,900の水溶性キトサンの添加によるマウス由来マクロファージの活性化について、活性化の指標としてNO（一酸化窒素）およびサイトカイン（TNF-α）産生量を測定した。水溶性キトサンの培養液への添加量は１〜1,000μg/mlとした。マウス由来マクロファージはRIKEN CELL BANKより購入のJ774.1細胞株を使用した。NO産生能は、Griess試薬を加え550nmの吸光度を測定した（Dennis J.Stuehr and Michael A.Marletta,Cancer Res,47,1987）。サイトカインは、BioSource International社製測定キットを使用した（Thomas, A. W. ed. The Cytokine Handbook p265-288,1994）。比較対照として、キトサンオリゴ糖（重量平均分子量2,500、５糖および６糖が22.0％および15.5％含有したもの）を用いた。
その結果、水溶性キトサンのＮＯ産生量は、陽性対照であるLPS（リボ多糖）に対し、濃度依存的に活性を示し、1,000μg/ml濃度におけるキトサンオリゴ糖の活性が約３％であるのに対し、水溶性キトサンは5.1％と高い免疫活性を示した（図５）。また、水溶性キトサンのサイトカイン（TNF-α）産生量は、キトサンオリゴ糖よりも１〜10μg/mlでコントロールの1.2倍の活性を示し、100μg/mlで２倍の活性を示した（図６）。
(3) Immunoreactivity of water-soluble chitosan Regarding the activation of mouse-derived macrophages by addition of water-soluble chitosan having a weight average molecular weight of 4,900 obtained in Preparation Example 3, NO (nitrogen monoxide) and cytokine (TNF- α) Production was measured. The amount of water-soluble chitosan added to the culture solution was 1 to 1,000 μg / ml. For mouse-derived macrophages, the J774.1 cell line purchased from RIKEN CELL BANK was used. The NO production ability was measured by adding Griess reagent and measuring the absorbance at 550 nm (Dennis J. Stuehr and Michael A. Marletta, Cancer Res, 47, 1987). For the cytokine, a measurement kit manufactured by BioSource International was used (Thomas, AWed. The Cytokine Handbook p265-288, 1994). As a comparative control, chitosan oligosaccharide (containing 22.0% and 15.5% of weight average molecular weight 2,500, 5 and 6 sugars) was used.
As a result, the amount of NO produced by water-soluble chitosan shows a concentration-dependent activity against LPS (ribopolysaccharide) which is a positive control, and the activity of chitosan oligosaccharide at a concentration of 1,000 μg / ml is about 3%. On the other hand, water-soluble chitosan showed a high immune activity of 5.1% (FIG. 5). In addition, the amount of cytokine (TNF-α) produced by water-soluble chitosan was 1 to 10 μg / ml higher than that of chitosan oligosaccharide, showing 1.2 times the activity of the control, and 100 μg / ml showing twice the activity (FIG. 6). ).
（４）水溶性キトサンの安全性
キチン、キトサン類は、既に食経験があって安全であることが既に実証されている。調製例３で得た重量平均分子量4,900の水溶性キトサンの安全性について調べた結果、変異原性（Eecherichia coli WP2 uvrA株およびsalmonella typhimuriumTA）は陰性、マウスを用いた急性毒性では単回投与で雌雄ともに5,000mg/kg以上で、食品安全面において問題がないことが確認された。
(4) Safety of water-soluble chitosan
Chitin and chitosans have already been proven to be safe because of dietary experience. As a result of investigating the safety of the water-soluble chitosan having a weight average molecular weight of 4,900 obtained in Preparation Example 3, mutagenicity (Eecherichia coli WP2 uvrA strain and salmonella typhimuriumTA) was negative. Both were 5,000mg / kg or more, and it was confirmed that there was no problem in food safety.
(５) 飲料配合による官能試験
実際に市販されている複数の機能性飲料に調製例３で得た重量平均分子量，900の水溶性キトサンを0.3〜1.0％配合（w/v）し、社内で募ったパネラーに試飲してもらった。その結果、10人中すべての人が従来の高分子キトサン（重量平均分子量20万、粘度22mPa・s/0.5％、脱アセチル化度88％、乳酸に溶かし使用）を配合したものと比べ、違和感がなく良好との評価が得られた。なお、高分子キトサンを配合した機能性飲料の一部に沈殿物が見られた。
(5) Sensory test by blending beverages Incorporate 0.3 to 1.0% of water-soluble chitosan with a weight average molecular weight of 900 obtained in Preparation Example 3 into a plurality of functional beverages that are actually marketed (w / v). The recruited panelists had a tasting. As a result, all of the 10 people felt uncomfortable compared to the conventional polymer chitosan (weight average molecular weight 200,000, viscosity 22mPa · s / 0.5%, deacetylation degree 88%, dissolved in lactic acid). The evaluation that it was good was obtained. In addition, the deposit was seen in a part of functional drink which mix | blended polymeric chitosan.
以上の結果より、優れた抗酸化性、免疫活性を有する、呈味の改善されたキトサン素材が提供できる。 From the above results, a chitosan material with excellent taste and improved taste can be provided.
従来の高分子キトサンは、希酸には溶けるものの粘性が高くて水に溶け難く、中性ないしアルカリでは不溶化し、タンパク質やアミノ酸、核酸、酸性多糖などと凝集沈殿を起こしたりすることもある。さらには呈味にも重大な問題があった。健康食品で利用する場合に、カプセルや錠剤の形態で摂取するのがほとんどであるが、中性域ないしアルカリ域で溶解安定性に優れ、抗酸化作用および免疫活性を有した、本発明の水溶性キトサンによれば、生活習慣病の予防や美容、老化予防などの目的とした機能性飲料への利用展開が十分に可能である。 Conventional polymer chitosan is soluble in dilute acid, but has high viscosity and is difficult to dissolve in water. It may be insoluble in neutral or alkali, and may cause aggregation and precipitation with proteins, amino acids, nucleic acids, acidic polysaccharides, and the like. In addition, there was a serious problem with taste. When used in health foods, it is mostly taken in the form of capsules and tablets, but it has excellent solubility stability in the neutral to alkaline range, has antioxidant activity and immunological activity, and is water-soluble. According to sexual chitosan, it can be fully used for functional drinks for the purpose of preventing lifestyle-related diseases, beauty, and aging prevention.
Claims (4)
A functional beverage comprising the chitosan material according to claim 1, 2 or 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2003309224A JP2005075957A (en) | 2003-09-01 | 2003-09-01 | Water-soluble chitosan |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2003309224A JP2005075957A (en) | 2003-09-01 | 2003-09-01 | Water-soluble chitosan |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2005075957A true JP2005075957A (en) | 2005-03-24 |
Family
ID=34411454
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2003309224A Abandoned JP2005075957A (en) | 2003-09-01 | 2003-09-01 | Water-soluble chitosan |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2005075957A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100679411B1 (en) | 2005-08-22 | 2007-02-07 | 김중열 | Method for preparing high molecular water-soluble weak alkaline chitosan using natural organic germanium |
| JP2007217304A (en) * | 2006-02-14 | 2007-08-30 | Yaizu Suisankagaku Industry Co Ltd | Ultraviolet absorbing agent, skin preparation for external use containing the same, and antioxidant |
| WO2013073644A1 (en) * | 2011-11-14 | 2013-05-23 | 塚原 順子 | Combination health and functional food product comprising multiple minerals |
| WO2019130724A1 (en) | 2017-12-27 | 2019-07-04 | サントリーホールディングス株式会社 | Chitosan-containing non-fermented tea beverage or semi-fermented tea beverage |
| WO2020100405A1 (en) | 2018-11-14 | 2020-05-22 | サントリーホールディングス株式会社 | Chitosan-containing beverage |
| CN111647531A (en) * | 2020-06-03 | 2020-09-11 | 青岛北方茶仓茶文化有限公司 | Siamese bacillus LBP for increasing antioxidant activity, and fermentation product and application thereof |
| CN114366816A (en) * | 2022-01-05 | 2022-04-19 | 重庆福美干细胞生物科技发展有限公司 | Chitosan nanoparticles with excellent freeze-drying redispersibility, freeze-drying preparation, and preparation method and application thereof |
-
2003
- 2003-09-01 JP JP2003309224A patent/JP2005075957A/en not_active Abandoned
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100679411B1 (en) | 2005-08-22 | 2007-02-07 | 김중열 | Method for preparing high molecular water-soluble weak alkaline chitosan using natural organic germanium |
| JP2007217304A (en) * | 2006-02-14 | 2007-08-30 | Yaizu Suisankagaku Industry Co Ltd | Ultraviolet absorbing agent, skin preparation for external use containing the same, and antioxidant |
| WO2013073644A1 (en) * | 2011-11-14 | 2013-05-23 | 塚原 順子 | Combination health and functional food product comprising multiple minerals |
| AU2018398427B2 (en) * | 2017-12-27 | 2021-10-28 | Suntory Holdings Limited | Chitosan-containing non-fermented tea beverage or semi-fermented tea beverage |
| WO2019130724A1 (en) | 2017-12-27 | 2019-07-04 | サントリーホールディングス株式会社 | Chitosan-containing non-fermented tea beverage or semi-fermented tea beverage |
| JP2019115325A (en) * | 2017-12-27 | 2019-07-18 | サントリーホールディングス株式会社 | Non-fermented tea beverage or semi-fermented tea beverage containing chitosan |
| TWI788441B (en) * | 2017-12-27 | 2023-01-01 | 日商三得利控股股份有限公司 | Non-fermented or semi-fermented tea beverages containing chitosan |
| JP2020078285A (en) * | 2018-11-14 | 2020-05-28 | サントリーホールディングス株式会社 | Beverage containing chitosan |
| JP7182432B2 (en) | 2018-11-14 | 2022-12-02 | サントリーホールディングス株式会社 | Beverage containing chitosan |
| WO2020100405A1 (en) | 2018-11-14 | 2020-05-22 | サントリーホールディングス株式会社 | Chitosan-containing beverage |
| CN111647531A (en) * | 2020-06-03 | 2020-09-11 | 青岛北方茶仓茶文化有限公司 | Siamese bacillus LBP for increasing antioxidant activity, and fermentation product and application thereof |
| CN111647531B (en) * | 2020-06-03 | 2022-03-11 | 青岛北方茶仓茶文化有限公司 | Siamese bacillus LBP for increasing antioxidant activity, and fermentation product and application thereof |
| CN114366816A (en) * | 2022-01-05 | 2022-04-19 | 重庆福美干细胞生物科技发展有限公司 | Chitosan nanoparticles with excellent freeze-drying redispersibility, freeze-drying preparation, and preparation method and application thereof |
| CN114366816B (en) * | 2022-01-05 | 2023-06-20 | 重庆福美干细胞生物科技发展有限公司 | Chitosan nanoparticle with excellent freeze-drying redispersibility, freeze-drying preparation, and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Freitas et al. | Structure and applications of pectin in food, biomedical, and pharmaceutical industry: A review | |
| Kim et al. | Enzymatic production and biological activities of chitosan oligosaccharides (COS): A review | |
| Zhang et al. | Preliminary characterization and anti-hyperglycemic activity of a pectic polysaccharide from okra (Abelmoschus esculentus (L.) Moench) | |
| Lodhi et al. | Chitooligosaccharide and its derivatives: Preparation and biological applications | |
| Muzzarelli et al. | Chitin and chitosan hydrogels | |
| JP5306537B2 (en) | Antibacterial adjuvant, antibacterial composition, and food and drink containing kelp extract as an active ingredient | |
| JP2004519220A (en) | Method for producing pectin hydrolyzate | |
| Qin et al. | Safety evaluation of short-term exposure to chitooligomers from enzymic preparation | |
| JPH04218501A (en) | Edible fiber, production thereof and physiologically active agent containing the edible fiber | |
| WO2007013717A1 (en) | High-quality water-soluble chitosan oligosaccharide, composition comprising the same and method for the preparation thereof | |
| JP2021500085A (en) | Pectic polysaccharide enzyme hydrolyzate for the treatment or prevention of infectious diseases | |
| Yang et al. | Recent developments in Moringa oleifera Lam. polysaccharides: A review of the relationship between extraction methods, structural characteristics and functional activities | |
| JP4917584B2 (en) | Processed solubilized bees, method for producing the same, antioxidant, ACE inhibitor, antihypertensive agent, dermal fibroblast proliferation promoter, fatigue recovery agent, blood flow improving agent, and solubilized bees Pharmaceuticals, cosmetics or food / drinks contained | |
| JP2005075957A (en) | Water-soluble chitosan | |
| WO2008032134A1 (en) | Method for obtaining yeast glucan by autolysis of saccharomyces cerevisiae baker's yeast cells | |
| JPH06256208A (en) | Immunopotentiator | |
| JP2000217546A (en) | Sweetener, pathogenic bacterial infection-inhibitory agent, intestinal function-controlling agent or aging- inhibitory agent for starch, consisting of mannose- containing hetero oligosaccharide, and food, beverage, medine or feed consisting of the same | |
| JP4981327B2 (en) | UV absorber | |
| JP6275341B2 (en) | Single-stranded β-glucan composition, method for producing the same, and liquid composition | |
| JP3523220B2 (en) | Skin application agent | |
| JP5185519B2 (en) | Highly dispersible chitosan and method for producing the same | |
| Panchal et al. | Chitosan as a natural polymer: an overview | |
| DE10104055A1 (en) | Use of carbohydrates to eliminate intestinal infections in animals | |
| JP6544503B2 (en) | Cosmetic composition and functional food containing collagenase inhibitor and collagenase inhibitor | |
| Kim et al. | Production of bioactive chitosan oligosaccharides and their potential use as nutraceuticals |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20060704 |
|
| A711 | Notification of change in applicant |
Free format text: JAPANESE INTERMEDIATE CODE: A712 Effective date: 20080428 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20080826 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20091127 |
|
| A762 | Written abandonment of application |
Free format text: JAPANESE INTERMEDIATE CODE: A762 Effective date: 20100115 |