JP2009510234A - Abalone polysaccharide extraction method - Google Patents
Abalone polysaccharide extraction method Download PDFInfo
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- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 145
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 145
- 150000004676 glycans Chemical class 0.000 title claims abstract description 144
- 238000000605 extraction Methods 0.000 title claims abstract description 59
- 102000004190 Enzymes Human genes 0.000 claims abstract description 121
- 108090000790 Enzymes Proteins 0.000 claims abstract description 121
- 229940088598 enzyme Drugs 0.000 claims abstract description 121
- 239000002244 precipitate Substances 0.000 claims abstract description 117
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 116
- 239000006228 supernatant Substances 0.000 claims abstract description 106
- 238000000034 method Methods 0.000 claims abstract description 58
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 45
- 108090000284 Pepsin A Proteins 0.000 claims abstract description 26
- 102000057297 Pepsin A Human genes 0.000 claims abstract description 26
- 210000000056 organ Anatomy 0.000 claims abstract description 26
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- 238000001556 precipitation Methods 0.000 claims abstract description 26
- 102000004142 Trypsin Human genes 0.000 claims abstract description 21
- 108090000631 Trypsin Proteins 0.000 claims abstract description 21
- 239000012588 trypsin Substances 0.000 claims abstract description 21
- 238000002791 soaking Methods 0.000 claims abstract description 12
- 230000002255 enzymatic effect Effects 0.000 claims abstract description 9
- 239000000047 product Substances 0.000 claims abstract description 6
- 239000012141 concentrate Substances 0.000 claims abstract description 5
- 238000002137 ultrasound extraction Methods 0.000 claims abstract description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 72
- 238000005119 centrifugation Methods 0.000 claims description 65
- 230000007515 enzymatic degradation Effects 0.000 claims description 43
- 239000002994 raw material Substances 0.000 claims description 37
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- 239000000843 powder Substances 0.000 claims description 30
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- 210000002966 serum Anatomy 0.000 claims description 16
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- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 8
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- 239000008103 glucose Substances 0.000 description 7
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 6
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 6
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 6
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 6
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- 230000005526 G1 to G0 transition Effects 0.000 description 4
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 description 4
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical group C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 4
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- 239000012159 carrier gas Substances 0.000 description 4
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- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 4
- 108091005658 Basic proteases Proteins 0.000 description 3
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 description 3
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- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 description 2
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 2
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 2
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 2
- 230000003187 abdominal effect Effects 0.000 description 2
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- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 2
- SRBFZHDQGSBBOR-QMKXCQHVSA-N alpha-L-arabinopyranose Chemical compound O[C@H]1CO[C@@H](O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-QMKXCQHVSA-N 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229930182830 galactose Natural products 0.000 description 2
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- 150000007523 nucleic acids Chemical class 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 229960001322 trypsin Drugs 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 229920001503 Glucan Polymers 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 208000001894 Nasopharyngeal Neoplasms Diseases 0.000 description 1
- 206010061306 Nasopharyngeal cancer Diseases 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 102000004139 alpha-Amylases Human genes 0.000 description 1
- 108090000637 alpha-Amylases Proteins 0.000 description 1
- 229940024171 alpha-amylase Drugs 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000003471 anti-radiation Effects 0.000 description 1
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- 210000004369 blood Anatomy 0.000 description 1
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- 150000001720 carbohydrates Chemical class 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
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- 230000006806 disease prevention Effects 0.000 description 1
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- 238000003379 elimination reaction Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 239000010903 husk Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- CTYRPMDGLDAWRQ-UHFFFAOYSA-N phenyl hydrogen sulfate Chemical compound OS(=O)(=O)OC1=CC=CC=C1 CTYRPMDGLDAWRQ-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Public Health (AREA)
- General Chemical & Material Sciences (AREA)
- Sustainable Development (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Materials Engineering (AREA)
- Polymers & Plastics (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Extraction Or Liquid Replacement (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
本発明は、あわびから殻を取除いて本体（内臓を含む）を取り出し、組織破砕機で破砕し、水を加えて均一に混合し、20〜80℃で2〜6時間漬けて成分抽出し、遠心分離し、沈殿に再び水を加え、1〜2回繰り返して成分抽出して、上清液を混合するあわび多糖類の抽出方法を提供する。成分抽出した液を糖含有量が1.5〜3.0％となるように濃縮し、体積で3〜4倍の95％アルコールを加え、0〜4℃で12〜16時間アルコール沈殿を行い、遠心分離して真空乾燥または噴霧乾燥により生成品を得る。脱蛋白・除糖原したのち、SephadexG-100、DEAE-celluroseを用いて分離精製して均一のあわび多糖類を得る。成分抽出はアルカリ加水抽出、超音波抽出、酵素抽出によりも行うことができる。酵素抽出は、単一酵素と、二重酵素と、複合酵素と、自己融解酵素と、複合法とにより行える。酵素は、ペプシンとトリプシンなどである。本発明は、あわび（特に腹足と臓器）からあわび多糖類を抽出する技術を創出し、あわび多糖類の抽出・分離精製を効率的に行うようにした。あわび多糖類の薬用用途についての研究がますます深まり、本発明はその研究のさらなる開発に技術的基礎を提供した。 In the present invention, the shell is removed from the abalone, the main body (including the internal organs) is taken out, crushed with a tissue crusher, water is added and mixed uniformly, and the components are extracted by soaking at 20-80 ° C for 2-6 hours. A method for extracting abalone polysaccharides is provided by centrifuging, adding water to the precipitate again, extracting the components repeatedly 1-2 times, and mixing the supernatant. Concentrate the component-extracted solution to a sugar content of 1.5-3.0%, add 3-4 times 95% alcohol by volume, perform alcohol precipitation at 0-4 ° C for 12-16 hours, and centrifuge. The product is obtained by vacuum drying or spray drying. After deproteinization and deglycosylation, separation and purification using Sephadex G-100 and DEAE-cellurose yields a uniform abalone polysaccharide. Component extraction can also be performed by alkaline hydrolysis extraction, ultrasonic extraction, and enzyme extraction. Enzymatic extraction can be performed by a single enzyme, a double enzyme, a complex enzyme, a self-melting enzyme, and a complex method. Enzymes include pepsin and trypsin. The present invention has created a technique for extracting abalone polysaccharides from abalone (especially gastropods and organs), so that abalone polysaccharides can be efficiently extracted, separated and purified. With more and more research on the medicinal uses of abalone polysaccharides, the present invention has provided a technical basis for further development of the research.
Description
本発明は多糖類の抽出及び分離精製方法に関するものであり、生物製品領域に属すものである。 The present invention relates to a method for extracting and separating and purifying polysaccharides, and belongs to the biological product area.
近年、分子生物学の進歩により、多糖類とタンパク質は、核酸（ヌクレイン酸）と同様に、生命活動の本質にかかわる三種類の生物大分子中の一つであると認識されてきた。多糖類は多聚糖とも称され、広い生物活性を有する一種類の生物大分子物質であり、ヒトの生命にとって必要な成分であるだけでなく、すべての細胞膜にも存在し、且つ多様の生命機能活動に寄与する。近頃、多糖類はヒトの免疫機能を向上させる機能を有すると共に、抗ガン、抗放射線、抗炎症、血糖値を低下させるなどの生理活性をも有するので、広く注目され、重要視されている。 In recent years, due to advances in molecular biology, polysaccharides and proteins have been recognized as one of three types of biological macromolecules related to the essence of life activity, as well as nucleic acids (nucleic acid). Polysaccharides are also called polysaccharides, and are one type of biological macromolecular substance with a wide range of biological activities. They are not only necessary for human life but also present in all cell membranes and are used in various life forms. Contribute to functional activities. In recent years, polysaccharides have attracted wide attention and importance because they have functions of improving human immune function and have physiological activities such as anti-cancer, anti-radiation, anti-inflammation and blood glucose level reduction.
現在、国内外で多く研究されているのは食用・薬用菌多糖類である。しかし、多糖類への研究が深まるにつれて、研究者も動物体多糖類へと関心を寄せ始めている。特に海洋動物体多糖類の研究は、科学研究者にとって日増しに研究のホットスポットと注目点になってきている。あわびは成長期が長く、海洋の高塩、高圧、低温という特殊な環境に生存して、体の中には大量の生理活性物質が豊富に含有している。近年、あわびから分離・精製され、鼻咽喉ガン細胞を効率よく抑制できる純多糖類についての研究がある。大量の研究論文報道でも、あわび多糖類が非常によい抗腫瘍活性及びヒトの免疫機能を向上させる機能を具備することを示している。中国医薬情報ウェブ（http://www.yy2000.com）に発表された文章の記載によると、あわび多糖類とのり多糖類は抗腫瘍と心臓血管疾病の予防・治療作用を有し、この薬用研究は今も進行中であり、さらに多く、良い効果が得られると期待されている。しかしながら、動物から多糖類を抽出する研究はまだ比較的少ない。海洋動物から多糖類を抽出して、分離・精製する研究がさらに少なく、あわびから効果的に多糖類を抽出する研究はいまだに報道されていないようである。 Currently, edible / medicinal polysaccharides are being studied extensively both in Japan and overseas. However, as research into polysaccharides deepens, researchers are beginning to be interested in animal polysaccharides. In particular, research on marine animal polysaccharides is increasingly becoming a research hot spot and attention for scientific researchers. Abalone has a long growth period and lives in a special environment of high salt, high pressure and low temperature in the ocean, and the body is rich in a large amount of physiologically active substances. In recent years, there has been research on pure polysaccharides that have been isolated and purified from abalone and can efficiently inhibit nasopharyngeal cancer cells. A large amount of research paper reports also shows that abalone polysaccharides have very good antitumor activity and the ability to improve human immune function. According to a statement published on the Chinese Medicine Information Web (http://www.yy2000.com), abalone and glue polysaccharides have antitumor and cardiovascular disease prevention and treatment effects. Research is still ongoing, and many more are expected to produce better results. However, there are still relatively few studies to extract polysaccharides from animals. There are still fewer studies to extract, separate and purify polysaccharides from marine animals, and research on extracting polysaccharides effectively from abalone has not yet been reported.
本発明は、殻が除かれたあわび本体、あわびの腹足、あわびの臓器を原料とし、効果的にあわび多糖類を抽出して、効率よく分離・精製する方法を提供することを目的とする。 An object of the present invention is to provide a method for efficiently extracting, separating and purifying abalone polysaccharides, using abalone body, abalone stomach, and abalone organs from which shells have been removed as raw materials. .
本発明は、あわびから原料処理・抽出・アルコール沈殿・乾燥の工程によりあわび粗多糖類を得ることを目的としている。また、得られた粗多糖類から蛋白質を取り除き、分離・精製してあわび精製多糖類を得ることも可能である。具体的な方法は、以下の如くである。 It is an object of the present invention to obtain abalone crude polysaccharide from abalone by raw material treatment, extraction, alcohol precipitation, and drying. It is also possible to remove a protein from the obtained crude polysaccharide and separate and purify it to obtain an abalone purified polysaccharide. A specific method is as follows.
一、原料処理
原料あわびは新鮮なものでも、冷凍品でも、または乾燥品でも用いることができる。また、あわびの腹足と臓器から、またはあわびの腹足と臓器のそれぞれから単独であわび多糖類を抽出することができる。新鮮なあわびまたは解凍したあわびを洗浄して殻を取り除いて、分離作業によりあわび腹足と臓器とを得る。それらを個別にまたは均一に混合して5〜10倍の水を加えて、組織破砕機で破砕・ホモジネートして次の工程に備える。乾燥品は、次のように準備する。殻付きの冷凍あわびを5〜10分間かけてゆっくりと解凍し、殻を取除いて、分離作業によりあわびの腹足と臓器とを得る。それらを個別にまたは均一に混合して冷凍乾燥または火干して、100目以下まで粉砕して次の工程に備える。
1. Raw material treatment Raw material abalone can be used fresh, frozen, or dried. Also, abalone polysaccharide can be extracted from abalone stomach and organ or from abalone stomach and organ alone. Fresh abalone or thawed abalone is washed to remove the shell, and abdominal stomach and organs are obtained by separation work. Mix them individually or uniformly, add 5 to 10 times the water, crush and homogenize with a tissue crusher to prepare for the next step. The dried product is prepared as follows. Thaw frozen abalone with a shell slowly over 5-10 minutes, remove the shell, and obtain abdominal stomach and organs by separation. They are individually or evenly mixed, freeze-dried or fire-dried, and pulverized to 100 or less to prepare for the next step.
二、あわび多糖類の抽出
あわび多糖類の抽出は、水漬抽出法と、アルカリ液抽出法と、超音波抽出法とのいずれか又はこれらの組合せを採用すればよい。さらに、前記三種類の方法に酵素を加え得率を高めることも可能である。
2. Extraction of Abalone Polysaccharide Extraction of the abalone polysaccharide may be carried out by any one of a soaking extraction method, an alkaline liquid extraction method, an ultrasonic extraction method, or a combination thereof. Furthermore, it is also possible to increase the yield by adding enzymes to the above three methods.
１ 水漬抽出法
漿液（ホモジネートして得られる液）または干し粉に、10〜50重量倍の水を加え、均一化するように混合し、20〜80℃で2〜6時間漬けて成分抽出を行ってから、遠心分離により上清液と沈澱とが得られる。そして、沈澱に10〜40重量倍の水を加え、20〜80℃で2〜6時間漬けて成分抽出を行ってから、再び遠心分離し、それぞれ得られる上清液を混合して次の工程に備える。
1 Submerged extraction method Add 10 to 50 times by weight of water to serum (liquid obtained by homogenization) or dried powder, mix to homogenize, and extract components by soaking at 20-80 ° C for 2-6 hours Then, a supernatant and a precipitate are obtained by centrifugation. Then, add 10 to 40 times by weight water to the precipitate, soak for 2 to 6 hours at 20 to 80 ° C., extract components, then centrifuge again, and mix the resulting supernatants to the next step Prepare for.
２ アルカリ液抽出法
漿液または干し粉に、10〜50重量倍の水を加え、均一化するように混合させ、0.5mol/LのNaOHまたはKOH溶液を加え、pH値を8〜9に調整し、20〜60℃で2〜6時間漬けて成分抽出を行ってから、遠心分離することにより上清液と沈澱とを得ることができる。そして、得られた沈澱に10〜40重量倍の水を加え、pH値を8〜9に保持しながら、前記作業を1〜2回繰り返し、得られた上清液を混合して酸で中和して次の工程に備える。
2 Alkaline liquid extraction method Add 10-50 times by weight water to serum or dried powder, mix to homogenize, add 0.5 mol / L NaOH or KOH solution, and adjust pH value to 8-9. After extracting the components by soaking at 20 to 60 ° C. for 2 to 6 hours, the supernatant and the precipitate can be obtained by centrifugation. Then, 10 to 40 times by weight of water was added to the resulting precipitate, and the above procedure was repeated once or twice while maintaining the pH value at 8 to 9, and the resulting supernatant was mixed and mixed with acid. Prepare for the next step.
３ 超音波抽出法
原料処理により得られたあわび漿液または干し粉にあわび量の10〜50倍の水を加え、20〜30kHzの周波数を有する超音波を10〜60分間照射する処理を行い、遠心分離により上清液と沈澱とを得ることができる。そして、得られた沈澱に10〜40倍の水を加え、再び超音波で10〜60分間処理を行い、遠心分離により上清液と沈澱とを得ることができる。それぞれで得られた上清液を混合して次の工程に備える。
3 Ultrasonic extraction method Add water 10-50 times the amount of abalone to the abalone serum or dried powder obtained by raw material treatment, and irradiate with ultrasonic waves having a frequency of 20-30 kHz for 10-60 minutes, then centrifuge A supernatant and a precipitate can be obtained by separation. Then, 10 to 40 times as much water is added to the obtained precipitate, treatment is again carried out with ultrasonic waves for 10 to 60 minutes, and a supernatant and a precipitate can be obtained by centrifugation. The supernatant liquid obtained in each is mixed and prepared for the next step.
４ 酵素抽出法
酵素抽出法は、あわび漿液または干し粉に水を加えて、単一酵素、二重酵素、複合酵素または自己融解酵素を用いて、所用酵素に適宜なpH値と温度などの条件で酵素分解を行う。また、前記他の抽出法と結合することも、抽出する目的に達成できる。前記単一酵素、二重酵素、複合酵素の例は、ペプシン、トリプシン、パパイン、中性プロテアーゼなどである。
4 Enzyme extraction method Enzyme extraction method is to add water to abalone serum or dried powder, and use single enzyme, double enzyme, complex enzyme or self-melting enzyme, conditions such as appropriate pH value and temperature for the required enzyme. Enzymatic digestion at Also, combining with the other extraction methods can be achieved for the purpose of extraction. Examples of the single enzyme, double enzyme and complex enzyme are pepsin, trypsin, papain, neutral protease and the like.
（１）単一酵素、二重酵素、複合酵素による酵素分解法
１）単一酵素による酵素分解法：処理された原料にあわび量の10〜50倍の水を加えて攪拌・混合する。6mol/LのHClを用いてpH値を1〜5に調整し、溶液重量の0.05〜3.00％のペプシンを加え、35〜50℃で2〜5時間攪拌して酵素分解を行う。加水分解するときに前記pH値を保持する。そして0.5mol/LのNaOHまたはKOH溶液を用いてpH値を中性に調整し、2〜5分間で90〜98℃まで昇温して10分間維持し酵素を失活させ、5分以内に室温まで冷却し、遠心分離することにより上清液と沈殿とを得ることができる。上清液は次の工程に備えるが、沈殿には10〜40倍の水を加えて同様の作業を1〜2回繰り返し、遠心分離により得た上清液を混合して次の工程に備えることも可能である。その他の品種の酵素（ペプシン、トリプシン、パパイン、中性プロテアーゼ、アルカリ性プロテアーゼなど）による酵素分解の作業は、前記と同じであるが、アルカリ液または酸性液で所用酵素に適宜な範囲まで調整する。
(1) Enzymatic degradation method using a single enzyme, double enzyme, or complex enzyme 1) Enzymatic degradation method using a single enzyme: Add 10 to 50 times the amount of water to the treated raw material, and stir and mix. The pH value is adjusted to 1 to 5 with 6 mol / L HCl, 0.05 to 3.00% pepsin of the solution weight is added, and the mixture is stirred at 35 to 50 ° C. for 2 to 5 hours to perform enzymatic degradation. The pH value is maintained when hydrolyzing. Then, adjust the pH value to neutral with 0.5 mol / L NaOH or KOH solution, raise the temperature to 90-98 ° C in 2-5 minutes and maintain for 10 minutes to inactivate the enzyme within 5 minutes. The supernatant and the precipitate can be obtained by cooling to room temperature and centrifuging. The supernatant is prepared for the next step, but 10-40 times water is added to the precipitate and the same operation is repeated once or twice, and the supernatant obtained by centrifugation is mixed to prepare for the next step. It is also possible. The enzymatic degradation with other varieties of enzymes (pepsin, trypsin, papain, neutral protease, alkaline protease, etc.) is the same as described above, but the alkaline enzyme or acidic solution is adjusted to the appropriate range for the enzyme of interest.
２）二重酵素による酵素分解法：処理された原料にあわび量の10〜50倍の水を加えて攪拌・混合する。6mol/LのHClを用いてpH値を1〜5に調整し、溶液重量の0.05〜3.00％のペプシンを加え、20〜60℃で1〜6時間攪拌して酵素分解を行う。加水分解するときに前記pH値を保持する。そして0.5mol/LのNaOHまたはKOH溶液を用いてpH値が7〜9となるように調整し、溶液重量の0.05〜3.00％のトリプシンを加え、20〜60℃の水浴において、1〜6時間攪拌して酵素分解を行う。加水分解するときに前記pH値を保持する。酵素分解後に酸でpH値を中性に調整し、2〜5分間で90〜98℃まで昇温して10分間維持して酵素を失活させ、5分間室温まで冷却し、遠心分離により上清液と沈殿とが得られる。上清液は次の工程に備えるが、沈殿には10〜40倍の水を加えて同様の作業を1〜2回繰り返し、遠心分離により得た上清液を混合して次の工程に備えることが可能である。その他の二重酵素複合アルカリ性プロテアーゼとパパインによる酵素分解作業は、前記と同じであるが、アルカリ液または酸性液で所用酵素に適する範囲まで調整する。 2) Enzymatic degradation method using double enzyme: Add 10 to 50 times the amount of water to the treated material and stir and mix. The pH value is adjusted to 1 to 5 with 6 mol / L HCl, 0.05 to 3.00% pepsin of the solution weight is added, and the enzyme is degraded by stirring at 20 to 60 ° C. for 1 to 6 hours. The pH value is maintained when hydrolyzing. Then adjust the pH value to 7-9 using 0.5 mol / L NaOH or KOH solution, add 0.05-3.00% trypsin of the solution weight, and in a water bath at 20-60 ° C for 1-6 hours Stir to perform enzymatic degradation. The pH value is maintained when hydrolyzing. After enzymatic degradation, adjust the pH to neutral with acid, raise the temperature to 90-98 ° C in 2-5 minutes and maintain for 10 minutes to deactivate the enzyme, cool to room temperature for 5 minutes, and increase by centrifugation A clear liquid and a precipitate are obtained. The supernatant is prepared for the next step, but 10-40 times water is added to the precipitate and the same operation is repeated once or twice, and the supernatant obtained by centrifugation is mixed to prepare for the next step. It is possible. Other double enzyme complex alkaline protease and papain enzymatic degradation work is the same as described above, but the alkaline solution or acidic solution is adjusted to a range suitable for the required enzyme.
３）複合酵素による酵素分解法：原料処理から得られたあわび漿液またはあわび干し粉にあわび量の10〜50倍の水を加え、0.5mol/LのNaOHまたはKOH溶液を用いてpH値が7〜9となるように調整し、パパインと中性プロテアーゼとトリプシンを加え、添加量は溶液重量の0.05〜3.00％で、三種類の酵素の比率は1.0:0.01〜1.0:0.01〜1.0（又は1.0：0.1〜1.0：0.1〜1.0）であり、20〜60℃で1〜6時間酵素分解を行ったのち、酸でpH値を7に調整し（中和し）、2〜5分間90〜98℃まで昇温して10分間維持して酵素を失活させ、5分間以内に室温まで冷却し、遠心分離により上清液と沈殿とが得られる。上清液は次の工程に備えるが、沈殿には10〜40倍の水を加えて同様な作業を1〜2回繰り返し、遠心分離により得られた上清液を混合して次の工程に備えることも可能である。 3) Enzymatic degradation method with complex enzyme: Add 10-50 times the amount of water to abalone serum or abalone dried powder obtained from raw material treatment, and use a 0.5 mol / L NaOH or KOH solution to adjust the pH value to 7 Adjusted to be ˜9, papain, neutral protease and trypsin are added, the addition amount is 0.05 to 3.00% of the solution weight, and the ratio of the three enzymes is 1.0: 0.01 to 1.0: 0.01 to 1.0 (or 1.0 : 0.1 to 1.0: 0.1 to 1.0), and after performing enzymatic degradation at 20 to 60 ° C for 1 to 6 hours, adjust the pH value to 7 (neutralize) with acid, and 90 to 98 for 2 to 5 minutes The enzyme is deactivated by raising the temperature to 0 ° C. and maintaining for 10 minutes, cooling to room temperature within 5 minutes, and a supernatant and a precipitate are obtained by centrifugation. The supernatant is prepared for the next step, but 10-40 times water is added to the precipitate and the same operation is repeated once or twice, and the supernatant obtained by centrifugation is mixed to the next step. It is also possible to provide.
（２）結合抽出法
１）自己融解と外因性酵素との結合抽出法：（１）自己融解：下に述べるいずれの方法を用いて自己融解を行うこと：(i)あわび漿液に重量の10〜50倍の水を加え、紫外線を10〜30分間照射し、0.06〜0.08mol/LのNaClを用いてpH値7.0〜7.5、30〜50℃でその自身の酵素により自己融解を行う。(ii)あわび漿液にあわび量の10〜50倍の水を加え、無菌酵素分解缶に入れて、pH6〜7.5、常温で6〜8時間自己融解を行う。(iii)あわび漿液にあわび重量の10〜50倍の水を加え、無菌酵素分解缶に入れて、pH6.0〜7.5、0〜4℃で24〜48時間自己融解を行う。
(2) Bond extraction method 1) Bond extraction method with autolysis and exogenous enzyme: (1) Self-melt: Perform auto-melt using any of the methods described below: Add ~ 50 times water, irradiate with UV for 10-30 minutes, and self-melt with its own enzyme at pH 7.0-7.5, 30-50 ° C. using 0.06-0.08 mol / L NaCl. (ii) Add 10-50 times the amount of water to the abalone serum and place it in a sterile enzyme digestion can and perform autolysis at pH 6-7.5 at room temperature for 6-8 hours. (iii) Add 10 to 50 times the abalone weight of water to the abalone serum and place it in a sterile enzyme digestion can and perform autolysis for 24 to 48 hours at pH 6.0 to 7.5 and 0 to 4 ° C.
（２）外因性酵素による酵素分解：前記自己融解の溶液を6mol/LのHClを用いてpH値が1〜5となるように調整し、溶液重量の0.3〜0.5％のペプシンを加え、40〜60℃で1〜2時間酵素分解を行ったのち、pH値が中性となるように調整し、2〜5分間90〜98℃まで昇温して10分間維持して酵素を失活させ、5分間室温まで冷却し、遠心分離により上清液と沈殿とが得られる。上清液は次の工程に備えるが、沈殿には10〜40倍の水を加えて同様の作業を1〜2回繰り返し、遠心分離により得た上清液を混合して次の工程に備えることも可能である。 (2) Enzymatic degradation by exogenous enzyme: The self-melting solution was adjusted to a pH value of 1 to 5 with 6 mol / L HCl, and pepsin at 0.3 to 0.5% of the solution weight was added. After enzymatic degradation at ~ 60 ° C for 1-2 hours, adjust the pH value to neutral, raise the temperature to 90-98 ° C for 2-5 minutes and maintain for 10 minutes to deactivate the enzyme Cool to room temperature for 5 minutes and obtain a supernatant and precipitate by centrifugation. The supernatant is prepared for the next step, but 10-40 times water is added to the precipitate and the same operation is repeated once or twice, and the supernatant obtained by centrifugation is mixed to prepare for the next step. It is also possible.
１）水漬抽出と二重酵素との結合抽出法：原料処理から得られたあわび漿液またはあわび干し粉にあわび量の10〜50倍の水を加え、20〜80℃で2〜6時間水に漬けて成分抽出を行う。6mol/LのHClを用いてpH値が1〜5となるように調整し、溶液重量の0.05〜3.00のペプシンを加え、20〜60℃で1〜6時間攪拌して酵素分解を行う。加水分解するときに前記pH値を保持する。そして、0.5mol/LのNaOHまたはKOH溶液を用いてpH値が7〜9となるように調整し、0.05〜3.00％のトリプシンを加え、20〜60℃の水浴において1〜6時間攪拌して酵素分解を行う。加水分解するときに前記pH値を保持する。酵素分解を行ったのち、酸でpH値が中性となるように調整し、2〜5分間で90〜98℃まで昇温して10分間維持して酵素を失活させ、5分間以内に室温まで冷却し、遠心分離により上清液と沈殿とが得られる。上清液は次の工程に備えるが、沈殿には10〜40倍の水を加えて同様の作業を1〜2回繰り返し、遠心分離により得た上清液を混合して次の工程に備えることも可能である。 1) Combined extraction method with soaking and double enzyme: Add 10-50 times the amount of abalone to abalone serum or abalone dried powder obtained from raw material treatment and water at 20-80 ° C for 2-6 hours Extract the ingredients by immersing them in The pH is adjusted to 1 to 5 with 6 mol / L HCl, 0.05 to 3.00 pepsin of the solution weight is added, and the mixture is stirred at 20 to 60 ° C. for 1 to 6 hours for enzymatic degradation. The pH value is maintained when hydrolyzing. Then, adjust the pH value to 7-9 using 0.5 mol / L NaOH or KOH solution, add 0.05-3.00% trypsin, and stir in a water bath at 20-60 ° C. for 1-6 hours. Enzymatic degradation is performed. The pH value is maintained when hydrolyzing. After enzymatic degradation, adjust the pH to neutral with acid, raise the temperature to 90-98 ° C in 2-5 minutes and maintain for 10 minutes to inactivate the enzyme within 5 minutes Cool to room temperature and obtain a supernatant and precipitate by centrifugation. The supernatant is prepared for the next step, but 10-40 times water is added to the precipitate and the same operation is repeated once or twice, and the supernatant obtained by centrifugation is mixed to prepare for the next step. It is also possible.
前記作業において、二種類の酵素は加水分解するときに要求するpH値が異なるため、前後二回にわけて酵素分解を行ったが、二種類の酵素が要求するpH値が同じである場合、同時に二種類の酵素を加えて、一括して酵素分解を行ってもよい。たとえば、パパイン、中性プロテアーゼとトリプシンは加水分解するときに要求するpH値が同じく7〜9であるので、このpH値でそのうち二種類の酵素を同時に加えることができる。 In the above work, since the two kinds of enzymes require different pH values when hydrolyzing, the enzyme degradation was performed twice before and after, but when the pH values required by the two kinds of enzymes are the same, Two kinds of enzymes may be added at the same time, and enzymatic degradation may be performed in a lump. For example, since papain, neutral protease and trypsin require the same pH value of 7 to 9 when hydrolyzing, two kinds of enzymes can be added simultaneously at this pH value.
２）超音波と単一酵素との結合抽出法：原料処理から得られたあわび漿液またはあわび干し粉にあわび量の10〜50倍の水を加え、20〜30kHzの周波数を有する超音波で10〜60分間処理を行い、遠心分離により上清液と沈澱とが得られる。さらに沈澱に10〜40倍の水を加え、0.5mol/LのNaOHまたはKOH溶液を用いてpH値が7〜9となるように調整し、パパインを加え、添加量は溶液重量の0.05〜3.00％であり、20〜60℃で1〜6時間酵素分解を行ったのち、酸でpH値を7に調整し（中和し）、2〜5分間で90〜98℃まで昇温して10分間維持して酵素を失活させ、5分間室温まで冷却し、遠心分離により上清液と沈殿とが得られる。上清液は次の工程に備えるが、沈殿には10〜40倍の水を加えて同様の作業を1〜2回繰り返し、遠心分離により得た上清液を混合して次の工程に備えることも可能である。 2) Combined extraction method of ultrasound and single enzyme: Add water 10-50 times the amount of abalone to abalone serum or abalone dry powder obtained from raw material treatment, and use ultrasound with a frequency of 20-30 kHz. Treatment is performed for ˜60 minutes, and a supernatant and a precipitate are obtained by centrifugation. Further, add 10 to 40 times water to the precipitate, adjust the pH to 7-9 using 0.5 mol / L NaOH or KOH solution, add papain, and add 0.05 to 3.00 of the solution weight. After enzymatic digestion at 20-60 ° C for 1-6 hours, adjust the pH value to 7 with acid (neutralize), raise the temperature to 90-98 ° C in 2-5 minutes and increase to 10 The enzyme is inactivated by maintaining for 5 minutes, cooled to room temperature for 5 minutes, and a supernatant and a precipitate are obtained by centrifugation. The supernatant is prepared for the next step, but 10-40 times water is added to the precipitate and the same operation is repeated once or twice, and the supernatant obtained by centrifugation is mixed to prepare for the next step. It is also possible.
前記作業からわかるように、超音波、アルカリ液、水漬けと単一酵素、二重酵素および複合酵素との結合は、いずれも可能であるが、酵素の最適なpH値と酵素の添加量を調整するだけである。前記すべての遠心分離は3000〜6000rpmで10分間位高速遠心操作である。 As can be seen from the above work, it is possible to combine ultrasonic, alkaline solution, water soaking and single enzyme, double enzyme and complex enzyme, but the optimum pH value of enzyme and the amount of enzyme added should be adjusted. Just adjust. All the above centrifugations are high-speed centrifugation operations at 3000 to 6000 rpm for about 10 minutes.
三 あわび多糖類抽出液の濃縮
前記のように遠心分離により得られた上清液を多糖類含有量1.5〜3.0%になるように真空濃縮する。
(3) Concentration of abalone polysaccharide extract The supernatant obtained by centrifugation as described above is vacuum concentrated to a polysaccharide content of 1.5 to 3.0%.
四 あわび多糖類のアルコール沈殿
濃縮液に体積で3〜4倍の95％アルコールを加え、0〜4℃で12〜16時間位アルコール沈殿する。
(4) Alcohol precipitation of abalone polysaccharide Add 3-4 times volume of 95% alcohol to the concentrate, and alcohol precipitate at 0-4 ° C for 12-16 hours.
五 あわび粗多糖類の遠心
アルコール沈殿後の多糖類を10分間位高速遠心することにより得られた沈殿は、あわび粗多糖類である。
(5) Centrifugation of Abalone Crude Polysaccharide The precipitate obtained by high-speed centrifugation of the polysaccharide after alcohol precipitation for about 10 minutes is abalone crude polysaccharide.
六 あわび粗多糖類の乾燥
あわび多糖類の乾燥は、以下のいずれの方法を採用しても目的を達成できる。
6. Drying of Abalone Polysaccharide The purpose of drying of Abalone polysaccharide can be achieved by adopting any of the following methods.
1、精製して得られた遠心沈澱を50〜60℃で真空乾燥して、乾燥のあわび粗多糖類干し物が得られる。 1. The centrifugal precipitate obtained after purification is vacuum-dried at 50-60 ° C. to obtain dried dried abalone polysaccharides.
2、精製して得られた遠心沈澱を水で比重が1.05〜1.10位になるまで希釈して、入り口温度は160〜180℃で、出口温度は50〜60℃である噴霧乾燥設備で噴霧乾燥して、乾燥のあわび粗多糖類が得られる。必要であれば、前記に得られたあわび多糖類を粉砕機で160目以下まで粉砕してよい。このとき、本発明にかかる方法で抽出したあわび多糖類は、粗多糖類で、ブラン粉末であり、多糖類抽出率は20％〜40％（干品比）で、多糖類含有量は6〜18％であり、温水に溶けやすく、冷水に微溶する。食品工業では幅広く応用されうる。さらに精製して脱蛋白すれば、多糖類含有量は40％〜60％またはさらに高くなることができ、薬品工業において幅広く応用されうる。前記作業により抽出したあわび粗多糖類は、以下の作業でさらに精製されうる。 2. Dilute the centrifugal precipitate obtained by purification with water until the specific gravity reaches 1.05 to 1.10, and spray dry with spray drying equipment with inlet temperature of 160-180 ° C and outlet temperature of 50-60 ° C Thus, dried abalone crude polysaccharide is obtained. If necessary, the obtained abalone polysaccharide may be pulverized by a pulverizer to 160 mesh or less. At this time, the abalone polysaccharide extracted by the method according to the present invention is a crude polysaccharide, a bran powder, a polysaccharide extraction rate of 20% to 40% (dry product ratio), and a polysaccharide content of 6 to 18%, easily dissolved in warm water, slightly soluble in cold water. It can be widely applied in the food industry. If further purified and deproteinized, the polysaccharide content can be 40% -60% or even higher and can be widely applied in the pharmaceutical industry. Abalone crude polysaccharide extracted by the above operation can be further purified by the following operation.
七 蛋白及び小分子物質の取り除き
（1）あわび腹足から抽出したあわび粗多糖類（以下あわび腹足多糖類と称する）を2〜10%の溶液に調製し、4℃で体積比5：1の比率で10%トリクロロ酢酸溶液をゆっくり加えて攪拌する。この温度で10分間振り混ぜ、4℃で10分間高速遠心して上清液を取り出し、フォリンフェノール法で多糖類溶液中の蛋白含有量が0.5%以下であると検出されるまで、上記脱蛋白の作業を数回繰り返す。溶液に体積で4.5倍の95%アルコールを加えて一夜アルコール沈殿する。高速遠心して沈殿を取り出して3〜5%の多糖類液に調製し、分画分子量7,000Daの透析袋内に入れ、48〜72時間透析してから、透析袋内の液を濃縮し、冷凍乾燥することにより白い粉末が得られる。
7. Removal of protein and small molecule substances (1) Prepare abalone crude polysaccharide extracted from abalone stomach (hereinafter referred to as abalone stomach polysaccharide) in a 2-10% solution, 4: 1 volume ratio 5: 1 Slowly add a 10% trichloroacetic acid solution at a ratio of Shake at this temperature for 10 minutes, centrifuge at 4 ° C for 10 minutes at high speed, remove the supernatant, and until the protein content in the polysaccharide solution is detected to be 0.5% or less by the forinphenol method, Repeat the work several times. Add 4.5 times the volume of 95% alcohol to the solution to allow alcohol precipitation overnight. Precipitate is removed by high-speed centrifugation to prepare a 3-5% polysaccharide solution, put in a dialysis bag with a molecular weight cut off of 7,000 Da, dialyzed for 48-72 hours, and then concentrated in the dialysis bag and frozen. A white powder is obtained by drying.
（2）あわび臓器から抽出したあわび粗多糖類（以下あわび臓器多糖類と称する）を2〜10%の溶液に調製し、溶液重量の0.05〜1%のペプシンを加え、6mol/LのHClを用いてpH値が1.5〜3.0となるように調整し、37℃で1〜6時間酵素分解を行ったのち、2~5分以内に90〜98℃まで昇温して10分間維持して酵素を失活させ、室温まで冷却し、NaOH溶液を用いて中性に調整する。10分間遠心して上清液を取り出して体積で3〜4.5倍の95%アルコールを加え、0〜4℃で12〜16時間位アルコール沈殿する。遠心して沈殿を取り出して2〜5%の溶液に調製し、体積比5：1の比率でSevag試薬(Vクロロホルム：Vｎ-ブタノール=5：1)を加え、20分間激しく振り混ぜ、30分間静置してから、遠心して沈殿を取り除き、上清液を取り出し、フォリンフェノール法で多糖類溶液中の蛋白含有量が0.5%以下であると検知されるまで上記脱蛋白の作業を数回繰り返す。溶液に体積で4.5倍の95%アルコールを加えて一晩アルコール沈殿する。高速遠心して沈殿を取り出して2〜5%の多糖類液に調製し、分画分子量7,000Daの透析袋内に48〜72時間透析してから、透析袋中の液を濃縮し、冷凍乾燥することによりライトブラン粉末が得られ、精製あわび臓器多糖類（AHP）である。 (2) Prepare abalone crude polysaccharide extracted from abalone organ (hereinafter referred to as abalone organ polysaccharide) in 2-10% solution, add 0.05-1% pepsin of solution weight, and add 6 mol / L HCl. After adjusting the pH value to 1.5 to 3.0 and performing enzymatic degradation at 37 ° C for 1 to 6 hours, the temperature is raised to 90 to 98 ° C within 2 to 5 minutes and maintained for 10 minutes. Is cooled to room temperature and adjusted to neutral with NaOH solution. Centrifuge for 10 minutes, remove the supernatant, add 3 to 4.5 times 95% alcohol by volume, and precipitate alcohol for 12 to 16 hours at 0 to 4 ° C. Centrifuge and remove the precipitate to prepare a 2-5% solution. Add Sevag reagent (V chloroform : Vn -butanol = 5: 1) at a volume ratio of 5: 1, shake vigorously for 20 minutes, and 30 minutes After standing, centrifuge to remove the precipitate, remove the supernatant, and repeat the above deproteinization several times until the protein content in the polysaccharide solution is detected to be 0.5% or less by the forinphenol method . Add 4.5 times the volume of 95% alcohol to the solution to allow alcohol precipitation overnight. High-speed centrifugation removes the precipitate, prepares it in a 2-5% polysaccharide solution, dialyzes it in a dialysis bag with a molecular weight cut off of 7,000 Da for 48-72 hours, concentrates the solution in the dialysis bag, and freeze-drys it. As a result, a light bran powder is obtained, which is a purified abalone organ polysaccharide (AHP).
八 あわび腹足多糖類から糖原（グリコーゲン）の取り除き
蛋白と小分子物質が取り除かれたあわび腹足多糖類を5％の溶液に調製し、7,000Daの透析袋内に入れ、0.05〜1％のα-アミラーゼ（澱粉酵素）を加え、37℃で0.9％のNaCl溶液中で、48時間透析しながら、加水分解したのち、48時間水中で再透析する。透析後、袋中の液を濃縮し、冷凍乾燥することにより白い粉末が得られ、精製あわび腹足多糖類（AGP）である。
Elimination of Glucogen (Glycogen) from Abalone Gastropod Polysaccharides Prepare Abalone Gastropod Polysaccharides from which proteins and small molecules have been removed in a 5% solution and place them in a 7,000 Da dialysis bag, 0.05-1% Α-amylase (starch enzyme) was added, hydrolyzed in 0.9% NaCl solution at 37 ° C. for 48 hours, and then dialyzed again in water for 48 hours. After dialysis, the liquid in the bag is concentrated and freeze-dried to obtain a white powder, which is purified abalone stomach polysaccharide (AGP).
九 分離精製
（1）AGPはSephadexG-100カラム・クロマトグラフィで分離を行い、溶離剤は0.9%のNaClを用いて、溶出速度は0.5ml/minで、フェノール硫酸法で追跡検出し、グラフを作成して唯一の溶出ピークが検出され、溶出ピークで収集する。混合・透析・濃縮・冷凍乾燥することにより均一のあわび腹足多糖類AGPが得られる。
9. Separation and purification (1) AGP is separated by SephadexG-100 column chromatography, eluent is 0.9% NaCl, elution rate is 0.5 ml / min, followed by detection by phenol sulfuric acid method, and graph is created. Only one elution peak is detected and collected at the elution peak. Uniform abalone stomach polysaccharide AGP is obtained by mixing, dialysis, concentration, and freeze-drying.
（2）AHPはSephadexG-100カラム・クロマトグラフィで分離を行い、溶離剤は0.9%のNaClを用いて、溶出速度は0.5ml/minで、フェノール硫酸法で追跡検出し、グラフを作成して二つの溶出ピークが検出され、それぞれ溶出ピークで収集し、透析・乾燥してから、DEAEcellulose 52で精製し、NaCl溶液を用いて勾配溶出（gradientelution）を行い、すべて単一の溶出ピークである。収集して混合し、透析・濃縮する。冷凍乾燥して均一のあわび臓器多糖類AHP-1とAHP-2とが得られる。 (2) AHP was separated by Sephadex G-100 column chromatography, the eluent was 0.9% NaCl, the elution rate was 0.5 ml / min, followed by detection with the phenol-sulfuric acid method, and a graph was created. Two elution peaks were detected, collected at each elution peak, dialyzed and dried, purified with DEAEcellulose 52, and gradient elution with NaCl solution, all in a single elution peak. Collect, mix, dialyze and concentrate. By freeze-drying, uniform abalone organ polysaccharides AHP-1 and AHP-2 are obtained.
十 ゲルクロマトグラフ法で分子量の測定
ゲルカラムはSepharoseCL-6Bカラムを用い、溶離剤は0.9%のNaCl溶液で、流速は0.24ml/minである。
10. Measurement of molecular weight by gel chromatography The Sepharose CL-6B column is used as the gel column, the eluent is 0.9% NaCl solution, and the flow rate is 0.24 ml / min.
（１）ゲルカラムの規格化（standardization）
規格液の調製：分子量がそれぞれ1,000、5,000、12,000、80,000、270,000のスタンダードポリグルコサン（standardpolyglucosan）をそれぞれ二回蒸留水（doubledistilledwater）に溶かして10mg/mlの溶液に調製する。小さい分子量から大きい分子量順でそれぞれサンプルを注入し、分離して収集する。フェノール硫酸法で糖含量を追跡監視して、490nmで検出して、OD値が最も大きい試験管数を記録する。試験管数の分子量に対する対数による直線回帰分析を行い、直線回帰等式を求める。
(1) Standardization of gel column
Preparation of standard solution: Standard polyglucosan having molecular weights of 1,000, 5,000, 12,000, 80,000 and 270,000, respectively, is dissolved in double distilled water to prepare a solution of 10 mg / ml. Samples are injected in order from low to high molecular weight, separated and collected. The sugar content is monitored by the phenol sulfate method, detected at 490 nm, and the number of tubes with the highest OD value is recorded. Perform linear regression analysis by logarithm of the number of test tubes with respect to molecular weight to obtain a linear regression equation.
（２）糖分子量の測定
精製多糖類をダブル蒸留水に溶かして10mg/mlの溶液に調製する。同様な条件で分析を行い、試験管数を記録して回帰等式に代入して分子量を計算する。
(2) Measurement of sugar molecular weight A purified polysaccharide is dissolved in double distilled water to prepare a 10 mg / ml solution. Analyze under similar conditions, record the number of test tubes and substitute in regression equation to calculate molecular weight.
十一 色層法（ガスクロマトグラフ）で多糖類の単糖組成の測定
完全に乾燥した10mgの多糖類サンプルAGP、AHP-1、AHP-2のそれぞれに2mlの無水HCl-メチルアルコールを加え、N2を入れて封管し、80℃で20時間メチルアルコール分解し、管容器を取り出して室温になるまで放置する。無水KOH-メチルアルコールでpH=6となるように中和して、40℃で減圧回転蒸し干して乾燥する。完全に乾燥したメチルアルコール分解産物に0.2mlの無水ピリジンを加え、75℃で30分間溶解させてから、0.3mlシリル化剤を加えて均一になるまで振り混ぜ、数分間静置して上清を取り出して色層分析（ガスクロマトグラフ）を行う。
11 Measurement of polysaccharide monosaccharide composition by color layer method (gas chromatograph) Add 2 ml of anhydrous HCl-methyl alcohol to each of 10 mg of polysaccharide samples AGP, AHP-1 and AHP-2 which are completely dried. Put 2 in a sealed tube, decompose with methyl alcohol at 80 ° C for 20 hours, take out the tube container and leave it to room temperature. Neutralize with anhydrous KOH-methyl alcohol to pH = 6, and dry by drying under reduced pressure at 40 ° C. Add 0.2 ml of anhydrous pyridine to the completely dried methyl alcohol degradation product, dissolve at 75 ° C for 30 minutes, add 0.3 ml silylating agent, shake until homogeneous, leave it for a few minutes and leave the supernatant Is taken out and color layer analysis (gas chromatograph) is performed.
クロマトグラフ条件は、ガスクロマトグラフ（USAgilent6890N）、HP-1クロマトグラフカラム、固定相Methylsiloxane、キャリヤーガスN2、流速45ml/minである。水素炎イオン検出器（FID）を用い、検出温度は300℃で、注入量は1μlである。注入口のガス化温度は300℃であり、カラム温度はプログラムで昇温を制御し、150℃に、1分間保持、10℃/minで182℃まで昇温して、2分間保持、1℃/minで188℃まで昇温して、1分間保持、8℃/minで230℃まで昇温することである。 The chromatographic conditions are gas chromatograph (USAgilent 6890N), HP-1 chromatographic column, stationary phase Methylsiloxane, carrier gas N 2 , flow rate 45 ml / min. A flame ion detector (FID) is used, the detection temperature is 300 ° C., and the injection volume is 1 μl. The gasification temperature of the inlet is 300 ° C, and the column temperature is controlled by a program to raise the temperature. The temperature is raised to 188 ° C at / min, held for 1 minute, and raised to 230 ° C at 8 ° C / min.
このとき、本発明にかかる方法で得られるAGPは、白い粉末で、分子量が5,000〜10,000dal（Dalton）であり、ブドウ糖の組成である。本発明にかかる方法で得られるAHP-1とAHP-2は、ともにライトブラン粉末である。そのうち、AHP-1は、分子量が5×105dal位であり、ブドウ糖とペクチンシュガーの組成であり、そのモル比はGlu:Ara＝1.0:1.5で、アミノ糖鑑定の結果、アミノ糖が含まれないが、AHP-2は、分子量が10,000〜15,000dal（Dalton）であり、ラムノース（rhamnose）と、フコース（fucose）と、キシロース（xylose）と、ガラクトース（galactose）とグルコース（glucose）の組成であり、そのモル比はRha:Fuc:Xyl:Gal:Glu＝2.7:1.0:1.0:4.3:4.2で、微量のN-アセチルグルコサミン（N-acetylglucosamine）が含まれる。 At this time, AGP obtained by the method according to the present invention is a white powder having a molecular weight of 5,000 to 10,000 dal (Dalton) and a glucose composition. Both AHP-1 and AHP-2 obtained by the method according to the present invention are light bran powders. Among them, AHP-1 has a molecular weight of about 5 × 10 5 dal and is composed of glucose and pectin sugar, and its molar ratio is Glu: Ara = 1.0: 1.5. Although AHP-2 has a molecular weight of 10,000-15,000 dal (Dalton), the composition of rhamnose, fucose, xylose, galactose, and glucose The molar ratio is Rha: Fuc: Xyl: Gal: Glu = 2.7: 1.0: 1.0: 4.3: 4.2, and a trace amount of N-acetylglucosamine is contained.
1．本発明の抽出方法は合理的であり、あわび体内（殻を除く）からあわび多糖類を抽出する技術を創出し、あわび多糖類を最大限に抽出できるようにした。
2．抽出技術については、多数の効率的抽出方法が幅広く研究され確定していた。
3．あわび腹足多糖類とあわび臓器多糖類のそれぞれについて、分離精製方法と鑑定方法を体系的に研究して確定し、精製した均一多糖類を得ることができ、海洋薬物を豊富にするために前期準備をした。
4．あわび多糖類の薬用用途についての研究がますます深まり、その薬用価値が次第に高まるため、本発明はその経済的効果と利益を高めるには技術的基礎を提供した。
5．殻を除くあわびボディのすべてを抽出原料とし、特にあわびの腹足と臓器を利用して多糖類を抽出することができるので、資源を十分に利用するだけではなく、廃棄物の排出もなくした。
6．本発明は海洋生物の自己融解技術を採用するため、外因性酵素（exogenousenzyme）の使用量を大いに節約でき、コストが低減される。
1． The extraction method of the present invention is rational, and a technique for extracting abalone polysaccharides from the abalone body (excluding the shell) has been created so that abalone polysaccharides can be extracted to the maximum.
2． Regarding extraction techniques, a number of efficient extraction methods have been extensively studied and established.
3． To systematically study and confirm the separation and purification method and appraisal method for abalone gastropod polysaccharides and abalone organ polysaccharides, to obtain purified uniform polysaccharides, and to enrich marine drugs Prepared for the previous term.
Four. As research on medicinal uses of abalone polysaccharides has deepened and its medicinal value gradually increases, the present invention has provided a technical basis for enhancing its economic effects and benefits.
Five. Abalone body excluding husks is used as an extraction raw material, and polysaccharides can be extracted using abalone stomachs and organs in particular, which not only makes full use of resources but also eliminates waste generation. .
6． Since the present invention adopts marine organism auto-melting technology, the amount of exogenous enzyme used can be greatly saved and the cost can be reduced.
1,000gのあわび本体に5,000gの水を加え、組織破砕機でホモジネートし、25,000ｇの水を加えて均一に攪拌し、50℃で5時間漬けて成分抽出を行ったのち、高速遠心して上清液と沈殿とを得た。沈殿に25,000ｇの水を加えて均一に攪拌し、80℃で3時間漬けて成分抽出を行ったのち、高速遠心して上清液と沈殿とを得た。得られた上清液を混合した。上清液を多糖類含有量が3％（フェノール硫酸法で検出）となるまで真空濃縮した。濃縮液に体積で3倍の95％アルコール21,000mlを加え、4℃で16時間アルコール沈殿したのち、10分位高速遠心して沈澱を得た。この沈澱を60℃で真空乾燥して、乾燥したあわび粗多糖類61.6ｇを得たのち、粉砕機で160目以下に粉砕した。フェノール硫酸法で測定を行い、その多糖類含有量はたとえば8.1%であった。 Add 5,000 g of water to 1,000 g of abalone body, homogenize with a tissue crusher, add 25,000 g of water, stir uniformly, soak for 5 hours at 50 ° C, extract components, and then centrifuge at high speed A clear liquid and a precipitate were obtained. 25,000 g of water was added to the precipitate and stirred uniformly, and the components were extracted by soaking at 80 ° C. for 3 hours, followed by high-speed centrifugation to obtain a supernatant and a precipitate. The resulting supernatant was mixed. The supernatant was concentrated in vacuo until the polysaccharide content was 3% (detected by the phenol-sulfuric acid method). The concentrated solution was added with 21,000 ml of 95% alcohol at a volume of 3 times, and precipitated with alcohol at 4 ° C. for 16 hours, followed by high-speed centrifugation for about 10 minutes to obtain a precipitate. The precipitate was vacuum dried at 60 ° C. to obtain 61.6 g of dried abalone crude polysaccharide, and then pulverized to 160 meshes or less with a pulverizer. The measurement was performed by the phenol sulfuric acid method, and the polysaccharide content was 8.1%, for example.
1,000gの冷凍乾燥したあわび本体粉に5,000gの水を加えて均一に攪拌し、その中に0.1NのNaOH溶液を加えてpH値を9に調整し、20℃で6時間漬けて成分抽出を行ったのち、6mol/LのHClを用いてpH値を7に中和し、高速遠心して上清液と沈殿とを得た。沈殿に30,000ｇの水を加えて均一に攪拌し、その中に0.5mol/LのNaOH溶液を加えてpH値を8に調整し、80℃で3時間漬けて成分抽出を行ったのち、6mol/LのHClを用いてpH値を7に中和し、高速遠心して上清液と沈殿とを得た。得られた上清液を混合した。実施例1と同様にして濃縮・アルコール沈殿・遠心を行った。得られた沈澱を50℃で真空乾燥して、乾燥したあわび粗多糖類298ｇを得た。粉砕機で160目以下に粉砕した。フェノール硫酸法で測定を行い、その多糖類含有量はたとえば9.3%であった。 Add 5,000g of water to 1,000g of freeze-dried abalone powder, stir uniformly, add 0.1N NaOH solution to adjust pH value to 9, and soak for 6 hours at 20 ° C to extract ingredients Then, the pH value was neutralized to 7 using 6 mol / L HCl, and high speed centrifugation was performed to obtain a supernatant and a precipitate. Add 30,000 g of water to the precipitate and stir uniformly. Add 0.5 mol / L NaOH solution to the precipitate to adjust the pH value to 8, soak it at 80 ° C for 3 hours, extract components, and then add 6 mol. The pH value was neutralized to 7 using / L HCl, and high speed centrifugation was performed to obtain a supernatant and a precipitate. The resulting supernatant was mixed. Concentration, alcohol precipitation, and centrifugation were performed in the same manner as in Example 1. The resulting precipitate was vacuum dried at 50 ° C. to obtain 298 g of dried abalone crude polysaccharide. The powder was pulverized to 160 or less with a pulverizer. The measurement was performed by the phenol sulfuric acid method, and the polysaccharide content was, for example, 9.3%.
1,000gのあわび腹足を実施例1と同様に処理し、20kHzの超音波で60分間処理を行い、高速遠心して上清液と沈殿とを得た。沈殿に25,000ｇの水を加え、27kHzの超音波で20分間処理を行い、高速遠心して上清液と沈殿とを得た。得られた上清液を混合した。実施例1と同様にして，濃縮・アルコール沈澱・遠心を行った。得られた沈澱を50℃で真空乾燥して、乾燥したあわび粗多糖類65.2ｇを得た。粉砕機で160目以下に粉砕した。フェノール硫酸法で測定を行い、その多糖類含有量はたとえば8.8%であった。 1,000 g of abalone stomach was treated in the same manner as in Example 1, treated with 20 kHz ultrasonic waves for 60 minutes, and centrifuged at a high speed to obtain a supernatant and a precipitate. 25,000 g of water was added to the precipitate, treated with 27 kHz ultrasonic waves for 20 minutes, and centrifuged at a high speed to obtain a supernatant and a precipitate. The resulting supernatant was mixed. In the same manner as in Example 1, concentration, alcohol precipitation, and centrifugation were performed. The resulting precipitate was vacuum dried at 50 ° C. to obtain 65.2 g of dried abalone crude polysaccharide. The powder was pulverized to 160 mesh or less with a pulverizer. The measurement was performed by the phenol sulfuric acid method, and the polysaccharide content was, for example, 8.8%.
1,000gのあわび腹足を実施例1と同様の処理を行い、その中に0.5mol/LのNaOH溶液を加えてpH値が8となるように調整し、900gのトリプシン（酵素活性2,500U/mg）を加え、37℃で5時間酵素分解を行ったのち、6mol/LのHClを用いてpH値を7位に調整し（中和し）、5分間100℃まで昇温して10分間維持して酵素を失活させ、5分間以内に室温まで冷却し、高速遠心して上清液と沈殿とを得た。沈殿に25,000ｇの水を加え、その中に0.5mol/LのKOH溶液を加えてpH値を8に調整し、150gのトリプシン（酵素活性2,500U/mg）を加え、50℃で2時間酵素分解を行ったのち、6mol/LのHClを用いてpH値を7位に調整し（中和し）、5分間90℃まで昇温して10分間維持して酵素を失活させ、5分間以内に室温まで冷却し、高速遠心して上清液と沈殿とを得た。得られた上清液を混合した。実施例1と同様に濃縮・アルコール沈澱・遠心を行った。遠心分離で得られた沈澱を水で比重が1.07になるまで希釈し、噴霧乾燥を行った。入り口の温度が160℃で、出口の温度が50℃であった。この結果、たとえば乾燥したあわび多糖類43.2ｇを得た。フェノール硫酸法で測定を行い、その多糖類含有量はたとえば14.8%であった。 1,000 g of abalone stomach was treated in the same manner as in Example 1, and 0.5 mol / L NaOH solution was added thereto to adjust the pH value to 8, and 900 g of trypsin (enzymatic activity 2,500 U / mg) was added, and after enzymatic degradation at 37 ° C for 5 hours, the pH value was adjusted to the 7th position using 6mol / L HCl (neutralized), and the temperature was raised to 100 ° C for 5 minutes and then 10 minutes. The enzyme was inactivated by maintaining, cooled to room temperature within 5 minutes, and centrifuged at high speed to obtain a supernatant and a precipitate. Add 25,000 g of water to the precipitate, add 0.5 mol / L KOH solution to adjust the pH value to 8, add 150 g of trypsin (enzyme activity 2,500 U / mg), and enzyme for 2 hours at 50 ° C. After degradation, adjust the pH value to 7th position with 6mol / L HCl (neutralize), raise the temperature to 90 ° C for 5 minutes and maintain for 10 minutes to inactivate the enzyme for 5 minutes Were cooled to room temperature and centrifuged at high speed to obtain a supernatant and a precipitate. The resulting supernatant was mixed. Concentration, alcohol precipitation, and centrifugation were carried out in the same manner as in Example 1. The precipitate obtained by centrifugation was diluted with water to a specific gravity of 1.07 and spray-dried. The inlet temperature was 160 ° C and the outlet temperature was 50 ° C. As a result, for example, 43.2 g of dried abalone polysaccharide was obtained. The measurement was performed by the phenol sulfuric acid method, and the polysaccharide content was, for example, 14.8%.
1,000gのあわび腹足干し粉に50,000gの水を加えて均一に攪拌し、6mol/LのHClを用いてpH値が3となるように調整し、1,500gのペプシン（酵素活性50U/mg）を加え、50℃の水浴において2時間攪拌して酵素分解を行い、pH値を維持しつつ加水分解した。そして、0.5mol/LのKOH溶液を用いてpH値が中性となるように調整し、2〜5分間で90〜98℃まで昇温して10分間維持し酵素を失活させ、5分間に室温まで冷却し、遠心分離により上清液と沈殿とを得た。沈殿に30,000ｇの水を加え、500gのペプシン（酵素活性50U/mg）を加え、40℃の水浴において4時間攪拌して酵素分解を行い、pH値を維持しつつ加水分解した。そして、0.5mol/LのKOH溶液を用いてpH値が中性となるように調整し、5分間で98℃まで昇温して10分間維持して酵素を失活させ、5分間に室温まで冷却し、遠心分離により上清液と沈殿とを得た。遠心分離により得た上清液を混合して次の工程に備えた。実施例1と同様に濃縮・アルコール沈澱・遠心分離を行った。遠心分離で得られた沈澱を水で比重が1.06になるまで希釈し、噴霧乾燥を行った。入り口の温度が160℃で、出口の温度が50℃であった。この結果、たとえば乾燥したあわび多糖類236ｇを得た。フェノール硫酸法で測定を行い、その多糖類含有量はたとえば13.9%であった。 Add 50,000 g of water to 1,000 g of abalone stomach dried powder, stir uniformly, and adjust the pH value to 3 with 6 mol / L HCl, and 1,500 g of pepsin (enzyme activity 50 U / mg) The mixture was stirred for 2 hours in a 50 ° C. water bath and hydrolyzed while maintaining the pH value. Then, adjust the pH value to be neutral with 0.5 mol / L KOH solution, raise the temperature to 90-98 ° C. in 2-5 minutes and maintain for 10 minutes to deactivate the enzyme, 5 minutes After cooling to room temperature, a supernatant and a precipitate were obtained by centrifugation. 30,000 g of water was added to the precipitate, 500 g of pepsin (enzyme activity 50 U / mg) was added, and the mixture was stirred in a water bath at 40 ° C. for 4 hours to carry out enzymatic decomposition, and hydrolyzed while maintaining the pH value. Then, adjust the pH value to be neutral with 0.5 mol / L KOH solution, raise the temperature to 98 ° C. for 5 minutes and maintain for 10 minutes to inactivate the enzyme to 5 minutes to room temperature After cooling, a supernatant and a precipitate were obtained by centrifugation. The supernatant obtained by centrifugation was mixed and prepared for the next step. Concentration, alcohol precipitation, and centrifugation were performed in the same manner as in Example 1. The precipitate obtained by centrifugation was diluted with water to a specific gravity of 1.06 and spray-dried. The inlet temperature was 160 ° C and the outlet temperature was 50 ° C. As a result, for example, 236 g of dried abalone polysaccharide was obtained. Measurement was performed by the phenol sulfuric acid method, and the polysaccharide content was, for example, 13.9%.
1,000gのあわび腹足干し粉に50,000gの水を加えて均一に攪拌し、1,000gの中性プロテアーゼ（酵素活性100U/mg）を加え、40℃の水浴において5時間攪拌して酵素分解を行い、2分間で90℃まで昇温して10分間維持して酵素を失活させ、5分間で室温まで冷却し、遠心分離により上清液と沈殿とを得た。沈殿に30,000ｇの水を加え、150gの中性プロテアーゼ（酵素活性100U/mg）を加え、40℃の水浴において3時間攪拌して酵素分解を行い、2〜5分間90〜98℃まで昇温して10分間維持して酵素を失活させ、5分間に室温まで冷却し、遠心分離により上清液と沈殿とを得た。得られた上清液を混合した。実施例1と同様に濃縮・アルコール沈澱・遠心分離を行った。遠心分離で得られた沈澱を水で比重が1.1になるまで希釈し、噴霧乾燥を行った。入り口の温度が180℃で、出口の温度が60℃であった。この結果、たとえば乾燥したあわび多糖類225.5ｇを得た。フェノール硫酸法で測定を行い、その多糖類含有量はたとえば14.2%であった。 Add 50,000 g of water to 1,000 g of abalone stomach dried powder, stir uniformly, add 1,000 g of neutral protease (enzyme activity 100 U / mg), and stir for 5 hours in a 40 ° C water bath for enzymatic degradation. The temperature was raised to 90 ° C. in 2 minutes and maintained for 10 minutes to inactivate the enzyme, cooled to room temperature in 5 minutes, and centrifuged to obtain a supernatant and a precipitate. Add 30,000 g of water to the precipitate, add 150 g of neutral protease (enzyme activity 100 U / mg), stir in a 40 ° C water bath for 3 hours to perform enzymatic degradation, and raise the temperature to 90-98 ° C for 2-5 minutes Then, the enzyme was inactivated by maintaining for 10 minutes, cooled to room temperature in 5 minutes, and a supernatant and a precipitate were obtained by centrifugation. The resulting supernatant was mixed. Concentration, alcohol precipitation, and centrifugation were performed in the same manner as in Example 1. The precipitate obtained by centrifugation was diluted with water to a specific gravity of 1.1 and spray-dried. The inlet temperature was 180 ° C and the outlet temperature was 60 ° C. As a result, for example, 225.5 g of dried abalone polysaccharide was obtained. The measurement was performed by the phenol sulfuric acid method, and the polysaccharide content was, for example, 14.2%.
1,000gのあわび腹足干し粉に40,000gの水を加えて均一に攪拌し、0.5mol/LのKOH溶液を用いてpH値が8.5となるように調整し、600gのパパイン（酵素活性1,200U/mg）を加え、50℃の水浴において4時間攪拌して酵素分解を行い、pHを維持しつつ加水分解した。そして、6mol/LのHCl液を用いてpH値が中性となるように調整し、5分間98℃まで昇温して10分間維持して酵素を失活させ、5分間に室温まで冷却し、遠心分離により上清液と沈殿とを得た。沈殿に35,000ｇの水を加えて均一に攪拌し、0.5mol/LのKOH溶液を用いてpH値が9.0となるように調整し、175gのパパイン（酵素活性1,200u/mg）を加え、35℃の水浴において3時間攪拌して酵素分解を行い、pHを維持しつつ加水分解した。そして、6mol/LのHCl液を用いてpH値が中性となるように調整し、5分間90℃まで昇温して10分間維持して酵素を失活させ、5分間に室温まで冷却し、遠心分離により上清液と沈殿とを得た。得られた上清液を混合した。実施例1と同様にして濃縮・アルコール沈澱・遠心分離を行った。遠心分離で得られた沈澱を水で比重が1.1になるまで希釈し、噴霧乾燥を行う。入り口の温度が170℃で、出口の温度が55℃であった。この結果、たとえば乾燥したあわび多糖類234ｇを得た。フェノール硫酸法で測定を行い、その多糖類含有量はたとえば15.2%であった。 Add 40,000 g of water to 1,000 g of abalone stomach dried powder, stir uniformly, adjust to pH 8.5 with 0.5 mol / L KOH solution, and add 600 g of papain (enzyme activity 1,200 U). / mg), and the mixture was stirred in a water bath at 50 ° C. for 4 hours to carry out enzymatic degradation, and hydrolyzed while maintaining the pH. Then, adjust the pH value to neutral with 6 mol / L HCl solution, raise the temperature to 98 ° C. for 5 minutes and maintain for 10 minutes to deactivate the enzyme, and cool to room temperature for 5 minutes. The supernatant and the precipitate were obtained by centrifugation. Add 35,000 g of water to the precipitate and stir uniformly. Adjust to pH 9.0 with 0.5 mol / L KOH solution, add 175 g of papain (enzyme activity 1,200 u / mg), 35 Enzymatic degradation was carried out by stirring for 3 hours in a water bath at 0 ° C., and hydrolysis was carried out while maintaining pH. Then, adjust the pH value to neutral with 6 mol / L HCl solution, raise the temperature to 90 ° C. for 5 minutes and maintain for 10 minutes to inactivate the enzyme, and cool to room temperature for 5 minutes. The supernatant and the precipitate were obtained by centrifugation. The resulting supernatant was mixed. Concentration, alcohol precipitation, and centrifugation were carried out in the same manner as in Example 1. The precipitate obtained by centrifugation is diluted with water to a specific gravity of 1.1 and spray-dried. The inlet temperature was 170 ° C and the outlet temperature was 55 ° C. As a result, for example, 234 g of dried abalone polysaccharide was obtained. The measurement was performed by the phenol sulfuric acid method, and the polysaccharide content was, for example, 15.2%.
1,000gのあわび臓器に5,000gの水を加え、組織破砕機でホモジネートし、25,000ｇの水を加えて均一に攪拌し、6mol/LのHClを用いてpH値が1となるように調整し、900gのペプシン（酵素活性50U/mg）を加え、35℃の水浴において2時間攪拌して酵素分解を行い、pHを維持しつつ加水分解した。そして、0.5mol/LのKOH溶液を用いてpH値が9となるように調整し、300gのトリプシン（酵素活性2,500U/mg）を加え、55℃の水浴において4時間攪拌して酵素分解を行い、加水分解するときに前記pH値を保持していた。酵素分解後に酸でpH値を中性に調整し、5分間98℃まで昇温して10分間維持して酵素を失活させ、5分間で室温まで冷却し、遠心分離により上清液と沈殿とを得た。沈殿に10,000ｇの水を加えて同様の作業を一回繰り返した。遠心分離により得た上清液を混合して次の工程に備えた。その後の作業は実施例7の様に行い、たとえば乾燥したあわび多糖類50.2ｇを得た。フェノール硫酸法で測定を行い、その多糖類含有量はたとえば8.4%であった。 Add 5,000 g water to 1,000 g abalone organ, homogenize with tissue disrupter, add 25,000 g water, stir uniformly, and adjust the pH value to 1 using 6 mol / L HCl. 900 g of pepsin (enzyme activity 50 U / mg) was added, and the mixture was stirred in a water bath at 35 ° C. for 2 hours to carry out the enzymatic decomposition and hydrolyze while maintaining the pH. Then, adjust the pH value to 9 with 0.5 mol / L KOH solution, add 300 g trypsin (enzyme activity 2,500 U / mg), and stir in a water bath at 55 ° C. for 4 hours for enzymatic degradation. The pH value was maintained during the hydrolysis. After enzymatic degradation, adjust the pH to neutral with acid, raise the temperature to 98 ° C for 5 minutes and maintain for 10 minutes to inactivate the enzyme, cool to room temperature in 5 minutes, and centrifuge the supernatant and precipitate And got. The same operation was repeated once by adding 10,000 g of water to the precipitate. The supernatant obtained by centrifugation was mixed and prepared for the next step. Subsequent operations were performed as in Example 7, for example, to obtain 50.2 g of dried abalone polysaccharide. The measurement was performed by the phenol sulfuric acid method, and the polysaccharide content was, for example, 8.4%.
1,000gのあわび本体を実施例1と同様の処理を行い、その中に0.5mol/LのKOH溶液を加えてpH値を7に調整し、300gのパパイン（酵素活性1,200U/mg）と、100gの中性プロテアーゼ（酵素活性100U/mg）と、100gのトリプシン（酵素活性2,500U/mg）とを加え、37℃で6時間酵素分解を行ったのち、5分間98℃まで昇温して10分間酵素減滅し、5分間以内に室温（20℃）まで冷却し、高速遠心して上清液と沈殿とを得た。沈殿に20,000ｇの水を加え、その中に0.5mol/LのKOH溶液を加えてpH値を7に調整し（中和し）、200gのパパイン（酵素活性1,200U/mg）と、50gの中性プロテアーゼ（酵素活性100U/mg）と、200gのトリプシン（酵素活性2,500U/mg）とを加え、50℃で4時間酵素分解を行ったのち、pH値を7位に調整し（中和し）、5分間98℃まで昇温して10分間維持して酵素を失活させ、5分間以内に室温（20℃）まで冷却し、高速遠心して上清液と沈殿とを得た。遠心分離により得た上清液を混合して次の工程に備えた。その後の作業は実施例7と同様に行い、たとえば乾燥したあわび多糖類50.9ｇを得た。フェノール硫酸法で測定を行い、その多糖類含有量はたとえば14.6%であった。 1,000 g of abalone body was treated in the same manner as in Example 1, 0.5 mol / L KOH solution was added to adjust pH value to 7, 300 g of papain (enzyme activity 1,200 U / mg), Add 100 g of neutral protease (enzyme activity 100 U / mg) and 100 g trypsin (enzyme activity 2,500 U / mg), perform enzymatic degradation at 37 ° C for 6 hours, then raise the temperature to 98 ° C for 5 minutes. The enzyme was depleted for 10 minutes, cooled to room temperature (20 ° C.) within 5 minutes, and centrifuged at high speed to obtain a supernatant and a precipitate. Add 20,000 g of water to the precipitate, add 0.5 mol / L KOH solution to adjust the pH value to 7 (neutralize), 200 g of papain (enzyme activity 1,200 U / mg), 50 g Neutral protease (enzyme activity 100 U / mg) and 200 g trypsin (enzyme activity 2,500 U / mg) were added, and after enzymatic degradation at 50 ° C for 4 hours, the pH value was adjusted to the 7th position (neutralization) The temperature was raised to 98 ° C. for 5 minutes and maintained for 10 minutes to inactivate the enzyme, cooled to room temperature (20 ° C.) within 5 minutes, and centrifuged at high speed to obtain a supernatant and a precipitate. The supernatant obtained by centrifugation was mixed and prepared for the next step. Subsequent operations were performed in the same manner as in Example 7. For example, 50.9 g of dried abalone polysaccharide was obtained. The measurement was performed by the phenol sulfuric acid method, and the polysaccharide content was, for example, 14.6%.
1,000gのあわび本体を実施例1と同様の処理を行い、得られた漿液に紫外線を30分間照射し、濃度0.06mol/LのNaClを用いてpH7.5、50℃で自己融解を行い、得られた自己融解後の溶液を6mol/LのHClを用いてpH値が5となるように調整し、150gのペプシン（酵素活性50U/mg）を加え、40℃で2時間酵素分解を行った。pH値を7に調整し（中和し）、5分間98℃まで昇温して10分間維持して酵素を失活させ、5分間以内に室温まで冷却し、高速遠心して上清液と沈殿とを得た。沈殿に25,000ｇの水を加え、6mol/LのHClを用いてpH値が5となるように調整し、80gのペプシン（酵素活性50U/mg）を加え、60℃で1時間酵素分解を行ったのち、0.5mol/LのKOH溶液を用いてpH値が7位となるように調整し（中和し）、5分間98℃まで昇温して10分間維持して酵素を失活させ、5分間以内に室温まで冷却し、高速遠心して上清液と沈殿とを得た。得られた上清液を混合した。その後の作業は実施例1と同様に行った。この結果、たとえば乾燥したあわび粗多糖類51.3ｇを得た。フェノール硫酸法で測定を行い、その多糖類含有量はたとえば14.6%であった。 1,000 g of abalone body was treated in the same manner as in Example 1, and the resulting serum was irradiated with ultraviolet rays for 30 minutes, and self-melted at pH 7.5, 50 ° C. using NaCl with a concentration of 0.06 mol / L. Adjust the pH of the resulting solution after self-melting to 6 with 6 mol / L HCl, add 150 g of pepsin (enzyme activity 50 U / mg), and perform enzymatic decomposition at 40 ° C for 2 hours. It was. Adjust pH value to 7 (neutralize), heat up to 98 ° C for 5 minutes and maintain for 10 minutes to inactivate enzyme, cool to room temperature within 5 minutes, centrifuge at high speed and precipitate supernatant and precipitate And got. Add 25,000 g of water to the precipitate, adjust the pH value to 5 with 6 mol / L HCl, add 80 g of pepsin (enzyme activity 50 U / mg), and perform enzymatic degradation at 60 ° C for 1 hour. After that, adjust the pH value to 7th position with 0.5 mol / L KOH solution (neutralize), heat up to 98 ° C for 5 minutes and maintain for 10 minutes to deactivate the enzyme, The solution was cooled to room temperature within 5 minutes and centrifuged at a high speed to obtain a supernatant and a precipitate. The resulting supernatant was mixed. Subsequent operations were performed in the same manner as in Example 1. As a result, for example, 51.3 g of dried abalone crude polysaccharide was obtained. The measurement was performed by the phenol sulfuric acid method, and the polysaccharide content was, for example, 14.6%.
1,000gのあわび本体を実施例1と同様の処理を行い、得られた漿液に紫外線を10分間照射し、濃度0.08mol/LのNaClを用いてpH7、30℃で自己融解を行った。その後の作業は実施例10と同様に行った。たとえば乾燥したあわび粗多糖類50.9ｇを得た。フェノール硫酸法で測定を行い、その多糖類含有量はたとえば14.4%であった。 A 1,000 g abalone body was treated in the same manner as in Example 1, and the resulting serum was irradiated with ultraviolet rays for 10 minutes, and self-melted at pH 7 and 30 ° C. using NaCl with a concentration of 0.08 mol / L. Subsequent operations were performed in the same manner as in Example 10. For example, 50.9 g of dried abalone crude polysaccharide was obtained. The measurement was performed by the phenol sulfuric acid method, and the polysaccharide content was, for example, 14.4%.
1,000gのあわび臓器を実施例1と同様の処理を行い、得られた漿液を無菌酵素分解缶に入れ、pH値を6に調整し、常温で8時間自己融解を行った。得られた自己融解後の溶液を6mol/LのHClを用いてpH値が3となるように調整し、30gのペプシン（酵素活性50U/mg）とを加え、40℃で2時間酵素分解を行った。pH値を7に調整し（中和し）、3分間で90℃まで昇温して10分間維持して酵素を失活させ、5分間以内に室温まで冷却し、高速遠心して上清液と沈殿とを得た。その後の作業は実施例10と同様に行った。この結果、たとえば乾燥したあわび粗多糖類66.2ｇを得た。フェノール硫酸法で測定を行い、その多糖類含有量はたとえば6.5%であった。 1,000 g of abalone organs were treated in the same manner as in Example 1. The obtained serum was placed in a sterile enzymatic digester, the pH value was adjusted to 6, and autolysis was performed at room temperature for 8 hours. Adjust the pH of the resulting solution after self-melting to 6 using 6 mol / L HCl, add 30 g of pepsin (enzyme activity 50 U / mg), and perform enzymatic degradation at 40 ° C for 2 hours. went. Adjust the pH value to 7 (neutralize), heat up to 90 ° C in 3 minutes and maintain for 10 minutes to inactivate the enzyme, cool to room temperature within 5 minutes, centrifuge at high speed and Got with precipitation. Subsequent operations were performed in the same manner as in Example 10. As a result, for example, 66.2 g of dried abalone crude polysaccharide was obtained. The measurement was performed by the phenol sulfuric acid method, and the polysaccharide content was, for example, 6.5%.
1,000gのあわび臓器を実施例1と同様の処理を行い、得られた漿液を無菌酵素分解缶に入れ、pH値を7.5に調整し、常温で6時間自己融解を行った。前記自己融解後の溶液を6mol/LのHClを用いてpH値が5となるように調整し、100gのペプシン（酵素活性50U/mg）を加え、40℃で1〜2時間酵素分解を行った。酵素分解後にpH値を中性に調整し、5分間で98℃まで昇温して10分間維持して酵素を失活させ、5分間で室温まで冷却し、遠心分離により上清液と沈殿とを得た。沈殿に水を加えて同様の作業を一回繰り返し、遠心分離により得た上清液を混合して次の工程に備えた。その後の作業は実施例10と同様に行った。この結果、たとえば乾燥したあわび粗多糖類64.8ｇを得た。フェノール硫酸法で測定を行い、その多糖類含有量はたとえば6.8%であった。 1,000 g of abalone organs were treated in the same manner as in Example 1. The obtained serum was placed in a sterile enzymatic digester, the pH value was adjusted to 7.5, and autolysis was performed at room temperature for 6 hours. Adjust the pH of the self-melted solution to 6 with 6 mol / L HCl, add 100 g of pepsin (enzyme activity 50 U / mg), and perform enzymatic degradation at 40 ° C for 1-2 hours. It was. Adjust the pH to neutral after enzymatic degradation, raise the temperature to 98 ° C for 5 minutes and maintain for 10 minutes to inactivate the enzyme, cool to room temperature in 5 minutes, and centrifuge the supernatant and precipitate. Got. Water was added to the precipitate and the same operation was repeated once, and the supernatant obtained by centrifugation was mixed to prepare for the next step. Subsequent operations were performed in the same manner as in Example 10. As a result, for example, 64.8 g of dried abalone crude polysaccharide was obtained. Measurement was performed by the phenol sulfuric acid method, and the polysaccharide content was, for example, 6.8%.
1,000gのあわび腹足を実施例1と同様の処理を行い、50℃で3時間漬けて抽出し、6mol/LのHClを用いてpH値を3に調整し、150gのペプシン（酵素活性50U/mg）を加え、40℃の水浴において5時間攪拌して酵素分解を行い、pHを維持しつつ加水分解した。そして0.5mol/LのKOH溶液を用いてpH値が7〜9となるように調整し、100gのトリプシンを加え、55℃の水浴において2時間攪拌して酵素分解を行い、pHを維持しつつ加水分解した。酵素分解後に酸でpH値を中性に調整し、5分間で98℃まで昇温して10分間維持して酵素を失活させ、5分間で室温まで冷却し、遠心分離により上清液と沈殿とを得た。沈殿に水を加えて同様の作業を一回繰り返し、遠心分離により得た上清液を混合して次の工程に備えた。実施例1と同様に濃縮・アルコール沈澱・遠心を行った。遠心分離で得られた沈澱を水で比重が1.08になるまで希釈し、噴霧乾燥を行う。入り口の温度が170℃で、出口の温度が55℃であった。この結果、たとえば乾燥したあわび多糖類50.3ｇを得た。フェノール硫酸法で測定を行い、その多糖類含有量はたとえば16.1%であった。 1,000 g of abalone stomach legs were treated in the same manner as in Example 1, extracted by soaking at 50 ° C. for 3 hours, adjusted to pH 3 with 6 mol / L HCl, 150 g of pepsin (enzyme activity 50 U). / mg) was added, and the mixture was stirred in a water bath at 40 ° C. for 5 hours for enzymatic degradation, and hydrolysis was carried out while maintaining the pH. Then, adjust the pH value to 7-9 using 0.5 mol / L KOH solution, add 100 g trypsin, stir in a water bath at 55 ° C. for 2 hours to perform enzymatic degradation, while maintaining the pH Hydrolyzed. After enzymatic degradation, adjust the pH to neutral with acid, raise the temperature to 98 ° C for 5 minutes and maintain for 10 minutes to inactivate the enzyme, cool to room temperature in 5 minutes, and centrifuge to remove the supernatant. Got with precipitation. Water was added to the precipitate and the same operation was repeated once, and the supernatant obtained by centrifugation was mixed to prepare for the next step. Concentration, alcohol precipitation, and centrifugation were carried out in the same manner as in Example 1. The precipitate obtained by centrifugation is diluted with water to a specific gravity of 1.08 and spray-dried. The inlet temperature was 170 ° C and the outlet temperature was 55 ° C. As a result, for example, 50.3 g of dried abalone polysaccharide was obtained. The measurement was performed by the phenol sulfuric acid method, and the polysaccharide content was, for example, 16.1%.
1,000gのあわび腹足を実施例1と同様に処理を行い、20kHzの超音波で60分間処理を行い、遠心分離により上清液と沈澱とを得た。さらに沈澱に25,000ｇの水を加え、0.5mol/LのKOH溶液を用いてpH値が8.0となるように調整し、500gのパパイン（酵素活性1,200U/mg）を加え、4時間酵素分解を行ったのち、6mol/LのHClを用いてpH値が7となるように調整し（中和し）、2〜5分間で90〜98℃まで昇温して10分間維持して酵素を失活させ、5分間に室温まで冷却し、遠心分離により上清液と沈殿とを得た。上清液を混合した。実施例1と同様に濃縮・アルコール沈澱・遠心を行った。遠心分離で得られた沈澱を水で比重が1.08になるまで希釈し、噴霧乾燥を行う。入り口の温度が180℃で、出口の温度が50℃であった。この結果、たとえば乾燥したあわび多糖類49.2ｇを得た。フェノール硫酸法で測定を行い、その多糖類含有量はたとえば15.8%であった。 A 1,000 g abalone stomach was treated in the same manner as in Example 1, treated with 20 kHz ultrasonic waves for 60 minutes, and a supernatant and a precipitate were obtained by centrifugation. Add 25,000 g of water to the precipitate, adjust the pH to 8.0 using 0.5 mol / L KOH solution, add 500 g of papain (enzyme activity 1,200 U / mg), and perform enzymatic degradation for 4 hours. After that, adjust the pH value to 6 with 6mol / L HCl (neutralize), raise the temperature to 90-98 ° C in 2-5 minutes and maintain for 10 minutes to lose the enzyme. The mixture was cooled to room temperature for 5 minutes, and a supernatant and a precipitate were obtained by centrifugation. The supernatant was mixed. Concentration, alcohol precipitation, and centrifugation were carried out in the same manner as in Example 1. The precipitate obtained by centrifugation is diluted with water to a specific gravity of 1.08 and spray-dried. The inlet temperature was 180 ° C and the outlet temperature was 50 ° C. As a result, for example, 49.2 g of dried abalone polysaccharide was obtained. The measurement was performed by the phenol sulfuric acid method, and the polysaccharide content was, for example, 15.8%.
1,000gのあわび腹足を実施例1と同様に処理を行い、30kHzの超音波で30分間処理を行い、遠心分離により上清液と沈澱とを得た。さらに沈澱に25,000ｇの水を加え、6mol/LのHClを用いてpH値を3に調整し、100gのペプシン（酵素活性50U/mg）を加え、40℃の水浴において5時間攪拌して酵素分解を行った。pHを維持しつつ加水分解した。そして0.5mol/LのKOH溶液を用いてpH値が7〜9となるように調整し、80gのトリプシンを加え、55℃の水浴において2時間攪拌して酵素分解を行い、pHを維持しつつ加水分解した。酵素分解後に酸でpH値を中性に調製し、5分間で95℃まで昇温して10分間維持して酵素を失活させ、5分以内に室温まで冷却し、遠心分離により上清液と沈殿とを得た。遠心分離により得た上清液を混合して次の工程に備えた。実施例1と同様に濃縮・アルコール沈澱・遠心を行った。遠心分離で得られた沈澱を水で比重が1.10になるまで希釈し、噴霧乾燥を行う。入り口の温度が180℃で、出口の温度が55℃であった。この結果、たとえば乾燥したあわび多糖類58.2ｇを得た。フェノール硫酸法で測定を行い、その多糖類含有量はたとえば17.4%であった。 A 1,000 g abalone stomach was treated in the same manner as in Example 1, treated with 30 kHz ultrasonic waves for 30 minutes, and a supernatant and a precipitate were obtained by centrifugation. Add 25,000 g of water to the precipitate, adjust the pH value to 3 with 6 mol / L HCl, add 100 g of pepsin (enzyme activity 50 U / mg), and stir in a water bath at 40 ° C. for 5 hours. Decomposition was performed. Hydrolysis was performed while maintaining the pH. Then, adjust the pH value to 7-9 using 0.5 mol / L KOH solution, add 80 g trypsin, stir in a water bath at 55 ° C. for 2 hours to perform enzymatic decomposition, while maintaining the pH Hydrolyzed. After enzymatic degradation, the pH is adjusted to neutral with acid, heated to 95 ° C for 5 minutes and maintained for 10 minutes to inactivate the enzyme, cooled to room temperature within 5 minutes, and centrifuged to obtain the supernatant. And got a precipitate. The supernatant obtained by centrifugation was mixed and prepared for the next step. Concentration, alcohol precipitation, and centrifugation were carried out in the same manner as in Example 1. The precipitate obtained by centrifugation is diluted with water to a specific gravity of 1.10 and spray-dried. The inlet temperature was 180 ° C and the outlet temperature was 55 ° C. As a result, for example, 58.2 g of dried abalone polysaccharide was obtained. The measurement was performed by the phenol sulfuric acid method, and the polysaccharide content was, for example, 17.4%.
1,000gのあわび臓器粉に25,000gの水を加えて均一に攪拌し、6mol/LのNaOHを用いてpH値が10となるように調整し、1,000gのアルカリ性プロテアーゼ（酵素活性40U/mg）を加え、45℃の水浴において3時間攪拌して酵素分解を行い、pHを維持しつつ加水分解した。酵素分解後に酸でpH値を中性に調整し、5分間98℃まで昇温して10分間維持して酵素を失活させ、5分間に室温まで冷却し、遠心分離により上清液と沈殿とを得た。さらに沈殿に10,000ｇの水を加え、同様の作業を一回繰り返し、遠心分離により得られた上清液を混合した。その後の作業は実施例7と同様に行い、たとえば乾燥したあわび多糖類235.4ｇを得た。フェノール硫酸法で測定を行い、その多糖類含有量はたとえば11.8%であった。 Add 25,000g of water to 1,000g abalone organ powder, stir uniformly, adjust to pH value 10 with 6mol / L NaOH, 1,000g alkaline protease (enzyme activity 40U / mg) The mixture was stirred for 3 hours in a 45 ° C. water bath and hydrolyzed while maintaining the pH. After enzymatic degradation, adjust the pH to neutral with acid, raise the temperature to 98 ° C for 5 minutes and maintain for 10 minutes to inactivate the enzyme, cool to room temperature for 5 minutes, and centrifuge the supernatant and precipitate And got. Furthermore, 10,000 g of water was added to the precipitate, the same operation was repeated once, and the supernatant obtained by centrifugation was mixed. Subsequent operations were performed in the same manner as in Example 7. For example, 235.4 g of dried abalone polysaccharide was obtained. The measurement was performed by the phenol sulfuric acid method, and the polysaccharide content was, for example, 11.8%.
実施例17により得られたあわび臓器粗多糖類5ｇに、245ｇの水を加え、0.125ｇのペプシン（酵素活性50U/mg）を加え、6mol/LのHClを用いてpH値が1.5となるように調整し、37℃で6時間酵素分解を行ったのち、2分間90℃まで昇温して10分間維持して酵素失活させ、室温まで冷却し、0.5mol/LのNaOH溶液を用いてpH値を中性に調整し、10分間遠心して上清液を取り出し、1125mlの95％アルコールを加え、4℃で12時間アルコール沈殿した。遠心して沈殿を取り出し、250ｇの水を加え、50mlのSevag試薬(Vクロロホルム：Vｎ-ブタノール=5：1)を加え、20分間激しく振り混ぜ、30分間静置し、遠心して沈殿を取り除き、上清液を取り出し、以上の脱蛋白の作業を6回繰り返した。このとき多糖類溶液中の蛋白含有量はたとえば0.35%であった。溶液の中に1125mlの95％アルコールを加えて一夜アルコール沈殿し、高速遠心して沈殿を取り出し、100ｇの水を加え、分画分子量7,000Daの透析袋に48時間透析してから、透析袋中の液を濃縮し、冷凍乾燥することによりブラン粉末2.1ｇを得た。得られた粉末は、精製あわび臓器多糖類（AHP）であった。AHPをSephadexG-200カラム・クロマトグラフィで分離を行い、溶離剤は0.9%のNaCl溶液で、溶出速度は0.5ml/minで、フェノール硫酸法で追跡検出し、グラフを作成して二つの溶出ピークが得られ、それぞれ溶出ピークで収集し、透析・乾燥してから、DEAEcellulose 52で精製し、NaCl溶液で勾配溶出を行い、すべて単一の溶出ピークであった。収集して混合し、透析・濃縮した。冷凍乾燥することにより均一のあわび臓器多糖類AHP-1とAHP-2とを得た。 To 5 g of abalone organ crude polysaccharide obtained in Example 17, 245 g of water is added, 0.125 g of pepsin (enzyme activity 50 U / mg) is added, and pH is adjusted to 1.5 using 6 mol / L HCl. After the enzyme digestion at 37 ° C for 6 hours, the temperature was raised to 90 ° C for 2 minutes and maintained for 10 minutes to inactivate the enzyme, cooled to room temperature, and 0.5 mol / L NaOH solution was used. The pH value was adjusted to neutral, centrifuged for 10 minutes, the supernatant was taken out, 1125 ml of 95% alcohol was added, and alcohol precipitation was performed at 4 ° C. for 12 hours. Remove the precipitate by centrifugation, add 250 g of water, add 50 ml of Sevag reagent (V chloroform : Vn -butanol = 5: 1), shake vigorously for 20 minutes, let stand for 30 minutes, centrifuge to remove the precipitate, The supernatant was removed and the above deproteinization operation was repeated 6 times. At this time, the protein content in the polysaccharide solution was, for example, 0.35%. Add 1125 ml of 95% alcohol to the solution and precipitate overnight with alcohol, centrifuge at high speed to remove the precipitate, add 100 g of water, dialyze into a dialysis bag with a molecular weight cut off of 7,000 Da for 48 hours, The liquid was concentrated and freeze-dried to obtain 2.1 g of bran powder. The resulting powder was purified abalone organ polysaccharide (AHP). AHP was separated by Sephadex G-200 column chromatography, the eluent was 0.9% NaCl solution, the elution rate was 0.5 ml / min, traced and detected by the phenol-sulfuric acid method, and a graph was created to show two elution peaks. Each obtained was collected as an elution peak, dialyzed and dried, purified with DEAEcellulose 52, and gradient elution was performed with a NaCl solution, all of which were single elution peaks. Collected, mixed, dialyzed and concentrated. Uniform abalone organ polysaccharides AHP-1 and AHP-2 were obtained by freeze-drying.
実施例17により得られたあわび臓器粗多糖類5ｇに、45ｇの水を加え、0.5ｇのペプシン（酵素活性50U/mg）を加え、6mol/LのHClを用いてpH値が3.0となるように調整し、37℃で1時間酵素分解を行ったのち、5分間90℃まで昇温して10分間維持して酵素を失活させ、室温まで冷却し、0.5mol/LのNaOH溶液を用いてpH値を中性に調整し、10分間遠心して上清液を取り出し、225mlの95％アルコールを加え、0℃で16時間アルコール沈殿した。遠心して沈殿を取り出し、100ｇの水を加え、20mlのSevag試薬(Vクロロホルム：Vｎ-ブタノール=5：1)を加え、20分間激しく振り混ぜ、30分間静置し、遠心して沈殿を取り除き、上清液を取り出し、以上の脱蛋白の作業を8回繰り返した。このとき多糖類溶液中の蛋白含有量はたとえば0.25%であった。溶液の中に450mlの95％アルコールを加えて一晩アルコール沈殿し、高速遠心して沈殿を取り出し、80ｇの水を加え、分画分子量7,000Daの透析袋に72時間透析してから、透析袋中の液を濃縮し、冷凍乾燥することによりたとえばライトブラン粉末1.7ｇを得た。精製あわび臓器多糖類（AHP）であった。 To 5 g of abalone organ crude polysaccharide obtained in Example 17, 45 g of water is added, 0.5 g of pepsin (enzyme activity 50 U / mg) is added, and the pH value becomes 3.0 using 6 mol / L HCl. After the enzyme digestion at 37 ° C for 1 hour, the temperature was raised to 90 ° C for 5 minutes and maintained for 10 minutes to inactivate the enzyme, cooled to room temperature, and 0.5 mol / L NaOH solution was used. The pH value was adjusted to neutral, centrifuged for 10 minutes, the supernatant was taken out, 225 ml of 95% alcohol was added, and alcohol precipitation was performed at 0 ° C. for 16 hours. Remove the precipitate by centrifugation, add 100 g of water, add 20 ml of Sevag reagent (V chloroform : Vn -butanol = 5: 1), shake vigorously for 20 minutes, let stand for 30 minutes, centrifuge to remove the precipitate, The supernatant was removed and the above deproteinization operation was repeated 8 times. At this time, the protein content in the polysaccharide solution was, for example, 0.25%. 450ml of 95% alcohol is added to the solution, and alcohol is precipitated overnight. After high-speed centrifugation, the precipitate is taken out, 80g of water is added, dialyzed in a dialysis bag with a molecular weight cut off of 7,000 Da for 72 hours, and then in the dialysis bag. The liquid was concentrated and freeze-dried to obtain 1.7 g of light bran powder, for example. It was a purified abalone organ polysaccharide (AHP).
実施例17により得られたあわび臓器粗多糖類5ｇに、95ｇの水を加え、0.5ｇのペプシン（酵素活性50U/mg）を加え、6mol/LのHClを用いてpH値が2.0となるように調整し、37℃で6時間酵素分解を行ったのち、5分間98℃まで昇温して10分間維持して酵素を失活させた、室温まで冷却し、0.5mol/LのNaOH溶液を用いて中性に調整し、10分間遠心して上清液を取り出し、300mlの95％アルコールを加え、0℃で16時間アルコール沈殿した。遠心して沈殿を取り出し、100ｇの水を加え、20mlのSevag試薬(Vクロロホルム：Vｎ-ブタノール=5：1)を加え、20分間激しく振り混ぜ、30分間静置し、遠心して沈殿を取り除き、上清液を取り出し、以上の脱蛋白の作業を3回繰り返した。このとき多糖類溶液中の蛋白含有量はたとえば0.45%であった。溶液の中に450mlの95％アルコールを加えて一晩アルコール沈殿し、高速遠心して沈殿を取り出し、100ｇの水を加え、分画分子量7,000Daの透析袋に72時間透析してから、透析袋中の液を濃縮し、冷凍乾燥することによりたとえばライトブラン粉末2.25ｇを得た。得られた粉末は、精製あわび臓器多糖類（AHP）であった。 95 g of water is added to 5 g of abalone organ crude polysaccharide obtained in Example 17, 0.5 g of pepsin (enzyme activity 50 U / mg) is added, and the pH value becomes 2.0 using 6 mol / L HCl. After the enzyme digestion at 37 ° C for 6 hours, the temperature was raised to 98 ° C for 5 minutes and maintained for 10 minutes to deactivate the enzyme, cooled to room temperature, and 0.5 mol / L NaOH solution was added. The mixture was neutralized, centrifuged for 10 minutes, and the supernatant was removed. 300 ml of 95% alcohol was added, and alcohol precipitation was performed at 0 ° C. for 16 hours. Remove the precipitate by centrifugation, add 100 g of water, add 20 ml of Sevag reagent (V chloroform : Vn -butanol = 5: 1), shake vigorously for 20 minutes, let stand for 30 minutes, centrifuge to remove the precipitate, The supernatant was removed and the above deproteinization operation was repeated three times. At this time, the protein content in the polysaccharide solution was, for example, 0.45%. 450ml of 95% alcohol is added to the solution, and alcohol is precipitated overnight. After high-speed centrifugation, the precipitate is taken out, 100g of water is added, dialyzed into a dialysis bag with a molecular weight cut off of 7,000 Da for 72 hours, and then in the dialysis bag. The liquid was concentrated and freeze-dried to obtain, for example, 2.25 g of light bran powder. The resulting powder was purified abalone organ polysaccharide (AHP).
実施例7により得られたあわび腹足粗多糖類5ｇに、45ｇの水を加え、4℃で10%トリクロロ酢酸溶液を体積比5：1の比率でゆっくり加えて攪拌した。この温度に10分間振り混ぜ、4℃で10分間高速遠心して上清液を取り出して多糖類溶液中の蛋白含有量が0.42%以下となるように上記脱蛋白の作業を3回繰り返した。溶液の中に225mlの95%アルコールを加えて12時間アルコール沈殿した。高速遠心して沈殿を取り出し、100ｇの水を加え、分画分子量7,000Daの透析袋に入れ、48時間透析してから、透析袋中の液を濃縮し、冷凍乾燥することによりたとえば白い粉末1.5ｇを得た。 45 g of water was added to 5 g of the abalone stomach crude polysaccharide obtained in Example 7, and 10% trichloroacetic acid solution was slowly added at a volume ratio of 5: 1 at 4 ° C. and stirred. The mixture was shaken at this temperature for 10 minutes, centrifuged at 4 ° C. for 10 minutes at high speed, the supernatant was taken out, and the above deproteinization operation was repeated 3 times so that the protein content in the polysaccharide solution was 0.42% or less. 225 ml of 95% alcohol was added to the solution and alcohol precipitation was carried out for 12 hours. The precipitate is removed by high-speed centrifugation, 100 g of water is added, put into a dialysis bag with a molecular weight cut off of 7,000 Da, dialyzed for 48 hours, and then the liquid in the dialysis bag is concentrated and freeze-dried, for example, 1.5 g of white powder. Got.
実施例7により得られたあわび腹足粗多糖類5ｇに、245ｇの水を加え、4℃で10%トリクロロ酢酸溶液を体積比5：1の比率でゆっくり加えて攪拌した。この温度に10分間振り混ぜ、4℃で10分間高速遠心して上清液を取り出して多糖類溶液中の蛋白含有量が0.27%以下となるように上記脱蛋白の作業を5回繰り返した。溶液の中に1125mlの95%アルコールを加えて12時間アルコール沈殿した。高速遠心して沈殿を取り出し、80ｇの水を加え、分画分子量7,000Daの透析袋に入れ、48時間透析してから、透析袋中の液を濃縮し、冷凍乾燥することによりたとえば白い粉末1.3ｇを得た。 To 5 g of the abalone stomach crude polysaccharide obtained in Example 7, 245 g of water was added, and a 10% trichloroacetic acid solution was slowly added at a volume ratio of 5: 1 at 4 ° C. and stirred. The mixture was shaken at this temperature for 10 minutes, centrifuged at 4 ° C for 10 minutes at high speed, the supernatant was taken out, and the above deproteinization operation was repeated 5 times so that the protein content in the polysaccharide solution was 0.27% or less. 1125 ml of 95% alcohol was added to the solution and alcohol precipitation was carried out for 12 hours. The precipitate is removed by high-speed centrifugation, 80 g of water is added, put into a dialysis bag with a molecular weight cut off of 7,000 Da, dialyzed for 48 hours, and the liquid in the dialysis bag is concentrated and freeze-dried, for example, 1.3 g of white powder. Got.
実施例22により得られたあわび腹足多糖類5ｇに、95ｇの水を加え、分画分子量7,000Daの透析袋に入れ、1ｇのα-澱粉酵素（10U/mg）を加え、37℃で0.9％のNaCl溶液の中で48時間かけて透析しながら、加水分解してから、さらに水中で48時間透析した。透析袋中の液を濃縮し、冷凍乾燥することによりたとえば白い粉末AGP1.2ｇを得た。 95 g of water is added to 5 g of abalone gastropod polysaccharide obtained in Example 22, put into a dialysis bag with a molecular weight cut off of 7,000 Da, 1 g of α-starch enzyme (10 U / mg) is added, and 0.9% at 37 ° C. The mixture was hydrolyzed while dialyzing in a NaCl solution of 48% for 48 hours, and then dialyzed in water for 48 hours. The liquid in the dialysis bag was concentrated and freeze-dried to obtain, for example, 1.2 g of white powder AGP.
実施例22により得られたあわび腹足多糖類5ｇに、95ｇの水を加え、分画分子量7,000Daの透析袋に入れ、0.05ｇのα-澱粉酵素（10U/mg）を加え、37℃で0.9％のNaCl溶液の中で48時間かけて透析しながら、加水分解してから、さらに水中で48時間透析した。透析袋中の液を濃縮し、冷凍乾燥することによりたとえば白い粉末AGP1.38ｇを得た。 95 g of water is added to 5 g of abalone gastropod polysaccharide obtained in Example 22 and placed in a dialysis bag with a molecular weight cut off of 7,000 Da, and 0.05 g of α-starch enzyme (10 U / mg) is added at 37 ° C. It hydrolyzed by dialyzing in 0.9% NaCl solution for 48 hours, and then dialyzed in water for 48 hours. The liquid in the dialysis bag was concentrated and freeze-dried to obtain, for example, white powder AGP 1.38 g.
実施例24により得られたAGP100mｇに、10mlの水を加えて溶解させ、SephadexG-100カラム・クロマトグラフィで分離を行い、溶離剤は0.9%のNaClで、溶出速度は0.5ml/minであった。フェノール硫酸法で追跡検出し、グラフを作成して唯一の溶出ピークを検出して収集した。混合・透析・濃縮・冷凍乾燥することにより均一のあわび腹足多糖類AGPを得た。 10 ml of water was added to 100 mg of AGP obtained in Example 24 and dissolved, and separation was performed by Sephadex G-100 column chromatography. The eluent was 0.9% NaCl and the elution rate was 0.5 ml / min. Follow-up detection with the phenol-sulfuric acid method was performed and a graph was created to detect and collect the only elution peak. Uniform abalone stomach polysaccharide AGP was obtained by mixing, dialysis, concentration and freeze-drying.
実施例18により得られた100mｇのAHP100に、10mlの水を加えて溶解させ、SephadexG-100カラム・クロマトグラフィで分離を行い、溶離剤は0.9%のNaCl溶液で、溶出速度は0.5ml/minであった。フェノール硫酸法で追跡検出し、グラフを作成して二つの溶出ピークを検出した。それぞれ溶出ピークで収集・透析・乾燥してから、DEAEcellulose 52で精製し、NaCl溶液で勾配溶出を行い、すべて単一の溶出ピークであった。収集して混合し、透析・濃縮した。冷凍乾燥することにより均一のあわび臓器多糖類AHP-1とAHP-2とを得た。 To 100 mg of AHP100 obtained in Example 18, 10 ml of water was added and dissolved, and separation was performed by Sephadex G-100 column chromatography. The eluent was a 0.9% NaCl solution and the elution rate was 0.5 ml / min. there were. Following detection by the phenol sulfuric acid method, two elution peaks were detected by creating a graph. Each was collected, dialyzed and dried at the elution peak, then purified with DEAEcellulose 52, and gradient elution was performed with a NaCl solution, all of which were single elution peaks. Collected, mixed, dialyzed and concentrated. Uniform abalone organ polysaccharides AHP-1 and AHP-2 were obtained by freeze-drying.
実施例25により得られた10mｇのAGPに、10mlの超純水を加えて20分間超音波で溶解促進して濾過を行った。ゲルクロマトグラフで検出した分子量は5,000-10,000dal（Dalton）であった。クロマトグラフ条件は、ゲルカラムはSepharoseCL-6Bカラムで、溶離剤は0.9％のNaCl溶液で、流速は0.24ml/minであった。 To 10 mg AGP obtained in Example 25, 10 ml of ultrapure water was added, and dissolution was promoted with ultrasonic waves for 20 minutes, followed by filtration. The molecular weight detected by gel chromatography was 5,000-10,000 dal (Dalton). The chromatographic conditions were that the gel column was a Sepharose CL-6B column, the eluent was a 0.9% NaCl solution, and the flow rate was 0.24 ml / min.
実施例26により得られた10mｇのAHP-1に、10mlの超純水を加えて20分間超音波で溶解促進して濾過を行った。ゲルクロマトグラフで検出した分子量は5×105dal（Dalton）位であった。クロマトグラフ条件は、ゲルカラムはSepharoseCL-6Bカラムで、溶離剤は0.9％のNaCl溶液で、流速は0.24ml/minであった。 To 10 mg of AHP-1 obtained in Example 26, 10 ml of ultrapure water was added, and dissolution was promoted with ultrasonic waves for 20 minutes for filtration. The molecular weight detected by gel chromatography was about 5 × 10 5 dal (Dalton). The chromatographic conditions were that the gel column was a Sepharose CL-6B column, the eluent was a 0.9% NaCl solution, and the flow rate was 0.24 ml / min.
実施例26により得られた10mｇのAHP-2に、10mlの超純水を加えて20分間超音波で溶解促進して濾過を行った。ゲルクロマトグラフで検出した分子量は10,000-15,000dal（Dalton）位であった。クロマトグラフ条件は、ゲルカラムはSepharoseCL-6Bカラムで、溶離剤は0.9％のNaCl溶液で、流速は0.24ml/minであった。 To 10 mg of AHP-2 obtained in Example 26, 10 ml of ultrapure water was added, and dissolution was promoted with ultrasonic waves for 20 minutes for filtration. The molecular weight detected by gel chromatography was about 10,000-15,000 dal (Dalton). The chromatographic conditions were that the gel column was a Sepharose CL-6B column, the eluent was a 0.9% NaCl solution, and the flow rate was 0.24 ml / min.
完全に乾燥した、実施例25により得られた10mgのAGPに2mlの無水HCl-メチルアルコールを加え、N2を入れて封管し、80℃で20時間メチルアルコール分解してから取り出し、室温になるまで放置した。無水KOH-メチルアルコールでpH=6となるように中和し、40℃で減圧回転蒸し干ししてから乾燥した。完全に乾燥したメチルアルコール分解産物に0.2mlの無水ピリジンを加え、75℃で30分かけて溶解させて、0.3mlのシリル化剤を加えて均一に振り混ぜ、数分間静置して上清を取り出して色層分析（ガスクロマトグラフ）を行い、単糖はブドウ糖からの組成と分かった。クロマトグラフ条件は、ガスクロマトグラフ（USAgilent 6890N）、HP-1クロマトグラフカラム、固定相Methylsiloxane、キャリヤーガスN2、流速45ml/minであった。水素炎イオン検出器（FID）を用い、検出温度は300℃で、サンプル注入量は1μlであった。注入口ガス化温度は300℃であり、カラム温度はプログラムで昇温を制御し、150℃に、1分間保持、10℃/minで182℃まで昇温して、2分間保持、1℃/minで188℃まで昇温して、1分間保持、8℃/minで230℃まで昇温した。 2 mg anhydrous HCl-methyl alcohol was added to 10 mg AGP obtained according to Example 25, dried completely, sealed with N 2 , decomposed with methyl alcohol at 80 ° C. for 20 hours, taken out, and brought to room temperature. I left it until. The mixture was neutralized with anhydrous KOH-methyl alcohol to pH = 6, dried at 40 ° C. under reduced pressure and dried. Add 0.2 ml of anhydrous pyridine to the completely dried methyl alcohol degradation product, dissolve at 75 ° C for 30 minutes, add 0.3 ml of silylating agent, shake evenly, leave it for a few minutes and leave the supernatant Was taken out and subjected to color layer analysis (gas chromatograph), and it was found that monosaccharides were composed of glucose. The chromatographic conditions were gas chromatograph (USAgilent 6890N), HP-1 chromatographic column, stationary phase Methylsiloxane, carrier gas N 2 , flow rate 45 ml / min. A flame ion detector (FID) was used, the detection temperature was 300 ° C., and the sample injection volume was 1 μl. The inlet gasification temperature is 300 ° C, and the column temperature is controlled by a program. The temperature is maintained at 150 ° C for 1 minute, raised to 182 ° C at 10 ° C / min, held for 2 minutes, 1 ° C / The temperature was raised to 188 ° C at min, held for 1 minute, and raised to 230 ° C at 8 ° C / min.
完全に乾燥した、実施例26により得られた10mgのAHP-1に2mlの無水HCl-メチルアルコールを加え、N2を入れて封管し、80℃で20時間メチルアルコール分解してから取り出し、室温になるまで放置した。無水KOH-メチルアルコールでpH=6となるように中和し、40℃で減圧回転蒸し干ししてから乾燥した。完全に乾燥したメチルアルコール分解産物に0.2mlの無水ピリジンを加え、75℃で30分かけて溶解して、0.3mlのシリル化剤を加えて均一に振り混ぜ、数分間静置して上清を取り出して色層分析（ガスクロマトグラフ）を行い、単糖はブドウ糖とペクチンシュガーからの組成であり、そのモル比はGlu:Ara＝1.0:1.5で、アミノ糖鑑定の結果、アミノ糖が含まれなかった。クロマトグラフ条件は、ガスクロマトグラフ（USAgilent 6890N）、HP-1クロマトグラフカラム、固定相Methylsiloxane、キャリヤーガスN2、流速45ml/minであった。水素炎イオン検出器（FID）を用い、検出温度は300℃で、サンプル注入量は1μlであった。注入口ガス化温度は300℃であり、カラム温度はプログラムで昇温を制御し、150℃に、1分間保持、10℃/minで182℃まで昇温して、2分間保持、1℃/minで188℃まで昇温して、1分間保持、8℃/minで230℃まで昇温した。 2 mg anhydrous HCl-methyl alcohol was added to 10 mg AHP-1 obtained according to Example 26, completely dried, and N 2 was sealed in a sealed tube. Leave to room temperature. The mixture was neutralized with anhydrous KOH-methyl alcohol to pH = 6, dried at 40 ° C. under reduced pressure and dried. Add 0.2 ml of anhydrous pyridine to the completely dried methyl alcohol degradation product, dissolve at 75 ° C for 30 minutes, add 0.3 ml of silylating agent, shake evenly, leave it for a few minutes and leave the supernatant The saccharide is composed of glucose and pectin sugar, and its molar ratio is Glu: Ara = 1.0: 1.5. As a result of amino sugar identification, amino sugar is included. There wasn't. The chromatographic conditions were gas chromatograph (USAgilent 6890N), HP-1 chromatographic column, stationary phase Methylsiloxane, carrier gas N 2 , flow rate 45 ml / min. A flame ion detector (FID) was used, the detection temperature was 300 ° C., and the sample injection volume was 1 μl. The inlet gasification temperature is 300 ° C, and the column temperature is controlled by a program. The temperature is maintained at 150 ° C for 1 minute, raised to 182 ° C at 10 ° C / min, held for 2 minutes, 1 ° C / The temperature was raised to 188 ° C at min, held for 1 minute, and raised to 230 ° C at 8 ° C / min.
完全に乾燥した、実施例26により得られた10mgのAHP-2に2mlの無水HCl-メチルアルコールを加え、N2を入れて封管し、80℃で20時間メチルアルコール分解してから取り出し、室温になるまで放置した。無水KOH-メチルアルコールでpH=6となるように中和し、40℃で減圧回転蒸し干ししてから乾燥した。完全に乾燥したメチルアルコール分解産物に0.2mlの無水ピリジンを加え、75℃で30分かけて溶解させて、0.3mlのシリル化剤を加えて均一に振り混ぜ、数分間静置して上清を取り出して色層分析（ガスクロマトグラフ）を行い、ラムノース（rhamnose）と、フコース（fucose）と、キシロース（xylose）と、ガラクトース（galactose）とグルコース（glucose）の組成であり、そのモル比はRha:Fuc:Xyl:Gal:Glu＝2.7:1.0:1.0:4.3:4.2で、微量のN-アセチルグルコサミン（N-acetylglucosamine）が含まれた。クロマトグラフ条件は、ガスクロマトグラフ（USAgilent 6890N）、HP-1クロマトグラフカラム、固定相Methylsiloxane、キャリヤーガスN2、流速45ml/minであった。水素炎イオン検出器（FID）を用い、検出温度は300℃で、サンプル注入量は1μlであった。注入口ガス化温度は300℃であり、カラム温度はプログラムで昇温を制御し、150℃に、1分間保持、10℃/minで182℃まで昇温して、2分間保持、1℃/minで188℃まで昇温して、1分間保持、8℃/minで230℃まで昇温した。
2 mg anhydrous HCl-methyl alcohol was added to 10 mg of AHP-2 obtained according to Example 26, completely dried, N 2 was put in a sealed tube, and the methyl alcohol was decomposed at 80 ° C. for 20 hours. Leave to room temperature. The mixture was neutralized with anhydrous KOH-methyl alcohol to pH = 6, dried at 40 ° C. under reduced pressure and dried. Add 0.2 ml of anhydrous pyridine to the completely dried methyl alcohol degradation product, dissolve at 75 ° C for 30 minutes, add 0.3 ml of silylating agent, shake evenly, leave it for a few minutes and leave the supernatant The color layer analysis (gas chromatograph) is taken out, and the composition of rhamnose, fucose, xylose, galactose and glucose is the molar ratio of Rha : Fuc: Xyl: Gal: Glu = 2.7: 1.0: 1.0: 4.3: 4.2 and a trace amount of N-acetylglucosamine was contained. The chromatographic conditions were gas chromatograph (USAgilent 6890N), HP-1 chromatographic column, stationary phase Methylsiloxane, carrier gas N 2 , flow rate 45 ml / min. A flame ion detector (FID) was used, the detection temperature was 300 ° C., and the sample injection volume was 1 μl. The inlet gasification temperature is 300 ° C, and the column temperature is controlled by a program. The temperature is maintained at 150 ° C for 1 minute, raised to 182 ° C at 10 ° C / min, held for 2 minutes, 1 ° C / The temperature was raised to 188 ° C at min, held for 1 minute, and raised to 230 ° C at 8 ° C / min.
Claims (19)
殻が除かれたあわび本体またはあわび腹足と臓器を組織破砕機で破砕して5〜10倍の水を加えてホモジネートする、或いは殻が除かれたあわび本体またはあわび腹足と臓器を乾燥して100目以下の干し粉に粉砕する原料処理ステップと、
水漬抽出法と、アルカリ液抽出法と、超音波抽出法と酵素抽出法のうちの一つまたは複数を利用して成分抽出を行う抽出ステップと、
抽出した清液を糖含有量が1.5〜3.0％となるように濃縮する濃縮ステップと、
濃縮液に体積でその3〜4倍の95％アルコールを加え、0〜4℃で12〜16時間放置し、遠心分離により沈殿となるあわび多糖類を得るアルコール沈殿ステップと、
アルコール沈殿により得たあわび多糖類を乾燥して生成品を得る乾燥ステップと、を含む。 An abalone polysaccharide extraction method comprising:
The abalone body or abalone legs and organs with the shell removed are crushed with a tissue crusher and homogenized by adding 5 to 10 times water, or the abalone body or abalone legs and organs with the shell removed are dried. Raw material processing step to pulverize into 100 or less dried powder,
An extraction step of performing component extraction using one or more of a soaking extraction method, an alkaline liquid extraction method, an ultrasonic extraction method and an enzyme extraction method;
A concentration step of concentrating the extracted clear liquid so that the sugar content is 1.5 to 3.0%;
An alcohol precipitation step of adding 95% alcohol by volume 3-4 times to the concentrate, leaving it at 0-4 ° C. for 12-16 hours, and obtaining an abalone polysaccharide that precipitates by centrifugation;
Drying abalone polysaccharide obtained by alcohol precipitation to obtain a product.
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| PCT/CN2006/002650 WO2007041951A1 (en) | 2005-10-11 | 2006-10-10 | Extraction method of abalone polysaccharide |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101955548A (en) * | 2010-09-26 | 2011-01-26 | 陕西农产品加工技术研究院 | Method for enzyme-assisted extraction of fructus tribuli polysaccharide |
| KR101257494B1 (en) * | 2010-05-27 | 2013-04-26 | 조선대학교산학협력단 | Composition for the treatment and the prevention of inflammatory diseases in immune system containing Abalone gastrointestinal digests |
| CN115025515A (en) * | 2022-06-10 | 2022-09-09 | 陈建 | Pleurotus tuber-regium polysaccharide extraction and separation equipment |
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| CN101503721B (en) * | 2008-11-27 | 2012-01-11 | 浙江珍世堂生物科技有限公司 | Method for extracting swan-mussel polysaccharide |
| CN102140141B (en) * | 2011-05-13 | 2012-10-03 | 陈锦权 | Process for preparing polysaccharide of abalones |
| CN102839149B (en) * | 2012-09-21 | 2013-11-13 | 中国海洋大学 | Application of abalone viscera polysaccharide-alternative serum in cell culture medium |
| CN103819575B (en) * | 2014-03-07 | 2016-03-02 | 潍坊医学院 | The purposes of a kind of extracting method of Ground Beetle polysaccharide, the polysaccharide of extraction and this polysaccharide |
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| CN104497164A (en) * | 2015-01-26 | 2015-04-08 | 吉林大学 | High-voltage pulsed electric field assisted enzymolysis based clam polysaccharide extracting process |
| CN105087713A (en) * | 2015-09-01 | 2015-11-25 | 集美大学 | Preparation method of abalone viscera polysaccharide |
| CN105272887B (en) * | 2015-10-20 | 2017-06-23 | 集美大学 | A kind of method for extracting taurine and polysaccharide in the internal organ from abalone simultaneously |
| CN111513269B (en) * | 2020-04-16 | 2022-07-19 | 自然资源部第三海洋研究所 | Abalone viscera extract and application thereof |
| CN113150178B (en) * | 2021-04-14 | 2023-05-02 | 大连工业大学 | Sulfated abalone polysaccharide and application thereof in inhibiting new coronavirus |
| CN114133459A (en) * | 2021-11-23 | 2022-03-04 | 福建师范大学 | Preparation method and application of abalone polysaccharide |
| CN114805623A (en) * | 2022-04-22 | 2022-07-29 | 福建大众健康生物科技有限公司 | Ultralow-temperature freeze-thawing pressurization extraction process for abalone polysaccharide |
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| JPH10289760A (en) * | 1997-04-11 | 1998-10-27 | Molex Inc | Metal shell connection means |
| CN1057776C (en) * | 1997-06-27 | 2000-10-25 | 吉林大学 | Combined extraction technology of ginseng and American ginseng polysaccharide saponin |
| CN1202749C (en) * | 2002-08-30 | 2005-05-25 | 大连轻工业学院 | Abalone food and its preparation method |
| CN1215796C (en) * | 2003-08-07 | 2005-08-24 | 大连太平洋海龙科技有限公司 | Nutrient solution of abalone, sea cucumber and sea chestnut and its producing method |
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|---|---|---|---|---|
| KR101257494B1 (en) * | 2010-05-27 | 2013-04-26 | 조선대학교산학협력단 | Composition for the treatment and the prevention of inflammatory diseases in immune system containing Abalone gastrointestinal digests |
| CN101955548A (en) * | 2010-09-26 | 2011-01-26 | 陕西农产品加工技术研究院 | Method for enzyme-assisted extraction of fructus tribuli polysaccharide |
| CN115025515A (en) * | 2022-06-10 | 2022-09-09 | 陈建 | Pleurotus tuber-regium polysaccharide extraction and separation equipment |
| CN115025515B (en) * | 2022-06-10 | 2023-11-14 | 陈建 | Pleurotus tuber-regium polysaccharide draws splitter |
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