JP2008523113A - Scallop polysaccharide extraction method - Google Patents
Scallop polysaccharide extraction method Download PDFInfo
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- 150000004676 glycans Chemical class 0.000 title claims abstract description 177
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 177
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 177
- 235000020637 scallop Nutrition 0.000 title claims abstract description 128
- 241000237509 Patinopecten sp. Species 0.000 title claims abstract description 84
- 238000000605 extraction Methods 0.000 title claims abstract description 50
- 230000007515 enzymatic degradation Effects 0.000 claims abstract description 69
- 238000000034 method Methods 0.000 claims abstract description 63
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 62
- 241000237503 Pectinidae Species 0.000 claims abstract description 44
- 102000057297 Pepsin A Human genes 0.000 claims abstract description 25
- 108090000284 Pepsin A Proteins 0.000 claims abstract description 25
- 229940111202 pepsin Drugs 0.000 claims abstract description 24
- 102000004142 Trypsin Human genes 0.000 claims abstract description 19
- 108090000631 Trypsin Proteins 0.000 claims abstract description 19
- 238000000926 separation method Methods 0.000 claims abstract description 19
- 239000012588 trypsin Substances 0.000 claims abstract description 19
- 238000001556 precipitation Methods 0.000 claims abstract description 14
- 235000013372 meat Nutrition 0.000 claims abstract description 12
- 230000008569 process Effects 0.000 claims abstract description 8
- 238000001035 drying Methods 0.000 claims abstract description 6
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- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 105
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- 108090000790 Enzymes Proteins 0.000 claims description 67
- 229940088598 enzyme Drugs 0.000 claims description 67
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 59
- 238000006243 chemical reaction Methods 0.000 claims description 55
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- 210000002966 serum Anatomy 0.000 claims description 49
- 230000000694 effects Effects 0.000 claims description 46
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- 239000006228 supernatant Substances 0.000 claims description 25
- 238000005520 cutting process Methods 0.000 claims description 22
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- 230000002255 enzymatic effect Effects 0.000 claims description 14
- 238000004108 freeze drying Methods 0.000 claims description 14
- 238000006731 degradation reaction Methods 0.000 claims description 12
- 244000063299 Bacillus subtilis Species 0.000 claims description 11
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 11
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 10
- 239000000047 product Substances 0.000 claims description 9
- 102000035195 Peptidases Human genes 0.000 claims description 8
- 108091005804 Peptidases Proteins 0.000 claims description 8
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 8
- 239000012452 mother liquor Substances 0.000 claims description 8
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 8
- 235000019833 protease Nutrition 0.000 claims description 8
- 238000005904 alkaline hydrolysis reaction Methods 0.000 claims description 7
- 239000007787 solid Substances 0.000 claims description 6
- 230000009278 visceral effect Effects 0.000 claims description 5
- 101000693530 Staphylococcus aureus Staphylokinase Proteins 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 4
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 4
- 238000002844 melting Methods 0.000 claims description 3
- 238000003815 supercritical carbon dioxide extraction Methods 0.000 claims description 3
- 230000000593 degrading effect Effects 0.000 claims 1
- 230000008018 melting Effects 0.000 claims 1
- 238000005406 washing Methods 0.000 claims 1
- 239000002994 raw material Substances 0.000 abstract description 16
- 108090000145 Bacillolysin Proteins 0.000 abstract description 10
- 235000015170 shellfish Nutrition 0.000 abstract description 6
- 235000013305 food Nutrition 0.000 abstract description 5
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- 230000001502 supplementing effect Effects 0.000 abstract 1
- 239000002244 precipitate Substances 0.000 description 49
- 239000000243 solution Substances 0.000 description 49
- OKKJLVBELUTLKV-UHFFFAOYSA-N methyl alcohol Substances OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 40
- 210000001519 tissue Anatomy 0.000 description 38
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 29
- 238000010828 elution Methods 0.000 description 19
- 238000000502 dialysis Methods 0.000 description 17
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 239000003480 eluent Substances 0.000 description 10
- 239000000706 filtrate Substances 0.000 description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 238000011160 research Methods 0.000 description 9
- 210000001835 viscera Anatomy 0.000 description 9
- 239000007789 gas Substances 0.000 description 7
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- 230000003544 deproteinization Effects 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
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- 229920005654 Sephadex Polymers 0.000 description 5
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- SRBFZHDQGSBBOR-QMKXCQHVSA-N alpha-L-arabinopyranose Chemical group O[C@H]1CO[C@@H](O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-QMKXCQHVSA-N 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
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- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 5
- 238000000194 supercritical-fluid extraction Methods 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 238000004440 column chromatography Methods 0.000 description 4
- 239000007857 degradation product Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 210000001596 intra-abdominal fat Anatomy 0.000 description 4
- 239000003513 alkali Substances 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 3
- 230000005526 G1 to G0 transition Effects 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- -1 Methylsiloxane Chemical class 0.000 description 2
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- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
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- 150000002500 ions Chemical class 0.000 description 2
- 150000002772 monosaccharides Chemical class 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
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- 241001474374 Blennius Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 240000000599 Lentinula edodes Species 0.000 description 1
- 235000001715 Lentinula edodes Nutrition 0.000 description 1
- 241001125046 Sardina pilchardus Species 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003471 anti-radiation Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
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- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000013505 freshwater Substances 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 210000002816 gill Anatomy 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
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- 235000019512 sardine Nutrition 0.000 description 1
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Classifications
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- 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
Abstract
【課題】本発明は原料処理、ホモジネート、超音波処理、酵素分解、分離、濃縮、アルコール沈殿、乾燥のステップを有する帆立貝多糖類抽出方法を提供する。
【解決手段】原料処理は帆立貝肉組織を直接、または火干し/冷凍乾燥した後加水してホモジネートすることであり、酵素分解はトリプシン、ペプシン、枯草菌中性プロテイナーゼのいずれか一つまたは二つを用いて行う。肉組織は、帆立貝柱、貝柱以外のオッファル、部分的なオッファル、帆立貝柱及び部分的なオッファル、または貝殻を除く全部肉組織である。本発明は帆立貝から多糖類を抽出するプロセスを提供し、特に帆立貝のオッファルから多糖類を抽出することにより、廃棄物を十分に利用できる。得られた製品は高純度、高得率である。抽出した帆立貝多糖類は、単独で製品にしてもよいが、基材にして別食品を補充して多効能食品の開発も可能である。
【選択図】なしThe present invention provides a scallop polysaccharide extraction method comprising steps of raw material treatment, homogenate, sonication, enzymatic degradation, separation, concentration, alcohol precipitation, and drying.
SOLUTION: The raw material treatment is scallop shellfish tissue directly or after being fire-dried / freeze-dried and then watered and homogenized. Enzymatic degradation is one or two of trypsin, pepsin, and Bacillus subtilis neutral proteinase. To do. Meat tissue is whole meat tissue excluding scallops, non-scallops offal, partial offal, scallops and partial offal, or shells. The present invention provides a process for extracting polysaccharides from scallops, and in particular, waste can be fully utilized by extracting polysaccharides from scallop offals. The obtained product has high purity and high yield. The extracted scallop polysaccharide may be used alone, but it is also possible to develop a multi-effect food by supplementing another food as a base material.
[Selection figure] None
Description
本発明は多糖類の抽出技術に関するものであり、特に貝類を原料とする多糖類の抽出技術に関するものである。 The present invention relates to a polysaccharide extraction technique, and more particularly to a polysaccharide extraction technique using shellfish as a raw material.
近年、多糖類についての研究は迅速に発展し、空前に活躍する段階に進入してきた。張倩などの研究者による研究（多糖類効能研究の進展「食品研究と開発」1998,9（19））によると、多糖類が多方面にわたって生物活性を具備し、免疫細胞をアクティブにしてヒトの免疫機能を高め、ウィルスとコンパウンドを形成することによりウィルスと細胞受体の結合を妨げることができる一方、正常細胞には副作用がないとされる。その抗腫瘤、免疫機能の向上、血糖値低下、血脂低下、解毒、抗ウィルス、抗細菌、抗放射などの生理機能は世界医薬界において注目されている。 In recent years, research on polysaccharides has developed rapidly and has entered an unprecedented stage. According to research by researchers such as Zhang Yun (Polysaccharide Efficacy Research Progress “Food Research and Development” 1998, 9 (19)), polysaccharides have biological activity in many areas and activate immune cells to humans By improving the immune function and forming a compound with the virus, the binding between the virus and the cell receptor can be prevented, while normal cells have no side effects. Physiological functions such as anti-tumor, improved immune function, decreased blood sugar level, decreased blood fat, detoxification, anti-viral, anti-bacterial and anti-radiation are attracting attention in the world pharmaceutical world.
多糖類は植物、微生物（細菌と真菌）及び海藻に広く存在する。食用菌多糖類についての研究は多くあり、特にしいたけ多糖類についての研究はよく知られている。多糖類への研究が深まるにつれて、研究者は動物体多糖類、特に水生動物体多糖類へと関心を寄せている。現在、貝類多糖類の研究においての報道は、「淡水真珠貝肉について」（「雲南大学学報（自然科学版）1998,20（3）：187〜189」）と海湾帆立貝のひもについて（「中国水産科学」第1巻第２期）しかない。 Polysaccharides are widely present in plants, microorganisms (bacteria and fungi) and seaweed. There are many studies on edible fungal polysaccharides, and research on shiitake polysaccharides is particularly well known. As research into polysaccharides deepens, researchers are interested in animal polysaccharides, especially aquatic animal polysaccharides. Currently, reports on shellfish polysaccharides include “About Freshwater Pearl Shell Meat” (“Yunnan University Journal (Natural Science Edition) 1998, 20 (3): 187-189”) and Sea Bay Scallop String (“ China Fisheries Science "Volume 1 second period" only.
帆立貝は低温海水域に生息する二枚貝類であり、その原産地が日本であり、二十世紀八十年代初期に中国に導入され、味がよく、栄養が豊富で、Ca、P、Zn、Seなどの微量元素と人体に必須なアミノ酸が多く含まれる。新鮮な帆立貝は蛋白質含有量が14.5%であるが、干帆立貝は蛋白質含有量が63.7%にも達している。その蛋白質には十数種のアミノ酸が含まれ、鮮味を出すグルタミン酸の含有量が水産品の中で最も高く、7.15%でもある。近代科学研究により、帆立貝は蛋白質を豊富に含有するほか、多糖類も豊富に含有することが発見された。しかし、この発見は重要視されておらず、更なる研究が行われていないままである。帆立貝については、その養殖技術に関する研究及び報道が多いにもかかわらず、その栄養価値に関する研究及び報道は極めて少ない。帆立貝多糖類の抽出方法及び工程についての報告例は、いまだにないようである。 Scallops are bivalves that inhabit low-temperature seawater, their origin is Japan, and they were introduced to China in the early 20s of the 20th century, have good taste and rich nutrition, Ca, P, Zn, Se, etc. It contains a lot of trace elements and amino acids essential to the human body. Fresh scallops have a protein content of 14.5%, while dried scallops have a protein content of 63.7%. The protein contains more than a dozen amino acids, and the content of glutamic acid that produces a fresh taste is the highest among fishery products, at 7.15%. Modern scientific research has found that scallops are rich in protein as well as polysaccharides. However, this discovery has not been emphasized and no further research has been done. Regarding scallops, there are very few studies and reports on their nutritional value, despite much research and reports on their aquaculture techniques. There are still no reports on scallop polysaccharide extraction methods and processes.
本発明は、帆立貝を原料とし、酵素法を利用して関連工程により高温処理を避けて多糖類の原料から特定の生物活性を保持しつつ、最大限に多糖類の抽出率を高める活性多糖類抽出新工程を提供することを目的とする。 The present invention uses scallops as a raw material, an active polysaccharide that maximizes the extraction rate of polysaccharides while maintaining specific biological activity from the raw materials of polysaccharides by avoiding high-temperature treatment by related processes using an enzymatic method The purpose is to provide a new extraction process.
本発明の技術案では、帆立貝を原料処理して多糖類の抽出・精製をし、多糖類製品が得られる。また、抽出過程からの残渣は従来技術に基づいて帆立貝ポリペプチド製品の製造に用いられる。 In the technical solution of the present invention, a scallop is processed as a raw material to extract and purify the polysaccharide, thereby obtaining a polysaccharide product. Also, the residue from the extraction process is used for the production of scallop polypeptide products based on the prior art.
一、原料と処理
本発明に用いられる原料である帆立貝の肉組織は、殻を取った帆立貝の全部肉組織（貝柱、鰓、性腺、内臓、ひもなど）であってもよく、貝柱、「オッファル」（鰓（えら）、性腺、内臓、ひもなどの全てまたはその部分）または貝柱と部分的な「オッファル」であってもよい。
1. Raw material and treatment The scallop meat tissue which is the raw material used in the present invention may be the whole meat tissue of a scallop shell (shell, sardine, gonadal, viscera, string, etc.). ”(All, or parts thereof, such as gills, gonadal glands, internal organs, strings, etc.) or scallops and partial“ offals ”.
1、新鮮な殻付き帆立貝を水洗して水を切り、殻を刃物で割って取り除き、異なる部位単位で肉組織を取り出してそのまま使用に用いる。または2、異なる部位単位で肉組織を取り出し、水分含有量が4%以下になるまで肉組織を20〜60℃で火干しまたは真空冷凍乾燥してから、50〜200目まで破砕して干し粉を得たものを用いる。なお、内臓組織の干し粉は、次のステップに移る前に超臨界二酸化炭素抽出設備で脂肪を抽出してもよい。抽出工程のフローは、原料注入――抽出温度制御――抽出圧力制御――CO2流量制御――抽出――分離温度及び圧力制御――分離となる。 1. Wash fresh scallops with shells, drain the water, split the shells with a blade, remove the meat tissue from different parts, and use them as they are. Or 2, take out the meat tissue in different parts, and dry the meat tissue at 20-60 ° C or vacuum freeze-dry until the water content is 4% or less, then crush to 50-200 meshes and dry Use the one obtained. In addition, you may extract fat from the supercritical carbon dioxide extraction equipment, before moving to the next step. The flow of the extraction process is raw material injection--extraction temperature control--extraction pressure control--CO 2 flow rate control--extraction--separation temperature and pressure control--separation.
具体的な実施方法について述べると、超臨界抽出設備を開け、抽出温度を30〜70℃に設定し、温度が上昇するのを待っている間に抽出釜に釜容積の1/3の原料を注入する。抽出温度が設定値に達した後、抽出圧力20〜40MPa、CO2流量10〜40L/hに制御して帆立貝内臓脂肪を超臨界抽出する。45〜120分後、分離温度30〜40℃、分離圧力6〜10MPaに制御して脂肪を分離して収集する。脱脂された帆立貝内臓を抽出釜から取り出して使用に備える。 To describe the specific implementation method, open the supercritical extraction equipment, set the extraction temperature to 30-70 ° C, and wait for the temperature to rise while adding 1/3 of the volume of the raw material to the extraction kettle. inject. After the extraction temperature reaches the set value, supercritical extraction of scallop visceral fat is performed by controlling the extraction pressure to 20 to 40 MPa and the CO 2 flow rate to 10 to 40 L / h. After 45 to 120 minutes, the fat is separated and collected by controlling the separation temperature at 30 to 40 ° C. and the separation pressure at 6 to 10 MPa. Remove the defatted scallop viscera from the extraction kettle and prepare for use.
二、ホモジネート
1.帆立貝の全部肉組織（貝柱と/または部分的な「オッファル」、または全部の「オッファル」であってもよい）にその質量の0.5〜5倍の水を加え、組織破砕機でホモジネートして一様な漿液が得られる。または2.帆立貝の全部肉組織（貝柱と/または部分的な「オッファル」、または全部の「オッファル」でもよい）干し粉にその質量の10〜50倍の水を加え、組織破砕機でホモジネートして一様な漿液が得られる。
Two, homogenate
1. Add 0.5 to 5 times the mass of water to whole scallop meat tissue (which may be scallops and / or partial “offal” or all “offal”) and homogenize with a tissue crusher. And uniform serum is obtained. Or 2. Add 10-50 times the mass of water to the whole meat tissue of scallops (which may be scallops and / or partial “offal” or all “offal”) and homogenize with a tissue crusher. And uniform serum is obtained.
三、超音波処理
次のステップにおける酵素分解の効果を高めさせるために、漿液を0.3〜1時間超音波処理（30〜60℃、50〜600W）する。
3. Sonication The sera is sonicated (30-60 ° C., 50-600 W) for 0.3-1 hour in order to enhance the effect of enzymatic degradation in the next step.
四、酵素分解
帆立貝多糖類の抽出は、酵素分解の方法を用いて、用いられる酵素に適宜なｐH値と温度等の条件下で帆立貝を酵素分解するものである。用いられる酵素は、単一酵素であってもよく、複合酵素であってもよい。酵素分解の前にアルカリ加水分解を行うか、自己融解酵素技術を用いて前処理を行うと抽出率を向上させることができる。
4. Enzymatic degradation The extraction of scallop polysaccharides involves enzymatic degradation of scallops under conditions such as the pH value and temperature appropriate for the enzyme used, using an enzymatic degradation method. The enzyme used may be a single enzyme or a complex enzyme. Extraction rate can be improved by carrying out alkaline hydrolysis prior to enzymatic degradation or by pretreatment using self-melting enzyme technology.
1.アルカリ加水分解：超音波処理された漿液に0.1〜1.0%の破砕の固体炭酸カリウムまたは炭酸ナトリウムを加え、30〜60℃で1〜5時間攪拌してアルカリ加水分解させる。
または2. 自己融解酵素技術：超音波処理された漿液を10〜60分間紫外線照射してから40〜60℃で2〜6時間加水分解させる。
1. Alkaline hydrolysis: 0.1-1.0% crushed solid potassium carbonate or sodium carbonate is added to sonicated serum and stirred at 30-60 ° C. for 1-5 hours for alkaline hydrolysis.
Or 2. Self-melting enzyme technology: Sonicated serum is irradiated with UV light for 10-60 minutes and then hydrolyzed at 40-60 ° C. for 2-6 hours.
3.単一酵素による酵素分解
（ｉ） トリプシン酵素分解：超音波処理された漿液を0.5mol/Lの水酸化カリウムでｐH7〜9に調整し、且つ酵素分解過程におけるｐH値をこの範囲に保持する。漿液に質量パーセント濃度0.1〜2.0%、酵素活性5×104u/gのトリプシンを加え、40〜60℃で保温し、1〜8時間攪拌して酵素分解させる。その後、急速に30℃以下まで冷却し、30分間保持してから反応を終止する。6mol/Lの塩酸でｐHを中性に調整する。または（ｉｉ） ペプシン酵素分解：超音波処理された漿液を6mol/Lの塩酸でｐH1〜5に調整し、且つ酵素分解過程におけるｐH値をこの範囲に保持する。漿液に質量パーセント濃度0.1〜2.0%、酵素活性7.8×105u/gのペプシンを加え、40〜60℃で保温し、1〜10時間攪拌して酵素分解させる。その後、急速に30℃以下まで冷却し、30分間保持してから反応を終止する。0.5mol/Lの水酸化カリウムでｐHを中性に調整する。
3. Enzymatic degradation with a single enzyme (i) Trypsin enzymatic degradation: Sonicated serum is adjusted to pH 7-9 with 0.5 mol / L potassium hydroxide, and the pH value in the enzymatic degradation process is kept within this range To do. Trypsin having a mass percent concentration of 0.1 to 2.0% and an enzyme activity of 5 × 10 4 u / g is added to the serum, kept at 40 to 60 ° C., and stirred for 1 to 8 hours for enzymatic degradation. Thereafter, it is rapidly cooled to 30 ° C. or lower, held for 30 minutes, and then the reaction is terminated. Adjust the pH to neutral with 6 mol / L hydrochloric acid. Or (ii) Pepsin enzyme degradation: sonicated serum is adjusted to pH 1-5 with 6 mol / L hydrochloric acid and the pH value in the enzymatic degradation process is kept in this range. Pepsin having a mass percent concentration of 0.1 to 2.0% and an enzyme activity of 7.8 × 10 5 u / g is added to the serum, kept at 40 to 60 ° C., and stirred for 1 to 10 hours for enzymatic degradation. Thereafter, it is rapidly cooled to 30 ° C. or lower, held for 30 minutes, and then the reaction is terminated. The pH is adjusted to neutral with 0.5 mol / L potassium hydroxide.
または（ｉｉｉ） 枯草菌中性プロテイナーゼ酵素分解：超音波処理された漿液を6mol/Lの塩酸でｐH6〜7に調整し、且つ酵素分解過程にｐH値をこの範囲に保持する。漿液に質量パーセント濃度0.1〜2.0%、酵素活性5×104u/gの枯草菌中性プロテイナーゼを加え、45〜55℃で保温し、1〜6時間攪拌して酵素分解させる。その後、急速に30℃以下まで冷却し、30分間保持してから反応を終止する。0.5mol/Lの水酸化カリウムでｐHを中性に調整する。 Or (iii) Bacillus subtilis neutral proteinase enzymatic degradation: sonicated serum is adjusted to pH 6-7 with 6 mol / L hydrochloric acid and the pH value is kept in this range during the enzymatic degradation process. Bacillus subtilis neutral proteinase having a mass percent concentration of 0.1 to 2.0% and an enzyme activity of 5 × 10 4 u / g is added to the serum and incubated at 45 to 55 ° C. and stirred for 1 to 6 hours for enzymatic degradation. Thereafter, it is rapidly cooled to 30 ° C. or lower, held for 30 minutes, and then the reaction is terminated. The pH is adjusted to neutral with 0.5 mol / L potassium hydroxide.
4.二重酵素を用いた酵素分解
（ｉ） 枯草菌中性プロテイナーゼとペプシンでの酵素分解：超音波処理された漿液を6mol/Lの塩酸でｐH6〜7に調整し、漿液に質量パーセント濃度0.05〜1.5%、酵素活性5×104u/gの枯草菌中性プロテイナーゼを加え、ｐH値をこの範囲に保持し、45〜55℃で1〜3時間攪拌して酵素分解させる。そして、6mol/Lの塩酸でｐH1〜5に調整し、漿液に質量パーセント濃度0.05〜1.5%、酵素活性7.8×105u/gのペプシンを加え、ｐH値をこの範囲に保持し、40〜60℃で1〜5時間攪拌して酵素分解させる。その後、急速に30℃以下まで冷却し、30分間保持してから反応を終止する。0.5mol/Lの水酸化カリウムでｐHを中性に調整する。
4. Enzymatic degradation using double enzyme (i) Enzymatic degradation with Bacillus subtilis neutral proteinase and pepsin: sonicated serum was adjusted to pH 6-7 with 6 mol / L hydrochloric acid, and mass percentage concentration in serum Bacillus subtilis neutral proteinase with 0.05 to 1.5% and enzyme activity of 5 × 10 4 u / g is added, and the pH value is kept within this range, and the mixture is stirred at 45 to 55 ° C. for 1 to 3 hours for enzymatic degradation. Then, the pH is adjusted to 1 to 5 with 6 mol / L hydrochloric acid, pepsin having a mass percent concentration of 0.05 to 1.5% and an enzyme activity of 7.8 × 10 5 u / g is added to the serum, and the pH value is maintained within this range. Stir at 60 ° C. for 1-5 hours to allow enzymatic degradation. Thereafter, it is rapidly cooled to 30 ° C. or lower, held for 30 minutes, and then the reaction is terminated. The pH is adjusted to neutral with 0.5 mol / L potassium hydroxide.
または（ｉｉ）枯草菌中性プロテイナーゼとトリプシンでの酵素分解：超音波処理された漿液を0.5mol/Lの水酸化カリウムでｐH7〜8に調整し、漿液に質量パーセント濃度0.05〜1.5%、酵素活性5×104u/gの枯草菌中性プロテイナーゼを加え、ｐH値をこの範囲に保持し、45〜55℃で1〜3時間攪拌して酵素分解させる。そして、0.5mol/Lの水酸化カリウムでｐH7〜9に調整し、漿液に質量パーセント濃度0.05〜1.5%、酵素活性5×104u/gのトリプシンを加え、ｐH値をこの範囲に保持し、40〜60℃で1〜4時間攪拌して酵素分解させる。その後、急速に30℃以下まで冷却し、30分間保持してから反応を終止する。6mol/Lの塩酸でｐHを中性に調整する。 Or (ii) Enzymatic degradation with Bacillus subtilis neutral proteinase and trypsin: sonicated serum is adjusted to pH 7-8 with 0.5 mol / L potassium hydroxide, mass percent concentration in serum is 0.05-1.5%, enzyme An active 5 × 10 4 u / g Bacillus subtilis neutral proteinase is added, the pH value is maintained within this range, and the mixture is stirred at 45 to 55 ° C. for 1 to 3 hours for enzymatic degradation. The pH is adjusted to 7-9 with 0.5 mol / L potassium hydroxide, and trypsin with a mass percent concentration of 0.05-1.5% and enzyme activity of 5 × 10 4 u / g is added to the serum to maintain the pH value within this range. Stir at 40-60 ° C. for 1-4 hours for enzymatic degradation. Thereafter, it is rapidly cooled to 30 ° C. or lower, held for 30 minutes, and then the reaction is terminated. Adjust the pH to neutral with 6 mol / L hydrochloric acid.
または（ｉｉｉ）ペプシンとトリプシンでの酵素分解：超音波処理された漿液を6mol/Lの塩酸でｐH1〜5に調整し、漿液に質量パーセント濃度0.05〜1.5%、酵素活性7.8×105u/gのペプシンを加え、ｐH値をこの範囲に保持し、40〜60℃で1〜5時間攪拌して酵素分解させる。そして、0.5mol/Lの水酸化カリウムでｐH7〜9に調整し、漿液に質量パーセント濃度0.05〜1.5%、酵素活性5×104u/gのトリプシンを加え、ｐH値をこの範囲に保持し、40〜60℃で1〜4時間攪拌して酵素分解させる。その後、急速に30℃以下まで冷却し、30分間保持してから反応を終止する。6mol/Lの塩酸でｐHを中性に調整する。 Or (iii) Enzymatic degradation with pepsin and trypsin: sonicated serum is adjusted to pH 1-5 with 6 mol / L hydrochloric acid, and the serum has a mass percent concentration of 0.05-1.5%, enzyme activity 7.8 × 10 5 u / g of pepsin is added, the pH value is kept within this range, and the mixture is stirred at 40-60 ° C. for 1-5 hours for enzymatic degradation. The pH is adjusted to 7-9 with 0.5 mol / L potassium hydroxide, and trypsin with a mass percent concentration of 0.05-1.5% and enzyme activity of 5 × 10 4 u / g is added to the serum to maintain the pH value within this range. Stir at 40-60 ° C. for 1-4 hours for enzymatic degradation. Thereafter, it is rapidly cooled to 30 ° C. or lower, held for 30 minutes, and then the reaction is terminated. Adjust the pH to neutral with 6 mol / L hydrochloric acid.
五、分離
1.遠心：帆立貝の酵素分解液を6000rpmで20分間遠心して上清液を収集する。収集されたものは多糖類母液である。
または2.ろ過：帆立貝の酵素分解液を200目スクリーンでろ過して多糖類母液が得られる。
Five, separation
1. Centrifugation: Centrifuge the scallop enzymatic digestion solution at 6000 rpm for 20 minutes to collect the supernatant. Collected is a polysaccharide mother liquor.
Or 2. Filtration: Polysaccharide mother liquor can be obtained by filtering scallop enzymatic decomposition solution through a 200-mesh screen.
六、濃縮
得られた多糖類母液を真空濃縮設備に入れ、真空度85〜98ｋPa、温度30〜60℃に制御して元の体積の1/5〜1/3になるまで濃縮する。
6. Concentration The obtained polysaccharide mother liquor is put in a vacuum concentration facility, and concentrated to 1/5 to 1/3 of the original volume by controlling the degree of vacuum to 85 to 98 kPa and the temperature of 30 to 60 ° C.
七、アルコール沈殿
攪拌しながら多糖類母液に2〜5倍の95%アルコールをゆっくり加え、2〜6℃で2〜18時間アルコール沈殿した後、遠心して多糖類沈殿を収集する。
7. Alcohol precipitation While stirring, 2-5 times 95% alcohol is slowly added to the polysaccharide mother liquor. After 2-18 hours of alcohol precipitation at 2-6 ° C, the polysaccharide precipitate is collected by centrifugation.
八、乾燥
1.多糖類沈殿を直接20〜60℃で火干しすることにより得られるものは帆立貝多糖類である。
または2.多糖類沈殿を真空冷凍乾燥設備に入れ、真空度70〜150Pa、板温度30〜60℃に制御して6〜15時間冷凍乾燥することにより帆立貝多糖類が得られる。これを製品としてもよいが、更にカプセル剤、粉剤、錠剤等の製品に加工してもよい。
Eight, dry
1. The scallop polysaccharide is obtained by directly drying the polysaccharide precipitate at 20-60 ° C.
Or 2. The scallop polysaccharide is obtained by placing the polysaccharide precipitate in a vacuum freeze-drying facility and controlling it at a vacuum degree of 70 to 150 Pa and a plate temperature of 30 to 60 ° C. to freeze and dry for 6 to 15 hours. Although this may be used as a product, it may be further processed into a product such as a capsule, powder, or tablet.
以上のステップを経て抽出された帆立貝粗多糖類は、以下のステップで更に精製・純化してもよい。 The scallop crude polysaccharide extracted through the above steps may be further purified and purified by the following steps.
九、多糖類の生成及び構造の初めての確定
1.蛋白及び小分子物質の取り除く
（1）粗多糖類を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して多糖類溶液中の蛋白含有量が0.5%以下になるまで、上記脱蛋白のステップを数回繰り返す。溶液に4.5倍体積の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7000Daの透析袋内に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純多糖類が得られる。
または（2）粗多糖類を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でpH1.5〜3.0に調整し、37℃で1〜6時間酵素分解させてから2〜5分間90〜100℃まで昇温して10分間酵素減滅し、室温まで冷却し、NaOH溶液で中性に調整する。10分間遠心して上清液を取り出して3〜4.5倍体積の95%アルコールを加え、0〜4℃で12〜16時間ぐらいアルコール沈殿させる。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、上清液を取り出してフォリン−フェノール法で多糖類溶液中の蛋白含有量0.5%以下と検知されるまで上記脱蛋白のステップを数回繰り返す。溶液に4.5倍体積の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2〜5%の多糖類液に調製し、分画分子量7000Da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純多糖類が得られる。
9. First confirmation of polysaccharide formation and structure
1. Removal of protein and small molecule substance (1) Prepare crude polysaccharide in 2-10% solution, slowly add 10% trichloroacetic acid solution at a volume ratio of 5: 1 at 4 ° C and stir. Shake at this temperature for 10 minutes, centrifuge at 4 ° C. for 10 minutes, remove the supernatant, and repeat the above deproteinization step several times until the protein content in the polysaccharide solution is 0.5% or less. 4.5 times volume of 95% alcohol is added to the solution and alcohol is precipitated overnight. Precipitate is removed by high-speed centrifugation, prepared into a 3-5% polysaccharide solution, dialyzed in a dialysis bag with a molecular weight cut off of 7000 Da for 48-72 hours, and then the solution in the dialysis bag is concentrated and freeze-dried To obtain a pure polysaccharide.
Or (2) Prepare crude polysaccharide in 2-10% solution, add 0.05-1% pepsin of solution weight, adjust to pH 1.5-3.0 with 6 mol / L HCl, 1-7 at 37 ° C After 6 hours of enzymatic degradation, warm to 90-100 ° C for 2-5 minutes to quench the enzyme for 10 minutes, cool to room temperature and adjust to neutral with NaOH solution. Centrifuge for 10 minutes, remove the supernatant, add 3-4.5 volumes of 95% alcohol, and allow alcohol precipitation at 0-4 ° C for 12-16 hours. Centrifuge and remove the precipitate to prepare a 2-5% solution. Add 5: 1 volume ratio of Sevag reagent (V chloroform : Vn -butanol = 5: 1), shake vigorously for 20 minutes, and leave for 30 minutes Then, the precipitate is removed by centrifugation, the supernatant is taken out, and the above deproteinization step is repeated several times until the protein content in the polysaccharide solution is detected to be 0.5% or less by the forin-phenol method. 4.5 times volume of 95% alcohol is added to the solution and alcohol is precipitated overnight. Precipitate is removed by high-speed centrifugation to prepare a 2-5% polysaccharide solution, dialyzed in a dialysis bag with a molecular weight cut off of 7000 Da for 48-72 hours, and then the solution in the dialysis bag is concentrated and freeze-dried. To obtain a pure polysaccharide.
2.分離精製
純多糖類はSephadex G-200カラム・クロマトグラフィで分離させ、溶離剤は0.9%のNaClで、溶出速度は0.5mL/minで、フェノール−硫酸法で追跡検出し、唯一の溶出ピークが検出され、収集・混合・透析・濃縮・冷凍乾燥することにより精製多糖類が得られる。
2. Separation and purification Pure polysaccharides were separated by Sephadex G-200 column chromatography, the eluent was 0.9% NaCl, the elution rate was 0.5 mL / min, followed by the phenol-sulfuric acid method, and the only elution peak Is detected, and purified polysaccharides are obtained by collection, mixing, dialysis, concentration, and freeze-drying.
3．分子量の測定
精製された多糖類はSephroce 6Bゲルカラム法で分離させ、溶離剤は0.9%のNaClで、溶出速度は0.25mL/minで、フェノール−硫酸法で追跡検出し、ピーク試験管数を測定して標準グラフに照らし合わせることにより当該多糖類分子量は6〜8万であることが分かった。
3． Molecular weight measurement Purified polysaccharides are separated by Sephroce 6B gel column method, eluent is 0.9% NaCl, elution rate is 0.25mL / min, followed by phenol-sulfuric acid method, and the number of peak test tubes is measured. Then, it was found that the polysaccharide molecular weight was 60,000 to 80,000 by comparing with a standard graph.
4.単糖組成の確定
完全に乾燥した精製多糖類10mg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させ、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）を行い、当該多糖類はペクチンシュガーの組成と分かった。
4. Determination of monosaccharide composition Add 2 mL of anhydrous HCl-methyl alcohol to 10 mg of fully dried purified polysaccharide, put N 2 in a sealed tube, decompose methyl alcohol at 80 ° C. for 20 hours, and take out the tube container. Leave to room temperature. Neutralize to pH = 6 with anhydrous KOH-methyl alcohol, dry under reduced pressure at 40 ° C., and dry. 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 Was taken out and subjected to color layer analysis (gas chromatograph), and the polysaccharide was found to be the composition of pectin sugar.
クロマトグラフ条件は、ガスクロマトグラフ（US Agilent 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 (US Agilent 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, hold at 150 ° C for 1 minute, increase to 182 ° C at 10 ° C / min, hold for 2 minutes, 1 ° C The temperature is raised to 188 ° C at / min, held for 1 minute, and raised to 230 ° C at 8 ° C / min.
1.本発明の抽出方法は合理的、且つ有効であり、帆立貝体内（殻を除く）から多糖類を抽出するプロセス（工程）を創出し、帆立貝多糖類を最大限で抽出するようにした。 1. The extraction method of the present invention is rational and effective, and a process (step) for extracting a polysaccharide from the scallop shell (excluding the shell) is created to extract the scallop polysaccharide to the maximum.
2.本発明における関連工程温度が全て60℃以下に制御されており、特に従来の高温酵素減滅の変わりに低温下活性抑制という方法を採用したことにより、抽出された多糖類の生物活性が確保され、最終製品に多糖類栄養性と効能性とを兼備せしめられる。 2. All the relevant process temperatures in the present invention are controlled to 60 ° C. or less, and the biological activity of the extracted polysaccharide is reduced by adopting a method of suppressing the activity under low temperature in place of the conventional high temperature enzyme decay. It is ensured that the end product combines polysaccharide nutrition and efficacy.
3.本発明の抽出プロセスは、帆立貝における比較的価値の高い貝柱を原料にして多糖類を抽出することを可能にしただけでなく、その殻を除く全ての組織、ひいてはオッファルまでを原料にして多糖類を抽出することもでき、コストダウン及び経済的効果・利益の向上には技術的保証を提供した。 3. The extraction process of the present invention not only enables extraction of polysaccharides from scallops of relatively high value in scallops, but also uses all tissues except shells and even offal as raw materials. Polysaccharides can also be extracted, providing technical guarantees to reduce costs and improve economic benefits and benefits.
4.本発明はアルカリ加水分解と酵素分解の結合方法で原料からより多くの多糖類を分解せしめられることを確立した。 4. The present invention has established that more polysaccharides can be decomposed from raw materials by a combined method of alkaline hydrolysis and enzymatic degradation.
5.本発明は自己融解酵素技術と酵素分解の結合方法で帆立貝原料自身の酵素体系を十分に利用しただけでなく、多糖類の抽出率を高めせしめられると確立した。 5. The present invention has established that not only the enzyme system of scallop raw material itself can be fully utilized, but also the extraction rate of polysaccharides can be enhanced by the combined method of autolytic enzyme technology and enzymatic degradation.
6.本発明の抽出過程からの残渣は従来技術により帆立貝ポリペプチド製品を製造するために用いることができる。 6. The residue from the extraction process of the present invention can be used to produce scallop polypeptide products by conventional techniques.
7.本発明で抽出される帆立貝多糖類は、単独で製品としてもよく、更にカプセル剤、粉剤、錠剤等の製品に加工してもよい。基材にしてほかの食品を補助して多効能食品を開発し、栄養補助食品の効能性を多元化させることもできる。 7. The scallop polysaccharide extracted in the present invention may be used alone, or may be further processed into products such as capsules, powders and tablets. It is also possible to develop multi-potency foods by assisting other foods as a base material, and to diversify the efficacy of nutritional supplements.
8.本発明において、乾燥後の貝内臓組織干し粉は、多糖類の抽出前に超臨界二酸化炭素抽出設備で脂肪の抽出を行ってもよい。脱脂後の貝内臓組織干し粉から多糖類を抽出すれば、廃棄物を宝物に変え、貝内臓を十分に利用せしめられる。 8. In the present invention, dried shellfish tissue dried powder may be subjected to fat extraction with a supercritical carbon dioxide extraction facility before polysaccharide extraction. Extracting polysaccharides from defatted shellfish internal organs can turn waste into treasures and make full use of shellfish internal organs.
9.本発明は、帆立貝多糖類抽出工程を確立した上で、さらに当該多糖類の純化技術を確立し、且つその単糖組成を始めて分析したため、今後の深究には良好な基礎を築いた。 9. The present invention established a scallop polysaccharide extraction process, further established a purification technology for the polysaccharide, and analyzed the monosaccharide composition for the first time, and thus laid a good foundation for future research.
新鮮な殻付き帆立貝を水洗して水を切り、殻を刃物で割って取り除き、異なる部位単位で全部組織を取り出した。1kgの帆立貝の全部組織に2kgの水を加え、組織破砕機でホモジネートし、漿液を0.3時間超音波処理（60℃、600W）し、6mol/Lの塩酸でｐH6に調整し、1.5g（0.05%）の酵素活性5×104u/gの枯草菌中性プロテイナーゼを加え、ｐHをこの範囲に保持し、45℃で3時間攪拌して酵素分解させた。6mol/Lの塩酸でｐH1に調整し、45ｇ（1.5%）の酵素活性7.8×105u/gのペプシンを加え、ｐH値をこの範囲に保持し、40℃で5時間攪拌して酵素分解させた。その後、急速に30℃以下まで冷却し、30分間保持してから反応を終止した。0.5mol/Lの水酸化カリウムでｐHを中性に調整した。6000rpmで20分間遠心して上清液を収集して真空濃縮設備に入れ、真空度85〜98ｋPa、温度30℃で制御し、1リットルになるまで濃縮した。攪拌しながら2リットルの95%アルコールをゆっくり加え、2℃で2時間アルコール沈殿し、多糖類沈殿を遠心により収集して直接20℃で火干しすることにより24.2ｇの多糖類干し粉を得た。フェノール−硫酸法で測定を行ったところ、多糖類含有量は17.1%であった。 Fresh scallops with shells were washed with water, drained, and the shells were removed by cutting with a blade. Add 2 kg of water to the whole tissue of 1 kg scallop, homogenize with a tissue crusher, sonicate the serum for 0.3 hours (60 ° C, 600 W), adjust to pH 6 with 6 mol / L hydrochloric acid, 1.5 g (0.05 %) Enzyme activity 5 × 10 4 u / g of Bacillus subtilis neutral proteinase was added, the pH was kept within this range, and the mixture was stirred at 45 ° C. for 3 hours for enzymatic degradation. Adjust to pH 1 with 6 mol / L hydrochloric acid, add 45 g (1.5%) of enzyme activity 7.8 × 10 5 u / g of pepsin, keep the pH value within this range, and stir at 40 ° C. for 5 hours for enzymatic degradation I let you. Thereafter, the reaction was rapidly cooled to 30 ° C. or lower, held for 30 minutes, and the reaction was terminated. The pH was adjusted to neutral with 0.5 mol / L potassium hydroxide. After centrifuging at 6000 rpm for 20 minutes, the supernatant was collected and placed in a vacuum concentrator, and concentrated at a vacuum of 85 to 98 kPa and a temperature of 30 ° C. until it was 1 liter. While stirring, 2 liters of 95% alcohol was slowly added, and alcohol precipitation was performed at 2 ° C for 2 hours. The polysaccharide precipitate was collected by centrifugation and directly dried at 20 ° C to obtain 24.2 g of polysaccharide dried powder. . When measured by the phenol-sulfuric acid method, the polysaccharide content was 17.1%.
新鮮な殻付き帆立貝を水洗して水を切り、殻を刃物で割って取り除き、異なる部位単位で全部組織を取り出した。1kgの帆立貝の貝柱に5kgの水を加え、組織破砕機でホモジネートし、漿液を0.5時間超音波処理（55℃、300W）し、0.5mol/Lの水酸化カリウムでｐH7に調整し、酵素分解過程においてｐH値をこの範囲に保持し、120g（2.0%）の酵素活性5×104u/gのトリプシンを加え、40℃で1時間攪拌して酵素分解させた。その後、急速に30℃以下まで冷却し、30分間保持してから反応を終止した。6mol/Lの塩酸でｐHを中性に調整した。200目スクリーンでろ過し、得られたろ過液を真空濃縮設備に入れ、真空度92ｋPa、温度55℃で制御し、1.5リットルになるまで濃縮した。攪拌しながら5.25リットルの95%アルコールをゆっくり加え、3℃で8時間アルコール沈殿し、多糖類沈殿を遠心により収集して真空冷凍乾燥設備に入れ、真空度150Pa、板温度30℃に制御して15時間冷凍乾燥することにより33.5ｇの帆立貝多糖類干し粉を得た。フェノール−硫酸法で測定を行ったところ、多糖類含有量は18.4%であった。 Fresh scallops with shells were washed with water, drained, and the shells were removed by cutting with a blade. Add 5 kg of water to the scallop of 1 kg of scallop, homogenize with a tissue crusher, sonicate the serum for 0.5 hours (55 ° C, 300 W), adjust to pH 7 with 0.5 mol / L potassium hydroxide, and enzymatic digestion During the process, the pH value was kept in this range, 120 g (2.0%) of enzyme activity 5 × 10 4 u / g of trypsin was added, and the mixture was stirred at 40 ° C. for 1 hour for enzymatic degradation. Thereafter, the reaction was rapidly cooled to 30 ° C. or lower, held for 30 minutes, and the reaction was terminated. The pH was adjusted to neutral with 6 mol / L hydrochloric acid. The mixture was filtered through a 200-mesh screen, and the obtained filtrate was put into a vacuum concentrating facility, and controlled at a vacuum degree of 92 kPa and a temperature of 55 ° C., and concentrated to 1.5 liters. While stirring, slowly add 5.25 liters of 95% alcohol, precipitate alcohol at 3 ° C for 8 hours, collect the polysaccharide precipitate by centrifugation, put it in a vacuum freeze-drying facility, and control the vacuum to 150 Pa and the plate temperature to 30 ° C. By freeze-drying for 15 hours, 33.5 g of scallop polysaccharide dried powder was obtained. When measured by the phenol-sulfuric acid method, the polysaccharide content was 18.4%.
新鮮な殻付き帆立貝を水洗して水を切り、殻を刃物で割って取り除き、異なる部位単位で全部組織を取り出した。1kgの帆立貝のオッファルに500gの水を加え、組織破砕機でホモジネートし、漿液を0.5時間超音波処理（50℃、200W）し、0.5mol/Lの水酸化カリウムでｐH7に調整し、22.5g（1.5%）の酵素活性5×104u/gの枯草菌中性プロテイナーゼを加え、ｐH値をこの範囲に保持し、45℃で1時間攪拌して酵素分解させた。0.5mol/Lの水酸化カリウムでｐH9に調整し、0.75ｇ（0.05%）の酵素活性5×104u/gのトリプシンを加え、ｐH値をこの範囲に保持し、60℃で4時間攪拌して酵素分解させた。その後、急速に30℃以下まで冷却し、30分間保持してから反応を終止した。6mol/Lの塩酸でｐHを中性に調整した。6000rpmで20分間遠心して上清液を収集して真空濃縮設備に入れ、真空度90kPa、温度50℃に制御して0.3リットルになるまで濃縮した。攪拌しながら0.9リットルの95%アルコールをゆっくり加え、4℃で10時間アルコール沈殿し、多糖類沈殿を遠心により収集して真空冷凍乾燥設備に入れ、真空度90Pa、板温度50℃に制御して10時間冷凍乾燥することにより18.8ｇの多糖類干し粉を得た。フェノール−硫酸法で測定を行ったところ、多糖類含有量は10.9%であった。 Fresh scallops with shells were washed with water, drained, and the shells were removed by cutting with a blade. 500 kg of water is added to 1 kg of scallop offal, homogenized with a tissue crusher, and the serum is sonicated for 0.5 hours (50 ° C, 200 W), adjusted to pH 7 with 0.5 mol / L potassium hydroxide, 22.5 g (1.5%) Enzyme activity 5 × 10 4 u / g of Bacillus subtilis neutral proteinase was added, the pH value was kept within this range, and the mixture was stirred at 45 ° C. for 1 hour for enzymatic degradation. Adjust to pH 9 with 0.5 mol / L potassium hydroxide, add 0.75 g (0.05%) of enzyme activity 5 × 10 4 u / g trypsin, keep the pH value within this range, and stir at 60 ° C. for 4 hours And then enzymatically decomposed. Thereafter, the reaction was rapidly cooled to 30 ° C. or lower, held for 30 minutes, and the reaction was terminated. The pH was adjusted to neutral with 6 mol / L hydrochloric acid. After centrifuging at 6000 rpm for 20 minutes, the supernatant was collected and placed in a vacuum concentrator, and concentrated to 0.3 liter by controlling the degree of vacuum at 90 kPa and the temperature at 50 ° C. Slowly add 0.9 liters of 95% alcohol with stirring, precipitate the alcohol at 4 ° C for 10 hours, collect the polysaccharide precipitate by centrifugation, put in vacuum freeze drying equipment, control the vacuum degree to 90Pa and the plate temperature to 50 ° C. By freeze-drying for 10 hours, 18.8 g of polysaccharide dried powder was obtained. When measured by the phenol-sulfuric acid method, the polysaccharide content was 10.9%.
新鮮な殻付き帆立貝を水洗して水を切り、殻を刃物で割って取り除き、異なる部位単位で全部組織を取り出した。1kgの帆立貝内臓に4kgの水を加え、組織破砕機でホモジネートし、漿液を0.5時間超音波処理（50℃、200W）し、6mol/Lの塩酸でｐH1に調整し、2.5g（0.05%）の酵素活性7.8×105u/gのペプシンを加え、ｐH値をこの範囲に保持し、60℃で5時間攪拌して酵素分解させた。0.5mol/Lの水酸化カリウムでｐH9に調整し、75ｇ（1.5%）の酵素活性5×104u/gのトリプシンを加え、ｐH値をこの範囲に保持し、60℃で1時間攪拌して酵素分解させた。その後、急速に30℃以下まで冷却し、30分間保持してから反応を終止した。6mol/Lの塩酸でｐHを中性に調整した。200目スクリーンでろ過し、得られたろ過液を真空濃縮設備に入れ、真空度90ｋPa、温度50℃に制御して1リットルになるまで濃縮した。攪拌しながら4リットルの95%アルコールをゆっくり加え、3℃で10時間アルコール沈殿し、多糖類沈殿を遠心により収集して直接45℃で火干しすることにより12.6ｇの帆立貝多糖類干し粉を得た。フェノール−硫酸法で測定を行ったところ、多糖類含有量は11.7%であった。 Fresh scallops with shells were washed with water, drained, and the shells were removed by cutting with a blade. Add 4kg water to 1kg scallop viscera, homogenize with tissue crusher, sonicate serum for 0.5 hours (50 ℃, 200W), adjust to pH1 with 6mol / L hydrochloric acid, 2.5g (0.05%) Enzymatic activity of 7.8 × 10 5 u / g of pepsin was added, the pH value was kept within this range, and the mixture was stirred at 60 ° C. for 5 hours for enzymatic degradation. Adjust to pH 9 with 0.5 mol / L potassium hydroxide, add 75 g (1.5%) of enzyme activity 5 × 10 4 u / g trypsin, keep the pH value within this range, and stir at 60 ° C. for 1 hour. The enzyme was decomposed. Thereafter, the reaction was rapidly cooled to 30 ° C. or lower, held for 30 minutes, and the reaction was terminated. The pH was adjusted to neutral with 6 mol / L hydrochloric acid. The mixture was filtered through a 200-mesh screen, and the obtained filtrate was put into a vacuum concentration facility, and concentrated to 1 liter by controlling the degree of vacuum at 90 kPa and the temperature at 50 ° C. Slowly add 4 liters of 95% alcohol with stirring, precipitate with alcohol at 3 ° C for 10 hours, collect the polysaccharide precipitate by centrifugation and directly dry at 45 ° C to obtain 12.6 g of scallop polysaccharide dried powder It was. When measured by the phenol-sulfuric acid method, the polysaccharide content was 11.7%.
新鮮な殻付き帆立貝を水洗して水を切り、殻を刃物で割って取り除き、異なる部位単位で全部組織を取り出した。1kgの帆立貝のひもに3kgの水を加え、組織破砕機でホモジネートし、漿液を0.5時間超音波処理（40℃、100W）した。6mol/Lの塩酸でｐH1に調整し、酵素分解過程にｐH値をこの範囲に保持し、4g（0.1%）の酵素活性7.8×105u/gのペプシンを加え、40℃で10時間攪拌して酵素分解させた。その後、急速に30℃以下まで冷却し、30分間保持してから反応を終止した。0.5mol/Lの水酸化カリウムでｐHを中性に調整した。6000rpmで20分間遠心して上清液を収集して真空濃縮設備に入れ、真空度90ｋPa、温度40℃に制御し、0.8リットルになるまで濃縮した。攪拌しながら3.2リットルの95%アルコールをゆっくり加え、4℃で14時間アルコール沈殿し、多糖類沈殿を遠心により収集して直接30℃で火干しすることにより多糖類干し粉が23.7ｇ得られた。フェノール−硫酸法で測定を行ったところ、多糖類含有量は6.8%であった。 Fresh scallops with shells were washed with water, drained, and the shells were removed by cutting with a blade. 3 kg of water was added to a 1 kg scallop string, homogenized with a tissue crusher, and the serum was sonicated (40 ° C., 100 W) for 0.5 hour. Adjust to pH 1 with 6 mol / L hydrochloric acid, maintain the pH value in this range during the enzymatic degradation process, add 4 g (0.1%) of enzyme activity 7.8 × 10 5 u / g pepsin, and stir at 40 ° C. for 10 hours And then enzymatically decomposed. Thereafter, the reaction was rapidly cooled to 30 ° C. or lower, held for 30 minutes, and the reaction was terminated. The pH was adjusted to neutral with 0.5 mol / L potassium hydroxide. The supernatant was collected by centrifugation at 6000 rpm for 20 minutes, placed in a vacuum concentrator, and concentrated to 0.8 liter by controlling the degree of vacuum at 90 kPa and the temperature at 40 ° C. While stirring, 3.2 liters of 95% alcohol was slowly added, and the alcohol was precipitated at 4 ° C for 14 hours. The polysaccharide precipitate was collected by centrifugation and directly dried at 30 ° C to obtain 23.7 g of polysaccharide dried powder. . When measured by the phenol-sulfuric acid method, the polysaccharide content was 6.8%.
新鮮な殻付き帆立貝を水洗して水を切り、殻を刃物で割って取り除き、異なる部位単位で全部組織を取り出した。1kgの内臓が取り除かれた帆立貝の全部組織に水4 kgを加え、組織破砕機でホモジネートし、漿液を0.5時間超音波処理（40℃、200W）した。6mol/Lの塩酸でｐH6に調整し、酵素分解過程にｐH値をこの範囲に保持し、4g（0.1%）の酵素活性5.0×104u/gの枯草菌中性プロテイナーゼを加え、45℃で6時間攪拌して酵素分解させた。その後、急速に30℃以下まで冷却し、30分間保持してから反応を終止した。0.5mol/Lの水酸化カリウムでｐHを中性に調整した。200目スクリーンでろ過し、得られたろ過液を真空濃縮設備に入れ、真空度90kPa、温度50℃に制御して1リットルになるまで濃縮した。攪拌しながら3リットルの95%アルコールをゆっくり加え、5℃で15時間アルコール沈殿し、多糖類沈殿を遠心により収集して真空冷凍乾燥設備に入れ、真空度90Pa、板温度50℃に制御して10時間冷凍乾燥することにより26.4ｇの帆立貝多糖類干し粉を得た。フェノール−硫酸法で測定を行ったところ、多糖類含有量は10.2%であった。 Fresh scallops with shells were washed with water, drained, and the shells were removed by cutting with a blade. 4 kg of water was added to the entire tissue of the scallop from which 1 kg of internal organs had been removed, homogenized with a tissue crusher, and the serum was sonicated (40 ° C., 200 W) for 0.5 hour. Adjust the pH to 6 with 6 mol / L hydrochloric acid, keep the pH value in this range during the enzymatic degradation process, add 4 g (0.1%) of enzyme activity 5.0 × 10 4 u / g of Bacillus subtilis neutral proteinase, 45 ° C The mixture was stirred for 6 hours for enzymatic degradation. Thereafter, the reaction was rapidly cooled to 30 ° C. or lower, held for 30 minutes, and the reaction was terminated. The pH was adjusted to neutral with 0.5 mol / L potassium hydroxide. The mixture was filtered through a 200-mesh screen, and the resulting filtrate was put into a vacuum concentration facility, and concentrated to 1 liter by controlling the degree of vacuum at 90 kPa and the temperature at 50 ° C. While stirring, slowly add 3 liters of 95% alcohol, precipitate the alcohol at 5 ° C for 15 hours, collect the polysaccharide precipitate by centrifugation, put it in the vacuum freeze drying equipment, and control the vacuum degree to 90Pa and the plate temperature to 50 ° C. By freeze-drying for 10 hours, 26.4 g of scallop polysaccharide dried powder was obtained. When measured by the phenol-sulfuric acid method, the polysaccharide content was 10.2%.
新鮮な殻付き帆立貝を水洗して水を切り、殻を刃物で割って取り除き、異なる部位単位で全部組織を取り出した。水分含有量が4%以下になるまで60℃で火干しし、200目まで破砕して干し粉を得た。100gの帆立貝の全部組織干し粉に5kgの水を加え、組織破砕機でホモジネートし、漿液を0.6時間超音波処理（35℃、80W）した。0.5mol/Lの水酸化カリウムでｐH9に調整し、酵素分解過程にｐH値をこの範囲に保持し、5.1g（0.1%）の酵素活性5.0×104u/gのトリプシンを加え、60℃で8時間攪拌して酵素分解させた。その後、急速に30℃以下まで冷却し、30分間保持してから反応を終止した。6mol/Lの塩酸でｐHを中性に調整した。200目スクリーンでろ過し、得られたろ過液を真空濃縮設備に入れ、真空度92ｋPa、温度45℃に制御して1.5リットルになるまで濃縮した。攪拌しながら3.75リットルの95%アルコールをゆっくり加え、4℃で14時間アルコール沈殿し、多糖類沈殿を遠心により収集して真空冷凍乾燥設備に入れ、真空度70Pa、板温度60℃に制御して6時間冷凍乾燥することにより12.1ｇの帆立貝多糖類干し粉を得た。フェノール−硫酸法で測定を行ったところ、多糖類含有量は17.6%であった。 Fresh scallops with shells were washed with water, drained, and the shells were removed by cutting with a blade. Dried at 60 ° C. until the water content was 4% or less, and crushed to 200 eyes to obtain dried powder. 5 kg of water was added to 100 g of scallop whole tissue dried powder, homogenized with a tissue crusher, and the serum was sonicated (35 ° C., 80 W) for 0.6 hours. Adjust to pH 9 with 0.5 mol / L potassium hydroxide, keep the pH value in this range during the enzymatic degradation process, add 5.1 g (0.1%) enzyme activity 5.0 × 10 4 u / g trypsin, 60 ° C The mixture was stirred for 8 hours for enzymatic degradation. Thereafter, the reaction was rapidly cooled to 30 ° C. or lower, held for 30 minutes, and the reaction was terminated. The pH was adjusted to neutral with 6 mol / L hydrochloric acid. The mixture was filtered through a 200-mesh screen, and the resulting filtrate was placed in a vacuum concentration facility, and concentrated to 1.5 liters while controlling the degree of vacuum at 92 kPa and the temperature at 45 ° C. Slowly add 3.75 liters of 95% alcohol with stirring, alcohol precipitate for 14 hours at 4 ° C, collect polysaccharide precipitate by centrifugation, put into vacuum freeze drying equipment, control to 70Pa vacuum and 60 ° C plate temperature. By freeze-drying for 6 hours, 12.1 g of scallop polysaccharide dried powder was obtained. When measured by the phenol-sulfuric acid method, the polysaccharide content was 17.6%.
新鮮な殻付き帆立貝を水洗して水を切り、殻を刃物で割って取り除き、異なる部位単位で全部組織を取り出した。水分含有量が4%以下になるまで60℃で火干しし、50目まで破砕して干し粉を得た。100gの帆立貝の貝柱干し粉に水1kgを加え、組織破砕機でホモジネートし、漿液を1時間超音波処理（30℃、50W）した。6mol/Lの塩酸でｐH7に調整し、16.5g（1.5%）の酵素活性5.0×104u/gの枯草菌中性プロテイナーゼを加え、ｐH値をこの範囲に保持し、55℃で1時間攪拌して酵素分解させた。6mol/Lの塩酸でｐH5に調整し、0.55g（0.05%）の酵素活性7.8×105u/
gのペプシンを加え、ｐH値をこの範囲に保持し、60℃で1時間攪拌して酵素分解させた。その後、急速に30℃以下まで冷却し、30分間保持してから反応を終止した。0.5mol/Lの水酸化カリウムでｐHを中性に調整した。6000rpmで20分間遠心して上清液を収集して真空濃縮設備に入れ、真空度98ｋPa、温度60℃に制御して0.3リットルになるまで濃縮した。攪拌しながら1.5リットルの95%アルコールをゆっくり加え、6℃で18時間アルコール沈殿し、多糖類沈殿を遠心により収集して直接60℃で火干しすることにより13.4ｇの多糖類干し粉を得た。フェノール−硫酸法で測定を行ったところ、多糖類含有量は18.1%であった。
Fresh scallops with shells were washed with water, drained, and the shells were removed by cutting with a blade. It was fire-dried at 60 ° C. until the water content was 4% or less, and crushed to 50 eyes to obtain dried powder. 1 kg of water was added to 100 g of scallop dried powder of a scallop, homogenized with a tissue crusher, and the serum was sonicated (30 ° C., 50 W) for 1 hour. Adjust pH to 7 with 6 mol / L hydrochloric acid, add 16.5 g (1.5%) of enzyme activity 5.0 × 10 4 u / g neutral proteinase of Bacillus subtilis, maintain pH value within this range, and maintain at 55 ° C for 1 hour The mixture was stirred for enzymatic degradation. Adjusted to pH 5 with 6 mol / L hydrochloric acid, 0.55 g (0.05%) of enzyme activity 7.8 × 10 5 u /
g pepsin was added, the pH value was kept within this range, and the mixture was stirred at 60 ° C. for 1 hour for enzymatic degradation. Thereafter, the reaction was rapidly cooled to 30 ° C. or lower, held for 30 minutes, and the reaction was terminated. The pH was adjusted to neutral with 0.5 mol / L potassium hydroxide. After centrifuging at 6000 rpm for 20 minutes, the supernatant was collected and placed in a vacuum concentration facility, and concentrated to 0.3 liter by controlling the degree of vacuum at 98 kPa and the temperature at 60 ° C. While stirring, 1.5 liters of 95% alcohol was slowly added, and alcohol precipitation was performed at 6 ° C for 18 hours. The polysaccharide precipitate was collected by centrifugation and directly dried at 60 ° C to obtain 13.4 g of polysaccharide dried powder. . When measured by the phenol-sulfuric acid method, the polysaccharide content was 18.1%.
新鮮な殻付き帆立貝を水洗して水を切り、殻を刃物で割って取り除き、異なる部位単位で全部組織を取り出した。水分含有量が4%以下になるまで真空冷凍乾燥し、150目まで破砕して干し粉を得た。100gの帆立貝オッファル干し粉に4kgの水を加え、組織破砕機でホモジネートし、漿液を0.5時間超音波処理（40℃、100W）した。6mol/Lの塩酸でｐH5に調整し、酵素分解過程にｐH値をこの範囲に保持し、82g（2.0%）の酵素活性7.8×105u/gのペプシンを加え、60℃で1時間攪拌して酵素分解させた。その後、急速に30℃以下まで冷却し、30分間保持してから反応を終止した。0.5mol/Lの水酸化カリウムでｐHを中性に調整した。6000rpmで20分間遠心して上清液を収集して真空濃縮設備に入れ、真空度90ｋPa、温度40℃に制御して1リットルになるまで濃縮した。攪拌しながら4リットルの95%アルコールをゆっくり加え、4℃で14時間アルコール沈殿し、多糖類沈殿を遠心により収集して直接30℃で火干しすることにより9.8ｇの帆立貝多糖類干し粉を得た。フェノール−硫酸法で測定を行ったところ、多糖類含有量は11.3%であった。 Fresh scallops with shells were washed with water, drained, and the shells were removed by cutting with a blade. Vacuum-freeze-dry until the water content was 4% or less, and crush to 150th to obtain dried powder. 4 kg of water was added to 100 g of scallop offal dried powder, homogenized with a tissue crusher, and the serum was sonicated (40 ° C., 100 W) for 0.5 hour. Adjust to pH 5 with 6 mol / L hydrochloric acid, maintain the pH value in this range during the enzymatic degradation process, add 82 g (2.0%) of enzyme activity 7.8 × 10 5 u / g pepsin, and stir at 60 ° C. for 1 hour And then enzymatically decomposed. Thereafter, the reaction was rapidly cooled to 30 ° C. or lower, held for 30 minutes, and the reaction was terminated. The pH was adjusted to neutral with 0.5 mol / L potassium hydroxide. After centrifuging at 6000 rpm for 20 minutes, the supernatant was collected and placed in a vacuum concentration facility, and concentrated to 1 liter by controlling the degree of vacuum at 90 kPa and the temperature at 40 ° C. Slowly add 4 liters of 95% alcohol with stirring, precipitate with alcohol at 4 ° C for 14 hours, collect the polysaccharide precipitate by centrifugation and directly dry at 30 ° C to obtain 9.8 g of scallop polysaccharide dried powder It was. When measured by the phenol-sulfuric acid method, the polysaccharide content was 11.3%.
新鮮な殻付き帆立貝を水洗して水を切り、殻を刃物で割って取り除き、異なる部位単位で全部組織を取り出した。水分含有量が4%以下になるまで40℃で火干しし、100目まで破砕して干し粉を得た。100gの帆立貝内蔵干し粉に2kgの水を加え、組織破砕機でホモジネートし、漿液を0.5時間超音波処理（40℃、200W）した。6mol/Lの塩酸でｐH7に調整し、酵素分解過程においてｐH値をこの範囲に保持し、42g（2.0%）の酵素活性5.0×104u/gの枯草菌プロテイナーゼを加え、55℃で1時間攪拌して酵素分解させた。その後、急速に30℃以下まで冷却し、30分間保持してから反応を終止した。0.5mol/Lの水酸化カリウムでｐHを中性に調整した。200目スクリーンでろ過し、得られたろ過液を真空濃縮設備に入れ、真空度90ｋPa、温度50℃に制御して0.5リットルになるまで濃縮した。攪拌しながら1.5リットルの95%アルコールをゆっくり加え、5℃で15時間アルコール沈殿し、多糖類沈殿を遠心により収集して真空冷凍乾燥設備に入れ、真空度90Pa、板温度50℃に制御して10時間冷凍乾燥することにより7.4ｇの帆立貝多糖類干し粉を得た。フェノール−硫酸法で測定を行ったところ、多糖類含有量は12.8%であった。 Fresh scallops with shells were washed with water, drained, and the shells were removed by cutting with a blade. Fire-dried at 40 ° C. until the water content was 4% or less, and crushed to 100 eyes to obtain dried powder. 2 kg of water was added to 100 g of scallop built-in dried powder, homogenized with a tissue crusher, and the serum was sonicated (40 ° C., 200 W) for 0.5 hour. Adjust to pH 7 with 6 mol / L hydrochloric acid, maintain the pH value in this range during the enzymatic degradation process, add 42 g (2.0%) enzyme activity 5.0 × 10 4 u / g Bacillus subtilis proteinase, The mixture was stirred for hours to allow enzymatic degradation. Thereafter, the reaction was rapidly cooled to 30 ° C. or lower, held for 30 minutes, and the reaction was terminated. The pH was adjusted to neutral with 0.5 mol / L potassium hydroxide. The mixture was filtered through a 200-mesh screen, and the obtained filtrate was put into a vacuum concentration facility, and concentrated to 0.5 liters while controlling the degree of vacuum at 90 kPa and the temperature at 50 ° C. Slowly add 1.5 liters of 95% alcohol while stirring, precipitate with alcohol at 5 ° C for 15 hours, collect the polysaccharide precipitate by centrifugation, put into vacuum freeze-drying equipment, control the vacuum degree to 90Pa and the plate temperature to 50 ° C. By freeze-drying for 10 hours, 7.4 g of scallop polysaccharide dried powder was obtained. When measured by the phenol-sulfuric acid method, the polysaccharide content was 12.8%.
新鮮な殻付き帆立貝を水洗して水を切り、殻を刃物で割って取り除き、異なる部位単位で全部組織を取り出した。水分含有量が4%以下になるまで真空冷凍乾燥し、120目まで破砕して干し粉を得た。100gの帆立貝ひも干し粉に3kgの水を加え、組織破砕機でホモジネートし、漿液を0.6時間超音波処理（50℃、200W）した。0.5mol/Lの水酸化カリウムでｐH8に調整し、1.55g（0.05%）の酵素活性5×104u/gの枯草菌プロテイナーゼを加え、ｐH値をこの範囲に保持し、55℃で3時間攪拌して酵素分解させた。0.5mol/Lの水酸化カリウムでｐH7に調整し、46.5g（1.5%）の酵素活性5×104u/gのトリプシンを加え、ｐH値をこの範囲に保持し、40℃で1時間攪拌して酵素分解させた。その後、急速に30℃以下まで冷却し、30分間保持してから反応を終止した。6mol/Lの塩酸でｐHを中性に調整した。6000rpmで20分間遠心して上清液を収集して真空濃縮設備に入れ、真空度90ｋPa、温度50℃に制御して0.8リットルになるまで濃縮した。攪拌しながら2.4リットルの95%アルコールをゆっくり加え、4℃で10時間アルコール沈殿し、多糖類沈殿を遠心により収集して真空冷凍乾燥設備に入れ、真空度90Pa、板温度50℃に制御して10時間冷凍乾燥することにより10.8ｇの帆立貝多糖類干し粉を得た。フェノール−硫酸法で測定を行ったところ、多糖類含有量は6.4%であった。 Fresh scallops with shells were washed with water, drained, and the shells were removed by cutting with a blade. It was vacuum-frozen and dried until the water content was 4% or less, and crushed to 120th to obtain dried powder. 3 kg of water was added to 100 g of scallop string dried powder, homogenized with a tissue crusher, and the serum was sonicated (50 ° C., 200 W) for 0.6 hours. Adjust to pH 8 with 0.5 mol / L potassium hydroxide, add 1.55 g (0.05%) of enzyme activity 5 × 10 4 u / g Bacillus subtilis proteinase, keep the pH value within this range, The mixture was stirred for hours to allow enzymatic degradation. Adjust to pH 7 with 0.5 mol / L potassium hydroxide, add 46.5 g (1.5%) of enzyme activity 5 × 10 4 u / g trypsin, keep the pH value within this range, and stir at 40 ° C. for 1 hour And then enzymatically decomposed. Thereafter, the reaction was rapidly cooled to 30 ° C. or lower, held for 30 minutes, and the reaction was terminated. The pH was adjusted to neutral with 6 mol / L hydrochloric acid. The supernatant was collected by centrifuging at 6000 rpm for 20 minutes, placed in a vacuum concentration facility, and concentrated to 0.8 liter by controlling the degree of vacuum at 90 kPa and the temperature at 50 ° C. Slowly add 2.4 liters of 95% alcohol with stirring, precipitate with alcohol at 4 ° C for 10 hours, collect the polysaccharide precipitate by centrifugation, put into vacuum freeze-drying equipment, control the vacuum degree to 90Pa and the plate temperature to 50 ° C. By freeze-drying for 10 hours, 10.8 g of scallop polysaccharide dried powder was obtained. When measured by the phenol-sulfuric acid method, the polysaccharide content was 6.4%.
新鮮な殻付き帆立貝を水洗して水を切り、殻を刃物で割って取り除き、異なる部位単位で全部組織を取り出した。水分含有量が4%以下になるまで40℃で火干しし、150目まで破砕して干し粉を得た。100gの内蔵が除かれた帆立貝の全部干し粉に2kgの水を加え、組織破砕機でホモジネートし、漿液を0.5時間超音波処理（50℃、200W）した。6mol/Lの塩酸でｐH5に調整し、31.5g（1.5%）の酵素活性7.8×105u/gのペプシンを加え、ｐHをこの範囲に保持し、40℃で1時間攪拌して酵素分解させた。0.5mol/Lの水酸化カリウムでｐH7に調整し、1.05g（0.05%）の酵素活性5×104u/gのトリプシンを加え、ｐHをこの範囲に保持し、40℃で4時間攪拌して酵素分解させた。その後、急速に30℃以下まで冷却し、30分間保持してから反応を終止した。6mol/Lの塩酸でｐHを中性に調整した。200目スクリーンでろ過し、得られたろ過液を真空濃縮設備に入れ、真空度90ｋPa、温度50℃に制御して0.5リットルになるまで濃縮した。攪拌しながら2リットルの95%アルコールをゆっくり加え、3℃で10時間アルコール沈殿し、多糖類沈殿を遠心により収集して直接50℃で火干しすることにより12.8ｇの帆立貝多糖類干し粉を得た。フェノール−硫酸法で測定を行ったところ、多糖類含有量は10.7%であった。 Fresh scallops with shells were washed with water, drained, and the shells were removed by cutting with a blade. Fire-dried at 40 ° C. until the water content was 4% or less, and crushed to 150 eyes to obtain dried powder. 2 kg of water was added to the whole dried scallops from which 100 g was removed, homogenized with a tissue crusher, and the sera was sonicated (50 ° C., 200 W) for 0.5 hour. Adjust to pH 5 with 6 mol / L hydrochloric acid, add 31.5 g (1.5%) of enzyme activity 7.8 × 10 5 u / g of pepsin, keep the pH in this range, and stir at 40 ° C. for 1 hour for enzymatic degradation I let you. Adjust to pH 7 with 0.5 mol / L potassium hydroxide, add 1.05 g (0.05%) of enzyme activity 5 × 10 4 u / g trypsin, keep the pH within this range, and stir at 40 ° C. for 4 hours. The enzyme was decomposed. Thereafter, the reaction was rapidly cooled to 30 ° C. or lower, held for 30 minutes, and the reaction was terminated. The pH was adjusted to neutral with 6 mol / L hydrochloric acid. The mixture was filtered through a 200-mesh screen, and the obtained filtrate was put into a vacuum concentration facility, and concentrated to 0.5 liters while controlling the degree of vacuum at 90 kPa and the temperature at 50 ° C. Slowly add 2 liters of 95% alcohol with stirring, precipitate with alcohol at 3 ° C for 10 hours, collect the polysaccharide precipitate by centrifugation and directly dry at 50 ° C to obtain 12.8 g of scallop polysaccharide dried powder It was. When measured by the phenol-sulfuric acid method, the polysaccharide content was 10.7%.
新鮮な殻付き帆立貝を水洗して水を切り、殻を刃物で割って取り除き、異なる部位単位で全部組織を取り出した。1kgのオッファルに4kgの水を加え、組織破砕機でホモジネートし、漿液を0.5時間超音波処理（30℃、100W）した。50gの破砕の固体炭酸カリウムを加え、30℃で1時間攪拌してアルカリ加水分解させた。6mol/Lの塩酸でｐH6.5に調整し、50g（1.0%）の酵素活性5.0×104u/gの枯草菌プロテイナーゼを加え、ｐH値をこの範囲に保持し、50℃で2時間攪拌して酵素分解させた。6mol/Lの塩酸でｐH4に調整し、50g（1.0%）の酵素活性7.8×105u/gのペプシンを加え、ｐH値をこの範囲に保持し、50℃で3時間攪拌して酵素分解させた。その後、急速に30℃以下まで冷却し、30分間保持してから反応を終止した。0.5mol/Lの水酸化カリウムでｐHを中性に調整した。6000rpmで20分間遠心して上清液を収集して真空濃縮設備に入れ、真空度90ｋPa、温度50℃に制御して1.25リットルになるまで濃縮した。攪拌しながら3.75リットルの95%アルコールをゆっくり加え、3℃で10時間アルコール沈殿し、多糖類沈殿を遠心により収集して直接50℃で火干しすることにより19.7ｇの帆立貝多糖類干し粉を得た。フェノール−硫酸法で測定を行ったところ、多糖類含有量は10.4%であった。 Fresh scallops with shells were washed with water, drained, and the shells were removed by cutting with a blade. 4 kg of water was added to 1 kg of offal, homogenized with a tissue crusher, and the serum was sonicated (30 ° C., 100 W) for 0.5 hour. 50 g of crushed solid potassium carbonate was added, and the mixture was stirred at 30 ° C. for 1 hour for alkali hydrolysis. Adjust pH to 6.5 with 6 mol / L hydrochloric acid, add 50 g (1.0%) of enzyme activity 5.0 × 10 4 u / g Bacillus subtilis proteinase, maintain the pH value within this range, and stir at 50 ° C. for 2 hours And then enzymatically decomposed. Adjust to pH 4 with 6 mol / L hydrochloric acid, add 50 g (1.0%) of enzyme activity 7.8 × 10 5 u / g pepsin, keep the pH value within this range, and stir at 50 ° C. for 3 hours for enzymatic degradation I let you. Thereafter, the reaction was rapidly cooled to 30 ° C. or lower, held for 30 minutes, and the reaction was terminated. The pH was adjusted to neutral with 0.5 mol / L potassium hydroxide. After centrifuging at 6000 rpm for 20 minutes, the supernatant was collected and placed in a vacuum concentration facility, and concentrated to 1.25 liters by controlling the degree of vacuum at 90 kPa and the temperature at 50 ° C. 3.75 liters of 95% alcohol is slowly added with stirring, alcohol precipitation is performed at 3 ° C for 10 hours, polysaccharide precipitate is collected by centrifugation and directly dried at 50 ° C to obtain 19.7 g of scallop polysaccharide dried powder It was. When measured by the phenol-sulfuric acid method, the polysaccharide content was 10.4%.
新鮮な殻付き帆立貝を水洗して水を切り、殻を刃物で割って取り除き、異なる部位単位で全部組織を取り出した。水分含有量が4%以下になるまで真空冷凍乾燥し、180目まで破砕して干し粉を得た。100gの内蔵干し粉に3kgの水を加え、組織破砕機でホモジネートし、漿液を0.8時間超音波処理（40℃、200W）した。3.1gの破砕の固体炭酸カリウムを加え、60℃で5時間攪拌してアルカリ加水分解させた。6mol/Lの塩酸でｐH6.5に調整し、酵素分解過程においてｐH値をこの範囲に保持し、46.5g（1.5%）の酵素活性5.0×104u/gの枯草菌プロテイナーゼを加え、50℃で5時間攪拌して酵素分解させた。その後、急速に30℃以下まで冷却し、30分間保持してから反応を終止した。0.5mol/Lの水酸化カリウムでｐHを中性に調整した。200目スクリーンでろ過し、得られたろ過液を真空濃縮設備に入れ、真空度95ｋPa、温度40℃に制御して1リットルになるまで濃縮した。攪拌しながら4リットルの95%アルコールをゆっくり加え、5℃で15時間アルコール沈殿し、多糖類沈殿を遠心により収集して真空冷凍乾燥設備に入れ、真空度85Pa、板温度45℃に制御して8時間冷凍乾燥することにより7.9ｇの帆立貝多糖類干し粉を得た。フェノール−硫酸法で測定を行ったところ、多糖類含有量は12.3%であった。 Fresh scallops with shells were washed with water, drained, and the shells were removed by cutting with a blade. Vacuum-freeze-dry until the water content was 4% or less, crush to 180, and obtain dried powder. 3 kg of water was added to 100 g of the internal dried powder, homogenized with a tissue crusher, and the serum was sonicated (40 ° C., 200 W) for 0.8 hour. 3.1 g of crushed solid potassium carbonate was added, and the mixture was stirred at 60 ° C. for 5 hours for alkali hydrolysis. Adjust the pH to 6.5 with 6 mol / L hydrochloric acid, maintain the pH value in this range during the enzymatic degradation process, add 46.5 g (1.5%) enzyme activity 5.0 × 10 4 u / g Bacillus subtilis proteinase, The mixture was stirred at 5 ° C. for 5 hours for enzymatic degradation. Thereafter, the reaction was rapidly cooled to 30 ° C. or lower, held for 30 minutes, and the reaction was terminated. The pH was adjusted to neutral with 0.5 mol / L potassium hydroxide. The mixture was filtered through a 200-mesh screen, and the resulting filtrate was put into a vacuum concentration facility, and concentrated to 1 liter by controlling the degree of vacuum at 95 kPa and the temperature at 40 ° C. Slowly add 4 liters of 95% alcohol with stirring, precipitate with alcohol at 5 ° C for 15 hours, collect the polysaccharide precipitate by centrifugation, put into vacuum freeze-drying equipment, and control the vacuum to 85Pa and plate temperature to 45 ° C. By freeze-drying for 8 hours, 7.9 g of scallop polysaccharide dried powder was obtained. When measured by the phenol-sulfuric acid method, the polysaccharide content was 12.3%.
新鮮な殻付き帆立貝を水洗して水を切り、殻を刃物で割って取り除き、異なる部位単位で全部組織を取り出した。1kgの内臓に3kgの水を加え、組織破砕機でホモジネートし、漿液を0.6時間超音波処理（45℃、400W）した。4g（0.1%）の破砕の固体炭酸ナトリウムを加え、30℃で5時間攪拌してアルカリ加水分解させた。6mol/Lの塩酸でｐH4に調整し、48g（1.2%）の酵素活性7.8×105u/gのペプシンを加え、ｐH値をこの範囲に保持し、45℃で2時間攪拌して酵素分解させた。0.5mol/Lの水酸化カリウムでｐH8に調整し、20g（0.5%）の酵素活性5×104u/gのトリプシンを加え、ｐH 値をこの範囲に保持し、50℃で2時間攪拌して酵素分解させた。その後、急速に30℃以下まで冷却し、30分間保持してから反応を終止した。6mol/Lの塩酸でｐHを中性に調整した。200目スクリーンでろ過し、得られたろ過液を真空濃縮設備に入れ、真空度95ｋPa、温度45℃に制御して1リットルになるまで濃縮した。攪拌しながら3リットルの95%アルコールをゆっくり加え、5℃で16時間アルコール沈殿し、多糖類沈殿を遠心により収集して直接40℃で火干しすることにより26.3ｇの帆立貝多糖類干し粉を得た。フェノール−硫酸法で測定を行ったところ、多糖類含有量は11.0%であった。 Fresh scallops with shells were washed with water, drained, and the shells were removed by cutting with a blade. 3 kg of water was added to 1 kg of the internal organs, homogenized with a tissue crusher, and the serum was sonicated (45 ° C., 400 W) for 0.6 hours. 4 g (0.1%) of crushed solid sodium carbonate was added, and the mixture was stirred at 30 ° C. for 5 hours for alkaline hydrolysis. Adjust to pH 4 with 6 mol / L hydrochloric acid, add 48 g (1.2%) of enzyme activity 7.8 × 10 5 u / g pepsin, keep the pH value within this range, and stir at 45 ° C. for 2 hours for enzymatic degradation I let you. Adjust to pH 8 with 0.5 mol / L potassium hydroxide, add 20 g (0.5%) of enzyme activity 5 × 10 4 u / g trypsin, keep the pH value within this range, and stir at 50 ° C. for 2 hours. The enzyme was decomposed. Thereafter, the reaction was rapidly cooled to 30 ° C. or lower, held for 30 minutes, and the reaction was terminated. The pH was adjusted to neutral with 6 mol / L hydrochloric acid. The mixture was filtered through a 200-mesh screen, and the resulting filtrate was put into a vacuum concentration facility, and concentrated to 1 liter by controlling the degree of vacuum at 95 kPa and the temperature at 45 ° C. Slowly add 3 liters of 95% alcohol with stirring, precipitate with alcohol at 5 ° C for 16 hours, collect the polysaccharide precipitate by centrifugation and directly dry at 40 ° C to obtain 26.3 g of scallop polysaccharide dried powder It was. When measured by the phenol-sulfuric acid method, the polysaccharide content was 11.0%.
新鮮な殻付き帆立貝を水洗して水を切り、殻を刃物で割って取り除き、異なる部位単位で全部組織を取り出した。1kgの帆立貝の全部組織に4kgの水を加え、組織破砕機でホモジネートし、漿液を0.8時間超音波処理（40℃、500W）した。50g（1.0%）の破砕の固体炭酸ナトリウムを加え、60℃で1時間攪拌してアルカリ加水分解させた。6mol/Lの塩酸でｐH6.7に調整し、10g（0.2%）の酵素活性5.0×104u/gの枯草菌プロテイナーゼを加え、ｐH値をこの範囲に保持し、50℃で2時間攪拌して酵素分解させた。6mol/Lの塩酸でｐH4に調整し、15g（0.3%）の酵素活性7.8×105u/gのペプシンを加え、ｐH値をこの範囲に保持し、50℃で3時間攪拌して酵素分解させた。その後、急速に30℃以下まで冷却し、30分間保持してから反応を終止した。0.5mol/Lの水酸化カリウムでｐHを中性に調整した。6000rpmで20分間遠心して上清液を収集して真空濃縮設備に入れ、真空度90ｋPa 、温度40℃に制御して1リットルになるまで濃縮した。攪拌しながら3リットルの95%アルコールをゆっくり加え、4℃で12時間アルコール沈殿し、多糖類沈殿を遠心により収集して直接30℃で火干しすることにより24.1gの帆立貝多糖類干し粉を得た。フェノール−硫酸法で測定を行ったところ、多糖類含有量は15.7%であった。 Fresh scallops with shells were washed with water, drained, and the shells were removed by cutting with a blade. 4 kg of water was added to all tissues of 1 kg of scallops, homogenized with a tissue crusher, and the serum was sonicated (40 ° C., 500 W) for 0.8 hours. 50 g (1.0%) of crushed solid sodium carbonate was added and stirred at 60 ° C. for 1 hour for alkali hydrolysis. Adjust pH to 6.7 with 6 mol / L hydrochloric acid, add 10 g (0.2%) of enzyme activity 5.0 × 10 4 u / g Bacillus subtilis proteinase, keep the pH value within this range, and stir at 50 ° C. for 2 hours And then enzymatically decomposed. Adjust to pH 4 with 6 mol / L hydrochloric acid, add 15 g (0.3%) of enzyme activity 7.8 × 10 5 u / g of pepsin, keep the pH value within this range, and stir at 50 ° C. for 3 hours for enzymatic degradation I let you. Thereafter, the reaction was rapidly cooled to 30 ° C. or lower, held for 30 minutes, and the reaction was terminated. The pH was adjusted to neutral with 0.5 mol / L potassium hydroxide. After centrifuging at 6000 rpm for 20 minutes, the supernatant was collected and placed in a vacuum concentration facility, and concentrated to 1 liter by controlling the degree of vacuum at 90 kPa and the temperature at 40 ° C. Slowly add 3 liters of 95% alcohol with stirring, precipitate with alcohol at 4 ° C for 12 hours, collect the polysaccharide precipitate by centrifugation and directly dry at 30 ° C to obtain 24.1 g of scallop polysaccharide dried powder It was. When measured by the phenol-sulfuric acid method, the polysaccharide content was 15.7%.
新鮮な殻付き帆立貝を水洗して水を切り、殻を刃物で割って取り除き、異なる部位単位で全部組織を取り出した。1kgの帆立貝の全部組織に2kgの水を加え、組織破砕機でホモジネートし、漿液を0.3時間超音波処理（60℃、600W）した。60分間紫外線照射し、60℃で2時間加水分解させた。6mol/Lの塩酸でｐH6に調整し、1.5g（0.05%）の酵素活性5.0×104u/gの枯草菌プロテイナーゼを加え、ｐH値をこの範囲に保持し、45℃で3時間攪拌して酵素分解させた。6mol/Lの塩酸でｐH1に調整し、30g（1.0%）の酵素活性7.8×105u/gのペプシンを加え、ｐH値をこの範囲に保持し、40℃で4時間攪拌して酵素分解させた。その後、急速に30℃以下まで冷却し、30分間保持してから反応を終止した。0.5mol/Lの水酸化カリウムでｐHを中性に調整した。6000rpmで20分間遠心して上清液を収集して真空濃縮設備に入れ、真空度85ｋPa 、温度30℃に制御して1リットルになるまで濃縮した。攪拌しながら2リットルの95%アルコールをゆっくり加え、2℃で2時間アルコール沈殿し、多糖類沈殿を遠心により収集して直接20℃で火干しすることにより25.6ｇの帆立貝多糖類干し粉を得た。フェノール−硫酸法で測定を行ったところ、多糖類含有量は17.4%であった。 Fresh scallops with shells were washed with water, drained, and the shells were removed by cutting with a blade. 2 kg of water was added to the entire tissue of 1 kg of scallops, homogenized with a tissue crusher, and the serum was sonicated for 0.3 hours (60 ° C., 600 W). It was irradiated with ultraviolet rays for 60 minutes and hydrolyzed at 60 ° C. for 2 hours. Adjust to pH 6 with 6 mol / L hydrochloric acid, add 1.5 g (0.05%) of enzyme activity 5.0 × 10 4 u / g Bacillus subtilis proteinase, keep the pH value within this range, and stir at 45 ° C. for 3 hours The enzyme was decomposed. Adjust to pH 1 with 6 mol / L hydrochloric acid, add 30 g (1.0%) of enzyme activity 7.8 × 10 5 u / g of pepsin, keep the pH value within this range, and stir at 40 ° C. for 4 hours for enzymatic degradation I let you. Thereafter, the reaction was rapidly cooled to 30 ° C. or lower, held for 30 minutes, and the reaction was terminated. The pH was adjusted to neutral with 0.5 mol / L potassium hydroxide. After centrifuging at 6000 rpm for 20 minutes, the supernatant was collected and placed in a vacuum concentration facility, and concentrated to 1 liter by controlling the degree of vacuum at 85 kPa and the temperature at 30 ° C. Slowly add 2 liters of 95% alcohol with stirring, precipitate with alcohol at 2 ° C for 2 hours, collect the polysaccharide precipitate by centrifugation and directly dry at 20 ° C to obtain 25.6 g of scallop polysaccharide dried powder It was. When measured by the phenol-sulfuric acid method, the polysaccharide content was 17.4%.
新鮮な殻付き帆立貝を水洗して水を切り、殻を刃物で割って取り除き、異なる部位単位で全部組織を取り出した。1kgの内臓に3 kgの水を加え、組織破砕機でホモジネートし、漿液を0.6時間超音波処理（45℃、400W）した。10分間紫外線照射し、40℃で6時間加水分解させた。6mol/Lの塩酸でｐH4に調整し、48g（1.2%）の酵素活性7.8×105u/gのペプシンを加え、ｐH値をこの範囲に保持し、45℃で2時間攪拌して酵素分解させた。0.5mol/Lの水酸化カリウムでｐH8に調整し、20g（0.5%）の酵素活性5×104u/gのトリプシンを加え、ｐH 値をこの範囲に保持し、50℃で2時間攪拌して酵素分解させた。その後、急速に30℃以下まで冷却し、30分間保持してから反応を終止した。6mol/Lの塩酸でｐHを中性に調整した。200目スクリーンでろ過し、得られたろ過液を真空濃縮設備に入れ、真空度95ｋPa、温度45℃に制御して1リットルになるまで濃縮した。攪拌しながら3リットルの95%アルコールをゆっくり加え、5℃で16時間アルコール沈殿し、多糖類沈殿を遠心により収集して直接40℃で火干しすることにより26.3gの帆立貝多糖類干し粉を得た。フェノール−硫酸法で測定を行ったところ、多糖類含有量は11.0%であった。 Fresh scallops with shells were washed with water, drained, and the shells were removed by cutting with a blade. 3 kg of water was added to 1 kg of the internal organs, homogenized with a tissue crusher, and the serum was sonicated (45 ° C., 400 W) for 0.6 hours. It was irradiated with ultraviolet rays for 10 minutes and hydrolyzed at 40 ° C. for 6 hours. Adjust to pH 4 with 6 mol / L hydrochloric acid, add 48 g (1.2%) of enzyme activity 7.8 × 10 5 u / g pepsin, keep the pH value within this range, and stir at 45 ° C. for 2 hours for enzymatic degradation I let you. Adjust to pH 8 with 0.5 mol / L potassium hydroxide, add 20 g (0.5%) of enzyme activity 5 × 10 4 u / g trypsin, keep the pH value within this range, and stir at 50 ° C. for 2 hours. The enzyme was decomposed. Thereafter, the reaction was rapidly cooled to 30 ° C. or lower, held for 30 minutes, and the reaction was terminated. The pH was adjusted to neutral with 6 mol / L hydrochloric acid. The mixture was filtered through a 200-mesh screen, and the resulting filtrate was put into a vacuum concentration facility, and concentrated to 1 liter by controlling the degree of vacuum at 95 kPa and the temperature at 45 ° C. While stirring, slowly add 3 liters of 95% alcohol, precipitate with alcohol at 5 ° C for 16 hours, collect the polysaccharide precipitate by centrifugation and directly dry at 40 ° C to obtain 26.3 g of scallop polysaccharide dried powder It was. When measured by the phenol-sulfuric acid method, the polysaccharide content was 11.0%.
新鮮な殻付き帆立貝を水洗して水を切り、殻を刃物で割って取り除き、異なる部位単位で全部組織を取り出した。水分含有量が4%以下になるまで50℃で火干しし、80目まで破砕して干し粉を得た。超臨界抽出設備を開け、抽出温度を30℃で設定して温度が上昇するのを待っている間に、抽出釜に30gの原料を注入した。抽出温度が設定値に達した後、抽出圧力20MPa、CO2流量40L/hに制御し、帆立貝内臓脂肪を超臨界抽出した。120分後に、分離温度30℃、分離圧力6MPaで脂肪を分離して収集した。脱脂された帆立貝内臓組織を抽出釜から取り出して、使用に備えた。 Fresh scallops with shells were washed with water, drained, and the shells were removed by cutting with a blade. It was fire-dried at 50 ° C. until the water content was 4% or less, and crushed to 80 eyes to obtain dried powder. The supercritical extraction equipment was opened and the extraction temperature was set at 30 ° C. While waiting for the temperature to rise, 30 g of raw material was injected into the extraction kettle. After the extraction temperature reached the set value, the extraction pressure was controlled to 20 MPa and the CO 2 flow rate was 40 L / h to supercritically extract scallop visceral fat. After 120 minutes, fat was separated and collected at a separation temperature of 30 ° C. and a separation pressure of 6 MPa. The degreased scallop visceral tissue was removed from the extraction kettle and prepared for use.
新鮮な殻付き帆立貝を水洗して水を切り、殻を刃物で割って取り除き、異なる部位単位で全部組織を取り出した。水分含有量が4%以下になるまで40℃で火干しし、120目まで破砕して干し粉を得た。超臨界抽出設備を開け、抽出温度を70℃で設定して温度が上昇するのを待っている間に、抽出釜に30gの原料を注入した。抽出温度が設定値に達した後、抽出圧力40MPa、CO2流量10L/hに制御して、帆立貝内臓脂肪を超臨界抽出した。45分後に、分離温度40℃、分離圧力10MPaで脂肪を分離して収集した。脱脂された帆立貝内臓組織を抽出釜から取り出して、使用に備えた。 Fresh scallops with shells were washed with water, drained, and the shells were removed by cutting with a blade. Fire-dried at 40 ° C. until the water content was 4% or less, and crushed to 120 eyes to obtain dried powder. The supercritical extraction equipment was opened, and the extraction temperature was set at 70 ° C. While waiting for the temperature to rise, 30 g of raw material was injected into the extraction kettle. After the extraction temperature reached the set value, scallop visceral fat was supercritically extracted by controlling the extraction pressure to 40 MPa and the CO 2 flow rate to 10 L / h. After 45 minutes, fat was separated and collected at a separation temperature of 40 ° C. and a separation pressure of 10 MPa. The degreased scallop visceral tissue was removed from the extraction kettle and prepared for use.
新鮮な殻付き帆立貝を水洗して水を切り、殻を刃物で割って取り除き、異なる部位単位で全部組織を取り出した。水分含有量が4%以下になるまで60℃で火干しし、200目まで破砕して干し粉を得た。超臨界抽出設備を開け、抽出温度を30℃で設定して温度上昇するのを待っている間に、抽出釜に30gの原料を注入した。抽出温度が設定値に達した後、抽出圧力25MPa、CO2流量20L/hに制御して、帆立貝内臓脂肪を超臨界抽出した。60分後に、分離温度30℃、分離圧力7MPaで脂肪を分離して収集した。脱脂された帆立貝内臓組織を抽出釜から取り出して、使用に備えた。 Fresh scallops with shells were washed with water, drained, and the shells were removed by cutting with a blade. Dried at 60 ° C. until the water content was 4% or less, and crushed to 200 eyes to obtain dried powder. The supercritical extraction equipment was opened, and while setting the extraction temperature at 30 ° C and waiting for the temperature to rise, 30 g of raw material was injected into the extraction kettle. After the extraction temperature reached the set value, scallop visceral fat was supercritically extracted by controlling the extraction pressure to 25 MPa and the CO 2 flow rate to 20 L / h. After 60 minutes, fat was separated and collected at a separation temperature of 30 ° C. and a separation pressure of 7 MPa. The degreased scallop visceral tissue was removed from the extraction kettle and prepared for use.
実施例19〜21のいずれか一例の100gの脱脂帆立内臓干し粉に2kgの水を加え、組織破砕機でホモジネートし、漿液を0.5時間超音波処理（40℃、200W）した。6mol/Lの塩酸でｐH7に調整し、酵素分解過程にはｐHをこの範囲に保持し、42g（2.0%）の酵素活性5.0×104u/gの枯草菌プロテイナーゼを加え、55℃で1時間攪拌して酵素分解させた。その後、急速に30℃以下まで冷却し、30分間保持してから反応を終止した。0.5mol/Lの水酸化カリウムでｐHを中性に調整した。200目スクリーンでろ過し、得られたろ過液を真空濃縮設備に入れ、真空度90ｋPa、温度50℃に制御して0.5リットルになるまで濃縮した。攪拌しながら1.5リットルの95%アルコールをゆっくり加え、5℃で15時間アルコール沈殿し、多糖類沈殿を遠心により収集して真空冷凍乾燥設備に入れ、真空度90Pa、板温度50℃に制御して10時間冷凍乾燥することにより7.4gの帆立貝多糖類干し粉を得た。フェノール−硫酸法で測定を行ったところ、多糖類含有量は12.8%であった。 2 kg of water was added to 100 g of the defatted scallop visceral dried powder of any one of Examples 19 to 21, homogenized with a tissue crusher, and the serum was sonicated (40 ° C., 200 W) for 0.5 hour. The pH was adjusted to 7 with 6 mol / L hydrochloric acid. During the enzymatic degradation process, pH was maintained within this range, and 42 g (2.0%) of enzyme activity 5.0 × 10 4 u / g of Bacillus subtilis proteinase was added. The mixture was stirred for hours to allow enzymatic degradation. Thereafter, the reaction was rapidly cooled to 30 ° C. or lower, held for 30 minutes, and the reaction was terminated. The pH was adjusted to neutral with 0.5 mol / L potassium hydroxide. The mixture was filtered through a 200-mesh screen, and the obtained filtrate was put into a vacuum concentration facility, and concentrated to 0.5 liters while controlling the degree of vacuum at 90 kPa and the temperature at 50 ° C. Slowly add 1.5 liters of 95% alcohol while stirring, precipitate with alcohol at 5 ° C for 15 hours, collect the polysaccharide precipitate by centrifugation, put into vacuum freeze-drying equipment, control the vacuum degree to 90Pa and the plate temperature to 50 ° C. 7.4 g of scallop polysaccharide dried powder was obtained by freeze-drying for 10 hours. When measured by the phenol-sulfuric acid method, the polysaccharide content was 12.8%.
実施例22で得られた粗多糖類を2%の溶液に調製し、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0.5%以下になるまで上記脱蛋白のステップを数回繰り返した。溶液に4.5倍体積の95%アルコールを加えて一夜アルコール沈殿させた。高速遠心して沈殿を取り出して3%の多糖類液に調製し、分画分子量7000Da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純多糖類を得た。次に、Sephadex G-200カラム・クロマトグラフィで分離した。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、唯一の溶出ピークが検出され、収集・混合・透析・濃縮・冷凍乾燥することにより精製多糖類を得た。精製された多糖類をSephroce 6Bゲルカラム法で分離した。溶離剤は0.9%のNaClで、溶出速度は0.25mL/minであった。フェノール−硫酸法で追跡検出し、溶出ピーク試験管数を測定して標準グラフに照らし合わせることにより当該多糖類分子量は6〜8万であることが分かった。完全に乾燥した精製多糖類10mgに無水HCl-メチルアルコールを2mL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させてから取り出し、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分間溶解させてから、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、当該多糖類はペクチンシュガー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（US Agilent 6890N）、HP-1クロマトグラフカラム、固定相メチルシロキサン（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。 The crude polysaccharide obtained in Example 22 was prepared as a 2% solution, 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 step was repeated several times until the protein content in the polysaccharide solution was 0.5% or less. 4.5 times volume of 95% alcohol was added to the solution to allow alcohol precipitation overnight. The precipitate is taken out by high-speed centrifugation and prepared into a 3% polysaccharide solution, dialyzed for 48 hours in a dialysis bag with a molecular weight cut off of 7000 Da, and the solution in the dialysis bag is concentrated and freeze-dried to concentrate the pure polysaccharide Obtained. Next, it was separated by Sephadex G-200 column chromatography. The eluent was 0.9% NaCl and the elution rate was 0.5 mL / min. Following detection by the phenol-sulfuric acid method, the only elution peak was detected, and purified polysaccharide was obtained by collection, mixing, dialysis, concentration, and freeze-drying. The purified polysaccharide was separated by Sephroce 6B gel column method. The eluent was 0.9% NaCl and the elution rate was 0.25 mL / min. The polysaccharide molecular weight was found to be 60,000 to 80,000 by tracking and detecting with the phenol-sulfuric acid method, measuring the number of elution peak test tubes and comparing them with a standard graph. To 10 mg of completely dried purified polysaccharide, 2 mL of anhydrous HCl-methyl alcohol was added, and N 2 was put in a sealed tube. After 20 hours of methyl alcohol decomposition at 80 ° C., the solution was taken out and left to reach room temperature. Neutralized to pH = 6 with anhydrous KOH-methyl alcohol, dried under reduced pressure at 40 ° C. and dried. Add 0.2 mL of anhydrous pyridine to the completely dried methyl alcohol decomposition product, dissolve at 75 ° C for 30 minutes, add 0.3 mL of silylating agent, shake evenly, leave it for a few minutes, and remove the supernatant. It was taken out and subjected to color layer analysis (gas chromatograph), and the polysaccharide was found to be a composition from pectin sugar. The chromatographic conditions were gas chromatograph (US Agilent 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.
実施例22で得られた粗多糖類を10%の溶液に調製し、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0.5%以下になるまで上記脱蛋白のステップを数回繰り返した。溶液に4.5倍体積の95%アルコールを加えて一夜アルコール沈殿させた。高速遠心して沈殿を取り出して5%の多糖類液に調製し、分画分子量7000Da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純多糖類を得た。次は、Sephadex G-200カラム・クロマトグラフィで分離させた。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、唯一の溶出ピークが検出され、収集・混合・透析・濃縮・冷凍乾燥することにより精製多糖類を得た。精製された多糖類をセファロース（Sephroce）6Bゲルカラム法で分離させた。溶離剤は0.9%のNaClで、溶出速度は0.25mL/minであった。フェノール−硫酸法で追跡検出し、溶出ピーク試験管数を測定して標準グラフに照らし合わせることにより当該多糖類分子量は6〜8万であることが分かった。完全に乾燥した精製多糖類10mgに無水HCl-メチルアルコールを2mL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させてから取り出し、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分間溶解させてから、0.3mL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、当該多糖類はペクチンシュガーからの組成と分かった。クロマトグラフ条件は、実施例23と同じであった。 The crude polysaccharide obtained in Example 22 was prepared into a 10% solution, 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 step was repeated several times until the protein content in the polysaccharide solution was 0.5% or less. 4.5 times volume of 95% alcohol was added to the solution to allow alcohol precipitation overnight. The precipitate is taken out by high-speed centrifugation and prepared into a 5% polysaccharide solution, dialyzed for 72 hours in a dialysis bag with a molecular weight cut off of 7000 Da, and the solution in the dialysis bag is concentrated and freeze-dried. Obtained. Next, separation was performed by Sephadex G-200 column chromatography. The eluent was 0.9% NaCl and the elution rate was 0.5 mL / min. Following detection by the phenol-sulfuric acid method, the only elution peak was detected, and purified polysaccharide was obtained by collection, mixing, dialysis, concentration, and freeze-drying. The purified polysaccharide was separated by Sepharose 6B gel column method. The eluent was 0.9% NaCl and the elution rate was 0.25 mL / min. The polysaccharide molecular weight was found to be 60,000 to 80,000 by tracking and detecting with the phenol-sulfuric acid method, measuring the number of elution peak test tubes and comparing them with a standard graph. To 10 mg of completely dried purified polysaccharide, 2 mL of anhydrous HCl-methyl alcohol was added, and N 2 was put in a sealed tube. After 20 hours of methyl alcohol decomposition at 80 ° C., the solution was taken out and left to reach room temperature. The mixture was neutralized with anhydrous KOH-methyl alcohol to pH = 6, dried under reduced pressure at 40 ° C. 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 silylating agent, shake evenly, leave for several minutes and remove the supernatant. A color layer analysis (gas chromatograph) was conducted, and the polysaccharide was found to be a composition from pectin sugar. The chromatographic conditions were the same as in Example 23.
実施例22から得られた粗多糖類を2%の溶液に調製し、溶液重量の0.1%のペプシンを加え、6mol/LのHClでpH3.0に調整し、37℃で6時間酵素分解させてから急速に30℃以下まで冷却し、30分間保持して反応を終止した。室温にNaOHで溶液を中性に調整した。10分間遠心して上清液を取り出して3倍体積の95%アルコールを加え、2℃で12時間ぐらいアルコール沈殿させた。遠心して沈殿を取り出して2%の溶液を調製し、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分間静置した。遠心して沈殿を取り除き、上清液を取り出してフォリン−フェノール法で多糖類溶液中の蛋白含有量0.5%以下と検知されるまで以上の脱蛋白のステップを数回繰り返した。溶液に4.5倍体積の95%アルコールを加えて一夜アルコール沈殿させた。高速遠心して沈殿を取り出して2%多糖類液に調製し、分画分子量7000Da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純多糖類を得た。次は、Sephadex G-200カラム・クロマトグラフィで分離させ、溶離剤は0.9%のNaClで、溶出速度は0.5mL/minで、フェノール−硫酸法で追跡検出し、唯一の溶出ピークが検出され、収集・混合・透析・濃縮・冷凍乾燥することにより精製多糖類を得た。精製された多糖類をSephroce 6Bゲルカラム法で分離させ、溶離剤は0.9%のNaClで、溶出速度は0.25mL/minで、フェノール−硫酸法で追跡検出し、溶出ピーク試験管数を測定して標準フラフに照らし合わせることにより当該多糖類分子量は6〜8万であるとが分かった。完全に乾燥した精製多糖類10mgに無水HCl-メチルアルコールを2mL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させてから取り出し、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に中和し、40℃で減圧回転蒸し干してから乾燥した。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分間溶解させてから、0.3mL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、当該多糖類はペクチンシュガーからの組成と分かった。クロマトグラフ条件は実施例23と同じであった。 The crude polysaccharide obtained from Example 22 was prepared in a 2% solution, 0.1% pepsin of the solution weight was added, pH was adjusted to 3.0 with 6 mol / L HCl, and enzymatic degradation was performed at 37 ° C for 6 hours. Thereafter, the reaction was rapidly cooled to below 30 ° C. and held for 30 minutes to terminate the reaction. The solution was adjusted to neutral with NaOH to room temperature. The supernatant was removed by centrifugation for 10 minutes, 3 volumes of 95% alcohol was added, and alcohol precipitation was carried out at 2 ° C. for about 12 hours. Centrifuge to remove the precipitate to prepare a 2% solution, add Sevag reagent (V chloroform: Vn-butanol = 5: 1) at a volume ratio of 5: 1, shake vigorously for 20 minutes, and let stand for 30 minutes . The precipitate was removed by centrifugation, the supernatant was taken out, and the above deproteinization step was repeated several times until the protein content in the polysaccharide solution was detected to be 0.5% or less by the folin-phenol method. 4.5 times volume of 95% alcohol was added to the solution to allow alcohol precipitation overnight. The precipitate is taken out by high-speed centrifugation and prepared into a 2% polysaccharide solution, dialyzed for 48 hours in a dialysis bag with a molecular weight cut off of 7000 Da, and then the solution in the dialysis bag is concentrated and freeze-dried to obtain a pure polysaccharide. It was. Next, separation by Sephadex G-200 column chromatography, eluent is 0.9% NaCl, elution rate is 0.5 mL / min, follow-up detection with phenol-sulfuric acid method, only elution peak is detected and collected -Purified polysaccharide was obtained by mixing, dialysis, concentration, and freeze-drying. The purified polysaccharide was separated by the Sephroce 6B gel column method, the eluent was 0.9% NaCl, the elution rate was 0.25 mL / min, followed by the phenol-sulfuric acid method, and the number of elution peak test tubes was measured. The polysaccharide molecular weight was found to be 60,000 to 80,000 by comparing with a standard fluff. To 10 mg of completely dried purified polysaccharide, 2 mL of anhydrous HCl-methyl alcohol was added, and N 2 was put in a sealed tube. After 20 hours of methyl alcohol decomposition at 80 ° C., the solution was taken out and left to reach 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 silylating agent, shake evenly, leave for several minutes and remove the supernatant. A color layer analysis (gas chromatograph) was conducted, and the polysaccharide was found to be a composition from pectin sugar. The chromatographic conditions were the same as in Example 23.
実施例22で得られた粗多糖類を10%の溶液に調製し、溶液重量の1%のペプシンを加え、6mol/LのHClでpH1.5に調整し、37℃で1時間酵素分解させてから急速に30℃以下まで冷却し、30分間保持して反応を終止した。室温にNaOHで溶液を中性に調整した。10分間遠心して上清液を取り出して4.5倍体積の95%アルコールを加え、4℃で16時間ぐらいアルコール沈殿させた。遠心して沈殿を取り出して5%の溶液を調製し、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分間静置した。遠心して沈殿を取り除き、上清液を取り出してフォリン−フェノール法で多糖類溶液中の蛋白含有量0.5%以下と検知されるまで以上の脱蛋白のステップを数回繰り返した。溶液に4.5倍体積の95%アルコールを加えて一夜アルコール沈殿させた。高速遠心して沈殿を取り出して5%の多糖類液に調製し、分画分子量7000Da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純多糖類を得た。次は、Sephadex G-200カラム・クロマトグラフで分離させ、溶離剤は0.9%のNaClで、溶出速度は0.5mL/minで、フェノール−硫酸法で追跡検出し、唯一の溶出ピークが検出され、収集・混合・透析・濃縮・冷凍乾燥することにより精製多糖類を得た。精製された多糖類をSephroce 6Bゲルカラム法で分離させ、溶離剤は0.9%のNaClで、溶出速度は0.25mL/minで、フェノール−硫酸法で追跡検出し、溶出ピーク試験管数を測定して標準グラフに照らし合わせることにより当該多糖類分子量は6〜8万であることが分かった。完全に乾燥した精製多糖類10mgに無水HCl-メチルアルコールを2mL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させてから取り出し、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分間溶解させてから、0.3mL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、当該多糖類はペクチンシュガーからの組成と分かった。クロマトグラフ条件は実施例23と同じであった。 Prepare the crude polysaccharide obtained in Example 22 in a 10% solution, add 1% pepsin of the solution weight, adjust to pH 1.5 with 6 mol / L HCl, and perform enzymatic degradation at 37 ° C for 1 hour. Thereafter, the reaction was rapidly cooled to below 30 ° C. and held for 30 minutes to terminate the reaction. The solution was adjusted to neutral with NaOH to room temperature. The supernatant was removed by centrifugation for 10 minutes, and 4.5 times volume of 95% alcohol was added, and alcohol precipitation was performed at 4 ° C. for about 16 hours. Centrifuge to remove the precipitate to prepare a 5% solution, add Sevag reagent (V chloroform: Vn-butanol = 5: 1) at a volume ratio of 5: 1, shake vigorously for 20 minutes, and let stand for 30 minutes . The precipitate was removed by centrifugation, the supernatant was taken out, and the above deproteinization step was repeated several times until the protein content in the polysaccharide solution was detected to be 0.5% or less by the folin-phenol method. 4.5 times volume of 95% alcohol was added to the solution to allow alcohol precipitation overnight. The precipitate is taken out by high-speed centrifugation and prepared into a 5% polysaccharide solution, dialyzed for 72 hours in a dialysis bag with a molecular weight cut off of 7000 Da, and the solution in the dialysis bag is concentrated and freeze-dried. Obtained. Next, separation with Sephadex G-200 column chromatograph, eluent is 0.9% NaCl, elution rate is 0.5 mL / min, follow-up detection with phenol-sulfuric acid method, only elution peak is detected, The purified polysaccharide was obtained by collection, mixing, dialysis, concentration, and freeze-drying. The purified polysaccharide was separated by the Sephroce 6B gel column method, the eluent was 0.9% NaCl, the elution rate was 0.25 mL / min, followed by the phenol-sulfuric acid method, and the number of elution peak test tubes was measured. The polysaccharide molecular weight was found to be 60,000 to 80,000 by comparing with a standard graph. To 10 mg of completely dried purified polysaccharide, 2 mL of anhydrous HCl-methyl alcohol was added, and N 2 was put in a sealed tube. After 20 hours of methyl alcohol decomposition at 80 ° C., the solution was taken out and left to reach room temperature. The mixture was neutralized with anhydrous KOH-methyl alcohol to pH = 6, dried under reduced pressure at 40 ° C. 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 silylating agent, shake evenly, leave for several minutes and remove the supernatant. A color layer analysis (gas chromatograph) was conducted, and the polysaccharide was found to be a composition from pectin sugar. The chromatographic conditions were the same as in Example 23.
Claims (17)
（2） 帆立貝肉組織の質量の0.5〜5倍の水を加え、前記処理ステップ（1）で得られた肉組織を組織破砕機でホモジネートして一様な漿液を得る、または干し粉の質量の10〜50倍の水を加え、前記処理ステップ（1）で得られた干し粉を組織破砕機でホモジネートして一様な漿液を得るホモジネートステップと、
（3） 作用温度が30〜60℃、出力が50〜600Wで、漿液を0.3〜1時間超音波処理する超音波処理ステップと、
（4） 超音波処理された漿液をトリプシン、ペプシン、中性プロテイナーゼのいずれか一つまたは複数を用いて酵素分解させる酵素分解ステップと、
（5） 帆立貝酵素分解液を遠心又はろ過し、上清液を収集して多糖類母液を得る分離ステップと、
（6） 得られた多糖類母液を真空濃縮設備に入れ、真空度85〜98ｋPa、温度30〜60℃に制御して元の体積の1/5〜1/3になるまで濃縮する濃縮ステップと、
（7） 多糖類母液を攪拌しながら、2〜5倍の95％アルコールをゆっくり加え、2〜6℃で2〜18時間アルコール沈殿し、多糖類沈殿を遠心により収集するアルコール沈殿ステップとを、含む、
帆立貝多糖類抽出方法。 (1) A processing step of washing fresh shelled scallops with water, draining the water, cutting and removing the shells with a blade, taking out the scallop shell tissue in different units, or further drying the scallop shell tissue to obtain dried powder. ,
(2) Add 0.5 to 5 times as much water as the scallop shell tissue, and homogenize the meat tissue obtained in the processing step (1) with a tissue crusher to obtain uniform serum, or the mass of dried powder 10 to 50 times as much water as above, and homogenate step to obtain uniform serum by homogenizing the dried powder obtained in the processing step (1) with a tissue crusher;
(3) an sonication step of sonicating the serum for 0.3 to 1 hour at an operating temperature of 30 to 60 ° C. and an output of 50 to 600 W;
(4) an enzymatic degradation step of enzymatically degrading the sonicated serum using any one or more of trypsin, pepsin, and neutral proteinase;
(5) A separation step of centrifuging or filtering the scallop enzymatic decomposition solution and collecting the supernatant to obtain a polysaccharide mother liquor;
(6) A concentration step in which the obtained polysaccharide mother liquor is placed in a vacuum concentration facility and concentrated to 1/5 to 1/3 of the original volume by controlling the degree of vacuum to 85 to 98 kPa and temperature of 30 to 60 ° C. ,
(7) While stirring the polysaccharide mother liquor, 2-5 times 95% alcohol is slowly added, alcohol precipitated at 2-6 ° C. for 2-18 hours, and the polysaccharide precipitation is collected by centrifugation, Including,
Scallop polysaccharide extraction method.
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| JP5313064B2 (en) * | 2009-07-03 | 2013-10-09 | 株式会社ノエビア | Neutral fat accumulation promoter, breast augmentation |
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| JP2005229990A (en) * | 2004-02-20 | 2005-09-02 | Aomori Prefecture | New protein and its gene |
| CN1273496C (en) * | 2004-06-07 | 2006-09-06 | 中国人民解放军第二军医大学 | Polysaccharose MF4 of mussel with enhancing immunity and anti-tumour activity |
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