CS274030B1 - Method of yeast /1-3/-beta-d-glucan purification - Google Patents
Method of yeast /1-3/-beta-d-glucan purification Download PDFInfo
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- CS274030B1 CS274030B1 CS3889A CS3889A CS274030B1 CS 274030 B1 CS274030 B1 CS 274030B1 CS 3889 A CS3889 A CS 3889A CS 3889 A CS3889 A CS 3889A CS 274030 B1 CS274030 B1 CS 274030B1
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- glucan
- beta
- alpha
- yeast
- purified
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- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 12
- 229920002498 Beta-glucan Polymers 0.000 title claims description 11
- 238000000034 method Methods 0.000 title claims description 4
- 238000000746 purification Methods 0.000 title abstract description 3
- 229920001503 Glucan Polymers 0.000 claims abstract description 19
- 108090000790 Enzymes Proteins 0.000 claims abstract description 12
- 102000004190 Enzymes Human genes 0.000 claims abstract description 12
- 239000004382 Amylase Substances 0.000 claims abstract description 3
- 108010065511 Amylases Proteins 0.000 claims abstract description 3
- 102000013142 Amylases Human genes 0.000 claims abstract description 3
- 235000019418 amylase Nutrition 0.000 claims abstract description 3
- 238000002360 preparation method Methods 0.000 claims description 10
- 150000004676 glycans Chemical class 0.000 claims description 9
- 229920001282 polysaccharide Polymers 0.000 claims description 9
- 239000005017 polysaccharide Substances 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 4
- 238000010521 absorption reaction Methods 0.000 claims description 4
- 230000003625 amylolytic effect Effects 0.000 claims description 4
- 238000002329 infrared spectrum Methods 0.000 claims description 4
- 239000008363 phosphate buffer Substances 0.000 claims description 4
- 239000011541 reaction mixture Substances 0.000 claims description 3
- 229920001353 Dextrin Polymers 0.000 claims description 2
- 239000004375 Dextrin Substances 0.000 claims description 2
- 239000007900 aqueous suspension Substances 0.000 claims description 2
- 235000019425 dextrin Nutrition 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 239000000725 suspension Substances 0.000 claims description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims 3
- 239000003344 environmental pollutant Substances 0.000 claims 2
- 231100000719 pollutant Toxicity 0.000 claims 2
- 108010021511 Aspergillus oryzae carboxyl proteinase Proteins 0.000 claims 1
- 206010011985 Decubitus ulcer Diseases 0.000 claims 1
- 208000004210 Pressure Ulcer Diseases 0.000 claims 1
- 208000025865 Ulcer Diseases 0.000 claims 1
- 239000002671 adjuvant Substances 0.000 claims 1
- 230000035876 healing Effects 0.000 claims 1
- 230000003308 immunostimulating effect Effects 0.000 claims 1
- 231100000397 ulcer Toxicity 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 4
- 108010001062 polysaccharide-K Proteins 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 9
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 8
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- FYGDTMLNYKFZSV-WFYNLLPOSA-N (2s,3r,4s,5s,6r)-2-[(2r,4r,5r,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,3s,4r,5r,6s)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](O[C@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-WFYNLLPOSA-N 0.000 description 3
- 108090000637 alpha-Amylases Proteins 0.000 description 3
- 102000004139 alpha-Amylases Human genes 0.000 description 3
- 229940024171 alpha-amylase Drugs 0.000 description 3
- 239000013049 sediment Substances 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 230000002519 immonomodulatory effect Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 108010019077 beta-Amylase Proteins 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
Description
Riešenie sá týká purifikácie kvasinkového (1—>3)-beta-D-glukánu, ktorý obsahuje aj nežiadúce alfa-D-glukány enzýmami s amylázovou aktivitou, pričom sa Specificky odstránia alfa-D-glukány bez atakovania a modifikácie připravovaného (1-*3)-beta-D-glukánu v purifikovanej formě.The solution relates to the purification of yeast (1-> 3) -beta-D-glucan, which also contains undesired alpha-D-glucans by enzymes with amylase activity, specifically removing alpha-D-glucans without attacking or modifying the prepared (1- *) 3) -beta-D-glucan in purified form.
cs 274030 Blcs 274030 Bl
Vynález sa týká sposobu purifikácie kvasinkového (1—>3)-beta-D-glukánu znečistěného alfa-D-glukánmi.The invention relates to a method of purifying yeast (1 → 3) -beta-D-glucan contaminated with alpha-D-glucans.
Kvasinkový (1—>3)-beta-D-glukán sa získává bud z nedezintegrovaných buniek / J. D. MANNERS, A. J. MASSON, J. C. PATTERSON, J. Gen. Microbiol. 80, 411 (1974); A. MISAKI,Yeast (1-> 3) -beta-D-glucan is obtained either from non-disintegrated cells / J. D. MANNERS, A. J. MASSON, J. C. PATTERSON, J. Gen. Microbiol. 80, 411 (1974); A. MISAKI,
J. JOHNSON, S. KIRKWOOD, J. V. SCALETTI, F. SMITH, Carbohydr. Res. 6, 150 (1968); CS 270103/ alebo z dezintegrovaných buniek /CS 243173/, ktoré sa viacnásobne extrahujú zriedenými alkáliami a zriedenými organickými i anorganickými kyselinami, připadne za použitia zmáčadiel. Získaný zvyšok představuje nerozpustný stěnový (1—»3)-beta-D-glukán kvasiniek.J. JOHNSON, S. KIRKWOOD, J.V. SCALETTI, F. SMITH, Carbohydr. Res. 6, 150 (1968); CS 270103) or from disintegrated cells (CS 243173) which are extracted several times with dilute alkali and dilute organic and inorganic acids, optionally using wetting agents. The residue obtained is an insoluble yeast (1 → 3) -beta-D-glucan.
Týmito sposobmi připravený beta-D-glukán máze v niektorých prípadoch - závislých od výběru kmeňa a spSsobu jeho kultivácie - obsahovat' alfa-D-glukány, ktoré v značnéj miere žnižujú jeho imunomodulačnú aktivitu.The beta-D-glucan prepared in this manner may, in some cases - depending on the strain selection and the way in which it is cultivated - contain alpha-D-glucans, which greatly reduce its immunomodulatory activity.
Zistilo sa, že taký polysacharidový preparát, ktorý okrem (1—^3)-beta-D-glukánu obsahuje aj nežiadúci alfa-D-glukán, je možné purifikovať sp&sobom podťa vynálezu. Podstata vynálezu spočívá v tom, že polysacharidový preparát sa tepelne upraví do homogenizovanej formy, ktorou je gél, event. v případe pólysacharidového preparátu, ktorý gél netvoří, vodná suspenzia, přidá sa k němu fosfořečňanový tlmivý roztok a amylolytický enzým. Zistilo sa, že ako amylolytický enzým sa máze použiť čistá alfa-amyláza, Specificky hydrolyzujúca (l->4)-alfa-D-glukány alebo zmes enzýmov (komerčně dostupné technické preparáty) súčastíou ktorej sú připadne enzýmy s beta-amylázovou aktivitou alebo s (1—>3)-alfa-D-glukozidázovou aktivitou. Amylolytický enzým sa pri teplote 25 až 50 *C nechá posobiť až do rozloženia nežiadúcich alfa-D-glukánových příměsí na rozpustné dextriny, ktoré sa odstránia filtráciou reakčnej zmesi a nerozpustný beta-D-glukán sa izoluje, premyje (vodou a organickým rozpúšťadlom) a vysuší. Přítomností, resp. nepřítomností znečistíujúcich polysacharidov (alfa-D-glukány) sa zisťuje IČ připadne ^C-NMR spektroskopiou.It has been found that such a polysaccharide preparation which, in addition to (1 -?) - beta-D-glucan, also contains the undesired alpha-D-glucan, can be purified according to the invention. The present invention is characterized in that the polysaccharide preparation is heat-treated into a homogenized form, which is a gel, optionally a gel. in the case of a non-gel-forming polysaccharide preparation, an aqueous suspension, phosphate buffer and an amylolytic enzyme are added thereto. It has been found that pure alpha-amylase, specifically hydrolyzing (1-> 4) -alpha-D-glucanes or a mixture of enzymes (commercially available technical preparations) may be used as an amylolytic enzyme, optionally including enzymes with beta-amylase activity or (1-> 3) -α-D-glucosidase activity. The amylolytic enzyme is maintained at 25 to 50 ° C until decomposition of undesirable alpha-D-glucan impurities into soluble dextrins, which are removed by filtration of the reaction mixture and the insoluble beta-D-glucan isolated, washed (water and organic solvent) and dried. Presence, respectively. in the absence of polluting polysaccharides (alpha-D-glucans), IR and @ 1 C-NMR spectroscopy were used.
Hlavnou výhodou postupu je špecifické odstránenie znečistí ujúcich alfa-D-glukánov bez atakovania a modifikácie připravovaného (1—»3)-beta-D-glukánu, ktorý v purifikovanej formě má vyššiu imunomodulačnú účinností ako povodný znečistěný (1—*3)-beta-D-glukán.The main advantage of the process is the specific removal of contaminating alpha-D-glucans without attacking and modifying the prepared (1 → 3) -beta-D-glucan, which in purified form has a higher immunomodulatory activity than the flooded (1 → 3) -beta -D-glucan.
Uvedené příklady ilustrujú, ale neobmedzujú predmet vynálezu.These examples illustrate but do not limit the scope of the invention.
Příklad 1Example 1
Získanie kvasinkového (1—>3)-beta-D-glukánuObtaining yeast (1-> 3) -beta-D-glucan
100 g pekárského droždia (Saccharomyces cerevisiae) sa suspenduje v 150 ml 6 % hmot. hydroxidu sodného a pri 60 C sa mieša 4 h. Suspenzia sa zriedi 100 ml 60 C teplej vody a odstředí. Sediment sa suspenduje v 150 ml 3 % hmot. hydroxidu sodného, mieša pri 80 až 100 *C 2 h a odstředí. Sediment sa trikrát premyje vodou a extrahuje trikrát 20 ml 4 S hmot. kyselinou chlorovodíkovou za miešania pri teplote miestnosti 2 h a odstředí. Sediment sa premýva vodou dovtedy, až supernatant je číry a reaguje neutrálně. Získaný stěnový glukán sa lyofilizuje. Výťažok 4,5 až 5,4 % hmot. počítané na sušinu kvasiniek. Glukán neobsahuje dusík a obsah popola je 0,3 až 0,6 % hmot. IČ spektrum obsahovalo okrem absorbčného pásu pri 890 cm“1 charakteristického pre beta-glykozidové vazby aj pásy pri 720 až 725 a 920 až 935 cm1 a v 1^C-NMR spektre boli signály s chemickým posunom 100,9 a 83,2 ppm charakteristické pre (l—>4)-alfa- príp. (1—>3)-alfa-D-glykozidové vazby.100 g of baker's yeast (Saccharomyces cerevisiae) is suspended in 150 ml of 6% by weight. sodium hydroxide and stirred at 60 ° C for 4 h. The suspension is diluted with 100 ml of 60 C warm water and centrifuged. The sediment is suspended in 150 ml of 3 wt. sodium hydroxide, stirred at 80-100 ° C for 2 h and centrifuged. The sediment is washed three times with water and extracted three times with 20 ml of 4 wt. hydrochloric acid with stirring at room temperature for 2 h and centrifuged. The sediment is washed with water until the supernatant is clear and neutral. The obtained wall glucan is lyophilized. Yield 4.5-5.4 wt. calculated on the dry matter of yeast. The glucan does not contain nitrogen and the ash content is 0.3 to 0.6% by weight. The IR spectrum contained, in addition to the absorption band at 890 cm -1 characteristic of beta-glycoside bonds, the bands at 720-725 and 920-935 cm -1 , and in the 1 C-NMR spectrum, chemical shift signals of 100.9 and 83.2 ppm were characteristic for (1-> 4) -alpha- or. (1-> 3) -alpha-D-glycoside bonds.
Příklad 2 g beta-D-glukánu z příkladu 1 sa zahrejú v 100 ml HjO na vriacom vodnom kúpeli tak, aby sa vytvořil gél. Po vychladnutí sa přidá 10 ml fosforečňanového tlmivého roztoku pH = 6,0, obsahujúceho 10 mg alfa-amylázy s pH optimom v oblasti 5,9 až 6,5. Enzým sa nechá pri laboratórnej teplote posobiť 72 h za občasného premiešania.Example 2 g of the beta-D-glucan of Example 1 are heated in 100 ml of H10 on a boiling water bath to form a gel. After cooling, 10 ml of phosphate buffer pH = 6.0 containing 10 mg of alpha-amylase with a pH optimum in the range of 5.9 to 6.5 are added. The enzyme is allowed to stand at room temperature for 72 h with occasional stirring.
Potom sa nerozpustný polysacharid oddělí na frite, trikrát premyje vodou 50 ‘c teplouThen the insoluble polysaccharide is separated on a frit, washed three times with 50 vodou C water
V«IN"
CS 274030 Bl a acetónom a nechá vysušiť na vzduchu. Výťažok 1,5 g. IČ spektrum produktu nevykazuje absorbčné pásy charakteristické pre alfa-D-glukány.CS 274030 B1 and acetone and allowed to air dry. Yield 1.5 g. The IR spectrum of the product does not show absorption bands characteristic of alpha-D-glucans.
Příklad 3Example 3
Gél polysacharidového preparátu, připravený ako v příklade 2 sa termostatuje na teplotu 35 'C, přidá sa fosforečňanový tlmivý roztok o hodnotě pH 4,5 až 6,5, ktoré vyhovuje pH optimu oboch použitých enzýmov, přidá sa roztok alfa-amylázy a gluko-amylázy v množstve, aby poměr sumy enzýmov ku substrátu bol 1 : 100. Enzýmy sa nechájú posobiť za miešania 2 dni a reakčná zmes sa spracuje ako v příklade 2. IČ spektrum produktu nevykazuje absorbčné pásy charakteristické pre alfa-D-glukány.The gel of the polysaccharide preparation, prepared as in Example 2, is thermostatized to a temperature of 35 ° C, a phosphate buffer of pH 4.5 to 6.5 is added, which satisfies the pH optimum of the two enzymes used, a solution of alpha-amylase and glucose is added. The enzymes were allowed to stir under stirring for 2 days and the reaction mixture was worked up as in Example 2. The IR spectrum of the product did not show the absorption bands characteristic of alpha-D-glucanes.
Přiklad 4Example 4
Claims (2)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS3889A CS274030B1 (en) | 1989-01-02 | 1989-01-02 | Method of yeast /1-3/-beta-d-glucan purification |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS3889A CS274030B1 (en) | 1989-01-02 | 1989-01-02 | Method of yeast /1-3/-beta-d-glucan purification |
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| Publication Number | Publication Date |
|---|---|
| CS3889A1 CS3889A1 (en) | 1990-08-14 |
| CS274030B1 true CS274030B1 (en) | 1991-04-11 |
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| Application Number | Title | Priority Date | Filing Date |
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| CS3889A CS274030B1 (en) | 1989-01-02 | 1989-01-02 | Method of yeast /1-3/-beta-d-glucan purification |
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| CS (1) | CS274030B1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| FI20080665A0 (en) | 2008-12-18 | 2008-12-18 | Glykos Finland Oy | Natural saccharide compositions |
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1989
- 1989-01-02 CS CS3889A patent/CS274030B1/en unknown
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| Publication number | Publication date |
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| CS3889A1 (en) | 1990-08-14 |
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