Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/12—Disaccharides

Definitions

• the invention pertains to the method for producing disaccharide ⁇ , ⁇ -trehalose ( ⁇ -D-glucopyranosyl- ⁇ -D-glucopy- ranoside) , which is an important raw material or intermediate in food industry, pharmacy and biochemistry.
• ⁇ , ⁇ -Trehalose may be obtained by isolation from a broad selection of natural substances, above all from Selaginella lepidophyla by origin from southwest of USA which is now grown in the countries of Middle East.
• the most suitable source of this disaccharide is baker's yeast, above all for its good accessibility, low price and a relatively high content of a , ⁇ -trehalose.
• the isolation of ⁇ , ⁇ -trehalose from baker's yeast was described for the first time already in 1936 (K. Myrback, B. Ortenblad: Biochem. Z . 288, 329 (1936)) and its modified form (Z.C. Stewart, N.K. icht yer, C.S. Hudson: J.Am.Cheia. Soc . 72, 2059 (1950)) is employed for the preparation of ⁇ , ⁇ -treh- alose up to the present time.
• the object of the present invention is a method for producing crystalline ⁇ , ⁇ -trehalose by isolation from baker's yeast, which method consists in the extraction of baker's yeast with ethanol of concentration 70 to 96 volume %, vacuum concentration of the obtained extract to the content of ⁇ , ⁇ -trehalose 2 to 30 weight %, after removing the solid portion by filtration, and in the following membrane filtra ⁇ tion process consisting of ultrafiltration through an ultrafiltration membrane with the separation limit of molecular masses higher than 500 and, if desirable, of the preceding microfiltration through a microfiltration membrane with the mean pore diameter ranging from 0.05 to 5 ⁇ m, preferably from 0.05 to 0.15 ⁇ , where the obtained clear solution, after decolourizing with activated charcoal, is brought into contact with an ion exchanger in H + cycle and then with an ion exchanger in OH " cycle and the resulting demineralized solution is evaporated in vacuum to the concentration of 40 to 85 weight % and ⁇ , ⁇ -trehalose, crystallizing after
• Another object of the method according to the inventa- tion consists in the fact that the continuous ultrafiltration is carried out through an ultrafiltration membrane with the separation limit of molecular masses higher than 500 and, if desirable, the preceding microfiltration is carried out through a microfiltration membrane with mean pore diameter ranging from 0.05 to 5 ⁇ m, preferably from 0.05 to 0.15 ⁇ m.
• a styrene-divinylbenzene copolymer with strongly acid sulfo groups as a cation exchanger and a styrene- divinylbenzene copolymer with quaternary dimethy- lhydroxyethyl- ammonium exchanging groups as an anion exchanger.
• the method according to the invention may be realized in such a way, that baker's yeast is extracted at ambient tempe ⁇ rature with 96% ethanol denatured with 2-propanol, the extracted yeast is filtered off and ethanol is removed from the filtrate by vacuum distillation.
• the obtained aqueous or water- ethanolic solution with the concentration of ⁇ , ⁇ -treh ⁇ alose 10 to 40 weight % is subjected to ultrafiltration through an ultrafiltration membrane with the separation limit of molar masses higher than 500, or may be first subjected to microfiltration through a microfiltration membrane with the mean pore diameter ranging from 0.05 to 5 ⁇ m and then to ultrafiltration through an ultrafiltration membrane with the separation limit of molar masses higher than 500.
• the clear aqueous solution after decolourizing with activated charcoal is deionized on the columns of cation exchanger and anion exchanger and concentrated in a vacuum evaporator to aqeous syrup with the concentration of ⁇ , ⁇ -trehalose 50 to 70 weight %.
• the disaccharide crystallizes at normal or suppressed temperature.
• Crystalline a, ⁇ -trehalose is isolated by filtration and dried at 40 to 60°C at reduced presure. If crystalline ⁇ , ⁇ -trehalose of purity higher than 99 weight % is produced, the crude substance has to be recrystaUized from ethanol after deionization realized in a similar way.
• the method according to the invention renders ⁇ , ⁇ -tre ⁇ halose of high quality in a satisfactory yield, thus enabling its further direct use, e.g., in pharmacy or in food produc ⁇ tion.
• Baker's yeast 1000 g is extracted under intense stirring at ambient temperature with 3000 ml 96 % ethanol denatured with 6 % 2-propanol for 45 min. The suspension is filtered and the filtration cake is washed with 250 ml ethanol and is further used for the isolation of ergosterol. The resulting filtrate is concentrated in a vacuum evaporator (20 mm Hg, bath temperature 60°C) to the volume of 400 ml and transferred into a filtration cell of capacity 450 ml provided with an efficient stirring of filtered liquid and microfiltration membrane Synpor" with the mean pore diameter 0.12 ⁇ m.
• Overpressure 0.08 MPa is exerted above the filtered liquid and membrane by nitrogen introduced from a pressure cylinder and 380 ml liquid is filtered. This filtrate is subjected to ultrafiltration in the same cell provided with an ultrafiltratiom membrane from polysulfone having the separation limit of molecular masses 20,000. In this operati ⁇ on, overpressure above the filtered liquid and membrane is 0.25 MPa and 360 ml filtrate is obtained.
• aqueous solution (360 ml) is decolourized by addition of 10 g activated charcoal, which is then filtered off, and the filtrate is deionized in a column of 300 ml Wofatit KPS in H + cycle and a column of 215 ml Wofatit SBK in OH " cycle.
• the ion exchangers are washed with 1500 ml water and the combined solutions are evaporated in vacuum (20 mm Hg) to aqeous syrup containing 71 weight % ⁇ , -trehalose. After addition of 50 ml ethanol, crystallization proceeds at ambient temperature for 16 hours.
• Crystalline ⁇ , ⁇ -trehalose is isolated by filtration of a fritted-glass filter G-2, the filtration cake is washed with 50 ml ethanol and dried in vacuum (50°C, 15 mm Hg) giving 31.5 g crystalline ⁇ , ⁇ -trehal ⁇ ose of purity 97.1 %. Another 2.7 g of the crystalline product is obtained by concentrating mother liquors and crystallization under above specified conditions.
• Dried baker's yeast 250 g is mixed at laboratory temperature with 580 ml demineralized water to a paste which is then extracted with 2000 ml 96 % ethanol denatured with 7 % 2-propanol for 30 minutes. The suspension is filtered and the filtration cake is washed with 2150 ml of 70 % ethanol denatured with 2-propanol.
• the filtrate obtained is concen ⁇ trated in a vacuum evaporator (20 mm Hg, temperature of water bath 60°C) to the volume of 400 ml, transferred into filtra- tion cell of capacity 450 ml with the continuous flow of filtered liquid over a membrane and with the ultrafiltration membrane from cellulose acetate having the separation limit of molecular masses 5000.

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• Organic Chemistry (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Zoology (AREA)
• Life Sciences & Earth Sciences (AREA)
• Wood Science & Technology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Microbiology (AREA)
• General Chemical & Material Sciences (AREA)
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• Biochemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Separation Using Semi-Permeable Membranes (AREA)

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• 1993
  • 1993-09-28 CZ CZ932025A patent/CZ281723B6/cs not_active IP Right Cessation
• 1994
  • 1994-09-23 WO PCT/CZ1994/000022 patent/WO1995009243A1/en not_active Application Discontinuation
  • 1994-09-23 JP JP7510039A patent/JPH08506248A/ja active Pending
  • 1994-09-23 CA CA 2150256 patent/CA2150256A1/en not_active Abandoned
  • 1994-09-23 SK SK697-95A patent/SK279610B6/sk unknown
  • 1994-09-23 EP EP94926769A patent/EP0674717A1/de not_active Withdrawn

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