Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1998-11-25
2001-12-11
Prats, Francisco (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S095000, C435S099000, C435S100000, C210S651000, C210S652000, C210S654000, C127S055000
Reexamination Certificate
active
06329182
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority under 35 U.S.C. 119 of Danish application 1356/97 filed Nov. 26, 1997, the contents of which are fully incorporated herein by reference.
TECHNICAL FIELD
The present invention relates to a method of producing oligosaccharide syrups, in particular to the production of syrups having a high concentration of saccharides with a degree of polymerization of at least 2.
Oligo- and disaccharide mixtures are used industrially, in particular in the food industry, such as in bakeries, confectioneries, soft drinks and saké production.
Oligosaccharide syrups with a low concentration of monosaccharides are advantageous in the field of low caloric additives in many food applications, such as sweeteners, dietary fibers or bulking agents, where they are employed in ice-cream and dairy products, chocolate preparations, chewing gum, biscuits, fruit preparations, pharmaceutical preparations and animal fodder. Due to the low content of monosaccharides the syrups do not cause caries or other dental diseases.
Furthermore, oligosaccharides find use in the drug industry, where for example interest is growing for saccharide adhesion molecules, such as bacterial or leucocyte adhesion.
Oligo- and disaccharide syrups may be produced by enzymatic conversion of starch into sugars followed by enzymatic reactions of the sugars. Enzymatic conversion of starch into sugars involves the steps of liquefaction and saccharification. By the subsequent enzymatic reactions it is possible to obtain a content of disaccharides of 10% to 30%, and of particular disaccharides, such as isomaltose, of 10% to 28% by weight calculated on the basis of dry substance (DS) of syrup.
Handbook of Amylases and Related Enzymes, Ed. by The Amylase Research Society of Japan, Pergamon Press, pp 215-217 discloses a process of producing oligosaccharide mixtures, wherein the content of monosaccharides is 40.5%, disaccharides is 28.3%, trisaccharides is 19.0%, the rest being higher saccharides.
Harder et al. (Kinetics of Isomaltose Formation by Amyloglucosidase and Purification of the Disaccharide by Fermentation of Undesired By-Products, Annals New York Academy of Sciences, Biochemical Engineering 3, vol. 413, pp. 340-351, 1983) suggested to increase the yield of isomaltose in a syrup, in laboratory scale, by fermentation of some of the by-products from the enzymatic reaction of glucose to isomaltose, i.e. glucose and maltose, by
Saccharomyces cerevisiae
and subsequent extraction of glycerol formed by the fermentation. The process involved several steps to achieve an isomaltose content of about 96.5% by weight on the basis of dry substance. This process, however, does not apply in an industrial scale.
Until now it has not been possible to obtain a syrup enriched in disaccharides and/or higher saccharides in industrial process lines.
It is an object of the present invention to provide a method of producing an oligosaccharide syrup comprising the steps of:
enzymatic reaction of a substrate at a temperature in the range of 50° C. to 100° C. obtaining a saccharide solution comprising monosaccharides and disaccharides, trisaccharides and higher saccharides,
nanofiltration of the saccharide solution at a temperature in the range of 60° C. to 100° C. obtaining a syrup essentially comprising disaccharides, trisaccharides and higher saccharides,
recovering said syrup,
optionally recycling the permeate resulting from the nanofiltration step to the enzymatic reaction, whereby it is possible to obtain an oligosaccharide syrup having a high content of saccharides of a degree of polymerisation of at least 2.
The permeate comprising monosaccharides may be used as substrate for the enzymatic reaction saving a considerable amount of substrate and reducing the total costs involved in the method. In a continuous method, this can be achieved by recycling the permeate material to the enzyme reactor directly by having a direct passage to the enzyme reactor from the nanofiltration unit. Alternative, the permeate may be stored for later use.
It is another object of the present invention to provide a system for producing oligosaccharide syrups comprising
an enzyme reactor having at least one inlet and at least one outlet and means for controlling the temperature in the tank,
means for passing saccharide solution from the reactor outlet(s) to at least one nanofiltration unit,
at least one nanofiltration unit having a nanofiltration membrane dividing the nanofiltration unit into an entrance side having at least one inlet and at least one outlet, and an exit side having at least one outlet, and having means for controlling the pressure applied to the membrane,
optionally means for recycling material from an outlet of the exit side of the nanofiltration unit to the enzyme reactor.
It is yet another object of the present invention to provide a disaccharide syrup obtainable by the method, wherein the ratio by weight of disaccharide to total dry substance is from at least 40%, preferably at least 50%, most preferably at least 70%, and the ratio by weight of monosaccharide to total dry substance is from 0.5% to 30%, preferably 0.5% to 20%, most preferably 0.5% to 10% the balance being trisaccharides and higher saccharides.
A further object of the present invention is an oligosaccharide syrup obtainable by the method, wherein the ratio by weight of monosaccharide to total dry substance is at most 10%, preferably at most 5% and the ratio by weight of saccharides having a polymerization degree from 2 to 6 to total dry substance is at least 90%.
Another object of the present invention is a method for using the permeate from the method, for example as a method of producing at least 55% pure fructose, comprising the steps of:
enzymatic reaction of glucose and fructose at a temperature in the range of 50° C. to 80° C. obtaining a saccharide solution comprising glucose and fructose, and disaccharides, trisaccharides and higher saccharides,
nanofiltration of the saccharide solution at a temperature in the range of 60° C. to 80° C. obtaining a syrup essentially comprising disaccharides, trisaccharides and higher saccharides,
recovering said syrup, and
recovering the permeate resulting from the nanofiltration step.
In the present context all percentages are calculated as percent by weight on basis of dry substance (DS) unless otherwise indicated.
In the present context “oligosaccharide syrup” means a syrup comprising monosaccharides (DP1), disaccharides (DP2), trisaccharides (DP3) and higher saccharides (DP4
+
). By the term a disaccharide syrup is meant that the major part of the saccharides are disaccharides.
The notation DP1, DP2, DPX refers to the degree of polymerization of the saccharide, i.e., DP1 is a polymerization degree of 1 (monosaccharide), DP 2 is a disaccharide, and DPX is a polymerization degree of X.
REFERENCES:
patent: 4254225 (1981-03-01), Tamura et al.
patent: 4511654 (1985-04-01), Rohrbach et al.
patent: 5130237 (1992-07-01), Thomas et al.
patent: 5869297 (1999-02-01), Binder et al..
patent: 0 301 522 A2 (1989-02-01), None
patent: 0 452238 A (1991-10-01), None
Handbook of Amylases and Related Enzymes, Their Sources, Isolation Methods Properties and Applications, The Amylase Research Society of Japan, pp. -214-217, (1988).
A. Harder et al., Kinetics of Isomaltose Formation By Amyloglucosidase and Purification of theDisaccharide by Fermentation of Undesired By-Products, (1983).
Hendriksen Hanne Vang
Pedersen Sven
Ambiris Elias J L
Garbell Jason I
Novozymes A/S
Prats Francisco
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