CARBOHYDRATE PROCESS

Abstract: Methods of producing glucosone which comprises enzymatically oxidizing glucose with glucose-2-oxidase in a first zone and separating the concomitantly produced hydrogen peroxide from said first zone through a semi-permeable membrane into a second zone, said membrane being permeable only to compounds of a molecular weight of less than about 100.

Description: CARBOHYDRATE PROCESS This invention is concerned with a new and useful process for the ' production of glucosone. and more particularly ■ for the production of glucosone. from which food-grade .fructose 5 can be obtained. Commercial methods for the production of fructose, a commerciall important sweetner, primarily involve a two-step process, the first, hydrolysis of a polysaccharide such as starch to produce glucose and the second, isomerization of the so-produced glucose to form fructose. The latter step, as is well-known, produces a mixture of glucose and fructose from which it is difficult to separate the desired product, fructose. The commercial separation method involves the use of crystallization techniques, which are costly and ' ime-consumin • t c - * ' More detailed description of the various methods of isomerizing glucose can be found in the literature, e.g., U.S. Patent 3,788,945, and 3,616,221. Glucose can also be. converted to fructose by the action of an enzyme, designated glucose-2-oxidase, to form 20 glucosone (D-arabino-2-hexosulose) which in turn can be re¬ duced to fructose with zinc and acetic acid [Folia Microbiol. 23, 292-298 (1978) and Czechoslovakian Patent No. 175897 to Vole et al. ] . The reaction of glucose-2-oxidase with glucose to <~ ^ produce glucosone also yields hydrogen peroxide in equimolar amount. The use of the so-produced hydrogen -peroxide in the conversion of alkenes to corresponding halohydrins and epoxides has been proposed in European Patent Application 7176. In the published application, the in situ formation of hydrogen ^ peroxide is proposed by inclusion of glucose-2-oxidase and glucose in the reaction mixture which includes- a halogenating enzyme and a source of inorganic halide into which the sele alkene is to be introduced. The disclosure of the European patent application further indicates that he glucosone pro duct of the enzymatic oxidation of glucose can be converted to fructose by simple chemical hydrogenation. However, fructose produced by the said process ca be contaminated with significant amounts of by-products fro both the enzymatic conversion of glucose and the alkene con version reaction. In particular, the latter reaction produ halohydrins and alkylene oxides, e.g. ethylene oxide, which are highly toxic materials even at levels in the region of parts per million. Thus, fructose produced by such a proces will require careful and costly purification to attain food grade purity. Further, the potential for contamination of fructose by virtue of secondary reactions during the initia processing stage is quite high due to the highly reactive products, halohydrins and alkyleneoxides, and substantial purification procedures are required to assure the high leve purity required for food grade fructose. SUMMARY OF THE INVENTION This invention provides a method for the productio of glucosone by enzymatic oxidation of glucose' to glucosone a reaction zone from which hydrogen peroxide is removed by use of a hydrogen peroxide-permeable membrane into a second reaction zone. In accordance with one embodiment of the invention the second reaction zone contains a reducing agent for the hydrogen peroxide which migrates through the semi-permeable membrane. The presence of the reducing agent in the second zone encourages a faster migration of the hydrogen peroxide out of the first zone. Reducing agents for this embodiment are well-known to those skilled in the art and include a "g TlE OMFI variety of systems such as organic reducing agents, anions, cations and enzymes. Organic reducing agents are exemplified by aldehydes which are readily oxidized to corresponding car- boxylie acids. Reducing anions include, for example, oxalate, sulfite, phosphite and iodide ions. Reducing cations include a wide variety of cations which can exist in variable valence states such as the transition metals Fe, Co, Ni, Cr and the like Reducing enzymes are readily available from a variety of natural products and include catalase and peroxidase. Catalase is found in yeast, eggs and blood while peroxidase is found in horseradish. The quantity of reducing agents in the second zone is not critical but it is preferably used at levels to significantl reduce the amount of hydrogen peroxide produced in the first reaction zone. Thus, stoichiometric quantities of reducing agent will assure more complete removal of hydrogen peroxide, from the first reaction zone and the use of even excess amounts over the stoichiometric will be practical particularly in those cases where the selected reducing agent is readily available and economical. Of course, the use of less than stoichiometric quantities of the reducing agent is also within -the scope of the invention but will be less efficient. The membranes employed in the present process are for the purpose of establishing two separate zones and per¬ mitting migration of hydrogen peroxide from the first to the second zone. The membranes therefore should be of suitable pore size to selectively permit hydrogen peroxide migration, but to preclude passage of larger molecules in the first reaction zone. Such membranes are readily available commercially and can be defined in terms of the molecular weight of solute OMPI particles to pass through the membrane. In the present inven¬ tion, membranes which permit substances of a molecular weight of less than about 100 are to be used and preferably less tha The migration or passage of hydrogen peroxide throug the aforesaid membrane is accomplished through establishment of an equilibrium predicated on the relative concentrations of H 2 0- on each side of the membrane. As the concentration of hydrogen peroxide in the first zone increases, the H_0_ tends to migrate to the second zone until equilibrium is rees- tablished. The inclusion of a reducing agent in the second zone increases the rate of flow of hydrogen peroxide through the membrane by offsetting the equilibrium in the direction of the second zone, for which reason the reducing agent embodimen is generally preferred. Employing the present process results in considerabl advantage particularly in the further processing of glucosone to fructose. The migration of hydrogen peroxide from the firs reaction zone of course ' affects the rate of the enzymatic oxi- dation of glucose so that the reaction tends to be more comple and the reaction times can be shorter than normally required. Further, the first reaction zone is essentially free of con¬ taminants that will accumulate primarily in the second reactio zone where the so. produced hydrogen peroxide is reacted. The glucosone solution produced in the first reaction zone can be used as such in the hydrogenation step or can be concentrated or otherwise processed as desired. The glucosone solution is substantially free of contaminants other than some unreacted glucose, or glucose di er or tri er, and whatever contaminants that may have been introduced in the original glucose charge. Usually, the glucose charge will be a hydrolysate of a natural product containing glucose units, most commonly starch, which will contain soluble contaminants such as other carbohydrates, le.g. maltose, formed in the starch hydrolysis. Accordingly, the reduction of the reaction product of the first zone wi l provide a product, fmnt sft, which will b comparatively free of contaminants that effect food grade 5 status for the product, the contaminants being derived only from the glucose natural sources, e.g. starches such as corn starch. PREFERRED EMBODIMENTS The membranes to be used in the present process 0 are any of those commonly employed in aqueous systems and include a wide variety. Most commonly, the membranes will be comprised of nylon, a styrene polymer, usually polystyrene teflon, or a cellulose ester such as cellulose acetate or propionate. In a first embodiment, the membrane is fitted 5 into a reactor to provide two zones in a manner to preclude unintended mixing of the contents of the two zones. In a second embodiment, separate reactors can be coupled with the selected membrane providing the requisite interface in the coupling. For maximum migration of hydrogen peroxide from 0 the first zone to the second zone, membranes of significant exposed surface area are of course preferred for which reason the first embodiment is more preferable. The glucose-2-oxidase enzyme can be provided in the form of the enzyme solution ' in water, immobilized enzyme or 5 immobilized cells or mycelium or the free cells or mycelium. Most commonly since the enzyme is intr cellular, the cells or mycelium of the selected microorganism are used by merely suspending them in the reaction solution. Promoters and pro¬ tectors for the enzyme can also be present. For example, as described in the aforesaid Folia Microbiol. 23, 292-298 (1978) , the presence of fluoride ion promotes the enzymatic oxidation 1 of glucose with 0. mucida. Protectors for enzymes can also be used, e.g. Co, Mn and Mg salts. The enzymatic oxidation reaction ia carried out unt substantially complete as can be determined by monitoring the 5 mixture using aliquots to test for glucose content, or by colorimetric determination of glucosone or by determination o of hydrogen peroxide. Usually, reaction periods of about 24 48 hours are sufficent, depending on enzyme potency or activi A wide variety of microorganisms can be used to pro 0 duce the glucose-2-oxidase employed in the present process. For example, the following organisms are described in the literature for this purpose: I Aspergillus parasiticus [Biochem. J. .31, 1033 (1937)] II Iridophycus flaccidum [Science 124, 171 (1956) ] 5 III Oudemansiella mucida [Folia Microbiol. 1_3, 334 (1968) ibid. _2_3, 292-298 (1978) J IV Gluconobacter roseus [J. Gen Appl. Microbiol. 1,152 (1955) V Polyporus obtusus [Biochem. Biophys. Acta 167, 501 (1968) VI Corticium caeruleum [Phytoche istry 1977 Vol. 16, p 1895- 0 The temperature for the enzymatic oxidation reactio is not critical. The reaction can be conducted at room tempe ture, or even somewhat higher than room temperature where the enzyme system employed is of reasonable heat stability. In 5particular, it is preferable to operate at 50°C. and above. with heat stable enzyme systems in which range bacterial infe of the reaction mixture is minimized. Alternatively, the jsnz matic reaction mixture can contain antibacterial agents to pr clude extensive bacterial growth. ■ Q The first reaction zone of course should contain no significant amounts of a reducing agent for hydrogen peroxide *- so that the beneficial results of the present process can be - ϋREX OMPI - — 1 realised. Thus, the system should be substantially free of reducing agents for H-,0-., i.e. a non reducing system. During the course of the present process, it is possible for some diffusion of material from the second reaction zone into the first zone, especially where anions, cations or low molecular weight reducing agents are present in the second zone. Therefore, it is usually preferred to use reducing agents which either do not diffuse, e.g. peroxidase and catalase enzymes particularly in the immobilized form, or which on oxidation form 10 food-acceptable products, e.g. ferric or sulfate which derive from ferrous and sulfite ions on oxidation by H-0 2 . By this preferred procedure, undesirable contamination of the first reaction zone is reasonably avoided. In a particular preferred form of the invention the sep 15 rated hydrogen peroxide is reacted with yeast, hole milk or eggs to effect pasteurization at room temperature with the obvious advantage of avoiding elevated temperature normally used for pasteurization. Thus, the yeast, milk and the eggs eac are capable of acting as the reducing agent in the second zone. 20 The reduction of glucosone to fructose is accomplished by known procedures including chemical reduction as with zinc and acetic acid as well as catalytic hydrogenation, with the usual metal catalysts. Of these, the preferred metal catalyst is Raney Ni since its use is compatible with the desired food 25 grade of fructose, i.e. no residues or contaminants are left by this catalyst. In the ususal procedure employed, the glucosone is hydrogenated at elevated pressure and temperature over the selected metal catalyst until the desired degree of hydrogenation 30has been achieved. Pressures can range from 100 to 700 atmos¬ phere and even higher while the temperature can range up to v. about 200°C. Preferred is 100° to 150°C. and a pressure of about 500 atmospheres. The following example further illustrates the 35 invention. OMPI Aλf- * W7PO 1 EXAMPLE Mycelium of 0. mucida are grown in accordance with Example 1 of Czechoslovakian patent 175897 and the equivalent of 15 g. (dry weight) of the mycelium is suspended in 3 L. of 2.5% glucose solution 0.05M NaF in one zone of a 10 L. reactor fitted with a hydrogen peroxide-permeable membrane to form two zones. In the second zone, catalase enzyme immo¬ bilized on DEAE-cellulose (Cellex-D, manufactured by Bio-Rad Laboratories) is suspended in 3 L. of water. 10 The suspension in the first zone is mixed at 25°C. and aerated with oxygen while the second zone is also mixed. After 24 hours the mycelium is then separated from the solu¬ tion in the first zone and the resulting clear solution is th hydrogenated over Raney Ni at 500 atmospheres hydrogen gas an 15100°C. The aqueous mixture is filtered clear of the catalyst decolorized with carbon, dionized with ' ion-exchange (anionic and cationic) , and concentrated to a fructose syrup at reduce pressure. Alternatively, the aqueous mixture is concentrated and fructose allowed to crystallize. 20 The fructose obtained as either syrup or crystallin product is of food grade quality. The membrane employed in this example permits passa of molecules of molecular weight of less than 50. Essentially the same results are obtained when O. mucida is replaced with the following organisms: Polyporus obtusus Radulum casearium Lenzites Trabea Irpex flanus 0 Polyporus versicolor Pellicularia filamentosa ** Ar illaria mellea Schizophyleum commune Corticium caeruleum

Claims: WHAT IS CLAIMED IS: 1. A method of producing glucosone which comprises U _ O C W U. l-.ll y j. U UO _ " ώ UΛO.UUO C _l Cl first zone and separating the concomitantly produced hydrogen peroxide from said first zone through a semi-permeable membrane into a second zone, said membrane being permeable only to com¬ pounds of a molecular weight of less than about 100. 2. The method according to Claim 1 wherein said second zone comprises a reducing agent for hydrogen peroxide. 3. The method according to Claim 2 wherein said reducing agent is an enzyme. 4. The method according to Claim 3 wherein said enzyme is peroxidase or catalase. 5. The method according to Claim 2 wherein said reducing agent is an anion or cation. 6. The method according to Claim 5 wherein said anion is sulfite. 7. The method according to Claim 5 wherein said cation is ferrous. 8. A method of producing fructose which comprises the steps of (a) producing glucosone which comprises enzymati¬ cally oxidizing glucose with glucose-2-oxida_ιe in a first zone and separating the concomitantly produced hydrogen per- oxide from said first zone through a semi-permeable membrane into a second zone, said membrane being permeable only to compounds of a molecular weight-of"less than about 100, and (b) reducing the so-produced glucosone to obtain fructose. 9. The method according to Claim 8 wherein, the reduction is effected by catalytic hydrogenation. 10. The ' method according to Claim 9 wherein the catalyst is Raney Ni. 11. The method according to Claim 8 wherein the filtered reaction mixture obtained in Step a is employed in Step b.

Inventors: HORWATH R, MASELLI J
Applicants: STANDARD BRANDS INC
Application year: 1981
Main classification: C12P7
IPC codes: C07H1, C07H3, C12N11, C12P1, C12P7, C12P19