ENZYMATIC PRODUCTION OF MALTOHEXAOSE-RICH COMPOSITIONS

Abstract: Malto-oligosaccharide compositions which contain up to about 40 % maltohexaose by weight are produced from starchy substrates and maltodextrins by a simple, one-step hydrolysis with certain thermostable alpha -amylase from Bacillus stearothermophilus. This process is particularly useful in the production of novel compositions with properties that will lead to new applications in both food and nonfood industries.

Description: ENZYMATIC PRODUCTION OF MALTOHEXAOSE-RICH COMPOSITIONS Bac-p-πreupd of the Invention Field of the Invention Maltcr-oligosaccharides, also referred to as __alt-_x_extrins and dextrose syrup solids, .are produced frαn starch by hydrolysis with α-amylases. These <_____ri-chydrates are used in adhesives and in food applications such as syrups, flavor encapsulation, texture control, binding agents, carriers for low-calorie sweeteners, and gels in reduced-calorie foods. This invention relates to the production of novel malto-oligosaccharide cαrpositions that contain a large proportion of maltohexaose. Description of the Prior Art Thermally stable α-aπylases have enabled a rapid advance in the commercial production of italto-olitgosaccharides by enzymatic hydrolysis of starch. Hcwever, the mode of the amylase action on starches is only partly understood because the fine st_n_cture of starches is still obscure. Furthermore, amylases from different scυrces behave differently, and their action patterns are dependent on reaction conditions. Certain amylases are nw known to yield distinctive patterns of malt^^oliα saccharide products that are at variance with the distribution of products that would be predicted on the basis of randan cleavage of starch molecules. Robyt et al. [Arch. Biochem. Biophys. 200: 451-467 (1963)] teach that α-amylase from Bacillus subcilis selectively forms _naltotri»ose and maltohexaose. NakaJ iki et al. [Cartxhydr. Res. 128 : 297-310 (1984)] report that the α-amylase from _ . licheniformis degrades short-chain amylose at 1% concentration and 40* C to give mainly maltopeπtaose and maltσtriose with slightly smaller quantities of maltose. In cαitrast, Inglett [J. Food Biochem. 11: 249-258 (1987) ] shows that this same enzyme, acting on higher substrate <_xιnceπtτations (20-30% starch) and at a higher teπperature (95* C) , yields increased quantities of maltose, essentially equivalent to or slightly hicjher than the other two oligcπ_ers. Slcinin'ska et al. [Starch Starke 33(6): 205-210 (1986)] show t t a 72-hr saα±arificaticn of liquified starch with a thermostable maltogenic amylase from B . stearothermophilus virtually eliminates the maltohesaose (G€) constituent, αitrup et al. [Starch Stajrias 35(12): 405-411 (1984) shows that treatment of aπylcpectin with a B . scearochermoph±lus amylase produces only traces of maltohexaose (G€) . αi —m-r-T Λ*ς the Invgction I have now surprisingly found that certain thermostable α-amylases have a unique and unexpected .action on starches and maltodextrins to produce large quantities of maltohexaose [degree of polymerization (DP) 6], with cα__paratively minor amounts of oligαneric constituents of DP greater than 6. These amyl.ases have utility in a process for converting starch into products with potentially unique and expanded markets. In accordance with this diseσvery, it is an object of the invention to provide a siπple, one-step method for converting starch into novel malto-oligosaccharide ccnpositions that contain a large proportion of maltohexaose. It is also an object of the invention to provide novel maltodextrins and dextrose .syrup solids containing maltohexaose as the dominant oligomer and with properties leading to potential new uses. Other objects and advantages of the invention will become readily apparent from the ensuing description. T*^ 1 * DβscriDtJ«» «? +- Invention .Suitable starting materials cαπteπplated for use in the invention include unmodified natural granular starches such, as regular cereal, potato, .and tapioca starch, as well as waxy starches and high-esπylose starches. These materials are prepared for enzyme treatment by gelatinization. Far purposes of this invention, gel tinizatian is acccB-plished preferably fay passage of an aqueous slurry of the starch through a steam-injection cooker at a temperature of about 120°-165" C to ensure thorough dispersion of the starch. Other methods of gelatinizatiαn are well-known in the art. Alternatively, pregelatinized starch and maltodextrins would serve as useful starting materials. The concentratica of substrate should be in the range of about 5-45% by weight. A suitable calcium salt is added to the aqueous dispersion of substrate in an aim.iunt sufficient to stabilize the subsequently added α-amylase (preferably .about 50 pp of calcium) .. The pH of the resulting starchy dispersion is adjusted to .about 6.0 with sodium hydroxide or other alkali, and the dispersion is treated at a tenperature in the range of 70°-100* C, preferably abcut 95* C, with a thermostable α-amylase. The thermostable α-amylases useful herein .are those referred to as 1,4-alpha-D-glucan glixanchydrolases and having the essential enzymatic characteristics of those produced by the B. scearothermophilus strains ATCC Nos. 31,195; 31,196; 31,197; 31,198; 31,199; and 31,783. These strains .are described in U.S. Patent No. 4,284,722, which is herein incorporated by reference. Other sources of this enzyme include organisms such as B . subtil ± s which have been genetically modified to express the thermostable α-amylase of B . scearochermophllus as described in U.S. Patent No. 4,493,893, herein incorporated by reference. These enzymes sure available cαanercially under the name "Enzeco Thermolase" (Enzyme Develcpnent, Div., Biddle Sawyer Corp., New York, N.Y.). The level of enzyme suitable for use in this process is generally in the range of about 3-25 units per g of starch or dextrin, where l unit of haπt p rial α-amylase activity is the f-mn*int. of enzyme required to hydrolyze 10 mg of starch per minute under specified conditions [Enzyme Development, Div., Biddle Sawyer Corp., New York, N.Y., Technical Bulletin No. 20 (Revised 7/86)]. Similarly, the duration of treatment depends on the prodtjct desired and will generally range from about 10-60 min; with 30-50 min being preferred for enzyme concentrations in the range of 10-20 units/g substrate; and 10-30 min being preferred for enzyme σoncentrati<_-ns in the range of 20-25 units/g substrate. After the desired conversion time, it is preferable to decolorize the resulting mixture with activated carbon and add a filter aid to facilitate subsequent recovery of the hydrolyzate. The pH is then adjusted to 3.5-4.0, such as with 0.2 N sulfuric acid, and the product is heated at about 95* C for 10 min to inactivate remaining enzyme. The pH is then adjusted to about 6.5, .such as with 1 N sodium hydroxide, and the product is separated by filtration and then dried by any of a variety of techniques as within the skill of the person in the art. The products of this invention differ from σcπmerσially available maltodextrins and dextrose syrup solids in that the latter products contain a fairly uniform distribution of oligosaccharides with no preponderance of any particular oligαner. It is therefore envisioned that the maltchexaose-rich products of this invention will have unique properties that will lead to new food applications in fields such as flavor encapsulation, texture control, and binding agents, as well as new industrial applications. These products might also serve as starting matf-ri l in new procedures for preparing carbohydrate coEopositions such as cyclodextrins. The following exaπples are presented only to further illustrate the invention and are not intended to limit the scope of the invention which is defined by the claims. All percentages herein disclosed are by weight unless otherwise specified. EXsφle 1 Standard Process Conditions. Two hundred g (dry basis) of high-amylose corn starch ("Aπylαnaize VH," American Maize-Products Co., Haxaicnd, IN) was slurried in 800 ml of water containing 50 ppm of calcium (0.185 g/1 CaCl 2 .2H 2 0) and passed through a steam-injectiαn cooker at 138'-143* C (30-40 psi of steam pressure) . The gelatinized starch paste was collected in a Dewar flask, and the pH was adjusted to 6.0 with 1.0 N sodium hydroxide. Thermostable α-amylase ("Enzeco Thermolase," supra) was adcted to the starch paste at 95" C in an amount sufficient to provide 16.5 units (supra) per g of starch. Samples of converted .starch were removed 20, 40, .and 60 min after addition of the enzyme. To each sample was added, with stirring, activated carbon ("Darσo G-60," E M Science, Div., E M Industries, Inc., Cherry Hill, NJ) sufficient to provide a concentration of 0.1%, and filter aid ("Hyflo Filter Cel," Maπville, Fitration & Minerals Div., Denver, CO) sufficient to provide a concentration of 2%. The pH was adjusted to 3.5-4.0 with 0.2 N sulfurlc acid, and the products were heated at 95* for 10 min to inactivate remaining enzyme. The pH was then raised to 6.5 with 1 iv sodium hydroxide, and the mixtures were filtered hot through a bed of filter aid (supra) on filter paper ("Whatman No. 1," Whatman Chemical Separation Inc., Clifton, NJ) on a Buchner funnel under vacuum. The filtrates were spray-dried (Rαlvis Mini Spray Dryer, Model GA-31, Yamato, Northbrook, IL) , and the carbchydrate ccπposition of the products was deteπnined by high-pressure liquid chrcmatography (Inglett, supra) . The analytical results in the Table, below, show that highest yields of maltohexaose were obtained with conversion times of 40 and 60 min. Sxaoples 2—3 Effect of Enzyme Laval. Coπpositions were prepared as described in Example 1 except that the α-amylase level was 11.0 units per g of starch in Exaπple 2.and 22.0 units per g in Exaπple 3. Bκ____ples 4—5 Effect of pH. Coπpositions were prepared as described in Exanple 1 except that the pH during enzyme conversion of the starch was 7.0 in Exanple 4 and 5.0 in Exanple 5. The results in the Table show that pH 6.0 (Exanple 1) is the preferred pH, but that slight variations above or below this value would not significantly affect the yield of maltohexaose. E-Qnple 6 Ooπveraion of Potato Λmyloβe. (Expositions were prepared as •described in Exanple 1 except that the starchy substrate was potato a ylose (Avebe America, Inc., Hopelawn, NJ) instead of high-amylose corn starch. The results in the Table shew that yields of maltohexaose from potato amylose were slightly less than those from high-amylose corn starch (Exaπple 1) . It is understood that the foregoing detailed description is given merely by way of illustration and that modification and variations may be made therein without departing from the spirit and scope of the invention. Table Enzywa oonoβntirat ion 0___nversi»__n Aaount of constituent 0 , wt. t (units/g time Exzuφle substrate) PH (-in) DP >9 DP-9 DP-8 Ur-1 DP-6 DP-5 0P-4 DP-3 DP-2 DP-1 1A 16.5 6 20 29. 7 0.2 0.8 12.3 22. .0 7.8 6.2 14.0 6.9 0 IB 16.5 6 40 10.9 0 0.2 0 38. , 7 12.6 8.9 17.5 10.6 0. 6 1C 16.5 6 60 8. 2 0 0.1 0 37, .1 14.1 9.1 18.1 12.3 1. .0 2λ 11.0 6 20 18. 8 0.1 0.7 10.7 25. .8 10.8 8.2 16.5 8.1 0. .3 2B 11.0 6 40 β. 2 0 0 0 34. .8 16.1 9.3 18.7 12.1 0. .9 2C 11.0 6 60 5. 4 0 0 0.1 31. .3 19.1 9.3 19.2 14.0 1. .5 3A 22.0 6 20 6. 2 0 0 0 34. .9 16.4 9.2 18.8 13.3 1. .2 3B 22.0 6 40 3. 6 0 0.1 0.1 28. .1 20.9 9.2 19.0 16.1 2. ,9 3C 22.0 6 60 2. .6 0 0.1 0.1 23. .2 21.5 9.2 19.1 18.5 5. .7 4Λ 16.5 7 20 14. 3 0 0.6 11.8 27. .4 11.0 8.8 16.2 9.3 0. .4 4B 16.5 7 40 6. 1 0 0 0 35. .7 15.4 9.6 18.3 13.5 1. .2 4C 16.5 7 60 4. ,5 0 0 0 30, .6 18.9 9.9 18.7 15.3 2. ,0 SA 16.5 5 20 10. .2 0 0 0.2 39. .7 12.4 8.5 17.8 10.7 0. 6 5B 16.5 5 40 6. .5 0 0 0 35. ,6 15.9 9.0 18.7 13.0 1. .2 5C 16.5 5 60 6. .1 0 0 0 34. ,8 16.5 9.1 19.0 13.5 1. 1 6A 16.5 6 20 1 .6 0 0 10.1 30. .2 15.2 9.5 20.3 12.2 0. .9 6B 16.5 6 40 1 .0 0 0 0 33. . 1 18.8 9.6 20.4 14.8 2. 3 6C 16.5 6 60 0 .9 0 0 0 30. .7 20.1 9.6 20.4 15.6 2. 8 a DP - degree of polymerization of da_rt_tx.ee , Oiete DP-2 ie di_«acx_ ar__de_ DP-3 1» trieaαc-harJ le. etc.

Claims: I claim: 1. A method for producing a πaltohexaose-rich coπposition from a substrate selected from the group of gelatinized starches and italtodextrins σcπprising treating an aqueous dispersion or solution of said substrate with a thermostable α-amylase having the essential enzymatic characteristics of the thermostable α-amylases produced by B . stearothermophllus strains AICC No. 31,195; 31,196; 31,197; 31,198; 31,199; and 31,783 for a period of 10-60 min and recovering said maltohexaose-rich coπpositiσn. 2. The method as described in Claim 1 wherein said starch is selected from the group consisting of regular cereal, potato, and tapioca starches, as well as waxy and high-amylose starches. 3. The method as described in Claim 1 wherein said substrate is a maltodextrin. 4. The method as described in Claim 1 wherein the amount of said α-amylase is sufficient to provide about 3-25 units of the enzyme per g of .starch. 5. The method as described in Claim 1 wherein said treatment of starch with enzyme is performed at a teπperature in the range of about 70*-100* C. 6. The method as described in Claim l wherein the amylase is present in the amount of 10-20 units/g substrate and the period of enzyme treatment is in the range of about 30-50 min. 7. A ooπposition produced by the method of Claim 1. 8. A ocnposition produced by the method of Claim 2. 9. A ocnposition produced by the method of Claim 3. 10. A ocnposition produced by the method of Claim 4. 11. A cαπposition produced by the method of Claim . 12. A ocnposition produced by the method of Claim 6.

Inventors: INGLETT GEORGE E, INGLETT, GEORGE, E.
Applicants: INGLETT GEORGE E, INGLETT, GEORGE, E.
Application year: 1989
Main classification: C12P19
IPC codes: C12N9, C12P19