United States Patent
`
`[191
`
`[11] Patent Number:
`
`5,266,467
`
`Nov. 30, 1993
`[45] Date of Patent:
`Inglett
`
`IllllllllllllllllllllllllllIlllllllllllllllliillllilIllllllllllllllllllllll
`US005266467A
`
`[note: only 451-460 fur-
`
`phys. 100: 451-460 (1963)
`nished].
`L. S1omin’ska ct al., “Studies on the Application of
`Maltogenic Amylase in the Production of Maltose Con-
`taining Syrup,” Starch/Starke 38(6): 205-210 (1986).
`H. Outtrup et 11]., “Properties and Application of a
`Thcrmostable Maltogenic Amylase Produced by a
`Strain of Bacillus Modified by Recombinant—DNA
`Techniques,” Starch/Starke 36(12): 4054]] (1984).
`G. E. Inglett, “Action Pattern of Bacillus Iichemformis
`Alpha-Amylase on Ordinary, Waxy and High»
`—Amylose Corn Starches and Their Hydroxypropyl
`Derivatives,” J. Food Biochem. 11: 249-258 (1987)
`(NRRC #5886).
`Teruo Nakakuki et al., “Action Patterns of Various
`Exo—Amy1ases and the Anomeric Configurations of
`Their Products,” Carbohyd. Res. 128: 297-310 (1984).
`
`Primary Examiner—G1ennon H. Hollrah
`Assistant Examt'ner—-—Shailendra Kumar
`Attorney, Agent, or Firm-—-M. Howard Silverstein; John
`D. Fado; Curtis P. Ribando
`
`ABSTRACT
`[57]
`Malto-oligosacchaiide 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 a-amy-
`lase from Bacillus stearotkermophilus. This process is
`particularly useful in the production of novel con-iposi~
`tions with properties that will lead to new applications
`in both food and nonfood industries.
`
`[54] ENZYMATIC PRODUCTION OF
`MALTOHEXAOSE-RICH COIWPOSITIONS
`
`[75]
`
`Inventor: George E. Inglett, Peoria, 111.
`
`[73] Assignee:
`
`The United States of America as
`represented by the Secretary of
`Agricultllre, Washington, D.C.
`
`[21] App1.No.: 649,348
`
`[22] Filed:
`
`Feb. 1, 1991
`
`Related U.S. Application Data
`
`[63]
`
`Continuation of Ser. No. 189,093, May 2, I988, aban-
`doned.
`
`Int. Cl.5 ............................................ .. C12P 19/22
`[51]
`
`[52] us. Cl. . . . . . .. . . . . . . . . . .
`. . . .. 435/99; 435/832
`[58] Field of Search ................................ .. 435/99, 832
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`....................... .. 435/99 X
`4,241,183 12/1980 Witt et al.
`4,284,722
`8/1981 Tamuri et al.
`435/94
`4,298,400 11/1981 Armbruster et al.
`435/99 X
`4,493,893
`1/1985 Mielenz et al.
`435/172.3
`4,603,110 7/1936 Morehouse et al.
`................ .. 435/96
`
`
`
`.
`
`FOREIGN PATENT DOCUMENTS
`
`189838
`252730
`
`8/1986 European Pat. Off.
`1/1988 European Pat. Off.
`
`............ .. 435/99
`............ .. 435/99
`
`OTHER PUBLICATIONS
`
`John Robyt et al., “Action Pattern and Specificity of an
`Amylase from Bacillus subtilis," Arch. Biochem. Bio-
`
`5 Claims, No Drawings
`
`TATE & LYLE AMERICAS LLC
`
`EXHIBIT 1012
`
`

`
`1
`
`5,266,467
`
`ENZYMATIC PRODUCTION OF
`MALTOHEXAOSE-RICH COMPOSITIONS
`
`This application is a continuation of application Ser.
`No. 07/189,093, filed May 2, 1988, now abandoned.
`BACKGROUND OF THE INVENTION
`
`1. Field of the Invention
`Malto~oligosaccharides, also referred to as ma1todex-
`trins and dextrose syrup solids, are produced from
`starch by hydrolysis with a-amylases. These carbohy-
`drates 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 compo-
`sitions that contain a large proportion of maltohexaose.
`2. Description of the Prior Art
`Thermally stable a-amylases have enabled a rapid
`advance in the commercial production of malto~
`oligosaccharides by enzymatic hydrolysis of starch.
`However, the mode of the amylase action on starches in
`only partly understood because the fine structure of
`starches is still obscure. Furthermore, amylases from
`different sources behave differently, and their action
`patterns are dependent on reaction conditions. Certain
`amylases are now known to yield distinctive patterns of
`malto-oligosaccharide products that are at variance
`with the distribution of products that would be pre-
`dicted on the basis of random cleavage of starch mole-
`cules. Robyt et al. [Arch Biochem. Biophys. 100:
`451-467 (1963)]
`teach that a-amylase from Bacillus
`subtilis selectively forms maltotriose and maltohexaose.
`Nakakuki et al. [Carbohydr. Res. 128: 297-310 (1984)]
`report that the a-amylase from B. Iichemformis degrades
`short-chain arnylose at 1% concentration and 40° C. to
`give mainly maltopentaose and maltotriose with slightly
`smaller quantities of maltose. In contrast, Inglett
`[J.
`Food Biochem. 11: 249-258 (l987)] shows that this
`same enzyme, acting on higher substrate concentrations
`(20-30% starch) and at a higher temperature (95° C.),
`yields increased quantities of maltose, essentially equiv-
`alent to or slightly higher than the other two oligorners.
`Slomin’ska et al. [Starch/Starke 38(6): 205-210 (l986)]
`show that a 72-hr saccharification of liquified starch
`with a thermostable maltogenic amylase from B. steam-
`thermophilus virtually eliminates the maltohexaose (G6)
`constituent. Outrup et
`al.
`[Starch/Starke
`36(12):
`405-411 (1984) shows that treatment of amylopectin
`with a B. stearothermophilus amylase produces only
`traces of maltohexaose (G6).
`SUMMARY OF THE INVENTION
`
`I have now surprisingly found that certain thermosta-
`ble a.-amylases have a unique and unexpected action on
`starches and rnaltodextrins to produce large quantities
`of maltohexaose [degree of polymerization (DP) 6],
`with comparatively minor amounts of oligomeric con-
`stituents of DP greater than 6. These amylases have
`utility in a process for convening starch into products
`with potentially unique and expanded markets.
`In accordance with this discovery, it is an object of
`the invention to provide a simple, one—step method for
`converting starch into novel malto-oligosaccharide
`compositions that contain a large proportion of mal-
`tohexaose.
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`65
`
`2
`It is also an object of the invention to provide novel
`maltodextrins and dextrose syrup solids containing mal-
`tohexaose 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.
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`Suitable starting materials contemplated 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-amylose starches. These
`materials are prepared for enzyme treatment by gelati-
`nization. For purposes of this invention, gelatinization is
`accomplished preferably by 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 gelatinization
`are well-known in the art. Alternatively, pregelatinized
`starch and maltodextrins would serve as useful starting
`materials. The concentration of substrate should be in
`the range of about 5—45% by weight.
`A suitable calcium salt is added to the aqueous disper-
`sion of substrate in an amount sufficient to stabilize the
`subsequently added a-amylase (preferably about 50
`ppm of calcium). The pH of the resulting starchy dis-
`persion is adjusted to about 6.0 with sodium hydroxide
`or other alkali, and the dispersion is treated at a temper-
`ature in the range of 70°-100° C., preferably about 95°
`C., with a thermostable a-amylase.
`The thermostable ct-amylases useful herein are those
`referred to as 1,4-alpha-D-glucan glucanohydrolases
`and having the essential enzymatic characteristics of
`those produced by the B. stearothermophilus strains
`ATCC Nos. 31,195; 31,196; 31,197; 31,198; 31,199; and
`31,783. These strains are described in U.S. Pat. No.
`4,284,722, which is herein incorporated by reference.
`Other sources of this enzyme include organisms such as
`B. subzilis which have been genetically modified to
`express the therrnostable a-amylase of B. .t'tearotherm0-
`philus as described in U.S. Pat. No. 4,493,893, herein
`incorporated by reference. These‘ enzymes are available
`commercially under the name “Enzeco Thermolase"
`(Enzyme Development, 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 1 unit of bacterial a-amylase activity
`is the amount 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)]. Simi-
`larly, the duration of treatment depends on the product
`desired and will generally range from about 10-60 min;
`with 30-50 min being preferred for enzyme concentra-
`tions in the range of 10-20 units/g substrate; and 10-30
`min being preferred for enzyme concentrations 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 O.2N 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 1N sodium hydroxide and the product is separated
`
`

`
`5,266,467
`
`3
`by filtration and then dried by any of a variety of tech-
`niques as within the skill of the person in the art.
`The products of this invention differ from commer-
`cially available maltodcxtrins and dextrose syrup solids
`in that the latter products contain a fairly uniform distri-
`bution of oligosaccharides with no preponderance of
`any particular oligomer. It is therefore envisioned that
`the maltohexaose-rich products of this invention will
`have unique properties that will lead to new food appli-
`cations in fields such as flavor encapsulation, texture
`control, and binding agents, as well as new industrial
`applications. These products might also serve as start-
`ing materials in new procedures for preparing carbohy-
`drate compositions such as cyclodextrins.
`The following examples 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.
`
`EXAMPLE 1
`Standard Process Conditions
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`4
`(“Whatman No. 1,” Whatman Chemical Separation
`1nc., Clifton, NJ.) on a Buchner funnel under vacuum.
`The filtrates were spray-dried (Pulvis Mini Spray
`Dryer, Model GA-31, Yamato, Northbrook, 111.), and
`the carbohydrate composition of the products was de-
`termined by high—pressure liquid chromatography (In-
`glett, supra). The analytical results in the Table, below,
`show that highest yields of maltohexaose were obtained
`with conversion times of 40 and 60 min.
`
`EXAMPLES 2-3
`
`Effect of Enzyme Level
`
`Compositions were prepared as described in Example
`1 except that the ct-amylase level was 11.0 units per g of
`starch in Example 2 and 22.0 units per g in Example 3.
`EXAMPLES 4-5
`
`Effect of pH
`Compositions were prepared as described in Example
`1 except that the pH during enzyme conversion of the
`starch was 7.0 in Example 4 and 5.0 in Example 5. The
`results in the Table show that pH 6.0 (Example 1) is the
`preferred pl-1, but that slight variations above or below
`this value would not significantly affect the yield of
`maltohexaose.
`
`EXAMPLE 6
`
`Conversion of Potato Amylose
`
`Compositions were prepared as described in Example
`1 except that the starchy substrate was potato amylose
`(Avebe America, Inc., 1-Iope1awn,_N.J.) instead of high-
`amylose corn starch. The results in the Table show that
`yields of maltohexaose from potato amylose were
`slightly less than those from high-amylose corn starch
`(Example 1).
`It is understood that the foregoing detailed descrip-
`tion is given merely by way of illustration and that
`modification and variations may be made therein with-
`out departing from the spirit and scope of the invention.
`
`Two hundred g (dry basis) of high-amylose corn
`starch (“Arnylomaize VII,” American Maize-Products
`Co., Hammond, Ind.) was slurried in 800 ml of water
`containing 50 ppm of calcium (0.185 g/l CaCl2.2H2O)
`and passed through a
`steam-injection cooker at
`138°—l43° C. (30-40 psi of steam pressure). The gelati-
`nized starch paste was collected in a Dewar flask, and
`the pH was adjusted to 6.0 with 1.0N sodium hydrox-
`ide. Thermostable at-amylase (“Enzeco Thermolase,"
`supra) was added 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
`(“Darco G-60,” E M Science, Div., E M Industries,
`Inc., Cherry Hill, N.J.) sufficient to provide a concen-
`tration of 0.1%, and filter aid (“Hyflo Filter Cel,” Man-
`ville, Fitration & Minerals Div., Denver, Colo.) suffi-
`cient to provide a concentration of 2%. The pH was
`
`TABLE
`Enzyme
`Conversion
`concentration
`(units/g
`time
`Amount of comituent“ , wt. %
`
`Example
`substrate)
`1:}!
`(min)
`DP >9 DP-9 DP-8 DP-7 DP-6 DP-5 DP-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
`1B
`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
`ZA
`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
`3.2
`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
`O
`0
`0
`34.9
`16.4
`9.2
`18.8
`13.3
`1.2
`313
`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
`4A
`16.5
`7
`20
`14.3
`0
`0.6
`11.8
`27.4
`11.0
`8.8
`16.2
`9.3
`04
`4B
`16.5
`7
`40
`6.1
`D
`0
`O
`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
`SE
`16.5
`5
`40
`6.5
`O
`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
`613
`16.5
`6
`40
`1.0
`0
`O
`O
`33.1
`18.8
`9.6
`20.4
`14.8
`2.3
`
`16.5 6 60 0.9 0 O 0 30.7 20.1 9.6 20.4 15.66C 2.8
`
`
`
`
`
`
`
`
`
`
`
`
`‘D1’ = degree of polymerization of dextrose, where D1’-2 is disuccharide, DP-3 is xrincchuide. etc.
`
`adjusted to 3.5-4.0 with 0.2N sulfuric acid, and the
`products were heated at 95° for 10 min to inactivate
`remaining enzyme. The pH was then raised to 6.5 with
`IN sodium hydroxide, and the mixtures were filtered
`hot through a bed of filter aid (supra) on filter paper
`
`65
`
`I claim:
`1. A method for producing a maltohexaose-rich com-
`position from a substrate selected from the group of
`gelatinized starches and maltodextrins comprising treat-
`
`

`
`5
`ing an aqueous dispersion or solution of said substrate
`with about 3—25 units per gram of starch of a thermosta-
`ble B. steamthermophilus <1-amylase having the mal~
`tohexaose—producing characteristics of the thermosta-
`ble a-arnyiases produced by B.
`stearathermophilus
`strains ATCC No. 31,195, 31,196, 31,197, 31,198,
`31,199, and 31,783 under conditions favorable to the
`production of maltohexaose as the dominant oligomer
`whereby said maltohexaose comprises at
`least about
`20% by weight of the composition, and recovering said
`maltohexaose-rich composition.
`2. The method as described in claim 1 wherein said
`starch is selected from the group consisting of regular
`
`6
`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 said
`treatment of starch with enzyme is performed at a tem-
`perature in the range of about 70"—lO0° C. and for a
`period of about l0~6O min.
`5. The method as described in claim 1 wherein the
`amylase is present in the amount of 10-20 units/g sub-
`strate and the period of enzyme treatment is in the range
`of about 30-50 min. 3
`¥
`3
`3
`I
`
`5,266,467
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`10
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`20
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`25
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`30
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`65