United States Patent
`
`[19]
`
`[11] Patent Number:
`
`5,266,467
`
`Nov. 30, 1993
`Inglett
`[45] Date of Patent:
`
`USOOSZ66467A
`
`[54] ENZYMATIC PRODUCTION OF
`MALTOHEXAOSE-RICH COMPOSITIONS
`
`[75]
`
`[73] Assignee:
`
`Inventor: George E. Inglett, Peoria, 111.
`The United States of America as
`represented by the Secretary of
`Agriculture, Washington, D.C.
`
`[21] App]. No.: 649,348
`
`[22] Filed:
`
`Feb.1, 1991
`
`Related US. Application Data
`
`[63]
`
`Continuation of Ser. No. 189,093, May 2, 1988, aban-
`cloned.
`
`[51]
`Int. Cl.5 .............................................. C12P 19/22
`
`[52] US. Cl. ............................. 435/99; 435/832
`[58] Field of Search .................................. 435/99, 832
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`4,241,183 12/1980 Witt et a1.
`......................... 435/99 X
`4,284,722 8/ 1981 Tamuri et al. ................ 435/94
`
`4,298,400 11/1981 Armbruster et a].
`...... 435/99 X
`.................. 435/172.3
`4,493,893
`1/1985 Mielenz et a1.
`4,603,110 7/1986 Morehouse et a].
`.................. 435/96
`
`FOREIGN PATENT DOCUMENTS
`
`189838
`252730
`
`8/1986 European Pat. Off.
`1/1988 European Pat. Off.
`
`.............. 435/99
`.............. 435/99
`
`OTHER PUBLICATIONS
`
`[note: only 451—460 fur-
`
`phys. 100: 451—460 (1963)
`nished].
`L. Slomin’ska et 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 al., “Properties and Application of a
`Thermostable Maltogenic Amylase Produced by a
`Strain of Bacillus Modified by Recombinant—DNA
`Techniques,” Starch/Starke 36(12): 405—411 (1984).
`G. E. Inglett, “Action Pattern of Bacillus licheniformis
`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-Amylases and the Anomeric Configurations of
`Their Products,” Carbohyd. Res. 128: 297—310 (1984).
`
`Primary Examiner—~Glennon H. Hollrah
`Assistant Examiner—Shailendra Kumar
`
`Attorney, Agent, or Firm--M. Howard Silverstein; John
`D. Fado; Curtis P. Ribando
`
`[57]
`
`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 a-amy-
`lase from Bacillus stearothermophilus. This process is
`particularlyuseful in the production of novel composi-
`tions with properties that will lead to new applications
`in both food and nonfood industries.
`
`John Robyt et al., “Action Pattern and Specificity of an
`Amylase from Bacillus subtilis," Arch. Biochem. Bio~
`
`5 Claims, No Drawings
`
`

`

`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 maltodex-
`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
`sublilis selectively forms maltotriose and maltohexaose.
`Nakakuki et al. [Carbohydr. Res. 128: 297—310 (1984)]
`report that the a-amylase from B. Iicheniformis degrades
`short-chain amylose at 1% concentration and 40° C. to
`give mainly maltopentaose and maltotriose with slightly
`smaller quantities of maltose. In contrast, Inglett
`[1.
`Food Biochem. 11: 249-258 (1987)] 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 oligomers.
`Slomin’ska et al. [Starch/Starke 38(6): 205—210 (1986)]
`show that a 72-hr saccharification of liquified starch
`with a thermostable maltogenic amylase from B. stearo-
`thermophilus virtually eliminates the maltohexaose (66)
`constituent. Outrup et
`a1.
`[Starch/Starke 36(12):
`405—411 (1984) shows that treatment of amylopectin
`with a B. steamthermophilus 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 maltodextrins 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 converting 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
`
`3O
`
`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°~l65° 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 a-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 US. Pat. No.
`4,284,722, which is herein incorporated by reference.
`Other sources of this enzyme include organisms such as
`B. subtilis which have been genetically modified to
`express the thermostable a-amylase of B. steamthermo-
`philus as described in US. 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 0.2N 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
`
`4
`(“Whatman No. 1,” Whatman Chemical Separation
`Inc., Clifton, NJ.) on a Buchner funnel under vacuum.
`The filtrates were spray-dried (Pulvis Mini Spray
`Dryer, Model GA-3l, 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 24
`
`Effect of Enzyme Level
`
`Compositions were prepared as described in Example
`1 except that the a-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 pH, but that slight variations above or below
`this value would not significantly affect the yield of
`maltohexaose.
`
`EXAMPLE 6
`
`Conversion of Potato Amylose
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`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 maltodextrins 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
`
`Two hundred g (dry basis) of high-amylose corn
`starch (“Amylomaize VII,” American Maize-Products
`Co., Hammond, Ind.) was slurried in 800 ml of water
`containing 50 ppm of calcium (0.185 g/l CaC12.2H20)
`and passed through a
`steam-injection cooker at
`l38°—143° 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 LON sodium hydrox-
`ide. Thermostable a-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, NJ.) 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
`
`Enzyme
`Conversion
`concentration
`(units/g
`time
`Amount of contituent" , wt. %
`
`Example
`substrate)
`pH
`(min)
`DP >9 DP-9 DP-S DP-7 DP-6 DP-5 DP-4 DP-3 DP-Z DP-l
`1A
`16.5
`6
`20
`29.7
`0.2
`0.8
`12.3
`22.0
`7.8
`6.2
`14.0
`6.9
`0
`18
`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
`2A
`11.0
`6
`20
`18.8
`0.1
`0.7
`10.7
`25.8
`10.8
`8.2
`16.5
`8.1
`03
`23
`11.0
`6
`40
`8.2
`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
`33
`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
`0.4
`43
`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
`5A
`16.5
`5
`20
`10.2
`0
`0
`0.2
`39.7
`12.4
`8.5
`17.8
`10.7
`0.6
`SB
`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
`O
`10.1
`30.2
`15.2
`9.5
`20.3
`12.2
`0.9
`63
`16.5
`6
`40
`1.0
`0
`O
`0
`33.1
`18.8
`9.6
`20.4
`14.8
`2.3
`
`6C 2.8 16.5 6 60 0.9 0 0 0 30.7 20.1 9.6 20.4 15.6
`
`
`
`
`
`
`
`
`
`
`
`
`‘DP = degree of polymerization of dextrose, where DP-2 is disaccharide. DP-3 is trisaccl'uride. etc.
`
`35
`
`Compositions were prepared as described in Example
`1 except that the starchy substrate was potato amylose
`(Avebe America, Inc., Hopelawn,.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-
`40 out departing from the spirit and scope of the invention.
`
` TABLE
`
`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
`1N 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-
`
`

`

`5,266,467
`
`6
`cereal, potato, and tapioca starches, as well as waxy and
`high-amylase 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
`
`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 a-amylase having the mal-
`tohexaose-producing characteristics of the thermosta-
`ble a-amylases produced by B.
`steamrhermophilus
`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
`
`treatment of starch with enzyme is performed at a tem-
`perature in the range of about 70°—100° C. and for a
`period of about 10—60 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.
`t
`t
`t
`‘
`t
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`65
`
`