3,654,082
`United States Patent Ofice,
`Patented Apr. 4, 1972
`
`1
`
`‘
`
`3 654 082
`PRODUCTION OF HIGlI lVlALTO'I‘ETRAOSE SYRUP
`Mukhtar Abdullah, Westmont, Ill., assignor to
`CPC International Inc.
`No Drawing. Filed Feb. 10, 1969, Ser. No. 798,151
`Int. Cl. C12b I/00
`
`US. Cl. 195--31 R
`
`
`31 Claims
`
`ABSTRACT OF THE DISCLOSURE
`
`_A process is described for converting starch which com-
`prises subjecting a solution of a gelatinized starch selected
`from the group consisting of acid-modified starch, partial
`hydrolyzates of starch obtained by acid and/or enzyme
`hydrolysis of starch, and mixtures thereof, to the action
`Of 311 amylase Which is produced by the microorganism
`Pseudomamzs stutzeri and recovering a starch conversion
`product. The prior or simultaneous use of a starch de-
`branching enzyme during the conversion process is also
`disclosed. A novel maltotetraose syrup and dried solids ob-
`tainable from said syrup are additionally revealed.
`
`
`This invention relates to a novel starch conversion prod-
`uct and a method of producing said starch conversion
`product. The product is a high maltotetraose hydrolyzate
`or syrup. Dried solids obtainable from said hydrolyzate
`or syrup are also revealed.
`The amount of corn syrup manufactured annually in
`the ‘United States is in excess of two billion pounds. Its
`primary use is in the food processing industry and the
`major application is in the confectionery field. Nearly all
`major countries of the world manufacture or import starch
`syrups.
`‘
`The properties desired in a finished candy generally de-
`termine the specific type of syrup to be used in formulating
`the candy. I-lard candies, which are essentially solid solu-
`tions of carbohydrates, contain corn syrup which con-
`tributes resistance to heat discoloration, prevention of
`sugar crystallization, and lack of stickiness. High maltose
`syrups have been found to provide less hygroscopicity and
`better color stability than regular acid hydrolyzed corn
`syrups.
`Corn syrup is also substantially used in the ice cream
`and irozen dessert industry. In these applications, dextrose
`and corn syrups are added in combination with cane or
`beet sugar as supplemental sweeteners. The primary pur-
`pose of the corn syrup is to provide maximum flexibility
`in adjusting flavor,
`texture, smoothness and body. The
`use of high maltose syrups generally permits the use of
`a higher oorn syrup-to~sucrose ratio than do other types
`of corn syrup. Hight maltose syrups are also useful in
`controlling crystal formation in frozen dessert formula
`tions.
`'
`The syrup product of the present invention is a high
`maltotetraose syrup. That-is, it is a syrup containing a
`large percentage of maltotetraose which is a tetramer of
`glucose comprised of four glucose residues connected
`through alpha-1, 4-glucosidic linkages. The syrup may be
`spray dried by conventional techniques to form a high
`maltotetraose syrup solid.
`The use of high rnaltotetraose syrup in hard candy for-
`mulation is especially promising due to its low hygro-
`scopicity. The use of high maltotetraose syrup in ice
`creams should permit the use of quite high corn syrup-to-
`sucrose ratios.
`The use of acid-modified starch or partial hydrolyzates
`of starch obtained by acid and/or enzyme hydrolysis of
`starch in the present process makes possible the treatment
`of high d.ry substance concentrations of starch, whereas,
`only low dry substance concentrations of unmodified
`
`2
`starch, up to about 5% by weight, can be treated, due to
`retrogradation problems. Thus, to achieve large quantities
`of maltotetraose syrup from unmodified starch it is neces-
`sary to handle large quantities of liquids with concurrent
`large costs in bringing these large volumes of liquids to
`temperature and with the expensive necessity of removing
`a large quantity of water to attain syrups of a truly high
`maltotetraose content.
`it is a principal object of this
`In view of the above,
`invention to provide an improved method for the produc-
`tion of a starch conversion product high in maltotetraose
`content.
`A further object is to provide an improved method for
`the production of high maltotetraose syrup in high yield
`from acid-modified starch and -from partial hy-drolyzates
`of starch obtained by acid and/or enzyme hydrolysis of
`starch.
`Still another object of the invention is to provide a
`process for converting acid-modified starch and partial
`hydrolyzates of starch obtained by acid and/or enzyme
`hydrolysis of starch to maltotetraose syrup with a maltote-
`traose content higher than those produced heretofore with-
`out the costly necessity of removing large quantities of
`water.
`Yet another object of the invention is to provide such
`a maltotetraose syrup for use in confectionery applica-
`tions and for the formulation of ice creams and frozen
`desserts.
`Other objects of the invention will be self-evident or
`will appear hereinafter.
`My method of high maltotetraose syrup production con-
`sists, generally, of incubating a water solution of an acid-
`rnodified starch, a partial hydrolyzate of starch obtained
`by acid and/or enzyme hydrolysis of starch, or mixtures
`thereof with an amylase of the type produced by the
`bacterium Pseudomonas stutzeri.
`An improved yield of maltotetraose is obtained if a
`starch debranching enzyme is also used in the Incubation
`process either prior to or simultaneously with the Pseu-
`domonas stutzeri.
`_
`The acid-modified starch and the partial hydrolyzate of
`starch obtained by acid and/or enzyme hydr_olys1s_ of
`starch, used as starting materials in the P1"€§<‘-T115 1U"‘3“t1°“
`may be obtained by acid-modifying or acid and/or en-
`zyme-hydrolyzing
`any
`conventional
`starch. Suitable
`starches include cereal starches such as corn, grain, sor-
`ghum, and wheat; waxy starches such as waxy H1110 and
`waxy maize; and root starches such as potato starch and
`tapioca starch. Crude starch sources may also be used
`such as ground cereals, macherated tubers, or the partially
`purified starches therefrom. Preferred starches include
`acid-modified waxy milo starch, partially enzyme 113'-
`drolyzed waxy milo starch, partially acid hydrolyzed corn
`starch, and partially enzyme hYdT01YZ6d ¢°m_ Starch-
`Before the starch can be converted to ‘a high maltotet-
`raose syrup, it must first be solubilized lIl water, prefer-
`ably to form a solution containing over 5% by weight of
`starch. Preferably, the Water solution should contain from
`about 10% to about 55% by Weight of starch. This solu-
`tion may be formulated by any of the methods common
`in the starch art.
`In the case of an acid-modified starch, the starch solu-
`tion is prepared by first modifying the starch by conven-
`tional methods, such as are disclosed in detail, for exam-
`ple, in volume 2, Chapters 9, 10 and 11 of Starch: Chem-
`istry and Technology, R. L. Whistler and "E. F. Paschal],
`Editors, Academic Press, Inc., New York and London
`_“5}§Z§;
`TATE & LYLE AMERICAS LLC
`ifititli
`EXHIBIT 101 1
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`
`United States Patent Ofice
`3,654,082
`Patented Apr. 4, 1972
`
`1
`
`'
`
`3 654,082
`PRODUCTION OF HIGlI MALTOTETRAOSE SYRUP
`Mukhtar Abdullah, Westmont, IIl., assignor to
`CPC International Inc.
`No Drawing. Filed Feb. 10, 1969, Ser. No. 798,151
`Int. Cl. C12b I/00
`
`US. Cl. 195--31 R
`
`
`31 Claims
`
`ABSTRACT OF THE DISCLOSURE
`
`A process is described for converting starch which com-
`prises subjecting a solution of a gelatinized starch selected
`from the group consisting of acid-modified starch, partial
`hydrolyzates of starch Obtained by acid and/or enzyme
`hydrolysis of starch, and mixtures thereof, to the action
`of an amylase which is produced by the microorganism
`Pseudomorzas stutzeri and recovering a starch conversion
`product. The prior or simultaneous use of a starch de-
`branching enzyme during the conversion process is also
`disclosed. A novel maltotetraose syrup and dried solids ob-
`tainable from said syrup are additionally revealed.
`__.m---...__.
`
`This invention relates to a novel starch conversion prod-
`uct and a method of producing said starch conversion
`product. The product is a high maltotetraose hydrolyzate
`or syrup. Dried solids obtainable from said hydrolyzate
`or syrup are also revealed.
`The amount of corn syrup manufactured annually in
`the ‘United States is in excess of two billion pounds. Its
`primary use is in the food processing industry and the
`major application is in the confectionery field. Nearly all
`major countries of the world manufacture or import starch
`syrups.
`The properties desired in a finished candy generally de-
`termine the «specific type of syrup to be used in formulating
`the candy. Hard candies, which are essentially solid solu-
`tions of carbohydrates, contain corn syrup which con-
`tributes resistance to heat discoloration, prevention of
`sugar crystallization, and lack of stickiness. High maltose
`syrups have been found to provide less hygroscopicity and
`better color stability than regular acid hydrolyzed corn
`syrups.
`Corn syrup is also substantially used in the ice cream
`and frozen dessert industry. In these applications, dextrose
`and corn syrups are added in combination with cane or
`beet sugar as supplemental sweeteners. The primary pur-
`pose of the corn syrup is to provide maximum flexibility
`in adjusting flavor,
`texture, smoothness and body. The
`use of high maltose syrups generally permits the use of
`a higher corn syrup-to-sucrose ratio than do other types
`of corn syrup. Hight maltose syrups are also useful in
`controlling crystal formation in frozen dessert formula-
`tions.
`’
`The syrup product of the present invention is a high
`maltotetraose syrup. That is, it is a syrup containing a
`large percentage of maltotetraose which is a tetramer of
`glucose comprised of four glucose residues connected
`through alpha-1, 4-glucosidic linkages. The syrup may be
`spray dried by conventional techniques to form a high
`Inaltotetraose syrup solid.
`The use of high maltotetraosc syrup in hard candy for-
`mulation is especially promising due to its low hygro-
`scopicity. The use of high maltotetraose syrup in ice
`creams should permit the use of quite high corn syrup-to-
`sucrose ratios.
`The use of acid-modified starch or partial hydrolyzates
`of starch obtained by acid and/or enzyme hydrolysis of
`starch in the present process makes possible the treatment
`of high dry substance concentrations of starch, whereas,
`only low dry substance concentrations of unmodified
`
`2
`starch, up to about 5% by weight, can be treated, due to
`retrogradation problems. Thus, to achieve large quantities
`of maltotetraose syrup from unmodified starch it is neces-
`SBFY I0 handle large quantities of liquids with concurrent
`large costs in bringing these large volumes of liquids to
`temperature and with the expensive necessity of removing
`a large quantity of water to attain syrups of a truly high
`maltotetraose content.
`it is a principal object of this
`In view of the above,
`invention to provide an improved method for the produc-
`tion of a starch conversion product high in maltotetraose
`content.
`A further object is to provide an improved method for
`the production of high maltotetraose syrup in high yield
`from acid-modified starch and from partial hydrolyzates
`of starch obtained by acid and/or enzyme hydrolysis of
`starch.
`Still another object of the invention is to provide a
`process for converting acid-modified starch and partial
`hydrolyzates of starch obtained by acid and/or enzyme
`hydrolysis of starch to maltotetraose syrup with a maltote—
`traose content higher than those produced heretofore with-
`out the costly necessity of removing large quantities of
`water.
`Yet another object of the invention is to provide such
`a maltot-etraose syrup for use in confectionery applica-
`tions and for the formulation of ice creams and frozen
`desserts.
`Other objects of the invention will be self-evident or
`will appear hereinafter.
`My method of high maltotetraose syrup production con-
`sists, generally, of incubating a water solution of an acid-
`modified starch, a partial hydrolyzate of starch obtained
`by acid and/or enzyme hydrolysis of starch, or mixtures
`thereof with an amylase of the type produced by the
`bacterium Pseudomonas Stu tzeri.
`An improved yield of maltotetraose is obtained if a’
`starch debranching enzyme is also used in the incubatlon
`process either prior to or simultaneously with the Pseu-
`domonas smtzeri.
`The acid-modified starch and the partial hydrolyzate of
`starch obtained by acid and/or enzyme hydr01Y31$_ Of
`starch, used as starting materials in the present 1nV6nt10I1
`may be obtained by acid-modifying OF 3915 and/OF 3”‘
`zyrne-hydrolyzing
`any
`conventional
`starch. Suitable
`starches include cereal starches such as corn, grain, sor-
`ghum, and wheat; waxy starches such as waxy milo and
`waxy maize; and root starches such as potato starch and
`tapioca starch. Crude starch sources may also be used
`such as ground cereals, macherated tubers, or the partially
`purified starches therefrom. Preferred starches include
`acid-modified waxy milo starch, partially enzyme hy-
`drolyzed waxy milo starch, partially acid hydrolyzed corn
`starch, and partially enzyme hydmlyled °°m,Sta‘°h°
`Before the starch can be converted to ‘a high maltotet—
`raose syrup, it must iirst be solubilized in water, prefer-
`ably to form a solution containing over 5% by wc1gh’£0f
`starch. Preferably, the water solution should contain from
`about 10% to about 55% “by weight of starch. This solu-
`tion may be formulated by any of the methods common
`in the starch art.
`In the case of an acid-modified starch, the starch solu-
`tion is prepared by first modifying the starch by conven-
`tional methods, such as are disclosed in detail, for exam-
`ple, in volume 2, Chapters 9, 10 and 11 of Starch: Chem-
`istry and Technology, R. L. Whistler and E. F. Paschall,
`Editors, Academic Press, Inc., New York and London
`(1967).
`The acid-modified starch is then solubilized by suspend-
`ing it in water and heating the solution to above the solu-
`bilization temperature of the starch. Alternatively, a com-
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`3,654,082
`
`4
`zyme may, of course, be used but this will only result in
`additional cost. Smaller quantities of the enzyinemay also
`be used but this may result in longer incubation times.
`PREPARATION OF ENZYMES
`
`3
`mercially available acid-modified starch may be solubilized
`in the same manner.
`A partial acid hydrolyzate of starch may be prepared by
`acid hydrolysis comprising heating a Water slurry of the
`parent starch above the gelatinization temperature of the
`starch in the presence of acid. Alternatively, a solution of
`a partial acid hydrolyzate of starch may be prepared by
`solubilizing in water a commercially available syrup solid
`made from a partial acid hydrolyzate of starch.
`-A partial enzyme hydrolyzate of starch, commonly
`known as an enzyme-thinned starch, may be formulated
`by making a solution of a non-thinned starch in water by
`heating a slurry of the non-thinned starch above the solu-
`bilization temperature of the starch and then incubating
`the starch solution by conventional procedures with an
`alpha-amylase such as bacterial alpha-amylase. A solution
`of an enzyme—thinned starch may alternatively be made by
`solubilizing a syrup made from an enzyme-thinned starch
`by the same method used for making a solution of an acid-
`modified starch or by other means known in the starch
`art. A solution of a mixture of the above starches may be
`for-mulated in the same manner.
`By conventional procedures, starches for use in this in-
`vention are generally partially hydrolyzed by acid or en-
`zyme hydrolysis to a D.E. value below about 25. Prefer-
`ably, the DE. falls within the range of from about 5 to
`about 16. D.E. is the abbreviation for dextrose equivalent
`which represents the conventional measure of the total
`reducing sugar content of a starch hydrolyzate, calculated
`as dextrose and expressed as percent by weight of the dry
`substance present. Thinning to these extents enables the
`starches to be handled as solutions as the preferred solids
`levels of from about 10% to about 55% by weight with-
`out introducing the problem of extensive retrogradation.
`The acid-modified starch, partial acid and/or enzyme
`hydrolyzate of starch, or mixture thereof,
`is then con-
`verted to a high maltotetraose hydrolyzate at a tem-
`perature falling within the range from about 40° C.
`to
`about 65° C. and at a pH value falling within the range
`from about 6 to about 9. It is preferred that the incubation
`be carried out at a temperature falling within the range
`from about 50° C. to about 60° C. and at a pH falling
`within the range about 6.5 to about 7.5. A starch conver-
`sion product produced in this manner will have a malto-
`tetraose content of the solids therein of at
`least 20%
`by weight and will have a D.E. of at least 13.
`The high maltotetraose hydrolyzate may then be con-
`verted to a high maltotetraose syrup by removal of Water
`by evaporation or other equivalent means. Since the malto-
`tetraose concentration in the hydrolyzate is higher than
`that attainable from unmodified starch considerably less
`‘water need be removed to arrive at a high maltotetraose
`syrup product.
`It is preferred that the enzyme which is utilized to form
`the high maltotetraose syrup be the amylase produced by
`the microorganism Pseudomonas stutzeri. Most preferably
`the enzyme is produced by Pseudomonas stutzeri strain
`NRRL B3389 obtainable from the Northern Regional Re-
`search Laboratory of the United States Department of
`Agriculture, located at Peoria, Ill.
`Improved yields of maltotetraose may be obtained if
`the starch is also incubated with a starch debranching
`enzyme. This can be done either prior to or simultaneous-
`ly with the incubation with the amylase of the type pro-
`duced by Pseudomonas smtzeri. Preferably, the starch con-
`version is simultaneously carried out in the presence of
`the starch debranching enzyme and the Pseuabmonas
`stutzerz‘ type amylase. Pullulanase is the preferred starch
`debranching enzyme. The preferred temperature, pH and
`concentration ranges are about the same as when a starch
`debranching enzyme is not used.
`According to my experience as little as 100 units (as
`later defined) of Pseudomonas stutzeri amylase per 100
`grams of starch is sufficient to produce a maximum malto-
`tetraose yield within 2 days. Larger quantities of the en-
`
`Pseudomorzas stutzeri amylase
`Pseudomomzs stutzeri amylase is produced from the
`microorganism Pseudomonas stutzeri and may be prepared
`as follows. A culture of Pseudomonas sturzeri strain
`NRRL B3389, was obtained from the Northern Regional
`Research Laboratory of the U.S. Department of Agricul-
`ture in Peoria, Ill. The organism was maintained on slants
`of Brain~I-Ieart-Infusion (BHI-Agar) containing 0.2%
`soluble starch. The BHI-Agar was obtained from Difco
`Laboratories, Detroit, Mich.
`For production of Pseudomanas szutzeri amylase, cells
`from a stock slant were aseptically transferred to 500
`milliliter Erlenmeyer flasks containing 100 milliliters each
`of a seed medium composed of 1% Tryptone (obtained
`from Difco Laboratories, Detroit, Mich.), 0.5% yeast ex-
`tract, 0.28% KZHPO4, 0.1% KI-l3PO4, 0.2% soluble starch
`and the remainder water. These cliasks were incubated at
`30° C. for 18 hours on a reciprocal shaker.
`Ten milliliter portions of seed medium were used to in-
`oculate 1,000 milliliter Erlenmeyer flasks containing 200
`milliliters of a production medium composed of 1% Tryp-
`tone, 0.5% yeast extract, 028% K21-IP04, 0.1% KHZJPO4,
`1% soluble starch, and the remainder water. The fermen-
`tation flasks were incubated for about 24 hours at 30° C.
`on a rotary shaker operated at 220 r.p.m. After fermenta-
`tion, the culture broth, which possessed an activity of 1-5
`units per milliliter, was harvested by centrifugation. The
`centrifugate was then used as the source of Pseudomonas
`stutzeri amylase.
`To further concentrate the Pseudomonas s/utzerz’ amy-
`lase,
`the culture centrifugate was mixed with a volume
`of 2-propanol equal
`to twice the volume of the cen-
`trifugate and which contained suspended therein 10 grams
`of diatomaceons earth per liter of 2-propanol. The enzyme
`was insoluble in the resulting solution. It was physically
`adsorbed onto the diatomaceous earth. The enzyme—diato-
`maceous earth precipitate was collected by filtration and
`dried to give a dried enzyme product which was 5 to 20
`times more active per unit weight than was the original
`culture broth.
`Pseudomonas stutzeri amylase activity was determined
`as follows: (1) a solution of 0.55 weight percent soluble
`starch (Merck, reagent grade) was dissolved in a 0.02
`molar glycerophosphatehydrogen chloride buffer (pH 6.9)
`which contained 0.02 molar calcium chloride; (2) rea-
`gents were prepared for the determination of reducing
`sugars as described by ‘N. Nelson, J. Biol. Chem. 153,
`375.;
`(1944);
`(3) 9 milliliters of starch solution was
`pipetted into a test tube, and the tube was placed in a
`25 " C. water bath for 15 minutes to equilibrate to tem-
`perature;
`(4)
`1 milliliter of the Pseudomonas stutzeri
`amylase solution to be assayed, was added to the test
`tube; (5) the resultant 10 milliliter solution was mixed
`and incubated at 25 ° C. for 10 minutes; (6) a 2 milliliter
`sample of the solution was withdrawn and the amount of
`reducing sugars produced was determined by the above-
`referenced method of Nelson.
`One unit of enzyme activity is defined as being equal
`to one micromole of reducing sugar produced, calculated
`as dextrose, per milliliter of enzyme solution per minute.
`PULLULANASE
`
`The debranching enzyme, pullulanase, used in the proc-
`ess of this invention,
`is produced by members of the
`bacterial species Aerobacter aerogenes when suitably in-
`cubated under conditions of aerobic culture.
`Characteristics by which members of the species Aero-
`bacter aerogenes may be distinguished are described by
`M. W. Yale, R. S. Breed et al.,
`in “Bergey’s Manual of
`Determi-native Bacteriology,” Seventh edition, p. 341-42,
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`3,654,082
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`5
`Williams and Wilkins Co., Baltimore, Md., 1967, although
`it is well recognized by those skilled in the science, that
`individual strains may be isolated from time to time which
`do not completely conform to this identical description.
`The method of producing the enzyme pullulanase is
`described by Hans Bender and Kurt Wallenfels in Bio-
`chemische Zeitschrift, 334, 79-95 (1961).
`The source of the Aerobacter aerogenes culture exem-
`plified in the present disclosure was the American Type
`Culture Collection, 12301 Parklawn Drive, Rockville,
`Md. The designation of the culture was Aerobacter aero-
`gerzes ATCC -8724. One method of obtaining the enzyme
`from the culture is as follows:
`A medium containing: 0.8% Difco ‘Bacto-peptone,
`0.5% maltose, 0.3% sodium nitrate, 0.05% dibasic po-
`tassium phosphonate, 0.05% potassium chloride, 0.001%
`ferrous sulfate heptahydrate with a pH adjusted to 7.2;
`is dispensed into 1,000 milliliter Erlenmeyer flasks, 200
`milliliters per flask. The flasks are stoppered with cotton
`plugs and sterilized.
`The culture inoculum is obtained by aseptically trans-
`ferring from an agar slant, cells of a pure culture of the
`microorganism Aerofacter aerogenes ATCC 8724, into a
`sterilized flask with the above medium. The flask is then
`placed on a reciprocal shaker in a constant temperature
`room operated at 20° C. The flask is shaken 24 hours
`after which time the cuture has prown abundantly and
`is ready to be used to inoculate flasks of the above de-
`fined medium. 10 milliliters are aseptically transferred
`to each of the enzyme production flasks. These flasks are
`then placed on a reciprocal shaker in a constant tempera-
`ture roorn operated at 29° C. They are shaken for a pe-
`riod of time from 66 to 72 hours. At the end of the fer-
`mentation the flasks are removed from the shaker, their
`contents pooled and the cells therein removed from the
`culture liquor by centrifugation. The supernatant liquor
`is then adjusted to pH 6.2 and preserved by the addition
`of toluene. An aliquot of the supernatant liquor is then
`assayed for enzyme activity. The amount of pullulanase
`enzyme produced may vary from about 0.05 to 015 unit
`per milliliter.
`A concentrated dried preparation of the pullulanase
`enzyme may be obtained by the following procedure:
`15-00 milliliters of chilled (4° C.) acetone is added to 1
`liter of chilled (4° C.) cell free culture liquor contain-
`ing 10 grams of diatomaceous earth. After complete mix-
`ing the suspension is vacuum filtered to recover the in-
`solubilized enzyme. Upon completion of filtration,
`the
`filter cake is recovered, spread, and allowed to dry over-
`night at room temperature. Once the filter cake is dried
`it is assayed for pullulanase enzyme activity. Preparations
`obtained by this procedure will have an activity from 3
`to 10 units per gram, depending on the activity of the
`culture liquor used and the efficiency of recovery.
`in
`The level of pullulanase enzyme activity present
`pullulanase preparations may be determined as follows:
`An aliquot of enzyme solution is adjusted to pH 5.5,
`and 1.0 milliliter is added to a digestion mixture composed
`of 2 milliliters of a 5% pullulan solution and 7 milli-
`liters of a M/50 phosphate buffer, pH 5.5. The reaction
`is carried out in test tubes placed in a 40° C. water bath
`and is allowed to proceed for 1 hour. At the end of the
`digestion period, the reaction is stopped by the addition
`of hydrochloric acid to lower the pH to 3.0. The reducing
`sugar content of the digestion mixture is determined, as
`well as that of the culture liquor and pullulan used, by a
`modification of the alkaline potassium ferricyanide meth-
`od described hereinafter and is experessed as rnicrograrn
`equivalents of dextrose. Pullulanase enzyme activity is
`calculated as follows:
`
`_T—(0+P)
`A" l80><60 X”
`
`where
`
`6
`
`A-=P~ullulanase enzyme activity, units per milliliter or
`gram of enzyme preparation.
`'Il=Tota1 reducing sugars in digestion mixture in micro-
`grams expressed as dextrose.
`C—-«Residual reducing sugars in culture liquor in micro-
`grams expressed as dextrose.
`P=Reducing value of the pullulan polysaccharide used in
`the digestion mixture in micrograms expressed as dex-
`trose.
`D=Dilution «factor of enzyme preparation.
`180=Red-ucing value of l micrornole of dextrose.
`60=Time (minutes) of reaction.
`One unit of pullulanase is defined as the amount of en-
`zyme required to produce 180 micrograms of reducing
`sugars, calculated as dextrose, per minute from -pullulan
`under the conditions specified above. The polysaccharide,
`pullulan, which is a polymer of maltotriose units connected
`to each other by alpha 1-6 linkages, may be obtained from
`Pztllularia pullulans ATCC 9348 by use of the procedure
`of S. Ueda, K. Fujita, K. Komatsu, and Z. Nakashitna, [Ap-
`plied Microbiology 11, 211-215 (1963). The modified
`potassium ferricyanide assay procedure used for determi-
`nation of reducing agents is assaying enzyme preparations
`is conducted as follows:
`Reagents.—Alkaline ferricyanide: Dissolve 1.170 g. of
`potassium ferricyanide and 19.5 g. of anhydrous sodium
`carbonate in water and dilute to 1 liter. Store in amber
`bottle. Standard dextrose solution, 0.1 mg./ml.: Weigh
`1.000 g. of pure anhydrous dextrose and dilute to 100 ml.
`Using a class A pipette, transfer 10.0 ml. of the solution
`to a 1-liter flask and dilute to mark..
`Procedure.-—Standardization: Pipette 0.5, 1.0, 2.0 and
`2.5 ml. aliquots of standard dextrose solution, 0.1 mg./ml,
`into respective 18—cm. test tubes. Then water is added in
`amounts to bring the total volume of the respective tubes
`to 2.5 ml. The reagent -blank contains 2.5 ml. of water. To
`each tube is then added 5 ml. of the alkaline ferricyanide
`solution. The mixture is then heated in a boiling water-
`bath for exactly 5 minutes, cooled immediately in a tap-
`watenbath, diluted to 12.5 ml. volume with water and
`mixed. Using water as reference solution at 0 absorbance,
`determine the absorbency of the blank and of each of the
`standard tubes at 373 my on a Beckman DU spectophoto-
`meter, using 1-cm. cuvettes.
`Analysis.———An aliquot of enzyme preparation is used
`which will -produce from 1 to 10 mg. reducing sugar per
`10 ml. digestion mixture. The sample of the digestion mix-
`ture assayed in this method will contain from 50 to 250
`micrograms of reducing sugar.
`Calculation.——-Plot absorbances of standard tubes cor-
`rected for -blank versus micrograms of dextrose per 12.5
`ml. on linear coordinate graph for standardization curve.
`The invention may be further understood by reference
`to the specific examples described in detail below. All
`percent figures are by weight unless otherwise particularly
`specified.
`In some of the examples, reference is made to the fluid-
`ity of acid-modified starches. The fluidity of a starch is de-
`fined as the number of milliliters of a standard alkaline
`starch paste delivered by a special funnel in the time re-
`quired by the same funnel to deliver 100 milliliters of
`water. The standard procedure for determining fluidity is
`described by P. Shildneck and C. E. Smith in Starch:
`Chemistry and Technology, vol. II, p. 219, R. L. Whistler
`and E. F. Paschall, Editors, Academic Press, New York
`and London, 1967.
`It is understood, of course, that the following examples
`are merely illustrative and that the invention is not to be
`limited thereto.
`Example 1.--—This example illustrates the use of Pseudo-
`monas srutzeri amylase in the conversion of acid-modified
`waxy milo starch of 80 fluidity to conversion products hav-
`ing high maltotetraose contents.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`70
`
`75
`
`

`
`3,654,082
`
`7
`10% and 20% solutions of acid-modified starch were
`prepared by dissolving the acid—mod1fied starch in boiling
`water. The resulting solutions were incubated with Pseztdo—
`monas stmzeri amylase at 50° C. at a pH of 6.5 for 20
`hours and ‘for 48 hours, respectively. At the end of this
`time, the starch hydrolyzates had the compositions shown
`in Table 1.
`
`TABLE I
`
`8
`TABLE III
`
`Percent
`Glucose ——————————————————————————————————-~ 4-5
`Maltose .................................. —_
`7-0
`5 Maltotriose .............. --...... -c......... M 8.6
`
`Maltotetraose
`...... -- 50.2
`Higher sacchartdes ..........................— 29.5
`
`Pxeudomonasstutzeri
`amylase (u/100 g.)
`1,000................ __
`200.................. _-
`
`Weight
`percent,
`starch D.E. Glucose Maltese
`10
`26
`1.1
`2. 3
`20
`21.1
`1.1
`1.9
`
`Saccharide distribution, weight percent, D.B.
`Ma1to-
`Higher
`tct«
`saccha-
`mose
`rides
`55
`35. 6
`56.2
`36.4
`
`Malto-
`triose
`5
`4.4
`
`Example 2.-—This example describes the use of Pseu-
`domonas stutzeri amylase to convert enzyme-thinned
`waxy milo starch at high solids concentrations to a high
`maltotetraose hydrolyzate. The hydrolyzate produced in
`accordance with this example is then converted to a high
`maltotetraose syrup by removing part of the water by
`
`Example 4.—-—The starches of Examples 1, 2, and 3
`were converted simultaneously with Pseudomorzas stutzeri
`amylase and pullulanase debranching enzyme from Aera-
`bacter aeragenes at 50° C. and pH 6.5. The resulting high
`maltotetraose hydrolyzates had the following composi-
`tions:
`
`20
`
`TABLE IV
`
`Weight
`percent, Pullulanase
`starch
`(u/100 g.) D .E. Glucose Maltese
`10
`300
`30
`1. 6
`4. 0
`20
`200
`26.8
`1.0
`5.7
`30
`200
`29. 4
`2. 5
`7.8
`35
`200
`31, 0
`4. 5
`8.1
`
`Saccharide distribution, weight. percent, D.B.
`Higher
`Saecliar-
`ides
`20. 2
`24.6
`24. 5
`25. 8
`
`Malto- Malto-
`triose
`tctraose
`7. 2
`67. 0
`7.7
`61.0
`11.5
`55. 7
`11. 6
`50. 0
`
`Sterch substrate
`Waxy Milo, 80 fluidity .
`Do ............. ..
`Waxy milo, enzyme-
`thinncd to 5 DE ....... _.
`Corn, acid-thinned to 16
`D.E .................... __
`
`
`
`evaporation or other equivalent means. The high malto-
`tetraose syrup produced in this manner may then be .
`utilized in candy and ice cream formulations. Alterna-
`tively, it may be spray dried to form a syrup solid which
`may be utilized in these and other applications.
`A 30% solution of waxy milo starch was thinned with
`Bacillus .mbtz'lz's alpha—amylase to 5 D.E. by conventional
`procedures. This
`thinned starch was
`incubated with
`Psediromonas stutzeri amylase (200 units/100 grams) at
`503 C. and pH of 6.5 for 48 hours. The resulting hydro-
`lyzate exhibited a D.E. of 22.7 and had the composition
`‘
`~~
`.
`.
`h
`.
`(weight Pelgeml) B ) S Own bebw
`TABLE II
`
`40
`
`This example illustrates the increased maltotetraose
`yields attainable when pullulanase enzyme as well as
`Pseudomonas stutzeri amylase is used during incubation
`of acid-modified starch, and of partial acid and enzyme
`hydrolyzate of starch. It also illustrates that solutions
`containing relatively high levels of solids may be success-
`fully treated by this procedure.
`Example 5.~Pseudomomzs smtzeri amylase was em-
`45
`.
`.
`.
`' ployed as described in the previous examples to convert
`20% solutions of enzyme-thinned corn starc