MULTILING CORPORATION
`
`180 NORTH UNIVERSITY AVE.
`suite 500
`PROVO, UT 84601
`
`VOICE (801) 377~2000
`FAX (801) 377-7085
`
`TRANSLATOR’S CERTIFICATE OF TRANSLATION
`
`TRANSLATION FROM Japanese into English
`
`We certify that we are a professional translation company ot‘./apmzese ofthe
`languages listed above, a Utah corporation, which has its principal office at l80 North
`University Avenue, Sixth Floor, Provo, UT 84601, USA; that we are competent in all the
`above listed languages; that we have carefully made and/or verified the translation from the
`original document to the translated document, which is indentiticd below; and that to the best
`ofour knowledge and beliefthe translations ofthat document are true, complete, and correct
`version of the original.
`
`Multiling Project Number: RATPIBIOOZ
`Document Identifier: JPl0I6596A
`
`Signed this 22”“ day of March, 2013.
`
`p,Tp_,,/
`
`mject Manager
`
`ACKNOWLEDGMENT BEFORE NOTARY
`
`State of Utah
`
`County of Utah
`
`}SS..
`
`20[?7 before me, the undersigned Notary
`day of MM ah A
`On this
`, who proved on the basis ofsatisfactory
`Public, personally appeared
`evidence to be the person whose name is subscribed to this ’I‘ranslator’s Certificate of
`Translation and who acknowledged that he or she executed the same for the purposes stated
`therein.
`
`IN WITNE S WHEREOF, I hereunto set my hand and ofticial seal.
`
`T
`otary PubIic
`
`Residing at:
`
`g ;
`
`TATE & LYLE AMERICAS LLC
`EXHIBIT I014
`
` ‘ E NOTARYPUBUC-STATE OF UTAH
`
`COMMlSSlON# 656494
`
`
`
`s
`
`I
`COMM. EXP. 05-309015
`
`
`
`

`
`Multii" »
`l""~5$k<
`
`180 NORTH UNIVERSITY AVE.
`Suite 600
`PROVO. UT 84601
`
`MULTILING CORPORATlON
`
`VOICE (801) 377-2000
`FAX (801) 377-7085
`
`TRANSLATOR’S CERTIFICATE OF TRANSLATION
`
`"l"l{ANSL/\TION FROM Japanese into English
`
`We certify that we are a professional translation company of./apcmese of the
`languages listed above, a Utah corporation, which has its principal office at 180 North
`University Avenue, Sixth Floor, Provo, UT 84601, USA; that we are competent in all the
`above listed languages; that we have carefully made and/or veritied the translation from the
`original document to the translated document, which is indentitied below; and that to the best
`otour knowledge and beliefthe translations ofthat document are true, complete, and correct
`version of the original.
`
`M ultiling Project Number: R/\TPl3lOO2
`Document Identifier: JPl0I659oA
`
`Signed this 22”" day oflvlarch, 2013,
`
`to/Tow,
`
`rojecl Manager
`
`ACKNOWLEDGMENT BEFORE NOTARY
`
`State of Utah
`
`County oi’ Utah
`
`}ss.
`
`2015 before me, the undersigned Notary
`On this day of MM M4
`, who proved on the basis ofsatisfactory
`Public, personally appeared
`evidence to be the person whose name is subscribed to this 'l‘ranslator’s Certificate of
`Translation and who acknowledged that he or she executed the same for the purposes stated
`therein.
`
`IN WITNF. S WHERl§iOF, I hereunto set my hand and otlicial seal.
`
`
`
`otary Public
`
`'
`
`7
`
`Residing an .2a2¢,i$,t2<§0“£?:3?rA¢W_l4iT5‘il40“
`
`
`
`
`ANN ROBERTSON
`NOTARYPUBUOSTATE or UTAH
`
`S
`:7
`COMMlSSlON# 656494
`COMM. EXP. O5-30~20t5
`
`
`
`
`_
`
`

`
`Case #3 JP S64—Ol 6596(A)
`
`(19) Japanese Patent
`Office (JP)
`
`(12) Publication of
`Unexamined Japanese
`Patent Application (A)
`
`(51) Int. Cl.“
`C12P
`19/14
`C13K 1/06
`
`ID Code
`
`(1 1) Publication Number‘!
`Unexamined Application S64—016596
`(43) Date of Publication:
`January I, 1989
`Agency Int. Ref. Code
`Z—7236—4B
`82l4—4B
`
`Examination Request Status: Not Yet Requested. No. of Claims: 1 (total 7 pages)
`(54) Title or the Invention:
`STARCH SUGAR CONTAINING MALTOTETRAOSE As A
`PRIMARY COMPONENT
`
`(21) Filing Number:
`(22) Filing Date:
`(72) Inventor:
`
`S62-171608
`July 9, 1987
`Teruo NAKAKUKI
`7 Karno Green Hill
`
`(72) Inventor:
`
`(72) Inventor:
`
`(71) Applicant:
`
`(74) Agent:
`
`57 Karno, Mishima—shi, Shizuoka~ken
`Seishiro KAINUMA
`A—201 Haikopo Kitsunegasaki
`241-2 Mabase, Shimizu-shi, Shizuoka—l<en
`
`Shunsaku NAKANO
`2-404 Nisshoku Kinomiya Company Housing
`2954 lmaizumi, Fuji~shi_, Shizuoka—ken
`Nihon Shokuhin Kako CO, LTD.
`3-4-1 Marunouchi, Chiyoda-ku, TOK)/O—lO
`Shigcru MATSUI, Attorney
`
`1. Title of the Invention
`
`Specification
`
`Starch sugar containing maltotetraose as a primary component
`
`2. Claims
`
`A novel starch sugar containing maltotetraose as a primary component obtained
`1.
`by performing fractionation and purification after or while subjecting a liquefied starch to
`the effects of a maltotetraoseforming enzyme, and a debranching enzyme as necessary,
`to effect a saccharification reaction, the starch sugar containing from 70 to 95 weight%
`maltotetraose, no more than 12 weight% of penta— or higher—order sugars such as
`maltopentaose or maltohexaose, and no more than 18 weight% of tri— or lower—order
`sugars comprising glucose, maltose, and maltotriose.
`2.
`The starch sugar containing maltotetraose as a primary component according to
`claim 1, wherein the content of glucose in the starch sugar is no more than I weight"o.
`3.
`'1“he starch sugar containing rnaltotetraose as a primary component according to
`claim l or 2, wherein the content of hexa— and higher order sugars such as maltohexaose
`in the starch sugar is no more than 8 weight%.
`
`

`
`Case #: JP S64—0l6596(A)
`
`3. Detailed Description of the Invention
`Technical Field
`The present invention relates to a novel starch sugar containing maltotetraose as a
`primary component obtained by performing fractionation and purification after or while
`subjecting a liquefied starch to the effects of a maltotetraose-forming enzyme, and a
`debranching enzyme as necessary, to effect a saccharification reaction.
`Conventional Technology and Problems Inherent Therein
`Starch sugars obtained by subjecting starch to the effects of various amylases,
`such as glucose, isomerized sugar, maltose, cyclic dcxtrin, and polymeric dextrin sugar
`powder. are produced at industrial scales. Various oligosaccharides, from trisaccharides
`to hexasaccharides and the like, are also known as sugars positioned between the
`abovementioned mono— and disaccharides and polymeric dextrin.
`Various oligosaccharide-forming amylases capable of specifically producing tri-
`to hexasaccharides have been discovered in recent years (see Keiji Kainuma, Journal of
`the Japanese Society of Starch Science, Vol. 28, p. 92 (1981)), and the production of
`various oligosaccharides is becoming easier. However, these various oligosaccharide—
`forming amylases are still inefficient to produce and costly to use, and tri— and hi gher—
`order oligosaccharides, being crystallization resistant, are difficult to produce via
`crystallization methods as in the case of, for example, glucose or maltose. These
`problems have led to delays in development.
`FIG. 6 shows the level of sweetness for various sugars from monosaccharides
`through heptasaccharides (G1—G7) compared to a sweetness of 100 for table sugar.
`It can
`be seen from the figure that sweetness generally decreases as the level of glucose
`polymerization increases. Moreover, FIG. 7 shows the inherent viscosities of various
`sugars from monosaccharides through decasaccharides (G;~—G10).
`It can be seen from the
`figure that viscosity increases as the level of glucose polymerization increases.
`When adding sugars as sweeteners to food products, it may actually be preferable
`to reduce the level of sweetness to a certain degree in order to improve flavor, as this will
`yield a richer flavor. Conventionally, various dextrins and other types of comparatively
`highly polymerized starch sugars have been used when attempting to reduce sweetness.
`However, the use of dextrins, which have high levels of glucose polymerization, has the
`problem ofincreasing viscosity, negatively affecting the physical properties of the
`finished product.
`For such reasons, there is a demand for the development of a sugar that has both a
`low level of sweetness and low viscosity.
`Object, of the Invention
`An object of the present invention is to provide a novel low~sweetness, low-
`viscosity starch sugar containing maltotetraose as a primary component.
`Description of the Invention
`The present invention is characterized in being a novel starch sugar containing
`maltotetraose as a primary component obtained by performing fractionation and
`purification after or while subjecting a liquefied starch to the effects of a maltotetraose
`forming enzyme, and a debranching enzyme as necessary, to effect a saccharification
`reaction, the starch sugar containing from 70 to 95 weight% maltotetraose, no more than
`12 weight% of penta— or higher—order sugars such as maltopentaose or rnaltohexaose, and
`
`

`
`Case f-7‘: JP S64—0l6596(A)
`
`no more than 18 weight% of the tri- or lower—order sugars including glucose, maltose,
`and rnaltotriose.
`Because the starch sugar according to the present invention contains maltotetraose
`as a primary component, as described above, it has a reduced level of sweetness, allowing
`it to yield a full, rich flavor.
`In addition, the starch sugar does not have an especially high
`level of glucose polymerization, and thus has been kept to a low viscosity. Thus, the
`starch sugar can be added to various food products to impart a rich, moderately sweet
`flavor while keeping down increases in viscosity arising from the addition of sugars,
`thereby maintaining good physical properties.
`Moreover, in the present invention, a maltotetraose content of less than 70
`weight"/o will not sufficiently yield the effects of reducing sweetness while keeping
`viscosity low, and a maltotetraose content of greater than 95 weight% will necessitate
`multiple steps in the process of producing the sugar liquid, and is thus impractical.
`Additionally, an amount of penta- and higher—order saccharides, such as maltopentaose
`and maltohexaose, greater than 12 weight% will make it difficult to keep viscosity low.
`An amount of the tri— or lower-order sugars including glucose, maltose, and maltotriose
`greater than 18 weight% will tend to yield too high a level of sweetness, with richness of
`flavor being lost.
`A preferred embodiment of the present invention contains 1 weight% or less of
`glucose, and contains 8 weight% or less of hexa~ and hi gher—order saccharides such as
`maltohexaose.
`
`Preferred Embodiments of the Invention
`The liquefied starch used as a raw material in the present invention can be
`obtained according to a normal method, such as, for example, heating and gelatinizing
`starch and adding 0c—amylase to cause a reaction.
`In the present invention, this liquefied starch is subjected to the effects of a
`maltotetraose-forming enzyme, and a debranching enzyme as necessary, to cause a
`saccharification reaction.
`
`The maltotetraose-forming enzyme cleaves the bonded glucose molecules in the
`starch into groups of four units, starting at a non—reducing terminal, to form maltotetraose.
`A Pseudomonas stulzerz'—derived extracellular enzyme (see J .F. Robyt and R1. Ackerman,
`Arch. Biochem. Biophys. 145 105 (1971)), Pseudomonas sacc//zar0phila~derived
`extracellular enzyme (see Okemoto et al., Japan Society for Bioscience, Biotechnology,
`and Agrochemistry, Addresses for 1985, p. 370), or the like is preferably used as the
`maltotetraose—forming enzyme.
`A debranching enzyme is necessary in order to cleave B—1,6 bonds within the
`starch molecule and form amylose. lfa debranching enzyme is not used, the reaction
`caused by the maltotetraose—forming enzyme will be either stopped or drastically retarded
`at the 163- 1 ,6 bonds, leading to a tendency toward reduced rnaltotetraose yield. Preferable
`debranching enzymes include, for example, Klebslellaprzeumoniwderived pullulanase
`(see 11. Bender and K. Wallenfels, Biochem. 21,3551 79(_1961)), Pseudomarzas
`amyloderamosa-derived isoamylase (see '1‘. Harada et al., Appl. Microbiol. 16, 1439
`(1968)), and the like. Pullulanas is more preferable from considerations ofthe ideal pH
`for the maltotetraose—forming enzyme.
`Moreover, the sources of the debranching enzyme and maltotetraose-forming
`enzyme of the present invention are not limited to the microorganisms mentioned above.
`
`

`
`Case #: JP S64~0l6596(A)
`
`In a saccharification reaction using the abovementioned enzymes, the liquefied
`starch may be subjected to the effects of the rnaltotetraose-forming enzyme after having
`been subjected to the effects of the debranching enzyme, or may be subjected to the
`effects of both the debranching enzyme and the maltotctraose—forming enzyme
`simultaneously; alternatively, the maltotetraose—forming enzyme may be used alone. The
`saccharification reaction may be performed using any method, such as a batch method, a
`bioreactor method using a multi—enzyme system containing both enzymes in solidified
`form, a membrane reactor method, or the like.
`If a membrane reactor method is employed, the formation of low—molecular-
`weight sugars must be prevented in order to obtain a purity of 70 weight”/o or higher for
`the maltotetraose in the transmitted liquid‘, to which end a maltotetraose~forming enzyme
`is preferably used alone. Moreover, the membrane used preferably has as small a
`molecular weight fractionation range as possible; for example, a limited f1ltration2
`membrane having a molecular weight fractionation range of no more than 8,000 or a
`reverse osmosis membrane having a sucrose impedance rate of 20 to 40% or greater can
`be effectively used.
`Furthermore, in a saccharification reaction in which both a maltotetraose—forming
`enzyme and a debranching enzyme are used, a saccharified liquid containing
`approximately from 50 to 65 weight% maltotetraose can be obtained. However, in order
`to further refine this saccharified liquid to obtain the starch sugar according to the present
`invention, which contains from 70 to 95 weight% maltotetraose, contains no more than
`12 weight% of penta— or higher—order sugars such as maltopentaose or maltohexaose, and
`contains no more than 18 weight% of the tri— or lower—order sugars including glucose,
`maltose, and maltotriose, various chromatographic refining methods can be employed,
`such as gel filtration chromatography, carbon column chromatography, strong acid cation
`exchange resin column chromatography, and the like.
`Examples of the Present Invention
`Example l
`Potato starch was liquefied according to a normal method to obtain a liquefied
`starch having a DB of no more than 1 and a concentration of 10 weight%. 2 units of a
`maltotctraose—forrning enzyme per substrate solid components was added thereto, pH was
`adjusted to 6.5, and the mixture was reacted at 55°C for 10 hours, after which
`diatomaceous earth filtration was performed to remove impurities.
`ln the present context,
`“one unit” refers to the amount of enzyme needed to perform an enzymatic reaction at
`pH 7 and temperature 40°C using reduced soluble starch as a substrate and hydrolyze l
`umol of glycosidic bonds in one minute. Next, a 0.36 m2 ultrafiltration membrane having
`a molecular weight cut—off of 2,000 was attached to a flat sheet membrane UF device
`(manufactured by DDS; product name “LaboModule”3), the reacted liquid was reduced at
`55°C for 10 hours, and both saccharification and membrane treatment were performed.
`The saccharide composition of the filtrate thus obtained (hereafter “G470”) is shown in
`(a) of Table 1, and a chromatogram obtained via high-speed liquid chromatography is
`shown in FIG. l.
`
`Example 2
`
`in the original. Possible error for i%ll:?t§”(“saccharitied liquid")?
`'
`ffififliléibi
`In the original. Possible error for |3l§15’t7.>if§ ("ultrafiltra1ion”)?
`’ Could not find official English name.
`
`4
`
`

`
`Case #: .11’ S64~0l6596(A)
`
`A 5 weight% aqueous suspension of waxy corn starch was prepared, pll was
`adjusted to 3.8 using hydrochloric acid, and autoclaving was performed at 121°C for 30
`minutes using a jar fermenter. After cooling the suspension to 55°C, 5 units isoamylase
`(produced by Hayashibara Biochemical Laboratories) per gram substrate was added, and
`the mixture was reacted for one day to perform amylopectin debranching. The mixture
`was then autoclaved at 121°C for 20 minutes, cooled to 55°C, and adjusted to a pH of 6.5,
`2 units maltotetraose—forming enzyme per gram substrate was added, and the mixture was
`reacted for 30 minutes. The saccharide composition of the obtained reacted liquid was
`G1: 0.5. G2: 40. G3: 5.0 G4: 705, G3: 3.0, G6: 05, G7: 0.2, and Gg and higher: 16.3 (all
`numbers in terms of weight%).
`In Gx, x indicates the level of glucose polymerization
`(likewise hereafter).
`The reacted liquid was further autoclaved to deactivate the enzymes, then
`decolorized using activated charcoal, deionized using an ion exchange treatment, and
`condensed to 8X 60.
`
`Next, a column of 13.5 cm inner diameter >< length 6.4 m was charged with
`sodium—type “Dowex 99” (product name; Dow Chemical Co.) as a carrier for the fraction,
`and a cation exchange ion resin chromatography column was constituted so that the liquid
`sugar load was 5.7% (v/V) with respect to the resin. The condensed liquid was passed
`through the chromatography column at conditions of a temperature of 75°C and a speed
`of SV 0.34 hr, and a maltotetraose—rich fraction was gathered. The saccharide
`composition of the liquid sugar (hereafter, G480”) so obtained is shown in (b) in Table 1,
`and the chromatogram obtained via high—speed liquid chromatography is shown in PK}. 2.
`Example 3
`After condensing the liquid sugar obtained in example 2 (G480) to 8><60, the
`liquid was again subjected to cation exchange resin column chromatography in a manner
`similar to that of example 2 to gather a maltose—rich fraction. The saccharide
`composition of the liquid sugar so obtained (hereafter, "‘G485”) is shown in (c) in Table 1,
`and the chromatogram obtained via hi gh—speed liquid chromatography is shown in FIG. 3.
`(rest ofcolumn blank)
`
`Table l (Saccharide compositions oflifliid sugars obtained in examples 1 to 3)
`Total for G8
`
`G1
`
`G2
`
`G3
`
`G4
`
`G5
`
`G6
`
`G7
`
`(6)0470
`(119480
`(6)9435
`
`0.5 L 3.3
`0.3 g 2.6
`0
`1.4
`
`13.3
`10.7
`8.6
`
`4.9
`71.2
`5.1
`73.5
`85.9 A 3.6
`
`0.6
`1.3
`0.3
`0.7
`0.2 1 0.1
`
`and higher-
`__order sugars
`4.9
`1.8
`0.2
`
`Test Example 1
`Sweetness, richness, and starch odor were measured for each of the liquid sugars
`(G470, G480, G485) obtained in examples 1 through 3, using table sugar, glucose,
`maltose, and sugar powder as references. Sweetness was evaluated against a value of 100
`for table sugar.
`In the richness evaluations, ~ indicates no richness, + indicates some
`richness. and ++ indicates a full, round rich flavor. Evaluation results are shown in Table
`2.
`
`

`
`Case #: JP S64—Ol6596(A)
`
`(Sweetness/richness/starch odor comparison)
`Table
`Specimen
`Sweetness
`Richness
`“G470
`20
`++
`MG480
`I8
`++
`_Z G485
`17
`++
`Ta_ble sugar
`100
`—~
`Glucose
`70
`~
`Maltose
`40
`+
`Sugar powder
`25
`+
`
`r__
`
`_
`
`_
`
`V _S_tarch odor
`N0
`No
`No
`No
`No
`No
`Yes
`
`J
`
`Test example 2
`’l‘he relationship between sugar concentration and viscosity was measured for the
`liquid sugars (G470, G480, G485) obtained in examples I through 3, using enzymatic
`starch syrup and table sugar as references. Results are shown in FIG. 4. As can be seen
`from the figure, the liquid sugar according to the present invention is somewhat more
`viscous than table sugar, but clearly less viscous than enzymatic starch syrup.
`Test example 3
`The relationship between heat treatment temperature and degree of coloration was
`measured for the liquid sugars (G470, G480, G485) obtained in examples I through 3,
`using enzymatic starch syrup, table sugar, and glucose as references. Each of the liquid
`sugars was adjusted to 8850 and pH 4.5, and heat—treated via autoclave for 1 hour.
`Degree of coloration was evaluated in terms of light absorption at OD 420 nm. Results
`are shown in FIG. 5. As can be seen from the figure, even at high temperatures the starch
`sugar according to the present invention exhibits very little coloration.
`Effectswof the Invention
`Because the starch sugar according to the present invention contains maltotetraose
`as a primary component, as described above, sweetness can be reduced, thereby allowing
`a full, rich flavor to be imparted.
`In addition, because the starch sugar has a relatively
`low degree of glucose polymerization, viscosity is kept low. The starch sugar can
`therefore be added to various food products to impart a rich, moderately sweet flavor
`while keeping down increases in viscosity arising from the addition of sugars, thereby
`maintaining good physical properties, Furthermore, the starch sugar also has the
`advantage of exhibiting very little coloration at high temperatures.
`
`4. Briet’ Explanation of the Drawings
`FIG.
`1
`is a chromatogram ofa liquid sugar obtained according to a first example,
`the chromatogram having been obtained using high—speed liquid chromatography;
`FIG. 2 is a chromatogram ofa second example, the chromatogram having been
`obtained using high—speed liquid chromatography;
`FIG. 3 is a chromatogram of a third example, the chromatogram having been
`obtained using high—speed liquid chromatography;
`1?‘ IG. 4 is a graph showing the relationship between sugar concentration and
`viscosity for various liquid sugars;
`
`

`
`Case #: JP S64—Ol6596(A)
`
`FIG. 5 is a graph showing the relationship between heat treatment temperature
`and degree of coloration for various liquid sugars;
`FIG. 6 is a graph showing sweetness levels for various sugars, from
`monosaccharides to heptasaccharides (G;—G7); and
`HG. 7 is a graph showing intrinsic viscosities for various sugars, from
`monosaccharides to decasaccharicles (G;—G,0).
`
`FIG.
`
`1
`
`
`
`FIG. 2
`
`

`
`Case "ff: JP S64-016596(A)
`
`FIG. 3
`
`
`
`Enzymatic
`stamh SYN”
`
`
`
`
`Specimen 470
`~485
`
`
`10
`20
`30
`1.0
`50
`60
`70
`
`Sugar Concentration (Bx)
`
`

`
`Case 151 JP S64—Ol6596(A)
`
`FIG. 5
`
`.0 no
`
`.1
`
`C) N
`
`absorptionatOD420nm) O
`Coloration(Light
`
`
`
`
`Specimen
`G470 - 485
`
`100
`
`110
`
`120
`
`130
`
`MO
`
`Heat Treatment Temperature
`(Autoclave, Bx 50, pH 4.5, 1 hour)
`
`FIG. 6
`
`Sweetness
`
`50
`
`100
`
`

`
`Case ii; JP S64—Oi6596(A)
`
`FIG. 7
`
`0.05
`
`0.01.
`
`C.)
`
`F“
`
`0.03
`
`0.02
`
`
`
`IntrinsicViscosity
`
`Gm
`G9
`
`6“
`
`G7
`G6
`
`55
`54
`
`52
`G3
`
`10