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`Original text full file name: JP04-0457941A1.
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`
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`' 20, 2017
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`

`

`(19) Japan Patent Office (JP)
`
`11) Japanese Unexamined Patent
`
`(12) Japanese Unexamined Patent
`
`Application Publication Number
`
`Application Publication (A)
`
`H04-45794
`
`(51) Int Cl 5
`'
`'
`19/20
`1/09
`19/14
`
`C 12 P
`A 23 L
`C 12 P
`
`”A 23 L
`1/236
`
`Identification
`codes
`
`Z
`
`A
`
`JPO
`numbers
`8214-48
`2121-48
`8214-48
`
`7823-48
`
`file
`
`(43)Pub|ication date February. 14 Heisei 4
`(1992)
`
`Request for examination Not Requested Yet
`Number of claims
`2
`(Total of 5 pages)
`
`
`Tsurumi—Ku, Yokohama
`
`City,
`
`(54) Title of the invention METHOD FOR MANUFACTUR‘NG MALTOPENTAOSE SYRUP
`(21) Application Number
`HO2—150048
`(22) Filing Date
`June 11, Heisei 2 (1990)
`(72) Inventor
`Takashi Okemoto
`6-5—28 Kitaterao,
`Kanagawa Prefecture
`Akihisa lwamoto
`
`(72)lnventor
`
`7—25—31 Kitaterao, Tsurumi-Ku, Yokohama City,
`
`(72) Inventor
`
`(72) Inventor
`
`(72)lnventor
`
`(71) Applicant
`
`(71) Applicant
`
`(71) Applicant
`(74) Agent
`
`Kanagawa Prefecture
`Kozo Hara
`2-6—3-104 Namiki, Kanazawa—Ku, Yokohama City, Kanagawa
`Prefecture
`
`HitoshiHashimoto
`Prefecture
`
`ShoichiKobayashi
`Prefecture
`
`4-31—10
`
`lmaizumidai, Kamakura City, Kanagawa
`
`2—9—2 Otsudominami,
`
`Tsuchiura
`
`City,
`
`lbaraki
`
`13—46 Daikoku-Cho, Tsurumi-Ku,
`
`Ensuiko Sugar Refining Co, Ltd.
`Yokohama City, Kanagawa Prefecture
`The President, Natural Food Research Institute, Ministry of Agriculture,
`Forestry and Fishery of Japan
`2-1-2 Kannondai, Tsukuba City, lbaraki Prefecture
`Research Development Corporation of Japan
`Fujio Kubota, Patent attorney
`
`Description
`1. Title of the invention
`FOR
`METHOD
`MALTOPENTAOSE SYRUP
`
`MANUFACTURIG
`
`,
`2. Claims
`(1) Math“ f°r mammmmg maltopentaose syrup which is
`characterized in that at least one material selected from
`
`a group consisting of starch, composing fractions of
`starch, and decomposition product of starch is reacted
`with maltopentaose forming enzyme, and then the
`resulting reaction product is reacted with q—amylase.
`(2) The method according to Claim 1, wherein the said
`maltopentaose
`forming
`enzymehas
`the
`following
`characteristics:
`
`i) The said enzyme reacts with amylose, soluble
`starch, potato starch, ocarina starch,
`rice starch,
`tapioca starch, corn starch, sugarcorn starch, sago
`starch and the like to form maltopentaose.
`ii) The optimum pH for the said enzyme is 6 to 7 at
`45°C, and the said enzyme is stable at pH range of 6.5
`to 9.0.
`
`iii) The optimum temperature for the said enzyme at
`pH 6.5 is 50 to 55°C, and the said enzyme will be
`deactivated after being left at 55C or higher for 15
`minutes.
`
`inhibited
`is
`said enzyme
`iv) The
`solution
`and
`p—chloromercuribenzoate
`monoiodoacetamide
`solution
`and
`the
`
`in 0.4leI
`in
`1mM
`inhibition
`
`percentage thereof is 40 to 50%.
`v) The molecular weight of the said enzyme is
`72500i2500 (by a disc gel electrophoresis method).
`vi) The isoelectric point of the said enzyme is pH 6.5
`(by an ampholine electrophoresis method).
`3. Detailed description of the invention
`[Field of invention]
`The present
`invention relates to a method for
`manufacturing maltopentaose (hereinafter, referred to
`as (35) syrup, and particularly relates to a method for
`manufacturing G5
`syrup
`containing
`quite
`small
`amounts
`of maltooligosaccharide
`having
`the
`polymerization degree of maltohexaose (hereinafter,
`referred to as G6) or higher and high—molecular—weight
`dextrin.
`
`[Prior Art and Problems to be Solved by the Invention]
`Recently, the research on maltooligosaccharide has
`been developed; an enzyme selectively producing
`maltooligosaccharide has been found or the usage of
`maltooligosaccharide has been developed. Accordingly,
`there have been many proposals of producing methods
`of maltooligosaccharide.
`
`559
`
`

`

`G5 syrup is expected to be used as a clathrate
`powderization base, a food improving agent, and
`a material for nutrient food for sick peopie, infant
`children, and the aged. Conventionally,
`the G5
`syrup has been produced by reacting G5 forming
`enzyme to a material, such as starch. And the
`enzyme derived from Genus Pseudomonas
`bacteria found by the inventors (See Japanese
`Examined Patent Application Publications Showa
`59-44609 and Showa 59—44070) has been used
`as the G5 forming enzyme.
`However,
`the G5 syrup produced by this
`method
`contains
`more
`than
`20% of
`
`maltooligosaccharide having the polymerization
`degree of G6 or higher or high—molecular—weight
`dextrin.
`Accordingly,
`when
`the
`sugar
`concentration is higher the turbidity becomes high
`and it is not preferred as a product.
`the present
`Therefore,
`the
`inventors
`of
`invention
`conducted
`a
`research
`on
`the
`development of methOdmmanamWw G5 starch which
`contains
`lowest
`possible
`amounts
`of
`maltooligosaccharide having the polymerization
`degree
`of
`G6
`or
`higher
`or
`high—molecular-weighted dextrin.
`[Means to Solve the Problems]
`As
`a
`result,
`the inventors
`
`found that
`
`the
`
`objective G5 syrup can be obtained by acting
`d-amylase on the reaction product produced by the
`reaction of G5 forming enzyme and a material,
`such as starch; thus the inventors could complete
`the present invention
`That is to say, the present invention provides a
`math“ “3’ manumur'r‘g maltopentaose syrup which is
`characterized in that at least one material selected
`
`from a group consisting of starch, composing
`fraction of starch, and decomposition product of
`starch is
`reacted with maltopentaose forming
`enzyme, and then the resulting reaction product is
`reacted with d~amylase.
`In the present invention a variety of starches
`may be used. For example, starches obtained from
`arbitrary materials, such as potato, ocarina, rice,
`tapioca, corn, sugarcorn, sago, barley, and wheat,
`may be used alone or combined with more than
`one other starch. Additionally, there are composing
`fractions of starch, such as, for example, amylose
`or
`amylopectin.
`Furthermore,
`there
`are
`decomposition products of starch,, such as white
`dextrin, yellow dextrin,
`roasted dextrin, such as
`British gum; modified starch,, such as oxidized
`starch, and low-viscosity modified (by treatment
`with an enzyme, acid or mechanical high-speed
`stirring etc.,) starch;
`
`starch ether represented by phosphate starch
`and acetate starch; starch derivatives, such as
`starch esters; physically treated starch, such as
`starch treated with radiation, neutron radiation,
`radio-frequency radiation or moist-heat treatment;
`and d-starch may be used. These starches may
`be used alone or combined with more than one
`other starch.
`
`Next, as the G5 forming enzyme, for example,
`the enzyme produced by Pseudomonas KO~894O
`(FERM P-7456, see Japanese Examined Patent
`Application Publications Showa 61—49955 for the
`mycological properties of this bacterium) may be
`used. The properties of this enzyme shall be
`shown below.
`
`i) The said enzyme reacts with amylose,
`soluble starch, potato starch, ocarina starch, rice
`starch,
`tapioca starch, corn starch, sugarcorn
`starch,
`sago starch and the
`like
`to
`form
`maltopentaose.
`ii) The optimum pH for the said enzyme is 6
`to7 at 45°C, and the said enzyme is stable at pH
`range of 6.5 to 9.0.
`the said
`iii) The optimum temperature for
`enzyme at pH 6.5 is 50 to 55°C, and the said
`enzyme will be deactivated after being left at 55°C
`for 15 minutes.
`
`inhibited
`is
`said enzyme
`iv) The
`solution
`and
`p-chloromercuribenzoate
`monoiodoacetamide
`solution
`and
`the
`
`0,4mM
`in
`1mM
`in
`inhibition
`
`percentage thereof is 40 to 50%.
`v) The molecular weight of the said enzyme is
`72500i2500
`(by
`a
`disc
`gel
`electrophoresis
`method).
`vi) The isoelectric point of the said enzyme is
`pH 6.5 (by an ampholine electrophoresis method).
`Additionally, as the d-amylase,
`the enzyme
`produced by Bacillus licheniformis (produced by
`Novo Nordisk
`Bio
`Industry,
`Product
`name:
`TERMAMYL) and the like may be used.
`In the
`present invention,
`in order to produce the G5 with
`higher
`efficiency,
`puilulanase which
`is
`a
`debranching enzyme may be used together.
`The starch source used for the production of the
`G5 syrup may be liquefied, as needed. For the
`liquefacation,
`the enzyme liquefacation method
`using an enzyme, such as d~amylase of liquefying
`type or physical method using acids or bases, may
`be used.
`
`Since the formation rate of G5 may be affected
`the
`enzyme
`concentration,
`substrate
`by
`concentration,
`reaction pH,
`reaction temperature
`and the like,
`the optimum conditions should be
`found.
`
`560
`
`

`

`The G5-forming enzyme is stable at the pH
`range of 6.5 to 9 and the formation rate of G5 is
`increased by performing the reaction at the pH
`range of 8 to 8.5. Additionally,
`the lower
`the
`substrate concentration is, the higher the G5 yield
`is;
`however,
`the
`substrate
`concentration
`is
`preferred to be 5 to 20% industrially. Regarding the
`reaction temperature, 45 to 50°C is appropriate;
`however, under the presence of calcium ion,
`the
`reaction can be carried out at 60°C or
`lower.
`
`Meanwhile, the amount of enzyme may be selected
`so that the reaction can be performed sufficiently
`and it is not necessary to use an excess amount of
`enzyme.
`At the same time, d-amylase (product name:
`TERMAMYL) is generally reacted at the pH range
`of 6 to 6.5; however, it may react well under the pH
`range of 5 to 8. The operative temperature of this
`enzyme is 95 to 100°C; however, 50"C was used in
`the present invention.
`reaction of G5
`formation
`Accordingly,
`the
`according to
`the present
`invention may be
`preferably carried out by reacting the G5 forming
`enzyme under 5 to 20% of substrate concentration
`at
`the pH range of 8 to 8.5 at 50°C;
`then the
`G5-forming enzyme is acted; then the enzyme is
`deactivated at an appropriate stage to terminate the
`reaction;
`then
`d—amylase
`(product
`name:
`TERMAMYL) is added thereto and the reaction is
`carried out at pH 6 and at 50°C. Meanwhile, when
`the debranching enzyme is added thereto, there is
`no limit for the amount and it may be added before
`
`The enzyme solution 0.1mL was added to
`reduced 2% soluble starch (produced by Merck
`KGaA) 0.5mL, and 01M phosphate buffer 0.4mL;
`then the reaction was carried out at 45°C and the
`amount
`of
`resulting
`reducing
`sugars were
`measured
`by
`Somogyi-Nelson method. The
`enzyme activity to cleave 1pmol of glucoside bond
`per 1 minute was defined as 1 unit (IU). The activity
`of d—amylase
`(product name: TERMAMYL)
`to
`decompose 5.269 of starch (produced by Merck
`KGaA) per 1 hour at 37:0.05°C, under calcium
`concentration of 0.0003M, and at pH 5.6 was
`defined as 1 Novo d—amylase activity (1NU). The
`activity of pullulanase to increase the amount of
`reducing power corresponding to 1uM glucose at
`40:0.1”C,
`at
`pH 6.0, and in 0.5% substrate,
`pullulan, aqueous solution in 1 minute was defined
`as 1 pullulanase potency unit (PUN).
`[Embodiment 1]
`Bacterial q—amylase of liquefying type 18.8mg,
`and water 150mL were added to corn starch 159,
`and the mixture was stirred in a boiling water bath
`to
`liquefy the starch;
`then the mixture was
`immediately autoclaved at 120°C for 15 minutes;
`then the mixture was cooled and the pH was
`adjusted to pH8.4 with aqueous sodium hydroxide
`
`or at the same time to the addition of G5-forming
`enzyme.
`The G5 syrup obtained according to the
`present invention contains 50% or more of G5,
`and the total of sugar less than G5, such as
`glucose (hereinafter, referred to as G1), maltose
`(hereinafter,
`referred to as G2), maltotriose
`(hereinafter,
`referred
`to
`as
`G3),
`and
`maltotetraose (hereinafter, referred to as G4) is
`40% or more;
`however,
`the
`amount
`of
`high—molecular-weight
`sugar
`having
`the
`polymerization degree
`of G6
`or more
`is
`suppressed to 10% or less.
`As a result, the characteristics of G5 syrup
`are improved, and clear and high-concentration
`G5 syrup even at
`low temperature may be
`obtained. Also, the sweetness thereof is slightly
`increased.
`
`[Embodiments]
`Next, the present invention shall be explained
`in more detail by referring to the embodiments.
`Meanwhile, the G5 forming enzyme used in the
`embodiment was
`prepared
`as
`follows:
`Pseudomonas KO-894O (FERM P-7456) was
`cultivated according to the method of Hashimoto
`etal.
`(Appl. Microbiol. Biotechnol, 1986 25:
`137—142); then the culture fluid is condensed by
`an ammonium sulfate
`salting-out;
`and the
`resulting condensed fluid is dissolved in a buffer
`solution to make the crude enzyme solution. The
`enzyme activity was measured according to the
`following method, and was 52lU/mL.
`
`solution. Subsequently, pullulanase 450PUN and
`G5 forming enzyme 45lU were added thereto, and
`the reaction was carried out at 50 degrees for 9
`hours. Then,
`the enzymes were deactivated by
`heating the reaction mixture at 100C for 30
`minutes. The sugar composition at this moment is
`shown as Sample 1
`in Table 1.
`One hundred fifty pL of an q—amylase (product
`name: TERMAMYL 60L) 150uL was added to the
`above—mentioned solution and the reaction was
`carried out at 50C for 2 hours. Immediately after
`the reaction was completed, the pH of the reaction
`mixture was adjusted to pH 4.5 with hydrochloric
`acid, and then the mixture was autoclaved at 120°C
`for 15 minutes, Subsequently,
`insoluble materials
`were removed by a diatomite filtration to obtain the
`G5 syrup. The composition of syrup is shown as
`Sample 2 in Table 1.
`Table 1
`
`Sugar composition (%)
`
`G4G1 G2 GS 66 G6< (35
`
`
`Sample 0
`5.3
`7.0
`5.6
`50.5
`4.9
`26.7
`
`
`1 Sample 14
`
`
`
`
`
`
`
`9.6
`
`16.1
`
`7.9
`
`55.4
`
`3.2
`
`6.4
`
`2
`
`561
`
`

`

`carried out at 50 degrees for 5 hours. Then, the
`enzyme was deactivated by heating the reaction
`mixture at 100°C for 30 minutes. The sugar
`composition at this moment is shown as Sample 3
`in Table 2.
`
`The pullulanase 2100PUN and d—amylase
`(product name: TERMAMYL 60L)
`100uL was
`added to the above-mentioned solution and the
`reaction was carried out at 50°C for 5 hours.
`
`immediately after the reaction was completed, the
`pH of the reaction mixture was adjusted to 4.5 with
`hydrochloric acid,
`and then the mixture was
`autoclaved at 120°C for 15 minutes. Subsequently,
`insoluble materials were removed by a diatomite
`filtration to obtain the G5 syrup. The composition of
`syrup is shown as Sample 4 in Table 2.
`Table 2
`
`Sugar composition (0/0)
`
`G1 G2
`(33
`G4 G5
`G6 G6<
`
`Sample 0
`
`0.9
`
`1.6
`
`1.8
`
`29.1
`
`0.7
`
`65.9
`
`
`
`
`
`
`
`
`
`
`ample 3.1l12.4 18.0‘86‘516 2.2
`
`4.4
`
`3 S
`
`4
`
`As clearly shown in the table, the total of the
`high—molecular—weight portion consisting of G6
`and more than G6 in the obtained G5 syrup was
`6.6% and low—molecular—weight sugar including
`G5 accounting for 51.3% comprises the majority.
`
`insoluble materials were removed by a diatomite
`filtration to obtain the G5 syrup. The composition of
`syrup is shown as Sample 6 in Table 3
`
`. Table 3
`
`Sugar composition (0%)
`
`I G1 | G2
`G3
`G4 GS
`G6 G6<
`Sample! 0
`I 6.2
`8.4
`5.3
`49.7 4.1
`26.3
`ample] 2.8 ) 12.0
`15.9
`7.8
`53.4
`1.7
`6.4
`
`
`
`5 S
`
`
`
`the total of the
`As clearly shown in tre table,
`high—molecular-weight portion consisting of G6 and
`more than G6 in the resultant G5 syrup was 8.1%
`and
`low—molecular—weight
`sugar
`including G5
`accounting for 53.4% comprises the majority,
`[Effect of the invention]
`the
`invention,
`present
`According
`to
`the
`maltopentaose containing quite small amounts of
`maltooligosaccharide having the polymerization
`degree
`of maltohexaose
`or
`higher
`and
`high—molecular—weight dextrin can be obtained,
`The resultant G5 syrup is expected to be used as a
`clathrate powderization base, a food improving
`agent, and a material
`for nutrient food for sick
`people, infant children, and the aged.
`
`562
`
`
`
`
`
`
`
`
`
`
`
` 6
`
`As clearly shown in the table, the total of the
`high—molecular—weight portion consisting of G6
`and more than G6 was 31.6% and it was reduced
`
`to 9.6% by the TERMAMYL reaction. Additionally,
`G5 was increased from 50.5% to 55.4%. The
`
`sugar composition of the resulting reaction liquid
`was analyzed by
`a high-performance liquid
`chromatography (hereinafter,
`referred to
`as
`HPLC), The analytical condition for the HPLC
`was as follows:
`
`Used apparatus
`
`Column
`
`Eluent
`
`: LC-4A manufactured
`by
`Shimadzu
`Corporation
`:
`
`Aminex
`
`Carbohydrate
`HPX-42A
`
`by
`manufactured
`Bio-Rad Laboratories
`
`lnc.7.8mm (diameter)
`x 300mm
`: distilled water
`
`: 80°C
`Temperature
`: Rl[Embodiment 2]
`Detection
`Bacterial q—amylase of liquefying type 17.5mg,
`and water 700mL were added to sugarcorn starch
`709, and the mixture was stirred in a boiling water
`bath to liquefy the starch;
`then the mixture was
`immediately autoclaved at 120°C for 15 minutes;
`then the mixture was cooled and the pH was
`adjusted to 8.45 with aqueous sodium hydroxide
`solution. Subsequently,
`the G5—forming enzyme
`140lU was added thereto, and the reaction was
`
`[Embodiment 3]
`liquefying type 32mg,
`Bacterial o—amylase of
`and water 400mL were added to sugarcorn starch
`40g, and the mixture was stirred in a boiling water
`bath to liquefy the starch;
`then the mixture was
`immediately autoclaved at 120°C for 15 minutes;
`then the mixture was cooled and the pH was
`adjusted to 8.4 with 100 mL of 0.05M Tris—HCl
`buffer solution. Subsequently, G5—forming enzyme
`200|U and pullulanase 1200PUN were added
`thereto, and the reaction was carried out at 50
`degrees for 4.5 hours. Then,
`the enzymes were
`deactivated by heating the reaction mixture at
`100°C for 30 minutes and the mixture was cooled.
`The sugar composition at this moment is shown as
`Sample 5 in Table 3. Additionally, the eluting curve
`by the HPLC is shown in Figure 1.
`To the above—mentioned reaction liquid, 3N
`hydrochloric acid was added to adjust
`the pH
`thereof
`to 6;
`then q-amylase (product name:
`TERMAMYL 60L) 200uL was added thereto and
`the reaction was carried out at 50°C for 2 hours.
`
`immediately after the reaction was completed, the
`pH of the reaction mixture was adjusted to 4.5 with
`hydrochloric
`acid,
`and then the mixture was
`autoclaved at 120C for 15 minutes, Subsequently,
`
`

`

`4. Brief description of drawings
`Figure l and Figure 2 are HPLC eluting curves of
`sugar solutions obtained by Embodiments l and 2,
`respectively.
`in Figures, G1 stands for glucose, G2 for maltose,
`GB for maltotriose,
`(34 for maltotetraose, G5 for
`maltopentaose, G6 for maltohexaose, and D for
`dextrin.
`
`Patentapplicant
`
`Agent
`
`Ensuiko Sugar Refining
`Co, Ltd.
`The President, Natural
`Food Research Institute,
`Ministry of Agriculture,
`Forestry and Fishery of
`Japan
`Research Development
`Corporation of Japan
`Fujio
`Kubota,
`Patent
`attorney
`[Seal of the agent]
`
`Figure 1
`
`Figure 2
`
`
`
`0
`
`E
`
`‘0
`
`15
`
`29
`
`Elution Time (min }
`
`Elution Time {min )=
`
`563
`
`