Physical and Chemical Properties of Oligosaccharidesl
`
`J. A. JOHNSON and R. SRISUTHEPZ, Kansas State University, Manhattan 66506
`
`ABSTRACT
`
`Maltooligosaccharides (G1 to G2) from partially hydrolyzed amylose starch were partially
`separated on a Celite-carbon column and further separated and purified by macro-paper
`chromatography. The fractions were shown to be pure, straight-chain molecules of a
`homologous
`glucose
`series. Certain
`physical
`and chemical
`properties of
`these
`oligosaccharides were determined. Reducing power agreed with theoretical values and with
`values ofsome found in the literature. Specific gravity of solutions increased with chain length
`and concentration. Refractive indices did not
`increase with chain length but did with
`increasing concentrations. Solubility decreased with chain length. Oligosaccharides (G9 and
`Gm) did not completely dissolve at 8 to 10% concentrations. Relative viscosity and
`hygroscopicity increased with molecular weight of the oligosaccharides.
`
`Many procedures have been developed to separate maltooligosaccharides
`from starch hydrolysates and to measure certain physical and chemical
`properties of the fractions. Unfortunately, probably because of great difficulty in
`obtaining pure fractions, the values reported for physical properties vary widely.
`To establish physical and chemical properties of these substances would have
`obvious value to scientists and industrial users.
`
`Many investigators have used carbon columns and ethanol solutions to
`separate the maltooligosaccharides (l-1l).They obtained pure fractions of the
`lower—molecular—weight sugars with ease, but experienced increasing difficulty as
`they attempted to separate the higher-polymer fractions. Carbon mixed with
`Celite (diatomaceous earth) with ethanol gradients has been used (3,6,l0,l3,l4).
`Celite aids
`in maintaining a uniform flow—rate. Paper chromatography,
`including cellulose columns, with a wide range in solvents, has been used to
`separate maltooligosaccharides (15-22). These separation methods, while
`precise, usually are applied to small quantities of sugars and for identification.
`Other procedures for isolating oligosaccharides have included forming of the
`borate complex (23), carbon-aluminum oxide columns (24), polyacrylamide gels
`(25), cross—linked starch (6), and gas chromatography (26). Derivatives of the
`sugars have limited value when physical and chemical properties of pure sugar
`polymers are to be measured.
`Numerous investigators have measured the physical and chemical properties
`of the maltooligosaccharides but their data do not agree well. Rt values for
`maltooligosaccharides using paper chromatography with various solvents have
`been reported by Jeanes et al. (l7) and by French and Wild (18). Hoover et al.
`(12) reported values for density, refractive index, viscosity, optical rotation,
`reducing power, and infrared spectra of the maltooligosaccharides from corn
`syrup. Some of their values for the higher—molecular—weight fractions were
`estimated by linear regression. Unusually high reducing values were reported.
`Generally, most measured values increased with chain length, but solubility
`decreased. Commerford and Scallet (27) found the dextrose equivalent to be
`higher than theoretical values for dextrose polymers, G1 to G6. They did not
`
`‘Contribution No. 840, Department of Grain Science and Industry, Kansas Agricultural Experiment Station,
`Manhattan. Condensed from thesis submitted by Rujira Srisuthep in partial fulfillment ofrequirementsfor Master
`of Science degree. Kansas State University.
`"Ri:spcctively: Professor and Graduate Research Assistant.
`
`Copyright © 1975 American Association of Cereal Chemists, Inc., 3340 Pilot Knob Road, St, Paul,
`Minnesota 55121. All rights reserved.
`
`70
`
`TATE & LYLE AMERICAS LLC
`
`EXHIBIT 1016
`
`

`
`January-February
`
`JOHNSON and SRISUTHEP
`
`71
`
`report on higher—molecular—weight polymers. Birch et al. (28) suggested that
`copper oxidation was associated with oxidation beyond the terminal reducing
`group of the polymer. Donnelly et al.
`(29) have shown that degree of
`polymerization has little relation to hygroscopicity.
`In view of the broad interest and wide variation in existing data on physical
`and chemical properties relating to the maltooligosaccharides, we attempted to
`obtain highly purified samples that could be used to measure the physical and
`chemical properties of the homologous glucose series.
`
`MATERIALS AND METHODS
`
`A partially hydrolyzed amylose starch (Morrex T. E)‘ was used as a crude
`source of maltooligosaccharides. Morrex is a dry powder consisting of glucose
`polymers from (31 to GM but mainly G6 and G7.
`The polymers of Morrex were crudely separated on a large Celite~carbon
`column (6 in. X 12 ft.) filled with a mixture of Darco G—60 carbon, granulated
`carbon (20 mesh), and Celite 535 (4:4:2). The carbon—Celite was blended and
`water was added to form a thick slurry which was packed in the Pyrex-column.
`After being packed, the column was washed with one gallon of40% hydrochloric
`acid, then with several gallons of distilled water until the eluant reached a pH of
`3.5. The Celite-carbon column was then loaded with 450 g. of Morrex dissolved
`in 1 liter of water. The column was further washed with 20 gal. ofdistilled water
`before beginning elution with 5% aqueous ethanol. An elution pressure was
`created by sealing the top of the column with an appropriate cap and elevating
`the eluant reservoir 6 ft. above the column cap. The flow rate was approximately
`0.3 gal. per hr. at first, but slowed to 0.08 gal. per hr. at the end ofthe elution with
`50% aqueous ethanol.
`Each gallon of eluant was analyzed for total carbohydrate by the method of
`Dubois et al.
`(30,31). Two milliliters of eluant of each gallon of the
`oligosaccharides was concentrated under reduced pressure and was spotted on
`No. 4 Whatman Chromatography paper, which was then irrigated with n-
`propanol—ethyl acetate—water (6: l:3) for 18 hr. After the paper was dried, the
`chromatogram was dipped successively in silver nitrate, sodium hydroxide in
`methanol and sodium thiosulfate solutions to detect and fix the sugar on the
`paper (32).
`After the eluant had been collected and analyzed for sugar content and type of
`oligosaccharides, the fractions of similar types were combined and concentrated
`in a vacuum evaporator (a dry milk evaporator operated at 26 in. of Hg vacuum
`and 110° F.). The concentrated solution represented a mixture of neighboring
`polymers, cations, and anions originating from the Celite-carbon column.
`The concentrated solutions were deionized by passing through Amberlite IR—
`100 (H+ form) and Dowex IX—8
`(CO3—form) columns. The deionized,
`concentrated solution was further concentrated in a rotary evaporator and
`finally dried to a white powder by lyophylization.
`The
`polymers were
`finally
`separated on washed 3MM Whatman
`Chromatography paper using the procedure of Commerford et al. (21). The
`paper was irrigated with rz—propanol, ethyl acetate, and water( l4:3:7); it took 3 to
`
`V “Corn Products International, Argo, Ill.
`
`

`
`7'3
`
`01.,I('i()53.f\C(j7¥v~l/\RID ESS
`
`Vol, 52
`
`to (ii:
`to 14 days to scpmatcs C}:
`to C}: and 8
`($3
`5 days to SCpE1l’I.l\‘;*
`01ig()S£1CCh€1I‘i(3(%:s. After sczparating and iclentifying the p<‘)'lyznci's, the I‘(tiI121iI‘m‘1g
`paper was cut into strips and the i'ndivid'ua1 poiyrm-:x"s eliltcd with S to 10 ml. of
`deioriizitd wzitazr, The water:m1Liti0n 0.!" the inciividual })(.)1ylTlt;‘1’S was 1‘1‘ee7.e~d ried
`and collected in small vials, which were desiccated over f.‘)h0$p1’10l”Lt5 peiitm<:ic1c=:.
`After collecting many sz1n‘1pless<.)feach aligns;-1cchm'id<: and cmnbining, them. they
`were di>::;c>.§wd in water, .fiILei‘c:.d and fm::ze~di‘ieci. The saimples were i'L1rt|x<:*rch‘ied
`under vacuum and stored in 21 dc,-isiczcamr with phosphoms pcritcuxidic imtii
`physical and chemical propcrtitrs could be c1<tw1'niiru=:<i.
`'Rec1L2c.i11g power was determined by the Smimugyi imzthmi (33) with rczzigcms
`prczpared .:=u’:cm‘dirig to Hudgc and Hoffeiter (34).
`(.72:1cu1zi1i0ri>; wcrc niaicie
`ziiccording, to C.‘0inrnc1'ford at 211. (21).
`
`arr.
`
`6?
`
`62
`
`as
`
`as
`
`as
`
`ea
`
`Fig. 1. Paper chrornatograph of maltooligosaccharides separated from Morrex.
`
`
`
`

`
`January—February
`
`JOHNSON and SRISUTHEP
`
`73
`
`Specific gravity was measured by dissolving samples in water and diluting to 1,
`2, 4, 8, and 10% concentrations. Specific gravities were measured in a 2—ml.
`pycnometer at 20°C. The specific gravities were corrected for buoyancy
`according to Hann (35).
`The homogeneity of the fractions was established by paper chromatography
`(Fig. I). The curvilinear nature of the Rf suggested a homogeneous glucose series
`of relatively high purity. In addition, linearity of the individual oligosaccharides
`was established by B—amylolysis (29). The even-numbered oligosaccharides,
`after 24 hr. of[3—amylolysis, produced only maltose when tested by TLC, whereas
`the odd—numbered oligosaccharides produced mainly maltose with traces of
`glucose and maltotriose.
`Hygroscopicity of the maltooligosaccharides was determined by placing a
`weighed quantity of each oligosaccharide in an aluminum dish that was placed in
`a desiccator containing sulfuric acid of known concentration to regulate the
`humidity at 60% r.h. (36). In addition, humidities were recorded with an Abbeon
`humidiscope and thermometer. Changes in weight of the dishes and polymers
`were determined periodically for 4.5 hr. Hygroscopicities were expressed as the
`percent increase in weight of the polymers by absorption of water.
`
`RESULTS AND DISCUSSION
`
`While glucose and maltose were present in Morrex in small quantities, they
`
`TABLE I. DEXTROSE EQUIVALENTS (D.E.) AND STOICHIOMETRY
`OF MALTOOLIGOSACCHARIDES
`
`Degree
`of
`Polymerization
`
`Hoover
`et al.‘
`(12)
`
`Commerford
`and
`Scailetz
`(27)
`
`Theoretical
`D. E. Value
`
`S$?lF(:2,i:,)::r:y
`Present ——j———?
`Stoichiometry
`Investigation
`D. E. Values
`of Glucose3
`
`G,
`
`G2
`
`G3
`
`G.,
`
`G5
`
`Ga
`
`G7
`
`Ga
`
`G9
`
`Gm
`
`G.,
`
`G”
`
`10008
`
`100.00
`
`100.00
`
`100.39
`
`58.10
`
`39.50
`
`29.80
`
`24.20
`
`20.80
`
`71.95
`
`61.63
`
`52.27
`
`53.97
`
`41.14
`
`38.04
`
`33.84
`
`31.83
`
`28.67
`
`52.63
`
`35.71
`
`27.03
`
`21.74
`
`18.18
`
`15.63
`
`13.70
`
`12.20
`
`10.99
`
`10.00
`
`9.17
`
`54.34
`
`37.83
`
`29.58
`
`23.19
`
`20.36
`
`15.69
`
`1365
`
`12.57
`
`10.91
`
`9.63
`
`8,42
`
`Terrlcyonide procedure (12).
`?Lane and Eynon procedure (27).
`3Stolchiometry of the copper—sugar reaction (27).
`
`1.00
`
`098
`
`0.99
`
`103
`
`1.01
`
`106
`
`0.93
`
`0.93
`
`0.95
`
`0.93
`
`0.89
`
`0 84
`
`

`
`74
`
`OLIGOSACCHARIDES
`
`Vol. 52
`
`TABLE ll. TRUE SPECIFIC GRAVITY‘ OF SOLUTIONS OF
`MALTOOUGOSACCHARIDES AT 20°C.
`
`1%
`
`1.0018
`
`1.0011
`
`1.0013
`
`1.0014
`
`1.0015
`
`1.0016
`
`1.0019
`
`1.0020
`
`1.0021
`
`True Specific Gravity
`4%
`
`8%
`
`2%
`
`1.0050
`
`1.0047
`
`1.0049
`
`1.0051
`
`1.0053
`
`1.0057
`
`1.0058
`
`1.0062
`
`1.0057
`
`1.0131
`
`1.0126
`
`1.0129
`
`1.0130
`
`1.0130
`
`1.0136
`
`1.0138
`
`1.0140
`
`1.0140
`
`1.0282
`
`1.0266
`
`1.0287
`
`1.0289
`
`1.0290
`
`1.0286
`
`1.0297
`
`1.0296
`
`Degree of
`Polymerization
`
`G1
`
`G;
`
`G3
`
`G4
`
`G5
`
`G5
`
`G7
`
`G8
`
`G9
`
`__(_5m
`‘Specific gravity in vacuo.
`
`1.0020
`
`1.0056
`
`1.0139
`
`10%
`
`1.0348
`
`1.0337
`
`1.0364
`
`1.0369
`
`1.0369
`
`1.0386
`
`1.0386
`
`were not collected when eluted by 10% aqueous ethanol. Both were obtained
`from commercial sources and purified with batch carbon treatment and
`crystallization. Maltotriose and maltotetraose were eluted together with 20%
`aqueous
`ethanol. Later
`fractions
`eluted with 20% ethanol contained
`maltotetraose, maltopentaose and maltohexaose. As
`the higher ethanol
`concentrations were used, higher members of the homologous glucose series
`were eluted but always as a mixture of the neighboring polymers.
`The proximities of the Rr values of the longer—chain polymers (Fig. 1) suggest
`why complete separation by Celite-carbon columns was impossible. The
`proximities also explain why good separation could be achieved with 3MM
`paper chromatographs irrigated 7 to 10 days to separate the G7 to G12 polymers.
`The dextrose equivalents of the 12 maltooligosaccharides calculated from the
`reducing power are compared with theoretical and literature values (1227) in
`Table 1. In general, the values agree well with those reported by Commerford and
`Scallet (27) for polymers up to G6 and with the theoretical values. High values
`reported by Hoover et al. (12) suggest a low degree of purity in separation or
`perhaps, oxidation by ferricyariide beyond the terminal reducing group. The
`stoichiometry was in agreement with values reported by Commerford and Scallet
`(27). Samples G1
`through G6 gave approximately equal relative response
`(approximately l.O) while G7 through G12 tended to be 10 to 15% less than an
`equivalent amount of glucose. Samples G7 through G12 may have needed longer
`time to react with copper than G1 through G6.
`The relationships between true specific gravity (corrected for buoyance) for I
`to 10% sugar solutions and degree of polymerization are shown in Table II. There
`is a general
`linear
`relationship between specific gravity and degree of
`polymerization. With larger polymers (Go through G2) the specific gravity
`
`

`
`January-February
`
`JOHNSON and SRISUTHEP
`
`75
`
`TABLE III. REFRACTIVE IND|CES OF MALTOOLIGOSACCHARIDE SOLUTIONS AT 20°C.
`
`Degree of
`Polymerization
`
`G,
`
`G;
`
`G3
`
`G4
`
`G5
`
`G6
`
`G7
`
`G8
`
`G9
`
`Gm
`
`1%
`
`1.3350
`
`13349
`
`1.3348
`
`1.3348
`
`1.3348
`
`1.3348
`
`1.3348
`
`1.3348
`
`1.3348
`
`1.3348
`
`I
`.o5oo[.
`i
`
`.0400
`
`Refractive Index
`
`Oligosaccharide concentration of solution
`2%
`4%
`8%
`
`1.3367
`
`1.3362
`
`1.3360
`
`1.3361
`
`1.3361
`
`1.3380
`
`1.3360
`
`1.3360
`
`1.3360
`
`1.3360
`
`1,3392
`
`1.3390
`
`1.3390
`
`1.3390
`
`1.3390
`
`1.3390
`
`1.3390
`
`1.3390
`
`1.3390
`
`1.3390
`
`1.3451
`
`1.3448
`
`1.3448
`
`1.3449
`
`1.3450
`
`1.3450
`
`1.3450
`
`1.3450
`
`10%
`
`1.3480
`
`1.3480
`
`1.3480
`
`1.3482
`
`1.3483
`
`1.3483
`
`1.3482
`
`6'0
`
`G9
`
`8
`
`67
`G6
`C5
`
`
`
`0300-
`
`.O 200
`
`2
`
`4
`
`6
`
`10
`
`2. The relationship of
`Fig.
`rnaltooligosaccharides.
`
`the ratio of specific viscosity to concentration of
`
`CONCENTRATION
`
`(W/V)
`
`

`
`76
`
`OLIGOSACCHARIDES
`
`Vol. 52
`
`TABLE lV. HYGROSCOPICWIES OF MALTOOLIGOSACCHAFUDES (G,
`AT 60 i 5% RELATIVE HUM|D|TY AT 26°C.
`
`to Gm)
`
`Degree of
`Polymerization
`
`15 min.
`
`Moisture, %
`90 min.
`
`270 min.
`
`(31
`
`G2
`
`G3
`
`G4
`
`G5
`
`G5
`
`G7
`
`Ga
`
`G9
`
`Gm
`
`1.72
`
`1.45
`
`3.82
`
`4.21
`
`6.21
`
`7.76
`
`4.01
`
`7.48
`
`9.62
`
`8.90
`
`0.52
`
`0.33
`
`7.93
`
`9.22
`
`9.94
`
`9.14
`
`9.67
`
`11.53
`
`12.73
`
`12.30
`
`0.43
`
`0.34
`
`11.27
`
`10.82
`
`1010
`
`10.96
`
`11.44
`
`13.23
`
`14.32
`
`13.92
`
`could not be measured because of the limited solubility of the polymers. As
`would be expected,
`the densities (Table II) of the solutions increased with
`concentration of solution.
`
`Refractive indices of maltooligosaccharides of l to 10% solution of G1 to Gm
`are summarized in Table Ill. These data indicate that refractive index did not
`
`increase as the size of the polymer increased but as expected increased with the
`concentration of the solution. Refractive indices of 8 and 10% concentrations of
`G9 and G10 could not be measured because of limited solubility.
`Viscosities of solutions of large polymers are defined as the ratio ofsheer stress
`per square centimeter to the velocity gradient produced as the solution flows.
`Viscosities of various sugar solutions are listed in the international Critical
`Tables (37) and Viscosities of maltooligosaccharides have been reported by
`Hoover et al. (12). Specific viscosity (nsp.) frequently is used to express the
`relationship of polymer size to viscosity since specific viscosity depends on the
`volume occupied by the polymers (38). Specific viscosity can be used to express
`intrinsic viscosity of the polymers as the concentration approaches zero.
`The relationship of intrinsic viscosity to concentration of oligosaccharides is
`shown in Fig. 2. Maltooligosaccharides G3 to Go and above had limited
`solubility. Therefore, few values were obtained for these maltooligosaccharides.
`The intrinsic viscosity increased linearly for
`the lower—molecular—weight
`oligosaccharides but for G7 to Go the intrinsic viscosity increased curvilinearly
`with increasing concentrations. The intrinsic viscosity values are generally higher
`than those reported by Hoover et al. (12) but lower than values reported for
`cellodextrins of equivalent chain length (39).
`Hygroscopicity is a characteristic of sugar polymers because of the many
`residual valence forces that attract water through hydrogen bonding. Table IV
`lists percentages of water absorbed by the maltooligosaccharides when exposed
`
`

`
`January-February
`
`JOHNSON and SRISUTHEP
`
`77
`
`to relative humidity of 60 i 5% at 26° C. These data indicate that hygroscopicity
`increased as the polymer became larger. Moisture increase was particularly large
`for polymers G3 to Gio.
`
`SUMMARY
`
`Maltooligosaccharides G1 to G2 were separated by Celite~carbon column and
`further separated and purified by macro—paper chromatography. They were
`proved to be free of any branched fractions.
`Reducing power of the maltooligosaccharides agreed closely with theoretical
`values. Specific gravity tended to increase with increasing chain length and
`concentration but refractive indices were identical for all oligosaccharides and
`increased with concentrations. Intrinsic viscosity increased as polymerization
`and concentration increased. Hygroscopicity likewise increased with length of
`the glucose polymer.
`
`Literature Cited
`
`octadea—0—
`
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`of carbohydrates on charcoal. Chem. Ind. (London) I956: 658.
`2. WHISTLER, R. L., and DUFFY,
`J. H. Maltopentaose and crystalline
`acetylmaltopentaitol. J. Amer. Chem. Soc. 77: I017 (1955).
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`I. The preparation and properties of maltodextrin substrates. J. Chem. Soc. 1953:
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`4. BAILEY, J. M., WHELAN, W. J., and PEAT, S. Carbohydrate primers in synthesis ofstarch. J.
`Chem. Soc. 1950: 3692.
`5. ALM, R. S., WILLIAMS, R. J. F., and TISELIUS, A. Gradient elution analysis. I. A general
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`7. WHISTLER, R. L., and HICKSON, J. L. Determination of some components in corn sirups by
`quantitative paper chromatography. Anal. Chem. 27: I514 (1955).
`8. WHISTLER, R.
`L.,
`and HICKSON,
`J.
`L. Maltotetraose
`pentadecacetylmaltotetraitol. J. Amer. Chem. Soc. 76:
`I671 (1954).
`9. WHISTLER, R. L., and MOY, B. F. Isolation of maltohexaose. J. Amer. Chem. Soc. 77: 5761
`(1955).
`I0. SALEM, A. E., and JOHNSON, J. A. Influence of various oligosaccharides on staling ofbread.
`Food Technol.
`I9: 849 (I965).
`I I. HOOVER, W. J., NELSON, A. L., MILNER, R. T., and WEI, L. S. Isolation and evaluation of
`the saccharide components of starch hydrolysates. I. Isolation. J. Food Sci. 30: 248 (I965).
`I2. HOOVER, W. J., NELSON, A. L., MILNER, R. T., and WEI, L. S. Isolation and evaluation of
`the saccharide components of starch hydrolysates. II. Evaluation. J. Food Sci. 30: 253 ( I965).
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`dextrins by charcoal chromatography. J. Chromatogr. 24: 68 (I966).
`15. PARTRIDGE, S. M. Application ofpaper partition chromatogram to the qualitative analysis of
`reducing sugars. Nature 158: 270 (1946).
`I6. HOUGH, L., JONES, J. K. N., and WADMAN, W. H. Quantitative analysis of mixtures of
`sugars by the method of partition chromatography. V. Improved methods for the separation
`and detection of the sugars and their methylated derivatives on the paper chromatogram. J.
`Chem. Soc. I950: I702.
`17. JEANES, A., WISE, C. S., and DIMLER, R. J. Improved techniquesin paperchromatography
`of carbohydrates. Anal. Chem. 23: 415 (I951).
`
`and
`
`crystalline
`
`

`
`OLIGOSACCHARIDES
`
`Vol. 52
`
`18.
`
`19.
`
`21.
`
`24.
`
`25.
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`27.
`
`29.
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`laJ'u-IL»)b-11.:-I
`
`[Received November 15, 1973. Accepted June 19, 1974]