Note: Descriptions are shown in the official language in which they were submitted.
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BACKGROUND O~ THE INVENTION
The present invention relates to the specific depolgrnerization
of a polysaccharide having a rod-like helical conformation.
Several viscous polysaccharides such as xanthan, lentinan,
5 schizophyllan, scleroglucan or curdlan, have been found to have two
or three stranded helical structures. (T. Norisuye, et. al;
J. Polymer Science, Polymer Physics Ed. 18, 547-558 (1980):
E.D.T. Atkins and K.D. Parker; J. Polymer Science Part C. 28,
69-81 (1969): T. L. Bluhm and A. Sarko; Can. J. Chem. 55,
10 293-299 (1977): R.H. Marchessault, et. al; Can. J. Chem. 55,
300-303 ~1977): E.R. Morris; A.C.S. Symposium Series, n, 45,
81 (1977): E.R. Morris, et. al; J. Mol. Biol. 110, 1 (1977)3.
Exploitation of the potentials of these polysaccharides has been
investigated and some were developed as thickening agents for foo-l
15 industry based on their high viscosities, and others were found to
have potent, host-mediated anti-tumor activities. But in some cases,
the extremely high viscosities of their solutions make their utilizations
difficult .
In order to reduce their viscosities properly, we invented
20 an ultrasonic method for depolymerization of the polysaccharides~
(Japanese Patent Laid open No. (Kokai) 57335/1977~
Our investigations on the mode of the ultrasonic depolymerization
confirmed that it is caused mainly by the cledvages of the main chain
of the polysaccharide and that neither side s~hain nor carbon-carbon
25 ~ond in glucose residue is cleaved during the sonic depolymellzation.
Thus, the resulting degraded polysaccharide consists of the same
repeating unit and also has the same helical conLormation, as those
of the original polysaccharide.
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Recently, we found that the ultrasonic depolymerization method
does not suit for industrial depolymerization of a large bulk of the
polysaccharide, because of its low efficiency. The ultrasonic
depolymerization method is also involved in high noise-level and
erosion of the vibrating rod of the ultrasonic oscillator.
The present invention was derived from a finding that the
treatment of a solution of the polysaccharide under a high shear
rate also depolymerizes the polysacchalide in a manner similar to that
o~ the ultrasonic treatment; i.e., only main chain of the polysaccharide
but neither other glucosidic linkage nor carbon-carbon bond in glucose
residue is cleaved during the present depolymerization treatment.
The helical-structural polysaccharide is known to disperse into
single chains in a specific condition; for example, beta-1,3-D-glucan
disperses to single chains in dimethyl sulfoxide or alkaIine solution.
~5 When the dimethyl sulfoxide solution is diluted with wa~er or the
alkaline solution is neutralized with acid, the helical structure of the
polysaccharide is not recoveredj but, by random association, large
aggregate is formed. The present invention is useful only for a
solution of the polysaccharide having a helical structure, but not for
that having single chain structure or aggregated conformation.
The present method was confirmed to be able to overcoroe the
foregoing disadvantages, such as low ef~lciency, high noise-level,
or erosion of the vibrating rod of the ultrasonic oscillator, as seen
in the ultrasonic depolymerization method.
Acid-hydrolysis and enzymic degradation of a polysaccharide
have been known. Acid-hydrolysis cleaves equally all glucosidic
linkages of the polysaccharide, while enzymic degradation causes
hydrolysis of a specific glucosidic linkage.
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But their depolymerization-modes are quite different from those of the
ultrasonication and the present method. They liberate very low
moleeular mono- or oligo-saeeharicles and can not afford quantitatively
clegraded polysaceharide, whieh has the same ehemical and eonforma-
5 tional structures as those of the original polysaeeharide.
SUMMARY OF THE IN~rENTION
It is an objeet of the present invention to depolymerize a poly-
saccha~de having a rod-like helical strueture by forcing its solution
to flow at a high shear rate.
Another objeet of the present invention is to provide a special
degraded polysaeeharide, which eonsists of the same repeating unit
and the helieal eonformation as those of the starting polysaeeharide.
A solution of the degraded polysaccharide exhibits extremely lower
viseosity in comparison with that of the starting polysaecharide, and
15 the polysaecharide still holds the ehemieal and physieal features sueh
as antitumor aetivity of the original polysaceharide except for the
molecular weight and viscosity. The reduetion in the viscosity of the
solution is helpful for its industrial utilization. For example, it makes
administration of the polysaeeharide easy in a case of its elinieal uses
20 as an antieaneer drug, or a thixotropie solution of the starting poly-
saceharide turns Newtonian fluid, improving the fluidity of a food
eontaining the polysaecharide.
The depolymerization according to the present invention is per-
formed by foreing ~ solution of the polysaceharide to pass through a
. 25 eapillary at a high shear rate,~~higher than 1 x 104 see 1
Examples of the polysaecharide used in the present method are beta-
1, 3-D-glueans and xanthan gum.
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The depolymerization-efffciency of the present method was found
to become higher, as the concentration of the polysaccharide-solution
increases~ especially when it is higher than 0.1 wt.%. The addition
of a solvent (solvent B) to the polysaccharide-solution was also found
5 to increase the efficiency of the depolymerization according to the
present method, where solvent B is miscible with the solvent (solvent
A) of the polysaccharide-solution and does not dissolve the polysac-
charide .
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The depolymerization of the polysaccharide is performed by forc-
ing a solution of the polysaccharide to pass through a capillary such
as nozzle, slit, or porous sintered plate or ceramics, using a high
pressure-driving force.
The depolymerization-velocity depends only on the value of the
15 shear rate applied, which results from the driving force, pressure,
diameter and length of the capillary used, and the viscosity of the
solution. While repetition of the passage of the solution through a
capiLlary in a certain condition, the molecular weight of the polysac-
charide gradually decreases, approaching a minimum value, from which
20 no further depolymerization occurs. The minimum molecular weight
also depends on the value of the shear rate applied; higher shear
rate gives lower minimum molecular weight.
Thus, the value of the shear rate applied is a dominant factor
for the present method. ~o substantial depolymerization occurs at
~3~ 25 too low shear rate. Generally, a shear rate~e~ higher than
1 x 104 sec 1 is necessary for the present depolymelization method.
In order to give such a high shear rate, the pressure applied
to the polysaccharide-solution and the cross-secl:ional area of the
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capi~lary are ~0-800 kglcm2 and 1 x 10 ~ -100 mm2, respectively.
But these values are not limited.
The present invention includes the finding that
1. Increase in the concentration of the polysaccharide-solution
5 increases the efficiency of the depolymerization.
2. Addition of a solvent (solvent B), which is miscible with the
solvent (solvent A) of the polysaccharide-solution and does not dissolve
the polysaccharide3 to the polysaccharide-solution also increases the
efficiency of the depolymerization.
The effect of the increase in the concentration of the polysac-
charide-solution reveals significantly, when it is higher than 0.1 wt.~6.
Although the concentration is desirable to be as high as passible, a
part of the polysaccharide tends to remain undissolved at too high
concentration, because of its low solubility. Thus9 practically, the
concentration is preferably 0.1 - 10 wt.~6.
The solvent B is exemplified by acetone, methanolJ ethanol,
iso- or n-propanol, tetrahydrofuran, etc. when the solvent A is water.
In most cases, water is useful as the solvent A, but, for a water-
insoluble derivative of the polysacchalide such as N-alkylol amide
20 derivative of the polysaccharide, that still has a helical conformation
similar to that of the original polysacchalide, acetone or benzene is
used as the solvent A., and water, as solvent B.
Although the depolymerization e~iciency increases as the amount
of the solvent B a.dded increases, its amount must be limited so that
25 no insoluble precipitate of the polysaccharide is formed.
The temperature gives no significant influence upon the result
of the present depolymeIization method. Therefore, the depolymeriza-
tion is usually performed at a temperature lower than 10ûC.
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In order to depolymerize the polysaccharide to a certain molecular
weight, its solution is forced through a capillary repeatedly until it8
molecular weight reaches the desired value.
Particles suspending in the polysaccharide-solution often cloggs
the capillary, leading to interruption of the operation. Thus, the
solution is preferably ~ltrated before its passage through the capillary.
Since a solution of the polysaccharide is viscous and adhesive,
a considerable amount of the solution remains in vessels or other
equipments used, after its discharge from them. In order to prevent
the adhesion of the solution on inner surfaces of the vessels or equip-
ments, it is desirable to agitate the solution moderately. The moderate
agitation also prevents retenffon of a part of the solution adhering on
the equipment-walls, resulting in uniform depolyme~ization of the
polysàccharide .
EXAMPLE 1:
Schizophyllan having 5.6 x 106 molecular weight was dissolved
in water, to prepare a 0.2 wt.% solution. The solution was forced
to pass through a no~.zle of 0.16 mm radius, by being driven by a
plunger pump. The flow rate of the solution was adjusted to each
0.23 cm3/sec., 0.90 cm31sec., 14.5 cm3/sec. and 35.4 cm3/sec. by
control of the speed of the pump. The shear rate computed ~r each
flow rate from the following ormula was 7.1 x 104 sec 1 for 0.23 cm3/
sec flow rate, 2.8 x 105 sec 1 for 0.90 cm3/sec flow rate, 4.5 x 10
sec 1 for 14. 5 cm3 /sec flow rate ~ and 1.1 x 107 sec 1 for 35. 4 C1113 /.
25 sec flow rate, respectively.
4 x flow rate
shear rate = 11 x ~radius of the capillary)3
~2~ 2
Eig. 1 shows the relationship between the retention time of the
solution in the nozzle and the molecular weight of the polysaccharide.
The starting schizophyllan and all the depolymerized schizophyllans
were methylated by the Hakomori method and then hydrolyzed with
5 formic acid and subsequently trifluoro acetic acid. The hydrolyzate
was acetylated with anhydrous acetic acid in pyridine. The sugar-
components in the product were analyzed by gas-liquid chromatography,
resulting in that it contained 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-
D-glucitol, 1,3,5-tri-O-acetyl-2,4,6-tri-O-methyl glucitol and 1,3,5,6-
10 tetra-O-acetyl-2,4-di-O-methyl-D-glucitol in a molar ratio 1: 2: 1.
The starting schizophyllan and all the depolymerized schizophyllans
were oxidized with 0.01N sodium meta-periodate and the amounts of
sodium meta-periodate consumed and formic acid formed were determined
by iodometry followed by the titration with sodium hydro~nde solution.
15 As the results, 0~48-0~55 mol sodium meta-peliodate was consumed and
0.21 - 0.27 mol formic acid was formed, per 1 mol glucose residue in
schi zophyllan .
The starting schizophyllan and all the depolymerized schizophyllans
were degraded with exo-beta~1,3-D-glucanase. The degraded product
20 was confirmed to contain glucose and gentiobiose in a molar ratio 2: 1.
The molecular weights of the starting schizophyllan and all the
depolymerized schizophyllans in water and also in dimethyl sulfoxide
were determined by the ultracentrifugal method. Each ratio of the
molecular weight in water to that in dimethyl sulfoxide was in the
25 range between 2.8 and 3;3.
These results indicated that each depolymerized poiysaccharide
had the same chemical and conformational structures as those of the
starting polysaccharide.
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EXAMPLE 2:
Scleroglucan having 5 x 106 molecular weight and xanthan gum
having 1.4 x 10 molecular weight were dissolved in water, to prepare
each 0. 5 wt . ~6 aqueous solution.
A pressure, 200 kg/cm2, was applied to each solution and it was
allowed to pass through a nozzle of 0.1 mm radius and 5 cm length.
The flow rates were 1.4 cm3/sec for scleroglucan and 7.4 x 10 1 cm3/
sec for xanthan gum, respectively. Thus, shear rates calculated from
the following formula were 1.8 x 10 sec 1 for scleroglucan cmd
9. 4 x 105 sec 1 for xanthan gum.
shear rate = 4 x flow rate
Il x (radius of the capillary)
Each solution was allowed to pass through the nozzle ten-times
in the foregoing condition. The depolymelized scleroglucan has mole-
cular weight, 8 x 105 and the depolymerized xanthan gum, 1. 05 x 106,
15 respectively.
The starting and resultlng polysaccharides were methylated and
subsequently acetylated as desclibed in Example 1, and then the
components in the products were analyzed by gas-liquid chromatography.
The analyses showed that each depolymerized polysaccharide had
20 essentially the same primary structure as that of the corresponding
starting polysaccharide. The ratios of the molecular weights in water
to those in dimethyl sulfoxide of the starting and depolymerized
scleroglucans were close to three, showing t}le resemblance between
the conformational structures of both scleroglucans. The intrinsic
25 viscosities of the starting xanthan gum and tlle depolymelized one
were 12000 dl/g and 1070 dllg, respectively. The relationship between
the intrinsic viscosity and the n~olecular weig~ht of ecach stc~ting and
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depolymerized xanthan gum was consistent with the relationship
determined by Holzwarth et. al (G. Holzwarth; Carbohydrate P~esearch
66, 173-186 (1978)), indicating that both xanthan gum had similar
helical structures.
5 EXAMPLE 3:
Schizophyllan having 2 x 106 molecular weight was dissolved in
water, a mixture of 20 wt.% acetone and 80 wt.% of water, and a
mixture of Z0 wt.% ethanol and 80 wt.% of water, in each 0.8 wt.%
concentration. Each solution was forced to pass through a sintered
10 plate having 1 cm thickness and 50 micron mean pore size, with
400 kg/cm2 pressure. It was ivident from the following formula that
the shear rate for each solution was higher than 2.5 x 106 sec 1.
(pressure) x (diameter of the capiliary)
Shear rate 2xIviscosity of the solution)x(length of the capillary)
The viscosity of each solution was lower than 38 c.p.
After 5-times passages of each solution through the sintered plate,
polysaccharide had the following molecular weight.
Molecular wei~ht
Aqueous ethanol solution 5. 3 x 105
Aqueous acetone solution 6. 0 x 105
Water 7.8 x 105
EXAMPLE 4:
Scleroglucan having 5. 2 x 106 molecular weight was dissolved in
water, to prepare each 0.1 wt.%, 0.45 wt.% and 0.90 wt.% aqueous
solution. Each solution was driven by 170 kglcm2 pressure to pass
25 through a nozzle of 0.16 mm radius. After 20-times passage, the
polysaccharide in each solution had the following molecular weight.
Concentration of
the solution Molecular wei~ht
~.1 wt.% 5.8x ~0~
0.45 wt.% ~.2 x 105
0.90 wt.~6 2.8 x 105
5 EXAMPLE S:
A 1.0 wt.% aqueous solution of the schizophyllan used in Example
1 was filtrated using a ceramic-fillter having 0.1 mm pores. A pressure
of 50 kg/cm2 was applied to a vessel filled with the filtrate, to force
it to pass through a nozzle of 0.15 mm radius and recirculated to the
10 vessel. The filtrate was a~,itated in the vessel at P~eynold's number=120.
The operation continued Ior 8 hours. The molecular weight of
schizophyllan was 3.7 x 106 after the operation. After the solution was
discharged from the veseel, 100 ml of the solution remained in the
vessel adhering on its inner surface.
The same schizophyllan solution was forced to pass through the
same nozzle with neither its filtration with the ceramic filter nor
agitation during its treatment. The operation was interrupted several
times due to the clogging of the nozzle \,v;th particles suspending in
the solution. After the solution was discharged from the vessel,
20 2,500 ml of the solution collected in the bottom of the veseel, running
down along the wall.
