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Bacterial polysaccharide from bacillus polymyxa No. 271

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Die Angewandte Makromolekulare Chemie 6 (1969) 179-185 (Nr. 72)
From the Research Institute, Meiji Sugar Mfg. Co., Ltd.
580, Horikawa-Cho, Kawasaki, J a p m
Bacterial Polysaccharide from Bacillus polymyxa
No. 271
By EIZININOMIYA
and TADASHICE
KIZAKI
(Eingegangen a m 2. Januar 1969)
SUMMARY :
The solution characteristics of the polysaccharide (D-glucose: D-mannose: Dgalactose: D-glucuronic acid = 3 : 3 : 1 : 2) which was formed from glucose by
Bacillus polymyxa No. 271 were studied. The aqueous solution of the polymer had
a high viscosity comparable with that of gum gum, and the viscosity was enhanced
still further in the presence of acids, salts, and sugars. The polymer had a high
water hold capacity, and formed a viscoelastic gel from ethanol solutions below
40 yo concentration.
ZUSAMMENFASSUNG :
Die Losungseigenschaften des Polysaccharides (Rausteine: D-Glucose : D-Mannose : D-Galactose : D-Glucuronskure = 3 : 3 : 1 : 2), das aus Glucose durch
Bacillus polymyxa Nr. 271 gebildet wird, wurden untersucht. Die waBrige Losung
des Polymeren hat, verglichen mit ,,gum guar“, eine hohe Viskositiit; die Viskositat erhoht sich noch in Gegenwart von Sauren, Salzen und Zuckern. Das Polymere
hat ein hohes Ruckhaltevermogen fur Wasser ; es gibt viskoelastische Gele aus
bthanol-Wasser-Mischungen(unter 40 V0l.-06 Alkohol).
Zntroduction
SANBORN~
published the review on the microbial polysaccharide, in which
it was suggested to be possible to use this for the food-additives, adhesives,
and chemical sources. Although the potentialities for the polysaccharide
production by microorganisms have been noted, the developments of such
industrial application were made in real only by the production of dextran
by Leuconostoc sp. for use as a blood plasma extender. I n recent years, a
concerted effort was made a t the “Northern Division of the U.S. Department
of Agriculture” t.0 find other microbial polysaccharidesz that might be industri179
E. NINOMIYA
and T. KIZAKI
ally useful. One such product is B-1459 gum produced by a strain of Xanthomonas campestris, NRRL B-1459. This polymer is developing as a dispersant
in oil-webl drilling fluids3. Extracellular polysaccharide from Bacillus polymyxa
has been also studied by several workem-9, but their investigations have
dealt with merely the polysaccharide production and the structure. This
paper describes some properties of the extracellular polysaccharide produced
in a high yield by Bacillus polymyxa No. 271 isolated in our laboratory. The
polymer can be expected to be used as industrial gum because of its unusual
properties in aqueous system.
Experimental
Preparation of plysaccharide
One hundred liters of medium containing 3.000g of commercial glucose, 200 g
of peptone, 50 g of urea, 100 g of magnesium sulfate, 100 g of monobasic potassium
phosphate, 500g of calcium carbonate, and a trace of manganese sulfate was
inoculated with 5 1 of inoculum precultured in a jar-fermentor for 8 hrs. Fermentation was e k e d out aerobically for 60 hrs. a t 28°C in a 200-1 fermentor equipped
anchor-type agitator. A typical course of the fermentation is shown in Fig. 1.
When fermentation was finished, culture fluids had a viscosity as high as 19.000 cp
and contained about 1.4% polymer. Cells and suspended impurities were removed
from the culture fluids through a SHARPLES
centrifuge and a filter-press after
decreasing the viscosity by heating for half an hour a t 80°C. A crude polymer
precipitated from the clear filtrate after adding 1% of potassium chloride as the
20r
I
Fig. 1. Changes during fermentation in 200-1-fermentor.
Time in hours
180
Polysacchride from Bacillus polymyxa
subsidiary precipitant and methanol (to 50 vol.-yo) was redissolved in a suitable
volume of water to purify further, then filtration and precipitation were repeated
twice. The fibrous polymer was dried in a vacuum oven after washing with methanol
and ground to a powder with a ball-mill.
General properties1Q
Refined polymer, which is called No. 271 gum, was colorless, tasteless, and
odorless, and it was soluble in water to give a clear solution, but insoluble in organic
solvents such as alcohols, ethers, acetone, and chloroform. On typical analysis,
it gave the following results: sulfate ash, 10.60~0;protein (N * 6.25), I.06y0;
lipid, O.OOC)!o ;carbohydrate (balance),88.34%. This polymer showed [a]:' = 125'
(C = 0.5wt.-yo in water), and its molecular weight, determined by the viscositymeasuring method, was found to be approximately 1 300 000. The pure polymer
was composed of D-glucose, D-mannose, D-galactose, and. D-glucoronic acid in a
molar ratio of 3 : 3 : 1 : 2 from the results of column chromatography of acid hydrolysates.
+
Analytical method
Polymers, others than locust bean gum, were dispersed in deionized water, then
the mixtures were agitated with magnetic stirrer a t room temperature for 18 hrs.
in order to obtain a homogeneous solution. Locust bean gum was dissolved by
heating for 10 minutes a t 86OC. No. 271 gum does not dissolve immediately in
water. Agitation is important for preventing the formation of clumps and for
moving the vicid mass of hydrated material from the surface of each particle,
since diffusion of such large molecules is extremely slow. Viscosities were measured
with rotational viscometer (Type BL, Tokyo Keiki Co.) a t 25°C and a t 30 rpm,
unless otherwise mentioned. Readings were made when the values had become
constant.
Results and discussion
1. viscosity
Fig. 2 shows that aqueous solution of No. 271 gum had a high viscosity
comparable with that of gum gum, i. e., 0.5y0, l.Oyo,and 1.5% solution
gave a viscosity of 100 cp, 1 800 cp, and 7 4-00 cp, respectively. On the other
hand, a dilute solution of crude No. 271 gum was decationized with Amberlite
C--125 (HO), and K, Na, and Ca form of No. 271 gum were prepared by the
addition of KC1, NaC1, and CaCl2 as the subsidiary precipitant to the decationized solution. One % solution of the polymer in a different salt form had
a viscosity of 1 560 cp (K form), 2 120 cp (Na form), and 6 200 cp (Ca form),
respectively. The viscosity of No. 271 gum was influenced by the valences of
cation combined to the carboxyl groups.
181
E. NINOMIYA
and T. KIZAKI
10000 c
Fig. 2.
Viscosity-concentration
curves: No. 271 gum compared
with some plant gums.
1. Locust bean gum
2. Gum gum
3. No. 271 gum
4. Gum karaya
5. Sodium carboxymethyl cellulose
6. Carrageenan
7. Tamarind
8. Sodium alginate.
Concentration,Yo
2. Effect of pH
The changes in viscosity of 0.5% solution of No. 271 gum a t different pH
values are shown in Fig. 3. Viscosities decreased slowly in the alkaline range
and increased markedly with a peak at pH 2.3 in the acidic range. Stability
Fig. 3. Viscosity-pH curves. The pH
value and the viscosity of original
solution containkg 0.6% of No. 271
gum was 7.2 and 95 cp, respectively.
182
Polysacchccride from Bacillus polymyxa
of No. 271 gum was fairly low in an acidic condition similarly to the other
plant gums, for example, the viscosities and pH values of 0.5% solution of
KO.271 gum changed to pH 2.9 from pH 7.4 and to 420 cp from 95 cp by the
addition of 0.3 yo citric acid, respectively. However, the viscosity decreased
to 270 cp after 14 days at 30°C and to 50 cp after 2 hrs. at 70°C. In the
case of neutral pH, the decomposition of No. 271 gum was not recognized
under the same temperature. The peculiar changes in viscosity at pH 2.3
are interpreted as follows. This polymer is a polyelectrolyte as it possesses
carboxyl groups derived from glucuronic acid residues, consequently the ion
exchange reactions occur to transform from salt-formed carboxyl groups to
salt-free ones in the presence of excess Heion (equivalent for pH 2.3), and the
viscosity decreases immediately with the dissociation of intermolecular ionic
bonds.
3. Effect of salt
Changes in viscosity of 0.5% solution of No. 271 gum are shown in Fig. 4.
The viscosities further increased in the presence of salts such as sodium
chloride, potassium chloride, monobasic sodium phosphate, and calcium
Viscosity in the presence of
various salts (0.5% solution of No. 271
gum)
1. CaCl2 * 2 HzO
3. NaCl
2. KCl
4. NaH2PO4
Fig. 4.
-
-0
a5
io
1.5
20
Salt concentration,%
183
E. NINOMIYA
and T. KIZAKI
chloride. The viscosities which once changed were stable, and the decreasing
of the viscosity was not observed even after 20 days. The individual polyanionic molecules appear to form a giant three-dimensional molecule in water
by additional intermolecular association through the intermediation of cation.
4. Effect of sugar
Viscosity of No. 271 gum solution also was found to be increased by the
addition of sugars. For example, when glucose, sucrose, and lactose were
added to 30 % in 0.5 % solution of No. 271 gum, the viscosities changed to
240 cp, 265 cp, and 295 cp, respectively. In these experiments it was found
that the larger concentration of sugar does not necessarily correspond to
the increase of the viscosity, because the viscosities of each 30 yo sugar solution
were below 2 cp. These results may occur by the increase of molecular interaction resulting from the intertwinement or the formation of bridge structure between No. 271 gum and sugars.
5. Gel formation in ethanol solution
Xo.271 gum dissolved homogeneously in a limited concentration of an
ethanol solution and formed a clear viscoelastic gel (see Fig. 5). No. 271 gum
did not dissolve in 50 vol.-yo ethanol solution but dissolves to the concentration of 2 yo in 40 vol.-yo ethanol solution and of 3 % in 5 v01.- Yo ethanol
solution. The mixture of 2 parts of polymer and 100 parts of 40 v01.- yo ethanol
solution was incubated over night at 50 "C in a closed container and was cooled
to 25°C to form a gel. Such gel was very stable at the neutral condition, the
coacervation did not occur for two years. The gelation was promoted in the
presence of salts and sugars, but the viscosity of gel diminished gradually
with a degradation of polymer in the acidic side.
Fig. 5. Gel formed from the mixture of
No. 271 gum and aqueous ethanol (40%
ethanol).
184
Polysaccharide from Bacillus polymyxa
6. Water hold capacity
Water hold capacity of No. 271 gum solution was high in comparison with
that of the other gums. An end of filter paper strip (TOyG Filter Paper No. 50,
2 . 40 cm) was dipped in 0.5 yosolution of gums for 1 hr. a t room temperature,
and distance of capillary ascension of solution was compared with that of
water. Calculation of water hold capacity was done by the following formula :
Water hold capacity = 100 -
Distance of capillary ascension of solution.
100.
Distance of capillary ascension of water
Water hold capacity was independent of the viscosity in comparison with
the solutions prepared from different gums, even if the viscosities of the
solutions are the same. As shown in Table 1, gum karaya had a lower water
hold capacity than sodium alginate and carrageenan despite its higher viscosity.
Such differences in water hold capacity appear to be attributable to the
structure of the polysaccharide.
Table 1. Comparison of water hold capacity in some plant gums.
GUm
No. 271 gum
Locust bean gum
Gum guar
Sodium alginate
Carrageenan
Sodium carboxymethyl cellulose
Gum karaya
Gum arabic
1
2
3
4
5
6
8
Water hold
capacity
Viscosity of 0.5%
solution (cp)
97.9
97.7
95.8
95.5
93.7
105
150
185
25
30
91.6
85.3
12.6
45
120
(2
J. R. SANBORN,
Ind. Engng. Chem. 28 (1936) 1189.
K. L. SMILEY,
Food Technol. 20 (1966) 1206.
Chem. Engng. News. 45, No. 32 (1967) 20.
W. G.C. FORSYTH
and D. M. WEBLEY,
Biochem. J. 44 (1949) 455.
K. H. CHAO,Ind. aliment. agric. 68 (1951) 13.
D. MURPHY, Canad. J. Chem. 30 (1952) 872.
A. MISAKI and I. HORI,J. Ferment. Tech. Japan 23 (1954) 147.
D. MURPHY, C. T. BISHOPand G. A. ADAMS,
Canad. J. Biochem. Physiol. 3$
(1956) 1271.
D. H. BALLand G.A. ADAMS,
Canad. J. Chem. 37 (1959) 1012.
lo E. NINOMIYA,
T. KIZAKIand K. HANADA,
J. agric. chem. Soc. Japan 42 (1968)
9
178, 431.
185
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