Polysaccharide, micro-organism and method for obtaining...

Details Polysaccharide, micro-organism and method for obtaining... Polysaccharide, micro-organism and method for obtaining...

2000-02-22

2002-02-05

Prats, Francisco (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S072000, C536S123000

Reexamination Certificate

active

06344346

ABSTRACT:

The invention relates to a novel polysaccharide, a micro-organism and method of obtaining it, a composition containing it and application thereof.
As is known, microbial populations constitute a wide reservoir for bringing new molecules into operation. Description of the bacteria in the rhizospheres of cereal plants was the first means of demonstrating the existence of nitrogen-fixing species, mostly associated with the roots of wheat, sunflower, rice and maize. In order to isolate these species, it was necessary to develop specific methods of isolation that is the “spermosphere model” for selecting the bacteria most adapted to the rhizosphere, and antibody trapping, using the specific antibodies of certain species. These bacteria are also involved in the mechanisms of attachment and colonisation of the roots.
For some years, research has concentrated on the capacity of most bacteria, present on the surface of roots and in the rhizosphere, to produce exopoly-saccharides (EPS). It has been amply demonstrated that these polymers play a part in colonisation of roots by bacteria and in aggregation of soil around the roots.
There has been recent work on the study of the capacity of strains to stimulate aggregation of soil around the roots of sunflower and wheat.
An aim of the invention is to provide a micro-organism which produces exopolysaccharides on a gelose medium, grows rapidly on culture medium requiring little in the way of agricultural raw materials (hydrolysed wheat bran, wheat peptides, glucose syrups, hydrolysed starch-factory co-products, for example), easy to use and of course non-pathogenic and genetically stable.
One strain discovered, for example, has a map and a gene sequence which codes for rRNA 16S, indicating that it belongs to the Rhizobiaceae family (the alpha sub-division of the proteobacteria). The percentage similarity to the nucleic acids of this region of the chromosome (rDNA 16S) of the strain YAS34 is 97.2% with Rhizobium etli and 96.2% with Rhizobium leguminosarum (strain LMG 9518). Hybridization on colonies of YAS34 with an oligonucleotide probe specific for Rhizobium (in the wide sense) in the nodD gene was found positive (personal communication from G. Laguerre, INRA, Dijon).
The presence of the nodD gene and the percentage similarity between the gene coding for rRNA 16S and species belonging to the Rhizobium genus are other factors indicating that the strain YAS34 is a Rhizobium.
The genotype imprint of the strain YAS34 by rep-PCR using three different primer sets (REP, ERIC and BOX) is available.
YAS 34 was deposited under number 1-1809 at the CNCM of the Institut Pasteur on Jan. 15, 1997.
The strain YAS 34 was isolated from the surface of the roots (rhizoplane) of a sunflower (Helianthus annuus cv Albena) taken in the four-leaf stage. The seeds used had not been treated with plant protectives and had been sterilised before sowing. The soil was alluvial, the main exchangeable cation being calcium.
The strain YAS 34 is a gram-negative, aerobic catalase-positive and oxidase-negative bacterium. It is a mobile rod bacterium which forms elastic, translucent, white colonies on an RCV-glucose medium (4 g/l).
The strain YAS 34 biosynthesises polysaccharide by fermentation in the presence of a culture medium containing a source of preferably assimilated carbon.
It has been shown that the most efficient carbonaceous sources in terms of growth and production of polysaccharides are glucose, fructose, saccharose and galactose.
This capacity to assimilate numerous glucides was a reason for studying the capacity of this strain to cause fermentation in various media of vegetable origin originating from fractionation of agricultural materials such as wheat, potato or grapes. Table I hereinafter shows the various media tested, and Table II shows the results obtained.
TABLE I
COMPOSITIONS
SUGARS AND
AGRICULTURAL
NITROGENOUS
PRODUCTS
MEDIA TESTED
MATERIALS
Potato
JCII ED:
electrodialysed
clear juice II
Wheat bran
JPAS: pressed
juice after
saccharification
FAM: filtrate
Glucose: 12-15
after micro-
g/L
filtration
Nitrogenous
matter: 5.25 g/L
Grape
JRB 033 ED LD:
Glucose: 10 g/L
pressed juice of
Fructose: 10 g/L
grape marc after
Nitrogenous
electrodialysis
material: traces
TABLE II
TOTAL
CONSUM.
OD at 600
VISCOSITY
OF
NATURAL
&mgr;max
nm
(cps at 25° C., 26 s-1)
GLUCOSE
SUBSTRATES
(h-1)
max
at 52 h
at 140 h
(g/L)
Potato
JCII ED
0.21
7.6
238
273
5.6
JCII
0.26
6.04
197
193
3.8
ED1/2
Wheat
bran
JPAS 2/3
0.17
3.9
267
374
9.5
FAM
0.47
2.9
280
356
5.6
Grape
marc
JRB 03 ED
nd
9
36
1.8
LD1/2
The medium called FAM, which originates from wheat bran and contains 12 g/L glucose and also 5.25 g/L nitrogenous material, is the most efficient with regard both to the growth of the strain (&mgr;max 0.47 h-1) and the biosynthesis of the polysaccharide (viscosity approaches 400 cps at end of cultivation).
Various culture media were studied, including those having the following composition:
RCVs
Glucose
20
g
Yeast extract
1.72
g
Buffer solution (2)
15
ml
Mineral solution (1)
50
ml
Osmosed water
qs ad 1 liter
DSM
Glucose
20
g
Corn steep
5
g
NaNO3
2
g
K
2
HPO
4
1
g
MgSO
4
, 7H
2
O
1.5
g
Solution E (3)
2.5
ml
Osmosed water
qs ad 1 liter
Composition of the mineral solutions (1), buffer (2) and E (3)
(1) Mineral solution
EDTA (tritriplex II)
0.4
g
MgSO
4
.7H
2
O
2
g
CaCl
2
.2H
2
O
2
g
FeSO
4
.7H
2
O
0.44
g
Elements in solution
20
ml
Osmosed water
qs ad 1 liter
Elements in solution
ZnSO
4
.7H
2
O
430
mg
MnSO
4
.7H
2
O
1300
mg
Na
2
MoO
4
.2H
2
O
750
mg
H
3
BO
3
2800
mg
CuSO
4
.5H
2
O
22.5
mg
CoSO
4
.7H
2
O
70
mg
Osmosed water
qs ad 1 liter
(2) Buffer solution
KH
2
PO
4
40
g
K
2
HPO
4
60
g
Osmosed water
qs ad 1 liter
(3) Solution E
CaCl
2
.2H
2
O
3
g
FeIII nitrate
1
g
MnSO
4
0.2
g
ZnCl
2
0.1
g
CuSO
4
.5H
2
O
0.025
g
Na
2
B
4
O
7
.10H
2
O
0.02
g
CaCl
2
0.004
g
Na
2
MoO
4
.2H
2
O
0.01
g
Osmosed water
qs ad 1 liter
NA
Meat extract
3
g
Peptone
5
g
Osmosed water
qs ad 1 liter
The results are shown in Table III:
TABLE III
Final
viscosity at
OD at 600 nm
&mgr;max
26s-1, 20° C.
Medium
C/N ratio
max
(h-1)
(cps)
RCVs
50
2.70
0.28
227
DSM+
8
6.20
0.38
254
NA
0.1
1.91
0.35
17
As Table III shows, media rich in nitrogen and low in carbon (low C/N) such as NA medium promote the growth of the strain but prevent production of polysaccharides. On the other hand RCV and DSM media, which are rich in nitrogenous and carbonaceous substrates, are the best compromise and give good growth of the strain combined with synthesis of polysaccharides. These tests have also shown that growth of the strain can be dissociated from production of polysaccharides.
An improved preculture medium for growth of the strain was thus chosen, the composition for a 7.5% inoculum being as follows:
Opt2_ns
Glucose (sterilised separately)
20
g
Yeast extract
2.5
g
Ammonium sulphate
1
g
Mineral solution (1)
70
ml
Buffer solution (2)
20
ml
Osmosed water
qs ad 1 liter
Inoculum
7.5%
Similarly an improved medium for production of exopoly-saccharide was developed in order to maximise the yield of polymer. The composition, hereinafter called MP1, was thus defined as the most efficient.
MP1
Glucose
20
g
Yeast extract
1.7
g
Mineral solution (1)
70
ml
Osmosed water
qs ad 1 liter
Table IV shows the results for growth and production using RCVs medium alone (reference) or the two optimised media hereinbefore—Opt2_ns and MP1.
TABLE IV
REFERENCE
IMPROVED
CYCLE
CYCLE
PRE-
Medium
RCVs
Opt2_ns
CULTURE
% inoculum
0.22
0.37
Final corrected
0.6
1.6
OD at 600 nm
Duration
20 hours
15 hours
PRO-
Medium
RCVs
MP1
DUCTION
&mgr;max (h-1)
0.28
0.29
Final corrected
8.9
9.2
OD at 600 nm
Final
10.1 g/l
10.2 g/l
concentration of
EPS
dS/dt
0.25 g/l.h
0.30 g/l.h
Consumption of
glucose
dP/dt
0.27 g/l.h
0.36 g/l.h
Yield of EPS
(Production
phase)
Viscosity at 25° C.
1500 cps at 26s
−1
1560 cps at 26s#1
Duration of
73 hours
63 hours
fermentation
Duration of
38 hours
28 hours
production phase
Total duration of
93 hours
78 hours
cycle
Two methods can be used for recovering the polysaccharide produced by the strain by fermentation as explained hereinbefore.
In a first metho

Affiliated with

Also associated with

Rating

Polysaccharide, micro-organism and method for obtaining... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Polysaccharide, micro-organism and method for obtaining..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Polysaccharide, micro-organism and method for obtaining... will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2959296