Fermentation process for the production of xanthane

The invention relates to a process for the fermentation of carbohydrates by bacteria of the genus Xanthomonas for the production of a polysaccharide of the xanthane type, in which the source of nitrogen consists of a gelatin with a molecular weight of less than 5000. Application: preparation of xanthane.

The present invention relates to a process for the fermentation of 
carbohydrates for the preparation of a polysaccharide of the xanthane type 
by means of microorganisms of the Xanthomonas type, and to the 
polysaccharide obtained by this process. 
It is known that the aerobic fermentation of bacteria of the Xanthomonas 
type in aqueous media with a pH of between 5.5 and 9, containing at least 
one source of carbon, a source of nitrogen, phosphate ions and trace 
elements, makes it possible to obtain a polysaccharide composition of the 
xanthane type. The source of carbon generally consists of carbohydrates, 
while the following have been described as sources of nitrogen: dry 
distillery draff in U.S. Pat. No. 3,000,790, peptones in TAPPI 5, p. 
442-445, by M. O. BAGBY, I. A. WOLFF and M. C. CADMUS, yeast extracts in 
Biotechnol. Bioeng. (1971) 13, p. 381, by R. A. MORAINE and P. ROGOVIN, 
maceration liquors in U.S. Pat. No. 3,335,447 or soya bean flours in U.S. 
Pat. No. 3,271,267; cereal flours, such as sorghum flours, provide both 
the source of carbon and the source of nitrogen, as described in U.S. Pat. 
No. 3,271,267. 
The products commonly used as sources of nitrogen introduce insoluble 
impurities into the medium, which appear in the polysaccharide isolated at 
the end of fermentation, this implying, for certain applications of the 
polysaccharide, treating the resulting product by various processes, for 
example filtration or flocculation, in order to remove these insoluble 
impurities which lead to the formation of cloudiness in aqueous 
polysaccharide solutions. 
Furthermore, on the strength of its thickening property which is virtually 
independent of temperature and pH, xanthane is used in the assisted 
recovery of petroleum, as an edible additive in pharmaceutical or cosmetic 
formulations, or in the preparation of textiles or explosives, and it is 
of particular importance to have a grade of xanthane having naturally a 
high thickening property i.e. having aqueous solutions of high viscosity. 
The present invention relates to a fermentation process which, through the 
use of a suitably chosen source of nitrogen, makes it possible to obtain a 
polysaccharide of xanthane type with a high thickening property, which 
contains few insoluble materials and whose aqueous solutions are clear and 
only very slightly colored, which is advantageous for numerous 
applications. 
According to the invention, gelatin, and more particularly a gelatin of low 
molecular weight, is used as the source of nitrogen soluble in aqueous 
media. Two types of gelatin are known to exist: type A gelatin, which is 
obtained by the acid hydrolysis, for example with sulfuric acid, of the 
collagen present in the skin or bones of pigs and cattle, and type B 
gelatin, which is obtained by the basic hydrolysis of collagen, especially 
with Ca(OH).sub.2. The common gelatins, with a molecular weight greater 
than or equal to 20000 are known sources of nitrogen but they do not make 
it possible to achieve the advantageous results of the invention. It is 
desirable to use gelatins with an average molecular weight of less than 
5000, having practically no gelling strength; the hydrolyzed gelatins 
with a molecular weight between 1500 and 2500 are particularly preferred. 
It is possible to introduce into the fermentation medium a gelatin which 
has been hydrolyzed beforehand either by heating in an aqueous acid medium 
at a pH&lt;4 and at a temperature above 100.degree. C., or through the 
enzymatic action of a protease in the manner described in French patent 
1.501.821. 
However, the gelatin can also be at least partially hydrolyzed during the 
sterilization of the culture medium, before the microorganisms are 
introduced, if the pH, temperature and heating time are suitably chosen; 
in this case, it is preferred initially to introduce gelatins whose 
molecular weight, before hydrolyzing sterilization, is no greater than 
10.000. 
When sterilization is carried out at an essentially neutral pH or at a 
slightly acid pH, for very short times of less than 5 minutes, the 
gelatin used must have a molecular weight of less than 5000, preferably 
less than 3000. 
The amount of gelatin introduced into the fermentation medium is chosen so 
as to give a total nitrogen content of 0.1 g/l to 1 g/l. 
The medium can also contain, in conventional manner, from 5 g/l to 50 g/l 
of carbohydrates such as glucose, native or hydrolyzed starches, sucrose, 
levulose, fructose, maltose, sugar beet molasses or sugar cane molasses, 
as well as 0.10 to 20 g/l, preferably 0.5 to 5 g/l, of phosphates such as 
K.sub.2 HPO.sub.4, and one or more trace elements, including magnesium at 
a concentration of 0.025 to 1 g/l of magnesium in the form of a soluble 
magnesium salt such as magnesium sulfate, acetate, chloride or nitrate. 
The bacteria which make it possible to obtain a xanthane by fermentation in 
these media are generally of the genus Xanthomonas and can belong to 
different species such as Xanthomonas begoniae, Xanthomonas incanae, 
Xanthomonas vesicatoria, Xanthomonas campestris and Xanthomonas phaseoli, 
and other species well known to those skilled in the art; strains of 
Xanthomonas campestris are generally used. 
Fermentation is carried out at a pH which can be between 5.5 and 9 and is 
preferably between 6.5 and 8. at a temperature between 25.degree. C. and 
35.degree. C. and preferably between 27.degree. C. and 32.degree. C. The 
medium is agitated and aerated in conventional manner; under these 
conditions, fermentation takes 1 to 6 days. 
In a conventional manner, the process used to obtain the polysaccharide 
according to the invention comprises several steps: 
1. Preparation of an inoculum from a strain of Xanthomonas which was 
previously quick-frozen or lyophilized; 
2. If appropriate, growing of the microorganisms in a prefermentation 
medium; 
3. Production of the polysaccharide in the fermentation medium, which has 
been inoculated with the media obtained in step 1 or 2 and; 
4. Isolation of the polysaccharide. 
The polysaccharide is generally isolated by precipitation through the 
introduction into the fermentation medium of a solvent in which the 
polysaccharide is insoluble. Among the solvents generally used, there may 
be mentioned lower alcohols such as methanol, ethanol, isopropanol and 
butanol, or acetone; isopropanol is preferred. Before the precipitation 
step, it is possible to heat the reaction medium at a temperature between 
80.degree. C. and 130.degree. C. for a few minutes in order to destroy the 
bacteria. 
A further possibility is to treat the medium before or after sterilization, 
with an aliphatic dialdehyde such as glyoxal, which is a known way of 
improving the dispersibility of polysaccharides in water; the xanthane is 
then precipitated in the medium, isolated by filtration, washed with the 
precipitating solvent and then dried and ground if necessary. In this 
case, 0.1 to 1% by weight of glyoxal is used, relative to the weight of 
dry xanthane; in general, about 2% is used. The xanthane can also be 
treated with a dialdehyde after isolation, if this operation has not 
already been carried out in the fermentation medium. An advantageous 
process consists in wetting the xanthane powder with a solution of glyoxal 
in aqueous isopropanol, prepared by mixing 5 to 10 ml of a 30% aqueous 
solution of glyoxal with 150 ml of isopropanol; after a contact time of 20 
to 40 minutes, the solvents are removed by heating and the dry 
polysaccharide can then be stored for subsequent use in its conventional 
applications. 
To improve the fermentation conditions and, in particular, to reduce the 
viscosity of the medium, fermentation on gelatin can also be carried out 
in the presence of an oil and a surfactant, by a process described in 
European patent application A-58364, which uses a water-in-oil emulsion as 
fermentation medium or a process described in European patent application 
A-187092, which uses an oil-in-water emulsion as fermentation medium. 
The invention further relates to the polysaccharide composition of the 
xanthane type which is obtained by the fermentation process according to 
the invention. 
This polysaccharide composition, like all products of this type, consists 
of saccharide units of the mannose, glucose and salified glucuronic acid 
type; some of the hydroxyl groups are esterified with acetic acid, and 
pyruvic acid is bonded via an acetal group to some of the mannose units. 
The polysaccharide composition according to the invention has a molecular 
weight of more than 2,000,000. 
Its aqueous solutions have a particularly high viscosity which is greater 
than that of solutions of the xanthane produced by fermentation in a 
medium containing soya bean flour or corn maceration liquors as the source 
of nitrogen. 
Thus an aqueous solution containing 1% by weight of KCl and 0.2% by weight 
of the polysaccharide of the invention has a viscosity of at least 200 
mPa.s, measured on a Brookfield viscometer at 24.degree. C., whereas, 
under the same conditions, that of an aqueous solution of the 
polysaccharide obtained by fermentation on soya bean flour is about 160 
mPa.s. 
The aqueous solutions of the invention polysaccharide have a low optical 
density. For example, the solutions containing 0.2% by weight of 
polysaccharide have an optical density, measured at 620 nm, less than or 
equal to 0.2. 
An Example of the way in which the invention is carried out is now 
described below.

EXAMPLE 
(a) Preparation of the inoculum: 
A revitalization culture medium MY consisting 
______________________________________ 
Glucose 10.0 g 
Pentone 0.5 g 
Yeast extract 0.3 g 
Malt extract 3.0 g 
Distilled water 1000 g 
______________________________________ 
sterilized beforehand by heating for 20 min at 120.degree. C., is 
inoculated with 3 ml of defrozen bacteria of the Xanthomonas campestris 
strain deposited under no. NRRL.B 1459. 
After inoculation, the medium is incubated at 30.degree. C. for 12 to 18 
hours, with agitation. 
(b) Prefermentation 
The microorganisms of the inoculum are then grown in a preculture fermentor 
containing 10 l of a medium consisting of: 
______________________________________ 
Glucose 150.0 g 
Gelatin 20.0 g 
K.sub.2 HPO.sub.4 8.0 g 
MgSO.sub.4 1.8 g 
Antifoam 0 to 3.0 g 
Water qs for 10 
l 
______________________________________ 
The glucose is sterilized beforehand by heating at pH 4, while the other 
constituents of the medium are sterilized as a mixture, for example by 
heating at 120.degree. C. for 30 minutes at pH 4.5. 
Prefermentation is continued for 20 hours before the inoculum is used to 
inoculate the fermentation medium. 
(c) Fermentation and production of the polysaccharide: 
The fermentation medium for a fermenter with a useful volume of 100 l 
consists of: 
______________________________________ 
Glucose 3 kg 
Gelatin 200 g 
K.sub.2 HPO.sub.4 
80 g 
MgSO.sub.4 18 g 
Antifoam 0 to 30 g 
Water qs for 100 
l 
______________________________________ 
The glucose is sterilized on its own at pH 4, as previously, and the other 
constituents are sterilized at 120.degree. C. for 30 minutes at different 
pH values, leading to gelatins with different molecular weights. 
Before inoculation with the inoculum prepared in step b, the pH of the 
medium is adjusted to 7 and kept around this value throughout 
fermentation. 
In a conventional manner, the medium is agitated and aerated throughout 
fermentation. 
Table I indicates the results obtained using two type A gelatins with a 
molecular weight of 5000, no gelling strength and an isoelectric point of 
6.3 or 6.6, hydrolyzed during sterilization at different pH values. 
Gelatin 1 is marketed by Rousselot under the reference ASF and gelatin 2 
under the reference HP50. 
By way of comparison, a fermentation was also carried out using soya bean 
flour as the source of nitrogen. 
The xanthane was precipitated at the end of the fermentation process by 
introducing 200 l of isopropanol into the medium; it was then filtered off 
and dried by heating in a stream of air. 
TABLE I 
__________________________________________________________________________ 
Weight of Viscosity 
xanthan of the 
Optical 
Source of 
Sterilization 
Molecular 
Fermentation 
g/kg of 
Productivity 
solution (a) 
density 
nitrogen 
pH weight 
time medium 
g/l/h mPa.s (b) 
__________________________________________________________________________ 
Soya bean 
7 47 h 19.2 0.40 160 0.35 
flour 
Gelatin 1 
7 5000 70 h 5 0.071 -- -- 
4 4000 63 h 20 0.32 230 0.2 
2 2000 55 h 22.6 0.41 205 0.194 
Gelatin 2 
4 2500 54 h 22.5 0.42 220 0.168 
2 2000 53 h 22.7 0.43 225 0.174 
__________________________________________________________________________ 
(a) concentration: 0.2% by weight in aqueous solution containing 1% by 
weight of KCl; measured on a Brookfield viscometer at T = 24.degree. C. 
(b) in aqueous solution containing 0.2% by weight and measured at a 
wavelength of 620 nm.