FR2543973A1 - BACTERIOLOGICAL PROCESS FOR THE PREPARATION OF POLYSACCHARIDES - Google Patents

Abstract

THIS PROCESS, WHICH IS CARRIED OUT WITH THE AID OF ZOOGLOEA BACTERIA RAMIGERA STRAIN 115 CONSISTS OF DEVELOPING THE ZOOGLOEA RAMIGERA IN A STERILE AQUEOUS MEDIUM CONTAINING A SOURCE OF CARBON, A SOURCE OF NITROGEN, NUTRITIVE SALTS AND AN IRON SALT, AT A CONCENTRATION OF 0.15 TO 45MGL, PREFERREDLY 0.5 TO 5MGL, A DENSE SUSPENSION OF THE SAID BACTERIA IS OBTAINED, AND TRANSFER TO THE SAID SUSPENSION IN A FERMENTER CONTAINING A STERILE AQUEOUS MEDIUM HAVING THE ABOVE COMPOSITION, OR ABANDONNETERIA FOR APPROXIMATELY 20 TO 60 HOURS, TO CONSUME MAINLY ALL SOURCE OF CARBON ADDED TO AN INITIAL CONCENTRATION OF ABOUT 10 TO 50G OF SOURCE OF CARBON PER LITER, A LOW VISCOUS PRODUCT CONTAINING 10 TO 35GL OF POLYMER OBTAINED IN THE FORM OF CAPSULES.

Description

Process for the preparation of polysaccharides by the bacteriological route. The

  The present invention relates to a process for the preparation of polysaccharides bacteriologically using the bacterium Zoogloea ramigera, strain 115. According to the literature, we already know how to prepare polysaccharides on a laboratory scale at

  using the bacteria Zoogloea ramigera, strain 115 Any-

  times, a very long culture time is required. In addition, a high concentration of polymers could not be obtained. There is also a problem with the high viscosity.

culture medium.

  The disadvantages due to a long culture time are, for example, infection of the culture container and

low productivity.

  The low concentration of polymers obtained with the previous processes has led, for example, to

high recovery.

  A high viscosity of the culture medium raises great difficulties, particularly with regard to the

  transfer of oxygen to the bacteria.

  This is extremely serious because the bacteria in

  question has an aerobic metabolism.

  Due to the above mentioned disadvantages

  of known prior processes, it quickly became

  likely to produce polysaccharides on an industrial scale

  with the bacteria Zoogloea ramigera, strain 115.

  According to the present invention, it has become possible

  in a completely surprising way to overcome the disadvantages

  mentioned mentions linked to known prior processes and to provide a process for the preparation of polysaccharides bacteriologically using the bacterium Zoogloea ramigera, strain 115 The process consists (A) in developing Zoogloea ramegera in a sterile aqueous medium containing a source carbon such as glucose, starch or potatoes, a

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  nitrogen source such as ammonium chloride, air nitrate-

  monium or potassium nitrate, nutritive salts such as phosphates and sulfates, and an iron salt such as Fe C 13 or Fe 2 (SO 4) 3 at a concentration of 0.15 to 45 mg / l, preferably 0 , 5 to 5 mg / l, a dense suspension of said bacteria being obtained, and (B) transferring said suspension into a fermenter containing a sterile aqueous medium with the above composition, fermenter in which the bacterium

  is abandoned for about 20 to 60 hours, preferably

  30 to 50 hours, so that it consumes mainly

  all of the carbon source added, at a concentration

  initial tration of approximately 10 to 50 g of carbon source per liter, a slightly viscous product containing 10 to g / l of polymer being obtained, said polymer being present

in the form of capsules.

  The known strain 115 of the bacteria Zoogloea ramigera used according to the present invention, is deposited at The American Type Culture Collection, USA under No ATCC

25935.

  The polysaccharide obtained by the process according to the invention usually consists, inter alia

  things, by glucose, galactose and pyruvic acid.

  The addition of iron salt to the culture medium has been found to be highly favorable for increasing the growth rate of the bacteria. Preferably, a sterile filtered iron solution is used. The amount of iron salt calculated as Fe 3 +, must be at least 0.15 mg / l and at most 45 mg / l of the culture medium; Preferably,

  it is at least 0.5 mg / l and at most 5 mg / l.

  The p H of the culture medium should normally be at least 6.0 and not more than 8.0, preferably it

is between 6.5 and 7.5.

  During the first stage of cultivation, the growth

  sance of the bacteria is stipulated Growth will stop when the amount of nitrogen initially added is consumed,

  since nitrogen is the element that limits growth.

  The next stage of the culture is to produce the polysaccharide which is stored in the form of capsules

  bulky surrounding the bacteria.

  A carbon source must be present

  in the culture medium at the time of production of the poly-

  saccharide, on the one hand to provide the organism with the starting material which is transformed into building blocks which

  are included in the polysaccharide, on the other hand, for satis-

  make the energy requirement of the bacteria.

  According to an embodiment of the present invention

  tion, the polymer concentration of the weakly viscous product from stage (B) above can still be

  increased by a successive controlled addition of a quanti-

  additional carbon source tee in a later stage

laughing (F).

  According to another embodiment of the present

  invention, the polymer concentration of the low-product

  viscous from stage (B) may be further increased

  supported by a successive controlled addition of additional amounts of carbon source and nitrogen source

in a later stage (G).

  When the carbon source initially added

  in stage (B) is consumed, the addition of additional quantity

  carbon source supplementation in stages (F) and (G), respectively, is conveniently carried out from a separate container using a pump which is controlled by a signal from a C analyzer 02 The analyzer continuously monitors the level of C 02 in the outgoing gas

of the fermenter.

  When the addition of the carbon source to the culture medium is carried out, the level of C 02 in the outgoing gas

  of the fermenter increases until the amount of

  trat sent to the fermenter by a pump is consumed.

  Then, the C 02 level decreases rapidly and the addition of the carbon source is repeated. Thanks to this unique process, the addition of the carbon source can be controlled,

  so the concentration of the carbon source will be main-

  held in a desired interval during the phase where the polysaccharide is synthesized. A suitable CO 2 level is at least 0.2 and at most 3.0%, preferably it is between 0.7 and

1.3%.

  To the above embodiments which include stages (F) and (G) respectively, where more carbon source, and more carbon source and nitrogen source are added respectively, the total concentration of carbon source in stages (B) plus (F) and (B) plus (G), respectively, is 15 to 75 g / l, preferably

20 to 50 g per liter.

  As the carbon source in stages (F) and (G), respectively, it is possible to use for example lactose,

  glucose, starch or potatoes.

  The stages (B) plus (F) and (B) plus (G) are respected

  preferably completed within 30 to 48 hours.

  Thanks to a combination of the addition of carbon source and nitrogen source in stage (G), an additional advantage of producing a polysaccharide is obtained.

  uniform and high throughout the stage (G).

  According to an appropriate embodiment of the invention

  tion, we use starch as carbon source, chloride

  ammonium as nitrogen source and Fe C 13 as iron salt.

  Normally, the recovery of the polysaccharide and

  of bacteria in stage (C) is carried out by precipitation

  tation by adding an alcohol such as ethanol or isopro-

  panol The precipitate obtained is then collected in an appropriate manner by centrigutation (D) then usual drying (E)

to have the finished product.

  Stages (C), (D) and (E) can however be carried out by other means, for example depending on the use

of the finished product.

  The invention will now be explained in detail with reference to the attached single figure, which shows a diagram of the process for the preparation of polysaccharides according to the present invention, and with the aid of the exemplary embodiments below, among which Examples 1 to 5 relate to comparative tests not belonging to the

  present invention, while Examples 6 to II relate to

  nent the process of the present invention.

  The single figure shows three different embodiments of the invention The different

  marks with letters in the figure have the significance

following tion: -

  A stage of development in which the bacteria are allowed to grow in an aqueous medium containing a carbon source, a nitrogen source, nutrient salts

  and an iron salt according to the present invention.

  B Culture stage in fermenter where is carried out

the production of polysaccharides.

  C Recovery stage including for example

  a precipitation stage where the polysaccharide and the bacteria

laughs are rushed.

  D Centrifugation stage o the precipitated product

is collected.

E Drying stage.

  F Culture stage in a fermenter where takes place

  an additional successive addition of carbon source.

  G Culture stage in a fermenter where an additional successive addition of carbon source takes place

and nitrogen source.

  The present invention is illustrated by the examples

  ples descriptive and not limiting below.

Example 1

  A colony of Zoogloea ramigera, strain 115, is transferred from an agar plate to a

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  Erlenmeyer flask fitted with a diameter reducer

  2 liters (A) containing 250 ml of sterile medium consisting

  killed per g / liter of Glucose 25 aqueous solution

K 2 HPO 4 2

KH 2 PO 4 1

NH 4 CI 1

  Mg SO 4 x 7 H 20 0.2 Yeast extract (Difco) 0.01 The flask is then placed for 18 hours on a rotary shaker (100 to 300 rpm) having

  a temperature of 260 ° C. The suspension of bacteria is used.

  Ries obtained for inoculating (B) a sterilized medium in a fermenter The medium has the same composition as that mentioned above. The air is blown into the fermenter, 0.5 liter of air per liter of medium per minute, while stirring about 800 revolutions / minute The temperature of the fermenter is 260 C The p H of the medium is set to 7.0

  by automatic titration with Na OH.

  Glucose is consumed after 95 hours of culture

  and the polysaccharide concentration is 11.5 g / l.

Example 2

  The process of Example 1 is repeated, except that in stage (B) a solution of Ca C 12 is added to the medium The total amount of calcium added is 3 mg / i of the culture medium The glucose is consumed after 95 hours and the

  polysaccharide concentration is 10.1 g / l.

Example 3

  The process of Example 1 is repeated, except that in stage (B) a solution of Mn SO 4 is added to the medium The total amount of manganese added is 3 mg / i of the culture medium The glucose is consumed after 85 hours

  and the polysaccharide concentration is 10.2 g / l.

Example 4

  The process of Example 1 is repeated, except that in stage (B) a solution of Cu C 14 is added. The total amount of copper added is 3 mg / l of the culture medium.

  ture Glucose is consumed after 88 hours and the concentration

  The polysaccharide tration is 11.0 g / l.

Example 5

  The process of example 1 is repeated, except that in stage (B) a solution of Zn C 12 is added. The quantity

  total zinc added is 3 mg / 1 of the culture medium.

  Glucose is consumed after 90 hours and the concentration

of polysaccharide is 9.2 g / l.

Example 6

  The process of Example 1 is repeated, except that in stages (A) and (B) a solution of Fe C 13 is added to the medium The total amount of iron added is 3 mg / l of the culture medium Glucose is consumed already after hours and the polysaccharide concentration is

  13.2 g / l The viscosity is 40 centipoise (0.04 Pa s).

Example 7

  The optimal C / N ratio of a culture medium for the production of extracellular polysaccharide is established with the strain 115 of Zoogloea ramig in separate discontinuous cultures without feeding system. A glucose concentration of approximately 25 is used for the tests. g / l By varying the amount of added nitrogen source (0.25-4 g NH 4 Cl / 1) in the middle for the

  different cultures, it has been established that opti-

  male of polysaccharides from the added glucose is usually obtained for 0.5 to 2 g of NH 4 Cl / l The duration

  total culture is often 36 to 60 hours.

Example 8

  The optimal C / N ratio of a culture medium, when polysaccharide is produced with Zoogloea strain 115 will ramigrate in a discontinuous system which, thanks to a feeding system, is supplied with an additional carbon source by the addition of glucose, has been established in general to be between 10 and 25 By tolerating a higher nitrogen content during the initial discontinuous process, a mass of cells develops

  higher than normal Once the initial glucose is consumed

  mé, the addition of glucose begins The existing cell mass efficiently transforms the added glucose into

  polymer The glucose dosage is controlled automatically-

  ment by means of a signal value from the analysis

  CO 2 sor which measures the level of Ca 2 in the gas leaving the fermenter The addition of glucose begins when the

  signal value decreases below a displayed value.

  The culture time required is generally around

36 to 54 hours.

Example 9

  The optimal level of 2 dissolved in a culture medium is established with the strain 115 of Zoogloea ramig in discontinuous cultures The speed is varied

  ventilation in separate tests The agitation which

  The rate of dissolved O 2 is also kept constant at 800 revolutions per minute. If the air flow rate passing through the fermenter is kept at 0.251.5 vvm (air volume per volume of medium and per minute) the best conditions for convert glucose into polymer are obtained The rate of dissolved 2 is never less than 20% of the saturation value for tests which give good

conversion of glucose to polymer.

Example 10

  According to the present invention, it has been established that iron is a trace element necessary for a

  efficient production of the polymer with Zoogloea ramigera.

  Iron must be added during the growth phase of the bacteria.

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  done by adding per dose in the middle of the farm-

  iron solution (about 0.01 M) at the start of the culture The level of iron salt in the fermenter medium should be 0.15 to 45 mg / l, preferably 0.5 to 5 mg / l, calculated as iron Examples 7 to 9 above are carried out by adding iron to the medium in the form of a solution of Fe C 13 at a concentration of 2.5 mg / l of medium of the fermenter. of iron gives the result that the total duration of the process can be reduced by the duration

  normal from around 100 hours to around 36-60 hours.

Example 11

  Description of the culture with glucose supply,

o the C / N ratio is established.

  Test 1: The initial glucose concentration is 25 g / l

  and the nitrogen concentration is 0.26 g / l.

  In addition, the medium also contains the following compounds (g / l):

K 2 HPO 4, 2

KH 2 PO 4, 1

  Mg SO 4 x 7 H 20 0.2 yeast extract (Difco) 0.01 Test 2: 2.5 mg / l of Fe C 13 x 6 H 20 O is added per dose

  Glucose added at the start is consumed in 50 hours.

  An additional addition of glucose (5 g / l)

  in the middle of the fermenter leads to a concen-

  increased tration of the polymer, but the synthesis of

polymer is slowed down.

  Final polymer concentration: 18 g / l Final cell mass: 2.3 g / l Viscosity: 250 c P (0.25 Pa s) The nitrogen concentration in the culture medium is 0.52 g / l, but the rest is in accordance with test 1 The production of the mass of cells is stopped after 14 hours The initial quantity

  glucose is consumed after 10 p.m. Ad-

  dition of fresh glucose results in a rapid conversion of glucose to polymer At the end of the culture (<48 hours), the concentration of the polymer is 32 g / l The concentration of the cell mass is 4.3 g / 1 The viscosity is of

225 c P (0.225 Pa s).

  Test 3 The nitrogen concentration of the culture medium

  is 1.04 g / l, but the rest is unchanged

  mement to test 1 The production of the mass of cells is stopped after 18 hours Glucose

  added at the start is consumed after 8 p.m.

  The addition of fresh glucose leads to a conversion

  very fast release of glucose into polymer but large quantities of glucose are lost in this case in the form of CO 2 At the end of the culture (45 hours) the concentration of the polymer is g / l The mass of cells is 9, 8 g / l La

  viscosity is 230 c P (0.23 Pa s).

  The above embodiments show that the absence of iron in the culture medium, or the use of other metal salts such as a calcium salt, a manganese salt, a copper salt or a salt of zinc, gives a much longer culture time than the process according to the invention, where a specific amount of iron salt is used.

  It should be understood that the description which

  above has been given for illustrative purposes only and is not limitative and that any variant or modification may be made without departing from the general framework of

  the present invention as defined in the claims

attached. 1 l

Claims (6)

  1 Process for the preparation of polysaccharides by the bacteriological route using the bacteria toogloea ramigera strain 115, which consists (A) in developing Zoogloea ramigera in a sterile aqueous medium containing a carbon source such as glucose, starch or apples   of earth, a source of nitrogen such as ammonium chloride   nium, ammonium nitrate or potassium nitrate, nutritive salts such as phosphates and sulfates, and an iron salt such as Fe C 13 or Fe 2 (SO 4) 3 at a concentration of 0.15-45 Mg / l , preferably 0.5 to 5 mg / l, a suspension   dense of said bacteria being obtained, and (B) to be transferred   rer said suspension in a fermenter containing a sterile aqueous medium having the above composition, fermenter in which the bacteria is left for about 20 to 60 hours, preferably 30 to 50 hours, so that it consumes mainly all the carbon source added, at an initial concentration of approximately 10 to 50 g of carbon source per liter, a slightly viscous product containing 10 to 35 g / l of polymer being obtained,   said polymer being present in the form of capsules.   2 Method according to claim 1, which consists in (C) recovering the product from stage (B), said recovery comprising precipitation with an alcohol, for example ethanol or isopropanol, (D) a separation stage implementing centrifugation in order to collect the precipitate obtained and (E) preferably, the   drying of the separated product to have a finished product.   3 Method according to claims 1 or 2, which   consists in further increasing the concentration of the polymer of the low-viscosity product originating from stage (B), by   (F) a successive controlled addition of carbon source.   4 Method according to claims 1 or 2, which   consists in further increasing the polymer concentration of the slightly viscous product originating from stage (B) by (G) a successive controlled addition of carbon source and nitrogen source.   Process according to any one of Claims 1 to 4, which consists in using starch as the carbon source, ammonium chloride as the nitrogen source and Fe C 13 as the salt 6 Process according to the fact that the stages mined in the space of 30 to 7 Process according to the fact that the stages mined in the space of 30 to 8 Process according to terized by the fact that the carbon in the stages 9 Process according to of iron. claim 3, characterized (B) and (F) are together ter- 48 hours. claim 4, characterized (B) and (G) are together ter- 48 hours. claims 3 or 4, charac- lactose is added as source (F) and (G) respectively. claims 3 or 4, charac-   terrified by the fact that the successive addition of source of   carbon in stages (F) and (G) is controlled, respec-   tively, by a signal from a CO 2 analyzer.