FR2671099A1 - Process for the production of trehalose by microbial fermentation under conditions which induce variation of the osmotic pressure - Google Patents

Abstract

The invention relates to a process for the production of trehalose by fermentation, characterised in that a microorganism which naturally produces trehalose is cultured on a culture medium containing at least one carbon source under conditions which induce variation of the osmotic pressure of the culture medium.

Description

 The present invention relates to a process for producing trehalose by fermentation.

 Trehalose in the present application is understood to mean the isomer of α, α-trehalose found in nature. It is a disaccharide consisting of two glucose molecules linked together by a bond (1-1).

Α, α-trehalose or α-D-glucopyranosyl-D-glucopyranoside has the formula

Trehalose was first isolated from ergot of rye by
WIGGERS in 1832. Since then, it has been found in a large number of different organisms.

 It has been found in many bacteria, especially the fungi-like bacteria actinomycetes. It is present in the spores and myceliums of streptomycetes (1) and in corynebacteria (2). Recently, trehalose has been found in a number of archaebacteria (3), which are halophilic, thermophilic and methanogenic.

 Finally, many cyanobacteria (4) accumulate trehalose.

 In most of these organisms, much of the trehalose is esterified with long-chain fatty acids. It seems in these cases that free trehalose is only a precursor of esterified trehalose (5).

 Much of the trehalose present in bacteria such as mycobacteria, nocardia and corynebacteria is esterified and becomes a component of the bacterial wall. Thus, the toxic compound of the cell membranes of Mycobacterium tuberculosis, called "cord factor" is a glycolipid, the α,--trehalose 6,6'-diester of mycolic acid.

If similar glycolipids have been found in other organisms,
Nocardia and Corynebacterium (5), trehalose is esterified with other fatty acids (nocardic and corynomycolic) in place of mycolic acid. In bacteria that could use hydrocarbons (6), the "cord-factor" would have a strong emulsifying effect and would allow microbial enzymes to access the substrate which has a very low solubility in water.

 The amount of trehalose produced in the world is less than one tonne and is used only as a laboratory product. Currently, trehalose is produced industrially mainly by fermentation and extraction from yeasts.

Yeast fermentation production processes are characterized by trehalose accumulation intracellularly followed by extraction of trehalose contained in the cells. It is also possible to make a chemical synthesis by treating 2,3,4,6 tetra-O-benzyl
D-glucopyranoside with two equivalents of the corresponding glycosyl chloride gives a, trehalose and α, ε-trehalose in yields of 18% and 2%, respectively. The problem of this chemical synthesis is the obtaining of the racemic mixture.

 There may also be mentioned an enzymatic method for synthesizing trehalose from maltose. The process uses two enzymes: maltose phosphorylase and trehalose phosphorylase, the conversion rate obtained is 60%.

 The enzymatic process however involves a more expensive raw material and especially enzymes whose cost is very high. In addition, the purification is made difficult because of the presence of a large amount of glucose or maltose in the final medium.

 The production of trehalose by bacterial fermentation is practically non-existent in the literature.

There are publications describing strains excreting trehalose in the external environment (including Nocardia, Brevibacterium,
Arthrobacter) but the conditions of production and the reported performances are not likely to allow a large industrial production in interesting economic conditions.

 The object of the present invention is to provide a process for the production of trehalose by fermentation of microorganism under economic conditions of interest for industrial production.

 The present invention indeed provides a process for the production of trehalose by fermentation, characterized in that a microorganism producing trehalose is naturally produced on a culture medium containing at least one carbon source under conditions inducing a variation of the osmotic pressure. from the culture medium.

 In the process according to the invention, the fermentation can be carried out batchwise, semi-continuously or continuously.

 Preferably, the culture medium contains potassium in an amount of at least 1 g / l.

 This potassium concentration is essential for the survival of the microorganism since the entry of potassium into the cell is the primary response of the microorganism to a rise in osmotic pressure.

 In a second step, potassium contributes to the induction of the secondary response. This secondary response consisting in increasing the intracellular concentration is either trehalose or another compound.

 The performance of the process according to the invention makes it possible to extract the trehalose produced using the standard techniques of the industry.

 In one embodiment, the change in osmotic pressure is controlled to create a pressure gradient during fermentation such that the osmotic pressure change between the initial osmotic pressure and the final osmotic pressure is between 1000 and 5000 mosmoles, and preferably 2000 mosmoles.

 According to one characteristic of the process according to the invention, the osmotic pressure variation of the medium is caused by addition of an osmoinductive compound such as a salt, in particular KCl, NaCl, (NH 4) 2 SO 4 or a sugar or a sugar-sugar compound. glycerol type alcohol, sorbitol or mannitol, or mixtures thereof.

According to another characteristic of the process according to the invention, the excretion of trehalose in the medium is triggered and / or amplified.
by performing an addition in the medium of cationic surfactant (s) or neutral (s) and / or of antiobiotics,
and / or causing a variation of the temperature between 1 and 200C, preferably 5 and 100C relative to the normal temperature of growth
and / or by adjusting the vitamin concentration, in particular biotin and thiobiotin, to the minimum necessary to ensure the growth of the microorganism.

 The originality of the process for producing trehalose according to the invention therefore lies in the control of the osmotic pressure of the culture medium coupled, if appropriate, to the natural or induced excretion of trehalose in the external medium.

 Any cell tends to establish and maintain in the intracellular medium an osmotic pressure greater than that of the exocellular medium. This "turgor pressure" is necessary for normal growth of bacteria. When the bacteria is subjected to a hyperosmotic shock, it initiates various processes of osmotic regulation in order to maintain its turgor pressure.

 In addition, it appears that trehalose has a function of protecting cells against natural stress, such as water or heat stress. The protective effect of trehalose would be at the membrane level maintaining their fluidity and integrity.

 Therefore, the stress conditions resulting in particular from the variation of the osmotic pressure of the medium and, where appropriate, the high excretion of trehalose which characterize the process according to the invention could result from a phenomenon of regulation of the cells.

The microorganisms useful in the process are particularly of the genus
Corynebacterium, Brevibacterium, Nocardia, Escherichia, Bacillus arthrobacterium, Streptomyces, Microbacterium, preferably Corynebacterium or Brevibacterium.

Strains are chosen
for their ability to produce trehalose
for their ability to excrete trehalose (with or without additive)
for their ability to grow and ferment on at least one sugar;
for their rapid growth
for their aerobic nature
for their ability to grow and ferment on an osmotic high pressure medium.

 A strain used to illustrate the invention is a Corbynebacterium glutamicum, Gram + bacterium, Corynebacterium melaseccola ATCC No. 17965.

It is auxotrophic for biotin.

In one embodiment of the method according to the invention
a) an aerobic preculture is carried out at 30 ° C. in a preculture medium containing at least one sugar, mineral salts, especially of phosphate and / or magnesium, vitamins such as biotin, thiobiotin
b) the midfoot is inoculated with the preculture medium.

The midfoot contains
- from 50 to 100 g / l of sugar,
inorganic salts, especially of phosphate and / or magnesium, and at least 1 g / l of potassium,
vitamins such as biotin and desthiobiotin,
a source of nitrogen, such as (NH4) 2SO4 or acid hydrolysates of proteins and extracts of bacteria or yeasts. In these concentration conditions, the osmotic pressure is between 400 and 2000 mosmoles, in particular between 600 and 2000 mosmoles.
c) the growth and fermentation stage is carried out with thorough stirring
at a pH of between 7 and 8 and a temperature of between 30 and 420 ° C., in particular between 32 ° and 380 ° C.,
the feed medium having a sugar concentration of 500 to 1000 g / l and,
the concentrations of osmoinductive compound and the concentrations of compounds which promote the excretion of trehalose, in particular the surfactants and vitamins, are adjusted in such a way that the osmotic pressure increases by at least 1000 mosmoles relative to the initial value and the osmotic pressure final reaches 1500 or even 5000 mosmoles.

The pH adaptation varies according to the bacterial strain for
Corynebacterium, the optimum extracellular growth pH of the strain is 7.8.

 In particular, in step c), the feed medium can contain a concentration of osminductive compounds controlled so as to obtain at the end of fermentation an osmotic pressure of between 1500 and 5000 mosmols, the osminductive salt concentration can be, for example from 100 to 500 g / l. When the vitamin is biotin, the concentration of biotin can be in particular of the order of 5 μg / l.

 The foot medium generally represents 40 to 70% of the final volume, the feed medium representing respectively 60 to 30% of the final volume.

 In the fermentation process according to the invention, the fermentation time is short: 20 to 40 hours; trehalose / sugar yield (w / w) 15-20%; the final concentration in the important medium 15 to 50 g / l; trehalose is present in the external environment; the concentration of sugar other than trehalose is very low at the end of fermentation (1%) The process can be carried out continuously or batchwise in fermentors with a capacity ranging from a few liters to a few hundred cubic meters .

 The raw material will be for example the sugar selected from glucose, fructose or sucrose or raw materials including, such as molasses, syrups, starch hydrolysates.

 Suitably, trehalose is extracted after about 40 hours of culture.

 The extraction can be carried out by any method known to those skilled in the art by separation of the bacteria by filtration, ultrafiltration and / or centrifugation, optionally by concentration, optionally by acidification (removal of contaminating organic acids), optionally by filtration, optionally by neutralization, by passage over anionic and / or cationic resin, optionally by concentration, optionally by crystallization, optionally in the cold state or possibly by the addition of alcohol, passage over cationic resin and then anionic, concentration (yield 90% , 90% purity) and / or cold crystallization with or without the addition of alcohol (30% yield, 96% purity).

Other advantages and features of the invention will emerge in the light of the following examples: 1. DEFINITIONS OF THE ENVIRONMENTS
1.1 - ENVIRONMENT A
This medium is used for the conservation of strains of the genus
corynebacterium in subculture on Petri dish and slant agar.

Beef extract 20 g / l
Agar 20 g / l
Bactopeptone 20 g / l
NaCl 5 g / l
1.2 - ENVIRONMENT B
This medium is used for the realization of precultures in medium
liquid.

Beet molasses (sugar equivalent) 40 g / l
H3PO4 3 g / l
MgSO4 0.5 g / l
Urea 8 g / l
Biotin 50 pg / l
1.3 - MIDDLE C1
This medium is used for the production of the foot of fermentations
in Examples 1 and 2.

Sucrose 70 g / l
H3PO4 2.1 g / l
Yeast extract 0.5 g / l
(NH4) 2SO4 1.3 g / l
MgSO4 1.3 g / l
Solution of trace elements * 2 ml / I
2 mineral solution ** 5 ml / l
Catechol 1.1 mg / l
K + 1 g / 1
Biotin 1 mg / l
Thiamine 0.2 mg / l
The pH of the medium is adjusted to 7.8 after autoclaving with sodium hydroxide.

(*) Solution 1 of trace elements
Na2B407.10H2O 88 mg / l
(NH4) 6Mo7024.4H2O 40 mg / l
ZnS04,7H20 10 mg / l
CuS04.5H2O 270 mg / l
MnCl2,4H2O 7.2 mg / l
FeC13.6H20 0.87 g / l (**) Mineral solution 2
FeS04,7H20 20 mg / l
MnS04.7H2O 2 mg / l
NaCl 25 mg / l 1.4 - medium C2
This medium is used to make the sugar supply solution in Examples 1 and 2.

Sucrose 550 g / l 1.5 - MIDDLE D1
This medium is used for making the foot of the fermentations for Examples 3 to 6.

*** Syrup (sucrose equivalent) 70 g / l
Yeast extract 0.5 g / l
H3P04 2.1 g / l
(NH4) 2SO4 1.3 g / l
MgSO4 1.3 g / l
Biotin I mg / l
K + 1 g / l
1.6 - MIDDLE D2
This basic medium is used as a sugar supply solution
for example 5.

*** Syrup (sucrose equivalent) 550 g / l
According to examples 3,4,6 it is completed with a salt playing the role
osmoinducer such as ammonium sulfate or potassium chloride.

1.7 - MIDDLE D3
(NH4) 2SO4 100 g / l
*** Syrup (sucrose equivalent) 550 g / l
1.8 - MIDDLE D4
KCI 112 g / l
*** Syrup (sucrose equivalent) 550 g / l
(***) Syrup is the purified, filtered and concentrated
beet chips.

2. GENERAL CONDITIONS OF FERMENTATION
2.1 - Preparation of preculture
The cells developed in 30 ml of physiological saline are recovered.
slant agar (medium A). This bacterial suspension is used to seed a preculture (medium B) at a rate of 3%. The preculture is incubated at 30 ° C. under aerobic conditions and immersed culture for 6 hours. A volume of 35 ml of this preculture is used to seed 750 ml of fermentation medium (medium C1 or D1).

2.2 - Fermentation F1
In the context of Examples 1, 2 and 3, the fermentation is carried out as follows: fermentation pH 7.3, stirring at 1200 rpm, aeration at 0.6 v / v.mn, coverage pressure at 0.degree. , 2 bars,
Temperature of 34.5 C.

When the biomass estimated by optical density of the medium (measured at 650 nm) reaches the value of 0.33, the first surfactant is added at a concentration of 0.6 g / l. This surfactant is of the nonionic type composed of a polyoxyethylene fatty acid ester mainly of palmitic and stearic acids.

When the optical density reaches 0.58, the second surfactant is added at a concentration of 0.3 g / l. This surfactant is a mixture of fatty amine acetate, mainly dodecylamine.

One hour later, the feed solution (medium D2, D3, D4) is added successively in pulses so that the residual concentration in the medium is maintained at 30 g / l.

After 20 hours of fermentation, the temperature is increased and maintained at 38 C.

The fermentation is continued until the sucrose is exhausted (residual sugar concentration below 0.1%).

2.3 - Fermentation F2
In the context of Examples 5 and 6, the fermentation is carried out as follows
fermentation pH of 7.8,
stirring of 1200 rpm,
aeration of 0.6 v / v.mn,
cover pressure of 0.2 bar,
Temperature of 34.5 C.

When the biomass estimated by optical density of the medium (measured at
650 nm) reaches the value of 0.33 the first surfactant is added to
a concentration of 0.6 g / l. This surfactant is of the nonionic type
composed of a polyoxyethylene fatty acid ester predominantly
of palmitic and stearic acids.

1 h 30 after the first surfactant, the temperature is increased to
41.5 C and it is added by successive pulses the feed solution
(medium C2) so that the residual concentration in the medium
maintained at 30 g / l.

Fermentation is continued until sucrose is exhausted
(residual sugar concentration below 0.1%).

3. PURIFICATION
Activated carbon (3% w / v) is added to the medium which is then ultrafiltered
on SCT membrane at 65 ° C to remove bacteria and then concentrate by
evaporation up to 30 brix.

Most of the salts are removed by acidification with acid
sulfuric acid (pH = 3.3) then filtration. After concentration the solution is
adjusted to pH 4 then passed successively over anionic resin and
cationic. The eluate is then concentrated.

4. SUMMARY TABLE
The following table summarizes for all the examples the
medium and process conditions.

Examples <SEP> Medium <SEP> Medium <SEP> Medium <SEP> Foot <SEP> Medium <SEP> Method <SEP> Process <SEP> Osmotic
<tb> Maintenance <SEP> Preculture <SEP> Fermentation <SEP> Food <SEP> Fermentation
<tb> EXAMPLE <SEP> 1 <SEP> A <SEP> B <SEP> C1 <SEP> C2 <SEP> F1
<tb> EXAMPLE <SEP> 2 <SEP> A <SEP> B <SEP> C1 <SEP> C2 <SEP> F1 <SEP> SHOCK <SEP> Osmotic <SEP> with <SEP> KCl
<tb> EXAMPLE <SEP> 3 <SEP> A <SEP> B <SEP> D1 <SEP> D4 <SEP> F1 <SEP>SEP> continuous <SEP> gradient <SEP> pressure
<tb> osmotic <SEP> with <SEP> KCl
<tb> EXAMPLE <SEP> 4 <SEP> A <SEP> B <SEP> D1 <SEP> D3 <SEP> F1 <SEP> Continuous <SEP> continuous <SEP> of <SEP> pressure
<tb> osmotic <SEP> with <SEP> (NH4) 2SO4
<tb> EXAMPLE <SEP> 5 <SEP> A <SEP> B <SEP> D1 <SEP> D2 <SEP> F2 <SEP> Initial <SEP> Osmotic <SEP> Shock <SEP> with
<tb> KCl
<tb> EXAMPLE <SEP> 6 <SEP> A <SEP> B <SEP> D1 <SEP> D4 <SEP> F2 <SEP> Continuous <SEP> Continuous <SEP> of <SEP> Pressure
<tb> osmotic <SEP> with <SEP> KCl
<tb> SUMMARY TABLE
EXAMPLE 1
Strain: Corynebacterium glutamicum ATCC No. 17965.

 Preculture conditions are made according to the scheme described in the general conditions.

 The characteristics of the media correspond to the medium C1 in foot and C2 in feed described in the general conditions.

The fermentation conditions correspond to the process FI described in the general conditions. The main elements being
- Use of two surfactants to trigger excretion,
- Minimum content in K + in the foot (1 g / l),
- initial osmotic pressure of 460 mosmoles,
- Final osmotic pressure of 1000 mosmoles,
- 42 hours fermentation time,
- Under these conditions, there is no production of trehalose.

EXAMPLE 2
Strain: Corynebacterium glutamicum ATCC No. 17965.

 The conditions are identical to those described in Example 1 for media and fermentation, except the osmotic shock which is caused by addition of a solution of KCl (330 g / l) which makes it possible to establish a KCl content. in the middle of 27 g / l. This osmotic shock is carried out half an hour after the addition of the first surfactant.

- initial osmotic pressure of 460 mosmoles,
- Final osmotic pressure of 1824 mosmoles,
- Duration of the fermentation of 42 hours,
- The trehalose content in the fermentation must is 17 g / l.

 After fermentation, the trehalose is purified according to the process described in the general conditions and after concentration 9.8 g of trehalose at 90 B purity are recovered.

EXAMPLE 3
Strain: Corynebacterium glutamicum ATCC No. 17965.

 Preculture conditions are made according to the scheme described in the general conditions.

 The characteristics of the media correspond to the medium D1 syrups in the foot and D4 in food described in the general conditions.

The fermentation conditions correspond to the process F1 described in the general conditions. The main elements being
- Use of two surfactants to trigger excretion,
- Minimum content in K + in the foot (1 g / l),
Initial osmotic pressure of 450 mosmoles,
- Final osmotic pressure of 3000 mosmoles,
- It is caused a continuous variation of the osmotic pressure throughout the duration of the fermentation by feeding with a mixed solution of sucrose and a salt of KCl type.

- The fermentation time is 40 hours,
- The trehalose content in the fermentation must is 40 g / l.

 After fermentation, the trehalose is purified according to the method described in the general conditions and after concentration, 23 g of trehalose at 90% purity are recovered.

EXAMPLE 4
Strain: Corynebacterium glutamicum ATCC No. 17965.

 The conditions of medium and fermentation are identical to those described in Example 3 above except the feed solution (D3) used which additionally contains sugar, an ammonium sulfate type salt.

The initial osmotic pressure is 450 mosmoles,
- The final osmotic pressure is 1500 mosmoles,
- The fermentation time is 42 hours,
- The trehalose content in the fermentation must is 25 g / l.

 After fermentation the trehalose is purified according to the method described in the general conditions and after concentration is recovered 14.4 g of trehalose at 90% purity.

EXAMPLE 5
Strain: Corynebacterium glutamicum ATCC No. 17965.

 Preculture conditions are made according to the scheme described in the general conditions.

 The characteristics of the media correspond to the media D1 syrups in the foot and D2 in feed described in the general conditions.

The fermentation conditions correspond to process F2 described in the general conditions. The main elements being
- Use of a single surfactant to trigger excretion,
- The composition of the foot medium is reinforced by adding a salt of
KC1 so that the content in the medium in this salt is 22 g / l.

Initial osmotic pressure of 1125 mosmoles,
- Final osmotic pressure of 2300 mosmoles,
- The fermentation time is 30 hours,
- The trehalose content in the fermentation must is 17 g / l.

 After fermentation, the trehalose is purified according to the process described in the general conditions and after concentration, 9.7 g of trehalose at 90% purity are recovered.

EXAMPLE 6
Strain: Corynebacterium glutamicum ATCC No. 17965.

 Preculture conditions are made according to the scheme described in the general conditions.

 The characteristics of the media correspond to the medium D1 syrups in the foot and D4 in food described in the general conditions.

The fermentation conditions correspond to process F2 described in the general conditions. The main elements being
- Use of a single surfactant to trigger excretion,
- It is caused a continuous variation of the osmotic pressure throughout the duration of the fermentation by feeding with a mixed solution (D4 medium) consisting of sucrose and a salt of KCI type.

The initial osmotic pressure is 600 mosmoles,
- The final osmotic pressure is 2050 mosmoles,
- The fermentation time is 30 hours,
- The trehalose content in the fermentation must is 16 g / l.

 After fermentation, the trehalose is purified according to the process described in the general conditions and after concentration, 9.2 g of trehalose at 90% purity are recovered.

BIBIOLOGICAL REFERENCES 1. MARTIN, MC, DIAZ, LA, MANZANAL, MB and HARDISSON, C. Role
of trehalose in the spores of Streptomyces FEMS Microbiol. Lett. 35
49-54 (1986).

2. BRENNAN, P. J. Free trehalose in Corynebacterium xerosis, Biochem.

 J.110, 9 p (1968).

3. NICOLAUS, B., GAMBACORTA, A., BASSO, AL, RICCIO, R., DE ROSA,
M. and GRANT, WD Trehalose in Archaebacteria. System. Appl.

 Microbiol. 10, 215-17 (1988).

4. MACKAY, MA, NORTON, RS and BOROWITZKA, LJ Organic
osmoregularoty solutes in Cyanobacteria, J. General Microbiol. 130
2177-2191 (1984).

5. ELBEIN, A.D., The metabolism of a, a-trehalose, Adv.Carbohydr. Chem.

 Biochem. 30, 227-256 (1974).

6. STEPHENS, GM, and DALTON, H. Is Toxin Production by Coryneform
do you know how to utilize hydrocarbons? Tibtech 5
(1987).

Claims (10)

 A process for the production of trehalose by fermentation, characterized in that a microorganism producing naturally trehalose is cultured on a culture medium containing at least one carbon source under conditions inducing a variation of the osmotic pressure of the culture medium.  2. Method according to claim 1, characterized in that triggers and / or amplifies the excretion of trehalose in the medium.  3. Method according to one of the preceding claims, characterized in that the medium contains potassium in a concentration of at least 1 g / l.  4. Method according to one of claims 1 to 3, characterized in that controlling the change in osmotic pressure so as to create a pressure gradient during fermentation such that the change in osmotic pressure between the initial osmotic pressure and the final osmotic pressure is between 1000 and 5000 osmoles and preferably 2000 mosmoles.  5. Method according to one of claims 1 to 4, characterized in that it causes the osmotic pressure variation of the medium by addition  an osmoinductive compound such as a salt, in particular KCl, Nazi, (NH4) 2SO4, or  - a sugar or,  - A sugar alcohol compound glycerol type, sorbitol or mannitol, and their mixture.  6. Method according to one of claims 1 to 5, characterized in that triggers and / or amplifies the excretion of trehalose in the medium during the fermentation phase,  by making an addition in the medium of cationic surfactant (s) or neutral (s) and / or of antiobiotics,  and / or causing a variation of the temperature between 1 and 20 ° C., preferably 5 and 100 ° C., with respect to the normal growth temperature,  and / or by adjusting the vitamin concentration, in particular biotin and thiobiotin, to the minimum necessary to ensure the growth of the microorganism.  7. Method according to one of claims 1 to 6, characterized in that the microorganism is of the genus Corynebacterium, Brevibacterium, Nocardia, Escherichia, Bacillus Arthrobacterium, Streptomyces, Microbacterium, and preferably of the genus Corynebacterium or Brevibacterium.  8. Method according to one of claims I to 7, characterized in that  a) an aerobic preculture is carried out at 30 ° C. in a preculture medium containing at least one sugar, mineral salts, in particular phosphate and / or magnesium salts, vitamins such as biotin and thiobiotin;  b) preculture medium is then inoculated into a medium containing  - from 50 to 100 g / l of sugar,  - inorganic salts including phosphate and / or magnesium and at least 1 g / l of potassium,  vitamins such as biotin, desthiobiotin, and a source of nitrogen, such as (NH4) 2SO4 or acid hydrolysates of proteins and extracts of bacteria or yeast, under conditions of concentrations such that the osmotic pressure is between between 400 and 2000 mosmoles, in particular between 600 and 2000 mosmoles  c) the growth and fermentation stage is carried out with thorough stirring  by increasing the pH to a pH remaining between 7 and 8 and the temperature between 30 and 420 ° C., in particular between 32 and 380 ° C.,  the feed medium having a sugar concentration of 500 to 1000 g / l and the concentrations of the osmoinductive compound and the concentrations of compounds promoting the excretion of trehalose, in particular the surfactants and vitamins are adjusted until the osmotic pressure increases by at least 1000 mosmoles with respect to the initial value and the final osmotic pressure reaches 1500 or even 5000 mosmoles.  9. The method of claim 8, characterized in that in step c), the feed medium has a osminductive salt concentration of 100 to 500 g / l.  10. Method according to one of claims 1 to 9, characterized in that the vitamin is biotin and the concentration of biotin of the order of 5 pg / l.