FR2691975A1 - Fed batch prodn. of sophorolipid(s) from Candida - including pre-culture step, giving prod. in acetylated acid form, useful as emulsifier e.g. in sec. oil recovery - Google Patents

Abstract

Fedbatch prodn. of a sophorolipid compsn. (A) contg. a major proportion of acids at least partly acetylated, comprises growing Candida bombicola or C. apicola in a medium contg. a N source and opt. sugar, then feeding the culture to reaction zone with a sugar and at least one substrate under appropriate conditions of aeration, temp. and pH. The culture is fed continuously at 0.01-4 g/hr. l. of initial reactor vol. such that the concn. of residual substrate is at most 18 g/l. during the feeding period. (A) is then recovered. The new feature is that before the culture step, at least one preculture stage is carried out in a medium contg. at least one N source and at least one C source, i.e. carbohyarate, 10-24C satd. or unsatd. fatty acid ester, 10-20C satd. or unsatd. aliphatic hydrocarbon, aliphatic alcohol or acid, or their mixt., in which carbohydrate is not over 20% of the preculture medium and the wt. proportion of ester, hydrocarbon, alcohol and/or acid is below 0.5%. The culture medium is then seeded with the preculture medium. USE/ADVANTAGE - (A), which are more hydrophobic than hydrophilic, are useful as emulsifiers for making oil in water emulsions esp. for sec. oil recovery. This method produces a product contg. a high (pref. over 60%) proportion of acetylated acids.

Description

The invention relates to a process for the production with continuous feeding of a sophorolipid composition comprising a major part of at least partially acetylated acids and the sophorolipid composition thus obtained. It has been mentioned in the patents US Pat. a quantity of sophorolipids was produced by a fermentation process involving a culture of Torulopsis bombicola, strain currently classified Candida bombicola.

 A process for the production of sophorolipids by fermentation in a reaction zone with a sugar feed and with a continuous feed has also been described in the applicant's French patent application under registration number 90/16211. as fatty acid esters or as oils.

The sophorolipids obtained are considered to be a mixture of compounds represented by the formulas (1) and (2), the formula (1) representing the acid form and the formula (2) the lactone form.

In these formulas, R1 represents hydrogen or an acetyl group and R2 hydrogen or an alkyl residue containing 1 to 9 carbon atoms, when R3 is a saturated hydrocarbon residue 2 containing 7 to 16 carbon atoms, or R2 represents hydrogen or a methyl group, when R3 is an unsaturated hydrocarbon residue with 13 to 17 carbon atoms.

Up to now, the processes for preparing the products describe a set of numerous homologs and the separation of one of the main forms (acid or lactone) requires, for example, extractions with alcohol (EP - B 209783) which are long and expensive. Furthermore, extraction with a specific solvent does not always give good results because the solubility of all of the counterparts in a solvent can differ significantly, which affects the quality of the products obtained.

On the other hand, the methanolysis reactions in the presence of an acid catalyst do not give better results and it is very often a mixture of acids or deacetylated esters which is obtained.

In addition, the prior art (US 4197166) mentions that it is extremely difficult to prepare by fermentation a desired product having a given ratio of homologs.

Finally, it is known that the acetyl bonds in sophorolipids are chemically unstable and are very easily hydrolyzed by heating or prolonged storage near neutrality or even at room temperature under weakly alkaline conditions, which leads to obtaining of the fully deacetylated acid form.

 It is therefore extremely difficult by fermentation or chemistry to obtain a single product and a fortiori an acetylated product.

Furthermore, in petroleum applications linked, for example, to enhanced oil recovery, it is necessary to be able to create water-in-oil emulsions and therefore to be able to have emulsifiers more hydrophobic than hydrophilic, which cannot be the case for deacetylated acid products.

 There is therefore a demand in the industry for products having a fatty acid function and a saccharide structure comprising acetylated functions, masking the very hydrophilic hydroxyl groups, in particular on products obtained by fermentation and comprising a saccharide structure such as sophorose. .

We have therefore discovered a process for producing a fedbatch of a sophorolipid composition comprising a major part of acids at least partly acetylated under particularly advantageous conditions and responding to the above drawbacks. More particularly, at least one Candida bombicola or Candida apicola strain is cultured in a culture medium comprising a source of nitrogen and optionally sugar under appropriate conditions such that the said strain is produced and the said strain is subjected cultivated in a reaction zone to a sugar supply and to a supply of at least one suitable substrate under adequate aeration, temperature, pH conditions and the following sequence is carried out at least once. continues feeding the strain into said substrate at a rate
feed in the reaction zone of between 0.01 and 4 grams per
hour and per liter of initial reaction volume and for a period
such as the residual concentration of said substrate in the area
reaction is maintained at a value at most equal to 18 grams per liter
initial reaction volume during said feeding time; and B. The composition of sophorolipids produced is recovered.

More precisely, before the cultivation step, at least one step of preculture of the strain is carried out under appropriate preculture conditions in a preculture medium containing
- at least one source of nitrogen, and
- at least one carbon source chosen from the group comprising at least one
carbohydrate, at least one saturated or unsaturated fatty acid ester from 10 to
24 carbon atoms, at least one saturated aliphatic hydrocarbon or
unsaturated from 10 to 20 carbon atoms, at least one aliphatic alcohol from 10 to
20 carbon atoms, at least one aliphatic acid of 10 to 20 atoms
carbon and their mixtures; the proportion of carbohydrate being at most
equal to 20% compared to the preculture medium and the weight proportion
ester, hydrocarbon, alcohol and / or acid being at most equal to 0.5%; and the culture medium is seeded with the preculture medium.

 Preferably, the preculture medium can comprise at least one carbohydrate and at least one other carbon source chosen from the group comprising at least one ester, a hydrocarbon, an alcohol, an acid defined above.

According to a characteristic of the process, the preculture step or steps can be carried out at a temperature of 18 to 40 "C and preferably 20 to 30" G, during a period of time of 12 to 72 hours each and more particularly 24 at 36 hours.

According to another advantageous characteristic of the process, the fatty acid ester,
Aliphatic hydrocarbon, aliphatic alcohol, aliphatic acid may be in a proportion by weight of 0.1 to 0.30% relative to the preculture medium and carbohydrate in a proportion by weight of 2 to 12% compared to the preculture medium. Preferably, the ethyl or methyl ester of rapeseed, sunflower, palm or soybean oil is recommended.

Particularly interesting results have been obtained with a weight concentration of carbohydrate of 6 to 10% in the preculture medium.

According to another advantageous characteristic of the process, the carbon source can be chosen from the group comprising said fatty acid esters of 16 to 18 carbon atoms, said hydrocarbons of 16 to 18 carbon atoms, said alcohols of 16 to 18 atoms carbon and acids from 16 to 18 carbon atoms.

The nitrogen source used in the preculture stage is generally a mineral nitrogen source in the form of ammonium ions and / or organic nitrogen, for example in the form of urea, of amino acids such as in particular the extract of yeast, soy peptone, casein hydrolysates, corn maceration liquor, wheat gluten hydrolysates, meat extracts.

The addition of mineral elements such as for example potassium, sodium, magnesium and trace elements such as iron, manganese, molybdenum in the form of their salts (sulfates, phosphates, chlorides) can also make it possible to improve still growth.

The culture medium can comprise at least one sugar such as glucose or sucrose and / or esters of aliphatic fatty acids such as those which are introduced in the preculturn stage.
The propagation of the strain is generally carried out by seeding a preculture medium in small volume from an agar tube used for the conservation of this same strain.

This preculture medium, after an incubation period on an agitation table, is then transferred to a production reactor in which the implementation of the process can be carried out according to the operating protocol already described in the Applicant's French patent application. (EN 90/16211).

The process for producing sophorolipids is generally carried out according to the fed-batch technique and implemented under the conditions described for example in the same French patent application: temperature 18-35 "C, pH 2.5 to 8, 0.

advantageously 3.0 to 4.0, start of aeration 0.2 to 2 VVM under an absolute pressure of 1 to 5 bar and preferably 1 to 2 bar (1 bar = 0.1 MPa).

According to another characteristic of the method according to the invention, the step of recovering the sophorolipid composition produced during the cultivation step can comprise the separation of the strain from the fermentation must containing the sophorolipid composition, the neutralization at a pH close to the neutrality of the must and the elimination of water by heating and under reduced pressure.

According to another variant, the step of recovering the sophorolipid composition produced during the cultivation step can comprise the separation of the strain from the fermentation must containing the sophorolipid composition and the elimination of water under pressure. scaled down. This removal of water under reduced pressure is preferably carried out by lyophilization.

 By the process according to the invention, generally more than 55% of acidic sophorolipids are obtained in acetylated form and preferably at least 60%, for example 60 to 70%.

The characterization of the structural forms of the sophorolipids is usually carried out by high performance liquid chromatography (HPLC) of the mixture obtained, equipped with a detector, for example refractometric.

The acetylation rate of the acid forms is generally determined by high performance liquid chromatography, equipped with a detector, for example with light diffusion.

EXAMPLE 1
This example illustrates the invention The strain used is the yeast Candida bombicola
CBS 6009 which is preserved by monthly subcultures on an agar medium of the following composition
Pepsic peptone (Biothione BIOMERIEUX) 1 0 g / l
Yeast extract 5 g / l
Rapeseed ethyl ester 15 g / l
Agar 30 g / l
The strain is propagated on the following preculture medium
Glucose 100 g / I
(NH4) 2SO4 4 g / l
KH2PO4 1 g / l
MgSO4,7H2O 0.5 g / l
Dried liqueur of corn maceration 5 g / l
Rapeseed ethyl ester 2 g / l
For the preparation of this preculture medium, the glucose, the ethyl rapeseed ester, MgSO4, 7H O in half of the water are sterilized together at 1200 ° C. for 25 minutes.
4 '2 necessary, while KH2PO4, (NH4) 2SO4 and the corn maceration liquor, are sterilized, with the same sterilization schedule, in the other half of water. The preculture medium is reconstituted, after cooling to room temperature, by mixing the two solutions.

A 200 ml Erlenmeyer flask with a capacity of 30 ml of preculture medium is inoculated. After 40 hours of incubation on a shaking table, at a temperature of 25 "C, the entire medium contained in the Erlenmeyer flask is transferred to a Fernbach flask with a capacity of 2 liters containing 200 ml of a medium whose composition is identical to that of the Erlenmeyer flask. This second preculture stage is carried out as the first stage with stirring, at a temperature of 25 ° C. The production fermentation is carried out with the culture medium in a reactor of four liters of capacity containing a reaction volume of two liters.

The agitation of the medium is caused by a RAYNERI turbine, the speed of rotation of which is fixed at 1000 rpm. The aeration is 0.5 VVM of air under atmospheric pressure. The culture medium has the same composition as that used for the preculture chain, with the exception of the rapeseed ethyl ester which is added continuously, from sowing, at a feed rate of 1.0 gram per hour and per liter of medium. The reactor is seeded with the entire contents of the Fernbach flask. After self-acidification of the culture, the pH of the medium is kept constant at the value of 3.5 by the addition of 4 N sodium hydroxide solution controlled with a solenoid valve controlled by a pH meter.

During fermentation, 4 additions of glucose in solid form are carried out, each of them being equal to 50 g per liter of medium. These additions are made after 24, 48, 72 and 96 hours of culture respectively. The culture is stopped after 120 hours. The cells of the yeast strain are removed by centrifugation and the liquid phase is lyophilized. 162 g of a solid containing 146 g of sophorolipids are thus recovered per liter of medium. The analysis of the structural forms of the sophorolipids is carried out by high performance liquid chromatography (HPLC) with a Hypersil C-18 column 15 cm in length (Interchim, Montluçon, France) and a refractometric detector. The solvent used for the elution is a mixture of acetonitrile and water in the ratio 70/30 (v / v) at a flow rate of 0.7 ml / min. The acid forms of acetylated or non-acetylated sophorides are eluted at the head, followed by the lactone forms. To determine the acetylation rate of the acid forms, a liquid chromatograph equipped with a column identical to the previous one and a light scattering detector are used. The resolution of the acid forms is obtained with an acetonitrile / water elution gradient. The eluent mixture contains 98% water at the start of the analysis. The intervention of the linear gradient brings the composition of the eluent to 30% water after 48 minutes. The composition of the eluent mixture is then kept constant for an additional 14 minutes. It is thus determined that the mixture of sophorolipids consists of 110 g / l of acid forms (i.e. 75.3% of the total sophorolipids) and that, among these, the acid acetylated forms represent 95 g per liter of initial culture medium (ie 86% of the total acids).

Comparative example 2
Example 1 is repeated, increasing the quantity of rapeseed ethyl ester used in the two stages of preculture. This is increased from 2 g / l to 10 g / l. Under these conditions, 170 g / l (quantity relative to the initial volume) of a solid containing 156 g / l of sophorolipids are obtained. The acid forms represent 48 g / l (30.7% of the total sophorolipids) and among these the acetylated forms are 39 g / l (81.2% of the total acids).

EXAMPLE 3
Example 1 is repeated, replacing the rapeseed ethyl ester used in the two preculture stages with the sunflower methyl ester. At the end of the test, 158 g / l of a solid containing 142 g / l of sophorolipids are obtained. The amount of sophorolipids in acid form is 106 g / l (i.e. 74.6% of sophorolipids) including 92 g / l of acetylated acids (i.e. 95% of acid forms).

EXAMPLE 4 (comparison of Example 3)
Example 3 is repeated, increasing the concentration of the sunflower methyl ester from 2 g / l to 8 g / l in the two stages of preculture. 160 g / l of a solid containing 145 g / l of sophorolipids are thus obtained. The acid forms represent 37 g / l (ie 25% of the total sophorolipids) including 19 g / l of acetylated forms (ie 51% of the acid forms).

EXAMPLE 5
The fermentation protocol described in Example 1 is repeated, replacing the rapeseed ethyl ester with rapeseed oil. At the end of fermentation, the must is centrifuged and the yeast cells are eliminated. The clarified must is neutralized to pH 7.0 with a 4N sodium hydroxide solution and evaporated to dryness under reduced pressure in a Rotavapor.

105 g / l of dry residue containing 85 g / l of sophorolipids are recovered in this way.

Among these, 68 g / l of acid forms (80% of the total sophorolipids) are dosed, of which 54 g / l are in acetylated forms (ie 79% of the acid forms).

EXAMPLE 6
Example 1 is repeated, replacing the rapeseed ethyl ester by a cut of nalcanes with chain lengths between 12 and 18 carbon atoms. 70 g / l of product containing 65 g / l of sophorolipids are recovered at the end of the test. Among these we dose 52 g / l of acid forms (i.e. 80% of total sophorolipids) including 37 g / l of acetylated forms (i.e. 71% of acid forms)

Claims (9)

 the culture medium is inoculated with the preculture medium.  of hydrocarbon, alcohol and / or acid being less than 0.5%, and  in the preculture medium and the proportion by weight of ester,  proportion of carbohydrate being at most equal to 20% relative to  aliphatic of 10 to 20 carbon atoms and their mixtures; the  aliphatic alcohol of 10 to 20 carbon atoms, at least one acid  saturated or unsaturated aliphatic of 10 to 20 carbon atoms, at least one  unsaturated from 10 to 24 carbon atoms, at least one hydrocarbon  at least one carbohydrate, at least one saturated fatty acid ester on  - at least one carbon source chosen from the group comprising at least  - at least one source of nitrogen, and  preculture appropriate in a preculture medium containing  performs at least one step of preculture of the strain under conditions of  the process being characterized in that, before the cultivation step, one  b.We recover the composition of sophorolipids produced  feeding time; and  equal to 18 grams per liter of initial reaction volume during said  substrate in the reaction zone is maintained at a value at most  a feeding time such as the residual concentration of said  grams per hour per liter of initial reaction volume and for  feed rate in the reaction zone between 0.01 and 4  a.We continuously feed the strain in said substrate to a  times the following sequence  adequate aeration, temperature, pH conditions and at least one  sugar and a diet of at least one suitable substrate in  subjects said strain grown in a reaction zone to a supply of  under appropriate conditions such that the said strain is produced and  a culture medium comprising a source of nitrogen and optionally sugar  at least one Candida bombicola or Candida apicola strain is cultured in  comprising a major part of at least partly acetylated acids, in which  CLAIMS 1. Method for producing a sophorolipid composition in fedbatch 2. Method according to claim 1 wherein the preculture is carried out at a  temperature from 18 to 40 "C, preferably from 20 to 30" C. 3. Method according to claim 1 or 2, wherein the preculture time is 12 to  72 hours and preferably 24 to 36 hours. 4. Method according to one of claims 1 to 3 wherein the fatty acid ester,  Aliphatic hydrocarbon, aliphatic alcohol, aliphatic acid is in a  weight proportion of 0.1 to 0.3% relative to the preculture medium and  carbohydrate in a weight proportion of 2 to 12% relative to the  preculture medium. 5. Method according to one of claims 1 to 4 wherein the fatty acid ester,  Aliphatic hydrocarbon, aliphatic alcohol, aliphatic acid contain  16 to 18 carbon atoms. 6. Method according to one of claims 1 to 5 wherein the recovery step  sophorolipid composition made during the cultivation step  includes the separation of the strain from the fermentation wort containing the  composition of sophorolipids, neutralization at a pH close to neutral  of the must and the removal of water by heating and under reduced pressure. 7. Method according to one of claims 1 to 5 wherein the recovery step  sophorolipid composition made during the cultivation step  includes the separation of the strain from the fermentation wort containing the  composition of sophorolipids and elimination of water under reduced pressure. 8. Method according to one of claims 1 to 7 wherein the preculture medium  includes a carbohydrate and at least one other selected carbon source  in the group of esters, hydrocarbons, alcohols and acids of the  claim 1. 9. Composition of sophorolipids containing a major part of acids at least in  acetylated parts, obtained by the process according to one of claims 1 to 8.  is greater than 55% and preferably greater than 60%.  1 0. The composition of claim 9 wherein the proportion of acetylated acids