MXPA01004081A - METHOD OF PRODUCING&ggr;-DECALACTONE - Google Patents

Abstract

The invention relates to a method of producing&ggr;-decalactone, using Yarrowia lipolytica containing cultures.

Description

PROCEDURE FOR PRODUCING GAMMA-DECAIACTONE DESCRIPTION OF THE INVENTION The present invention relates to a new process for preparing? -decalactone. ? -decalactone due to its organoleptic properties is an important flavoring compound that has a fruity flavor and aroma similar to peach. In principle,? -decalactone can be produced from fruits. However, it is present in them in such small amounts that it can not be economically isolated by means of extraction or distillation. Therefore in recent years there have been numerous attempts to prepare? -decalactone by means of biotechnological processes. Most processes operate with the use of different yeasts. In this case the castor oil or the netilo ester of ricinoleic acid isolated from it, are converted by the yeasts. The yields obtained in this process vary between a few milligrams per liter up to 9.4 g / 1 in 75 hours (FR 2 734 843). The process has in addition the disadvantage that the uracil auxotrope material is employed and thus two separate steps are required for the formation and production of the biomass. The task of the present invention is therefore REF: 128475 to provide a procedure that makes possible high yields. This objective is achieved by the fact that a culture consisting of Yarrowia lipolytica is used. According to the invention it is possible to use Yarrowia lipolytica in a mixture with other microorganisms. However, Yarrowia lipolytica is preferably used as a pure culture. Particular preference is given according to the invention to the cultivation of Yarrowia lipolytica strain HR145 (DSM 12397). As a substrate for the culture used according to the invention, synthetic, semi-synthetic or complex culture media can be used. These comprise carbon and nitrogen compounds, inorganic salts with or without trace elements and vitamins. As carbon compounds, carbohydrates, hydrocarbons or basic organic chemicals can be preferably used. Examples of compounds that can be used preferably are sugars, alcohols and / or sugar alcohols, organic acids or complex mixtures. Preference is given to oils. As sugar, glucose is preferably used. The usable alcohols preferably include glycerol and mannitol. The organic acids that can be used are preferably citric acid. Complex mixtures include for example malt extract, yeast extract, casein or casein hydrolyzate. As the oil, castor oil can be used in particular. In these cases, mixtures of two or more of these compounds can be used according to the invention. As substrates containing nitrogen, inorganic compounds can be used. Examples of these are nitrates and ammonium salts. Likewise, organic sources of nitrogen can be used. These include yeast extracts, soybean meal, cottonseed meal, casein hydrolyzate, wheat gluten and corn steep liquor. It is also possible to use two or more of the mentioned compounds in the form of a mixture. Inorganic salts that may be used include, for example, borates, carbonates, chlorides, molybdates, nitrates, phosphates and sulfates. As metals contained in the mentioned salts, calcium, iron, potassium, cobalt, copper, magnesium, manganese, sodium or zinc are preferred. A mixture of two or more of these salts can also be used according to the invention. The temperature for the culture is preferably in the range of 10 to 40 ° C. Particular preference is given to the range of 20 to 35 ° C, a very high preference is given to the range of 25 to 30 ° C.

The pH of the medium is preferably 4 to 9. Particular preference is given to the range of 5 to 8. Sufficient ventilation is required during the production process. The reactors that can be used according to the invention must be designed accordingly. In principle according to the invention, all bioreactors suitable for aerobic processes and known to those skilled in the art can be used. Preferably, all suitable apparatuses can be used for any submerged process. This means that containers with or without mechanical mixing devices can be used according to the invention. The foregoing include, for example, stirrers, bubble or circuit column reactors. The latter preferably include all known apparatuses that have agitators with any design. The process according to the invention can be carried out continuously or discontinuously. The fermentation time until a maximum amount of product is reached, is in the range of 36 to 72 hours preferably in the range of 48 to 66 hours, calculated from the inoculation of the crop. According to the invention, the substrates can be added at the start of incubation, during growth or after growth. This can be achieved by simple addition of substrates or by continuous successive addition during the process. However, preference is given to the continuous addition over a period of a plurality of hours after the inoculation of the culture. Using the processes described according to the invention it is surprisingly possible to produce more than llg / 1 of? -decalactone in less than 70 hours. The invention and the surprising findings are described in more detail by means of the following examples. EXAMPLES 1. Preparation of a preliminary culture 1.7 g of malt extract are dissolved in 100 ml of water in a 500 ml Erlenmeyer flask having a side insert and the solution sterilized in an autoclave at 121 ° C for 15 minutes. After cooling to room temperature, the flask with the malt broth is inoculated using an inclined culture inoculum of Yarrowia lipolytica HR 145. The flask is incubated for 24 hours in a rotary agitator machine at 27 ° C and 100 rpm. Two 500 ml Erlenmeyer flasks with a lateral insert are charged with medium (1461 g of Na2HPO_xl2H20; 0.352 g of KH2P04: 0.53 g of urea; 0.07 g of Tween 80; 5g of baking powder; 1 g of castor oil and 100 ml of water) and sterilized in an autoclave at 121 ° C for 15 minutes. After cooling to room temperature, each of the flasks is inoculated with 500 μl of a malt extract broth of Yarrowia lipolytica HR 145. The flasks are incubated for 24 hours on a rotary agitator machine at 27 ° C and 100 rpm. 2. Production of? -decalac ona in the fermenter of 10 1. 9.8 1 of water are loaded in the fermenter and 14.61 g of Na2HP0.12H20 are added. 53 g of urea, 50 g of MgSO4.7H20, 0.04 g of riboflavin, 500 g of baking powder, 7.0 g of Tween 80, 100 g of castor oil and 5 g of antifoaming agent. The medium is sterilized in situ at 121 ° C for 30 minutes. In addition, 500 g of diluted solution of sodium hydroxide and sulfuric acid and castor oil are sterilized in the autoclave. After cooling, the antifoam probe and the injection attachment for NaOH are connected. The pH after sterilization is approximately 7.9. A pH of 7.0 is established by dilute sulfuric acid. The stirring speed is 400 rpm; the aeration is 3 l / min of compressed air; the temperature is 27 ° C. The fermenter is inoculated with the preliminary culture by means of a sterile injection attachment. During fermentation, more sodium hydroxide solution is added to maintain the pH at a value of 7.0. The antifoam is added automatically as required. 14 hours after the inoculation, the addition of the substrate is started. 500 g of castor oil in the course of 4 hours. After a fermentation time of approximately 52 hours, the fermentation is finished. The time to stop the fermentation is reached when no more sodium hydroxide solution has been added for one hour. To stop the fermentation, the contents of the fermenter are adjusted to a pH of 2.0 using concentrated sulfuric acid and heated at 80 ° C for 30 minutes.

After cooling, the contents are emptied for subsequent processing. The final concentration of y-decalactone, according to CLAP is 11,500 to 12500 ppm. The ratio of 3-hydroxy -? - decalactone to y-decalactone is less than 0.2. For the resulting culture broth, if appropriate after centrifugation, the? -decalactone is then isolated by the known physical process (distillation, extraction, etc.) 3. Production on a scale of 300 1 200 ml of preliminary culture is prepared accurately as described in Example 1 and used to inoculate a 300 1 fermenter. 140 1 of water were pre-charged in the fermenter and 742 g of urea, 49 g of KH2P0, 12 g of H20, were added, 7 g of yeast extract and 98 g of Tween 80. The medium is sterilized in situ at 121 ° C for 320 minutes. After cooling, the antifoam probe and the NaOH injection fitting are placed. The pH after sterilization is about 7.0. The stirrer speed is adjusted to 180 rpm, the aeration to l / min and the temperature at 27 ° C. The fermenter is inoculated under sterile conditions with 200 ml of the preliminary culture. During fermentation the pH is kept constant at 7.0 using sodium hydroxide solution. After 16 hours after the inoculation, the addition of the castor oil (7.4 kg) is started and it is completed after another 4 hours. After a fermentation time of 69 hours, fermentation is completed. The final concentration of β-decalactone is 12.3 g / 1. The ratio of 3-hydroxy-β-decalactone to β-decalactone is 0.125, since only 1.53 g / 1 of 3-hydroxy-β-decalactone are formed. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (15)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1.- Procedure for preparing? -decalactone in bioreactors, characterized in that cultures containing Yarrowia lipolytica are used.
2. 2 . - Process according to claim 1, characterized in that a pure culture consisting of Yarrowia lipolytica is used.
3. 3. Method according to one of claims 1 or 2, characterized in that Yarrowia l ipolytica HR 145 (DSM 12397) is used.
4. 4. Method according to any of claims 1 to 3 characterized in that synthetic, semi-synthetic or complex substrates are used.
5. 5. - Process according to one of claims 1 to 4, characterized in that substrates are used that contain carbon or nitrogen compounds, inorganic salts, trace elements and / or vitamins.
6. 6. - Process according to claim 5, characterized in that the carbon compounds present in the substrate are sugars, sugar alcohols, alcohol, organic acids, complex mixtures, oil or mixtures of two or more of these substances.
7. 7- Process according to claim 6, characterized in that glucose, glycerol, mannitol, citric acid, malt extract, yeast extract, casein, casein hydrolyzate and castor oil or mixture of two or more are present in the substrates used. more of those substances.
8. 8. Method according to one of claims 1 to 7, characterized in that substrates containing organic and / or inorganic components are used.
9. 9. Process according to claim 8, characterized in that nitrates, ammonium salts, yeast extract, soybean meal, cottonseed meal, casein, casein hydrolyzate, wheat gluten or corn steeping liquor are used. .
10. 10. Process according to one of claims 1 to 9, characterized in that sulphates, nitrates, chlorides, carbonates or phosphates of the metals sodium, potassium, magnesium, manganese, calcium, zinc or iron or mixtures of two or more are used. Substrates containing these compounds.
11. 11. Process according to one of claims 1 to 10, characterized in that the temperatures are in the range of 10 to 40 ° C, preferably 20 to 35 ° C.
12. 12. Process according to one of the claims 1 to 11, characterized in that the pH is in the range of 4 to 11, preferably 5 to 8.
13. 13. Yarrowia lipolytica HR 145 (DSM 12397).
14. 14. Use of Yarrowia lipolytica according to claim 13 to prepare? -decalactone.
15. 15. Process for preparing? -decalactone, characterized in that the ratio of hydroxy -? - decalactone to? -decalactone is less than or equal to 0.25 in the fermentation solution. PROCEDURE TO PRODUCE GAMMA-DECALACTONE SUMMARY OF THE INVENTION The invention relates to a method for producing? -decalactone, using cultures containing Yarrowia lipolytica.