GB1584194A - Process for reducing the content of lipids in cell masses - Google Patents

Abstract

The lipid and nucleic acid content in microbial cell masses is decreased by treating the cell masses with an extraction mixture of ammonia or ammonium hydroxide and an organic solvent of the formula I. After the removal of the extraction mixture, the residue of the cell mass is washed with water and the aqueous phase is removed. The residue is then dried - optionally after extraction with an organic solvent of the formula I. The substituents in the formula I have the meanings given in Claim 1. Instead of a compound of the formula I, isopropanol can be used as organic solvent. R1-(CH2)n-OR2 (I)I

Description

(54) PROCESS FOR REDUCING THE CONTENT OF LIPIDS IN CELL MASSES (71) We,' HOECHST AKTIENGES ELLSCHAFT, a body corporate organised according to the laws of the Federal Republic of Germany, of 6230 Frankfurt/Main 80, Postfach 80 03 20, Federal Republic of Germany, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: The present invention relates to processes for reducing the quantities of lipids and nucleic acids present in the cells of microorganisms.

Every cell contains carbohydrates, proteins, lipids and nucleic acids, and the suitability of microbial-cell masses for food and feed purposes is impaired by nucleic acids and lipids.

The content of nucleic acid is critical, since nucleic acids may cause pathological effects, for example arthritis or urinary calculus. The presence of lipids reduces the stability during storage as they become rancid and produce an unpleasant taste.

Lipids may be separated from the cell wall and membrane by treatment with an organic solvent, or they may be saponified by treating them with an aqueous alkali. They may also be separated from microorganisms using a mixture of methanol and chloroform; this process, however, is complicated, and may lead to toxic by-products. Lipid extractions by means of alcohol/water mixtures (according to German Offenlegungsschrift 24 05 593, or German Offenlegungsschrift 21 37038) are generaly not suitable for bacteria, and in addition their execution is complicated and costly.

Further processes for reducing the content of nucleic and lipidic substances in microbial cell masses involve their separation by alkaline substances at elevated temperatures. These processes have the disadvantage that the free fatty acids formed upon hydrolysis are set free and split off together with protein.

Moreover, certain of the essential amino acids, for example lysine, change to a state in which they can no longer be used by the organism.

The present invention provides a process for reducing the content of lipids in a cell mass which comprises treating the cell mass with ammonia and an organic solvent of the general formula R1 H2n)OR2 (I) wherein either (i) R1 and R2 each represents a hydrogen atom and n represents 1, 2, or 3, or (ii) Rl represents a hydroxy group, R2 represents a hydrogen atom, a methy group or represents a hydrogen atom, a methyl group, or an ethyl group, and n represents 2 or 3.

the total amount of water present in the reaction mixture being within the range of from 0 to 10% by weight, calculated on the weight of the solvent of the general formula I.

The ammonia may be introduced into the reaction mixture as a gas; it may be used in the form of an aqueous solution, especially as a concentrated solution (for example 33% solution); or it may be dissolved in the organic solvent of the general formula I prior to treatment of the cell mass.

After the cell mass has been treated with ammonia and an organic solvent of the general formula I, the liquid may be separated from the cell mass, which is then preferably treated with water in order to reduce the content of water-soluble components, especially nucleic acids. The aqueous phase may then be removed, the remaining cells may, if desired, be further extracted with an organic solvent, for example a solvent of the general formula I, and the residue is subsequently dried.

The cell mass is suitably a microbial cell mass, preferably that of a microorganism which has been produced, for example, by cultivation on an alcohol or an n-paraffin in the presence of an aqueous nutrient medium and a gas containing free oxygen. Preferably bacteria, yeast and fungi are used as microorganisms.

Examples of such microorgaisms include methanol-utilizing bacteria of the genus Methylomonas, for example, Methylomonas clara ATCC 31226, or yeasts, for example Candida lipolytica ATCC 20383, which may be obtained by cultivation on an n-paraffin in the presence of an aqueous nutrient medium.

The cell mass may be in the form of a desiccated fungus mycelium. This cell material may be produced from substrates originally used for the production of antibiotics, for ex ample of a penicillin by fermentation, after having extracted the antibiotic.

Suitable solvents of the general formula I are alcohols, for example methanol, ethanol, n-propanol or isopropanol, preferable metha nol or ethanol, especially methanol; and glycols and monoethers thereof, especially glycol or monomethyl glycol. A mixture of solvents may be used.

Ammonia is admixed with the solvent of the general formula I either as a gas (NH,) or as a concentrated aqueous solution (NH40H).

Whether the gas or the solution is more suit ably used depends on the water content of the cell mass, for example the residual water content of the desiccated cell material, and the quantity and water content of the solvent.

NO4 OOH is suitable for cell masses with a low water content (for example 0 to 15%), while NH3 is more appropriate for cell masses with a higher water content (for example 10 to '30%).

The quantity of lipid which is removed by extraction with ammonia and a solvent of formula I, depends on the total water content calculated'on or the quantity of solvent (in weight %), and on the NH, concentration (in weight %), calculated on the solvent.

Especially good results are obtained with a cell mass/solvent ratio in the range of from 1: 3 to 1:6 when using methanol or ethanol, and in the range of from 1: 8 to 1:12 when using propanol, a glycol, especially glycol, or a monoether thereof. The ammonia concentra tion, calculated on the quantity of solvent, is suitably in the range of from 1 to 10 weight %, preferably 1 to 5 weight %. The total quantity of water, provided by the cell mass, the solvent, and possibly by aqueous ammonia, is in the range of from 0 to 10 weight %, calculated on the quantity of solvent.

When operating on an industrial scale, it may not be necessary to dry the cells after fermentation to a residual water content of less than about 4%; it is sufficient to bring the cell mass to a desiccated cell weight such that the total water content after addition of the required quantity of solvent is within the required range. (Ammonia may be used in the form of gaseous NH,).

In order to extract the lipids, the microbial cell mass is suitably suspended in an organic solvent of the general formula I and NH, is introduced or NO4 OOH added. It is desirable to mix the suspension thoroughly by agitation.

In general, the processing temperature is suitably in the range of from 200 to +60"C, preferably from + 5 c to +50 C and especially from +10 to +30 C. The processing time generally varies from 5 to 120 minutes, pre ferably 25 to 35 minutes. The process is suit ably carried out under normal pressure.

At the end of the treatment with solvent and ammonia, the resulting cell material (pro tein) is separated from the solvent by any suitable method, for example, centrifugation, filtration or sedimentation, preferably filtration.

The solid cell material thus obtained may be treated once more with a solvent of the general formula I in order to remove the lipids as completely as possible.

The residue may be dried for further re moval of residual solvent and ammonia, suit ably under reduced pressure, preferably from 80 to 150 mm Hg, and at an elevated tem perature, preferably from 40 to 50"C. The dried product thus substantially freed from lipids is generally odourless.

The liquid phase separated~from the solid cell material as described above contains ammonia and dissolved lipids. The solvent may be separated from the fats by distillation under reduced pressure, and then recycled.

Subsequently the resulting cell mass is pre ferably added to water to extract nucleic acids. Preferred ratios of the cell mass (in weight %) to water added are in the range of from 1:1 to 1:30, especially 1:5 to 1:15.

Sufficient water should be'added to allow the suspension to be agitated.

The pH value during the water treatment should generally be within the range of from 5 to 8.5, preferably 6 to 7.5 and is, if 'necessary, adjusted accordingly, especially when the cell masses resulting from the first process stage are not completely dry and containing residual quantities of ammonia, which may lead to too high a pH.

This extraction with water of nucleic acids, salts, polysaccharides and water-soluble secondary metabolites is generally carried out at a temperature in the range of from 30 to 95"C under normal pressure, preferably at a temperature in the range of from 40 to 70"C, especially from 50 to 60"C. The ex traction time, depending on the extraction temperature and the quantity of water, generally varies from 5 to 120 minutes, good results generally being obtained with an ex traction time from 25 to 45 minutes. For separating solid and liquid components, the suspension is centrifuged at a temperature of preferably from 10 to 30"C. Other suitable separation processes include sedimentation and filtration.

In order to facilitate the separation of the cell mass, there may be added to the water used in this second extraction step up to 20 weight % calculated on the weight of water, preferably from 5 to 15 weight % of a hydrophilic solvent, for example a lower alcohol, for example ethanol or methanol, preferably methanol.

The solid phase may be freed from residual liquid by any suitable method, for example vacuum freeze drying, vacuum drying or spray drying. The product has an agreeable smell, a lighter colour than the starting material and a particularly good water-binding ability. Due to the removal of lipids and nucleic acids, the products is especially suitable for preparing food and feed mixtures. The product according to the invention generally contains a residue of from 0.5 to 3.5 weight % of lipids and 0.5 to 4.5 weight % of nucleic acids.

The process according to the invention avoids the disadvantages of other processes, for example treatment with toxic solvents or separation by alkaline substances, and no chlorinated hydrocarbons are required. The removal of fats, especially when using bacteria, with mixtures of ammonia and a solvent of the general formula I is more complete than the removal achieved by treating the cell mass with mixtures of other solvents and water. Extreme temperature and pH ranges, which reduce the usability of the product, are avoided. The need for energy is lower than that of other processes involving separation of alkali substances. The protein content can be increased without producing large quantities of salts by neutralization.

The aqueous phase separated from the solid cell material contains, as well as other watersoluble components, nucleic acids, which may be recovered by any suitable method, for example precipitation in an acid medium, ultrafiltration, dialysis or enzyme treatment.

The following Examples illustrate the invention. The results of the Examples are shown in Tables Ia and Ib.

EXAMPLE 1.

Methylomonas clara ATCC 31226 was cultivated under aerobic conditions in a nutrient medium containing methanol as the only carbon source, ammonia as the only nitrogen source, phosphate, iron salts, magnesium salts and other commonly used trace elements. The bacteria cell mass produced during this cultivation was separated from the solution and spray dried. 100 g of this cell mass were added to 300 g of methanol. 10 g of gaseous NH, were introduced into the suspension while stirring, and thus dissolved.

The temperature was maintained at 25" to 35"C by cooling. The mixture of methanol, ammonia and cell mass was then agitated at 20"C for 30 minutes.

The solid and the liquid phases were then separated by filtration and the solid residue was washed once with 200 ml of methanol and refiltered. The two filtrates were combined: this brown solution contained lipids from the starting material. Methanol and ammonia were removed by distillation under reduced pressure (100 mm Hg, 40 C). The residue, representing 9.5 weight % of the original cell material, was a dark brown, unpleasant-smelling paste, contained free fatty acids, glycerides, phospholipids and secondary metabolites.

The solid residue of the extracted cell mass, which had been obtained by filtration, was dried under reduced pressure (100 mm Hg) at 40"C for 5 hours. 90 g of degreased cell mass, being odourless and having a lighter colour shade than the starting material, were obtained.

This cell mass was suspended in 900 ml of water in order to reduce the nucleic acid content. The suspension was homogenized by agitation and had a pH of 6.9.

After having raised the temperature to 55"C, agitation was continued for another 20 minutes; the mixture was then cooled to 30"C and the solid and liquid phases were separated by centrifugation. The sediment thus obtained was added to 900 ml of water and agatited at 20"C for 10 minutes. Centrifugation was then repeated and the sediment was dried under reduced pressure.

A yield of 68 g of cell mass was obtained.

The nucleic acid content had decreased from originally 11.2% to a mere 1.5%. The dry product was odourless; after moistening with water, it had a pleasant smell.

EXAMPLE 2.

The starting material was a bacteria cell mass as described in Example 1. The procedure of Example 1 was repeated exactly, with the exception, however, that the gaseous NH, was replaced by 30 ml of concentrated NH4OH (33%).

EXAMPLE 3.

The process according to Example 2 was repeated except that methanol was replaced by ethanol as solvent.

EXAMPLE 4.

The process according to Example 2 was repeated except that methanol was replaced by iso-propanol as solvent.

EXAMPLE 5.

A cell mass of Methylomonas clara as described in Example 1 was used as starting material. 100 g of the spray-dried material was separated and degreased using 15 g of NH, according to the description of Example 1, with the difference, however, that the residue was not subjected to complete desiccation. The filter cake was sucked off thoroughly and had a solid matter content of 85%. It was then suspended in 900 ml of water. The pH was 8.9, due to ammonia retained in the moist biomass.

The temperature of the suspension was raised to 65"C while agitating, and after 5 minutes the pH was adjusted to 7.2 by adding dilute hydrochloric acid. Agitation was then continued for another 15 minutes at 65 CC, then cooling to 40"C and centrifugation took place. The sediment obtained was dried.

EXAMPLE 6.

A yeast type Candida lipolytica ATCC 20383 utilising hydrocarbons, was cultivated on n-paraffins in the presence of an aqueous nutrient medium and an oxygen-containing gas. The yeast cell mass was separated from the nutrient solution and dried.

100 g of the dry yeast cell mass were sus pended at room temperature under normal pressure in 300 g of methanol and 10 g of gaseous NH, were added to this mixture over 15 minutes, the temperature of the suspension being maintained at 15"C by cooling. After having introduced the gas, agitation was continued for another 20 minutes at 22"C, followed by filtration through a suction frit.

The filter cake was mixed thoroughly on the frit with 300 ml of methanol, then sucked off.

The two filtrates were combined. The solution had a yellow colour and contained lipids from the starting cell material. Methanol and NH3 were removed under reduced pressure (14 mm Hg).

The residue after the second filtration, comprising the destroyed and degreased cells of the microorgans, was dried in a vacuum drying cabinet (100 mm Hg) at 40"C for 5 hours. The product obtained was a lighter colour than the yeast cell mass originally used, and was odourless.

Degreased and dried yeast were suspended in a solution of in the proportions ig yeast: 10 ml distilled water and methanol water and methanol water 1 ml methanol, in order to reduce the nucleic acid content (originally 7.5 weight %, calculated on the starting material). The mixture was agitated and, at the resulting pH of 6.8, was heated to 50"C for 15 minutes. It was then converted by centrifugation to a sediment containing the yeast protein, and separated to form a liquid phase containing nucleic acid. The sediment was subjected to vacuum freeze drying having been washed once more at room temperature.

The nucleic acid content of the dry material had diminished from 7.5 weight % to 0.4 weight %.

EXAMPLE 7.

Penicillium chrysogenum ATCC 10238 was cultivated aerobically according to known methods in a nutrient solution containing lactose, cornsteep fluid, phosphate, carbonate and magnesium sulphate. The mycelium remaining after separation of the penicillin produced was dried and used as starting material.

The process was carried out according to the description of Example 6, except that NH, was replaced by NHOH (33%). The degreased dry mycelium was washed with water at 30"C.

EXAMPLE 8.

The starting material was a cell mass as described in Example 7, and the procedure of Example 10 was followed except that methanol was replaced by ethanol, and the temperature during the water extraction was increased to 85"C and maintained at this level for 15 minutes.

The following Tables Ia and Ib show the results of Examples 1 to 8.

TABLE Ia Removal of lipids

100 g of cell mass Fat nucleic- NH3 (g) or weight acid solvent NH4 OH Example type % weight % 300 g (33 %, ml) 1 Methylomonas 7 11. 2 methanol NH3 10 2 " 7 11.2 methanol NH4OH 30 3 " 7 11.2 ethanol NH4OH 30 4 " 7 11.2 isopropanol NH4OH 30 5 " 9.6 15.4 methanol NH3 15 6 Candida- 8.2 7.5 methanol NH3 10 lipolytica 7 desiccated 3.5 2.6 methanol NH4OH 30 myce lium 8 " 3.5 2.6 ethanol NH4OH 30 TABLE Ib Removal of nucleic acids

Washing water Cell mass obtained quantity temperature quantity Fats weight Nucleic acid Example (1) pH ( C ) (g) % weight % 1 0.9 6.9 55 68 0.8 1.5 2 0.9 6.9 60 67 1.3 1.2 3 0.9 6.9 50 67 2.2 1.5 4 0.9 7.0 60 64 3.4 4.0 5 0.9 7.2 65 62 1.2 1.4 6 0.8 6.8 50 68 1.4 0.4 7 0.9 6.5 30 78 1.1 0.5 8 0.9 6.5 85 79 1.3 0.8

Claims (29)

WHAT WE CLAIM IS:-

1. A process for reducing the content of lipids in a cell mass, which comprises treating the cell mass with ammonia and an organic solvent of the general formula R1 - (C0H20) - OR2 (I) wherein either (i) Rl and R2 each represents a hydrogen atom and n represents 1, 2, or 3, or (ii) R1 represents a hydroxy group, R2 represents a hydrogen atom, a methyl group or an ethyl group, and n represents 2 or 3, the total amount of water present in the reaction mixture within the range of from 0 to 10% by weight, calculated on the weight of solvent of the general formula I.

2. A process as claimed in claim 1, wherein the ammonia is introduced into the reaction mixture in the form of a gas or an aqueous solution or is dissolved in the organic solvent of the general formula I prior to treatment of the cell mass.

3. A process as claimed in claim 1 or claim 2, wherein the cell mass is a microbial cell mass.

4. A process as claimed in claim 3, wherein the cell mass is derived from a bacterium, a yeast or a fungus.

5. A process as claimed in claim 4, wherein the cell mass is Methylomonas clara ATCC 31226.

6. A process as claimed in claim 4, wherein the cell mass is Candida lipolytica ATCC 20383.

7. A process as claimed in claim 4, wherein the cell mass is Penicillium chrysogenum ATCC 10238.

8. A process as claimed in claim 4, wherein the cell mass is desiccated fungus mycelium.

9. A process as claimed in any one of claims 1 to 8, wherein the organic solvent of the general formula I is methanol, ethanol, n-or iso-propanol, glycol or monomethyl glycol.

10. A process as claimed in claim 9, wherein methanol or ethanol is used in a cell mass to solvent ratio in the range of from 1: 3 to 1 : 6.

11. A process as claimed in any one of claims 1 to 9, wherein a propanol, a glycol or a monoether thereof is used as the solvent of the general formula I in a cell mass to solvent ratio in the range of from 1: 8 to 1: 12.

12. A process as claimed in any one of claims 1 to 11, wherein there is used from 1 to 10% by weight of ammonia based on the weight of solvent of the general formula I.

13. A process as claimed in claim 12, wherein there is used from 1 to 5% by weight of ammonia based on the weight of solvent of the general formula I.

14. A process as claimed in any one of claims 1 to 13, wherein the process is carried out at a temperature in the range of from -20 to 60"C.

15. A process as claimed in claim 14, wherein the process is carried out at a temperature in the range of from 5 to 50"C.

16. A process as claimed in claim 15, where in the process is carried out at a temperature in the range of from 10 to 30"C.

17. A process as claimed in any one of claims 1 to 16, wherein after treatment with ammonia and the solvent of the general formula I, the resulting cell mass is treated with water to reduce the content of water-soluble components.

18. A process as claimed in claim 17, where in the ratio of cell mass to water is in the range of from 1:'1 to 1:30.

19. A process as claimed in claim 18, where in the ratio of cell mass to water is in the range of from 1:5 to 1:15.

20. A process as claimed in any one of claims 17 to 19, wherein the pH during the treatment with water is in the range of from 5 to 8.5.

21. A process as claimed in claim 20, wherein the pH during the treatment with water is in the range of from 6 to 7.5.

22. A process as claimed in any one of claims 17 to 21, wherein the treatment with water is carried out at a temperature in the range of from 30 to 95 cm.

23. A process as claimed in claim 22, wherein the treatment with water is carried out at a temperature in the range of from 40 to 70"C.

24. A process as claimed sin claim 23, wherein the treatment with water is carried out at a temperature in the range of from 50 to 60"C

25. A process as claimed in any one of claims 17 to 24, wherein there is present in the reaction mixture during the treatment with water up to 20% by weight, calculated in the weight of water, of a hydrophilic solvent.

26. A process as claimed in any one of claims 17 to 25, wherein after separation of the aqueous phase the resulting cell mass is extracted with an organic solvent of the general formula I.

27. A process as claimed in claim 1, carried out substantially as described in any one of the Examples herein.

28. A cell mass which has been treated for the extraction of lipids by a process as claimed in any one of claims 1 to 27.

29. A process for reducing the content of lipids and nucleic acids in a microbial cell mass, which comprises treating the cell mass with ammonia or ammonium hydroxide and an organic solvent having the general formula I R1-(CH2)0-0R2 (I) wherein either (i) R1 and R2 each represents a hydrogen atom and n stands for 1, 2, or 3, or (ii) Rl represents a hydroxy group, R2 represents a hydrogen atom, a methyl group or an ethyl group, and n stands for 2 or 3, the total amount of water present in the reaction mixture being within the range of from O to 10% by weight, calculated on the weight of the solvent of the general formula I and, after separation of the liquid, washing the residue with water, separating the aqueous phase, and drying the residue.