FI57443B - FOERFARANDE FOER NEDSAETTANDE AV LIPID- OCH NUKLEINSYRAHALTEN I MIKROBIELLA CELLMASSOR - Google Patents

Description

M (11) ANNOUNCEMENT 5 744T Ma lJ '} UTLÄCGN INGSSKRIFT 0 1 * H ° C <4S> ^ (51) Kv.ik.3 / Incci.3 c 12 N 1/08, A 23 J 3/00 ENGLISH —EN N LAN D (21) PtMftttlhelwmu # - PttwttanaMcnlng 772272 (22) Hak «ml * ptlvi - An * ttkning * d« f 25 · 07 · 77 (23) Alkupllvi — Glltlfh * t * U | 25.07.77 (41) Translators | utktMksl - Bllrlt offMtllg 28.01.78

Pstolttti. J. raki.terih.IHtU, Nlhtivlkslptnon J. heard on the date_

Patent · och regirteiftyrelten Arrtksn utl «gd oct.« Krift «n public ·» * 30.0l +. 80 (32) (33) (31) Requested «tuolktu * —Begird priori ** 27.07.76 Federal Republic of Germany-Förbundsrepubliken

Germany (DE) P 2633666.3 (71) Hoechst Aktiengesellschaft, Postfach 80 03 20, 6230 Frankfurt (Main) 80, Federal Republic of Germany-Förbundsrepubliken Germany (DE) (72) Merten Schlingmann, Kelkheim, Laslo Vertesy, Eppstein / Taunus, Federal Republic of Germany Förbundsrepubliken Tyskland (DE) (7 ^) Oy Kolster Ab (5 ^) Method for reducing the lipid and nucleic acid content of the cell mass of microorganisms - Förfarande för nedsättande av lipid- och nukinsyrahalten i mikrobiella cellmassor

The invention relates to a method for reducing the lipid and nucleic acid content of the cell mass of microorganisms by extracting the cell mass with solvents and ammonia, separating the extract and washing and drying the cell mass. Each cell contains carbohydrates, proteins, lipids, and nucleic acids. The content of nucleic acids and lipids adversely affects the usefulness of the cell mass obtained from microorganisms as a nutrient and feed.

Nucleic acid is harmful because it causes diseases (gout, urinary stones). Lipids limit storability by rotting and adversely affect taste.

It is therefore an object of the present invention to reduce the lipid and nucleic acid content of a microbial cell mass. By known methods, lipids are dissolved from the cell wall and cell membrane with organic solvents or saponified with aqueous alkalis.

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One known method for separating lipid from microorganisms is by working with mixtures of methanol and chloroform. The method is costly and can lead to toxic products. Lipid extraction with alcohol / water mixtures (DE applications 2 h05 593 or 2 137 038) is poorly suited for bacteria and is technically expensive.

FI publication 52 99 ^ discloses a method for extracting certain microorganisms with a lower alcohol, which may contain up to $ 60 of water. However, the concentration of lipids and nucleic acids in the cell mass is not sufficiently reduced by this method. According to FI publication 53 590, cell mass is treated with aqueous alkali, whereby proteins and nucleic acids are dissolved. A disadvantage of this method is that alkali treatment alters the original structure of the proteins, thereby reducing their value as a nutrient. U.S. Patent No. 3,775,393 describes a method for reducing the nucleic acid content of microbial cells by treating them at high temperature and elevated pressure with aqueous ammonia. The disadvantage of this method is the alteration of cellular proteins, which produces, among other things, toxic by-products.

Other known methods for reducing the nucleic acid and lipid content of a microbial cell mass use alkaline leaching at elevated temperatures. The disadvantage in this case is the separation of the free fatty acids formed in the hydrolysis together with the protein.

In addition, some of the essential amino acids contained in proteins, e.g. lysine, become unsuitable for the body.

A method for reducing the lipid and nucleic acid content of a microbial cell mass has now been invented. The process according to the invention is characterized in that in the first step the cell mass is extracted with an extraction mixture containing ammonia or ammonium hydroxide and an organic solvent of formula V (CVn-OE2 1 wherein R1 and R2 represent a hydrogen atom and n is 1, 2 or 3, including iso-propanol, or wherein R 1 is a hydroxy group and R 1 is a hydrogen atom or a methyl or ethyl group and n is 2 or 3, at normal pressure and temperature between -20 ° C and + 60 ° C for up to 120 minutes, the total water content during extraction being 0 -30% by weight based on the amount of solvent used, and when the solvent is methanol or ethanol is a weight ratio of cell mass, solvent to ammonia of 1: 3: 0.03-1: 6: 0.6, preferably 1: 3: 0.03-1 : 6: 0.3, and when the solvent is propanol, glycol or monoglycol ether 1: 8: 0.08-1: 12: 1.2, preferably 1: 8: 0.08-1: 12: 06, and that in the second step the residue of the cell mass after separation of the extraction mixture 3 57443 is washed with water at pH 5-8.5, at a temperature of 30-95 ° C and at normal pressure for 5 to 120 minutes, the weight ratio of water to cell mass being 1: 1 to 1:30, after which the aqueous phase is separated, the cell mass is optionally extracted as an organic solvent of the formula I, and the cell mass is dried.

The microbial cell mass is preferably obtained from microorganisms which are produced, for example, by culturing them in alcohol or n-paraffins in the presence of an aqueous nutrient medium and a gas containing free oxygen. Preferred microorganisms are bacteria, yeasts and fungi.

Examples of such microorganisms are methanol-utilizing bacteria of the genus Methylomonas, e.g. Methylomonas Clara ATCC 31226, or yeasts such as Candida lipolytica ATCC 20.383, obtained by culturing with n-paraffins in the presence of an aqueous nutrient,

Dried fungal mycelium can also be used. This cellular material is obtained from the preparation of antibiotics such as penicillin after antibiotic extraction,

Preferred solvents of formula I are methanol and ethanol, especially methanol. In addition to the alcohols mentioned, glycols and their monoethers of the formula I, namely glycol and monomethyl glycol, are also suitable.

Ammonia can be added to said solvents as a gaseous (NH 4 OH) or concentrated aqueous solution (NH 4 OH). The choice is made according to the water content of the dry cell mass and the water content of the solvent used. NH 2 OH is more suitable for a cell mass with a low moisture content (0-15 #), whereas NH 2 OH is better for a cell mass with a higher water content (10-30 #).

The amount of lipid removed by extraction with ammonia and solvent depends on the total water content and the NH 4 concentration, which are calculated as a percentage by weight of the amount of solvent used.

The ammonia content, based on the amount of solvent, is 1-10% by weight # preferably 1-5 #. The amount of water derived from the cell mass, the solvent and possibly aqueous ammonia is 0 to 30% by weight, preferably 0 to 20% and especially 0 to 10% by weight, based on the amount of solvent used.

When working on a technical scale, post-fermentation drying of cells to a low water content of 4 # or less can be dispensed with. It is sufficient to bring the cell mass to such a water content that, after the addition of the required amount of solvent, the total water content is in the optimum range, possibly gaseous NH 4 is added to the solvent.

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The dissolution of fats from the microbial cell mass is carried out by suspending the cell mass in an organic solvent of the formula I and introducing NH 4 H into the suspension or adding NH 4 OH. Suitably the suspension is thoroughly mixed mechanically. The treatment temperatures are between -20 and + 60 ° C, the preferred range being +5 to + 50 ° C and especially + 10 to + 30 ° C. The processing time is 5 to 120 minutes, preferably 25 to 35 minutes.

At the end of the solvent / ammonia treatment, the cell mass (protein) obtained is separated from the solvent by any method such as centrifugation, filtration or sedimentation.

Preferably, filtration is used. The resulting cell mass can be further treated with one of said organic solvents to remove lipids as completely as possible. The residue can be dried to remove residual solvent and ammonia. Suitably this is carried out under reduced pressure, preferably at a pressure of 11-20 kPa and at an elevated temperature, e.g. L0 ~ 50 ° C. The degreased product is odorless.

The liquid phase separated from the solid cell mass by the above method contains ammonia and dissolved lipids. The solvent can be separated from the fats by vacuum distillation and reused. The defatted cell pulp, which may have been dried, is then mixed with water. Preferably the weight ratio of water to cell mass is 1: 1 to 1:30, especially 1: 5 to 1:15. The amount of water should be at least such that mixing of the suspension is possible.

The pH during the water treatment should be between 5-8.5, preferably 6-7.5, and if necessary the pH is adjusted to this range. This is especially necessary when using undried or incompletely dried cell mass from the first step, which still contains ammonia, which can lead to too high a pH.

This extraction of nucleic acids, salts, polysaccharides and water-soluble secondary metabolites with water is generally carried out at a temperature in the range of 30-95 ° C under normal pressure. Temperatures between U0 and 70 ° C are particularly preferred, particularly between 50 and 60 ° C. The extraction time can be 5-120 minutes, depending on the extraction temperature and the amount of water, good results are achieved in 25-1 + 5 min. extraction. To separate the solid and liquid components, the suspension is preferably centrifuged at temperatures of 10-30 ° C. Other suitable separation methods are sedimentation and filtration.

In order to simplify the separation of the cell mass, a second alcohol, such as ethanol, methanol, preferably methanol, is preferably added to the water in a second step up to 20% by weight, preferably from 5 to 15% by weight.

Liquid residues are removed from the solid phase by conventional methods such as freeze, vacuum or spray drying. The product has a pleasant odor, is lighter than the starting material and binds water particularly well. Thanks to the removal of lipids and nucleic acids, it is particularly suitable for the production of nutrients and feeds.

By the method of the invention, the lipids of the product can be removed to a residual content of 0.5-3.5 wt% and the nucleic acids to a residual content of 0.5-5.5 wt%.

The process according to the invention avoids the disadvantages of known processes, such as solvent treatment with chlorinated hydrocarbons or alkali leaching. Degreasing with ammonia / solvent mixtures is substantially more complete, especially in the case of bacteria, than with cell / solvent mixtures. This also avoids the deteriorating effect of high temperature and pH ranges on product quality. Energy consumption is lower than in known temporal dissolution methods. Protein enrichment does not produce large amounts of neutralizing salts.

The aqueous phase separated from the solid cell mass contains, among other water-soluble components, nucleic acids which can be recovered by known methods, e.g. by mixing in acidic media, ultrafiltration, dialysis or enzyme treatment.

The invention is illustrated by the following examples.

Example 1

Methylomonas clara and ATCC 31226 were cultured under aerobic conditions in a nutrient solution containing methanol as the sole carbon source, ammonia as the sole nitrogen source, phosphate, iron, magnesium salts and other common trace elements. The bacterial cell mass thus produced was separated from the solution and spray-dried.

100 g of this cell mass was mixed with 300 g of methanol. 10 g of NH 4 gas were introduced into the suspension with stirring, which dissolved in it. By cooling, the temperature of the suspension was maintained at 25-35 ° C during the introduction of NH 4. A mixture of methanol, ammonia and cell mass was stirred for 30 minutes at 20 ° C,

To separate the solid and liquid phases, the mixture was filtered and the resulting solid was washed once with 300 ml of methanol. After re-filtration, both filtrates were combined. The resulting brown solution contained the lipids of the starting material used. Methanol and ammonia were removed by vacuum distillation (13 kPa, Lo ° C). The residue with 9.5% by weight of the cell mass used was a dark brown, malodorous paste consisting of free fatty acids, glycerides, phosphorus lipids and secondary metabolites.

The solid extracted cell mass obtained by filtration was dried under vacuum (13 kPa) at 1 + 0 ° C for 5 hours. In this way, 90 g of defatted cell mass were obtained, which was odorless and lighter in color than the starting material.

To reduce the nucleic acid content, this cell mass was suspended in 900 ml of water. The pH of the stirred homogenized suspension was 6.9.

The temperature was raised to 55 ° C, the suspension was stirred for a further 20 minutes, cooled to 30 ° C and centrifuged. The resulting sediment was re-mixed with 900 ml of water, and the mixture was stirred for 10 minutes at 20 ° C. The mixture was then centrifuged again and the sediment was dried under reduced pressure.

The yield was 65 g. The nucleic acid content had decreased from the original 11.2% to 1.5%. The product was odorless when dry, moistened with water and had a pleasant odor.

The results of this and other examples are summarized in Tables Ia and Ib below.

Example 2 The starting material was the same bacterial cell mass as in Example 1. It was subjected to the same process steps and conditions, except that 30 ml of concentrated NH 1 + OH (33%) was used as reagent instead of gaseous NH 4.

Example 3

The procedure was as in Example 2, but 60 ml of concentrated NH 4 OH (from 33%) was used.

Example * +

In the procedure of Example 2, ethanol was used as the solvent instead of methanol.

Example 5

The procedure was otherwise the same as in Example 3, but using glycol monomethyl ether as solvent instead of methanol.

Example 6

The procedure was as in Example 2. Instead of methanol, isopropanol was used as the solvent.

Example 7 The same cell mass (Methylomonas Clara) as in Example 1 was used as starting material. 1 g of the spray-dried product was dissolved as described in Example 1 and degreased (15 g of NHg). Complete drying of the residue was not performed. The thoroughly filtered suction filter cake with a dry matter content of 85% was suspended in 900 ml of water. The pH was adjusted to 8.9 under the influence of ammonia remaining in the moist biomass.

The temperature of the suspension was raised to 65 ° C with stirring and after 5 minutes the pH was adjusted by adding HCl to 7.2. The suspension was then stirred for a further 15 minutes at 65 ° C, cooled to 40 ° C and centrifuged. The resulting sediment was dried.

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Example 8

The 3-strain Candida lipolytica ATCC 20.38 3 strain of edible hydrocarbons was cultured with 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 dry yeast solution was suspended in 300 g of methanol at room temperature under normal pressure, and 10 g of NH 4 gas was introduced into this mixture over a period of 15 minutes. The temperature of the suspension was maintained by cooling to 15 ° C. After the gas was introduced, it was stirred for a further 20 minutes at 22 ° C and then filtered through suction. The filter cake was sintered once with 300 ml of methanol and then suction filtered. Both filtrates were combined. The solution was yellow and contained the lipids of the cell mass used. Methanol and NH 4 were removed under reduced pressure.

After the second filtration, the residue consisting of the decomposed cells of the microorganism, defatted, was dried in a vacuum oven at 40 ° C (1.9 kPa) for 5 hours.

The product thus obtained was odorless and lighter in color than the yeast cell mass originally used.

To reduce the nucleic acid content from the original 7.5% by weight of the starting material, 100 g of defatted dried yeast were suspended in a solution of 1 liter of distilled water and 1000 ml of methanol. The pH of the mixture was 6.8 with stirring, the mixture was heated at 50 ° C for 15 min and then centrifuged. The sediment contained yeast protein, and the liquid phase contained solubilized nucleic acid. The sediment was washed once at room temperature and lyophilized.

The nucleic acid content of the dried material had decreased from the original 7.5% by weight to 0.4% by weight.

Example 9

In the procedure of Example 8, ethanol was used instead of methanol and 60 ml of NH 4 OH (33%) was used instead of NH 4 gas (10 g).

Example 10

Penicillium chrysogenum ATCC 10,238 was aerobically cultured according to standard methods in a nutrient solution containing lactose, corn solubilizer, phosphate, carbonate and magnesium sulfate. After separation of the penicillin produced, 8,547,443 of the mycelium obtained were dried and used as starting material.

The procedure was performed as described in Example 8.

NH 2 OH (33%) was used instead of NH 4. The defatted dry mycelium was washed at 30 ° C with water,

Example 11 The starting material was the same cell mass described in Example 10. Ethanol was used instead of methanol. During the water extraction, the temperature was raised to 85 ° C and held for 15 minutes.

The following Tables Ia and Ib show the results of Examples 1-11.

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Table Ia Lipid removal

Eg 100 g of used cell mass Solvent 300 g NH3 (g) or

Micro- Fat Nucleii- NH 4 OH (33%, ml) organism (% by weight) nitric acid (% by weight) 1 Methylo- 7 11.2 Methanol NH- 10 monas 2 "7 11, 2 Methanol ΝΗμ0Η 30 3 "7 11.2 Methanol NH 2 H 60 H" 7 11.2 Ethanol ΝΗ, ^ ΟΗ 30 5 "7 11.2 Glycol mono- NH 4 OH 60 methyl ether 6" 7 11.2 Isopropanol NH ^ OH 30 7 "9.6 15, U Methanol NH3 15 8 Candida 8.2 7.5 Methanol NH, 10 lipolysic acid 9" 8.2 7.5 Ethanol ΝΗμ0Η 60 10 Penicyl- 3.5 2.6 Methanol NHuOH 30

lium H

(dry mycelium) 11 "3.5 2.6 Ethanol NH 4 OH 30 10 57443

Table Ib

Removal of nucleic acids

Wash water Obtained in cell mass

Eg · Amount Heat ”Amount Fat Nucleic acid (1) pH (° C) (g) (% by weight) and (% by 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 7.0 65 63 2, 0 1.1 4 0, 9 6, 9 50 67 2.2 1.5 5 0.9 7.1 55 63 2.8 2.3 6 0.9 7.0 60 64 3.4 4.0 7 0.9 7.2 65 62 1.2 1.4 8 0.8 6.8 50 68 1.4 0.4 9 0.8 6.5 45 67 1.8 0.7 10 0, 9 6.5 30 78 1.1 0, 5 11 0.9 6.5 85 79 1.3 0.8

Claims (1)

Claim: A method for reducing the lipid and nucleic acid content of the cell mass of microorganisms by extracting the cell mass with solvents and ammonia, separating the extract and washing and drying the cell mass, characterized in that in the first step the cell mass is extracted with an extraction mixture containing ammonia or ammonium hydroxide and organic the formula is R- (CH-) -OR- 1 n 2 n 2 wherein R 1 and R 2 represent a hydrogen atom and n is 1, 2 or 3, including isopropanol, or wherein R 1 is a hydroxy group and R 1 is a hydrogen atom or methyl - or an ethyl group and n is 2 or 3, at normal pressure and temperature between -20 ° C and +60 ° C for up to 120 minutes, the total water content during the extraction being 0-30% w / w based on the amount of solvent used and wherein, when the solvent is methanol or ethanol, the weight ratio of cell mass, solvent to ammonia is 1: 3: 0.03 to 1: 6: 0.6, preferably 1: 3: 0.03 to 1: 6: 0, 3 * and when the solvent is propanol, glycol or monoglycol ether 1: 8: 0.08-1: 12: 1.2, preferably 1: 8: 0.08-1: 12: 0.6, and that in the second step the residue of the cell mass in the extraction mixture after separation, wash with water at pH 5-8.5, at a temperature of 30-95 ° C and at normal pressure for 5-120 minutes, the weight ratio of water to ringing being 1: 1-1: 30, after which the aqueous phase is separated, optionally extracted with ringing as an organic solvent of formula I, and dried in a ring.