DK146511B - PROCEDURE FOR REDUCING LIPID AND NUCLEIC ACID CONTENTS IN MICROBIAL CELL MASS - Google Patents

Description

(19) DENMARK (W)

§ "g PRESENTATION," 146511 B

DIRECTORATE OF THE PATENT 06 BRAND

(21) Patent Application No: 3376/77 (51) Irrt.CI.3: C12N 1/08, „. . C12 N 1/32 (22) Date of filing: 26 Jul 1977 (41) Aim. available: 28 Jan 1978 (44) Submitted: 24 Oct 1983 (86) International Application No :- (30) Priority: 27 Jul 1976 DE 2633666 (71) Applicant: 'HOECHST AKTIENGESELLSCHAFT; 6230 Frankfurt / Main 80, DE.

(72) Inventor: Merten * Schlingmann; DE, Laslo * Vertesy; THE.

(74) Plenipotentiary: The engineering firm Budde, Schou & Co_ (54) Procedure for reducing the lipid and nucleic acid content of microbial cell mass

The present invention relates to a method for reducing the lipid and nucleic acid content of microbial cell mass. Each microorganism cell contains carbohydrates, proteins, lipids and nucleic acids. However, the usefulness of microbial cell mass as a food source in feed is adversely affected by the presence of

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holdings of nucleic acids and lipids.

T · Nucleic acid content is questionable as it leads to L |) (O pathological conditions (arthritis, stone formation). Lipids limit la-

Jpl provides durability because they become rancid and destroy the taste.

It is therefore an object of the present invention to lower the lipid and nucleic acid content of microbial cell mass. In known processes, the lipids from cell wall and membrane are either dissolved by organic solvents or hydrolyzed by treatment with aqueous alkalis.

A known method for dissolving lipids from microorganisms works with mixtures of methanol and chloroform. The process is costly and can lead to toxic products. Lipid extractions with alcohol / water mixtures, according to German Patent Specification No. 2,405,593 or 2,137,038, are poorly suited for bacteria and are also costly in technical design.

Other known methods for lowering microbial cell mass on nucleic acids and lipids employ alkaline uptake at elevated temperatures. A disadvantage of this is the release of the free fatty acids resulting from the hydrolysis which are excreted together with the protein.

In addition, some of the essential amino acids contained in the protein, e.g. lysine, in a form that cannot be utilized by the organism.

A method has now been found for reducing the lipid and nucleic acid content of microbial cell mass, characterized in that in a first step the cell mass is treated at a temperature between -20 ° C and 60 ° C with an extraction mixture of ammonia or ammonium hydroxide. and an organic solvent selected from alcohols of formula ROH wherein R is an alkyl group and glycols and their monoethers of formula

H0- (CH9) -OR1 I

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where R is hydrogen, methyl or ethyl and n is 2 or 3, the total water content during the uptake being 0-30% by weight and the ammonia concentration being 1-10% by weight based on the amount of solvent used, and in a second step after separation The extraction mixture treats the cell mass with water at a pH of 5-8.5 cf. Claim 1 separates the aqueous phase and, optionally after extraction with one of the above solvents, dries the cell mass.

As microbial cell mass, microorganisms which are e.g. are prepared by growing on alcohol or n-paraffins in the presence of an aqueous nutrient medium and a gas containing free oxygen. As microorganisms, bacteria, yeasts and fungi are preferably used.

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

Also, fungal dry mycelia can be used. This cellular material is obtained as a by-product of antibiotic extraction, e.g. from penicillin fermentations after the antibiotic is extracted.

As solvents of the above formulas, alcohols of the former formula such as methanol, ethanol, n-propanol and isopropanol are considered. Preferred are methanol and ethanol, especially methanol. In addition to said alcohols, glycols and their monoethers of the latter formula are particularly suitable for glycol and monomethyl glycol.

Ammonia can be added in gaseous form (NH 2) or as concentrated aqueous solution (NH 2 OH) to said solvents. The choice is determined by the water content of the cell dry material and by the amount and water content of the solvent used. NH 2 OH is suitable for low moisture cell masses (0-15%), while NH 2 is better for higher water cell masses (10-30%).

The amount of lipids removed by ammonia and solvent extraction depends on the total water content, calculated as a percentage by weight of the amount of solvent used, and by the NH4 concentration, by weight, of the solvent used.

Particularly good test results are obtained with a weight ratio of cell mass to solvent of 1: 3 to 1: 6 with methanol and ethanol, and 1: 8 to 1:12 with propanol, glycol and monoglycol ethers. The ammonia concentration calculated on the amount of solvent according to the invention is conveniently 2-8% by weight, preferably 3-5% by weight. The sum of the amounts of water derived from cell mass, solvent and optionally the aqueous ammonia amounts to 0-30 wt.%, Preferably 0-20, and particularly preferably 4 1465 11 0-10 wt.% Based on the amount of solvent used, cf. claim 2.

When working on a technical scale, after drying, the cells can be desiccated to a low residual water content of 4% or less. It is sufficient that the cell mass has such a cell dry weight that the total water content after the addition of the required amount of solvent is within the optimum range; optionally, the solvent can be added gaseous NH 2.

The dissolution of fats from the microbial cell masses takes place by suspending the cell material in one or more of the aforementioned solvents and adding NH 2 or adding NH 2 OH. Preferably, the mixture of the suspension occurs by stirring. The treatment temperatures are, as mentioned, in the range of -20 to + 60 ° C, with a range of 5-50 and especially of 10-30 ° C being preferred. The duration of treatment is 5-120 minutes, preferably 25-35 minutes. The treatment is usually done at normal pressure.

After the solvent / ammonia treatment is completed, the cellular material (protein) obtained is separated by a process such as centrifugation, filtration and sedimentation from the solvent.

Filtration is preferred. The solid cell material obtained can, in order to remove the lipids as completely as possible, be treated again with one of said organic solvents.

The residue can be dried to remove solvent and ammonia residues. This drying is carried out under reduced pressure, preferably 80-150 torr, and at elevated temperature, preferably 40-50 ° C. The product thus degreased and dried is odorless.

The liquid phase separated liquid phase material mentioned above contains ammonia and dissolved lipids. The solvent used can be separated from the fats by vacuum distillation and used again.

Then, the degreased and possibly dried cell masses are taken up in water. The preferred weight ratio of water volumes to cell mass is 1: 1 to 1:30, especially 1: 5 to 1:15. The amount of water must be at least metered so that a suspension of the suspension is possible.

The pH value during the water treatment should be in the range 5-8.5, preferably 6-7.5, and optionally adjusted to this range. This is especially necessary if non-fully dried cell masses from the first stage are to be used, which then also still contain residual ammonia, which can lead to a high pH.

This extraction of nucleic acids, salts, polysaccharides and water-soluble secondary metabolites with water usually takes place in a temperature range of 30-95 ° C at normal pressure. Preferably, the temperatures are 40-70 ° C, especially preferably 50-60 ° C. Depending on the extraction temperature and the amount of water, the duration of the extraction can be 5-120 minutes, which results in an extraction time of 25-45 minutes. To separate solid and liquid components, the suspension is centrifuged, preferably at temperatures of 10-30 ° C. Other suitable separation methods are sedimentation and filtration.

In order to more easily separate the cell mass, it is conveniently added to the water in the second step up to 20% by weight, preferably 5-15% by weight of a lower alcohol such as ethanol or methanol, preferably methanol.

The solid phase is freed by conventional methods such as freeze, vacuum or spray drying for liquid residues. The product has pleasant odor properties, a lighter color than the starting material and a particularly good water-bonding ability. Due to the removal of lipids and nucleic acids, it is particularly suitable for the preparation of feed.

Thus, in the process of the invention, the lipids are removed to a residual content of the product of 0.5-3.5% by weight and the nucleic acids to a residual content of 0.5-4.5% by weight.

In the process according to the invention, the disadvantages of the known methods such as treatment with solvents and alkaline absorption are avoided. Chlorinated hydrocarbons are not needed. The removal of mixtures of ammonia and solvent is, in particular for bacteria, considerably more complete than in treating the cell mass with mixtures of solvent and water. Extreme temperature and pH ranges that limit the utility of the product are avoided. The energy consumption is less than the known alkaline opening methods. Protein enrichment occurs without the presence of large amounts of neutralization salts.

146511 6

The aqueous phase separated from the solid cell material contains, in addition to other water-soluble constituents, nucleic acids which can be recovered by known methods, e.g. precipitation in acidic media, ultrafiltration, dialysis or enzyme treatment.

The method according to the invention will now be described in more detail by way of example.

Example 1

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

100 g of this cell mass is added to 300 g of methanol. While stirring the suspension, 10 g of NH 2 gas is introduced and dissolved therein. On cooling, the temperature is maintained during the supply of 25-35 ° C. The mixture of methanol, ammonia and cell mass is stirred for 30 minutes at 20 ° C.

To separate the solid and liquid phase, filter and wash the solid residue with 300 ml of methanol. After repeated filtration, both filtrates are combined. This brown solution contains the lipids from the starting substance used. Methanol and ammonia are removed by vacuum distillation (100 torr, 40 ° C). The residue, which constitutes 9.5% by weight of the cellular material used, is a dark brown, smelly paste consisting of free fatty acids, glycerides, phospholipids and secondary metabolites.

The solid residue obtained by filtration of the extracted cell mass is dried in vacuo (100 torr) at 40 ° C for 5 hours. Thereby 90 g of degreased cell mass is obtained which is odorless and has a lighter color than the starting material.

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

After the temperature is raised to 55 ° C, stir for another 20 minutes, cool to 30 ° C and separate in solid and liquid phase by centrifugation. The resulting sediment is again mixed with 900 ml of 7 U6511 water and stirred for 10 minutes at 20 ° C. Then centrifuge again and the sediment is dried under reduced pressure.

The yield weighs 65 g. The nucleic acid content has dropped from 11.2% to 1.5%. The product is odorless in dry condition, moistened with water it has a pleasant smell.

The results of this and the following examples are set forth in the following Tables 1a and 1b.

Example 2

As the starting material, the same bacterial cell mass is used as in Example 1. It is subjected to the same treatment steps and conditions, however, instead of gaseous NHg 30 ml of concentrated 33% NH 2 OH is used as reagent.

Example 3

Proceed as described in Example 2, however, 60 ml of concentrated 33% NH 2 OH are used.

Example 4

Proceed as described in Example 2 but use methanol ethanol as a solvent instead.

Example 5

Proceed as described in Example 3, but instead use methanol glycol monomethyl ether as the solvent.

Example 6

Proceed as in Example 2, but instead use methanol i-propanol as the solvent.

Example 7

As a starting material, a cell mass of Methylomonas clara is used as described in Example 1. 100 g of the spray-dried product is dissolved as described in Example 1 and degreased (15 g NHg). However, no complete drying of the residue is performed. The carefully drained filter cake with a solids content of 85% is suspended in 900 ml of water. The pH is adjusted to 8.9, depending on the ammonia remaining in the moist biomass.

The temperature of the suspension is raised with stirring to 65 ° C and after 5 minutes the pH is adjusted by adding HCl to 7.2. Then stir for an additional 15 minutes at 65 ° C, then cool to 40 ° C and centrifuge. The resulting sediment is dried.

Example 8

A hydrocarbon-consuming strain of the yeast species Candida lipolytica ATCC 20,383 is grown on n-paraffins in the presence of an aqueous nutrient medium and an oxygen-containing gas. The yeast cell mass is separated from the nutrient solution and dried.

100 g of the dry yeast cell mass is suspended at room temperature under normal pressure in 300 g of methanol and this mixture is fed 10 g of NH This keeps the temperature of the suspension cooled to 15 ° C. After the gas is fed, stir for another 20 minutes at 22 ° C and then filtered over a suction fryer. Once filtered, mix the filter cake with 300 ml of methanol on the frit and then suck it dry. The two filtrates are combined. The solution is yellow and contains the lipids from the cell material used. Methanol and NH 2 are removed at reduced pressure (14 torr).

The residue remaining after the second filtration, consisting of broken and degreased microorganism cells, is dried in a vacuum dryer at 40 ° C (100 torr) for 5 hours. The resulting product has a lighter color than the yeast cell mass used initially and is odorless.

To reduce the nucleic acid content from the original 7.5% by weight based on the starting material, 100 g of the degreased and dried yeast is suspended in a solution of 1 liter of distilled water and 100 ml of methanol. With stirring, the temperature of the mixture is brought to a temperature of 6.8 which is adjusted to 15 ° C for 15 minutes and separated by centrifugation in a sediment containing the yeast protein and a liquid phase containing soluble nucleic acid. The sediment is subjected to washing again at room temperature freeze-drying.

The nucleic acid content of the dried material has dropped from originally 7.5 to 0.4% by weight.

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Example 9

Proceed as described in Example 8 but use instead of methanol ethanol and instead of 10 g of gaseous NH4 60 ml of 33% nh4oh.

Example 10

Penicillium chrysogenum ATCC 10.238 is grown in a nutrient solution containing lactose, corn starch, liquid, phosphate, carbonate and magnesium sulfate aerobically in conventional manner. After separation of the produced penicillin, the dried mycelium remains, which acts as a starting material.

Proceed as described in Example 8, but 33% of NH 2 OH is used instead of NH 2. The degreased dry mycelium is washed with water at 30PC.

Example 11

As the starting material, the same cell mass is used as described in Example 10. Instead of methanol, ethanol is used. The temperature during the water extraction is raised to 85 ° C and kept here for 15 minutes.

The following Table I a + b lists the results of Examples 1-11.

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Table 1a

Removal of the lipids 100 g of cell mass used

Nuclein-NH 2 (g) el.

Fatty Acid Solvent NH.OH

Ex. Species wt% wt% 300 g (33%, ml) 1 Methylomona 7 11.2 Methanol NH3 10 2 "7 11.2 Methanol NH4OH 30 3" 7 11.2 Methanol NH4 OH 60 4 "7 11.2 Ethanol NH4OH 5 "7 11.2 Glycol monomer NH4OH 60 thyl ether 6" 7 11.2 i-Propanol NH4OH 30 7 "9.6 15.4 Methanol NH4 8 Candida lipo 8.2 7.5 Methanol NH4 10 lytica 9 "8.2 7.5 Ethanol ΝΗ ^ ΟΗ 60 10 Dry Mycelium 3.5 2.6 Methanol NH4OH 30 11" 3.5 2.6 Ethanol NH4OH 30

Table Ib

Removal of the Nucleic Acids _Washing Water_ Obtained Cell Mass_ Quantity Temp. Amount of Fats Nucleic Acid

Ex. Liters pH ^ (£) _% 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 1.0 6.8 50 68 1.4 0.4 9 1.0 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