NO147034B - PROCEDURE FOR REDUCING LIPID AND NUCLEIC ACID CONTENTS IN MICROBIAL CELL MASSES - Google Patents

Description

The invention relates to a method for reducing the amount of lipid and nucleic acid contained in micro-organism cells. Each cell contains carbohydrates, proteins, lipids and nucleic acids. The applicability of microbial cell masses as food and fodder is negatively affected

tive by the content of nucleic acids and lipids.

A nucleic acid content is undesirable as it leads

to pathological conditions (gout, urinary stones). Lipids limit shelf life by becoming rancid and affecting taste.

The task of the invention is therefore to lower the lipid

and the nucleic acid content of microbial cell masses. According to known methods, the lipids are either released from the cell wall and membrane by organic solvents or saponified by treatment with aqueous alkalis.

A known method for releasing the lipids from microorganisms works with mixtures of methanol and chloroform. The procedure is cumbersome and can lead to toxic products. Lipid extractions with alcohol-water mixtures (according to DOS 2 405 593 or 2 137 038) are not suitable for bacteria and are likewise cumbersome in the technical execution.

Furthermore, known methods for making microbial cell masses nucleic acid- and lipid-poor use alkaline digestion at elevated temperatures. Unfortunate is the release of the free fatty acids formed during the hydrolysis, which are secreted together with the protein.

In addition, part of the essential amino acids contained in the protein, e.g. lysine, left in a state that cannot be used by the organism.

The invention relates to a method for reducing the lipid and nucleic acid content in microbial cell masses by extracting the cell mass with solvent and ammonia, separating the extract and washing and drying the cell mass,

in that the method is characterized by the fact that in a first step the cell mass is extracted with an extraction mixture containing ammonia or ammonium hydroxide and an organic solvent with the formula

in which R1 and R2 denote a hydrogen atom and n means 1, 2 or 3 including isopropanol, or in which R-^ means a hydroxy group and R2 means a hydrogen atom or a methyl or ethyl group, and n is 2 or 3, under normal pressure and at a temperature between

-20 and +60°C for a maximum of 120 minutes, the total water content during the extraction being 0-30% by weight calculated on the amount of the solvent used, and if the solvent is methanol or ethanol, the weight ratio between cell mass, solvent and ammonia is 1:3:0.03-1:6:0.6, preferably 1:3:0.03-1:6:0.3, and if the solvent consists of 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 another step after the extraction mixture has been separated, the cell mass residue is washed with water with a pH value of 5-8.5 and a temperature of 30-95°C and normal

pressure for a time of 5-120 minutes, with the weight ratio of water quantity and cell mass being 1:1-1:30, after which the water phase is separated, possibly extracting the cell mass with the organic solvent

the agent of formula I, and dries the cell mass.

As microbial cell masses are preferably used

show microorganisms such as is produced by cultivation on alcohol or n-paraffins in the presence of an aqueous nutrient medium and a free oxygen-containing gas. Preferred micro-organisms are bacteria, yeast and fungi.

Examples of such microorganisms are methanol-utilizing bacteria of the genus Metylomonas, e.g. Methylomonas clara ATCC 31226, or yeasts such as Candida lipolytica ATCC 20383 which can be obtained by cultivation on n-paraffins in the presence of an aqueous nutrient medium.

Dried mushroom mycelia can also be used. This cell material can be taken from antibiotic production, for example from penicillin fermentation after the antibiotic has been extracted.

As solvent with formula I, alcohols such as methanol, ethanol, n-propanol and isopropanol come into consideration. Preferred are methanol and ethanol, especially methanol. In addition to the alcohols mentioned, glycols and their monoethers of formula I, namely glycol and monomethylglycol, are suitable.

Ammonia can be added to the aforementioned solvents in gaseous form (NH-) or as a concentrated aqueous solution (NH^OH). The selection takes place according to the water content of the cell dry material as well as the amount and water content of the solvent used. NH^OH is suitable for cell masses with a low moisture content (0-15%), NH^, on the other hand, is better for cell masses with a higher water content (10-30%).

The amount of lipids removed by extraction with ammonia solvents depends on the total water content which refers to the weight* of the amount of solvent used.

de and of the NH^ concentration in % by weight refer to the solvent used.

Particularly good test results can be achieved

at cell mass/solvent weight ratios of 1:3 to 1:6

with methanol and ethanol, and 1 : 8 to 1 : 12 with propanol, glycol and monoglycols after this. The ammonia concentration referred to the amount of solvent was 1-10% by weight, preferably 1-5% by weight. The sum of the amounts of water originating from cell mass solvent and possibly aqueous ammonia amounted to 0-30% by weight, preferably 0-20%, especially 0-10% by weight, referred to the amount of solvent used.

For work on a technical scale, it can be disregarded

from a drying of the cell after fermentation until a minor residual water content of 4% or lower. It is sufficient to

bring the cell mass to such a cell dry weight that, after adding the required amount of solvent, the total water content is in the optimum range, optionally adding gaseous NH 2 to the solvent.

The dissolution of fat from the microbial cell masses is carried out in such a way that the cell material is suspended in the organic solvent of formula I and NH 3 is introduced or NH 3 OH is added. It is appropriate to thoroughly mix the suspension by means of stirring. The treatment temperature is •

in the range from -20° to +60°C, with a range from +5° to +50°C and especially from +lo° to +30°C being preferred. The treatment duration is up to 120 minutes and is preferably 25 - 35 minutes. The treatment is carried out at normal pressure.

After completion of the solvent/ammonia treatment, the formed cell material (the protein) is separated

following desirable methods such as centrifugation, filtration and sedimentation from the solvent.

Filtering is preferred. The formed solid cell material allows for the best possible complete removal of lipids again. treated with one of the aforementioned organic solvents.

The residue can be dried to remove solution residues and ammonia. Appropriately, this is carried out under reduced pressure, preferably 80 - 150 torr and elevated temperature, fortrd/rin"s vi' s'• *»0° "- -5'0°C'. The thus defatted and dried product 'ér-llfK t-1'est'; • ;'-'-- "

- TV' ';<;>>:ir' The liquid phase separated from the solid cell material by the above-mentioned method contains ammonia and dissolved lipids. The solvent used can be separated by vacuum distillation, from ' fat tene and" again ' is used."'

<:> •*.'■>. The degreased and possibly dried cell masses are then taken up in ■ : .'~ : amounts of water which to the cell- '' mass hsts in - weight ratio 1' :: 1't'i'l • 1 : 30-^'especially in :' 5til ' ' ' ';

1 :■■ 1-5-'Wanrimenden- 'damfensj:cn-ér¥s3mih'st':'såiedés-• ate it' is possible with a - stirring'-' of- suspension j bhen"! ' "-v '•''

• - '.' •■' p'H-v'érdien during the-'''water treatment: must be 'in the range of 5 - 8 .5, preferably^ 6''-;'-7 .5^'bg'set'if necessary'' in this - area -. ■" This is particularly necessary when: it is "not used or not completely dried cell masses from the first step which then contain residual amounts of ammonia, which can lead to a pH value too high. - -. The extraction: of nucleic acid, salts, polysaccharides and water-soluble secondary metabolites with water is carried out in a temperature range of 30°--95°C - at normal pressure. Preferred is a temperature of 40° - 70°C/especially preferred 50° - :-6..0°C. according to extra'riération temperature -'-and'.quantity of water (make up 5:''-'"l'20 minutes. -Good tea sul-tates ."achieved -one 'with '"-25 •• -' :4'5' 'minutes extraction time. For' "•" separation"<v>a"v- solid:"e:"e" and liquid constituents' are centrifuged' "'''' suspension'','-'' triri- ricewise' at temperatures of 10° -'30°C<L>:' Other' suitable 'ski llef remgairgsmater :'er sedimentas j on and filtration-.-' .'»*.' • For a > !<*i 'e r e -' a d s 1 < i 1 i e l's e ! å v'' ' c' e' l 1 e rna s se n 'is preferably added to the water at ar.net steps up to 20 weight'-*' for - ' tri no.s vi?-'-'5 -'" ]'=*"•• vÆ:-'7 :'av'"a lower alkbhol' 'as-ethanol, methanol, f crtr.'.- hn's'V-ib'-'niev anbl'.' "■ "' ■ -■'' : 1 ' -<:>

-.<" -" - Den' f as te: "f ase' :b é f r'£e s''"e 11 e v "ordinary procedure

such as freezer, vacuum or f cr s t^ vhihgs t'ø r'k ni ng f or liquid residues.' The product'- -h!year- gbd'e- odor<;>segéncreas'v'''eri lighter -color;r;eri' than starting-material%-t bg—én" srieM el;t'v gb'd: water absorption capacity.' For the removal of lipids and ucléanic acid raising agents, it is particularly important to produce ; • ■' ■'

Thus, in the product, the lipids have been removed up to a residual content of from 0.5 to 3.5% by weight nucleic acid up to a residual content of 0.5 - 4.5% by weight.

The method according to the invention avoids the disadvantages

by the known methods such as solvent, treatment or alkaline digestion. Chlorinated hydrocarbons are not required. The degreasing with ammonia/solvent mixtures is, especially with bacteria, significantly more complete than when treating cell masses with solvent/water mixtures. Extreme temperature and pH ranges that reduce the product's usability are avoided. The energy consumption is less than by the known alkaline digestion methods. Protein enrichments succeed without the formation of large amounts of neutralization salts.

The aqueous phase separated from solid cell material contains, in addition to other water-soluble constituents, the oleic acids, which can be recovered according to known methods, for example, by precipitation in an acidic medium, ultrafiltration, dialysis or en-z/m treatment .

The invention .shall. is explained in more detail, with the help of-.-.. some examples.

Example 1 ..

Methylomonas cia ra ATCC 31226 was grown in a stirring solution containing methanol as the sole carbon source. ammonia son sole source of nitrogen.,. f osf that, ■ j ern-, magnesium- . salts and other common trace elements under aerobic conditions. The resulting bacterial cell mass was separated from the solution and subjected to dust drying.

100 g of this cell mass was mixed with 300 g of methanol. While stirring the suspension, 10 g of NK2 gas was introduced and dissolved. During cooling, the temperature during the introduction was kept at 25 - 3.5°Cr: The mixture of methanol, ammonia,

and cell mass was stirred for 30 minutes at 20°C........

For separation of the solid, and.. f ly tend.e phase was

the filtrate and the solid residue were washed once with 300 ml of methanol. After repeated filtration, both filtrates were combined..--This brown solution contained the lipids of the starting material used. Methanol and ammonia were removed by vacuum distillation (100 torr, 40°C). The residue, which constituted 9.5% by weight of the cell material used, was a dark brown paste, with a bad smell, consisting of free fatty acids. , .glycerides.,. phospholipids and secondary metabolites.

The solid residue formed by the filtration of the extracted cell mass was dried in vacuum (100 torr) at 40°C for 5 hours. Thus, 90 g of defatted cell mass could be obtained which was odorless and had a lighter color than the starting material.

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

After raising the temperature to 55°C, it was further stirred for 20 minutes, cooled at 30°C and separated into solid and liquid phases by centrifugation. The formed sediment was again mixed with 900 ml of water and stirred for 10 minutes at 20°C. It was then centrifuged again and the sediment 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 in the dry state, moistened with water it had a sympathetic odor.

The result of this and the further examples is

summarized in the tables Ia and IB below.

Example 2.

The same bacterial cell mass as was used in example 1 served as starting material. It was subjected to the same process steps and conditions, however, instead of gaseous NH 3 30 ml of concentrated NH 3 OH (33%-ig) was used as reagent.

Example 3-

The procedure was the same as in example 2, however, 60 ml of concentrated NH 3 OH (33% strength) was used.

Example 4.

In a method as in example 2, ethanol was used as solvent instead of methanol.

Example 5-

In otherwise the same procedure as in example 3, glycol monomethyl ether was used as solvent instead of methanol.

Example 6.

The procedure was as in example 2. Instead of methanol, i-propanol was used as solvent.

Example 7•

As starting material, a cell mass of Metylomonas clar-a was used as described in Example 1. 100 g of this spray-dried product was digested, defatted (15 g NHy) as described in Example 1. However, the complete drying of the residue was disregarded . The thoroughly aspirated filter cake with a solids content of 85% was suspended in 900 ml of water. The pH value settled at 8.9, due to the ammonia remaining in the moist biomass.

The temperature of the suspension was increased while stirring

to 65°C and after 5 minutes the pH value was adjusted by the addition of HCl to 7.2. It was then further stirred for 15 minutes at 65°C, then cooled at 40°C and centrifuged. The sediment formed was dried.

Example 8.

_A hydrocarbon-utilizing strain of the yeast Candida. lipolytica ATCC 20 383 was cultured 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 was suspended at room temperature under normal pressure in 300 g of methanol and 10 g of NH 2 gas was added to this mixture within 15 minutes. Thereby, the suspension's temperature during cooling was kept at 15°C. After the introduction of the gas, it was further stirred for another 20 minutes at 22°C and then filtered over a suction frit. The filter cake was thoroughly mixed once with 300 ml of methanol on the frit and then aspirated. The two filtrates were combined. The solution was yellow and contained the lipids of the cell material used. Methanol and NH 3 were removed at reduced pressure (14 torr).

The residue remaining after second filtration, which consisted of the destroyed and degreased cells of the microorganism, was dried in a vacuum drying cabinet at 40°C (100 torr) for 5 hours. The product thus formed had a lighter color than that to start with the yeast cell mass used and was odorless.

To reduce the nucleic acid content from the original 7.5% by weight referred to the starting material, 100 g of the defatted and dry yeast were suspended in a solution of 1 liter of distilled water and 1000 ml of methanol. While stirring, the mixture was brought to 50°C at a pH value of 6.8 for 15 minutes and separated by centrifugation into a sediment containing yeast protein and a liquid phase containing dissolved nucleic acids. The sediment was after repeated washing

at room temperature subjected to freeze drying.

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

Example 9-

In a method as in example 8, ethanol was used instead of methanol and instead of 10 g NH 3 gaseous 60 ml NH 3 OH (33%-ig)'.

Example 10.

Penicillium crysogenum ATCC 10 238 was grown in a nutrient solution containing lactose, liquid grain cast, phosphate, carbonate and magnesium sulphate according to usual methods. After separation of the penicillin formed, mycelium remained, which, when dried, served as starting material.

The procedure was carried out as in example 8. Instead of NH 2 , NH 2 OH (33%-ig) was used. The defatted dry mycelium was washed at 30°C with water.

Example 11.

The starting material was the same cell mass as in example 10. Instead of methanol, ethanol was used. The temperature during the water extraction was increased to 85°C and held here for 15 minutes.

The following table III lists the results of examples 1-11.

Claims (1)

Method for reducing the lipid and nucleic acid content in microbial cell masses by extracting the cell mass with a solvent and ammonia, separating the extract and washing and drying the cell mass, characterized in that in a first step the cell mass is extracted with an extraction mixture containing ammonia or ammonium hydroxide and an organic solvent with formula in which and R~ denotes a hydrogen atom and n means 1, 2 or 3 including isopropanol, or in which R^ means a hydroxy group and R 2'' is a hydrogen atom or one methyl or ethyl group and n means 2 or 3, under normal pressure and at a temperature between -20 and +60°C for a maximum of 120 minutes, the total water content during the extraction being 0-30% by weight calculated on the amount of the solvent used and if the solvent is methanol or ethanol, the weight ratio between cell mass, solvent and ammonia is 1:3:0.03-1:6:0.6, preferably 1:3:0.03-1:6:0.3, and if the solvent consists of 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 another step, after the extract mixture has been separated, washes the cell mass residue with water at a pH value of 5-8.5 and a temperature of 30-95°C and normal pressure for a time of 5-120 minutes, the weight ratio of amount of water to cell mass being 1:1-1: 30, after which the water phase is separated, optionally the cell mass is extracted with the organic solvent of formula i., and the cell mass is dried.