KR810001964B1 - Process for reducing the contents of lipids and nucleic acid in mocrobial cell masses - Google Patents

Abstract

Process for reducing the lipid and nucleic acid content in microbial cell masses was described. Thus, dry cell masses of Methylomonas clarus was mixed with MeOH. While stirring, NH3 was added and dissolved. The mixt. was filtered and washed with MeOH. The cell mass was dried under vaccum and suspended in the water. The mixt. was held at 55≰C for 20 min., cooled, and centrifuged and the solids were mixed with water, stirred for 10 min. at 20≰C, and dried under reduced pressure to give title materials.

Description

Reduction of Lipids and Nucleic Acids in Microbial Cell Masses

The present invention relates to a method for reducing the amount of lipids and nucleic acids present in microbial cells.

Every cell contains carbohydrates, proteins, lipids and nucleic acids. The usefulness of microbial cell masses in food and feed is reduced due to nucleic acids and lipids.

The amount of nucleic acid is important because it can cause disease (arthritis, urolithiasis). The presence of lipids leads to rancidity, which reduces storage and also gives an unpleasant taste.

It is therefore an object of the present invention to a method for reducing the amount of lipids and nucleic acids contained in microbial cells.

According to known methods, lipids are separated from the cell walls and cell membranes by organic solvents or treated with alkaline aqueous solution to saponify.

The known method of separating lipids from microorganisms is carried out with a mixture of methanol and chloroform. This process is complex and produces toxic byproducts. Lipid extraction methods by alcohol / water mixtures (according to German Publication No. 2,405,593 or German Publication No. 2,137,038) are rarely suitable for bacteria and are also complex and expensive in technical methods.

In addition, a known method of reducing nucleic acids and lipids contained in microbial cell masses is to separate them by alkaline substances at elevated temperatures. This process releases the free fatty acids produced by hydrolysis and breaks down or breaks down with the protein.

Moreover, essential amino acids, such as lysine, are changed to a state that is no longer available by the organism.

The cell mass was treated with an organic solvent of structural formula (I) such as ammonia or ammonium hydroxide to separate lipids, the residual cell mass was washed with water, the aqueous layer was separated, and the remaining cell mass was extracted with an organic solvent of the following structural formula (I) and then dried. This method has been found to reduce the amount of lipids and nucleic acids in microbial cell masses.

R _1- (CH ₂ ) n-OR ₂ (I)

In the above structural formula

As the microbial cell mass, it is preferable to use a microorganism capable of growing by culturing with alcohol or n-paraffin in the presence of a water-soluble nutrient medium and a gas containing free oxygen. It is preferable to use bacteria, enzymes and fungi as microorganisms.

Examples of such microorganisms include Candida lopolytica, which can be obtained by culturing with n-paraffins in methylomonas-type bacteria, such as Methylomonas clara ATCC 31226, which can be grown using methanol, or in aqueous nutrient media. ) There is yeast such as ATCC 20383.

Dried fungal mycelium can also be used. This cellular material is used after extracting the antibiotic material from the cell material used in the production of antibiotics such as penicillin fermentation method.

Suitable solvents of formula (I) are alcohols such as methanol, ethanol, n-propanol and isopropanol, methanol and ethanol are preferred, and methanol is better. In addition to the alcohols mentioned above, they are also suitable as glycols and monoethers of formula (I), in particular glycols and monoethyl glycols.

Ammonia is added to the solvent in the form of ammonia gas (NH ₃ ) or concentrated ammonia solution (NH ₄ OH). In the selection, the residual moisture and the amount of solvent contained in the dried cell material and the water content in the solvent are important. NH ₄ OH is suitable for low water content (0-15%) cell masses and for NH ₃ high water content (10-30%).

The amount of lipids that can be extracted and removed by ammonia and the solvent of formula (I) depends on the total moisture content calculated for the amount of the solvent (weight percent) and the NH ₃ concentration (percentage) calculated for the solvent.

Particularly good results were obtained when using methanol and ethanol with a cell mass / solvent ratio of 1: 3-1: 6 and when using propanol, glycol and monoglycol ether with 1: 8-1: 12 Get The ammonia concentration calculated for the amount of solvent is 1-10% (weight ratio), and preferably 1: 5% (weight ratio). When calculating the amount of solvent produced by the cell mass, the solvent and the aqueous ammonia solution, the total moisture content is 0-30% (by weight), preferably 0-20% (by weight), in particular 0-10% (by weight). good.

On an industrial scale it is not necessary to dry the cells to 4% residual moisture after fermentation. When the cell mass is at this dry cell weight, it is enough to add the desired amount of solvent to maintain the total moisture content in an optimal state, and optionally, gaseous ammonia can be added to the solvent.

To extract lipids, microbial cell masses are suspended in an organic solvent of formula (I) and introduced NH ₃ or NH ₄ OH. It is useful to mix the suspension with complete stirring. Generally the process temperature is -20 °-+ 60 ° C, preferably + 5 °-+ 50 ° C, in particular + 10 °-+ 30 ° C. The process time is 5-120 minutes and it is desirable to finish within 25-35 minutes. In general, the process proceeds at atmospheric pressure.

It is preferable that the cell mass (protein) obtained after treatment with the solvent and ammonia is separated from the solvent by a suitable method such as centrifugation, filtration or precipitation and then separated by filtration.

The solid cell mass thus obtained can be treated with one of the solvents mentioned above to remove lipids as completely as possible at least once.

Xana may be dried to further remove residual solvent and ammonia at a moderately reduced pressure, preferably at 80-150 mmHg and at elevated temperature, preferably at 40 ° -50 ° C. The dried product, free of lipids, is odorless.

The liquid layer separated from the solid cellular material by the above-mentioned method contains ammonia and dissolved lipids. The solvent can be separated from the fat by distillation under reduced pressure and then recycled.

The cell mass thus obtained is then added to water. The ratio of the cell mass (weight percentage) to water is preferably 1: 1-1: 30, in particular, 1: 5-1: 15. A minimum amount of water must be present to stir the suspension.

The pH value during the treatment with water should be 5-8.5, preferably 6-7.5 and if necessary, especially when using the cell mass obtained from the first process, i.e. residuals that are not yet completely dry or not very dry and become very high pH. If it contains ammonia amount, adjust accordingly.

Extraction of nucleic acids, salts, polysaccharides and water-soluble secondary metabolites by water generally proceeds at atmospheric pressure of 30-50 ° C., preferably at temperatures of 40 ° -70 ° C., in particular at 50 ° -60 ° C. It is good to proceed. Depending on the extraction temperature and the amount of water, the extraction time can be changed between 5 and 120 minutes. Good results are obtained when the extraction time is 25 to 45 minutes. The suspension is preferably centrifuged at 10 ° -30 ° C. to separate the solid and liquid components. Other suitable separation methods are precipitation and filtration.

In order to facilitate the separation of the cell mass, in this second extraction process, it is appropriate to add 20% water (by weight), preferably 5-15% lower alcohol, such as ethanol or methanol, preferably methanol.

Residual liquid components are removed in the solid layer by conventional methods such as vacuum lyophilization, vacuum drying or spray drying. The product has a decent odor and is lighter in color than the starting material, in particular water bondable. Because of the removal of lipids and nucleic acids, the product is particularly suitable for preparing food and feed mixtures. The product according to the invention thus contains only 0.5-3.5% (by weight) of lipids and 0.5-4.5% (by weight) of nucleic acid.

The preparation according to the invention avoids the disadvantages of known methods such as treatment with toxic solvents or separation with alkaline substances and does not require chlorinated hydrocarbons in particular. Particularly in the case of bacteria, the removal of fat with ammonia and a solvent of structural formula (I) is far more complete than the removal results obtained by treating the cell mass with a mixture of other solvents and water. Avoid extreme temperature and pH categories that reduce the usefulness of the product. The need for heat is much lower than that required for known methods implemented by alkaline separation methods. The protein content can be increased without producing a large amount of salt by neutralization.

The aqueous layer from which the solid cellular material has been separated contains, in addition to other water-soluble substances, nucleic acids that can be dilute by known methods such as precipitation by acid medium, ultrafiltration, dialysis or enzyme treatment.

The following examples illustrate the invention.

Example 1

Methylonomonas Clara ATCC 31226 is incubated under aerobic conditions in a nutrient medium containing methanol as a carbon source, ammonia, phosphate, iron salts, magnesium salts and other trace elements as a nitrogen source. The bacterial cell mass produced during fermentation is separated from the solution and spray dried. 100 g of this cell mass is added to 300 g of methanol. 10 g of ammonia gas is injected into the suspension with stirring to dissolve. The temperature is cooled and kept at 25 ° -35 ° C. The methanol, ammonia and cell mass mixture is shaken at 20 ° C. for 30 minutes.

The solid phase and the liquid phase are separated by filtration and the solid substance is washed once with 300 ml of methanol. Then wash again and combine the filtrates. This brown solution contains the lipid of the starting material used initially. Methanol and ammonia are removed by steam distillation under reduced pressure (100 mmHg, 40 ° C.). The residue with 9.5w% of the cell material initially used is a dark brown odorous plaque with free fatty acids, glycerides, phospholipids and secondary metabolites.

The solid residue of the extracted cell mass was obtained by filtration and dried at 40 ° C. for 5 hours under reduced pressure (100 mmHg). This gives 90 g of cell mass which is free of odor and is lighter in color than the starting material.

The cell mass is suspended in 900 ml of water to reduce the nucleic acid content. The pH of the suspension homogenized by shaking is 6.9.

Raise the temperature to 55 ° C, shake for another 20 minutes, and cool to 30 ° C. After that, the precipitate obtained by centrifugation of the solid phase and the liquid phase is added again to 900 ml of water and shaken at 20 ° C. for 10 minutes. After centrifugation and the residue was dried under reduced pressure to give a yield of 65 g of cell mass. Nucleic acid content is reduced from the first 11.2% to 1.5%. The dried product is odorless and smells good when it contains water.

The results of those detailed above and of the following examples are summarized in Tables 1a and 1b.

Example 2

The starting material is the same bacterial cell mass as described in Example 1 above. Instead of the ammonia gas reaction solution, 30 ml of concentrated ammonium hydroxide (33%) was used and reacted according to the same procedures and conditions as in Example 1 above.

Example 3

60 ml of concentrated ammonium hydroxide (33%) was used according to the preparation method of Example 2.

Example 4

It is carried out according to the preparation method of Example 2 using ethanol instead of metaol as a solvent.

Example 5

The reaction was carried out according to the preparation method of Example 3, using glycol monomethyl ether instead of methanol as a solvent.

Example 6

Reaction was carried out according to the preparation method of Example 2, using i-propanol instead of methanol as a solvent.

Example 7

The methyllomonas clara cell mass used in Example 1 is used as a starting material. 100 g of sprayed material are separated according to the method described in Example 1 above and the lipids are removed. (15 g of ammonia) provided that the residue is not completely dried. The fully aspirated filtrate, with 85% solids, is suspended in 900 ml of water. Ammonia is added to pH 8.9 to maintain a watery biomass.

The temperature of the suspension is raised to 65 ° C with shaking, and after 5 minutes, diluted hydrochloric acid is added to pH 7.2. Thereafter, shaking was continued at 65 ° C. for 15 minutes, cooled to 40 ° C., and the precipitate obtained by centrifugation was dried.

Example 8

Yeast Kanda Lipolitica ATCC 20383 is incubated on n-paraffins using hydrocarbons and water soluble medium and oxygen containing gas. Yeast cell mass is separated from nutrient solution and dried.

100 g of the dried yeast cell mass is suspended in 300 g of methanol at room temperature and atmospheric pressure, and 10 g of ammonia gas is injected within 15 minutes. The temperature of the suspension is cooled and kept at 15 ° C. It is then shaken again at 22 ° C. for 20 minutes and filtered with suction glass. In addition, the filtrate is thoroughly mixed with 300 ml of methanol, followed by suction filtration. Collect two filtrates. The solution is a yellow solution containing the substrate of the cellular material used initially. Methanol and ammonia are removed under reduced pressure (14 mmHg).

The second filtered residue contains cells of microorganisms that have been destroyed and lipid removed and dried at 40 ° C. for 5 hours in a vacuum dryer (100 mmHg). The product thus obtained is lighter and odorless than the yeast cell mass used initially. In order to reduce the nucleic acid content of the original 7.5w% calculated based on the starting material, 100g of dry yeast without lipids is dissolved in 1L of distilled water and 1L of methanol. The mixture is shaken, adjusted to pH 6.8 and heated at 50 ° C. for 15 minutes. Centrifugation is then carried out to obtain a precipitate containing yeast protein and separated to form a liquid phase containing nucleic acid. The precipitate is washed once more at room temperature and then lyophilized in vacuo.

The nucleic acid content of the dry matter is reduced from 7.5 (wt%) to 0.4 (wt%).

Example 9

The reaction was carried out according to the preparation method of Example 8 using ethanol instead of methanol and 60 ml of ammonium hydroxide (33%) instead of 10 g of ammonia gas.

Example 10

Penicillin chrysogenium ATCC 10238 is cultured in aerobic state in a nutrient solution containing lactose, corn liquor, phosphate, carbonate and magnesium sulfate according to a known method. The resulting penicillin is isolated and the remaining mycelium is dried and used as a starting material.

Ammonium hydroxide (33%) instead of ammonia was reacted according to the preparation method of the above example. The dry mycelium from which lipids have been removed is washed in water at 30 ° C.

Example 11

The same cell mass was used as a starting material and reacted according to the preparation method of Example 10 using ethanol instead of methanol. When extracted with water, the temperature was raised to 85 ° C and maintained at the same temperature for 15 minutes.

Table 1a + 1b below summarizes the results of Examples 1-11 above.

TABLE 1a

TABLE 1b

Claims (1)

The content of lipids and nucleic acids in the microbial cell mass is characterized by treating the microbial cell mass with ammonia or ammonium hydroxide and an organic solvent of the following structural formula (I), separating the liquid phase, treating the residue with water, separating the aqueous layer, and drying the residue. How to reduce it. R _1- (CH ₂ ) n-OR ₂ (I) In the above structural formula When R ₁ and R ₂ are hydrogen n is 1,2 or 3, or R ₁ is hydroxy When R ₂ is hydrogen, methyl or ethyl n is 2 or 3.