Process for the purification of microbial protein isolates.

FOR PURIFICATION OF PROTEIN ISOLATES

MICROBIAN COS.

THE CAM.

Descriptive Memory

From German Published Memory 2 137 03 # The process of extracting unpleasant taste and aroma substances from cellular protein products, especially yeast and bacteria, by extraction with an aqueous solution containing 60 to 80% by volume of an aliphatic alcohol bottom.

From German Patent No^fi. 26 The process of reducing the lipid and nucleic acid content in microbial cell masses is known by first treating this cell mass with an extractive mixture consisting of ammonia and a polar solvent of the lower alkanols, lower glycols and 0 methyl or ethyl ester of a lower glycol, the total water content being at most 30 wt.% by weight of the solvent mixture, and the ammonia concentration preferably being 1 to 10 wt.%, also referred to

-1 solvents, then treating the residue of the cell mass with water. In this way the lipids and a large part of the flavoring and flavoring substances are extracted by the extractive mixture, while by treating the water, which can be carried out in one or more steps, the nucleic acids thus obtained are eliminated. it is practically odorless and tasteless and can be used directly for many applications as well as for human consumption.

In practice, however, it is not always possible to prevent some protein isolates from still having an unpleasant aroma and / or taste which is particularly detrimental to special applications. In these cases extraction with an aqueous aliphatic alcohol according to German Published Specification 2 137 03 $ is ineffective.

It has now been found that undesirable aromas and palates can be eliminated from microbial protein isolates by treating these isolates with aqueous solutions containing a phosphate and / or a low molecular weight hydrophilic aliphatic compound containing at least two serie functional groups. hydroxy, formyl, keto and carboxy. Preferred embodiments of this invention are described in more detail below.

As a low molecular weight aliphatic compound, it is of interest primarily to those containing a carbon chain having 2 to 6 carbon atoms, or in the case of disaccharides and oligosaccharides two or more of these units.

Since protein isolates or perfunctional derivatives prepared therefrom are intended for human consumption, low toxic compounds are used as extraction aids according to the invention.

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Physiological harmless substances are therefore preferred, and among the aliphatic compounds, therefore, are preferred innocuous acids, sugars and polyols such as glycerine and sugar alcohols. If however the substance can be separated from the protein isolate easily and practically quantitatively then other auxiliary substances such as oxalic acid can be used, since then the remaining residues fall well below the limits of harmlessness.

As phosphates are of interest to orthophosphates, oligosaccharides and polyphosphates, the polyphosphates may be long chain and cyclic. Orthophosphates and diphosphates, especially in the form of easily soluble sodium and potassium salts, are preferable.

The aliphatic acid reaction compounds are advantageously used in the form of their alkaline earth salts and especially alkaline salts. In addition to the aforementioned oxalic acid, glycolic, glyoxylic acid, piravic acid, fumaric acid, succinic acid, tartaric acid, ascorbic acid, especially lactic acid, citric acid and malic acid, as well as sugars derived from sugars are preferable. aldonic, aldric and uronic series, as well as the corresponding lactones, especially β-lactone of gluconic acid.

Advantageous means of extraction are also sugars, namely from the monosaccharide series, aldoses and ketoses of the pentose and hexoses series, especially glucose and fructose, and from disaccharides in particular saccharide, and from oligosaccharides in particular hydrolysis products. starches like starch syrup

As aliphatic compounds which have only the hydroxy group as functional groups, in addition to the aforementioned glycerin, erythrite, pentaerythrite and especially sugar alcohols, especially sorbite, are also suitable.

Also advantageous are mixtures of these extraction aids, for example a phosphate in connection with an aliphatic compound such as sucrose or a mixture of aliphatic compounds, for example an oxalic acid-containing glyoxylic acid, or a sugar-sugar alcohol mixture.

The purification according to the invention may be carried out following the process according to German patent 26 33 666. It is however especially advantageous that the extraction aid is used in the extraction of nucleic acids. In a multi-step extraction of the nucleic acids, the extraction aid according to the invention may be used in the first step, a later step or in several steps.

The treatment according to the invention may be carried out in a range of from about 0 to 100 ° C, but preferably about 20 to 90 ° G, especially from 40 to 0 ° C. In multi-step processes the temperature in each step may be the same or different.

Depending on the intended use of the protein product, smaller or larger residual amounts of the extraction aid, for example a sugar, may remain in the protein residue if the protein is incorporated into sweet food media such as beverages, confectionery or desserts, or phosphate if the protein is incorporated into meat or sausage products, cheese or cheese products or (other) bread spreads.

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The content of aqueous solutions in phosphate and / or aliphatic compound is limited inferiorly by efficacy, and superior in principle only by solubility, as - as already mentioned - in many cases residues of the extraction aid may remain in the product of the product. extraction. Concentrations of medium au are generally chosen. from 0.1 to 3% by weight, preferably from 0.2 to 2%, especially from 0.5 to 1%.

It is of course also possible to apply the purification process according to the invention to other protein substances, especially to chemically purified microbial proteins.

In the following examples the invention will be eluted. addressed in more detail. Percentage data refer in these cases to weight percent.

Example 1

To a solution of 5 g sucrose in 1 L of water, 100 g of a spray-dried protein isolate according to Example 1 of German Patent 26 33 666 is added under stirring. The suspension is heated to 80 ° C and stirred continuously for half an hour at this temperature. Then the protein isolate is separated from the extraction solution by centrifugation and dried.

The microbial protein isolate thus obtained is characterized by an improved taste and aroma. The composition of the protein constituent is the same as before extraction.

Example 2

Example 1 is repeated with the modification that the solid substance prepared by centrifugation is again triturated with one L of water to remove adherent sucrose,

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After further centrifugation the dry protein isolate. It also has an improved taste and aroma and has the same amino acid composition as before extraction.

Example 3 Starting product is a cell mass degreased according to Example 1 of German patent 26 33 666 and further containing the nucleic acid. 9θ g of this cell mass is suspended in 900 ml of a 0.5% saccharide solution if the suspension is heated at 55 ° C at this stirred temperature for 20 min. After cooling to 30 ° C, centrifuge.

The pellet thus obtained is mixed with 900 ml of water and stirred 10 min at 20 ° C. Then it is centrifuged again and the sediment is removed. dry at reduced pressure.

Unlike a product according to example 1 of German patent 26 33 666, the product is distinguished by an improved aroma and taste. The nucleic acid content and amino acid composition are identical.

Example 4

Example 2 is repeated using a 0.5% disodium orthophosphate solution in the two extraction steps. An improved flavor and taste product with no modification of the amino acid composition is also obtained.

Example 5

Example 1 is repeated but sucrose is replaced by sorbite. A product of comparable quality is obtained.

Examples 6-8

Work according to Example 1, but replace sucrose with lactic acid, malic acid or tartaric acid to give qualitatively comparable products.

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Examples 9-11

Work according to Example 3 but substitute sucrose for lactic acid, starch syrup or glycerin to obtain qualitatively comparable products.

Example 12

Work as in Example 3, but a 0.5% solution of sodium diphosphate is substituted for both extraction steps, thereby also obtaining a product with improved aroma and taste and unchanged amino acid composition. From.

Example 13

Work as in Example 2, but lactic acid and caustic soda are used in the first step, the pH is adjusted to 6-7, the sucrose solution is then used in the second step and thus a product is obtained. with improved aroma and taste and unchanged amino acid composition.

Example 14

Work as in Example 3 but use the extractive medium described in Example 13 to give a comparable product.

ANNEX: German Patent Example 1 26 33 666: Methylomonas clara ATCC 31 226 was grown in a nutrient solution containing methanol as the sole carbon source, ammonia as the sole source of nitrogen, phosphates, iron and magnesium salts, and other standard trace elements. , in aerobic conditions. The bacterial cell mass thus produced was separated from the solution and spray dried.

100 g of this cell mass were mixed with 300 g of methanol

Under agitation of the suspension, 10 g of dissolved ammonia was bubbled into it and dissolved. Upon cooling the temperature was maintained at 25-35 ° C during gas passage. The mixture of methanol, ammonia and cell mass was stirred for 30 minutes at 20 ° C,

For the separation of the solid and liquid phases, the solid was filtered and the solid residue was washed once with 300 ml of methanol. After further filtration both filtrates were pooled. This brown solution contained the lipids of the starting substance used. Methanol and ammonia were removed by vacuum distillation (100 Torr, 40 ° C). 0 The residue, which was 9.5% by weight of the cell product used, was a brown, phosphate-free paste consisting of free fatty acids, glycerides, phospholipids and secondary metabolites.

The solid residue from the extracted cell mass obtained by filtration was dried under vacuum (100 Torr) at 40 ° C for 5 hours. In this way 90 g of odorless degreased cell mass which had a lighter color than the starting product could be obtained.

To decrease the nucleic acid content this cell mass was suspended in 900 ml of water. The pH of the homogenized suspension by stirring was 6.9%.

After raising the temperature to 55 ° C, it was stirred for a further 20 minutes, cooled to 30 ° C and then centrifuged into a solid phase and a liquid phase. The pellet thus obtained was again mixed with 900 ml of water. and stirred 10 minutes at 20 ° C. It was then centrifuged again and the latter pellet was dried under reduced pressure.

yield was 65 g. Nucleic acid content decreased from initial 11.2% to 1.5 β and fat content decreased from 7% to 0.0%. The product in the dry state was odorless, and moistened with water had a pleasant aroma.