Process for the purification of microbial protein isolates.

The present invention relates to a process for purifying microbial protein isolates used in the food industry.

It is known (patent, RFG, no. 5 2137038) that there are substances with unpleasant odor and taste, due to^renites from unicellular protein materials, especially bacteria and yeasts, which are extracted with an aqueous solution containing between 60 and 80% in the world from lower aliphatic alcohol.

It is also known (patent, RFG, no. 2633666) that the content of lipids and nucleic acids in microbial cell masses can be reduced by treating cell masses with an extraction mixture consisting of ammonia and a polar solvent, from the series of lower alkanols, lower glycols and methyl- or ethyl ethers of a lower glycol, contain ²⁰the total water length of the extraction mixture being at most 30% by weight relative to the amount of solvent and the ammonia concentration being advantageously from 1 to 10% by weight also relative to the solvent and then by treating the residue with water. Through this process, the lipids and most of the substances are extracted using the extraction mixture³⁰ smell and taste, while water treatment, which can occur in one or more stages, removes nucleic acids. The protein isolate thus obtained is mainly odorless and tasteless and can be used for many purposes directly, including in human food. The disadvantage of the procedures described above is that it cannot always be avoided that such albumin isolates still retain an unpleasant odor and / or taste and this is particularly disturbing to particular applications. An extraction with aqueous aliphatic alcohol, according to the RFG patent, no. 2137038 has no effect in these cases.

The aim of the present invention is to obtain microbial protein isolates with pleasant odor and taste, which can be used for various purposes, including in human food.

The problem solved by the invention is to find a mixture of extraction of substances with unpleasant odor and taste from microbial protein isolates and how to treat the latter with the respective mixture.

The process according to the invention removes the disadvantages mentioned above by the fact that the cellular isolates degree 90644

The price of LEI 14.10 villages is treated at 0 ... 10Q ° C with aqueous solutions containing at least one compound selected from corn- or alkaline diphosphates, hydrophilic aliphatic substances with 2 ... 6 carbon atoms, which possess at least two functional groups selected from hydroxyl, formyl, keto and carboxyl, disaccharides and oligosaccharides, the treatment being carried out in one or more steps, and in the latter case the extraction agent may be the same or different in each step.

15 examples of carrying out the process according to the invention are given below.

Example 1. Methylomonas clara ATCC 31226 was grown in a nutrient solution containing methanol as the sole source of hydrocarbon, ammonia as the sole source of nitrogen, phosphate, iron salts, magnesium and other trace elements, being the usual conditions. aerobics (patent, RFG, no. 2633666). The manufactured bacterial cell mass was separated from the solution and subjected to spray drying. 100 g of this cell mass were treated with 300 g of methanol. Under stirring, 10 g of ammonia gas was added to the suspension and dissolved. By cooling, the temperature was maintained during feeding to 25 ... 35 ° C. The mixture of methanol, ammonia and cell mass was stirred for 30 minutes at 20 ° C. To separate the solid phase from the liquid phase the suspension was filtered; the solid residue was washed once with 300 ml of methanol. After a new filtration, the two filtrates were combined. This control solution contains the lipids of the substance initially introduced. The methanol and ammonia were removed by vacuum distillation (133 mmHg, at a temperature of 40 ° C). The residue, which was 9.5% by weight of the introduced cellular material, was a dark brown, odorless brown paste, consisting of free fatty acids, glycerides, phospholipids and secondary metabolites. The solid residue obtained by filtration of the extracted cell mass was dried under vacuum (133 mmHg) at a temperature of 40 ° C for 5 h. Thus, 90 g of skinned cell mass, odorless and with a lighter color than the original material, were obtained. To reduce the nucleic acid content, this cell mass was suspended in 900 ml of water. The pH value of the homogenized suspension by stirring was 6.9. After raising the temperature to 55 ° C, it was further stirred for a further 20 minutes, then cooled to 30 ° C and by centrifugation the solid phase was separated from the liquid phase. The obtained sediment was again mixed with 900 ml of water and stirred for 10 min at 20 ° C. Then, it was centrifuged again and the sediment dried under reduced pressure. The yield was 65 g. The nucleic acid content was reduced from the initial 11.2% to 1.5%, and the fat content from 7% to 0.8%. The product obtained did not have a dry odor, and water-moistened had a pleasant odor.

Example 2. In a solution of 5 g sucrose in 1 1 water, 100 g spray-dried protein isolate is stirred, according to example 1. The suspension is heated to 80 ° C and further stirred for 30 minutes at this temperature. . Further, the protein isolate is separated by centrifugation from the extraction solution and dried. The microbial pioteic isolate thus obtained is characterized by an improved odor and taste. The composition of the protein part is the same as before extraction.

Example 3. Repeat example 2 with the modification that the solid removed by centrifugation is stirred again with 1 1 of water to remove the adhering sucrose thereon. After further centrifugation, the protein isolate is dried. It also has an improved odor and taste and has the same amino acid composition as before extraction.

Example 4. The starting material is the degreased cell mass, according to example i, which still contains nucleic acids. 90 g of this cell mass is suspended in 900 ml of a 0.5% sucrose solution, the suspension is heated to 55^JC and at this temperature is stirred for a further 20 minutes. After cooling to 30 ° C, centrifuge, add 900 ml of water to the obtained sediment and stir for 10 minutes at 20 ° C. It is then centrifuged again and the sediment is dried under reduced pressure. Compared to the product obtained according to example 1, the product obtained has an improved odor and taste. Nucleic acid content and amino acid composition are identical.

Example 5. Repeat example 3, but in both extraction phases a solution of 0.5% dinatrioortophosphate is introduced. An improved odor and taste product and unmodified amino acid composition are also obtained.

Example 6. Repeat example 1, but sucrose is replaced by sorbit. A product of comparable quality is obtained.

Examples 7 ... 9. Proceed according to example 1, but the sucrose is replaced by lactic acid, apple acid or tartaric acid; products of comparable quality are obtained.

Examples 10 ... 12. Proceed, according to example 3, but replace sucrose with lactic acid, starch syrup or glycerin; products of comparable quality are obtained.

Example 13. Proceed, according to example 4, but introduce in both extraction stages a solution 0,5% dc sodium diphosphate; an improved odor and taste product and unmodified amino acid composition are also obtained.

Example 14. Proceed, according to example 3, but in the first stage of extraction lactic acid is introduced and the ρ la is adjusted to 6 ... 7 with sodium hydroxide, and in the second step sucrose solution is used, · get a product with improved odor and taste and an unmodified amino acid composition.

Example 15. Proceed, according to example 3, but the extraction agents from example 14 are used; a comparable quality product with previous ones is also obtained.

Generally, by the process according to the invention, the undesirable odor and taste substances from microbial protein isolates are removed by treating these isolates with aqueous solutions containing a phosphate and / or a micmolecular hydrophilic aliphatic substance, which contains at least two hydroxyl functional groups. -, formyl-, keto- and carboxy-. By "aliphatic substances" micmolecular means, first of all, those substances having a hydrocarbon chain of 2 ... 6 carbon atoms, respectively in the case of oligosaccharides, they contain two or more of these units. Because the albumin isolates or functional derivatives prepared therefrom are intended for human consumption, toxic compounds will not be prepared as auxiliary substances for extraction according to the present invention, but only those compounds with the lowest toxicity. Therefore, physiologically non-hazardous substances, for example from the aliphatic group, will be preferred; tasting acids, sugar and polyols, such as glycerin and sugar alcohols. If the substance in the protein isolate can be easily removed, other auxiliary substances, such as oxalic acid, can be used, as the remaining quantities will be well below the permissible limit of non-hazardousness. As phosphates, ortho-, oligo- and polyphosphates can be used, the polyphosphates used can be long chain and cyclic. Ortho- and diphosphates are preferred, especially in the form of slightly soluble sodium and potassium salts,

Aliphatic compounds that react with acids are preferably introduced in the form of their alkaline-earth salts and, in the species], in the form of alkaline salts. In addition to the aforementioned oxalic acid, glycolic acid, glycoside acid, racemic pyrotartric acid, fumaric acid, succinic acid, tartaric acid, ascorbic acid, especially lactic acid, citric acid and apple acid, as well as the sugars of the addon series are preferred. , aldar- and uronics, like lactonelc? appropriate, especially gluconic acid beta-lactone. Advantages of extraction are also sugars, from the series of monosaccharides, aldoses and ketones, from the series of pentoses and hexoses glucose and fructose, from sucrose disaccharides and from oligosaccharides hydrolysis products of starch, such as, for example, starch syrup. As aliphatic, containing only hydroxyl groups as functional groups, it is suitable in addition to the aforementioned glycerin and erythritis, pentaerythritis and, in particular, sugar alcohols, especially sorbitite. Advantageous are also mixtures of these auxiliary extraction substances, such as phosphate, in combination with aliphatics such as sucrose, or a mixture of aliphatic compounds, for example glyoxylic acid containing oxalic acid or a sugar-sugar alcohol mixture. The purification of the product, according to the present invention, can be carried out following the process, according to the patent, RFG, no. 2633666. The process is particularly advantageous when adding extraction aids to the extraction of nucleic acid. To an extraction of nucleic acid in several steps, the extraction auxiliary, according to the invention, can be added in the first step or phase, in a later stage or in several stages of the process. The treatment according to the invention can be carried out at a temperature of 0 to 100 ° C, preferably 20 ... 9CPC, especially at 40 ... 80 C. In multi-step processes, the temperature may be in each phase identical or different; depending on the purpose of use of the protein material, there may be higher or lower residual quantities of the auxiliary, ie extraction in the protein residue, such as sugar, when the protein is processed into sweet foods, such as beverages, bakery materials and desserts, a phosphate, when the protein should be introduced into meat preparations, or sausages, cheese or cheese-like products, as well as other bread preparations. The content of aqueous solutions in phosphate and / or aliphatic is limited in the lower sense by activity, and in the upper sense, in principle only by solubility, because

9 (5fe-44 as mentioned above - in many cases residual quantities of the auxiliary extraction agent may remain in the extracted material. In general, concentrations of the auxiliary agent are chosen from 0.1 to 3, preferably between 0.2 and 2, in particular 0.5 ... 1% by weight, It is possible to apply the purification process according to the present invention to other albumin, for example on chemically modified microbial albumin. The percentage indications refer to weight.

A taste test was performed, whereby the purified protein, extracted according to Example 2 at different temperatures, was compared with the unpurified protein. The individual products were evaluated by points by 15 test persons.

The weight of 1 point at 6 points could be given, the more the point, the better the taste.

From the results indicated in the table it appears that the extracted protein, according to the invention, at all temperatures has an improved taste compared to the unpurified protein. Particularly good results were achieved when the extraction was carried out at 25, 55 and 80 ° C.

// ^h Appreciation of protein samples

<td rowspan="2">The person of test · **.</td><td> J Ϊ</td><td colspan="4">Purified protein</td><td rowspan="2">Protein; nepuri- your daughter</td>

<td>2 ° C</td><td>25 ° C</td><td>55 ° C</td><td>80 ° C</td><td>100 ° C</td>

<td> 1</td><td> 3</td><td> 4</td><td> 5</td><td> 4</td><td> 4</td><td> 1</td>

<td>of</td><td> 3</td><td> 6</td><td> 6</td><td> 5</td><td> 4</td><td> 1</td>

<td> 3</td><td> 2</td><td> 5</td><td> 6</td><td> 6</td><td> 3</td><td> 1</td>

<td> 4</td><td> 4</td><td> 4</td><td> 4</td><td> 3</td><td> 3</td><td> 1</td>

<td> 5</td><td> 3</td><td> 4</td><td> 5</td><td> 5</td><td> 4</td><td> 3</td>

<td> 6</td><td> 4</td><td> 5</td><td> 5</td><td> 3</td><td> 3</td><td> 2</td>

<td> 7</td><td> 4</td><td> 5</td><td> 4</td><td>of</td><td> 4</td><td> 2</td>

<td>β</td><td> 4</td><td> 5</td><td> 4</td><td> 3</td><td> 4</td><td> 2</td>

<td> 9</td><td> 3</td><td> 5</td><td> 5</td><td> 6</td><td> 4</td><td> 1</td>

<td> 10</td><td> 3</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td><td> 1</td>

<td> 11</td><td> 3</td><td> 6</td><td> 5</td><td> 4</td><td> 5</td><td> 3</td>

<td> 12</td><td> 3</td><td> 6</td><td>ό</td><td> 5</td><td> 5</td><td> 1</td>

<td> 13</td><td> 5</td><td> 6</td><td> 6</td><td> 6</td><td> 5</td><td> 2</td>

<td> 14</td><td> 5</td><td> 3</td><td> 6</td><td> 5</td><td> 4</td><td> 3</td>

<td> 15</td><td> 4</td><td> 5</td><td> 5</td><td> 4</td><td> 5</td><td> 1</td>

Points l to β: l - unpleasant taste 6 = pleasant taste

The advantage of the process, according to the invention, is that it allows obtaining microbial protein isolates, with pleasant odor and taste, which can be used for various purposes, including in human food.