FI58156B - FOERFARANDE FOER SAENKNING AV NUCLEINSYRAHALTEN HOS MIKROORGANISMPRODUCERAT RAOPROTEIN - Google Patents

Description

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Patent raoddeint (51) Kv.ik? /Int.a.3 C 12 N 1/08, 0 07 G 7/00 FINLAND —FINLAND (21) 771621 (22) Hukumhptlv * —AmMuilnpd *, 20.05.77 (23) Alkvpthrt — GlHlfh «tsd» f 20.05-77 (41) Tullut | ulkMal - Bltvlt offumll, 22.11.77

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Patent- och registerstyrelaen 'Amöku utlagd oeh utUkrtfun publkurad 29.00.00 - (32) (33) (31) Pyydetty «tuolkaut —8« ^ rd prlorlcut 21.05-76

Federal Republic of Germany Förbundsrepubliken Tyskland (DE) P 2622982.3 (71) Boehringer Mannheim GmbH ,, Sandhofer Strasse 112-132, D-6800 Mannheim-Waldhof, Federal Republic of Germany Förbundsrepubliken Tyskland (DE) (72) Erich Haid, Tutzing, Michael Nelb Hochstetter, Tutzing, Gotthilf Näher, Tutzing, Gunter Weimann, Tutzing, Federal Republic of Germany (DE) (71 *) Berggren Oy Ab (5U) Method for reducing the nucleic acid content of a crude protein produced by microorganisms - Förfar hos mikroorganismproducerat räprotein

It is known that the world's population is growing rapidly and that, as a result, severe nutrient shortages are to be expected in a few decades. In several countries, this situation has already been reached. The main problem in this case is the lack of proteins. The daily human protein requirement is about 70-100 g. In underdeveloped countries, this protein requirement is mainly met by vegetarianism. However, unlike animal-derived protein, vegetable protein contains very little vital lysine and methionine. When only vegetable proteins are available, severe deficiency diseases can occur. For this reason, efforts have been made for several years to introduce microbial protein in addition to animal and vegetable proteins in order to meet the protein needs of the population. A microbial-derived protein has the advantage of having a similar amino acid composition to an animal protein. When microbial protein is used in food, there is no fear of the occurrence of deficiency diseases that are possible with the use of vegetarian food alone.

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Attempts have been made for decades to use microorganisms to produce nutrients or feed. The advantage of these microorganisms is that they grow much faster than plants or animals under favorable conditions. The following table describes this feature.

Doubling times for some organisms

Organism Biomass doubling time

Yeast and bacteria for 20 to 120 minutes

Microorganisms such as algae for 1 to 48 hours

Grass 1 - 2 weeks -

Chicken 2 - 4 weeks

Pig 4 to 6 weeks

Cattle 1 to 2 months „

The rapid proliferation of microorganisms indicates that more protein can be obtained in 1-2 hours than in the fattening of pigs or cattle in 4-8 weeks.

However, there are some difficulties with such protein production: 1. The cultivation of microorganisms requires very high technical equipment (fermentors) as well as costs; 2. the separation of organisms from the culture broth causes technical difficulties when carried out on the scale in question; 3. Harmful additives, such as nucleic acids and toxins, must be removed from the micro-organism.

Despite these difficulties, microorganisms are already grown in large numbers for feeding to domestic animals (feed yeast). However, so far, protein derived from micro-organisms has not been used at all or only in very limited quantities for human consumption. Namely, the high ribonucleic acid content of this protein poses a difficult problem. Ribonucleic acid (RNA) is broken down in the body of a mammal into uric acid, which in turn can cause gout. The RNA content of the microorganism protein should therefore be less than 2%, preferably less than 0.5%.

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Thus, one of the main objectives is to produce a microbial protein that does not contain ribonucleic acid. So far, it has not been satisfactorily achieved. Attempts have been made to grow microorganisms with a particularly low RNA content. However, these microorganisms have the disadvantage that they grow much more slowly. It is known that microorganisms with a high RNA content grow faster than those with a lower RNA content. This causes a fundamental contradiction; the desired rapid growth of microorganisms is always associated with a high concentration of RNA. If slower production is satisfied, microorganisms with a low RNA content can be grown.

Another possibility to remove RNA from a microorganism protein, commonly referred to as a Single Cell Protein (SCP), is based on the extraction of ribonucleic acid followed by separation of the insoluble protein. Several possibilities for the extraction of F.NA have already been presented: a) alkaline extraction - German application 16 70 110.3 b) acid extraction c) treatment with high salt concentration (NaCl, KCl, NH4Cl) d) treatment with detergents (dodecyl sulphate, etc.).

DE-A-1 670 110 discloses a method of heating living yeast cells to 90-100 ° C, where they die and their cell walls break. The ribonucleic acid is extracted from the disrupted cells by alkaline addition until the pH is 12.5-13.5 and the RNA is digested with malt binding into 5 'nucleotides.

All of these methods have the disadvantage that the microorganisms have to be extracted in a rather complicated way and that the protein is partially hydrolysed, racemized or otherwise altered by treatment and various additives, making it unfit or less suitable for nutritional purposes. Thus, for example, alkaline extraction changes the structure of the protein so that a colloidal solution is formed. The separation then becomes possible only after neutralization, which requires the removal of the neutral salts formed. In addition, dissolved RNA is partially denatured and can no longer be quantitatively degraded by enzymes into soluble mononucleotides.

U.S. Pat. No. 3,720,585 discloses a process in which two specific types of yeast are subjected to a short-term heat treatment comprising one or two heating sessions of 60-70 ° C for 20 seconds followed by 4 58156 heating sessions of 45-50 ° C for about 20 minutes. , with a pH between 4.5 and 7.

This method has the disadvantage that it releases proteases which degrade a useful (digestible) protein, and thus a significant reduction in protein production. The remaining RNA content is only reduced and its concentration always remains higher than the maximum value of 2% of the target range.

Accordingly, it is an object of the invention to provide a method for producing a unicellular protein which does not have the above-mentioned disadvantages of the previously known methods. In particular, it is an object of the invention to provide a simple method which can be carried out in a large-scale production method without the addition of expensive equipment and foreign admixtures, which method does not require any prior treatment of microorganisms to kill and degrade them and is environmentally friendly.

This object is achieved according to the invention by a method for reducing the nucleic acid content of a crude protein produced by microorganisms by heat treatment, which method is characterized in that an aqueous slurry of microorganisms, optionally with ribonuclease-containing substance, e.g. grain sticks or extract, is maintained at pH 7-7. 8.5, e.g. using aqueous solutions of sodium hydroxide, potassium hydroxide or ammonia, is heated at 63-67 ° C without breaking the cell walls of the microorganisms until the 5'-nucleotide content in the medium has reached the desired value and the slurry is then boiled, and insoluble crude protein separated from the suspension.

When using the method according to the invention, the nucleic acid decomposes only into 5 'nucleotides present in the medium, and at the same time no 2' and / or 3 'nucleotides are formed. Thus, depending on the nucleic acid content of the starting microorganism and the target final nucleic acid content, it is easy to determine in advance at which 5'-nucleotide content in the medium the nucleic acid content has decreased to the target value and to terminate the heat treatment at 63-67 ° C. It is crucial that the temperature is kept strictly between 63 and 67 ° C and that the pH remains constant in said range. Since the pH tends to decrease as the nucleotides are released during the heat treatment, a base, preferably sodium hydroxide, potassium hydroxide or ammonia, is added to the slurry to maintain it in said region.

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Using the method of the invention, the cell walls of the microorganisms are not disrupted and endogenous nucleases are activated, causing nucleic acid degradation to form highly desired 5 'nucleotides as by-products, namely adenosine monophosphate (AMP), guanosine monophosphate (GMP) and cytosine (GMP), cytosine. The heating time required depends on the microorganism used in each case, in particular the concentration of endogenous nucleases in it. Because the nuclease concentration can be very different, the heating time is also very variable, and is usually between about 20 minutes and about 20 hours.

Already during the heat treatment according to the invention, the protein material becomes largely insoluble. Protein precipitation is then completed by boiling, i.e. by heating between 90 and 100 ° C at normal pressure or higher at elevated pressure. The insoluble crude protein is then separated from the suspension, e.g. by centrifugation or filtration, and can now be used as a nutrient or further processed. as desired.

Suitable for use in the method of the invention are all species of microorganisms or other unicellular species, such as, for example, yeasts, bacteria, fungi and the like. and Bacillus subtilis, Streptococcus faecalis from streptococci, Pseudomonas fluorescens from pseudomonas and Aspergillus niger from fungi. As mentioned above, the microorganisms are most preferably used in a counterproductive viable form, i.e., as obtained from the culture medium. However, it is also possible to use unicellular organisms which have been treated according to known methods of preserving living microorganisms, for example gently dried or lyophilized unicellular organisms.

According to a particular embodiment of the method according to the invention, it is possible to shorten the heating time by adding a ribonuclease-containing substance to the slurry. By such a substance is meant both purified ribonuclease preparations and also pre-purified or crude biological preparations containing ribonuclease. The most suitable ribonuclease-containing substances are sprouts of grasses and cereals, especially malt sprouts or extracts thereof. In this embodiment, the method of the invention can also be applied to ribonuclease-free microorganisms, such as the E. coli MRE 600 mutant (deposited with the American Type Culture Collection No. 29,417 and the National Collection of Type Culture, London No. 8,164).

In general, the method according to the invention makes it possible to reduce the RNA content of the monocyte protein to 0.02% by weight. Protein losses are small, and in particular the degradation and racemization of the protein is negligible.

The method according to the invention achieves at least 90% protein yield. In particular, the method has the advantage that the ribonucleic acid is in fact degraded quantitatively into 5'-nucleotides, which are in themselves valuable substances. Thus, after separation of the crude protein, it is possible to preparatively recover the nucleotides from the filtrate. However, it is very advantageous to reuse the filtrate to grow microorganisms. Again, the microorganisms construct the necessary nucleic acid from the nucleotides, so that in practice in this way the circulation of the nucleotides is effected. At the same time, the microorganisms also utilize other dissolved cellular constituents for new growth, e.g. protein remaining in solution. Therefore, biotin or yeast extracts otherwise normally used in the growth of microorganisms may be omitted in whole or in part.

The process of the invention provides a purer and more digestible protein that does not contain detergents, salts or ribonucleic acid. In contrast, in conventional cell extracts prepared with alkalis or acids, the various amino acids in the protein are destroyed or racemized.

In addition, the method according to the invention has the special effect that the odor characteristic of proteins, which is particularly strong in yeasts, disappears at the same time. This, too, tends to make the unicellular protein more suitable for nutrient use.

Removal of an RNA compound is quite simple and inexpensive. 5 'nucleotides can be obtained as by-products. Returning 7 58156 rack filtrates to cultivation equipment is moreover achievable in culture medium savings. The method is also environmentally friendly because the cell extract can be easily incinerated, which is not possible when using a salt, acid or alkali-containing substance for extraction.

The following examples further illustrate the invention.

Examples 1-7

Each of the microorganisms listed in Table 1 is made into a 200 liter aqueous slurry having a dry matter content of about 15-20%, and each batch of slurry is heated to 65 ° C and maintained at this temperature for 15 hours with gentle agitation. The pH is constantly maintained between 7-8.5 by the addition of sodium hydroxide solution. The batch of slurry is then boiled, and the crude protein is separated by separators and dried. The crude protein yield ranged from 25 to 32 kg. Table 1 shows the RNA content of the starting materials and the degradation of the RNA compound achieved.

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As shown in Table 1, the yield of 5 'nucleotides in the method according to the invention is very good when using fresh microorganisms. When the microorganisms were stored for a long time, the yield was lower due to the reduced ribonuclea content.

Examples 8-14 a) 3 kg of malt bars are finely ground and mixed with 30 liters of water. The mixture is kept at 65 ° C for 40 minutes. The resulting suspension is treated either immediately or after centrifugation of the insoluble malt fractions as follows.

b) The same microorganisms as in Examples 1-7 are each made into a 200 liter slurry having the same dry matter content as above. Each slurry is heated to about 65 ° C and the malting suspension or equivalent extract obtained in a) is added, followed by heating for 2-15 hours at 65 ° C and the pH is adjusted as shown in Examples 1-7 to 7-. 8.5. Further work-up is carried out in the same way as in Examples 1-7. The crude caprotein yield ranged from 25 to 35 kg, with an average pure protein content of about 70%.

Table 2 shows the achieved degradation of the RNA compound. With an RNA content of about 10% of the dry matter, the yield of each of the four different 5 'nucleotides was about 7-800 g, or correspondingly less, with a lower RNA content of the starting material.

Example 15

As described in the previous examples, a slurry containing 100 g of yeast Saccharomyces cerevisiae as dry matter, with a protein content of about 45% and an RNA content of 1.5-3% in the dry matter, without the addition of ribonuclease-containing substance, is heated at 65 ° C for 2-15 hours at the same pH as above, after which the slurry is boiled and the crude protein is separated. The filtrate and crude protein compositions were determined and are shown in Table 3 below. The reported results show a 45% increase in crude protein to 56%.

When microorganisms with an initially higher protein content are used, the crude protein yield can increase up to 85.

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