DE2423762A1 - Proteins of low nucleic acid contents - prepd. by bacterial fermentation of Bacillus, Pseudomonas and Flavobacterium species on methanol-contg. nutrient media - Google Patents

Abstract

Protein prepn. from bacteria with MeOH as C-source and an N-source, under aerobic conditions, at pH 4-9, takes place by fermenting bacteria strains of Bacillus, Pseudomonas and Flavobacterium species, their mutants or variations, in a way such that MeOH concn. in logarithmic growth phase of bacterial cells is (10-150, esp. 50-100 p.p.m., w.r.t. culture suspension. Used as protein-source in food and animal feedstuffs. Prodt. contains 60-80 wt. % crude protein contg. (a) 90-95wt. % aminoacids, rich in essential and semi-essential aminoacids, and (b) only 5-10wt. % undesired nucleic acids (compared with 8-16wt. % by other processes).

Description

Process for the fermentative production of protein They are biosynthetic Process known in which microorganisms, e.g. certain bacterial strains, in a synthetic nutrient medium, which is the only carbon source methanol contains, fermentatit are increased. Such processes can be discontinuous as well as continuously. The bacterial cells obtained in this way contain in the so-called "crude protein" (calculated by multiplying dos by elemental analysis found value for nitrogen with the expressive factor 6.25) usually 40-75% by weight of amino acids and 8-16% by weight of nucleic acids. Such a high one However, the proportion of nucleic acids is undesirable because they are derived from them in particular in human The organism produces uric acid, which causes gout. In addition, the flora of the Gastrointestinal tract adversely affected.

There has now been a method of making bacteria using methanol as a carbon source and a nitrogen source under aerobic conditions found a pH between 4.0 and 9.0, which is characterized in that one bacterial strains of the genera Bacillus, Pseudomonas and Flavobacterium, whose Mutants or variants fermented so that the concentration of methanol in the Phase of logarithmic growth of the bacterial cells between 5 and 200 ppm, based on the culture suspension.

A concentration of methanol between 10 and 150 ppm.

The process is expediently carried out in fermentation reactors, which contain a nutrient medium, in addition to the aforementioned methanol as a nitrogen source Salts such as potassium nitrate, ammonium sulfate, ammonium phosphate or ammonia, urea or Contain soy flour. It also contains phosphates such as potassium dihydrogen phosphate or Disodium hydrogen phosphate as well as magnesium and potassium salts and trace elements, as they e.g.

are also contained in tap water. For example, iron, copper Molybdenum salts present in traces.

It can be useful, especially the precultures such as nutrients Add yeast extract, meat extract, liver extract, glucose or others.

The process is expediently carried out at temperatures between 200 and 450C, preferably carried out between 300 and 370C.

Bacillus species are used as bacterial strains, in particular well-known Bacillus strains such as B, subtilis or B.polymyxa, furthermore Pseudomonas species, such as Pseudomonas aeruginosa. Flavobacteria can also be used.

The culture suspension in the reactor is 0.1 to 1.5 1 air per liter Culture solution and minute (vvm), preferably with 0.3 to 0.6 tower, aerated. To a good uptake of oxygen through the cells to ensure can be agitated intensively; emulsifiers can also be added.

In other reactor systems than stirred kettles must be accordingly for a Sufficient amount of air can be provided, with the volatility of the methanol a Recirculation of the air is favorable. In conventional systems it has been found to be beneficial proved to have one or even better two or more stirrers in a fermentation kettle to use. It is also advantageous to have two stirrers, in particular perforated disc stirrers, should be arranged in the fermentation tank so that the stirrer distances are approximately one stirrer diameter. " be. The ratio of the stirrer diameter to the bowl diameter is appropriate between 0.35 and 0.65, preferably between 0.45 and 0.55. The speed of rotation the culture suspension is expediently more than 7 m / s If foam formation occurs, chemical or mechanical foam control, for example through Addition of 0.01 to 0.1% by weight of a defoamer such as octanol or oleic acid ester and lauric acid init glycol, glycerin or sorbitol, alcoholic cholesterol solution or silicones such as polydimethylsiloxane.

The methanol concentration between 5 and 200 ppm, preferably between 10 and 150 ppm, can be continuously controlled by various measures, e.g. by measuring the nitrogen consumption, the cell mass fraction or preferably by measuring carbon dioxide emissions. Here is a fine dosage of the addition of methanol with a quick reaction to the lower carbon dioxide emissions in the case of carbon deficiency possible.

This control is preferably based on the measurement of the gaseous Methanol using a flame ionization detector.

When the pH of the culture suspension is below the prescribed value drops, it is raised again by adding alkali, e.g. caustic soda or potassium hydroxide brought the prescribed value. Likewise, an excessively high pH value is caused by adding regulated by acid, e.g. hydrochloric acid or sulfuric acid.

To control the process, samples are taken from the culture suspension taken to find the dry weight and nitrogen content in the bacterial cell mass to determine. After that, the growth rate and the doubling time of the cell mass to be determined. If the dry weight of the cells in consecutive samples withdrawn no longer increases logarithmically, the fermentation in batch mode completed. In the case of continuous operation, the biomass production is controlled via the dilution rate controlled in the reactor.

The bacterial mass is separated off in the usual way Centrifuge, washing several times with water.

Processes have also proven themselves, whereby by flocculation with salts at a certain pH the cell mass can be concentrated. You get one like that paste-like bacterial cell mass which still contains 75 to 90% by weight of water. The drying can be done in different ways, e.g. by roller drying, Fluidized bed drying or spray drying. The product dried in this way only contains 2 - 5% by weight of water and 60 to 80% by weight of crude protein. In this crude protein the proportion of amino acids is 90 to 95% by weight.

It is noteworthy that not only this proportion of amino acids in general, but especially the content of essential and semi-essential Amino acids are higher than in comparable products according to the state of the art.

In addition, the crude ash content of the dried ones obtained according to the invention lies Cell mass significantly lower than that of known products. The obtained according to the invention Crude protein still contains only 5 to 10 weight nucleic acids, while known ones Products contain 8 to 16 or more nucleic acids by weight.

The dry bacterial mass obtained by the method according to the invention is therefore particularly suitable as a protein source in food and animal feed to serve.

Example t: The strain Pseudomonas sp. FH-B-5163, which is on agar slants of the composition 2.5% meat extract 1.0% methanol 5.0% yeast extract 0.2% NaN03 10.0% meat peptone 0.02% MgSO4 # 7H2O 10% 10.0% glucose + 90% 0.02% Ma2HPO4 10.0% NaCl 0.009% NaH2PO4 2.5% agar 0.004% KCl 0.00015% CaCl2 0.0001 % FeS04 7H20 traces of CuSO4 # 5H2O "H3BO3" MnSO4 # 5H2O sI ZnSO4 "7H20" Na2HOO4 # 2H2O (pH 6.6) is maintained with 4 ml of distilled water or physiological Saline solution washed off and sterile inoculated into a 2 1 Erlenmeyer flask, of the 250 ml preculture nutrient solution contains the following composition: 0.53% (NH4) 2SO4 0.4% KH2PO4 0.2% Na2HPO4 12 H20 0.02% MgSO4. 7 H20 0.02% KCl 1.5% methanol and 0.1% trace elements (pH 6.8) The inventive method is carried out in a 14 liter fermentation kettle, the 10 liter the following nutrient solution is charged: 1% (NH4) 2So4 0.4% KHSPOi 0.2% NazHPO4 12 H2O 0.02% MgSO4 # 7H2O 0.02% KC1 The pH value of the nutrient solution is 6.8. she is sterilized for 45 minutes at 1210C. In addition, 50 g (0.5 percent by weight) Methanol added to the nutrient solution before inoculating the main culture.

To inoculate the nutrient solution, 2 x 250 ml of the preculture, which was shaken at 300C for 24-48 hours, was used. The nutrient solution inoculated in this way is stored for 24 - 36 hours at 30 0C with aeration with 0.6 vvm air with a turbine stirrer.

If necessary, the pH value is corrected by adding 2 N sterile HCl. During the logarithmic phase of batch fermentation as well as during continuous Driving mode is a concentration of methanol between 5 to 150 ppm weight-5 ' adhered to, in addition to a reduction of the CO2.

When the cells are ejected, further proportions of methanol are automatically added, until the CO emissions rise again. This can also be done by measuring the gaseous Methanol content in the exhaust air is done by an FID (flame ionization detector).

The progress of the procedure is determined by taking samples the dry weight and nitrogen of the bacterial cell mass are controlled.

After the logarithmic growth phase has ended, the work-up in the usual way by centrifugation or flocculation, washing the cells with Water and subsequent spray drying made. The product thus obtained has a crude protein content of 71.87% by weight, an amino acid content of 66.55% by weight and a nucleic acid content of 9.8% by weight. The essential and semi-essential Amino acids alone make up 46.47% by weight (with glycine).

Detailed information can be found in the following table i.

TABLE t Product bacteria FH-B-5163 Crude protein (N x 6.25)% 71.87 Water content% Raw ash % Crude fat% Crude fiber Amino acid content 5 '66.55 Amino acids g / 16 g NN Asp 9.50 Thr 4.63 Ser 3.08 Glu 11.57 Per 4.15 Gly 6.05 Ala 8.41 Cys nb Val 6.19 Met 3.85 Ile 3.92 Leu 7.45 Tyr 2.09 Phe 3l55 His 1.89 Lys 5.90 Arg. 7.33 Try nb Nucleic acid content% 9.8 Example 2: The strain Pseudomonas aeruginosa FH-N-845 is grown as in Example 1. In addition, 9 l nutrient solution of the composition given in Example 1 are placed in a 14 l fermentation kettle and 0.5% by weight of methanol is added. The nutrient solution is inoculated as indicated in Example 1 and stirred at 30 ° C. with aeration with 0.6 VVm air with a turbine stirrer at 210 rpm. The pH is kept at 8.0 by adding sterile HCl if necessary.

During the logarithmic phase of fermentation there is a concentration of methanol between 50 and 100 ppm.

The regulation of this concentration, the control of the process as well the cell mass obtained is separated off and worked up as in the example 1 described. After spray drying, a product with a crude protein content is obtained of 75.8% by weight, an amino acid content of 67.0% by weight and a nucleic acid content of 8.5% by weight.

Example 3: The Klavobacterium Sp. FH-B-5108 strain is made as in Example 1 described initially grown in a 10 liter fermentation kettle. The culture suspension thus obtained is then placed in a 200 l fermentation kettle transferred, which is loaded with 150 l of the following nutrient solution: 1 5 '(NH4) 2S04 0.4% KH2PO4 0.2 5 'K2HP04 0.2% Na2HPO4 # 12 H2O 0.02% NaCl 0.02% MgSO4 # 7 H2O 0.5% methanol The pH of the nutrient solution is half-concentrated Phosphoric acid adjusted to 6.5. It is then sterilized at 1200C and 1.4 atü for 20 minutes. After sterilization, the pH is 6.7. Methanol is separated admitted.

The culture is then kept at 35 C for 24 to 36 hours Aeration of 1.1 vvm at a pressure of 0.3 atü while stirring with 2 stirrers Fermented at 250 rpm. During the logarithmic phase of fermentation, a Concentration of methanol between 50 and 120 ppm complied with, in which at a To reduce the carbon dioxide emissions, further proportions of methanol are added, until the carbon dioxide emissions rise again. The pH becomes more sterile by adding HC1 held between 7.9 and 8.4. The progress of the procedure and the timing its completion are determined as indicated in Example 1.

The culture suspension thus obtained is used as an inoculum for a 2000 1 fermentation kettle used. The nutrient solution has the same composition and the same pH as above for the last fermentation described specified. The fermentation reactor is under for 20 minutes at 1200C and 1.4 atm Stirring sterilized at 210 rpm.

After sterilization, the pH is 6.8.

The subsequent fermentation is carried out at 35 ° C, aeration at 0.6 vvm, carried out a pressure of 0.3 atü and 2 stirrers at 210 rpm. The pH will be Maintained between 7.9 and 8.4 during the fermentation with sterile HC1.

The progress of the process is indicated by taking samples for testing sterility, pH measurement, determination of dry weight and nitrogen the bacterial cell mass monitored.

The concentration of methanol is during the logarithmic growth phase kept between 10 and 150 ppm and as described above in accordance with the Carbon dioxide emissions supplemented.

After the logarithmic growth phase has ended, the culture suspension becomes Heated to SOC for a short time and then cooled to 180C. The pH is between 8.0 and 8.5.

2000 1 of the culture suspension thus obtained with a content of 20 g per 1 dry matter are in a tube centrifuge at 15,000 g with a throughput centrifuged at 400 l / h.

The moist bacterial cells separated in this way are then desalinated Water to a suspension with a content of 10 to 15 weight-0o 'dry matter Suspended and stirred vigorously in a suitable container at 15 to 180C. Accompanying substances and components of the nutrient solution are removed for about an hour washed out. After another centrifugation, a 20 up to 25 weight 5'ige bacterial suspension prepared, these under the same Conditions stirred and centrifuged again. The bacterial cells washed in this way are aufgeschweirinit to a 40 to 50% by weight suspension and spray drying subject.

This gives 37 kg of a light-colored, odorless cell product which is 73.7 % By weight of crude protein, 61.0% by weight of amino acids - 49.0 of which are 5 'essential by weight Amino acids - also contains 10.5% by weight nucleic acids.

Claims (9)

Patent claims: Process for the production of protein from bacteria with methanol as a carbon source and a nitrogen source under aerobic conditions a pH value between 4.0 and 9.0, characterized in that one bacterial strains of the genera Bacillus, Pseudomonas and Flavobacterium, their mutants or variants fermented so that the concentration of methanol in the phase of logarithmic Growth of the bacterial cells between 10 and 150 ppm, preferably between 50 - 100 ppm, based on the culture suspension. 2. The method according to claim 1, characterized in that the concentration of methanol is between 10 and 150 ppm. 3. Process according to Claims 1 and 2, characterized in that the culture suspension with 0.3 to 0.6 volumes of air per volume suspension per minute is ventilated. 4. Process according to Claims 1 to 3, characterized in that the culture suspension is stirred at 50 to 210 revolutions per minute. 5. Process according to Claims 1 to 4, characterized in that the fermentation is carried out in a kettle with at least 2 perforated disc stirrers contains, where the ratio of guide diameter to cup diameter is between 0.35 and 0.65 and the distance between 2 stirrers being one stirrer diameter amounts to. 6. Process according to Claims 1 to 5, characterized in that the fermentation is carried out in reactors that are both mechanical, hydrodynamic and can be circulated pneumatically. 7q method according to claims 1 to 6, characterized in that Pseudomonas species, the mutants or variants of which are used. 8. The method according to claims 1 to .6, characterized in that Bacillus species, the mutants or variants of which are used. 9. Process according to Claims 1 to 6, characterized in that Flavobacterium species, the mutants or variants of which are used.