NO751734L - - Google Patents

Description

Procedure for fermentative

production of protein.

Biosynthetic methods are known, in which microorganisms, e.g. certain bacterial strains, are propagated fermentatively in a synthetic nutrient medium, which contains methanol as the only carbon source. Such methods can be carried out discontinuously as well as continuously. The bacterial cells produced in this way contain the so-called "crude protein"

(calculated by multiplying the elemental analytically found value for nitrogen by the empirical factor 6.25) usually 40 - 75% by weight of amino acids and 8 - 16% by weight of nucleic acids. However, such a high amount of nucleic acid is undesirable, as from them, especially in the desirable organism, uric acid is produced, which causes gout. In addition, the flora of the gastrointestinal tract is adversely affected.

A method has now been found for the production of bacteria with methanol as a carbon source and a nitrogen source under aerobic conditions at a pH value between 4.0 and 950,

as the method is characterized by fermenting bacterial strains of the genus Bacillus, Pseudonomas and Flavobacterium, its mutants and variants so that the concentration of methanol in the phase of logarithmic growth of the bacterial cells is between 5 and 200 ppm, referred to the culture suspension.

Preferably, a concentration of methanol is preferred, which lies between 10 and 150 ppm.

The process is conveniently carried out in fermentation reactors which contain a nutrient medium which, in addition to said methanol as a nitrogen source, contains salts such as potassium nitrate, ammonium sulphate, ammonium phosphate or ammonia, urea or soy flour. It also contains phosphates such as potassium dihydrogen phosphate or disodium hydrogen phosphate as magnesium and potassium salts and trace elements, as they e.g. is also contained in tap water. Thus, e.g. iron, copper, molybdenum salts present in traces.

It may be appropriate, especially for the pre-cultures, to add nutrients such as yeast extract, meat extract, liver extract, glucose or others.

The method is conveniently carried out at temperatures between 20 and 45°C, preferably between 30 and 37°C.

Bacillus types are used as bacterial strains, especially such known Bacillus strains as e.g. Bacillus subtilis or Bacillus polymyxa, further Pseudomonas types, such as Pseudomonas aeruginosa. Flavobacteria can also be used.

The culture suspensions in the reactor are aerated with 0.1

to 1.5 liters of air per liters of culture solutions and minute (vvm), preferably with 0.3 to 0.6 vvm. To ensure a good absorption of the oxygen by the cells, it can be stirred well, emulsifiers can also be added. In reactor systems other than stirring vessels, a sufficient amount of air must be provided accordingly, since in the case of volatility of methanol, a return of air is beneficial.

In normal systems, it has proven advantageous to use one or better also two or more stirrers in a fermentation vessel. Furthermore, it is advantageous to arrange two stirrers, especially perforated disc stirrers, in the fermentation vessel, so that the stirrer distance amounts to approximately one stirrer diameter. The ratio between stirrer diameter and boiler diameter is suitably between 0.35 and 0.65, preferably between 0.45 and 0.55. The circulation speed of the culture suspension is suitably more than 7 meters per second.

If foaming occurs, chemical or mechanical foam control is worthwhile, e.g. by adding 0.01 to 0.1% by weight of a defoamer such as octanol, esters of oleic acid and lauric acid with glycol, glycerol 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 controlled continuously by various precautions, e.g. by measuring the nitrogen consumption, the cell mass part or preferably by measuring the carbon dioxide emission. This makes it possible to finely dose the methanol addition with a rapid reaction to the decreasing carbon dioxide emission due to lack of carbon. Preferably, this control can take place over the measurement of gaseous methanol by means of a flame ionization detector.

When the culture suspension's pH value drops below

the prescribed value is brought by the addition of alkali, e.g. caustic soda or lye again to the prescribed value. Likewise, a too high pH value when adding acid, e.g. hydrochloric or sulfuric acid.

As a control part of the procedure, samples are taken from the culture suspension to determine the dry weight and nitrogen content of the bacterial cell mass. The growth rate and doubling time of the cell mass can then be determined. When the "dry weight" of the cells in successive sampling no longer increases logarithmically, the fermentation is terminated by batch methods. During continuous operation, the biomass production in the reactor is controlled over the degree of dilution.

The separation of the bacterial mass takes place in the usual way by centrifugation while washing several times with water. Suitable methods have also been found, as the cell mass can be concentrated by flocculation with acids at a pH value between 2 and 6. It may also be appropriate first of all to make the culture suspension alkaline, preferably at a pH value between 8 and 11, preferably between 8 and 9} then to heat for a short time at 60 to 95°C after cooling and acidify as stated above. Furthermore, de-flocking can be supported by the addition of iron or aluminum salts or de-flocking aids of natural origin, such as e.g. starch or glue or synthetic origin, such as polyacrylamides, polyacrylates, polyethyleneimines or polyethylene oxides. You thus get a paste-like bacterial cell mass which also contains 75 to 90% by weight of water. The drying can take place

in different ways, e.g. by roller drying, fluidized bed drying or spray drying. The thus dried product contains only 2-5% by weight of water and 60-80% by weight of crude protein. In this crude protein, the amount of amino acids is 90 - 95% by weight. Thereby, it is noteworthy that not only this part of amino acids in general, but especially the content of essential and semi-essential amino acids is higher than in comparable products according to the state of the art. Moreover, the raw ash content of the dried cell mass formed according to the invention is clearly lower than for known products. The crude protein obtained according to the invention further contains only 5 to 10% by weight of nucleic acids, while known products contain 8 to 16

or more wt% nucleic acid.

The dry bacterial masses obtained by the method according to the invention are therefore particularly suitable to serve as a source of egg white in foodstuffs and animal feed. Example 1.

The strain Pseudomonas sp. ATCC 31061 (FH-B-5163) maintained on compound agar slants

rinse with 4 ml of distilled water or physiological saline solution and inoculate sterilely into a 2 liter Erlenmeyer flask containing 250 ml of pre-culture nutrient solution of the following composition: 0.53$ (NH4)2S04

0.4% KH2POii

0.2% Na 2 HPO 1 . 12 H 2 O

0.02$ MgSO^. 7 H 2 O

0.02$ KC1

1.5% methanol and

0.1% trace elements

(pH 6.8).

The method according to the invention is carried out in a 14 liter fermentation vessel which is coated with 10 liters of the following nutrient solution: 1.0056 (NHi|)2S04

0.4% KH2P0I(

0.2% Na^HPO^. 12 H 2 O

''0,.02.JK MgSO^ . 7 H 2 O

0.02$ KC1

The nutrient solution's pH value is 6.8. It is sterilized for 45 minutes at 121°C. In addition, 50 g (0.5% by weight) of methanol is added to the nutrient solution before inoculation to the main culture.

For inoculation the nutrient solution is used

under sterile conditions 2 x 250 ml of the pre-culture, which was shaken for 24 - 48 hours at 30°C. The thus inoculated nutrient solution is stirred during batch production for 24 - 36 hours at 30°C under aeration with 0.6 vvm air with a turbine stirrer.

A pH-value correction takes place if necessary by adding 2 N sterile HC1. During the logarithmic phase of batch fermentation and during continuous operation, a concentration of methanol between 5 and 150 ppm by weight is observed. To this, when the CC^ emission is reduced by the cells, further amounts of methanol are added automatically until the CC^ emission increases again. This can also be done by measuring the gaseous methanol portion in the exhaust air with a flame ionization detector.

The progress of the process is checked by taking samples to determine the dry weight and nitrogen of the bacterial cell mass.

After the end of the logarithmic growth phase, processing is carried out in the usual way by centrifugation or flocculation, washing the cells with water and subsequent spray drying. The product thus formed has a crude protein content of 71.87% by weight, an amino acid content of 66.55% by weight and a nucleic acid portion 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 1.

Example-, 2.

The strain Pseudomonas aeruginosa ATCC 21996 (FH-N-845) works as in example 1. Furthermore, in a 14 liter fermentation vessel, 9 liters of nutrient solution of the composition specified in example 1 are added and 0.5% by weight methanol is added. The nutrient solution is inoculated as indicated in example 1 and stirred at 3°C under aeration with 0.6 vvm air with a turbine stirrer at 210 revolutions per minute. minute. The pH value is possibly maintained by adding sterile HC1 at pH 8.0. During the logarithmic phase of the fermentation, a methanol concentration between 50 and 100 ppm is observed. The regulation of this concentration, control of the method, as well as separation and processing of the formed cell mass takes place as indicated in example 1. After atomization and drying, a product is obtained with a crude protein content of 75.8% by weight, an amino acid content of 67.0 wt% and a nucleic acid content of 8.5 wt%

Example l 5.

The strain Flavobacterium sp. ATCC 31062 (FH-B-5108) is grown as in example 1 first in a 10 liter fermentation vessel. The culture suspension thus formed is then transferred into a

200 liter fermentation vessel, which is covered with 150 liters of the following nutrient solution: 1 , 0035 (NHl4)2S0lj

0.4% KH2POi|

0.2% K2HP0lj

0.2% Na2HPO3. 12 H 2 O

0.02% NaCl.

0.02$ MgSO^. 7 H 2 O

0.5% methanol.

The nutrient solution's pH value is set to 6.5 with semi-concentrated phosphoric acid. It is then sterilized at 120°C and 1.4 ato for 20 minutes. After sterilization, the pH is 6.7. Methanol is added separately.

The culture is then fermented for 24 to 36 hours

at 35°C under aeration of 1.1 vvm at a pressure of 0.3 ato under stirring with 2 stirrers at 250 revolutions per minute. During the logarithmic phase of the fermentation, a concentration of methanol between 50 and 120 ppm is observed, as further amounts of methanol are added when the carbon dioxide emission decreases, until the carbon dioxide emission increases again. The pH value is kept between 7.9 and 8.4 by the addition of sterile HC1. The progress of the procedure and the time of termination are determined as indicated in example 1.

The culture suspension thus formed is used as inoculation material for a 2000 liter fermentation vessel. The nutrient solution has the same composition and the same pH value as above for the last mentioned fermentation. The fermentation reactor is sterilized for 20 minutes at 120°C and 1.4 ato while stirring at 210 revolutions per minute. minute. After sterilisation, the pH value is 6.8.

The subsequent fermentation is carried out

at 35°C, aeration with 0.6 vvm, a pressure of 0.3 ato and 2 stirrers with 210 revolutions per minute.. The pH value is maintained during the fermentation with sterile HC1 between 7.9 and 8.4.

The progress of the process is monitored by taking samples to investigate sterility, to pH measurement to determine dry weight and the nitrogen of the bacterial cell mass.

The concentration of methanol is kept during the logarithmic growth phase between 10 and 150 ppm as indicated above applied according to the degree of carbon dioxide emission.

After completion of the logarithmic growth phase, the culture suspension is heated for a short time at 55°C and then cooled to 18°C. The pH value is between 8.0 and 8.5.

2000 liters of the thus formed culture suspension with a content of 20 g per liter of dry mass is centrifuged in a tube centrifuge at 15,000 g with a performance of 400 liters per hour. The thus separated moist bacterial cells are slurried in salted water to a suspension with a content of 10 to 15 wt/? dry mass and stir vigorously in a suitable container at 15 to 18°C. This washes out in the course of approx. one hour byproducts and constituents of the nutrient solution. After repeated centrifugation, a 20 to 25% by weight bacterial suspension is prepared with desalted water, this is stirred under the same conditions and centrifuged again. The thus washed bacterial cells are floated to a 40 to 50% by weight suspension and subjected to spray drying.

You thus get 37 kg of a bright, odorless cell product, which contains 7 3 3 7 weight/? crude protein, 6l.0 wt/» amino acids - of which 49.0 wt/? essential amino acids - further 10.5 weight/J nucleic acids.

Claims (10)

1. Process for the production of protein from bacteria with methanol as a carbon source and a nitrogen source under aerobic conditions at a pH value between 4.0 and 93 0, characterized by fermenting bacterial strains of the genera Bacillus, Pseudomonas and Flavobacterium, their mutants and variants so that the concentration of methanol in the logarithmic growth phase of the bacterial cells is between 10 and 150 ppm, preferably between 50 and 100 ppm, referred to the culture suspension. 2. Method according to claim 1, characterized in that the concentration of methanol is between 10 and 150 ppm. 3. Method according to claims 1 and 2, characterized in that the culture suspension is aerated with 0.3 to 0.6 volume of air per volume suspension and minute. 4. Method according to claims 1 to 3, characterized in that the culture suspension is stirred at 50 to 210 revolutions per minute. 5. Method according to claims 1 to 4, characterized in that the fermentation is carried out in a vessel containing at least 2 perforated disc stirrers, the ratio between stirrer diameter and boiler diameter being between 0.35 and 0.65 and the distance between two stirrers being one stirrer diameter. 6. Method according to claims 1 to 5, characterized in that the fermentation is carried out in reactors which can be rerolled both mechanically, hydrodynamically and pneumatically. 7. Method according to claims 1 to 6, characterized in that Pseudomonas species, their mutants or variants are used. 8. Method according to claims 1 to 6, characterized in that Bacillus species, their mutants and variants are used. 9. Method according to claims 1 to 6, characterized in that Flavobacterium species, their mutants and variants are used. 10. Method according to claims 1 and 2, characterized in that the methanol concentration is controlled by measuring the carbon dioxide emission.