JPS608114B2 - Methanol-based single cell protein production method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The increasing demand for protein for human and animal nutrition necessitates the development of processes for producing high value proteins.

Therefore, in recent years protein production from microorganisms that can utilize gaseous hydrocarbons such as methane or liquid hydrocarbons from petroleum distillates as carbon sources has been investigated. However, gaseous hydrocarbons have the disadvantage of low solubility in water and the formation of explosive gas mixtures under aerobic culture conditions. Liquid hydrocarbons also have the disadvantage of minimal solubility in water and the dispersion of both liquid phases requires high energy demands. In addition, cell aggregates grown in liquid hydrocarbons must be extracted at least once using an organic solvent. Methanol has been discussed for several years as a particularly advantageous carbon and energy source for the cultivation of large scale microorganisms.

This substrate is available in chemically defined, pure form and at low cost from synthesis gas, which has the advantage that it can be produced from a variety of raw materials, such as natural gas, petroleum distillates, coal or lignite. From DT-OS 2040358, the production of single-cell proteins using alcohols, aldehydes, ketones, carboxylic acids and their derivatives resulting from the oxidation of liquid alkanes as carbon sources is known.

From DT-AS2152039, Protamino
NezumiCterm戊r var. machi ship n melon
ATCC21611, ATCC21612, ATC
It is known to use bacterial species such as C21613 or ATCC21614 to obtain bacterial cell clusters in culture soot containing methanol. From DT-OS2311006, aerobic microorganism Me
thylomonas methanolicaNRRL
-Protein production methods for culturing B-5458 are known. From DT-OS2059277, a microbial protein production method using the following bacterial species under aerobic conditions in a suitable culture medium with methanol as the only carbon source is known.
Pseudonis methani
ca , Pseudomonas sp. ATCC21
438, Pseudomonas sp. ATCC214
39, Pseudomonas sp. PRL-W-4
, Cohinebacterium sp. ATC
C21232, Cohinebacterium s
p. ATCC21235 and Cohinebacte
riumsp. ATCC212360DT-OS240
7740, a microbial culture method using a mixed soot consisting of any methanol-utilizing bacteria and some non-methanol-utilizing bacteria is known.

Methanol-utilizing bacteria are not mobile and, in addition to methanol, can also utilize other carbon substrates as carbon sources, such as glucose or glycerin. DT-OS241838
From 5 onwards, microorganism Pseudomonase round orqu
A method for producing a protein-rich product is known using a strain derived from P. ens (NCIB No. 9399) without the addition of a pink pigment.

For the cultivation of proteins from methanol, according to the state of the art, methyl-uptake microorganisms are optionally used, rather than bacteria that indispensably utilize methanol.

This state of the art is surpassed by the method of the present invention which utilizes essentially methyl uptake bacteria for the cultivation of single cell proteins. Rhein in the city of Ludwigshafen
Bacteria that indispensably utilize methanol were isolated from riverbank soil samples.

Soil sample 1 evening is KH2P043.75 evening; Na2
HP042509; (N ratio) 2S044.00 evening; M Mori 04/7th 200.5 evening; Ca (N03) 2nd/4th 20
0.025 evening; FeS04/7 ratio 00.005 evening; Zn
An aqueous inorganic culture medium of pH 7.0 containing 1% (volume/volume) methanol as the sole carbon and energy source, dissolved in 100% distilled water. The mixture was turbidly placed in a 500° Erlenmeyer flask and incubated at 3000° C. on a shaker (100 times/min). After 5 days, 0.1 ml of each culture medium was applied to the petri dish.

The dishes contained the same medium containing 2% agar for fixation. After incubation in these dishes for an additional 3 days at 30 qo, individual colonies were probed from these and diluted and smeared in blood with the same medium to obtain a pure culture of the bacterial species with 5 liquid replicates.
This bacterial species is MeilomoMssp. and numbered DSM580 (see German Patent No. 245 Patent No. 51). This is a method for obtaining single-cell proteins using methanol as a substrate, with or without mechanical lighting means.Methyl bacteria that indispensably utilize methanol in a gas-supplied reactor.
omonassp. A viable submerged culture containing DSM580 is created under aerobic conditions, with methanol as the sole carbon and energy source, inorganic nutrients and optionally growth prime; Alternatively, oxygen-enriched air is introduced at a reaction temperature of 20% and 45%, after which the cell clumps are separated from the resulting three-phase system and dried, with a crude protein content of at least 60% by weight. A cell agglomerate with a nucleic acid content of 2-10% by weight, an ash content of 3-6% and a fat content of 3-8% by weight is obtained, and the liquid phase from which this is separated is recycled, in whole or in part, to the operating process. A device has been found that is characterized by the ability to Nutrients are compounds that, in addition to carbon and energy sources, contain anions and cations that are absorbed by microorganisms and necessary for their growth.

Growth elements are natural and synthetic compounds that are necessary for microorganisms but cannot be synthesized in sufficient quantities by the microorganisms themselves.

The method for obtaining single-cell proteins of the present invention is based on Methylo, a bacterium that has the following characteristics and that uses methanol indispensably.
moMssp. Producing DSM580: 1. Cell morphology: Small camphor-shaped in the range of 0.3-0.6 x 1.0-1.8 cells, movable with unipolar flagella 2. Colony morphology: transparent, white, round and smooth, about 1-2 sides in diameter after 2-3 days3. Bacterial species characteristics: Gram negative, dry cell aggregates are slightly pink 4. Physiology: Strong aerobic, positive for catalase, positive for methanol dehydrogenase, positive for hexose-phosphate dilutetase, negative for hydroxypyruvate redactase5. Growth characteristics: Minimum Optimum Maximum temperature (℃) 2
0 33-36 45PH 4.5
6.5-7.5 9.5 Methanol concentration% (V/V
) 0.5-1.55.0 bacterial Methylomo capes
sp. was deposited with the number DSM580 in the German Collection of Microorganisms in Japan.

Methylomonass sp. As can be seen from the table, DSM580 can grow on methanol as a carbon and energy source, whereas it does not grow on methane, methylamine, ethanol or glucose.

Methylomo female ssp. with various carbon sources in aqueous mineral culture medium under aerobic conditions. DSM580 growth substrate Growth 1) Substrate Growth methane
Brovanol (1) Methanol + Flobanol (2) Methylamine - Acetic acid Formaldehyde - Lactic acid Salt Formate Biruvate Ethanol Lacinate Metric acid Fructose Glucose Serine 1) Ten growth
No growth and Methylomonass sp. DSM580 has an initial methanol concentration of 0.5-5. % by volume, in particular 2-3% by volume, and in which case the methanol concentration can be controlled to 0.01-2. It has been found that the total methanol conversion rate can be maintained at 2% by volume of the insects when cultured at a constant feed volume%.

Also Mehilomo Misaki ssp. It has been found that DSM580 is obtained when working continuously under chemically or turbiditically static conditions with a flow rate of 0.1-0.5 vol/vol/hr.

In addition, during growth, by adding alkali or acid, 4
．． It has been found that substantially constant pH values can be adjusted between 5 and 9.0, preferably between 6.5 and 7.5.

Furthermore, Methylomonass sp. It has been found that culturing of DSM580 is carried out at temperatures between 20°C and 45°C, preferably between 33°C and 36°C. Also, during culture, air or oxygen-enriched air is supplied to the reactor at 0.00%.
5-1. The oxygen content of the gas mixture introduced was found to be between 20 and 6% by volume, delivered with an air flow rate of &volume/volume/min.

In addition, the nutrient aqueous solution contains ammonium salts and/or nitrates and/or urea as a nitrogen source and cations necessary for growth, such as sodium, potassium, magnesium, calcium,
It was found to contain iron, zinc, manganese and anions, phosphates, sulfates, nitrates, chlorides and trophins.

The method of the present invention is illustrated in detail by the following example: Example 1 (NH4)2S04200 Evening, KH2P041
50 Yu, Na2HP04125 Yu, MgS04
・7th 2025 Evening, Ca (N03) 2, Cape 201.
25 evening, FeS04 17th 200.25 evening, ZnS04
・Sunday 200.25 evening, Kclo. Sterilize an 80 microorganism reactor filled with 50% of a nutrient solution of 25% water dissolved in 50% water for 10 minutes at 120°C, cool to 3500°C, add 1000% methanol under sterile conditions,
ship s sp. The DSM spot 0 inoculum is inoculated with 500' and suspended for 28 hours at 350°C using a turbo machine (300 times/min) and incubated at an air flow rate of 0.7 vol/vol/min.

During cultivation, a constant pH value of 7.0 is maintained by automatically adding 6% by volume ammonia solution to the submerged culture. After 22 hours, the reactor is cooled to 1500 ℃, the pH value is adjusted to 3.0 by adding sulfuric acid, and the sedimented cell agglomerates are separated from the furnace, washed with water and dried (dried tethered cell agglomerates 324 hours). .

Cell composition: crude protein 71%, nucleic acid 9%, ash 4%, fat 7%
% (based on dry cell mass). Example 2 Biologische Vehahren, Basel
Nutrient salt solution 200 μm (composition: (
NH4)2S04500 evening, NH4N03500
Evening, KH2P04600 Evening, Na2HP045
00 Evening, MgS047th 20140 Evening, C
a (N08) 24th 2015 evening, FeS04/7th 20th
6th night, charge Kc12th night (dissolved with 200% tap water),
Adjust the pH value to 6.8 and sterilize at 121 °C for 15 minutes.
Cool to 0.5 ml, add 2,000 ml of methanol under sterile conditions, and
ethylomonass sp. A two-time preparatory culture of DSM 580 is inoculated with 4000 ml and incubated at 3300 ml with an air flow rate of 0.5 vol/vol/min and a rotational speed of 180 sub/min.

During growth, the methanol concentration in the submerged culture is automatically determined, and the gas phase concentration of methanol in the exhaust gas corresponding to the liquid phase concentration is continuously determined by a flame ionization detector, and the necessary methanol addition is performed via a limit value transmitter. By operating , it is always 0. Maintain the customer service volume%, and use a pH controller to add ammonia to 12%.
The pH value is automatically maintained at 6.81 by adding volume % solution. After reaching the cell concentration of a dry cell mass of 14 evenings already in the second period, oxygen-enriched air with an oxygen content of 4% by volume is introduced while keeping the air flow constant.

After 60 hours, it is cooled to 15oC, and the resulting cell clumps are separated and dried in a 1000 eaves continuous centrifuge.

Under these operating conditions, the cell yield is approximately 0.000 dry cell clumps per night of methanol. The composition was 76% crude protein, 6.5% nucleic acid, 5% ash, and 4.5% fat based on the dry cell mass.
It is. Example 3 Same as Example 2, “ln to nso”
Cultivation of Cape Methylomo ssp.
00 fence inoculated, 35oo, air flow rate 0. Cultivation is carried out in static conditions at a constant pH value of 7.0 at a receiving volume/volume/min and a rotational speed of 1200 revolutions/min.

Continuous culture was started at a flow rate of 0.05 at 1 tidal hour, and after another 48 hours the flow rate was increased to 0.1, then stepwise to 12
Increase to 0.35 within 0 hours. Under these conditions, an equilibrium is maintained with a cell yield coefficient of 0.46 (dry cell agglomerate/methanol) and a productivity of 10.8/h/h. The culture solution generated is 5% by volume after reaching an equilibrium state.
of the nutrient solution is returned to the reactor and the feed of the nutrient solution is correspondingly reduced. The composition of the cells is 72% crude protein, 4.5% nucleic acid, 3.5% ash and 5.5% fat based on the dry cell mass.

Claims (1)

[Claims] 1. In a method for obtaining single-cell proteins using methanol as a substrate, the bacterium Methylomonas sp., which indispensably utilizes methanol, is present in a gas feed reactor with or without a mechanical stirring device. A vegetative submerged culture of DSM580 was created in which the only carbon and energy sources were methanol, inorganic nutrients and optionally growth factors, and in addition to the system, air or oxygen-enriched air was added at a reaction temperature of 20°C. After delivery at -45°C, the cell clumps are separated from the resulting three-phase system and dried, with a crude protein content of at least 60-76% by weight and a nucleic acid content of 2-17% by weight.
, a cell agglomerate with an ash content of 3-6% by weight and a fat content of 3-8% by weight (based on the dry cell agglomerate) is obtained, from which the liquid phase is separated, in whole or in part, as the case may be, during the reaction process. A method characterized by recycling. 2 Methylomonas sp. Process according to claim 1, characterized in that DSM 580 is obtained in a batchwise operation with an initial concentration of methanol of 0.5-50% by volume, in particular 2-3% by volume. 3 Methylomonas sp. Claim 1, characterized in that DSM580 is cultivated to a total conversion of 25% by weight of methanol when controlled in a batch operation to maintain a constant methanol concentration of 0.01-2.0% by volume. the method of. 4 Methylomonas sp. Process according to claim 1, characterized in that the DSM 580 is obtained under chemically or turbidly static conditions with a throughput of 0.1-0.5 vol/vol/h in continuous operation. 5.4.5-9 by adding alkali or acid during growth.
．． 5. A method according to claims 1 to 4, characterized in that the pH value is adjusted to a substantially constant pH value of 0. 6 Methylomonas sp. 6. The method according to claims 1 to 5, wherein the cultivation of DSM580 is carried out at a temperature of 20 to 45°C. 7 During cultivation, air or oxygen-enriched air is introduced into the reactor at an air flow rate of 0.5-1.5 vol/vol/min, and the oxygen content of the introduced gas mixture is 20-60 vol%. The method according to any one of claims 1 to 6, characterized in that: 8 During cultivation, air or oxygen-enriched air is fed into the reactor at an air flow rate of 0.1-0.2 volume/volume/min, and the oxygen content of the introduced gas mixture is 20-60% by volume. The method according to any one of claims 1 to 7, characterized in that: 9. The nutrient aqueous solution contains ammonium salts and/or nitrates and/or urea as nitrogen sources and the cations necessary for growth, sodium, potassium, magnesium, calcium, iron, zinc, manganese and anions, phosphates, sulfates,
The method according to any one of claims 1 to 8, characterized in that the method contains nitrate, chloride, and a growth element.