CS241075B2 - Method of microbial protein preparation - Google Patents

Abstract

The strain is Methylomonas nov. spec. No. 7-3 which has been deposited in the collection of strains at the Hungarian Hygiene Institute under No. 00196 and at the DSM, Göttingen, under No. DSM 2937, and the process for preparing microbial protein entails cultivation of this strain in the presence of methanol as sole source of carbon and energy in a nutrient solution containing inorganic salts under aerobic conditions, where appropriate treating the resulting biomass with heat and/or alkali, and isolating the biomass from the fermentation broth. The Methylomonas strain provides high yields of cells with a short generation time.

Description

The invention relates to a process for the production of a microbial protein in a nutrient medium comprising methanol and inorganic salts to obtain a protein-rich cell mass. Protein-rich cell mass is mainly used in animal feeding, but it can also be used for human nutrition after adequate cleaning, which is of great importance.

The problems associated with worldwide protein deficiency in the context of demographic explosion and their dependence clearly indicate that protein is a substance of strategic importance. That is why research has been carried out in an extremely broad and intensive way in recent years to identify new protein sources and to produce them. New perspectives have also been found again for the production of unicellular proteins by fermentation: on the one hand, by microbiological conversion of cellulosic substances, which are available in large quantities and are constantly renewed, on the other hand by using unconventional substrates, ie hydrocarbons and derivatives production.

Based on experimental experience, it is now clear that the use of unicellular hydrocarbon-based proteins and their derivatives for feeding purposes is by no means restricted by toxicological limits or other requirements. . that their future depends entirely on the economy of technology and overall interest.

The economy of the production of unicellular proteins by fermentation is influenced by many factors: the most important are the physiological properties of the micro-organism used, its cultivation properties, its nutritional value, the type of carbon and energy used, ie the substrate. This concerns primarily oxygen measures, heat exchange and processing issues.

Of the hydrocarbon-based substrates and one hydrocarbon derivative, methanol is the most preferred for fermentation. Compared to methanol and liquid hydrocarbons, the oxygen consumption as well as the heat generated are lower when fermented using methanol; furthermore, methanol is mixed indefinitely with water, can be easily separated from the product and, last but not least, can be produced with high purity.

Of the microorganisms, fungi, yeasts and bacteria are suitable using methanol as the sole carbon source. However, in fungi and yeast, the growth rate, the number of cells relative to methanol, and the protein content of the product are substantially lower than those of bacteria.

Several types of bacteria are known to be used to produce unicellular proteins using methanol as a carbon source, including strains of Pseudomonas sp. ATCC No. 21439 and ATCC No. 21438, Corynebacterlum sp. ATCC No. 21232 and ATCC No. 21236 (German Patent No. 2,059,277), Pseudomonas rosea strains (Austrian Patent No.

324 997), strain Methylomonas methanolica

NRRL B-5458 (British Pat

420 264), Methylomonas espexii (British Patent Specification 1,480,684), Pseudomonas utilis strains FERM-P 1690 and FERM-P 1691, as well as Pseudomonas inaudita FERM-P 1693, FERM-P 1694 (German Patent No.

402 217).

The disadvantages of using the above mentioned strains of bacteria are that their generation time is long and the possible yield is low, or. most strains of bacteria are not obligatory methylotropic bacteria.

German Patent Specification No. 2,458,851 discloses a strain of Methylomonas sp. For the production of unicellular proteins. DSM 580 utilizing the necessary methanol. The disadvantage of using this strain is that the yield of unicellular proteins is low (Y = 0.40 to 0.46), as well as the formation of a slightly pink pigment, which weakens digestibility when used as a nutrient.

For the production of unicellular protein from methanol as a carbon source, the strain Methylomonas probus FERM-P 3193 (German Patent No. 2 708 112) has so far proved to be the best. The generation time of the above-mentioned strain is short, but the yield obtained is small (Y = 0.45 to 0.49).

Efforts have been made to use a strain that utilizes methanol and, in addition to having exceptionally good growth properties (short generation time), produces a high protein product and in large yield, further, the resulting cell mass can easily be isolated from the fermentation broth. and the amino acid composition of the product complies with FAO regulations.

Several methylotropic bacterial strains were isolated from natural sources, one of which corresponds to the above-mentioned target. The isolated bacterium represents a hitherto unknown, unwritten obligatory methylotropic bacterial strain which is designated Methylomonas nov. spec. No. 7 - 3 and is deposited in Hungarian. National collection of tribes. at the Hungarian Institute of Hygiene under number '00196.

Thus, according to the invention, the method for producing the microbial protein is carried out by cultivating a strain of Methylomonas. The cultivation is carried out in the presence of methanol as the sole source of carbon and energy, in a nutrient solution containing inorganic salts under aerobic conditions.

The principle of the method according to the invention consists in using the strain Methylomonas nov. spec. No. 7-3, which is deposited with the Hungarian Institute of Hygiene under number 00196. The cultivation is carried out at a temperature between 30 and 37 ° C and the pH of the fermentation broth is maintained between 6 and 8.

Tribe Methylomonas nov. spec. No. 7 - 3 has the following characteristics:

Morphological properties

In a liquid MSM nutrient solution (Methods in Microbiol. Vol. 3 / b) containing 1 percent by volume of methanol, rod cells with an average size of 0.5 - 1 X 1 - 2 can be observed for 24 hours at 30 ^° C. μ. Cells move by means of a polar flagellum, usually alone, but often also in two. Often three strands form a tangled string. The strain is gram-negative. Colonies developing on solid MSM broth had a smooth surface at two days of age, 0.5 to 1 mm in diameter, colorless and serrated. Three to five days old colonies have a large colorless smooth backyard, its diameter is about 2 to 2.5 mm, the edge is "fjordov", the center of the colony is slightly brownish. their surface is bluish.

Physiological and growth-affecting properties

Methylomonas nov. spec, no. 7 - 3 grows only on methylotropic substrates: very well on methanol, dimethylamine and formaldehyde only slightly, methane is not utilized. No growth was observed on other substrates containing a carbon-carbon bond such as various sugars, organic acids, amino acids, alcohols or hydrocarbons.

From nitrogen sources this strain nt • grows on ammonium salts; it grows weaker on nitrates, m <b> b, casamino acid and yeast 1 tract.

Strain growth is not stimulated by growth carriers (biotin, thiamine or yeast extract).

The optimum temperature is between 33 and 36 ° C and multiplication can also be observed at 25 or 39 ° C. PH optimum for growth j

6.5 to 7.5, but also at values. pH 5.5, resi 9 still multiplies the strain.

The maximum propagation rate can be achieved when the methanol concentration is below 0.6 percent by volume.

The highest specific growth rate of the strain is according to experiments carried out on the principle of continuous chemostat, ... - 0.691 · ¹ (generation time about 1 hour), on the other hand, the best yield obtained was Y = 0.55 g cells / g methanol.

In eubrrrrr culture, the base color of the cultivated product is yellow-white, greenish at the oxygen limit, and in a nutrient-free nutrient solution, after a few hours of starvation, it has a gray color.

The effect of the strain on the growth of various antibiotics is shown in the following table (Table 1).

Table 1

Active substance Amount of active substance Effect on growth Brake zone diameter cm Penicillin 3 NO ’0 Oxacillin 10, ug 0 Methilcilin 20 μξ, 0 Chloramphenicol 30 ^ g 0 Oleandomycin 30 ^ g 0 Streptomycin 30 μ% 0 Tetracycline 30 µg - 2.5 Oxytetracycline 30 ig - 3 Chíortetracycline 30 μ§ - 5 Neomycin 100 gg - Polymycin B 15 j «g - 1 Erythromycin 10 μ§ - 1 Vancomycin 50 0 Kanamycin 30 fig - 1.3 Spiramycin 30 0 Novobiocin Ampicillin 20 μ% 0 Kolistin 20 μ% 0 Linkomycin 10 µg 0 Nevígramon 30; - 1.5 Gentamycin 20 μ§ - 1 Carbenicillin 50 µg 0

growth is not affected - growth is inhibited

Biochemical properties of strain Methylomonas nov. spec. No. 7 - 3 are characterized by the results of the following biochemical tests:

Hexulosephosphate synthetase (HPSj, activity + HydroxypirLivat reductase (HPR), activity - Nitrate assimilation +

Reduction of nitrate to nitrite +

Indole production on MeOH—

Starch hydrolysis—

Production HZS—

Gelatin melting—

Acetone + production

The need for growth factors—

Production of urease +

Production of catalase +

Halotolerance 3%

The aforementioned bacterial strain which forms the subject of the present invention is based on its morphological, mild, physiological, and / or biochemical properties according to Bergey's Manual of Determinative Bacteriology, 8th Edition (published by Buchana, Gibbons, Cowan, Holt, Liston). Murray, Niven, Ravim, and Starmer; The Williams and Wilkins Gompany), to the genus Methylomonas. According to Bergeye's handbook, three populations belong to the genus Methylomonas: Methylomonas mechanica, Methylomonas' methanooxidans and Methylomonas methanitrificans. The bacteria forming the subject of the present invention differ from the above three populations of Methylomonas as well as the bacteria described to date in the literature that utilize methanol in morphological properties, in the use of carbon sources, pigment formation, extracellular mucus formation, at optimal growth temperature and in the kinetic characteristics of growth. The bacterium according to the invention is, in terms of its generation time, almost identical to the previously described strains of bacteria which assimilate methanol, but its yield based on methanol is better than the strains described so far.

Treatment of the fermentation broth and recovery of the cellulosic mass can be accomplished by filtration, separation, flocculation, flotation, preferably a combination thereof. Particularly preferred is a process wherein the fermentation solution is subjected to an alkaline and heat treatment prior to obtaining a cellulosic mass therefrom, thereby reducing the nucleic acid content of the product from 14-15% to about 9%. This improves digestibility.

The process of the present invention is illustrated by the following examples, which are not intended to limit the invention in any way.

The composition of the fermentation broth in each case corresponded to the following salt concentration:

(NH4) 2SO4 5 _g

CaC12. H2O 0.1 g

MgSOu. 7H2O OA ₈

KH2PO4 4.7 g

NazHPOd. 12H2O 13.0 g and trace elements.

The amount of bacteria shall in any case be:

percent by volume (in an inoculum containing 4 g / l cells).

I · &>.

Example 1

Discontinuous fermentation in biofermentor

Conditions:

Useful content Ventilation intensity

RPM 'liters

0.5 v / v min.

(1 volume of air per volume of fermentation solution per minute)

700

Under conditions determined from the above parameters and measured by the sulfite oxidation technique, the oxygen transfer rate in said fermenter is 90 mmol Oaai.h. The initial methanol concentration is 1% by volume.

Temperature: 30 ° C.

The pH was maintained between 6.7 and 7.2 by the addition of 10% NH 3 OH.

Results:

1% methanol by volume was consumed in 9 hours. The fermentation solution was heated at a pH between 7 and 8 for 15 minutes at a temperature between 45 and 65 ° C, after which the pH was adjusted to 3.5. The secreted cell mass was decanted, sprayed in a dryer and dried. 3.69 g / l of cell dry matter was formed. The yield calculated on total fermentation was 0.4659 g cells / g methanol. The shortest measurable generation time in fermentation was 1.4 hours.

Example 2

Discontinuous fermentation in so - called submerged - flow fermenter (Jet - type

Conditions:

Useful 130 liters

Ventilation ratios: under given parameters in the method of sulphide oxidation they are characterized by an oxygen transfer rate of 45 mmol Oz / l.h

PH: maintained between 6.6 and 6.9 with NH 4 OH

Temperature: between 29 and 35 ° C

Initial methanol concentration: 6 g / l

Y - 0.52 g cells / g MeOH.

The fermentation solution was treated as described in Example 1.

Results:

Methanol added in one portion was consumed after 3.5 hours. 381.4 g of cells were produced. The yield calculated for the total fermentation was: Y - 0.48 g cells / g MeOH.

Example 3

Continuous fermentation experiments

Continuous fermentation experiments were carried out in a Biofer fermenter with a useful content of 6 liters. The fermentation solution was treated as in Example 1.

Ventilation conditions: 1 vol / vol min. ventilation doses

Speed: 900 rpm

The composition of the nutrient solution corresponds to the above, the methanol concentration was 2 g / l.

Results of continuous cultivation experiments performed at different pH and temperature values:

3.1.1 pH = 6.5; mp = 30 ° C;

= 0.683 h ^_1

D-0.587 h-f

At this dilution rate, the cell concentration in the stationary state was x = 1.2 g / l

Yield value:

Y = 0.55 g cells / g MeOH;

3.1.2

D = 0.66 h ^-1

At this dilution rate, the cell concentration in the stationary state was x - 1.07 g / l, the yield was

PH = 7;

mp = 30 ° C;

0.565 h ^-1 ;

D = 0.51 h ¹

At this dilution rate, the cell concentration in the stationary state was x - 1.06 g / l, yield

Y = 0.49 g cells / g MeOH.

3.2.2

O = 0.56 h ¹

At this dilution rate, the cell concentration in the stationary state x = 0.25 g / l, yield

Y - 0.53 g cells / g MeOH.

3.3.1 pH = 7;

t = 33 ° C;

(Uiain = 0.691 ^h-1;

D = 0.575 1V ¹

At this dilution rate, the cell concentration in the stationary state was x - 0.96 g / l, yield

Y = 0.44 g cells / g MeOH.

3.3.2

D = 0.66 h ^-1

At this dilution rate, the cell concentration in the stationary state was n = 0.56 g / l, yield

Y = 0.43 g cells / g MeOH.

Example 4

Comparison of the cell composition of the product of Example 2 with that of ICI Pruteen and Hoechst Probion.

Lysine Methionine Cystine Threonine Tryptophan Leuzine Valine Tyrosine Phenylalanine Histidine

ICI

4.6

1,8

0.5

3.5

1.0

5.3

3.5

4.2

2.7

1.3% of amino acids in the product

BME HOECHST 5.0 4.9 1,8 2.1 0.9 - 3.6 3.5 not measured 1.6 5.8 5.6 3.3 3.5 5.0 - 3.4 3.2 1,8 1.4

% amino acids in the product

ICI BME HOECHST Arginine 3.5 4.4 2.5 Alanin 5.1 5.9 5.4 Asparagic acid 6.5 9.4 7.0 Glutamic acid 7.7 9.3 6.8 Glycine 4.0 2.9 4.2 Proline 2.4 4,5 3.1 Serin 2.3 4.2 2.7 62.2 69.0 60.0 Nucleic acids 12 9 not measured

The object of the invention

Claims (1)

The object of the invention A process for producing a microbial protein by culturing a Methylomonas strain in the presence of methanol as the sole carbon and energy source in a nutrient solution containing inorganic salts under aerobic conditions, characterized in that the strain is not Methylomonas nov. spec. No. 7-3, deposited with the Hungarian Institute of Hygiene under No. 00196, cultivation is carried out at a temperature between 30 and 37 ° C and the pH of the fermentation broth is maintained between 6 and 8.