DD219503A5 - PROCESS FOR THE PRODUCTION OF MICROBIAL PROTEIN - Google Patents

Abstract

The invention relates to a method for the production of microbial protein, in which the strain deposited in the Hungarian Hygiene Institute under the number 00196 Methylomonas nov. spec. No. 7-3 in the presence of methanol as the sole source of carbon and energy, in a nutrient solution containing inorganic salts, grown under aerobic conditions, the resulting egg white cell mass optionally subjected to a heat and / or alkali treatment and the cell mass is isolated from the fermentation broth.

Description

15 948 55

yerfahren for the production of microbial protein

Field of application is Srf indun *: · . · ^:;: · - ^; /.

The invention relates to a process for the production of microbial protein. Such protein, more precisely such protein-rich Zeilmassen, v / earth primarily in the animal feed, after appropriate cleaning but also used in human nutrition.

Characteristic of the known technical solutions:

The meaning of the with the. Demographic explosion of global protein deficiencies clearly indicates that protein is a substance of the utmost importance. Therefore, in recent years, numerous and extremely intensive research work has been carried out to develop and produce new sources of protein. Heue prospects were also opened up again for the production of Sinzellereiweiß by fermentation: on the one hand by the micro-. 'biological conversion of the cellulose base available and replenished in large quantities, on the one hand, and the use of unconventional substrates, on the other hand,

 .-Else

in Fora of non-agricultural hydrocarbons and hydrocarbon derivatives. - .- ..

On the basis of the investigations so far, it is already certain that the use of single celled whiteness based on hydrocarbons and coal derivatives for fodder purposes has no toxicological and nutritional limits, therefore its future is essential. depends solely on the economic viability of the technology and the general interest.

The economics of producing single celled white by fermentation are influenced by numerous factors: most important are the physiological and breeding characteristics of the finished microorganism, its intrinsic value, the nature of the carbon and energy source used, i. of the substrate, as well as the technical aspects of the large-scale fermentation process determined thereby, which relate primarily to oxygen supply, heat exchange and processing issues.

Of the substrates based on hydrocarbons and hydrocarbon derivatives, the use of methanol is most advantageous from the standpoint of fermentation. In contrast to methane and the liquid hydrocarbons, the fermentation on the base of methanol reduces the oxygen demand and the heat produced, and the methanol is also immiscible with water. Finally, it can be easily isolated from the product and made in great purity.

Yon the microorganisms are mold, yeast and the bactericidal en ι to iJutzung of methanol as the sole carbon source suitable. In mold and. Yeast, however, are the 'speedy

of the growth, the number of cells calculated on methanol, and the protein content of the product are significantly lower than those of the bacteria. ,

Several bacterial species are known which use methanol as a carbon source for single cell white production, eg, the strains Pseudononss sp "1" ¹ CC nr, '21439 and ATCC No. 21438, Corynebacterium sp. ATCC No. 21232 and ATCC • Ffr. 21236 (DE-PS 2 059 277), the strains Pseudomonas rosea (AT-PS 324 997), the strain Methylomonas methanolica NRRL B-5458 (GB-PS 1 420 264), the strain Methyl omonas espezii (GB PS 1 480 684), the strains Pseudomonas utilis PERM-P 1690 and PERM-P 1691 and Pseudomonas inaudita PERM-P ^: 1693, PEREI-P 1694 (DS-PS 2 402 217),

The disadvantage of using the above bacterial strains is that their generation time is long and the possible yield low, or that most of the bacterial strains are not obligate methylotrophic bacteria.

DE-PS 2 458 851 describes for the production of. Single cell white the obligate methanol-using strain Methylomonas sp. DSM 580 'The drawback of using this strain is that the yield of single cell white production is low (Y = 0.40-0.46) and a pale pink pigment is formed which, when used as a nutrient, produces the. Digestibility, weakens.

Pur. The production of single cell white on a methanol carbon source is the best used strain of the obligate methanol methylomonas probus PERM-? 3193 (DE-PS 2 708 112). Although the generation time of the above strain is short, the achievable yield is low (Y = 0.45-0.49).

Δ. -

Aim of ^: invention:. '. ''

The aim of the invention is to find an obligate methanol-using strain which produces on methanol in addition to extremely good growth characteristics * (short generation time), a product with high protein content in high yield, whereby the resulting cell mass can be isolated from the fermentation broth and the amino acid composition of the product complies with PAO regulations.

Explanation of the essence of the invention:

From natural sources, several methylotrophic bacterial strains were isolated, one of which corresponded to the above objective. The isolated bacterium is a previously unknown, not described, obligate methylotrophic bacterial strain, which was named Methylomonas nov. spec. I 7-3 at the Hungarian Hygiene Institute in the Hungarian Illegal Master Collection under the ITummer; 00196 was deposited. · ^:

The strain Methylomonas nov. spec. ITr. 7-3 is characterized by the following properties: · -.

IJorOholoijische properties

In a liquid MSIvI broth / Hethods in IIicrobiol, Vol. 3 / b7, with an Ivlethanolgehalt of 1 Yolumenpröaent, rod-shaped cells grown at a temperature of 30 ⁰ C for 24 hours with an average size of 0.5-1x1-2 -u are observed. The cells move with the help of a polar plagellum, usually alone, but often in twos, sometimes forming a chain with three interwoven ones. The strain is GRAM negative. Colonies developing on solid MSM-x soil had a smooth surface at the age of two days.

flat, her diameter was. 0.5- 1 inm, they were colorless, their edge ragged. The 3. to 5 'days old colonies' have a large colorless smooth yard ·' their diameter is about 2-2.5 now, their margin is' fjord-like ', the, middle of the colony is pale beige-brown, their. Surface bluish. "

Physiological and of w;., Cü5tua, ο ^ χΓβΓί ^ ΐΰθ properties

The isolated strain Methylomonas nov, spec. Ή ?. 7-3. grows only on methylotrophic substrates: very good on methanol, weak on dimethylamine and formaldehyde, methane is not suitable «. Other CC-containing substrates, such as various sugars, organic acids, amino acids, alcohols, hydrocarbons, were not. Growth observed. ,

Of the nitrogen sources, it grows best on ammonium salts; Nitrates, urea, casamino acid and yeast extract are used less.

The growth of the strain is not stimulated by growth factors (biotin, thiamine, yeast extract).

The optimum growth temperature is between 33 and 36 C, but even at 25 or 39 ⁰ C, growth can still be observed. The optimum pH for growth lies between 6.5 and 7.5, but also at pH Values of 5) 5 or * 9 the strain is still multiplying.

The maximum growth rate can be achieved at a metbanol concentration below 0.6% by volume.

The highest specific growth rate of the strain is based on experiments on the principle of

continuous chemostats / ¹ L = 0.691 h (Generamax

1 hour), while the best yield achieved was Y = 0.55 g cell / g methanol.

In a submerged culture, the color of the breed is yellowish-white, la oxygen limit greenish, in a liährlösung without carbon source starvation after a few hours it has a brownish ^tone:

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

Actually s Table 1 Effect on. diameter The wax the inhibitor active substance toffmenge tum: Zone .0 10 .0 penicillin .20 MJ-J 0 oxacillin 30: / Ug 0. methicillin 30 / Ug 0 chloramphenicol 30 / ^ug 0. oleandomycin 30 / Ug - ·. 2.5 cm streptomycin '30 / Ug · - 3 cm tetracycline .30 / Ug - 5 cm. oxytetracycline too / Ug 0 C hl tetracycline ¹ 5 / Ug - T cm neomycin 10 / Ug - 1 cm' Polymycin B 50 / Ug 0 ' erythromycin 30 / Ug - i, 3 cm Vancomycih 30 / Ug 0 Kanamycin. / Ug spiramycin 20 / Ug 0 novobiocin 20 0 ' ampicillin    ίο / Ug 0 Colistin, 30 / Ug. - 1, 5 cm Linpomycin 20 / Ug - 1 cm Nevigramon · 50 / Ug 0 gentamicin / Ug carbenicillin / Ug

0 growth is not affected - growth is inhibited ..

The biochemical! Properties of the 'tribe. Methylomonas' nov. spec. Ήτ ", 7-3 are characterized by the results of the following biochemical test:

Hexulose phosphate synthetase (HPS) activity: + hydroxypiruvate reductase (HPR) activity. liitratassimilation · -: '.. +

Reduction of nitrate to nitrite '. ^! - +

Indole production on MeOH. -

Starch hydrolysis. '-

HpS production. -:

Gelatin distilled. ·: '. -

Acetone production. +

Growth factor requirement,

Urease production. +

Catalase production. ' , +

Halotolerance _: .3 / 5

The invention blended bacterial strain described above belongs to C-about its morphological, growth, physiological or biochemical properties according to BERGEY'S MAWAL of DETERMILIATLYE BACTERIOLOGY, 8, edition (Buchana, Gibbons, Cov / an, Holt, Listpn, Murray '5Ti ν en, Ravim and Starmer; The Williams and Wilkins Gompany) on Methylomonas genus. According to the handbook of BERGEY, there are three populations of the genus Methylomonas genus: Methylomonas mechanica, Methylomonas methanooxidans and Methylomonas methanitrif.icans. The bacterium used in the invention differs from the above three methylomonas populations and also from the literature of the art. previously described, methanol-utilizing bacteria in their morphological properties, in the utilization of the carbon source, in the formation of pigment, in the formation of extracellular mucus, in the optimal value of the growth temperature, and in the kinetic character. of, growth * ^; The wrong one

 In terms of its generation time, the bacterium is almost identical to the known bacterial strains in terms of its ".! ethanol-based yield better than the previously described methanol-assimilating bacterial strains."

Processing of the fermentation broth and recovery of the mass can be accomplished by filtration, separation, flocculation or flotation, conveniently by a combination of these methods. It is particularly advantageous if the fermentation broth is subjected to an alkali and heat treatment before recovering the cell mass, and the nucleic acid content of the product drops from 14-15 % to about 9 % . This improves the digestibility. , -

Export information:. , '

The process according to the invention is illustrated by the following examples, without limiting the invention thereto,

The composition of the fermentation broth corresponded in each FAIL _1, the following salt concentration:

fmx "j ς λ" ς σ ·

v - ^^ / L ^ 2 4 - ⁷ p

CaCl ₂ · H ₂ O '0.1 g

LIgSO ₁ · 7HpO 0.1 g

KH ₂ PO ₄ '. , 4,7 g

NapHP0 _A · 12HpO. 13.0g and trace elements.,

The amount of the bacterium is in each case: · 2 volume percent (in an inoculum with a cell content of 4 g / l),

- 10 -

, : '' '- 10 -. '

Example 1 '. , Batch fermentation in a BIOPER fermentor

Conditions:. ':

•. ^: . /. , . ·

! Tut ζ content:. 6 liters ventilation rate: 0.5, vol. / Yol, min.

'.-. , /1. Volume air / 1. ' ^:

Volume of Perment broth per minute

 Number of revolutions: 700 n / min,

(Revolutions / minute)

.Bn given sermentor is at the. The oxygen transfer rate was measured by the conditions described above and measured by superfine oxide dehydration technique: 90 mmol Op / l-h / m. Initial methane concentration: 1 v. v. 7 ···. '...

Temperature: 30 ^° C ' _e -.

pH: It was kept between 6.7 and 7.2 by adding 10% HH.QH.

Results: _ ',

The 1 VpI.% B / Iethanol ^^ was consumed in 9 hours. The ferment broth was 15 minutes unterv / orfen at a pH value between 7 and _T 8 at a temperature between 45 and 65 ⁰ C to a heat treatment, then the pH value to 3 "5 is set. The excreted cell mass was decanted and sprayed on a dryer. 3? 69 g / l of cell dehydrated substance developed. The yield calculated on the total fermentation was 0.4659 g cell / g methanol. The shortest C-energy side measurable in the fermentation was 1.4 hours. ,

Example 2

Discontinuous fermentation in a so-called Tauohstrahlfermentor (jet type) '

Conditions:

Irätζinhalt:. , 130 liters:

Ventilation conditions: under the given parameters with

a SuIfidOxydationsmethode characterized by an oxygen transfer rate of 450 mmol O _p / l.fa

pH: maintained between 6.6 and 6.9 with Wd.OE. Temperature: between 29 and 35 ⁰ C

Initial methanol concentration: 6 g / l. The fermentation broth was as described in Example 1

processed. . ,

Results:. ,

The one-dose methanol was consumed in 3.5 hours. 381.4 g of cells were created. The yield calculated for the total fermentation was: Y = 0.48 g cell / g

Example 3 "

Continuous fermentation experiments

The continuous fermentation experiments were carried out in a BIOFER fermenter with a Mutz content of 6.1. The fermentation broth was processed as described in Example 1, ...

Ventilation ratios: 1 vol./vol., Min. Ventilation rate Number of revolutions: '' 900 revolutions / minute '' The composition of the nutrient solution corresponded to that given above, the ethanol concentration was 2 g / l. Results of continuous breeding trials carried out at different pH and temperature values:

3.1.1 pH. = 6.5; t. = 00 /% D = 0.537 h ""

= 0.633 h ^-1 ;

At this rate of dilution, the cell concentration was ^:. stationary state χ = 1.2 g / l, yield: Y = 0.55 g cell / g MeOH; , _:

3.1.2 D = 0.66 h

-1

At this rate of dilution, the cell concentration was in the steady state ''; Έ = 1.07 g / l. Yield: 0.52 g of cell / g of MeOH. '-

3.2.1 pH =

7; t = 30 0.51. h " ¹

yield

3.2.2 D = 0.56. H

-1

Yield:

3.3.1- · pH

  D

7-Ϊ

0.575 h

=. 33

yield

^C '/ ^u max = ^ü ' ^ * 'f.

At this rate of dilution

the cell concentration was steady state

x = 1.06 g / l ..

Y = 0.48 g cell / g MeGH,

At this dilution rate, the 'cell concentration in the steady state ^·; i = 0.25 g / l. , :. ',.

Y = 0.53 g cell / g MeCH '

³ CY / U _77121x = 0.691 hrs. " ¹ : At this dilution rate the cell concentration was steady state - -. ' χ = 0.96 g / l

 Y = 0.44 g cell / g MeOH,.

^: '- 13

3.3.2 D = 0.66 h

Yield:

At this rate of thinning

was the cell concentration in the

stationary condition

Ξ = 0.5.6 g / 1,

Y = 0.43 S cell / g MeQH,

Example '4

Comparison, the cell composition of the product prepared by the method of Example 2 with the cell composition of the products Pruteen IGI and Probion Hoechst.

% Amino acid in the product

ICI 3ΜΞ MAXIMUM lysine 4.6 5.0 4.9 methionine 1.8 -. 1.8 .. 2.1. cystine 0.5 0.9 threonine 3.5 3.6 3.5 tryptophan 1.0 not measured 1.6 Leuζin 5.3 5.8 5.6 Valine, 3.5 3.3 3.5 tyrosine 4 _} 2 5.0 phenylalanine 2.7 '3,4 3.2 histidine '· 1,3 1.8 1.4. arginine 3 * 5   4.4 2.5 alanine 5,1 5.9 5.4 aspartic acid 6.5 9.4 7.0 glutamic acid 7.7 9.3 6.8 Glycine, • 4 ₅ O .4,5 4.2 Proline · - '2,4 2.9 3.1 serine 2.3 .-. 4.2 2.7

Juki one aur e

62.2

12

69.6 60.0

, 9 not eat

Claims (3)

' ^s - 14 - -.' · ·. Sri "indungs ana Drue h .: .: · .T. Process for the production of microbial protein, characterized in that the strain deposited at the Hungarian Institute of Hygiene under the Hummer 00196. Methylomonas nov, spec. ITr. .7-3 in the presence of methanol as sole carbon and energy source, in an inorganic salts containing. Broth solution bred under aerobic conditions, the resulting protein cell mass, if desired, subjected to a heat and / or alkali treatment and the cell mass is isolated from the fermentation broth. 2. The method according to item 1, characterized in that the cultivation takes place at a temperature between 30 and 37 ⁰ C and the pH of the fermentation broth is maintained between 6 and 8. 3. The method according to item 1 and 2, characterized in that ₍ the processing of the fermentation broth and the recovery of the cell mass by filtering, separating, flocculation, flotation _K conveniently carried out by 'a combination of these methods and by a heat and alkali treatment ,