DE2631047C3 - Process for the production of biological proteins. Microorganisms of the species Prototheca - Google Patents

Description

The invention relates to a method for producing biological proteins by the action of Microorganisms of the Prototheca species that oxidize low molecular weight aliphatic alcohols.

The microorganisms used for the process according to the invention belong to the Prototheca group.

The invention also relates to protein products derived from microorganisms which are used as food additives for humans and animals can.

There are numerous (fermentation) processes in the literature indicated for the cultivation of microorganisms which supply proteinaceous substances. There are z. B. Method for growing yeasts on carbohydrates as contained in molasses, sulphite baths from paper mills and n-alkane fractions from light gasoline. Similar procedures are also available for Generation of biomass with the help of bacteria described.

During the progress of yeast and bacterial growth (fermentation) a considerable heat build-up occurs due to the oxidation of the substrate, which is all the more is stronger, the lower the degree of oxidation of the substrate itself.

The use of partially oxidized substrates reduces oxygen consumption and heat generation. Furthermore, the cooling costs are significantly reduced through the use of microorganisms, that grow at high temperatures.

Many methods of biomass formation may require extraction with organic solvents be to remove unwanted substrate residues or harmful compounds, such as high molecular weight To remove hydrocarbons or polycyclic hydrocarbons and the like.

Alcohols are substrates that are very well suited for the formation of bioproteins. They are water soluble, already partially oxidized and are produced on an industrial scale in high purity. Of the Aliphatic alcohols are primarily metha

nol and ethanol applied

Economic considerations make it necessary that the substrates for the formation of bioproteins are cheap.

Compared to waste products, the cost of ethanol is relatively high. It is therefore necessary that the substrate is fully utilized.

It is an object of the invention to breed strains of heterotrophic algae of the species Prototheca which

ίο Use ethanol as a substrate. When using using ethanol as a substrate avoids numerous disadvantages of other substrates. Of course there is ethanol Easily accessible and can be used as a nutrient. There are no toxicity problems as it is found in high purity is produced, and in addition, ethanol is volatile and may still be present Remnants can easily be removed while the cells are drying. Furthermore, there is no problem with the distribution of the substrate in the nutrient medium, as in the case of polysaccharides and hydrocarbons.

The strains according to the invention employed have the additional advantage that they at temperatures up to 40 ⁰ C, preferably at 33 to 37 ° C at a broad pH range (pH 1.0 to 8.0), preferably pH 3.0 to 5.0 grow, a fact that reduces the risk of bacterial infection during growth. Many microorganisms do not grow in substrates containing alcohol, others only grow when the concentrations are low. In contrast, the strains used according to the invention grow well even at high concentrations, and only concentrations above 4% inhibit growth.

The Prototheca strains according to the invention were isolated and selected from soil samples that were taken have been at Harcourt, Nigeria, and are designated by the numbers 855 A, 855 B and 855 C. The culture and physiological characteristics are summarized in Table I.

Table I.
Breeding and physiological characteristics

A. Breeding characteristics

Solid medium (24 h) (peptone glucose):
Colonies diameter: 2 to 3 mm,

brittle, coarse, opaque, without pigments
Liquid medium (24 h) (peptone glucose):

Sediment, thin film

B. Characteristics of the vegetation cycle

Formation of 2 to 8 aplanospores (endospores)

After breaking the mother cell, the aplanospores repeat the cycle and some form Hypnospores ("resting cells")

C. Characteristics of the vegetative cells

Spheroid or ellipsoid with a size of 15 to 25 χ 20 to 30 μm. Formation of aggregates. core and storage granules are clearly visible.

D. Gender

No sex cycle was observed.

E. Physiological property

1) Utilization of various carbon sources a. fermentative utilization (for growth)

Glucose Saue. *,

<^ s without gas,

slow

Galactose negative

Sucrose neeactive

Maltose negative Lactose negative Raffinose negative Melibiosis negative Inulin negative strength negative α-methyl-D-glucoside negative

b. assimilative utilization of carbon sources

D-glucose positive

D-galactose negative

Sucrose negative

Fructose positive

Maltose negative

Lactose negative

Raffinose negative

Melibiosis negative

Inulin negative

soluble starch negative

Λ-methyl-D-glucoside negative

L-sorbose negative

Salicin negative

Arbutin negative

Cellobiose negative

Trealose negative

Melicitis negative

D-xylose negative

D-arabinose negative

L-arabinose negative

D-ribose negative

L-rhamnose negative

Methanol negative

Ethanol positive

n-propanol positive

Isopropanol negative

n-butanol positive

tert-butanol negative

Cyclohexanol negative

Glycerin positive

Erythritol negative

Ribit negative

Dulcit negative

D-mannitol negative

Sorbitol negative

Inositol negative

DL-lactic acid negative

Succinic acid negative

Citric acid negative

η-paraffin positive

2) Utilization of various nitrogen sources ammonium, nitrate and urea

3) growth factors
requires thiamine

4) Growth at different temperatures 4 ° C negative to 42 ° C positive

54 ° C negative

Arnold and Ahearn (Mycologia, Vol. 64, p. 265, 1972) some time ago had a key to the Classification proposed by Prototheca after the strains according to the invention belong to the group of P. Zopfii species include.

However, the stems designated with the symbols 855 A, B and C differ from the (15th Standard description of P. Zopfii and of the strains present in the collections (such as of Prof. Liferri, Pavia, see Table II).

Table II 855 A P. Zopfii P. Zopfii
(Literature) negative
negative
positive positive
negative
negative positive
positive D-galactose
iso-pentanol
Growth at 42 ° C

The trunk 855 A (wild type) can be used as stable and defined variant of the species P. Zopfii. It becomes the name Prototheca Zopfii, Var. Harcourt, suggested in the standard description given in Table I. The trunk was at Centraalbureau voor Schimmelcultures Baarn and received the number CB 5 26 p. 75.

The cells of Prototheca Zopfii, var. Harcourt 855 A according to the invention for the formation of Bioproteins are grown in culture media that contain up to 4% ethanol as a carbon source, Contain nitrogen sources, mineral salts and growth factors. Thiamine can be used as such or in In the form of corn water or molasses or yeast extract added to pasture. The ethanol as a carbon source can be added all at once before fermentation (growth) or in small portions during fermentation will. However, it is preferred to use ethanol continuously while fermentation is in progress add to enable a more complete recovery.

The fermentation is carried out at temperatures in the range of 15 to 40 ⁰ C, preferably 30 to 38 ° C at a pH in the range of 1 to 8, preferably from 3.0 to 5.0.

A yield of 60 to 70% (g dry biomass / g Ethanol) obtained at a cell concentration in the culture of 20 to 60 g / l.

An exceptional advantage of this procedure is the use of heterotrophic algae of the species Prototheca. Because of their size, they are of course easy to win.

In the case of batch work, the cells are recovered by sedimentation or filtration complete fermentation. The same process is applied to the biomass at continuous Winning procedure.

The biomass obtained and dried in this way contains approximately 50 to 60% raw proteins along with polysaccharides, lipids and nucleic acids.

According to the invention it is possible to produce biomasses with high protein contents by breeding of heterotrophic algae of the species Prototheca in a simple culture medium, ethanol as the only one Contains carbon source.

The invention is explained in more detail by the following, non-limiting examples:

example 1

A culture medium of the following composition was prepared:

(NH ₄ I ₂ SO ₄ ■ H ₂ O 5 g / l NaH ₂ PO ₄ IO g / l KH ₂ PO ₄ H ₂ O 10 g / l MgSO ₄ · 7 H ₂ O 0.2 g / l FeSO ₄ 7 2 mg / 1

ZnSO ₄ - 7 H, O 2 mg / 1 CaCI ₂ 2 mg / 1 Yeast extract 500 mg / 1 Solution of traces elements
I ** JO I 1 ml Solution of the trace elements
CuSO ₄ · 5 H ₂ O nente besta
5 mg / 1 H ₃ BO ₃ 500 mg / 1 MnSO ₄ · H ₂ O 500 mg / 1 KJ 10 mg / 1 CaCi ₂ · 6 H ₂ O 10 mg / 1 MoO ₃ 10 mg /!

After the pH had been adjusted to 5.0, the medium was added in quantities of 100 ml each to 500 ml flasks and after sterilization at 116 ° C. for 30 minutes, 1% ethanol (volume / volume) was added. The flasks were then inoculated with the strain of a 3 day old culture (30 ⁰ C) 855 A of a slant containing the g'eiche medium and 2% agar, and then with stirring (orbital stirrer) at 220 rpm 30 ⁰ C incubated. After incubation for 24 hours, a further 2% ethanol was added.

The dry cell yield after 33 hours of incubation was 18.7 g / l.

Example 2

A fermentation was ^{carried out at 40 °} C., the other conditions of Example I not being changed. The dry cell yield after incubation for 48 hours was 13.5 g / l.

Example 3

A culture medium was prepared as in Example 1, but instead of yeast extract 1.5 g / l Corn water (C. S. L.) contained. The yield, as dry weight, after fermentation for 34 hours under the conditions given in Example 1 at 35 ° C. was 17.5 g / l.

ben, inoculated and incubated for 24 hours with stirring (800 rpm) at 35 ° C while introducing 0.5 vol./ Vol. Χ min. Air.

Two laboratory fermenters of the type described in Example 4, each containing 101 of the same culture medium and 1% ethanol, each with 1.5! inoculated in this preliminary course. The fermenters were using 1000 rpm and stirred at 0.5 vol / vol. χ min. ventilated. The fermentation temperature of one fermenter was 30 ° C, that of the other 35 ° C. To keep the pH on the To maintain the desired value (approximately 4) a mixture consisting of 32% aqueous Ammonia and 95% ethanol in a ratio of 20:80. By adding this mixture was not only the pH is kept at an optimal level for growth but also the carbon source automatically added.

After 20 hours of incubation the concentration betrüg of biomass 20.6 g / l (30 ⁰ C) and 21.3 g / l (35 ° C).

The agitation speed was then set to 1500 rpm increased and with 0.75 V0I./V0I. χ min ventilated. To For a further 25 hours, the biomass concentrations were 53.2 g / l (30 ° C.) and 58.1 g / l (35 ° C.), respectively.

With 2 l of the 35 ° C culture, a third fermenter of the same type, containing 10 l of the same culture medium inoculated and also at 35 ° C with stirring at 1500rpm and an air supply of 0.75 vol./ Incubated min by volume. After 8 hours of incubation, the biomass concentration increased from 10.3 g / l to 33.1 g / l.

Example 6

In a 16 l fermenter with a turbine stirrer and 4 (antisloshing) baffles and contained 7 l of medium, a culture of the strain 855 A produced according to example 4.

For the continuous fermentation, a nutrient medium (feed solution) was prepared in the following Composition:

Example 4

in small laboratory fermenters, which were provided with a turbine stirrer and 4 (antisloshing) baffle plates and had a volume of 16 l, 10 l of the medium according to Example 1 were added, with the only difference that the concentration of phosphate salts 2 g / l instead of 10 g / l. After 30 minutes, a long time sterilization at 116 ⁰ C the fermenter with 10% of a 21 hour old culture of the strain 855 A in a flask containing the same medium were inoculated as in Example 1, of 35 ° C. The cultivation was carried out at 35 ° C. with stirring at 1000 rpm and an aeration of 0.5 v / v. χ min. carried out. The pH was kept at 4.0 by adding soda throughout the fermentation period. Ethanol was added at the beginning (1% by volume) and after 24 hours (2% by volume).

After fermentation for 43 hours, the cells were filtered off on paper and, after washing with water, ^{dried at 90 °} C. in vacuo. The yield of dry cells was 50.5 g / l, containing 52.56% crude proteins (N χ 6.25) and 4.73% lipids.

At sp ie! 5

A laboratory fermenter with a volume of 20 l containing 121 of that used in Example 4 Culture medium, as indicated in this example

Na ^ HPO ₄ · H ₂ O 2.0 g / l KH ₂ PO ₄ 2.0 g / l (NH ₄ I ₂ SO ₄ 5.0 g / l MgSO ₄ · _{7H 2} O 0.2 g / l FeSO ₄ · _{7H 2} O 22 mg / 1 ZnSO. ■ 7 H ₂ O 20 mg / 1 CaCl: 20 mg / 1 Solution of traces elements 2 ml / 1 Yeast extract 0.5 g / l Ethanol (S ₀ ) 37.6 g / l

The medium (without ethanol) was adjusted to pH 4.0 and 30 minutes at 116 ° C sterilized.

With a dilution rate (D = μ) of 0.190 h- ', 2000 rpm and an aeration of 1.5 v / v χ min, the following results were obtained:

S = concentration of ethanol

in the culture 0.067 g / l
X = concentration of biomass

in the culture 25.12 g / l

Yiub = yield (g biomass / g substrate) 0.66

P = productivity 4.8 g / l h

The biomass was centrifuged off and after washing with water it was placed in a vacuum oven

dried. The composition of the biomass is given in Table III.

Table III

Analysis data of the biomass from Prototheca Zopfii, Var. Harcourt 855 A

Humidity 0.60%

Ashes 7.04%

N (Kjeldahl) 8.45%

Raw proteins (N χ 6.25) 52.62%

Protein (biuret) 45.70%

Raw lipids 4.82%

Fatty acids 3.08%

Soluble carbohydrates 11.96%

Raw fibers 2.50%

RNA 15.17%

DNS 0.42%

Elemental analysis:

C = 46.10%
H = 6.71%
N = 8.32%

Percentage composition of fatty acids:
nC _M 1.38%

n-C, 6 29.12%

n-Ci ": 1 *)

n-C, 8

n-Ci »: I.

nC _ls : 2

*) Double bonds

0.90% 4.44% 28.46% 35.70%

Percentage composition of amino acids (g / 100 g dry biomass)

Lysine 1.88 Histidine 0.779 Arginine 5.11 Aspartic acid 3.03 Threonine 1.73 Serine 1.56 Glutamic acid 4.85 Proline 3.13 Glycine 1.98 Alanine 2.26 Valine 2.11 Methionine 0.83 Isoleucine 1.12 Leucine 2.95 Tyrosine 1.32 Phenylalanine 1.55 Tryptophan 0.23

Claims (5)

Patent claims: 1. Process for the production of biological proteins by microorganisms of the species Prototheca, characterized in that they are grown in a culture medium, ethanol contains as the only source of carbon 2. The method according to claim 1, characterized in that it is grown in a culture medium are, in which ethanol is contained in an amount up to 4 wt .-% 3. The method according to claim 1 or 2, characterized in that the fermentation at a Temperature of 15 to 40 ° C, preferably 30 to 38 ° C, performs. 4. The method according to claim 3, characterized in that the fermentation at a pH between 1 and 8, preferably between 3 and 5. 5. Use of the microorganisms according to claim 1 or 2 for the production of bioproteins.