GB1578200A - Production of proteinaceous material and microorganism useful in that production - Google Patents

Abstract

The new microbial strain of the Candida genus was registered on 6 January 1977 at the Northern Regional Research Laboratory of Peoria, Illinois, USA under registration number NRRL-Y 11062. This strain is cultured to produce biomasses with a high protein content. The reaction is carried out on a growth medium comprising a saline solution containing as a source of energy and of carbon exclusively methanol.

Description

(54) PRODUCTION OF PROTEINACEOUS MATERIAL, AND NOVEL MICROORGANISM USEFUL IN THAT PRODUCTION (71) We, SNAMPROGETTI S.p.A., an Italian company, of Corso Venezia, 16, Milan, Italy, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- This invention relates to the production of proteinaceous material (which can be regarded as a microbial bio-mass) by culturing a novel microorganism capable of exploiting methanol as a sole source of carbon and energy, and to the microorganism itself.

On account of the high velocity of reproduction of many microorganisms and of their proteinic content, the production of proteinaceous material is a very quick protein-producing method.

For such production, there have been exploited in the past scrap carbohydrates such as molasses from sugar works or spent sulphite-cooking liquors from paper mills.

In more recent times, on the basis of the considerable availability and the low price of petroleum, the production of proteinaceous material has been based on the use as substrate of raw fractions of petroleum or highly purified mixtures of normal paraffins.

The use of such petroleum-based substrates involves a few shortcomings from a technological standpoint, which are attributable to their insolubility in water, the considerable amount of oxygen required for their assimilation by the microorganisms and the large build-up of heat during fermentation. In addition, the running costs in the production of the proteinaceous material are increased by the need to purify the substrate thoroughly and/or carefully wash the resulting proteinaceous material in order to remove potentially harmful petroleum components.

Such difficulties are not experienced if the production of proteinaceous material is carried out using the lower alcohols such as methanol or ethanol as the substrates. As a matter of fact, their complete solubility in water, their volatility and their availability in a high degree of purity make it possible to obtain a proteinaceous material which is substantially exempt from undesirable residues.

Their miscibility with water offsets the mixing problems which are encountered with the petroleum fractions; furthermore, on account of the fact that they already contain oxygen in their molecules, the oxygen demand for their assimilation is thereby reduced: for this reason, there is a further advantage, namely that the production of the proteinaceous material is accompanied by a reduced heat buildup, the result being a reduction of the cooling costs.

Ethanol can be utilized by a large number of micro-organisms and would be the ideal substrate for the production of proteinaceous materials, but its price is comparatively high. In contrast, methanol can be produced cheaply and at a high degree of purity. This explains the endeavours which have been made in order to find microorganisms which are capable of efficiently exploiting methanol. The technical and patent literature discloses many bacteria which fulfil these requirements, whereas the efficiency of known yeasts is rather low. By enriching a culturing medium and isolation in continuous culture, we have succeeded in isolating a novel yeast strain (which is herein referred to as SP M 180 cc) which is one aspect of the present invention and is capable of exploiting methanol as a sole source of carbon and energy in the production of proteinaceous material.

The novelty of this strain will become apparent from the following description of its characteristics.

The strain SP M 180 cc is reproduced by multipolar germination and forms discrete ovoidal cells, or tufts made up by a number of elongate cells (pseudomycelium).

In a liquid culturing medium it forms a sediment, whereas in a solid medium there are either smooth and glossy colonies, or opaque and wrinkled colonies. By subculturing the strain on a solid medium, the strain takes on more and more a smooth and glossy appearance, which corresponds, in a liquid medium, to the discrete cells, whereas, by culturing the strain in a liquid medium under certain conditions, the pseudomyceliar form predominates, which corresponds to the wrinkled and opaque cells. No spores, of any type, have ever been observed. On the basis of such morphological characteristics, it is thought that the strain belongs to the genus Candida, according to the classification suggested in "The Yeasts: a taxonomic Study" edited by J. Lodder, 1970.

The physiological characteristics of the strain SP M 180 cc are the following: 1. Fermentative Utilization of a Few Carbon Sources: D-glucose + D-galactose Saccharose Maltose Trehalose Lactose 2. Growth D-glucose + D-galactose l-sorbose Saccharose Maltose Cellobiose + Trehalose Lactose Melibiose Raffinose Melezitose Inulin Starch D-xylose + I-Arabinose + D-Arabinose D-Ribose + (weak) 1-Rhamnose Ethanol + Glycerol Erythritol Ribitol + Galactitol D-Mannitol + D-Glucitol + Lactic acid Succinic acid Citric acid Inositol Nitrate No vitamine at 37"C + (weak) A comparison of the physiological characteristics of the strain SP M 180 cc with those reported in the work edited by Lodder, quoted above, as well as with those reported in the book "A new Key to the Yeasts", by J. A. Barrett and R. J.

Pankhurst, 1974, has shown that the strain SP M 180 cc differs from all of the yeast species described in those books.

The technical and the patent literatures have described hitherto many yeasts capable of utilizing methanol (e.g. C. L. Cooney and D. W. Levine in "Single-Cell Protein" MIT Press, 1975), but the characteristics of the strain SPM 180 cc differ from those of all the strains known to us.

A "sui generis" characteristic of this strain is its capacity of assimilating methanol more efficiently than ethanol. The strain can be cultured both in discontinuous and continuous cultures, but its properties are better exploited in continuous cultures.

In addition, by virtue of its ability to form a pseudomycelium, a culture can be obtained which settles very easily: such a property can be exploited for conducting the continuous culture with a partial feed back or recycle of proteinaceous material, a fact which enables a higher hourly output to be obtained. The ease of settling, in addition, makes the collection of the proteinaceous material more convenient.

Strain SP M 180 cc was deposited at Northern Regional Research Laboratory, Peoria, Illinois, United States of America and accorded the accession No. NRRL- Y 11062.

One aspect of the present invention provides a microorganism belonging to the genus Candida, as has been deposited at the Northern Regional Research Laboratory where it has been accorded the accession No. NRRL-Y llC62; the microorganism being capable of using methanol as the sole source of carbon and energy, the microorganism being reproducible by multipolar germination to give flocks formed by a plurality of elongate cells, the microorganism being capable of forming a pseudomycelium sediment in a liquid culturing medium and forming colonies in a solid medium, the microorganism using positively in fermentation only glucose and using positively in growth, as sugars, only D-glucose, cellobiose, D-xylose and L-arabinose.

Another aspect of the present invention provides a process for producing a proteinaceous material, which comprises culturing in a culture medium the microorganism of the present invention, and recovering the proteinaceous material from the culture medium.

In practice, the process consists in inoculating with the strain SP M 180 cc a culturing medium which contains the essential elements (N, P, K, Mg, Fe, Ca), the growth-factors (yeast extracts and biotin), mineral trace elements, and methanol as the source of carbon and energy. The broth is incubated with stirring preferably at a temperature ranging from 200C to 350C, more preferably from 300C to 330C, the pH preferably being maintained in the range from 2.5 to 6.5, more preferably from 4 to 4.5, by providing a continuous supply of oxygen or a gaseous mixture which contains oxygen, such as air.

The yeast cells which multiply at the expense of the nutrients which are supplied, can be collected by sedimentation and filtration, washed with water and dried by heating.

The resulting proteinaceous material can be used as such as a proteinic integrator for foods and animal feeds, or nobler products can be extracted therefrom, such as proteins, aminoacids and nucleic acids.

The present invention is illustrated by the following Examples.

EXAMPLE 1 Into each of a plurality of 500-ml Erlenmeyer flasks were introduced 50 ml of a culturing medium which had the following composition: KH2PO4 2.0 grams per litre NaH2PO4.H2O 2.0 (NH4)2SO4 5.0 MgSO4.7H2O 0.2 FeSO4.7H2O 2.0 milligrams per litre CaC12 2.0 ZnSO4.7H2O 2.0 Yeast extract 200.0 Biotin 25.0 micrograms per litre Trace elements (in solution)l millilitre per litre The solution of trace elements, prepared in diluted HCI (1 millilitre of conc.

HCI in one litre of water) had the following composition: CuSO4.5H2O 200 milligrams per litre H3BO3 500 MnSO4.H2O 500 KI 10 CoCl2.6H2O 10 milligrams per litre MoO3 10 The pH of the medium in each flask was brought to 5.0 and the flasks were then sterilized at 1160C for 20 minutes. To each of two flasks there was added 0.75 ml (l.9V0 volume by volume ratio) of methanol, and these flasks were then inoculated with a slant of the strain SP M 180 cc, after which these flasks were incubated for 72 hours on a rotary stirring device (220 rpm, with the diameter of the displacement being 3.5 centimetres) at a thermostatically controlled temperature of 32.5"C, to give a pre-culture.

Other flasks, which contained the same culturing medium and to which there had been added 1% (volume/volume) of methanol, 2% (v/v) of ethanol or 2% g/dl of glucose, were each inoculated with 5 ml of the pre-culture specified above. After 24 hours of incubation, an additional 1% volume/volume of methanol was added to each flask. After a total of 48 hours of incubation, the content of the proteincontaining material in the flasks was measured, and the following results were obtained Dry material Optical density (1:10) (grams/ Proteins Substrate at 660 nm litre) ( /O) Glucose 0.340 3.99 54.5 Methanol 0.270 3.68 51.0 Ethanol 0.095 1.18 54.1 The protein content was determined by the biuret method.

EXAMPLE 2 A culturing medium as described in Example 1 which was contained in a fermentation vessel which had an effective volume of about 8 litres was inoculated with a suspension of the strain SP M 180 cc. To the vessel, which was thermostatically controlled at 32.50 C, there was added methanol, making sure that the residual concentration of methanol in the broth never exceeded 1% on a volume by volume basis. As the culture grew satisfactorily, the continuous addition of a sterile broth to the fermenter was started; the sterile broth contained 29.6 grams of methanol per litre, while an identical volume of broth-culture was simultaneously withdrawn. The volumes of added medium and of withdrawn brothculture were increased until there was attained a dilution velocity, D, (where D=incoming rate of flow/volume of the culture) of 0.166 h-'. Under these conditions the outgoing stream contained 10.28 grams per litre of proteinaceous material (calculated as dry material) and 146 parts per million of residual methanol, with a yield of 35% and an hourly production of 1.72 grams per litre of proteinaceous material. This material contained 55.6 of proteins (as determined by the biuret test).

EXAMPLE 3 To a fermentation vessel of the kind described in Example 2 was connected a settling tank for the outgoing stream, and a portion of the broth-culture enriched with proteinaceous material was regularly fed back to the fermentation vessel. By adding fresh culturing medium to the fermentation vessel, which medium contained 24 grams of methanol per litre, at a rate of flow such as to achieve a dilution velocity of 0.267 h-l, there was obtained in the non-recycled portion emerging from the settling tank a broth-culture containing 7.80 grams per litre of proteinaceous material and 120 parts per million of residual methanol, with a yield of 32.5% and a hourly output of 2.08 grams per litre. The proteinaceous material thus obtained contained 53.0% of proteins (as determined by the biuret test).

WHAT WE CLAIM IS: 1. A microorganism belonging to the genus Candida, as has been deposited at

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (8)

**WARNING** start of CLMS field may overlap end of DESC **. The solution of trace elements, prepared in diluted HCI (1 millilitre of conc. HCI in one litre of water) had the following composition: CuSO4.5H2O 200 milligrams per litre H3BO3 500 MnSO4.H2O 500 KI 10 CoCl2.6H2O 10 milligrams per litre MoO3 10 The pH of the medium in each flask was brought to 5.0 and the flasks were then sterilized at 1160C for 20 minutes. To each of two flasks there was added 0.75 ml (l.9V0 volume by volume ratio) of methanol, and these flasks were then inoculated with a slant of the strain SP M 180 cc, after which these flasks were incubated for 72 hours on a rotary stirring device (220 rpm, with the diameter of the displacement being 3.5 centimetres) at a thermostatically controlled temperature of 32.5"C, to give a pre-culture. Other flasks, which contained the same culturing medium and to which there had been added 1% (volume/volume) of methanol, 2% (v/v) of ethanol or 2% g/dl of glucose, were each inoculated with 5 ml of the pre-culture specified above. After 24 hours of incubation, an additional 1% volume/volume of methanol was added to each flask. After a total of 48 hours of incubation, the content of the proteincontaining material in the flasks was measured, and the following results were obtained Dry material Optical density (1:10) (grams/ Proteins Substrate at 660 nm litre) ( /O) Glucose 0.340 3.99 54.5 Methanol 0.270 3.68 51.0 Ethanol 0.095 1.18 54.1 The protein content was determined by the biuret method. EXAMPLE 2 A culturing medium as described in Example 1 which was contained in a fermentation vessel which had an effective volume of about 8 litres was inoculated with a suspension of the strain SP M 180 cc. To the vessel, which was thermostatically controlled at 32.50 C, there was added methanol, making sure that the residual concentration of methanol in the broth never exceeded 1% on a volume by volume basis. As the culture grew satisfactorily, the continuous addition of a sterile broth to the fermenter was started; the sterile broth contained 29.6 grams of methanol per litre, while an identical volume of broth-culture was simultaneously withdrawn. The volumes of added medium and of withdrawn brothculture were increased until there was attained a dilution velocity, D, (where D=incoming rate of flow/volume of the culture) of 0.166 h-'. Under these conditions the outgoing stream contained 10.28 grams per litre of proteinaceous material (calculated as dry material) and 146 parts per million of residual methanol, with a yield of 35% and an hourly production of 1.72 grams per litre of proteinaceous material. This material contained 55.6 of proteins (as determined by the biuret test). EXAMPLE 3 To a fermentation vessel of the kind described in Example 2 was connected a settling tank for the outgoing stream, and a portion of the broth-culture enriched with proteinaceous material was regularly fed back to the fermentation vessel. By adding fresh culturing medium to the fermentation vessel, which medium contained 24 grams of methanol per litre, at a rate of flow such as to achieve a dilution velocity of 0.267 h-l, there was obtained in the non-recycled portion emerging from the settling tank a broth-culture containing 7.80 grams per litre of proteinaceous material and 120 parts per million of residual methanol, with a yield of 32.5% and a hourly output of 2.08 grams per litre. The proteinaceous material thus obtained contained 53.0% of proteins (as determined by the biuret test). WHAT WE CLAIM IS:

1. A microorganism belonging to the genus Candida, as has been deposited at

the Northern Regional Research Laboratory where it has been accorded the accession No. NRRL-Y 11062; the microorganism being capable of using methanol as the sole source of carbon and energy, the microorganism being reproducible by multipolar germination to give flocks formed by a plurality of elongate cells, the microorganism being capable of forming a pseudomycelium sediment in a liquid culturing medium and forming colonies in a solid medium, the microorganism using positively in fermentation only glucose and using positively in growth, as sugars, only D-glucose, cellobiose, D-xylose and L-arabinose.

2. A process for producing a proteinaceous material, which comprises culturing in a culture medium the micro-organism claimed in claim 1, and recovering proteinaceous material from the culture medium.

3. A process according to claim 2, wherein the culturing is conducted with an aqueous culturing medium containing inorganic salts and containing, as the only or principal source of carbon and energy, methanol.

4. A process according to claim 2 or 3, wherein the culturing is carried out at a temperature in the range from 200C to 350C.

5. A process according to claim 2, 3 or 4, wherein the culturing is carried out at a pH in the range from 2.5 and 6.5.

6. A process according to any one of claims 2, 3, 4 or 5, wherein there is supplied to the culture medium a supply of oxygen or a gaseous mixture including oxygen.

7. A process according to claim 2, substantially as described in any one of the foregoing Examples.

8. A proteinaceous material whenever produced by a process according to any one of claims 2 to 7.