DK143765B - METHOD FOR PRODUCING YEAR CELL MASS WITH HIGH PROTEIN CONTENT - Google Patents

Description

(19) DENMARK (W)

| J | (12) PUBLICATION NOTICE and 143765 B

DIRECTORATE OF THE PATENT AND TRADEMARKET SYSTEM

(21) Application No. 5604/77 (51) | nt.CI.3 C 12 N 1/32 (22) Filing Day 15 · Dec. 1977 (24) Race day 15 · dec. 1977 (41) Aim. available Jun 24 1978 (44) Presented 5 · Oct. 1 g8l (86) International application no. "(86) International filing day (85) Transfer day" (62) Master application no. "

(30) Priority 23 · Dec. 1976, 30798/76, IT

(71) Applicant SNAMPROGETTI S.P.A., Milan, IT.

(72) Inventor Pasquale Zaffaroni, IT: Antonio _Senni, IT: Paolo

Branduzzi, IT: Lamterto Formiconi, IT.

(74) International Patent Bureau.

(54) Process for producing high protein yeast cell mass.

The invention relates to a process for producing high protein yeast cell mass by growing a strain of the genus Candida on a culture medium comprising a salt solution containing methanol as the sole carbon and energy source, at a temperature from 20 ° C to 35 ° C and a pH value between 2.5 and 6.5.

Due to the high rate of reproduction of the microorganisms and their protein content, the production of microbial biomasses is a very fast protein producing method.

<£) For the production of biomasses, waste carbohydrates such as e.g. molasses from sugar mills or -d "used sulphite cooking liquids from paper mills.

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Recently, due to the availability of petroleum *

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2 1Λ3765 and low cost adapted to biomass production methods using as substrate either the crude fractions of petroleum or highly purified mixtures of normal paraffins.

The use of such petroleum-based substrates exhibits a few shortcomings from a technological standpoint, due to their insolubility in water, the considerable amount of oxygen required for their assimilation of microorganisms and the high heat generation during fermentation. Further, the ongoing cost of biomass production is increased because it is necessary to thoroughly and thoroughly clean the substrate to wash the biomass thus produced to remove potential harmful petroleum components.

Such difficulties are not seen if the production of the biomasses is carried out using the lower alcohols such as methanol or ethanol as a substrate. Indeed, because of their complete solubility in water, their volatility, and their availability in such purity, it is possible to obtain a biomass where the undesirable residues are not found. The miscibility with water solves the mixing problems that exist with the petroleum fractions, while the oxygen demand, because they already contain oxygen in their molecules, is reduced by their assimilation: therefore, there is an additional advantage that the production of the biomass is accompanied by less heat generation, thereby reducing the cost of cooling.

Ethanol is used by a large number of microorganisms and will be the ideal substrate for biomass production, but the cost is relatively high. In contrast, methanol can be produced cheaply and with high purity. This is why efforts have been made to find microorganisms capable of efficiently utilizing methanol. In the technical literature and patent literature, many bacteria capable of meeting these requirements have been described, while the efficacy of the yeast species described has so far been rather low. The foregoing disadvantages are avoided by the method of the present invention, which is of the prior art in which a strain of the genus Candida is grown on a culture medium comprising a saline solution containing methanol as the sole carbon and energy source, at a temperature of 20 ° C to 35 ° C and a pH between 2.5 and 6.5. The process is characterized in that as the strain of the genus Candida the new strain NRRL-Y 11062 (SP M 180 cc) is used. Thereby a high protein yeast cell mass can be obtained which can be included as the protein part in animal feed or from which more noble products such as proteins, amino acids or nucleic acids can be recovered.

The strain in question was deposited on January 6, 1977, in the Northern Regional Research Laboratory, Peoria, Illinois, USA, under the designation NRRL-Y 11062.

The characteristics of the novel strain are described below.

The SP M 180 cc strain is reproduced by multipolar germination, forming separate egg-shaped cells or clumps consisting of a plurality of elongated cells (pseudomycelium).

In liquid culture media, it forms a precipitate, while in solid medium it forms either smooth and shiny colonies, or opaque and wrinkled colonies. When growing the strain on a solid medium, the strain becomes more and more smooth and shiny, which in a liquid medium corresponds to the separated cells, whereas when cultured in a liquid medium under certain conditions, the pseudomycelial form predominates, corresponding to the wrinkled and opaque cells. No traces of any kind have ever been seen.

Based on such morphological characteristics, it is assumed that the strain belongs to the species Candida according to the classification proposed by J. Lodder, The Yeasts: a taxonomic Study, 1970.

The physiological characteristics of the strain SP M 18 cc are as follows: 1. Fermentation utilization of a few carbon sources: D-glucose + D-galactose

sucrose

maltose

Trealose

Lactose 2. Growth: D-glucose + D-galactose 1-sorbose

sucrose

maltose

Cellobiose +

Trealose

Lactose, 143765 4

melibiose

raffinose

melezitose

Inulin -

Starch D-xylose + 1-arabinose + D-arabinose - D-Ribose + (weak) 1-Rhamnose -

Ethanol +

glycerol

erythritol

Ribitol +

Galacticol D-Mannitol + D-Glucitol + Lactic Acid Acetic Acid Citric Acid Inositol Nitrate

No vitamin at 37 ° C + (weak)

A comparison of the physiological characteristics of the SP M 180 cc strain with the characteristics described in the work by Lodder quoted above as well as compared with the information discussed in the book "A new Key to the Yeasts" by JA Barrett and RJ Pankhurst, 1974 has shown that the strain SP M 180 cc is different from all the yeast species described in these books.

The technical literature and patent literature have so far described many yeast species capable of using methanol (cf. CL Cooney and DW Levine in "Single-Cell Protein II - MIT Press, 1975), but the characteristics of the strain SP M 180 cc are different from all the known tribes.

A "sui generis" property of this strain is its ability to assimilate methanol more efficiently than ethanol. The strain can be grown in both discontinuous and continuous cultures, but its properties are better utilized in continuous cultures.

Furthermore, a culture that can very easily fall to the ground can be obtained because of its ability to form pseudomycelium. Such a feature can be utilized to perform continuous culture with a partial biomass feed back or recycling, a capability that allows a higher yield per capita to be obtained. hour. Furthermore, the light precipitation allows the collection of the biomass to be more convenient.

In practice, the process consists of inoculating a culture medium with strain SP M 180 cc, the culture medium containing the necessary elements (N, P, K, Mg, Fe, Ca), growth factors (yeast extracts and biotin), mineral trace elements, and methanol as carbon and energy source. The builion is grown with stirring at a temperature from 20 ° C to 35 ° C, preferably between 30 ° C and 33 ° C, maintaining the pH between 2.5 and 6.5, preferably between 4 and 4.5, supplies a continuous amount of a gas mixture containing oxygen, e.g. air.

The yeast cells, which multiply at the expense of the nutrients being supplied, are collected by bottom phaling and filtration, washed with water and dried by heating.

The biomass thus obtained can be used as the protein portion of animal feed, or more noble products may be extracted from it such as proteins, amino acids and nucleic acids.

The present patent does not cover the use of the process in the manufacture of foodstuffs.

The invention is further illustrated below by the following examples.

Example 1

500 ml Erlenmeyer flasks were used, each containing 50 ml of a culture medium of the following composition: KH 2 PO 4 2.0 g / ml liter

2.0 "(NH 4) 2 SO 4 5.0"

MgS04.7H20 0.2 "

FeSO4.7H2O 2.0 mg per liter

CaCl3 2.0 "

2.0 "Yeast Extract 200.0"

Biotin 25.0 micrograms per liter

Tracers (solution) 1 ml. liter c 143765 6

The solution of the trace elements was prepared in dilute HCl (1 ml of concentrated HCl in 1 liter of water) and had the following composition:

CuS04.5H20 200 mg per liter H3B03 500 "

MnS04.H20 500 "

10 o clock "

CoC12.6H20 10 "

Mo03 10 "

The pH of the substrate was adjusted to 5.0 and the flask sterilized at 116 ° C for 20 minutes. To two flasks, 0.75 ml (1.9% v / v) of methanol was added and the flasks were sown with a bevel graft of strain SP M 180 cc. The flasks were grown for 72 hours on a rotary device (220 rpm, with the movement diameter 3.5 cm) and a temperature of 32.5 ° C, thermostatically controlled.

Other flasks containing the same medium to which 1% (v / v) methanol, 2% (v / v) ethanol and 2% w / v glucose were added were added with 5 ml of the preculture described above. After 24 hours of culture, an additional 1% v / v methanol was added to each flask. After a total of 48 hours of culture, the contents of biomass in the flasks were measured, the results being as follows:

Substrate Optical density Dry biomass _ (1:10) at 660 nm g / liter Proteins

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 sample.

Example 2

A fermentation vessel that had an effective volume of approx. 8 liters and containing the culture medium of Example 1 were seeded with a suspension of strain SP M 180 cc. To the fermentation vessel, which was thermostatically controlled at 32.5 ° C, was added methanol, ensuring that the residual concentration in the builion never exceeded 1% v / v. When the culture had grown satisfactorily, the continuous addition of sterile builion to the fermentation vessel began, with the sterile builion containing 29.6 g of methanol per ml. liters, while at the same time removing part of the buillon culture, corresponding to the amount added to the culture substrate. The amount of added