CS214802B2 - Method of making the biomass of high contents of proteins - 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

The present invention relates to a method of producing high protein microbial biomass by culturing a yeast strain of the Candida genus which is capable of utilizing methanol as the sole source of carbon and energy.

Due to the high reproductive rate of microorganisms and their protein contents, the formation of microbial biomasses represents a very fast method of protein production.

To date, waste sugars have been used, such as molasses from sugar refineries or waste sulphite liquors from paper mills.

Recently, based on considerable availability and low oil prices, biomass production methods have been adapted using either crude oil fractions or highly purified mixtures of normal paraffins as a substrate.

The use of such petroleum-based substrates has several technological disadvantages due to their insolubility in water, the considerable amount of oxygen needed by the microorganisms to assimilate, and the considerable amount of heat generated during fermentation. In addition, the current cost of biomass production is increased by the density of thorough substrate cleaning and / or by careful washing of the biomass produced to remove potentially hazardous oil components.

Such problems do not occur when biomass production is carried out using lower alcohols such as methanol or ethanol as substrates. Because of their excellent water solubility, volatility and the fact that they are available in high purity, it is possible to obtain biomass that is free of undesirable residues. Miscibility with water removes the mixing problems encountered in oil fractions, while the fact that oxygen is already present in their molecules reduces the oxygen requirement for their assimilation: this has the added benefit of biomass production being accompanied reduced heat generation resulting in reduced cooling costs.

Ethanol is used by a large number of microorganisms and should be an ideal substrate for biomass production, but its cost is relatively high. Methanol, on the other hand, can be produced cheaply and in a high degree of purity. This has become the basis for the efforts that have been made to find microorganisms that are able to efficiently use methanol. It is known from the technical and patent literature that, although numerous bacteria meet these requirements, the efficacy of the yeast described so far is rather low.

By enriching the culture medium and isolating the obtained yeast cells in continuous culture, the present invention has succeeded in isolating a new preferred yeast strain (NRRL Y-11062) which is able to use methanol as the sole source of carbon and energy.

An improved method for producing high protein biomass by culturing a yeast Candida strain carried out at a temperature in the range of 20 to 35 ° C and at a pH of 2.5 to 6.5 on a nutrient broth consisting of a salt solution and which contains methanol as the sole carbon and energy source, and by isolating the yeast cells obtained, the present invention consists in using a strain of Candida NRRL Y-11062 as the yeast strain.

The new strain is deposited in the public collection of cultures of microorganisms NRRL (Northern Regional Research Laboratory in Peoria, Illinods, United States) under the deposit registration number Y-11062.

The properties of this strain are apparent from the following specification of its features.

The NRRLY-11062 strain is reproduced by multipolar germination and produces discrete egg cells or clusters formed by a plurality of elongated cells (pseudomycelium).

In a liquid culture medium they form a sediment, while in a solid medium they are either smooth and shiny colonies or opaque and wrinkled colonies. In the preparation of strain subcultures on a solid medium, the strain becomes more and more smooth and shiny, which corresponds to separate cells in a liquid medium, while the cultivation of the strain under certain conditions in a liquid medium predominates in a pseudomycelial form corresponding to wrinkled and opaque cells. Disputes of any kind were not observed. Based on such morphological features, it was concluded that the strain belongs to the genus Candida according to the classification proposed by J. Lodder in "The Yeasts; A Taxonomy Study ', 1970.

The physiological properties of said new strain are as follows:

1. Fermentation of certain carbon sources:

D-glucose4D-galactose - sucrose - maltose - trealose - lactose—

2.Get:

D-glucose +

D-galactesis—

L-sorbose - sucrose - maltose - cellobiose + trehalose - lactose - malibiosis - raffinose - melezitose - inulin - starch -

D-xyloza +

L-arabinose—

1. Fermentation use of carbon i: some resources D-arabinose __ D-ribose + (weak) L-rhamnose - ethanol + glycerol - erythrit - D-glucit + lactic acid - succinic acid - citric acid - inosít. - nitrate - without vitamins - at 37 ° C + (weak)

A comparison of the physiological properties of the new strain with those presented in the Lodder study as well as those of J. A. Barret and R. J. Pankhurst's "A New Key to the Yeasts" showed that this strain differs from all the yeast species described in these books.

Many yeasts capable of using methanol have been described in the technical and patent literature (see CL Cooney and DW Levine: "Singlo-cell protein" II-Press, 1975), but the properties of strain SP M 180 differ from all strains known to the inventors .

This strain is characterized by its ability to assimilate its species. more effectively methanol than ethanol. The strain can be cultivated in discontinuous and continuous cultures, but its properties are better utilized in continuous cultures.

In addition, due to its ability to form pseudomycelium, a culture can be obtained which is very easy to sediment: this property can be used for continuous cultivation with partial removal or recycling of biomass, a fact which. enables higher hourly production. Easy sedimentation also makes biomass insulation more convenient.

In practice, the method comprises inoculating the soil with the NRRL Y-11062 strain which contains the soil. essential elements (nitrogen, phosphorus, potassium, magnesium, iron, calcium), growth factors (yeast extracts and biotin), mineral trace elements and methanol as a source of carbon and energy. The broth was incubated under agitation at a temperature in the range 20 ° C to 30 ° C, preferably between 30 ^DEG C. and 33 DEG C. while the pH was maintained between 2.5 and 6.5, preferably between 4 and 4, 5, wherein an oxygen-containing gas mixture, for example air, is continuously supplied.

Yeast cells, which multiply to expend the nutrients supplied, are isolated by sedimentation and filtration, washed with water and dried by heating.

The biomass thus obtained can be used as such as a protein summary for food and feed, or can be isolated therefrom from refined products such as proteins, amino acids and nucleic acids.

The following examples illustrate the invention but do not limit its scope.

He did

500 ml Erlenmeyer flasks each contained 500 ml of medium containing the following composition:

potassium dihydrogen phosphate KH2PO4 sodium dihydrogen phosphate NaH ₂ PO / ammonium sulphate (NH4J.2SO4 magnesium sulphate MgSO ₄ .7 H2O ferrous sulphate FeSO ₄ .7 H2O calcium chloride CaCl ₂ zinc sulphate ZnSO / .7 H ₂ O

Yeast extract

Biotin

Trace Elements (Solutions)

2.0 g per liter

2.0 g per liter

5.0 g per liter

0.2 g per liter

2.0 mg

2.0 g per liter

2,0 g. . v. liter 200,0 in liter 25,0 μξ in liter ml in liter

Trace element solution. prepared in dilute hydrochloric acid (1 ml of concentrated hydrochloric acid in 1 liter of water), had the following composition:

copper sulphate. H2O boiling acid H3BO3 sulphate, MnSO4). H2O potassium iodide KJ cobalt (II) chloride CoCl2.6 H2O molybdenum oxide MoO3

200 mg per liter

500 mg per liter

500 mg per liter mg per liter mg per liter mg per liter

The pH of the medium was adjusted to 5.0 and the flask was then sterilized for 20 minutes at 116 ° C. The two flasks were then charged with 0.75 mL (1.9% v / v) methanol and the flask was then seeded with NRRL Y-11062 slanted agar. The flasks were incubated for 72 hours on a rotary mixer (220 rpm, 3.5 cm diameter) while maintaining a temperature of 32.5 ° C thermostatically.

Other flasks containing the same medium and to which 1% (v / v) methanol, 2% (v / v) ethanol and 2% (w / v) glucose were added were inoculated individually with 5 ml of the above subculture. After 24 hours of incubation, an additional 1% / volume of methanol was added to each flask. After a total of 43 hours incubation, the biomass content of the flask was measured and the following results were obtained:

Substrate Optical Density (1: 10) at Dry Biomass

660 nm grams per liter of protein

glucose 0.340 3.99 54.5 methano! 0.270 3.63 51.0 ethanol 0,095 1.18 54.1

Protein content was determined by the biuret method.

Example 2

A suspension of NRRL Y-11062 strain was seeded into a fermenter with an effective volume of about 8 liters and containing the culture medium of Example 1. Methanol was added to a fermenter whose temperature was controlled by a thermostat at 32.5 ° C to ensure that the residual culture medium concentration did not exceed 1% by volume, expressed in volume.

When the culture subsequently grew, the continuous addition of sterile culture medium to the fermenter was started, with the sterile culture medium containing

29.6 g of methanol per liter, while a quantity of nutrient culture equal to the amount added to the culture medium was simultaneously collected. The amount of medium added and the culture culture collection were increased until they reached a dilution rate (D - feed inflow to culture volume ratio) of 0.166 hr ^-1 . Under these conditions, the effluent contained 10.28 g dry biomass per liter and 146 ppm residual methanol, with a yield of 35 why, and an hourly production of 1.72 g biomass per liter. The biomass thus obtained contained 55.6% protein (according to the biuret method).

Example 1 a d 3

A settling tank for the outgoing stream was connected to a fermenter of the type described in Example 2, and a portion of the biomass-enriched culture medium was regularly returned to the fermenter. By adding fresh broth to the fermenter, the broth containing 24 g of methanol per liter, at a flow rate such that a dilution rate of P, 267 h ¹ was achieved, was recovered in non-recycled form. a fermentation culture containing 7.80 g of biomass per liter, and 120 ppm of residual methanol, with a yield of 32.5% / h and an hourly production of 2.08 g per liter. The biomass thus obtained contained 53.0% protein (according to the biuret method).

Claims (1)

P R E DMÉ T - V Y N E L Z Z A method for producing high protein biomass by culturing a yeast Candida strain at a temperature in the range of 20 to 35 ° C and at a pH of 2.5 to 6.5 on a nutrient broth consisting of a salt solution and which as the sole carbon source and The energy comprises methanol, with the isolation of the obtained yeast cells, characterized in that the yeast strain used is a Candida strain NRRL Y-11062.