RO116203B1 - Polysaccharide-producing microorganism - Google Patents

Abstract

The invention relates to a pullulan polysaccharide-producing microorganism, which is a strain of Aureobasidium pullulans ICCF-83, registered in the Institute of Chemical-Pharmaceutical Research Institute under no. 211, which are in the form of elongated, perilate, hyaline cells with no pigmentation potential, with smooth-type colonies that totally ferment fructose, glucose, xylose, able to rapidly consume the carbon source at initial concentrations up to 10 % and with a superior capacity to synthesize pullulan.

Description

RO 116203 B

The invention relates to a polysaccharide-producing microorganism that synthesizes, through fermentation on an appropriate medium and under optimal conditions, a pullulan-type polysaccharide, with wide uses, especially in the food, chemical and pharmaceutical industries.

Different strains of Aureobasidium pullulans with superior pullulan biosynthesis traits are known. Thus, in order to increase the productive potential of a parental strain, mutagenesis with gamma radiation [RO 90 438/1989) or chemical (FEMS Micro biology Letters 124, 167-172, 1994) was applied, for the same purpose and to reduce the degree of pigmentation over time of the fermentation medium, with negative consequences both on the separation and purification process and on the quality of the polysaccharide obtained, new strains of Aurebasidium are isolated from nature [Korean J.Appi. Microbiol. Biotechnol,Ș/\_, 494-503 [1993],

Although superior results have been achieved, the productive strains obtained so far with a large volume of work and costs, present some disadvantages such as:

- initial concentration of the carbon source reaching max. 8%, thus limiting the final concentration of the synthesized polysaccharide;

- slow capacity for consumption of the carbon source and, as a result, for polysaccharide synthesis, requiring a long duration of the fermentation process [5...7 days):

- the impossibility, in most cases, of preventing the formation of brown pigment (melanin type), creating difficulties in isolating a high-quality product.

The microorganism according to the invention is a strain of Aureobasidium pullulans ICCF-83, registered in the collection of the Institute of Chemical-Pharmaceutical Research under no. 211, which appears in the form of elongated, pearly, hyaline cells, with absent pigmentation potential, with smooth-type colonies that completely ferment fructose, glucose, xylose, capable of rapidly consuming the carbon source in initial concentrations of up to 10% and with a superior capacity to synthesize pullulan.

The invention has the advantage of creating a microorganism capable of synthesizing polysaccharides with a high yield.

An example of an embodiment of the invention is given below.

Example 1. From the Aureobasidium pullulans ICCF-68 strain, stored under laboratory conditions (+4°C) on a liquid static medium, 0.1 ml is taken and dispersed in serial dilutions on agarized nutrient medium, distributed in Petri plates.

The agarized nutrient medium contains (%]: glucose 5; ammonium sulfate 0.2; sodium nitrate D.2; monopotassium phosphate 0.5; sodium chloride 0.2 and magnesium sulfate 0.05.

Incubation is done at 28°C, 48 h.

A selection of isolated colonies is made according to the criteria: smooth colonies, creamy white color, convex, shiny, smooth surface.

The selected colony is dropped again onto the agarized nutrient medium, with incubation at 28°C, static, for 48 h, which constitutes the pre-inoculum.

The culture developed in the plate from the selected colony is suspended in sterile distilled water, 5 ml/tube, and an Erlenmayer flask is inoculated with inoculum medium at a ratio of 2% (v/v).

The inoculum medium is obtained as described in example 1, containing (%]: glucose 2; bactopeptone 0.2; KH ₂ P0 ₄ 0.2; NaCl 0.2.

The optimal culture, as described above, is inoculated at a ratio of 1 5% [v/v] in the described biosynthesis medium.

RO 116203 B

The work scheme is as follows:

Static liquid environment +4°C 0.1 ml in _r Nutrient static inoculation medium 28 ^q C isolated colonies Aureobasidium serial dilutions agarized -+ typical ⁵⁰ pullulans ICCF-68 in Petri dishes

Replication of 55 colony on inoculation medium 28°C, stirring nutrient 48 h agarized in -+ Pre-inoculation 5 ml distilled water/ _tube inoculum biosynthesis test tubes, suspension 2 ml liquid -> 28°C culture in Shaking 48 60

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7-14 days

Pre-inoculums that had the characteristics: constant and absolute depigmentation in 65 for 7-14 days; rapid consumption of carbon mass in the biosynthesis phase (48 h); increased productivity in pullulan - were transferred to liquid storage medium, to preserve these characteristics and resume the selection/production cycle. Over time [approx. 5 years) from the ICCF-68 strain, the ICCF-83 strain was selected, having superior biochemical characteristics. 7o

The Aureobasidium pullulans strain ICCF-83, registered in the ICCF collection under no. 211, presents the following characteristics:

a) Morphological: round, hyaline, pearly cells with thin walls that become thickened, with a yeast-like appearance with ballastospores that, through division, develop a true mycelium, forming chiamidospores. 75

b) Cultural: on static liquid media it presents a translucent deposit, which, over time, becomes pigmented with melanin; the liquid is clear, viscous and becomes opalescent upon agitation; on agitated liquid media, the culture presents an opalescent appearance with uniform turbidity that becomes dark green-black, after 80 h of agitation.

Colonies on peptone agar media, with a 48 h cycle, are of the smooth 8o type, creamy white in color. Their surface is convex, glossy, and smooth.

With aging, the colonies do not change their smooth morphological appearance, they are shiny, mucoid and remain totally depigmented regardless of age.

On gelatin, the microorganism grows on the surface, with an opaque appearance.

c) Physiological: aerobic strain, optimal pH for multiplication 5.5...7: for 85 biosynthesis 5...7.5 with a limit of 4.5...8, optimal development temperature 25...28°C. Does not reduce nitrates and nitrites; does not hydrolyze starch; reactions to methyl red, VogesProscauer, bend, urease and hydrogen sulfide are positive; ferments glucose completely, ferments maltose, cellobiose, lactose, xylose, arabinose and fructose moderately at neutral pH; does not ferment galactose, sucrose, raffinose, melibiose, dulcitol, inositol, 90 salicin, adonit and inulin.

RO 116203 B

The procedure for selecting the microorganism is as follows:

A vial of lyophilized (of a totally depigmented suspension, with maximum productivity in pullulan or a vial cryopreserved in liquid nitrogen [with the same suspension] is suspended in 5 ml of sterile physiological serum and inoculated onto a nutrient agar medium, containing glucose, yeast extract, malt extract and bactopeptone. Incubation is done at 25,..28°C for 48 h.

The culture with the appropriate appearance, according to the description of the smooth mucoid colonies on the nutrient medium, is suspended with 5 ml of sterile distilled water and, simultaneously, a tube with static preservation medium is inoculated, as well as a tube with nutrient agar medium - according to the description above, which constitutes the pre-inoculum to test the capacity of pullulan productivity on a specific biosynthesis medium.

The static liquid medium (RO 88881/1985} preserves the microorganism for an average period of time (6-12 months) at a temperature of +4°C, with the inhibition of metabolic processes that form the slow pigmentation of the microorganism's cells, which, once returned to development conditions on nutrient media and biosynthesis media, have the ability to resume the evolutionary and synthesis cycle of pullulan, without producing melanin.

The working method for testing productive capacity is as follows:

The pre-inoculum obtained as described above [nutrient medium inoculated with 0.1 ml of culture from the liquid static medium) is suspended with 5 ml of distilled water and, from this, an inoculum medium is inoculated, at a ratio of 2%, into Erienmayer-type flasks.

The inoculum medium contains glucose, bacto peptone and mineral salts at a pH within the range of 4.5...7: it is incubated at 25...28°C with rotary shaking of 200...300 rpm for 18...24 h.

The optimal culture, according to the description above, is inoculated in a ratio of 1 ... 5 % in the biosynthesis medium containing (%): glucose 5 -10 [srJ; KH ₂ P0 ₄ 0.5; (NH ₄ ) ₂ S0 ₄ 0.2; NaNOg 0.2; NaCl 0.2; MgS0 ₄ .7H ₂ 0 0.08, initial pH adjusted to 5 - 6.5 with 10 N NaOH solution.

The cultures that showed the highest pullulan productivity were further selected according to the described procedure, outlined as follows;

Powder suspension in_ Nutrient medium Agarized physiological serum Tn suspension culture _ Static medium cryconservative liquid +4°C Culture in Preinocul suspension____

(agarized nutrient medium (25-28°C] Inoculum suspension 2% Biosynthesis

2% Stirring 25-28°C 25-28°C h stirring

RO 116203 B Table 1 gives, in comparison, the characteristics of similarity and differentiation between the parental strain and the one newly obtained through selection.

140

Table 1

Features Aureobasidium pullulans ICCF-68 (M ₅ . _a ) ICCF - 83 0 1 2 145 I. Morphological characters 1. Dimensions - radulaspores 3-3.5/1.5-2 hours 3.5/1.5 - 2 units - mycelium 10-20/1.5-2.5 u 10-20/2.5 u 150 2, Mobility does not present does not present 3. Sporulation 3-3.5/1.5-2 hours 3.5/2 - 2.5 u 4. Morphological form Round, pearly hyaline cells with thin walls that become thickened with a yeast-like appearance; blastospores by division form true spores Pearly elongated cells with thin hyaline walls that have lost the ability to form mycelium and melanin-pigmented blastospores 155 II. Cultural aspects 1. Appearance on liquid media 160 - static translucent deposit growing on the walls of the tube in the shape of a ring that becomes pigmented with age; the liquid is clear and becomes opalescent upon shaking. translucent deposit growing on the walls of the tube in the shape of a ring that remains completely depigmented with age; the liquid is clear and becomes opalescent upon shaking. 165 - agitated opalescent with uniform turbidity, slightly viscous, yellowish-white in color that becomes dark green-black after 48 h through the release of pigment (melanin). opalescent with uniform turbidity, slightly viscous, yellowish-white in color that does not change after 48 h. 170

RO 116203 B

Table 1 (continued)

Features Aureobasidium puliulans ICCF-68 [M ₅ . _b ) ICCF - 83 0 1 2 2, Appearance of colonies on peptone agar media shows growth at 48 h; smooth and rough type colonies of creamy white color: the convex, glossy, smooth surface becomes rough with an adherent appearance to the medium and with the release of melanin. on peptone agar media it shows growth at 48h; smooth glossy colonies, with a white mucoid appearance; convex, smooth surface; the described macroscopic appearance does not change with aging: it is easily removed from the medium; melanin is not formed. ICCF-68 [M ₅ . _b ] ICCF - 83 0 1 2 3. Gelatin development growth on the surface with a shiny appearance growth on the surface with a shiny mucoid appearance. III. Morphological characters 1, Unchristians of 0 ₂ aerobic aerobic 2. Optimal pH - multiplication - biosynthesis - pH limit 5.5-7 5-7.5 4.5-8 5.5-7 4.5-7.5 4 - 8 3. Optimal development temperature 25 - 28°C 25 - 28°C 4. Nitrate reduction - - 5. Starch hydrolysis - 6. Methyl red reaction - - 7. Voges Proscauer reaction - - 8. Reaction to bending [Kovacs] - - 9. Urease reaction - - 1D. Hydrogen sulfide - -

RO 116203 B

Table 1 [continued]

Characteristics Aureobasidium pullulans ICCF-68 (M ₅ . _b ) ICCF - 83 0 1 2 11. Fermentation of sugars A] total with acid production fructose, xylose fructose, xylose, glucose B) moderate [neutral pH) maltose, cellobiose, lactose, arabinose, glucose maltose, cellobiose, lactose, arabinose, sucrose C) does not ferment galactose, sucrose, raffinose, melibiose, dulcitol, inositol, salicin, adonit, inulin galactose, raffinose, melibiose, dulcitol, inositol, salicin, adonit, inulin IV. Nutritional requirements prototrophic strain grows on minimal medium prototrophic strain grows on minimal medium V. Carbon source consumption capacity (glucose(% (sr) - batch fermentation 5-8 (48-60 h] 5-10 (48-60 h] - fedbatch fermentation 14 [82 h] 19 [90 h) VI. Capacity to produce pullulan - Final conc. g/l - batch fermentation in metal bioreactor (150 1) 25-50 30-60 - fedbatch fermentation [90 h) in microfermentor (14 1] 65 103 Characteristics of the polysaccharide (pullulan) produced: - molecular mass Kda 200 - 300 150-300 - apparent viscosity of aqueous sol. 2% [rotary viscometer 1300 ș- ¹ , 23 ^Q C] 3 - 6 2.5-6

Claims (1)

RO 116203 B 260 Claim Microorganism producing pullulan-type polysaccharides, characterized by being a strain of Aureobasidium pullulans ICCF-83, registered in the collection of the Institute of Chemical-Pharmaceutical Research under no. 211, which presents itself in the form of 2 65 elongated, pearly, hyaline cells, with absent pigmentation potential, with smooth-type colonies that completely ferment fructose, glucose, xylose, capable of rapidly consuming the carbon source in initial concentrations of up to 1 □% and with a superior capacity to synthesize pullulan. President of the examination committee; chemist Hăulică Mariela Examiner: biochemist Crețu Adina Computerized editing and proofreading - OSIM Printed at: State Office for Inventions and Trademarks