RU2567672C1 - FUNGAL STRAIN Eremothecium ashbyi - PRODUCER OF ESSENTIAL OIL SIMILAR TO ROSE "OTTO" OIL - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: strain Eremothecium ashbyi Guill. 810-ssb-III is a mutant, it was selected from the strain population Eremothecium ashbyi RNCIM F-36 (NRRL Y-1363) and deposited in the RNCM under the number RNCM F-4566D.

EFFECT: possibility to use the strain as a producer of essential oil similar to rose oil of Bulgarian origin.

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Description

The invention relates to biotechnology, applied microbiology and can be used for use as a producer of essential oils, similar to pink oil of Bulgarian origin. The aim of the invention is to obtain a new strain of the fungus Eremothecium ashbyi Guill. 810-ssb-III (registration number VKM F-4566D) synthesizing an essential oil close to that of Bulgarian rose oil regulated by the international standard ISO 9842: 2003. Strain Eremothecium ashbyi Guill. 810-ssb-III is a mutant selected in the laboratory of Penza State University from a population of the Eremothecium ashbyi strain VKPM F-36 (NRRL Y-1363) obtained from the All-Russian collection of industrial microorganisms.

The present invention relates to a biologically pure culture of a strain of the fungus Eremothecium ashbyi Guill. 810-ssb-III, which can be used as a producer of essential oil, similar to rose oil of Bulgarian origin.

Bulgarian rose oil is one of the most valuable oils in the world, widely used in the food, pharmaceutical, perfume and cosmetics industries [1, 2]. Its yield is on average 0.025%, so to get 1 kg of oil you have to collect it manually and process about 4 tons of petals. In the world, rose oil is produced that complies with ISO 9842: 2003, in just 4 countries (the volume of which is currently about 600 kg / year): Saudi Arabia (Taif), Bulgaria (Kazanlak), Turkey (Istanbul) and Uzbekistan (Tashkent region) [1, 3, 4]. Its quality significantly depends on environmental factors, the area in which essential oil roses are cultivated, in addition, plantation cultivation is characterized by seasonality [1].

These problems can be solved using biotechnological approaches. It should be noted that obtaining rose oil in the culture of isolated plant cells and tissues was unattainable, since it was found that the amount of synthesized oil in the cell culture of the rose is an order of magnitude lower than in the petals of an intact plant; the composition of the extracted oils was different from the rose oil obtained from the plant [5]. However, in the 90s of the last century, the possibility of obtaining an aromatic product with a rose smell based on strains of Eremothecium ashbyi Guilliermond 1935 and E. gossypii Kurtzman 1995 [6–9] was shown.

The proposed strain of the fungus Eremothecium ashbyi 810-ssb-III is new, not described in the scientific and patent literature. Closest to the invention in technical essence and the achieved positive effect are the strain of the fungus Eremothecium ashbyi BKM F-3009 - producer of essential oil (A. S. 1454845 USSR) [9]. However, compared with the prototype under the same conditions of cultivation and isolation (extraction with hexane), the proposed strain is characterized by higher productivity in relation to the synthesis of essential oil and its composition, which is closer to the Bulgarian rose oil regulated by international standard ISO 9842: 2003 (table. 1 )

The strain was identified by the main cultural and morphological characters, physiological and biochemical properties according to the Kurtzman’s determinant [10].

To check the purity of the cultures, a collection sample was seeded on Saburo agar and incubated for 120 hours.

Morphology. The microorganism has a dichotomously branched septic mycelium, consisting of multinucleated cells; the diameter of the hyphae varies between 2.3 ... 17.1 microns; sporangia oblong multispore, conidia fusiform; sizes of ascospores 2.4 ... 2.9 × 19.8 ... 27.1 microns. On a solid agarized medium it forms creamy flat matte colonies, which later acquire bright yellow staining and shallow radial folding. The shape of the colonies is round, the diameter after 120 hours of growth at 29 ° C on rice agar - 4 ... 5 mm, on Saburo agar - 7 ... 12 mm, on potato-glucose agar - 8 ... 13 mm.

Physiological and biochemical properties. Aerobe. The optimum growth temperature is 25-28 ° C. The optimum pH lies in the range of 5.5 ... 6.5. It uses glucose, fructose, raffinose, sucrose, glycerin, acetate and citrate as carbon and energy sources; it does not absorb starch, cellulose, ethanol, and inositol; coagulates milk, slightly dilutes gelatin.

The strain synthesizes an essential oil, which includes geraniol (26.5-64.9%), citronellol (9.4-27.0%), nerol (2.6-11.9%), β-phenylethanol (6 , 8-24.0%), linalool (0.1-0.4%), geranial (0.1-0.2%). The resulting strain differs in the composition of the synthesized aromatic product from the original strain of E. ashbyi VKPM F-36 and the previously known strain of E. ashbyi BKM F-3009 (Table 2) and is closer in terms of the ratios of the main components to the performance of rose oil regulated by the international standard ISO 9842: 2003.

Storage conditions of the strain.

Storage methods: in test tubes on a solid, slanted agar medium for up to 1 month at a temperature of 24 ° C, up to 3 months at a temperature of 5 ° C, up to 5 years under liquid paraffin.

Storage media: Saburo agar (g / l: glucose - 40.0; peptone - 10.0; agar-agar - 18.0-20.0); glucose-peptone agar with yeast extract (g / l: glucose - 10.0-30.0; peptone - 2.0-10.0; yeast extract - 0.5-3.0; agar-agar - 18.0 -20.0); malt-peptone agar (g / l: malt extract - 15.0-30.0; peptone - 1.0-4.0; agar-agar - 18.0-20.0); malt agar (g / l: malt wort (3.5 ° B) - 1.0 l; agar agar - 18.0-20.0); potato glucose agar (g / l: potato 200.0; glucose 1.0-10.0; agar agar 18.0-20.0); soy-sucrose agar (g / l: sucrose - 10.0-30.0; soy flour - 10.0-40.0; agar-agar - 18.0-20.0).

Cultivation media:

Solid: Saburo agar (g / l: glucose 40.0; peptone 10.0; agar agar 18.0-20.0); glucose-peptone agar with yeast extract (g / l: glucose - 10.0-30.0; peptone - 2.0-10.0; yeast extract - 0.5-3.0; potassium hydrogen phosphate - 0.0- 0.5; agar-agar - 18.0-20.0); malt-peptone agar (g / l: malt extract - 15.0-30.0; peptone - 1.0-4.0; agar-agar - 18.0-20.0); malt agar (g / l: malt wort (3.5 ° B) - 1.0 l; agar agar - 18.0-20.0); potato glucose agar (g / l: potato 200.0; glucose 1.0-10.0; agar agar 18.0-20.0); soy-sucrose agar (g / l: sucrose - 10.0-30.0; soy flour - 10.0-40.0; agar-agar - 18.0-20.0); rice agar (g / l: dehydrated rice flakes - 20.0; glucose - 10.0-20.0; agar-agar - 15.0-20.0);

Liquid: soy-sucrose (g / l: soy flour - 10.0-30.0, sucrose - 10.0-35.0); malt broth with yeast extract (g / l: malt extract - 5.0-15.0, maltose - 5.0-10.0, yeast extract - 0.5-2.0, glucose - 7.5-15, 0, peptone - 3.0-10.0, potassium hydrogen phosphate - 0.0-0.5); glucose-peptone (g / l: glucose - 7.5-15.0, peptone - 2.0-10.0, sodium succinate - 1.0-2.5, potassium hydroorthophosphate - 0.0-1.0, inositol - 0.0-0.5).

The temperature and time of cultivation: 25-28 ° C, 7 days. Frequency of transfers: 12 times a year when stored at 24 ° C, 4 times a year at 5 ° C, up to 5 years under liquid paraffin.

The use of the strain is illustrated by the following examples.

Example 1. The strain was grown for 6 days at 28 ° C in test tubes on a beveled agar medium of the following composition (g / l): glucose - 10.0, peptone - 3.0, yeast extract - 0.5, potassium hydrogen phosphate - 0 5, agar-agar - 20.0, distilled water to 1 liter. For the formation and accumulation of essential oil, a cell suspension was added in an isotonic solution corresponding to the turbidity standard 2, in an amount of 1% in flasks with a liquid nutrient medium of the following composition (g / l): malt extract - 6.0, maltose - 6.0, yeast extract - 1.2, glucose - 13.5, peptone - 4.0, potassium hydrogen phosphate - 0.5, distilled water to 1 liter. Incubation was carried out at 28 ° C on a shaker (150 rpm) for 67 hours. The essential oil was extracted with hexane. The amount of aromatic product was 122.9 mg / L. The composition of aromatizing compounds, wt. %: geraniol 64.84, citronellol 9.38, nerol 2.55, β-phenylethanol 6.84, linalool 0.33, geranial 0.10, nonral 0.08.

Example 2. The strain was grown for 6 days at 28 ° C in test tubes on a beveled agar medium of the following composition (g / l): glucose - 10.0, peptone - 3.0, yeast extract - 0.5, potassium hydrogen phosphate - 0 5, agar-agar - 20.0, distilled water to 1 liter. The culture was used to obtain the inoculum. Inoculation was carried out in a liquid nutrient medium of the following composition (g / l): malt extract - 6.0, maltose - 6.0, yeast extract - 1.2, glucose - 13.5, peptone - 4.0, potassium hydrogen phosphate - 0 , 5, distilled water to 1 liter. Cultivated on a shaker at 28 ° C for 72 hours. For the formation and accumulation of essential oil, the inoculum in an amount of 5% was introduced into flasks with a liquid nutrient medium of the following composition (g / l): soy flour - 20.0, sucrose - 20.0, distilled water to 1 liter. Incubation was carried out at 28 ° C on a shaker (150 rpm) for 96 hours. The essential oil was extracted with hexane. The amount of aromatic product reached 220.0 mg / L. The composition of aromatizing compounds, wt. %: geraniol 26.43, citronellol 26.99, nerol 11.89, β-phenylethanol 17.41, linalool 0.08, geranial 0.18, neral 0.03.

Example 3. The strain was grown for 24 hours at 28 ° C in test tubes on beveled potato-glucose agar. The culture was used to obtain the inoculum. Sowing was carried out in a liquid nutrient medium of the following composition (g / l): glucose - 7.5, peptone - 4.0, sodium succinate - 2.0, potassium hydrogen phosphate - 0.5, inositol - 0.14, distilled water to 1 l Cultivated on a shaker at 28 ° C for 24 hours. For the formation and accumulation of essential oil, the inoculum in an amount of 5% was introduced into flasks with a liquid nutrient medium of the following composition (g / l): soy flour - 20.0, sucrose - 20.0, distilled water to 1 liter. Incubation was carried out at 28 ° C on a shaker (220 rpm) for 72 hours. The essential oil was extracted with hexane. The amount of aromatic product reached 293.1 mg / L. The composition of aromatizing compounds, wt. %: geraniol 53.16, citronellol 12.20, nerol 10.67, β-phenylethanol 23.97.

Bibliography

1. Kovacheva, N. Industrial cultivation of oil bearing rose and rose oil production in Bulgaria during 21 ^{st century, directions and challenges / N.} Kovacheva, K. Rusanov, I. Atanassov // Biotechnol. & Biotechnol. Eq. - 2010. - No. 2. - P. 1793-1798.

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3. Erdogan, G. Turkey rose oil production and marketing: a review on problem and opportunities / G. Erdogan // Journal of Applied Sciences. - 2005. - Vol. 5, No. 10. - P. 1871-1875.

4. Research and current profile of Iranian production of damask rose {Rosa damascena Mill.) / M. Haghighi, A. Tehranifar, A. Nikbakht, M. Kafi // Acta horticulturae. - 2008. - Vol. 769. - P. 499-455.

5. Egorova, N.A. Some results and prospects of biotechnological studies of essential oil plants / N.A. Egorova, I.V. Stavtseva // Essential oil plants and medicinal plants. Scientific works of the Institute of essential oil and medicinal plants of the Ukrainian Academy of Agricultural Sciences. - 2006. - Issue 26. - S. 19-26.

6. A.S. 1794948 USSR Strain of the mushroom Ashbya gossypii - producer of essential oil / Semenova E.F., Bugorsky P.S., Radzimovskaya S.B. (THE USSR). Claim 08.23.90 (application No. 4862283 with the priority date of the invention on 08.23.1990). It is registered in the State register of inventions of the USSR on 08.10.1992, Publ. 02/15/93, BI No. 6.

7. A.S. 1421765 USSR A method for producing a mixture of fragrant substances with a rose smell / Bugorsky P.S., Semenova E.F., Rodov B.C., Mironov V.A. (USSR) Decl. 05.22.87 (application No. 4246910 with the priority date of the invention on 05.22.1987). Registered in the State Register of Inventions of the USSR 05/08/1988 Publ. 09/07/88, BI No. 33.

8. A.S. 1698276 USSR A method for producing an aromatic product with the smell of a rose / Semenova E.F., Bugorsky P.S. (THE USSR). Claim 07.20.88 (application No. 4600171 with the priority date of the invention on 07.20.1988). It is registered in the State register of inventions of the USSR on August 15, 1991. Publ. 12/15/91, BI No. 46.

9. A.S. 1454845 USSR Strain of the fungus Eremothecium ashbyi - producer of essential oil / Semenova E.F., Rodov B.C., Bugorsky P.S. (USSR) Decl. 07/28/87 (application No. 4291537 with the priority date of the invention 07/28/1987). It is registered in the State register of inventions of the USSR on 01.10.1988. Publ. 01/30/89, BI No. 4.

10. The yeast, a taxonomic study. Ed. by Kurtzman C.P., Fell J. W. Fourth Edition. - Elsevier Science, 1998 .-- 1055 p.

Claims (1)

The strain of the fungus Eremothecium ashbyi BKM F-4566D is a producer of essential oil, similar to Bulgarian rose.