WO2009082365A1 - The strain of fungus blakeslea trispora tkst culture pht 1+, pht 1- producer of phytoene - Google Patents

The strain of fungus blakeslea trispora tkst culture pht 1+, pht 1- producer of phytoene Download PDF

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WO2009082365A1
WO2009082365A1 PCT/UA2008/000069 UA2008000069W WO2009082365A1 WO 2009082365 A1 WO2009082365 A1 WO 2009082365A1 UA 2008000069 W UA2008000069 W UA 2008000069W WO 2009082365 A1 WO2009082365 A1 WO 2009082365A1
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phytoene
pht
strain
producer
fungus
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Galyna Dmytrenko
Alla Stenko
Margaryta Gorna
Yuriy Turiyansky
Aleksey Tyurenkov
Vladimir Tyurenkov
Iaroslav Soroka
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'research And Production Enterprise 'vitan' Ltd.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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    • A23L33/155—Vitamins A or D
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • This invention relates to biotechnology, specifically, to the production of natural carotenoid phytoene with microbiological synthesis that can be used in medicine, food, and cosmetic industries.
  • Phytoene is the natural carotenoid with an expressed biotechnological activity, possessing a set of multiple valuable properties, a considerable pharmaceutical potential (treatment and prevention of diseases of respiratory tract, gastrointestinal tract, cardiovascular system, vision; photo-protecting and anti-carcinogen antioxidant properties), and widely used in food and cosmetic industries. Specifically, in cosmetic industry, it can be used as a factor preventing deep UV rays penetration in skin, skin burns, contributes to skin rejuvenation [1-4] .
  • Phytoene (7, 8, 11, 12, 7', 8 1 , II 1 , 12 ' -octahydro- ⁇ - ⁇ -carotene) is a colorless carotenoid, having acyclic isoprenoid chain C 40 H 64 . It is a lycopene and R-carotene biosynthesis precursor: phytoene - phytophluene - C- carotene - neurosporene - lycopene - R-carotene.
  • Phytoene is synthesized of two geranyl-geranyl pyrophosphate (GGPP) molecules with the use of phytoene synthetase; geranyl-geranyl pyrophosphate is formed through mevalonic acid conversion or pyruvate and glyceraldehyde-3- phosphate condensation.
  • GGPP geranyl-geranyl pyrophosphate
  • Phytoene unlike lycopene and ⁇ -carotene, is light- and oxidation-resistant, which provides for certain advantages in its use.
  • Phytoene can be produced from higher plants and algae. Of them, the most readily available source is Dunaliella species.
  • Dunaliella sp. can be cultivated in open salt lakes with intensive insolation, and in conditions of cultivation in fermenters . It synthesizes 1 to 30 mg/1 of phytoene.
  • the Israeli scientists succeeded in chemical mutagenesis of white modifications of Dunaliella sp. capable of synthesizing more phytoene [5], but only together with phytophluene .
  • the purpose of this invention is to obtain phytoene producer strain with filamentous fungus Blakeslea trispora, which presently is a lycopene and R-carotene producer .
  • Phtl + and Phtl by means of the fungus Blakeslea trispora selection using diphenylamine (5-10 mg/ml ) . Characteristics of Phtl + and Phtl " strains
  • the fungus Blakeslea trispora belongs to mucoraceous fungi of Mucoraceae class (Choanephora re-identified to Blakeslea trispora species, based on research by Kirka in 1984 described in indicator "A monograph of the Choanephoraceae . Mycological paper, Kew, England, 1984, No.152, p. 1-61) .
  • the strain was deposited in the Collection of industrial microorganisms of D. Zabolotny Institute for Microbiology and Virology of the National Academy of Sciences of Ukraine under No. F-100053
  • Pht I + mating form is a well-developed aerial mycelium of grayish-white to grayish-yellow color.
  • the substrate mycelium is of the same colour, well-developed, dense, with a poor spore production.
  • Pht 1 mating form is a developed white aerial mycelium with massive asexual spore production, solely sporangial, presented by stylosporangia and sporangia. Sporangia are 12x16 - 10x12 ⁇ m elliptic-spherical, trisporic, multiple. The substrate mycelium grows into the medium, is sticky and hard to separate from the agarized medium.
  • wort agar the optimal nutritive medium for the fungus, the fungi grow well on 6-5% corn agar, beef-extract agar, and grow worse on Czapek's medium.
  • the optimal temperature is 28 °C.
  • the fungus is cultivated on wort agar slants or on Petri dishes during 7-10 days. To preserve the fungus culture, specially treated and sterilized soil is used. For deep cultivation (in flasks) the following inoculation and fermentation media are used.
  • the media are poured in 750 ml flasks, 100 ml in each. Both media are sterilized in an autoclave during 40 min at a temperature of 120-122 0 C.
  • the strain is prototroph.
  • the phytoene concentration is determined as follows: a 4-8 mg portion of phytoene biomass is put into a flask with 0,5-0,6 g of anhydrous Na 2 SO 4 , 5 cm of hexane is added, the biomass is thoroughly grinded with a glass stick, the flask is plugged with a cork or polypropylene plug and left for extraction in a dark place for 1 hour. After that, the biomass suspension is re-grinded with anhydrous sodium sulphate and stirred on a vortex mixer.
  • V the volume of the dilution, cm 3 ;
  • m the sample portion mass, g;
  • a 1% - 1% solution extinction, for phytoene dissolved in hexane it is 915 ( ⁇ 286 nm) .
  • the biomass moisture is determined: a 0,15-0,20 g biomass portion is weighed accurate to 5 th digit and dried in a drying cabinet during 40-45 min. After drying, the sample is weighed and the moisture is determined.
  • the phytoene concentration is depended on a proportion between two mating forms and the fermentation time.
  • the ratio of 1:5 at 7-day fermentation proved to be optimal.
  • the yield of phytoene was 1,5 to 2,2 g/1 of the fermentation broth, or 10-15 % concentrations.

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Abstract

The strain of fungus Blakeslea trispora - a producer phytoene is selected; are investigated its morphological, cultural, physiological and biochemical properties. The strain is deposited in depository Institute of microbiology and virology by it. D. K. Zabolotnogo NAS Ukraine No.F-100053.

Description

THE STRAIN OF FUNGUS BLAKESLEA TRISPORA STRAIN PHT I+, PHT 1" PHYTOENE PRODUCER
This invention relates to biotechnology, specifically, to the production of natural carotenoid phytoene with microbiological synthesis that can be used in medicine, food, and cosmetic industries.
Phytoene is the natural carotenoid with an expressed biotechnological activity, possessing a set of multiple valuable properties, a considerable pharmaceutical potential (treatment and prevention of diseases of respiratory tract, gastrointestinal tract, cardiovascular system, vision; photo-protecting and anti-carcinogen antioxidant properties), and widely used in food and cosmetic industries. Specifically, in cosmetic industry, it can be used as a factor preventing deep UV rays penetration in skin, skin burns, contributes to skin rejuvenation [1-4] .
Phytoene (7, 8, 11, 12, 7', 81, II1, 12 ' -octahydro-γ- γ-carotene) is a colorless carotenoid, having acyclic isoprenoid chain C40H64. It is a lycopene and R-carotene biosynthesis precursor: phytoene - phytophluene - C- carotene - neurosporene - lycopene - R-carotene. Phytoene is synthesized of two geranyl-geranyl pyrophosphate (GGPP) molecules with the use of phytoene synthetase; geranyl-geranyl pyrophosphate is formed through mevalonic acid conversion or pyruvate and glyceraldehyde-3- phosphate condensation. Phytoene, unlike lycopene and β-carotene, is light- and oxidation-resistant, which provides for certain advantages in its use.
Phytoene can be produced from higher plants and algae. Of them, the most readily available source is Dunaliella species.
Dunaliella sp. can be cultivated in open salt lakes with intensive insolation, and in conditions of cultivation in fermenters . It synthesizes 1 to 30 mg/1 of phytoene. The Israeli scientists succeeded in chemical mutagenesis of white modifications of Dunaliella sp. capable of synthesizing more phytoene [5], but only together with phytophluene .
The microorganisms' capability of synthesizing carotenoids was known, but this was only synthesis of a complex set of carotenoids. Over 600 different carotenoids and sources of their origin are known, but only a small part of the said sources can be used as a producer of biologically active substances.
Presently, no phytoene producers among microorganisms are known.
The purpose of this invention is to obtain phytoene producer strain with filamentous fungus Blakeslea trispora, which presently is a lycopene and R-carotene producer .
We produced Phtl+ and Phtl" by means of the fungus Blakeslea trispora selection using diphenylamine (5-10 mg/ml ) . Characteristics of Phtl+ and Phtl" strains
The fungus Blakeslea trispora belongs to mucoraceous fungi of Mucoraceae class (Choanephora re-identified to Blakeslea trispora species, based on research by Kirka in 1984 described in indicator "A monograph of the Choanephoraceae . Mycological paper, Kew, England, 1984, No.152, p. 1-61) . The strain was deposited in the Collection of industrial microorganisms of D. Zabolotny Institute for Microbiology and Virology of the National Academy of Sciences of Ukraine under No. F-100053
Morphological Properties
Pht I+ mating form is a well-developed aerial mycelium of grayish-white to grayish-yellow color. The substrate mycelium is of the same colour, well-developed, dense, with a poor spore production.
Pht 1" mating form is a developed white aerial mycelium with massive asexual spore production, solely sporangial, presented by stylosporangia and sporangia. Sporangia are 12x16 - 10x12 μm elliptic-spherical, trisporic, multiple. The substrate mycelium grows into the medium, is sticky and hard to separate from the agarized medium.
Joint cultivation of (+) and (-) fertile forms yields no zygospore formation zone, but a wide white zone is created.
Physiological properties: wort agaris the optimal nutritive medium for the fungus, the fungi grow well on 6-5% corn agar, beef-extract agar, and grow worse on Czapek's medium. The optimal temperature is 28 °C. The fungus is cultivated on wort agar slants or on Petri dishes during 7-10 days. To preserve the fungus culture, specially treated and sterilized soil is used. For deep cultivation (in flasks) the following inoculation and fermentation media are used.
Inoculation medium, g/1: corn flour - 47,0; soya flour -28,0; KH2PO4 - 0,5; piped water - 1,0 1; pH=6,3- 6,4.
Fermentation medium, g/1: corn flour - 14,0; soya flour - 33,0; oil - 26,5; piped water - 1,0 1; pH=6,3- 6,4.
The media are poured in 750 ml flasks, 100 ml in each. Both media are sterilized in an autoclave during 40 min at a temperature of 120-1220C. The strain is prototroph.
Obtaining a target product - phytoene - is illustrated by the following examples.
Example 1
Producing phytoene through cultivation of Blakeslea trispora fungi Phtl+ and Phtl" under laboratory conditions .
For joint cultivation of 7 -day old agar discs with (+) and (-) fertile forms are applied polarIy on Petri dishes and cultivated during 7 days. A wide white strip without zygospore formation is created on the contact area of 2 fertile forms. The 5-7 mg portions were taken from this zone, extracted into hexane, and phytoene was detected using a spectophotometer .
The phytoene concentration is determined as follows: a 4-8 mg portion of phytoene biomass is put into a flask with 0,5-0,6 g of anhydrous Na2SO4, 5 cm of hexane is added, the biomass is thoroughly grinded with a glass stick, the flask is plugged with a cork or polypropylene plug and left for extraction in a dark place for 1 hour. After that, the biomass suspension is re-grinded with anhydrous sodium sulphate and stirred on a vortex mixer. The optical density at a wave length of λ=286 nm is measured with a spectophotometer (if the optical density exceeds 2, the solution shall be diluted with hexane, and the volume of dilution shall be taken into account when calculating the concentration) . Pure hexane is used for the control .
Results processing
% concentration (C) is calculated by the formula:
✓"1 D -V
Figure imgf000006_0001
in which:
D - the optical density of the solution under investigation;
V - the volume of the dilution, cm3; m - the sample portion mass, g;
1 - the measuring cuvette thickness, cm;
A1% - 1% solution extinction, for phytoene dissolved in hexane it is 915 (λ=286 nm) . Simultaneously, the biomass moisture is determined: a 0,15-0,20 g biomass portion is weighed accurate to 5th digit and dried in a drying cabinet during 40-45 min. After drying, the sample is weighed and the moisture is determined.
Similar portions of each mating form cultivated separately were used for the control. If the phytoene concentration in separately cultivated (+) and (-) fertile forms was 0,35-0,38 %, the joint cultivation concentration was 6 to 11-15%. That is, the joint cultivation phytoene concentration was 30 times higher than that of the separate cultivation of the forms.
The phytoene absorption spectrum in our research was compared to the data presented by G. Britton et al .
The phytoene spectra obtained when scanning extracts from the 'biomass of the strains Phtl+ and Phtl" on a Helios spectophotometer γ, λτax: 276; 286; 297 (Fig. 1) and the spectrum presented by G. Britton et al . [6] (Fig. 2) .
Example 2
Production of phytoene when cultivating Pht I+ and Pht 1~ in flasks and on shakers. The spore-mycelium suspension washed off the 7-day old slants was used to inoculate the maternal flasks with the medium described above. Cultivation was continued on shakers at 180- 200 rpm and t°=28°C. The biomass accumulation was determined. The flasks with the fermentation medium were inoculated with different proportions of (+) and (-) mating forms - 1:1,5; 1:3; 1:5; 1:7, respectively, and cultivated during 3 to 7 days.
The phytoene concentration is depended on a proportion between two mating forms and the fermentation time. The ratio of 1:5 at 7-day fermentation proved to be optimal. The yield of phytoene was 1,5 to 2,2 g/1 of the fermentation broth, or 10-15 % concentrations.
References
1. Villadsen I. A method of generating high carotenoid-producing microorganisms, microorganisms obtained by the method and a process for producing carotenoid containing cells or cell parts or purified carotenoid. WO 92/22648, 1992.
2. Harris A., Zelkha M., Paran E. Carotenoids as anti-hypertension agents. WO 02/058683 A2. 2002.
3. Zelkha ML, Nir Z., Sedlov T. Carotenoid compositium and method for protection skin. WO 03/041678 Al. 2002.
4. Zelkha M., Sedlov T., Nir L., An anti- atherosclerosis compositium containing carotenoids and method for inhibiting LdI oxidation. WO 02/058682 A2. 2002
5. Sondant E., Schickier H. et al . A carotenoid preparation. WO 00/13654. 2000.
6. Britton G., Liaaen-Jensen S., Pfander H. Carotenoids. - Basel: Birkhaeser Verlag, 329 p. - 2004.

Claims

1. The mucoraceous fungus Blakeslea trispora strain Phtl+, Phtl" - phytoene producer, characterized by deposition at D. Zabolotny Institute for Microbiology and Virology of Ukraine NAS under No. F-I 00053.
2. The mucoraceous fungus according to the claim 1 characterized by a phytoene yield of up to 10-15%.
3. The mucoraceous fungus according to the claim 1 characterized by a colorless substrate mycelium.
4. The mucoraceous fungus according to the claim 1 characterized by a massive asexual spore production.
5. The mucoraceous fungus according to the claim 1 characterized by a white zone without zygospore formation at a joint cultivation on a dish.
PCT/UA2008/000069 2007-12-20 2008-11-28 The strain of fungus blakeslea trispora tkst culture pht 1+, pht 1- producer of phytoene WO2009082365A1 (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105018410A (en) * 2015-06-15 2015-11-04 浙江汇能动物药品有限公司 Method for inducing Blakeslea trispora aging strain to rapidly produce a large number of spores
EP2935562A4 (en) * 2012-12-19 2016-08-10 Lycored Ltd Preparation for radio protection
WO2023062640A1 (en) * 2021-10-17 2023-04-20 Lycored Ltd. Phytoene-containing oleoresin and composition

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000013654A2 (en) * 1998-09-04 2000-03-16 I.B.R. Israeli Biotechnology Research, Ltd. A carotenoid composition containing phytoene and phytofluene
RU2177505C2 (en) * 2000-02-15 2001-12-27 ЗАО "Роскарфарм" Two strains of heterothallic fungus blakeslea trispora kp74+ and kp 86- producing beta-carotene
WO2003038064A2 (en) * 2001-10-29 2003-05-08 Dsm Ip Assets B.V. Blakeslea trispora producing high yield of lycopene in a suitable medium in the absence of an exogenous carotenogenesis inhibitor
UA55331C2 (en) * 2002-12-23 2006-07-17 Товариство З Обмеженою Відповідальністю "Науково-Виробниче Підприємство "Вітан" A STRAIN (ТКSТ) BLAKESLEA TRISPORA ІМВ F-100022 PRODUCER OF 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--CAROTENE

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000013654A2 (en) * 1998-09-04 2000-03-16 I.B.R. Israeli Biotechnology Research, Ltd. A carotenoid composition containing phytoene and phytofluene
RU2177505C2 (en) * 2000-02-15 2001-12-27 ЗАО "Роскарфарм" Two strains of heterothallic fungus blakeslea trispora kp74+ and kp 86- producing beta-carotene
WO2003038064A2 (en) * 2001-10-29 2003-05-08 Dsm Ip Assets B.V. Blakeslea trispora producing high yield of lycopene in a suitable medium in the absence of an exogenous carotenogenesis inhibitor
UA55331C2 (en) * 2002-12-23 2006-07-17 Товариство З Обмеженою Відповідальністю "Науково-Виробниче Підприємство "Вітан" A STRAIN (ТКSТ) BLAKESLEA TRISPORA ІМВ F-100022 PRODUCER OF 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--CAROTENE

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2935562A4 (en) * 2012-12-19 2016-08-10 Lycored Ltd Preparation for radio protection
CN105018410A (en) * 2015-06-15 2015-11-04 浙江汇能动物药品有限公司 Method for inducing Blakeslea trispora aging strain to rapidly produce a large number of spores
CN105018410B (en) * 2015-06-15 2019-02-19 甘肃汇能生物工程有限公司 A method of the mould aging bacterial strain rapid, high volume of three spore cloth Laplaces of induction produces spore
WO2023062640A1 (en) * 2021-10-17 2023-04-20 Lycored Ltd. Phytoene-containing oleoresin and composition

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