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WO2004057012A1 - BLAKESLEA TRISPORA TKST (+) AND (-) HIGH-YIELD STRAIN-PRODUCER OF ß-CAROTENE - Google Patents

BLAKESLEA TRISPORA TKST (+) AND (-) HIGH-YIELD STRAIN-PRODUCER OF ß-CAROTENE Download PDF

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Publication number
WO2004057012A1
WO2004057012A1 PCT/UA2003/000024 UA0300024W WO2004057012A1 WO 2004057012 A1 WO2004057012 A1 WO 2004057012A1 UA 0300024 W UA0300024 W UA 0300024W WO 2004057012 A1 WO2004057012 A1 WO 2004057012A1
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Prior art keywords
strain
tkst
carotene
producer
milliliter
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/UA2003/000024
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French (fr)
Inventor
Aleksey A. Turenkov
Yuriy D. Turiyanskiy
Eugenia A. Kunschikova
Inna S. Kunschikova
Alla S. Sten'ko
Nadiya K. Bezkorovayna
Margaryta S. Gorna
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Research And Production Enterprise 'vitan' Ltd
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Research And Production Enterprise 'vitan' Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
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Application filed by Research And Production Enterprise 'vitan' Ltd filed Critical Research And Production Enterprise 'vitan' Ltd
Priority to AU2003265218A priority Critical patent/AU2003265218A1/en
Publication of WO2004057012A1 publication Critical patent/WO2004057012A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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

  • the invention is applicable in medicine, various branches of the food processing industry, and farming.
  • the most active natural antioxidants are carotinoid pigments with unique ⁇ -carotene among them. This antioxidant number 1 is the most biologically active.
  • the condition of the respiratory and cardiovascular systems, liver, and kidneys is in accordance with norms; blood condition is as follows: quantity of gamma globulin, erythrocytes, leucocytes, White Blood Count (WBC) , blood coagulation during the time of fibrin formation, content of total blood protein (serum protein) in Serum of blood are in accordance with the norm. Cumulative effect, teratogen and embryo toxic effects are absent .
  • WBC White Blood Count
  • strain VSD-1 was obtained by means of 30% oil suspension of the crystal ⁇ - carotene and riphampicin in concentration of 500 microgram/milliliter.
  • the strain had higher activity of ⁇ - carotene on the background of some technological changes during the fermentation.
  • the traditional estimation of the strain effectiveness is its biosynthetically activity expressed in weight units of the target product per 100 milliliter of the cultured liquid in gram/liter or in microgram/100 milliliter.
  • This indicator does not fully reflect an objective character of the ' effectiveness and some other significant biotechnological indicators. In particular an ability of biomass accumulation and the duration of the biosynthesis are not taken into consideration, which crucially affects final technical and economic indices of the industrial production. Taking into account everything said above and also the increasing requirements of the international market, the effectiveness evaluation of the new strains is shifted towards the strain productivity.
  • the productivity indicator was considered to be the foreground criterion for the new strain evaluation expressed in kilograms (kg) of the target product, obtained per unit of the geometrical volume of the fermenter (m 3 ) per ⁇ unit of time (an hour) : (kg/m 3 /hr) .
  • This can be expressed in a formula:
  • the strain possesses following cultural-morphological and physiological-biochemical qualities :
  • (+) form has well developed air mycelium ranging from gray to yellow color, substrate mycelium is well developed, compact, yellow with intensive spore-bearing (stylo sporangium - 50-100 mkm; sporangium - elliptic-spherical, 12- 16 - 10-14 mkm, three-spore; sporangium spores - elliptical, 14-18 mkm, light-snuff color with prolonged stripes.
  • (-) form has developed yellow air mycelium; solid substrate mycelium, which in tern has an intensive color ranging from yellow to orange. Spore bearing is less pronounced than in (+) sexual form.
  • mycelium has a high number of fruit sprouts, well-developed with typical apical growth (physiologically active culture) .
  • Mycelium is filled with ⁇ -carotene in crystals, and also with ⁇ -carotene yellow drops dissolved in fat.
  • Mycelium reacts well to methylene blue: young culture turns blue, old culture turns indigo.
  • Optimal vivifying environments are wort-agar and 5-6% corn-steep agar. The growth is worse in mold agar, Chapek environment, and MPA.
  • Sterilization period of the environment in autoclave at 120-122°C is 40 minutes . Sowing environments are poured into 750 milliliter retorts - 150 milliliter per ( ⁇ ) form - sowed and grown during 48 hours at the rockers (200-240 turn/minute) at 28°C. Then the biomass accumulation is determined, and subject to this, fermentation environments are sowed in 1:1,5 and 1:9 ratios.
  • the biomass has been filtered, rinsed, and dried.
  • the productivity has been determined from the earlier formula.
  • results obtained in the industrial production from the TKST strain indicate that its productivity ranges from 0,032 to 0,041 kg/m 3 /hr, which exceeds the productivity of the prototype strain (0,011 - 0,018 kg/m 3 /hr) 3 - 3,5 times .

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
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  • General Engineering & Computer Science (AREA)
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  • Tropical Medicine & Parasitology (AREA)
  • Biomedical Technology (AREA)
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  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
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Abstract

The invention is applicable in medicine, various branches of the food processing industry, and farming. The objective of invention is a selection of high-yield strain - producer of (ß - carotene, with more effective biomass accumulation and biosynthesis duration characteristics (kg/m3/hr). This reflects a genuine situation of the bioengineering processes. Strain TKST (+) and TKST (-) selection was obtained by means 10 of 20 % diphenylamine and sodium of the nicotinic acid 1000-­1300 microgram/milliliter. On average, obtained TKST strain productivity equals 0,036, which exceeds that of the strain­prototype 3-3,5 times (0,011). This conforms to the requirements of the international market of bioengineering products.

Description

BLΑKESLEA TRISPORA TKST (+) AND (-) HIGH-YIELD STRAIN-PRODUCER OF β - CAROTENE
The invention is applicable in medicine, various branches of the food processing industry, and farming.
The most active natural antioxidants are carotinoid pigments with unique β-carotene among them. This antioxidant number 1 is the most biologically active. The presence of the coupled binary ties C=C determines the absorption of the various free radicals, protecting cell from mutagens and cancerogens (carcinogens) , ionizing radiation, and chemical compounds, reduces oxidation of the membrane lipids, and evades the damage of the membrane-connected ferments, system of the endoplasm reticulum, cytochrome-450, β-carotene- immunomodulator, has an anti-inflammatory effect and vitamin properties .
It is widely applied in the following branches of the food processing industry; confectionary, meat and dairy, perfumery, bakery and macaroni as an additive to child' s nourishment. It is used in poultry, fish, and fur farming and in agriculture as an additive to the ration of the dairy and meat cattle. β-carotene of microbiological origin is biologically justified (96% - 98% β-carotene has the most active trans- formula) . It is not toxic, which has been tested on animals of different classes. The condition of the respiratory and cardiovascular systems, liver, and kidneys is in accordance with norms; blood condition is as follows: quantity of gamma globulin, erythrocytes, leucocytes, White Blood Count (WBC) , blood coagulation during the time of fibrin formation, content of total blood protein (serum protein) in Serum of blood are in accordance with the norm. Cumulative effect, teratogen and embryo toxic effects are absent .
Industrial production of β-carotene started with Blakeslea trispora 64 (+) TΛ 490 (-) strains. Activity of the present strain (according to the certificate) equaled 70000 - 100000 units/100 milliliter of the culture liquid.
Rolling into beads was distinguishing in the strain's growth nature, which worsened mass exchange and reduced activity to a considerable extent. Later strain 8A (+) and 8A (-) has been obtained, whose activity was 24% higher than that of the prototype. Then there were selection strains Kl (+) - Kl (-) and K2 (+) - K2 (-) , which considerably differed from the strain-prototype. Their activity was 1,5 - 2 times more than that of 8A(-) and 8A (+) , and the duration of the biosynthesis process has been reduced from 112 to 72 hours. Furthermore, a cell wall of the present strains possessed less chitin and was more open to the activity of the β-carotene extracting substances; hence, they were more technological. Finally, strain VSD-1 was obtained by means of 30% oil suspension of the crystal β- carotene and riphampicin in concentration of 500 microgram/milliliter. The strain had higher activity of β- carotene on the background of some technological changes during the fermentation. The traditional estimation of the strain effectiveness is its biosynthetically activity expressed in weight units of the target product per 100 milliliter of the cultured liquid in gram/liter or in microgram/100 milliliter. This indicator does not fully reflect an objective character of the ' effectiveness and some other significant biotechnological indicators. In particular an ability of biomass accumulation and the duration of the biosynthesis are not taken into consideration, which crucially affects final technical and economic indices of the industrial production. Taking into account everything said above and also the increasing requirements of the international market, the effectiveness evaluation of the new strains is shifted towards the strain productivity.
At present, the productivity indicator was considered to be the foreground criterion for the new strain evaluation expressed in kilograms (kg) of the target product, obtained per unit of the geometrical volume of the fermenter (m3) per ■ unit of time (an hour) : (kg/m3/hr) . This can be expressed in a formula:
Vτ '
Q - quantity of β-carotene in dried biomass, kilogram; V - geometrical volume of the fermenter, m3; τ - fermentation duration, hour.
The strains TKST (+) and TKST (-), descending from Kl (+) and Kl (-) , have been obtained from the fungus selection by means of 20% diphenylamine (m.m. 169, 2 Sigma ) with the further treatment by sodium of the nicotinic acid 1000-1300 microgram/milliliter (m.m. 145,1 Sigma -Aldrich) . The selection has been carried out simultaneously on two (+) and (-) sexual forms. The strain possesses following cultural-morphological and physiological-biochemical qualities :
(+) form has well developed air mycelium ranging from gray to yellow color, substrate mycelium is well developed, compact, yellow with intensive spore-bearing (stylo sporangium - 50-100 mkm; sporangium - elliptic-spherical, 12- 16 - 10-14 mkm, three-spore; sporangium spores - elliptical, 14-18 mkm, light-snuff color with prolonged stripes.
(-) form has developed yellow air mycelium; solid substrate mycelium, which in tern has an intensive color ranging from yellow to orange. Spore bearing is less pronounced than in (+) sexual form.
During the concurrent culturing of (+) and (-) sexual forms on Petri plates substrate mycelium in the contact points has a wide 3 - 3,5 centimeter zone ranging in color from intensive orange to crimson, with well pronounced features of (Car+) , and absent phenotype features of sexual interaction (Zyg~) .
During the deep-laid culturing mycelium has a high number of fruit sprouts, well-developed with typical apical growth (physiologically active culture) . Mycelium is filled with β-carotene in crystals, and also with β-carotene yellow drops dissolved in fat. Mycelium reacts well to methylene blue: young culture turns blue, old culture turns indigo. Optimal vivifying environments are wort-agar and 5-6% corn-steep agar. The growth is worse in mold agar, Chapek environment, and MPA.
For deep-laid culturing the sowing and fermentation environments of the following content, are used (%) :
Soy flour - 2,3 Green treacle - 2,5-4,5
Corn flour - 4,7 Corn extract - 4,0-8,0
KH2 P04 - 0,05 Sunflower seed oil - 3,0-5,0 •Vitamin Bi (triamine) - 0,0002
Tap water pH = 6,2 - 6,4 Tap water pH = 6,2 - 6,4
Sterilization period of the environment in autoclave at 120-122°C is 40 minutes . Sowing environments are poured into 750 milliliter retorts - 150 milliliter per (±) form - sowed and grown during 48 hours at the rockers (200-240 turn/minute) at 28°C. Then the biomass accumulation is determined, and subject to this, fermentation environments are sowed in 1:1,5 and 1:9 ratios.
Fermentation environments are also poured into 750 milliliter retorts in quantity of 50 milliliter and are brought up under the same conditions. Activities have been determined on FEK - 56 M, KFL - UHL - 42, and KFK - 3 with a blue optical filter (λ =451 nm) , in 1-centimeter cuvettes.
The biomass has been filtered, rinsed, and dried. The productivity has been determined from the earlier formula.
Results obtained in the industrial production (data from 10 operations) , q, kg/m3/hr.
Strain-prototype Strain TKST
0,01 0,034 0,0089 0,035
0,011 0,039
0,013 0,041
0,016 0,036
0,018 0,037 0,005 0,039
0,008 0,037
0,012 0,032
0,011 0,036
Thereby, results obtained in the industrial production from the TKST strain indicate that its productivity ranges from 0,032 to 0,041 kg/m3/hr, which exceeds the productivity of the prototype strain (0,011 - 0,018 kg/m3/hr) 3 - 3,5 times .

Claims

Blakeslea trispora, strain TKST - a high-yield strain- producer of β-carotene with productivity of 0,032 - 0,041 kg/m3/hr.
PCT/UA2003/000024 2002-12-23 2003-07-07 BLAKESLEA TRISPORA TKST (+) AND (-) HIGH-YIELD STRAIN-PRODUCER OF ß-CAROTENE Ceased WO2004057012A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003265218A AU2003265218A1 (en) 2002-12-23 2003-07-07 BLAKESLEA TRISPORA TKST (+) AND (-) HIGH-YIELD STRAIN-PRODUCER OF ss-CAROTENE

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
UA20021210432A UA55331C2 (en) 2002-12-23 2002-12-23 A STRAIN (ТКSТ) BLAKESLEA TRISPORA ІМВ F-100022 PRODUCER OF 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--CAROTENE
UA20021210432 2002-12-23

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7851199B2 (en) 2005-03-18 2010-12-14 Microbia, Inc. Production of carotenoids in oleaginous yeast and fungi
US8691555B2 (en) 2006-09-28 2014-04-08 Dsm Ip Assests B.V. Production of carotenoids in oleaginous yeast and fungi

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
UA85489C2 (en) * 2007-12-20 2009-01-26 Товариство З Обмеженою Відповідальністю "Науково Виробниче Підприємство "Вітан" Mucoraceous mold blakeslea trispora strain pht 1+, pht 1- - the producer of phytoine

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1059664A (en) * 1964-08-17 1967-02-22 Rhone Poulenc Sa Process for the production of -ß-carotene
RU2053301C1 (en) * 1993-12-15 1996-01-27 Государственный научно-исследовательский институт генетики и селекции промышленных микроорганизмов Pair strains of heterothallic fungus blakeslea trispora f - 674(+) and f-551 (-) producing beta-carotene
RU2177505C2 (en) * 2000-02-15 2001-12-27 ЗАО "Роскарфарм" Two strains of heterothallic fungus blakeslea trispora kp74+ and kp 86- producing beta-carotene

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1059664A (en) * 1964-08-17 1967-02-22 Rhone Poulenc Sa Process for the production of -ß-carotene
RU2053301C1 (en) * 1993-12-15 1996-01-27 Государственный научно-исследовательский институт генетики и селекции промышленных микроорганизмов Pair strains of heterothallic fungus blakeslea trispora f - 674(+) and f-551 (-) producing beta-carotene
RU2177505C2 (en) * 2000-02-15 2001-12-27 ЗАО "Роскарфарм" Two strains of heterothallic fungus blakeslea trispora kp74+ and kp 86- producing beta-carotene

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7851199B2 (en) 2005-03-18 2010-12-14 Microbia, Inc. Production of carotenoids in oleaginous yeast and fungi
US8288149B2 (en) 2005-03-18 2012-10-16 Dsm Ip Assets B.V. Production of carotenoids in oleaginous yeast and fungi
US9909130B2 (en) 2005-03-18 2018-03-06 Dsm Ip Assets B.V. Production of carotenoids in oleaginous yeast and fungi
US8691555B2 (en) 2006-09-28 2014-04-08 Dsm Ip Assests B.V. Production of carotenoids in oleaginous yeast and fungi
US9297031B2 (en) 2006-09-28 2016-03-29 Dsm Ip Assets B.V. Production of carotenoids in oleaginous yeast and fungi

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UA55331A (en) 2003-03-17
UA55331C2 (en) 2006-07-17

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