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ARACHIDONIC ACID ANDOTHER FATTY ACID FROM
MICROORGANISM
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Arachidonic acid
Is a polyunsaturated fatty acid (PUFA) that ispresent in the phospholipids (especiallyphosphatidylethanolamine, phosphatidylcholineand phosphatidylinositides) ofmembranes of thebody's cells, and is abundant in the brain,muscles, liver.
in the human body usually comes from dietaryanimal sourcesmeat, eggs, dairyor issynthesized from linoleic acid.
essential fatty acids required by most mammals
http://en.wikipedia.org/wiki/Phospholipidhttp://en.wikipedia.org/wiki/Phosphatidylethanolaminehttp://en.wikipedia.org/wiki/Phosphatidylcholinehttp://en.wikipedia.org/wiki/Phosphatidylinositidehttp://en.wikipedia.org/wiki/Cell_membranehttp://en.wikipedia.org/wiki/Cell_%28biology%29http://en.wikipedia.org/wiki/Brainhttp://en.wikipedia.org/wiki/Muscleshttp://en.wikipedia.org/wiki/Liverhttp://en.wikipedia.org/wiki/Essential_fatty_acidhttp://en.wikipedia.org/wiki/Mammalhttp://en.wikipedia.org/wiki/Mammalhttp://en.wikipedia.org/wiki/Essential_fatty_acidhttp://en.wikipedia.org/wiki/Liverhttp://en.wikipedia.org/wiki/Muscleshttp://en.wikipedia.org/wiki/Brainhttp://en.wikipedia.org/wiki/Cell_%28biology%29http://en.wikipedia.org/wiki/Cell_membranehttp://en.wikipedia.org/wiki/Phosphatidylinositidehttp://en.wikipedia.org/wiki/Phosphatidylcholinehttp://en.wikipedia.org/wiki/Phosphatidylethanolaminehttp://en.wikipedia.org/wiki/Phospholipid7/28/2019 Arachidonic Oil
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Arachidonic acid (cont.)
Some mammals lack the ability to or have
a very limited capacity to convert linoleic
acid into arachidonic acid
need supplement commercial source of
arachidonic acid has been derived, from
the fungus Mortierella alpina
http://en.wikipedia.org/wiki/Linoleic_acidhttp://en.wikipedia.org/wiki/Linoleic_acidhttp://en.wikipedia.org/w/index.php?title=Mortierella_alpina&action=edit&redlink=1http://en.wikipedia.org/w/index.php?title=Mortierella_alpina&action=edit&redlink=1http://en.wikipedia.org/wiki/Linoleic_acidhttp://en.wikipedia.org/wiki/Linoleic_acid7/28/2019 Arachidonic Oil
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Similarity of ARA (top) with prostaglandin hormone
(bottom)
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The importance of Arachidonic acid
in body
repair and growth of skeletal muscle tissue
one of the most abundant fatty acids in thebrain (similar quantities to DHA
docosahexaenoic acid)
http://en.wikipedia.org/wiki/Docosahexaenoic_acidhttp://en.wikipedia.org/wiki/Docosahexaenoic_acid7/28/2019 Arachidonic Oil
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Microorganism for commercial
productionMortierellaalpina
Colony formation of filamentous fungi Mortierella. GenusMortierella could be classified into two subgenera Mortierella (A) and
Micromucor(B). Strains of subgenus Mortierella form rose-like colony
on agar plate. While all the strains of subgenus Mortierella can
produce C20 fatty acids, strains of subgenus Micromucorcan only
produce fatty acids up to C18.
(A) Mortierellaalpina 1S-4: (B) Mortierellaisabellina CBS 194.28.
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Growth ofMortierella fungi on agar plate containingtetrazolium salt. (A) When M. alpina 1S-4 grew on a agar medium
containing triphenyltetrazolium chloride (TCC), oils formed could be
stained red. (B) M. isabellina CBS 194.28 grown on the same
medium.
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Oil drops accumulated in the cells ofMortierella alpina 1S-
4. A large number of oil drops could be observed, when M.
alpina 1S-4 grown in a liquid medium
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Mortierella alpina
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Mortierella alpina
Oil
globule
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Other microorganisms
Genetic engineered Yarrowia lipolytica
capable of producing greater than 10%
arachidonic acid (ARA, an omega.-6
polyunsaturated fatty acid) in the total oilfraction (Patent 7588931 )
Mucor circinelloides (gamma linoleic acid)
Rhizopus sp.(gamma linoleic acid)
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Rhizopus spp Mucor circinelloides
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Mucor circinelloides colonies incubated in varioustemperature
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Biosynthesis of fat in
microorganism
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Production of ARA by
microorganisms
Submerged fermentation and Solid
substrate Solid substrate using rice bran, wheat
bran, peanut meal residue, and sweet
potato residue
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Production step
1. Culture Media and Conditions
2. Submerged Fermentation and Solid
state fermentation
3. Downstream processing
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Ad 1. Culture Media and Conditions
Mortierella was grown at 20C in a culture
containing (mg /l): glucose, 10; yeast
extract, 5 and agar, 20 at pH 6.5.
Mycelia were harvested from culture and
blended with a micro-Waring blender for
mycelial suspension.
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Ad 2. Submerged Fermentation
Submerged basal medium contained(mg/l) soluble starch, 20; Bacto yeastextract, 5; KNO3, 10; KH2PO4, 1 and
MgSO47H2O, 0.5 at pH 6.5. The broth was inoculated with 5% (v/v)
mycelial suspension and shaken at 200rev min-1 and at 20C for 2 to 10 days.Each ml of mycelial suspension contained1.0-1.5 106 mycelial fragments.
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Ad 2. solid state fermentation
Media contained (g) solid substrate (rice
bran, wheat bran, peanut meal residue,
sweet potato residue, or a mixture of
sweet potato residue and rice bran) 100;Bacto yeast extract (Difco, Michigan) 2.5;
KNO3 5; KH2PO4 0.5 and MgSO47H2O
0.25.
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Factors affecting ARA production
1. Initial moisture content
2. Initial pH3. Incubation temperature
4. Supplement of nitrogen
5. Supplement of oil
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Analyzing of fatty acids
The lipids were extracted with a 5 times volumeof chloroform/methanol (2:1, v/v) by anultrasonicator for 2 h and concentrated by rotaryevaporator at 50C.
The residue was dissolved in 1 ml of 0.5 MKOH-methanol solution, and methylated with 1ml of 20% (w/v) of BF3-methanol complex.
The methylated fatty acids were separated fromthe water layer by adding saturated NaCl andanhydrous Na2SO4, and then dissolving in n-hexane.
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Downstream Process
Cells were homogenized to break the cells
walls so that the products can be extracted
Extraction by butane crude fatty acids
are obtained
Purification of fatty acids by hexane and
citric acid followed by bleaching,
deodorization and filtration
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Procedure to
analyze fatty
acids from
microorganisms
to selectpotential
microorganisms
that produced
highest fatty acid
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Figure 1. Time course of biomass, pH, cell protein, and PUFA production in
submerged fermentation with Mortierella alpina ATCC 3222. Culture medium
was incubated at 20C with orbital shaking at 200 rev min-1.
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Figure 2. Time course of moisture content, pH, cell protein, and PUFA production in rice bran
solid substrate fermentation with Mortierella alpina ATCC 3222. Solid substrate with an initial
moisture content of 65% and an initial pH of 6.5 were statically incubated at 20C.
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Lipid formation and -linolenic acid production by
Mucor circinelloides and Rhizopus sp., grown on
vegetable oil The submerged cultivation were carried out in 250 mL Erlenmeyer
flasks with 50 mL of medium containing 1 - 4% oils (palm, canola,soybean oil that had been used for frying, sesame, or sunflower) or1 - 4% carbohydrates (galactose, maltose, malt extract, sorbitol) and1% yeast extract as nitrogen source.
Each flask was inoculated with 1 ml of freshly prepared spore
suspension. ForM. circinelloides, malt extract was also usedreplacing maltose in the medium.
The cultures were agitated continuously for 72 hours at 150 rpm, at25C and allowed to stand for 48 hours without agitation at 12C.
The biomass produced was separated using vacuum filtration in No.1 Whatman filter paper. The wet biomass was placed in pre-weighed
beakers at 105C for 48h to determine the dry weight. After part of the resulting biomass was set aside for later extraction
of the fatty acids; it was dried by storage for 5 days in an oven at55C.
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Downstream process (cont.)
Approximately 100 mg of mycelia were
used to extraction of lipid using
chloroform: methanol: water (2:1:0,8) and
the solvent removed in a nitrogenatmosphere.
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GLA = gamma linoleic acid, AA= arachidonic acid
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Docosahexaenoic acid (DHA)
is a long-chain polyunsaturated omega-3
fatty acid
important for brain, eye and heart health
throughout the lifecycle
Can be produced by algae vegetarian
DHA
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DHA's structure
a carboxylic acid(~oic acid) with a 22-carbon chain (docosa- is Greek for 22)and six (Greek "hexa") cisdouble bonds (-
en~); the first double bond is located at thethird carbon from the omega end.
Its trivial name is cervonic acid,its systematic name is all-cis-docosa-4,7,10,13,16,19-hexa-enoic acid,
shorthand name is 22:6(n-3)
http://en.wikipedia.org/wiki/Carboxylic_acidhttp://en.wikipedia.org/wiki/Carbon_chainhttp://en.wikipedia.org/wiki/Greek_languagehttp://en.wikipedia.org/wiki/Greek_languagehttp://en.wikipedia.org/wiki/Cis-trans_isomerismhttp://en.wikipedia.org/wiki/Double_bondhttp://en.wikipedia.org/wiki/Trivial_namehttp://en.wikipedia.org/wiki/Systematic_namehttp://en.wikipedia.org/wiki/Systematic_namehttp://en.wikipedia.org/wiki/Trivial_namehttp://en.wikipedia.org/wiki/Double_bondhttp://en.wikipedia.org/wiki/Cis-trans_isomerismhttp://en.wikipedia.org/wiki/Greek_languagehttp://en.wikipedia.org/wiki/Greek_languagehttp://en.wikipedia.org/wiki/Carbon_chainhttp://en.wikipedia.org/wiki/Carboxylic_acid7/28/2019 Arachidonic Oil
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Applications and function
infant formulas, products for pregnant and
nursing women, food and beverage
products and dietary supplements.
DHA possesses a variety of immune
modulating effects.
DHA was found to inhibit growth of human
colon carcinoma cells, more than otheromega-3 PUFAs.
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Source of DHA from microbial
process
microalgae; Crypthecodinium cohniiand
another of the genus Schizochytrium
(example : Schizochrytium limacinum)
Pavlova lutheriseawater media atau
synthetic medium
Isochrysis galbana
Phytium irregulare
etc
http://en.wikipedia.org/wiki/Crypthecodinium_cohniihttp://en.wikipedia.org/w/index.php?title=Schizochytrium&action=edit&redlink=1http://en.wikipedia.org/w/index.php?title=Schizochytrium&action=edit&redlink=1http://en.wikipedia.org/wiki/Crypthecodinium_cohnii7/28/2019 Arachidonic Oil
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Schematic representation of
Crypthecodinium cohnii cell
drawn from Perret et al. (1991). A Ventralview. B Dorsal view. E
episome, H hyposome, L.F. longitudinal
flagellum, T.F. transverse
flagellum, C cingulum. Bar 5 m.
Reproduced with permission of the
Company of Biologists
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Scanning electron micropgraph of aCrypthecodinium species
showing the ventral view (from Parrow et
al. 2006). Reproduced with
permission of the Editor-in-Chief of the Afr.
J. Mar. Sci.
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Production of DHA from algae
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Example of DHA media
composition produced by algae
Glucose or glycerol 10 g/l
Yeast extract 1 g/l Pepton 1 g/l
Diluted inartificial
seawater
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Composition of artificial
seawater/liter 18 g Na Cl 2.44 g MgSO4.7 H2O
0.16 g KCl
1 g Tris buffer
1 g NaNO3
0.3 g CaCl2.2H2O 0.005 g KH2PO4
0.0027 g NH4Cl
15 x 10-8 vit B12
3 ml chelated iron solution
10 ml trace element (Boron, Cobalt, Managanese, Zinc,Molybdenum)
pH 7.5-8.0
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Fermentation condition
20oC
170 rpm
10 % inoculum
pH 7.5 8.0
DHA is produced in early stage of stationaryphase
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Products of microbial fatty acids
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