arachidonic oil

7/28/2019 Arachidonic Oil

1/42

ARACHIDONIC ACID ANDOTHER FATTY ACID FROM

MICROORGANISM


2/42

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/Phospholipid


3/42

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_acid


4/42

Similarity of ARA (top) with prostaglandin hormone

(bottom)


5/42

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_acid


6/42

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.


7/42

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.


8/42

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


9/42

Mortierella alpina


10/42

Mortierella alpina

Oil

globule


11/42

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)


12/42

Rhizopus spp Mucor circinelloides


13/42

Mucor circinelloides colonies incubated in varioustemperature


14/42

Biosynthesis of fat in

microorganism


15/42

Production of ARA by

microorganisms

Submerged fermentation and Solid

substrate Solid substrate using rice bran, wheat

bran, peanut meal residue, and sweet

potato residue


16/42

Production step

1. Culture Media and Conditions

2. Submerged Fermentation and Solid

state fermentation

3. Downstream processing


17/42

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.


18/42

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.


19/42

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.


20/42

Factors affecting ARA production

1. Initial moisture content

2. Initial pH3. Incubation temperature

4. Supplement of nitrogen

5. Supplement of oil


21/42

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.


22/42

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


23/42

Procedure to

analyze fatty

acids from

microorganisms

to selectpotential

microorganisms

that produced

highest fatty acid


24/42

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.


25/42

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.


26/42

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.


27/42

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.


28/42

GLA = gamma linoleic acid, AA= arachidonic acid


29/42


30/42

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


31/42

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_acid


32/42

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.


33/42

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_cohnii


34/42


35/42

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


36/42

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.


37/42

Production of DHA from algae


38/42

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


39/42

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


40/42

Fermentation condition

20oC

170 rpm

10 % inoculum

pH 7.5 8.0

DHA is produced in early stage of stationaryphase


41/42

Products of microbial fatty acids


42/42

arachidonic oil

Documents