Microbial Production of Oils and Fats

Course: Food Microbiology 2014Lecturer: Dr. Vu Thi Lam AnGroup 5: Nguyen Quynh Hai Yen BTFTIU11009

Ho Quang Loc BTFTIU12011

Contents

1. Introduction to Single Cell Oils (SCO)

2. SCO production process

3. Applications

4. The significance and safetyness of SCO

1. Introduction to Single Cell Oils.» What is it?

» Condition for lipid accumulation

» Principles of lipid accumulation

• Single Cell Proteins?

• Single Cell Oils (SCO): triglyceride fats generated by

microorganisms.– Lipid components are similar to those found in plant and animal.

→ Applications: animal feeds, aqua feeds, biodiesel.

What is Single Cell Oils?

What is Single Cell Oils?

Why not for human?

Is it safe for human consumption?

• Microorganism → a human food source from ancient time. – Can you name some?

Oleaginous microorganism

• Microorganism that gathers >20-25% of their biomass as oil.

• Many yeast, fungi and several algae are maximum producers. – Algae → biodiesel– Yeast and fungi → edible oil• They are also capable of producing high levels of

nutritionally important polyunsaturated fatty acids (PUFAs).

Advantages of Oleaginous microorganism

• Growth on various substrates → utilizing by-product = reduce cost

• Ability to synthesize a divertarray of fatty products → many useful applications.

• Able to be genetic manipulation → selection for highest productivity.

Condition for lipid accumulation

• Lipid accumulation is trigger when – Nutrients in the growth medium (usually Nitrogen

source) is exhausted – Surplus of Carbon source (usually glucose)

→ Cells stop multiplying.→ Cells convert Carbon source to Storage oils or fats.

Condition for lipid accumulation

Stages of Lipid accumulation in Batch culture

...

Cells reach limit of obesity → stop accumulating.

Lipid accumulation → cells expand

Cells convert C → storage lipid (intracellular)

Exhaustion of N, cells stop divide

Microorganisms grow and multiply until a certain time

Prepare medium: high Carbon, low Nitrogen

Biochemistry of Lipid accumulation

Cellular respiration of microorganism

TCA

Biochemistry of Lipid accumulation

Acetyl-CoA →(fatty acid biosynthesis)→ triacylglycerols

Enzyme ATP-citrate lyase: Citric acid → acetyl-CoA (Oleaginous microorganisms only)

Citric acid in mitochondrion → cytosol

TCA is disrupted

[AMP] is diminished;Isocitrate dehydrogenase in mitochondrion stop working

Exhaustion of N

Biochemistry of Lipid accumulation

2. Production of Commercial SCO.

Production of commercial oils and fats from SCO

• Only if cost of production of SCO could compete against plant oil.

• In fact, production cost of SCO is much higher.

Plant oil = 400-800$/ton

- 4 tons of sugar (300$ each) → 1 ton SCO + biomass- Cost of extraction and refining → food graded oil- Cost of selling, waste disposal...

Production of commercial oils and fats from SCO

• High production cost

→ Apply for production of specialty oils and fats only.

→ Choose fast growing microorganisms, high oil yield, simple

growth requirements.

→ Focusing on the use of by-products of agriculture and industry

as substrates

Production Process

Harvesting the cells

Cultivation Drying the cells Extraction

Production Process

Commercial Production Process

• It is important to harvest SCO before Carbon substrate is completely depleted.

• As the cells will use the lipid that they have accumulated for their own survival.

• Consequences:1. Low Oil yield2. Cells forming lipase → degrade quality of SCO

• Alter flavor, color, functional characteristics.

– How to avoid lipase? → Answer: heat denaturation <60ᴼC

Commercial Production Process

• SCO usually contain natural antioxidant

→ A clear, bright oil with a minimum of coloring → favorable.

→ Very stable to oxidation

→ Good for consumption

SCO by Cryptococcus curvatus

SCO by Mortierella alpina

3. Applications of Single Cell Oils.» Cocoa butter equivalent fat

» Polyunsaturated fatty acids (PUFAs)

• DHA

• ARA

Cocoa Butter Equivalent Fat• Cocoa Butter: triacylglycerol, consists of: - palmitic acid (16:0) - oleic acid (18:1) - stearic acid (18:0)

Cocoa Butter Equivalent Fat

• Cocoa Butter: very expensive

• SCO = Cocoa Butter Equivalent Fat = cheaper substitute

• However, SCO are low in stearic acid→ genetic modification is necessary

Cacao Butter Substitutes

Cocoa Butter Equivalent Fat

* Henk Smit (Netherlands):– Using the yeast Cryptococcus curvatus D – Delete the gene that codes for the Δ9−desaturase, which converts

stearate into oleate→ Fail, not stable products

* Julian Davies (New Zealand)– Culturing in O2 deficiency → stop enzyme desaturase from working →

more stearate→ But uneconomical

* Chance for you!

Polyunsaturated fatty acids

• Omega-3 and omega-6: cannot synthesize by human body.– Omega-6: from plant food.– Omega-3 (EPA, DHA):

• Are long chain PUFAs• Most plants are unable to synthesize• Must obtain from animal food.

Dietetic significant of PUFAs

• Long chain PUFAs: – Key components of cell

membrane– Inflammation response

• Arachidonic acid (n-6) and DHA (n-3): essential for brain and neural development

• In general, our diet contains too much n-6 and too little n-3

SCO as source of Omega-3• Animal source of Omega-3: – Oily fish → distinct fishy taste– Animal organs (esp. liver)→ Unfavorable + shortage of supply + extensive purification→ Costly→ Why not SCO?

• In this presentation, we will also briefly introduce to you:– Arachidonic Acid (ARA) production process.– Docosahexaenoic acid (DHA) production process.

Arachidonic Acid (ARA)Processes for the production of various PUFAs

Arachidonic Acid is a long chain polyunsaturated fatty acid with twenty carbon atoms and four double bonds. Its systematic name is (all-cis)- 5,8,11,14-eicosatetraenoic acid(ETA)

Arachidonic Acid (ARA)Processes for the production of various PUFAs

Biosynthetic pathway of polyunsaturated fatty acids in Mortierella alpina

ARA is synthesized throughmany enzymatic steps of fatty acid synthesis, elongation of fatty acid and desaturation of fatty acid

Microorganism ARA yield/cultivation

period

Scale

Submerged cultureMortierella alpina 1S-4 13 g/L/10d 10-kL fermentor

M. alpina ATCC 32222 11 g/L/11d 250-mL flask

M. alpina ATCC 32221 11 g/L/16d 500-L fermentor

M. alpina UW-1 5.5 g/L/6d 20-L fermentor

M. alpina LPM 301 4.5 g/L/8d 30-L fermentor

Mortierella alliacea YN-15 7.1 g/L/6d 50-L fermentor

Mortierella schmuckeri S12 2.3 g/L/3d 14-L fermentor

Mortierella sp. S-17 0.96 g/L/7d 1-L flask

Mortierella elongata SC-208 0.49 g/L/5d 250-mL flask

Pythium irregulae ATCC 10951

3.1 g/L/8d 250-mL flask

Solid-state culture

M. alpina IFO 8568 13 g/kg-medium/20d

M. alpina CCF 185 36 g/kg-medium/21d

Arachidonic Acid (ARA)

The fatty acid compositionof 50 Mortierella subgenus isolates was analyzed Amanoet al

They found that ARA composition of alpinawas higher than those of any

Processes for the production of various PUFAsDocosahexaenoic acid (DHA)

Docosahexaenoic acid (DHA, 22:6) is a particularly important ω 3‐PUFA, with a 22 carbon chain ‐and six double bonds.

Processes for the production of various PUFAsDocosahexaenoic acid (DHA)

Microbial PUFAs on market

ARA for muscle stimulation ARA-rich SCO and DHA-

rich SCO in infant formulae in many

countries of Europe, Australia, and the Far East

Advantages and Disadvantages of SCOADVANTAGES: 1. Simple fatty acid profile → easy for extraction and

purification.

2. Both quality and quantity of the product can be guaranteed.

3. SCO is not affected by geographical or environmetal factors (e.g pollution etc.)

4. Rapid production: increase cells in a short time and independence from climatic conditions.

5. SCO act as a novel source of polyunsaturated fatty acids, such as DHA and AA for nutritional supplementation.

Advantages and Disadvantages of SCO

DISADVANTAGES:

1. Limited production capacity.2. Consumer acceptance.3. DHA from fish oil are linked to growth retardation in infant

→ also DHA of SCO

Understanding 2 routes for safety evaluation of a food ingredient

1. Petition for FDA approval as a food additive.– FDA will review before products are available on the market. – FDA is responsible for their decision.

2. Manufacturer can determine that a substance is GRAS if there is scientific consensus among qualified experts about its safety under the intended condition of use.

→ SCO is only FDA approval.

Safety of SCO

• SCO is only FDA approval. Why?→ For some years, high level of SCO-ARA in the human diet was

thought to induce potential adverse effects on blood clotting.

• However, later intensive and prolong studies demonstrate the lack of evidence of toxicity in volunteers.

• Recently, microbial oils have been shown to be no more toxic than oils from traditional sources.

• More researches are needed!

Summary

• SCO are now used to produce high value oils rich in arachidonic acid (ARA) and docosahexaenoic acid (DHA).

• These oils are used in infant nutrition and also as nutriceuticals and food additives appropriate for adults.

• Potential therapeutic applications are very promising.

• Production of SCO:– SCO can be produced in large scale.– To ensure stabilization of SCO, must harvest the cells before

substrates are totally depleted.– SCO extraction is much the same as used for plant seeds.

References • James, P.W., Colin, R. Microbial production of fats and oils. Food Biotechnology.

2006, pp. 443-472

• http://www.ukessays.com/essays/education/microbial-production-of-fats-and-oils.php

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