© Fraunhofer IGB

Alexander Beck

SuperBIO Workshop Biosurfactants, Gent, Belgium

User Group Meeting Biosurfactants Carbosurf, 3rd April 2017

Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB

MANNOSYLERYTHRITOL LIPIDS (MEL) AS ADDITIVES IN COSMETIC FORMULATIONS

© Fraunhofer IGB

...the next promising biosurfactants produced by microorganisms

Literature available for lab scale synthesis and applications

Large gap towards process optimization and piloting scale

Addressed in the Carbosurf Project as one of four product linestogether with Sophorolipids, Rhamnolipids and Xylolipids

MicrobialBiosurfactants

Origin Properties and PotentialApplications

Titers

Mannosyl-erythritol lipids(MEL)

Pseudozymasp.

• Self assembling properties• Emulsifier• Cosmetics and personal care• Pharmaceuticals• Cleaning agents

160 g/L

Cellobiose lipids(CL)

Ustilago sp. • Antimicrobial effects• Preservatives• Cleaners

33 g/L

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Current research on MEL production at IGB

Strain selection

Evaluation of renewable resources

Metabolic pathway analysis

Bioprocess engineering

Medium composition

Reactor design

Process strategies

Controllability

Downstream processing

Aim

High space-time-yield

Scale-up engineering

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Chemical structure of Mannosylerythritol Lipids

Glycolipids – contain hydrophilic sugar moiety and fatty acid residues

Microorganisms produce a mixture of different MEL structures

R6 = Ac- or H-

R4 = Ac- or H-

C2 - C18

C2 - C18

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Different MEL variants lead to different properties

MELs clustered according to polarity on TLC (Kitamoto et al., 1990)

MEL-A

MEL-B

MEL-C

MEL-D

Oil

Fatty acid

TLC result

pola

rity

MEL-B MEL-C

MEL-D

MEL-A

Kitamoto, D., Akiba, S., Hioki, C., Tabuchi, T., (1990) Extracellular Accumulation of Mannosylerythritol Lipids by a Strain of Candida antarctica. Agricultural and Biological Chemistry 54, 31-36.

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How to tailor MEL production?

Different approaches possible (simultaneously):

Use of different strains

Use of different (renewable) feedstocks

Post-modification of produced MEL-mixture

Modification of metabolic pathway

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Selection of production strain

Use of different strainsp

ola

rity

(Morita et al., 2008)

Morita, T., Konishi, M., Fukuoka, T., Imura, T., Kitamoto, D., (2008) Production of glycolipid biosurfactants, mannosylerythritol lipids, by Pseudozyma siamensis CBS 9960 and their interfacial properties. Journal of bioscience and bioengineering 105, 493-502.

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Selection of production strain

Pseudozyma strain 2 produces all main MEL derivatives and a high titer of hydrophilic MEL-D

P. strain 3 produces mainly MEL-B and MEL-C

Different Pseudozyma strains

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Variation of MEL composition induced by carbon source

Use of different (renewable) feedstocks

Hydrophilic: Molasses, Lignocellulosic sugars, Glycerol, …

Used for biomass growth

Hydrophobic: Soybean oil, Olive oil, Coconut oil, Castor oil, …

Necessary for MEL synthesis

Influences side-chain composition

Oleic acid

Lauric acidRicinoleic acidDifferent oils

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Post-modification of produced MEL-mixture

Enzymatic treatment with lipase at 60°C

Increases percentage of hydrophilic MEL variants

Mono-acylated„MEL-X“

MEL-Dp

ola

rity

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How to set up a scalable fermentation process?

Strategies for a controllable fermentation process

MediaComponents

MetabolismOperating Conditions

C-Sources

Mineral MediaGenetics /

BiosynthesisStirring, Foam,

O2 SupplyOil, Fatty Acids, Hydrophilic C

Seed Culture Media C- and N-source Amino Acids Vitamins Trace Elements

Un-/saturated Chain Length

(C12-C18) Water-soluble C-

Sources: Glycerol, Glucose

Expression Level of Target Genes

Regulatory Controls Repression (C or N) Inhibition Induction Overflow

Metabolites

Antifoam/ Defoamers

Feeding Strategies Aeration and

Stirring Strategy pH Control Seed Culture

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How to set up a scalable fermentation process?

Strategies for a controllable fermentation process

Repeated-Batch strategy with oil addition after growth phase

Control of pH, stirring and pO2

Growth phase

Productionphase

Growth phase

Productionphase

Repeated oil addition

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Physico-chemical properties of MEL

Measurement of surfactant properties

Different foaming ability of MEL structures

StrainSubstrate

(oils )CMC

[mg/L]σmin

[mN/m]

Pseudozyma 2

Oil A 1.33 25.5

Oil B 2 25

Oil C 4 26.5

Oil D 1.4 27

Oil E 1.5 26.4

Pseudozyma 1 Oil A 1.99 30.8

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Application of MEL in cosmetics

Reported potential applications in literature:

Moisturizing effect on dry skin (ceramide-like properties of MEL-A)

Repair of damaged hair (MEL-A and B)

Activation of fibroblasts and papilla cells

Antioxidant activities – anti-aging ingredients?

Work performed by Croda:

High throughput screening for formulationsin personal care

Extended functionalities

Currently fermentation running to supply samples

Morita, T., Fukuoka, T., Imura, T., Kitamoto, D., (2013) Production of mannosylerythritol lipids and their application in cosmetics. Appl MicrobiolBiotechnol 97, 4691-4700.

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Great structural variety of MELs

Tailoring of MEL variants already possible

Development of a scalable production process

Properties of MEL make them suitable for cosmetic applications

Summary and Outlook

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Acknowledgement

Key researchers:

Dr. Michael Günther

Paula Carrillo

Head of Department:

apl. Prof. Dr. Steffen Rupp

Contact

Alexander Beck, M.Sc.

Dr. Susanne Zibek

Nobelstrasse 12, 70569 Stuttgart, Germany

alexander.beck@igb.fraunhofer.de

susanne.zibek@igb.fraunhofer.de

www.igb.fraunhofer.de

mailto:alexander.beck@igb.fraunhofer.demailto:susanne.zibek@igb.fraunhofer.dehttp://www.igb.fraunhofer.de/