Philippe J.

BLANC
Laboratoire Ingnierie des Systmes Microbiens et des Procds INSTITUT NATIONAL DES SCIENCES APPLIQUEES 135 Avenue de Rangueil 31077 TOULOUSE Cdex 04

FRANCE

Primary identification of food (tomato = red; lemon = yellow; carrot = orange)

Protection against consumption of spoiled food

Experience, tradition, education, environment

Negative assessment of synthetic food dyes

Strong interest in natural colouring alternatives

An example of traditional food colorant

Cochineal carmine (red)

OH O5H12C6

CH3 COOH

HO OH O

OH

Others traditional food colorant

Red from paprika, beetroots, berries or tomatoes
Yellow from saffron or marigold

Orange from annatto

Green from leafy vegetables

Acceptability on the market

Regulatory approval

Capital investment size

Carotenoids Melanins Flavins Quinones

Azaphilones Violacein Indigo

Monascus pigments: an old story in Asia but still forbidden in Europe and the USA

The fungus Monascus

Ang-Kak = Red Rice Origin = China, Indonesia, Korea, Japan Discovered in France by Philippe van Tieghem in 1884 Lack of toxicity studies LD50(oral) = 33.3 g/kg (mice) Not approved in Europe and USA

The chemical structure of the pigments

C5H11 O CH3 O O H3C O Monascine M = 358 O

C7H15 O CH3 O O H3C O Ankaflavine M = 386

C7H15 O CH3 O

C5H11 O CH3 O O H3C O Rubropunctatine M = 354 O

O H3C O Monascorubrine M = 382

C5H11 O CH3 O O H3C O Rubropunctamine M = 353 NH

C7H15 O CH3 O O H3C O Monascorubramine M = 381 NH

Structure of the free pigments of Monascus

C5H11 O CH3 O O H3C O COOH N COOH

Glutamylrubropunctatine M = 483

C7H15 O CH3 O O H3C O COOH N COOH

Glutamylmonascorubrine M = 511

Structure of the complexed pigments of Monascus

The production in various modes of culture

Red Rice (solid culture)

Liquid culture

The applications

Some advertisements in China (Ningxia R.D. Natural pigment) Patents in Japan - Cosmetic containing pigment derived from Monascus (Kanebo Lt) - Monascus culture extract for skin cleansing and protect effect (Pentel KK) - Monascus pigment for inhibiting melanin formation (Hokuto Kasei KK, Ichimaru Pharmacos Inc.)

Coloration of textiles as silk, wool, linen (Kasetsart University, Bangkok)

Coloration of biodegradable materials (ATO/DLO Netherlands)

Others pigments from fungi

Arpink RedTM

an anthraquinonyl derivative from Penicillium oxalicum

Ascolor Biotech s.r.o. produced it in Czech Republic

Fermentation performances : 2 g/L

Riboflavin from Ashbya gossypii

the vitamin B2 but also a yellow food colorant

Riboflavin

Ashbya gossypii was originally isolated from cotton as a pathogen !

It produces more than 2 g/L riboflavin .

b-carotene from various fungi

b-carotene from Blakeslea trispora

DSM was the 1st company to produce b-carotene , today 2 others productions in Russia and Spain.

Others fungi produce b-carotene

Mucor circinelloides
Phycomyces blakesleeanus Fusarium sporotrichiodes

Astaxanthin from Xanthophyllomyces dendrorhous

Astaxanthin
Contributes as a flesh pigmenter to the orange-red coloration of crustaceans and salmonids
Salmon and trout business is now a huge business Fed batch production: 17 g/L biomass

Coloration of crustaceans
Feed non supplemented with any pigment

Feed supplemented with astaxanthin

Melanin from Saccharomyces neoformans var. nigricans

Resarch project presently

Partial conclusion:
Some fermentative food grade pigments are on the market, many are at the development stage or research project

Microbial production of pigments (Industrial production)

Molecule

Colour

Microorganism

Status

Ankaflavin
Anthraquinone Monascorubrami n Rubropunctatin b-carotene Riboflavin

Yellow
Red Red

Monascus
Penicillium oxalicum Monascus

IP
IP IP

Purple Yellow-orange Yellow

Monascus Blakeslea trispora Ashbya gossypii

IP IP IP

Microbial production of pigments (Development stage)

Molecule Astaxanthin Lycopene Rubrolone Torularhodin

Colour Pink-red Red Red Orange-red

Microorganism Xanthophyllomyces dendrohous Blakeslea trispora Streptomyces echinoruber Rhodotorula sp.

Status DS DS DS DS

Zeaxanthin
b-carotene

Yellow
Yellow-orange

Flavobacterium sp.
Mucor circinelloides

DS
DS

Microbial production of pigments (Research project)

Molecule
Astaxanthin Canthaxanthin Lycopene Melanin Naphtoquinone Zeaxanthin b-carotene

Colour
Pink-red Dark red Red Black Deep blood-red Yellow Yellow-orange

Microorganism
Agrobacterium aurantiacum Paracoccus carotinifaciens Bradyrhizobium sp. Fusarium sporotrichoides Saccharomyces neoformans var. nigricans Cordyceps unilateralis Paracoccus zeaxanthinifaciens Fusarium sporotrichiodes Neurospora crassa Phycomyces blakesleeanus

Status
RP RP RP RP RP RP RP

Carotenoids from microalgae: a well established business

Chlorophyceae (green colour)

Rhodophyceae (red colour)

Cyanophyceae (blue green) Pheophyceae (brown colour)

Dunaliella species for carotenoids

Production: 400 mg b-carotene /m2 of cultivation area GRAS status

Main companies for carotenoids

Parrys Agro Ltd (India) ABC Biotech Ltd. (India) Betatene Ltd. (Australia) Western Biotechnology Ltd. (Australia)

Cyanotech Corp., Hawaii (USA) Inner Mongolia Biological Eng. Co. (China) Tianjin Lantai Biotechnology (China) Nature beta Technologies (Israel)

Haematococcus for astaxanthin

Approved by USFDA

Main companies for astaxanthin

Parrys Pharmaceuticals (India) Mera Pharmaceuticals, Hawaii (USA) Cyanotech Co., Hawaii (USA) BioReal Inc., Hawaii (USA)

Phycoerythrin (fluorescent pink) from Porphyridium

Production: 200 mg /L of culture Not yet approved

Phycocyanin (marine blue) from Spirulina or Porphyridium

Production: 100 mg/L of culture Not yet approved

Important aspects before selling a new food colorant

Presentation of the microorganism (natural, not GMO)

Lobbying by other colorants producers (nature-identical)

Safety of the fermentation process (HPLC, genotoxicity)

Guidelines for labelling

Conclusions
- Traditional Monascus pigment in Asia - Success of b-carotene produced by Dunaliella salina

- Success of b-carotene produced by Blakeslea trispora

- Success of Arpink red produced by Penicillium oxalicum - Exploration of biodiversity to get water-soluble pigments - Biosynthesis of niche pigments not found in plants