Fermentation and industrial

microbiologyFIM 202

MICROBIAL PIGMENTS

Introduction

• A number of different kinds of pigments are produced bymicroorganisms. The pigments vary in colour from red, yellow, orange, purple etc., these pigments play important roles in photosynthesis, photo protection , pathogenesis etc.• Microbial pigments have numerous beneficial properties

like anticancer, anti proliferative, immunosuppressive, antibiotic, biodegradability etc.

In food industry• β –Carotene production• β –Carotene is a yellowish carotenoid pigments

also known as pro-vitamin A. It acts asantioxidant and has potential positive propertiesagainst certain diseases.• Following microbes are

mainly using for β –Carotene production:• Blakesleatrispora• Mucorcircinelloides• Phycomyces blakesleeanus

Blakesleatrispora: Some of strains of this mould produce high level of β –Carotene. B. trispora strains are of two types: (+)mating type and (-) mating type. Strains obtained by the specific ratio mating of above two mating types produce β –Carotene14.Today B. trispora fungal β –Carotene is produced by two industries, the first in Russia and Ukraine, and the second in Leone, Spain.

Mucorcircinelloides: Wild strains of M.circinelloides, when exposed to the light impulses of blue light, get activated due to significant change in structural genes of β–Carotene, hence produce it to high level

Phycomyces blakesleeanus: Phycomycesis mainly used for the production of various chemicals like β Carotene. They have enhanced carotenogenic potential when grown on solid substrates or in liquid media

• Arpink Red ProductionIt is the red pigment produced by the strain Penicillium oxalicum obtained from the soil. It contains chromophore of anthraquinone type14 .The amounts of red pigment Arpink Red in various food products was amount recommended by Codex Alimentarius Commmision.• Codex Alimentarius Commission (Rotterdam meeting, March

11-15, 2002)S. No Food product Amount of Arpink Red (in mg/kg)1. Meat products 1002. Meat and meat products analogues 2003. Milk products 1504. Ice cream 1505. Confectionery 300

• Riboflavin (Vitamin B2) ProductionIt is the yellow food colourant and its use is permitted in many countries. Because of it specific affinity, it is used mainly for cereal-based products. Applications of riboflavin somewhere limited due to its bitter taste and naturally slight odour.

• Different level of Riboflavin production from microbes• S.No. Producer microorganism Amount produced

1. Weak overproducer 100 mg/L or less Clostridium acetobutylicum2. Moderate overproducer Upto 600mg/L Candida guilliermundiior Debaryomyces subglobosus3. Strong overproducer Over 1g/L Eremothecium ashbyiiand, Ashbyagossypi

Fermentation with Ashbyagossypi is preferred because of higher yield and greater genetic stability; riboflavin levels of over 15g/L have been reported

• Monascus pigments productionMonascus spp. belongs to the group of Ascomycetes and particularly to the family of Monascaceae. The genus Monascus can be dividedinto four species: M. pilosus, M.purpureus, M ruberand M. froridanus, which account for the majority of strains isolated from traditionaloriental food.

• The common names of this fungal product are Red Yeast Rice (RYR), red rice, angkak, red leaven, benikoji (Japanese), hung-chu, hongqu, zhitai (Chinese), rotschimmelreis (Europe), red mould (USA) and MFR (Monascus fermented rice).

• Monascus spp. produce many pigments of industrial importance and thesepigments are mainly of three types i.e. red colorants, orange colorants and yellowish colorants.

• Monascus fungi, organisms produce angkak can convert starchysubstrates into several metabolites such as alcohols, antibiotic agents, antihypertensives enzymes, fatty acids, flavor compounds, flocculants, ketones, organic acids, pigments andvitamins. Thus, the implementation of Monascus pigment as a coloring agent in food provided anadditional advantage of specific flavor in theproducts.

• Lycopene Production It is a red open-chain unsaturated carotenoid, acyclic isomer of beta-carotene, and longer than any other carotenoid .• Lycopene, also known as psi-carotene, is very sensitive to

heat and oxidation and is insoluble in water, Because of the abundance of double bonds in its structure.• Genetically modified fungus

Fusarium sporotrichioides was used by Jones etal to manufacture the colourant and antioxidant lycopene. They used the cheap corn fiber material as the substrate.• Cultures in lab flasks produced 0.5 mg (lycopene)/g of dry

mass within 6 days and such a production will be increased within the next years.

Microorganism(s) Pigments/Molecule Colour/appearanceBacteriaAgrobacterium aurantiacum Astaxanthin Pink-redParacoccus carotinifaciens Astaxanthin Pink-redBradyrhizobium sp. Canthaxanthin Dark- redFlavobacterium sp., Paracoccus zeaxanthinifaciens Zeaxanthin yellowAchromobacter CreamyBacillus BrownBrevibacterium sp. Orange yellowCorynebacterium michigannise Greyish to creamishCorynebacterium insidiosum Indigoidine BlueRugamonas rubra , Streptoverticillium rubrireticuli,Vibriogaogenes, Alteromonas rubraProdigiosin RedRhodococcus maris Bluish- redXanthophyllomyces dendrorhous Astaxanthin Pink -redHaloferax alexandrinus Canthaxanthin Dark RedStaphylococcus aureus Staphyloxanthin Zeaxanthin Golden YellowChromobacterium violaceum Violacein PurpleSerratia marcescens, Serratia rubidaea, Prodigiosin RedPseudomonas aeruginosa Pyocyanin Blue-greenXanthomonas oryzae Xanthomonadin YellowJanthinobacterium lividum Violacein Purple

AlgaeDunaliella salina β-carotene RedChlorococcum LuteinHematococcus CanthaxanthinFungiAspergillus sp. Orange-redAspergillus galucus Dark -redBlakeslea trispora β -carotene CreamHelminthosporium catenarium RedHelminthosporium avenae BronzePenicilllum cyclopium OrangePenicilllum nalgeovensis YellowFusarium sporotrichioides Lycopene RedHaematococcus Pluvialis Astaxanthin RedMonascus sp. Monascorubramin Rubropunctatin Red OrangeMonascus purpureus Monascin Ankaflavin Red-yellowMonascus roseus Canthaxanthin Orange-PinkMonascus sp. Ankaflavin YellowPenicillium oxalicum Anthraquinone RedBlakeslea trispora Lycopene RedCordyceps unilateralis Naphtoquinone Deep blood-redAshbya gossypi Riboflavin YellowMucor circinelloides, Neurospora crassa and Phycomycesblakesleeanusβ-carotene Yellow-orange

• Benefits and Applications of Microbial pigments • Pigments produced by microorganisms are of traditional use in oriental

countries and have been a subject of intense research in the present decades because of its potential for applications. Use of microorganisms and biotechnology would offer solutions to the problems of various industries especially food colorant industry.

• Fermentative production of colorants has a number of benefits that include: cheaper production, readily available raw materials, high yields and no seasonal variations . In contrast to higher plants, single cell algae and fungi are more appropriate for biotechnological production because they can be grown using existing culture techniques. Many fungi produce pigments which have application in both textile and food industries .

• Fungal pigments are routinely utilized as colorants for both foodstuffs and materials. Some of the more importantly utilized fungal pigments come from the water soluble orange/red pigments produced by Monascus sp., frequently used in rice wines in eastern countries .Pigments produced from Monascus purpureus Piedallu are used in wool dying , and an anthraquinone pigment obtained from Penicillium oxalicum Currie & Thom is currently being developed for use as a ‘natural’ food additive that may have some anticancer effects.

• Mycelial extracts of some promising mushrooms are Chroogomplus vinicolor(which gives red tints), Bankera violascens (which gives greens) and Collybia iocephala(which gives blues), they have a remarkable potential for dyeing wool and silk fabrics.Carotenoids such as β-carotene and lycopene have been known to be produced by fungal cell factories.

• For example, β-carotene by Blakeslea trispora, Mucor circinelloides, andPhycomyces blakesleeanus and lycopene from Fusarium sporotrichioides, Blakeslea trispora are already in use as for food colorants . Vitamin, riboflavin (vitamin B2) is a yellow.

• food colorant that is fermentatively produced by the fungi Eremothecium ashbyii and Ashbyagossypi and has huge commercial potential . Microalgae such as Haematococcus (astaxanthin), Spirulina (phycocyanin), and Dunaliella (ß-carotene and other carotenoids) are already explored as sources of natural food colorants with some limitations of low productivity and contamination in the open culture system, where they are grown [76]. Newly, a blue-green polyphenolic antioxidant pigment termed marennine from the Diatom Haslea ostrearia has been reported . This may aid as a promising food coloring additive. Among yeasts, the marketable success has been with the case of astaxanthin production by Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) and is widely in use as food additive. Additional example of production of carotenoids is by the yeast Rhodotorula spp., R. glutinis, R. gracilis, R. rubra, and R. graminis. The chief carotenoids produced by these yeasts are torulene and torularhodin, with minute quantity of β-carotene .

• The pigments obtained from bacteria offer the following benefits and advantages ; • they are progressively attractive to science because of broad ranging activities,

easy propagation and wide-ranging strain selection, high versatile and productive compared to other sources, fermentation is integrally faster and more productive production when compared to any other chemical process, simple and fast culturing methods allowing continuous bioreactor operation, quite easy to manipulate genes, structural complexity suits for industrial requirements, Cheap substrates used for bulk production and their pigments extracted using simple liquid-liquid extraction technique minimizing operation cost.

• Some important functions of bacterial pigments of commercial potential are • 1) Riboflavin synthesized by Bacillus subtilis and used in foods, vitamin enriched milk products and

energy drinks, 2) β-carotene usually produced by Flavobacterium, Agrobacterium aurantiacum and widely used as food supplement for humans and as food additives for animals and fish,

• 3) Prodigiosin by Serratia marcescens, Pseudomonas, Pseudoalteromonas, Alteromonas denitrificans, Hahella, Vibrio species used as antibacterial, antifungal, antimalarial, antibiotic, immunosuppressive, anticancer etc.,

• 4) Phycocyanin by Cyanobacteria used as dietary supplement rich in proteins, and • 5) Violacein by Chromobacterium violaceum,Alteromonas luteoviolacea was used as Antiviral,

antibacterial, antiulcerogenic, antileishmanial, and anticancer properties, potent cytotoxic effects against U937 and HL60 Leukemia cell lines

Novel practices of microbial pigment production

• Strain improvement• Fermentation• Low-cost substrates