Indian Journal of BiotechnologyVol 2, July 2003, pp 362-369

Microbial Pigments

V K Joshi*, Devender Attri, Anju BaJa and Shashi BhushanDepartment of Post-harvest Technology, Dr Y S Parmar University of Horticulture and Forestry, Nauni, Solan 173230, India

Received 9 December 2002; accepted 10 February 2003

The microorganisms such as Monascus, Rhodotorula, Bacillus, Achromobacter, Yarrowia and Phil/fia produce alarge number of pigments. An ideal pigment-producing microorganism should be capable of using a wide range of Cand N sources, have tolerance to pH, temperature and minerals, and give reasonable colour yield. Non-toxic and non-pathogenic nature of pigment-producing microorganisms coupled with easy separation from the cell biomass isstressed. The various advantages of producing pigments from microorganisms include independence from weatherconditions, easy and fast growth, colours of different shades and growth on cheap substances. Studies revealedunstable, largely degradable and sensitive to heat, light, acidity and water activity as characteristics of microbialcolour. Improvement in stability, safety and solubility can certainly make widespread use of microbial pigments inthe food industry.

Keywords: microorganisms, pigment, food, toxicity, carotenoid, solid-state fermentation

IntroductionColour is the most pleasing attribute of any article;

red colour exudes warmth, increases pulse rate andrespiration, whereas, blue or green colour suggestscool and peaceful environment and encouragesrelaxation. The colour is associated with quality andsensory properties of food. The first characteristicperceived by our senses is appearance or colour offood, which not only determines its acceptance, buteven helps in its recognition. Acceptability of colourin a given food, in turn, is influenced by cultural,geographical and sociological aspects of thepopulation. Indeed, colour and other eating habits areviewed as a type of culinary anthropology andindigenous to specific region. However, certain foodgroups are acceptable only if they fall within certainrange of colour gamuts and their acceptability isreinforced by their economic worth judged purely onthe colour value. A well-textured food, rich innutrients and flavour, cannot be eaten unless it has theright colour. Due to the consumer awareness andconcern for healthful and perfectly balanced food, anincreasing interest in the food colourants of naturalorigin has been developed. Consequently, theconsumer wants to see the food looking natural as the

*Author for correspondence:Tel.: 01792-252410; Fax: 01792-252242E-mail: vkjoshipht@rediffmail.com

synthetic colours have been proved to be carcinogenicto humans. Natural pigments possess anticanceractivity, contain pro-vitamin A and have somedesirable properties like stability to light, heat and pH.

The colour of a food is dependent upon the type ofpigment and reflection or scattering of light. Thus, itdenotes the human eye's perception of colouredmaterials such as red, green or blue. The 'colourant',however, referes to any chemical compound thatimparts colour, while the pigment indicates normalconstituents of cells or tissues giving colour. Plants,animals and microbes are the sources of naturalpigments. When the microbial cells are used toproduce the colour the term refers to 'MicrobialPigments'. Different types of pigments extracted frommicrobes, their production and characteristics formthe subject matter of this review article.

HistoryAddition of colour to the processed foods is an old

practice but microbial pigments production is a recentphenomenon (Table 1). Saffron, turmeric andvegetable dyes had been used to colour food (Singhal& Kulkarni, 1999). Nata, a microbial coloured food,is one of the traditional holiday foods in Philippinesand is also popular in Indonesia, Japan and Taiwan.

Sources of Natural PigmentsMost of the natural pigments are extracted from

plants and animals like annato, grapes, beet, paparika,

JOSHI et al: MICROBIAL PIGMENTS 363

Year

Table I-Historical developments in colour

Development

Perkin's mauve pigment was discovered and coaltar dyes were synthesized.Monascus sp. was traditionally cultivated and utilized in the orient for making red rice wine, redshaohsing wine and red Chinese rice.The first carotenoid pigment from Cryptococcus was marketed.Production of carotenoid pigments from Rhodotorula sp. started.Astaxanthin was isolated from Phaffia rhodozyma, (in honour of Prof. Herman Jan Phaff) grown onexudates of deciduous trees in Japan and Alaska.

Production of (3-carotene from Dunaliella salina took place.Betatene Limited Corporation was established for cultivation of D. salina on large scale for producingnatural (3-carotene products.

18561884

19541963Early 1970's

Late 1970's and early 80's

1985

Table 2-Food-based natural pigments and their stability

Pigment Source Solubility Effect of acid/alkali StabilityHeat Light Oxygen pH

Anthocyanins Fruits Water soluble Redness/gray High High High LowBetataines Beet root Water soluble Moderate High High HighBixin Seed coat of Fat soluble Moderate to low Low High

Bixa orellanaCanthaxanthin Fat soluble Moderate Moderate Moderate ModerateCaramel Heated sugar Water soluble High High High HighCarotenes Leaves Fat soluble Moderate to low Low Low HighChlorophylls Leaves Water soluble Darkness/yellow High High High LowCusavmin Low Low LowNorbixin Seed coat of Water soluble Moderate to low Low High

Bixa orellanaPolyphenols Tea leaf Water soluble High High High HighCavinones High Moderate ModerateXanthophylls Fruits Water soluble Moderate High High LowerCurcumin Turmeric Water solubleFlavone Vegetables Water soluble BrightnesslbrownsQuinones Roots, bark Water solubleCusavminCavinones

Fellows, 1988; Prescott &Dunn, 1959

female insects (Coccus cacti) and microorganismslike Monascus, Rhodotorula, Bacillus, Achromo-bacter, Yarrowia, Phaffia, etc. (Table 2). Pigmentslike carotenoids, anthoquinone, chlorophyll have beenproduced from yeast, fungi, bacteria and algae. Thereis growing interest in microbial pigments due to theirnatural character and safety to use, medicinalproperties; nutrients like vitamins, production beingindependent of season and geographical conditions,and controllable and predictable yield. Microbialpigment like B-carotene gradually shifts its colour

towards orange red, which is more attractive and thisunique property is absent in plant-derived pigments.Some of the microbial pigments can be producedfrom waste material (starch and juice industry), hencereducing water and environmental pollution.

Some microbial pigments are water insoluble; havepoor stability (Table 2), show changes in colour underdifferent pH conditions, lack standards or exhibit off-flavour and are hygroscopic. Limited range of shadesand low usefulness in specific foods, low pigmentcontent and bulk handling is also a problem

364 INDIAN J BIOTECHNOL, JULY 2003

Table 3--Microbial pigments and their colour shades

Microorganism Pigments

1) BacteriaAchromobacterBacillus sp.Brevibacterium sp.Corynebacterium michigannisePseudomonas sp.Rhodococcus marisStreptomyces sp.

2) MoldsAspergillus sp.A. glaucusBlakeslea trisporaHelminthosporium catenariumH. gramineumH. cynodontisH. avenaeH. catenarinMonascus purpureusP. cyclopiumP. nalgeovensis

3) YeastsCryptococus sp.Phaffia rhodozymaRhodotorula sp.Yarrowia lipolytica

4) AlgaeDunaliella salina

CreamyBrownOrange, yellowGreyish to creamishYellowBluish redYellow, red, blue

Orange, redDark redCreamRed colourRedBronze colourBronze colourDark maroonYellow, orange, redOrangeYellow

RedRedRedBrown

Red

associated with microbial pigments. However,problems can be minimized, using suitable technologyand quality assurance measures.

Pigments Producing MicroorganismsA large number of bacteria, molds, yeasts and algae

produce pigments (Table 3). Suitable species shouldsatisfy following criteria: 1) Capability to use a widerange of C and N sources; 2) Should have tolerance topH, temperature, mineral concentration and possessmoderate growth conditions; 3) Reasonable colouryield; 4) Should be non-toxic and non-pathogenic;and 5) Must be easily separable from the cell mass.

BacteriaBacteria are. a good source of pigments, which are

mostly carotenoids, especially f3-carotene in nature.Streptomyces chrestomyceticus subsp. rubescens hasbeen employed to producce lycopene whilezeaxanthin and lutein production from

Flavobacterium sp. is gaining importance. A mutantstrain of Flavobacterium produced zeaxanthin in basemedium by supplementing glucose and corn steepliquor with palmitic esters, methionine pyridoxine andferrous salt, at a reduced temperature.Brevibacterium KY -431 '3, originally isolated from

the petroleum field, contained canthaxanthin withsmaller amount of echinenone and f3-carotene. InJapan, it is considered as a model organism for thecanthaxanthin biosynthetic study. Corynebacteriumsp. and Rhodococcus maris are also adopted forcanthaxanthin production. The canthaxanthinproduction has been optimized in a mediumcontaining hydrocarbons, ammonium phosphate,minerals, vitamin B 12, malt extract and a smallamount of non-ionic detergent. Using TLC, 5components of brown pigment were resolved andproduced in the culture broth by Bacillus subtilis.Removal of cells from the medium at the end oflogarithmic phase did not alter the type or amount ofpigment production indicating that the pigments wereproduced in the medium during growth.

FungiThe various species of fungi, belonging to

Phycomycetes, order mucorales in particular, are theprominent source of f3-carotene, and accumulate it tounusual degree. Valuable sources of f3-carotene usedearlier were either Ascomycetes (Penicilliumsclerotiorum and Neurospora crassa), orDeuteromycetes (Rhodotorula aurantiaca), yieldinginferior f3-carotene. Mating of sexual forms resultedin a spectacular increase in f3-carotene. Mixed culturesof strains Of opposite sex usually contain 5-15 timesmore pigment than the separate culture. Trisporicacid, having hormonal activity and produced duringmating, has been shown to mediate the stimulation off3-carotene biosynthesis. Though f3-carotene is themajor pigment produced by fungi, lycopene is anotherpigment produced by Blakeslea trispora.

The traditional fermented product ang-khak (redrice), made by fermenting rice with Monascuspurpureus, is ground and used as a colourant.Pigments, produced from immobilized cultures ofMonascus, are a mixture of six major related pigments(red, yellow and purple pigmented polyketides) andinsoluble in acid. The red pigments, rubropunctatinand monoscorubin with different side chains on a 20-lactone ring, are nitrogen analogue of the orangepigment. Monascin or monoscoflavin and ankaflavin

JOSHI et al: MICROBIAL PIGMENTS

are yellow and are the reduced form of two redpigments. Though these pigments have been isolatedfrom both the solid and liquid cultures of fungus, theformer gives ten folds more yield than the latter. Forthe production of pigment, the molds are grown in thesolid state culture using rice, cassava or corn, oat,wheat and barley as a substrate. Aspergillus sp. hasbeen grown successfully on starch medium usingdifferent C sources wherein dextrin and maltoseproduced the highest colour intensity. Undersubmerged conditions, factors like N, pH andtemperature affect the pigment production.

YeastsMany yeasts (Rhodotorula, Yarrowia lipolytica,

Cryptococcus sp., Phaffia rhodozyma) are goodsource of microbial pigments. Astaxanthin, a redpigment, is found in animals but rarely found inmicroorganisms like P. rhodozyma. The culturalconditions play an important role in producingcarotenoid from P. rhodozyma (Andrews et al, 1976),which produces a variety of carotenoids as follows: ~-carotene, 2-2.5; v-carotene, 0.01; neurosporene, 0.01;lycopene, 0.01; echimenone, 2-4; 3-hydroxyechinone,3-4; phoenicoxanthin, 5-7; astaxanthin, 83-87% oftotal carotenoids. Out of these carotenoids,astaxanthin is mainly produced during the exponentialphase of growth. The yeast belonging toBasidiomycetes has the ability to utilize urea, which isless common in Ascomycetous yeasts. While growingP. rhodozyma as a source of pigment for aquacultureanimals, astaxanthin provides proper colour for aquaculture feeds.

365

AlgaeAlgae produce a number of pigments (Table 4).

Dunaliella salina, which occurs singly in marineenvironment and belongs to class Chlorophyta,produced .~-carotene (ovoid in shape, 4-10 urn wideand 6-15 urn long). The ~-carotene production isfavoured by high light intensity and high salinity. MisBetatene Limited, founded in 1985, is the firstcompany associated with manufacturing and sellingof natural ~-carotene. Another red microalgae,Rhodophyta, contains phycocyanins andphycoerythrins (collectively known asphycobiliproteins) apart from the main pigmentchlorophyll, which are red or blue and also present inspecies Cyanophyta and Cryptophyta. Such pigmentsconstitute a major proportion of the algae cell proteine g, phycobiliproteins account upto 30% of the totalcell protein (5-10% of the dry biomass) in a numberof red microalgae and can go even up to 50% of thetotal protein in P. cruentum. However, only about10% of the soluble cell proteins in Gracileratikvahiae are phycobiliproteins, which absorb light inthe visible region of 450-650 J..iM.

Production of Microbial PigmentsTo produce microbial pigments,

microorganisms are as follows:major

1)Monascus purpureusMonascus pigments are used in traditional oriental

foods as a natural colouring agent. These are extractedfrom M. purpureus grown on steamed rice by solidstate fermentation (Fig. 1, Table 5).

Table 4-Carotenoids produced by algae

Pigment

~-caroteneLutein

Algae

Dunaliella salinaChlorococcumChlamydomonasSpongiococcumChlorella pyrenoidosaHematococcusChlorellaChlamydomonasScenedesmusAnkistrodesmusDictyococcus cinnanarinusHemacoccus pluvialis

Canthaxanthin

Astaxanthin

Nelis & Leenheer, 1991

Use

Food colourantPoultry feed

Poultry feed and fish feed

Fish feed

366 INDIAN J BIOTECHNOL, JULY 2003

Medium(broth)

+(maltose, yeast extract, glucose,KH2P04, MgS04, CaCh)

SterilizationMedium

(yeast extract, sucrose, agar)

. 1 +Plates (5 mm disc)

+Inoculation <llll1IIIf------

••Incubation(28°e for 14 days in darkness)

••Harvesting of mycelium

••Mycelium coloured

••Drying

••••Grinding

••Extraction with solvent(methanol)

••Shaking(1 min.)

••Filtration

••Drying(80oC for 24 hrs.)

••Pigment

••Packaging

Fig. I-Flowsheet for the production of Monascus pigment(Broder & Koehler, 1980)

To improve the quality (water solubility) ofMonascus pigments, chemical modificationsinvolving reaction of the native pigments withproteins, amino acids, nucleic acids, laccaic acid,chitosan, amino alcohols etc, were made. But thesemethods are costly, so two inexpensive chemicals likeamino acetic acid and p-amino benzoic acid havebeen employed. The flow sheet of the process is givenin Fig. 2 (Wong & Koehler, 1983).

2) RhodotorulaRhodotorula has been successfully used for the

production of pigments. To grow Rhodotorula, therecommended medium is too complex and requires anumber of chemicals. Apple pomace, a waste fromapple juice processing industry, which is cheap, rich

in sugar, acid and minerals, has been employed inpigment production (Table 6).Rhodotorula is rich in fat and vitamins besides

carotenoids. It can be incorporated in feeds to enhancethe nutritional value and prevent fungal contami-nation. Rhodotorula pigment has maximum absor-ption at 200-400 nm with only small absorptionbetween 400-600 nm. The absorption pattern showsthat the pigment is not single rather a mixture of twomajor compounds. The production of Rhodotorulaglutinis by batch fermentation in rotatory shaker hasresulted in good yield of carotenoids. TheRhodotorula cells are grown at 30°C for 5 days and,after centrifugation, washed with distilled water .Further, the use of small fermenter (1 litre) and fedbatch fermentation using the co-cultures ofRhodotorula glutinis and Debaromyces castelliiincreases the biomass during the initial time of 96-120hrs only (Buzzini, 2001). The optimum conditions inthe fermenter for the carotenoid production are:fermenter capacity, 1 litre; temperature, 30°C; airflow rate, 1 min-I; magnetic stirrer, 220 rev/min; and,antifoaming agent, 0.3% w/v.

3) Phaffia rhodozymaPhaffia rhodozyma ferments glucose and other

sugars to produce pigment, astaxanthin. The pigmentis produced in shake flask at 21DC and an increase in

Add 2 mg amino acetic acid p-amino benzoicacid to 50 ml of 50% ethanol

.().

Addition ofIN NaOH solution.().

Reflux (lOO°C for 2 hrs).().

Cooling.().

Filtration.().

Drying under vacuum.().

Redissolved in 50 ml of 0.01 M solution citrate,phosphate buffer at pH 3,7,9.2

.().

Addition of pigment (112 g)in 2 g gelatin

.().

Boiling 30 min.().

Freeze drying

Fig. 2-Flow sheet of chemical modification of the Monascuspigment (Wong & Koehler, 1983)

JOSHI et al: MICROBIAL PIGMENTS

Table 5-- Growth conditions for different species ofMonascus

ParametersSolvent

M. purpurensMethanol

M. ruberMethanol:chloroformRed28°C80 hrs6.5

ColourTemperatureTime of incubationpHCarbon sourceNitrogen sourceWavelengthType of fermentation

Red28°C14 days6.3MaltoseYeast extract500nmSolid state

MSG

Submerged

Wong & Koehler, 1983

Table 6-Optimum conditions for Rhodotorula pigmentproduction

Microorganism Rhodotorula sp.

Incubation temperatureIncubation periodMedium usedComposition

30±I°C72 hrsApple pomace agarApple pomace + ferrousammonium sulphate (0.3%)+ agar (2%)

Sandhu & Joshi, 1997

glucose concentration in the medium gave higheryield of pigment rather than growth only.

4) Bacillus subtilisB. subtilis, on sporulation, produces pigment. The

liquid medium for optimum pigment formationcontained: Spizizen's salt, - glucose, 27.7; L-tryptophan, 0.25; L-tyrosine, 0.25; L-histidine, 0.055;and MnS04. 0.67 mM. The salt, amino acid andglucose were autoclaved separately before inoculationwith microorganisms.

5) Aspergillus oryzaeA. oryzae var. effuses produces an orange-red

pigment belonging to anthraquinone group. The starchmedium used includes: MgS04.7H20 sol, 1 ml(24.658 g/100 ml); eaCh, 1 ml (14.79 gllOO ml);FeCh, 0.5 ml (16.221 gllOO ml); ZnS04, 1 ml (28.756gllOO ml); NH4H2P04, 0.173; sodium citrate, 0.1176and carbon source 100 g/l.

6) Yarrowia lipolyticaY. lipolytica produces brown pigments using

tyrosine. The medium used for growing micro-organism contained: peptone, 5; yeast extract, 5; and,

367

glucose 2 g r'. The pigments were assessed intyrosine medium, which. contained: KH2P04, 4;MgS04.7H20, 2.5; thiamine-hydrochloride, 0.01;L-asparagine, 1; glycine, 1; and L-glutamine, 19 r':biotin, 20 ug r': MnS04.5H20, 1 and L-tyrosine 1.5m M r', and pH, 5.5. Lactic acid promotes browningactivity of yeast. During exponential phase, reddishbrown colour develops and changes to deep brownduring stationary phase. Exponential phase occurredby increasing glucose from 0.5 to 10 g r'. It dis-appears in the absence of Mn. The glucose (>10 g rl)delayed browning during stationary phase.

Factors affecting Microbial Pigment Production1) Temperature

The production of microbial pigments is greatlyaffected by the temperature of incubation, dependingupon the type of microorganism. The growth ofMonascus sp. requires 25-28°C for the production ofpigment, whereas Pseudomonas requires 35-36°C forits growth and pigment production.

2)pHThe growth and type of pigment produced is

affected by the pH of the medium (Table 7) in whichmicroorganisms are grown. It differs from onemicroorganism to another and slight change in pHmay change the shade of colour. Optimum pH forMonascus sp. and Rhodotorula is 5.5-6.5 and 4.0-4.5,respectively. Neutral to slight alkaline pH favourslycopene formation whereas acidic pH favours B-carotene synthesis.

3) Carbon SourceThe mycelial growth of pigment producing

microorganism is affected by the type of carbonsource like glucose, fructose, maltose, lactose,galactose, etc. Glucose and its oligosaccharides arebetter carbon sources for growth and pigmentproduction. For the Monascus sp., the volumetricpigment formation is best on starch and dextrin,

Table 7--Effect of pH on astaxanthin formation fromPhaffia rhodozyma

pH of medium Astaxanthin (!!g/g yeast")

6.55.54.53.5

Johnson & Lewis, 1979

325336387212

368 INDIAN J BIOTECHNOL, JULY 2003

Table 8--Effect of carbon source on growth and pigmentation of P. rhodozyma

Carbon source Growth rate Astaxanthin yield1.1(h-1) (ug ml") [Ilg (g yeast)"]

1.10 2.27 6520.14 1.86 5120.19 1.89 5080.16 1.80 4890.20 1.62 4.21

D-cellobioseD-maltoseSucroseD-mannitolD-glucoseJohnson & Lewis, 1979

Medium Solid fermentation

Table 9--Comparison of solid and submerged fermentation using identical media

Submerged fermentationA 400 nm A 500 nm

Glucose-nitrate 0.28Glucose-peptone 0.19Maltose-nitrateMaltose-peptone 0.17

Johns & Stuart, 1991

A 400 nm A 500 nm

0.10 0.57 0.Q7

0.09 0.69 0.180.61 0.10

0.10 0.69 0.20

moderate on glucose and maltose but poor onfructose. In M. purpureus, fermentation with maltoseand glucose as carbon sources gave very dark liverpigment, whereas sucrose produced a light anduneven red pigment. For pigment production fromPhaffia rhodozyma, cellobiose supported morepigmentation than others. D-mannitol also supportedpigmentation, whereas glucose promoted both growthand pigmentation (Table 8). The sugar-type alsoinfluences the shade of pigment.

4) Nitrogen Source

The production of microbial pigments is alsoaffected by the nitrogen source depending upon themicroorganism. Ammonium chloride is the best forproduction of Monascus pigment followed byammonium nitrate and then glutamate. Potassiumnitrate is the poorest nitrogen source, while glutamateproved outstanding for the pigment production. Useof peptone plays an important role in pigmentproduction from Monascus sp. In M purpureus, 1.5%MSG medium produced an appealing red colour,whereas other nitrogen sources produced faint orfoggy red pigment. For pigment production fromAspergillus sp., ammonium phosphate provedessential for the growth of the organism. The pigmentproduction in Bacillus subtilis has also beenstimulated by incorporation of tyrosine and histidinein the medium.

5) Type of FermentationFermentation (solid or submerged fermentation)

affects production of microbial pigment. The solid-state fermentation yields 3 folds more pigment thansubmerged fermentation. In M purpureus, yields aresuperior in solid cultures than submerged, thoughmedia composition, pH and agitation also affectpigment production. Solid culture of M purpureusgave greater production of red and yellow pigmentsunder similar conditions i.e. media composition andincubation temperature in solid and submergedfermentation (Table 9).

6) MineralsMinerals play an important role in pigment

production (Table 10). Zn (2xlQ-3 M and 3xlQ-3 M)stopped the growth in liquid medium whereas in solidmedium vigorous growth and pigmentation wasobserved. Sometimes Zn acts as a growth inhibitor

Table 1000Effect of minerals on pigment production byA. oryzae var. effusus

Minerals Pigment

Deep redNo growthPinkLight pink

KCl, csci, or ZnS04NH4H2P04MgS04 or FeCl3Sodium citrateManonmani & Sreektaniah, 1984

JOSHI et al: MICROBIAL PIGMENTS

and increases glucose uptake for the synthesis ofpigments. Mn stimulates pigment production fromLactobacillus plantarum and Streptococcus lactis.

Stability of Microbial PigmentsMicrobial pigments are sensitive to heat, light,

acidity, air and water activity and, therefore, unstableand largely degradable, which is a major disadvantagefor their use in foods. The pH plays an important rolein the pigment production (Table 2). Chemicalmodifications, which increase solubility of microbialpigments, improve stability.

Conclusions and Future ThrustUse of microbial pigments in processed foods is

promising with large economic potential. However,microbial pigments offer challenges due to high cost,lower stability and variation in shades due to changesin pH. At present, none of the microbial pigment canreplace synthetic pigments. It is hard to predict thatthe genetic engineering of microorganisms will resultin commercial products or not, nevertheless, X-rayand fast neutron irradiations are being used to induceseveral strains of M. purpureus differing inpigmentation and 'morphology. Genetic engineering ofmicroorganisms has improved the fermentationprocess but further research for non-toxic microbialpigments is required.

369

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