Research Trends in Molecular Biology, 2016: 85-98 ISBN ... · bacteria, fungi, yeast, ......

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Research Trends in Molecular Biology, 2016: 85-98 ISBN: 978-81-308-0564-1

Editors: Nidhi Gupta and Koushik Biswas

4. Microbial pigment as a potential natural

colorant for contributing to mankind

Vivek Prakash Pankaj1 and Roshan Kumar2 1Center for Energy and Environmental Engineering, NIT-Hamirpur (H.P)

2Vellore Institute of Technology, University, Vellore, Tamil Nadu, India

Abstract. Colors are important for every organism as they are

inherent to all organisms. Living systems have natural colors in the

form of various pigments that helps in proper functioning of life

processes. Due to safety and benefits of natural colors over

synthetic colors there is a great demand in the market for the

natural pigments; especially microbial pigments as of widely used

synthetic pigments have harmful effects on the ecosystem and

living organisms. Industry workers and consumer have profound

dangers of the synthetic colors as they are in direct contact. Use of

microbial pigments have increased due to their numerous beneficial

properties like anticancer, ant proliferative, immune suppressive,

antibiotic, biodegradability etc. Various microorganisms, like

bacteria, fungi, yeast, mould and algae etc. are used for the

production of various pigments by using fermentation technology.

These microbial pigments have wide use and application, especially

in food industries, pharmaceutical industries and textile

industries. Food grade pigments such as β-carotene, are pink

red, Riboflavin lycopene and monascus pigments are used in food

industry. In pharmaceutical industry pigments like Anthocyanin,

Correspondence/Reprint request: Dr. Roshan Kumar, Vellore Institute of Technology University, Vellore,

Tamil Nadu, India. E-mail: [email protected]

Vivek Prakash Pankaj & Roshan Kumar

86

Prodigiosin and Violacein are widely used to treat diseases. Several microbial

pigments are also used in textile industry. Advancement in technologies like

recombinant technology, fermentation technology, bioreactors etc. have made easy

extraction and production of microbial pigments.

Introduction The most pleasurable feature of any article is its color. Various colors

are associated with variety of feelings such as red color shows warmth,

increases pulse rate and respiration, blue or green suggests cool and

peaceful environment and encourages relaxation [1]. Naturally colors are

obtained from color imparting compounds in living systems known as

pigments which play an important role in physiology and molecular

processes of microorganisms e.g. help to adapt at in extreme environment,

protection from solar radiation and photosynthesis. Since several decades,

taxonomists have been using pigments as a taxonomic tool for the

identification and classification of bacteria [2]. Pigments diversity depends

on the differences in their chemical structures and the presence of specific

chromophores [3].Since long time, we get pigments from natural sources,

and their use has increased due to the side effects caused by toxic nature of

the synthetic pigments. In this way the microbial pigments are a good

substitute. Synthetic colors are widely used in foodstuff, dyestuff, beauty

products and drugs making processes, comprise various lethal effects.

Many limitations of synthetic pigments are synthetic in nature,

carcinogenic hazardous. They are also non-environment friendly and non-

biodegradable [4]. Microbial pigments are a promising alternative

source for natural food grade pigments and have huge potential for

applications in foodstuff due to their natural color and safety to use; these

have medicinal properties, nutrients like vitamins. Their production is

independent of season and graphical conditions with controllable and

predictable yield [5]. In nature, a great number of microorganisms e.g.

yeast, fungi, algae, protozoa and bacteria produce pigments (Fig. 1).

Nevertheless, appropriate species should satisfy some criteria: i) ability to

use a variety of C and N sources, ii) tolerance to growth conditions (e.g.

pH, temperature, nutrient concentration), iii) providing expected color at

reasonable yield, iv) non - toxic and non-pathogenic product, and v)

simple extraction of pigment from cell biomass if not excreted [2].

Microbes as source of natural color: current trends and future perspectives

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Figure 1. Representation of various colors producing microorganisms on a Petri dish

[6, 7].

Microbes as a source of natural pigment: Basics and brief

idea

Pigment producing microorganisms like, bacteria offers certain

distinctive advantages, owing to their short life cycle, non-sensibility to

season and climate, potentiality to produce pigments of different colors and

shades, easier scale-up production of pigments, etc. Some examples of the

pigment producing bacterial species include Flavobacterium sp. which

produces the yellow pigment zeaxanthin, Agrobacterium aurantiacum (pink-

red pigment, astaxanthin), Micrococcus sp. (various colored pigments,

carotenoids), and Pseudomonas aeruginosa (blue-green pigment),

Serratiamarcescens (red pigment), Chromobacterium sp. (violet pigment)

and Rheinheimera sp. (blue pigment) [8].

Factors affecting microbial pigment production

Stress condition Pigment Microorganism References

Temperature

25-28°C Monascus pigment β-Carotene

Carotenoids

Monascus sp. Rhodutorula

R. rubra

R. gultinis Pseudomonas sp.

[4, 5, 9, 10]

5-26 °C

42 °C

29-32 °C

35-36°C


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Continued

pH

5.5-6.5, β-Carotene

Carotenoids

Monascus sp.

Rhodotorula R. rubra

[4, 9, 10, 11]

4.0-4.5

2.5 -9.5

Carbon source

Glucose Carotenoids

Monascus pigment

Phaffia rohodozyma

R. lectis

Rhodutorula Lactobacillus helveticus

R. gultinis

[1, 4, 10]

Fructose

Maltose

Lactose

Galactose

Salt source

10-350 mg/ml Metarhodopsin Halorubrum sodomense ATCC 33755

[12]

Mineral

Zn (2×10-3M and

3× 10-3M)

Monascus pigment

Rubropunctatin

Monascorubrin Monascin

Monascus sp. [11]

Nitrogen source

Aluminium chloride Ammonium nitrate

Glutamate

Potassium nitrate

Rubropunctamine Monascorubramine

Rubropunctatin

Monascorubrin Monascin

Ankaflavin

Carotenoids

Monascus sp. R. gultinis

[4, 10]

Moisture content

70% Carotenoids Monascus ruber [4]

Light condition

Dark

Sunlight

Carotenoids R. gultinis

Phaffia rohodozyma

[4, 10, 13]

Incubation Time

7 days

12 Days

Carotenoids Phaffia rohodozyma

Lactobacillus helveticus R. gultinis

[10, 14]


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Some functions of microbial pigments

Protection from UV rays

Act as antioxidant

Protect from extreme heat and cold

Functions as antimicrobial and anticancer

Acquisition of nutrients like iron, nitrogen and carbon

Types and classes of microbial pigments

i. Microbial pigments can be classified on the basis of origin, color, and

solubility.

ii. On the basis origin:

a) Bacterial pigments: It includes bacterium Flavobacter produce

zeaxanthin, Rhodococcus produce canthaxanthin.

b) Fungal pigments: It includes Penicillium, Neurospora and

Rhodotorula yield β-carotene.

c) Algal Pigments: It includes Dunaliella of Chlorophyta produce

β-carotene, Rhodophyta contains phycoerythrins and phycocyanin

iii. On the basis of color: On the basis of color pigments are grouped as in

three pigments as depicted in fig. 2.

iv. Based on solubility microbial pigments can be classified as:

a) Water soluble: These pigments are soluble in water, such as

anthocyanins, xanthophylls etc.

b) Fat soluble: These pigments are soluble in fat, such as canthaxanthin

and carotenes etc.

Figure 2. Classification of pigment on color basis.


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Source of pigments

A number of algae, yeasts, molds and bacteria are excellent source of

pigments. Following criteria should be fulfilled by suitable species for

production of pigments: a) ability to use a wide range of C and N sources; b)

should have tolerance to pH, temperature, mineral concentration and possess

moderate growth conditions; c) reasonable color yield; d) should be non-toxic

and non-pathogenic; e) easily separate from cell mass [1] and potential

microbial sources used for biopigment production as shown in table 1.

Table 1. Potential microbial sources used for bio-pigment production [15, 16]

Name of the microorganism Pigment Color

Bacteria

Bacillus subtilis Riboflavin Yellow

Bacillus thuringiensisH-14 Melanin Dark Brown

Brevibacterium sp. Canthaxanthin Orange red

Chromobacterviolaceum Violacein Violet

Dietziamaris Canthaxanthin Red

Janthinobacteriumlividum Violacein Violet

Pseudomonas aeruginosa Pyocyanin Blue green

Serratia sp. Prodigiosin Red

Streptomyces sp. Prodigiosin Red

Streptomyces virginiae Melanin Dark brown

Streptoverticilliumrubrireticuli Prodoginine Red

Mold

Ashbyagossypi Riboflavin Yellow

Aspergillusruber Physcion Yellow

Blaksleatrispora Lycopene Red

Fusariumsp. Naphthoquinone Brownish Yellow

Monascus sp. Monascin Yellow

Rubropunctatin Orange

Monascorubramine Red

Penicilliumoxalicum Arpink Red Dark red

Penicilliummelinii Atrovenetin Yellow

Yeast

Cryptococcus sp. Melanin Black

Phaffiarhodozyma Astaxanthin Orange red


91

Table 1. Continued

Rhodotorularubra Carotenoids Red

Saccharomyces neoformans

var. nigricans

Melanin Black

Yarrowialipolytica Melanin Brown

Algae

Dunaliellasalina Carotenoid Red

Chlamydomonas Lutein yellow

Chlorella sp.

Hemacoccuspluvialis Astaxanthin Pinkish red

Actinomycetes

Streptoverticilliumrubrireticuli Prodigiosin Red

Streptomyces echinoruber Rubrolone Red

Generation/development of microbial pigment in support of

stress condition and genetic engineering approach

Microbial pigments are produced in variety of conditions such different

range of temperature, humidity, pH and source of nitrogen and carbon. For

example Monascus growth occurs rapidly glucose, fructose, dextrines and

starch. Red pigment produced when there is low initial moisture (26-32%).

Genus Dunaliella accumulates enormous amount of β-carotene, mainly in

response to high light intensity [17]. In recent years Dunalliell sp. mainly

cultivated for carotenoids. Under ideal conditions it can yield 400mg

β-carotene/m2

of cultivation area.

Carotenoids production involve following steps:

i. Selection of the appropriate substrate: The raw materials utilized

might or not be pretreated depending on the fermentative capacity

of the microorganism and the type of enzymes produced.

ii. Bioreactor: The bioreactor configuration and operational variables are

crucial for the maximum yields of the process.

iii. Downstream processing: Production of intracellular pigments is limited

by the complexity of “downstream” processing. Cell disruption is a

critical step to recover intracellular compounds and it affects recovery

yield and carotenoids properties.


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Different microbial pigment and their applications

Microbial pigments have wide applications in day-to-day life, they can be

used as colorants, nutraceutical and food supplements. They are also used in

cosmetics, medicines and biotechnological processes. Various roles of

pigments are:

Pigments as indicators: Violet pigment bacteria (Flexibacter and

Sporocytophaga) indicates polluted drinking water. Blue pigment bacteria

(Vogesellaindigofera) indicates chromium contaminated sites. Phototropic

brown bacteria Chlorobiump haeovibroides indicates the hypoxic condition.

Pigments in food and nutrition: β-Carotene is a precursor of vitamin A. it is

also have applications in beverages such as orange drinks, as solution or

suspensions in vegetable oils, baked products. Application of microbial

pigments used in food industry as shown in table 2.

Pigments in disease control and human health: Eating of carotene rich yellow and green leafy vegetables improves the vitamin A pool size and hemoglobin content in the body. Lutein, zeaxanthin and xanthophyll in corn, kale and spinach believed act as protectant of age related macular degeneration. Astaxanthin helps in cardiovascular disease prevention and immune system boosting. Prodigiosin from Serratiamarcescens, Vibrio psychroerythrous, and Pseudomonas magneslorubra have been reported as antifungal, immunosuppressive, and antiproliferative agents. Pigment producing microorganisms and their proposed bioactivities as shown in table 3.

Table 2. Microbial pigments and their use in food industry [15, 18, 19].

Microorganisms Pigment Application in food

Xanthophyllomycesdendrorhous Astaxanthi Feed supplement for salmons, crabs,

shrimps, chickens, and egg production

Neospongiococcumexcentricum Zeaxanthin Colorant for poultry and fish

Flavobacterium Zeaxanthin As an additive in poultry feed to increase

yellow color of animal’s skin and egg yolk Colorant in cosmetic and food industry

Blakesleatrispora Lycopene Food colorant

Spongiococcumexcentricum Lutein Poultry feeds

Monascus sp. Ankaflavin Color supplement

Pseudomonas aeruginosa Colorant in beverages, cakes,

confectionaries, pudding, decoration of food items


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Pigments and the immune system: Carotenoids is known to modulate the

immunological reactions. Pigments enhance specific and nonspecific immune

functions. Prodigiosin act as immunosuppressant, Phyocyanin from

Pseudomonas sp. helps in cytotoxicity, neutrophil apoptosis.

Table 3. Pigment producing microorganisms and their proposed bioactivities [18].

Srno. Pigment Color Microorganism Activities

Bacteria

1 Astaxanthin

Pink-red

Haematococcuspluvialis,

Agrobacterium aurantiacum

Antioxidant,

photoprotectant, Anti-cancer,

Anti-inflammatory

2 Canthaxanthin Orange Bradyrhizobiumsp. Antioxidant,

Anti-cancer

3 Cycloprodigiosin Red Pseudoalteromonasdenitrificans Anti-plasmodial,

Anti-cancer

4 Granadaene Orange–red Streptococcusagalactiae Antioxidant,

detoxifyROS

5 Heptylprodigiosin Red α-Proteobacteria Anti-plasmodial

6 Indigoidine Blue Corynebacteriuminsidiosum Anti-microbial,

Phaeobactersp

7 Prodigiosin Red Serratiamarcescens,

Pseudoalteromonasrubra

Anti-cancer,

DNA cleavage,

Immunosuppressant

8 Pyocyanin

Blue,green Pseudomonassp.

Cytotoxicity,

Neutrophilapoptosis,

Ciliarydysmotility,

Pro-inflammatory

9 Rubrolone Streptomycesechinoruber

10 Scytonemin Cyanobacteria Anti-inflammatory,

Anti-proliferative

11 Staphyloxanthin Golden Staphylococcusaureus Antioxidant,

detoxifyROS

12 Tryptanthrin Cytophaga/

FlexibacteriaAM13,1Strain

–

13 Undecylprodigiosin Red Streptomycessp. Anti-bacterial,

anti-oxidative

14

Violacein

Purple

Janthinobacteriumlividum,

Pseudoalteromonastunicate,

Pseudoalteromonassp.,

Chromobacteriumviolaceum,

Antioxidant,

detoxifyROS

15 Xanthomonadin Yellow Xanthomonasoryzae Protection

againstphotodamage

16 Zeaxanthin Yellow Staphylococcusaureus,

Flavobacteriumsp.,

ParacoccusZeaxanthinifaciens,

SphingobacteriumMultivorum


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Table 3. Continued

Fungi

17 Ankaflavin Yellow Monascusspp. Anti-tumor,

Anti-inflammatory

18 Anthraquinone Red Penicilliumoxalicum Anti-fungal,

virucidal

19 Canthaxanthin Orange,Pink Monascusroseus Antioxidant,

Anti-cancer

20 Lycopene Red FusariumSporotrichioides

,Blakesleatrispora

Antioxidant,

Anti-cancer

21 Monascorubramin Red Monascusspp. Anti-micrbial,

Anti-cancer

22 Naphtoquinone Deepbloodred Cordycepsunilateralis Anticancer,Anti-

bacterial,Trypanocidal

23 Riboflavin Yellow Ashbyagossypi Anti-cancer,anti-

oxidant,protection

24 Rubropunctatin Orange Monascusspp. Anti-cancer

25 β-carotene Yellow-orange Blakesleatrispora,

Fusariumsporotrichioides,

Mucor,circinelloides,

Neurosporacrassa,

Phycomyces,Blakesleeanus

Anti-cancer,

Antioxidant,

suppression

of cholesterol

synthesis

Algae

26 Astaxanthin Red Haematococcuspluvialis Antioxidant,

photoprotectant,

Anti-cancer

27 β-carotene Orange Dunaliellasalina Anti-cancer,

Antioxidant

Yeast

28 Astaxanthin Red,Pink-red Phaffiarhodozyma,

Xanthophyllomyces,

Dendrorhous

Antioxidant,

photoprotectant,

Anti-cancer,

Anti-inflammatory

29 Melanin Black Saccharomyces,Neoformans –

30 Torularhodin Orange-red Rhodotorulaspp. Antioxidant,

Anti-microbial

Archea

31 Canthaxanthin Orange HaloferaxAlexandrines Antioxidant,

Anti-cancer

Protozoan

32 Hemozoin Brown–black Plasmodiumsp. –


95

Recent trends of microbial pigment

Pigments are inseparable component of human life as they are involve in

every aspect such as health, disease control and aesthetics etc. In recent

times, demand for natural pigment substance especially from microbes have

been increased. They have many industrial applications, coloring materials

in food industries, in pharmacy and aqua culture industries, also used as

fluorescent markers in biochemical assays.

There is significant growths seen in demand of naturally derived color

due to improvements are seen in technology and industrial setups. A study

on dyes and organic pigments, the worldwide organic colorants market share

is projected to increase from 4.9% in 2003 to $10.6 billion in 2008. A report

from Mintel and Leatherhead Food Research the global market for food

color was approx. $1.55 billion in 2011 [20].

The market share of microbes and microbial products seems to have a

significant and have huge commercial potential in the future demand for safe

and naturally occurring (edible) coloring agents has tremendously increased

in response to carcinogenic and teratogenic nature of synthetic coloring

agents. There are no reliable published statistics on the size of the color

market, however, on a global scale a reasonable estimate would be US$940

million. Presently, the cost of the natural colors is higher than that of

synthetic colors, but this barrier can be overcome by the mass biotechnical

production of the natural colors, in that way, the cost can be bring down.

(Commission on Genetic resources for Food and Agriculture,

2013) [19].

Future perspective and scope of advancement of microbial

pigments

There is serious need of alternate colorants that are natural, cheaper

and biodegradable and without production of unmanageable intermediates

when they enter the ecosystem. There is a growing interest in

microorganisms as a potential alternate source of colorants used in food

industry. In this way, biotechnology may play an important role for large

fermentation of natural bio-colorants. Recent studies prominently projected

the value of natural colorants over synthetic colors. 2011 saw a global sale

of $600 million, an increase of 29 % from 2007. Many microbial pigments

not only used as coloring agents in food and cosmetic industry but also act

as anticancer, anti-inflammatory anti-microbial and anti-oxidant. There is a


96

huge demand for colorants in textile, plastic, paint, paper and printing

industry [17].

Using microbial pigments in food processing have huge economic

potential. But using it has challenges due to its high cost, lesser stability

and change in shades due to pH change. In recent times genetic

engineering of microorganisms have improved the fermentation

technology but extensive research is needed for the production of nontoxic

microbial pigments [20]. Generation of various from industrial and

domestic wastes is very promising as it can meet ever -increasing demand

for safe and natural colors. Microbial pigments have huge potential for

meeting the consumer demand as they are independent on weather

conditions, have fast growth.

Conclusion

Pigments are commonly used in numerous products, for example,

food, coloring paint, animal feed, plastic, fabric, cosmetics, and medicine.

Accordingly, synthetic and natural pigments have been immensely used in

various industries. Pigments are responsible for the wide spectrum of

colors in various objects. They are often associated with quality, attractive

appearance, and sensory properties. In recent years, the utilization of

natural pigments in manufacturing sectors e.g. animal feed, food, dye,

textile, cosmetic and pharmaceutical has been increasing due to the

demands by the consumers to replace synthetic dyes. The various benefits

of producing pigments from microorganisms comprises of independence

from weather conditions, easy and fast growth, colors of different shades

and growth on cheap substances and have no negative effect on

environment i.e., eco-friendly. Advancement in stability, safety and

solubility can definitely make extensive use of microbial pigments in the

textile, paper and food industry.

Acknowledgement

All authors want to say thanks of their respective departments or

institutes.

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