1. INTRODUCTION AND LITERATURE SURVEY - …shodhganga.inflibnet.ac.in/bitstream/10603/4573/10/10... · · 2015-12-041. INTRODUCTION AND LITERATURE SURVEY ... called biocolors since

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G. SIVARANJANI, PhD THESIS, 2011 INTRODUCTION

1. INTRODUCTION AND LITERATURE SURVEY

Since ancient times Man has always been fantasized by colours

and started extracting natural colours from environment and

succeeded in using it as colouring agent in various food, textile and

cosmetics. The historic evidences prove that the pre-Vedic Indians

used turmeric, saffron, indigo etc as natural dyes.

Dye is an intensely colored complex organic substance used for

colouring other materials. They can be synthesized artificially

(Table1.1) or obtained from natural products. Natural colours are also

called biocolors since they are extracted from biological materials like

fruits, vegetables, seeds, roots, insects and microorganisms. Until 19th

Century natural colours were in usage but few disadvantages like

stability, availability, price and application methods lead to the

synthesis of artificial dyes.

Artificial dyes like azodye, nitrodye, nitrosodye etc are toxic and

recalcitrant and cause environmental pollution. They are also known

to cause many health hazards Eg. Azorubin and tartrazine are known

to cause allergies, Sunset yellow causes kidney tumours, Erythrosine

may inhibit iodine intake, which in turn may cause goitre and in

recent studies proved that maternal exposure to certain hair dyes lead

to the development of brain tumours in children (Bluhm et. al. 2006).

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Product Food, Drug & Cosmetic Colour No. (US)*

Allura Red AC Red 40

Amaranth Red 2

Brilliant Blue FCF Blue 1

Carmoisine -

Chocolate Brown HT -

Eosine DC Red 22

Erythrosine Red 3

Fast Green FCF Green 3

Green S -

Indigo Carmine Blue 2

Napthol Blue Black -

Ponceau 4R -

Quinoline Yellow WS DC Yellow 10

Red 2G -

Sunset Yellow FCF Yellow 6

Tartrazine Yellow 5

Table1.1. Different certified synthetic food colors extensively used in pharmaceuticals and cosmetics, as per I.S.I. (Bureau of Indian Standards) and also confirming to B.S.(British Standards) and F.D.A. (Food and Drug Administration). Data source: http://www.mentholcrystals.com/food-colors.html

http://www.mentholcrystals.com/food-colors.htm

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Increase in the awareness of environmental pollution led to the

resurgence of demand for non toxic and non polluting natural and

mineral colours. Most of the natural pigments are extracted from

plants like annatto, grapes, beet, paparika and animals like female

insects (Coccus cacti) and microorganisms like Monascus, Rhodotorula,

Bacillus, Achromobacter, Yarrowia, Phaffia etc. (Fellows, 1988; Prescott

and Dunn, 1959). Pigments like anthoquinone, carotenoids,

chlorophyll have been produced from yeast, fungi, bacteria and algae.

The biocolors produced from microorganisms have Pro-vitamin A and

other medicinally important properties a apart from being natural and

safe to use. also, the production of biocolors is independent of season

and medicinal properties geographical conditions due to which there

has been increasing interest in using microorganisms as a colour

source. Microbial pigments already in use as natural food colorants

are listed in Table 1.2.

Through microbial fermentations higher pigment yields can be

achieved on tons of substrate per batch within short periods as

compared with plants in which the growth and harvest (for use in

dyes) takes months. Some of the microbial pigments can be produced

from waste materials like starch and juice industry, hence reducing

water and environmental pollution. Monascus pigment is extracted

from Monascus purpureus grown on steamed rice by solid state

fermentation (Wong and Koehler, 1983). Rhodotorula has been

successfully used for the production of pigment on apple pomace, a

waste from apple juice processing industry (Sandhu and Joshi, 1997)

The type of fermentation is one of the major factor which influences

the microbial pigment production. Solid state fermentation yields

more pigment than submerged fermentation (Monascus pigments).

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Table 1.2. Microbial production of pigments (already in use as

natural food colorants or with high potential in this

field).

Molecule Colour Microorganism

Ankaflavin yellow Monascus sp. (fungus)

Anthraquinone red Penicillium oxalicum (fungus)

Astaxanthin

pink-red

Anthophyllomyces dendrorhous (yeast)

Agrobacterium aurantiacum (bacteria)

Paracoccus carotinifaciens (bacteria)

Canthaxanthin dark red Bradyrhizobium sp. (bacteria)

Lycopene

red

Blakeslea trispora (fungus)

Fusarium sporotrichioides (fungus)

Melanin black Saccharomyces neoformansvar. nigricans (yeast)

Monasco rubramin red Monascus sp. (fungus)

Naphtoquinone deep blood-red Cordyceps unilateralis (fungus)

Riboflavin yellow Ashby a gossypi (fungus)

Rubrolone red Streptomyces echinoruber (bacteria)

Rubropunctatin orange Monascus sp. (fungus)

Torularhodin orange-red Rhodotorula sp. (yeast)

Zeaxanthin

yellow

Flavobacterium sp. (bacteria)

Paracoccus zeaxanthinifaciens (bacteria)

β-carotene

yellow-orange

Blakeslea trispora (fungus)

Fusarium sporotrichioides (fungus)

Mucor circinelloides (fungus)

Neurospora crassa (fungus)

Phycomyces blakesleeanus (fungus)

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1. Solid state fermentation:

All microbial processes leading to the formation of commercially

important products are called fermentations or fermentation can be

defined as any process for the production of a product by the mass

cultivation of microbial cells. Microbial fermentations are practiced in

variety of ways based on type of growth environment, type of

substrate, number/variety of microorganism(s) involved and others.

Microbial fermentations are also categorized based on the

process in which they are produced. These include surface and

submerged fermentations with soluble substrates. Certain

fermentations are carried out with insoluble substrates under solid

state or submerged, solid or semi-solid substrate conditions. Solid-

state fermentation (SSF) process can be defined as “the growth of

microorganisms on moist solid materials in the absence of free-flowing

water” [Moo Young et. al., 1983; Pandey, 1992].

In most SSF processes, the solid matrix serves as both support

and nutrient source. In such cases, usually substrate is pre-treated

but in few cases solid matrix serves only as inert substrate for support

in fermentation process.

In general, a variety of substrates used in SSF bioprocess are

obtained from agriculture (by-products of agriculture, food processing

industries) such as rice bran, wheat bran, red gram husk, Bengal

gram, etc. some of these substrates such as rice and wheat bran

provide a nutritionally complete medium for microbial growth while

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the others require addition of nutrients. Most of the substrates are

insoluble or sparingly soluble complex, polymeric, heterogeneous

macromolecules.

Starchy substrates can suffer from problems of stickiness which

can cause substrate particles to agglomerate during the SSF process

(Farzana et. al., 2007). Lignocellulosic substrates such as wheat bran,

wood etc. usually require significant pre-treatment to disrupt the

structure of cellulose and lignin molecules within the substrate

(Banarjee & Bhattacharya 2003).

Wheat bran is considered as universal suitable substrate due to

presence of sufficient nutrients and even in moist state it remains less

compact by providing a large surface area (Feniksova et. al., 1960). In

wheat bran some vital nutrients necessary for growth and product

formation may be present at suboptimal levels hence supplementation

with other nutrients (solid/water soluble) was required to enhance the

product formation in SSF (Kumar and Lonsane, 1990).

SSF technique is influenced by different parameters like

physical conditions (particle size and shape, porosity, consistency

etc.), environmental parameters (water activity, moisture content,

temperature, pH, oxygen level and nutrient concentration) and finally

by products (Pandey et al. 2000). Natural substrates generally need

preparation (chopping, grinding, etc.) or pre-treatment (cooking,

chemical hydrolysis, autoclaving moist substrate) to reduce size,

enhance the availability of interior particles, bound nutrients and also

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to destroy the contaminant (normal flora) growth (Papagianni 2004; de

Vrije et. al., 2001) and amylase inhibitors (oxidizes SH groups) which

are generally present in cereals.

For the production of secondary metabolites, it is essential to

sterilize the substrates to avoid chances of contamination. During

sterilization by heat, there are 2 steps; heat transfer to the particle

surface and intraparticle heat transfer (Bigelis et. al., 2006).

Particle size and shape that affect the surface area to volume

ratio of the particle is considered as an important factor in SSF

process. Particle sizes less than 1mm to almost 1cm have been often

used in SSF (Mitchell et. al., 1992). Exact characterization of particle

size may be difficult but its length determination can be done by

passing through the series of meshes with different aperture sizes

(Michele et. al., 2002).

Moistening agent is one of the important parameter in SSF

process. Too much of moistening agent may influence the bacterial

growth, product formation, porosity, development of stickiness and

difficulty in oxygen transfer. Low availability of water in the system is

important feature of SSF. Amount of water needed for moistening the

substrates is in the range of 12%-80% for SSF (Mitchell et. al., 2000).

During fermentation process, moisture content may change due to

evaporation and metabolic activities (Nishio et. al., 1979) and hence it

is essential to control moisture content of the fermenting medium.

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This can be achieved on a large scale by humidification of the

fermentation chamber (Ghildyal et. al., 1981).

Inoculum density is also an important aspect in SSF. Too low

density results in insufficient biomass and permits the growth of

undesirable contaminants (Uyar and Baysal, 2003). The inoculum

may be added as dry powder/vegetative cells for bacteria and yeasts

or vegetative mycelium or spores for fungi (Kumar et. al., 2003).

SSF is being applied to large scale industrial processes mainly

in Japan and Thailand (Lonsane, 1991). In India, medium scale

productions of pigments, such as β-carotenes and riboflavin’s are

reported (Pandey et. al., 1999). Table 1.3 and 1.4 presents a list of SSF

processes in economical sectors and advantages and disadvantages of

SSF over SmF respectively.

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Table1.3. Main application of SSF processes in various

economic sectors (source: Pandey et. al., 2000).

Sector Application Examples Agro-food Industry Environmental control Industrial fermentation

Biotransformation of crop residues Food additives Bioremediation and biodegradation of hazardous compounds Biological detoxification of agro-industrial wastes Enzyme production Bioactive products Organic acid production Biofuel Miscellaneous compounds

Traditional food fermented (Koji, sake, ragi,), protein enrichment and single cell protein production, mushrooms production. Aroma compounds, dyestuffs, essential fat and organic acids. Caffeinated residues, pesticides, polychlorinated biphenyls (PCBs) Coffee pulp, cassava peels, canola meal, coffee husk Amylases, lipase, amyloglucosidase, cellulases, proteases, pectinases, xylanases, glucoamylases Mycotoxins, gibberellins, alkaloids, antibiotics, hormones Citric acid, fumaric acid, itaconic acid, lactic acid Ethanol production Pigments, bio surfactants, vitamines

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Advantages :

1. Similar or higher yields than those obtained in the corresponding

submerged cultures.

2. Low availability of water reduces the possibilities of contamination. This

allows working in aseptic conditions in some cases.

3. Similar environmental conditions to those of the natural habitats of

microorganisms used in SSF.

4. Higher levels of aeration especially adequate in those processes

demanding an intensive oxidative metabolism.

5. Inoculation with spores (in those processes that involve fungi) facilitates

its uniform dispersion through the medium.

6. Culture media are quite simple. The substrate usually provides most of

the nutrients necessary for growth.

7. Simple design reactors with few spatial requirements can be used due

to the concentrated nature of the substrates.

8. Low energetic requirements (in some cases autoclaving or vapour

treatment, mechanical agitation and aeration are not necessary.

9. Small volume of polluting effluents. Fewer requirements of dissolvent

are necessary for product extraction due to their higher concentration.

10. Low moisture availability may favour the production of specific

compounds that may not be produced or may be poorly produced in

SmF.

11. In some cases, the products obtained have slightly different properties

(e.g more thermo tolerance) when produced in SSF in comparison to

SmF.

12. Due to concentrated nature of the substrate, smaller reactors in SSF

with respect to SmF can be used to hold the same amounts of

substrate. So, volumetric productivity more.

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Disadvantages

1. Biomass determination is difficult.

2. Agitation may be very difficult. For this reason static conditions are

preferred.

3. Frequent need of high inoculum volumes.

4. Many important basic scientific and engineering aspects are yet poorly

characterized.

5. Information about the design and operation of reactors on a large scale

is scarce.

6. Mass transfer is limited to diffusion.

7. In some SSF, aeration can be difficult due to the high solids

concentration

8. Cultivation times are longer than SmF.

Table 1.4. Advantages and disadvantages of solid state

fermentation bioprocess (Source: Pandey et. al., 2001; Leka and Lonsane, 1994; Pandey 1994; Pandey et. al., 2008).

1.1 Scale up process in SSF:

Tray process has long history in the production of traditional

fermented foods such as soya sauce, koji and more recently used for

the production of enzymes and secondary metabolites. Tray bioreactor

consists of chamber which may be small as an incubator or as large

as a room within which a number of trays are located. Individual trays

may be constructed of plastic, wood or metal. Plastic bags are also

employed for the purpose. The environment is usually controlled by

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controlling the temperature and humidity of the chamber. The most

easily manipulated design and operating variable is the bed depth.

The bed of the tray is left static or may be mixed aseptically. The scale

up of the tray fermentation bioprocess is feasible by just increasing

the number of trays and making the surface area more.

1.2 Recovery of product

The most crucial step in the solid substrate fermentation is

recovery of products. Two important criteria such as percentage of the

extracted product and its concentration after extraction shows effect

on the economic performance of the final product in SSF bioprocess.

In developing nations most of the research activity on SSF is

being done as a possible alternative for conventional submerged

fermentations. Bacteria, yeast and fungi can grow on solid substrates

and find application in the SSF processes. There is a marked trend

towards natural colors used by food and textile industries. In SSF for

pigment production fungi are the most adequate microorganisms. On

the other side, bacteria may grow in solid substrates but usually show

a better development in liquid media. The use of cyanobacterial

cultures in SSF is limited because of poor light penetration on the

substrates which is required for the stimulation of photo-pigments

production.

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At present, yellow and red dyes are already being produced on

an industrial scale from vegetable raw materials. However, in the food,

pharmaceutical, cosmetic and textile industries a large demand for

natural blue and violet dyes still exists. The legal prohibition of the

red-blue dye Monasine (isolated from Monascus purpureus) in foods

because of allergological problems has gained particular significance

for a search of other dyes from this spectrum. Indigoidine has been

officially approved as a food dye (E 132). A microbiologically produced

dye may thus help to close this gap and to open economically highly

interesting paths.

Pigments from the blue-violet spectrum are azaquinone,

indigoidine, which are formed by several organisms such as

Pseudomonas indigofera, Corynebacterium insidiosum, Arthrobacter

arthrocyaneus and Arthrobacter polychromogenes. Another pigment of

this spectrum, violacein and deoxyviolacein, produced from a bacterial

strain of Janthinobacterium has been reported to be used for dying

textiles. Good dyeing has not only been obtained in connection with

natural fibres such as cotton, wool and silk but also with synthetic

fibres such as nylon and vinylon. Using the mordent thiourea, dyeing

stability of violacein has been improved against detergent washings

and sunlight drying (shirata et. al., 1999). To increase the biological

activity of violacein and to reduce its toxicity, a small alteration in the

structure of the molecule resulted in the non toxicity (Bromberg and

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Duran 2001) which can be used as additives in the food, cosmetics

and toy making industries.

2. VIOLACEIN

Violacein is made up of 3 subunits i.e. the 5-hydroxyindole, an

oxindole and 2-pyrrolidone moieties (fig 1.1).

Fig 1.1. Structure of violacein

Chemically violacein is characterized as 3-[1,2-dihydro-5-(5-

hydroxy-1H-indol-3-yl)-2-oxo-3H-pyrrol-3-ylidene]-1,3-dihydro-2H-

indol-2-one, of the summation formula C20-H13-N3-O3 (molecular

weight 343.33). It can be easily identified spectrophotometrically

which shows absorbance in the range of 550-580 nm indicating the

existence of large conjugation C=C in violacein (Renee and Kendall,

2000; Yuan et al, 2009; Rettori and Duran, N. 1998). Violacein can

also be identified by the following properties (1) Violacein is insoluble

in water, but it is soluble in acetone, ethanol and dioxane [Gillis and

De Ley, 1992]. In ethanolic solution it has an absorption maxima at

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579nm (Fig 1.2); (2) On addition of 10% (v/v) H2SO4, the solution

turns green with an absorption maximum at 700nm and (3) When

NaOH is added to an ethanolic solution, the solution turns green and

afterwards reddish brown [Ballantine et. al., 1958].

Fig. 1.2. UV-Vissible absorption spectrum of violacein in ethanolic

solution (Ballantine et al 1958).

It is suggested that violacein production is regulated by

tryptophan (Ballantine et. al., 1960). The synthesis of violacein

pigment through condensation of two modified tryptophan molecules

by decarboxylation process has been shown in the figure 1.3. To

determine the violacein biosynthesis pathway several efforts have been

made through studies on the role of tryptophan and other indole

derivatives in the stimulation of violacein biosynthesis (De Moss and

Evans, 1960; Hoshino et. al., 1987a, b). Using 14C labeled molecules

and feeding experiments with 2-12 C, 2-13 C labeled tryptophan

(Hoshino et. al., 1986; Momen and Hoshino, 2000) it was shown that

all the carbon, nitrogen and hydrogen atoms of the violacein molecule

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come from L-tryptophan molecules while all the oxygen originated

from molecular oxygen (De Moss and Evans 1959). The hydroxylation

of tryptophan to form hydroxyl tryptophan suggested the importance

of molecular oxygen in an oxidation reaction (Hoshino et. al., 1987a,

b). The 1, 2-shift of the indole ring takes place through an

intramolecular rearrangement process during the formation of the left

part of the 5-hydroxyindole side of the violacein skeleton. Duran et.

al., 2001 suggested in a study with radioisotope, that besides L-

tryptophan, C. violaceum is capable of synthesizing violacein starting

from indole-3-acetic acid as metabolite precursor of L-tryptophan.

In the Chromobacterium violaceum proteins of violacein

biosynthesis are codified by one cluster of only 4 genes denominated

vio ABCD, found in a fragment of DNA of about 8Kbp, probably

arranged in a single operon. The inactivation of the vioA or vioB gene

completely blocks the formation of the pigment, while vioC or vioD

inactivation results in formation of violacein precursors (August et. al.,

2000).

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Fig 1.3. Path way of biosynthesis of violacein an indole derivative

produced via the oxidation of L-tryptophan (Ballantine et. al., 1960).

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Violacein is produced by Chromobacterium sp. (Skerman et al.,

1980), Janthinobacterium (Lincoln et al., 1999), Alteromonas

luteoviolacea (Gauthier et al., 1995) and Iodobacter fluviatile (Logan

1989). Although violacein is produced by only a few groups of

bacteria, its presence does not necessarily indicate a close

relationship between these organisms (Gauthier et. al., 1976) and also

the yield and conditions of violacein production are very variable.

The genus Chromobacterium was placed under the group-β, the

order Nesseriales and the family Neisseriaceae. Chromobacterium

consists of gram negative, oxidase positive, facultative anaerobes,

motile rods producing violet colonies on solid media, and a violet ring

is formed in liquid media at the surface with a fragile pellicle. Growth

is best at 30-35 ºC; the minimum growth temperature is 10-15 ºC and

the maximum is 40ºC. It can grow easily on common laboratory media

such as nutrient agar or GYCA (1% glucose, 0.5% yeast extract, 3%

CaCO3 and 2% agar in distilled water). Six different species identified

till now in this genus are C. violaceum, C. psuedoviolaceum, C.

piscinae, C. subtsugae, C. heamolyticum and C. aquaticum. In general,

C. violaceum is the most studied bacterium in the violacein production

field.

Different variable yields and conditions of violacein production are

reported from different bacterial strains. Compared with C. violaceum

ATCC 553 strain (DeMoss and Happel, 1958), the Amazonian strain of

C. violaceum B78 (Riveros et. al., 1998) was shown to produce high

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yields. The production of violacein was optimized by using Brazilian

strain (C. violaceum CCT 3496) (Rettori and Duran 1997; Mendes

et.al., 2001), Pseudoalteromonas DQ504310 (Yang et. al., 2007),

Dunganella sp. B2 (Wang et. al., 2009). Violacein production from

various strains is listed in the Table 1.5. The nutritional and physical

parameters required to increase violacein production have been

reported in previous studies, such as glucose [Kimmel and Maier

1969, Duran et. al., 1994], yeast extract, DL-methionine, vitamine B12,

peptone [DeMoss & Evans 1959, Kimmel & Maier 1969], temperature,

agitation and pH [DeMoss & Evans 1959, Riveros et. al., 1989] among

others. Violacein production in Chromobacterium violaceum is

specifically induced by N-hexanoyl-L-homoserine lactone (HHL), a

quorum sensing molecules and this pigments extraction allows a

quantitative bioassay for detecting the lactones (Blosser and Gray,

2000). Apart from its application as bio colorant, violacein pigment is

having much biotechnological potential which are listed in table 1.6.

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Table 1.5 Violacein production from various microorganisms

Strain Yields References

Psychrotropic bacterium RT102 20 ˚C; 30 h; pH 6.0 3.5 g/L

Nakamura et al (2002, 2003)

Marine sediment bacterium Pseudomonas

25 ˚C;96 h 0.52 g/L

Tan et al. (2002)

Psychrotrophic bacterium, XTI 15 ˚C pH 8 0.8 g/L

Lu et al. (2009)

Chromobacterium violaceum 27 ˚C, 144 h pH 6.2 0.025 g/L

Tobie (1934)

Alteromonas luteoviolacea 22 ˚C; 72h 0.002 g/h

Laatsch and Thomson (1984)

C. violaceum CCT 3496 30 ˚C; 24 h Semisolid fermentation 2 g/L

Rettori and Duran (1997), Rettori et al. (1998)

C. violaceum B78 32 ˚C; 36 h; pH 6.8; Semisolid fermentation 2.6 g/L

Riveros et al. (1989)

C. violaceum B78 28 ˚C; 24 h; pH 7.2 Liquid fermentation 3.4 g/L

Riveros et al. (1989)

C. violaceum ATCC 553 30 ˚C; 72 h; pH 7.2 0.002 g/L

De Moss and Happel (1959)

C. violaceum CCT 3496 30 ˚C; 36 h; pH 7.2 0.43 g/L

Mendes et al. (2001a, b)

Pseudoalteromonas luteoviolacea 20 ˚C; 240 h 0.013 g/L

Yada et al. (2008)

J. lividum strain DSMI 522 25 ˚C 0.016 g/L

Pantanella et al. (2007)

Dunganella sp B2 25 ˚C; 40 h; pH 8.4

1.62 g l-1

Wang et al. (2009)

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Violacein has antibacterial activity and protects against the

predation by protozoa and also lethal to some ciliated protozoa

(anti protozoal) (Duran et. al., 1989).

Violacein has antimalarial activity (Stefanie et al, 2009).

Violacein has anti bacterial activity and inhibitory to both Gram-

positive and negative bacteria (Duran et. al., 1989).

Violacein displayed an antimicrobial activity against

phytopathogenic fungi like Rosellinia necatrix, which causes

white root rot of mulberry and it could also be used as a

fungicide (Duran et. al., 1989).

Violacein exhibited in vitro antimycobacterial activity against

Mycobacterium tuberculosis (H37Ra) [De Souza et. al., 1999].

Violacein has also a trypanocidal activity against Trypanosoma

cruzi [Duran et. al., 1989] and antileishmanial activity (Leon et.

al., 2001).

Violacein (with 10% deoxyviolacein) also presented anti viral

activity against Herpes simplex virus (HSV) and poliovirus [May,

Brummer and Ott, 1991].

Violacein also has other properties like tumoricidal activity

[Duran et. al. 1996; Duran and Haun 1997] cytotoxic activity

[Duran et al 1989; Haun et. al., 1992] and apoptotic induction

capacity [Melo et. al., 2000].

It has an antioxidant property (Azevedo et. al., 2000) and

antichagasic activity (Momen and Hoshino, 2000).

Table1.6. Different biotechnological potential of violacein

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3. Patents:

1. Cosmetic lotion containing violacein was formulated (Aoki and

Nomura, 1998) as violacein showed a strong antibiotic activity

against Staphylococcus aureus and an antioxidant effect on

linoleic acid.

2. Violacein and its derivatives can be used as an insecticide as it

contains insecticidal and antifungal properties. It was effective

on plant parasitic nematode diseases such as watermelon

Meloidogyne sp. diseases and prevents plant mycosis, such as

sclerotinia stem rot, grass pythium blight, and bean sprout

seedling blight (Baek et al., 2007).

3. Violacein was used as a dye in cosmetics like lipsticks, eye

makeup and also in other hair and skin preparations

(antiperspirant, leave-on systems, rinse-off systems) (Meiring et

al., 2007).

4. Violacein from Pseudoalteromonas sp. (DSM 13623), was

proposed for economical use in large amounts for consumer and

environmental-friendly products, especially in the food, textile,

and toy industries (Tan et al., 2002).

5. Violacein was reported to be used for dying fibrous material and

nylon cloth (Shirata et. al., 2000).

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DEFINITION OF THE PROBLEM

Till now to the best of our knowledge, most of work reported on

the violacein production was through broth cultivation using synthetic

and complex media by different bacterial species. None have used low

cost substrates for the production of the same through solid state

fermentation. So the present study was concentrated on violacein

production in more economical way using low cost substrates like

agricultural wastes by a newly isolated bacterium, Chromobacterium

sp. JCI.

OBJECTIVES

Isolation, purification and characterization of a violacein

producing bacterium using polyphasic taxonomic approach.

Screening and selection of various agricultural by-products as

solid substrate for the production of violacein.

Optimization, enhancement and scaling up of violacein

production in the selected substrate.

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1. INTRODUCTION AND LITERATURE SURVEY - …shodhganga.inflibnet.ac.in/bitstream/10603/4573/10/10... · · 2015-12-041. INTRODUCTION AND LITERATURE SURVEY ... called biocolors since