«AKRAM NESHATI A Dissertation Submitted To The Faculty Of Science In Partial Fulfillment Of The Requirement For The Award Of The Degree In Masters of ...»
Saffron, known also as CI natural yellow 6, safran, crocin, crocetin, and crous, is the dried stigma of Crocus sativus, a plant indigenous to the orient but also grown in North Africa, Spain, Iran and France. It is a reddish, brown or golden yellow odoriferous powder with a slightly bitter taste. The stigma of approximately 165,000 blossoms is required to make 1 kg of colorant (Daniel, 1983). The coloring principles of saffron are crocin (Figure 2.1) and crocetin (Figure 2.2).
Crocin is a yellow-orange glycoside that is freely soluble in hot water, slightly soluble in absolute alcohol, glycerine, and propylene glycol and insoluble in vegetable oils. Crocetin is a dicarboxylic acid that forms brick-red rhombs from acetic anhydride that melts with decomposition at about 285°C. It is very sparingly soluble in water and most organic solvents (Daniel, 1986).
As a food colorant, saffron shows good overall performance. In general it is stable toward light, oxidation, microbiological attack, and changes in pH. Its tinctorial strength is relatively high, resulting in use levels of 1-260 ppm (Daniel, 1986).
When D-xylose and glycine were reacted at low temperature (2-26.5°C), the reaction mixture produced yellow, red and blue pigment. The isolated blue pigment reveals its novel chemical structure as shown in Figure 2.3. Blue melanoidin has two pyrrolopyrrole ring coupled with Methane Bridge. The UV-VIS spectrum of blue melanoidin shows a large peak at 625 nm and a small peak at 283, 322 and 365 nm.
The infinite variety of animal colors certainly suggests that coloration plays a significant role in the life of animals. Humans use animal colors as a way of differentiating one species from another, and this also happens among the animals themselves. In closely related species, coloration may be the initial signal for species identification (Martha, 2002).
Color also provides a way for animals to determine the sex of another individual. In Ruby-throated Hummingbirds, for example, only the adult male has throat feathers that form a red gorget; females and young males have a white throat.
When a territorial male ruby-throat encounters another hummer, he can quickly determine if the intruder is an adult male that he needs to chase away, or a female that he might like to woo (Martha, 2002).
A third function of animal coloration is evident in the juvenile stage, or eft (small lizard), of the Red-spotted Newt (a common South Carolina salamander). The juvenile's skin is fire-orange in color, with two rows of small red dots down its back.
In its eft stage, this newt wanders the forest floor pursuing earthworms; its striking color is a warning to predators that its skin is loaded with toxins. As an adult, the newt retains its spots but the rest of its skin becomes dark green just at the time when it returns to the safe haven of a small pond to mate and live out the rest of its days as an aquatic organism (Martha and Daniel. 2003).
If someone leaves a plate of nutrient agar exposed to the air for about 30 min, or makes a spread plate of an appropriate dilution of river water, and incubates at 25°C for a few days, a number of colored colonies of bacteria will usually appear (Austin and Moss, 1986).
In general, bacteria contain many pigments that are similar, if not identical, to those of more complex organisms, particularly plants. Bacterial chlorophylls differ from plant chlorophylls in the reduction of one double bond (Chapman and Hall, 1996).
The yellow and pink colonies from the air exposed plate will usually be Gram-positive micrococci whereas the much wider range of colors from the river water will often be Gram-negative rods such as Flavobacterium, Cytophaga, chromobacteria, Serratia and pseudomonads (Logan, 1994).
Perhaps the most familiar examples of colored colonies seen in the routine soil, water and medical laboratory are those of Pseudomonads such as the blue-green colonies of Pseudomonas aeruginosa or the yellow fluorescent colonies of Pseodomonas fluorescens and related species. An example of a water soluble, nonfluorescent blue-green pigment produced by Pseodomonas aeruginosa is pyocyanin which crystallises as beautiful blue needles and may have a role in respiration (Austin and Moss, 1986).
One group of pigments apparently confined to bacteria is the phenazines based on dibenzopyrazine skeleton. Among these often intensely colored compounds, are the purple iodinin from species of Chromobacterium and the dark blue (in acid solution) pyocyanine isolated from Pseudomonas aeruginosa. Many of the several dozen phenazines so far described have potential commercial intrest particularly as antibiotics (Chapman and hall. 1996).
12 Pigment can be produced either as primary or secondary metabolites of bacterial growth. Primary metabolite is the one which forms pigment during the growth phase of the microorganism. The production of pigments is not significant and pigments are not essential for growth and reproduction of bacteria because cell still can maintain normal growth rate after all the pigment have been removed (Nur Zulaikha, 2006).
Numerous microorganisms synthesize small molecular weight compounds that have no verified function in the cell. Maximum production generally occurs after cellular multiplication has finished. Since the substances are not required to the primary metabolism of cellular growth and multiplication, they are called secondary metabolites. Secondary metabolites are the ones forming near the end of the growth phase, frequently at or near the stationary phase (Nur Zulaikha, 2006).
Bacterial pigments can be extracted from the bacterial cells and be used in industries as drug or dye etc. Table 2.1 shows some examples of pigmented bacteria
and the pigment they produce:
Table 2.1: Examples of different bacteria and their pigment.
2.1.2 Synthetic pigments A huge number of dyes have been synthesized and used mainly for dying textiles. According to their chemical structure they are generally classified into six classes : Azo, indigoid, anthracene, azobenzene, phtalocyanine, triphenylmethan (trityl).
However the structural characteristic of dye sometimes overlaps, uniting in the molecule more than one structural element, making impossible the unambiguous classification. Besides their use in textile industry, various dyes have found application in a wide variety of other fields of up-to-date research and industrial activity (Heinrich, 2003).
220.127.116.11 Azo dyes
Azo colors (Figure 2.4) comprise the largest group of certified colorants. The compounds bear the functional group R-N=N-R', in which R and R' can be either aryl or alkyl. The N=N group is called an azo group, although the parent compound, HNNH, is called diimide. The more stable derivatives contain two aryl groups. The name azo comes from azote, the French name of nitrogen that is derived from the Greek a (not) zoe (to live) (Daniel, 1986).
Figure 2.4 : Structure of Azo dye 14
Azo pigments are important in a variety of paints including artist's paints.
They have excellent coloring properties, again mainly in the yellow to red range, as well as lightfastness. The lightfastness depends not only on the properties of the organic azo compound, but also on the way they have been adsorbed on the pigment carrier. Many azo pigments are non-toxic (Heinrich, 2003).
18.104.22.168 Indigoid A series of water-soluble sulfonated indoxyl derivatives have been prepared, including their base salts with pharmacologically acceptable cations. These particular compounds are useful as food dyes or as cosmetic colorants.
Figure 2.5 (2,2'-(1,4-Phenylenedimethylidyne)-bis(2,3-dihydro-3-oxo-1Hindole-5-sulfonic acid)) represents a typical and preferred member compound.
Methods for preparing these compounds are provided.
Azobenzene compound (Figure 2.6) is an important and valuable multifunctional dye with pure chromophoric properties, high molar extinction coefficient, and fine staining qualities.
2.2 Chromobacterium violaceum Chromobacterium violaceum (C. violaceum), (Figure 2.8), a bacteria belonging to the Rhizobiaceae (Soilborne Phytopathogen) family is found in soil and water in tropical and subtropical areas (Natalia and Nelson, 2001). Chromobacterium violaceum is a Gram negative, facultatively anaerobic, rod-shaped bacterium that is generally considered being non-pathogenic (Rettori and Duran, 1998).
Figure 2.8: Purple colonies of Chromobacterium violaceum Its colonies are lightly convex, not gelatinous, regular and violet, although irregular and non-pigmented colonies can also be found (in anaerobic conditions as violacein is produced only in the presence of oxygen (Marlon et al.
As in all chemoheterotrophic bacteria, C. violaceum is able to grow in minimal medium that includes simple sugars, such as glucose, fructose, galactose, or ribose. However, C. violaceum is not able to synthesize glucose through gluconogenesis, since, based on genome analysis, it lacks the gene that codes for glucose-6-phosphatase (Tania and Regina, 2004).
Violacein consist of three structural units: 5-hydroxyindol, 2-oxoindol, and 2pyrolidone which are poorly water soluble and during formation rapidly precipitates either as discrete particles or on cells or cell clumps (DeMoss and Happle, 1958).
Figure 2.9: General structure of violacein (3-(1,2-dihydro-5-(5-hydroxy-1H-indol-3yl)-2-oxo-3H-pyrrol-3-ilydene)-1.
Experiments have been carried out by researchers to find the significant carbon source for production of violacein. Labeled substrates such as: Succinate, Ribose, Glucose, DL-alanine and L-tryptophan have been tested for this purpose (DeMoss and Evans, 1959).
The low 14C dilution observed with tryptophan-2- 14C suggests that at least a protein of the tryptophan side chain enters pigment directly and without dilution from other carbon sources. Since alanine, lactate and acetate do not contribute either directly or indirectly to pigment synthesis, it is probable that the tryptophan molecule, with the possible carboxyl carbon, is incorporated intact into pigment (DeMoss and Evans, 1959).
It is clear that the carboxyl carbon of tryptophan is eliminated during pigment synthesis, and it is quite probable that all other carbon atom of the tryptophan molecule is incorporated as a unit (Figure 2.11). These results may be expected from a consideration of the pigment’s structure, although no consideration can be formed concerning the synthetic pathway (DeMoss and Evans, 1959).
2.2.1 Application of Violacein Violacein has attracted much attention in literatures lately due to its broad applications in various industries such as pharmaceutical industries. Some activities
of violacein are as follows:
2.3 Violacein Production Due to the vast applications of violacein, researches have been working on growth of C. violaceum in order to ease the process and enhance production of pigment. It is well known that C. violaceum is a very selective bacterium in terms of conditions of growth (Common growth culture for C. violaceum is Nutrient broth and Nutrient agar). It has been reported that the optimum growth conditions should be obtained to achieve the desired product.
Growth of bacteria using soil extract agar (SEA) was reported by Innis and Mayfield at 20°C and 0°C. The results showed that the colonies which developed at 20°C were totally pigmented where as colonies grown at 0°C were non-pigmented.
When colonies grown at 0°C were incubated at 20°C pigments started to appear indicating optimum temperature for pigment production was at 20°C (Inniss and Mayfield, 1979).
The same experiment was carried out with using soil extract broth (SEB), at 0°C, 15°C, 20°C, 25°C. Growth was measured spectrophotometrically at 650 nm.
Since results show that violacein production was lacking in SEB at 0°C, experiment were performed in which various concentration of tryptophan, a known precursor of violacein, were added to SEB in flasks and SEA in plates, growth and pigment production determined. The optimum and suitable pH for this experiment reported around 7.4 where the temperature of medium was 20°C (Inniss and Mayfield, 1979).
22 A summary of the growth media used for production of violacein is shown in Table 2.3.
Table 2.3: Possible growth media for production of C.
2.3.1 Growth Profile A spectrophotometer is an instrument that measures the amount of light that is able to pass through a bacterial culture. It shines a constant beam of light on the sample that is being tested. If the light hits the bacterial cell, then it will bend and bounce off the cell.
The more cloudy a culture is, the more bacterial cells are present within the culture allowing less light to penetrate through and more light is bounced back to the register within the spectrophotometer. This is the instrument used for measuring bacterial production which aims to draw the growth profile of bacteria. Growth
profile consists of four phases (Figure 2.13):
Lag phase is an adjustment period when the bacteria are switching on or off different machinery necessary to break down the energy source within the immediate environment. Log phase is the rapid growth of bacteria at an accelerated pace.
2.4 Violacein Extraction Natalia and Nelson, (2001), extracted the violacein from reaction mixture with ethyl acetate and evaporated the solvent under reduced pressure while Walter, (1934), filtered out the pigment, dried it and extracted it with alcohol.