Munawer Khan Mohammed et al.

/ International Journal of Engineering Science and Technology (IJEST)

PRODUCTION OF CAROTENOIDS (ANTIOXIDANTS/ COLOURANT) IN SPIRULINA PLATENSIS IN RESPONSE TO INDOLE ACETIC ACID (IAA)
Munawer Khan Mohammed
Department of Microbiology, Mumtaz Degree & P.G. College, Malakpet, Hyderabad 500036, Andhra Pradesh ( India ) munawerkhan79@gmail.com + 91- 9989713246

Mazharuddin Khan Mohd.

Prof. & Head, Dept. of Microbiology & Biotechnology Mumaz Degree & PG College, Malakpet, Hyderabad 500036, Andhra Pradesh ( India ) dr_mazhar_khan@yahoo.co.in + 91 9885218310 Abstract : The Carotenoid compounds (antioxidants) synthesis in spirulina platennsis was studied in vitro under the influence of the Indole Acetic Acid (IAA). Spirulina platensis is grown in Zarrouks medium supplemented with different concentration of Indole Acetic Acid ( 1g/ml10g/ml ). The gradual increase in the total Carotenoids content was recorded from 1- 6 g/ml of IAA. Inhibition in the synthesis of Carotenoids compounds was noticed in 7g/ml-10g/ml the similar trend was also observed with synthesis of chlorophyll a .Growth of Spirulina was also inhibited at higher level of IAA .Maximum production of Carotenoid compounds noticed at 6g/ml IAA. Keywords: Spirulina platensis, Antioxidants , colourant,Carotenoids, IndoleAceticacid ,chlorophyll- a , Growth INTRODUCTION Spirulina platensis is a blue green fast growing microalga that is relatively large size(2x110m).it is multicellular filamentous cyanobacterium belongs to cyanophyta. It is capable of growing in high alkalinity with the presence of carbonate bicarbonates and inorganic nitrogen (1) .it is the natures richest and most complete source of organic nutrition. becoming a health food world wide.it is ideal for those preferring a whole food supplement to artificial nutrient sources. The biomass of Spirulina platensis has been recognized to b a wonderful food health since it contain high proteins (2) . and various bioactive compounds. Such as essential Fatty Acids (linolenic and -Linolenic acid ) ( 3,4) essential amino acid, B complex vitamins ( Riboflavin , cyanocobalamin, thiamin,nicotinic acid ) Bio pigments (phycocyanin and chlorophyll-a ) (5) and also it contains antioxidant compounds Carotenoids (6) . Carotenoids are natural pigments which are synthesized by plant and algae . there are several dozen Carotenoids in the food and most of these Carotenoids have antioxidants activity.The -carotene is a member of Carotenoids content in biomass is important for food health, pharmaceutical, cosmetics industeries and food industeries and is thus potential to be produced in large scale (7) In 1982 the famous monograph Diet, Nutrition and Cancer published by the National research council reviewed this overwhelming literature. It concluded the epidemiological evidences sufficient to suggest that food rich in carotenes or Vitamin A are associated with a reduced risk of cancer The study recommended a diet including Beta carotene rich Vegetables to reduce cancer risks (8).

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Munawer Khan Mohammed et al. / International Journal of Engineering Science and Technology (IJEST)

Israeli study (1987) demonstrated natural beta carotene is more effective than Synthetic. Natural beta carotene is better assimilated by the body because it contain the 9-cis carotene isomer ,lacking in synthetic carotene molecules . Thus foods rich in Carotenoids in this case beta carotene may not only be able to prevent but also reverse cancers (9) Another study showed that beta carotene reduces the size of tumors that were already present in hamsters and slowed new tumor growth, extending the hamsters survival time (10) One of the largest studies to determine the relationship between beta carotene and cancer is the ten year study at Harvard medical School involving 22000 physicians. Natural beta carotene is chemically and physically different from the synthetic form and although there is evidence that the body absorbes natural beta carotene ten times more easily than it absorbs the synthetic form (11). Anti oxidants in particular Carotenoids ,help to prevent the free radicals damage associated with the aging process itself. There is strong evidence that beta carotene also enhance many aspects of immune fuction (12) .it stimulate immunocompetence in healthy individuals and enhances immune function in people who have tested HIV positive (13) . the cells of the immune system are among the most sensitive to oxidation Carotenoids are natural colourants :- two types of colours are used in food industries natural and synthetic . Synthetic ones are mainly coal tar derivatives made from chemicals which are by products of coal distillation and they are banned in many countries because of the health risks. Thus there is an increasing demand for natural colours represent an apparently more sustainable sources of colourants than synthetic counterpart which are derived from non renewable sources. Synthetic colours are in more use compared to natural ones due to the fact that yield of natural colours from plant sources is low as compared to the yield from modern synthetic processes. Agreater amount of plant biomass is needed for the Extraction of natural colours would require large scale cultivation of plant species and the disposal of large volumes of crop residue may be another problem. Besides colours from higher plant origin, Carotenoids and phycobiliproteins from microalgae are good alternatives from natural colour .Production of colours from microalgae has a number of advantages such as cheaper and easy production ,easier extraction, higher yields, no lack of raw materials and no seasonal variation.(14) Keeping all these advantages of Carotenoids i.e as antioxidants and natural colourants we have selected carotenoids rich Spirulina platensis to carry out the experiments to enhance production of natural Caratenoid compounds, in spirulina which is the richest beta carotene food known having over ten times more beta carotene than any other food ,including carrots.

Materials & Methods : Spirulina platensis was obtained from the stock available in Centre for Food Technology and Research Institute ( CFTRI ) Mysore India. The strain was maintained in Zarrouks medium (15) standard synthetic medium in 500 ml Erlenmeyer flasks maintenance & culture of the spirulina was carried out in continues Illumination at 32+/-1oC temperature the illumination provided with 6 cool white florescent tube lights 40 watts each further shaking of culture were carried out manually thrice a day. The monitoring of Spirulina growth was measured spectrophotometrically at 540nm. This culture was used for further experiments. Experimental design : Ten ( 10 )1000 ml Erlenmeyer flask were used in the experiments. In all the flasks 500ml of Zarrouks media was taken and under aseptic condition 10 % v/v Pure culture of spirulina platensis having an optical density of 0.34 was inoculated in all the flask . After completion of inoculation Indole Acetic Acid ( IAA ) was added in these flasks in the following manner. (Table-I)

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Munawer Khan Mohammed et al. / International Journal of Engineering Science and Technology (IJEST)
TABLE-1: Flasks with Zarrouks medium + different concentration of Indole Acetic acid(IAA)

Flask No

Zarrouks medium in ml 500ml 500ml 500ml 500ml 500ml 500ml 500ml 500ml 500ml 500ml 500ml

Concentration of IAA g/ml --1 g/ml 2 g/ml 3 g/ml 4 g/ml 5 g/ml 6 g/ml 7 g/ml 8 g/ml 9 g/ml 10 g/ml

Control Flask -1 Flask -2 Flask -3 Flask -4 Flask -5 Flask -6 Flask -7 Flask -8 Flask -9 Flask-10

Flasks were incubated under artificial light provided in growth chamber and illuminated by two 4ft white fluorescent tubes which provide estimated light intensity of 15 Em2S-1(16). Samples were collected every 4th day from the flasks (control and 1-10 NO) to estimate the Carotenoids , chlorophyll-a and Growth measurement Analytic procedure : 1. Estimation of total carotenoids : a) Extraction of carotenoids :- The total Carotenoids are extracted by cell lysis (sonication )and partitioned in organic solvent (acetone) on the basis of their solubility .Carotenoides that are bound as esters are hydrolysed using aqueous 60% KOH .the amount of Carotenoids present in sample is estimated colorimetrically at 450nm using -carotene as a standard. (17,) b) Estimation of Caratenoid compounds: Total Carotenoids were estimated by spectrophototmetric method at 450 nm and -Carotene served as a standard compound was used for preparing the calibration curve. (18) 2. Chlorophyll-a : content was determined spectrophotometerically after acetone extraction (19). 3. Growth : Growth of spirulina was measured spectrophotometerically at absorbance maximum 540 nm (20). Results & Discussion: Significant enhancement in production of Carotenoids was recorded when nutrient medium supplemented with Indole Acetic Acid (IAA).positive correlation between Indole Acetic Acid (IAA) concentration and synthesis of higher amounts of Carotenoids in Spirulina plantensis was observed in the cells and this increased Carotenoids content was also related to increase in other cellular compounds such has Chlorophyll-a. (Table- 2) Modified culture media with Indole Acetic acid( IAA) in appropriate quantities was supporting in increasing the antioxidants potential of Spirulina plantensis biomass which is used as a nutrient supplement. The gradual enhancement in Carotenoids production recorded from 4th day to 8th day in the flask which are supplemented with 1 to 6g/ml IAA ,then accelerated production of Carotenoids noticed in the same flasks up to 20th day.The highest quantities of Carotinoids obtained in the flask which supplemented with 6g /ml IAA (fig -1).In contrast to this accelerated production of Carotenoids was noticed in initial days in the flasks which are having higher concentration of IAA i.e 7 to 10g /ml IAA from 4th day to 8th day then gradual decline was noticed after 8th day as a result lowest yield of Carotenoids recorded in these flasks from 8th to 20th day .The trend of synthesis of Carotenoid compounds were presented in the Fig-I

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Munawer Khan Mohammed et al. / International Journal of Engineering Science and Technology (IJEST)
TABLE- 2: Effect of Indole Acetic Acid (IAA) on synthesis of carotenoid compounds

Concentration of IAA g / ml

Concentration of Carotenoid content of Spirulina platensis ( g/5ml ) 4th Day 8th Day 46 72 72 54 32 90 99 62 60 79 64 12th Day 50 108 118 119 106 93 144 110 82 112 110 16th Day 69 104 140 142 158 150 200 160 108 152 144 20thDay 92 170 118 180 138 122 210 176 130 140 165

500ml Zarrouks media + spirulina culture (control ) 500ml Zarrouks media + spirulina culture + 1 g/ml (IAA) 500ml Zarrouks media + spirulina culture + 2 g/ml (IAA) 500ml Zarrouks media +spirulina culture + 3 g/ml (IAA) 500ml Zarrouks media + spirulina culture + 4 g/ml (IAA) 500ml Zarrouks media + spirulina culture + 5 g/ml (IAA) 500ml Zarrouks media +spirulina culture + 6 g/ml (IAA) 500ml Zarrouks media +spirulina culture + 7 g/ml (IAA) 500ml Zarrouks media +spirulina culture + 8 g/ml (IAA) 500ml Zarrouks media +spirulina culture + 9 g/ml (IAA) 500ml Zarrouks media +spirulina culture + 10 g/ml (IAA)

5 8 9 10 13 16 22 10 15 11 16

Fig 1 -:: Effect of Indole Acetic Acid (IAA) on carotenoid content of spirulina platensis

Effect of IAA on Carotenoid content of Spirulina platensis ( g/5ml )

250 C oncentration of C arotenoid Concentration of Carotenoid content of Spirulina platensis ( g/5ml ) 4th Day Concentration of Carotenoid content of Spirulina platensis ( g/5ml ) 8th Day Concentration of Carotenoid content of Spirulina platensis ( g/5ml ) 12th Day Concentration of Carotenoid content of Spirulina platensis ( g/5ml ) 16th Day Concentration of Carotenoid content of Spirulina platensis ( g/5ml ) 20th Day

200

150

100

50

0
C on tro l

Conce ntration IAA g / ml

From the table-3 it is clear that from 4th day to 20th day the production of Chlorophyll a also enhanced in the flasks when concentration of IAA increased . Maximum chlorophyll-a content was recorded in 6 g/ml IAA containing flask as Carotenoids . Maximum production observed at 9 g/ml and decline in chlorrphyl-a was noticed in flask containing 10 g/ml IAA.up to 12th day . after 12th day inhibition in production of chlorophyll-a was noticed in the flasks containing 8 g/ml, 9 g/ml, 10 g/ml.

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TABLE-3:

Effect of Indole Acetic Acid (IAA) on synthesis of Chlorophyll a

Concentration of IAA g / ml

500ml Zarrouks media + spirulina culture

(control )

Concentration of chlorophyll a of Spirulina platensis (mg/10ml) 4th Day 8th Day 12th Day 16th day 20th Day 0.41095 1.30133 2.83238 3.56294 4.14748 0.9932 0.56736 0.59028 0.75717 0.91129 0.98732 0.94857 1.07481 1.02528 1.07762 1.88614 1.87819 1.87964 1.71368 1.71948 3.4438 2.42954 1.6517 1.58519 1.94301 4.3367 4.11135 5.15851 5.26571 6.23469 7.47455 6.06928 6.45487 3.30676 4.89103 4.73391 5.09506 7.55959 8.33313 9.0735 9.31214 7.55044 7.3697 5.6379 5.31274 5.67772 6.11336 8.59474 9.63276 10.65262 11.4557 8.65508 8.3267 6.9799 6.37874

500ml Zarrouks media + spirulina culture + 1 g/ml (IAA) 500ml Zarrouks media + spirulina culture + 2 g/ml (IAA) 500ml Zarrouks media +spirulina culture + 3 g/ml (IAA) 500ml Zarrouks media + spirulina culture + 4 g/ml (IAA) 500ml Zarrouks media + spirulina culture + 5 g/ml (IAA) 500ml Zarrouks media +spirulina culture + 6 g/ml (IAA) 500ml Zarrouks media +spirulina culture + 7 g/ml (IAA) 500ml Zarrouks media +spirulina culture + 8 g/ml (IAA) 500ml Zarrouks media +spirulina culture + 9 g/ml (IAA) 500ml Zarrouks media +spirulina culture + 10 g/ml (IAA)

Fig-2 Trend of Chlorophyll a Synthesis

Trend of Chlorophyll - a Synthesis
C n e t aio o C o r p y - am /m o c nr t n f h loo h ll g l 14 12 10 4th Day 8 6 4 2 0
co nt ro l

8th Day 12th Day 16th day 20th Day

Concentration of Indole Acetic Acid ( IAA ) g/ml

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TABLE- 4: Effect of Indole Acetic Acid on Growth of Spirulina platensis

Concentration of IAA g/ml 4 500ml Zarrouks media + spirulina culture (control ) 500ml Zarrouks media + spirulina culture + 1 g/ml(IAA) 500ml Zarrouks media + spirulina culture + 2 g/ml(IAA) 500ml Zarrouks media + spirulina culture +3 g/ml(IAA) 500ml Zarrouks media + spirulina culture + 4 g/ml(IAA) 500ml Zarrouks media + spirulina culture + 5 g/ml(IAA) 500ml Zarrouks media + spirulina culture + 6 g/ml(IAA) 500ml Zarrouks media + spirulina culture +7 g/ml(IAA) 500ml Zarrouks media + spirulina culture + 8 g/ml(IAA) 500ml Zarrouks media + spirulina culture +9 g/ml(IAA) 500ml Zarrouks media + spirulina culture +10g/ml(IAA)
th

Effect of IAA on growth of Spirulina platensis at 540nm Day 8th Day 0.468 0.586 0.742 0.81 0.815 0.89 0.885 0.893 0.742 0.772 0.688 12th Day 0.59 0.901 0.951 0.788 0.92 0.998 0.747(1:1) dil 0.622(1:1) dil 0.588(1:1) dil 0.622(1:1) dil 0.547(1:1) dil 16th day 0.526(1:1)dil 0.814(1:1)dil 0.849(1:1)dil 0.996(1:1)dil 0.955(1:1) dil 0.620(1:2)dil 0.736(1:2)dil 0.687(1:2)dil 0.681(1:2)dil 0.723(1:2)dil 0.632(1:2)dil 20th Day 0.434(1:2)dil 0.670(1:2)dil 0.680(1;2)dil 0.731(1:2)dil 0.867(1:2)dil 0.846(1:2)dil 0.923(1:2)dil 0.871(1:2)dil 0.836(1:2)dil 0.833(1:2)dil 0.800(1:2)dil

0.141 0.187 0.242 0.221 0.274 0.294 0.366 0.322 0.287 0.27 0.269

The results presented in the table -4 indicates that there is a concentration dependent increased in growth as Carotenoid contents and chlorophyll-a . Further from 4th day to last day of experiment i.e 20th day, maximum growth recorded in the flasks which were containing 6 g/ml and 7 g/ml of IAA. Then concentration dependent decline in growth was noticed in the flasks which are having 8 , 9 , 10 g/ml. In these flask growth decreased as concentration of IAA increased .indicates that the higher concentration of IAA i.e more than 7 g/ml were inhibiting the growth . The results indicates that addition of appropriate quantities of Indole Acetic Acid (IAA) shows the significant positive effect on production of Carotenoid compounds, chlorophyll-a and growth in Spirulina . Thus its seems to be a physiological effect of IAA on Spirulina .As it is well established that IAA stimulate the elongation of the cells, elongation occur because of increase osmotic solutes of the cells, reducing the wall pressure increasing the permeability of the cells to water and cell wall synthesis. Increasing the synthesis of specific DNA, new mRNA and specific enzymatic proteins which leads to stress relaxation as it is happen in the plants. These developed novel features in the Spirulina enhance the uptake rate of the nutrients from the medium leads more production of biomass. Rapid intake of nutrients leads to increase in photosynthesis in turn leading synthesis of amino acids, proteins and other cellular components. Therefore the biomass of algal cell increased in IAA containing flask, when compared with the control .These results further reveled that maximum amount of nitrogen entered in to cells of Spirulina and utilized for the synthesis of Aromatic Amino Acids (AAA) which are the precursors for the synthesis of Carotenoid compounds . . Accelerated production of Carotenoids up to 8 day and Inhibition of in production of Carotenoid after 8th day in the flask containing higher concentration of IAA i.e 7,8,9 and 10 g/ml. Similar trend also observed in growth and chlorophyll-a.These results indicates that decline of required nutrients in the media due to rapid uptake rate of nutrients in presence of higher quantities of IAA i.e 7,8,9,10 g/ml within a short span of time i.e. with in 8 days which created scarcity and disequilibrium in demand and supply of the required nutrients in the flasks

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Based on the results obtained , we can conclude that Indole Acetic Acid (IAA) phytohormone can be used in appropriate quantities as a potential stimulator as it has been observed with the production of phenolic compounds (21) to enhance the production of Carotenoid compounds in Spirulina. Reference :
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