Indian Journal of Biotechnology Vol. 4, July 2005, pp. 353-357 Influence of growth factors on carotenoid pigmentation of Rhodotorula glutinis DFR-PDY from natural source B V Latha, K Jeevaratnam*, H S Murali and K S Manja Defence Food Research Laboratory, Siddarthanagar, Mysore 570 011, India Received 31 July 2003; revised 2 July 2004; accepted 15 July 2004 A red yeast (Rhodotorula glutinis DFR-PDY) strain, isolated from a contaminated Potato Dextrose Agar (PDA) plate, was evaluated for its growth and pigment production. The yeast was able to grow and produce carotenoid pigments over a wide range of ph from 2.5 to 9.5 (optimum ph 5.5) and at ambient temperature of 29-32 o C in a modified Czapek Dox broth after 12 days of incubation. However, light was not a limiting factor for the growth and pigmentation of the red yeast. Among the various nitrogen and carbon sources examined, maximum growth and pigmentation was observed in sodium nitrate as nitrogen source and glucose and fructose as carbon sources. Thin layer chromatography of the extracted carotenoid pigments revealed the presence of four colour components. Keywords: carotenoids, fermentation, Rhodotorula glutinis IPC code: Int. Cl. 7 A01N63/02; B01D15/08; C12P23/00; CR1:645 Introduction In one form or another, colour has been added to our foods for centuries. The mineral and metal-based colours were added to disguise low quality and adulterated foods. The bulk of chemically synthesized colours were derived from aniline, a toxic petroleum product. Over the last 25 years, although many advances in the developments of food colours have been made, particularly in terms of processing and formulation technology as well as harmonized legislation, there is still scope for future developments. The overall forecast for colour market is to grow in line with the technological and social changes, leading to an increase in the availability and acceptability of processed foodstuffs. It is thought that the natural colour market will grow globally at a greater pace than synthetic colours, owing to a continued consumer pressure to go natural 1. Among the pigments of natural origin, carotenoids play primary role, since their presence in the human diet have been considered positive because of their action as pro-vitamin 2, antioxidant or possible tumour-inhibiting agent 3. Despite the availability of a variety of natural and synthetic carotenoids from the plants, there has currently been a renewed interest in *Author for correspondence: Tel: 91-821-2473949; Fax: 91-821-2473468 E-mail: dfoodlab@sancharnet.in microbial sources of pigments for the problem of seasonal and geographical variability in plant origin 4. Moreover, industrial interest is now gradually shifting away from the yellow carotenoids, such as β-carotene and lutein, towards the considerably more valuable orange-red keto-carotenoids, such as torularhodin and torulene, for which at present no commercially exploitable plant or animal sources exist 5. Rhodotorula and Rhodosporidium produce the major carotenoid pigments, viz. carotene, torulene, torularhodin 6. However, the type, concentration and yield of various carotenoids depends on the species of microorganism and their culture conditions. The biosynthesis of carotenoid pigments was studied in Rhodotorula glutinis by co-cultivation with Lactobacillus helveticus in cheese ultrafiltrate 7 and in raw materials of agro-industrial origin 8. Whereas, use of apple pomace after drying and pulverising into powder, supplemented with ferrous ammonium sulphate, was reported as a cheaper medium for the pigment production by Rhodotorula 9. Besides carotenoids, Rhodotorula is a rich source of fats and vitamins and can be incorporated in feeds to enhance the nutritional value and prevent fungal contamination 10. The present work describes the optimization of growth conditions for the R. glutinis DFR-PDY (local strain) in Czapek Dox synthetic media.
354 INDIAN J BIOTECHNOL, JULY 2005 Materials and Methods Culture A local red yeast strain was isolated from a contaminated Potato Dextrose Agar (PDA) plate in the laboratory by plating method. Its identity was confirmed by IMTECH, Chandigarh as R. glutinis DFR-PDY. The culture was maintained on PDA slants, transferred to fresh slants every three weeks and stored at 4 C until needed. Inoculum Preparation The culture medium used was a modified Czapek Dox broth having the following composition: glucose, 30; NaNO 3, 2; K 2 HPO 4, 1; MgSO 4, 0.5; KCl, 0.5; and FeSO 4, 0.01 g/l supplemented with yeast extract, 3 g/l, where the C/N ratio was maintained at 40:1. R. glutinis DFR-PDY was grown in 250 ml Erlenmeyer flask containing 100 ml of culture medium with 0.3% yeast extract, at an ambient temperature 29-32 C for 48 h on a rotary shaker (200 rpm). These cultures were used as inoculum at 4.0% (v/v) for all the experiments performed in 500 ml shake flasks with a working volume of 150 ml. Optimization of Growth Conditions The total biomass yield and pigmentation of R. glutinis DFR-PDY were studied in a culture medium as described above having glucose (30 g/l) as carbon source and different nitrogenous compounds as nitrogen source at a C:N ratio of 40:1. Yeast extract (2.8 g/l), peptone (2.4 g/l), casein acid hydrolysate (4.4 g/l), urea (0.64 g/l), sodium nitrate (2 g/l), ammonium nitrate (0.8 g/l) and ammonium tartarate (1.9 g/l) were the different nitrogen sources evaluated. Xylose, ribose and arabinose (pentoses); glucose, fructose and galactose (monosaccharides); and sucrose, lactose and maltose (disaccharides) were evaluated as carbon sources (30 g/l) with sodium nitrate (2 g/l) as nitrogen source at 40:1 C:N ratio. The growth conditions for R. glutinis DFR-PDY were optimized with different C:N ratios by having various concentrations of glucose: 8 (10:1), 24 (30:1), 30 (40:1) and 40 g/l (50:1), as carbon source and a single concentration of NaNO 3 (2 g/l) as nitrogen source in above mentioned culture medium. Nitrogen sources at different concentrations were not studied as pigmentation occurred only in stationary phase. Subsequently, the influence of incubation time (3, 5, 7, 9, 11 and 13 days), initial ph (2.5, 3.5, 4.5, 5.5, 6.5, 7.5 and 9.5) and incubation temperature (15, ambient 29-32, 37 and 42 C) were studied at the optimized culture medium, comprising glucose (30 g/l) as carbon source and sodium nitrate (2 g/l) as a nitrogen source at a constant agitation speed of 200 rpm. To study the effect of light on growth and pigmentation, the culture was grown in triplicate in modified Czapek Dox broth in dark (flasks wrapped in black polythene cover) and in the presence of sunlight. Harvest Method The cells were harvested after 12 days of culture growth by centrifuging at 10,000 g and washing twice with distilled water. The cells were centrifuged again and freeze-dried. Red yeast biomass was expressed as cell mass (dry wt) per litre of broth (g/l). Analyses The initial and the final glucose concentrations were determined by dinitro salicylic acid method 11. Total protein content was calculated from the total nitrogen content (N 6.25) determined by the micro- Kjeldhal 12. Fractionation of the crude carotenoid pigment from R. glutinis DFR-PDY was done by thin layer chromatography (TLC). Silica gel G was used as an adsorbent and two different developing solvent systems, viz. benzene: petroleum ether (95:5) and petroleum ether : benzene (95:5), were used for separation of the polar and nonpolar components, respectively. All the chemicals used were of AR grade. Carotenoid Extraction A known amount of freeze-dried red yeast was hydrolyzed with 1N HCl in a water bath at 70 C for 1½ h 13. The acid free cells were soaked overnight in acetone:methanol (1:1) solution. The pigment was extracted with acetone and transferred to lightpetroleum (40-60 C) in a separating funnel and washed thrice thoroughly with distilled water 7. The absorbance of the light-petroleum phase was read at 474 nm (λ max ) and the concentration of the carotenoids was determined using the absorption coefficient 14 as A 1% 1cm = 1600. Carotenoid yield is reported on the basis of cell mass (μg/g cell dry wt) and also on the basis of culture broth volume (mg/l culture broth). All the observations were made in triplicate and the values represented in figures are mean + SD.
LATHA et al: CAROTENOID PIGMENT FROM NATURAL SOURCE 355 Results and Discussion Effect of Different Carbon Sources Assimilation of different sugars as carbon source by the red yeast for its growth and pigmentation shows (Fig. 1) that among the monosaccharides, the carotenoid yield was higher on fructose followed by glucose and galactose, even though galactose supported maximum cell growth compared to glucose and fructose. On disaccharides, maximum growth and pigmentation was observed on sucrose, while very little yield was noticed on maltose. Further, lactose supported no growth to the yeast. It may be noted here that lactose-assimilating yeasts are rarely found in natural conditions and R. lactis is the only species that could assimilate lactose among 163 strains of Rhodotorula 7. However, it was reported that carotenogenesis of R. glutinis in whey ultrafiltrate could be achieved when it was co-cultivated with Lactobacillus helveticus 7. Among the pentoses, it was observed that L-arabinose was a poor substrate for growth and pigment production. D-xylose supported a high rate of growth but only a moderate carotenoid yield, whereas D-ribose resulted in higher rate of both growth and pigmentation (3.1 mg/l). Effect of Different Nitrogen Sources Among the various nitrogen sources (Fig. 2), sodium nitrate was the best nitrogen source for production of carotenoids (3.3 mg/l), followed by casein acid hydrolysate (2.4 mg/l), urea (2.2 mg/l) and yeast extract (2.1 mg/l), while the others gave poor carotenoid production. Effect of Dextrose Concentration R.glutinis cultivated on different dextrose concentrations, with fixed amount of sodium nitrate as nitrogen source, revealed the highest yield of pigmentation (3.5 mg/l) at dextrose concentration of 30 g/l (Fig. 3). Effect of Incubation Time Effect of various incubation periods on the growth and carotenoid production of R. glutinis (Fig. 4) showed that the pigmentation of this red yeast was not growth related because as the carotenoid content in the cell mass reached their maximum, the cell growth had practically stopped, i.e. reached to the stationary phase. In contrast, Bhosale and Gadre 15 reported that the pigment production in the yeast was growthassociated. The maximum cell mass concentration (10.2 g/l culture broth) was obtained on the 7 th day Fig. 1 Effect of different carbon sources on cell dry weight and Fig. 2 Effect of different nitrogen sources on cell dry weight and Fig. 3 Effect of dextrose concentration on cell dry weight and
356 INDIAN J BIOTECHNOL, JULY 2005 accompanied by complete utilization of glucose, whereas maximum carotenoid yield (3.1 mg/l culture broth) was obtained on 12 th day on the modified Czapek Dox broth. Maintaining cells in stationary condition after 12 days of incubation till 20 th day did not further increase the carotenoid content. Therefore, a period of 12 days was selected as the optimum time of incubation in contrast to 10 days reported for R. glutinis 22P when cultivated with L. helveticus 12A 7. Effect of ph The red yeast was able to grow and form pigments under a wide range of initial ph conditions from 2.5 to 9.5. The cell dry weight increased gradually with an increase in the ph of the broth, while the highest carotenoid yield (3.3 mg/l) was obtained at ph 5.5 (Fig. 5) and can be considered as the optimum ph for pigment production in this yeast. R. rubra also had the same optimum ph but failed to grow at ph 3.0 16. Effect of Temperature R. glutinis DFR-PDY was able to grow and produce pigments at all the temperatures examined with the exception of 42 o C, at which a negligible growth took place without any pigmentation (Table 1). A considerably increased cell dry weight (11.3 g/l) with less pigmentation yield (2.5 mg/l) was observed at 15 o C; whereas, the maximum pigmentation yield (3.5 mg/l) was recorded at the ambient temperature 29-32 o C. Ideally, Rhodotorula spp. can be cultivated over a wide range of temperatures from 5 to 26 o C, whereas the optimum temperature for R. rubra has been reported to be 22 o C 16. The effect of temperature on yeast carotenogenesis is dependent on the species specificity and strain characteristics of the microorganism 17. Effect of Light The cultivation of the yeast in the dark and under sunlight conditions showed that light was not a limiting factor for growth and pigmentation of R. glutinis DFR-PDY. In contrast, light stimulated carotenogenesis in many other microorganisms, such as Phaffia rhodozyma, where cultures exposed to light synthesized more astaxanthin with a redder hue 18. Nature of Pigment Thin layer chromatography separation of the crude extract of pigments from R. glutinis DFR-PDY revealed the presence of multiple components. In the developing solvent system petroleum-ether : benzene Fig. 4 Effect of incubation time on glucose utilization, cell dry weight and pigmentation in R.glutinis DFR-PDY culture Fig. 5 Effect of initial medium ph on cell dry weight and pigmentation in R.glutinis DFR-PDY culture Table 1 Effect of temperature on cell dry weight and Temperature ( o C) Cell dry weight (g/l) Carotenoid yield (mg/l) 15 11.3 ± 0.69 2.5 ± 0.22 R.T. 29-32 9.8 ± 0.07 3.5 ± 0.24 37 6.7 ± 0.18 1.4 ± 0.50 42 Negligible Nil (95:5) two major fractions, one yellow and one orange, were observed with R f values 0.9 and 0.7, respectively; while in benzene:petroleum-ether (95:5) multiple spots were observed, of which two prominent fractions were pink and orange-red with the R f values 0.4 and 0.94, respectively. Further studies are needed to identify these components.
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