การเพาะเลีย้ งยีสต์ Rhodotorula rubra ในถังปฏิกรณ์ ชีวภาพแบบอากาศยก
Cultivation of Rhodotorula rubra in Airlift Bioreactor
Asst. Prof. Dr. Nuttaporn Chanchay1/
ผศ, ดร. ณัฐพร จันทร์ ฉาย1/
Abstract: Carotenoids are used as additives in food and animal feed industries. They can be obtained by
chemical synthesis or from natural sources. Chemical process is costly, therefore, biochemical production of
carotenoids by microorganisms or plants is preferable. This study aimed to optimum the culture conditions
simultaneously for maximum carotenoid productions by red yeast (Rhodotorula rubra).
It has been found that R. rubra cultivate in airlift bioreactor conditions with glucose as a carbon
source at 10.0% (w/v), ammonium sulfate as a nitrogen source at 1.5% (w/v), pH 7.0, temperature at 35 ๐ C
and Yeast extract 1 g/l as growth factor. Using 2.5 inches of aquarium air stone at the air volume with the
pressure of 0.5 kg/bar to 96 hr. could produce carotenoid to a maximum of 150.818 μg/g cell dry weight.
Moreover, had the ability of antioxidants using DPPH method maximum equal to 99.54%.
Keywords: Rhodotorula rubra, Airlift Bioreactor, carotenoid, antioxidation
บ ท คั ด ย่ อ : แ ค โร ที น อ ย ด์ เป็ น ส า ร เติ ม แ ต่ ง ที่ นิ ย ม ใ ช้ ใ น อุ ต ส า ห ก ร ร ม อ า ห า ร แ ล ะ อ า ห า ร สั ต ว์
โด ย แ ค โรที น อ ย ด์ ที่ ได้ จ าก ก ระ บ ว น ก า รท า งเค มี ต้ อ งน า เข้ า จ า ก ต่ า งป ระ เท ศ ซึ่ งมี รา ค า ค่ อ น ข้ า งสู ง
ดั ง นั ้ น แ ค โ ร ที น อ ย ด์ ที่ ไ ด้ จ า ก ธ ร ร ม ช า ติ จ า ก ก ร ะ บ ว น ก า ร ท า ง ชี ว เ ค มี จ า ก พื ช
แ ล ะ จุ ลิ น ท รี ย์ จึ ง เ ป็ น ท า ง เ ลื อ ก ห นึ่ ง ที่ ก า ลั ง ไ ด้ รั บ ค ว า ม ส น ใ จ
การศึกษาในครัง้ นีม้ ีวัตถุประสงค์ เพื่อศึกษาสภาวะที่เหมาะสมต่อการผลิตแคโรทีนอยด์จ ากยีสต์ สีแดง (Rhodotorula
rubra)
จ า ก ก า ร ศึ
ก ษ
า พ
บ
ว่ า
R. rubra
เมื่ อ เลี ย้ งในถั ง หมั ก แบบอากาศยกในสภาวะที่ มี น า้ ตาลกลูโคสเป็ นแหล่ ง คาร์ บ อนที่ ป ริ ม าณ 10.0กรั ม ต่ อ ลิ ต ร
มี แ อ ม โ ม เ นี ย ม ซั ล เ ฟ ต เ ป็ น แ ห ล่ ง ไ น โ ต ร เ จ น ที่ ป ริ ม า ณ 1.5
กรั ม ต่อ ลิ ต รมี สารสกัด จากยี สต์ เป็ น ปั จ จัย ส่ง เสริ ม การเจริ ญ เติ บ โตที่ ป ริ ม าณ 1 กรั ม ต่อ ลิต ร ท าการเลีย้ ง 96 ชั่ว โมง
ที่ ค วามเป็ นกรด-ด่ า ง เริ่ ม ต้ นที่ 7.0 ภายใต้ อุ ณ หภู มิ 35 องศาเซลเซี ย ส มี ก ารให้ อากาศจากปั ้ม ด้ วยแรงดัน 0.5
กิ โล ก รั ม ต่ อ บ า ร์ ด้ ว ย หั ว ท รา ย ข น า ด 2.5 นิ ้ว ส า ม า รถ ผ ลิ ต แ ค โรที น อ ย ด์ ได้ ป ริ ม า ณ ม า ก ถึ ง 150.818
ไมโครกรั ม ต่ อ กรั ม น า้ หนัก แห้ ง นอกจากนี ย้ ัง มี ผ ลต่ อ ความสามารถต้ านอนุ มูล อิ ส ระด้ วยวิ ธี DPPH เท่ า กับ 99.54
เปอร์ เซ็นต์
คาสาคัญ: Rhodotorula rubra, ถังปฏิกรณ์ชีวภาพแบบอากาศยก, แคโรทีนอยด์, ความสามารถต้ านอนุมลู อิสระ
1/
1/
สาขาวิชาเทคโนโลยีชีวภาพ มหาวิทยาลัยแม่โจ้ -แพร่ เฉลิมพระเกียรติ จ. แพร่ 54140
Department of Biotechnology, Faculty of Maejo University Phrae Campus, Maejo University, Phrae, 54140 Thailand.
Introduction
Carotenoids are a group of bioactive compounds and are responsible for bright yellow/orange
colours of various plants, microorganisms and animals (Wilhelm and Helmut, 1996). It has been found that
carotenoids can inhibit various types of cancer and guard one from other important ‘‘lifestyle-related’’
diseases, such as cardiovascular disease and age-related macular degeneration due to their antioxidant
activity and provitamin A function. (Andrew and Young, 2001 ; Benemann, 1992 ; Krinsky, 1998 ; Sotirios, and
Vassiliki, 2006 ; Steven et al., 2000). It has been increasingly used as a feed and food pigment in the
aquaculture industry, and has also been regarded as a potential functional food and pharmaceutical
supplement, because of its excellent antioxidant activity (Johnson and Schroeder, 1995 ; Guerin et al., 2003).
However, most of the carotenoids sold in the market are derived from chemical synthesis and cannot
meet consumers’ desire for natural carotenoids. Thus, researchers shifted attention from chemical synthesis
to isolation of carotenoids from biological sources such as Chlorella zofingiensis (Po-Fung, and Feng, 2005)
and Haematococcus pluvialis (Garcia-Malea et al., 2005) for green microalgae study, Rhodotorula
mucilaginosa (Aksu, and Eren, 2005), Rhodotorula rubra (Parajo, 1996) and Phaffia rhodozyma (Fontana et
al., 1996 ; Parajo et al., 1998) for yeast study, Gibberella fujikuroi (Garbayo et al., 2003) for filamentous fungi
study, and Rhodospirillum rubrum (Goodwin, and Osman, 1954), and Rhodobacter sphaeroides for bacteria
study. Effective methods for carotenoids extraction from biological sources have been investigated (MaciasSanchez et al., 2005 ; Sachindra, and Mahendrakar, 2005).
Rhodotorula rubra was originally considered a high-carotenoid producing strain, its carotenoid
content is still too low for commercial application. Recently, a few carotenoid hyper producing yeast strains
have been attained through conventional and modern strain development methods, which can accumulate
carotenoids at much higher levels and have a greater commercial potential. (Chanchay et al., 2012). This
study aims to optimize the major nutrients and culture conditions simultaneously for maximum carotenoid
production and antioxidant activity in airlift bioreactor to much higher levels for animal feed industry
Materials and Methods
Microorganism and culture conditions
The Rhodotorula rubra was used in this study, which was kindly provided from Maejo University.
It was later identified, to be Rhodotorula rubra by the Faculty of Associated Medical Sciences, Chiangmai
University. The basal medium for routine liquid culture contained 10.0 g glucose, 1.0 g (NH4)2SO4, 2.0 g
KH2PO4, 1.0 g MgSO4.7H2O and 1.0 g yeast extract (YE) (per litre), with the pH adjusted to 5.5
For stock culture, the yeast was sub cultured on agar slop (basal medium + 15 g/l agar) and
incubated at 30 0C for 72 h. It was kept in the refrigerator. The continuous sub culture was done in every
3 months.
For starter culture, one loop of the yeast was transferred from the culture slop into 100 ml of basal
medium contained in 250 ml Erlenmeyer. The flask was rotated on the rotary shaker at 250 rpm, and 30 0C for
72 h.
To investigate optimal condition (pH, carbon sources, nitrogen sources, time, temperature, growth
factor, Air volume and Head size sand) the liquid culture was done in 250 ml Erlenmeyer shake flasks, each
was filled up with 100 ml medium and incubated on the rotary shaker at 250 rpm, 30 oC. Each of the flasks
was inoculated with 5 per cent starter culture. The culture broth was centrifuged at 5,000 rpm, 10 min,
washed with deionized water and centrifuged at 5,000 rpm for 10 min. The cells of Rhodotorula rubra were
analyzed for carotenoid content by Foss method (Foss et al.,1984) and antioxidant activity modified by Foti et
al (2004). Three replication experiments were carried out.
Measurement of carotenoid content
Yeast cells were separated from the liquid medium by centrifugation and rinsed twice with double
distilled water, and then freeze dried. The carotenoid content was extracted from the yeast was determined
for carotenoid contents by Foss method.
Measurement of antioxidation characteristic of Rhodotorula rubra
The antioxidation characteristics was extracted from the yeast was determined for antioxidation
characteristics were as 2,2-Diphenyl-1-picrylhydrazyl (DPPH free radical scarvenging assay) is used to
determine antioxidant activity of carotenoids produced by Rhodotorula rubra that is modified by Foti et al.
(2004).
Statistical Analysis
Experimental data were subjected to analysis of variance using the Completely Randomized Design
(CRD). Duncan, s New Multiple Range Test was used to identify significant differences among mean of
treatments.
Results and Discussion
Rhodotorula rubra in cultivation in airlift bioreactor conditions with glucose as a carbon source at
10.0% (w/v), ammonium sulfate as a nitrogen source at 1.5% (w/v) for 96 hr., pH 7.0, temperature at 35 ๐C
and Yeast extract 1 g/l as growth factor. (Figure 1). The air volume from pump to the 0.5 kg/bar with aquarium
air stone size 2.5 inch can produce carotenoid increases to a maximum equal to 150.818 μg/g cell dry weight
and the ability of antioxidants using DPPH maximum equal to 99.54%. (Table 1-5).
Rhodotorula rubra was cultured in the fermentor at 23.77 g glucose, 3.19 g (NH4)2SO4, 3.19 g yeast
extract (YE), pH 6.69, and 37 0C (per litre). Dissolved Oxygen was constant maintained by aeration and
agitation. The cells of Rhodotorula rubra were collected, analyzed for carotenoid contents, while it was
239.72± 0.86 µg/g cell dry weight in practical, DPPH = 96.58 per cent, ABTS = 99.84 per cent, and MDA =
85.71 µmol/l (Chanchay et al., 2013)
Rhodotorula rubra grew well in yeast malt extract medium (Glucose 10 g/l) and produced 30.679
g/ g (cell dry weight) of carotenoids. The optimal ratio of molasses to water for carotenoid production by
Rhodotorula rubra in molasses medium, was 1 to 20. The supplementation of 5 per cent (w/v) sucrose in the
medium provided the better yield of carotenoid content of 164.54 g/g cell dry weight (Aksu and Eren,
2005). In general, increase in sugar concentration in the growth medium increased the growth of yeast and
carotenoids formation. The ability of R. mucilaginosa yeast for growing on a variety of carbon sources, such
as glucose, sucrose, and lactose is a remarkable advantage. When compared with the results obtained with
other yeasts in the literature, the high carotenoid productivity of the yeast also suggests a feasible process
(Bhosale and Gadre, 2001 ; Buzzini and Martini, 2000). Thus, the yeast R. mucilaginosa will be one of the
most promising microorganisms for the commercial production of carotenoids by the use of agricultural
wastes as a cheap carbon source. The highest carotenoid concentration (125.0 mg total carotenoids per liter
of fermentation broth) was obtained when 20 g/l molasses sucrose was used as the carbon source while the
highest product yield based on the maximum cell concentration (35.5 mg total carotenoids per gram of dry
cells) was achieved when 13.2 g/l whey lactose was the carbon source in the broth. (Aksu and Eren, 2005).
Compare to another,s work (Aksu and Eren, 2007). An initial ammonium sulfate concentration of 2 g/l gave the
maximum carotenoids production by Rhodotorula mucilaginosa. The highest carotenoid concentration
(89.0 mg total carotenoids per liter of fermentation broth.
Rhodotorula glutinis was cultivated aerobically on Yeast-extract–glucose–chloramphenicol agar
(Merck, Germany) for 9 days at 25 °C under diffuse light. (Kaiser et al., 2007). Many yeast fermentation
companies in Korea used ammonium sulfate and urea as nitrogen source. Yeast accumulated dark pigments
of the molasses in the presence of ammonium sulfate but not in the presence of urea. Yeast extract is the
water-soluble portion of autolyzed yeast. The autolysis is carefully controlled to preserve naturally occurring Bcomplex vitamins. Yeast extract is prepared and standardized for bacteriological use and cell cultures, and is
an excellent stimulator of bacterial growth. Yeast extract is generally employed in the concentration of 0.3% 0.5%. Yeast extract is typically prepared by growing baker’s yeast, Saccharomyces spp., in a carbohydraterich plant medium. Yeast extract has been successful in culture media for microorganism studies in milk and
other dairy products. (Chan et al.,1998) Several media containing Yeast extract have been recommended for
cell culture applications. Yeast extract provides vitamins, nitrogen, amino acids, and carbon in
microbiological and cell culture media. (Ikonomou and Agathose, 2001)
Glucose, ammonium sulfate and Yeast extract have been screened out as the significant factors
affecting on carotenoid biosynthesis of R. rubra in shake-flask cultures. The optimal levels of the two major
nutrients for cell growth are very different from those for carotenoid biosynthesis. The cell growth required
relatively high concentrations carbon and nitrogen source of carotenoid production, while carotenoid
biosynthesis required much lower concentrations of glucose and ammonium sulfate. In addition, the optimal
growth factor (Yeast extract) for cell growth was slightly higher than that peptone for carotenoid biosynthesis.
A plausible explanation for the low optimal nutrient concentrations required for carotenoid biosynthesis is that
carotenoids as the secondary metabolites of R. rubra are mainly synthesized when the cells are under stress
(such as nutrient limitation) and the cell growth (primary metabolism) is suppressed. (Yuan and Jia, 2006).
Carotenoids belong to isoprenoids synthesized through the mevalonate (MVA) pathway, which is one of the
major secondary metabolism pathways in microbial cells (Bailey and Ollis, 1986). Although, most of the
secondary metabolites such as antibiotics are mainly produced during the stationary phase (non-growth
associated), some may be produced during the growth phases. (Shuler and Kargi, 2002).
Sugars such as glucose in the culture medium provides both the major energy source for cell
metabolism and the carbon element for biosynthesis of biomolecules. However, excessive glucose has been
found to repress the carotenoid synthesis due to the so-called Crabtree effect (Yuan and Jia, 2006 ; Reynders
et al., 1997). Nitrogen source, such as ammonium sulfate, is another major nutrient which has been shown to
affect the growth and carotenoid production of several Xanthophyllomyces dendrorhous mutant strains (An et
al., 1989). Yeast extract is another significant factor affecting on the carotenoid production in R. rubra cultures
found in this and many previous studies. Most previous studies have chosen and identified yeast extract 1 g/l
for cell growth and carotenoid biosynthesis in the cultures of wild R. rubra strains (Yuan and Jia, 2006 ;
Johnson and Lewis, 1979).
Figure 1 Optimal condition for carotenoids production and antioxidant activity by R. rubra
Table 1 The effect of growth factor on the production of carotenoids and antioxidant activity by R. rubra
Growth factor (g/l)
Yeast extract
Peptone
Carotenoid (µg /g cell dry weight)
139.453a±1.765
98.743b±1.542
Antioxidant activity (%)
98.87a±0.44
96.34b±0.31
Table 2 The effect of temperature on the production of carotenoids and antioxidant activity by R. rubra.
Temperature (๐C)
25
30
35
40
45
50
Carotenoid (µg /g cell dry weight)
88.653bc
89.962c
99.983a
91.243b
88.519bc
75.025d
Antioxidant activity (%)
93.56bc
93.61bc
97.88a
95.65b
83.67d
81.21e
Table 3 Optimal conditions for carotenoids production and antioxidant activity by R. rubra
Optimal conditions
pH 7.0
Glucose 10.0 % (w/v)
Ammonium sulfate 1.5 % (w/v)
Time 96 h.
Temperature 35 ๐C
Yeast extract ที่ 1 g/l
Carotenoid (µg /g cell dry weight)
Antioxidant activity (%)
145.673±1.782
98.93±0.34
Table 4 Air volume used to produce carotenoids and antioxidant activity by R. rubra in airlift bioreactors
Air volume (kg/bar)
0.25
0.5
0.75
Carotenoid (µg /g cell dry weight)
142.152 b±1.136
146.442 a±1.343
146.528a±1.236
Antioxidant activity (%)
96.83b±0.13
98.46 a±0.16
98.19 a±0.15
Table 5 Size of aquarium air stone for carotenoids production and antioxidant activity by R. rubra in airlift
bioreactors
Aquarium air stone (inch)
1.0
1.5
2.5
Carotenoid (µg /g cell dry weight)
143.553 c±1.456
147.872 b±1.043
150.818a±1.556
Antioxidant activity (%)
97.73c±0.23
98.99 b±0.46
99.54 a±0.19
Conclusion
Carotenoid microbial products into the feed industry is increasing year after year. Efforts
have been made in order to reduce the product costs of fermentation pigments compared to those of
systhetic pigments or pigments extracted from natural sources. Innovations will improve the economy
of pigment production by isolating new or creating better microorganisms, by improving the
processes.
This study aimed to screen out and to optimize the major nutrients (Carbon and nitrogen sources)
and growth factor source (Yeast extract and Peptone) simultaneously for maximum carotenoid production by
Rhodotorula rubra. The optimal culture conditions for carotenoid biosynthesis by R. rubra in airlift bioreactor
cultures with 2.5 inches of aquarium air stone under the pressure from air pump 0.5 kg/bar. It was found that
supplementary of 10 g/l glucose as carbon source, supplementary of 1.5 g/l ammonium sulfate as nitrogen
source for 96 hours, pH 7.0 temperature at 35 ๐C and supplementary of 1 g/l yeast extract as growth factor
source in the medium provided the better yield of carotenoid content of 150.818 µg/g cell dry weight and the
ability of antioxidants using DPPH maximum equal to 99.54%.
Airlift bioreactor, could accumulate carotenoids at much higher levels and had a greater commercial
potential for animal feed industry.
Acknowledgement
This study was financially supported by research and development private sector commercial.
Commission on higher education and partnership Vision Bio Aqua Culture, Thailand. I would like to express
profound appreciation and deep gratitude to all my supervisors for providing of laboratory facilities,
convenience and valuable advice and suggestions on this research work.
References
An, G. H, D. B. Schuman, and E. A. Johnson. 1989. Isolation of Phaffia rhodozyma mutants with increased
astaxanthin content. Appl. Environ. Microbiol. 55: 116–121.
Andrew, J. and G. M. Young. 2001. Antioxidant and prooxidant properties of carotenoids. Archives of
Biochemistry and Biophysics. 385 (1): 20–27.
Aksu, Z. and A. T. Eren. 2005. Carotenoids production by the yeast Rhodotorula mucilaginosa: Use of
agricultural wastes as a carbon source. Process Biochemistry. 4: 2985–2991.
Aksu, Z. and A. T. Eren. 2007. Production of carotenoids by the isolated yeast of Rhodotorula glutinis.
Biochemical Engineering. Journal. 35: 107–113.
Bailey, J. E. and D. F. Ollis. 1986. Biochemical Engineering Fundamentals", McGraw-Hill, New York.
Benemann, M. A. 1998. Microalgae aquaculture feeds. Journal of Applied Phycology. 4: 233–245.
Bhosale, P. and R. V. Gadre. 2001. Carotene production in sugarcane molasses by a Rhodotorula glutinis
mutant", J. Ind. Microbiol. Biotechnol. 26: 327–332.
Buzzini, P. and A. Martini. 2000. Production of carotenoids by strains of Rhodotorula glutinis cultured in
rawmaterials of agro-industrial origin, Bioresour. Technol. 71: 41–44.
Chan, L., P. F. Greenfield, and S. Reid. 1998. Optimizing fed-batch production of recombinant proteins using
the baculovirus expression vector system. Biotechnology Bio Engineering. 59: 178-188.
Chanchay, N., S. Sirisansaneeyakul, C. Chaiyasut and N. Poosaran. 2012. Optimal conditions for carotenoid
production and antioxidation characteristics by Rhodotorula rubra. World Academy of Science,
Engineering and Technology 68: 1934-1938.
Chanchay, N., S. Sirisansaneeyakul, C. Chaiyasut and N. Poosaran. 2013. Optimal Condition for Growth of
Rhodotorula rubra and Antioxidation Characteristics of Its Carotenoids. Doctor of Philosophy. Thesis.
: Chiang Mai University, 124 p.
Fontana, J. D., B. Czeczuga, and T. M. B. Bonfim. 1996. Bioproduction of carotenoids: The comparative use
of raw sugarcane juice and depolymerized bagasse by Phaffla rhodozyma. Bioresource Technology.
58: 121–125.
Foss, P., T. Storebakken, K. Schiedt, K. Liaaen-Jensen, E. Austreng, and K. Streiff. 1984. Carotenoids in diets
for salmonids I: Pigmentation of rainbow trout with the individual optical isomers of astaxanthin in
comparison with canthaxanthin. Aquaculture. 41: 213-226.
Foti, M., C., Daquino, C. and Geraci, C. 2004. Electron-transfer reaction of cinnamic acids and their methyl
esters with the DPPH radical in alcoholic solutions. J. Org. Chem. 69: 2309-2314.
Garbayo, I., C. Vilchez, and J. E. Nava-Saucedo. 2003. Nitrogen, carbon and light-mediated regulation
studies of carotenoid biosynthesis in immobilized mycelia of Gibberella fujikuroi. Enzyme and Microbial
Technology. 33: 629–634.
Garcia-Malea, M. C., C. Brindley, and E. Del Rio 2005. Modelling of growth and accumulation of carotenoids
in Haematococcus pluvialis as a function of irradiance and nutrients supply. Biochemical Engineering
Journa. 26: 107–114.
Goodwin, T. W. and H. G. Osman. 1954. Studies in carotenogenesis. 10. Spirilloxanthine synthesis by washed
cells of Rhodospirillum rubrum. Biochemistry Journal. 53: 222–230.
Guerin, M., M. E. Huntley and M. Olaizola 2003. Haematococcus astaxanthin: applications for human
health and nutrition.Trends Biotechnol. 210–216.
Ikonomou, B. and S. Agathose. 2001. Design of efficient medium for insect cell growth and recombinant
protein production, in vitro Cell Dev. Biol. Anim. 37: 549-559.
Johnson, E. A. and M. J. Lewis,. 1979. Astaxanthin formation by the yeast Phaffia rhodozyma, J. Gen.
Microbiol.115: 173–183.
Johnson, E. A. and W. A. Schroeder 1995. Microbial carotenoids production. Adv. Biochem. Eng, 53:119–
178.
Kaiser, P, P. Surmann, G. Vallentin, and H. Fuhrmann. 2007. A small-scale method for quantitation of
carotenoids in bacteria and yeasts. Journal of Microbiological Methods. 70: 142–149.
Krinsky, N. I. 1998. The antioxidant and biological properties of the carotenoids. Annals of the New York
Academy of Sciences. 854(20): 443–447.
Macias-Sanchez, M. D., C. Mantell, and M. Rodriguez. 2005. Supercritical fluid extraction of carotenoids from
Nannochloropsis gaditan. Journal of Food Engineering. 66: 245–251.
Parajo, J. C., V. Santos, and M. Vazquez. 1996. Production of carotenoids by Rhodotorula rubra from
Sauerkraut Brine. Journal of Fermentation and Bioengineering. 29: 570–572.
Parajo, J. C., V. Santos, and M. Vazquez. 1998. Optimization of carotenoid production by Phaffia rhodozyma
cells grown on xylose. Process Biochemistry. 33(2): 181–187.
Po-Fung, I, and C. Feng. 2005. Production of astaxanthin by the microalga Chlorella zofingiensis in the dark.
Process Biochemistry. 40: 733–738.
Reynders, M. B., D. E. Rawlings and S. T. L. Harrison 1997. Demonstration of the Crabtree effect in Phaffia
rhodozyma during continuous and fed-batch cultivation. Biotechnol. Lett. 19: 549–552.
Sachindra, N. M. and N. S. Mahendrakar. 2005. Process optimization for extraction of carotenoids from
shrimp waste with vegetable oils. Bioresource Technology. 96: 1195–1200.
Shuler, M. L. and F. Kargi. Bioprocess Engineering: Basic Concepts, Prentice Hall, Englewood Cliffs, NJ.
Sotirios, K. and O. Vassiliki. 2006. Antioxidant properties of natural carotenoid extracts against the AAPHinitiated oxidation of food emulsions. Innovative Food Science and Emerging Technologies 7: 132–139.
Steven, M., B. Carla, and Y. Ayako. 2000. -Carotene and selenium supplementation enhances immune
response in aged humans. IntegrativeMedicine. 2(2/3): 85–92.
Wilhelm, S. and S. Helmut. 1996. Lycopene: A biologically important carotenoid for humans. Archives of
Biochemistry and Biophysics, 336 (1): 1–9.
Yuan, S. H. and Jia. 2006. Optimization of cell growth and carotenoid production of Xanthophyllomyces
dendrorhous through statistical experiment design. Biochemical Engineering Journal. 36: 182-189.