Plackette-Burman 설계법을 이용한 카로테노이드생산의 최적화

박평규, 김의용

서울시립대학교 화학공학과

Optimization of production carotenoid by Plackette-Burman Design

Pyoung-Kyu Park, Eui-Yong, Kim

Department of Chemical Engineering, University of Seoul, Seoul, Korea

INTRODUCTION

Carotenoid was ealier detected in some algae, fungi, and bacteria, and also in small crustaceans. these organism initiate a food chain that leads to pigmentation of the muscles of salmonid fishies, the muscles and shells of crustaceans, and the plumage of some birds. This has resulted in considerable interest in commercial culturing of the yeast R. glutinis as a source of natural pigment in aquaculture[1]. These carotenoids are an important antioxidant, photoprotective agent and source of provitamin-A[2]. the carotenoid yield and the type and concentration of various pigments that form carotenoids depend on the species of the microorganism and on the cultural conditions. The conventional medium optimization is usually time-consuming - a single dimensional search which involves changing one variable while fixing the others at a certain level is laborious and time consuming, especially when the number of variables is large[3].

In this study is a Plackette-Burman design carried out to study the carotenoids produced by the R. glutins.

MATERIAL AND METHODS

Growth of the yeast

R. glutinis was cultivated in YM(glucose;1%, peptone;0.5%, yeast extract; 0.3%, malt extract; 0.3%) medium. this was incubated on a rotary shaker(125rmp) for 24hr at 22□2□, which served as the initial inoculum. A calculated amount of 20hr old inoculum was incubated into the flasks containing the carotenoid production medium(CPM); glucose 1.5, (NH4)2SO4 0.2, KH2PO4 0.1, MgSO4·7H2O 0.05, CaCl2, 0.01, NaCl, 0.01at pH 6.0 was used throughout the studies. Flask cultures were performed in 500mL Erlenmeyer flasks containing 100mL of medium. The flasks were inoculated with 2%(v/v) of a 20hr precultured broth and incubated at 22□ on a shaking incubator.

Extraction and estimation of carotenoids

Cell mass in flask culture was measured by the optical density at 660nm with UV-spectrophotometer. Total carotenoids content of the cells was determined as the value for torularhodin whose the absorbance maximum and extinction coefficient([eqn: { E}`_{1cm} ^{1% }] ) in petroleum ether are 501nm and 2040, respectively[4]. Composition of carotenoids was measured by HPLC. The conditions of HPLC for determination of carotenoids composition were as follows; column, □Bondapak C18 3.9□300mm; elution solvent, acetonitile-tetrahydrofuran-water 5:3:1; flow rate, 1.5ml/min; detector, UV detector; wavelength, 436nm.

Design of experiments

When the problem involves data that are subject to exprimental errors, statistical methods measure the effects of change in operating variables and their mutual interactions on the process performance through Plackette-Burman design[4]. The data analysis is carried on the basis of unreplicated Plackette-Burman design which includes 12 experiments of 8 variable at 2 level(-1, +1). The important factors considered are maltose, valine, KH2PO4, MgSO4, CaCl2, NaCl, pH, Temp, Working volume. The response variable was the final concentration of volumetric carotenoid express as mg/L. The culture conditions were varied according to the experimental design.

RESULT AND DISCUSSIONS

Table 1 shows the coded and actual levels of the variables employed in the design matrix. Table 2 shows the data biomass and carotenoid production. The first-order model adjusts well to the experimental data, only 16.4% of the total variation was not explained by the model(R2=0.9366). Valine, maltose concentration and CaCl2 were the most important effects in the carotenoid production, as well as the interaction between maltose-valine(P<0.05).

Table 1. Coded levels and actual values of the variables empolyed in the design of experiment

Coded level	Maltose	Valine	KH2PO4	MgSO4	CaCl2	NaCl	pH	Temp	Working volume
-1	1	0.02	0.1	0.01	0.01	0.01	4	20	100
+1	2	0.25	0.6	0.1	0.06	0.1	6	30	200

Table 2. Experimental carotenoid yield and predicted yield

Exp.	maltose	valine	KH2PO4	MgSO4	CaCl2	NaCl	pH	Temp	Working volume	Cell mass (g/L)	Carotenod yield(mg/L)
											Experimental	Predicted
1	-1	-1	-1	+1	+1	+1	-1	-1	+1	1.69	1.63	2.29
2	+1	+1	-1	+1	+1	-1	+1	-1	-1	10.06	9.94	8.52
3	+1	-1	+1	+1	-1	+1	-1	-1	-1	2.32	1.50	0.50
4	-1	-1	-1	-1	-1	-1	-1	-1	-1	3.46	2.46	2.40
5	+1	+1	+1	-1	+1	+1	-1	+1	-1	8.11	7.05	7.52
6	+1	-1	+1	-1	-1	-1	+1	+1	+1	2.34	0.20	0.50
7	-1	-1	+1	+1	+1	-1	+1	+1	-1	2.01	0.46	0.49
8	-1	+1	-1	-1	-1	+1	+1	+1	-1	8.01	2.51	2.16
9	+1	+1	-1	+1	-1	-1	-1	+1	+1	6.20	3.26	4.20
10	+1	-1	-1	-1	+1	+1	+1	-1	+1	2.55	2.11	2.82
11	-1	+1	+1	+1	-1	+1	+1	-1	+1	6.33	0.56	1.16
12	-1	+1	+1	-1	+1	-1	-1	-1	+1	5.94	5.73	5.48

The remaning main effects were not significant, as well as the rest of the interaction effects. As shown in Table 2, the highest carotenoid production and biomass were observed at 0.25g/L valine concentration, 0.06g/L CaCl2 and  2.00g/L maltose concentration. On the other hand, the lowest cell mass formation was obtained when maltose and valine concentration were high. The volumetric carotenoid formation is known to be increased by low C/N ratio and by 1.41 times as pH increase and Temperature decreased. The final predictive equation developed is

[eqn: Y~=~3.12~+~0.89A~+~1.72B~+~1.16E~+~0.63AB]

The reponse equation developed indicated linear relationship between maltose, valine concentration and calcium chloride on carotenoid production.

Table 3. ANOVA for response of dependent variable carotenoid production

Effect	Sum of squares	d.f.*	Mean square	F-ratio
A: Maltose	9.54	1	9.54	2.60
B: Valine	35.64	1	35.64	9.71
E: CaCl2	14.33	1	14.33	3.90
AB	4.21	1	4.21	1.15
Total error	25.70	7	3.67

* d.f.= degrees of freedom.

REFERENCE

1. Andrew Young and George Britton, Carotenoids in Photosynthesis,            Chapman & Hall, (1993)

2. Helga Gerster, Internat. J. Vit. Nutr. Res., 63, 93-121 (1993)

3. Box, G.E., Hunter, W.G., Hunter, J.S., Statistics for Experimenters. Wiley,      New York, NY.(1978)

4. Montagomery, D.C., Design and Analysis of Experiments. Wiley, New         York,(1991)