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ABSTRACT
Production of Lactobacillus salivarius i 24, a probiotic strain for chicken, was studied in batch fermentation using 500 mL
Erlenmeyer flask. Response surface method (RSM) was used to optimize the medium for efficient cultivation of the
bacterium. The factors investigated were yeast extract, glucose and initial culture pH. A polynomial regression model
with cubic and quartic terms was used for the analysis of the experimental data. Estimated optimal conditions of the
factors for growth of L. salivarius i 24 were; 3.32 % (w/v) glucose, 4.31 % (w/v) yeast extract and initial culture pH of
6.10.
Keywords: Lactobacillus salivarius, optimization, response surface method, probiotic for chicken.
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Mal. J. Microbiol. Vol 3(2) 2007, pp 41-47
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Mal. J. Microbiol. Vol 3(2) 2007, pp 41-47
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Mal. J. Microbiol. Vol 3(2) 2007, pp 41-47
that they were significant. The larger the R2, the more > 0.05 were forced into the final equation due to the fact
accurately the values of the response can be predicted that its corresponding higher-order terms were chosen for
by the model. Furthermore, the lack of fit was inclusion. The intercept b0 is the estimated response at
2
insignificant (P = 0.0903). However, X1, X2 and X2 with p the center point (X1, X2, X3) = (0, 0, 0).
Table 4: Analysis of variance in the regression model selected through variable selectiona
Total 19 0.597145
a
R2 = 0.9393, Coefficient of variance = 0.64746
The optimum value of X1 that maximized f1 (X1) was
Table 5: Coefficient estimates in the regression model found through differentiation. X2 and X3 that maximized
selected through variable selection f23 (X2, X3) were maximized through calculation and
sorting of f23 (X2, X3) values on a grid of points for X2 and
Variable Coefficient Standard t value P X3. The search was done with computer programs written
estimate error value in SAS (data not shown).
The optimum point obtained through this study was
Intercept 9.404500 0.02458758 382.490 0.0001 (X1, X2, X3) = (-0.147, 0.54, 0.15). By encoding the coded
levels back to the original levels, the following results
X1 -0.008134 0.01629649 -0.499 0.6285 were obtained: glucose = 33.24 g/L, yeast extract =
43.1g/L and pH = 6.1. The estimated maximum response
X2 0.038193 0.03572549 1.069 0.3102 corresponding to the optimum factor levels was 9.440
log10CFU/mL, which was slightly higher than the center
X3 0.048076 0.01629649 2.950 0.0145 factor levels, 9.404 log10 CFU/mL. This was a slight
improvement claimed by the regression model. A
X12 -0.027747 0.01738176 -1.596 0.1415 validation experiment would ascertain whether there was
a real improvement.
2
X2 0.015734 0.04772295 0.330 0.7484
Assessment of Factor Effects with the Partial-effects
3 Plot
X2 0.060682 0.01808672 3.355 0.0073
X24 -0.044730 0.01842177 -2.428 0.0356 The partial-effect functions and plots were used to
determine the effect of each factor graphically. The
X3
2
-0.059382 0.01738176 -3.416 0.0066 partial-effect function of a certain factor is a function that
describes how the response moves as the level of that
X2X3 -0.068625 0.0219347 -3.223 0.0091 factor changes when the other factors are fixed at their
optimum levels (Liew, 2005). Let Y = f (X1, X2, X3) denote
the response surface model described in Tables 5.4 and
Note: The variables which gave P values higher than 0.5, 5.5 and (X1*, X2*, X3*) denote the optimum points of the
which are not significant, are not presented in this table. factors which are, (-0.147, 0.54, 0.15), in this study.
Hence, the partial-effect function of X1 is defined as
Determining the Optimum Point of the Factors
Y (X1) = f (X1, X2*, X3*)
This response surface model can be written as: [equation 3]
Y= b0+ f1 (X1) + f23 (X2, X3) Similarly, the partial-effect functions of X2 and X3 are
[equation 2] defined as:
Where Y (X2) = f (X1*, X2, X3*)
f1 (X1) = b1X1 + b11X12 [equation 4]
f23 (X2, X3) = b2X2 + b22X22 + b222X23 + b2222X24 + b3X3 + Y (X3) = f (X1*, X2*, X3)
2
b33X3 b23X2X3 [equation 5]
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Mal. J. Microbiol. Vol 3(2) 2007, pp 41-47
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Mal. J. Microbiol. Vol 3(2) 2007, pp 41-47
10.5
10.0
9.5
Log 10 CFU/m L
9.0
8.5
8.0
7.5
7.0
0 5 10 15 20
Time (h)
Figure 4: Response surface for the effects of glucose Figure 5: Growth curves of L salivarius i 24 in MRS broth
and pH on the growth of L salivarius i 24 at (▲), optimum-point ( ) and center point (ο) media as
yeast extract = 4.31% obtained from the validation experiment
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Mal. J. Microbiol. Vol 3(2) 2007, pp 41-47
Table 6: Compositions of three media for the growth of L Gilliland, S.E. (1985). Concentrated starter cultures. In
salivarius i 24 Bacterial starter cultures for foods, (Gilliland, S.E.
Ed). pp 145-157. Florida: CRC Press Inc.
Composition Amount of component (%) in Ha, M.Y., Kim, S.W., Lee, Y.W., Kim, M.J. and Kim, S.J.
Optimum- Center- MRS (2003). Kinetics analysis of growth and lactic acid
point point production in pH-controlled batch cultures of
Lactobacillus casei KH-1 using yeast extract/ corn
Glucose 3.324 3.5 2.0 steep liquor/ glucose medium. Journal of Bioscience
Yeast extract 4.31 3.5 0.5 and Bioengineering 96: 134 – 140.
Jin, L.Z., Ho, Y.W., Abdullah, N. and Jalaludin, S.
Tween 80 0.1 0.1 0.1 (1997). Probiotics in poultry: modes of action.
Proteose - - 1.0 World's Poultry Science Journal 53: 351 – 368.
peptone Jin, L.Z., Ho, Y.W., Abdullah, N., Ali, A. M. and
Beef extract - - 1.0 Jalaludin, S. (1996). Antagonistic effects of
Ammonium - - 0.2 intestinal Lactobacillus isolates on pathogens of
citrate chickens. Letters in Applied Microbiology 23: 67–71.
Sodium citrate - - 0.5 Liew, S.L. (2004). Large-scale production of lactic acid
Magnesium 0.002 0.002 0.01 bacteria for human probiotics. Ph.D Dissertation,
sulfate Universiti Putra Malaysia, Malaysia.
Manganese - - 0.005 Liew, S.L., Ariff, A.B., Raha, A.R. and Ho, Y.W. (2005).
sulfate Optimization of medium composition for the
Dipotassium 0.2 0.2 0.2 production of a probiotic microorganism,
phosphate Lactobacillus rhamnosus, using response surface
pH 6.1 6 6.20 methodology. International Journal of Food
Microbiology 102: 137 – 142.
Lim, C.M. (2006). Optimization of fermentation and
CONCLUSIONS freeze-drying processes to enhance the productivity
and stability of a probiotic, Lactobacillus salivarius I
This study demonstrated that RSM was used 24. Ph.D Dissertation, Universiti Putra Malaysia,
successfully in designing, analyzing, finding the optimum Malaysia.
point and assessing the effects of factors leading to a Logothetis, N and Wynn, H.P. (1989). Quality through
higher growth rate of L. salivarius i 24, which in turn, design. New York: Oxford University Press.
improve the overall productivity of the cultivation process. Montgomery, C.D. (1991). Design and analysis of
The optimum conditions of the factors for the growth of L. experiments. New York: John Wiley and Sons, Inc.
salivarius i 24 are as follows: glucose = 33.24 g/L, yeast Oh, S.J., Rheem, S.S., Sim, J.H. and Kim, S.K. (1995).
extract = 43.1 g/L and pH = 6.1. Even though the Optimization condition for the growth of Lactobaillus
optimum levels of glucose and yeast extract were higher casei YIT 9018 in Typtone-Yeast Extract-Glucose
than the centre point level, the composition of the medium by using response surface methodology.
medium was less complicated than the commercial MRS Applied and Environmental Microbiology 45: 3809 –
medium. Hence, it could imply a reduction in the cost of 3814.
production, which would translate to an economic gain. Rao, A.V., Jayaraman, K. and Lakshamanan, C.M.
Another advantage of using the optimized medium is (1993). Production of lipase by Candida rugosa in
significant increase in the productivity as compared to for solid state fermentation 2: media optimization and
the MRS medium. effect of aeration. Process Biochemistry 28: 391 –
395.
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