Optimization of Oligosaccharide Production from Leuconostoc lactis Using a Response Surface Methodology and the Immunostimulating Effects of These Oligosaccharides on Macrophage Cells
Abstract
:1. Introduction
2. Results and Discussion
2.1. Optimization of Oligosaccharide Production Using a Statistical Technique
2.2. Effect of Oligosaccharides on RAW264.7 Cell Viability and NO Secretion
2.3. Effect of Oligosaccharides on Cytokine and iNOS Secretion
3. Materials and Methods
3.1. Bacterial Strains and Culture Conditions
3.2. Response Surface Methodology Using Central Composite Design
3.3. Analysis of Oligosaccharides by HPAEC-PAD
3.4. Purification of Oligosaccharides
3.5. Cell-Based Immunostimulatory Effect Test
3.5.1. Cell Culture
3.5.2. Cell Viability
3.5.3. NO Assay
3.5.4. Expression of Cytokines
3.5.5. Statistical Analysis
4. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Sample Availability: Not available. |
Run | Coded Variable Levels | Oligosaccharide Production (Relative Peak Area) | ||
---|---|---|---|---|
Temperature, °C (X1) | Sucrose, % (X2) | Maltose, % (X3) | ||
1 | 27.5 (−1) | 5.0 (−1) | 10.0 (+1) | 212.9 ± 13.1 * |
2 | 32.5 (+1) | 5.0 (−1) | 5.0 (−1) | 145.1 ± 20.5 ** |
3 | 30 (0) | 12.5 (+2) | 7.5 (0) | 312.6 ± 19.4 * |
4 | 27.5 (−1) | 10.0 (+1) | 10.0 (+1) | 248.9 ± 1.1 ** |
5 | 35 (+2) | 7.5 (0) | 7.5 (0) | 97.8 ± 12.8 |
6 | 30 (0) | 7.5 (0) | 7.5 (0) | 270.3 ± 1.9 *** |
7 | 30 (0) | 2.5 (−2) | 7.5 (0) | 139.0 ± 10.7 ** |
8 | 30 (0) | 7.5 (0) | 7.5 (0) | 248.1 ± 26.7 ** |
9 | 32.5 (+1) | 5.0 (−1) | 10.0 (+1) | 186.8 ± 14.8 ** |
10 | 25 (−2) | 7.5 (0) | 7.5 (0) | 227.0 ± 13.5 * |
11 | 27.5 (−1) | 10.0 (+1) | 5.0 (−1) | 234.1 ± 15.7 ** |
12 | 30 (0) | 7.5 (0) | 7.5 (0) | 244.7 ± 19.5 ** |
13 | 30 (0) | 7.5 (0) | 2.5 (−2) | 231.1 ± 10.9 * |
14 | 32.5 (+1) | 10.0 (+1) | 10.0 (+1) | 304.1 ± 18.6 * |
15 | 30 (0) | 7.5 (0) | 7.5 (0) | 259.6 ± 23.0 ** |
16 | 30 (0) | 7.5 (0) | 7.5 (0) | 233.5 ± 17.0 * |
17 | 30 (0) | 7.5 (0) | 7.5 (0) | 245.0 ± 23.4 ** |
18 | 30 (0) | 7.5 (0) | 12.5 (+2) | 252.3 ± 12.9 ** |
19 | 27.5 (−1) | 5.0 (−1) | 5.0 (−1) | 183.8 ± 7.6 * |
20 | 32.5 (+1) | 10.0 (+1) | 5.0 (−1) | 270.3 ± 12.1 * |
Variables | DF | Adj SS | Adj MS | F-Value | p-Value |
---|---|---|---|---|---|
Model | 9 | 151,743 | 16,860.3 | 10.78 | 0.000 |
Linear | 3 | 61,162 | 20,387.5 | 13.03 | 0.000 |
Quadratic | 3 | 77,970 | 25,990.1 | 16.61 | 0.000 |
Cross-product | 3 | 12,610 | 4203.4 | 2.69 | 0.056 |
X1 | 1 | 7750 | 7750.0 | 4.95 | 0.031 |
X2 | 1 | 50,631 | 50,631.1 | 32.37 | 0.000 |
X3 | 1 | 2781 | 2781.3 | 1.78 | 0.188 |
X12 | 1 | 75,158 | 75,157.8 | 48.04 | 0.000 |
X22 | 1 | 10,678 | 10,678.1 | 6.83 | 0.012 |
X32 | 1 | 1653 | 1653.5 | 1.06 | 0.309 |
X1X2 | 1 | 7159 | 7159.0 | 4.58 | 0.037 |
X1X3 | 1 | 5436 | 5436.4 | 3.48 | 0.068 |
X2X3 | 1 | 15 | 15.0 | 0.01 | 0.922 |
Residual | 50 | 78,217 | 1564.3 | - | - |
Lack of fit | 5 | 13,947 | 2789.5 | 1.95 | 0.104 |
Pure error | 45 | 64,270 | 1428.2 | - | - |
Cor Total | 59 | 229,960 |
Gene | Sequence | |
---|---|---|
GAPDH | Forward | 5′―ATC CCA TCA CCA TCT TCC AG―3′ |
Reverse | 5′―CCT GCT TCA CCA CCT TCT TG―3′ | |
TNF-α | Forward | 5′―ATG AGC ACA GAA AGC ATG ATC CG―3′ |
Reverse | 5′―CCA AAG TAG ACC TGC CCG GAC TC―3′ | |
IL-1β | Forward | 5′―ATG GCA ACT GTT CCT GAA CTC AAC T―3′ |
Reverse | 5′―CAG GAC AGG TAT AGA TTC TTT CCT T―3′ | |
IL-6 | Forward | 5′―CAA GAG ACT TCC ATC CAG TTG C―3′ |
Reverse | 5′―TTG CCG AGT TCT CAA AGT GAC―3′ | |
iNOS | Forward | 5′―AAT GGC AAC ATC AGG TCG GCC ATC ACT―3′ |
Reverse | 5′―GCT GTG TGT CAC AGA AGT CTC GAA CTC―3′ |
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Lee, S.; Park, G.-G.; Jang, J.-K.; Park, Y.-S. Optimization of Oligosaccharide Production from Leuconostoc lactis Using a Response Surface Methodology and the Immunostimulating Effects of These Oligosaccharides on Macrophage Cells. Molecules 2018, 23, 2118. https://doi.org/10.3390/molecules23092118
Lee S, Park G-G, Jang J-K, Park Y-S. Optimization of Oligosaccharide Production from Leuconostoc lactis Using a Response Surface Methodology and the Immunostimulating Effects of These Oligosaccharides on Macrophage Cells. Molecules. 2018; 23(9):2118. https://doi.org/10.3390/molecules23092118
Chicago/Turabian StyleLee, Sulhee, Gwi-Gun Park, Jae-Kweon Jang, and Young-Seo Park. 2018. "Optimization of Oligosaccharide Production from Leuconostoc lactis Using a Response Surface Methodology and the Immunostimulating Effects of These Oligosaccharides on Macrophage Cells" Molecules 23, no. 9: 2118. https://doi.org/10.3390/molecules23092118