LIST OF TABLES
Table 1.1. HoPS produced by LAB 7
Table 1.2. EPS repeating unit structure in some LABs 9
Table 1.3. Monosaccharide composition in EPS of some L. plantarum strains .12
Table 2.1. Experimental layout of CO2 enhanced culture 42
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Table 2.2. Primers used for Real-time qPCR analysis 47
Table 3.1. Morphological and biochemical characteristics of LAB strains isolated from fermented foods 49
Table 3.2. Effects of different environmental stress conditions on EPS yield, density and survival rate after freeze-drying of L. plantarum VAL6 64
Table 3.3. Probiotic bacteria density cultured in sugar-free MRS medium supplemented with EPS of L. plantarum VAL6 68
Table 3.4. Changes in mRNA expression of EPS synthesis-related genes under environmental challenges compared to non-stress conditions 89
LIST OF IMAGES
Figure 1.1. Phylogenetic position of L. plantarum compared to some other related LAB based on 16S rRNA 4 sequences
Figure 1.2. Cell wall structure of Gram-positive bacteria 5
Figure 1.3. The 6-membered (pyranose) ring structure of glucose and the 5-membered (furanose) ring structure of fructose 8
Figure 1.4. Biological activity of EPS produced by LAB 15
Figure 1.5. Antibacterial mechanisms of EPS 16
Figure 1.6. Detailed diagram of EPS production by LAB through the conversion of lactose, galactose and glucose in the cytoplasm 19
Figure 1.7. Structure of nucleotide-sugar 20
Figure 1.8. Schematic diagram of EPS biosynthesis in L. lactis NIZO 21
Figure 1.9. Dextran synthesis by glycosyltransferase (dextran sucrase) 22
Figure 1.10. Genetic organization diagram of the eps gene cluster in L. plantarum 28
Figure 1.11. Genetic organization diagram of the eps gene cluster in different LABs 28
Figure 1.12. Environmental stress stimulates EPS production 33
Figure 1.13. Transcriptional regulation mechanism against environmental stress in bacteria 37
Figure 3.1. Cell shape of bacterial strain L6 under optical microscope 49
Figure 3.2. Colony characteristics of bacterial strain L6 50
Figure 3.3. EPS production ability of LAB strains isolated from fermented foods 50
Figure 3.4. BLAST SEARCH results for classifying strain L6 51
Figure 3.5. Phylogenetic tree of L. plantarum strain VAL6 constructed by comparing 16S rRNA sequences 52
Figure 3.6. PCR amplification product of recA gene 52
Figure 3.7. EPS yield produced by L. plantarum VAL6 under heat stress conditions 53
Figure 3.8. Changes in L. plantarum VAL6 density under heat stress conditions 54
Figure 3.9. Freeze-drying survival rate of heat-stressed L. plantarum VAL6..55 Figure 3.10. EPS yield produced by L. plantarum VAL6 under pH 56 stress conditions
Figure 3.11. Changes in L. plantarum VAL6 density under stress conditions of pH 57
Figure 3.12. Lyophilization survival rate of pH-stressed L. plantarum VAL6
................................................................ ................................................................ ..............................57
Figure 3.13. EPS yield produced by L. plantarum VAL6 under NaCl stress conditions 59
Figure 3.14. Changes in L. plantarum VAL6 density under NaCl stress conditions 59
Figure 3.15. Lyophilization survival rate of L. plantarum VAL6 stressed under NaCl60 Figure 3.16. EPS yield produced by L. plantarum VAL6 under CO2 enhanced culture conditions 61
Figure 3.17. Changes in L. plantarum VAL6 density under CO2-enhanced culture conditions 62
Figure 3.18. Survival rate after freeze-drying of L. plantarum VAL6 cultured in CO2-enhanced conditions 62
Figure 3.19. Antioxidant capacity of EPSs produced by L. plantarum VAL6 under environmental stress conditions 65
Figure 3.20. Protein content of EPS produced by L. plantarum VAL6 under environmental stress conditions 66
Figure 3.21. The ratio of monosaccharides: (A) mannose; (B) glucose; (C) galactose;
(D) arabinose; (E) rhamnose and (F) xylose in EPS produced by L. plantarum VAL6 under heat stress conditions of 70
Figure 3.22. Chromatogram of monosaccharide composition in EPS produced by L. plantarum VAL6 after 3 hours of stress at 42 oC 71
Figure 3.23. Proportions of monosaccharides: (A) mannose; (B) glucose; (C) galactose; (D) arabinose; (E) rhamnose and (F) xylose in EPS produced by L. plantarum VAL6 under stress conditions of pH 73
Figure 3.24. Proportions of monosaccharides: (A) mannose; (B) glucose; (C) galactose; (D) arabinose; (E) rhamnose and (F) xylose in EPS produced by L. plantarum VAL6 under 74 NaCl stress conditions
Figure 3.25. The ratio of monosaccharides: (A) mannose; (B) glucose; (C) galactose and
(D) rhamnose in EPS produced by L. plantarum VAL6 under CO2-enhanced culture conditions 76
Figure 3.26. Agarose gel electrophoresis of PCR mRNA products of genes involved in EPS synthesis under heat stress conditions 79
Figure 3.27. mRNA expression of genes involved in EPS synthesis under heat stress conditions of 80
Figure 3.28. Agarose gel electrophoresis of PCR mRNA products of genes involved in EPS synthesis under stress conditions of pH 81
Figure 3.29. mRNA expression of genes involved in EPS synthesis under pH 82 stress conditions
Figure 3.30. Agarose gel electrophoresis of PCR mRNA products of genes involved in EPS synthesis under NaCl stress conditions 83
Figure 3.31. mRNA expression of genes involved in EPS synthesis under NaCl stress conditions 84
Figure 3.32. Agarose gel electrophoresis of PCR mRNA products of genes involved in EPS synthesis under CO2-enhanced culture conditions 85
Figure 3.33. mRNA expression of genes involved in EPS synthesis under increased CO2 concentration 86
Figure 3.34. Expression of genes involved in EPS synthesis under the influence of environmental stress 90
Figure 3.35. Proposed model for enhancing EPS production by L. plantarum VAL6
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Figure 3.36. Correlation between total EPS and survival of L. plantarum
VAL6 in freeze drying process 94
SUMMARY
The thesis "Research on environmental stress conditions on the ability to synthesize exopolysaccharides of Lactobacillus plantarum " was conducted to determine the appropriate stress conditions to stimulate the ability to strongly synthesize exopolysaccharide (EPS) in Lactobacillus plantarum. The study isolated L. plantarum strains with high EPS synthesis ability from fermented foods, evaluated the effects of environmental stress conditions such as temperature, pH, NaCl and increased CO 2 concentration on the EPS production process, on changes in monosaccharide composition and the expression of genes related to EPS synthesis.
From traditional fermented food sources in An Giang (Vietnam), a native Lactobacillus strain with high EPS production capacity was isolated and identified as Lactobacillus plantarum VAL6.
Performing culture under the influence of environmental stress conditions, the study determined the appropriate stress conditions that stimulate the strong EPS biosynthesis ability of L. plantarum . The highest EPS yield (50.44 g/L) was obtained under pH 3 stress conditions for 3 hours.
Determination of the correlation between EPS production and survival showed that increased EPS production under environmental stress conditions significantly increased bacterial survival during freeze-drying. Corresponding to the highest EPS yield, the freeze-drying survival rate of L. plantarum VAL6 under pH 3 stress conditions also reached the highest (30.72%), 1.536 times higher than that under non-stress conditions.
Analysis of monosaccharide composition of EPS by gas chromatography with flame ionization detector. The results showed that environmental stress changes the monosaccharide composition of EPS with more accumulation of some common sugars (mannose, galactose, xylose, etc.) and rare sugars (rhamnose, fucose, etc.). Specifically, EPS produced by L. plantarum VAL6 under normal culture conditions is a heteropolysaccharide consisting of mannose (83.44%), glucose (14.01%), galactose (1.15%), arabinose (0.00%), rhamnose (0.71%) and xylose (0.67%). After stress, the monosaccharide composition changes to include mannose (69.13-80.34%), glucose (12.55-23.60%), galactose (1.87-6.50%), arabinose (0.00-
8.96%), rhamnose (0.38-8.00%) and xylose (0.00-6.55%). Under heat stress conditions, the presence of fucose was also found in the EPS composition.
Analysis of mRNA expression of genes involved in EPS biosynthesis by Real-time qPCR technique showed that environmental stress has an impact on increasing or decreasing the expression of genes involved in EPS biosynthesis ( glm U, pgm B1, cps 4E, cps 4F, cps 4J and cps 4H), resulting in changes in EPS yield and monosaccharide composition.
These results suggest that environmental stress can alter EPS biosynthesis in lactic acid bacteria (LAB). In addition, environmental stress can be used to stimulate LAB to produce novel EPS with high biological activity for industrial applications.
ABSTRACT
The thesis, "Study on environmental stress conditions on the ability to synthesize exopolysaccharides of Lactobacillus plantarum " was carried out with the aim to determine the appropriate stress conditions that stimulate strong exopolysaccharide (EPS) biosynthesis in Lactobacillus plantarum . In this study, we isolated L. plantarum strain with high EPS biosynthesis ability from fermented foods and evaluated the effects of environmental stress conditions such as temperature, pH, NaCl, and increased CO 2 concentration, on EPS production, changes in monosaccharide composition, and the expression of genes involved in EPS synthesis.
From traditional fermented foods in An Giang (Vietnam), a native strain of Lactobacillus with high EPS production capacity was isolated and identified as Lactobacillus plantarum VAL6.
Carrying out the culture under the influence of environmental stresses, the study discovered the appropriate stress condition that stimulates the strong EPS biosynthesis of L. plantarum . active, the maximum EPS yield (50.44 g/L) was obtained after 3 hours of stress at pH 3.
Observation of the correlation between the EPS production and survival indicates that increased EPS production under environmental stresses markedly increased bacterial survival during freeze-drying. Corresponding to the maximum amount of EPS produced, the freeze-dried survival of L. plantarum VAL6 stressed at pH 3 was also the highest (30.72%), 1,536 times higher than the normal culture condition.
Analysis of EPS monosaccharide composition was performed using gas chromatography with a flame ionization detector. The analysis demonstrated that environmental stresses alter the monosaccharide composition of EPS, resulting in greater accumulation of some common sugars (mannose, galactose, xylose, etc.) and rare sugars (rhamnose, fucose, etc.). specifically, the EPS produced by L. plantarum VAL6 under normal culture conditions are heteropolysaccharides consisting of sugars such as mannose (83.44%), glucose (14.01%), galactose (1.15%), arabinose (0.00%), rhamnose (0.71%) , and xylose (0.67%). After stress, the monosaccharide composition changed including mannose (69.13-80.34%),
glucose (12.55-23.60%), galactose (1.87-6.50%), arabinose (0.00-8.96%), rhamnose (0.38-8.00%), and xylose (0.00-6.55%). Under heat stress conditions, the presence of fucose was also found in the EPS component.
Analyzing the mRNA expression of genes involved in EPS biosynthesis via Real-time qPCR, the results show that environmental stresses could increase or decrease the expression of genes involved in EPS biosynthesis ( glm U, pgm B1, cps 4E, cps 4F, cps 4J, and cps 4H), resulting in changes in the yield and monosacchride composition of EPS.
These results suggested that environmental stresses could alter EPS biosynthesis in lactic acid bacteria (LAB). Furthermore, environmental stresses could be used to stimulate LAB to produce novel EPS which is highly bioactive for industrial applications.