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More efficient malolactic fermentation by directed evolution of Lactobacillus plantarum
Acknowledgements: This work is supported by Wine Australia (Project # UA1302) Wine Microbiology and Microbial Biotechnology Laboratory (University of Adelaide1) 1The University of Adelaide is a member of the Wine Innovation Cluster
Krista Sumby*, Paul R Grbin, Vladimir Jiranek
University of Adelaide Department of Wine and Food Science, PMB 1, Glen Osmond, South Australia 5064, Australia
Introduction: • Lactobacillus plantarum (Figure 1) is the LAB most
typically used as an alternative to O. oeni in winemaking to carry out MLF.
• If a homofermetative strain is chosen Lb. plantarum
will not increase volatile acidity and has a large potential to contribute positively to wine aroma.
• An important requirement of MLF is that the process is reliably completed in a timely manner.
• When an LAB starter strain is added to wine, it encounters multiple stressors, including low pH and high ethanol concentrations.
• Directed evolution (DE) was used to improve Lb. plantarum for more efficient and reliable MLF.
• The best isolates from the micro-plate screen (Figure 4) were selected for further screening in larger 50 ml cultures in Red FCDGJM (15% ethanol, pH 3.5) (Figure 5).
Figure 4: Screening of adapted strains, micro-plate scale.
Figure 5: Screening of best isolates from micro-plate screen in 50 ml FCDGJM (lab-scale).
Hypothesis: • Lb. plantarum will stabily adapt to its environment
through evolution when placed under continuing and increasing stress conditions.
Method: • DE by continuous culture
of improve Lb. plantarum (Lallemand) in fermented Chemically Defined Grape Juice Media (FCDGJM) (Figure 2).
• Over time ethanol was increased and pH decreased, for 300 generations.
DE mixed culture
Isolate single clones
Identify the best strains by micro & larger-scale screening
0 24 48 72 96 120 144 168 1920
1
2
3
Time (h)
L-m
alic
aci
d (g
/L)
Mix continious cultureParent
Continuous culture isolates
Figure 2: Continuous culture of Lb. plantarum.
Figure 1: Lb. plantarum 1000x magnification.
Figure 3: Screening for improved isolates.
Conclusion: DE has been used as a tool to improve Lb. plantarum and thereby improve its ability to complete MLF under difficult wine conditions. Isolates capable of faster MLF were found through a 2-step screening process.
Evaluation of strain improvement: • Screening of isolates from the continuous culture was
performed in Red FCDGJM (14.5% ethanol, pH 3.3) at micro-plate scale and then 50ml scale to select for evolved strains (Figure 3).
0 24 48 72 96 120 144 168 1920
1
2
3
Time (hour)
L-m
alic
aci
d (g
/L)
ParentContinuous culture isolates
Improved isolates
