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Contact: Name Phone: +61 8 8313 0402 Email: [email protected] www.adelaide.edu.au 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 Adelaide 1 ) 1 The 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 96 120 144 168 192 0 1 2 3 Time (h) L-malic acid (g/L) Mix continious culture Parent 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 96 120 144 168 192 0 1 2 3 Time (hour) L-malic acid (g/L) Parent Continuous culture isolates Improved isolates

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Contact: Name Phone: +61 8 8313 0402 Email: [email protected]

www.adelaide.edu.au

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