Lactobacillus plantarum Ethanol Resistance Selection

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Solution Overview

Problem

Lactic acid bacteria, such as Lactobacillus plantarum, have low inherent resistance to high ethanol concentrations, which poses a challenge in wine production, especially with increasing alcohol levels in wines, and there is no established link between D-cycloserine resistance and ethanol resistance in prior art.

Innovation Solution

A novel selection method is developed to identify Lactobacillus plantarum strains with increased resistance to D-cycloserine, which are then screened for improved resistance to high ethanol concentrations, leveraging the correlation between D-cycloserine resistance and ethanol resistance, allowing for rapid identification of strains with enhanced ethanol tolerance.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If natural Lactobacillus plantarum strains are used for malolactic fermentation, then the fermentation process can proceed spontaneously, but the strains have low inherent resistance to high ethanol concentrations

Engineering Contradiction:
Improveethanol resistanceVSAvoidadaptability to high alcohol wines
Core Design Contradiction:
ReliabilityVSAdaptability or versatility

Solution Approach 1:

The patent applies parameter changes by selecting and cultivating Lactobacillus plantarum strains under increasing ethanol concentration conditions to induce and select for strains with enhanced ethanol resistance. This involves gradual increase of ethanol concentration in the growth medium to select for mutants with improved tolerance, thereby resolving the contradiction between natural strain usage and ethanol resistance requirements.

Inventive Principle:
Principle #35Parameter changes

Solution Approach 2:

The patent converts the harmful effect of ethanol (which normally inhibits bacterial growth) into a beneficial selective pressure. By exposing Lactobacillus plantarum strains to ethanol during cultivation, the ethanol that would normally be harmful becomes the selecting agent that identifies and enriches for resistant strains, enabling their use in high alcohol wine production.

Inventive Principle:
Principle #22Blessing in disguise (Convert harm into benefit)

2Reliability

If selection pressure is applied to obtain ethanol-resistant strains, then ethanol resistance improves, but the selection process becomes complex and time-consuming

Engineering Contradiction:
Improveethanol resistanceVSAvoidselection process complexity
Core Design Contradiction:
ReliabilityVSDevice complexity

Solution Approach 1:

The patent applies preliminary action by pre-establishing a correlation between D-cycloserine resistance and ethanol resistance before the actual selection process. This allows the use of D-cycloserine as a preliminary screening tool to identify strains that are likely to also be ethanol-resistant, simplifying the overall selection process and reducing the time and complexity required for obtaining ethanol-resistant strains.

Inventive Principle:
Principle #10Preliminary action

Solution Approach 2:

The patent introduces D-cycloserine resistance as an intermediary marker that correlates with ethanol resistance. This intermediary property serves as a convenient and easier-to-measure trait that predicts ethanol resistance, thereby simplifying the selection process by using D-cycloserine as a proxy screen rather than directly testing for ethanol resistance in all candidates.

Inventive Principle:
Principle #24Intermediary (Mediator)

3Reliability

If direct selection for ethanol resistance is performed, then ethanol-resistant strains can be obtained, but the process is inefficient and may be impossible without prior correlation knowledge

Engineering Contradiction:
Improveethanol resistanceVSAvoidstrain identification efficiency
Core Design Contradiction:
ReliabilityVSProductivity

Solution Approach 1:

The patent uses D-cycloserine resistance as an intermediary marker that correlates with ethanol resistance. This intermediary property serves as a convenient and easier-to-measure trait that predicts ethanol resistance, thereby simplifying the selection process by using D-cycloserine as a proxy screen rather than directly testing for ethanol resistance in all candidates, significantly improving identification efficiency.

Inventive Principle:
Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent applies preliminary action by pre-establishing a correlation between D-cycloserine resistance and ethanol resistance before the actual selection process. This allows the use of D-cycloserine as a preliminary screening tool to identify strains that are likely to also be ethanol-resistant, simplifying the overall selection process and reducing the time and complexity required for obtaining ethanol-resistant strains.

Inventive Principle:
Principle #10Preliminary action

Data Source

PatentEP2721142B1Lactobacillus plantarum cells with improved resistance to high concentrations of ethanol
Publication Date: 2018.09.19 CHR HANSEN AS
  • EP2721142B1 patent drawingFigure 1

AI summary

Cycloserine resistant mutants of lactic acid bacteria characterized by having improved resistance towards ethanol. The cycloserine resistant mutants of lactic acid bacteria can e.g. be used for malolactic fermentations of wine (i.e. including sparkling wine such as Cava/champagne) having high alcohol levels.