Enzymatic Steviol Production via Aspergillus niger Hydrolysis
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Solution Overview
Problem
Current methods for producing steviol from steviosides are inefficient and not suitable for large-scale commercial production due to issues such as rearrangement under acidic conditions, high costs associated with sodium periodate use, and limited enzyme availability from traditional sources like snails or commercial pectinases that are no longer commercially available.
Innovation Solution
A process using a mixture of enzymes from a selected GRAS strain of Aspergillus niger, including pectinases, hemicellulases, β-D-glucosidase, β-glucopyranosidase, and α-rhamnosidase activities, in the presence of a yeast culture, to cleave stevioside and produce steviol, which overcomes enzyme inhibition by glucose through yeast fermentation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If acid hydrolysis is used to cleave stevioside, then steviol can be produced, but steviol rearranges into isosteviol under acidic conditions
Solution Approach 1:
The patent changes the chemical environment from acidic to neutral/alkaline conditions by using enzymatic hydrolysis instead of acid hydrolysis. This parameter change allows steviol to be produced without rearranging into isosteviol, as the enzymes (pectinases, hemicellulases, β-D-glucosidase, β-glucopyranosidase, and α-rhamnosidase) function optimally at neutral to slightly alkaline pH, preventing the acid-catalyzed rearrangement reaction.
2Productivity
If sodium periodate is used to cleave stevioside, then steviol can be produced, but large excess of expensive reagent is required
Solution Approach 1:
The patent replaces the chemical oxidation method (sodium periodate) with a biological catalytic method (enzymatic hydrolysis). The enzymes pectinases, hemicellulases, β-D-glucosidase, β-glucopyranosidase, and α-rhamnosidase catalyze the cleavage of stevioside glycosidic bonds under mild conditions, eliminating the need for large excesses of expensive chemical reagents like sodium periodate while maintaining high productivity.
3Productivity
If traditional enzyme sources are used (snail diastase or commercial pectinases), then steviol can be produced, but enzyme availability is limited or no longer commercially available
Solution Approach 1:
The patent uses a multi-enzyme system from Aspergillus niger that combines the functions of pectinases, hemicellulases, β-D-glucosidase, β-glucopyranosidase, and α-rhamnosidase in a single microbial source. This universal enzyme preparation from a readily available fungal strain replaces the need for multiple specialized enzyme sources (snail diastase, commercial pectinases like Pectinol 59L) that are either unavailable or difficult to obtain, ensuring continuous commercial production.
4Productivity
If glucose accumulates during enzymatic cleavage, then stevioside can be converted to steviol, but glucose inhibits further enzymatic reaction
Solution Approach 1:
The patent removes the inhibitory glucose product from the reaction system by employing yeast fermentation to convert accumulated glucose into ethanol and carbon dioxide. This extraction of the inhibitory substance (glucose) from the enzymatic reaction environment maintains high enzymatic activity throughout the process, allowing complete conversion of stevioside to steviol without reaction inhibition.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method enables efficient and commercially viable large-scale production of steviol by maintaining enzymatic activity and reducing glucose inhibition, allowing for high yields and scalability.
Implementation Method 1
Process for the enzymatic preparation of steviol from stevioside... cleaving glycoside residues from stevioside to produce steviol using a mixture of enzymes
Implementation Method 2
The reaction takes place in the presence of yeast... The yeast can remove the resultant glucose, and therefore the enzymatic reaction can proceed unhindered
Data Source
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AI summary
This invention is directed to a process for cleaving glycoside residues from stevioside to produce steviol using a commercially available enzyme mixture containing pectinase, CYTOLASE PCL5®. In preferred methods, the reaction takes place in the presence of a yeast culture. Also described are combinations of Helix pomatia enzymes and CYTOLASE PCL5®.