Malolactic fermentation (MLF) is a crucial process in winemaking where bacteria convert L-malic acid into L-lactic acid, impacting wine aroma, taste, color stability, and microbial control. Precise monitoring of both acids throughout the 20-40 day MLF process is essential for quality control. Current standard methods, based on chromatography and colorimetry, are time-consuming, expensive, and require skilled personnel in decentralized laboratories, hindering real-time on-site monitoring and corrective actions. This necessitates the development of miniaturized, portable, and cost-effective analytical tools for real-time, in-situ monitoring of MLF. Miniaturization offers advantages such as multiplexed analysis, low reagent consumption, and fast response times in portable devices. The use of polymers like polymethyl methacrylate (PMMA) for flow systems is cost-effective and easy to manufacture. Electrochemical biosensors, easily integrated into portable systems, are ideal for monitoring such processes. Previous research developed individual amperometric biosensors for L-lactate and L-malate with long-term stability, paving the way for this integrated flow system. This research aims to create a miniaturized, cost-effective flow system integrating these biosensors for simultaneous, real-time, and remote determination of L-lactic and L-malic acids in red wine during MLF, providing on-site and timely corrective actions.

Existing methods for determining L-lactic and L-malic acids in wine often involve separate analyses using techniques like chromatography and colorimetry or enzymatic approaches based on absorbance detection of NADH. These methods, while accurate, are typically performed in centralized laboratories, leading to delays and increased costs. The need for rapid, on-site analysis has driven research into miniaturized analytical systems. Several studies explored the use of flow injection analysis (FIA) with enzyme reactors or immobilized enzymes on membranes, aiming for simultaneous determination of L-lactic and L-malic acids. While some achieved acceptable LODs, the complexity of the systems, or the use of independent sensors for each analyte, remained challenges to the development of a simple and easily deployable system for this specific application. This paper directly addresses these limitations with the proposed compact flow system.

This study involved the design and fabrication of a compact analytical flow system integrating two electrochemical biosensors. A silicon chip with four platinum microelectrodes (counter, pseudo-reference, and two working electrodes) was fabricated using photolithographic techniques. The working electrodes were sequentially modified with electropolymerized polypyrrole (PPy) membranes entrapping the necessary enzymes and redox mediators for selective detection of L-lactate and L-malate. For the L-lactate biosensor, lactate oxidase (LOX) and horseradish peroxidase (HRP) were entrapped, using ferrocyanide as a redox mediator. The L-malate biosensor contained malate dehydrogenase (MDH), diaphorase (DP), and hexaammineruthenium (III) (HAR) as redox mediator, with NAD+ as a cofactor. The chip was integrated into a PMMA flow cell fabricated by laser cutting. Chronoamperometry was employed for analytical characterization. The system's performance was evaluated using wine samples collected during the MLF process of three different red wines. The obtained results were compared with those from standard enzymatic methods based on spectrophotometric measurement of NADH at 340 nm. The experimental procedure included electrode cleaning and activation, electropolymerization of PPy membranes, overoxidation to stabilize baseline signals, and chronoamperometric measurements to obtain calibration curves. Selectivity and long-term stability were also assessed. Wine samples were diluted appropriately before analysis.

The fabricated bi-parametric compact analytical flow system demonstrated excellent performance. The L-lactate biosensor exhibited a linear range from 5 × 10⁻⁷ to 1 × 10⁻⁴ M with a limit of detection (LOD) of 3.2 ± 0.3 × 10⁻⁶ M. The L-malate biosensor showed a linear range from 1 × 10⁻⁷ to 1 × 10⁻⁶ M and a LOD of 6.7 ± 0.2 × 10⁻⁸ M. Both biosensors showed high long-term stability, retaining more than 90% of their initial sensitivity after more than 30 days. The system's accuracy was validated using red wine samples collected during MLF. Excellent agreement was observed between the results obtained with the developed flow system and the standard enzymatic method, with absolute errors below 0.15 g L⁻¹ and all values within the 95% uncertainty range of the standard method. The complete analysis of each sample took approximately 5 minutes. The system successfully tracked the expected decrease in L-malic acid and increase in L-lactic acid concentration during MLF. A single system was used for over 80 assays (including calibrations) demonstrating its robust nature. Compared to other bi-parametric systems, the developed system showed superior LOD, especially for L-malate, and simpler design. The overall performance highlights the system’s suitability for real-time, on-site monitoring of MLF.

The findings demonstrate the successful development of a compact, robust, and accurate analytical flow system for simultaneous determination of L-lactic and L-malic acids in red wine during MLF. This addresses a significant need in the winemaking industry for real-time monitoring and control of this crucial fermentation process. The system's miniaturization, low cost, and ease of use offer substantial advantages over traditional methods. The high sensitivity and excellent agreement with standard methods validate its reliability for practical applications. The long-term stability of the biosensors is particularly crucial for monitoring the extended duration of MLF. The simplicity of the system allows for easy integration into winery workflows and provides opportunities for automated operation, further streamlining the monitoring process. The reduced reagent consumption and minimized sample volume contribute to enhanced sustainability.

This research presents a novel bi-parametric compact analytical flow system for the simultaneous determination of L-lactic and L-malic acids in red wine. The system’s high sensitivity, accuracy, long-term stability, and ease of use make it suitable for real-time, on-site monitoring of MLF. The miniaturized design and automated potential make it a significant advancement for the winemaking industry, facilitating efficient process control and cost savings. Future work could focus on fully automating the sample handling and reagent delivery for fully autonomous operation and exploring the system’s applicability to other types of wine and fermentation processes.

While the system demonstrates high accuracy and stability, potential limitations include the need for initial calibration and the possible impact of wine matrix components other than L-lactic and L-malic on the biosensors, even though the high dilution employed and the design of the electrochemical cell would minimize such interference. The current system uses a peristaltic pump, and future integration with more sophisticated fluid handling systems might improve efficiency and automation. Further testing on a larger variety of wines and under different fermentation conditions would strengthen the generalizability of the findings.