Sugarcane juice, a nutritious and energetic beverage, has a short shelf life due to rapid microbial and enzymatic deterioration. Traditional heat treatments used for preservation can negatively impact sensory and nutritional quality. This study explores the use of supercritical carbon dioxide (SC-CO₂) technology as a non-thermal alternative for preserving sugarcane juice. SC-CO₂ treatment involves exposing the juice to high pressure (above 74 bar), effectively inactivating spoilage microorganisms and enzymes while minimizing damage to the juice's quality. The critical temperature and pressure for carbon dioxide are 31.1 °C and 73.8 bar, respectively. The main advantage of this non-thermal technique is the preservation of sensory attributes, unlike conventional heat treatments which can lead to nutritional and sensory losses. Previous studies have shown the efficacy of SC-CO₂ in inactivating microorganisms and enzymes in various fruit juices, but its application to sugarcane juice remains unexplored. This research aims to evaluate the combined effect of mild temperatures and SC-CO₂ on microbial and enzymatic inactivation in sugarcane juice, ultimately assessing its potential for juice preservation.

Several studies have demonstrated the effectiveness of supercritical carbon dioxide (SC-CO2) in preserving the quality of fruit juices. These studies show that SC-CO2 can effectively inactivate microorganisms and enzymes responsible for spoilage and deterioration, while maintaining desirable sensory characteristics and nutritional components. Research on the inactivation kinetics of microorganisms, such as *Escherichia coli* and *Saccharomyces cerevisiae*, under high CO2 pressure has shown promising results. The energy efficiency of SC-CO2 processing has also been studied, highlighting its potential advantages over conventional thermal methods. However, a significant gap exists in research focused on applying SC-CO2 technology to sugarcane juice processing. While heat treatments are currently used, the potential for damage to quality motivates exploration of non-thermal techniques such as SC-CO2. Previous research on sugarcane juice preservation has mainly concentrated on traditional methods like heat pasteurization, acidification, chemical preservatives, and refrigeration. While these offer some preservation, they may compromise the sensory attributes or introduce unwanted chemicals.

Fresh sugarcane juice was obtained from a local vendor and transported to the laboratory on ice. The juice was divided into two portions: a control (untreated) and a treatment group. The SC-CO₂ treatment was conducted using a supercritical fluid system (Thar Technologies SFE-500). A central composite rotatable design (CCRD) was employed to investigate the combined effects of pressure (74–351 bar), temperature (33–67 °C), and holding time (20–70 min) on the juice. Seventeen trials, including three replicates at the central point, were performed. Physicochemical analyses (pH and soluble solids using AOAC methods), microbiological assays (mesophiles, molds and yeasts, lactic acid bacteria, and coliforms at 45 °C using methods from the Compendium of Methods for the Microbiological Examination of Foods), enzymatic activity determinations (polyphenol oxidase and peroxidase using adapted protocols), and instrumental color measurements (L*, a*, b*, chroma, and hue using a Hunterlab UltraScan colorimeter) were conducted on both raw and processed samples. Data analysis involved an analysis of effects to identify significant variables, followed by regression analysis (1st and 2nd order) and ANOVA to evaluate the significance of the models. Response surfaces and contour curves were generated to visualize the optimal ranges for the processing parameters. Statistical analyses were performed using Protimiza Experimental Design software.

The pH of the sugarcane juice ranged from 4.6 to 6.3, with treatments causing reductions up to 0.4 pH units. Soluble solids (measured as °Brix) varied from 18.2 to 25.3, exhibiting small changes after treatment (Δ ≤ 0.4). Microbial reduction varied significantly depending on treatment conditions, achieving reductions of up to 2.5 log CFU/mL for coliforms, 3.9 log CFU/mL for mesophiles, 2.1 log CFU/mL for lactic acid bacteria, and 4.1 log CFU/mL for molds and yeasts. Polyphenol oxidase (PPO) reduction ranged from 3.51% to 64.18%, while peroxidase (POD) reduction ranged from 0.27% to 41.42%. Color variations between fresh and processed samples varied widely, between 2.0 and 12.3 in total color difference (ΔE*). Statistical analysis showed that holding time (t) significantly affected PPO reduction, while temperature (T), holding time, and their interaction significantly affected POD reduction. Pressure (P) and the interaction of temperature and holding time significantly affected pH variation. Pressure, temperature, holding time, and the interaction between temperature and holding time significantly affected total color difference. The first-order models for POD reduction (R² = 0.86) and total color difference (R² = 0.90) were the best fit for the experimental data. Response surface analysis identified optimal ranges for temperature and holding time for POD reduction and for pressure, temperature, and holding time for minimizing color difference.

The results demonstrate the potential of using a combination of SC-CO₂ and mild temperatures for sugarcane juice preservation. While achieving complete microbial inactivation wasn't consistently observed for all microbial groups, significant reductions were obtained. The extent of reduction varied based on the specific treatment conditions (pressure, temperature, and time), highlighting the importance of optimizing these parameters. Partial inactivation of PPO and POD enzymes was achieved, potentially limiting enzymatic browning and maintaining better color and flavor. The lack of significant effects on some parameters (mesophiles, molds, yeasts, and soluble solids) may be attributed to the inherent variability of the raw material, processing conditions, or limitations in the detection methods. The study's findings provide valuable data for optimizing the SC-CO₂ process for sugarcane juice preservation, balancing the need for effective microbial inactivation and enzyme reduction with the preservation of sensory and nutritional qualities. The optimal ranges identified for various parameters in this study offer a guide for practical implementation.

This research demonstrates the potential of supercritical carbon dioxide (SC-CO₂) treatment combined with mild temperatures as a non-thermal method for preserving sugarcane juice. Significant reductions in microbial loads and partial enzyme inactivation were achieved, offering a promising alternative to traditional heat treatments. The study highlights the importance of optimizing pressure, temperature, and holding time to achieve desired levels of preservation while minimizing changes in sensory and physicochemical qualities. Future research could focus on scaling up the process, optimizing it for specific sugarcane varieties, and conducting extensive sensory evaluations to assess consumer acceptance.

The study used sugarcane juice from a single source, potentially limiting the generalizability of the findings to other sources or varieties. The relatively small scale of the experiments may limit the direct application to industrial settings. The microbiological analyses were performed immediately after processing, without simulating long-term storage conditions, and this could influence the microbial loads observed. Further research should include long-term storage stability tests to assess the long-term efficacy of the SC-CO₂ treatment and complete sensory analysis to assess the changes in flavor and aroma.