The potential use of supercritical carbon dioxide in sugarcane juice processing

Sugarcane juice is a low-acid, high–water activity beverage whose composition depends on cultivar and growing conditions. Its shelf life is limited by rapid microbiological and enzymic deterioration, particularly browning catalyzed by polyphenol oxidase (PPO) and peroxidase (POD), and spoilage by organisms such as Leuconostoc mesenteroides, molds and yeasts. Conventional heat treatments used to stabilize juice can impair sensory, nutritional, and functional quality. Supercritical carbon dioxide (SC-CO2) is a non-thermal preservation technique in which foods are exposed to CO2 above its critical point (31.1 °C, 73.8 bar). SC-CO2 can inactivate enzymes and microorganisms while better preserving fresh-like quality and potentially reducing energy consumption compared with thermal processing. Despite promising results in other juices, no prior studies have specifically targeted stabilization of cane juice using SC-CO2. This study investigates the combined effect of mild temperatures and SC-CO2 on endogenous microorganisms and enzymes in sugarcane juice, and examines impacts on pH, soluble solids, and color.

Prior work indicates that non-thermal technologies often better preserve nutrients and sensory attributes and can be energetically favorable. CO2 is advantageous among supercritical fluids for its low toxicity, low cost, and moderate critical conditions. Studies applying SC-CO2 with direct injection to fruit juices have demonstrated effective microbial and enzymatic inactivation, with improved retention of thermolabile compounds (phenolics, flavonoids, anthocyanins). However, the literature on SC-CO2 for cane juice is scarce to non-existent, preventing direct comparisons. Thermal pasteurization studies in cane juice report substantial microbial reductions (e.g., 4–5 log), but with potential quality trade-offs. SC-CO2 has also been reviewed for spore inactivation with better preservation of quality than high hydrostatic pressure or high-temperature methods. The literature further highlights that enzyme inactivation by pressure and temperature depends on enzyme source, matrix, and water interactions, and that energy requirements of pressure-based processes can be lower than those of indirect thermal processing.

• Raw material and preparation: Fresh sugarcane juice was obtained from a local vendor in Pirassununga/SP, Brazil. Juice was kept on ice and transported to the University of São Paulo laboratory. Aliquots were placed into sterilized glass bottles; one served as raw control and the other was processed.
• SC-CO2 processing: Direct injection in a 100 mL reactor of a supercritical fluid system (Thar Technologies SFE-500). Each 100 mL sample was processed under preset conditions, then depressurized rapidly into a sterile flask.
• Experimental design: Central composite rotatable design (CCRD) with three variables: pressure (P, 74–351 bar), temperature (T, 33–67 °C), and holding time (t, 20–70 min). Seventeen trials were conducted, including three central-point replicates. Mild temperatures were chosen to preserve quality and the CO2 pressure range exceeded critical pressure. Actual and coded levels: P (74, 130, 213, 295, 351 bar), T (33, 40, 50, 60, 67 °C), t (20, 30, 45, 60, 70 min). Three central-point replicates assessed process repeatability.
• Analyses (all in triplicate):
  • Physicochemical: pH (Analyzer model 300 M) and soluble solids in °Brix (Reichert AR 200 refractometer) per AOAC (2010).
  • Microbiological: counts of aerobic mesophiles, molds and yeasts, lactic acid bacteria, and coliforms at 45 °C per Compendium of Methods for the Microbiological Examination of Foods.
  • Enzymatic: PPO and POD activities using adapted spectrophotometric assays; one unit defined as the amount causing an absorbance increase of 0.001 units/min at 425 nm (PPO) or 470 nm (POD) under specified conditions.
  • Color: L*, a*, b* measured by Hunterlab Ultra-Scan (D65 illuminant, 10° observer). Chroma (C*) and hue calculated; total color difference (TCD, ΔE*) computed between raw and processed.
• Statistics: Effect analysis (significance at p ≤ 0.1) identified significant variables among P, T, and t for each response (microbial and enzymatic reductions, pH and °Brix variation, color/TCD). First- and second-order regression models were fit; non-significant terms were removed (re-parameterized). ANOVA assessed model significance and R². Response surfaces and contour plots were generated for significant models. Modeling validity was restricted to the coded ranges corresponding to P: 130–295 bar, T: 40–60 °C, and t: 30–60 min for first-order models.

• Physicochemical:
  • pH: Raw 4.6–6.0; processed 4.4–6.3; maximum decrease 0.4 units; some trials unchanged. Pressure (P) and T×t interaction significantly affected pH variation.
  • Soluble solids (°Brix): Raw 18.5–25.3; processed 18.2–25.0; Δ ≤ 0.4 across trials; no significant effects of P, T, t on °Brix variation.
• Microbiological reductions (maximum observed across design):
  • Coliforms: up to ~2.5 log reduction (some counts below detection prevented full statistical analysis).
  • Aerobic mesophiles: up to 3.9 log reduction.
  • Lactic acid bacteria: up to 2.1 log reduction (some below detection prevented full statistical analysis).
  • Molds and yeasts: up to 4.1 log reduction.
  • For mesophiles and molds/yeasts reductions, none of P, T, t or their interactions were statistically significant in Pareto analysis.
• Enzymes:
  • PPO reduction ranged ~3.3%–64.5%; only holding time (t) significantly affected PPO reduction.
  • POD reduction ranged ~0.0%–40.9%; temperature (T), time (t), and their interaction (T×t) significantly affected POD reduction.
  • First-order POD reduction model (coded): Y1 = 18.56 + 4.46x2 + 7.09x3 + 8.30x2x3; R² = 0.86; significant by ANOVA.
  • Optimal region for POD reduction: T ≈ 57–60 °C and t ≈ 56–60 min (within 130–295 bar).
• Color:
  • L*, a*, b*, chroma, hue showed wide variability due to raw material variability.
  • Total color difference ΔE* between raw and processed ranged 2.0–12.3; ΔE* < 3 in 6 of 17 trials (not easily perceptible); ΔE* > 12 indicates distinct color spaces.
  • Significant factors for ΔE*: P, T, t, and T×t.
  • First-order TCD model (coded): Y2 = 5.15 + 1.36x1 + 2.06x2 + 2.09x3 + 1.39x2x3; R² = 0.90; significant by ANOVA.
  • Conditions minimizing ΔE*: approximately P 130–150 bar, T 40–43 °C, t 30–35 min.
• Overall: SC-CO2 with mild temperatures achieved meaningful microbial reductions and partial enzyme inactivation with minimal changes in pH and soluble solids; parameter significance differed by response.

The study demonstrates that SC-CO2 combined with mild temperatures can reduce microbial loads in sugarcane juice while partially inactivating endogenous browning enzymes, addressing the need to stabilize cane juice without intensive heat treatment. The lack of significant effects of P, T, and t on mesophiles and molds/yeasts reductions in the Pareto analysis suggests complex inactivation dynamics and potential variability in raw material contamination and process diffusion effects; nevertheless, up to 3.9–4.1 log reductions were achieved. Enzymatic inactivation responses were more systematically governed by process variables: PPO responded primarily to holding time, while POD was influenced by temperature, time, and their interaction, enabling predictive modeling with high R² and identification of practical operating windows. Quality attributes showed small changes in pH and °Brix, but color differences varied; modeling identified conditions to minimize ΔE*, allowing trade-offs between enzyme inactivation and visual quality. Compared with thermal pasteurization reports, SC-CO2 offers a non-thermal alternative that preserves fresh-like attributes and can be energy-efficient, though enzyme inactivation may be partial and color impacts must be managed via process optimization.

This work is the first to systematically evaluate SC-CO2 with mild temperatures for sugarcane juice stabilization. Across a CCRD of P (74–351 bar), T (33–67 °C), and t (20–70 min), SC-CO2 achieved up to 3.9 log reductions in mesophiles and 4.1 log in molds/yeasts, with partial inactivation of PPO (to ~64.5%) and POD (to ~40.9%), and minimal changes in pH and soluble solids. Statistical analyses identified key variable effects: time for PPO; temperature, time, and their interaction for POD; pressure and T×t for pH; and P, T, t, plus T×t for color difference. First-order coded models with high R² predicted POD reduction and total color difference, enabling identification of robust operating windows for maximizing POD reduction (T ~57–60 °C, t ~56–60 min) and minimizing ΔE* (P ~130–150 bar, T ~40–43 °C, t ~30–35 min). Overall, SC-CO2 at mild temperatures shows promise for cane juice preservation. Future research should include scale-up studies, continuous-flow processing, validation on diverse cultivars and seasons, optimization to balance enzyme inactivation and color preservation, shelf-life and sensory evaluation during storage, and assessment of pathogen inactivation and spore control.

• No prior SC-CO2 studies on cane juice for direct comparison; results are exploratory.
• Small-scale, batch reactor (100 mL) may limit generalizability; scale-up effects not assessed.
• High variability of raw material (different stalks/days) likely contributed to variability in microbial counts, enzymatic activity, and color parameters.
• Some microbial counts were below detection limits, preventing full statistical modeling for coliforms and lactic acid bacteria.
• Storage stability, shelf-life, and sensory evaluations post-processing were not conducted.
• Enzyme inactivation was partial; color differences were perceptible in many trials, indicating trade-offs that require further optimization.
• Model validity is restricted to coded ranges (approximately P 130–295 bar, T 40–60 °C, t 30–60 min).