Efficient removal of allicin from the stalk of *Allium fistulosum* for dietary fiber production

Allium fistulosum (green onion) is widely consumed and contains bioactive compounds including polyphenols with antioxidant activity. Its stalk is rich in polysaccharides (~16%) with complex monosaccharide compositions and glycosidic linkages, and more complex dietary fiber structures can support higher gut microbiota richness, suggesting nutritional value for functional foods and dietary supplements. Despite reported health benefits of allicin (antioxidative, antimicrobial, gut flora regulation, anticancer), its strong characteristic odor makes Allium-derived fibers less acceptable. Allicin is enzymatically generated (alliinase acting on alliin upon tissue disruption) and is volatile/reactive. Prior work has explored thermal decomposition kinetics of allicin, blanching and freezing effects in other Allium matrices, tea polyphenols’ potential deodorizing effects, and citric acid’s influence on garlic processing, but a systematic comparison of allicin-removal methods specifically for A. fistulosum stalk is lacking. This study aims to optimize allicin extraction/determination conditions and to comprehensively compare chemical (tea polyphenols, citric acid) and physical (freezing, blanching) strategies for efficient, practical allicin removal to enable production of acceptable, green dietary fiber from A. fistulosum stalk.

Several studies inform approaches to managing allicin in Allium. Accurate allicin quantification has used HPLC-UV at 242 nm, but simpler spectrophotometric assays based on allicin–cysteine reaction and DTNB detection are common. Thermal behavior shows allicin degrades with first-order kinetics and can thermally transform to larger thiosulfinates such as ajoene. Processing methods (freezing, blanching) affect sensory and bioactive composition in leeks, onions, and garlic, though effects in A. fistulosum stalk are underexplored. Anecdotal and clinical evidence suggests tea-based rinses can reduce oral malodor and tea polyphenols interact with diverse food constituents, indicating potential to react with or sequester allicin. Citric acid, a GRAS organic acid used as antioxidant and for mycotoxin control, has shown variable effects in other systems (e.g., promoting melanin synthesis in certain cell lines; affecting garlic processing), but its direct influence on allicin content in A. fistulosum was previously unknown. These findings motivate testing tea polyphenols and citric acid, alongside physical treatments, for allicin mitigation.

Materials: A. fistulosum was sourced from Shandong Province, China; stalks (white parts) were separated for experiments. Reagents included absolute ethanol, cystine, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), citric acid (analytical grade; Sinopharm, Shanghai), and tea polyphenols (Red Star Pharmaceutical, Anhui). Allicin determination: Approximately 5 g chopped stalk was mixed with 5 mL water and incubated at 26 °C (baseline 15 min; optimization varied 5–35 min). Allicin was then extracted with ethanol (baseline 50 min at 26 °C; optimization varied ethanol 45–95%) and centrifuged (6000 rpm, 10 min). Supernatants were reacted in 50 mM HEPES buffer with 1 mM cystine and 2 mM DTNB for 15 min in the dark, and absorbance at 412 nm was measured (DeNovix DS-11 FX+). Optimization identified 20 min water incubation and 75% ethanol as optimal for maximal extraction yield. Chemical removal – tea polyphenols: - Concentration test: Tea polyphenol aqueous solutions at 4, 6, 8, 10, and 12 mg/mL were mixed with chopped stalk at a 1:3 solid–liquid ratio, incubated 5 min at 25 °C, then discarded; residual allicin was extracted/determined. - Processing time test: 6 mg/mL solution, 1:3 ratio, incubations of 5, 10, 15, 20, 25 min. - Solid–liquid ratio test: 6 mg/mL solution at ratios 1:1, 1:1.5, 1:2, 1:2.5, 1:3; 5 min incubation. - Multivariate optimization: OPLS-DA (SSPSAU) evaluated three factors with three levels: time (10, 15, 20 min), ratio (1:1, 1:1.5, 1:2), concentration (6, 8, 10 mg/mL). Nine experimental combinations were run to rank factor importance and select optimal settings. Chemical removal – citric acid: Citric acid solutions at 0.125, 0.25, 0.5, 1.0, and 2.0 mg/mL were added to chopped stalk (5 min contact), solution discarded, and residual allicin measured. Physical removal: - Freezing/chilling: Chopped stalk was held at 4 °C, −20 °C, or −80 °C for 0.5 h, 1.0 h, or 1.5 h before allicin extraction/determination. - Blanching: Chopped stalk was blanched at 60 °C for 1, 2, 3, 4, or 5 min; or at 100 °C for 4, 8, 12, 16, 20, 24, 28, or 32 s, followed by allicin extraction/determination. Statistics: Data were plotted with GraphPad Prism 8.0; one-way ANOVA with Duncan test (SPSS v17) assessed significance.

- Optimized extraction/determination: Incubation of chopped A. fistulosum stalk with water for 20 min followed by extraction with 75% ethanol provided maximal allicin extraction yield; longer water incubation (35 min) reduced yield, likely due to volatility. Increasing ethanol from 45% to 75% increased extraction; 75–95% gave no further gain. - Tea polyphenols (chemical removal): Clearance increased as concentration rose from 4 to 8 mg/mL, with no further improvement beyond 8 mg/mL. Clearance increased with processing time up to 15 min, with little/no gain thereafter. Higher solid–liquid ratios improved clearance from 1:1.5 to 1:3. OPLS-DA ranked factor influence as processing time > concentration > solid–liquid ratio. The optimal combination (20 min, 6 mg/mL, 1:1.5) yielded an average allicin clearance of 51.43%. - Citric acid (chemical removal): Varying concentrations (0.125–2.0 mg/mL) affected allicin, with a maximal clearance rate of 54% under tested conditions. - Freezing/chilling (physical removal): At 0.5 h, all temperatures (4, −20, −80 °C) gave ~38% clearance. At 1 h, clearance decreased and became negative at −20 and −80 °C (indicating apparent allicin increase vs. control). At 1.5 h, clearance slightly recovered but remained below 0.5 h levels. Reported maximum clearance by freezing was 43%. - Blanching (physical removal): • 60 °C: 1 min gave 13.4% clearance; prolonged times reduced clearance to negative values, reaching −36.3% at 4 min. • 100 °C: 4 s gave 12.1% clearance; 20 s reached 25%; 32 s achieved 73.3% clearance (maximum). - Overall ranking by maximum clearance: blanching at 100 °C (73.3%) > citric acid (54%) ≈ tea polyphenols (51.4%) > freezing (43%) > blanching at 60 °C (13%).

The study addressed the practical need to remove allicin from A. fistulosum stalk to enable its use as a dietary fiber supplement without strong odor. By first optimizing allicin extraction/detection, the authors ensured reliable comparison across treatments. Chemical treatments showed moderate efficacy: tea polyphenols likely react with or sequester allicin, with process time being the dominant factor; citric acid achieved a slightly higher maximum clearance, suggesting acid-mediated reactions or stability changes. Physical treatments behaved distinctly: short-time blanching at moderate temperature (60 °C) initially reduced allicin slightly but longer exposure led to increased measured allicin (negative clearance), consistent with ongoing enzymatic generation from alliin/alliinase before substantial thermal inactivation or conversion. In contrast, brief high-temperature blanching (100 °C) rapidly achieved substantial clearance (up to 73.3%), likely via accelerated thermal degradation/transformations of allicin (e.g., to ajoene) and/or enzyme inactivation limiting further formation. Freezing provided limited, transient reductions and could even increase apparent allicin after 1 h at lower temperatures, potentially due to cellular damage enhancing substrate-enzyme contact upon thawing. Collectively, results indicate that short, high-temperature blanching is the most efficient and effective approach for allicin mitigation in chopped A. fistulosum stalk while avoiding chemicals, aligning with a green processing objective.

This work provides a comprehensive comparison of chemical (tea polyphenols, citric acid) and physical (freezing, blanching) methods for allicin removal from A. fistulosum stalk, underpinned by an optimized allicin extraction/detection protocol (20 min water incubation; 75% ethanol extraction). The most effective treatment was brief high-temperature blanching at 100 °C, achieving a maximal clearance rate of 73.3% within 32 s, outperforming tea polyphenols (51.4%), citric acid (54%), freezing (up to 43%), and 60 °C blanching (~13%). These findings provide a practical, chemical-free strategy to prepare odor-reduced, green dietary fiber from A. fistulosum. Future research could evaluate process scalability and energy use; impacts on dietary fiber structure, functionality, and microbiota interactions; sensory outcomes and consumer acceptance; retention or transformation of other bioactives; and optimization for whole-stalk or industrial continuous processing.