Effect of Thermal Processes on S-Allyl Cysteine Content in Black Garlic

{"Background": "Black garlic (BG) is produced by aging raw garlic at 30\u201390\u00b0C with 50\u201390% relative humidity for 10\u201390 days without additives, resulting in a product with a unique flavor and gelatinous texture. S-allyl cysteine (SAC) is a key bioactive compound in BG that is synthesized through hydrolysis of \u03b3-glutamyl bond in \u03b3-L-glutamyl-S-allyl-L-cysteine (GSAC) by \u03b3-glutamyl transpeptidase (GGT), with optimal GGT activity achieved at 70\u00b0C and 80% RH. SAC is useful in reducing oxidative stress and inflammation, and BG has been associated with decreased risk of diabetes, hypertension, hypercholesterolemia, hyperlipidemia, arteriosclerosis, and cancer. In Thailand, commercially available BG must meet a SAC requirement of >160 mg/kg fresh weight. While many studies have focused on single-clove garlic, no data existed on the optimum aging process for multi-clove garlic, which is the main economical local crop of Thailand. This study aimed to investigate the optimum thermal conditions for developing BG from multi-clove garlic with high SAC content and to compare total phenolic contents, total flavonoid contents, and antioxidant activities of fresh garlic, BG, and their powder forms.", "Methods": "Multi-clove Thai garlic was purchased from an organic farm in Si Sa Ket Province, Thailand (cultivated January\u2013March 2020 and December 2020\u2013February 2021), while Chinese single-clove garlic was purchased from Talaad-Thai wholesale market. BG was produced in a fermentation chamber (1.50 m \u00d7 1.43 m \u00d7 1.85 m, capacity 100 kg) using two methods: Method A (70\u00b0C, 80% RH for 42 days) and Method B (stepwise: 75\u00b0C/85% RH for 9 days, 70\u00b0C/80% RH for 12 days, 60\u00b0C/80% RH for 21 days). After analysis, Method A was selected for large-scale production (chamber size 4.50 m \u00d7 2.25 m \u00d7 2.1 m, capacity 1800 kg) at 70\u00b0C and 80% RH for 12 days. SAC content was analyzed by HPLC with photodiode array detection (\u03bb=250 nm) after derivatization with dansyl chloride. Total phenolic contents (TPCs) were determined using Folin-Ciocalteu reagent, total flavonoid contents (TFCs) using aluminum chloride colorimetric assay, and phenolic profiles by LC-ESI-MS/MS with 27 authentic standards. Antioxidant activities were assessed using FRAP, DPPH radical scavenging, and ORAC assays. All experiments were conducted in triplicate to pentaplicate measurements (n=3\u20135), with results expressed as mean \u00b1 standard deviation. Statistical analysis used paired samples test and one-way ANOVA with Tukey's post-hoc test (p<0.05).", "Key Results": "The initial moisture content of Thai garlic was 67.30\u201368.14% versus 58.04\u201358.63% for Chinese garlic. Under Method A, water evaporation rates until day 41 were 58.26% and 56.25% for Thai and Chinese garlic, respectively. SAC content increased rapidly at the initial step and then decreased significantly with prolonged heat treatment. Thai BG samples subjected to Method A had significantly higher SAC content than Chinese BG samples. Under the optimum condition (70\u00b0C, 80% RH for 12 days), SAC content increased approximately 139-fold in Thai BG and 122-fold in Chinese BG compared to fresh garlic. The SAC contents of Thai BG and Chinese BG were 3.29 and 2.91 times higher than the Thai FDA requirement of 160 mg/kg fresh weight, respectively. No significant difference was found in SAC content between different fermentation chamber sizes (p=0.68 and 0.05). During storage (2\u2013162 days at 4\u00b0C), SAC content in Thai BG decreased by 14.64% (from 781.98\u00b117.23 to 667.51\u00b110.30 mg/kg DW), and in Thai BG powder decreased by 12.53% (from 626.52\u00b19.20 to 547.99\u00b13.87 mg/kg DW). SAC content (mg/g DW), TPCs (mg gallic acid/g DW), and TFCs (mg QE/g DW) of Thai BG increased 176-fold, 11-fold, and 2-fold from fresh garlic, respectively. None of the 24 phenolic compounds were detected by LC-ESI-MS/MS in any sample. Antioxidant activities of Thai BG were significantly higher than fresh garlic: FRAP increased by 97%, DPPH by 84%, and ORAC by 65%. The IC50 values for DPPH scavenging were 5.56 \u00b5g/mL for vitamin C, 713.23 \u00b5g/mL for Thai BG, and 491.26 \u00b5g/mL for Thai BG powder.", "Clinical Implications": "This study establishes a standardized, reproducible thermal processing method (70\u00b0C, 80% RH for 12 days) for producing BG with high SAC content from both Thai multi-clove and Chinese single-clove garlic, meeting and exceeding Thai FDA requirements. The substantial increases in SAC content (139-fold in Thai BG) and antioxidant activities (34-fold in FRAP, 6-fold in DPPH, 3-fold in ORAC) support the potential of BG as a functional food for reducing oxidative stress and inflammation. The finding that SAC content decreases significantly with prolonged heat treatment (>14 days) and during storage (14.64% decrease over 162 days at 4\u00b0C) has practical implications for manufacturing timelines and storage recommendations. The inability to detect phenolic profiles despite increased TPC suggests that SAC and other organosulfur compounds, rather than phenolics, may be the primary contributors to the antioxidant capacity of BG produced under these specific conditions. These findings support the commercial utilization of Thai multi-clove garlic for BG production, potentially increasing the economic value of this local crop, though further studies are needed to confirm functional and health-related benefits in clinical settings."}