5-hydroxymethyl-2-furfural and acrylamide formations in black garlic during aging period and their correlations with browning degree

Abstract

Solo-bulb black garlics (SBBG) are novel foods rich in nutritional and functional ingredients. However, certain process contaminants formed during thermal processing of SBBG raise safety concerns. This study investigated the formations of 5-hydroxymethyl-2-furfural (5-HMF) and acrylamide, as well as the browning degree of SBBG during a 12-day aging period. Both 5-HMF and acrylamide contents exhibited exponential increases, with their formations well simulated by pseudo-first-order kinetic models. 5-HMF formation showed a significant positive correlation with fructose content (p < 0.05), while acrylamide formation was strongly associated with asparagine content (p < 0.01). Browning degree correlated significantly with acrylamide accumulation and asparagine content (p < 0.01), but not with 5-HMF (p > 0.05). These results indicate that fructose and asparagine accumulations play critical roles on 5-HMF and acrylamide formations in SBBG, respectively, and acrylamide accumulation promotes SBBG browning. Notably, acrylamide level in SBBG exceeded those reported in some commonly consumed foods, which is a concern considering the daily consumption of this food.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13197-025-06315-x.

Introduction

Black garlics (BG) are very popular in China and other Asian countries dues to their nutritional and therapeutic properties (Afzaal et al. 2021; Kilic-Buyukkurt et al. 2023). BG are produced by aging fresh white garlics under high-temperature (60–90 °C) and high-humidity (70–90% relative humidity, RH) conditions for over 30 days (Afzaal et al. 2021; Chua et al. 2022). The characteristic colour, aroma and flavour of BG are formed mainly based on the Maillard reaction and caramelisation during thermal processing (Kilic-Buyukkurt et al. 2023; Nam et al. 2023). Meanwhile, many bioactive compounds such as polyphenols, polysaccharide, organosulfur compounds, and melanoids, are formed or enriched during the aging process, endowing BG with antioxidant, antibacterial, anti-inflammatory, and anti-cardiovascular properties (Jing 2020; Utama et al. 2024). However, certain toxic compounds, such as 5-hydroxymethyl-2-furfural (5-HMF) and acrylamide, can also be generated (Chan et al. 2022; Chua et al. 2022; Ma et al. 2025). It is reported that 5-HMF and acrylamide are potentially probable carcinogens, hepatotoxins, nephrotoxins, and gentoxins to humans (Mesías et al. 2024). 5-HMF was determined as 0.10 mg/g when BG were processed at 65 °C and 70% RH for 24 days, while as 2.82 mg/g at 84 °C and 85% RH for 50 days (Sasmaz et al. 2023). Chan et al. (2022) found that the 5-HMF contents in BG were rapidly increased to 3.8, 5.0, 5.8, and 6.3 mg/g for the control, ultrasound, high hydrostatic pressure (HHP), and freezing pretreatment groups during 30 days of thermal processing, respectively. The similar increases in 5-HMF contents during BG processing have also been reported (Li et al. 2020; Ríos-Ríos et al. 2021). An exponential increment of 5-HMF content in BG was observed after being aged at 70–75 °C and 80–90% RH for 150 h (Chua et al. 2022). Notably, Nam et al. (2023) not only detected the 5-HMF in BG, but also found its main metabolite 5-hydroxymethyl-2-furoic acid (5-HMFA), which reached tenfold the 5-HMF content during 40 days of aging process. Our previous study also showed that the maximum level of 5-HMF in solo-bulb black garlics (SBBG) reached 1.37 mg/g after being aged at 70–75 °C and 80–90% RH for 9 days (Ma et al. 2025). However, the acrylamide level in BG has hardly been reported yet, which is often detected in many baked, fried, and roasted carbohydrate-rich foods (Augustine and Bent 2022; Basinci et al. 2022). Recently, the changes of acrylamide content in SBBG after solo-bulb fresh garlics (SBFG) being pretreated by electron beam irradiation (EBI) were investigated, and detected its maximum level of 34.01 μg/g (Ma et al. 2025). In addition, it is found that the accumulation of 5-HMF is closely related to the blackening rate of BG, and 5-HMF also causes bitterness of BG when it accumulates to a certain extent (Li et al. 2020). Therefore, it is of practical importance to investigate the 5-HMF and acrylamide formations, as well as their correlations with the browning degree of BG during aging process. Simultaneously, the changes in 5-HMF, acrylamide, and the browning degree of SBBG during aging were simulated with regression models, which could be used in predicting the quality and safety of SBBG. These findings provide a comprehensive insight into the correlations among 5-HMF and acrylamide formations with browning degree of SBBG.

Materials and methods

Materials and chemicals

Solo-bulb fresh garlics (SBFG) with 3.0 to 3.5 cm of diameter were purchased from a local market in the Dali city, Yunnan province, China. Both 5-hydroxymethyl-2-furfural (5-HMF, purity > 99.5%), acrylamide (purity > 99.9%), fructose (purity > 99.0%), glucose (purity > 99.0%), and asparagine (purity > 99.0%) were all standards, and purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Methanol and acetonitrile were of HPLC grade, were purchased from Shanghai Sangon Biotech Co., Ltd.

Preparation of solo-bulb black garlics (SBBG)

The SBBG were prepared by aging the SBFG at 70–75 °C and 80–90% RH for 12 days using a 7.5 L garlic fermenter (AZK186-80, Guangdong, China) with a three-layer tray. The aging temperature of SBFG was set at 75 °C for the first 3 days and then at 70 °C for the remaining 9 days with the same RH of 80–90%. The samples were taken out with an interval of three days during 12 days of aging period, and cooled to the room temperature, then refrigerated at 4 °C until further analysis.

Colour measurement

Colour changes of the SBFG and SBBG were measured using a chromameter (CR-400, Konica Minolta, Japan) calibrated with a white plate (colour parameter: L/84.6, a/0.3155, and b/0.3213). The L (lightness), a (redness), b (yellowness) values were measured at different points of garlic samples to control possible non-homogeneous colour distributions. The delta components (ΔL, Δa, and Δb) were calculated by subtracting the corresponding colour parameters of white plate. The total colour differences (ΔΕ) were used to judge the browning degree of SBBG during 12 days of aging period, which were calculated according to the following equation: $\mathrm{\Delta }\mathrm{E}=\sqrt{\mathrm{\Delta }{L}^2+\mathrm{\Delta }{a}^2+\mathrm{\Delta }{b}^2}$.

Determination of 5-HMF and acrylamide during SBBG processing

The contents of 5-HMF and acrylamide in SBFG and SBBG were simultaneously determined using the HPLC–DAD method previously described by Shi et al. (2019). Garlic sample was finely triturated and weighed about 2.0 g into 15 mL centrifuge tube containing 5.0 mL of methanol, then vortex vigorously for 2 min. The mixed solution was centrifuged at 4 °C and 10,000 rpm for 10 min. The supernatant was transferred into a centrifuge tube and added subsequently with Carrez I (0.25 mL, 15% of Potassium ferrocyanide solution, w/v) and Carrez II (0.25 mL, 30% of Zinc sulfate solution, w/v) solutions, and then centrifuged at 4 °C and 10,000 rpm for 10 min. 1.0 mL of supernatant was dried with low-pressure evaporator at 40 °C, and the residue was redissolved with 1.0 mL of ultrapure water, which was filtered through 0.22 µm aqueous membrane for quantitation by HPLC–DAD (Agilent 1260 Infinity II). The ODS-3 C18 column (250 mm × 4.6 mm ID, 5 µm) was used to separate 5-HMF and acrylamide; the mobile phase was acetonitrile and water with a ratio of 5:95 (v/v) at a flow rate of 1.0 mL/min. The column temperature was set at 40 °C, and the injection volume was 10 µL, as well as the detector wavelength was set at 210 nm for acrylamide and at 284 nm for 5-HMF. The peak identifications were based on the retention times of the standard 5-HMF and acrylamide.

Determination of reducing sugars and asparagine during SBBG processing

Fructose and glucose contents in SBFG and SBBG were measured according to the method described by Ma et al. (2025). Triturated garlic sample was weighed about 2.0 g into 100 mL beaker, and added with 30 mL of ultrapure water, then sonicated for 2 min. The mixed solution was transferred into 100 mL volumetric flask, and made up to volume with ultrapure water. After being centrifuged at 5000 rpm for 10 min, 50 µL of the supernatant was derived with 100 µL of derivative reagent (0.7 g p-aminobenzoic acid, 1.0 g glacial acetic acid, and 0.1 g sodium cyanoborohydride were dissolved with methanol and diluted to 10 mL) at 70 °C for 60 min in water bath. The derivant was cooled and diluted to 2 mL with ultrapure water, and then filtered through 0.22 µm aqueous membrane for quantitation by HPLC-FLD (Agilent 1260 Infinity II). The ODS-3 C18 column (250 mm × 4.6 mm ID, 5 µm) was used to separate fructose and glucose; the mobile phase was 0.1 mol/L phosphate buffer (containing 20 mmol/L tetrabutylammonium hydrogen sulfate, pH 2.5) and acetonitrile with a ratio of 95:5 (v/v) at a flow rate of 0.5 mL/min. The column temperature was set at 30 °C, and the injection volume was 20 µL. The excitation wavelength was set at 313 nm and the emission wavelength was set at 358 nm. The peak identifications were based on the retention times of the standard fructose and glucose.

The asparagine content was also determined by HPLC–DAD method as described by Ma et al. (2025). 1.0 g of sample was hydrolyzed by HCl solution (6 mol/L) in a hydrolysis tube at vacuum state and 95 °C for 60 min. The hydrolysate was diluted to 50 mL with ultrapure water and then filtered using a vacuum pump. The filtrate was derived with phenyl isothiocyanate and passed through 0.22 µm organic membrane for quantitation by HPLC–DAD (Agilent 1260 Infinity II). The ODS-3 C18 column (250 mm × 4.6 mm ID, 5 µm) was used to separate asparagine; the mobile phase consisted of (A) sodium acetate/triethylamine solution (1.64 g/L, pH 6.20) and (B) acetonitrile solution (80%, v/v) with a gradient elution program (92% A for 2 min, decreased to 81% A within 10 min, and then decreased to 74% A within 7 min). The column temperature was set at 40 °C, and the injection volume was 10 µL, as well as the detector wavelength was set at 254 nm. The peak identification was based on the retention time of the standard asparagine.

Statistical analysis

All samples were performed in triplicate and the data were expressed as means ± standard deviations. Statistical analysis was carried out using one-way ANOVA and Duncan’s multiple range test with the SPSS 18.0 software (SPSS 18.0, USA), and statistical significance was set at p < 0.05. Pearson correlations among 5-HMF, acrylamide, reducing sugars, asparagine, and browning degree of SBBG were analyzed using the software Origin2021 (OriginLab Corporation, USA).

Results and discussions

Formation of 5-HMF in SBBG during aging period

It is well known that the 5-HMF is formed from the Maillard reaction and caramelisation (Karadeniz et al. 2024). The amount of 5-HMF depends on the temperature, time, water activity, type and concentration of reducing sugars and amino acids, as well as pH environment (Li et al. 2019). 5-HMF is also produced from the dehydration of fructose and glucose based on a cyclic or an acyclic pathway under acidic conditions (caramelisation) (Liu and Kerton 2021). So it is widely produced and detected in heated foods rich in carbohydrates (Karadeniz et al. 2024; Li et al. 2019). Similarly, 5-HMF is also generated and detected in BG during thermal processing (Chan et al. 2022; Chua et al. 2022; Li et al. 2020; Ma et al. 2025; Ríos-Ríos et al. 2021; Sasmaz et al. 2023). Figure 1 shows the changes of 5-HMF in SBBG during 12 days of aging period. Seen from the Fig. 1, 5-HMF was not detected in SBFG, indicating no 5-HMF in fresh garlics. While it was significantly produced in SBBG after being aged for 9 days at 70–75 °C and 80–90% RH (p < 0.01). 5-HMF was gradually increased to 46.06 μg/g at the aging time of 9 days, and then rapidly reached 217.81 μg/g at the aging time of 12 days (p < 0.01). An exponential increment of 5-HMF content in SBBG was also observed during 12 days of aging time, which is consistent with the results reported by Chua et al. (2022). Therefore, increasing the aging time significantly promoted the formation of 5-HMF in SBBG (p < 0.01), and a pseudo-first-order kinetic model could well simulate its changes in SBBG during the aging period (Fig. 1). The regression equation between the aging time (x) and 5-HMF yield (y) is y = − 1.29902 + 0.42468 e^0.52055x (R² = 0.9994).

Fig. 1.

5-HMF formation in SBBG during 12 days of aging time

To investigate the reasons for the increase of 5-HMF level in SBBG, the levels of reducing sugars including fructose and glucose in SBFG and SBBG were measured during 12 days of aging period. The fructose was the main reducing sugar, which was also exponentially increased from 3.18 mg/g in SBFG to 209.93 mg/g in SBBG at the aging time of 12 days, which improved by more than 66 folds (Fig. 2). However, the glucose level in SBBG was slowly decreased during the first 9 days of aging time (p > 0.05), and then obviously increased to 5.42 mg/g at the aging time of 12 days (p < 0.05), which was only one-39ths of fructose level (Fig. 2). Thus, the increase of fructose promoted the rapid production of 5-HMF in SBBG during aging period (Ríos-Ríos et al. 2021). Liu and Kerton (2021) pointed out that fructose was easier to form 5-HMF by dehydration based on the cyclic pathway than glucose, and even glucose needed to be firstly isomerized to fructose and then to produce 5-HMF. In addition, our previous study revealed that the total acid contents in SBBG were increased during aging period (Ma et al. 2025), and the acidic condition and low moisture content of SBBG were more conducive to the formation of 5-HMF even at the low temperature (Lee and Nagy 1990). So the increase of fructose in SBBG is the main reason for the rapid increase of 5-HMF during SBBG aging process. Additionally, in the late stage of SBBG aging, the rapid increase of glucose content may be contributed to the hydrolysis of sucrose and polysaccharides present in SBBG due to the high temperature (70 °C) and high relative humidity (80–90%) conditions (González-Ramírez et al. 2022; Ríos-Ríos et al. 2021).

Fig. 2.

Changes of fructose (○) and glucose (●) contents in SBBG during 12 days of aging time

To extract some latent information about the effects of reducing sugars on the formation of 5-HMF during SBBG aging process, Pearson correlation analysis was carried out between them (Table 1). Seen from the Table 1, a significant positive correlation was observed between fructose concentration and 5-HMF formation (r = 0.9384, p < 0.05), while glucose and asparagine contents showed no significant correlation with 5-HMF formation (p > 0.05). Kuster (1990) found that the main sources of 5-HMF were sugars with different types, especially fructose. Many researchers also considered that the fructose and glucose were the basic substrates for 5-HMF production, which obtained from the acid/enzymatic hydrolysis of oligo- or polysaccharides or disaccharides (sucrose) (Kowalski et al. 2013). Some literatures also reported the pH decrease of BG during thermal processing (Ma et al. 2025; Najman, Sadowska, and Hallmann 2020; Nam et al. 2023), which favored the production of monosaccharides (fructose and glucose). So the increase of fructose content significantly promoted the formation of 5-HMF in SBBG.

Table 1.

Pearson correlations among 5-HMF, acrylamide, reducing sugars, asparagine, and browning degree of solo-bulb black garlics (SBBG)

Correlation coefficient (r)	Fructose	Glucose	Asparagine	Browning degree
5-HMF	0.9384*	0.8223	0.7197	0.4589
Acrylamide	0.7588	0.0576	0.9737**	0.9874**
Browning degree	0.6475	− 0.0618	0.9286*	1

^** Extremely significant correlation (p < 0.01), ^* significant correlation (p < 0.05)

Considering the toxicity of 5-HMF, some international organizations and countries have established the limit levels of 5-HMF only for honey and fruit juice (Martins et al. 2022). For example, Codex Alimentarius Commission (CAC) and European Commission (EC) set the limit levels of 5-HMF in honey between 40 and 80 μg/g depending on the country of origin (Codex Alimentarius Commission, 2001; European Commission, 2001); a maximum concentration of 10 μg/mL in fruit juice and 25 μg/mL in concentrated one were recommended by the International Federation of Fruit Juice Processors (Habibi et al. 2018). While the maximum level of 5-HMF in other foods has not been established, including the BG. Various animal experiments revealed that no adverse effect levels of 5-HMF were in the range of 80–100 mg/kg body weight per day (Abraham et al. 2011). According to the limit levels of 5-HMF in honey, the 5-HMF contents in SBBG exceeded 40 μg/g at the aging time of 9 days, which reached 46.06 μg/g. Thus, the 5-HMF content in SBBG should be strictly controlled by developing some migration measures or optimizing their processing technology (such as aging temperature, time, relative humidity, type of raw garlics, pH, and water activity, etc.). In addition, the limit level of 5-HMF in BG also should be established based on the safety assessment and existing processing level.

Formation of acrylamide in SBBG during aging period

According to the reported literature, acrylamide is mainly generated through the Maillard reaction between free amino acid asparagine and the reducing sugars during thermal processing at temperatures above 120 °C (Augustine and Bent 2022). So it is often detected in carbohydrate-rich foods processed at high temperature. For example, the mean acrylamide content in roasted almonds was 149 μg/kg (Mesías et al. 2024), and it was formed higher than 600 μg/kg in the roasted malts at 200 °C (Basinci et al. 2022). However, acrylamide is difficult to generate when the processing temperature less than 60 °C (Basinci et al. 2022). In this study, it is also observed an exponential increment of acrylamide content in SBBG during 12 days of aging period. Thus, the aging time obviously affected the acrylamide formation, especially for the first 9 days (p < 0.05). Similarly, acrylamide accumulation in SBBG also followed the pseudo-first-order kinetic model (Fig. 3). The regression equation between the aging time (x) and acrylamide yield (y) is y = 7.31711–7.242 e^–0.30322x (R² = 0.98375). While the increasing trend of acrylamide was very slow due to the low aging temperature of 70–75 °C during 12 days of aging period. Like the 5-HMF, acrylamide was also not detected in fresh garlics, while it was increased to 6.76 μg/g in SBBG aged for 9 days (p < 0.05), and then slowly increased to 7.43 μg/g in SBBG aged for 12 days (p > 0.05). Compared with the acrylamide contents in roasted almonds (120–195 °C for 10–30 min) and malts (150–200 °C for 2 h), it was relative higher in SBBG due to the longer aging time of 12 days at 70–75 °C (Basinci et al. 2022; Mesías et al. 2024). The similarly higher acrylamide contents were also detected in potato chips (4.87–9.01 μg/g), French fries (0.005–1.999 μg/g), crispbreads (0.01–2.542 μg/g), and roasted coffees (0.135–1.139 μg/g) (Ghiasvand and Hajipour 2016; Yan et al. 2023).

Fig. 3.

Acrylamide formation in SBBG during 12 days of aging time

The formation of acrylamide is closely associated with the reducing sugars and asparagine (Augustine and Bent 2022), and even many researchers consider that the major pathway for acrylamide formation is the Maillard reaction with free asparagine as main precursor (Capuano and Fogliano 2011). Therefore, we also investigated the changes of asparagine in SBBG during 12 days of aging period (Fig. 4). Meanwhile, we explored the influences of reducing sugars and asparagine on the acrylamide formation by Pearson correlation analysis (Table 1). It can be seen that the changing trend of asparagine with an exponential increment is very similar to that of acrylamide formation during SBBG aging (Figs. 3, 4). It increased from 0.50 mg/g for the SBFG to 0.73 mg/g for the SBBG aged for 12 days, which increased by 46% (p < 0.05). So an extremely significant positive correlation was also observed between asparagine concentration and acrylamide formation (r = 0.9737, p < 0.01) (Table 1). It can be supported by one of the formation mechanisms of acrylamide, i.e. asparagine can thermally decompose to acrylamide by decarboxylation and deamination (Capuano and Fogliano 2011; Lingnert et al. 2002). However, fructose content did not display significant positive correlation with acrylamide formation (r = 0.7588, p > 0.05), and even glucose content was almost unrelated to the production of acrylamide (r = 0.0576, p > 0.05), which was contradictory to the reported conclusions (Augustine and Bent 2022). The reason maybe that the lower aging temperature (70–75 °C) of SBBG is not benefit for the degradation of reducing sugars to acrolein, which is a very probable precursor of acrylamide (Lingnert et al. 2002). Therefore, the asparagine is a very important factor influencing the formation of acrylamide during SBBG aging period.

Fig. 4.

Changes of asparagine content in SBBG during 12 days of aging time

Presently, no official maximum limited level is established for acrylamide in foods. A reference value of 0.47 μg/g in roasted almonds is proposed to implement control or mitigation measures within the manufactures (Mesías et al. 2024), and European Commission recommends that the maximum permitted intake of acrylamide from food is 0.5 mg/kg body weight per day (European Commission, 2019). In this study, the acrylamide content in SBBG is tenfold higher than the reference value set in roasted almonds. Therefore, acrylamide in SBBG faces the same problems as the 5-HMF, which needs to be addressed in future studies.

Colour change of SBBG during aging period

Browning is an important symbol of BG aging, which affects its quality and consumer perception. The black colour of BG is mainly obtained from the Maillard reaction and caramelisation during thermal processing (Nam et al. 2023). The changes in browning degree (ΔΕ) of SBBG during 12 days of aging period are shown in Fig. 5A, and the corresponding appearance images are displayed in Fig. 5B. The colour of SBFG developed gradually from white to brown, then to completely black as the increase of aging time. An exponential increment of browning degree (ΔΕ) was observed, which was very similar to that of acrylamide formation. ΔΕ was rapidly increased from 21.55 of the SBFG to 63.48 of the SBBG aged at 9 days (p < 0.01), and then gently improved to 64.49 of the SBBG aged at 12 days (p > 0.05), which was consistent with the previously reported results (Chan et al. 2022; Ríos-Ríos et al. 2021). Pearson correlation analysis further confirmed the extremely significant correlation between acrylamide formation and browning degree (r = 0.9874, p < 0.01) (Table 1). The reason might be that acrylamide was an important intermediate in the non-enzymatic browning reaction, which eventually could convert to melanoidins by complicated browning reactions (Liu et al. 2020).

Fig. 5.

Colour changes of SBBG during 12 days of aging time A browning degree, B appearance images

Many literatures also reported that the browning degree of food was closely related to the 5-HMF accumulation (Karadeniz et al. 2024; Li et al. 2020). However, Pearson correlation analysis in this study cannot support this conclusion. There was no significant positive correlation between browning degree and 5-HMF accumulation in SBBG (r = 0.4589, p > 0.05) (Table 1). The reasons need to be deeply investigated in the next work. In addition, we found that the browning degree of SBBG was significant positive correlation with the asparagine content (r = 0.9286, p < 0.05), which further verified that asparagine could promote the browning reaction during SBBG aging.

Conclusion

Thermal processing (aging) is the main method to produce BG, which induces the white garlics to gradually turn black colour, as well as produces a jelly-like texture and sour–sweet taste based on the Maillard reaction and caramelization. While it also generates 5-HMF and acrylamide simultaneously, which may be toxic to human health. In this study, both 5-HMF and acrylamide contents were significantly increased, and the pseudo-first-order kinetic model could well simulate their changes in SBBG during the 12 days of aging period. The formation of 5-HMF was significant correlation with fructose content in SBBG, while acrylamide formation was closely related to the asparagine content in SBBG. The browning degree was extremely significant correlation with the acrylamide formation while no significant correlation with the 5-HMF formation. Presently, no maximum levels of 5-HMF and acrylamide in BG were established, while they should be strictly controlled by developing some migration measures or optimizing their processing technology based on the safe consideration. In addition, their limit levels in BG should also be established as soon as possible based on the various safety assessments and existing processing technology.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Shutian Ma: Conceptualization, Formal analysis, Methodology, Writing—original draft. Zhuang Shi: Data curation, Methodology, Validation. Yi Yang: Formal analysis, Methodology, Project administration. Jinglin Zhang: Data curation, Formal analysis. Hongyu Zou: Conceptualization, Investigation, Methodology. Enjie Diao: Funding acquisition, Investigation, Writing—review & editing. Liming Zhang: Resources, Supervision.

Funding

This work was supported by the Team Research Project from Jiangsu Collaborative Innovation Center of Regional Modern Agriculture & Environmental Protection (No. HSXT30413).

Data availability

The experimental data are available from supplementary materials.

Code availability

Not applicable.

Declarations

Conflict of interest

The authors declare that they have no competing interest.

Ethical approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.