Structural Variations of Broccoli Polyphenolics and Their Antioxidant Capacity as a Function of Growing Temperature

Polyphenolics in plants exist in free, soluble-bound, and insoluble-bound structural forms. The concentration of these structural forms depends on the plant's developmental stage, tissue type, soil water availability, and food preparation methods. In this study, for the first time, the effects of growth temperature (RT-room temperature-23 °C day/18 °C night, HT-high temperature-38 °C day/33 °C night, LT-low temperature-12 °C day/7 °C night) on variations of polyphenolic structural forms-free, soluble-bound (esterified and glycosylated), and insoluble-bound-in broccoli (Brassica oleracea L. convar. botrytis (L.) Alef. var. cymosa Duch.) microgreens were investigated. Using spectrophotometric, RP-HPLC, and statistical analyses, it was found that the highest amount of total phenolics (TP) in broccoli microgreens was present in the esterified form, regardless of the temperature at which they were grown (63.21 ± 3.49 mg GAE/g dw in RT, 65.55 ± 8.33 mg GAE/g dw in HT, 77.44 ± 7.82 mg GAE/g dw in LT). LT significantly increased the amount of free (from 13.30 ± 2.22 mg GAE/g dw in RT to 18.33 ± 3.85 mg GAE/g dw) and esterified soluble TP (from 63.21 ± 3.49 mg GAE/g dw in RT to 77.44 ± 7.82 mg GAE/g dw), while HT significantly increased the amount of TP glycosylated forms (from 14.85 ± 1.45 mg GAE/g dw in RT to 17.84 ± 1.20 mg GAE/g dw). LT also enhanced free and esterified forms of total flavonoids, tannins, hydroxycinnamic acids, and flavonols. HT, on the other hand, increased glycosylated forms of TP, flavonoids, tannins, hydroxycinnamic acids, flavonols, and phenolic acids, and decreased insoluble-bound tannins. According to the ABTS method, HT induced antioxidant potential of free and glycosylated forms, while LT increased antioxidant capacity of free forms only. According to the FRAP method, LT increased antioxidant potential of free and esterified polyphenolic forms. Also, based on ABTS and FRAP assays, esterified polyphenolics showed significantly higher antioxidant capacity than any other form. Principal component analysis showed that structural form had a greater impact than temperature. Hierarchical clustering showed that RT-, HT- and LT-broccoli microgreens were most similar in their glycosylated polyphenolics, but differed the most in esterified forms, which were also the most distinct overall. In conclusion, HT and LT induced specific shifts in the structural forms of broccoli polyphenolics and their antioxidant capacity. Based on the results, we recommend applying LT to increase the amount of free and esterified polyphenolics in broccoli microgreens, while HT may be used to enhance glycosylated forms.