Influence of high-pressure processing on the profile of polyglutamyl 5-methyltetrahydrofolate in selected vegetables

Abstract

In plants, folate occurs predominantly as 5-methyltetrahydrofolate (5MTHF) polyglutamyl forms. Differences in stability and bioavailability of food folate compared to synthetic folic acid have been attributed to the presence of the polyglutamyl chain. High-pressure processing (HPP) was tested for whether it might shorten polyglutamyl chains of 5MTHF species in fresh vegetables by enabling action of native γ-glutamylhydrolase (GGH). A validated ultrahigh-performance reversed-phase liquid chromatography-tandem mass spectrometry method using stable isotope as internal standard was applied for characterizing 5MTHF polyglutamyl profiles. HPP conditions included 300, 450, and 600 MPa at 30 °C for 0 or 5 min, and vegetables were vacuum-packed before treatment. Investigated vegetables included cauliflower (Brassica oleracea), baby carrots (Daucus carota), and carrot greens (D. carota). HPP treatment caused conversion of polyglutamyl 5MTHF species to short-chain and monoglutamyl forms. Maximal conversion of polyglutamyl folate to monoglutamyl folate occurred at the highest pressure/time combination investigated, 600 MPa/30 °C/5 min. Under this condition, cauliflower monoglutamyl folate increased nearly 4-fold, diglutamyl folate 32-fold, and triglutamyl folate 8-fold; carrot monoglutamyl increased 23-fold and diglutamyl 32-fold; and carrot greens monoglutamyl increased 2.5-fold and the diglutamyl form 19-fold. Although some folate degradation was observed at certain intermediate HPP conditions, total 5MTHF folate was largely preserved at 600 MPa/5 min. Thus, HPP of raw vegetables is a feasible strategy for enhancing vegetable monoglutamate 5MTHF.

Figure 1

Chemical structures of folate species and polyglutamyl folate. Folate consists of a pteridine moiety, p-aminobenzoate, and a glutamyl tail of variable length. Folates can differ in the oxidation state of the pteridine and C1 substituent. Oxidation of reduced pteridine can give dihydrofolate (double bond C7–N8) and folic acid (double bonds C7–N8 and C6–N5).

Figure 2

Distribution of folate species in raw vegetables: (A) carrot (Daucus carota); (B) cauliflower (Brassica oleracea); (C) carrot greens (D. carota). Folate species were determined by external calibration with authentic standards using HPLC-MS/MS. The mobile phase included formic acid, and thus 5,10-CH⁺THF represents (5,10-CH⁺THF + 10-formyltetrahydrofolate (10-CHOTHF)) and THF represents (THF + 5,10-methylenetetrahydrofolate (5,10-CH₂THF)). Data represent the mean of three replicates.

Figure 3

Effect of steaming time on the polyglutamyl 5-methyltetrahydrofolate (5MTHF-Glu_n) species of carrot (D. carota): (A1) no steaming; (A2) steaming for 10 min; (A3) steaming for 20 min. The folate levels were determined by HPLC-MS/MS based on the methods reported by Wang et al. Data represent the mean of three replicates.

Figure 4

Effect of pressure and holding time on the distribution of polyglutamyl 5MTHF of carrot: (A) raw carrot; (B1) 300 MPa/0 min; (B2) 300 MPa/5 min; (C1) 450 MPa/0 min; (C2) 450 MPa/5 min; (D1) 600 MPa/0 min; (D2) 600 MPa/5 min. The scale for panels B1–D2 is matched to that of panel A, that is, 0–100%. Data represent the mean of six replicates.

Figure 5

Effect of pressure and holding time on the distribution of polyglutamyl 5MTHF of cauliflower: (A) raw cauliflower; (B1) 300 MPa/0 min; (B2) 300 MPa/5 min; (C1) 450 MPa/0 min; (C2) 450 MPa/5 min; (D1) 600 MPa/0 min; (D2) 600 MPa/5 min. The scale for panels B1–D2 is matched to that of panel A, that is, 0–100%. Data represent the mean of six replicates.

Figure 6

Effect of pressure and holding time on the distribution of polyglutamyl 5MTHF of carrot greens: (A) raw carrot greens; (B1) 300 MPa/0 min; (B2) 300 MPa/5 min; (C1) 450 MPa/0 min; (C2) 450 MPa/5 min; (D1) 600 MPa/0 min; (D2) 600 MPa/5 min. The scale for panels B1–D2 is matched to that of panel A, that is, 0–100%. Data represent the mean of six replicates.