High hydrostatic pressure (HHP) has drawn considerable attention because of its potential application in food industry. Ferritin, an iron storage protein, is widely distributed in food made from legume seeds, which is highly stable due to its shell-like structure. Therefore, it is of special interest to know whether or not high HHP treatment has effect on this protein. In this study, the structure and activity of soybean seed ferritin (SSF) were examined by circular dichroism spectrum (CD), UV–VIS and fluorescence spectrophotometry in conjunction with stopped-flow light scattering upon treatment with HHP at 400 MPa for 10 min. Results revealed that such treatment has little effect on the primary and secondary structure of SSF, but pronouncedly altered its tertiary and quaternary structure. As a result, the protein aggregation property and iron release activity were dramatically changed, while its activity of iron oxidative deposition was kept unchanged.Highlights► HHP treatment had little effect on the primary and secondary structure of SSF. ► HHP greatly changed its tertiary and quaternary structure around Trp residues. ► Protein aggregation properties were markedly altered after HHP treated. ► The rate of iron release from holo-SSF became much slower. ► The catalytic activity of SSF remained unchanged.

It was established that green pigment(s) responsible for garlic greening is composed of yellow and blue species, and pyruvic acid (a product from 1-PeCSO or 2-PeCSO under the action of alliinase) reacted with pigment precursor (PP) model compounds, 2-(1H-pyrrolyl) carboxylic acids to produce yellow pigments. However, the structure of the yellow pigments is unknown. In present paper, we identified three yellow pigments (Y1, Y2 and Y3) from three reaction systems containing pyruvic acid and 2-(1H-pyrrolyl) acetic acid (P-Gly) or 1-(2′-methyl-1′-carboxy-propyl) pyrrole (P-Val) or 1-(2′-methyl-1′-carboxy-butyl) pyrrole (P-Ile), respectively, by LC-ESI MS/MS and IT-TOF mass spectrometry. The three pigments have a UV/visible maximum absorbance between 400 and 434 nm and might be formed by dimerisation of the three corresponding PP under participation of pyruvic acid, molecular formula of which are C16H16N2O4 (Y1), C22H28N2O4 (Y2) and C24H32N2O4 (Y3), respectively.

Six model compounds having a 2-(1H-pyrrolyl)carboxylic acid moiety and a hydrophobic R group were synthesized to study their effects on garlic greening, the structures of which are similar to that of 2-(3,4-dimethyl-1H-pyrrolyl)-3-methylbutanoic acid (PP-Val) (a possible pigment precursor for garlic greening). The puree of freshly harvested garlic bulbs turned green after being soaked in solutions of all these compounds, and with both increasing concentrations and incubation time the green color of the puree became deeper. In contrast, neither pyrrole alone nor pyrrole combined with free amino acids had the ability to discolor the puree. The compounds exhibited a good relationship between structure and activity of garlic greening, namely, the smaller the size of the R group, the larger the contribution. Also, it was found that the unidentified yellow species can be produced by reacting the model compounds with pyruvic acid at room temperature (23–25 °C). Moreover, blue species were formed by incubation of the model compounds with di(2-propenyl) thiosulfinate at room temperature. On the basis of these observations, a pathway for garlic greening was proposed.

It was established that storage at low temperature (less than 10 °C) was required for garlic greening occurring either during processing or in the course of “Laba” garlic preparation while storage at high temperature (higher than 20 °C) inhibited its occurrence. However, the reason for this observation is unclear. To obtain insights into a tie connected between storage temperature and garlic greening, it was detected if the γ-glutamyl transpeptidase (GGT) activity correlated with garlic greening because the activity of this enzyme is very sensitive to storage temperature. Results showed that garlic puree (which was prepared from fresh garlic) turned green upon addition of GGT but the color of garlic puree remained unchanged when either water or heat-treated GGT (which has no activity due to heat treatment) was used, a result giving a positive answer to the above proposal. Subsequently, to further clarify the relationship between the GGT activity and garlic greening, the GGT activity, the degree of garlic greening, and the concentration of total thiosulfinates in garlic bulbs were determined respectively after the garlic bulbs had been stored at 4 °C for up to 59 days followed by storage at 35 °C for up to 22 days. It was found that cold storage facilitated the GGT activity whereas warm storage inhibited the activity of this enzyme, just like the effect of storage temperature on greening, indicating that the increase of GGT activity could be a direct factor resulting in garlic greening. Consistent with this conclusion, the concentration of total thiosulfinates (the color developers) in garlic purees likewise exhibited a reversible change by moving garlic bulbs from one low storage temperature to a higher one; namely, it increased with increasing storage time during storage at 4 °C while decreasing as storage time increased during storage at 35 °C. The present study provided direct evidence that the GGT is involved in garlic greening.