Purification, caractérisation et essais d’inhibition de la polyphénol-oxydase de l’abricot par des combinaisons

Abstract

Enzymatic browning generally is an undesirable phenomenon in the food industry. It is considered to be one of the biggest problems during apricot handling, storage, preservation and transformation. Browning reactions in fruits and vegetables are mainly initiated by polyphenol oxidase (PPO). Therefore, the objective of this thesis is extract, purify, characterize and then inhibit apricot PPO (PaPPO). Thus, herein describes a new method to inhibit as well as inactivate PaPPO by combinations of plant proteases (papain, calotropain, ficin and bromelain) and ascorbic acid. Our results show that PaPPO is present in the fruit in its latent form (L-PaPPO). The purified PaPPO was characterized with a molecular weight of 63 kDa on SDS-PAGE, a pH and a temperature optimum at pH and 45 °C for catechol as substrate. The activity was enhanced by low concentrations (≤ 2 mM) of SDS. It showed diphenolase activity and highest affinity toward 4-methylcatechol (Km = 2.0 mM) and chlorogenic acid (Km = 2.7 mM). L-PaPPO was found to be spontaneously activated during storage at 4 °C, creating a new band at 38 kDa representing the activated form (A-PaPPO). Surprisingly the active form showed a weak monophenolase activity. The mass of A-PaPPO was determined by mass spectrometry as 37 455.6 Da (Asp102 → Leu429). Both L-PaPPO and A-PaPPO were identified as PPO corresponding to the known PaPPO sequence (UniProt O81103) by means of peptide mass fingerprinting. The inactivation of PaPPO with proteases showed that the selected proteases were able to inactivate PaPPO at pH 4.5, with the degree of inactivation proportional to incubation time and protease concentration. Papain was the most effective protease, with 50 μg completely inactivating PaPPO in less than one hour. AA prevented browning reactions that occur before or during PaPPO inactivation by protease. The combinations of ascorbic acid/proteases (AA/P) were highly effective in vitro, where 2 mM AA/500 μg P inhibited PaPPO activity completely over 24 h. The combination of AA/P was also effective in vivo, as treated apricot purees preserved their color (p < 0.0001, compared to untreated samples after 10 days of storage). The results demonstrate that AA/P combinations constitute a promising practical anti-browning method with feasible application in the food industry that can help control enzymatic browning in fruits and vegetables.