Peptídeo do hidrolisado protéico de RuBisCO e sua aplicação na preservação de carne bovina

The objective of this work was to purify the small (705 Da) and hydrophilic antimicrobial Arg-Asp-Arg-Phe-Leu peptide from RuBisCO protein hydrolysate and to evaluate its effect on the microbiological and oxidative stability of beef mince during refrigeration. RuBisCO was obtained from alfafa green juice. The peptide extract was fractionated using RP-HPLC, and the active fractions were analyzed by liquid chromatography, electrospray ionization, and tandem mass spectrometry (LC-ESI-MS). Beef mince was analyzed in the following treatments: negative control, meat with two different BHT concentrations of 0.1 and 0.5% (w/w), and meat with two different Arg-Asp-Arg-Phe-Leu peptide concentrations of 0.1 and 0.5% (w/w). Lipid oxidation using the thio-barbituric acid-reactive substance (TBARS) values were significantly affected by the storage period and the concentration of bioactive peptide. Arg-Asp-Arg-Phe-Leu, a small antibacterial peptide from RuBisCO, can be isolated and purified by HPLC from alfafa green juice with retention time between 10 and 50 min, which corresponds to antimicrobial peptides. RuBisCO peptide Arg-Asp-Arg-Phe-Leu 0.5% increases oxidative stability of beef mince during refrigeration. RuBisCO peptide Arg-Asp-Arg-Phe-Leu inhibit microbial growth under refrigeration for 11 days.O objetivo deste trabalho foi purificar peptídeo antimicrobiano pequeno (705 Da) e hidrofílico Arg-Asp-Arg-Phe-Leu a partir do hidrolisado proteico RuBisCO e avaliar seu efeito na estabilidade microbiológica e oxidativa de carne bovina moída durante refrigeração. RuBisCO foi obtida a partir do suco verde de alfafa. O extrato peptídico foi fracionado utilizando RP-HPLC, e as frações ativas foram analisadas por cromatografia líquida, ionização por eletrospray e espectrometria de massa em tandem (LC-ESI-MS). A carne bovina moída foi analisada nos seguintes tratamentos: controle negativo, carne com duas concentrações diferentes de BHT de 0,1 e 0,5% (p/p) e duas concentrações de peptideo Arg-Asp-Arg-Phe-Leu de 0,1 e 0,5% (p/p). Os valores de oxidação lipídica utilizando a substância reativa ao ácido tiobarbitúrico (TBARS) foram significativamente afetados pelo período de armazenamento e pela concentração do peptídeo bioativo. Arg-Asp-Arg-Phe-Leu, um pequeno peptídeo de RuBisCO, pode ser isolado e purificado por HPLC de suco verde de alfafa com tempos de retenção de 10 e 50 min, o que corresponde ao peptídeo antimicrobiano. O peptídeo Arg-Asp-Arg-Phe-Leu 0,5% aumenta a estabilidade de oxidação da carne moída durante a refrigeração. O peptídeo Arg-Asp-Arg-Phe-Leu de RuBisCO inibe o crescimento microbiano sob refrigeração durante 11 dias

Peptide from RuBisCO protein hydrolysate and its application in beef meat preservation

Abstract The objective of this work was to purify the small (705 Da) and hydrophilic antimicrobial Arg-Asp-Arg-Phe-Leu peptide from RuBisCO protein hydrolysate and to evaluate its effect on the microbiological and oxidative stability of beef mince during refrigeration. RuBisCO was obtained from alfafa green juice. The peptide extract was fractionated using RP-HPLC, and the active fractions were analyzed by liquid chromatography, electrospray ionization, and tandem mass spectrometry (LC-ESI-MS). Beef mince was analyzed in the following treatments: negative control, meat with two different BHT concentrations of 0.1 and 0.5% (w/w), and meat with two different Arg-Asp-Arg-Phe-Leu peptide concentrations of 0.1 and 0.5% (w/w). Lipid oxidation using the thio-barbituric acid-reactive substance (TBARS) values were significantly affected by the storage period and the concentration of bioactive peptide. Arg-Asp-Arg-Phe-Leu, a small antibacterial peptide from RuBisCO, can be isolated and purified by HPLC from alfafa green juice with retention time between 10 and 50 min, which corresponds to antimicrobial peptides. RuBisCO peptide Arg-Asp-Arg-Phe-Leu 0.5% increases oxidative stability of beef mince during refrigeration. RuBisCO peptide Arg-Asp-Arg-Phe-Leu inhibit microbial growth under refrigeration for 11 days

Purification of RuBisCO from Alfalfa, enzymatic hydrolysis, identification, structure-function of bioactive peptides and their valorisation in food products

La luzerne est la plante la plus cultivée dans le monde et est une excellente source de protéines. Cependant, la RuBisCO a montré le plus d'intérêt. Cette protéine a été étiquetée comme la protéine la plus abondante sur terre, elle constitue environ 65% (p/p) des protéines solubles de la luzerne. Dans ce travail une nouvelle méthode a été mise en place pour la purification de la RuBisCO à partir de la poudre de luzerne 10% (p/v) en utilisant deux solvants différents et l’effet du pH. Premièrement, des méthodes d’analyse qualitative et quantitative ont été utilisées pour confirmer l’efficacité de la méthode de purification qui pourrait remplacer certains procédés industriels classiques. Puis, une hydrolyse pepsique a été réalisée sur la RuBisCO purifiée qui aboutit à une forte population peptidique bioactive. Les peptides finaux de 24h d’hydrolyse ont montré une meilleure activité antibactérienne ou antioxydante par rapport aux autres hydrolysats. 9 nouveaux peptides antibactériens ont été identifiés et caractérisés par SM et qui ont un CMI entre 2-6mM contre quatre espèces de bactéries: B subtilis, E coli, L innocua et M luteus. En plus, des fractions peptidiques antioxydantes ont également été identifiées dans ce travail. Et leur activité antioxydante a été évaluée par différents tests in vitro et in vivo sur l’huile de Colza. Enfin, l’addition du peptide RDRFL issu de l’hydrolyse pepsique de la RuBisCO a montré un effet positif sur la prolongation de la durée de conservation de la viande hachée et de la purée de tomate.Alfalfa is an excellent source of protein. However, RuBisCO proteins showed most interest. Indeed, this protein has been labelled the most abundant on earth; it constitutes about 65% (w/w) of soluble leaf protein of Alfalfa. In this work, a new method was introduced for the purification of RuBisCO from alfalfa powder 10% (w /v), using two different solvents and pH effect. In a first step, the performance of the proposed RuBisCO recovery method was evaluated through qualitative and quantitative analysis and the results obtained showed that this new method could replace some conventional industrial processes. In a second step, enzymatic hydrolysis was carried out on the purified RuBisCO, which resulted in a large bioactive peptide population. The final peptides after 24h of hydrolysis showed better antibacterial or antioxidant activity compared to the other peptide hydrolysates. Nine new antibacterial peptides have been identified and characterized by MS and have a MIC of 2-6 mM against four species of bacteria: B subtilis, E coli, L innocua and M luteus. In addition, antioxidants peptide fractions were identified in this work, their antioxidant activity was evaluated by various in vitro and in vivo tests on oil of Colza. Finally, the addition of peptide RDRFL derived from the peptic hydrolysis of RuBisCO has a positive effect on the prolongation of the shelf life of minced meat and of tomato puree

Physicochemical and biological properties of eel by-products protein hydrolysates: Potential application to meat product preservation

European eel by-products have been hydrolysed in order to produce novel bioactive peptides with antibacterial and antioxidant activity. Protein hydrolysates from Anguilla by-products obtained by treatment with Savinase® (PHAB-s) have high protein content than that produced with Protamex® (PHAB-p). Furthemore, both protein hydrolysates had a high percentage of essential amino acids. In addition, results indicated that PHAB-s and PHAB-p exhibited different degrees of antioxidant activities, evaluated by several antioxidant assays. Thus, PHAB-s displayed high antioxidant activity, in terms of DPPH-radical scavenging effect (IC50 = 5.2 mg/ml), β-carotene bleaching inhibitory effect (CI50 = 4.4 mg/ml) and metal chelating activity. At 10 mg/ml, PHAB-p showed the highest reducing power. Antibacterial activities of PHAB-s and PHAB-p were also evaluated. PHAB-s showed the most important inhibitory effects against seven bacterial species. The application of PHAB-s and PHAB-p on minced meat as preservative reduced the lipid oxidation and inhibited the microbial growth during chilled storage for 11 days.This work was funded by the Ministry of Higher Education and Scientific Research, Tunisia and the Spanish Ministry of Economy, Industry and Competitiveness under project AGL2014-52825-R.Peer reviewe