Synergism Of Licorice Extract And Pea Protein Hydrolysate In The Inhibition Of Lipid Oxidation In Food Emulsions

Enzymatic hydrolysis of protein can not only improve the digestibility, but also thebioactivities, especially the antioxidant activity. However, due to size reduction and increasedcharges, peptides are distributed preferentially in the aqueous phase, therefore more effectivein the aqueous phase than in the lipid phase where hydroperoxides and peroxide radicals aregenerated. Hence, other antioxidants were required to work with protein hydrolysates toobtain ideal inhibitory effect on lipid oxidation in oil-in-water (O/W) emulsions. In this study,radical-scavenging pea protein hydrolysate (PPH) with low-allergenicity was used toinvestigate its cooperative inhibitory effect with lipophilic licorice extract (LE) on lipidoxidation in O/W emulsions. The mechanism of synergistic antioxidative effect waselucidated. Saccharides with various lengths (glucose, maltodextrin, and dextran) wereintroduced into pea peptides via Maillard reaction to try to improve the emulsifying propertiesof PPHs in emulsions. The effect of LE on glycosylation of PPH as well as the cooperativeeffect of glycated PPH and LE on lipid oxidation in O/W emulsions were also studied.Pea protein isolates (PPIs) with or without heating (90C,5min) were hydrolyzed usingseven proteases (trypsin, chymotrypsin, papain, pepsin, Alcalase, Flavourzyme and Protamex)to produce pea protein hydrolysates (PPHs). Antioxidant activity and emulsifying property ofPPHs were assessed. Results showed all PPHs exhibited stronger radical scavenging activitythan intact proteins, but weaker emulsifying property. Those prepared with Flavourzymeand Protamex were most effective in inhibiting lipid oxidation in a liposomal system, and alsoshowed relatively good emulsifying property. Overall, PPHs from heated PPIs exhibited betterantioxidant activity and emulsifying property than those from native PPIs.The cooperative effect of PPHs in combination with LE for the inhibition of lipidoxidation in a liposomal model system was assessed. Degree of oxidation was expressed asformation of TBA reactive substances (TBARS) per liter of liposome suspension. Resultsshowed that most PPHs, especially those prepared from heated protein with Flavourzyme andProtamex (Fla-PPH and Pro-PPH) exhibited remarkable synergistic inhibitory effects with LE.The synergism was attributed to the abundant charged amino acid side chain groups and thedominating presence of hydrophobic N-terminus of constituting peptides in the proteinhydrolysates and the preponderance of phenolic compounds in licorice extract. Afterseparation and purification of peptide fractions that exhibited excellent TBARS inhibition,10peptides were identified, of which, LQEEDNVISQ, ITPERTLQLQDLDIFVN andLANRDDNEDLVGVL matched the sequence of pea vicilin. While self-assembled networksformed around lipid droplets appeared to be a prevalent physical mechanism by whichpeptides diminished the potency of oxidants, the peptides’ association with phenolic and otheractive components from licorice extract afforded a more compact and rigid protective network,providing liposomes with a stronger defense against oxidative stress. Both chemical andphysical mechanisms exerted by PPH and LE were responsible for the antioxidativeprotection.The cooperative inhibitory effect of PPH with LE on lipid oxidation in an O/W emulsionwas further determined. Fla-PPH and Pro-PPH, which exhibited the strongest inhibition on liposome oxidation, and LE were subjected to soybean O/W emulsions (oil phase10%, pH7)co-stabilized by Tween20. The suppression of lipid oxidation (POV and TBARS) wasassessed during storage at37°C. Results showed that both PPHs significantly retardedoxidation of the emulsions. Electron microscopy revealed an interfacial peptidyl membranearound oil droplets which afforded steric restrictions to oxidation initiators. When LE wasalso used in emulsion preparation, a remarkable synergistic oxidation inhibition was observedwith both PPHs. The combination of PPH and LE, on one hand, allowed the efficient removalof radicals in both aqueous and lipid phases; on the other hand, reduced the surface charge andparticle size of oil droplets, thereby facilitating the formation of thick and rigid interfacialmembrane which offered superior defense against oxidative stress. This result furtherconfirmed that both chemical and physical mechanisms exerted by PPH and LE wereresponsible for the antioxidative protection. Liquiritin apioside, neolicuroside, glabrene and18β-glycyrrhetic acid were the predominant compounds in LE partitioning at the oil–waterinterface and were likely responsible for the strong antioxidant activity of LE in O/Wemulsions and the notable synergism when coupled with PPHs.Although PPH exerted synergistic antioxidative effects with LE in O/W emulsions, thepoor emulsifying property hindered PPH from making stable emulsions and high-performancesynergistic antioxidative effects with LE in these emulsions. Hence, Maillard-typeglycosylation was employed to improve the emulsifying propery of PPH. Results showed thatinitial stage products Schiff base was generated when incubated PPHs and saccharides(glucose, maltodextrin, and dextran) in aqueous solutions at pH7.0,60C for48h. Despitethe low degree of glycation relative to glucose and maltodextrin, dextran exhibited the highestefficacy on improving emulsifying property and maintaining the antioxidant potential of PPHs.Pro-PPH–dextran conjugate showed the highest improvement in emulsifying capacity.Therefore, controlled Maillard reaction with dextran, can be used as an effective tool toimprove the emulsifying property of antioxidative pea protein hydrolysates.Next, apply Pro-PPH–dextran conjugates into soybean O/W emulsions to furthercharacterize their emulsifying property. Results showed that Pro-PPH–dextran conjugateswere effective emulsifiers and offered superior defense against oxidative stress. This can beattributed to their highly interactive and crosslinked network structure, which facilitated theformation of thick and rigid interfacial membrane and the improvement of viscosity. As aresult, the collision or aggregation of oil droplets was restricted, and the diffusion and transferof oxidative substrates were also hindered. Meanwhile, the stabilization of structure andfunction of PPHs in these emulsions towards environmental stresses were also strengthened.PF1and PF2, the first two largest gel filtration fractions of Pro-PPH, showed the highestMaillard reaction reactivity. N-termini of peptide chains were more accessible to saccharides.MS/MS analyses detected the glycated peptides IISPPEK and LSIISPPEK from pea protein11S, and KLP, NVIVK and LSPGDVF from pea protein7S.Ultimately, study the effect of LE on glycosylation of PPH as well as the cooperativeinhibitory effect of Pro-PPH–dextran conjugates with LE on lipid oxidation in O/W emulsions.Results showed that LE promoted the covalent conjugation between Pro-PPH and dextran. It can act as a bridge to enhance the reactivity between Pro-PPH and dextran, thus furtherimproving the emulsifying property of Pro-PPH–dextran conjugates. On the other hand,synergistic antioxidative effects were still observed on Pro-PPH–dextran conjugates and LE inO/W emulsions. Hence, the addition of LE either before of after glycosylation of PPH, canbring positive improvements to the properties of emulsion-type food products containing PPH.Overall, pea protein hydrolysates when used in combination with licorice extract, canproduce a synergistic inhibition on lipid oxidation in O/W emulsions. Glycosylation of PPHshowed little affect on its synergism with LE, but enhaced the reliability of synergism inemulsions. Hence, enzymatic pea protein hydrolysates and licorice extract can be appliedtogether to develop superior antioxidant ingredient and to maximize the stability of emulsion-type food products, both physically and chemically.