Milk-clotting properties on bovine caseins of a novel cysteine peptidase from germinated Moringa oleifera seeds

Xuefeng Wang,Qiong Zhao,Li He,Yanan Shi,Jiangping Fan,Yue Chen,Aixiang Huang

doi:10.3168/jds.2021-21415

Abstract

A cysteine peptidase was previously identified from germinated Moringa oleifera seeds, but its milk-clotting properties on bovine caseins was still unclear. In this study, this novel cysteine peptidase (MoCP) showed preferential activity on κ-casein (κ-CN), with greater hydrolytic activity compared with calf rennet, whereas weak hydrolysis of α-casein and β-casein made MoCP suitable for application in cheesemaking and may yield various functional peptides. All 3 evaluated caseins were hydrolyzed to form relatively stable peptide bands within 3 h of proteolysis with MoCP. Cleavage sites were determined by gel electrophoresis, liquid chromatography mass spectrometry/mass spectrometry, and peptide sequencing, which revealed that cleavage of κ-CN by MoCP occurred at residue Ile129-Pro130 and generated a 14,895.37-Da peptide. The flocculation reaction between MoCP and κ-CN determined by 3-dimensional microscopy with super-depth of field revealed that the initial 30 min of reaction were key for milk coagulation, which may affect curd yield. Overall, the findings presented herein suggest that the cysteine peptidase from germinated M. oleifera seeds can be considered a promising plant-derived rennet alternative for use in cheese manufacture.

Highlights

Coagulation of milk is one of the key steps in cheese manufacture, and milk-clotting enzymes play an important role in this process
Germination of plant seeds can improve the activity of plant proteases for the mobilization of proteins stored in the seed, which serve as the main source of nutrients, and certain plant proteases with hydrolytic activity can potentially be used as industrial biomaterials (Simpson, 2001; Dong et al, 2015; Szewińska et al, 2016)
This indicated that MoCP might hydrolyze κ-CN first and lead to an effective accumulation of hydrolysates, an evidence that is in line with the cleavage pattern of an an extracellular metalloprotease (ACPS) isolated from Termitomyces clypeatus MTCC 5091 and a cysteine protease from the latex of Ficus johannis (Majumder et al, 2015; Afsharnezhad et al, 2019)

Summary

Introduction

Coagulation of milk is one of the key steps in cheese manufacture, and milk-clotting enzymes play an important role in this process. Rennet is the most widely used complex of milk-clotting enzymes in cheesemaking, which is obtained from the abomasum of unweaned calves (Alihanoğlu et al, 2018) and whose main com-. Moringa oleifera is well known for its high protein, calcium, and vitamin content, as well as for its nutritional and health properties (Saini et al, 2016; Falowo et al, 2018). Previous reports show that proteolytic activity and milk-clotting specific activity of protease extracted from M. oleifera increase during seed germination (Wang et al, 2020). In a recent study conducted by our group, a novel cysteine peptidase (MoCP) obtained from germinated M. oleifera seeds was characterized and exhibited good milk-clotting activity, high stability at acidic pH, and

Methods

Results

Conclusion