Artichoke leaf extracts: Proteolytic activity, coagulant and HPLC analysis

Abstract

The search for origin plant-based proteases increases gradually due to their diversity and stability over a wide range of pH and temperature. Artichoke (Cynara scolymus) flowers are a proteolytic vegetable source already studied, but their leaves are scarce in this respect. Thus, the objective of this research was to obtain extracts of artichoke leaves with different buffers and extraction methods as an alternative proteolytic source and plant coagulant, as well as the separation and comparison of the protein profile of these extracts. The methodology used was based on extraction with sodium citrate buffer (pH 3), sodium acetate (pH 5) and Tris-HCl (pH 7) by mechanical stirrer (MS) and ultrasound (US); protein determination; proteolytic activity (PA) and specific activity (SA); milk clotting activity (MCA) and rennet substitution potential (RSP); high- performance liquid chromatography analysis (HPLC) with UV-Vis detector and principal component analysis (PCA). Extracts of Cynara scolymus leaves showed high results with Citrate-US for the parameters PA (14.38), SA (19.71), MCA (440) and RSP (30.60) compared to other treatments. The extracts with citrate and acetate presented a quick coagulation time (max 3 min). The HPLC analysis enabled the separation of the different protein compounds present in the extracts and most expressive peaks in the samples with Citrate-MS and Acetate-MS; and isolated peaks for Citrate-US. It was concluded that extracts of artichoke leaves with citrate and acetate buffer attributed satisfactory results to act as plant coagulant, as well as to carry out further studies for the purification of proteolytic enzymes and application in cheeses.

Future Perspective and Technological Innovation in Cheese Making Using Artichoke (Cynara scolymus) as Vegetable Rennet: A Review

“…As previously mentioned, the coagulation of milk by enzymatic means is usually related to protease enzymes. Regarding vegetable extracts, enzymes derived from aspartate, serine, and cysteine proteases are capable of performing coagulating functions in milk, according to reports ( Table 2 ) [ 1 , 2 , 30 ].…”

“…As previously mentioned, various artichoke organs presents enzymatic activity in milk, but research has focused above all on extracts from mature artichoke flower due to its greater coagulant activity and probably due to its possible comparison with the extract of the cardoon flower, which has been more investigated and utilised [ 2 , 5 , 7 , 45 ]. This research reveals that artichoke and cardoon flower extracts have a coagulating capacity similar and comparable to the more commonly used animal or microbial rennets.…”

“…As was exposed by several authors [ 2 , 7 , 38 , 70 , 75 ], much of the artichoke enzymatic activity is given by protease enzymes, positioning the artichoke as a possible source of these enzymes. Moreover, as Llorente explained [ 38 ], the artichoke presents this type of activity in several of its organs, unlike similar plants such as the cardoon, where it is only possible to find this enzymatic activity of proteases in the flower [ 58 , 59 ], positioning artichoke above other plants as a good source of this type of enzyme.…”

“…This causes the milk to destabilise and separate into two parts: one consisting mainly of water and the other consisting mainly of fat and proteins present in the milk, constituting a new three-dimensional mass. The latter forms the basis of cheeses and other dairy products [ 1 , 2 , 3 ].…”

“…As Silva¨ [ 2 ] and her teamdescribed, the new generation of research on enzymes capable of coagulating milk is largely focused on the formation of plant-derived rennets due to their high availability and their generally simpler extraction and purification processes compared to other rennets. Her work highlighted the use of various types of plants, such as Asteraceae .…”