Extraction and immobilization of vegetable aspartic proteases for cheese making

Abstract

The worldwide increase in cheese production and consumption, together with the reduced supply and consequent price increase of calf has led to the search of new milk coagulant agents. Much research interest has been thus directed towards discovering milk clotting enzymes which would satisfactory replace calf rennet in cheese manufacture. Microbial rennet produced by genetically engineered bacteria has proven to be a suitable substitute even though the consumer constraints on the use of animal rennet either for religion reasons (Judaism and Islam) or diet habits (vegetarianism) or consumer concerns regarding genetically engineered foods (Germany, Netherlands and France forbid the use of recombinant calf rennet) have led to a growing interest toward coagulants. Therefore, several sources of milk-clotting enzymes have been investigated. Unfortunately, most of the plant rennets were found unsuitable because they produced extremely bitter dairy products due to a strong proteolysis. However, an exception to this general rule is represented by the extract of flowers of the Cynara genus. In particular, flowers of Cynara scolymus and Cynara cardunculus contain two different aspartic proteases named cynarase and cardosin, respectively. The latter enzyme was successfully used for cheese manufacture in several areas of Portugal and Spain. Regarding the cynarase, its use is limited since the flowers of globe artichoke (Cynara scolymus) represent an edible part before the blooming. In fact, in Mediterranean countries, the globe artichoke plays an important economic role, so that Italy is the first producer with ~475 kt. However, the artichoke processing industry generates large amounts of agricultural solid wastes of which ~60% is represented by the leaves. Therefore, I found of great interest to investigate the presence of aspartic proteases, possessing milk clotting properties, in the Cynara scolymus leaves as well as in the flowers of Carduus defloratus, as unproductive and/or intensely exploited agricultural lands. The obtained results showed the presence of clotting enzyme both in the leaves of Cynara scolymus and in the flowers of Carduus defloratus. In the first case, the extraction of proteins from tissue resulted difficult due to the presence of interfering compounds such as cell wall cellulose, lipids and other polysaccharides. To optimize protein extraction an enzymatic pretreatment with a mixture of carbohydrases was successfully carried out. However, the yield was always very low and, therefore, this protocol would be hardly suitable for an industrial application. For these reasons, in the second part of the work, I have studied the possibility to immobilize the enzyme extracted from the flowers of Carduus defloratus by using a commercial rennet from Mucor meihei as model enzyme. The immobilization of the enzyme extracted from both sources was successfully carried out by using an epoxy support and, in particular, the results showed that the Carduus defloratus enzyme exhibited higher stability than the commercial rennet one. All these results suggest the possibility to use an immobilized vegetable rennet, extracted from the flowers of Carduus defloratus, for the production of novel dairy products.