Milk coagulation is the primary step for producing cheese and used in cheese making for thousands of years. They seem to the oldest known application of enzymes. Almost all the enzymes used as milk coagulants belong to aspartic proteases, but enzymes from other groups such as cysteine and serine protease also use [1]. The chemical rennin enzyme extracted from the calf stomach of young ruminants uses to coagulate milk and form curds. It is highly essential in the cheese-making process. But its supply had become less available because instead of slaughtering calves to get rennet, they are preferably used to provide meat for human consumption [3]. The shortage of Calf's rennet also highly increased because of religious restrictions and ethnic regulations. These paved the way for the replacement of rennet from more convenient sources [4].
The most important alternative enzyme substitutes fulfilling the requirements of cheese manufacturers include microbial, recombinant, and plant-based enzymes. A plant-derived substitute has a particular value because of its essentially unrestricted availability [5]. Certain plants reported to yield promising rennet activity, such as Withania coagulants; Ficus carica; Pumpkin; seeds of Moringa oleifera; seeds of Ricinis communis; dried Papaya latex, Pineapple, cucumber; Benincasa cerifere, Galium verum; Pinquicule Vulgaris; Drosera rotindifolia; and Ranunculus ligua. However, in Ethiopia, in tradition, they use the plant, but little work reports before [6].
Solanum incanum is one of the most energetic traditional medicinal plants belongs to the Solanaceae family. The fruit contains many small and soft edible seeds but is bitter because they have an insignificant amount of nicotinic alkaloids and cyanogenic glycosides [2]. Leaf and fruits use for washing painful areas. Ash of burnt plants is mixed with fat and applied to treat a sore throat, angina, stomach pain, colic, headache, painful menstruation, and liver pain. Even if the wide variation in toxicity makes it dangerous to transfer specific uses from one region to another, it extensively uses for antibacterial and antitumor activities [7]. Its anti-microbial activities also use for milk clotting activity of cheese production in rural communities and certain dairy farmlands [8].
Calotropis procera is soft-wooded, evergreen, perennial shrubs with broad fleshy leaves that grow wild in the tropics and warm temperate regions of the family Apocynaceae and Asclepiadoideae subfamily (milkweeds). It uses to treat pain, inflammation, digestion, antidysenteric, antirheumatic, diaphoretic, an expectorant, bronchial asthma, and skin conditions [9]. They used roots and Dry latex as an antidote for snake poisoning. They also used it as an abortifacient to treat piles and intestinal worms [10].
Plant extracts used as milk coagulants in cheese making for long times. Cheeses made with vegetable coagulant found mainly in Mediterranean, West African, and southern European countries [1]. However, the excessive proteolytic nature of most vegetable coagulants has limited their use in cheese manufacturing due to lower cheese yield and defects in flavor and texture [11]. Therefore, searching for new potential milk-clotting enzymes from plants is undergoing to make them useful for industry, to go with the increasing global demand for diversified and high-quality cheese production [12].
Several studies performed using plant-derived enzymes for cheese making. Jacob [13] reviewed the importance of milk-clotting enzymes, including animal rennet, microbial coagulants, recombinant coagulants, and plant-derived clotting enzymes. Yegin and Dekker [14] reported the progress of animal, plant, and microbial origin aspartic proteinases with a specific emphasis on structures, functions, catalytic mechanism, inhibition, and engineering.
In Ethiopia, the commercial calf rennet enzyme used to coagulate milk in cheese making is too costly, and the need for calf sacrifice makes it complex to use it at commercial and small-scale dairy production levels in Ethiopia. Very little research performs to use plant-derived protease for cheese making.
These studies have a great significance by solving the problems facing the society in day-to-day life to cheese production, creating awareness for the scientific community in the cheese-making process using natural products, and giving a basic knowledge about the application of calotropis latex, solanum incanum, and lemon juice as it will satisfactorily replace the calf rennet. Therefore, the aim of this study focused on the extraction, purification, and characterization of a milk-clotting enzyme, which was assumed to replace calf rennet produced from animals.