| Cheese has played an important role in the human diet since ancient times. In prehistoric era, cheese was generally regarded as the concentrated form of milk with an advantage of extended storage life. The high content of fat and protein in cheese made it an energy-rich and wholesome food product. These days, production and consumption of cheese is widely expanded around the globe. Milk coagulating enzymes are the main factors in cheese development. Rennet from the abomasal tissues of the suckling calf was the first, and still is the most extensively utilized, milk-coagulating enzyme preparation.A global increase in cheese production and consumption coupled with limited availability of calf rennet, religious reasons (e.g., Islam and Judaism), diet (vegetarianism) and restriction on recombinant version of calf rennet (in France, Germany and the Netherlands), has contributed to the seek for suitable alternative sources of milk clotting enzymes. Microbial proteinases obtained from fungi and other microorganisms have been attempted as substitutes for calf rennet, but most of them have proved unsuitable for cheese manufacturing.Natural milk coagulants from plant sources are of growing worldwide attention. The application of plant rennets is helpful in improving the nutritional intake of people who can not use animal rennet-based products. Despite of the fact that numerous plant-derived proteases are able to clot milk, most of the plant coagulants are not very successful for cheese production owing to unwarranted proteolytic nature, which significantly influence the sensory attributes as well as yield of the cheese. Subsequently, the search for novel and potential plant-originated milk coagulants still continues to cope with ever-increasing worldwide demand for good quality cheese production. This study reports about:(1) Optimization of extraction conditions for milk coagulating activity of ginger protease, (2) Purification and characterization of new milk coagulating cysteine protease from ginger rhizomes, (3) Impact of ginger protease, papain and calf rennet on the coagulation of bovine milk, (4) Proteolysis in cow milk cheese manufactured with calf rennet and ginger protease, and (5) Evaluation of physicochemical, microbiological and sensorial characteristics of Peshawari cheese manufactured with ginger protease and calf rennet.(1) The impact of three processing parameters namely homogenization time (1-3 min, X1) pH of buffer (6-8, X2), amount of buffer (50-100 mL, X3), involved in the liquid extraction of proteinases from ginger rhizomes, was investigated on milk coagulating activity by response surface methodology. An empirical quadratic model was applied to experimental data pertaining to the average enzymatic activity and equations describing the optimal conditions were obtained. The maximum milk clotting activity (274 U/mL) was obtained by homogenizing the ginger rhizomes in 76 mL of extraction buffer with a pH of 7.0 for 2.4 min. No significant (P> 0.05) difference was observed between the experimental and predicted values. These results are useful in the optimization of extraction procedures, which are of significant relevance to the production of standardized plant coagulants suitable for the cheese industry.(2) A milk coagulating protease was purified-10.2 fold to apparent homogeneity from ginger rhizomes in 34.9% recovery using ammonium sulfate fractionation, ion exchange and size exclusion chromatographic techniques. The molecular mass of the purified protease was computed to be～36 kDa by SDS-PAGE, and exhibited a pI of 4.3. It is a glycoprotein with 3% carbohydrate content. The purified enzyme showed maximum activity at pH 5.5 and at a temperature of～60℃. Its protease activity was strongly inhibited by iodoacetamide, E-64, PCMB, Hg2+ and Cu2+. Inhibition studies and N-terminal sequence classified the enzyme as a member of the cysteine proteases. The cleavage capability of the isolated enzyme was higher forαs-casein followed byβ-andκ-casein. The purified enzyme differed in molecular mass, pI, carbohydrate content, and N-terminal sequence from previously reported ginger proteases. These results indicate that the purified protease may have potential application as a rennet substitute in the dairy industry. (3) The coagulation of bovine milk was investigated by using different coagulants namely calf rennet, ginger protease and papain. Coagulation properties of the three coagulants were compared and evaluated by means of turbidimetric method. Ginger protease and calf rennet caused sigmoidal increment in turbidity to the milk with three discrete phases. Comparatively shorter gelation time and longer time for restructuration (end of coagulation) was observed in case of ginger protease and papain as coagulants. The curd produced by the action of ginger protease exhibited the highest yield. Most of the sensory attributes, with exception to taste and bitterness, did not differ significantly among different coagulants.(4) The impact of type of coagulant, calf rennet (CR) or ginger protease (GP), on the proteolysis of bovine milk cheese was evaluated throughout the ripening period. The proteolysis was more pronounced in cheese manufactured using GP and values of 29.19%, 9.86%,0.43% and 0.59% for water-soluble nitrogen (WSN) at pH 4.6, non-protein nitrogen (NPN), ammonia nitrogen (NH3N) and amino acid nitrogen (AAN), were obtained after 60 days of ripening as compared to values of 18.23%,8.64%,0.46% and 0.73% for WSN, NPN, NH3N and AAN in cheese made with CR. The extent of degradation of a-casein,β-casein and y-casein was greater in cheese produced with GP than those produced with CR. However, pre-a-casein was more intensively broken down in cheese made using CR throughout the ripening period. The formation of hydrophobic peptides, hydrophilic peptides and the ratio of hydrophobic/hydrophilic peptides throughout the ripening were higher in cheeses manufactured with GP in relation to the cheeses produced with CR. These outcomes were further verified by Principal component analysis (PCA).(5) The influence of ginger protease and calf rennet on the physicochemical, microbiological and sensory characteristics of Peshawari cheese manufactured from cow milk was examined. For most of the physicochemical parameters (fat, protein, lactose, acidity, pH), and the main groups of microorganisms (total viable, enterobacteria, Lactobacilli, molds and yeasts) studied, no significant (P> 0.05) differences were observed between the two cheeses made by using different coagulants. However, significantly lower (P<0.05) levels of moisture and higher levels of soluble nitrogen (SN) were observed in the cheese produced by ginger protease compared to that made by calf rennet. The main sensory attributes (appearance, body-texture, and flavor) were significantly enhanced (P< 0.05) in Peshawari cheese prepared with ginger protease. Importantly, no bitterness was noted by the sensory panel in the Peshawari cheese made with ginger protease. The results reveal that the ginger protease may have potential application for the manufacture of Peshawari cheese. |
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