Abstract:
The majority of cheeses produced around the world are manufactured using an enzyme coagulant, rennet, traditionally extracted from the abomasum or fourth stomach of 10-30 day old, milk fed calves. The worldwide increase in demand of cheese alongside with the reduced supply of calf rennet and higher prices, have led to an increase in search for alternative sources of milk coagulants. The present study was carried out with an aim to investigate milk clotting ability of kiwifruit extract and tamarillo fruit extract under different temperature conditions and different amounts of CaCl2 in comparison the rennet. The results showed that both kiwifruit extract and tamarillo extract possess milk clotting activity. It was observed that milk clotting activity increased and milk clotting time decreased with increase in temperature (32, 40 and 50°C) and CaCl2 concentration (0, 2.5, 5, 10, 15, 20 and 25 mM) with all the three milk coagulants. Kiwifruit extract showed low or no milk clotting activity at lower levels of extract, whereas tamarillo extract exhibited milk clotting activity even at lower concentrations and milk clotting time was comparable to that of rennet. Purification of kiwifruit extract by DEAE sepharose fast flow column chromatography gave three prominent peaks which when run on SDS PAGE showed the presence of three distinct proteins of molecular mass ~ 26, 20 and 17kDa. Protein of 26kDa, is a major protein in kiwifruit, belongs to cysteine protease group. The 20kDa and 17 kDa proteins are kiwellin and thaumatin-like protein respectively. Inhibition experiment conducted using protease inhibitors (PCMB, PMSF and EDTA) showed that the enzyme present in tamarillo extract was inhibited by PMSF indicating that it belongs to group of serine protease. The exact molecular mass of protein couldn‘t be ascertained from SDS PAGE electrophoresis of tamarillo skin, pulp and seed extract. It was recorded that tamarillo pulp and seeds both possess milk clotting enzyme whereas it is absent in skin of tamarillo fruit. Rheological measurement demonstrated that the gel formed with rennet and tamarillo possesses similar viscoelastic properties in the absence of added CaCl2. On addition of CaCl2, Gʹmax for rennet was higher than that of tamarillo gels and the difference in Gʹ value between rennet and tamarillo gels increased with increase in CaCl2 concentration. Course of gelation in kiwifruit was remarkably different from that of rennet resulting from a high non-specific proteolytic activity of extract after gelation point. The results in this study provide a fundamental understanding of milk clotting ability of tamarillo and kiwifruit extract. Although, tamarillo extract shows potential of an alternative to rennet but a detailed and more elaborate study needs to be done on mechanism of action of tamarillo extract on casein milk protein and whey produced.