Dual effects in curdling milk and providing antioxidant, antidiabetic and antimicrobial activities by a mixed extract of noni, pineapple and mango plants / Rupbansraaj Bathmanathan

Faculty of Industrial Sciences and Technology

Thesis (Master of Science ) -- Universiti Malaysia Pahang – 2021

Includes bibliographical references

The dairy products of cheese and yogurt require enzymes to coagulate milk, but many issues plague the industry with the current enzymes used. The present study focused on finding a plant-based enzymic coagulant with dual effects of coagulating the milk to form curd and at the same time giving the curd medicinal properties. A total of 14 Malaysian plants with a literature of biological activities were chosen and tested to coagulate milk, and among them, three were selected (Morinda citrifolia L., Ananas comosus L. cv. Sarawak, and Mangifera indica L. cv. Apple) based on their ability to rapidly coagulate milk. The three plants were mixed to form super-coagulant (a milk coagulant mixture of extracts at a ratio of 1:1:1), and the presence of enzymic milk-coagulation proteins within it was determined through ammonium sulphate precipitation followed by size exclusion chromatography. Bicinchoninic Acid Assay (BCA) showed a protein content of 16.6 ± 1.2%. The super-coagulant had a milk-coagulating time of 32 ± 2.74 s at 50 mg/ml concentration and the Milk-Clotting time/Proteolytic Activity (MCA/PA) was determined to be at a ratio of 950.71 ± 216.79. The capabilities of the super-coagulant to form curd were demonstrated through centrifugation, light microscope, SEM and ICPOES analysis. Then, for the purpose of testing for biological activities, the optimum parameters for the super-coagulant were determined to be 120 mg/ml coagulant concentration, pH 4.5, 50 ℃ and 30 mM CaCl2 concentration and used to scale up the production of fortified curd. The antioxidant activity was determined through DPPH and FRAP assays, whereby the curd had an IC50 value of 26.53 ± 0.14 µg/ml for DPPH and FRAP value of 85.35 ± 2.38 mg GAE/g. The antidiabetic activity was determined through α-glucosidase and α-amylase inhibitory assays where the curd had IC50 values of 4.04 ± 0.01 mg/ml and 3.42 ± 0.03 mg/ml, respectively. The antimicrobial determination was done, and the curd exhibited no inhibition on bacteria and fungi strains. Nevertheless, once the curd was re-fortified with super-coagulant of 100 mg, it managed to show antimicrobial activity. Consequently, the in-vivo study was done on STZ diabetesinduced Sprague Dawley rats and super-coagulant curd was fed for a period of 20 days. The super-coagulant-treated diabetic rats (SC) managed to lower the weight loss in the rats to 201 ± 10.23 g on Day 20 compared to diabetic control rats (DC). The elevated fasting blood glucose (FBG) level of the SC rats was found to be reduced to 15.0 ± 1.31 mmol/L on Day 20 compared to DC rats. Liver function test on SC for AST, ALT, GGT and ALP levels revealed to be 214 ± 78.8 U/L, 91 ± 12.57 U/L, 3 ± 0.00 U/L and 509.75 ± 138.32 U/L, respectively while total protein test and renal function test showed levels of Albumin, Globulin, Serum creatinine and Urea to be 37.25 ± 1.89 g/L, 30.25 ± 2.22 g/L, 42.25 ± 2.75 µmol/L and 11.05 ± 1.08 mmol/L, respectively. These levels were found to be close to levels of the normal rats (NC). Furthermore, histopathological studies on the liver and kidney of the rats found no significant damage to the organs for the duration of 20 days. This study proves that super-coagulant has potential milkcoagulating capabilities. Its derived curd managed to contain good antioxidant and antidiabetic activities and, hence, has the potential to be used as raw material for functional dairy food products.