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Lactoferrin is the part of whey proteins, which does not become the part of curd and is lost in the whey. The objective of the current investigation was to develop lactoferrin-supplemented cheddar cheese. Cheddar cheese was supplemented with lactoferrin at 5, 10, 15, 20, and 0 mg/100 g concentrations (T1, T2, T3, T4, and control) and matured for 90 days. The supplementation of lactoferrin at all four concentrations did not affect the compositional attributes of cheddar cheese. In mature cheddar cheese, values of log-colony forming units in T1, T2, T3, T4, and control per mL were 7.82, 7.77, 6.51, 6.21, and 7.95, respectively. Lactoferrin contents of T1, T2, T3, T4, and control were 4.98, 9.89, 14.91, 19.88, and 0.11 mg/100 g cheese. In vitro total antioxidant capacity of mature T1, T2, T3, T4, and control were 32.47%, 47.68%, 65.37%, 78.82%, and 45.79%. High-performance liquid chromatography (HPLC) analysis revealed that concentrations of citric acid, lactic acid, acetic acid, and propionic acid in mature T4 were 2389, 16,468, 126, and 141 mg/kg. Analysis of cheese samples on gas chromatography–mass spectrometry (GC-MS) showed that fatty acids composition was not influenced by the supplementation of lactoferrin. Peroxide value of matured T1, T2, T3, T4, and control was 0.31, 0.30, 0.28, 0.35, and 0.28 (meqO2/kg) with no variation in color, flavor and texture score. The results of the current investigation proved that lactoferrin can be used for the supplementation of cheddar cheese. [ABSTRACT FROM AUTHOR]