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Document Type

Thesis - University Access Only

Award Date


Degree Name

Master of Science (MS)

Department / School

Dairy Science

First Advisor

Vikram V. Mistry


The objectives of this study were to develop a method for the estimation of vitamin D3 in pasteurized Process cheese using HPLC, and to develop a technique for the fortification of pasteurized Process cheese with vitamin D3. The method for vitamin D3 estimation utilized high temperature (70°C for 30 min), alkaline saponification followed by extraction of unsaponifiable matter using petroleum ether: diethyl ether mixture (90: 10 v/v). One-ml silica cartridges were used for intermediate sample clean-up before the extracts were injected into HPLC. The HPLC system included a C10 liquid chromatographic column (4.6 mm i.d. x 15 cm, 5 μm particle size) with UV detection at 254 nm, mobile phase: methanol: acetonitrile (70:30 v/v), injection volume of 100 μI and a flow rate of 1-ml/min. The retention time for vitamin 0 3 was approximately 9 min. A standard curve was prepared for quantification of vitamin D3 in unknown samples. This curve was prepared by adding standard solutions, at six levels of vitamin D3, to approximately 5 g of unfortified Process cheese samples, which were analyzed using the above procedure. Analysis for each level of vitamin D3 was replicated 5 times. The areas obtained in each case were adjusted for the area in the control (cheese with no added vitamin D3) and then plotted against known concentrations of vitamin D3. The r2 obtained for the curve was 0.9722. In the second phase of the study, pasteurized Process cheeses were manufactured with vitamin 03 added at a level of 100 IU per serving (28 g). Vitamin fortificants were commercially available water and fat dispersible forms of vitamin D3. Suitable dilutions of fortificants were made and added in the cheese cooker with other ingredients. Fortified Process cheeses were evaluated for recovery of vitamin D3 during cheese manufacture and distribution of vitamin D3 in the cheese mass. It was found that there was no loss of vitamin D3 during manufacture of Process cheese and vitamin D3 was uniformly distributed in the cheeses. Fortified Process cheeses, stored at 21 to 29°C and 4 to 6°C, did not exhibit any loss in vitamin D3 activity over a 5 mo time period. As Process cheeses are often used as food ingredients and are heat treated to high temperatures for considerable time, stability of vitamin D3 under such circumstances was also studied. Twenty-five to thirty percent of vitamin D3 was destroyed under severe heat treatment of 232°C for 5 min. Sensory analysis showed that added vitamin D3 did not exhibit any off-flavor to the Process cheeses.

Library of Congress Subject Headings

Process cheese
Vitamin D
High performance liquid chromatography


Includes bibliographical references (pages 56-75)



Number of Pages



South Dakota State University


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