The free fatty acids of milk fat are believed to be involved in imparting flavor properties to milk and other dairy products. In the past the free fatty acids have largely been related to quality deterioration and hence the methods for measurement have been devised to determine the changes in the free fatty acid content and to relate these data with quality. No method has been reported to determine the quantities of individual free fatty acids. The purpose of this investigation was to evaluate procedures and adapt methods for isolation and measurement of the free fatty acids of milk fat and then to evaluate adapted methods by quantitative measurement of the individual acids in fresh cream fat samples and in butter made therefrom. The ion exchange method of Hornstein et al. (50) was modified to isolate and esterify the free fatty acids from milk fat. The free fatty acids were adsorbed on Amberlite IRA-400 resin, the resin was made fat free and the bound acids were simultaneously esterified and eluted with anhydrous methanol-HCl. The methyl esters were extracted from the reaction mixture with ethyl chloride (b.p. 12.3°C.). The ethyl chloride was evaporated and the esters weighed. The methyl esters were then separated by gas-liquid chromatography using the thermal conductivity detector. The recovery of each saturated even numbered fatty acid from 4:0 to 18:0 was checked. The percent recoveries obtained were: 4:0, 71.4; 6:0, 86.5; 8:0, 66.6; 10:0, 75.1; 12:0, 94.3; 14:0, 100.2; 16:0, 99.5 and 18:0, 92.5. The ion exchange resin was checked for its fat hydrolysing capacity, for retention of fatty acids when used in successive analyses and for leachings of brown polymers during each analysis. The resin did not show detectable hydrolysis of triglycerides nor did it retain or exchange fatty acids from previous use. It was necessary to pretreat the resin with stearic acid and anhydrous methanol-HCl to avoid interference of a leached polymer with quantitative results. An average of 5.0 mg of residue leached from the resin during every analysis, but this did not interfere with the quantitative determination of free fatty acids. Twenty samples of milk fat; ten from pasteurized sweet cream, nine from cultured butter and one from sweet cream butter were analyzed for free fatty acids. The results obtained were compared with the esterified fatty acid content of milk fat. The percent composition of free fatty acids was similar to that of the esterified fatty acids in milk fat. Also the manufacturing process of butter had little or no effect upon the free fatty acid composition of the fat. The values obtained for volatile fatty acids, especially 4:0, were not consistent. One reason for this probably was that evaporation of the ethyl chloride from the solution of the methyl esters was carried out at room temperature and the evaporation rate was not controlled. It is believed that the results would be more consistent if the evaporation of ethyl chloride were carried out under controlled and standardized conditions and if internal standards are employed for quantitative references rather than weighing the ester mixture. Samples of autoxidized milk fat, sweet cream fat and rancid cream fat were analyzed for further evaluation of the method. / Graduation date: 1963

Graduation date: 1961

Methods were developed to quantitatively measure resazurin reduction and the reducing capacity of milk. The method for resazurin determination involved butanol extraction of the dye from milk and measurement of the optical density of extracts at 582 mμ and 615 mμ after saturation with sodium bicarbonate; that for reducing capacity was based on reaction of 2,6-dichloro-phenolindophenol with milk at its normal pH. For the latter determination excess indophenol was added and the quantity of dye remaining estimated spectrophotometrically at 660 mμ in butanol extracts saturated with sodium bicarbonate. This assay could be applied in the presence of resazurin or resorufin, since these compounds had negligible absorbance at 660 mμ. Both of these methods were reproducible in milk and their respective accuracies estimated at greater than 90 percent. The quantitative assay for resazurin was employed in order to study the behavior of resazurin in milk and the systems in fresh milk responsible for reduction of the dye. Results obtained on aging whole and skim milk were used to demonstrate that the resazurin reduction dealt with in the present investigation was due to the reducing systems of milk rather than to bacterial activity. These results showed that the reducing system was less stable and more sensitive to temperature of aging in skim than in whole milk. Measurement of rates of resazurin reduction by various fractions of normal milk showed that the major portion of the reducing ability of whole milk was associated with the cream. The aqueous phase from centrifuged warm gravity cream had a greater ability to reduce resazurin than did the whole milk from which the fractions were derived. The rate of resazurin reduction by milk decreased with incubation at 37°C, however, in the presence of sufficient numbers of bacteria, an acceleration of rate with incubation was noted. The point at which washed suspensions of added bacteria became significant in reduction was demonstrated as a change in slope of logarithmic plots of dye reduction rates. Resazurin was shown to have a destabilizing influence on the reducing capacity of milk. This Influence was catalytic and dependent on total concentration of dye; rate of inactivation being constant for a given dye concentration. Evidence was presented to show that the component of the reducing system that was inactivated was ascorbic acid. The influence of fractions, obtained from passage of resazurin through a silicic acid column, suggested that this catalytic effect was probably due to the dye as such, rather than to artifacts in the commercial dye preparation used. Examination of the reducing capacity of milk fractions before and after treatment with ascorbic acid oxidase indicated that resazurin reduction was brought about by that part of the reducing capacity that could not be accounted for as free ascorbic acid. In mastitic samples, this element of the reducing capacity was concentrated completely in the fat and centrifuged sediment. It was concluded from these investigations that the reducing system of milk existed as a measurable entity at any given time rather than as a continuous evolution of electrons from some slow enzymatic reaction. This system consisted of the measurable ascorbic acid of the milk, which occurred in the plasma, and some reducing agent bound to structural components of the cream and sediment. The measurable ascorbic acid accounted for approximately 80 percent of the reducing capacity but was concluded to have little influence on resazurin reduction. It was concluded that the bound reducing agent depended on structural elements in the milk for its ability to reduce resazurin, and that it lost this ability on dissociation from whatever particle it occurred on. It was postulated that this reducing agent was ascorbate and that it occurred bound to leucocytes and other cellular debris in the milk in situations analogous, to its reported occurrence in blood. Attempts to identify this reducing agent as ascorbate were unsuccessful in this investigation, but the techniques employed were probably inadequate. / Land and Food Systems, Faculty of / Graduate

The main objects of this investigation were to study fat and solids-not-fat levels in cow's milk, the relationship between them and to calculate heritability estimates. During the period of four months in 1955-1956 the milk of twenty Ayrshire cows was sampled and tested for butterfat and solids-not-fat. In the same period of 1956-1957 eighteen Ayrshires and ten Holsteins were similarly tested. Ayrshire cows were used for heritability determinations and represent a group of inbred animals with a high average relationship. A detailed calculation of heritability for fat and solids-not-fat and resultant estimates of 21.2% and 10.9% respectively are presented. Highly significant correlation coefficients between fat and solids-not-fat of + 0.680 in 1955-1956 and + 0.618 in 1956-1957 for Ayrshires and a non-significant value of + 0.570 for Holsteins, were calculated. The question of testing milk for solids-not-fat with respect to achieving genetical improvement is explored and the usefulness of correlation coefficients and regression equations for estimating solids-not-fat is discussed. The effects of mastitis on the level of solids-not-fat are indicated. / Land and Food Systems, Faculty of / Graduate

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Call number: LD2668 .T4 1956 B37 / Master of Science

The purpose of this investigation was to modify the procedure of Bills, Khatri and Day for use in the development of a suitable method for determining the quantitative distribution of the free fatty acids (FFA) in normal, heated, and rancid milk and milk fractions The method consists of extracting the FFA from lyophilized milk, separating the FFA from neutral fat, converting the FFA to methyl esters, and analyzing the methyl esters by gas-liquid chromatography (GLC). The salts of FFA present in lyophilized milk or milk fractions were released by lowering the pH of the sample with H₂SO₄, and subsequently extracted with ethyl ether. The extracted FFA were simultaneously isolated from the extract and methylated by using the one-step procedure of Bills, Khatri and Day. A specially designed concentration flask was employed with a reflux system to concentrate the methyl esters. The esters were then separated by GLC. Quantitative calculations were made from the GLC peak areas using internal standards. The major esterified fatty acids of milk are n-saturated, evennumbered 4:0-18:0 and unsaturated 18:1 and 18:2 acids. The distribution of major FFA in whole milks was found to be essentially the same as that of the esterified fatty acids of milk fat. Heat treatments of milk, whether pasturization or extended holding at 100°C, effect a progressive reduction in total FFA. Decreases in long chain fatty acids are also characteristic of extended heating. Milks determined to be rancid by acid degree value and organoleptic analysis showed high levels of FFA, 1.5-3.6% of the fat content of the sample. The increases in 6:0-12:0 resulting from lipase hydrolysis approximate the amounts shown by Al-Shabibi and co-authors to produce rancid flavor when added to good quality milk. Milk triglycerides, fat globule membrane, skim milk, and buttermilk show characteristic FFA patterns which appear to be related to the solubility properties of the individual acids. Seventy-one percent of the total FFA in a given portion of 40% cream was found in the triglyceride fraction, 26% in the crudie fat globule membrane preparation and 3% in the skim milk fraction. The total concentrations of FFA were found to be: 1% of the fat in whole milk or 0.04% of the fluid milk weight; 0.16% of the total weight of 40% cream; 0.28% of centrifuged triglycerides; 1.7% of the fat globule membrane; and 0.008% of the fluid weight of skim milk. / Graduation date: 1965

Call number: LD2668 .T4 1957 T47 / Master of Science

Thesis (BTech (Food Technology))--Cape Technikon, 1999. / The Dairy industry, one of the larger food industries in South Africa processes probably the most perishable and possibly the most regulated foodstuff, namely mille The unique combination of vitamins, proteins, carbohydrates, lipids, moisture and near neutral pH, offers a suitable environment for the proliferation of microbes. Milk is therefore highly susceptible to microbiological activity resulting in the irreversible spoilage of this food (Frazier & Westhoff, 1988). The coliform group of organisms comprises all aerobic and anaerobic, gram-negative, non-spore-forming rods that are able to ferment lactose with the production of acid and gas at 32°C within 48 hours (Richardson, 1985). The primary purpose of the coliform detection test is to measure the quality of the practices used to minimise bacterial contamination of processed dairy products (Richardson, 1985). IDF Standard 132A: (1991) defines psychrotrophic organisms as organisms forming countable colonies when incubated aerobically at 6.5°C for 10 days under the conditions specified in IDF standard 101A. Shelf-life tests conducted in the fresh milk laboratory of a processing plant, revealed significant growth of coliforms in samples stored at 5°C. Luch, (1985) reported that other contaminating psychrotrophs together with the coliforms reduce the shelf-life of the milk when the storage temperature thereof is above 10°C.

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