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Studies on human milk : effect of heat treatment and ultrasonication, and separation of E. coli 0111: B4 LPS specific IgA Dhar, Jyoti


Human milk banks have been established in children's hospital's around the world to provide a steady supply of expressed donor human milk (EHM) to premature or sick infants. There are, however, two major technical concerns regarding feeding of EHM to premature neonates: a) possibility of disease transmission and b) problem of fat separation during tube feeding the babies. A method using combination of ultrasonic waves and heat was developed for simultaneous pasteurization and homogenization of human milk. By using a sonicator which produced 600 W/ in2 energy at output control setting of 10 with a 3/8th inch disrupter horn, 80 ml_ of human milk in a bottle submerged in a hot water bath (93°C) could be heated to 72° C in 90 sec. The process was capable of inactivating 107 CFU7 ml_ of Staphylococcus aureus while retaining 80±2% of immunoglobulin A activity. The processed milk was adequately homogenized based on recovery of fat seen after mechanical infusion of milk, used as a simulation of tube feeding in clinical practice. In addition, the size of fat globules in human milk was reduced after thermo-ultrasonication to < 2.5um which could further explain the stable fat recovery observed upon infusion. Microwave heating patterns of 60 ml_ of water were studied to evaluate feasibility for pasteurization of human milk. Differences of ~7°C between the side and center of the sample bottle were observed. Immersing the sample bottle in a water bath to prevent surface heating did not reduce nonuniformity in heating. Constant agitation of the sample during heating was the best way of ensuring homogeneous temperatures and also resulted in rapid heating. A continuous flow, small scale, economical heat processing device was also studied for rapid and efficient pasteurization of human milk . Human milk inoculated with Escherichia, coli (106 CFU/mL) or S. aureus (107CFU/ml_) was heated at 71 °C at flow rates of 5.9, 12.3 and 18.9 mL/min, corresponding to holding times of 18.5, 9.0 and 5.75 sec. All conditions inactivated more than 106 CFU/mL of S. aureus and more than 105 CFU/mL of E. coli and also resulted in negative alkaline phosphatase activity indicating complete pasteurization. Pasteurization at 71 °C for holding times mentioned above, resulted in retention of 63-83% of IgA, 58-79% IgG and 49-72% IgM which are comparable to batch pasteurization procedures used in human milk banks. A 30% residual activity of y-glutamyl transpeptidase (GGTP) was found in bovine milk pasteurized at 71 °C. Lower residual activity could therefore be used as an indicator of overpasteurization. An affinity chromatography technique was developed for separation of enteropathogenic E. coli 0111:B4 LPS specific IgA from human milk. The affinity column was prepared by immobilizing LPS on porous chitosan beads using gluteraldehyde activation. The stability of immobilized LPS was confirmed upon repeated uses of the column with negligible leaching of the ligand. The column was able to bind 0.22 mg of LPS-specific IgA per mL. The affinity purified specific IgA accounted for 12.3% of total IgA and was found to cross react highly with E. coli 0128:B12, and Klebsiella pneumoniae using LPS-ELISA. The anti-LPS Ab separated might have aplications for prevention or treatment of gram-negative infections in high risk individuals. The technique might be used for separation of LPS specific antibodies for treatment or diagnosis of gram-negative bacteremia.

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