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Authors: PARKHA, O. A.
Keywords: Lactose intolerance
Enzyme characterization
Hydrolysis effect
β-galactosidase production
Lactic acid bacteria
Issue Date: Jan-2014
Abstract: Lactose intolerance (a condition in which man elucidates an immune reaction towards the presence of lactose due to inability to produce enzyme lactase) is a major nutritional deficiency among some adult consumers of milk and other dairy products worldwide. β–galactosidase hydrolysis of milk is one of the promising enzymatic applications in dairy industries for reducing lactose intolerance of milk products. However, plant and animal sources cannot meet the high demand of the enzyme in food industries. Hence, the aim of this study was to characterize β–galactosidase production by Lactic Acid Bacteria (LAB) isolated from locally fermented milk products. Raw milk from Sokoto Gudali was collected from Fulani settlement in Ojoo, Ibadan along with some fermented milk products (‘’Nono’’ and ‘’Wara’’). LABs were isolated from them and identified using conventional methods. The ability of the isolates to hydrolyze 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) was used to screen for β–galactosidase production. Isolates with the best β-galactosidase production were selected. The enzyme was extracted and optimization of growth conditions (temperature, pH, nitrogen, carbon sources, inoculums size and inoculums age) for β-galactosidase production was carried out using o-nitrophenyl-β-D-galactopyranoside (ONPG). The enzyme produced was characterized using pH, temperature, metal and non-metal ions, and inhibitors. Purification of the enzyme was carried out using dialysis and chromatographic methods. Hydrolytic effects of the purified β–galactosidase were determined in different concentrations of lactose using standard method. Data were analyzed using descriptive statistics. The isolated bacteria were identified as Lactobacillus plantarum (G11, E13 and E36), L. brevis, L. casei, L. lactis, Leuconostoc lactis, Streptococcus sp, and Bacillus subtilis. Lactobacillus plantarum (G11) had optimum growth value of 4.2 at 20°C and pH 7.0 with maximum enzyme value of 6.2U/mL at 30 hrs. The optimal β-galactosidase production occurred at neutral pH and 6% inoculum size. The best inoculums age varied between 18 hrs and 36 hrs. The best carbon source for enzyme production was raffinose with maximum value of 0.3U/mL while minimum activity was found in fructose with 0.2U/mL. The best nitrogen source was NH4NO3 with maximum value of 0.5U/mL and yeast extract had minimum value of 0.1U/mL. β–galactosidase activity increased with increase in molar concentration of the mono-valent chloride ions in which the highest was recorded in KCl at maximum value of 0.08U/mL while the minimum value of 0.001U/mL was obtained by NaCl at a concentration of 0.2 mmol respectively. The best sulfate ion was CuSO4 with maximum activity value of 0.2 U/mL at 0.2 mmol concentration and minimum value of 0.007 U/mL at 0.1 mmol by ZnSO4. The best enzyme inhibitor was KCN with maximum activity of 0.2U/mL at 0.2 mmol. The specific activity of β-galactosidase was 292.5 U/mg, 104.2 U/mg and 585.46 U/mg for G11, E13 and E36 respectively. The hydrolytic effects of the purified β-galactosidase showed a maximum yield of 35.8% glucose, 19.3% galactose and 35.3% glucose and 18.5% galactose at 80% and 60% lactose concentration respectively. β-galactosidase produced by Lactobacillus plantarum strain achieved lactose hydrolysis and could be of potential application for production of low lactose dairy products for consumption by lactose intolerant people.
Description: A Thesis in the Department of MICROBIOLOGY Submitted to the Faculty of Science in partial fulfillment of the requirements for the award of the Degree of DOCTOR OF PHILOSOPHY (Ph.D) of the UNIVERSITY OF IBADAN, NIGERIA
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