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Title: The enzymatic removal of lactose from skimmed milk using a membrane reactor
Author: Ratanapongleka, Karnika
Awarding Body: Swansea University
Current Institution: Swansea University
Date of Award: 2007
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Many people are lactose intolerant. The aim of this project was to produce suitable alternative on milk products with low lactose. The hydrolysis of lactose in skimmed milk was investigated using an enzyme reaction employing beta-galactosidase in membrane bioreactor (MBR). This process with suitable membrane technology can produce low lactose milks. Lactobacillus delbrueckii ssp bulgaricus NCTC 11778 was selected to be the source of beta-galactosidase production since as it already used in fermented foods and produces relatively high levels of intracellular beta-galactosidase. Initially, the effects on nutrient composition of growth medium for L. delbrueckii were studied in test tube to fonmilate a medium for high cell productivity. The suitable growth medium consisted of (g/1) yeast extract 10, soy peptone 10, lactose 20, KH2PO4 2.5, sodium acetate 5, triammonium citrate 2, MgS04.7H20 0.2, MnS04.H20 0.05 and tween80 1 ml/1. The optimum pH for cell growth conducted in 5-1 batch fermenter at control temperature 37 °C was range from 5.0 to 5.5. From this study, the pH value at 5.5 was selected for cultivation in 140-1 pilot femienter because growth time to stationary phase was shorter than pH at 5.0. Harvested the cells from pilot fermenter were washed buffer in microfiltration (MF) membrane system, the cells were disrupted to release the enzyme by using high pressure homogenisation. The pressure at 25 kpsi (172 MPa) was used based on optimal release in high protein and enzyme contents. The enzyme was separated and purified from cell debris by membrane separation apparatus. A 0.2 mum MF membrane was used to remove soluble protein from cell debris and this was dialyzed to remove additional enzyme from debris. The penneate was then concentrated in ultrafiltration (UF) membrane system with MWCO 50 kDa to form the partially purified enzyme used for hydrolysis. The properties of beta-galactosidase from both crude and partially purified enzyme showed good activity on ONPG and lactose at neutral pH range from 6.0 to 7.0. The optimum pH value for both substrates was at 6.5. The activity of beta-galactosidase increased with the rising temperature from 20 to 65 °C. However, the stability of the enzyme at temperature above 40 °C decreased rapidly. The calculated Vmax and Km values based on the Michaelis-Menten equation and of Lineweaver-Burk transfomiation plots in two substrates, ONPG and lactose were investigated. The Vmax values from crude enzyme were 1.25 and 0.91 mumol/min/ml and Km values were 12.63 mM and 23.23 mM respectively. While the Vmax values from purified fonn were 1.04 and 1.34 mumol/min/ml and Km values were 17.62 mM and 27.58 mM when ONPG or lactose were applied as substrate respectively. The results also indicate that glucose and galactose acted as non-competitive inhibitor for beta-galactosidase. Additions of Na2+ in lactose hydrolysis reduced the enzyme activity while Ca2+ also strongly inhibited the activity. Only the presence of K+ seemed to promote beta-galactosidase activity. The enzymatic lactose hydrolysis reaction in synthetic and skimmed milk preparation was carried out in both batch bioreactor and membrane bioreactor (MBR) with 50 kDa MWCO UF membrane. Lactose concentrations and amount of enzyme activity influenced the hydrolysis. The results from batch bioreactor were in agreement with that observed in MBR. High concentration of lactose and enzyme increased the productivity. Low penneate flow rate during operating MBR resulted in greater hydrolysis yield (the obtained product was 67.7 mumol/ml at flow rate 8 ml/min) than when at high permeate flow rate (the obtained product was 44.7 mumol/ml at flow rate 25 ml/min). But in terms of productivity, high flow rates gave greater productivity (1117 mumol/min at flow rate 25 ml/min whereas 541mumol/min at flow rate 8 ml/min). The kinetics of lactose hydrolysis in batch bioreactor could be predicted by mathematical model based on reversible reaction from Haldane equation while in MBR did not correlate well with the model. Using the results of the work, a basic unoptimised design study showed that 1.5-1.75% lactose milk could be prepared for a cost of 15 pence per litre which suggest that process may be viable.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available