Characterisation of proteases and lipases produced by Paenibacillus spp. and analysis of heat stability of these enzymes : a thesis presented in partial fulfilment of the requirements for the Master of Food Technology at Massey University, Manawatū, New Zealand
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Date
2025
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Massey University
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Abstract
The aim of this study includes the isolation of Paenibacillus species from raw milk, investigation of Paenibacillus species that produces protease and lipase, investigation of the effect of temperature, growth medium and inoculum conditions (fresh and chilled) on the production of protease and lipase by Paenibacillus species and the heat stability of protease and lipase produced by Paenibacillus species. The isolation of Paenibacillus species from raw milk was carried out by using milk plate count agar and the isolated species were identified using Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysisThere were none Paenibacillus species isolated from raw milk therefore pre-isolated Paenibacillus species obtained from Massey University microbiology lab culture collection were used for further experiments. The production of protease and lipase were tested using milk agar and tributyrin agar plates respectively. Seven out of 26 isolates were strong positive for protease at 37 °C. Only 2 isolates were positive for proteolysis at 30 °C and 2 isolates were strongly positive for proteolysis at 30 °C. The effects of inoculum conditions (fresh and chilled) were tested by comparing the ability of isolated Paenibacillus species at day 1 (fresh) and day 31 (chilled) of storage and there were no obvious differences. The effect of medium was tested by growing Paenibacillus in TSB, 10% TSB and 1% skim milk. Paenibacillus produced more proteolysis in full strength Tryptic Soy Broth (TSB), 10% TSB and 1% skim milk. The enzyme activities were further investigated by using azocasein and p-nitrophenol palmitate assays. Seven isolates produced obvious amounts of proteolysis, and the highest amount of proteolysis produced was 72 U/mL. There was no proteolysis and lipolysis found after heat treatment at 100 °C for 10 minutes which indicated no production of heat-stable enzymes by the Paenibacillus isolates. In conclusion, the investigation of enzyme activities of species provides new insights for the dairy industry with no heat-stable enzymes produced by the Paenibacillus isolates tested. Further studies are required for medium effect on enzyme production and heat stability of enzyme produced by testing different medium of growth and different temperature conditions for the enzyme produced.