Kesinai (Streblus Asper) Protease as a Potential Milk Coagulating Enzyme
Leaf extract of plant kesinai (Streblus asper) contains a milk coagulating protease, which could be a potential rennet substitute. However, its potential has not been investigated and the protease has not been purified and characterised. Preparation of the crude leaf extract results in an undesir...
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| Format: | Thesis |
| Language: | English English |
| Published: |
2000
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/8418/1/FSMB_2000_6_IR.pdf |
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| Summary: | Leaf extract of plant kesinai (Streblus asper) contains a milk coagulating
protease, which could be a potential rennet substitute. However, its potential has not
been investigated and the protease has not been purified and characterised. Preparation
of the crude leaf extract results in an undesirable, very dark brown colour and inhibition
of this browning may enhance the use of the leaf extract.
The browning inhibitors, citric acid, ascorbic acid, L-cysteine and sodium
metabisulphite were used for prevention of browning and to obtain a crude extract with
an acceptable colour. Metabisulphite was found to be an effective inhibitor of the
enzymatic browning of the leaf extract. At 2 mM concentration it has inhibited browning
and the extract obtained resulted in a white milk coagulum compared to the brown
coloured coagulum of the brown extract. It is thermostable up to 85°C, with an optimum
temperature at 70°C and its optimum pH is 7.2. Six mM added calcium chloride was
optimum for its milk coagulation activity.Microstructure, texture and syneresis of the milk coagulum of the crude extract
were assessed by Scanning electron microscopy (SEM), Transmission electron
microscopy (TEM), the Texture Analyser, and measurement of whey volume,
respectively and were compared with that of calf rennet and Fromase. Kesinai coagulum
appeared as a sponge-like when examined under SEM, while calf rennet and Fromase
coagulum appeared as a fibrous network. Quantification results showed that porosity of
kesinai coagulum is low, and significantly different from both of calf rennet and
Fromase coagulum (P<0.05) and (P<0.01), respectively. Kesinai coagulum was soft,
and its strength is significantly lower than that of calf rennet and Fromase coagulum (P<
0.01). Syneresis of its coagulum was low, and the whey volume as per cent of milk
volume was 34.75% compared to 46.75% and 48.79%, for calf rennet and Fromase,
respectively.
The ratio of milk coagulation activity to proteolytic activity of the extract was
very low (0.02) and the protein profile of the milk coagulum and whey on sodium
dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the
protease was more proteolytic than calf rennet, and Fromase.The protease was purified by ultrafiltration (UF), Fast protein Liquid
Chromatography (FPLC) gel filtration with Superose 6, FPLC ion exchange using
MonoQ HR 5/5 and lsoelectric Focusing (IEF) using the Rotofor system, with a
puritification fold of 25, and 18% recovery. The purified protease appeared as a single
band on SDS-P AGE with a molecular weight of 31.3 kDa. Characterisation of the purified protease showed that it could be a serine protease with optimum pH of 7.2,
stable in the pH range 5.0 -9.5, and its pI is 5.2. It is thermostable up to 85°C, with
optimum temperature at 70°C. Zymogram analysis showed that protease activity is
associated with milk coagulation activity.
It is concluded that kesinai protease could be used in the production of short
ripened cheese varieties. |
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