Remarkable PRACTICAL rennet BIOTECHNOLOGY Practical details CHEESE PRODUCTION has for thousands of years relied upon the action of enzymes which coagulate the 1. Dispense 10 cm3 of milk into test tubes. proteins in milk, forming solid curds (from which cheese 2. Add 1 cm3 of rennet enzyme. 3. Record the time taken for the milk to is made) and liquid whey. The process hass two stages. coagulate. It may be possible to distinguish Firstly, the enzyme breaks specific bonds in a between a ‘partial set’ i.e. when particles glycopeptide on the surface of soluble calcium caseinate adhere to the side of the tube when it is gently rocked from side to side, and a ‘full set’ i.e. particles in the milk. Relatively insoluble calcium when a solid curd is formed. paracaseinate is formed, and in the presence of calcium ions this coagulates to form a continuous curd. Safety Traditional cheese-making has mainly involved the use The products of this investigation should not be consumed (some daring home economics students have of animal enzymes, although some cheeses in the UK been known to try this!) were made with plant proteases. Today there are several enzymes available to the manufacturer — from animals, Further activities naturally-occurring fungi and animal-identical products from genetically-modified microbes. 1. Investigate the effect of temperature on the time taken for the curd to form. Here we suggest investigations which can be done using 2. Compare the activity of different types of enzymes. one or several of these enzymes. 3. Add calcium chloride to the milk, to investigate the effect of calcium ion concentration on the setting rate e.g. Maxiren requires a slightly greater calcium ion concentration than some other enzymes. Materials 4. Alter the pH of the milk, using 1M hydrochloric or citric acid, 1M sodium hydrogen carbonate or sodium hydroxide. 1 Rennet enzyme cm3 of any of these reagents, added to 10 cm3 e.g. calf rennet essence; Novo Nordisk of Pasteurized milk will give approximate Rennilase® (a fungal protease); or Gist- pHs of 1, 5, 8 and 14, respectively. brocades Maxiren (pure calf chymosin from 5. Dilute the enzyme with distilled water to a genetically-modified yeast). Rennet investigate the effect of enzyme essence is usually sold in supermarkets or concentration. ADDITIONAL health food shops. The two microbial INFORMATION enzymes are available from the NCBE. Pasteurized milk 1 cm3 syringes (for measuring out enzymes) Madden, D. (1991) 3 10 cm syringes (for measuring out milk) Below: ‘Milk-coagulating Test tubes Chymosin converts enzymes by Stopclock milk protein (casein) accident and Water bath (optional) into a solid curd. design’ NCBE Newsletter Summer 1991. pp. a aaa a a aa aa a a a a a a a a a a a a a a a a aaaa a a a a a aa a a a a a a a a 1–5. a a a a a a a a a a a a a a a a aaaa a a a a a a aaProtease a a a a a a a a a a a a a a a a a a a a a a aa a a a a a a a aaa a a a enzyme a aaaa a a a a a aaa a aa 2+ a a a a a a a a e.g. chymosin a a a a a a a aa a a a a aa Ca a a a a a a a a a Gill, J. and a a a aa a a a a a a a a a a a a a a a a a a a a a a a a a a a aa a a a a aaa a a a aa a a a a a a a aa a a a a aa a a aa a a a aa a a a a a a a a a a aa a a a a a aa aaaaa a a a a a a aa Saunders, T. a a a a aaa a a a a a aa a a a a a aa aaaa a aaaa a a a a a aaaa a a a aaa a aa aa a a a a a a a a a a a a a (1987) ‘Rennin - a a aa a a a a a Surface a a a a a a a a a a a a a a a a a a glycopeptides a aa a a a a a a a a a aaa a neglected a a a a a a a a a a removed a a a a a a aa a a aa aaa a enzyme?’ Journal of Biological Soluble calcium Unstable, denuded Aggregated calcium Education 21, (4) caseinate particles casein particles paracaseinate particles in milk in milk coagulum 248–250. Remarkable rennet Use 1 cm3 of enzyme to 10 cm3 of Pasteurized milk in a test tube Record how long it takes for the milk to set : a) partially; and b) completely (You will need to think carefully about how you decide when these two points have been reached) En zy lle me Co s for Schools and N ovo nilase Ren 3 M ilk- cm coa 00 gula 1 ting protease Sto eze. N re at 4°C. Do not fre A N TI TIO ON A D A UC g ep L D in a CE Y E d rtm NT LOG ea e RE FO HNO f R nt R BIOTEC y o of M ersit icrobiology, Univ Extend the investigation by trying: Different temperatures; (use a water bath) E n e zy oll Different enzymes; mes d C for Schools an N e ovo Rennilas (for example, compare shop-bought calf rennet (for 3 M ilk- cm coa 00 gula 1 ting protease Sto eze. re at 4°C. Do not fre E N N n AT IO z le IO AT y ol D N C m C e AL DU ng es nd pa C Y E di for ls a rt EN G ea Schoo making junket), fungal enzyme and pure chymosin from m TRE OLO f R e FO HN nt R BIOTEC y o of M ersit icrobiology, Univ G n ist- xire brocades Ma 3 Pu genetically-modified yeast) re cm c hym 100 osin from a ge ast netica ified ye N lly-mod Sto ze. N A re at 4°C o not free TI . D TIO ON A D A UC g ep L D in a CE Y E d rtm NT LOG ea e RE FO HNO f R nt R BIOTEC y o of M ersit icrobiology, Univ Different pH values; (1 cm3 of 1M hydrochloric acid added to 10 cm3 of milk plus enzyme gives a pH of about 1; 1 cm3 of 1M citric acid, pH 5; 1M 1 M Sodium hydroxide sodium hydrogen carbonate, pH 8; 1M sodium hydroxide, pH 1 M 14 and distilled water, pH 7) Hydrochloric acid © National Centre for Biotechnology Education, 1995.