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A Passion Avenue For Science


Cheese is traditionally made by mixing milk with rennet. Rennet itself is curdled milk from the stomach of an unweaned calf which is a by-product of calves killed for veal, making it extremely unusual for an animal to be killed only for the production of rennet [2]. Not only that, there are other various factors such as limited availability of rennet, religious concerns such as Islam and Judaism, and diet restrictions (vegetarianism) or ban on recombinant calf rennet in several European countries (e.g. France, Germany and the Netherlands) that may encourage further research on alternative coagulants for cheese.

In addition, as rennet is merely an additive, not a food, it has no nutritional value. Unlike proteases from fruits which allow them to act as a coagulant, while still retaining some of its nutritional value, such as noni and papaya. The use of the crude extract in this study, instead of the purified one, may allow the presence of other compounds with nutritional values in the extracts. Noni itself has been known as a popular health supplement, though it isn’t as frequently used in Indonesia. Rather, it is found discarded on the streets and is known as a stinky fruit instead of for its health benefits. So, by using fruits such as noni as an alternative cheese coagulant, incorporate less well-known and eaten fruits into more functional benefits.

Extraction of Protease Enzyme from fruits and vegetables: Papaya

1.Fruits and vegetables were blended with phosphate buffer.

2.The mixture is stirred constantly for 1 hour using a magnetic stirrer, and placed in an ice bath to maintain cold temperature.

3.Centrifuge the mixture for 30 minutes at different speed for different extracts.

Parameter for Analysis: Milk-clotting Activity (MCA)

MCA was determined using the method described by Arima, Yu, and Iwasaki (1970) with slight modification [3]

  1. 1 ml of extract is added to 10 ml of low-fat (1%) pasteurized milk (containing 0.02% CaCl2)

  2. Place the mixture in the water bath until the coagulation was observed.

Once the plant extracts containing milk clotting properties have been found. Cheese production is carried out using these extracts.


Ideally, extraction of protein use 4℃ to optimally retain the functional properties as well as the enzymatic activities. After several trials, it has been found that the use of room temperature for the extraction including the process of centrifugation can still retain the milk clotting activity of the crude extracts obtained. This use of crude extracts may have benefits such as allowing for more easier application. All cheese made during this research was made using extracts that were centrifuged at room temperature (±24°𝐶). From all the fruits and vegetables that was sampled, noni was shown to be the most effective in milk-clotting, as shown by the high average SU of 222,5589. Followed by kiwi and papaya with an average SU of 11,6064 (R), 18,6551 (N) and 17,1149 (R), 9,7177 (N), respectively. 

This can also be seen visually throughout the milk clotting process in which noni is shown to have the most separation and fastest clotting time compared to papaya and kiwi. But the slower rate of milk clotting for papaya is still an achievement compared to other extracts that did not show any form of milk-clotting. Though further research with cheese making was only done with noni and papaya as kiwi has been used to make cheese in other scientific research. Results from mozzarella cheese. production with papaya showed to be crumbly and had no stretchy properties, in contrast to mozzarella. 

On the other hand, results using noni extracts were very soft and barely forming a solid. Both extracts dissolved when placed in hot water and did not show any properties of mozzarella, such as stretch. Results for cream cheese production, only done with papaya extract, showed very similar properties to manufactured cream cheese as well as cream cheese trial using rennet. There is a slight difference in texture, cream cheese made using rennet had a flaky texture compared to cream cheese made using papaya extract. Furthermore, papaya cream cheese showed slight fruit after taste, though not one that affected the overall taste of the cheese.


In conclusion, this research found that a simplified procedure still allows for extracts to show protease activity. Extracts do not need to be centrifuged at refrigerated temperature for it to show protease activity. Furthermore, out of the several plant extracts that were tested for milk-clotting analysis, noni, papaya and kiwi have shown milk-clotting activities, with noni having the highest milk-clotting activity. Research also found that substitution of rennet can be achieved in the production in cream cheese using papaya extracts.

In this work, Tiffany determined to research plant-based proteases for use as coagulants in the production of cheese.

Plant Based Milk-Clotting Proteases Investigation


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