Potential antioxidant activity and physical of cottage cheese using bromelain enzyme pineapple[Ananas comusus] as a natural coagulant

The proteolytic enzyme bromelain, which can aid in hydrolyzing proteins into amino acids, is found in pineapple fruit. The bromelain enzyme can also aid in the coagulation of milk, which makes it useful in the cottage cheese-making process. The numerous phenolic compounds and flavonoids found in pineapple fruit serve as a source of antioxidants. Antioxidants function by scavenging free radicals. This study used the pineapple fruit’s bromelain enzyme as a natural coagulant to ascertain the cottage cheese’s physical characteristics and antioxidant activity. Each treatment (K1 = 0%, K2 = 3%, K3 = 4%, and K4 = 5%) was repeated four times as part of the research methodology. The outcomes demonstrated that the bromelain enzyme’s inclusion had a very substantial impact [p≤ 0.01] on the cottage cheese’s physical characteristics and antioxidant activity. The results of the study concluded that bromelain concentrations up to 5% produced good antioxidant activity and physical properties of cottage cheese with a very significant difference P≤0.01. This indicated that the bromelain enzyme from pineapple extract could be used as a cheap substitute for rennet in making cheese.


Introduction
Cheese is a dairy-based food that is prepared by coagulating milk to separate the solid components from the liquid.Curd forms and milk serum separates as a result of the natural coagulation process, which is carried out by the fermentation stage of lactic acid bacteria or by utilizing the rennet enzyme.[1].Bromelain, an enzyme found in pineapple (Ananas comosus), is frequently used to cook meat by dissolving the protein and resulting in a more soft texture.The bromelain enzyme has not, however, been used in the creation of cheese curd.The fruit pineapple is widely recognized as a source of the readily available and reasonably priced bromelain enzyme.
There are several benefits to using pineapple as a source of rennin replacement enzymes, including the fact that it comes from plants rather than animals, making it exempt from animal welfare laws, having a relatively low cost potential due to cheaper pineapple prices, and being natural.which, when compared to synthetic enzymes on the market, can lower production costs.[2] One of the proteolytic enzymes that can aid in hydrolyzing proteins into amino acids is bromelain The bromelain enzyme can also aid in the coagulation of milk, which makes it useful in the manufacture of cheese.
According to several studies, pineapple fruit contains a variety of physicochemical phenolic compounds and flavonoids, which are sources of antioxidants.Antioxidants function by snatching free radicals, which enables them to stop the growth of cancer cells and fight cancer.Finding alternatives to the usage of rennet enzyme in cheese IOP Publishing doi:10.1088/1755-1315/1341/1/012037 2 manufacturing is important to lower the cost of cheese production because rennet enzyme is still imported into Indonesia for the process of creating cheese from nations on the European continent.Use of the bromelain enzyme, which is present in pineapple plants, can be used as an alternative to rennet.The physico-chemical properties of cottage cheese have been studied in the past with the inclusion of bromelain enzyme extract at various pH levels.By looking at the protein profile of cottage cheese, research has also been done on the bromelain enzyme as a natural coagulant .The bromelain enzyme, which has been used in this study to assess the microstructural diversity.The quality of cottage cheese will be determined by testing the total solids, calcium levels, and curd cheese produced using bromelain enzymes, which is an enzyme derived from pineapple fruit and used to make cheese, which acts as a natural coagulant.[3,4,5]

Materials
Sisteen [16] liters of fresh milk, The pineapple fruit must first be juiced to obtain the juice before the bromelain enzyme 530 ml, is extracted.

Procedure
The research was conducted as a laboratory experiment with a completely randomized design, consisted of 4 treatments and 4 replications.The research treatments were different bromelain enzyme concentration levels (K1= 0% K2 = 3%; K3 = 4.% K4= 5%), The pineapple fruit must first be juiced to obtain the juice before the bromelain enzyme 530 ml, is extracted.16 liters of fresh milk must be pasteurized for 15 minutes at 70 0 C in order to make cottage cheese [HTST].Bromelain extract was added at 3%, 4%, or 5% [w/w] with each treatment, and the mixture was then cooled to 40 0 C. It was then incubated for 35 minutes, filtered through a gauze filter, and 1% salt [w/w] was added.After being kept at -17 0 C for 24 hours, the curd was then examined.

Data analysis
All of the data was evaluated by using analysis of variance [Anova] and followed with the loast Significant Difference [LSD] test to find any significant differences by using SPSS version 16.0.[6]

Parameters
The parameters of this research are antioxidants, cheese curd, cheese solids, calcium.Antioxidant activity testing was carried out using the descriptive method DPPH Absorbance -Sample absorbance/DPPH absorbance x 100.Curd was obtained from the weight of curd/initial milk volume x 100%.Total Solids Value is obtained from constant weightinitial weight/sample weight -initial weight x 100%.To test calcium levels, the homogenized solution is put into a cuvette and the absorbance is read at the maximum wave.

Results and discussion
Research data on antioxidant activity, cheese curd , cheese solids and calsium [ca] can be seen in Table 1.Description: Different superscripts on each row indicateds high significant (P≤0.01)

3.1Antioxidant activity.
[The results of statistical analysis showed that differences in bromelain enzyme concentrations had a very significant effect [P 0< .01] on cottage cheese antioxidants.The value of antioxidant activity increased with increasing concentrations of the bromelain enzyme in the form of ascorbic acid.The increase in antioxidant activity is due to the bromelain enzyme having antioxidant and anthocyanin components.The antioxidant value of cottage cheese ranges from 21.70 to 38.12%.The higher the concentration of bromelain extract in the cottage cheese formulation, the higher the antioxidant activity produced.Antioxidant activity is the ability of oxidants in a food to capture free radicals.The percentage of the results of testing the antioxidant activity with the DPPH method shows the ability of antioxidants in food ingredients to capture free radicals by 50% (IC50).The higher the antioxidant activity and the higher the antioxidant levels, the less food is needed to capture free radicals .[7]

Cheese curd
The results of statistical analysis showed that differences in bromelain enzyme concentrations had a very significant effect [P< 0.01] on cottage cheese antioxidants.The value of antioxidant activity increased with increasing concentrations of the bromelain enzyme in the form of ascorbic acid.The increase in antioxidant activity is due to the bromelain enzyme having antioxidant and anthocyanin components.The antioxidant value of cottage cheese ranges from 8.08 to15.17%.One of the most important parameters in assessing the enzyme alternatives for cheese making is the yield by weight of the produced cheese.Optimal cheese yield is important for cheese production, as it would determine the cost and benefit.The total cheese yield during processing is affected by several factors, such as enzyme concentration, temperature, and pH during processing.In this research, the total cheese yield was significantly different between each treatment, which is 8.08 ± 0.02% (K1); 10.15± 0.03% (K2); 15.17± 0.06% (K3), and 9.34±0.37%.It can be seen that the usage of bromelain enzyme as much as 4.5% produced lower cheese yield, while 5% bromelain had higher yield compared to the 3% and 5% bromelain enzyme concentration.This showed that 4% of bromelain enzyme concentration would produce more optimum cheese yield compared to other concentrations.
Explained that an optimum pH and enzyme concentration could initiate efficient milk coagulation, resulting in a compact curd and higher cheese yield.[8]

Total solids
The average total solids of cottage cheese can be seen in Table 1.Statistical analysis showed that differences in bromelain enzyme concentrations had a very significant effect (P 0.01) on total cottage cheese solids.The results obtained ranged from 20.25 to 60.60%; the highest mean value of total solids in cottage cheese was in treatment K4, at 4%, and the lowest was in treatment K1, at 0%.From these results, it can be explained that the total solids are affected by the bromelain enzyme.The average total cheese solids produced ranged from 20.25% to 60.60%; [9]  The table1 below shows the average calcium level.According to statistical analysis, variations in the levels of the bromelain enzyme had a significant impact [P0≤01] on the total calcium.With the addition of the bromelain enzyme, calcium levels rose.The inclusion of the bromelain enzyme had an impact on cottage cheese's calcium levels, as seen by the treatment with the highest calcium level-5% concentration-of 5.73% and the lowest calcium level-K1 concentration-of 30.34%.With the exception of K1 without the addition of the bromelain enzyme, all treatments that included it raised calcium levels.Calcium functions as a bridge between negatively charged phosphate groups and helps to balance the negative charge of casein micelles.reduces the time needed for coagulation and boosts curd hardness during the manufacturing of cheese.[10] .Research, the level of acidity during the cheese-making process and fluctuations in pH during ripening had an impact on the calcium levels of cheddar cheese.[11]

Conclusion
The results of the study concluded that bromelain concentrations up to 5% produced good antioxidant activity and physical properties of cottage cheese with a very significant difference P≤0.01.This indicated that the bromelain enzyme from pineapple extract could be used as a cheap substitute for rennet in making cheese.

Table 1 .
Antioksidan Activity and Physical of Cottage Cheese the total cheese solids ranged from 45.51 to 84.70%.