Amino Acid, Fatty Acid and Curd Profile of Cottage Cheese using Crude Extract of Bromelain Enzyme (Ananas comosus) as Coagulant Source

The nutritional value, functional characteristics and bioactive potential of dairy products are significantly influenced by their amino acid and fatty acid profiles. Amino acids serve as the fundamental building blocks of proteins, whereas fatty acids, the primary constituents of lipids, play various physiological roles, including serving as energy sources, hormone precursors and structural components of cell membranes. Fatty acids are generally categorized into two groups: saturated fatty acids (SFAs) and unsaturated fatty acids (UFAs). While SFAs are associated with elevated blood cholesterol levels, UFAs are known to confer health benefits, particularly in supporting cardiovascular health (Fox et al., 2017; Sartika, 2008).
       
In cheese production, the type of coagulant enzyme used can influence the final nutritional composition, particularly the amino acid and fatty acid profiles. Traditionally, rennet-an enzyme complex derived from the stomachs of ruminant animals-has been widely used in cheese making. However, the use of animal-derived rennet raises concerns regarding halal compliance and sustainability. Additionally, the high cost and limited local availability of rennet in countries such as Indonesia contribute to elevated cheese production costs (Mijan et al., 2010; Kayagil, 2006).
       
Previous studies have demonstrated the coagulating potential of plant/fruit extracts, such as artichoke (Fguiri et al., 2024), papain enzyme (Malaka et al., 2024) and kiwi extract (Fguiri et al., 2023) as a rennet substitute in cheese making. Bromelain, a proteolytic enzyme extracted from pineapple (Ananas comosus), presents a promising natural alternative to animal-derived rennet. Bromelain has been widely studied for its protein-degrading capabilities and is commonly applied in the food, pharmaceutical and biotechnology industries (Pavan et al., 2012; Arshad et al., 2014). In the context of cheese making, bromelain may facilitate milk coagulation and influence the release of peptides and amino acids from milk proteins. Moreover, its proteolytic activity may also assist in the separation and profiling of lipids, thus affecting the fatty acid composition of cheese.
       
Cottage cheese is a fresh, unripened soft cheese known for its mild flavor, soft texture and favorable nutritional profile. In Indonesia, commercially available cheeses are predominantly hard cheeses that require long ripening times and higher production costs. Developing cottage cheese using bromelain as a milk coagulant offers a cost-effective and halal-compliant alternative while potentially enhancing nutritional quality.
               
There is limited information on how the use of bromelain as a coagulant affects the amino acid and fatty acid composition of cheese. Therefore, this study aims to evaluate the effects of bromelain enzyme from pineapple on the amino acid, saturated fatty acid and unsaturated fatty acid profiles in cottage cheese. The findings are expected to support the development of high-quality, nutritionally balanced and economically viable cheese products that meet consumer preferences and dietary requirements.

Sample preparation and cheese making process
 
The preparation of cottage cheese and curd sample and also the analysis, were conducted in May to June 2024 at the Animal Products Laboratory of the Faculty of Animal Husbandry, Sam Ratulangi University, Manado. In this study, four treatments of bromelain enzyme (as the milk coagulant) concentration levels were used, which are K1 = 0%, K2 = 3%, K3 = 4% and K4 = 5%. At first, the bromelain enzyme was extracted by blending and pressing the juice from the pineapple fruit. The fresh milk (16 L) was pasteurized using the high temperature short time (HTST) method for 15 minutes at 70^oC before being used to make the cottage cheese. After pasteurization, the bromelain enzyme was added with different levels of treatment to be tested and cooled to 400^oC. When it is cooled down, the mixture is then incubated for 35 minutes, filtered through a gauze filter and then 1% salt is added. After being kept at -17^oC for 24 hours, the curd was then examined.
 
Analysis procedure
 
Total curd yield
 
Curd is a parameter to determine the amount of curd formed after the milk casein is coagulated and has been separated from the whey. Total curd can be calculated using the following formula:

Amino acid content

 

The identification of amino acids was carried out by analyzing their chemical reactions with functional groups common to all amino acids, particularly the amino (-NH‚ ) and carboxyl (-COOH) groups, which produce distinctive reactions (Lehninger et al., 2008). A 5-gram sample was weighed and placed into a beaker, then mixed with 100 mL of acid orange reagent while stirring. After the solution became homogeneous, 10 mL of phosphate buffer was added. A chromatography paper measuring 3 cm in width and 10 cm in length was prepared and a sample spot was marked 3 cm from the top. The chromatography tube was filled with approximately 2 cm of carrier solution and the filter paper was inserted such that the spot remained 1 cm above the liquid level, allowing capillary action to separate the sample components based on molecular size and interaction with the carrier. This process was left overnight. The following day, the paper was sprayed with a dye reagent to reveal colored spots corresponding to different amino acids. Standard solutions of essential amino acids were prepared and spotted similarly for comparison. Each visible spot was then cut out and chemically processed to convert the amino acids into ammonium compounds. The absorbance of these compounds was measured and compared with the standards to determine the concentration of each amino acid in the sample.

 

 

The fatty acid content was measured using the gas chromatography (GC) method. To begin the extraction process, 10 grams of the sample were placed into a 100 mL test tube, followed by the addition of 1.25 mL of concentrated ammonium hydroxide (NH₄OH). The mixture was homogenized, then 10 mL of ethanol (C₂H₅OH) was added and mixed thoroughly. The sample was subsequently extracted with 25 mL of diethyl ether and then with 25 mL of petroleum benzine (boiling point range 40-60^oC). The mixture was shaken for one minute and allowed to stand until phase separation occurred, forming two distinct upper layers. These upper layers were carefully pipetted into a new tube and evaporated using a water bath at 45^oC with a flow of nitrogen gas until dry. The remaining residue represented the fat or oil content of the sample. To prepare fatty acid methyl esters (FAMEs), the fat residue was reacted with 1.5 mL of a 20% boron trifluoride (BF₃) methanol solution in a sealed tube. The mixture was heated in a water bath at 45^oC for 30 minutes, then cooled. The resulting methyl esters were extracted with 0.5-1 mL of n-hexane, forming two layers. The upper layer, containing the methyl ester-n-hexane mixture, was transferred to a bottle containing anhydrous sodium sulfate (Na₂SO₄) and allowed to stand for 5-10 minutes to remove any residual moisture. Finally, the clear hexane layer was transferred into a GC vial, flushed with nitrogen gas, sealed tightly and either stored in a freezer or directly analyzed by gas chromatography.

Total cheese curd
 
The results of statistical analysis showed that the difference in bromelain enzyme concentration had a very significant effect [P≤0.01] on cheese curd. One of the most important parameters in assessing enzyme alternatives for cheese making is based on the weight of the cheese produced. Optimal cheese yield is important for cheese production because it will determine the costs and benefits. The total cheese yield during the process is influenced by several factors, such as enzyme concentration, temperature during the process. In this study, the total cheese curd was very significantly different between each treatment, namely 9.07% [K1], 11.16% [K2], 16.18% [K3] and 10.12% [K4], (Fig 1) . The use of 5% bromelain enzyme produced low cheese curd; this was due to the high proteolytic activity of bromelain at that concentration, which could cause excessive breakdown of milk protein. As a result, the curd structure became less dense and its quality decreased. Optimal enzyme concentration can initiate efficient milk coagulation, resulting in compact curd and higher cheese yield (Myagkonosov et al., 2023; Cai et al., 2024). The use of bromelain enzyme at too high a concentration, such as 5%, can cause over-hydrolysis of milk proteins, resulting in a less dense curd and decreased cheese quality (Walstra et al., 2006).


 
Amino acid profile
 
Amino acids are basic components of proteins that play an important role in various biological functions, including the formation and maintenance of body tissues. Amino acid profile is the type and composition of amino acids that make up cottage cheese, which includes various types of essential and non-essential amino acids that contribute to the nutritional value of a product (Geantaresa et al., 2010). Cottage cheese also contains non-essential amino acids, making it a good source of protein. For example, the total amino acid content in cottage cheese can vary depending on the manufacturing method and raw materials used (Arifiansyah, 2020).
       
Table 1 shows the amino acid composition of cottage cheese made with varying concentrations of bromelain enzyme (0%, 3%, 4% and 5%) used as a coagulant. Overall, the results demonstrate that the addition of bromelain significantly increases the concentration of most amino acids. For instance, L-Glutamic Acid rises from 0.70 at 0% bromelain to 2.08 at 5%, while L-Leucine and L-Lysine also show sharp increases. Even amino acids present in smaller amounts, such as L-Histidine and L-Arginine, exhibit consistent upward trends. Bromelain is a proteolytic enzyme, meaning it breaks down proteins into smaller peptides and free amino acids (Komansilan et al., 2021; 2024). As the concentration of bromelain increases, it facilitates more extensive hydrolysis of milk proteins like casein and whey, making amino acids more accessible and measurable.


       
The highest increase of amino acid content at 5% bromelain enzyme was the glutamic acid (C₅H₉NO₄) because glutamate is one of the amino acids that is most easily produced through the hydrolysis process by this enzyme. The bromelain enzyme has a high affinity for proteins containing glutamine, producing glutamate in greater amounts compared to other amino acids, such as L-glutamine (C₅H₁₀N₂O₃) (Anggraini et al., 2013). This enzymatic activity improves the nutritional profile of the cheese by increasing its amino acid content and potentially enhancing its digestibility and flavor. Glutamic acid is known for providing umami, one of the five basic tastes (along with sweet, sour, bitter and salty). Cottage cheese contains glutamic acid, which contributes to its savory taste, which is often one of the main attractions of cheese products. As well as improving the quality of the taste of cottage cheese (Abdelmontaleb et al., 2021). Research shows that the use of bromelain enzyme from pineapple fruit can affect the casein coagulation process and increase proteolytic activity, which in turn affects the physical and chemical quality of cheese (Walther et al., 2008).
 
Saturated fatty acid profile
 
The results presented in Table 2 indicate that the addition of bromelain enzyme from pineapple extract leads to a progressive reduction in the content of saturated fatty acids in cottage cheese. As the enzyme concentration increases from 0% to 5%, the levels of nearly all saturated fatty acids, including butyrate, hexanoate, octanoate, decanoate and notably palmitic acid (C16:0), decrease consistently. Palmitic acid (C₁₆H₃₂O₂) remains the most abundant saturated fatty acid across all treatments, although its concentration drops from 35.93% at 0% bromelain to 35.20% at 5%. Meanwhile, three types of long-chain saturated fatty acids, namely docosanoate (C₂₃H₄₆O₂), tricosanoate (C₂₃H₄₆O₂) and lignocerate (C₂₄H₄₈O₂) acids, remain below detectable levels (<0.1%) in all samples.


       
The decrease in saturated fatty acid content with increasing bromelain concentration is likely due to enzymatic lipid degradation that occurs during the cheese-making process. Bromelain, being a proteolytic enzyme, may indirectly influence fat breakdown by disrupting protein-fat interactions and weakening fat globule membranes, facilitating the release and potential hydrolysis of fatty acids. This aligns with previous findings that fat degradation can occur with enzymatic assistance during cheese production (Kayagil, 2006). Additionally, the variation in fatty acid levels may be influenced by the structural composition of milk fat and its membrane, which affects fat retention or release during curd formation (Collins et al., 2003). Overall, the results suggest that the use of bromelain as a coagulant not only impacts protein hydrolysis but also contributes to the modification of fat composition in cottage cheese, potentially improving its nutritional profile by reducing saturated fat content.
 
Unsaturated fatty acid profile
 
As shown in Table 3, the addition of bromelain enzyme from pineapple extract led to a progressive increase in the content of unsaturated fatty acids in cottage cheese. The most substantial increase was observed in methyl trans-9-elaidate acid, which rose from 13.03% in the control to 13.15% at 5% bromelain concentration, making it the predominant unsaturated fatty acid across all treatments. Other unsaturated fatty acids, such as cis-9-oleate, palmitoleate and linoleate, also showed incremental increases with rising enzyme levels. In contrast, several long-chain unsaturated fatty acids (cis-11-eicosenoate, 11,14-eicosadienoate, cis-11,14,17-eicosatrienoate and docosahexaenoate) remained below detectable levels (<0.1%) across all samples.


       
The increased presence of unsaturated fatty acids, particularly methyl trans-9-elaidate, is attributed to the proteolytic activity of bromelain, which enhances the release of triglyceride-bound lipids during protein hydrolysis. As bromelain breaks down casein and other milk proteins, it disrupts fat-protein complexes, promoting the liberation and transformation of lipids into free fatty acids (Shah et al., 2014). The enzymatic hydrolysis reaches optimal efficiency at 5% concentration, facilitating the formation of more stable and readily formed unsaturated fatty acids. The chemical structure of methyl trans-9-elaidate may favor greater interaction with bromelain, allowing it to accumulate at higher levels than other fatty acids (Shingfield et al., 2013).
       
The use of bromelain as a milk coagulant significantly influences the physicochemical and nutritional properties of cheese compared to traditional rennet and other alternative enzymes. Bromelain, being a broad-spectrum proteolytic enzyme, tends to produce a softer curd structure and may accelerate protein hydrolysis, leading to higher levels of free amino acids in the final product. This can enhance the nutritional value and bioactivity of the cheese, but may also affect textural integrity and yield. In contrast, rennet-particularly chymosin-has a highly specific cleavage site in κ-casein, resulting in a firmer curd, better moisture retention and more consistent sensory attributes (Fox et al., 2017). However, rennet’s animal origin poses limitations in terms of cost, availability and religious or ethical acceptability.
       
When compared to other plant-derived enzymes, such as papain (from papaya), ficin (from figs), or cardosins (from thistle), bromelain exhibits comparable or superior coagulation ability, but its higher proteolytic activity can lead to excessive proteolysis if not carefully controlled. Over-hydrolysis may result in bitterness or poor curd formation (Shah et al., 2014). Microbial coagulants, often produced from genetically modified strains of Rhizomucor miehei or Aspergillus niger, offer high yield and process stability, but may also vary in specificity and performance. Compared to these enzymes, bromelain stands out for its natural origin, environmental friendliness and potential cost-effectiveness, especially in tropical regions where pineapple is abundantly available. However, optimizing its concentration and activity remains critical to balance curd firmness, yield and nutritional quality, making it a viable but still underutilized coagulant in cheese technology.
       
Overall, these findings suggest that bromelain not only functions effectively as a milk coagulant but also contributes to a favorable modification of the lipid profile in cottage cheese. The increase in unsaturated fatty acids, particularly trans-9-elaidate, indicates an improvement in the nutritional quality of the product through enzymatic enhancement of lipid metabolism during cheese manufacture (Anggraini et al., 2013; Mahmood and Usman, 2010).

It can be concluded that the concentration of bromelain enzyme up to 5% produced a profile of amino acids, saturated fatty acids and unsaturated fatty acids contained in cottage cheese. The higher the concentration of bromelain enzyme, the higher the content of amino acids and unsaturated fatty acids, while saturated fatty acids were lower in cottage cheese. Meanwhile, the 4% bromelain enzyme produced the highest total cheese yield (curd). Bromelain enzyme from pineapple extract can be used as a cheap substitute for rennet in making cottage cheese.

The present study was supported by the authors gratefully thank the Rector of Universitas Sam Ratulangi Manado and the Chairman of LPPM Universitas Sam Ratulangi Manado. This research was financially supported by the RDUU Cluster 1. Number SP DIPA - 023.17.2.677519/2024.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

There was no conflict of interest associated with this research by any of the authors.