1 1

1OPTIMIZATION OF PINEAPPLE CROWN VARIETIES IN

2ISOLATION AND PURIFICATION OF BROMELAIN
3
4Siti Susanti*, Heni Rizqiati, Hendriy Rinaldi Setiawan, Fahmi Arifan
5
6Food Technology Division, Department of Agriculture, Faculty of Animal Science and
7Agriculture Diponegoro University, Semarang, Indonesia, 50275
8Diploma Program of Chemical Engineering, Faculty of Vocational School, Diponegoro
9University, Semarang 50275, Indonesia
10*Corresponding Author e-mail: sitisusanti@live.undip.ac.id
11

121. Abstract
13There were 8 varieties of pineapple are commonly commercialized in the world, but in Indonesia
14only the Queen, Cayenne, and Honi varieties are commonly found in the market. The high level
15of consumption of the 3 varieties resulted in abundant post-harvest waste. Pineapple crown is a
16waste that has been known to contain high concentration of bromelain content. This study aims
17to determine the optimization of pineapple crowns from 3 different varieties as a source of
18bromelain. Pineapple crowns of Queen, Cayenne, and Honi varieties were dried under the sun
19until the moisture content was less than 8% then isolated the bromelain. The optimum variety of
20pineapple crown was determined based on the characteristics of crude bromelain (protein
21content, activity unit, and specific activity). Crude Bromelain from the best varieties was further
22purified with ammonium sulfate solution (20, 40, 60, and 80%). The optimization of ammonium
23sulfate concentration was determined based on the characteristics of the bromelain extract
24produced. The highest protein content of crude bromelain was obtained from Queen variety
25while the highest unit activity and specific activity was obtained from Cayenne variety. The
26highest protein content and unit activity of pure bromelain was obtained from 60% ammonium
27sulfate but the highest specific activity was obtained from 40% concentration. Pineapple crown
28of Cayenne variety is the best source of bromelain with specific activity of 0.82 U/mg for crude
29bromelain and 1.94 U/mg for pure bromelain.
30Keywords: Bromelain, Optimization, Pineapple Crown, Varieties.
 1 2

12. Introduction
2 Pineapple is a very popular fruit consumed in the world with a production capacity that
3continues to increase every year (Abu et al., 2013). It is not only consumed as fresh fruit and
4processed products such as jam, probiotic drinks, fried pineapple, and sweetmeat but also a
5major source of natural bromelain (Hossain et al., 2015). Bromelain (EC 3.4.22.32) is a protease
6enzyme group which has the ability to catalyze the breakdown of proteins into amino acids
7through hydrolysis reactions or the decomposition of large molecules into smaller molecules
8with a combination of water (Chaurasiya and Hebbar, 2013). The protease enzyme itself is a type
9of hydrolase enzyme that dominates the world market share, which is about 60% of the total
10other enzyme groups (Feijoo-siota and Villa, 2011). The application of bromelain in the food
11sector itself is widely used as a bread developer, tenderization agent in the hydrolysis of
12myofibrils in meat (Hage et al., 2013), anti-browning agent to inhibit browning in fruit and
13phenol oxidation (Sarkar et al., 2017), regulates beer stability and reduces foam formation in
14alcoholic beverages (Benucci et al., 2011), and as an agent for producing protein hydrolysates
15from fish or meat (Elavarasan et al., 2014). In fact, some parts of the pineapple such as the stem,
16crown, and skin which are considered as waste, instead contain high ammount of bromelain
17(Nurhidayat et al., 2018). Based on the characteristics of the bromelain enzyme produced, the
18pineapple crown obtained from the Bogor area showed greater value of enzyme activity than
19other parts such as the skin and core which could reach 29.76% of the total bromelain enzyme
20activity unit in all parts of the pineapple (Mulyono et al., 2013). Pineapple crown waste itself
21was also abundant, very easy to handle and utilize. Pineapple crown can weigh up to 23.50% of
22the total pineapple weight. By looking at pineapple production capacity in Indonesia what could
23reach 1.73 million tons with an average weight of 1.8 kg, it was meant that the pineapple crown
24waste produced per harvest could reach 731,790 tons (Mohdali et al., 2020).
25 One of the important considerations in choosing pineapple crowns for producing bromelain is
26the type of variety used. There were more than 100 varieties of pineapple in the world but only 8
27varieties are mass-produced for commercial (Steingass et al., 2020). In Indonesia, there are 3
28types of pineapple varieties that are commonly found in the market, namely Queen, Cayenne,
29and Honi. The selection of varieties need to be carried out to produce high quality bromelain
30with uniform characteristics. Based on previous studies conducted in Ghana and Nigeria on
31bromelain isolated from 2 varieties and 3 species of pineapple crown, it showed that there were
 1 3

1differences in sensitivity to certain pH and temperature which impacted on the characteristics in

2terms of protein content which describes the quantity of enzymes, activity units that describe the
3ability to break down proteins, then specific activities that describe enzymes purity (Benefo and
4Issac., 2018). The sensitivity of bromelain to temperature and pH determines the final quality of
5bromelain considering the drying process and the use of various solutions during extraction to
6purification. Differences in sensitivity of each variety could be caused by differences in
7environmental conditions in the planting area which include climate and weather, soil conditions,
8humidity levels, and rainfall (Kusuma et al., 2015). Differences in harvesting methods and
9standards of maturity level also affect the characteristics of the bromelain enzyme of each variety
10of pineapple crown. Pineapple crowns from fruits harvested at a young age tend to have high
11protease activity compared to pineapple crowns harvested at an ripe stage for consumption
12purposes (Bresolin et al., 2013). The proportion of bromelain in each part on some varieties also
13varies according to the number and type of constituent enzymes that dominate in certain parts,
14such as the proportion between ananin and comosain which dominates in the fruit and stem
15(Kumar et al., 2011).
16 Previous studies only focused on explaining the characteristics of bromelain from several
17varieties without an in-depth explanation of the factors causing those differences. Previous
18studies also have not involved the drying process before the extraction, whereas the high water
19content of the pineapple crown waste causes it to be easily damaged due to decay which affect
20the quality of the bromelain produced. This study aimed to find the optimum variety
21optimization and compared the protein content, unit activity, and specific activity of the
22bromelain enzyme isolated from 3 local varieties of pineapple crown in Indonesia consisting of
23Cayenne, Honi, and Queen.

243. Experimental Section

253.1. Materials
26 The materials used were pineapple crown waste of cayenne, queen, honi variety obtained
27from PT Lion Super Indo, Ngesrep and Banyumanik Traditional Market Semarang, distilled
28water, tissue, aluminium foil, muslin cloth, solution of sodium citrate buffer pH 6 (Merck,
29Germany), BSA (Sigma-Aldrich, USA), Lowry reagent (Thermo, USA), foline solution (Merck,
30Germany), trichloroacetic acid 10% (Merck, Germany), casein (Sigma-Aldrich, USA), standard
 1 4

1bromelain enzyme (Life Extension, USA), phosphate buffer (Merck, Germany).

23.2. Methods
3 3.2.1 Preparation of Making Tofu
4 Pineapple crowns of each variety were sorted, cut into small pieces, and blended. The
5pineapple crown was then dried for 2-3 days under the sun. The temperature was checked
6periodically 3 times a day every morning, afternoon and evening for monitoring. Drying was
7stopped when the water content of the pineapple crown slury had reached <8% (Hanifah et al.,
82017). The dried pineapple crown was then ground with a grinder for 4 minutes and sieved with
9a 40 mesh sieve. The pineapple crown powder was homogenized in a cold citrate buffer pH 6
10then filtered with a muslin cloth. The ratio of powder and solvent was 1:9. The filtered solution
11was then centrifuged at 5000 rpm for 15 minutes (Kaumang and Kamu, 2011). The centrifuged
12supernatant was separated into a tube and stored at -25 °C for preparation of parameter testing.
13The best variety according to the test result was purified by dissolving 10 ml of sample with
14ammonium sulfate until it reached saturation of 20%, 40%, 60%, and 80% (Hartesi et al., 2020).
15The sample solution was incubated for 24 hours at 4℃ then centrifuged at 3500 rpm for 25
16minutes. The precipitate resulting from the centrifugation was dissolved in 1 ml of citrate buffer
17solution pH 6. The dissolved precipitate was put into a dialysis plastic, tied, and put into a plastic
18tube filled with sodium citrate buffer solution to immerse the entire surface of the sample in the
19dialysis plastic. Dialysis was carried out for 6 hours and sodium citrate buffer in a plastic tube
20was replaced every 3 hours twice (Priya, 2012).
21
22 3.2.2 Protein Content
23 Protein content was determined by the Lowry method (Jayanti, 2013). Standard solution of
24BSA 1.00 mg/mL was reacted with Lowry's reagent and incubated for 10 minutes. The solution
25mixture was then mixed with Folin-Ciocalteau reagent and incubated for 30 minutes. The
26solution was then read for absorbance using a spectrophotometer at a wavelength of 650 nm to
27form a standard BSA curve. A total of 0.5 ml of bromelain extract was added to 5 ml of Lowry's
28solution, vortexed, and incubated for 10 minutes at room temperature. The extract was then
29added with 0.5 mL of 0.5 mL of Folin reagent and incubated for 30 minutes at room temperature.
30The absorbance of the extract solution was then measured with a spectrophotometer at the same
31length as BSA. The protein content of the extract solution was determined based on linear
 1 5

1regression on the BSA standard curve.

2
3 3.2.3 Enzyme Activity Unit
4 The activity unit of bromelain enzyme crude extract isolated from pineapple crown was
5determined based on its ability to catalyze casein substrates (Suhartono and Artika, 2017). 0.5 ml
6of 0.5% (w/v) casein substrate was added to 0.5 ml of bromelain extract which had been diluted
715 times. 0.5 ml of phosphate buffer pH 6 was added to the solution and incubated at 40°C in a
8water bath for 20 minutes. The reaction between the bromelain extract and casein was stopped by
9adding 1 ml of 10% trichloroacetic acid and incubated for 10 minutes at room temperature. The
10solution was centrifuged at 4000 rpm for 20 minutes to get the supernatant. The absorbance of
11the supernatant was read at a wavelength of 280 nm using UV-vis spectrophotometry. The unit
12activity value is the result of division between the absorbance value of the sample and the blank
13with the absorbance of the standard and blank multiplied by the dilution factor and incubation
14time (Sumardi et al., 2020).
15
16 3.2.4 Enzyme specific activity
17 Specific activity measurement was carried out to determine the purity level of the bromelain
18enzyme produced. The specific activity value was determined by dividing the unit activity value
19by the protein content of the sample (Tazkiah et al., 2017).

204. Results and Discussion

214.1. Protein Content of Bromelain Crude Extract

22 The results of protein content measurement on bromelain crude extract isolated from 3
23varieties of pineapple crown can be seen in Illustration 1.
24
25
26
27
28
29
30
31
 1 6

1 Illustration 1. Protein content of pineapple crown from 3 different varieties

2 Based on Illustration 1. obtained the result that the highest protein content was found in the
3queen pineapple variety, which was 3.524 mg/mL, followed by honi and cayenne varieties.
4Enzymes are proteins, so by determining the overall protein content, it describes the amount of
5protein that functions as an enzyme through its ability to convert the substrate into the desired
6product (Effaliza et al., 2019). Each pineapple crown variety was obtained from a different areas.
7Cayenne varieties from Subang, Queen from Bogor and Palembang (Mulyono, 2013) and Honi
8from East Lampung (Sunpride, 2021). Differences in planting and harvesting areas are related to
9differences in soil characteristics and productivity. Areas with low contours which bordering the
10coast, connected access of the river to the sea, and inadequate irrigation have an impact on high
11soil salinity levels. High levels of salinity cause the phenomenon of salt stress due to ion
12poisoning, lack of nutrients and water supply in plants that inhibit protein synthesis (Vilanova et
13al., 2013). The salt stress phenomenon is characterized by the low supply of potassium through
14the plant roots which disrupts the physiological balance due to the low mineral raw materials for
15protein synthesis (Wang et al., 2018). If the stress condition continues for a long time, it will
16cause a drastic decrease in protein levels. The topographical condition of the Bogor area which
17has an average contour height of 100-500 meters above sea level (MASL) supported by adequate
18soil nutrients is one of the causes of the high protein content in Queen variety pineapple crown
19grown in Bogor area. Communication, Informatics, and Public Relations Office of Bogor stated
20that the mountainous area of Bogor can even reach 1000-2000 masl compared to the average
21height of the Palembang plain which only reaches 4-21 masl and the Subang plain as high as 0-
2250 masl (Diskominfo Kabupaten Bogor, 2021). Protein content in the crown of ripe pineapple
23tends to increase due to degradation during the ripening process for the formation of certain
24compounds (Detris et al., 2013). Queen and Honi Pineapple are commonly sold in the market in
25a ripe condition with a sweet taste for consumption. Therefore, both varieties contain higher
26levels of protein than Cayenne. Determining the protein content is not sufficient to describe the
27proteolytic activity of the bromelain enzyme because there is still the possibility of other types of
28protein being calculated as total protein (Silvestre et al., 2012).
29 4.2. Enzyme Activity Unit
30 The results of enzyme activity unit measurement on bromelain crude extract isolated from 3
31varieties of pineapple crown can be seen in Illustration 2.
 1 7

1.800 1.760
1.750

Activity Unit (U/ml)

1.700
1.650 1.630
1.600
1.550 1.533
1.500
1.450
1.400
Cayenne Queen Honi
Variey of Pineapple Crown
1
2 Illustration 2. Enzyme activity unit of pineapple crown from 3 different varieties
3
4 Based on Illustration 2.it is known that the highest value of activity unit was found in the
5Cayenne variety, which was 1.760 U/mL, followed by the Honi and Queen varieties. The high
6enzyme activity in the Cayenne pineapple crown is to be closely related to the standard of
7maturity level during the harvesting process. The Cayenne varieties are commonly harvested
8half-ripe with green skin while the Queen and Honi varieties are harvested when they reach
9perfect maturity. Based on observations, Cayenne pineapple is indeed sold in a young and half-
10ripe condition for certain use such as meat tenderizer agent and for health purposes. During the
11fruit development before reaching 100% maturity, protease activity of bromelain in pineapple
12crown tends to be high as a form of defensive expression against pathogens and predators (Pang
13et al., 2020). Higher proteolytic activity in young pineapple is a form of gene expression to
14produce unwanted sensations in the tongue tissue of mammals to prevent them consuming it and
15to increase anti-pathogenic properties against insect and fungal pests such as Cyanophora
16paradoxa, Diaspis bromeliea, and mealybugs. The high expression of the pineapple crown gene
17at the early stage of ripening is a unique characteristic of bromelain belonging to the CA1
18protease group. During the ripening process, high bromelain activity was triggered by the
19expression of several genes such as AccCEP3 and AccPAP25 in the fruit and AcbPAP10 in the
20pineapple crown tissue which showed a dynamic expression pattern with high activity at certain
21ripening stages (Chen et al., 2019). It still has not been known for sure about the mechanism of
22this gene in increasing bromelain activity, but it was estimated that the gene inhibited and
23inactivated bromelain inhibitor genes, namely AccBI1 and AccBI2 during the maturation process
24which was characterized by the increasing of bromelain content, tissue proteolysis, softening and
 1 8

1protein degradation. When it reached the maximum maturity level, the expression of these genes
2decreased with the initial value of reads per kilobase per million mapped reads (RPKMs) of
3443.814 to 0 RPKMs. This phenomenon is in line with the results of the previous research which
4explained that the pineapple ripening phase consists of 3 main stages, namely the young, old, and
5ripe phases (Omotonyibo and Sani., 2017). Bromelain activity increases exponentially from the
6young phase to the old phase and drops dramatically from the old to mature or ripe phase.
7Decreasing in activity during fruit ripening occurs due to the formation of certain compounds
8which involving the use of enzyme as ingredient that causes the enzyme damage by breaking
9disulfide bonds, peptides, or changes in the active site of the enzyme which inactivate it (Nor et
10al., 2015).
11 4.3. Enzyme Specific Activity
12 The results of enzyme specific activity measurement on bromelain crude extract isolated
13from 3 varieties of pineapple crown can be seen in Illustration 3.

14
15 Illustration 3. Enzyme specific activity of pineapple crown from 3 different varieties.
16
17 It is known that the highest specific activity value was found in the cayenne pineapple crown
18which reached the value of 0.817 U/mg followed by honi and queen varieties. The specific
19activity measurement is parameter to determine the level of purity of an enzyme because the high
20concentration of protein is not always directly proportional to its proteolytic activity due to the
21presence of other proteins besides the targetted enzyme which was also measured in the total
22protein concentration measurement (Feng et al., 2017). This result is in line with the previous
23study which explained that the mixture of pineapple waste of smooth cayenne variety consisting
24of crown, stem, and pineapple peel has also been shown to have greater specific activity than the
 1 9

1Red queen and Bogor queen varieties with a difference in specific activity value of 0.14. -0.16
2U/mg (Charlena et al., 2013). The main key of specific activity measurement is the balance
3proportion between the value of the activity unit and the protein content. There were several
4cases where the specific activity measurement results using spectrophotometer were too high due
5to the absorbance value in protein content measurement which was too low. The error reading in
6spectrophotometer happens due to the inhibition of color absorption by brown pigments from
7polyphenols contained in the enzyme extract (Dwi et al., 2011). Therefore, the addition of a
8polyphenol binding agent such as silica needs to be added to increase absorbance readings in
9measuring protein content, separating polarized components such as aromatic hydrocarbons and
10mixtures of less polar dissolved components such as esters, phenols, and aliphatic ethers.
11Another thing that needs to be considered is to ensure that the drying process for samples is not
12carried out for too long and must be protected from rain to maintain the value of the activity unit.
13The constraint of the drying process using sunlight is the intensity of heat which depends on the
14weather. Samples that have been exposed to rainwater should not be re-dried and not used
15because the repeated drying process that is too long has clearly damaged the enzyme activity in
16the pineapple crown.
17 4.4. Optimization of Ammonium Sulfate Concentration in Bromelain purification
18 The characteristics of purified bromelain enzyme isolated from pineapple crown of Cayenne
19variety can be seen in Table 1.

20Table 1. The effect of different levels of ammonium sulfate saturation in the purification to protein level,
21 activity unit, and specific activity of cayenne pineapple crown extract.
22
23

Saturation (%) Protein (mg/mL) Activity Unit Specific Activity

(U/mL) (U/mg)

20 0.75 1.03 1.37

40 0.84 1.63 1.94
60 1.63 2.02 1.24
80 1.44 1.62 1.12
24
25 Tabel 1 shows that the evaluation about the effect of differences ammonium sulfate saturation
26level in purification of the cayenne pineapple crown crude extract was based on protein content,
 1 10

1activity unit, and specific activity measurement. The highest protein content and bromelain
2enzyme activity units were found at the 60% saturation level which reached the value of 1.627
3mg/mL and 2.021 U/mL. The highest protein content in purified pineapple crown extract was
4instead lower than the crude extract but the unit activity value was higher. The lower value of the
5protein content measurement after purification is caused by the length of the purification stage
6causing protein deposits which are not collected maximally due to sticking to the glass tools
7(Azmy and Wardani, 2014). The lower value of bromelain activity unit in crude extract with
8higher protein content indicates the bromelain damage due to degradation or denaturation during
9the extraction. Measurement of protein levels could not distinguish between active and inactive
10bromelain enzyme. The two types of protein are considered the same so that the enzyme that has
11been damaged and inactive will still be counted in the measurement of protein content but is not
12detected in the enzyme activity test. The higher post-purification activity unit value is also a
13parameter of the effectiveness and success of the purification. Purification involves 2 main steps
14which consist of precipitation using ammonium to remove a mixture of interfering compounds
15contained in the extract such as carbohydrates, fiber, vitamins, fats, nucleic acids and a dialysis
16process to remove ammonium sulfate salts at the precipitation stage and metal ions which can
17interfere the proteolytic activity. The activity of the protease enzyme can be increased by the
18addition of ascorbic acid to prevent oxidation and the formation of disulfide bonds which
19inactivate the protease enzyme, the addition of EDTA to bind metal ions and enzyme inhibitors,
20addition of some activators from the metal ions of Zn2+, Fe2+, and Mg2+ to increase protease
21catalytic activity and mantain protease conformational stability (Warsono et al., 2019). The
22saturation level of ammonium sulfate which was considered the most effective was 40% based
23on the specific activity value of 1,929 U/mg. Enzyme purity increased by 136% compared to the
24specific activity of crude extract. This result was in line with the previous study which explained
25that solubility of each type of protein varies under certain conditions and at a concentration of
2640% could produce the highest protein content and spesific activity because as the concentration
27of ammonium sulfate salt increased it binded more water which caused the stronger attraction
28between protein molecules so that the protein was precipitated (Nadzirah et al., 2013).
29Ammonium sulfate salt has a high solubility in water and does not react with protein so that there
30is competition between salt and protein to bind water. The ions in proteins bind many water
31molecules very strongly but salt ions have a greater charge density than proteins so that salt can
 1 11

1bind more water molecules (Amid et al., 2011). At a higher concentration of ammonium sulfate
2salt, the specific activity of the enzyme decreases because it has reached the saturation point or
3isoelectric point (Coelho et al., 2013). The most important consideration in measuring specific
4activity is the accuracy of measuring protein levels because the value of specific activity is
5determined from the comparison between the unit value of enzyme activity and protein content.

65. Conclusion
7 Pineapple crown of Cayenne variety is the best source of bromelain because it produces
8crude bromelain extract with the highest specific activity of 0.82 U/mg. Ammonium sulfate 40%
9is the optimum concentration to produce pure bromelain with a specific activity of 1.94 U/mg.
10Further research on the characteristics of powdered bromelain from Cayenne pineapple crown by
11freeze drying method needs to be done to increase the practical value and sustainability of this
12study.

136. Acknowledgement
14 This research was funded by the Program of Penelitian Terapan Unggulan Perguruan Tinggi
15(PTUPT). On this occasion, the author would like to thank the Department of Agriculture, for
16providing laboratory facilities so that the author could complete this research project
17successfully.

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