A heavy-metal assay has been developed using bromelain, a protease. The enzyme is assayed using casein as a substrate with Coomassie dye to track completion of hydrolysis of casein. In the absence of inhibitors, casein is hydrolysed to completion, and the solution is brown. In the presence of metal ions such as Hg2+ and Cu2+, the hydrolysis of casein is inhibited, and the solution remains blue. Exclusion of sulfhydryl protective agent and ethylenediaminetetraacetic in the original assay improved sensitivity to heavy metals several fold. The assay is sensitive to Hg2+ and Cu2+, exhibiting a dose-response curve with an IC50 of 0.15 mg 1(-1) for Hg2+ and a one-phase binding curve with an IC50 of 0.23 mg 1(-1) for Cu2+. The IC50 value for Hg2+ is found to be lower to several other assays such as immobilized urease and papain assay, whilst the IC50 value for Cu2+ is lower than immobilized urease, 15-min Microtox, and rainbow trout.
Pineapple has been used as part of traditional folk medicine since ancient times and it continues to be present in various herbal preparations. Bromelain is a complex mixture of protease extracted from the fruit or stem of the pineapple plant. Although the complete molecular mechanism of action of bromelain has not been completely identified, bromelain gained universal acceptability as a phytotherapeutic agent due to its history of safe use and lack of side effects. Bromelain is widely administered for its well-recognized properties, such as its anti-inflammatory, antithrombotic and fibrinolytic affects, anticancer activity and immunomodulatory effects, in addition to being a wound healing and circulatory improvement agent. The current review describes the promising clinical applications and therapeutic properties of bromelain.
Cysteine proteases in pineapple (Ananas comosus) plants are phytotherapeutical agents that demonstrate anti-edematous, anti-inflammatory, anti-thrombotic and fibrinolytic activities. Bromelain has been identified as an active component and as a major protease of A. comosus. Bromelain has gained wide acceptance and compliance as a phytotherapeutical drug. The proteolytic fraction of pineapple stem is termed stem bromelain, while the one presents in the fruit is known as fruit bromelain. The amino acid sequence and domain analysis of the fruit and stem bromelains demonstrated several differences and similarities of these cysteine protease family members. In addition, analysis of the modelled fruit (BAA21848) and stem (CAA08861) bromelains revealed the presence of unique properties of the predicted structures. Sequence analysis and structural prediction of stem and fruit bromelains of A. comosus along with the comparison of both structures provides a new insight on their distinct properties for industrial application.
Fruit bromelain is a cysteine protease accumulated in pineapple fruits. This proteolytic enzyme has received high demand for industrial and therapeutic applications. In this study, fruit bromelain sequences QIM61759, QIM61760 and QIM61761 were retrieved from the National Center for Biotechnology Information (NCBI) Genbank Database. The tertiary structure of fruit bromelain QIM61759, QIM61760 and QIM61761 was generated by using MODELLER. The result revealed that the local stereochemical quality of the generated models was improved by using multiple templates during modelling process. Moreover, by comparing with the available papain model, structural analysis provides an insight on how pro-peptide functions as a scaffold in fruit bromelain folding and contributing to inactivation of mature protein. The structural analysis also disclosed the similarities and differences between these models. Lastly, thermal stability of fruit bromelain was studied. Molecular dynamics simulation of fruit bromelain structures at several selected temperatures demonstrated how fruit bromelain responds to elevation of temperature.
Knowledge about the thermal and storage behavior of produced protein is important for the purpose of storage, transport and shelve life during industrial application. Recombinant bromelain thermal and storage stability were measured and compared to the commercial bromelain using Differential Scanning Calorimetry (DSC). Recombinant bromelain is more stable than commercial bromelain at higher temperature but the stability was reduced after 7 days of storage at 4oC. Higher energy is needed to break the bond between amino acid chains in recombinant bromelain as shown by the enthalpy obtained, suggesting that recombinant bromelain has good protein structure and conformation compared to commercial.
Angelwing clam (Pholas orientalis) hydrolysate was prepared by hydrolysis using bromelain. The hydrolysate named as bromelain hydrolysate (BH) was then treated with β-cyclodextrin in the ratio of 1:0.8 (v/w) by physical mixing and kneading methods producing the physical mixed hydrolysate (PMH) and kneaded method hydrolysate (KMH), respectively. The masking effect of β-cyclodextrin on bitterness was evaluated based on sensory analysis, amino acid analysis and determination of flavor compound by gas chromatography- mass spectrometry (GC-MS) and field emission scanning electron microscope (FESEM). Sensory analysis showed that KMH has least bitter taste compared to BH. Amino acids analysis showed that hydrophobic amino acids content that contributed to the bitter taste were lower in KMH and PMH compared to BH. GC-MS analysis also showed that benzothiazole compounds were present in KMH. The absence of benzene, 1-phenyl-4-2-(2-cyano-2-phenylethyl) in KMH and PMH indicated that phenylalanine in BH had been masked by β-cyclodextrin. FESEM showed that the new solid phase formed by kneading method has a crystal structure which was completely different from the original morphology of BH and β-cyclodextrin. Therefore, the bitterness in BH had successfully been masked by β-cyclodextrin, thus indicates its potential to be used as food ingredient..
About 60% of world’s commercial enzyme products are proteases, giving promising opportunity
to derive such enzymes sustainably from waste sources. Bromelain is a crude protease occurring
naturally in pineapple, and it possesses properties of benefit for pharmaceutical, medical and food products. The production of bromelain involves a purification stage, normally performed by small-scale conventional operations which lead to high operating cost and low product recovery, while being difficult to scale up and produce polluting by-products. Membrane-based technology offers an alternative to produce high quality purified bromelain in a more efficient and sustainable process. This review identified the current state and future needs for utilising membrane processes for sustainable bromelain production at larger scales. It was found that declining membrane flux due to fouling have been reported, but may be effectively overcome with more appropriate (and advanced) membrane types and/or processing conditions. For example, interactions between macromolecules present in the pineapple derived bromelain mixture (particularly polysaccharides) and the membrane may cause performance limiting fouling, but can be overcome by enzymatic pre-treatment. Membrane fouling can be further reduced by the employment of ceramic membrane filters operating at optimised trans-membrane pressure, cross-flow velocity, feed pH and temperature. Two-stage ultrafiltration together with diafiltration or gas sparging was suggested as a means to reduce fouling and improve enzyme purity. Despite these promising technical findings, the review identified the need for a valid economic assessment to properly guide further work towards purifying bromelain from pineapple waste for sustainable production of commercial proteases.
Bromelain is one of the vegetal proteases found in pineapple plant. It has numerous applications in food and pharmaceuticals. This review discussed different bromelain purification techniques which will assist in determining the effect of processing conditions on the purification efficacy. There are four purification techniques to be discussed, namely; reverse micellar system, aqueous two phase extraction, cation exchange chromatography and ammonium sulphate precipitation. Of the four techniques, cation exchange chromatography had shown the best bromelain purification technique with purification fold of 10.0 followed by reverse micellar system containing CTAB/ isooctane/ hexanol/ butanol, ATPE containing PEG polymer, ammonium sulphate precipitation and ATPE containing PEO-PPO-PEO with purification fold of 5.2, 4.0, 2.81 and 1.25, respectively.
Bromelain, a cysteine protease with various therapeutic and industrial applications, was expressed in Escherichia coli, BL21-AI clone, under different cultivation conditions (post-induction temperature, L-arabinose concentration and post-induction period). The optimized conditions by response surface methodology using face centered central composite design were 0.2% (w/v) L-arabinose, 8 hr and 25°C. The analysis of variance coupled with larger value of R2 (0.989) showed that the quadratic model used for the prediction was highly significant (p < 0.05). Under the optimized conditions, the model produced bromelain activity of 9.2 U/mg while validation experiments gave bromelain activity of 9.6 ± 0.02 U/mg at 0.15% (w/v) L-arabinose, 8 hr and 27°C. This study had innovatively developed cultivation conditions for better production of recombinant bromelain in shake flask culture.
The stone fish is an under-utilized sea cucumber with many nutritional and ethno-medicinal values. This study aimed to establish the conditions for its optimum hydrolysis with bromelain to generate angiotensin I-converting enzyme (ACE)-inhibitory hydrolysates. Response surface methodology (RSM) based on a central composite design was used to model and optimize the degree of hydrolysis (DH) and ACE-inhibitory activity. Process conditions including pH (4-7), temperature (40-70 °C), enzyme/substrate (E/S) ratio (0.5%-2%) and time (30-360 min) were used. A pH of 7.0, temperature of 40 °C, E/S ratio of 2% and time of 240 min were determined using a response surface model as the optimum levels to obtain the maximum ACE-inhibitory activity of 84.26% at 44.59% degree of hydrolysis. Hence, RSM can serve as an effective approach in the design of experiments to improve the antihypertensive effect of stone fish hydrolysates, which can thus be used as a value-added ingredient for various applications in the functional foods industries.
Phospholipase A2 (Pla2) is an enzyme that induces inflammation, making Pla2 activity an effective approach to reduce inflammation. Therefore, investigating natural compounds for this Pla2 inhibitory activity has important therapeutic potential. The objective of this study was to investigate the potential in bromelain-phytochemical complex inhibitors via a combination of in silico and in vitro methods. Bromelain-amenthoflavone displays antagonistic effects on Pla2. Bromelian-asiaticoside and bromelain-diosgenin displayed synergistic effects at high concentrations of the combined compounds, with inhibition percentages of more than 70% and 90%, respectively, and antagonistic effects at low concentrations. The synergistic effect of the bromelain-asiaticoside and bromelain-diosgenin combinations represents a new application in treating inflammation. These findings not only provide significant quantitative data, but also provide an insight on valuable implications for the combined use of bromelain with asiaticoside and diosgenin in treating inflammation, and may help researchers develop more natural bioactive compounds in daily foods as anti-inflammatory agent.
Natural products with immunomodulatory activity are widely used in treatment of many diseases including autoimmune diseases, inflammatory disorders in addition to cancer. They gained a great interest in the last decades as therapeutic agents since they provide inexpensive and less toxic products than the synthetic chemotherapeutic agents. Immunomodulators are the agents that have the ability to boost or suppress the host defense response that can be used as a prophylaxis as well as in combination with other therapeutic modalities. The anticancer activity of these immunomodulators is due to their anti-inflammatory, antioxidant, and induction of apoptosis, anti-angiogenesis, and anti-metastasis effect. These natural immunomodulators such as genistein, curcumin, and resveratrol can be used as prophylaxis against the initiation of cancer besides the inhibition of tumor growth and proliferation. Whereas, immunostimulants can elicit and activate humoral and cell-mediated immune responses against the tumor that facilitate the recognition and destruction of the already existing tumor. This review represents the recent studies on various natural immunomodulators with antitumor effects. We have focused on the relationship between their anticancer activity and immunomodulatory mechanisms. The mechanisms of action of various immunomodulators such as polyphenolic compounds, flavonoids, organosulfur compounds, capsaicin, vinca alkaloids, bromelain, betulinic acid and zerumbone, the affected cancerous cell lines in addition to the targeted molecules and transcriptional pathways have been review and critically analyzed.
Background: This study aimed to evaluate the antitumor enhancing effect of bromelain consumption on 4T1-challenged mice treated with cisplatin. Methods: Mice challenged with 4T1 triple-negative breast cancer cells received water, bromelain, cisplatin, or bromelain + cisplatin treatment for 28 days. Tumor size was measured, and lung metastasis was evaluated by clonogenic assay. Expression of tumor inflammatory genes of the harvested tumor was quantified by polymerase chain reaction array and ELISA (enzyme-linked immunosorbent assay). Results: All treatments significantly reduced the size of tumor and lung metastasis, with combination treatment showing the best effect. Also, bromelain alone and combination treatment showed downregulation of the expression of tumor inflammatory genes (Gremlin [GREM1], interleukin 1β [IL-1β], interleukin-4 [IL-4], nuclear factor κB subunit 1 [NFκB1], and prostaglandin-endoperoxide synthase 2 [PTGS2]), tumor nitric oxide level, and serum IL-1β, and IL-4 levels. On the other hand, cisplatin treatment increased the expression of selected inflammatory markers. Conclusion: This study suggests that bromelain treatment could potentiate the antitumor effect of cisplatin on triple-negative breast cancer 4T1 cells through modulating the tumor environmental inflammation.
Little is known about the efficacy of cysteine proteinases (CP) as anthelmintics for cestode infections. We examined the effects of CPs on two rodent cestodes, Hymenolepis diminuta and H. microstoma in vitro. Our data showed that naturally occurring mixtures of CPs, such as those found in papaya latex, and relatively pure preparations of fruit bromelain, papain and stem bromelain, were active in vitro against both juvenile, artificially excysted scoleces, as well as against adult worms of both rodent cestodes. Significant dose-dependent reduction in motility, ultimately leading to death of the worms, was observed with both species, and against both freshly excysted scoleces and 14-day old pre-adult worms. The most effective was fruit bromelain (after 30 min of incubation of juvenile H. diminuta and H. microstoma IC50=63 and 74 μM, respectively, and for pre-adult worms=199 and 260 μM, respectively). The least effective was stem bromelain (after 30 min of incubation of juvenile H. diminuta and H. microstoma IC50=2855 and 2772 μM, respectively, and for pre-adult worms=1374 and 1332 μM, respectively) and the efficacies of papaya latex supernatant and papain were between these extremes. In all cases these values are higher than those reported previously for efficacy of CPs against intestinal nematodes, and in contrast to nematodes, all CPs were effective against cestodes in the absence of exogenous cysteine in incubation media. The CPs appeared to attack the tegument resulting in generalised erosion mainly on the strobila. The scolex was more resistant to CP attack but nevertheless some damage to the tegument on the scolex was detected.
A study was carried out to determine the effect of enzyme concentration, temperature and incubation time of bromelain on nitrogen content (NC) and degree of hydrolysis (DH) of hydrolysate from cockle (Anadara granosa) meat wash water. Protein precipitation of cockle meat wash water was conducted at pH 4. The precipitate was then hydrolyzed using bromelain at concentrations of 0.5, 1.5 and 2.5% (enzyme/substrate). The best enzyme concentration was subsequently used to study the effect of incubation temperature at 30, 45 and 60°C. The best temperature was then used to determine the effect of incubation time at 0, 24 and 48 hours. Increasing bromelain concentration from 0 to 2.5% produced an increase in NC and DH. Similarly, increasing the incubation time from 0 to 48 hours also increased the value of NC and DH. However, while the increasing of incubation temperature from 30 to 60°C produced an increase in NC, no significant difference was observed for DH.
Cockle (Anadara granosa) meat wash water precipitate was hydrolyzed using bromelain. Experiments were carried out to determine optimum conditions for temperature, enzyme concentration and hydrolysis time using response surface methodology (RSM) based on a central composite rotatable design (CCRD) to obtain the highest value of nitrogen content (NC) and degree of hydrolysis (DH). Results revealed that the optimum conditions for temperature, enzyme concentration and hydrolysis time were 33.7°C, 1.45% (E/S) and 28.42 hrs, respectively. At the optimum condition, hydrolysis of cockle meat wash water precipitate using bromelain resulted in a NC of 0.6% and DH of 48%. The NC and DH were significantly influenced by temperature, enzyme concentration and hydrolysis time. When the bromelain concentration, hydrolysis time and temperature were increased, the values of NC and DH also increased. The hydrolysate produced contained flavor compounds found in clam and oyster which were 3-methylbutanol and 1-pentanol. The compound 3-MCPD was not found in the hydrolysate.
As a protein-rich, underutilized crop, green soybean could be exploited to produce hydrolysates containing angiotensin-I converting enzyme (ACE) inhibitory peptides. Defatted green soybean was hydrolyzed using four different food-grade proteases (Alcalase, Papain, Flavourzyme and Bromelain) and their ACE inhibitory activities were evaluated. The Alcalase-generated green soybean hydrolysate showed the highest ACE inhibitory activity (IC50: 0.14 mg/mL at 6 h hydrolysis time) followed by Papain (IC50: 0.20 mg/mL at 5 h hydrolysis time), Bromelain (IC50: 0.36 mg/mL at 6 h hydrolysis time) and Flavourzyme (IC50: 1.14 mg/mL at 6 h hydrolysis time) hydrolysates. The Alcalase-generated hydrolysate was profiled based on its hydrophobicity and isoelectric point using reversed phase high performance liquid chromatography (RP-HPLC) and isoelectric point focusing (IEF) fractionators. The Alcalase-generated green soybean hydrolysate comprising of peptides EAQRLLF, PSLRSYLAE, PDRSIHGRQLAE, FITAFR and RGQVLS, revealed the highest ACE inhibitory activity of 94.19%, 99.31%, 92.92%, 101.51% and 90.40%, respectively, while their IC50 values were 878 μM, 532 μM, 1552 μM, 1342 μM and 993 μM, respectively. It can be concluded that Alcalase-digested green soybean hydrolysates could be exploited as a source of peptides to be incorporated into functional foods with antihypertensive activity.
Actinopyga lecanora, as a rich protein source was hydrolysed to generate antibacterial bioactive peptides using different proteolytic enzymes. Bromelain hydrolysate, after 1 h hydrolysis, exhibited the highestantibacterial activities against Pseudomonas aeruginosa, Pseudomonas sp., Escherichia coli and Staphylococcus aureus. Two dimensional fractionation strategies, using a semi-preparative RP-HPLC and an isoelectric-focusing electrophoresis, were applied for peptide profiling. Furthermore, UPLC-QTOF-MS was used for peptides identification; 12 peptide sequences were successfully identified. The antibacterial activity of purified peptides from A. lecanora on P. aeruginosa, Pseudomonas sp., E. coli and S. aureus was investigated. These identified peptides exhibited growth inhibition against P. aeruginosa, Pseudomonas sp., E. coli and S. aureus with values ranging from 18.80 to 75.30%. These results revealed that the A. lecanora would be used as an economical protein source for the production of high value antibacterial bioactive peptides.
The contributing molecular pathways underlying the pathogenesis of breast cancer need to be better characterized. The principle of our study was to better understand the genetic mechanism of oncogenesis for human breast cancer and to discover new possible tumor markers for use in clinical practice. We used complimentary DNA (cDNA) microarrays to compare gene expression profiles of treated Michigan Cancer Foundation-7 (MCF-7) with recombinant bromelain and untreated MCF-7. SpringGene analysis was carried out of differential expression followed by Ingenuity Pathway Analysis (IPA), to understand the underlying consequence in developing disease and disorders. We identified 1,102 known genes differentially expressed to a significant degree (p<0.001) changed between the treatment. Within this gene set, 20 genes were significantly changed between treated cells and the control cells with cutoff fold change of more than 1.5. These genes are RNA-binding motif, single-stranded interacting protein 1 (RBMS1), ribosomal protein L29 (RPL29), glutathione S-transferase mu 2 (GSTM2), C15orf32, Akt3, B cell translocation gene 1 (BTG1), C6orf62, C7orf60, kinesin-associated protein 3 (KIFAP3), FBXO11, AT-rich interactive domain 4A (ARID4A), COPS2, TBPL1|SLC2A12, TMEM59, SNORD46, glioma tumor suppressor candidate region gene 2 (GLTSCR2), and LRRFIP. Our observation on gene expression indicated that recombinant bromelain produces a unique signature affecting different pathways, specific for each congener. The microarray results give a molecular mechanistic insight and functional effects, following recombinant bromelain treatment. The extent of changes in genes is related to and involved significantly in gap junction signaling, amyloid processing, cell cycle regulation by BTG family proteins, and breast cancer regulation by stathmin1 that play major roles.