Fruit Bromelain Research Articles

Experimental Design of the Interaction Effect of Independent Variables on Pineapple (Ananas comosus) Fruit Bromelain Activity

Background: Certain factors are known to influence the rate of enzyme activity. It is paramount to study such independent factors to obtain the ideal conditions required to maximize enzyme activity. The time-consuming traditional one-factor-at-a-time (OFAT) approach of evaluating the effect of independent variables on enzyme activity does not consider the interaction effects of the independent variables on dependent responses. Methods: The impact of four independent variables on fruit bromelain activity was evaluated using the complete factorial design of the experiment approach. Results: The highest actual fruit bromelain activity (76.83 U/ml) and predicted activity (74.81 U/ml) were recorded at an optimum pH of 7.5, temperature of 50oC, and incubation period of 10 minutes using azocasein substrate. All tested variables except temperature; the interaction between substrate type and incubation time significantly affected fruit bromelain activity (p < 0.05). Conclusion: The design model analysed the main and interaction effect of the variables on bromelain activity. Therefore, complete factorial design is a better approach than OFAT to determine the effect of independent variables on fruit bromelain activity.

Enhancing Bromelain Recovery from Pineapple By-Products: A Sustainable Approach for Value Addition and Waste Reduction.

Pineapple by-products are good sources of bromelain, a complex enzyme with commercial applications. This study evaluated the feasibility of producing bromelain powders from pineapple waste using an organic solvent-free approach. Pineapple by-products (from var. MD2), including cores, peels, crowns, stems, and basal stems, were homogenized with deionized water, and the pH of the mixture was adjusted to 4.5 and 9 (isoelectric points of fruit bromelain and stem bromelain), clarified, ultra-filtered, and freeze-dried to produce bromelain powders. The enzymatic activity of the bromelain powders was measured using the gelatin and casein digestion methods. The bromelain powders from the crowns did not show significant enzymatic activity (p < 0.05). Meanwhile, bromelain powders produced from the cores and peels had an enzymatic activity of 694 gelatin digesting units (GDU)/g and 124 casein digesting units (CDU)/mg, and 1179 GDU/g and 217 CDU/mg, respectively. Bromelain powders from the basal stems showed the highest enzymatic activity (2909 GDU/g and 717 CDU/mg). Increasing the pH of the liquid mixture before the purification and freeze drying significantly (p < 0.05) reduced the enzymatic activity of the bromelain powders. Using a practical and organic solvent-free approach, this study demonstrates the feasibility of producing bromelain powders with high enzymatic activity from pineapple waste.

Enzymatic Analysis and Characterization of Bromelain from Two Varieties of Pineapple (Ananas comosus) Fruit and Stem Extracts

This study was designed to examine the presence of enzymatic activity of fruit and stem bromelain extracted from two varieties of pineapple (Ananas comosus): Morris and Sarawak. The bromelain crude extract obtained from fruits and stems were tested for their protein content via Bradford’s assay, and both crude extract from Morris variety showed the highest value. Further analysis was done to investigate the proteolytic activity of the crude fruit and stem bromelain, resulting in Morris variety having the highest activity for fruit bromelain, while Sarawak variety having the highest activity for stem bromelain. The Gelatin Digestion Unit (GDU) analysis performed revealed both fruit and stem bromelain from Morris variety exhibited the highest activity. Furthermore, the temperature optimization showed that both fruit bromelain of Morris and Sarawak varieties were optimum at 35 ℃, while stem bromelain from both pineapple varieties were optimum at 45 ℃. Meanwhile, the optimum value of the pH for Sarawak variety extract of fruit and stem bromelain were found at pH 5.6, while Morris variety were optimum at pH 6.6. These results indicated both varieties of pineapple extracts of fruits and stems were having bromelain enzyme that can be further developed for application at industrial level.

Isolation of Proteolytic Enzyme from Pineapple Crown

The pineapple waste from the pineapple industry has contributed to an increase in waste in Malaysia and worldwide every year. A major type of endopeptidase enzymes found in pineapple is fruit bromelain, stem bromelain, ananain, and comasain. This study aims to extract and purify protease from the crown of MD2 pineapple. Protease was extracted and purified using anion exchange chromatography, gel filtration, and desalting before being identified using liquid chromatography-mass spectrometry (LC-MS). Proteolytic activity was determined using the well diffusion method and Casein Digestion Unit. In the present study, the proteolytic assay showed that 1 kg crown of MD2 cultivar produced an activity of 126.0 ± 3.86 U/ml, a specific activity of 3937.50 U/mg. In the present study, the proteolytic assay showed that 1 kg crown of MD2 cultivar produced an activity of 126.0 ± 3.86 U/mL, a specific activity of 3937.50 U/mg and the total activity of 3.94 × 109 U. The molecular weight of the purified enzyme was in the range of 25 to 35 kDa under the optimum condition of pH 7 and 37°C. Purification of the extract yielded a band at the molecular weight of 20–25 kDa at the optimum pH of 3 and 9 at 60°C. From LC-MS analysis, the purified enzyme from the crown extract was similar to ananain under accession number A0A199VSS3 (according to Uniprot). It had five unique peptides and covered 97/356 amino acids (44.9% coverage). The ananain (EC 3.4.22.31) is classified in the subfamilies of cysteine protease C1A (clan CA, family C1), a peptidase family related to papain. In conclusion, protease was extracted and identified as an ananain-like protease from the crown.

Manufacturing of Bromelain based PVA support structure for 3D printing

There has been a critical need for the development of cost effective Poly-vinyl Alcohol based wire to be used as support material filament for fused deposition modelling (FDM) machines. But hitherto, very little work has been reported for the development of support material for acrylonitrile butadiene styrene (ABS) and Poly Lactic Acid (PLA) based wire which is presently used in most FDM machines. The present research work is focused on development of Poly-vinyl Alcohol wire which can be employed in support structures for modelling intricate shapes, production of bandages or wipes for inflammation wounds without changing any hardware or software of the machine. For the present study, polyvinyl alcohol is tailored with Bromelain (Pineapple enzyme) which is used as an additive in this process and was analyzed in different stoichiometric ratios. Glycerine was employed as plasticizer to improve its flexibility and thus enabling it to be extruded. The tests carried out on the tailored feed stocks are namely tensile strength test, FTIR analysis, and observation under optical microscopy and SEM for studying its fibrous nature. Therefore, the tailored feedstock was tested and analyzed to understand the variation in the properties of the feedstock with the commercially produced filaments. Thus we were successfully able to manufacture Bromelain based PVA feedstock which satisfies the conditions of a filament produced in conventional methods. The work represents a major development in reducing the cost and the time in dissolving the support structure.

IN VITRO EVALUATION OF FUNGICIDAL ACTIVITY OF BROMELAIN FROM ANANAS COMOSUS AGAINST FUNGAL PHYTOPATHOGENS INFECTING MUSA PARADISIACA

Bromelain from Ananas comosus is a mixture of proteolytic enzymes which are cysteine protease inhibitors containing thiol endopeptidases employed as a phytomedical compound. The present study focussed on extraction and characterization of bromelain and to determine its protein concentration and protease enzyme activity. The effect of temperature and pH on the extracted enzyme was also determined. The extracted enzyme was determined for its molecular weight by SDS and characterized for the presence of their functional groups by FT-IR spectroscopy. The purified bromelain of stem showed highest concentration of protein with maximum protease activity when compared to that of fruit bromelain and on characterization by FT-IR was found to contain thiol containing amino groups. The extracted and characterized bromelain was treated against isolated fungal pathogens of Musa paradisiaca. The isolated bromelain was assessed for their enzyme activity and protein concentration in vitro against the isolated fungal spores and determined for its activity. The determined two-way ANOVA indicated that there were significant differences at different concentrations after incubation at varying time period and the effective concentration and time were determined through Turkey post-hoc analysis which showed stem bromelain possess more effective antifungal activity in vitro against fungal phytopathogens of Musa paradisiaca.

Bromelain, a Group of Pineapple Proteolytic Complex Enzymes (Ananas comosus) and Their Possible Therapeutic and Clinical Effects. A Summary.

Bromelain is a complex combination of multiple endopeptidases of thiol and other compounds derived from the pineapple fruit, stem and/or root. Fruit bromelain and stem bromelain are produced completely distinctly and comprise unique compounds of enzymes, and the descriptor “Bromelain” originally referred in actuality to stem bromelain. Due to the efficacy of oral administration in the body, as a safe phytotherapeutic medication, bromelain was commonly suited for patients due to lack of compromise in its peptidase efficacy and the absence of undesired side effects. Various in vivo and in vitro studies have shown that they are anti-edematous, anti-inflammatory, anti-cancerous, anti-thrombotic, fibrinolytic, and facilitate the death of apoptotic cells. The pharmacological properties of bromelain are, in part, related to its arachidonate cascade modulation, inhibition of platelet aggregation, such as interference with malignant cell growth; anti-inflammatory action; fibrinolytic activity; skin debridement properties, and reduction of the severe effects of SARS-Cov-2. In this paper, we concentrated primarily on the potential of bromelain’s important characteristics and meditative and therapeutic effects, along with the possible mechanism of action.

Protein extract of Bromelia karatas L. rich in cysteine proteases (ananain- and bromelain-like) has antibacterial activity against foodborne pathogens Listeria monocytogenes and Salmonella Typhimurium.

Bromelia karatas L. is a plant species from the Americas. The presence of proteases in fruits of B. karatas has been reported but scarcely studied in detail. Proteolytic enzymes from Ananas comosus have displayed antifungal and antibacterial activity. Thus, novel proteases present in B. karatas may be useful as a source of compounds against microorganisms in medicine and food production. In this work, the protein extract from the fruits of B. karatas was characterized and its antibacterial activity against Salmonella Typhimurium and Listeria monocytogenes was determined for the first time. Proteins highly similar to ananain and the fruit bromelain from A. comosus were identified as the main proteases in B. karatas fruits using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The soluble protein extract (SPE) at a concentration of 2.0mg/mL displayed up to 80% of antibacterial activity against S. Typhimurium. Complete inhibition of L. monocytogenes was reached with up to 1.65mg/mL of SPE. Plant protease extract containing ananain-like enzyme inhibited up to 90% against S. Typhimurium and up to 85% against L. monocytogenes using only 10μg/mL of the partial-purified enzyme.

Soluble Expression and Catalytic Properties of Codon-Optimized Recombinant Bromelain from MD2 Pineapple in Escherichia coli.

Bromelain, a member of cysteine proteases, is found abundantly in pineapple (Ananas comosus), and it has a myriad of versatile applications. However, attempts to produce recombinant bromelain for commercialization purposes are challenging due to its expressibility and solubility. This study aims to express recombinant fruit bromelain from MD2 pineapple (MD2Bro; accession no: OAY85858.1) in soluble and active forms usingEscherichia colihost cell. The gene encoding MD2Bro was codon-optimized, synthesized, and subsequently ligated into pET-32b( +) for further transformation intoEscherichia coliBL21-CodonPlus(DE3). Under this strategy, the expressed MD2Bro was in a fusion form with thioredoxin (Trx) tag at its N-terminal (Trx-MD2Bro). The result showed that Trx-MD2Bro was successfully expressed in fully soluble form. The protein was successfully purified using single-step Ni2+-NTA chromatography and confirmed to be in proper folds based on the circular dichroism spectroscopy analysis. The purified Trx-MD2Bro was confirmed to be catalytically active against N-carbobenzoxyglycine p-nitrophenyl ester (N-CBZ-Gly-pNP) with a specific activity of 6.13 ± 0.01 U mg-1 and inhibited by a cysteine protease inhibitor, E-64 (IC50 of 74.38 ± 1.65nM). Furthermore, the catalytic efficiency (kcat/KM) Trx-MD2Bro was calculated to be at 5.64 ± 0.02 × 10-2µM-1s-1 while the optimum temperature and pH were at 50°C and pH 6.0, respectively. Furthermore, the catalytic activity of Trx-MD2Bro was also affected by ethylenediaminetetraacetic acid (EDTA) or metal ions. Altogether it is proposed that the combination of codon optimization and the use of an appropriate vector are important in the production of a soluble and actively stable recombinant bromelain.

Comparative modelling studies of fruit bromelain using molecular dynamics simulation.

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.

Gene Expression Analysis of Fruit Bromelain in Ripening of &lt;i&gt;Ananas comosus&lt;/i&gt; Cultivar MD 2

Fruit bromelain is a proteolytic enzyme harbouring cysteine catalytic residue found abundantly in pineapple fruit. The expression of cysteine proteases is usually regulated during fruit ripening. In the present study, we aimed to study the expression and proteolytic activity level of fruit bromelain during the ripening stage of A. comosus cultivar MD 2. The gene expression of fruit bromelain was investigated via relative gene expression analysis using qPCR while the proteolytic activity of fruit bromelain was analysed via enzymatic assay using casein as a substrate. The qPCR analysis revealed that the expression of fruit bromelain was down-regulated 10-fold in ripe pineapple fruits. Besides that, the unripe pineapple fruits [1.9101 ± 0.0831 U/mL] had a higher proteolytic activity than the ripe MD 2 pineapple fruits [1.1333 ± 0.0896 U/mL]. This result showed that the function of fruit bromelain may be related to the protection of young pineapple fruits during the fruit development stage.

Fractionation and activity profiling of fruit bromelain from pineapples of Phuket variety growing in Thailand.

Bromelain is a type of protease found in both fruits and stems of pineapples. Stem bromelain has been extensively studied and is commercially available for applications in various industries. In contrast, studies of fruit bromelain are quite limited since most of pineapples have been consumed freshly, canned or juiced. Nowadays, the consumption of canned fruits, including canned pineapples has decreased greatly. Fruit bromelain could be a new growth point for pineapple industry. In this study, fruit bromelain was extracted from the pineapple juice of Phuket variety and some of its properties were studied. The enzyme was purified by precipitation using ammonium sulfate fractionation followed by ion-exchange and gel filtration chromatography. Consequently, the protease purification level was increased by 95.2 fold. The final specific activity was getting to 448,590 U/mg on average, dominated by cysteine proteases, with optimal activity at 45°C and pH ranging from 6 to 8. The study facilitates the molecular and application research of fruit bromelain. PRACTICAL APPLICATIONS: The research has been carried out at Funong Food Technology Co., Ltd., Guangdong, China, which produces primarily pineapple chunks and juice. As plenty of by-products, like peels and cores of pineapples, are produced, the techniques are employed to extract bromelain from the by-products. The techniques reported in this work are not new or advanced, however, they are applicable during the manufacturing process and the related equipment is easy to set up and operate. What's more, the practical application of the techniques is cost-effective for the manufactories. Take Funong Food Technology Co., Ltd. as an example, they was using 80% saturation ammonium sulfate to precipitate protein from pineapple juice and obtained a bromelain with activity of approximately 8,000 U/mg and yield of 1.7kg per ton of juice. With the application of the techniques reported in this work, bromelain was first extracted by ammonium sulfate gradient precipitation, and then purified through ion-exchange and gel filtration chromatography. Each step of precipitation and purification generates a different level of activity and recovery of bromelain, ranging from around 2,506 to 448,590 U/mg, which allows for the production of bromelain according to the requirement of the market and brings more profits.

Effect of high pressure processing on fibrinolytic activity of fruit bromelain in vivo

AbstractAiming at exploring how high pressure processing (HPP) affects the fibrinolytic activity of fruit bromelain (FB) in vivo, the obtained FB was subjected to HPP treatment (300 MPa, 20°C for 20 min) before advanced structure was determined; then six groups of rats were fed with various dosages of enzymes (0.5–1.5 mL/kg·bw) and six target parameters of blood plasma were analyzed. Structural measurements revealed that, by comparison with FB, HPP treated FB (HPP‐FB) showed an increase (7.67%) in α‐helix and its fluorescence intensities were increased by 42.5%, 78.7% at the excitation wavelength of 295 nm, 280 nm, respectively. As to the fibrinolytic activity, compared with the blank control, activated partial thromboplastin time (APTT), prothrombin time (PT) and tissue‐type plasminogen activator (t‐PA) of Group 8 (highest HPP‐FB dosage) were markedly increased (p &lt; .05) by 18.40%, 16.57%, and 10.68% separately, while fibrinogen (FIB) was significantly decreased (p &lt; .05) by 13.88%. Besides, compared with the positive control, the highest HPP‐FB dosage performed almost the same function as aspirin. The results indicated that, compared with FB, the fibrinolytic activity of HPP‐FB was obviously increased which might be attributed to the increase of α‐helix and the exposure of tryptophan residues and tyrosine residues.Practical applicationsThe results in this research suggested that HPP treatment could effectively enhance the fibrinolytic activity of fruit bromelain in vivo and increase the added‐value as well as market competitiveness of pineapple‐derived products. HPP‐FB could potentially be used in the development of a new functional food that can prevent thrombosis.

The Utilization of Pineapples Waste Enzyme for the Improvement of Hydrolysis Solubility in Aquaculture Sludge

High volumes of sludge discharge from the aquaculture industry have relatively high pollutant content that may lead to severe local environmental problems. Anaerobic digestion is one of the technologies for sludge treatment that might be an efficient method to reduce salty aquaculture sludge production load. However, hydrolysis solubility is becoming a limitation step during the anaerobic digestion process when the occurrence of intermediate accumulation resulted from the conversion of non-soluble biopolymers to soluble organic compounds. Thus, pretreatment is required to increase the solubilization of pollutant parameters from aquaculture sludge before it is further treated in the anaerobic treatment. Therefore, this study focuses on the production of biocatalytic enzyme from the fermentation of pre-consumer supermarket waste such as pineapple dregs to increase the solubility of aquaculture sludge. The fermentation of the pineapples waste was produced via a three-month fermentation of the mixture of molasses, pineapple dregs and water, with the ratio of 1:3:10 in a tight plastic container. Apart from that, analyses showed that the enzyme possessed lipase, amylase and protease activity. The sludge solubilization pretreatment was performed at different pH values, with treatment time for 120 hours and the solubilization was evaluated by determination of Chemical Oxygen Demand (COD) and Total Ammonia Nitrogen (TAN) solubilization; the solubilization of COD and TAN increased by 80% and 50%, respectively. This finding showed that the pineapple enzyme has the capability to solubilize organic compound, which has the potential to improve hydrolysis in further anaerobic digestion process.

Alternation of aroma and flavor profiles in biscuits by an enzymatic approach

Bromelain is a complex protease existing in both the fruits and the stems of pineapple plants. Bromelain from these two parts of pineapple plants exhibits different characteristics. Stem bromelain has been extensively investigated and is commercially available for applications in various industries. On contrast, studies on fruit bromelain are quite limited. Here we investigated the effects of fruit bromelain on production of biscuits aroma and flavor compounds. The fruit bromelain was first isolated from pineapple fruit, activity assayed, purity of enzyme determined and then added into dough. Biscuits aroma and flavor compounds were identified by HS-SPME combined with GC-MS. Results showed that aroma and flavor compounds of biscuit varied in terms of amount and varieties.

Comparative structural analysis of fruit and stem bromelain from Ananas comosus

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.

Structural Properties of Macrodontain I, a Cysteine Protease from Pseudananas macrodontes(Morr.) Harms (Bromeliaceae).

The primary structure of macrodontain I, a peptidase from Pseudananas macrodontes fruits, was determined using Edman's degradation. The enzyme is a non-glycosylated peptidase composed by 213 amino acids with a calculated molecular weight of 23,486.18Da, pI value 6.99, and a molar extinction coefficient at 280nm of 61,685M-1cm-1. The alignment of the sequence of macrodontain I with those cysteine peptidases from species belonging to the family Bromeliaceae showed the highest identity degree (87.74%) against fruit bromelain. A remarkable fact is that all these peptidase sequences show two Met contiguous residues (Met121 and 122) and the nonapeptide VPQSIDWRD located in the mature N-terminal region. Residues Cys26 and His159, which constitute the catalytic dyad in all cysteine peptidases, as well as active site residues Gln20 and Asn176, characteristic of Clan C1A, are conserved in macrodontain I. The 3-D model suggests that the enzyme belongs to the α + β class of proteins, with two disulfide bridges (Cys23-Cys63 and Cys57-Cys96) in the α domain, while the β domain is stabilized by another disulfide bridge (Cys153-Cys201). Further, we were able to establish that the cysteine peptidases from P. macrodontes are involved in the anti-inflammatory activity.

Corrigendum: Inhibition of Epithelial TNF-α Receptors by Purified Fruit Bromelain Ameliorates Intestinal Inflammation and Barrier Dysfunction in Colitis.

[This corrects the article on p. 1468 in vol. 8, PMID: 29176974.].

Inhibition of Epithelial TNF-α Receptors by Purified Fruit Bromelain Ameliorates Intestinal Inflammation and Barrier Dysfunction in Colitis.

Activation of the TNF-α receptor (TNFR) leads to an inflammatory response, and anti-TNF therapy has been administered to reduce inflammation symptoms and heal mucosal ulcers in inflammatory bowel disease (IBD). Bromelain, a complex natural mixture of proteolytic enzymes, has been shown to exert anti-inflammatory effects. This study aimed to investigate the effect of purified fruit bromelain (PFB)-induced inhibition of epithelial TNFR in a rat colitis model. Colitis was established by intracolonic administration of 2, 4, 6-trinitrobenzene sulfonic acid. Expression of TNFR1 and TNFR2 was measured by quantitative RT-PCR and western blotting. The effect of PFB on colitis was evaluated by examining the inflammatory response and intestinal epithelial barrier function. Our results showed that both TNFR1 and TNFR2 expression were significantly increased in a colitis model, and the increase was significantly reversed by PFB. Colitis symptoms, including infiltration of inflammatory cells, cytokine profiles, epithelial cell apoptosis, and epithelial tight junction barrier dysfunction were significantly ameliorated by PFB. Compared with fruit bromelain and stem bromelain complex, the inhibition of TNFR2 induced by PFB was stronger than that exhibited on TNFR1. These results indicate that PFB showed a stronger selective inhibitory effect on TNFR2 than TNFR1. In other words, purification of fruit bromelain increases its selectivity on TNFR2 inhibition. High expression of epithelial TNFRs in colitis was significantly counteracted by PFB, and PFB-induced TNFR inhibition ameliorated colitis symptoms. These results supply novel insights into potential IBD treatment by PFB.

Destabilization of Human Insulin Fibrils by Peptides of Fruit Bromelain Derived From Ananas comosus (Pineapple).

Deposition of insulin aggregates in human body leads to dysfunctioning of several organs. Effectiveness of fruit bromelain from pineapple in prevention of insulin aggregate was investigated. Proteolyses of bromelain was done as par human digestive system and the pool of small peptides was separated from larger peptides and proteins. Under conditions of growth of insulin aggregates from its monomers, this pool of peptides restricted the reaction upto formation of oligomers of limited size. These peptides also destabilized preformed insulin aggregates to oligomers. These processes were followed fluorimetrically using Thioflavin T and 1-ANS, size-exclusion HPLC, dynamic light scattering, atomic force microscopy, and transmission electron microscopy. Sequences of insulin (A and B chains) and bromelain were aligned using Clustal W software to predict most probable sites of interactions. Synthetic tripeptides corresponding to the hydrophobic interactive sites of bromelain showed disaggregation of insulin suggesting specificity of interactions. The peptides GG and AAA serving as negative controls showed no potency in destabilization of aggregates. Disaggregation potency of the peptides was also observed when insulin was deposited on HepG2 liver cells where no formation of toxic oligomers occurred. Amyloidogenic des-octapeptide (B23-B30 of insulin) incapable of cell signaling showed cytotoxicity similar to insulin. This toxicity could be neutralized by bromelain derived peptides. FT-IR and far-UV circular dichroism analysis indicated that disaggregated insulin had structure distinctly different from that of its hexameric (native) or monomeric states. Based on the stoichiometry of interaction and irreversibility of disaggregation, the mechanism/s of the peptides and insulin interactions has been proposed. J. Cell. Biochem. 118: 4881-4896, 2017. © 2017 Wiley Periodicals, Inc.