Production Of Bioactive Peptides Research Articles

High Stability and Reusability of Papain Immobilized on the Non‐Toxic Magnetic Dialdehyde Starch Nanoparticles

AbstractPapain is widely used in food, drug, and bioactive peptide production and must be immobilized onto carriers with biocompatibility. Dialdehyde starch (DAS) can be a good biocompatible cross‐linker according to its active aldehyde groups. In the present study, the magnetic nanoparticles dialdehyde starch (MDASN), synthesized by DAS and Fe3O4 magnetic nanoparticles (Fe‐MNP), are successfully used to immobilize papain to improve the enzymic activity. The structure and morphology of DAS, MDASN, and immobilized papain onto magnetic dialdehyde starch nanoparticles (papain‐MDASN) are characterized detailly. The morphology of DAS is like a flat ball, and that of Fe‐MNP and papain‐MDASN are spherical and clumpy. The particle size of Fe‐MNP and papain‐MDASN are small, resulting in a large surface electrostatic effect and partial agglomeration. Enzymic activity studies of papain‐MDASN exhibit that the immobilized papain on MDASN represents better temperature resistance, alkaline resistance, thermal stability, and reusability, and its activity recovery is up to 68.21%. Papain onto magnetic dialdehyde starch nanoparticles (MDASN) may enhance its potential application in production processes.

Bioactive Peptides from Fermented Foods: Production Approaches, Sources, and Potential Health Benefits.

Microbial fermentation is a well-known strategy for enhancing the nutraceutical attributes of foods. Among the fermentation outcomes, bioactive peptides (BAPs), short chains of amino acids resulting from proteolytic activity, are emerging as promising components thanks to their bioactivities. Indeed, BAPs offer numerous health benefits, including antimicrobial, antioxidant, antihypertensive, and anti-inflammatory properties. This review focuses on the production of bioactive peptides during the fermentation process, emphasizing how different microbial strains and fermentation conditions influence the quantity and quality of these peptides. Furthermore, it examines the health benefits of BAPs from fermented foods, highlighting their potential in disease prevention and overall health promotion. Additionally, this review addresses the challenges and future directions in this field. This comprehensive overview underscores the promise of fermented foods as sustainable and potent sources of bioactive peptides, with significant implications for developing functional foods and nutraceuticals.

Research Progress on Food-Derived Bioactive Peptides: An Overview of the 3rd International Symposium on Bioactive Peptides.

Interest in food-derived bioactive peptides is on the rise. In 2023, the 3rd International Symposium on Bioactive Peptides (ISBP) was held in Niagara Falls, Canada, to provide a platform for knowledge exchange, networking, and collaboration among researchers in this field. This article aims to provide a high-level overview of the key progress and emerging trends in bioactive peptides based on the 3rd ISBP. This review highlights the production of bioactive peptides from sustainable sources through the integration of artificial intelligence and wet-lab research, the emerging roles of bioactive peptides in cognitive function, and the ability of peptides to act as taste modifiers. The emerging research trend in bioactive peptides focuses on utilizing novel processing technologies, understanding peptide-receptor interactions, applying omics in mechanistic studies, conducting clinical trials, and facilitating product development and commercialization.

Production of Bioactive Peptides from Tartary Buckwheat by Solid-State Fermentation with Lactiplantibacillus plantarum ATCC 14917.

Buckwheat is a valuable crop that contains various nutrients and functional components. Tartary buckwheat peptide is a protease-hydrolyzed protein with a wide range of physiological functions. Tartary buckwheat peptide produced through microbial fermentation can decrease the enzymatic digestion of buckwheat protein, which contributes to the bitter taste, and improve both the flavor and texture of buckwheat peptide products. In this study, microbial fermentation using probiotics was employed to prepare Tartary buckwheat peptides, and the preparation process was optimized. Based on single-factor experiments, the polypeptide content in the fermentation solution initially increased and then decreased with varying water content, inoculum concentration, glucose addition, fermentation temperature, fermentation time, and potassium dihydrogen phosphate addition. According to the response surface methodology, the maximum peptide content was achieved under fermentation conditions of 60.0% moisture content, 12.87% inoculum ratio, 2.0% glucose, and a fermentation temperature of 30.0 °C, with an actual value of (22.18 ± 1.02) mg/mL. The results show that fermentation with Lactiplantibacillus plantarum produces higher peptide levels and is safer than other microbial fermentation methods.

Unveiling the Brazilian kefir microbiome: discovery of a novel Lactobacillus kefiranofaciens (LkefirU) genome and in silico prospection of bioactive peptides with potential anti-Alzheimer properties

BackgroundKefir is a complex microbial community that plays a critical role in the fermentation and production of bioactive peptides, and has health-improving properties. The composition of kefir can vary by geographic localization and weather, and this paper focuses on a Brazilian sample and continues previous work that has successful anti-Alzheimer properties. In this study, we employed shotgun metagenomics and peptidomics approaches to characterize Brazilian kefir further.ResultsWe successfully assembled the novel genome of Lactobacillus kefiranofaciens (LkefirU) and conducted a comprehensive pangenome analysis to compare it with other strains. Furthermore, we performed a peptidome analysis, revealing the presence of bioactive peptides encrypted by L. kefiranofaciens in the Brazilian kefir sample, and utilized in silico prospecting and molecular docking techniques to identify potential anti-Alzheimer peptides, targeting β-amyloid (fibril and plaque), BACE, and acetylcholinesterase. Through this analysis, we identified two peptides that show promise as compounds with anti-Alzheimer properties.ConclusionsThese findings not only provide insights into the genome of L. kefiranofaciens but also serve as a promising prototype for the development of novel anti-Alzheimer compounds derived from Brazilian kefir.

Antioxidative, Glucose Management, and Muscle Protein Synthesis Properties of Fish Protein Hydrolysates and Peptides.

The marine environment is an excellent source for many physiologically active compounds due to its extensive biodiversity. Among these, fish proteins stand out for their unique qualities, making them valuable in a variety of applications due to their diverse compositional and functional properties. Utilizing fish and fish coproducts for the production of protein hydrolysates and bioactive peptides not only enhances their economic value but also reduces their potential environmental harm, if left unutilized. Fish protein hydrolysates (FPHs), known for their excellent nutritional value, favorable amino acid profiles, and beneficial biological activities, have generated significant interest for their potential health benefits. These hydrolysates contain bioactive peptides which are peptide sequences known for their beneficial physiological effects. These biologically active peptides play a role in metabolic regulation/modulation and are increasingly seen as promising ingredients in functional foods, nutraceuticals and pharmaceuticals, with potential to improve human health and prevent disease. This review aims to summarize the current in vitro, cell model (in situ) and in vivo research on the antioxidant, glycaemic management and muscle health enhancement properties of FPHs and their peptides.

Bioactive Milk Peptides as a Nutraceutical Opportunity and Challenges.

The biotechnology field has witnessed rapid advancements, leading to the development of numerous proteins and peptides (PPs) for disease management. The production and isolation of bioactive milk peptides (BAPs) involve enzymatic hydrolysis and fermentation, followed by purification through various techniques such as ultrafiltration and chromatography. The nutraceutical potential of bioactive milk peptides has gained significant attention in nutritional research, as these peptides may regulate blood sugar levels, mitigate oxidative stress, improve cardiovascular health, gut health, bone health, and immune responses, and exhibit anticancer properties. However, to enhance BAP bioavailability, the encapsulation method can be used to offer protection against protease degradation and controlled release. This article provides insights into the composition, types, production, isolation, bioavailability, and health benefits of BAPs.

Production of Bioactive Peptides from Microalgae and Their Biological Properties Related to Cardiovascular Disease

Microalgae are a substantial group of unicellular prokaryotic and eukaryotic marine organisms. Due to their high protein content of 50–70%, microalgae have the potential to become a sustainable alternative protein source, as well as aiding in the development of bioactive peptide-based nutraceuticals. A series of major steps are involved in the production of peptides from microalgae, which include the disruption of the microalgal cell wall, the hydrolysis of proteins, and the extraction or isolation of peptides derived from hydrolysis. Physical methods of cell wall disruptions are favored due to the ability to obtain high-quality protein fractions for peptide production. Bioactive peptides are protein fragments of two to twenty amino acid residues that have a beneficial impact on the physiological functions or conditions of human health. Strong scientific evidence exists for the in vitro antioxidant, antihypertensive, and anti-atherosclerotic properties of microalgal peptides. This review is aimed at summarizing the methods of producing microalgal peptides, and their role and mechanisms in improving cardiovascular health. The review reveals that the validation of the physiological benefits of the microalgal peptides in relation to cardiovascular disease, using human clinical trials, is required.

A Novel Workflow for In Silico Prediction of Bioactive Peptides: An Exploration of Solanum lycopersicum By-Products.

Resource-intensive processes currently hamper the discovery of bioactive peptides (BAPs) from food by-products. To streamline this process, in silico approaches present a promising alternative. This study presents a novel computational workflow to predict peptide release, bioactivity, and bioavailability, significantly accelerating BAP discovery. The computational flowchart has been designed to identify and optimize critical enzymes involved in protein hydrolysis but also incorporates multi-enzyme screening. This feature is crucial for identifying the most effective enzyme combinations that yield the highest abundance of BAPs across different bioactive classes (anticancer, antidiabetic, antihypertensive, anti-inflammatory, and antimicrobial). Our process can be modulated to extract diverse BAP types efficiently from the same source. Here, we show the potentiality of our method for the identification of diverse types of BAPs from by-products generated from Solanum lycopersicum, the widely cultivated tomato plant, whose industrial processing generates a huge amount of waste, especially tomato peel. In particular, we optimized tomato by-products for bioactive peptide production by selecting cultivars like Line27859 and integrating large-scale gene expression. By integrating these advanced methods, we can maximize the value of by-products, contributing to a more circular and eco-friendly production process while advancing the development of valuable bioactive compounds.

Bioactivities of black soldier fly larvae protein hydrolysate

One of the potential insects for feed and food sources is the Black Soldier Fly (BSF) (Hermetia illucens). The BSF originated in South America and has now spread to many tropical countries. The BSF larvae (BSFL) had attracted much attention due to their good nutrient content and were recently reported as having useful bioactivities. BSFL has been widely used as feed and has been found to reduce bad smells associated with the growth of pathogenic microbes. The high protein content in BSFL is advantageous in the production of bioactive peptides. This research aimed to extract and hydrolyze the protein from BSFL with protease enzyme to produce protein hydrolysate or peptides with antioxidant and antibacterial activities. The antioxidant activity was measured using DPPH and FRAP, while the antibacterial was analyzed using the agar well diffusion method. BSFL was hydrolyzed with bromelain extracted from pineapple or commercial enzyme (Enzyplex). The IC50 shown by protein hydrolysate was 9.43±0.84% (v/v) or 0.72 mg/mL. The FRAP value was 521.21±264.53 µM for BSFL protein without enzyme addition, 792.14±291.02 for BSFL with the addition of bromelain, and 5,352.114±861.99 µM for BSFL with the addition of Enzyplex. The addition of bromelain increased the FRAP value 1.2 times, while the addition of Enzyplex increased the FRAP value 10.2 times. The antibacterial activity indicated that protein hydrolysate with phosphate buffer pH 8 at 50℃ and 24 hrs incubation showed the highest antibacterial activity to the lowest are Vibrio cholerae, Bacillus cereus and Staphylococcus aureus, respectively. The results suggest that BSFL has antibacterial and antioxidant bioactivities using bromelain extracted from pineapple and Enzyplex, a commercial enzyme that contains protease, lipase and amylase. Using Enzyplex as an enzyme to hydrolyze BFSL shows a very high antioxidant capacity compared to bromelain as an enzyme. This suggests that more protein and other macromolecules contribute to the antioxidant capacity. Using bromelain as an enzyme to hydrolyze BSFL, shows that BSFL peptide has higher antibacterial activity in gramnegative bacteria.

Production of bioactive peptides during yogurt fermentation, their extraction and functional characterization

This study aimed to evaluate the antioxidant, antibacterial, antidiabetic and angiotensin-converting enzyme (ACE)-inhibitory effects of different fractions of bioactive peptides produced as a result of milk fermentation with yogurt starters and probiotics. In addition to yogurt starters, milk was fermented separately and together with Lacticaseibacillus rhamnosus GG and L. paracasei, then passed through 5 and 10 kDa (kilodalton) filters. A significant difference was observed in the fermentation time between the samples. The antioxidant, antibacterial, antidiabetic and ACE-inhibitory activities of the bioactive peptides were significantly affected by the probiotic strains and the molecular weight of the peptide (p < 0.05). ≤5 kDa fractions of Lactobacillus delbrueckii subsp. bulgaricus + Streptococcus thermophilus + L. rhamnosus GG + L. paracasei had the highest antioxidant activity. The addition of L. rhamnosus GG and L. paracasei to yogurt starter significantly increased ɑ-amylase activity, ɑ-glucosidase activity and ACE inhibitor activity. All samples showed antibacterial activity to Escherichia coli, Staphylococcus aureus, and Salmonella Typhimurium microorganisms and the molecular weight of peptides and sample types were significantly exhibited in antibacterial activity (p < 0.05). Cytotoxic activity showed that bioactive peptides had cytotoxic effect against Caco-2 adenocarcinoma cell and the presence of probiotics increased the cytotoxic effect. The results of the study suggest that the use of L. rhamnosus GG and L. paracasei, together with yogurt starters, could improve the bioactive properties of peptides formed in dairy products.

Fermentation of Soy-based dairy substitutes by lactic acid bacteria: Focus on the process of soy protein hydrolysis

Soy-based dairy substitutes (SBDS) made from soybeans are becoming increasingly popular due to the nutritional benefits of soybeans. The use of lactic acid bacteria (LAB) fermentation to enhance the quality of SBDS is a reasonable strategy, in which protein hydrolysis is one of the key biochemical reaction processes in the fermentation process. However, a systematic analysis is yet to be conducted to comprehensively assess the current impact and potential mechanisms of LAB hydrolyzed soy protein on the quality of SBDS. This review summarizes the effects of LAB hydrolyzed soy proteins on the flavor, texture, nutrient digestion and absorption, bioactive peptide production, and allergenicity of SBDS. In addition, measures to improve the ability of LAB to hydrolyze soy protein were presented, including pretreatment of raw materials, screening of suitable strains, optimization of fermentation conditions, and an outlook on future research directions. From the perspective of protein hydrolysis, this review effectively highlights the significant role of LAB in shaping the quality of SBDS during fermentation. It offers an invaluable reference for those involved in the development of high-caliber SBDS production.

Cryptic enzymatic assembly of peptides armed with β-lactone warheads

Nature has evolved biosynthetic pathways to molecules possessing reactive warheads that inspired the development of many therapeutic agents, including penicillin antibiotics. Peptides armed with electrophilic warheads have proven to be particularly effective covalent inhibitors, providing essential antimicrobial, antiviral and anticancer agents. Here we provide a full characterization of the pathways that nature deploys to assemble peptides with β-lactone warheads, which are potent proteasome inhibitors with promising anticancer activity. Warhead assembly involves a three-step cryptic methylation sequence, which is likely required to reduce unfavorable electrostatic interactions during the sterically demanding β-lactonization. Amide-bond synthetase and adenosine triphosphate (ATP)-grasp enzymes couple amino acids to the β-lactone warhead, generating the bioactive peptide products. After reconstituting the entire pathway to β-lactone peptides in vitro, we go on to deliver a diverse range of analogs through enzymatic cascade reactions. Our approach is more efficient and cleaner than the synthetic methods currently used to produce clinically important warhead-containing peptides.

Introducing Bacillus natto and Propionibacterium shermanii into soymilk fermentation: A promising strategy for quality improvement and bioactive peptide production during in vitro digestion

Herein, the texture properties, polyphenol contents, and in vitro protein digestion characteristics of soymilk single- or co-fermented by non-typical milk fermenter Bacillus natto (B. natto), Propionibacterium freudenreichii subsp. shermanii (P. shermanii), and traditional milk fermenter were evaluated. Co-fermenting procedure containing B. natto or P. shermanii could raise the amounts of gallic acid, caffeic acid, and GABA when compared to the unfermented soymilk. Co-fermented soymilk has higher in vitro protein digestibility and nutritional protein quality. Through peptidomic analysis, the co-work of P. shermanii and Lactobacillus plantarum (L. plantarum) may release the highest relative percentage of bioactive peptides, while the intervention of B. natto and Streptococcus thermophilus (S. thermophilus) resulted in more differentiated peptides. The multi-functional bioactive peptides were mainly released from glycine-rich protein, β-conglycinin alpha subunit 1, and ACB domain-containing protein. These findings indicated the potential usage of B. natto/S. thermophilus or P. shermanii/L. plantarum in bio-enhanced soymilk fermentation.

Protein identification and potential bioactive peptides from pumpkin (Cucurbita maxima) seeds.

Pumpkin is an economically important crop all over the world. Approximately, 18%-21% of pumpkins, consisting of peels and seeds by-products, are wasted during processing. In addition, the seeds are rich in protein and have the potency of bioactive peptide production. This study aims to recognize the proteins and investigate the potential bioactive peptides from pumpkin (Cucurbita maxima) seeds. Pumpkin seeds were subjected to hot air drying (HAD) at 55°C for 12 h and freeze-drying (FD) at -80°C for 54 h before they were powdered, analyzed, and precipitated by isoelectric point to obtain pumpkin seed protein isolates (PSPI). PSPI comprised 11S globulin subunit beta, 2S seed storage albumin, and chaperonin CPN60-1. To generate hydrolysate peptides, PSPI was hydrolyzed using papain, pepsin, and bromelain. FD group pepsin hydrolysates had the highest peptide content of 420.83 mg/g. ACE inhibition and DPP-IV inhibition activity were analyzed for each enzymatic hydrolysate. The pepsin hydrolyzed sample exhibited the highest ACE inhibition of 70.26%, and the papain hydrolyzed sample exhibited the highest DPP-IV inhibition of 30.51%. The simulated gastrointestinal digestion (SGID) conducted by pepsin and pancreatin increased ACE inhibitory activity from 76.93% to 78.34%, and DPP-IV inhibited activity increased from 58.62% to 77.13%. Pepsin and papain hydrolysates were fractionated using ultrafiltration to measure ACE and DPP-IV inhibition activity. The highest free radical scavenging abilities were exhibited by the <1 kDa hydrolysate fractions with 78.34% ACE inhibitory activities and 79.55% DPP-IV inhibitory activities. This research revealed that pumpkin seeds had the potency to produce bioactive peptides.

Sustainable Valorization of Coffee Silverskin: Extraction of Phenolic Compounds and Proteins for Enzymatic Production of Bioactive Peptides.

Coffee silverskin (CS), a by-product of the coffee roasting process, has high protein content (16.2-19.0%, w/w), making it a potential source for plant protein and bioactive peptide production. This study aims to develop innovative extraction methods for phenolic compounds and proteins from CS. The conditions for hydrothermal (HT) extraction of phenolic compounds from CS were optimized by varying CS loading (2.5-10%, w/v), temperature (110-130 °C), and time (5-30 min) using a one-factor-at-a-time (OFAT) approach. The highest TPC of 55.59 ± 0.12 µmole GAE/g CS was achieved at 5.0% (w/v) CS loading and autoclaving at 125 °C for 25 min. Following hydrothermal extraction, CS protein was extracted from HT-extracted solid fraction by microwave-assisted alkaline extraction (MAE) using 0.2 M NaOH at 90 W for 2 min, resulting in a protein recovery of 12.19 ± 0.39 mg/g CS. The CS protein was then subjected to enzymatic hydrolysis using protease from Bacillus halodurans SE5 (protease_SE5). Protease_SE5-derived CS protein hydrolysate had a peptide concentration of 0.73 ± 0.09 mg/mL, with ABTS, DPPH, and FRAP values of 15.71 ± 0.10, 16.63 ± 0.061, and 6.48 ± 0.01 µmole TE/mL, respectively. Peptide identification by LC-MS/MS revealed several promising biological activities without toxicity or allergenicity concerns. This study's integrated approach offers a sustainable and efficient method for extracting valuable compounds from CS, with potential applications in the food and pharmaceutical industries.

Nutrition and Cardiovascular Disease: The Potential Role of Marine Bioactive Proteins and Peptides in Thrombosis Prevention.

Thrombus and cardiovascular diseases pose a significant health threat, and dietary interventions have shown promising potential in reducing the incidence of these diseases. Marine bioactive proteins and peptides have been extensively studied for their antithrombotic properties. They can inhibit platelet activation and aggregation by binding to key receptors on the platelet surface. Additionally, they can competitively anchor to critical enzyme sites, leading to the inhibition of coagulation factors. Marine microorganisms also offer alternative sources for the development of novel fibrinolytic proteins, which can help dissolve blood clots. The advancements in technologies, such as targeted hydrolysis, specific purification, and encapsulation, have provided a solid foundation for the industrialization of bioactive peptides. These techniques enable precise control over the production and delivery of bioactive peptides, enhancing their efficacy and safety. However, it is important to note that further research and clinical studies are needed to fully understand the mechanisms of action and therapeutic potential of marine bioactive proteins and peptides in mitigating thrombotic events. The challenges and future application perspectives of these bioactive peptides also need to be explored.

Characterization, Semirational Design for pH Robustness, and the Application in Bioactive Peptide Production of a X-Prolyl Dipeptidyl Aminopeptidase from Lactococcus lactis MY-3.

PepXLcMY-3, an X-prolyl dipeptidyl aminopeptidase derived from Lactobacillus lactis MY-3, was screened and recombinantly expressed in Escherichia coli. The enzyme could exhibit about 40% activity within the pH range of 6.0-10. To further improve the pH robustness, site E396 located in the active pocket was discovered through alanine scanning. The mutant E396I displayed both developed activity and kcat/Km. The optimal pH of E396I shifted from 6.0 to 10 compared to WT, with the relative activity within the pH range of 6.0-10 significantly increased. The site K648 was then proposed by semirational design. The activity of mutant E396I/K648D reached 4.03 U/mg. The optimal pH was restored to 6.0, and the pH stability was further improved. E396I/K648D could totally hydrolyze β-casomorphin 7 within 30 min. The hydrolysate showed 64.5% inhibition on angiotensin I converting enzyme, which was more efficient than those produced by E396I and WT, 23.2 and 44.7%, respectively.

Black Soldier Fly Larvae Grown on Hemp Fiber: Nutritional Composition and Production of Potential Bioactive Peptides

Black soldier fly larvae (BSFL) represent a way of converting organic substrates into valuable biomolecules, and are potentially exploitable as feed and food. In the present work, BSFL grown on retted hemp fiber were chemically analyzed to evaluate their nutritional profile. Chemical analysis revealed BSFL biomass to be an interesting source of proteins (40% on dry matter) rich in essential amino acids. In addition, larval biomass contained 12% fat, mainly composed of saturated fatty acids, and β-sitosterol and campesterol were found to be the most abundant among sterols. A total of 9% of the larval biomass was composed of chitin. The investigation extended to the enzymatic hydrolysis of proteins, leading to the identification of potential bioactive peptides. Peptidomics analysis coupled with in silico tools unveiled promising antioxidant, ACE-inhibitory, and DPP-IV-inhibitory properties within the protein hydrolysates. These findings revealed the potential of BSFL grown on retted hemp fiber as a source of dietary compounds as well as bioactive molecules which can be exploited as functional ingredients in the feed and food sectors.

An Integrated Comprehensive Peptidomics and In Silico Analysis of Bioactive Peptide-Rich Milk Fermented by Three Autochthonous Cocci Strains.

Bioactive peptides (BPs) are molecules of paramount importance with great potential for the development of functional foods, nutraceuticals or therapeutics for the prevention or treatment of various diseases. A functional BP-rich dairy product was produced by lyophilisation of bovine milk fermented by the autochthonous strains Lactococcus lactis subsp. lactis ZGBP5-51, Enterococcus faecium ZGBP5-52 and Enterococcus faecalis ZGBP5-53 isolated from the same artisanal fresh cheese. The efficiency of the proteolytic system of the implemented strains in the production of BPs was confirmed by a combined high-throughput mass spectrometry (MS)-based peptidome profiling and an in silico approach. First, peptides released by microbial fermentation were identified via a non-targeted peptide analysis (NTA) comprising reversed-phase nano-liquid chromatography (RP nano-LC) coupled with matrix-assisted laser desorption/ionisation-time-of-flight/time-of-flight (MALDI-TOF/TOF) MS, and then quantified by targeted peptide analysis (TA) involving RP ultrahigh-performance LC (RP-UHPLC) coupled with triple-quadrupole MS (QQQ-MS). A combined database and literature search revealed that 10 of the 25 peptides identified in this work have bioactive properties described in the literature. Finally, by combining the output of MS-based peptidome profiling with in silico bioactivity prediction tools, three peptides (75QFLPYPYYAKPA86, 40VAPFPEVFGK49, 117ARHPHPHLSF126), whose bioactive properties have not been previously reported in the literature, were identified as potential BP candidates.