Antifungal properties and α-amylase inhibitory activity of lipid transfer proteins (LTPs) partially purified from Capsicum chinense Jacq. seeds

Antifungal properties and α-amylase inhibitory activity of lipid transfer proteins (LTPs) partially purified from Capsicum chinense Jacq. seeds | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Antifungal properties and α-amylase inhibitory activity of lipid transfer proteins (LTPs) partially purified from Capsicum chinense Jacq. seeds Arielle Pinheiro Oliveira, Larissa Maximiano Resende, Marciele Souza Silva, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4985077/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In this study, we identified and partially purified antimicrobial peptides belonging to the family of lipid transfer proteins (LTPs) from Capsicum chinense seeds (UENF 1751 accession). Fractions rich in LTPs were obtained via ion exchange chromatography and subsequently purified via reverse-phase chromatography in an HPLC system. Therefore, two fractions were revealed: C1 (the nonretained fraction) and C2 (the retained fraction). Fraction C1 was subjected to reverse-phase chromatography via a C18 column on an HPLC system, and ten fractions were obtained (P1–P10), all of which significantly inhibited the growth of C. albicans , except for P4 and P9. The viability analysis of the active fractions at a concentration of 100 µg.mL -1 against C. albicans revealed that they did not exhibit fungicidal activity but rather exhibited fungistatic activity. Fractions P3, P4, P7, and P10 inhibited Tenebrio molitor larvae α-amylase. The P10 fraction presented protein bands in its electrophoretic profile with a molecular mass between 6.5 kDa and 14.2 kDa and reacted positively to an antibody produced against a protein from the LTP family by Western blotting. The results of the analysis of amino acid residues from the P10 fraction revealed similarity between type I LTPs and type II LTPs. The ultrastructural aspects of C. albicans cells exposed to the P10 fraction were evaluated via transmission electron microscopy (TEM), with significant differences in their morphology being evident compared with those of the control. In summary, our results demonstrated the presence of LTPs in C. chinense seeds with inhibitory effects on the growth of yeasts of the genus Candida , which exhibited fungistatic effects and structural changes in C. albicans cells, in addition to exhibiting inhibitory effects on the larval insect T. molitor α-amylase. Antimicrobial peptide Capsicum Candida LTP α-amylase Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Plant antimicrobial peptides share several common characteristics, such as their small size, 12 to 100 amino acid residues, and molecular mass between 3 and 10 kDa. Most of the charge is net positive, being amphipathic with a hydrophilic and a hydrophobic region that allows association with negative charges in the microbial membrane. Thus, lipid transfer proteins (LTPs) are known to be involved in the transfer of lipids between membranes [ 1 ]. They can bind different lipids, such as sterols, coenzyme A acid (CoA) derivatives and fatty acids. Some LTPs are specific, as they have specific binding sites, whereas others are nonspecific (nsLTPs), displaying interactions with a variety of polar lipids. LTPs can be classified on the basis of amino acid sequence alignment, phylogeny, and biochemical structural analysis, among other factors. This categorization made it possible to classify them into distinct groups (types I–IX) that are applicable only to angiosperms [ 2 ]. Because this new classification system evaluates multiple characteristics of LTPs, it is more robust than previous classification systems and is suggested for new characterizations. Although it is a new system, the traditional classification of LTP1 and LTP2 types is still used. Because of these classifications, a nomenclature format for identifying LTPs in the literature was proposed by Edqvist et al. [ 3 ] to avoid confusion for researchers when considering different families. The best characterized groups within the family of LTPs are the LTPI and LTPII, with most of the information available in the scientific literature specifying these groups. In general, LTPs are cationic peptides of 70–100 amino acid residues with molecular masses between 7 and 10 kDa, high isoelectric points (pl), and 8–10 cysteine residues with 4–5 disulfide bonds. Type I LTPs have a molecular mass of 9–10 kDa, are basic, have a pl between 9 and 10, have 90–95 accessory units, and have 4 α-helices in parallel, forming a tunnel with large and small entrances. Type II LTPs have a molecular mass of approximately 7 kDa at high pH, are composed of an average of 70 amino acids, and have structures with 3 α-helices that form a cavity with a triangular structure [ 4 , 2 , 1 ]. In addition to recent innovations in the classification of LTPs, understanding the impact of these proteins in clinical contexts is crucial since, according to the World Health Organization [ 5 ], invasive fungal diseases are exacerbated by the rapid emergence of resistance to antifungal agents and contribute to increased mortality. Yeasts of the genus Candida are recognized as emerging pathogens and are responsible for the majority of invasive and systemic fungal infections in immunocompromised patients [ 6 ]. Candida albicans and other nonalbicans species, such as C. tropicalis , C. parapsilosis , and C. glabrata , are particularly prevalent in hospital settings, especially in intensive care units, making them the third most common infection in the United States [ 7 ]. In this context, antimicrobial peptides (AMPs), which are specific proteins involved in the defense of plants and the immune system of animals, are emerging as promising alternatives in the fight against microorganisms because of their broad-spectrum activity, low toxicity and low propensity for resistance of target cells [ 8 ]. In the genus Capsicum , some LTPs have already been isolated and identified, making this genus promising for the search for new peptides [ 9 ]. In peppers of the species Capsicum annuum , Ca -LTP1, which is localized in seed vesicles, was characterized by Diz et al. [ 10 , 11 ]. This LTP showed antifungal activity against C. albicans and C. tropicalis and inhibited mammalian α-amylase activity in vitro . The species Capsicum chinense has great antimicrobial potential, as noted by Oliveira et al. [ 9 ] however, the diversity of AMPs in this species has not been extensively studied. In this context, this plant family is of great importance and interest because of its medicinal and biotechnological properties, especially against yeasts of the genus Candida. The main objective of this work was to identify and characterize the inhibitory activities of antimicrobial peptides that are similar to LTPs from C. chinense seeds. 2. Materials and methods 2.1. Plants Capsicum chinense Jacq. seeds (UENF 1751 accession) were provided by the Laboratório de Melhoramento Genético Vegetal from Centro de Ciências e Tecnologias Agropecuárias, Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Campos dos Goytacazes, Rio de Janeiro, Brazil. 2.2. Microorganism The yeast Candida albicans (CE022) was obtained from the Departamento de Biologia, Universidade Federal do Ceará, Fortaleza, Brazil. The yeast was maintained on Sabouraud agar (1% peptone, 2% glucose and 1.7% agar) (Merck) at the Laboratório de Fisiologia e Bioquímica de Microrganismos at Centro de Biociências e Biotecnologia, UENF, Campos dos Goytacazes, Rio de Janeiro, Brazil. 2.3. Extraction of peptides from C. chinense seeds The extraction of peptides from C. chinense seeds was performed according to the methodology described by Diz et al. [ 10 ], with modifications. Initially, the seeds were macerated in liquid nitrogen until a fine-grained flour was obtained. Five grams was subsequently added to extraction buffer (0.01 M Na 2 HPO 4 , 0.015 M NaH 2 PO 4 , 0.1 M KCl, and 1.5% EDTA) at a pH of 5.4 at a ratio of 1:10 (seeds (g): extraction buffer (mL)) and agitated for 2 h at 4°C. The homogenized mixture was subsequently centrifuged at 7830 × g for 45 min at 4°C. The precipitate was discarded, and the supernatant was filtered (qualitative filter paper) and added to ammonium sulfate at 0–70% saturation. The obtained material was kept for 16 h at 4°C. The homogenate was subsequently centrifuged at 7830 × g for 45 min at 4°C, after which the supernatant was discarded. The precipitate was redissolved in distilled water (20 mL) and heated at 80°C for 15 min. Subsequently, the supernatant was dialyzed in distilled water for 72 h and then concentrated by lyophilization. The extract obtained at the end of the extraction process was referred to as the seed protein extract (SPE). 2.4. Partial purification of the antimicrobial peptides 2.4.1. Anion exchange chromatography SPE was applied to a glass column (3 cm × 7 cm) packed with 50 mL of diethylaminoethanol (DEAE) Sepharose resin (Sigma‒Aldrich) and equilibrated with 100 mM Tris HCl buffer, pH 8.0. For sample preparation, 50 mg of EPS was added to 10 mL of Tris HCl buffer. Fractions of 3 mL were collected in a continuous flow of 60 mL.h -1 . In the first tube, the nonretained proteins were collected, and from tubes 36 to 60, a saline solution of 100 mM Tris HCl + 1 M NaCl was used to elute the acidic proteins that remained bound to the resin. The absorbance was read at 280 nm. 2.4.2. Reverse-phase chromatography The peptides obtained through ion exchange chromatography were purified through reverse-phase chromatography in an HPLC system (high-performance liquid chromatography) using a C18 column (HIBAR LiChrosorb® RP 18, 5 µm particle size, L × I.D. 25 cm × 4.6 mm, MERCK) coupled to a C18 guard column (Pelliguard LC-18 Replacement Cartridges 2 cm, Supelco). The nonretained fraction (C1) was solubilized (≈ 1 mg.mL − 1 ) in ultrapure water and injected into the column. Chromatography was performed via 100% solvent A (8,77 mM trifluoroacetic acid (TFA) diluted in water) for the first 15 min, during which sample elution was performed via a gradient of propanol with 8,77 mM of TFA (solvent B), as follows: the concentration of solvent B gradually increased from 0% at 15 min to 50% at 115 min. Then, the concentration of solvent B was reduced to 0% and maintained at this concentration until the end of the run at 120 min. Protein elution from the column was monitored via absorbance readings at 220 nm via a diode array detector with a flow rate of 500 µL.min − 1 at 40°C. 2.5. Biochemical characterization 2.5.1. SDS‒PAGE Tricine Visualization of the SPE and chromatographic profile obtained at different purification steps was performed via an electrophoresis technique described by Schägger and Von Jagow et al. [ 12 ]. An ultralow-range molecular weight marker (MW 1,060–26,600 Da) (Sigma‒Aldrich) was used. After SDS‒PAGE, the gel was removed from the glass plates and immersed in a staining solution containing Coomassie blue G (0.025% in distilled water containing 10% acetic acid) with stirring for approximately 1 h. After staining, the gel was transferred to a destaining solution of 10% acetic acid in distilled water. 2.5.3. Western blotting Western blotting was performed for immunodetection of LTP(s) as described by Maracahipes et al. [ 13 ]. After separation of the proteins by SDS‒PAGE‒Tricine, the protein bands were transferred to nitrocellulose membranes at a constant current of 1 mA/cm 2 for 2 h in the gel‒membrane direction. The membrane was then stained with Ponceuau (0.1%) to check the efficiency of the process. After the bands were detected, the membrane was washed in ultrapure water and incubated with a blocking solution of PBS (10 mM NaH 2 PO 4 .2H 2 O, 3 mM KCl, 1.5 mM KH 2 PO 4 .2H 2 O-, 140 mM NaCl) with 2% skim milk (Molico) and 20–0.05% Tween 20vfor 1 h. Then, the membrane was immersed in blocking buffer (without Tween 20) containing pepper anti-LTP primary antibody (1:2000) and left overnight (16 h at 4°C). The next day, the membrane was washed 10× for 5 min each with PBS and then immersed in blocking buffer (without Tween 20) containing an anti-rabbit IgG secondary antibody conjugated with peroxidase (1:2000) for 2 h at room temperature. Afterward, the membrane was washed again in PBS 10× 5 min each time. Then, the membrane was visualized by reaction with diaminobenzidine (DAB). DAB was applied by immersing the membrane in a solution containing 40 mM Tris-HCl, pH 7.5, 1 mg.mL -1 DAB, 100 mM imidazole and 0.03% hydrogen peroxide until the bands were visible. The reaction was stopped with distilled water. 2.5.4. Mass spectrometry The sequencing was carried out in partnership with the Marine Biochemistry Laboratory (BioMar-Lab) of the Department of Fisheries Engineering at the Federal University of Ceará (UFC), Ceará, Brazil. For sequencing of the peptides present in the P10 fraction, the protein bands of interest were extracted from the gel after separation by SDS‒PAGE Tricine as described by Resende et al. [ 14 ]. The bands were extracted, and then, peptides were obtained according to Shevchenko et al. [ 15 ] and subjected to LC‒MS/MS analysis. The instrument used was a hybrid mass spectrometer (ESI-Q-ToF) (Synapt HDMS, Waters Corp, MA, USA). The data collection and processing parameters were adjusted as described by Carneiro et al. [ 16 ]. The interpretation of the spectra was performed via the Mascot program, and the sequenced peptides were submitted for alignment via the BLASTp tool [ 17 ]. Sequences with a high percentage of identity were chosen and subjected to multiple alignments via CLUSTAL multiple sequence alignment using the MUSCLE program (3.8). 2.6. Biological and statistical analyses 2.6.1. Yeast growth inhibition assay To analyze the antimicrobial activity, cells from C. albicans (1 × 10 4 cells.mL − 1 ) were incubated in 100 µL of Sabouraud broth (10 g/L peptone, 2 g/L glucose) (Merck) with fractions at a concentration of 100 µg.mL − 1 . The assay was performed in cell culture plates (96 wells) (NUNC, Nunclon Surface) at 28°C for 24 h. After the incubation period, the optical density was determined with a microplate reader (EZ Read 400, Research, Biochrom) at a wavelength of 620 nm. The entire assay procedure was carried out under aseptic conditions in a laminar flow hood following a methodology adapted from Broekaert et al. [ 18 ]. To monitor the effect of the treatment on yeast growth, the bottom of each well was photographed after 24 h of incubation. The percentages of inhibition were determined considering that the control group represented 100% growth, using the formula [100 - (tABS620 × 100/cABS620)]. Here, tABS620 refers to the average absorbance measured at 620 nm in the sample concentrated with the fraction after 24 h, whereas cABS620 represents the average absorbance measured at 620 nm in the control sample after 24 h. 2.6.2. Analysis of cell viability after peptide incubation After the C. albicans growth inhibition assay, the analysis of cell viability was performed following a methodology adapted from Gebara et al. [ 19 ]. The cells retrieved from the assay were washed and diluted 1000-fold. An aliquot of 100 µL from the dilution mixture was spread onto the surface of a Petri dish containing Sabouraud agar via a Drigalsky loop and incubated at 30°C for 36 h. At the end of this period, the number of colony-forming units was determined, and the Petri dishes were photographed. The experiments were performed in triplicate, and the results are presented under the assumption that the control represents 100% cell viability. 2.6.3. Transmission electron microscopy (TEM) To analyze the ultrastructure of C. albicans cells, after treatment with the P10 fraction as described in Section 2.6.1 ., the cells were fixed for 1 h in sodium cacodylate buffer (pH 7.4) containing 2.5% glutaraldehyde and 4% formaldehyde freshly prepared at 0.1 mol/L. After these procedures, the cells were washed twice with phosphate-buffered saline (PBS) for 10 min and postfixed for 1 h in the dark with a solution containing 1% osmium tetroxide (OsO4) and 1.6% ferrocyanide in 0.1 M sodium cacodylate buffer. The cells were subsequently washed in the same buffer, dehydrated in acetone (30%, 50%, 70%, 90%, 100% and 100% superdry) and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and observed under a Jeol JEM 1400 Plus transmission electron microscope. 2.6.4. α-Amylase activity inhibition assay The inhibition of α-amylase activity was determined by measuring the residual α-amylase hydrolytic activity of the intestine of T. molitor (yellow wheatworm) as described by Silva et al. [ 20 ], with modifications. To determine the enzyme inhibition activity, fractions from C . chinense seeds (P1–P10), at a concentration of 50 µg.mL − 1 , were incubated at 37°C for 30 min with 1 µL of intestinal α-amylase extracted by Resende et al. [ 14 ] (bulk determined by performing an enzyme curve, 10 U of amylase) and 25 µL of 1% starch (Sigma‒Aldrich) adjusted with water to a final volume of 100 µL. Then, 200 µL of DNS solution (4.5% sodium hydroxide, 3,5-dinitrosalicylic acid, 1% sodium double tartrate, 45 g of potassium and 2 g of crystalline phenol) was added, and the mixture was heated at 100°C for 5 min. At room temperature, the samples were read in a spectrophotometer (Spectroquant Pharo 100 Merck) at 540 nm to detect the hydrolysis of the substrate by the enzyme. This technique was performed in triplicate, and the standard deviation (SD) was calculated for each sample. One amylase unit is defined as a 0.1 change in absorbance at 540 nm per 30 min of reaction, as described by Franco et al. [ 21 ]. To calculate the percentage of inhibition, a positive control was performed with 5 mM ethylenediaminetetraacetic acid (EDTA) as a parameter, considering 100% inhibition of activity. Bovine serum albumin (BSA) was used as a negative control at a concentration of 10 µg.mL − 1 . 2.6.5. Statistical analysis The data from the experiments were obtained in triplicate. For the evaluation, one-way analysis of variance (ANOVA) was used. Differences in the means were considered significant at p < 0.05. The statistical analyses were performed via GraphPad Prism software (version 6.0 for Windows). 3. Results 3.1. Characterization of antimicrobial peptides: electrophoretic profiling and Western blotting The seed protein extract (SPE) displayed bands between 6.5 kDa and 26.6 kDa. This material was used as the starting material for the isolation of antimicrobial peptides from C. chinense. Immunodetection of an LTP of approximately 9 kDa in the SPE was performed to confirm the presence of the group of AMPs of interest. Therefore, we continued with the chromatography steps to obtain a homogeneous fraction with the presence of LTPs. When chromatographed by anion exchange, SPE revealed two fractions, C1 (nonretained fraction) and C2 (retained fraction) (Fig. 1 A). The electrophoretic profile of the chromatography fractions (C1 and C2) revealed protein bands ranging from 6.5 to 14.2 kDa (Fig. 1 B). Figure 1 C shows the Western blotting results, which revealed immunostaining for LTP in the C1 fraction, which was close to the expected 9 kDa for LTP. Reverse-phase chromatography was used to separate proteins from the C1 fraction (Fig. 2 A). Ten fractions rich in low-molecular-weight peptides were obtained and named according to the elution time on the column (Fig. 2 B). The method used did not reveal any protein bands in the electrophoretic profiles of fractions P1, P2, P3, and P4. Protein bands can be seen in fractions P5 to P10, with significant bands at approximately 6.5 and 14.2 kDa in P5, P6, and P7. P8 displayed two main bands ranging in size from 6.5 to 14.2 kDa. P9 had a single band at approximately 6.5 kDa, whereas P10 had two main bands with molecular masses ranging from 6.5 to 14.2 kDa and 14.2 to 17.0 kDa. LTP in the resulting fractions was detected by Western blotting (Fig. 2 C). As a positive control, SPE was utilized, which revealed a molecular band of approximately 9 kDa. Immunodetection revealed LTP with a molecular mass of 6.5 to 14.2 kDa in the P10 fraction. 3.2. Analysis of amino acid fragments obtained from the P10 fraction The alignment of the amino acid fragments (TINSLAK-TACNCLK-CGVQLSVPISR) from the P10 fraction revealed similarity with type I LTPs (Fig. 3 ). C. chinense (sequence ID: PHU05228.1) and C. annuum (sequence IDs: PHT70689.1, XP_016545892.2, KAF3624163.1, XP_016543253.1, PHT70835.1, XP_016567772.1, XP_016545932.1, and PHT70185.1) had 54% identity, and C. annuum (sequence ID: PHU04792.1) and C. annuum (sequence ID: PHT70186.1) had 50% identity. Furthermore, 46% identity with C. annuum was detected (sequence IDs: KAF3674251.1, XP_016545939.2, and KAF3674250.1). The alignment of amino acid fragments (QYVNSPNAR) revealed similarity with type II LTPs (Fig. 3 ), with 100% identity with C. annuum (sequence ID: KAF3615994.1), C. baccatum (sequence IDs: PHT56658. 1, PHT56659.1), and C. chinense (sequence ID: PHU06864.1). The colors in the figure highlight the conserved amino acids and the cysteine residues in the sequences in gray. 3.3. Effects of peptide fractions on the growth of C. albicans C. albicans was inhibited by treatment with 100 µg.mL − 1 C1 or C2. C1 was the most significant fraction, with 75% inhibition, whereas C2 inhibited 56% of the cell growth (Fig. 4 A). Because of the presence of LTP observed by Western blotting and its greater ability to inhibit C. albicans growth, the subsequent studies were carried out with the C1 fraction. The values are the means (standard deviations) of three replicates. The ten fractions obtained via reverse-phase chromatography were subjected to a C. albicans growth inhibition test at a concentration of 100 µg.mL − 1 . According to the statistical analysis, fractions P1, P2, P3, P5, P6, P7, P8, and P10 demonstrated growth inhibition, with P1 showing the most significant values (Fig. 4 B) with 91.3% inhibition. P2 resulted in 68% inhibition, P5 resulted in 66.6% inhibition, and P8 resulted in 49.9% inhibition. P3, P6, P7, and P10 inhibited fungal growth by 34.3%, 44.2%, 49.2%, and 40.9%, respectively. P4 and P9 showed no significant inhibition. Below the graph are images of the bottom of the plate showing the comparisons of cell growth of the yeast C. albicans . 3.4. Effects of peptide fractions on cell viability To verify the viability of the yeast cells, we used ten fractions from C. chinense seeds at a concentration of 100 µg.mL − 1 . After 36 h, fractions P1 and P5 presented the greatest reduction in colony-forming units (CFUs). At this concentration, the fractions P1, P3, P4, P5, P6, P7 and P10 caused 53.64, 38.43, 14.64, 81.89, 25.86, 34.2 and 8.79% cell death, respectively. Fractions P2, P8 and P9 did not induce the loss of viable cells (Fig. 5 ). 3.5. Analysis of ultrastructural changes in C. albicans treated with the P10 fraction from C . chinense seeds TEM observations (Fig. 6 ) revealed the integrity of membranes and organelles in control cells (Fig. 6 A, B and C), in contrast to treated cells (Fig. 6 D, E, F, G, H and I), which exhibited a loss of structural integrity in the plasma membrane, membrane invagination and intermembranous particle formation (Fig. 6 H and I). The tonoplast was disorganized, whereas the cytosol was reduced and had disappeared, indicating the presence of dense granular material. Disorganization and degradation of the nucleus were observed (Fig. 6 G), along with the destruction of intracellular organelles. In particular, the mitochondria were significantly degraded, highlighting the complete disorder of the mitochondrial cristae (Fig. 6 F). The vacuole expanded due to the accumulation of amorphous material (Fig. 6 E), and a notable increase in budding occurred, with morphological changes evident only in the budding regions (Fig. 6 D). The peptide did not appear to cause changes in the cell wall (Fig. 6 H and 6 I). The treatment induced destructive effects on several membrane components, as evidenced by the presence of osmiophilic sites and holes, as well as the formation of membrane fragments in other areas. 3.6. Effects of peptide fractions from reversed-phase chromatography on α-amylase activity Figure 7 shows the results of the inhibition of α-amylase activity from T. molitor . According to the results of the Dunnett test, fractions P3, P4, P7 and P10 significantly inhibited amylase activity (p < 0.05) by 78.7%, 85.8%, 77% and 99.3%, respectively. However, the other fractions did not show inhibition potential. 4. Discussion According to the World Health Organization (WHO), by 2022, the treatment of invasive fungal diseases will face major challenges due to limited access to quality diagnosis and treatment, as well as increasing resistance to antifungal drugs. In the WHO report, a list of the most dangerous pathogens was drawn up, highlighting two fungi of the genus Candida as a critical priority: C. auris and C. albicans . Therefore, it is crucial to develop new alternatives with antifungal potential [ 5 ]. AMPs are emerging as promising solutions because of their unique ability to control fungal infections, their broad activity against different microorganisms, their diverse mechanisms of action that minimize resistance, and their potential for structural modification to increase their efficacy and selectivity and reduce their environmental impact. These characteristics position them as robust candidates for the future development of more effective and sustainable antifungal therapies. In our research, the C1 fraction rich in AMPs of the LTP type obtained after purification from C. chinense seeds showed significant potential for inhibiting the growth of C. albicans (Fig. 4 A). After reverse-phase chromatography, LTP was detected in fraction 10 (P10), whose band presented a molecular mass consistent with that of LTPs described in the literature [ 22 , 10 , 23 , 24 ]. Given the presence of LTP and its notable inhibitory activity, the aim was to classify and sequence the amino acids of the fragments of this fraction. The LTP fragments detected in the P10 fraction presented high similarity with type I LTPs and type II LTPs from different Capsicum species (Fig. 3 ). The identification of more than one LTP is significant, as it indicates the presence of different groups (type I and type II). Literature searches have also identified these families of LTPs in different organs and pepper species [ 25 , 26 , 10 , 23 , 27 , 28 ]. To efficiently separate these proteins, more refined steps are necessary, where characteristics such as isoelectric point and hydrophobicity can help in the separation and purification of the different LTPs. Scientific evidence has shown that seed LTPs have antifungal activity, with values ranging from 2–400 µg.mL − 1 depending on the fungal species tested [ 29 ]. For example, the LTP from sunflower seed ( Helianthus annuus ) Ha-AP10, at a concentration of 6.5 µg, showed significant antimicrobial activity, with 50% inhibition when tested against the fungus Fusarium solani . However, the inhibitory effect was not significant when the fungus Alternaria alternata was tested [ 30 , 31 ]. In another study using LTP, LjAMP2 from Leonurus japonicus Houtt (a Chinese herb) seeds was shown to have activity against several fungi and bacteria, but some pathogens tested, such as Agrobacterium radiobacter and Escherichia coli , were not inhibited [ 32 ]. LTP Ca-LTP1 in C. annuum seeds inhibited the growth of C. lindemunthianum , C. tropicalis and S. cerevisiae at a concentration of 400 µg.mL − 1 . Compared with other yeasts, the yeast C. lindemunthianum was the most susceptible [ 11 ]. These results support the findings presented here and confirm that LTPs have the potential to inhibit a variety of fungi at different concentrations, indicating their promising application as alternative antifungal agents. This study revealed a potential antimicrobial effect of the P10 fraction against C. albicans cells. MET images suggested a cytoplasmic-directed effect that caused cellular stress and disorganization of internal structures without significantly affecting cell wall integrity (Fig. 6 ). This finding suggests that the mechanism of action of the peptide in microbial cells involves specific targets, highlighting the need to investigate these mechanisms in detail. This observation suggests an anticandidal effect of specific targets in the cytoplasmic region, which can be studied for the development of new antimicrobial agents. The disorganization and deformation of C. albicans cytoplasmic structures observed in response to the P10 fraction suggest the possibility of direct interference with vital cellular processes, leading to compromised cellular integrity and function. These effects can be exploited to develop therapeutic strategies to treat fungal infections, especially those caused by strains resistant to conventional treatments. Similar results have already been reported by Seyedjavadi et al. [ 32 ], who reported displacement of the plasma membrane from the cell wall and destruction of the cytoplasm and organelles without changes in the cell wall in C. albicans cells treated with the MCh-AMP1 peptide. Another study conducted by Basma et al. [ 33 ] using Euphorbia hirta seed extract on C. albicans cells revealed a decrease in cytoplasmic volume after 24 h, with significant structural disorganization in the cell cytoplasm. Although direct membrane damage is an important mechanism of action of AMPs, some studies have already demonstrated the importance of considering other targets and indirect effects that may contribute to the antimicrobial activity of these peptides [ 34 ]. In addition to their antimicrobial activity, LTPs have the ability to inhibit α-amylase activity, a property that helps characterize this family of peptides [ 35 , 36 , 14 ]. Diz et al. [ 10 ] were pioneers in suggesting that LTP in Capsicum could inhibit human salivary α-amylase, suggesting a role for LTP in plant defense against insects. In addition, Silva et al. [ 20 ] reported that when Vigna unguiculata LTP, termed Vu -LTP, was tested with the human salivary α-amylase enzyme, the positively charged amino acids interacted with the active site of α-amylase and were responsible for the inhibition. In our assays, the fractions from P1 to P10 were tested for their ability to inhibit α-amylase activity in the intestine of T. molitor larvae, an insect model with easy extraction of α-amylase. According to the results of this assay, the P10 fraction showed 99% inhibition (Fig. 7 ). Fractions such as P3, P4 and P7 also significantly inhibited α-amylase activity, indicating that other peptides present in these fractions may have inhibitory effects that deserve future investigation. The high value of α-amylase enzymatic activity in the P10 fraction reinforces the characteristics of this protein family. This characteristic reinforces the multipotency of these peptides, which have vast biotechnological potential, expanding their possible applications in industry and medicine [10 ]. 5. Conclusion This study represents a significant advance in understanding the LTPs found in pepper seeds, highlighting their remarkable antifungal activity with inhibitory efficacy on the growth of C. albicans yeast. We also observed ultrastructural changes in yeast cells treated with the P10 fraction, especially displacement of the plasma membrane and cell wall. Additionally, we explored their inhibitory effect on insect α-amylase, expanding the biotechnological prospects of these proteins. This report contributes significantly to the development of the biotechnological potential of LTPs, reinforcing their role as promising candidates for future applications in agriculture and medicine. Declarations Acknowledgments This work was performed at the Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF). We wish to thank L.C.D. Souza and V.M. Kokis for their technical assistance. Funding We acknowledge the financial support of the Brazilian agencies CNPq (307590/2021-6) and FAPERJ (E-26/200567/2023; E-26/210353/2022; E-26/200.127/2023). Data availability All the data generated or analyzed during this study are included in this published article. Conflict of interest The authors declare no conflicts of interest. Ethical Approval This article does not contain any studies with human or animal subjects. Author Contribution The study was conceived by APBFO, AOC and VMG. Experimental procedures were carried out by LMR, LAS, MSS, RPC, FFM. Data analyses were performed by CSN, SHS, MC, RR. The paper was written by APBFO, GBT, EOM, VMG. All authors reviewed the manuscript. 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17:20:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":127952,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Chromatogram of the C1 and C2 fractions obtained by ion exchange chromatography on DEAE-Sepharose resin. The C1– nonretained fraction was eluted in 100 mM Tris HCl, pH 8.0. The C2– retained fraction eluted in 100 mM Tris HCl, pH 8.0, plus 1 M NaCl. The samples were collected at a continuous flow rate of 60 mL.h\u003csup\u003e-1\u003c/sup\u003e. The absorbance was read at 280 nm. \u003cstrong\u003e(B)\u003c/strong\u003e Electrophoretic visualization of the extract and protein fractions obtained from \u003cem\u003eC\u003c/em\u003e. \u003cem\u003echinense\u003c/em\u003e seeds via SDS‒PAGE. M– molecular mass markers (kDa); EPS– protein extract from \u003cem\u003eC\u003c/em\u003e.\u003cem\u003e chinense\u003c/em\u003e seeds; LTP– 9.0 kDa lipid transfer protein obtained from \u003cem\u003eC\u003c/em\u003e. \u003cem\u003eannuum\u003c/em\u003e (control); C2– retained fraction and C1– nonretained fraction obtained by ion exchange chromatography on DEAE-Sepharose resin. All the samples were treated with 5% β-mercaptoethanol. \u003cstrong\u003e(C)\u003c/strong\u003e \u003cem\u003eWestern blotting\u003c/em\u003e of transient fractions by ion exchange chromatography.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4985077/v1/8c3419c7af81b4138d684748.png"},{"id":65944384,"identity":"802c8662-c1a1-45d3-b489-7beb777f9c93","added_by":"auto","created_at":"2024-10-04 17:12:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":127196,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eReversed-phase chromatography on an HPLC system (µRP C18 column) of the nonretained fraction (C1) obtained by ion exchange chromatography of the protein extract from \u003cem\u003eC. chinense seeds\u003c/em\u003e. The column was previously equilibrated, the run was performed with 0.1% trifluoroacetic acid (TFA) (solvent A), and the sample was eluted via a linear propanol gradient (solvent B). The flow rate was 0.5 mL.min\u003csup\u003e-1.\u003c/sup\u003e\u003cstrong\u003e (B) \u003c/strong\u003eElectrophoretic visualization of the SDS‒PAGE‒Tricine fractions obtained by reversed-phase chromatography; M– Molecular mass marker in kDa; LTP‒Lipid Transfer Proteins; P1‒P10‒ Fractions obtained by chromatography in an HPLC system. All the samples were treated with 5% β-mercaptoethanol. \u003cstrong\u003e(C)\u003c/strong\u003e \u003cem\u003eWestern blotting \u003c/em\u003eof the P10 fraction.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4985077/v1/67bae6f7a7f11ca96c9bcea9.png"},{"id":65944391,"identity":"5971c7a4-2d14-4f77-bc03-e68fdf079673","added_by":"auto","created_at":"2024-10-04 17:12:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":792455,"visible":true,"origin":"","legend":"\u003cp\u003eAlignment of the amino acid residues of the \u003cem\u003eCapsicum chinense\u003c/em\u003e seed peptide (UENF 1751 accession) from sample P10. The sequences were obtained via BLAST and aligned via Clustal Omega and Jalview. The identities indicate the percentage of identical residues and are written in italics. Positives indicate the percentage of positive residues (which have the same physicochemical characteristics) and are written in gray. Gaps (-)\u003cstrong\u003e \u003c/strong\u003ehave been introduced for better alignment.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4985077/v1/b7ec58f12b667d71091652f1.png"},{"id":65944385,"identity":"a8a7b645-5b0d-4160-905d-414e450c3085","added_by":"auto","created_at":"2024-10-04 17:12:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":84069,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eEffects of the C1 and C2 fractions (100 µg/mL) obtained via ion exchange chromatography of protein extracts from \u003cem\u003eC. chinense seeds\u003c/em\u003e on the growth of \u003cem\u003eC. albicans\u003c/em\u003e. \u003cstrong\u003e(B)\u003c/strong\u003e Effects of the P1–P10 fractions (100 µg.mL\u003csup\u003e-1\u003c/sup\u003e) obtained via reversed-phase chromatography on the growth of \u003cem\u003eC. albicans\u003c/em\u003e after 24 h. (%)– Percentage of inhibition caused by fractions. (*)- The asterisks indicate differences (p\u0026lt;0.0001) between the experimental treatment group and the control group according to the Dunnett test.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4985077/v1/bffd8c7a52457e600fe9294b.png"},{"id":65944386,"identity":"01c67873-f109-4021-9cec-48f62e7efc62","added_by":"auto","created_at":"2024-10-04 17:12:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":207691,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of the \u003cem\u003eC\u003c/em\u003e. \u003cem\u003echinense\u003c/em\u003e fractions (P1--P10) obtained via reversed-phase chromatography on the viability of \u003cem\u003eC. albicans\u003c/em\u003e yeast cells. The test was performed at a concentration of 100 µg.mL\u003csup\u003e-1\u003c/sup\u003e. After 36 h, cell viability was measured via direct counting of colony-forming units (CFUs). The percentage of cell death was calculated in relation to the control (cell viability 100%). The results are presented as average values obtained throughout the experiment, which were carried out in triplicate.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4985077/v1/43c0f5305e6cdda080fbdec4.png"},{"id":65944388,"identity":"397a510e-675b-44e0-b6dc-d4828216468a","added_by":"auto","created_at":"2024-10-04 17:12:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1029611,"visible":true,"origin":"","legend":"\u003cp\u003eTransmission electron microscopy of \u003cem\u003eC. albicans\u003c/em\u003ecells treated with or without the P10 fraction. Control cells (A, B and C); treated cells (D, E, F, G, H and I). Abbreviations: (CW)- cell wall; (PM)-plasma membrane; (N)- nucleus; (V)- vacuole; (M)- mitochondria; (Mb)-microbodies; (IP)- intramembranous particle; (MI)-membrane invagination.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4985077/v1/6f4e50ec860e0c21f7e3b0e3.png"},{"id":65944389,"identity":"7a2c1f61-2107-4b1a-a3f2-a51611398b0d","added_by":"auto","created_at":"2024-10-04 17:12:14","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":35340,"visible":true,"origin":"","legend":"\u003cp\u003eInhibitory effects of the \u003cem\u003eC\u003c/em\u003e. \u003cem\u003echinense\u003c/em\u003e fractions (P1--P10) obtained via reversed-phase chromatography on α-amylase activity. The test was performed at a concentration of 50 µg/mL. Control- α-amylase from \u003cem\u003eT. molitor\u003c/em\u003e; EDTA- positive control 5 mM; BSA-negative control\u003cstrong\u003e (\u003c/strong\u003e10 µg); (%)- percentage of enzymatic activity caused by the fractions. \u003cstrong\u003e(****)-\u003c/strong\u003e Asterisks indicate a significant difference (p\u0026lt;0.0001) according to the Dunnett test.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4985077/v1/b81dbc05e18f6071e933eba4.png"},{"id":68851881,"identity":"0a598c9b-5e01-4588-af84-61d83c0da108","added_by":"auto","created_at":"2024-11-12 17:31:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3331344,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4985077/v1/56b23dca-066a-4883-af51-a683674a2156.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antifungal properties and α-amylase inhibitory activity of lipid transfer proteins (LTPs) partially purified from Capsicum chinense Jacq. seeds","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePlant antimicrobial peptides share several common characteristics, such as their small size, 12 to 100 amino acid residues, and molecular mass between 3 and 10 kDa. Most of the charge is net positive, being amphipathic with a hydrophilic and a hydrophobic region that allows association with negative charges in the microbial membrane. Thus, lipid transfer proteins (LTPs) are known to be involved in the transfer of lipids between membranes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. They can bind different lipids, such as sterols, coenzyme A acid (CoA) derivatives and fatty acids. Some LTPs are specific, as they have specific binding sites, whereas others are nonspecific (nsLTPs), displaying interactions with a variety of polar lipids. LTPs can be classified on the basis of amino acid sequence alignment, phylogeny, and biochemical structural analysis, among other factors. This categorization made it possible to classify them into distinct groups (types I\u0026ndash;IX) that are applicable only to angiosperms [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBecause this new classification system evaluates multiple characteristics of LTPs, it is more robust than previous classification systems and is suggested for new characterizations. Although it is a new system, the traditional classification of LTP1 and LTP2 types is still used. Because of these classifications, a nomenclature format for identifying LTPs in the literature was proposed by Edqvist et al. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] to avoid confusion for researchers when considering different families.\u003c/p\u003e \u003cp\u003eThe best characterized groups within the family of LTPs are the LTPI and LTPII, with most of the information available in the scientific literature specifying these groups. In general, LTPs are cationic peptides of 70\u0026ndash;100 amino acid residues with molecular masses between 7 and 10 kDa, high isoelectric points (pl), and 8\u0026ndash;10 cysteine residues with 4\u0026ndash;5 disulfide bonds. Type I LTPs have a molecular mass of 9\u0026ndash;10 kDa, are basic, have a pl between 9 and 10, have 90\u0026ndash;95 accessory units, and have 4 α-helices in parallel, forming a tunnel with large and small entrances. Type II LTPs have a molecular mass of approximately 7 kDa at high pH, are composed of an average of 70 amino acids, and have structures with 3 α-helices that form a cavity with a triangular structure [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition to recent innovations in the classification of LTPs, understanding the impact of these proteins in clinical contexts is crucial since, according to the World Health Organization [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], invasive fungal diseases are exacerbated by the rapid emergence of resistance to antifungal agents and contribute to increased mortality. Yeasts of the genus \u003cem\u003eCandida\u003c/em\u003e are recognized as emerging pathogens and are responsible for the majority of invasive and systemic fungal infections in immunocompromised patients [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. \u003cem\u003eCandida albicans\u003c/em\u003e and other \u003cem\u003enonalbicans\u003c/em\u003e species, such as \u003cem\u003eC. tropicalis\u003c/em\u003e, \u003cem\u003eC. parapsilosis\u003c/em\u003e, and \u003cem\u003eC. glabrata\u003c/em\u003e, are particularly prevalent in hospital settings, especially in intensive care units, making them the third most common infection in the United States [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In this context, antimicrobial peptides (AMPs), which are specific proteins involved in the defense of plants and the immune system of animals, are emerging as promising alternatives in the fight against microorganisms because of their broad-spectrum activity, low toxicity and low propensity for resistance of target cells [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the genus \u003cem\u003eCapsicum\u003c/em\u003e, some LTPs have already been isolated and identified, making this genus promising for the search for new peptides [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In peppers of the species \u003cem\u003eCapsicum annuum\u003c/em\u003e, \u003cem\u003eCa\u003c/em\u003e-LTP1, which is localized in seed vesicles, was characterized by Diz et al. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This LTP showed antifungal activity against \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eC. tropicalis\u003c/em\u003e and inhibited mammalian α-amylase activity \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe species \u003cem\u003eCapsicum chinense\u003c/em\u003e has great antimicrobial potential, as noted by Oliveira et al. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] however, the diversity of AMPs in this species has not been extensively studied. In this context, this plant family is of great importance and interest because of its medicinal and biotechnological properties, especially against yeasts of the genus \u003cem\u003eCandida.\u003c/em\u003e The main objective of this work was to identify and characterize the inhibitory activities of antimicrobial peptides that are similar to LTPs from \u003cem\u003eC. chinense\u003c/em\u003e seeds.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Plants\u003c/h2\u003e \u003cp\u003e \u003cem\u003eCapsicum chinense\u003c/em\u003e Jacq. seeds (UENF 1751 accession) were provided by the Laborat\u0026oacute;rio de Melhoramento Gen\u0026eacute;tico Vegetal from Centro de Ci\u0026ecirc;ncias e Tecnologias Agropecu\u0026aacute;rias, Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Campos dos Goytacazes, Rio de Janeiro, Brazil.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Microorganism\u003c/h2\u003e \u003cp\u003eThe yeast \u003cem\u003eCandida albicans\u003c/em\u003e (CE022) was obtained from the Departamento de Biologia, Universidade Federal do Cear\u0026aacute;, Fortaleza, Brazil. The yeast was maintained on Sabouraud agar (1% peptone, 2% glucose and 1.7% agar) (Merck) at the Laborat\u0026oacute;rio de Fisiologia e Bioqu\u0026iacute;mica de Microrganismos at Centro de Bioci\u0026ecirc;ncias e Biotecnologia, UENF, Campos dos Goytacazes, Rio de Janeiro, Brazil.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Extraction of peptides from \u003cem\u003eC. chinense\u003c/em\u003e seeds\u003c/h2\u003e \u003cp\u003eThe extraction of peptides from \u003cem\u003eC. chinense\u003c/em\u003e seeds was performed according to the methodology described by Diz et al. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], with modifications. Initially, the seeds were macerated in liquid nitrogen until a fine-grained flour was obtained. Five grams was subsequently added to extraction buffer (0.01 M Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 0.015 M NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.1 M KCl, and 1.5% EDTA) at a pH of 5.4 at a ratio of 1:10 (seeds (g): extraction buffer (mL)) and agitated for 2 h at 4\u0026deg;C. The homogenized mixture was subsequently centrifuged at 7830 \u0026times; \u003cem\u003eg\u003c/em\u003e for 45 min at 4\u0026deg;C. The precipitate was discarded, and the supernatant was filtered (qualitative filter paper) and added to ammonium sulfate at 0\u0026ndash;70% saturation. The obtained material was kept for 16 h at 4\u0026deg;C. The homogenate was subsequently centrifuged at 7830 \u0026times; \u003cem\u003eg\u003c/em\u003e for 45 min at 4\u0026deg;C, after which the supernatant was discarded. The precipitate was redissolved in distilled water (20 mL) and heated at 80\u0026deg;C for 15 min. Subsequently, the supernatant was dialyzed in distilled water for 72 h and then concentrated by lyophilization. The extract obtained at the end of the extraction process was referred to as the seed protein extract (SPE).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Partial purification of the antimicrobial peptides\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. Anion exchange chromatography\u003c/h2\u003e \u003cp\u003eSPE was applied to a glass column (3 cm \u0026times; 7 cm) packed with 50 mL of diethylaminoethanol (DEAE) Sepharose resin (Sigma‒Aldrich) and equilibrated with 100 mM Tris HCl buffer, pH 8.0. For sample preparation, 50 mg of EPS was added to 10 mL of Tris HCl buffer. Fractions of 3 mL were collected in a continuous flow of 60 mL.h\u003csup\u003e-1\u003c/sup\u003e. In the first tube, the nonretained proteins were collected, and from tubes 36 to 60, a saline solution of 100 mM Tris HCl\u0026thinsp;+\u0026thinsp;1 M NaCl was used to elute the acidic proteins that remained bound to the resin. The absorbance was read at 280 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2. Reverse-phase chromatography\u003c/h2\u003e \u003cp\u003eThe peptides obtained through ion exchange chromatography were purified through reverse-phase chromatography in an HPLC system (high-performance liquid chromatography) using a C18 column (HIBAR LiChrosorb\u0026reg; RP 18, 5 \u0026micro;m particle size, L \u0026times; I.D. 25 cm \u0026times; 4.6 mm, MERCK) coupled to a C18 guard column (Pelliguard LC-18 Replacement Cartridges 2 cm, Supelco). The nonretained fraction (C1) was solubilized (\u0026asymp;\u0026thinsp;1 mg.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in ultrapure water and injected into the column. Chromatography was performed via 100% solvent A (8,77 mM trifluoroacetic acid (TFA) diluted in water) for the first 15 min, during which sample elution was performed via a gradient of propanol with 8,77 mM of TFA (solvent B), as follows: the concentration of solvent B gradually increased from 0% at 15 min to 50% at 115 min. Then, the concentration of solvent B was reduced to 0% and maintained at this concentration until the end of the run at 120 min. Protein elution from the column was monitored via absorbance readings at 220 nm via a diode array detector with a flow rate of 500 \u0026micro;L.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 40\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Biochemical characterization\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1. SDS‒PAGE Tricine\u003c/h2\u003e \u003cp\u003eVisualization of the SPE and chromatographic profile obtained at different purification steps was performed via an electrophoresis technique described by Sch\u0026auml;gger and Von Jagow et al. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. An ultralow-range molecular weight marker (MW 1,060\u0026ndash;26,600 Da) (Sigma‒Aldrich) was used. After SDS‒PAGE, the gel was removed from the glass plates and immersed in a staining solution containing Coomassie blue G (0.025% in distilled water containing 10% acetic acid) with stirring for approximately 1 h. After staining, the gel was transferred to a destaining solution of 10% acetic acid in distilled water.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.5.3. Western blotting\u003c/h2\u003e \u003cp\u003eWestern blotting was performed for immunodetection of LTP(s) as described by Maracahipes et al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. After separation of the proteins by SDS‒PAGE‒Tricine, the protein bands were transferred to nitrocellulose membranes at a constant current of 1 mA/cm\u003csup\u003e2\u003c/sup\u003e for 2 h in the gel‒membrane direction. The membrane was then stained with Ponceuau (0.1%) to check the efficiency of the process. After the bands were detected, the membrane was washed in ultrapure water and incubated with a blocking solution of PBS (10 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO, 3 mM KCl, 1.5 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO-, 140 mM NaCl) with 2% skim milk (Molico) and 20\u0026ndash;0.05% Tween 20vfor 1 h. Then, the membrane was immersed in blocking buffer (without Tween 20) containing pepper anti-LTP primary antibody (1:2000) and left overnight (16 h at 4\u0026deg;C). The next day, the membrane was washed 10\u0026times; for 5 min each with PBS and then immersed in blocking buffer (without Tween 20) containing an anti-rabbit IgG secondary antibody conjugated with peroxidase (1:2000) for 2 h at room temperature. Afterward, the membrane was washed again in PBS 10\u0026times; 5 min each time. Then, the membrane was visualized by reaction with diaminobenzidine (DAB). DAB was applied by immersing the membrane in a solution containing 40 mM Tris-HCl, pH 7.5, 1 mg.mL\u003csup\u003e-1\u003c/sup\u003e DAB, 100 mM imidazole and 0.03% hydrogen peroxide until the bands were visible. The reaction was stopped with distilled water.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.5.4. Mass spectrometry\u003c/h2\u003e \u003cp\u003eThe sequencing was carried out in partnership with the Marine Biochemistry Laboratory (BioMar-Lab) of the Department of Fisheries Engineering at the Federal University of Cear\u0026aacute; (UFC), Cear\u0026aacute;, Brazil. For sequencing of the peptides present in the P10 fraction, the protein bands of interest were extracted from the gel after separation by SDS‒PAGE Tricine as described by Resende et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The bands were extracted, and then, peptides were obtained according to Shevchenko et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and subjected to LC‒MS/MS analysis. The instrument used was a hybrid mass spectrometer (ESI-Q-ToF) (Synapt HDMS, Waters Corp, MA, USA). The data collection and processing parameters were adjusted as described by Carneiro et al. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The interpretation of the spectra was performed via the Mascot program, and the sequenced peptides were submitted for alignment via the BLASTp tool [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Sequences with a high percentage of identity were chosen and subjected to multiple alignments via CLUSTAL multiple sequence alignment using the MUSCLE program (3.8).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Biological and statistical analyses\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1. Yeast growth inhibition assay\u003c/h2\u003e \u003cp\u003eTo analyze the antimicrobial activity, cells from \u003cem\u003eC. albicans\u003c/em\u003e (1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were incubated in 100 \u0026micro;L of Sabouraud broth (10 g/L peptone, 2 g/L glucose) (Merck) with fractions at a concentration of 100 \u0026micro;g.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The assay was performed in cell culture plates (96 wells) (NUNC, Nunclon Surface) at 28\u0026deg;C for 24 h. After the incubation period, the optical density was determined with a microplate reader (EZ Read 400, Research, Biochrom) at a wavelength of 620 nm. The entire assay procedure was carried out under aseptic conditions in a laminar flow hood following a methodology adapted from Broekaert et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. To monitor the effect of the treatment on yeast growth, the bottom of each well was photographed after 24 h of incubation. The percentages of inhibition were determined considering that the control group represented 100% growth, using the formula [100 - (tABS620 \u0026times; 100/cABS620)]. Here, tABS620 refers to the average absorbance measured at 620 nm in the sample concentrated with the fraction after 24 h, whereas cABS620 represents the average absorbance measured at 620 nm in the control sample after 24 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2. Analysis of cell viability after peptide incubation\u003c/h2\u003e \u003cp\u003eAfter the \u003cem\u003eC. albicans\u003c/em\u003e growth inhibition assay, the analysis of cell viability was performed following a methodology adapted from Gebara et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The cells retrieved from the assay were washed and diluted 1000-fold. An aliquot of 100 \u0026micro;L from the dilution mixture was spread onto the surface of a Petri dish containing Sabouraud agar via a Drigalsky loop and incubated at 30\u0026deg;C for 36 h. At the end of this period, the number of colony-forming units was determined, and the Petri dishes were photographed. The experiments were performed in triplicate, and the results are presented under the assumption that the control represents 100% cell viability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.6.3. Transmission electron microscopy (TEM)\u003c/h2\u003e \u003cp\u003eTo analyze the ultrastructure of \u003cem\u003eC. albicans\u003c/em\u003e cells, after treatment with the P10 fraction as described in Section \u003cspan refid=\"Sec14\" class=\"InternalRef\"\u003e2.6.1\u003c/span\u003e., the cells were fixed for 1 h in sodium cacodylate buffer (pH 7.4) containing 2.5% glutaraldehyde and 4% formaldehyde freshly prepared at 0.1 mol/L. After these procedures, the cells were washed twice with phosphate-buffered saline (PBS) for 10 min and postfixed for 1 h in the dark with a solution containing 1% osmium tetroxide (OsO4) and 1.6% ferrocyanide in 0.1 M sodium cacodylate buffer. The cells were subsequently washed in the same buffer, dehydrated in acetone (30%, 50%, 70%, 90%, 100% and 100% superdry) and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and observed under a Jeol JEM 1400 Plus transmission electron microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e2.6.4. α-Amylase activity inhibition assay\u003c/h2\u003e \u003cp\u003eThe inhibition of α-amylase activity was determined by measuring the residual α-amylase hydrolytic activity of the intestine of \u003cem\u003eT. molitor\u003c/em\u003e (yellow wheatworm) as described by Silva et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], with modifications. To determine the enzyme inhibition activity, fractions from \u003cem\u003eC\u003c/em\u003e. \u003cem\u003echinense\u003c/em\u003e seeds (P1\u0026ndash;P10), at a concentration of 50 \u0026micro;g.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, were incubated at 37\u0026deg;C for 30 min with 1 \u0026micro;L of intestinal α-amylase extracted by Resende et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] (bulk determined by performing an enzyme curve, 10 U of amylase) and 25 \u0026micro;L of 1% starch (Sigma‒Aldrich) adjusted with water to a final volume of 100 \u0026micro;L. Then, 200 \u0026micro;L of DNS solution (4.5% sodium hydroxide, 3,5-dinitrosalicylic acid, 1% sodium double tartrate, 45 g of potassium and 2 g of crystalline phenol) was added, and the mixture was heated at 100\u0026deg;C for 5 min. At room temperature, the samples were read in a spectrophotometer (Spectroquant Pharo 100 Merck) at 540 nm to detect the hydrolysis of the substrate by the enzyme. This technique was performed in triplicate, and the standard deviation (SD) was calculated for each sample. One amylase unit is defined as a 0.1 change in absorbance at 540 nm per 30 min of reaction, as described by Franco et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. To calculate the percentage of inhibition, a positive control was performed with 5 mM ethylenediaminetetraacetic acid (EDTA) as a parameter, considering 100% inhibition of activity. Bovine serum albumin (BSA) was used as a negative control at a concentration of 10 \u0026micro;g.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e2.6.5. Statistical analysis\u003c/h2\u003e \u003cp\u003eThe data from the experiments were obtained in triplicate. For the evaluation, one-way analysis of variance (ANOVA) was used. Differences in the means were considered significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The statistical analyses were performed via GraphPad Prism software (version 6.0 for Windows).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Characterization of antimicrobial peptides: electrophoretic profiling and \u003cem\u003eWestern blotting\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe seed protein extract (SPE) displayed bands between 6.5 kDa and 26.6 kDa. This material was used as the starting material for the isolation of antimicrobial peptides from \u003cem\u003eC. chinense.\u003c/em\u003e Immunodetection of an LTP of approximately 9 kDa in the SPE was performed to confirm the presence of the group of AMPs of interest. Therefore, we continued with the chromatography steps to obtain a homogeneous fraction with the presence of LTPs. When chromatographed by anion exchange, SPE revealed two fractions, C1 (nonretained fraction) and C2 (retained fraction) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The electrophoretic profile of the chromatography fractions (C1 and C2) revealed protein bands ranging from 6.5 to 14.2 kDa (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC shows the \u003cem\u003eWestern blotting\u003c/em\u003e results, which revealed immunostaining for LTP in the C1 fraction, which was close to the expected 9 kDa for LTP.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eReverse-phase chromatography was used to separate proteins from the C1 fraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Ten fractions rich in low-molecular-weight peptides were obtained and named according to the elution time on the column (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The method used did not reveal any protein bands in the electrophoretic profiles of fractions P1, P2, P3, and P4. Protein bands can be seen in fractions P5 to P10, with significant bands at approximately 6.5 and 14.2 kDa in P5, P6, and P7. P8 displayed two main bands ranging in size from 6.5 to 14.2 kDa. P9 had a single band at approximately 6.5 kDa, whereas P10 had two main bands with molecular masses ranging from 6.5 to 14.2 kDa and 14.2 to 17.0 kDa. LTP in the resulting fractions was detected by Western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). As a positive control, SPE was utilized, which revealed a molecular band of approximately 9 kDa. Immunodetection revealed LTP with a molecular mass of 6.5 to 14.2 kDa in the P10 fraction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Analysis of amino acid fragments obtained from the P10 fraction\u003c/h2\u003e \u003cp\u003eThe alignment of the amino acid fragments (TINSLAK-TACNCLK-CGVQLSVPISR) from the P10 fraction revealed similarity with type I LTPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). \u003cem\u003eC. chinense\u003c/em\u003e (sequence ID: PHU05228.1) and \u003cem\u003eC. annuum\u003c/em\u003e (sequence IDs: PHT70689.1, XP_016545892.2, KAF3624163.1, XP_016543253.1, PHT70835.1, XP_016567772.1, XP_016545932.1, and PHT70185.1) had 54% identity, and \u003cem\u003eC. annuum\u003c/em\u003e (sequence ID: PHU04792.1) and \u003cem\u003eC. annuum\u003c/em\u003e (sequence ID: PHT70186.1) had 50% identity. Furthermore, 46% identity with \u003cem\u003eC. annuum\u003c/em\u003e was detected (sequence IDs: KAF3674251.1, XP_016545939.2, and KAF3674250.1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe alignment of amino acid fragments (QYVNSPNAR) revealed similarity with type II LTPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), with 100% identity with \u003cem\u003eC. annuum\u003c/em\u003e (sequence ID: KAF3615994.1), \u003cem\u003eC. baccatum\u003c/em\u003e (sequence IDs: PHT56658. 1, PHT56659.1), and \u003cem\u003eC. chinense\u003c/em\u003e (sequence ID: PHU06864.1).\u003c/p\u003e \u003cp\u003eThe colors in the figure highlight the conserved amino acids and the cysteine residues in the sequences in gray.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Effects of peptide fractions on the growth of \u003cem\u003eC. albicans\u003c/em\u003e\u003c/h2\u003e \u003cp\u003e \u003cem\u003eC. albicans\u003c/em\u003e was inhibited by treatment with 100 \u0026micro;g.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e C1 or C2. C1 was the most significant fraction, with 75% inhibition, whereas C2 inhibited 56% of the cell growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Because of the presence of LTP observed by Western blotting and its greater ability to inhibit \u003cem\u003eC. albicans\u003c/em\u003e growth, the subsequent studies were carried out with the C1 fraction. The values are the means (standard deviations) of three replicates.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe ten fractions obtained via reverse-phase chromatography were subjected to a \u003cem\u003eC. albicans\u003c/em\u003e growth inhibition test at a concentration of 100 \u0026micro;g.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. According to the statistical analysis, fractions P1, P2, P3, P5, P6, P7, P8, and P10 demonstrated growth inhibition, with P1 showing the most significant values (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) with 91.3% inhibition. P2 resulted in 68% inhibition, P5 resulted in 66.6% inhibition, and P8 resulted in 49.9% inhibition. P3, P6, P7, and P10 inhibited fungal growth by 34.3%, 44.2%, 49.2%, and 40.9%, respectively. P4 and P9 showed no significant inhibition. Below the graph are images of the bottom of the plate showing the comparisons of cell growth of the yeast \u003cem\u003eC. albicans\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Effects of peptide fractions on cell viability\u003c/h2\u003e \u003cp\u003eTo verify the viability of the yeast cells, we used ten fractions from \u003cem\u003eC. chinense\u003c/em\u003e seeds at a concentration of 100 \u0026micro;g.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. After 36 h, fractions P1 and P5 presented the greatest reduction in colony-forming units (CFUs). At this concentration, the fractions P1, P3, P4, P5, P6, P7 and P10 caused 53.64, 38.43, 14.64, 81.89, 25.86, 34.2 and 8.79% cell death, respectively. Fractions P2, P8 and P9 did not induce the loss of viable cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.5. Analysis of ultrastructural changes in\u003c/b\u003e \u003cb\u003eC. albicans\u003c/b\u003e \u003cb\u003etreated with the P10 fraction from\u003c/b\u003e \u003cb\u003eC\u003c/b\u003e. \u003cb\u003echinense\u003c/b\u003e \u003cb\u003eseeds\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTEM observations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) revealed the integrity of membranes and organelles in control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B and C), in contrast to treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, E, F, G, H and I), which exhibited a loss of structural integrity in the plasma membrane, membrane invagination and intermembranous particle formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH and I). The tonoplast was disorganized, whereas the cytosol was reduced and had disappeared, indicating the presence of dense granular material. Disorganization and degradation of the nucleus were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG), along with the destruction of intracellular organelles. In particular, the mitochondria were significantly degraded, highlighting the complete disorder of the mitochondrial cristae (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). The vacuole expanded due to the accumulation of amorphous material (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), and a notable increase in budding occurred, with morphological changes evident only in the budding regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). The peptide did not appear to cause changes in the cell wall (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). The treatment induced destructive effects on several membrane components, as evidenced by the presence of osmiophilic sites and holes, as well as the formation of membrane fragments in other areas.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Effects of peptide fractions from reversed-phase chromatography on α-amylase activity\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e shows the results of the inhibition of α-amylase activity from \u003cem\u003eT. molitor\u003c/em\u003e. According to the results of the Dunnett test, fractions P3, P4, P7 and P10 significantly inhibited amylase activity (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) by 78.7%, 85.8%, 77% and 99.3%, respectively. However, the other fractions did not show inhibition potential.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAccording to the World Health Organization (WHO), by 2022, the treatment of invasive fungal diseases will face major challenges due to limited access to quality diagnosis and treatment, as well as increasing resistance to antifungal drugs. In the WHO report, a list of the most dangerous pathogens was drawn up, highlighting two fungi of the genus \u003cem\u003eCandida\u003c/em\u003e as a critical priority: \u003cem\u003eC. auris\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e. Therefore, it is crucial to develop new alternatives with antifungal potential [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAMPs are emerging as promising solutions because of their unique ability to control fungal infections, their broad activity against different microorganisms, their diverse mechanisms of action that minimize resistance, and their potential for structural modification to increase their efficacy and selectivity and reduce their environmental impact. These characteristics position them as robust candidates for the future development of more effective and sustainable antifungal therapies. In our research, the C1 fraction rich in AMPs of the LTP type obtained after purification from \u003cem\u003eC. chinense\u003c/em\u003e seeds showed significant potential for inhibiting the growth of \u003cem\u003eC. albicans\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eAfter reverse-phase chromatography, LTP was detected in fraction 10 (P10), whose band presented a molecular mass consistent with that of LTPs described in the literature [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Given the presence of LTP and its notable inhibitory activity, the aim was to classify and sequence the amino acids of the fragments of this fraction. The LTP fragments detected in the P10 fraction presented high similarity with type I LTPs and type II LTPs from different \u003cem\u003eCapsicum\u003c/em\u003e species (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The identification of more than one LTP is significant, as it indicates the presence of different groups (type I and type II).\u003c/p\u003e \u003cp\u003eLiterature searches have also identified these families of LTPs in different organs and pepper species [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. To efficiently separate these proteins, more refined steps are necessary, where characteristics such as isoelectric point and hydrophobicity can help in the separation and purification of the different LTPs. Scientific evidence has shown that seed LTPs have antifungal activity, with values ranging from 2\u0026ndash;400 \u0026micro;g.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e depending on the fungal species tested [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. For example, the LTP from sunflower seed (\u003cem\u003eHelianthus annuus\u003c/em\u003e) Ha-AP10, at a concentration of 6.5 \u0026micro;g, showed significant antimicrobial activity, with 50% inhibition when tested against the fungus \u003cem\u003eFusarium solani\u003c/em\u003e. However, the inhibitory effect was not significant when the fungus \u003cem\u003eAlternaria alternata\u003c/em\u003e was tested [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In another study using LTP, LjAMP2 from \u003cem\u003eLeonurus japonicus Houtt\u003c/em\u003e (a Chinese herb) seeds was shown to have activity against several fungi and bacteria, but some pathogens tested, such as \u003cem\u003eAgrobacterium radiobacter\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e, were not inhibited [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. LTP Ca-LTP1 in \u003cem\u003eC. annuum\u003c/em\u003e seeds inhibited the growth of \u003cem\u003eC. lindemunthianum\u003c/em\u003e, \u003cem\u003eC. tropicalis\u003c/em\u003e and \u003cem\u003eS. cerevisiae\u003c/em\u003e at a concentration of 400 \u0026micro;g.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Compared with other yeasts, the yeast \u003cem\u003eC. lindemunthianum\u003c/em\u003e was the most susceptible [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These results support the findings presented here and confirm that LTPs have the potential to inhibit a variety of fungi at different concentrations, indicating their promising application as alternative antifungal agents.\u003c/p\u003e \u003cp\u003eThis study revealed a potential antimicrobial effect of the P10 fraction against \u003cem\u003eC. albicans\u003c/em\u003e cells. MET images suggested a cytoplasmic-directed effect that caused cellular stress and disorganization of internal structures without significantly affecting cell wall integrity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This finding suggests that the mechanism of action of the peptide in microbial cells involves specific targets, highlighting the need to investigate these mechanisms in detail. This observation suggests an anticandidal effect of specific targets in the cytoplasmic region, which can be studied for the development of new antimicrobial agents. The disorganization and deformation of \u003cem\u003eC. albicans\u003c/em\u003e cytoplasmic structures observed in response to the P10 fraction suggest the possibility of direct interference with vital cellular processes, leading to compromised cellular integrity and function. These effects can be exploited to develop therapeutic strategies to treat fungal infections, especially those caused by strains resistant to conventional treatments.\u003c/p\u003e \u003cp\u003eSimilar results have already been reported by Seyedjavadi et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], who reported displacement of the plasma membrane from the cell wall and destruction of the cytoplasm and organelles without changes in the cell wall in \u003cem\u003eC. albicans\u003c/em\u003e cells treated with the MCh-AMP1 peptide. Another study conducted by Basma et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] using \u003cem\u003eEuphorbia hirta\u003c/em\u003e seed extract on \u003cem\u003eC. albicans\u003c/em\u003e cells revealed a decrease in cytoplasmic volume after 24 h, with significant structural disorganization in the cell cytoplasm. Although direct membrane damage is an important mechanism of action of AMPs, some studies have already demonstrated the importance of considering other targets and indirect effects that may contribute to the antimicrobial activity of these peptides [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition to their antimicrobial activity, LTPs have the ability to inhibit α-amylase activity, a property that helps characterize this family of peptides [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Diz et al. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] were pioneers in suggesting that LTP in \u003cem\u003eCapsicum\u003c/em\u003e could inhibit human salivary α-amylase, suggesting a role for LTP in plant defense against insects. In addition, Silva et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] reported that when \u003cem\u003eVigna unguiculata\u003c/em\u003e LTP, termed \u003cem\u003eVu\u003c/em\u003e-LTP, was tested with the human salivary α-amylase enzyme, the positively charged amino acids interacted with the active site of α-amylase and were responsible for the inhibition.\u003c/p\u003e \u003cp\u003eIn our assays, the fractions from P1 to P10 were tested for their ability to inhibit α-amylase activity in the intestine of \u003cem\u003eT. molitor\u003c/em\u003e larvae, an insect model with easy extraction of α-amylase. According to the results of this assay, the P10 fraction showed 99% inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Fractions such as P3, P4 and P7 also significantly inhibited α-amylase activity, indicating that other peptides present in these fractions may have inhibitory effects that deserve future investigation. The high value of α-amylase enzymatic activity in the P10 fraction reinforces the characteristics of this protein family. This characteristic reinforces the multipotency of these peptides, which have vast biotechnological potential, expanding their possible applications in industry and medicine [10 ].\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study represents a significant advance in understanding the LTPs found in pepper seeds, highlighting their remarkable antifungal activity with inhibitory efficacy on the growth of \u003cem\u003eC. albicans\u003c/em\u003e yeast. We also observed ultrastructural changes in yeast cells treated with the P10 fraction, especially displacement of the plasma membrane and cell wall. Additionally, we explored their inhibitory effect on insect α-amylase, expanding the biotechnological prospects of these proteins. This report contributes significantly to the development of the biotechnological potential of LTPs, reinforcing their role as promising candidates for future applications in agriculture and medicine.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003eThis work was performed at the Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF). We wish to thank L.C.D. Souza and V.M. Kokis for their technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e We acknowledge the financial support of the Brazilian agencies CNPq (307590/2021-6) and FAPERJ (E-26/200567/2023; E-26/210353/2022; E-26/200.127/2023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003eAll the data generated or analyzed during this study are\u0026nbsp;included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e The authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u0026nbsp;\u003c/strong\u003eThis article does not contain any studies with human or animal subjects.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eThe study was conceived by APBFO, AOC and VMG. Experimental procedures were carried out by LMR, LAS, MSS, RPC, FFM. Data analyses were performed by CSN, SHS, MC, RR. The paper was written by APBFO, GBT, EOM, VMG. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMissaoui K, Gonzalez-Klein Z, Pazos-Castro D, Hernandez-Ramirez G, Garrido-Arandia M, Brini F, Diaz-Perales A, Tome-Amat J (2022) Plant non-specific lipid transfer proteins: An overview. Plant Physiology and Biochemistry 171:115\u0026ndash;127. https://doi.org/10.1016/j.plaphy.2021.12.026\u003c/li\u003e\n\u003cli\u003eAmador VC, Santos-Silva CA, Vilela LMB, Oliveira-Lima M, Santana MR, Roldan-Filho RS, De Oliveira-Silva RL, Lemos AB, De Oliveira WD, Ferreira-Neto JRC, Crovella S, Benko-Iseppon AM (2021) Lipid transfer proteins (Ltps)\u0026mdash;structure, diversity and roles beyond antimicrobial activity. 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Probiotics and Antimicrobial Proteins 13:862\u0026ndash;872. https://doi.org/10.1007/s12602-020-09739-3\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Antimicrobial peptide, Capsicum, Candida, LTP, α-amylase","lastPublishedDoi":"10.21203/rs.3.rs-4985077/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4985077/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this study, we identified and partially purified antimicrobial peptides belonging to the family of lipid transfer proteins (LTPs) from \u003cem\u003eCapsicum chinense\u003c/em\u003e seeds (UENF 1751 accession). Fractions rich in LTPs were obtained via ion exchange chromatography and subsequently purified via reverse-phase chromatography in an HPLC system. Therefore, two fractions were revealed: C1 (the nonretained fraction) and C2 (the retained fraction). Fraction C1 was subjected to reverse-phase chromatography via a C18 column on an HPLC system, and ten fractions were obtained (P1\u0026ndash;P10), all of which significantly inhibited the growth of \u003cem\u003eC. albicans\u003c/em\u003e, except for P4 and P9. The viability analysis of the active fractions at a concentration of 100 \u0026micro;g.mL\u003csup\u003e-1\u003c/sup\u003e against \u003cem\u003eC. albicans\u003c/em\u003e revealed that they did not exhibit fungicidal activity but rather exhibited fungistatic activity. Fractions P3, P4, P7, and P10 inhibited \u003cem\u003eTenebrio molitor\u003c/em\u003e larvae α-amylase. The P10 fraction presented protein bands in its electrophoretic profile with a molecular mass between 6.5 kDa and 14.2 kDa and reacted positively to an antibody produced against a protein from the LTP family by Western blotting. The results of the analysis of amino acid residues from the P10 fraction revealed similarity between type I LTPs and type II LTPs. The ultrastructural aspects of \u003cem\u003eC. albicans\u003c/em\u003e cells exposed to the P10 fraction were evaluated via transmission electron microscopy (TEM), with significant differences in their morphology being evident compared with those of the control. In summary, our results demonstrated the presence of LTPs in \u003cem\u003eC. chinense\u003c/em\u003e seeds with inhibitory effects on the growth of yeasts of the genus \u003cem\u003eCandida\u003c/em\u003e, which exhibited fungistatic effects and structural changes in \u003cem\u003eC. albicans\u003c/em\u003e cells, in addition to exhibiting inhibitory effects on the larval insect \u003cem\u003eT. molitor\u003c/em\u003e α-amylase.\u003c/p\u003e","manuscriptTitle":"Antifungal properties and α-amylase inhibitory activity of lipid transfer proteins (LTPs) partially purified from Capsicum chinense Jacq. seeds","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-04 17:12:09","doi":"10.21203/rs.3.rs-4985077/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cf85dad0-62e8-46d7-b42c-4e68544bddeb","owner":[],"postedDate":"October 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-12T17:23:28+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-04 17:12:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4985077","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4985077","identity":"rs-4985077","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}