Angiotensin-I converting enzyme (ACE) inhibitors are widely used to control hypertension. In this study, protein hydrolysates from shiitake mushroom were hydrolyzed to prepare ACE-inhibitory peptides. Optimum process conditions for the hydrolysis of shiitake mushrooms using Alcalase were optimized using response surface methodology. Monitoring was conducted to check the degree of hydrolysis (DH) and ACE inhibitory activity. In the results, the optimum condition with the highest DH value of 28.88% was 50.2 °C, 3-h hydrolysis time, and 1.16 enzyme/substrate ratios. The highest ACE inhibitory activity (IC50 of 0.33 μg/mL) was under 47 °C, 3 h 28 min hydrolysis time, and 0.59 enzyme/substrate ratios. The highest activity was fractionated into 5 ranges of molecular weight, and the fraction below 0.65 kDa showed the highest activity with IC50 of 0.23 μg/mL. This fraction underwent purification using RP-HPLC, meanwhile the peak which offered a retention time of about 37 min showed high ACE inhibitory activity. Mass spectrometry identified the amino acid sequence of this peak as Lys-Ile-Gly-Ser-Arg-Ser-Arg-Phe-Asp-Val-Thr (KIGSRSRFDVT), with a molecular weight of 1265.43 Da. The synthesized variant of this peptide produced an ACE inhibitory activity (IC50) of 37.14 μM. The peptide KIGSRSRFDVT was shown to serve as a non-competitive inhibitor according to the Lineweaver–Burk plot findings. A molecular docking study was performed, which showed that the peptide binding occurred at an ACE non-active site. The findings suggest that peptides derived from shiitake mushrooms could serve either as useful components in pharmaceutical products, or in functional foods for the purpose of treating hypertension.
Hypertension is known as a chronic health problem which affects patients through high blood pressure which harms their wellbeing. Hypertension can be a factor in many other conditions, such as heart disease, stroke, aneurysm and renal failure. Many factors can indicate a predisposition for hypertension, including obesity, stress, or a sedentary way of life, all of which can arise in younger people as well as those of greater years (Bhagani et al. 2018). There is a vital role played by the angiotensin I-converting enzyme (ACE, EC.188.8.131.52) when regulating blood pressure via the Renin-Angiotensin System (Sayer and Bhat 2014), which is able to influence arterial vasoconstriction and extracellular volume, which amounts to the combined volume of lymph, blood plasma, and interstitial fluids. ACE acts as a catalyst in converting the decapeptide angiotensin I to the potent vasoconstrictor angiotensin II, while in addition degrading bradykinin, which results in the arteries undergoing systemic dilation and hence a reduction in arterial blood pressure. When certain ACE inhibitor (ACEI) peptides are administered, this can lead to a decline in the formation of angiotensin II and accordingly a drop in blood pressure. However, there are side effects reported with such synthetic drugs, such as coughing, rashes on the skin, and altered sense of taste, or angioneurotic edema. It is believed that all of these conditions can be caused by synthetic ACEIs. Accordingly, it would be highly beneficial to develop a novel, economical and non-toxic type of ACEI in order to treat or reduce the incidence of hypertension (Li et al. 2004).
There are normally between 3 and 20 amino acid residues contained within each molecule of a bioactive peptide. Studies have indicated that it is possible for bioactive peptides to generate effects which have antimicrobial, antioxidative, immunomodulatory, and antihypertensive consequences (Erdmann et al. 2008). The most common means of producing ACE inhibitory proteins and peptides is through the process of enzymatic hydrolysis (Kim and Wijesekara 2010). Through this approach, the conditions associated with the protease, the protein substrate, and the hydrolysis process will have a significant effect upon the ACE inhibitory peptide release. A number of commercial enzymes are commonly used to generate protein hydrolysates offering ACE inhibitory activity, such as Alcalase, pepsin, papain, trypsin, and Protamex (Moller et al. 2008; Wang et al. 2017). The process parameters can be assessed using response surface methodology (RSM) which provides statistical analysis in cases where a number of factors interact to influence the outcomes of experiments. The industrialization of active peptides derived from proteolysis is one process which makes heavy use of RSM (Turan et al. 2015). It is possible for the proteolysis method to be optimized so that peptides can be generated which offer high potency or high yields. To do so requires analysis of the conditions, including the E/S (enzyme–substrate) ratio, the time for proteolysis, the temperature, pH value, and the current degree of hydrolysis, since all of these factors affect yield and potency. At present, ACE inhibitory peptides are derived from proteins found in stone fish (Auwal et al. 2017), whey proteins (Guo et al. 2009), lizardfish muscle proteins (Wu et al. 2012), and a range of other sources involving RSM.
Mushrooms have been shown to have profound health-promoting benefits and studies have confirmed their medical use and many of the bioactive molecules present in mushrooms have been identified. The many and diverse species of mushrooms provide a rich source of bioactive molecules, which have recognized potential in drug discovery and development (El Sheikha and Hu 2018). Consumption levels for shiitake mushrooms (Lentinula edodes) in Thailand are rather high, since the mushroom is an excellent source of proteins, fiber, carbohydrates, and a range of vitamin types (Thetsrimuang et al. 2011). Moreover, the properties of this particular mushroom type are known to promote health, and can also act to provide antioxidant, anticancer, antiviral, anti-inflammatory and antimicrobial effects, while lowering the levels of plasma glucose with induced diabetes, and reducing cholesterol and triglyceride levels (Elmastas et al. 2006; Bisen et al. 2010; Rahman et al. 2018). In our current understanding, earlier studies have emphasized the need to optimize the related variables in order to achieve a suitable the degree of hydrolysis (DH) in the production of protein isolates from shiitake mushrooms. Therefore, the aim of this study was to prepare shiitake-protein hydrolysates for the development of angiotensin-I-converting peptides (ACE)-inhibitory and then explores the inhibitory capabilities of the resulting hydrolysates for ACE. To investigate the optimal condition for generating the hydrolysis of shiitake mushrooms, RSM was used for the optimization of the conditions under which hydrolysis takes place at difference pH values, temperature, and E/S on DH and ACE-inhibitory activity. Those ACE inhibitory peptides which had been prepared using Alcalase then underwent evaluation. The purification of the ACE inhibitory peptides was performed using ultrafiltration along with reversed-phase high-performance liquid chromatography (RP-HPLC). The quadrupole time-of-flight (Q-TOF) mass spectrometer (LC–MS/MS) was then used to determine the peptide sequence, while molecular docking experiments produced findings which suggested that the potential binding interaction of the purified.
Materials and methods
Materials, equipment and chemicals
The shiitake mushrooms (L. edodes) were obtained from Tawan Produce Co., Ltd. (Samutprakarn, Thailand) during July–August 2017, and were kept at room temperature in a desiccator until required. The shiitake mushrooms are identified based on morphological characteristics, such as the size, shape, form, gills, basidium, and basidiospores. The past taxonomic studies were based mainly on morphological features (Alexopoulos et al. 1996; Hibbett and Donoghue 1996). The microscopic characteristics was followed the method as described by Largent et al. (1977). The identification was attempted using the available lilerature listed in the reference section. The specimens were submerged in 70% ethyl alcohol and deposited at the herbarium of mycology laboratory, Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Bangkok, Thailand.
Alcalase 2.4 L is a microbial protease of Bacillus licheniformis, was obtained from Novo Nordisk (Bagsverd, Denmark), and was kept at a temperature of 4 °C until required. The required chemicals, namely ACE (E.C. 184.108.40.206) obtained from rabbit lung, bovine serum albumin (BSA), hippuric acid, hippuryl-l-histidyl-l-leucine (HHL), captopril, acetonitrile, and 2, 4, 6-trinitrobenzenesulfonic acid (TNBS) were all supplied by Sigma Chemical Co. (St. Louis, MO, USA), and all were of analytical grade.
Enzymatic hydrolysis preparation of shiitake mushroom protein hydrolysate
The shiitake mushroom fruiting bodies were prepared by the method of He et al. (2012) with a slight modification. Briefly, the fruiting bodies were placed whole into a hot air oven where they were dried for 24 h at a temperature of 80 °C, then subsequently ground to form a fine powder. This powder was then sieved using an 80-mesh sieve with a 177 µm opening, before storage at room temperature in a vacuum-sealed polypropylene bag placed in a desiccator until required.
Hydrolysis conditions used for single-factor analysis involved temperatures in the range of 45 °C to 60 °C, while the time taken ranged from 30 min up to 5 h. In this process, 1 g of shiitake mushroom powder was suspended in 50 mL of phosphate buffer (PBS; 20 mM phosphate buffer, pH 7.2). The pH value for the resulting mixture was altered using 1 M sodium hydroxide (NaOH) to reach 7.2. Hydrolysis was then carried out with Alcalase (3.018 U/mL) using a shaker incubator at 180 rpm. Termination of the hydrolysis process was accomplished by maintaining a temperature of 90 °C for 20 min before centrifugation at 15,900×g for 30 min at a temperature of 4 °C. The resulting supernatant was then gathered and placed in storage at a temperature of − 20 °C until required.
Experimental design to optimize the preparation of shiitake mushroom protein hydrolysate using RSM
To find the optimal conditions to obtain the required responses for DH and ACE inhibitory activity, RSM was used. Having first conducted a number of single-factor experiments, RSM was then employed in order to establish the effects of the various independent variables, such as time (A), temperature (B), and E/S (C) upon DH and ACE inhibitory activity (Y). A central composite design (CCD) comprising three factors and five levels was employed to assess the influence of temperature (30 to 70 °C), hydrolysis time (30 to 360 min), and E/S (0.1 to 2%). The overall design comprised 17 combinations, which can be observed in Table 1. In this case the DH and ACE inhibitory activity served as the response values. The design of the experiments and the data analysis were carried out with the assistance of Stat-Ease software (Design Expert Version 7.0.0 Trial).
The shiitake mushroom hydrolysates were examined in order to establish the DH level using a modified version of an approach proposed by Nielsen et al. (2001). Measurement of the free amino groups of the hydrolysates was accomplished through the use of o-phthaldialdehyde (OPA solution). Following the hydrolysis phase, 3 mL of OPA reagent and 400 μL of shiitake mushroom hydrolysate were mixed for 5 s, before allowing the mixture to stand for 2 min. A reading was then obtained at 340 nm using a microplate spectrophotometer (Multiskan GO; Thermo Fisher Scientific, Waltham, MA, USA). The standard used was serine, while every assay was carried out in triplicate and the calculation of DH was based on the formula given as: %DH = (h/htot) × 100, in which htot denotes the total number of peptide bonds per protein equivalent, and h indicates the number of hydrolyzed bonds. In this equation, the htot factor depends upon the amino acid content in the raw material used.
Soluble protein determination
The Bradford assay (1976) was used to establish the soluble protein content, during which the standard used was bovine serum albumin. The sample used underwent mixing with Bradford working buffer to a ratio of 1:20 (v/v) and was then placed in a 96-well plate prior to incubation for 20 min. A microplate spectrophotometer was then used to measure the absorbance at 595 nm.
ACE inhibitory activity assay
ACE inhibitory activity was examined using the method slightly modified by Ibrahim et al. (2017). This assay was carried out in a 96-well plate, and began with the addition of 5 μL of 200 mU/mL ACE to an aliquot of the sample (10 μL), before adding 13 μL of the substrate (5 mM HHL) and incubating at a temperature of 37 °C for 1 h. In the next step, a further 50 μL of 0.68 mM TNBS was added before incubation at a temperature of 37 °C for 1 h and measurement of the absorbance at 420 nm to determine the ACE inhibitory activity. The tests were conducted in triplicate, while the positive control used was Captopril. The following equation was used to determine the ACE inhibitory activity percentage, which is equal to [(C − Bi) − (S − Bs)/(C − Bi)] × 100, in which C, S, Bs, and Bi are used respectively to indicate the absorbances of the control (100% activity), the sample (inhibitor peptide), the blank sample (only peptide sample), and the blank inhibitor (only HHL). IC50 is defined as the hydrolysate or peptide concentration capable of inhibiting 50% of ACE activity in the stated assay conditions; the calculation of the IC50 value is carried out through non-linear regression making use of GraphPad Prism Version 6 (GraphPad Software Inc., La Jolla, CA, USA).
ACE inhibitory peptide purification
The fractionation of ACE inhibitory shiitake mushroom peptides first requires the preparation of 500 mL of shiitake mushroom hydrolysate which is obtained via filtration using filter paper (Whatman No. 1). An ultrafiltration unit (Pellicon XL Filter; Merck Millipore, Billerica, MA, USA) is used for the process of fractionation performed through four ultrafiltration membranes with cut-off values for molecular weight (Mw) of 10, 5, 3, and 0.65 kDa. The soluble protein content and the ACE inhibitory activity of each fraction were evaluated. The fraction with the highest ACE inhibitory activity was concentrated and stored at –20 °C before further purification.
RP-HPLC (Spectra System, Thermo Fisher Scientific, San Jose, CA, USA) made use of a Luna C18 column (4.6 mm × 250 mm, Luna 5 µM, Phenomenex, Torrance, CA, USA). Filtration of a 1 mL aliquot of the concentrated fraction which had been previously collected via ultrafiltration was performed using a 0.45 μm nylon membrane (Whatman, GE, Buckinghamshire, UK). The HPLC system then received 200 microliters of this particular fraction delivered by injection. Separation was accomplished through gradient elution involving solvent A: 0.1% (v/v) trifluoroacetic acid (TFA) in distilled water, and solvent B: 0.05% (v/v) TFA in acetonitrile (ACN). The process used a flow rate of 0.7 mL/min and employed ultraviolet absorbance of 280 nm (A280) in order to check the eluate condition. All peaks deemed high and clear were gathered, concentrated, and further examined for ACE inhibitory capability. The fraction which showed the greatest activity was then dried using a freeze-drying technique, and MS was used to identify the peptide sequence.
Determination of the amino acid sequence for the purified peptide using liquid chromatography-quantitative time-of-flight tandem mass spectrometry (LC-Q-TOF–MS/MS)
The purified ACE inhibitory peptide was characterized through the use of a Q-TOF mass spectrometer combined with an electrospray ionization source mass spectrometer (Model Amazon SL, Bruker, Germany). The positive mode was used for the ionization, while separation used a flow rate of 100 mL/min with a 5–80% B gradient for a duration of 50 min. The solvent systems involved included solvent A, comprising 0.1% formic acid in water, and solvent B, comprising 100% ACN. The mass spectral data from 300 to 1500 m/z were gathered in the positive ionization mode, while HyStar 3.2 software (Bruker Daltonics Inc., Billerica, MA, USA) was employed for the purpose of interfacing the RP-HPLC and MS systems. Data analysis was performed using de novo sequencing.
Synthesis of peptides
The ACE inhibitory peptide which was identified as having particularly useful potential and was derived from the shiitake hydrolysate could be chemically synthesized using the technique of Fmoc solid phase synthesis, which takes place at Bootech Bioscience & Technology Co., Ltd., in Shanghai, using an Applied Biosystems Model 433A Synergy peptide synthesizer. Verification of the purity of the peptide was achieved via analysis through the MS system (Thermo Mod. FinniganTM LXQTM) connected to a Surveyor HPLC. ACE assay was used to establish the ACE inhibitory activity of the peptide, while the peptide sequence identified for the synthetic peptide was KIGSRSRFDVT, with 98% purity and a molecular weight of 1265.43 Da.
The ACE inhibition pattern can be estimated by examination of the various synthesized peptide concentrations (0.8, 0.6, 0.4, and 0 mM) which were incubated in ACE solution with varying HHL concentrations (1, 2, 3, 4, and 6 mM). An investigation of the inhibition kinetics when peptide is present, or absent, was performed using Lineweaver–Burk plots, in which the independent variable on the x-axis was the reciprocal of the HHL concentration, while the dependent variable on the y-axis was the absorbance at 420 nm (HL + TNBS complex). The Dixon plot representing the slope of the Lineweaver–Burk plot against inhibitor concentration was used to determine the inhibitor constant (Ki).
Molecular KIGSRSRFDVT docking at the ACE binding site
Studies of molecular docking were carried out with Hermes 1.10.1 software. The receptor used for this study was a three-dimensional crystal structure of a human ACE-lisinopril complex (1O8A.pdb) which was obtained by downloading from the RCSB PDB Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). The peptide structure was produced using Discovery Studio 20 software. Prior to docking, the removal of all hetero atoms from the ACE model was required, including water molecules and the inhibitor, lisinopril. The polar hydrogens were then introduced to the ACE model before an assessment of the molecular docking was carried out in line with a number of scoring categories, such as ChemScore, ChemPLP, ASP, and Goldscore. The scores collected for binding energy suggest that the ideal pose for each of the residues was achieved. Identification of the hydrogen bonds was accomplished using Discovery Studio 20 software, while hydrophobic, hydrophilic, coordination, electrostatic, and van der Waals interactions were also determined when taking place between the ACE molecule and the peptide residue. The optimal pose for the peptide was also sought in this phase.
The experiments were performed in triplicate, and findings presented in the form of mean ± standard deviation. SPSS 11.5 statistical software was used to perform all analyses. Hypothesis testing was conducted using one-way analysis of variance (ANOVA) along with the test of least significant difference (LSD), where P < 0.05 was used as the significance level.
Results and discussion
Shiitake mushroom protein hydrolysis optimization
The RSM experiments had their basis in single-factor experimental results, where the three hydrolysis factors underwent variation at five levels within a CCD, and analysis of the DH response surface was performed with Design-Expert 8.0.5. Time, temperature, and E/S served as the variable independent factors, while the experimental design can be seen in Table 1, which lists the 17 experiments conducted along with the responses. It is clear from the table that DH varied between 0 and 28.88% at its peak, DH of 28.88% was achieved at 3 h, 50 °C, and E/S of 1%. A second order polynomial model derived from multiple regression was used to assess the impact of the various conditions upon DH, as shown below:
in which A, B, and C denote the respective coded values for time, temperature, and E/S, and Y gives the DH value.
The model predicted optimal conditions to carry out hydrolysis as follows: time of 2.99 h, temperature of 50.21 °C, and E/S of 1.16%. DH was anticipated to be 28.244%. In such ideal conditions, DH was found to be 28.992% with the error reported to be 2.648%, which closely matches the value predicted. Accordingly, the RSM was able to generate reliable, valid, and accurate values in the context of protein hydrolysate preparation from shiitake mushrooms. Evaluation of the linear (A, B, C), quadratic (A2, B2, C2), and interaction terms (AB, AC, BC) of the variable effects upon the DH of shiitake protein hydrolysate was carried out to assess the fitness, adequacy, and significance using ANOVA as indicated in Table 2. ANOVA analysis of the DH data revealed that the results obtained by the second-degree polynomial model were highly significant (P < 0.0001) while the determination coefficient was satisfactory (R2 = 0.9866), indicating that accurate predictions could be generated by this model when considering the relationship between the response and the independent variables examined. The high value recorded for the adjusted determination coefficient (adjusted R2 = 0.9694) also suggests the model was very accurate. The model did not show significant lack of fit (P = 0.6177, > 0.05), and accordingly was capable of providing accurate DH predictions. P values were employed to show significance in the context of regression coefficients, whereby a low P value would indicate greater significance of the coefficient. Regression coefficient analysis showed the significance of the linear coefficients (C) (P < 0.0001) while the significance of A2, B2, and C2 was also noted (P < 0.0001) in the context of the response DH. Figure 1a–c shows the response surfaces when affected by different variables in terms of the DH values. The findings indicate that E/S has the greatest effect upon DH, and this value can in turn govern the reaction between Alcalase and the substrate. Figure 1a–c presents the 3D response surface plots which serve as graphical forms of the regression equation. It is possible to use these plots to better comprehend the nature of the interactions which occur between factors, and to find each factor’s optimal level in order to achieve the best response. A surface response is plotted for each of the DH values, representing a novel combination of the variables involved for each test, while all other variables are held constant at the zero level.
Optimization of the peptide ACE inhibitory activity
Table 1 presents the IC50 values which represent the ACE inhibitory activity produced by the shiitake mushroom hydrolysates. These IC50 values were shown to fall in the range of 0.39–11.90 μg/mL. The greatest activity levels occurred under hydrolysis conditions of 4 h, a temperature of 45 °C, and E/S of 0.5%. Table 3 displays the ANOVA results of ACE inhibitory activity of the shiitake mushroom hydrolysates. The regression model showed a high degree of significance (P < 0.0001), but the lack of fit (P = 0.0588, > 0.05) was considered not to be significant. The model produced an R2 value which suggested that the model could explain 98.65% of the ACE inhibitory activity produced by the shiitake mushroom hydrolysate. The adjusted R2 value measured 0.9743 while the coefficient of variation (CV) was 11.46%. These values suggest that the model is an accurate predictor of ACE inhibitory activity for shiitake mushroom hydrolysate. In order to examine the parameters for the conditions governing ACE inhibitory activity, it was necessary to create a second order polynomial model using multiple regression analyses as indicated below:
in which Y denotes the real value of the predicted response (the IC50 value for ACE inhibition), A indicates the code value for the time variable, B denotes the code value for the temperature variable, while C represents the code value of the E/S variable.
To perform the verification examination, an experiment was conducted to determine the model adequacy using a time of 2.06 h, E/S of 1.5%, and a temperature of 52.78 °C. According to the results, the experimental IC50 value for ACE inhibitory activity was 0.32 μg/mL, which was similar to the prediction of 0.399 μg/mL. The model showed that the most important factor affecting ACE inhibitory activity was time (P < 0.0001). The influence of treatment temperature and E/S was shown to have no significant effect upon the ACE inhibitory activity. In the equation, the interaction terms AC and BC and the quadratic terms A2 and B2 showed a high degree of significance (P < 0.0001) but the other terms were not found to be significant (P > 0.05). Figure 1d–f presents the response surfaces of each of the effects of the variables upon ACE inhibitory activity.
From this study, the optimal conditions for both DH and ACE inhibitory capacity when the hydrolysates from shiitake mushrooms are used were different from those reported in earlier works. For instance, Guo et al. (2009) used RSM to examine the influence of process conditions upon the ACE inhibitory activity of hydrolyzed whey protein concentrates. While whey protein hydrolysate was shown to have a DH ranging from 3.0 to 49.5%, the optimized conditions for the achievement of high ACE inhibitory activity were 92.2% with a DH of just 18.8%. The findings suggested that and increased DH level was not indicative in itself of greater ACE inhibitory activity. It may be the case that ACE activity is dependent upon the amino acids in the peptide sequence, whereby the process of hydrolysis can increase the quantity of amino acids released, thereby contributing to the activity of the peptides in ACE inhibition.
ACE inhibitory peptide purification
The shiitake mushroom sample producing the greatest ACE inhibitory activity in optimized conditions was identified and duly fractionated. The initial separation was carried out with a 10 kDa ultrafiltration membrane, followed by a series of 5, 3, and 0.65 kDa membranes. Sequential fractionation of the hydrolysate followed, to create five further fractions of > 10, 10–5, 5–3, 3–0.65, and < 0.65 kDa. The fraction identified as < 0.65 kDa produced the greatest inhibitory action against ACE (IC50 value was 0.23 μg/mL), whereas the least effective fraction was that for which MW > 10 kDa, where the IC50 value was 8.17 μg/mL and the 10–5, 5–3, and 3–0.65 kDa fractions produced IC50 values measured respectively at 0.71, 0.55, and 0.26 μg/mL. The findings indicated that the peptide ACE inhibitory activity is associated with the MW. Moreover, the specific activity of the fractions was enhanced by the ultrafiltration of the shiitake hydrolysates. Peptides which have a smaller MW can be more readily absorbed by the body, and thus the < 0.65 kDa fraction underwent additional separation to achieve the isolation of the ACE inhibitory peptides.
The molecular weight of peptides also has a strong influence upon ACE inhibitory activity. The study showed that the IC50 value for the shiitake mushroom hydrolysate fraction < 0.65 kDa, a result which exceeded that of fractions which offered greater MW values according to earlier research (Salampessy et al. 2017; Wang et al. 2017). The findings therefore confirm that smaller peptides can create superior ACE inhibition, while short peptides tend to be the most efficient in ACE inhibition since it is the longer chains which usually bind less readily to ACE, leading to lower levels of inhibition. It is reasonable to expect that bioactive peptide will be found in the low molecular weight fraction of protein hydrolysate. Especially, food bioactive peptides consist of short amino acid sequences, usually 2–20 residues can be absorbed into the hollow of the small intestine throughout the digestion process and then be carried to the blood circulatory system. However, the useful impacts of bioactive peptides depend basically on their capacities to reach the target organ (Onuh and Aluko 2019).
Purification of the fraction which had molecular weight < 0.65 kDa was carried out by RP-HPLC as presented in Fig. 2a. Following the process of gradient elution over 55 min, fractionation was then performed to separate the sample to obtain seven parts. These seven parts were then assessed for ACE inhibitory activity with an in vitro ACE inhibitory assay in order to determine which of the parts proved most effective. In particular, the ACE inhibitory activity of fractions F1–7 produced IC50 values measured respectively at 171.25, 255.23, 159.14, 167.48, 227.84, 295.47 and 132.87 μg/mL. Of the seven parts, the highest ACE inhibitory activity came from F7, which had a retention time of 36.96 min. This fraction was duly taken to determine the amino acid sequence.
Peptide sequence identification
The amino acid sequence for the ACE inhibitory peptide of the enriched fractions (F7) which is identified by LC-Q-TOF–MS/MS as Lys-Ile-Gly-Ser-Arg-Ser-Arg-Phe-Asp-Val-Thr (KIGSRSRFDVT) which has a molecular weight of 1265.43 Da (Fig. 2b). The NCBI GenBank and UniProt databases (de novo deducing) were used to align the fragment in order to determine the homologous region. The peptide sequence of KIGSRSRFDVT was shown to have 63% (7/11) amino acid sequence similarity to the AFG1-like ATPase (L. edodes), but did not exactly match any other known protein type. Synthesis of the peptide was accomplished through the use of the sequence as indicated for the purified peptide. The synthesized peptide produced an IC50 value for ACE inhibitory activity of 37.14 μM.
Peptide ACE inhibitory properties are governed by the structure, size, composition, sequence, and configuration of the peptide, with certain amino acids having a particularly powerful influence upon ACE inhibitory activity, namely Trp, Tyr, Phe, Pro, and aliphatic amino acids. Chul et al. (2006) performed an analysis of a hydrolysate of Pholiota adipose which used distilled water, and discovered the peptide sequence Gly-Glu-Gly–Gly-Pro which had an IC50 value of 0.25 μM. Meanwhile, Wang, et al. (2017) were able to isolate one peptide from a rice bran source which produced an ACE inhibitory activity IC50 value of 76 mM, with a Mw of 395.0 Da, and an amino acid sequence of Tyr-Ser-Lys. A number of research studies have argued that it is the amino acid sequence of the ACE inhibitor which influences the binding to ACE. It is possible that Phe, Pro, Trp, Tyr, and hydrophobic amino acids affect ACE inhibitory activity. This study notes that when the peptide sequence is isolated from shiitake hydrolysate, the residues are Ile, Phe, and Val, which have previously been shown to influence ACE inhibitory activity. Accordingly, the peptide KIGSRSRFDVT may prove to be a useful bioactive peptide capable of treating hypertension.
Establishing the kinetic properties of ACE inhibition by KIGSRSRFDVT
Analysis was carried out to better understand ACE inhibition of KIGSRSRFDVT by varying ACE velocity and the HHL substrate concentration while using fixed peptide concentrations. The ACE inhibition pattern is presented in Fig. 2c, d for the purified peptide obtained from the shiitake mushroom hydrolysate when examined through a Lineweaver–Burk plot depicting the differing KIGSRSRFDVT concentrations. From the findings it was apparent that the peptide KIGSRSRFDVT served as a non-competitive inhibitor. KIGSRSRFDVT had a Ki value of 12.9 μM, indicating that any bond between the substrate and ACE would have no influence upon the peptide in terms of its inhibitory activity. The ACE inhibitor derived from shiitake mushrooms is able to bind to the ACE at sites which differ from the active sites at which the substrate can bind. Accordingly, the KIGSRSRFDVT peptide can join with ACE molecules to generate a dead-end complex, and for this it is of no consequence whether or not binding of a substrate molecule has taken place. The peptide causes enzyme inhibition through conformational change, which exerts its influence no matter whether the substrate concentration is high or low.
Recent research has indicated that ACE inhibitory peptides display various different inhibition patterns: competitive, uncompetitive, non-competitive, and mixed-competitive patterns. A number of competitive ACE inhibitors are well-known, including WESLSRLLG derived from ostrich egg white (Asoodeh et al. 2016), VVSLSIPR which comes from pigeon peas (Nawaz et al. 2017), and TVGMTAKF and QLLLQQ obtained from horse gram flour (Bhaskar et al. 2019). A smaller number of non-competitive ACE inhibitor patterns have also been studied. Lee et al. (2011) investigated the ACE inhibitory activity of skate skin protein hydrolysates by isolating the peptides PGPLGLTGP (975.38 Da) and ELGFLGPR (874.45 Da), for which the respective IC50 values were 95 and 148 μM were non-competitive ACE inhibitors. Meanwhile, other ACE inhibitory peptides have also been shown to be non-competitive, such as PAFG from Enteromorpha clathrata (Pan et al. 2016); YAP, VIIF, and MAW from cuttlefish (Balti et al. 2010); AHIII from Styela clava flesh tissue (Ko et al. 2012); and VWDPPLFA from salmon (Ahn et al. 2012).
Molecular docking of KIGSRSRFDVT at the ACE binding site
Predictions of the enzyme interaction with small molecules is Predictions of the enzyme interaction with small molecules is achieved through molecular docking. It may become easier to design or synthesize novel ACE inhibitors on the basis of improved knowledge of the molecular mechanisms underpinning ACE and inhibitory peptide interactions. The flexible docking tool of the Hermes 1.10.1 software was used to perform a docking simulation between ACE and the KIGSRSRFDVT peptide. In this study, it was revealed that at the ACE non-active site, ligand A5 presented the best performance through an interaction score ASP fitness 62.7322 (RMSD 7.0137). The scoring outcomes from ligand–receptor combinations can show the extent to which such interactions are stable. Figure 3a presents the 3D interaction between ACE and the peptide KIGSRSRFDVT, while Fig. 3b presents a 2D diagram which reveals the interactions between ACE residues and the peptide molecule. Seven hydrogen bonds were established by the peptide KIGSRSRFDVT with the various ACE residues after docking: Arg479, Asn31, Ala315, Glu345, Lys468, Tyr477, and His344. Reports have detailed the residues at the ACE active site (Pan et al. 2012). ACE offers three principal active site pockets; S1, S2, and S1’, which are associated with the residues Ala354, Glu384, Tyr523, Gln281, His353, Lys511, His513, Tyr520, and Glu162. Accordingly, the bond between KIGSRSRFDVT and ACE is not linked to any of the active site pockets of ACE.
This study successfully identified an ACE inhibitory peptide produced from shiitake hydrolysate using Alcalase. To achieve the desired DH and ACE inhibitory activity, the RSM based on CCD was used to evaluate and optimize the effects of temperature, hydrolysis time, and enzyme/substrate ratios. The optimal conditions were found to be 50.2 °C, 3 h, and 1.16 enzyme/substrate ratios, which resulted in the highest DH (28.88%). However, the optimum conditions in which the hydrolysate exhibited the highest ACE inhibitory activity (IC50 of 0.33 μg/mL) was under 47 °C, 3 h 28 min, and 0.59 enzyme/substrate ratio. Subsequently, the hydrolysate with the highest ACE inhibitory activity was then fractionated using molecular weight cut-off membranes, and the MW < 0.65 kDa showed the highest activity with IC50 of 0.23 μg/mL. After fractionation through ultrafiltration and purification by RP-HPLC and MS, an ACE inhibitory peptide KIGSRSRFDVT with an IC50 value of 37.14 μM was identified. These results suggest that the inhibition of ACE by KIGSRSRFDVT is a non-competitive inhibitor determined by Lineweaver–Burk plots. Finally, a molecular docking examination was used to predict the binding site for the purified peptide KIGSRSRFDVT with ACE. The result show that the KIGSRSRFDVT binding site with ACE was a non-active site. In summary, the results presented in this study suggest that the peptide derived from shiitake mushroom protein hydrolysate has the potential to be utilized as a bioactive peptide in the treatment of hypertension, and in the future it may become part of industrial efforts to produce a functional food product capable of naturally mediating hypertension. Other properties of the peptide, such as the cytotoxicity, allergenicity, and its overall safety should be studied in greater detail prior to considering any particular peptide for application in either the pharmaceutical or food sectors.
Ahn CB, Jeon YJ, Kim YT, Je JY (2012) Angiotensin I converting enzyme (ACE) inhibitory peptides from salmon. Process Biochem 47:2240–2245. https://doi.org/10.3390/ijms150814077
Alexopoulos CJ, Minis CW, Blackwell M (1996) Introductory mycology, 4th edn. Wiley, New York, p 868
Asoodeh A, Tabrizi MH, Shabestarian H, Emtenani S, Emtenani S (2016) Biochemical characterization of a novel antioxidant and angiotensin I-converting enzyme inhibitory peptide from Struthio camelus egg white protein hydrolysis. J Food Drug Anal 24:332–342. https://doi.org/10.1016/j.jfda.2015.11.010
Auwal SM, Zarei M, Abdul-Hamid A, Saari N (2017) Optimization of bromelain-aided production of angiotensin I-coverting enzyme inhibitory hydrolysates from stone fish using response surface methodology. Mar Drugs 15:104. https://doi.org/10.3390/md15040104
Balti R, Arroume N, Bougatef A, Guillochon D, Nasri M (2010) Three novel angiotensin I-converting enzyme (ACE) inhibitory peptides from cuttlefish (Sepia officinalis) using digestive proteases. Food Res Int 43:1136–1143. https://doi.org/10.1016/j.foodres.2010.02.013
Bhagani S, Kapil V, Lobo MD (2018) Hypertension. Nat Rev Dis Prim 46:509–515. https://doi.org/10.1016/j.mpmed.2018.06.009
Bhaskar B, Ananthanarayan L, Jamdar S (2019) Purification, identification, and characterization of novel angiotensin I converting enzyme (ACE) inhibitory peptides from alcalase digested horse gram flour. LWT Food Sci Technol 103:155–161. https://doi.org/10.1016/j.lwt.2018.12.059
Bisen PS, Baghel RK, Sanodiya BS, Thakur GS, Prasad GB (2010) Lentinus edodes: a macrofungus with pharmacological activities. Curr Med Chem 17:2419–2430. https://doi.org/10.2174/092986710791698495
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254. https://doi.org/10.1016/0003-2697(76)90527-3
Chul KK, Lee DH, Kim JH, Yu HE, Par JS, Lee JS (2006) Production and characterization of antihypertensive angiotensin I-converting enzyme inhibitor from Pholiota adipose. J Microbiol Biotechnol 16:757–763
El Sheikha AF, Hu DM (2018) How to trace the geographic origin of mushrooms? Trends Food Sci Technol 78:292–303. https://doi.org/10.1016/j.tifs.2018.06.008
Elmastas M, Turkekul I, Ozturk L, Gulcin I et al (2006) Antioxidant activity of two wild edible mushrooms (Morchella vulgaris and Morchella esculanta) from North Turkey. Comb Chem High Throughput Screen 9:443–448. https://doi.org/10.2174/138620706777698544
Erdmann K, Cheung BW, Schröder H (2008) The possible roles of food-derived bioactive peptides in reducing the risk of cardiovascular disease. J Nutr Biochem 19:643–654. https://doi.org/10.1016/j.jnutbio.2007.11.010
Guo YX, Pan DD, Tanokura M (2009) Optimisation of hydrolysis conditions for the production of the angiotensin-I converting enzyme (ACE) inhibitory peptides from whey protein using response surface methodology. Food Chem 114:328–333. https://doi.org/10.1016/j.foodchem.2008.09.041
He JZ, Ru QM, Dong DD, Sun PL (2012) Chemical characteristics and antioxidant properties of crude water soluble polysaccharides from four common edible mushrooms. Molecules 17:4373–4387. https://doi.org/10.3390/molecules17044373
Hibbett DS, Donoghue MJ (1996) Implications of phylogenetic studies for conservation of genetic diversity in Shiitake mushrooms. Conserv Biol 10:1321–1327. https://doi.org/10.1046/j.1523-1739.1996.10051321.x
Ibrahim HR, Ahmed AS, Miyata T (2017) Novel angiotensin-converting enzyme inhibitory peptides from caseins and whey proteins of goat milk. J Adv Res 8:63–71. https://doi.org/10.1016/j.jare.2016.12.002
Kim SK, Wijesekara I (2010) Development and biological activities of marine-derived bioactive peptides: a review. J Funct Foods 2:1–9. https://doi.org/10.1016/j.jff.2010.01.003
Ko SC, Lee JK, Byun HG, Lee SC, Jeon YJ (2012) Purification and characterization of angiotensin I-converting enzyme inhibitory peptide from enzymatic hydrolysates of Styela clava flesh tissue. Process Biochem 47:34–40. https://doi.org/10.1016/j.procbio.2011.10.005
Largent DL, Johnson D, Watling R (1977) How to identify mushrooms to genus, III. Microscopic features. Mad River Press, Eureka
Lee JK, Jeon JK, Byun HG (2011) Effect of angiotensin I converting enzyme inhibitory peptide purified from skate skin hydrolysate. Food Chem 125:495–499. https://doi.org/10.1016/j.foodchem.2010.09.039
Li GH, Le GW, Shi YH (2004) Shrestha S, Angiotensin I-converting enzyme inhibitory peptides derived from food proteins and their physiological and pharmacological effects. Nutr Res 24:469–486. https://doi.org/10.1016/S0271-5317(04)00058-2
Moller NP, Scholz-Ahrens KE, Roos N, Schrezenmeir J (2008) Bioactive peptides and proteins from foods: indication for health effects. Eur J Clin Nutr 47:171–182. https://doi.org/10.1007/s00394-008-0710-2
Nawaz KA, David SM, Murugesh E, Thandeeswaran M, Kiran KG, Mahendran R, Palaniswamy M, Angayarkanni J (2017) Identification and in silico characterization of a novel peptide inhibitor of angiotensin converting enzyme from pigeon pea (Cajanus cajan). Phytomedicine 36:1–7. https://doi.org/10.1016/j.phymed.2017.09.013
Nielsen PM, Petersen D, Dambmann C (2001) Improved method for determining food protein degree of hydrolysis. J Food Sci 66:642–646. https://doi.org/10.1111/j.1365-2621.2001.tb04614.x
Onuh JO, Aluko RE (2019) Metabolomics as a tool to study the mechanism of action of bioactive protein hydrolysates and peptides: a review of current literature. Trends Food Sci Technol 91:625–633. https://doi.org/10.1016/j.tifs.2019.08.002
Pan D, Cao J, Guo H, Zhao B (2012) Studies on purification and the molecular mechanism of a novel ACE inhibitory peptide from whey protein hydrolysate. Food Chem 130:121–126. https://doi.org/10.1016/j.foodchem.2011.07.011
Pan S, Wang S, Jing L, Yao D (2016) Purification and characterisation of a novel angiotensin-I converting enzyme (ACE)-inhibitory peptide derived from the enzymatic hydrolysate of Enteromorpha clathrata protein. Food Chem 211:423–430. https://doi.org/10.1016/j.foodchem.2016.05.087
Rahman MA, Abdullah N, Aminudin N (2018) Lentinula edodes (shiitake mushroom): an assessment of in vitro anti-atherosclerotic bio-functionality. Saudi J Biol Sci 25:1515–1523. https://doi.org/10.1016/j.sjbs.2016.01.021
Salampessy J, Reddy N, Phillps M, Kailasapathy K (2017) Isolation and characterization of nutraceutically potential ACE Inhibitory peptides from leatherjacket (Meuchenia sp.) protein hydrolysates. LWT Food Sci Technol 80:430–436. https://doi.org/10.1016/j.lwt.2017.03.004
Sayer G, Bhat G (2014) The renin-angiotensin-aldosterone system and heart failure. Cardiol Clin 32:21–32. https://doi.org/10.1016/j.ccl.2013.09.002
Thetsrimuang C, Khammuang S, Chiablaem K, Srisomsap C, Sarnthima R (2011) Antioxidant properties and cytotoxicity of crude polysaccharides from Lentinus polychrous Lév. Food Chem 128:634–639. https://doi.org/10.1016/j.foodchem.2011.03.077
Turan D, Capanoglu E, Altay F (2015) Investigating the effect of roasting on functional properties of defatted hazelnut flour by response surface methodology (RSM). LWT Food Sci Technol 63:758–765. https://doi.org/10.1016/j.lwt.2015.03.061
Wang X, Chen H, Fu X, Li S, Wei J (2017) A novel antioxidant and ACE inhibitory peptide from rice bran protein: biochemical characterization and molecular docking study. LWT Food Sci Technol 75:93–99. https://doi.org/10.1016/j.lwt.2016.08.047
Wu SG, Sun JH, Tong ZF, Lan XD, Zhao ZX, Liao DK (2012) Optimization of hydrolysis conditions for the production of angiotensin I-converting enzyme inhibitory peptides and isolation of a novel peptide from lizard fish (Saurida elongata) muscle protein hydrolysate. Mar Drugs 10:1066–1080. https://doi.org/10.3390/md10051066
The authors would like to thank the Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, for their support and providing access to their facilities. We acknowledge the financial support from the Grant for Research: The Research Assistantship Fund, Faculty of Science, Chulalongkorn University (RAF_2561_009), and The Ratchadapisek Sompoch Endowment Fund (2019), Chulalongkorn University (762008) for providing the financial support for this research.
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Paisansak, S., Sangtanoo, P., Srimongkol, P. et al. Angiotensin-I converting enzyme inhibitory peptide derived from the shiitake mushroom (Lentinula edodes). J Food Sci Technol 58, 85–97 (2021). https://doi.org/10.1007/s13197-020-04517-z
- Angiotensin-I converting enzyme (ACE)
- Shiitake mushroom
- Response surface methodology (RSM)
- Molecular docking