Maolin Tu,Xinyu Qiao,Cong Wang,Hanxiong Liu,Shuzhen Cheng,Zhe Xu,Ming Du

School of Food Science and Technology,National Engineering Research Center of Seafood,Collaborative Innovation Center of Seafood Deep Processing,Dalian Polytechnic University,Dalian 116034,China

Keywords:

Casein

Anticoagulant

ACE inhibitory

Dual-function

NanoLC-Q-TOF-MS/MS

ABSTRACT

There is no study on food-derived peptide with both anticoagulant and angiotensin I-converting enzyme inhibitory(ACEI)activities yet.In this work,the anticoagulant and ACEI activities of the casein hydrolysates released by pepsin digestion were evaluated for the first time to the best of our knowledge.Results indicated that the casein hydrolysate exhibited potent anticoagulant activity by prolonging the thrombin time(TT)and the activated partial thromboplastin time(APTT).Compared with control samples,at 10mg/mL,the TT and APTT of casein hydrolysate were(186.0±6.6)% and(163.5±7.4)%,respectively.The casein hydrolysate also showed a strong ACEI activity with an IC50 value of 1.775mg/mL.The components of the bioactive casein hydrolysate were analyzed by nanoscale liquid chromatography quadrupole time-of- flight tandem mass spectrometry(NanoLC-Q-TOF-MS/MS).Total of 115 peptides were identified,among which 34,9,55 and 17 peptides were derived from αs1-,αs2-,β-,and k-casein,respectively.The results of PeptideRanker and PepSite 2 analysis showed that 6 peptides(FRQFYQL,NENLLRF,NPWDQVKR,PVVVPPFLQ,PVRGPFPIIV,and ARHPHPHLSF)have both ACEI and anticoagulant activities by binding to the active sites of ACE and thrombin.This study indicated that casein is a potential functional food supplement that can be used for medical purposes.

1.Introduction

Cardiovascular diseases(CVDs)have become the leading cause of mortality worldwide and is expected to lead to more than 22.2 million deaths by 2030[1–3].Thrombosis is highly related to 3 major cardiovascular disorders,contains ischemic heart disease,stroke,and venous thromboembolism[1].Moreover,thrombosis is playing a key role in the mortality incidence in cancer patients[4].Despite the availability of the clinical anticoagulant drugs,most of them are exhibiting health hazards and can generate bleeding[5,6].Therefore,the exploration of more effective and safer anticoagulant agents with minimal side effects is increasingly important.

Hypertension is not only one of the main risk factors for CVDs[7],but also associated with metabolic syndromes,such as obesity[8].Angiotensin I-converting enzyme(ACE,EC 3.4.15.1)is a milestone enzyme in controlling the blood pressure,as ACE is involved in both the kallikrein-kinin system(KKS)and the renin-angiotensin system(RAS).ACE will release the potent vasopressor angiotensin II and inactivate the bradykininin vivo[9].Similar to the anticoagulant drugs,some of the reported ACE inhibitors have several side effects including cough,rashes,and dizziness[10].

Casein is a primary and safe milk protein.Casein peptides are known with various biological activities,such as anti-inflammatory[11],immunomodulatory[12],anticoagulant[13],and angiotensin I-converting enzyme inhibitory(ACEI)activities[14].Thus,casein peptides are promising entities to explore their anticoagulant and ACE inhibitory effects.Moreover,exploring functional dietary supplements ingredients is a meaningful strategy to face the global burden of CVDs.The study of dual-function food products to combat both thrombosis and hypertension is absolutely a new sight for the development of the future medicinal food.

Recently,bioinformatics is widely applied to screen bioactive functional compounds,as well as peptides[15,16].Quantitative structure-activity relationship(QSAR)and molecular docking are used to predict the activity of the target molecules.The basic properties of the compounds,such as peptides could be analyzed by using the online tools.For instance,Expasy-Compute pI/Mw (https://web.expasy.org/compute_pi/) [17],ProtParam (https://web.expasy.org/protparam/) [17], Pepdraw (http://www.tulane.edu/～biochem/WW/PepDraw/),and ToxinPred(http://crdd.osdd.net/raghava/toxinpred/)[18].

Therefore,this study aims to investigate the anticoagulant and ACE inhibitory activities of the casein hydrolysate,and to identify the potential casein bioactive peptides.NanoLC-Q-TOF-MS/MS was adopted to sequence the casein peptides.Anticoagulant and ACEI activities of the hydrolysate were determined by thrombin time(TT),activated partial thromboplastin time(APTT),and high-performance liquid chromatography(HPLC)methods.The potential bioactive peptides were analyzed by combinedin vitroandin silicomethods.

2.Materials and methods

2.1.Materials and chemicals

Bovine casein powder was purchased from Beijing Solarbio Science&Technology Co.Ltd(Beijing,China).ACE(0.25 UN,from rabbit lung),Hippuryl-histidyl-leucine(HHL),pepsin(P7125-100G),acetonitrile(ACN),and trifluoroacetic acid(TFA)were obtained from Sigma-Aldrich Co.(St.Louis,MO,USA).TT and APTT kits were from Shanghai Sun Bio Biotech Co.,Ltd(Shanghai,China).

2.2.Preparation of casein hydrolysate

Bovine casein powder was dissolved in Milli-Q water with a final concentration of 35mg/mL(pH=2.0)and digested by pepsin(1:100,m/m).The hydrolysis reaction was maintained at 37?C(90min)and the solution was boiled in water for 10min to terminate the reaction.When the temperature of the sample was decreased below 25?C,the casein solution was adjusted to neutral pH,and centrifuged at 9190 ×gfor 20min(4?C).Then,the supernatant was collected,freeze-dried,and stored at?80?C.

2.3.Determination of molecular weight distribution

The method used to determine the molecular weight(MW)distribution of the casein hydrolysate was described by Xu et al.[19],with modifications.In this study,the temperature of the TSKgel G2000SWXL column(300mm×7.8mm)was set to 30?C.According to the retention time(RT)calculated from the standard curve,the MW range was divided into 6 parts(≥ 10kDa,7–10kDa,5–7kDa,3–5kDa,1–3kDa,and ≤ 1kDa).

2.4.Determination of anticoagulant and ACEI activities

The method described by Liu et al.[13]was adopted with modifications to determine thein vitroanticoagulant activity(TT and APTT assays).Merlin MC4 coagulometer(Merlin,Lemgo,Germany)was employed and Tris?HCl was used as a control buffer.The ACEI activity of the casein hydrolysate was evaluated by a reported highperformance liquid chromatography(HPLC)method[14].

2.5.NanoLC-Q-TOF-MS/MS analysis

The digested casein solution was analyzed by the Thermo NanoLC system coupled to a Synapt Mass Quadrupole Time of Flight Mass Spectrometer(Bruker Daltonik,Bremen,Germany).The mobile phase B and A was 0.1%FA in 80% ACN and water,respectively.The PepMapC18column(2μm,50μm×15cm)was adopted to separate the peptides in the sample for 90min with a constant flow rate(20μL/min).During 0–82min,the elution gradient(mobile phase B)increased from 9% to 90%.During 82–90min,the elution gradient decreased from 90% to 2%.Then,the data was processed by using Data Analysis 4.0 software(Bruker Daltonic GmbH,Bremen,Germany)and Mascot search engine 2.6.0(Matrix Science,London,UK).The Mascot search parameters were set as:below 1% of the false-positive discovery rate(FDR)of peptide identification,2 missed cleavage sites,no enzyme,10ppm of the peptide mass tolerance,oxidation as variable modifications,where the peptide with Mascot scores at least 20 was considered.

2.6.In silico analysis

Thein silicomethods have been popularly used to screen and analyze the bioactive peptides.In this study,the probability of the peptide with bioactivity was predicted by PeptideRanker,available at http://distilldeep.ucd.ie/PeptideRanker/,where the higher the score,the higher probabilities of the peptide to be bioactive[20,21].PepSite 2 program(http://pepsite2.russelllab.org/)were employed to analyze the interaction of casein peptides with ACE(PDB:108A)and thrombin(PDB:4BAN).The isoelectric point(pI)of the peptides was analyzed by Expasy-pI/Mw tool,available at https://web.expasy.org/compute_pi/.The PepDraw server(http://www.tulane.edu/～biochem/WW/PepDraw/)was used to evaluate the hydrophobicity and the net charge of the peptides.ToxinPred,available at http://crdd.osdd.net/raghava/toxinpred/,was used to predict the toxicity of the casein peptides[18].

2.7.Data analysis

The experiments were conducted at least 3 times and presented as the mean±SD.Origin Pro 8.6 software(OriginLab Corporation,Northampton,MA,USA)was used to analyze the data.The statistical significance was evaluated by using SPSS 26 software(SPSS Institute,Inc.,Cary,NC,USA).The statistical analyses were performed by the one-way ANOVA test and theP-value less than 0.05 was considered as statistically significant.

Fig.1.The molecular weight distribution of the casein hydrolysate released by pepsin digestion.(A)HPLC chromatogram.(B)Molecular weight distribution.Casein solution was digested by pepsin,with 1% enzyme/substrate(E/S)ratio on a m/m basis,for 90min at 37?C.

3.Results and discussion

3.1.Molecular weight distributions

In most cases,the bioactivity of the peptide strongly relied on its length[22].For instance,to pass through the membrane barrier,the antimicrobial peptides would contain 20–46 amino acids[22].The length of the peptide decides its molecular weight.Thus,it is significant to analyze the molecular weight distribution of the hydrolysate.In this study,the MW distribution of the casein hydrolysate was evaluated by a previously reported method[19].Fig.1 showed that 22.12% of the casein hydrolysate compounds have MW≤1kDa and 29.05% of the compounds display MW of 1–3kDa.Meanwhile,17.45% of casein hydrolysate showed MW of more than 10kDa.

3.2.In vitro anticoagulant and ACE inhibitory activities

Fig.2.In vitro anticoagulant activity of the casein hydrolysate.(A)TT assay of the casein hydrolysate.(B)APTT assay of the casein hydrolysate.Column bar with different letters(a–f)are significantly different(P＜ 0.05).

Fig.3.The ACE inhibitory activity of the casein hydrolysate.The ACEI activity was determined by an HPLC method.Data are expressed as mean±SD.Bars with different letters(a–e)are significantly different(P＜ 0.05).

Till now, there are few studies showed that protein hydrolysates,as well as casein hydrolysates,are having both anticoagulant and ACEI effects.Here,anticoagulant and ACE inhibitory activities of casein hydrolysate treated by pepsin were evaluated.As shown in Fig.2A and 2B,the casein hydrolysate demonstrated a significant anticoagulant activity.TT assay showed that the higher of the casein hydrolysate concentration,the longer of the clotting time.The casein hydrolysate showed about 1 time of prolongation of the thrombin clotting time at the concentration of 10mg/mL.APTT assay result exhibited that casein hydrolysate is also with the ability to prolong the APTT.Similar to the TT assay,the casein hydrolysate showed 3 folds increase of APTT compared with the control at the concentration of 20mg/mL.The prolongation of TT means that the thrombin activity or fibrin polymerization was inhibited[23].The prolongation of APTT means that the common and/or intrinsic pathway was inhibited[24].Compared to the TT and APTT assays of oyster(Crassostrea gigas)peptides reported by Cheng et al.[25],casein seems a more suitable option for exploring anticoagulant peptides.Moreover,the anticoagulant effect of casein hydrolysate is stronger than the reported anticoagulant casein peptide YQEPVL-GPVR[13]and lactoferrin peptide LRPVAAEIY[26],which indicated some peptides with high anticoagulant activity could exist in the casein hydrolysate and it deserves further analysis.

Fig.4.Casein peptides identified in the casein hydrolysate.(A)Peptide length distribution of the identified casein peptides.(B)The molecular weight distribution of the identified casein peptides.αs1-Casein(),αs2-Casein(),β-Casein(),k-casein().

Table 1Potential bioactive peptides screened from casein hydrolysate.

Table 2Potential binding sites between bioactive peptides and ACE were analyzed by using PepSite 2.

Apart from the anticoagulant activity of casein hydrolysate,Fig.3 indicated that the casein hydrolysate has a strong ability to inhibit the activity of ACE with an IC50value of 1.775mg/mL.Those data indicated that some ACE inhibitory peptides were released from the casein following the pepsin treatment.To find out which component of the casein hydrolysate is responsible for this effect,NanoLC-Q-TOF-MS/MS analysis was performed,and the casein peptides were ensured by using the Mascot server.A total of 115 peptides were identified,in which 34,9,55 and 17 peptides were from αs1-,αs2-,β-,and k-CN,respectively.Moreover,further analysis showed that the casein hydrolysate contains the ACE inhibitory peptides:YQEPVLGPVRGPFPIIV[27]and YQKFPQY[28].Meanwhile,5 antibacterial peptides,VQVTSTAV[29],SDIPNPIGSENSEK[30],RPKHPIKHQGLPQEVLNENLLRF[31],KTVYQHQKAMKPWIQPKTKVIPYVRYL[32],and VYQHQKAMKPWIQPKTKVIPYVRYL[33]were identified from the hydrolysate.Equally interesting,the peptides FRQFYQL and NLHLPLPLL were also identified from the mouse stomach after 0.5h ofin vivodigestion[23].Fig.4 and Table S1 showed that the identified peptides consist of 7–47 amino acids,which is consistent with the result as shown in Fig.1.

3.3.Possible mechanisms and physicochemical characters of the identified bioactive peptides

The findings of this study showed that casein hydrolysates have high anticoagulant and ACEI activities.However,as shown in Table S1,115 peptides were totally identified from the hydrolysate.Obviously,it is time-consuming and expensive to synthesize every peptide to confirm the activity.Fortunately,in silicomethods are able to screen the potential bioactive peptides with computational methodologies[34,35].

As most of the ACEI peptides contain no more than 10 amino acids,the casein peptides with≤10 amino acids were taken into consideration to save time and costs.PeptideRanker(http://distilldeep.ucd.ie/PeptideRanker/)was used to evaluate the probability of peptides to be bioactive by scoring each peptide.In the present study,peptides with the PeptideRanker score above 0.5 were selected for further investigations.As shown in Table 1,9 peptides were with PeptideRanker score more than 0.5 and less than 11 amino acid residues.Further,the interaction between the peptides and ACE/thrombin was analyzed by PepSite 2 server.PepSite 2 is a web server,functioning on the prediction of the binding sites of the peptide with receptors by giving theP-value,the reactive residue in peptide,and the interacting receptor residues[36].The server is widely used to analyze the binding mechanisms of bioactive peptides[37,38].Table 2 reveals that the 9 peptides(FRQFYQL,NENLLRF,VLNENLLRF,NPWDQVKR,NLHLPLPLL,PVVVPPFLQ,PVRGPFPIIV,SRYPSYGLN,and ARHPHPHLSF)have potent interaction with ACE,with Pepsite 2 ofP＜0.05.The peptide ARHPHPHLSF generated the lowestP-value(2.509×10?5).Similarity,Table 3 reveals that 6 peptides(FRQFYQL,NENLLRF,NPWDQVKR,PVVVPPFLQ,PVRGPFPIIV,and ARHPHPHLSF)have potent interactions with thrombin with Pepsite 2 ofP＜0.05,where the FRQFYQL presented the lowestP-value(0.002834).Therefore,those 6 peptides could have both ACE inhibitory and anticoag-ulant activities.Furthermore,the basic characteristics of those 6 peptides were analyzed as shown in Table 1.Interestingly,the peptide ARHPHPHLSF is a fragment of peptide ARHPHPHLSFM which showed antioxidative activity[39].The peptide PVVVPPFLQ overlapped partly with the ACE inhibitory peptide TPVVVPPFLQP[39].The toxicity of those 6 peptides were predicted as“non-toxin”by ToxinPred webserver,which indicated that all the peptides are non-toxicity based on the algorithm of ToxinPred webserver.Several studies showed that the charge and hydrophobicity of the casein peptides will influence their biological properties,such as transepithelial transport and bioavailability[40].Table 1 demonstrated that the net charge of those peptides ranged from 0 to 1.Hydrophobicity of those peptides was analyzed by PepDraw,and the results showed that those peptides are with hydrophobicity between 4.75 and 15.36kcal/mol.Those results provide the theoretical and practical significance of promising bioactive peptides with anticoagulant and ACE inhibitory activities.

Table 3Potential binding sites between bioactive peptides and thrombin were analyzed by using PepSite 2.

4.Conclusions

The bioactivity and peptides profile of casein hydrolysate released by pepsin digestion was evaluated in this study.A total of 115 peptides from the casein hydrolysate were identified by NanoLC-Q-TOF-MS/MS.Six peptides showed both anticoagulant and ACE inhibitory activities analyzed by usingin silicomethods.This study indicated the presence of dual-function peptides derived from casein hydrolysate,and casein is a promising source of functional food supplement.

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgements

The China Postdoctoral Science Foundation(2019M661072),the Basic Research Program of Liaoning Education Department(2017J080),and the National Natural Science Foundation of China(31771926)funded this study.

Appendix A.Supplementary data

Supplementary material related to this article can be found,in the online version,at doi:https://doi.org/10.1016/j.fshw.2020.08.014.