| China is reported to be among the lucrative markets for the sea cucumber(Phylum Echinodermata:Holothuroidea)trade.Sea cucumber,commonly called"??:haishen",is valued and widely consumed by Chinese and other Southeast Asians owing to its rich medicinal and therapeutic properties.It is a unique resource that has attracted the interest of nutritionists and pharmacologists.The preparation of bioactive peptides from sea cucumber has remained conventional for years,notwithstanding its socio-economic significance.In conventional enzymolysis,sticky protein samples like sea cucumber(with mutable collagenous tissue)result in low productivity because the viscous substrate impedes free mobility of enzymes.Therefore,a high liquid to solid ratio of the reaction solution must be employed to control protein viscosity to expedite enzyme reaction rate.As a result,industrial peptide production will inevitably face the severe challenge of separating the large amount of water(liquid)used,resulting in increased energy consumption.So,this research capitalized on the viscosity-reduction effect of ultrasonic shear force to control the protein substrate viscosity during enzymatic proteolysis.Additionally,the changes in the molecular structure of the main chemical components in the traditional polypeptide production process are detected by chemical analysis instruments(destructive chemical analysis),which are inefficient(too slow)to be used in the development of modern production technology.Therefore,in-situ real-time detection technology for the enzymatic hydrolysis process based on miniature-infrared spectroscopy was studied.Other challenges of the traditional enzymatic hydrolysis method,however,include the extensive use of enzymes,excessive enzymatic hydrolysis reaction,and product inhibition effects,all of which have a significant impact on production cost reduction,product activity improvement,and protein conversion rate increase.Therefore,the coupling technology of enzymatic hydrolysis and membrane separation with gradient dilution feed developed by our research group was adopted in the preparation of bioactive peptides from sea cucumber(A.japonicus)in this study.The following are the main contents and findings of the research:[1]Protein isolates from the various body parts of sea cucumber(A.japonicus)were assessed for their distinctive characteristics.The protein yield from the viscera,the whole-body,and the body wall ranked at 75.49%>55.07%>50.01%,respectively.The whole-body protein isolate(WBPIs)recorded the highest emulsion stability(50.61min)and had the strongest ability to induce hypocholesterolemic effects owing to its low Lys/Arg(0.30±0.01)ratio.The highest emulsion activity(148.22 m~2/g)and lowest bulk density were recorded by the body wall protein isolate(BWPIs).The protein isolate from the viscera(VPIs),on the other hand,recorded the highest foaming capacity/stability,water absorption capacity,essential and antioxidative amino acid scores.Glutamine(Glu)was the abundant amino acid found in WBPIs and VPIs,while glycine(Gly)was abundant in BWPIs.The peculiar features among the three sea cucumber protein isolates resulted from differences in their tissue structure and composition.Thus,these distinct physicochemical,techno-functional,and structural characteristics may influence their selection and applications as functional foods,nutraceuticals,and pharmaceuticals.[2]Ultrasonication parameters were optimized to facilitate the enzymatic proteolysis of sea cucumber protein.Under the conditions of a 20/68 k Hz slit dual-frequency ultrasound(SDFU)and a 10 g/L substrate concentration,the optimized treatment parameters of 150 W/L,30°C,and 17min for power density,ultrasonic temperature,and treatment time respectively,were obtained.Ultrasound enhanced the protein conversion degree(PCD),degree of hydrolysis(DH),DPPH radical scavenging activity(DRSA),and hydroxyl scavenging activity(HRSA)by 16.30%,27.43%,11.14%,and 12.68%,respectively,over the conventional enzymolysis method.In comparison to the control,ultrasound caused a continuous decrease in slurry apparent viscosity as shear rate increased,enhancing functional properties and bioactive component release.The increase in sulfhydryl(SH),decrease in disulfide(SS)content,strong intensity of UV-vis spectra,decrease in particle size,and shifting of FT-IR spectra(amide I,II,and III bands)all evinced the effects of ultrasound on the structure of sea cucumber protein.[3]The effect of SDFU shear thinning on the kinetics and thermodynamic mechanism of sea cucumber protein substrate enzymatic proteolysis was investigated.The impact of ultrasonic mechanical shear force on the viscosity of the substrate increased the average kinetic energies of the protein molecules,which shortened the enzyme-protein distance and exposed the enzyme-driven bonds for easy protease accessibility,resulting in significant enzymolysis efficiency.Sonication also ensured the development of a non-spontaneous reaction(+?G)with a significant decrease in enthalpy(?H),entropy(?S),and activation energy(Ea)(28.30±0.63k Jmol-1,-205.70±1.72 Jmol-1k-1 and 31.01±0.27 k Jmol-1 respectively),indicating a minimum energy required for an efficient reaction to occur.The kinetics results showed that the ultrasonic viscosity reduction resulted in a significant reduction in the Michaelis constant(km)and activation energy(Ea)by 19.37%and 38.78%respectively over the control,indicating that a substantial affinity between enzyme and substrate was formed.The substrate viscosity and sea cucumber protein conversion degree had a negative significant correlation(r=-0.865,p<0.01).The treated sample showed significant modification in its?-helix(reduction),?-turn(reduction),?-sheet(increase),and random coil(increase).The extent of structural modifications caused by ultrasonication was shown by the intrinsic fluorescence spectra intensity,atomic force microscopy(AFM),scanning electron microscopy(SEM),and surface hydrophobicity(H0).[4]The feasibility of monitoring the in situ and real-time enzyme proteolytic reaction of ultrasonic viscosity-reduced sea cucumber protein by miniature NIR spectroscopy coupled with a fiber-optic probe was explored.A synergy interval Partial Least Square(si PLS)algorithm with regression model calibration was used to select the variables.The calibration si PLS module developed between the chemical data and the NIR spectra resulted in a significantly high correlation coefficient(Rp)of the prediction set of 0.9189,0.9189,and 0.9522 and a root mean square error(RMSEP)of 2.38%,0.321 mg/m L,and 4.91%for protein conversion degree(PCD),peptide concentration,and ACE-inhibitory activity,respectively.Also,a root mean square error of cross-validation(RMSECV)of 1.85%,0.249 mg/m L,and 3.95%and a correlation coefficient(Rc)of 0.9379,0.9379,and 0.9651were recorded for PCD,peptide concentration,and ACE-inhibitory activity,respectively.The optimal spectral regions for both PCD and peptide concentration were 946.95-999.07,1849.02-1899.98,2147.6-2198.24,and 2242.5-2293.05 nm,while the optimal spectral region for ACE-inhibition activity fell within 1708.59-1798.01,1887.24-1976.3,2153.93-2242.5,and 2330.93-2400 nm.The developed model was able to monitor the change of PCD,peptide concentration,and ACE-inhibitory activity of the enzyme proteolytic reaction of sea cucumber in situ and in real time.[5]The composition characteristics,antioxidant and ACE inhibitory activities of sea cucumber peptides with different molecular weights were studied,and the molecular weight cut-off of enzymatic hydrolysis coupled with membrane separation in the next step was determined.In comparison to fractions of 5-3,3-1,and<1 k Da(80.81,76.15,and 75.83%respectively),the ferrous-ion chelating was lower in peptide fractions from the molecular weight cut-off of 100-30,30-10,and 10-5 k Da(71.74,73.37,and 75.42%respectively).The treated sample with molecular weight-cut off of<5k Da had stronger ACE-inhibitory activity,IC50(0.117-0.115 mg/m L).As the molecular weight of the peptides decreased,the hydrophobic amino acids(Val,Ala,Ile,Leu,Thr,Phe,Gly,Pro,and Met)increased,with a substantially greater increase observed in sonicated samples.The SDFU treated sample with a molecular weight of 5-3k Da had the greatest antioxidant amino acid(Met,Tyr,His,Lys)and ACE-inhibitory amino acid(Lys,Arg,Pro)levels of 11.27and 20.71mg/100mg,respectively.Based on the above results,a molecular weight cut-off of 5 k Da was selected for enzymatic hydrolysis coupled with membrane separation in the next step.[6]The enzymatic hydrolysis coupled with membrane separation(EC-MS)for preparation of sea cucumber peptides was studied.The reaction conditions of the EC-MS(nonfeeding,NF)were optimized.The optimized reaction conditions were used for the three feeding modes:gradient dilution feeding(GDF),feeding before water replenishment(FBW)and constant concentration feeding(CCF).The EC-MS-GDF,FBW,and CCF modes resulted in a significant(p<0.05)increase in protein conversion degree(PCD)over the conventional(EH offline-MS)by 60.39%,46.69%,and 23.33%,respectively.The EC-MS-GDF selected also increased peptide yield,ACE-inhibition activity,and free amino acid by 21.93%,36.54%,and 4.96%,respectively,and had a 7-fold increase in peptide output per unit enzyme compared to the conventional method.These results were influenced by the real-time separation of active peptides(products)from the reactor before the degradation of the target product with time.The EC-MS-GDF allowed for a 7-hour continuous generation of high yield and quality(high ACE-inhibitory activity)peptides from sea cucumber protein without exceeding the safe transmembrane pressure limit(TMP)of 0.15MPa.After nanofilter purification,the sodium content of EC-MS-GDF peptides could be reduced by 58.11%.The desalinated EC-MS-GDF peptides had a higher percentage(83.77%)of the peptides within the molecular weight range of1000-500 Da and 500 Da in comparison to the conventional method,EH-offline-MS(81.6%).At high temperatures,the antioxidative stability of the produced peptides was least affected.Consequently,integrating ultrasonic-assisted enzymatic coupling with membrane separation(EC-MS-GDF)and nanofilter-purification techniques into the production of highly stable desalinated bioactive peptides from materials with a peculiarly high viscosity(or high solid-to-liquid ratio)for industrial applications would be a feasible alternative. |
PDF Full Text Request