Antihypertensive Activity And Structural Analysis Of Soy Oligopeptides And Bitterness Masking Of Peptides By Double Emulsion

Soy peptides are complex proteolytic mixture that can be produced from enzymatic hydrolysis of soy protein.Soy peptides have high solubility,heat stability,better absorbility than amino acids and may exhibit important biological functions.Therefore,the development of soy peptides is of great significance for promoting in-depth utilization of soybean and increasing the added value of soybean products.However,it is still challenging to apply soy peptides in commercial products due to their intense bitter taste from enzymatic hydrolysis process.In industry point of view,separation and further enzymatic treatment have been used to remove the bitterness of soy peptides.However,it is difficult to remove bitterness without compromising the bioactivity and nutrition value of soy peptides.In this study,soy protein hydrolysates were produced by using commercial proteases,and their hydrolysis characteristics,bitterness intensity and antihypertensive activity were further studied with a special focus on bitter peptides and peptide sequence.Furthermore,this study aims to develop a double emulsion system with a high soy peptide loading in order to provide effective protection to these bioactive peptides against external environmental stresses,as well as masking their unpleasant bitter taste.Screening of proteases for preparation of soy peptides and bitterness evaluation:soy protein hydrolysates were prepared using 6 different commercial proteases and the degree of hydrolysis,molecular weight distribution,amino acid composition were investigated under various hydrolysis conditions.A trained sensory panel and electronic tongue were combined to assess the bitterness in different hydrolysates.Hydrolysate bitterness strongly depended on different proteases and bitter hydrolysates were produced by alkaline proteases than neutral proteases.Hydrolysate samples produced using different proteases could be characterized and distinguished by the taste sensor system.However,in the aspect of quantitative assessment of bitterness intensity,gustatory bitterness and bitterness score from electronic tongue did not show reasonable correlation,indicating that sensory evaluation of bitterness is still the“golden standard”for complex proteolytic mixture.Alkaline protease(Food Pro Alkaline)and neutral protease(Protin SD-NY10)were selected for the followed study.Preparation of soy oligopeptides and assessment of antihypertensive potency:Food Pro Alkaline,Protin SD-NY10 and trypsin which has high enzyme specificity were chosen as single enzyme or combination to prepare soy protein hydrolysates.The optimal enzymatic hydrolysis condition of soybean protein with highest in vitro ACE inhibition rate were combination of Food Pro Alkaline and Protin SD-NY10 and 4 hours of hydrolysis at 55°C.The molecular weight distributed below 1000 Da was 76.87%in this hydrolysate and belong to soy oligopeptides.After 30 days of feeding different doses of soy oligopeptides in SHR,high-dose group significantly reduced blood pressure(systolic,diastolic,and mean blood pressure)and angiotensin II levels in SHR at 4 weeks.It is speculated that soy oligopeptides exerts antihypertensive effect by inhibiting ACE activity.Fractionation,structural analysis and antihypertensive activity prediction of soy oligopeptides:soy oligopeptides were separated by ultrafiltration and reverse phase chromatography respectively.Bitterness of fraction with lower than 1000 Da separated by ultrafiltration was the highest and this fraction was further fractionated by chromatography into 9 fractions.Among them,3 fractions were of higher bitterness intensity and peptide sequences were identified by using LC-MS/MS.17 peptides with the characteristics of bitter peptides were identified and were mainly tetrapeptide and pentapeptide from soy7S and 11S protein with a molecular weight ranged from 400 to 1800 Da.Based on online tool of AHTpin and Peptide Ranker,among 17 identified bitter peptides,14 peptides were found to be potentially biologically active and 12 peptides were potentially antihypertensive.Preparation of water-in-oil-in-water(W/O/W)emulsion to encapsulate soy oligopeptides:soy oligopeptides were encapsulated in double emulsion using a two-step emulsification process.The effect of processing factors such as inner emulsifier type and concentration at W₁/O interface,the inner aqueous phase to oil ratio,peptide concentration in the inner aqueous phase,and homogenization processing parameters were investigated.Under optimal conditions,the double emulsion with the highest encapsulation efficiency(>80%)was prepared when:i)PGPR was selected as the hydrophobic emulsifier at 8%in MCT oil;ii)40%(w/w)soy peptides solution was used as W₁ phase;iii)W₁:O ratio was controlled at 3:7(w/w)and iv)12500 rpm for 2 min and high pressure homogenization of 250 bar in first emulsification step and 10000 rpm for 3 min in second emulsification step to produce the double emulsion.Bitterness masking effect of double emulsion and dried microcapsules and their release characteristics during in vitro digestion:the double emulsion was further spray-dried or freeze-dried to prepared microcapsules and the microcapsules could successfully mask the bitter taste of soy oligopeptides.Compared with spray-dried microcapsule powder,the freeze-dried one showed higher encapsulation efficiency(>78%).The in vitro digestion model was used to study the digestibility of both fresh prepared double emulsion and reconstituted emulsion from freeze-dried microcapsule.The particle size distribution and microstructure of the emulsions were analyzed using mastersizer and CLSM,and the release of soy peptides and free fatty acids were investigated.Fresh prepared double emulsion as well as the reconstituted emulsion have good stability in stimulated gastric stage.The digestion of double emulsions occur mainly in stimulated intestinal stage with a slow release of soy oligopeptides.