Preparation,Functional And Delivery Properties Of Soybean Bioactive Proteins

The storage protein is generally obtained in the processing of soybean protein.However,there are always some bioactive proteins in the processing by-products.They posess high value in the prevention and control of chronic diseases as well as biological medicine although they are low abundance.Therefore,to achieve the high-value utilization of soybean processing by-products,bioactive proteins have been prepared from soy whey and soy hull by a scale method,and the biological activities such as anti-inflammatory and antimicrobial activities as well as delivery properties of soybean bioactive proteins have been investigated.The main results are as follows:(1)A low cost and simplify operation method was developed based on the principles of salting out and solid-liquid separation to recover soybean Kunitz trypsin inhibitor(KTI)from soy whey.The anti-inflammatory effects of KTI on LPS-treated RAW264.7 macrophages were evaluated.The results indicated that the yield and purity of KTI were 18.66% and 90.03%,respectively under the optimal conditions(adding 15%(w/w)sodium sulfate mass to 12%(w/v)soy whey solution to precipitate protease inhibitor(including KTI and Bowman-Birk inhibitor(BBI)),then KTI was separated from KTI and BBI mixture by solid-liquid separation method).In anti-inflammation experiment,KTI could inhibit effectively NO and PGE2 production,which was probably due to KTI could suppress iNOS and COX-2 activities and pro-inflammatory cytokines(TNF-?,IL-6 and IL-1?)expression.These findings suggest that KTI possess favorable anti-inflammatory activity.(2)Soybean Kunitz trypsin inhibitor nanoparticles(KTIP)were prepared by heating KTI in the presence of sodium sulfite,the formation mechanism and properties of KTIP were studied.The results indicated that this simple treatment not only inactivated most trypsin inhibitor activity(TIA)of KTI,but also resulting in the formation of KTI nanoparticles(about 85.92 nm)with good monodispersity(PDI: 0.18).SDS-PAGE analysis revealed that the formation of KTIP was mediated by-SH/-SS-exchange reaction.The delivery capacity of KTIP for curcumin as model bioactives was evaluated.The results showed that the co-assembly of KTIP and curcumin actually greatly enhanced the dispersibility,stability and bioaccessibility of curcumin in aqueous solution.Moreover,in vitro anti-proliferative activity on tumor cells assay showed that nanoparticulate curcumin was more effective than free curcumin in solution by controlling the tumor cell growth with time.(3)Soybean Bowman-Birk inhibitor(BBI)was recovered from soy whey by a novel strategy based on the principles of salting out,solid-liquid separation and coacervation.A novel self-assembly nanoparticle delivery carrier was developed by using BBI to improve the solubility,bioaccessibility and oral absorption of curcumin.The results indicated that an abundant of BBI with desired purity was obtained,the encapsulation efficiencies(EE)and the loading efficiencies(LE)of curcumin in the curcumin-loaded BBI nanoparticles(Cur-BBI-NPs,size: 90.09 nm,PDI: 0.103)were 86.17 and 10.31%,respectively.The in vitro bioaccessibility of Cur-BBI-NPs was superior to that of curcumin-loaded sodium caseinate(SC)nanoparticles(Cur-SC-NPs)(as control).Moreover,Cur-BBI-NPs significantly enhanced bioavailability of curcumin in rats compared with Cur-SC-NPs.And the clathrin-mediated endocytosis pathway was probably contributed to the favorable bioavailability of Cur-BBI-NPs,as revealed by the cellular uptake inhibition study.(4)Phytosterols(PS)-loaded BBI nanoparticles(PS-BBI-NPs)were prepared by Nab technology to improve the bioaccessibility of PS.The basic properties of PS-BBI-NPs were characterized.The results indicated that the size of PS-BBI-NPs was about 80 nm except at pH around p I(4.2)of BBI,the encapsulation efficiencies(EE)and the loading efficiencies(LE)of PS in the PS-BBI-NPs were 95.86% and 11.63%,respectively.The dissolution of the PS-BBI-NPs in the reducing environment,together with the stability in ethanol solutions and phosphate buffer solution,suggested that the formation of the intermolecular disulfide bonds is the key factor in stabilizing the obtained PS-BBI-NPs.The XRD pattern of PS-BBI-NPs showed the formation of amorphous PS within the particle matrix.In vitro digestion revealed that nanoparticulate formulation significantly improved the bioaccessibility of PS as compared with free PS.(5)Soybean hull,an underutilized by-product of soybean processing,was investigated as a source of bioactive proteins.A Viscozyme L-assisted extraction method was developed to improve the yield of extracted proteins.The extracted proteins were identified by MALDI-TOF-TOF MS,and the most abundant disulfide-rich protein among identified proteins was purified and the enzymatic properties were evaluated.The results indicated that the Viscozyme L-assisted extraction method extracted significantly(p < 0.05)more proteins(39.01%)than did the aqueous method(4.52%).The yield of the purified aspartic proteinase nepenthesin-1-like [Glycine max](GmAPN1K)(the most abundant disulfide-rich protein)is 570 mg/Kg.The specific activity of GmAPN1 K was 62.1U/mg at pH 3.0 and 37 oC.The enzyme was optimally active at pH 3.0 and 55 oC.It was stable within pH range 3.0-10.0 and up to 55 oC,respectively,and was specifically inhibited by pepstatin A.(6)An alkaline isoform of the PR-5 protein(designated GmOLPc)has been purified from soybean hulls and identified by MALDI-TOF/TOF-MS.The antimicrobial activity and antimicrobial mechanism of GmOLPc were studied.The results indicated that GmOLPc effectively inhibited in vitro the growth of Phytophthora soja spore and Pseudomonas syringae pv.glycinea.The antimicrobial activity of GmOLPc should be mainly ascribed to its high binding affinity with vesicles composed of DPPG,(1,3)-?-D-glucans and weak endo-(1,3)-?-D-glucanase activity.From the 3D models,predicted by the homology modeling,GmOLPc contains an extended negatively charged cleft.The cleft was proved to be a prerequisite for endo-(1,3)-?-D-glucanase activity.Molecular docking revealed that the positioning of linear(1,3)-?-D-glucans in the cleft of GmOLPc allowed an interaction with the Glu83 and Asp101 that were responsible for the hydrolytic cleavage of glucans.Interactions of GmOLPc with model membranes indicated that GmOLPc possess good surface activity which could contribute to its antimicrobial activity,as proved by the behavior of perturbing the integrity of membranes through surface hydrophobic amino acid residues(Phe89 and Phe94).