Goat Milk Whey Protein Preparation And Its Application In Whey Protein-Soy Isoflavones Delivery System

Goat milk whey protein has high nutritional value and functional properties.Currently,goat milk whey protein was prepared by concentrating and spray-drying the by-products of cheese,which has high cost and residual solvent,and is limited by the volume of goat milk cheese production.Membrane separation technology is an efficient separation method and widely used in milk protein preparation.Goat milk whey protein can be separated by membrane technology directly from milk,which can retain its natural physicochemical and functional properties.The ?-lactoglobulin in goat milk is not easily digested by pepsin,making it an ideal carrier for biologically active substances.Therefore,goat milk whey protein could be used to construct a nano system for embedding hydrophobic active substances such as soy isoflavones to improve its solubility,stability and bioavailability.In this study,goat milk whey protein concentrate was prepared by membrane separation technology,and effects of heat treatments on the physicochemical and functional properties of goat milk whey protein were investigated.Soy isoflavones nano-delivery system was prepared based on polymerized goat milk whey protein,and its physicochemical stability and the simulated in vitro release properties were studied.The potential cell toxicity of soy isoflavones nanoparticles and absorption mechanism by Caco-2 cells were evaluated.The main results are shown as follows:(1)The nonfat goat milk was used as a raw material to prepare Goat Milk Whey Protein(GWP).GWP was subjected to microfiltration,ultrafiltration/diafiltration and electrodialysis.During microfiltration process,the whey protein removal rate reached the maximum value of92.1% with a cycle temperature of 50 ? and a transmembrane pressure(TMP)of 0.09 MPa.For ultrafiltration process,the goat milk whey protein concentrate had the protein content of76.4% with the cycle temperature of 45 ?,the TMP of 0.11 MPa,the volume concentration factor of 10 and the number of diafiltration 3 times.The demineralization rate of goat milk whey protein concentrate by electrodialysis was over 85%.The goat milk whey protein prepared by membrane separation technology were consisted of 80.99% protein(w/w),18.67%lactose content(w/w),and 0.34% ash content(w/w).Results showed that goat milk whey protein concentrate(80.99%)could be obtained by membrane separation technology.(2)Polymerized Goat Milk Whey Protein(PGWP)was prepared by heating GWP.Effects of heating temperature(69-78 ?),heating time(15-30 min)and p H(7.5-7.9)on the physicochemical and functional properties of PGWP were studied.Compared with native goat milk whey protein,the particle size,Zeta potential and the surface hydrophobicity of PGWP significantly increased.However,the content of free sulfhydryl groups was significantly decreased.The apparent viscosity characteristics of samples were analyzed by rotary rheometer.It was found that the sample had a larger viscosity after heat treatment.Fourier transform infrared spectroscopy revealed that the secondary structure of the sample was changed after heat treatment,causing the decrease in content of ?-helical structure,while the content of ?-sheet structure increased.Transmission electron microscope images suggested that PGWP was evenly distributed.The emulsion stability index and the foaming properties of PGWP were significantly increased,the emulsion activity index decreased after heat treatment.It can beconcluded that heat treatment can improve the physicochemical and functional properties of goat milk whey protein for potential industrial applications.(3)The nanoparticles prepared with Polymerized Goat Milk Whey Protein(PGWP)and Soy Isoflavones(SIF)were investigated.SIF nanoparticles with different levels of SIF were prepared using PGWP as wall material.The nanoparticle reached the highest encapsulation efficiency(89%)when the concentration of SIF was 2.4 mg/m L.The particle size,PDI and surface potential values of SIF nanoparticles were measured by dynamic light scattering technology.The nanoparticles showed larger particle size and lower zeta potential compared with PGWP.The apparent viscosity of the nanoparticles was analyzed by rotary rheometer.All samples showed shear thinning behavior.Differential Scanning Calorimetry analysis suggested that SIF was amorphous in the nanoparticles.Fourier Transform Infrared Spectroscopy indicated that the secondary structure of PGWP was changed after interacting with SIF,with transformation of ?-helix and ?-sheet to disordered structures.Raman spectroscopy studies found that the contents of ?-helical structure and ?-sheet structure of PGWP in the nanoparticles decreased after adding SIF,while the content of ?-turn structure increased.The nanoparticles had spherical microstructure revealed by Transmission Electron Microscope images.The effects of interactions between PGWP and SIF were studied by fluorescence spectroscopy.It was found that SIF had the quenching effect on PGWP.The Stern-Volmer curves slope of PGWP and SIF decreased with the temperature increased,which suggested that the quenching method was static quenching.The surface hydrophobicity and emulsification activity of the nanoparticles decreased(P < 0.05),but increased(P < 0.05)the emulsification stability.The data suggested that PGWP prepared directly from goat milk was suitable to encapsulate SIF and may also be for other fat soluble antioxidants.(4)The effects of ionic strength(0-200 m M),p H(2-7)and the storage temperature(4,25 and 37 ?)on the stability of PGWP-SIF nanoparticles were investigated.The storage stability revealed that PGWP-SIF nanoparticles had high ionic strength and p H stability.Low temperature storage was more conducive to improve the physical stability of SIF in the nanoparticles.During 14 days of storage,the retention rate of SIF in the nanoparticles was still above 80% at 4 °C,indicating that the nanoparticles prepared with PGWP as wall material had high chemical stability.The digestion behavior was investigated using an in vitro simulated gastrointestinal digestion model.Results suggested that the particle size of SIF nanoparticles increased(P < 0.05),and the absolute value of zeta potential decreased(P < 0.05)at the end of the gastric digestion.Results of in vitro release characteristics of gastrointestinal digestion showed that the release rate of SIF nanoparticles was low in vitro gastric digestion,which suggested that the using of PGWP as wall material can retain more SIF to the intestinal tract.These results indicate that PGWP can be used as a promising wall material for SIF biologically active substances to regulate the stability and digestibility of nanoparticles.(5)In order to investigate the cellular uptake behavior of SIF loaded in nanoparticles after digestion,a Caco-2 cells model was constructed.The nanoparticles showed no cytotoxicity and had good biocompatibility.The cellular uptake of SIF in the nanoparticles increased with increasing incubation time,suggesting a time-dependent cellular uptake behavior.Within 4-hour incubation,the uptake of SIF increased after digestion,which suggested that the digeation behavior improved the cellular uptake of SIF.The permeability rate(Papp)of SIF encapsulated PGWP based nanoparticles were increased by 1.5 2 times compared with those before digestion,suggesting that the absorption efficiency of SIF loaded in nanocarriers had been improved after digestion.The absorption mechanisms of nanoparticles by Caco-2 cells were caveolinmediated endocytosis and clathrin-mediated endocytosis,and clathrin-mediated endocytosis played an important role in the uptake process.Results of the absorption experiment showed that the SIF nanoparticles prepared with PGWP as the wall material help to improve the bioavailability of SIF,and the absorption efficiency of SIF were improved after digestion.