Preparation,Characterization And Application Of Iron-Bound Whey Protein Nanoparticles

Iron is an essential trace element for the human body.The form of iron and the correct combination with food carriers and the dietary environment are essential for iron absorption.Combining iron with a bioavailability enhancer and avoiding interaction with iron inhibitors can improve iron absorption.At present,the research on iron supplements mostly focuses on iron chelate peptide,iron chelate polysaccharide and iron chelate amino acid.There are few studies on the preparation,structural characterization and bioavailability of iron chelate protein nanoparticles.Whey protein isolate(WPI)has mineral-bound functional nutritional properties and has advantages in increasing the solubility,absorption and bioavailability of metal ions.In this paper,Fe SO₄·7H₂O was used as the iron source,common solvent and ultrasonic assisted method were used to chelate iron with WPI to prepare whey protein iron nanoparticles(WPI-Fe)and ultrasonic whey protein iron nanoparticles(UWPI-Fe).Using the amount of iron chelated as an indicator,the reaction conditions of the solvent method were optimized.Exploring the influence of WPI-Fe interaction,particle characteristics,structural changes and ultrasonic amplitude and other factors,reveals the formation mechanism of WPI-Fe and UWPI-Fe and the stability mechanism of in vitro gastrointestinal digestion.It provides a new idea for the preparation of WPI nanoparticles with high loading of mineral bioactive substances,and lays a theoretical foundation for its application in food fortification.The specific research content of this article is as follows:1.In the preparation method of common solvents,the feed ratio(quality)of iron and WPI(1:5,1:10,1:20,1:30,1:40,1:50)on the effect of WPI-Fe has been studied.The influence of iron chelation amount,structure and thermodynamic stability.The results showed that the feed ratio of iron to WPI is directly proportional to the amount of iron chelation.As the amount of iron chelation of WPI-Fe increased,iron was mainly concentrated on the surface of WPI-Fe.When the feed ratio of iron to WPI was 1:50-1:30,the particle size of WPI-Fe was significantly reduced compared to WPI.As the feed ratio increased(1:20-1:5),the particle size of WPI-Fe increased.The analysis of the iron valence state showd that as the feed ratio of iron to WPI increased,the content of divalent iron in WPI-Fe first increased and then decreased.When the feed ratio was 1:10,the chelated amount of ferrous iron reached the highest value of66.73%.In addition,the feed ratio had a certain effect on the morphology of the freeze-dried WPI-Fe nanoparticles:as the feed ratio of iron to WPI increased,the spherical WPI and iron chelated into flat flakes and tubular nanoparticles first,and then formed to honeycomb structure,finally the honeycomb structure disappeared.Circular dichroism studies have shown that iron induced changes in the structure of WPI.Compared with WPI,the content of?-helical structure of WPI-Fe nanoparticles reduced,and the?-sheet increased.Infrared spectroscopy and thermogravimetric analysis showed that iron mainly interacted with the carboxyl O,hydroxyl O and amino N atoms of WPI to form thermodynamically stable WPI-Fe nanoparticles.Isothermal titration analysis showed that the?H produced by the interaction between Fe²⁺and WPI was-61.95 k J/mol,?G was-30.68 k J/mol,and the binding constant K was 2.37×10^-5,indicating that Fe²⁺has a high affinity for WPI and can be performed spontaneously.Exothermic reaction.The conformational dissociation experiments showed that the interaction between Fe and WPI was mainly electrostatic interaction,which affected the hydrogen bond and hydrophobic interaction of the protein at the same time.2.The feed ratio of iron and WPI(1:5,1:10,1:20,1:30,1:40,1:50),p H value(2,3,4,5)were studied through single factor experiments.,6,7,8,9,10)and reaction time(5 min,15 min,40 min,65 min,90 min,120 min)on the particle size,Zeta potential and iron chelation amount of WPI-Fe nanoparticles.Based on the results of single factor experiments,response surface methodology was used to optimize the reaction conditions for preparing WPI-Fe nanoparticles in common solvents.Taking the ratio of iron to WPI,reaction time,and p H as the response factors,and the amount of iron chelation of WPI-Fe as the response value,a quadratic regression equation was established:Y=121.87+0.81A+1.39B+0.52C+0.83AB-0.88AC-0.56BC-6.70A²-4.88B²-5.43C².The regression equation of this model had a high degree of fit.The optimal preparation parameters of the iron chelation reaction obtained by the response surface software were:WPI:Fe feed ratio is 5.8,reaction time is 44.35 min,and p H value is6.04.The highest WPI-Fe iron chelation determined by the regression equation is122.01 mg/g,and the reliability is 99.5%,which has good simulation.3.On the basis of the common solvent method,UWPI-Fe was prepared by ultrasonic auxiliary means,and the effect of ultrasonic amplitude(20%,30%,40%,50%,60%)on the iron chelation of UWPI-Fe was investigated.The influence of structure,appearance and thermodynamic stability.The results showed that with the increase of ultrasonic amplitude,the iron chelation amount and solubility of UWPI-Fe first increased and then decreased,and the particle size first decreased and then increased.CD,FTIR and fluorescence spectroscopy showed that the secondary and tertiary structure of UWPI-Fe prepared by ultrasound was changed,the?-sheet content was reduced,the protein structure became loose,and the tryptophan group was exposed.Smaller ultrasonic amplitude(20%,30%)could unfold the UWPI-Fe structure,destroy the hydrogen bond and hydrophobic effect,increased the amount of iron chelation and solubility,and reduce the particle size.Larger ultrasonic amplitudes(50%,60%)reduced the local hydrophobic effect,leading to agglomeration of protein molecules,reducing the contact area of WPI and Fe,thereby reducing the amount of iron chelation and solubility,and increasing the particle size.Ultrasound-assisted preparation of UWPI-Fe had good thermal stability.The apparent morphology of UWPI-Fe under 20%amplitude showed small spherical particles,which were uniformly dispersed on the surface of the sheet.The UWPI-Fe spherical particles under 60%amplitude increased obviously and agglomerate together to present a porous structure.4.The in vitro gastrointestinal digestion stability of WPI-Fe and UWPI-Fe was studied.From the analysis of bioavailability,it could be seen that compared with inorganic iron salts,the in vitro gastrointestinal digestion stability of WPI-Fe and UWPI-Fe were both greater than that of Fe SO₄·7H₂O.Because WPI is a macromolecule,it can alleviate the enzymatic hydrolysis of proteases and has antioxidant activity.Chelating with iron is more conducive to protecting the transport of trace element iron to the small intestine from being oxidized,and it is more suitable for those who need to release nutrients slowly in the intestine transport.By comparing the digestion process of WPI-Fe and UWPI-Fe,it could be found that the particle size of UWPI-Fe simulated gastrointestinal digestion was smaller than that of WPI-Fe,and the bioavailability was higher than that of WPI-Fe.