Study About Allergenicity Reducing Of Special Soy Protein Isolate By High Hydrostatic Pressure Assisting Enzymatic Approach

The incidence of cow milk allergy (CMA) ranges from1%to2%in Chinese infants andchildren.The ratio of infants with varing degrees of lactose intolerance reached30%. Based onthe experience of developed countries, soy-based infant formula was applied to these twotypes of children. If infants and children indicate IgE mediated allergic response,53-63%ofwhom can have adverse reactions to soy-based infant formula. The soy allergies performanceincludes atopic dermatitis, gastrointestinal disorders, and life-threatening. Therefore,development of hypoallergenic soy protein isolate (SPI) special for infant formula andprotection the safety of soybean allergic infants have important practical significance. How todecrease the allergenicity of SPI and relieve its dangers to infants and children, is an urgentscientific problem to be solved.By comparison domestic SPI with foreign special SPI for infant formula, the differencesof structure and function were ascertained. Western blotting of domestic SPI and soy allergicchildren’s serum basing on one-dimensional electrophoresis and two-dimensionalelectrophoresis, combining with tandem mass resolution identified the allergens and allergicprobability relating to domestic children. Physical desensitization methods were screened.The mechanism of eliminating allergenicity was explored via the effects of HPP on surfacehydrophobicity, free sulfhydryl content, zeta potential, secondary structure, subunit andtertiary and quaternary structure. Basing on processing curve of enzymes on degree ofhydrolysis and allergenicity decrease, optimal enzyme types and enzymatic combinationroutine were screened. The effects of multi-enzymatic method on allergenicity wereinvestigated.The effects of HPP on multi-enzymatic method reducing allergenicity rate,Michaelis constant, molecular weight distribution and reversed-phase high performance liquidchromatogram were utilized to explore synergistic action of HPP and enzymatic method, thusproviding theoretical and experimental basis for special SPI application in infant formula. Themain findings were as follows:(1) By comparison domestic SPI for infant formula with foreign special SPI in the aspects ofSDS-PAGE, molecular weight distribution, scanning electron microscopy, allergen content，in vitro digestibility and thixotropy, the results show that protease enzymatic hydrolysis is aneffective approach to reduce the allergenicity of SPI for infant formula and to improve itsnutritional value and dietary quality.(2) The western blotting of domestic SPI (made from Zhonghuang35and Beidou10) andserum from14soy allergic children, basing on one-dimensional electrophoresis, indicated thatthe proportion of each allergic subunit from high level to low level were as follows:53~55kDsubunit,20~24kD subunit and33~34kD subunit,18kD subunit,43kD subunit,28kD subunit,69~72kD and95~100kD,60~62kD subunit,40kD,83kD and130kD subunit,9kD subunit.The western blotting basing on two-dimensional electrophoresis, combining with tandemmass spectrometry showed that the main allergens relating to chinese soy allergic childreninclude chain A1of11S globulin G1, chain A2of G2, chain A3of G5; α, α’ and β subunit of7S globulin; chain A of crystal structure of proglycinin C12g mutant, chain A of crystal structure of proglycinin mutant C88s and chain A of soybean agglutinin complexed with2,6-pentasaccharide. Chain A of soybean agglutinin complexed with2,6-pentasaccharidebelongs to L-type lectin superfamily, and the other allergens belong to cupin superfamily.Chain A of Crystal structure of proglycinin C12g mutant, chain A of crystal structure ofproglycinin mutant C88s and chain A of soybean agglutinin complexed with2,6-pentasaccharide are new allergens. Chain A1of G1and Chain A1of G1A1ab1b are theapproved allergen Gly m6.0101, and the rest of the allergens are new vairants of approvedallergens.(3) High hydrostatic pressure (HHP) showed the highest allergenicity reducing rate, comparedwith microwave, ultrasonic and high pressure homogenization. The parameters of HHPpressure and time were closely related to allergenicity reducing rate. In the ranges of200~300MPa and5~15min, the free SH content, hydrophobicity and peak value of extrinsicemission fluorescence spectra of SPI significantly increased (P＜0.05). Meanwhile at thelevels above300MPa and15min, the above three factors progressively decreased (P＜0.05).Whatever HHP pressure and time, the maximum emission wavelength indicated blueshifts.After HHP treatment, the helix1and turns content significantly increased and the strand1andunordered content considerably decreased(P＜0.05); whereas the amount of the helix2andstrand2did not indicate any obvious change. The average length of helices significantlyincreased(P＜0.05), while the helices (per100residues) did not strikingly change after HHPmodification. However, both the strand (per100residues) and the average length of strandsclearly decreased(P＜0.05). HHP can unfold helices and compress strands. The near-UVcircular dichroism spectra showed that HHP caused the tyrosine and phenylalanine peakdifferences of quaternary structure. The epitopes of SPI allergens identified in Chapter3couldbe closely related to the secondary structure of α-helix and β-sheet. Dimensionalelectrophoresis profiles, immunoblotting patterns showed that HHP can decrease theallergenicity of SPI, resulting from the conformational changes of SPI. The optimal HHPparameters using response surface method were1%(w/v) of SPI concentration,18min ofHHP time,369MPa of HHP pressure. The allergenicity reducting rate was49.33%at theoptimal levels.（4）Due to their restriction sites, different enzymes exhibited various mechanisms of actionon the allergens of SPI. Basing on the distributions and structures of the allergens identified inChapter3, while considering hydrolysis efficiency, cost, and bitterness of the hydrolyzate, theoptimal mass ratio of the neutrase and flavourzyme followed1:1, and synchronized enzymaticroute was determined. The optimal set of variables was initial substrate concentration of8%(w/v), pH of7.4, hydrolysis temperature of46℃, time of2.5h and enzyme adding dose of2.5%(g/100g protein). The allergenicity reducting rate reached76.05%at the optimal levelsof the tested factors.(5) The allergencity determined by commercial Elisa kit and antibody titers showed that HHPpretreatment significantly improved the allergenicity reducing rate of multi-enzymaticprocessing. The former increased by19.33%and the latter increased by17.24%. At theoptimal HHP parameters, Kmvalues significantly decreased compared with a single enzyme and multi-enzymes; However, Vmaxsignificantly increased compared with a single enzymeand multi-enzymes. HHP pretreatment caused higher content of low molecular weightfractions of SPI multi-enzymatic hydrolysate. HHP did not affect the types of SPI peptidesand increased their content. These data verified that HHP pretreatment exhibited synergisticaction on multi-enzymatic processing about the reducing allergenicity rate of SPI for infantformula. The domestic SPI was treated by composite enzymatic processing assisted by HHP.Compared with Solae products, the security and in vitro digestibility of hydrolysates assistedby HHP increased, but the thixotropy and viscosity should be further improved.