摘要
牛乳蛋白质过敏症和不同程度乳糖不耐症婴幼儿的患病率在我国分别约占1%～2%和30%。根据发达国家的经验，豆基婴幼儿配方粉适用于上述两类患儿，如果该婴幼儿的牛乳蛋白过敏是IgE介导的过敏，其对大豆蛋白过敏的几率高达53%～63%。大豆过敏症状主要表现为过敏性皮炎、胃肠道紊乱，严重者甚至危及生命。因此，开发低致敏性婴幼儿配方食品专用大豆分离蛋白，保护大豆过敏婴幼儿的消费安全，具有重要的现实意义；如何降低大豆分离蛋白致敏性，减轻其对婴幼儿的危害是迫切需要解决的科学问题。
本论文通过国产大豆分离蛋白与国外婴幼儿配方食品专用大豆分离蛋白的比较，明确结构与功能方面的差异；通过国产大豆分离蛋白与我国大豆过敏患儿血清的一维电泳及双向电泳的免疫印迹，结合串联质谱解析，鉴定出引起我国婴幼儿大豆过敏的致敏原及其致敏概率；通过物理方法的筛选，明确了物理脱敏方法，然后根据超高静压对游离巯基、表面疏水性、Zeta电位、二级结构、亚基、高级结构的影响，探明其降低致敏性的机理；根据酶对水解度和致敏性的影响，筛选出合适的酶制剂及复配路线；研究复合酶法对致敏性的影响规律。根据超高静压对复合酶法降低致敏性反应速率和米氏常数、分子量分布及反相超高效液相色谱图的影响，探明其协同作用，为其在婴幼儿配方食品中的应用提供理论和试验依据。主要研究结果如下：
（1）将国产大豆分离蛋白与国外婴幼儿配方食品专用大豆分离蛋白在SDS-PAGE、分子量分布、扫描电镜、过敏原含量、体外消化率、触变性等方面进行了比较，结果表明蛋白酶酶解是降低婴幼儿配方食品专用大豆分离蛋白致敏性，改善其营养价值和食用品质的有效途径。
（2）将由中黄35和北豆10制备而成的国产大豆分离蛋白一维电泳（SDS-PAGE）与我国14例大豆过敏患儿血清的免疫印迹结果表明，各亚基致敏比例自高至低依次为：53～55kD亚基、20～24kD亚基及33～34kD亚基、18kD亚基、43kD亚基、28kD及69～72kD亚基、95～100kD、60～62kD亚基、40kD、83kD及130kD亚基、9kD亚基。二维电泳免疫印迹及串联质谱结果表明，引起我国婴幼儿大豆过敏的致敏原主要包括11S球蛋白G1的A1链、G2的A2链、G5的A3链；7S球蛋白α、α'和β亚基；11S突变体C88s晶体结构A链与11S突变体C12g的A链；大豆凝集素2,6-戊糖复合体A链。除大豆凝集素2,6-戊糖复合体A链属于外源凝集素L型超家族之外，其他的致敏原全部为Cupin超家族。11S突变体C12g的A链、11S突变体C88s晶体结构A链与大豆凝集素2,6-戊糖复合体的A链为新过敏原，11S球蛋白G1中的A1链、G1A1ab1b中的A1a链为已批准的过敏原Gly m6.0101，其余的过敏原均为已批准过敏原的新变体。
（3）相对于微波、超声波、高压均质等物理方法，超高静压使大豆分离蛋白致敏性降低率最大。超高静压处理的压强、时间参数与致敏性降低率密切相关。在200～300MPa与5～15min之间，随着压强的增大和时间的延长，游离巯基的含量、表面疏水性、荧光光谱的峰值显著增加，而后显著下降（P＜0.05）。超高静压处理使大豆分离蛋白最大发射波长蓝移。超高静压处理后，螺旋1和转角的含量显著增加，折叠1和无规则卷曲的含量显著下降（P＜0.05），而螺旋2和折叠2的含量未发生显著变化。螺旋的平均长度显著增加（P＜0.05），而每100个残基中螺旋的个数变化不显著。折叠的平均长度与每100个残基中折叠的个数均显著减少（P＜0.05）。超高静压能够展开螺旋，压缩折叠。近紫外圆二色图谱表明超高静压处理后大豆分离蛋白三、四级结构的差异主要体现在酪氨酸与苯丙氨酸峰值的不同。第三章鉴定出的大豆分离蛋白过敏原与α螺旋、β折叠的二级结构密切相关。双向电泳图谱、免疫印迹图谱表明超高静压可降低专用大豆分离蛋白致敏性，其原因是蛋白空间构象发生了改变。响应面法优化超高静压的最佳工艺参数为：大豆分离蛋白浓度1%（w/v）、超高静压处理时间18min、压强369MPa；在最适参数下，致敏性降低率为49.33%。
（4）不同酶由于其酶切位点不同，对大豆分离蛋白中的过敏原呈现出不同的作用机制。根据第三章鉴定出的引起我国婴幼儿大豆过敏的致敏原分布及结构，同时综合考虑其水解效率、成本、水解物的苦味等多方面因素确定了中性蛋白酶与风味蛋白酶按照1∶1的质量比复配，确定了同步酶解路线，复合酶法的最佳工艺参数为：初始底物浓度8%（w/v）、pH7.4，酶解温度46℃，时间2.5h，加酶量2.5%（g/100g蛋白质）；在最适参数下，致敏性降低率为76.05%。
（5）采用商用试剂盒和抗体滴度方法测定大豆分离蛋白致敏性，结果表明超高静压预处理均可显著提高复合酶法致敏性降低率，前者提高了19.33%，后者提高了17.24%。在优化的超高静压处理参数下，Km值相对单一酶及复合酶而言均显著下降；然而，Vmax值相对单一酶及复合酶而言均显著提高；超高静压预处理使得大豆分离蛋白酶解物中低分子量组分含量提高，而且增加了大豆分离蛋白酶解物中肽的含量，不影响其种类；表明超高静压对复合酶法水解大豆分离蛋白具有协同增效作用。超高静压协同复合酶法处理的国产大豆分离蛋白，其安全性和体外消化率较Solae产品提高了，但触变性与粘度有待进一步改善。
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.
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