本文選題：大米蛋白 + MG轉(zhuǎn)變�。� 參考：《江南大學(xué)》2017年博士論文

【摘要】：大米蛋白是一種廉價的高營養(yǎng)蛋白,也是一種低致敏性的蛋白資源,可作為嬰兒及敏感人群食品配方。但是低溶解性成為限制大米蛋白作為食品功能性成分及商業(yè)化應(yīng)用的主要瓶頸。盡管目前已有多種溶解性改良手段,但是大米蛋白的改性存在著分子修飾不合理、產(chǎn)物破壞嚴(yán)重、處理效率差等多方面問題。針對上述問題,本研究從相變態(tài)水分子及蛋白質(zhì)拓?fù)浣Y(jié)構(gòu)入手,構(gòu)建一種全新的物理處理方法—物理相變耦聯(lián)機(jī)械脅迫(PTMS),全面分析大米蛋白結(jié)構(gòu)改性的分子學(xué)機(jī)理,并從納米科學(xué)及界面科學(xué)角度開發(fā)改性大米蛋白納米載體,拓展大米蛋白及其他谷物蛋白應(yīng)用的新領(lǐng)域�；谌苋趹B(tài)(MG)的PTMS大米蛋白的分子學(xué)溶解機(jī)制研究。結(jié)構(gòu)學(xué)表征表明,大米蛋白在MG轉(zhuǎn)變條件下,三級結(jié)構(gòu)解體而維持原有的一、二級結(jié)構(gòu);溶劑相變使得水分子氫鍵發(fā)生重排而產(chǎn)生自由水,并在相變溫度下滲透到蛋白質(zhì)內(nèi)部結(jié)構(gòu),進(jìn)一步形成冰晶體;機(jī)械脅迫借冰晶體媒介作用改變大米蛋白的二級及高級結(jié)構(gòu),導(dǎo)致其聚集度下降、蛋白分子展開、極性基團(tuán)暴露,形成20~80 nm的極性納米顆粒。此外,大米蛋白在PTMS過程中可維持原有的一級結(jié)構(gòu)。在pH12.5的MG條件下,經(jīng)三次PTMS,大米蛋白溶解度達(dá)到50.4%。因此,基于MG的PTMS溶解大米蛋白的分子學(xué)機(jī)制是,在維持大米蛋白一級機(jī)構(gòu)的基礎(chǔ)上實(shí)現(xiàn)二級結(jié)構(gòu)的折疊-轉(zhuǎn)角轉(zhuǎn)變,最終形成聚集度較低的極性蛋白體顆粒�；趦�(yōu)化PTMS的改性大米蛋白(MRPs)理化及功能特性研究。采用中心組合實(shí)驗(yàn),在pH12.5的MG轉(zhuǎn)變條件下,分析料液比、相變時間及相變溫度對MRPs得率的影響。響應(yīng)面分析表明,在料液比1:40、相變時間40 h及相變溫度-13°C條件下,大米蛋白經(jīng)PTMS后,MRPs得率達(dá)到40.50%,并在此條件下制備MRPs并噴霧干燥和冷凍干燥分別得到SD-RP和FD-RP。SDS-PAGE實(shí)驗(yàn)表明,SD-RP和FD-RP與對照具有相似度95%以上的一級結(jié)構(gòu)。水溶性實(shí)驗(yàn)表明,SD-RP和FD-RP在pH≤6.0時溶解度均10%,而當(dāng)pH≥7.0時溶解度均90%。由于乳化性及起泡性的發(fā)揮依賴于蛋白質(zhì)的水合能力,在中性條件下(pH7.0),SD-RP和FD-RP乳化性從25.18 m2/g(對照組)分別提高至29.43 m2/g和46.99 m2/g;起泡性從7.65 cm3(對照組)分別提高至18.2 cm3和16.55cm3。此外,形態(tài)學(xué)研究結(jié)果表明,對照組結(jié)構(gòu)致密,FD-RP結(jié)構(gòu)疏松,而SD-RP呈分散的顆粒狀結(jié)構(gòu);SD-RP和FD-RP在溶劑中聚集程度的降低是功能性質(zhì)提高的原因。基于MRPs-油乳液載體的β-胡蘿卜素結(jié)構(gòu)學(xué)控釋機(jī)制研究。在pH從9.0至6.2~7.0降低過程中,MRPs發(fā)生反溶劑沉淀并在油滴表面結(jié)構(gòu)化形成滴粒徑為300~400 nm的乳滴(Em6.2~Em7.0)。利用乳液殼層(載體)的對pH的高敏感度,導(dǎo)致其結(jié)構(gòu)密度可控,實(shí)現(xiàn)在模擬胃腸道消化過程中載體中β-胡蘿卜素的控釋。結(jié)構(gòu)分析及釋放動力學(xué)結(jié)果表明,Em6.2之間通過蛋白質(zhì)交聯(lián)發(fā)生疏水聚集,致使MRPs與油的結(jié)合程度減弱。因而,Em6.2在胃腸道模擬消化過程中,β-胡蘿卜素釋放速率最高。Em6.4~Em7.0在模擬胃液消化過程中β-胡蘿卜素釋放量均10%(即釋放過程被抑制),在模擬腸道消化過程中β-胡蘿卜素釋放過程均符合零級動力學(xué)且釋放速率隨pH升高而增加。此結(jié)果與乳滴表面蛋白質(zhì)的堆積密度隨pH升高而降低有關(guān)。因此,MRPs可應(yīng)用于制備具有控釋功能新型乳液運(yùn)載系統(tǒng)。同時,這項(xiàng)研究也大大提高了MRPs乳液穩(wěn)定性,為MRPs作為功能性成分提供新的解決方案。基于MRPs-精油自乳化行為的控釋機(jī)理研究。發(fā)現(xiàn)溶解在油相中的活性物質(zhì)可完成自乳化,這不僅是一種有趣的現(xiàn)象,且對功能活性物質(zhì)及藥物的運(yùn)載具有重要的意義。為此,瞄準(zhǔn)MRPs-精油內(nèi)源性結(jié)合反應(yīng),開發(fā)具有控釋功能的油核-蛋白殼復(fù)合材料。丁子香酚在與1%MRPs低速攪拌過程中去質(zhì)子化,并通過電荷轉(zhuǎn)移降低體系pH,造成MRPs結(jié)構(gòu)改變而堆積在油滴表面形成復(fù)合乳滴。蛋白質(zhì)-精油反應(yīng)導(dǎo)致油-水界面張力發(fā)生極顯著下降,相同剪切速率下油滴表面Laplace張力大大減小,最終油滴平均粒徑250 nm。同時,掃描透射電鏡及聚焦電子束掃描電鏡實(shí)驗(yàn)表明,丁子香酚濃度的變化造成蛋白質(zhì)在油滴表面堆積強(qiáng)度發(fā)生改變。乳液在體外透析階段精油呈現(xiàn)一級動力學(xué)釋放且釋放速率常數(shù)k隨蛋白質(zhì)-精油比的增加而降低。因此,作者利用這種現(xiàn)象進(jìn)行生物活性物質(zhì)的載運(yùn)及控釋研究。結(jié)果表明,在0.2μg/mL及2μg/mL劑量濃度下,裝載于蛋白質(zhì)-精油比為1:4載體內(nèi)的咖啡酸苯乙酯(CAPE)與溶解在DMSO的CAPE對HCT-116癌細(xì)胞抑制率相似,兩種濃度下抑制率分別為5%和35%。而在0.2μg/mL及2μg/mL劑量濃度下,裝載于蛋白質(zhì)-精油比為1:20載體內(nèi)的CAPE對HCT-116的抑癌率分別達(dá)到50%和70%。以上實(shí)驗(yàn)表明,具有自乳化特性的MRPs不僅能夠大大提高生產(chǎn)效率,還能提高生物活性物質(zhì)的生物利用度,因而在脂溶活性物質(zhì)載運(yùn)實(shí)踐中具有可觀的應(yīng)用前景。
[Abstract]:Rice protein is a cheap high nutrient protein and also a kind of low sensitizing protein resource, which can be used as food formula for infants and sensitive people. But low solubility becomes the main bottleneck for limiting rice protein as a functional component and commercial application of food. Although there are many methods of dissolved improvement, rice protein has been found at present. There are many problems in the modification, such as irrational molecular modification, serious damage and poor treatment efficiency. In this study, a new physical treatment method, physical transformation coupled mechanical stress (PTMS), is constructed to analyze the structural modification of rice protein. The mechanism is to develop the modified rice protein nanoscale from the perspective of nanoscience and Interface Science, and expand the new field of rice protein and other grain protein applications. The molecular dissolution mechanism of the PTMS rice protein based on melting state (MG) is studied. The structural characterization shows that the rice protein is disintegrated and maintains the original three stage structure under the condition of MG transformation. The first, two stage structure, the solvent phase change causes the hydrogen bond of water molecules to rearrange and produce free water, and permeate into the internal structure of protein at the temperature of phase change, and further form the ice crystal. The mechanical stress changes the two grade and the advanced structure of rice protein by the action of ice crystal medium, and leads to the decrease of the aggregation degree, the expansion of protein molecules and the polar group storm. In addition, the rice protein can maintain the 20~80 nm polar nanoparticles. In addition, the rice protein can maintain the original first order structure in the PTMS process. Under the MG condition of pH12.5, the solubility of rice protein reaches 50.4%. after three times PTMS, so the fractional mechanism of the PTMS dissolved rice protein based on MG is to realize the two level knot on the basis of maintaining the first grade mechanism of rice protein. The folding and turning angle of the structure was changed to form the polar protein body particles with low aggregation. The physicochemical and functional properties of the modified rice protein (MRPs) based on the optimized PTMS were studied. The effect of the ratio of material to liquid, the time of phase change and the temperature of phase change on the yield of MRPs was analyzed by the central combination experiment under the MG transformation condition of pH12.5. When the liquid was at 1:40, the phase transition time was 40 h and the phase transition temperature was -13 C, the yield of MRPs was 40.50% after PTMS, and the preparation of MRPs, spray drying and freeze-drying under the conditions of SD-RP and FD-RP.SDS-PAGE, respectively, showed that SD-RP and FD-RP had a first grade structure with similar degree similar to 95%. Water solubility experiment showed SD-RP The solubility of FD-RP and FD-RP at pH < 6 is 10%, while the solubility of the solubility of 90%. depends on the hydration ability of the protein when the emulsification and foaming ability is equal to 7. Under neutral conditions (pH7.0), the emulsification of SD-RP and FD-RP increases from 25.18 m2/g (control group) to 29.43 m2/g and 46.99 m2/g respectively, and the foaming property is increased to 1 from 7.65 cm3 (control group), respectively. 8.2 cm3 and 16.55cm3. in addition, the morphological study showed that the structure of the control group was dense, the FD-RP structure was loose, and the SD-RP was dispersed in granular structure. The decrease of the aggregation degree of SD-RP and FD-RP in the solvent was the reason for the improvement of the functional properties. The study on the structural release mechanism of beta Hu Luo based on MRPs- oil emulsion carrier was studied in pH from 9 to 6.2~7.. During the 0 reduction, MRPs precipitated and formed a droplet of 300~400 nm on the surface of the oil droplet (Em6.2~Em7.0). Using the high sensitivity of the emulsion shell (carrier) to pH, the structure density was controlled, and the controlled release of beta carotene in the carrier of simulated gastrointestinal tract, structure analysis and release power were realized. The results showed that the binding of Em6.2 through protein crosslinking resulted in a decrease in the binding of MRPs to oil. Thus, the highest beta carotene release rate of.Em6.4~Em7.0 in the simulated gastrointestinal digestion process was 10% (the release process was inhibited) during the digestive process of the simulated gastric juice (i.e. the release process was suppressed), and in the simulated intestinal tract. In the process of digestion, the release process of beta carotene conforms to zero order kinetics and the release rate increases with the increase of pH. The accumulation density of the protein on the milk drop surface decreases with the increase of pH. Therefore, MRPs can be applied to the preparation of a new emulsion delivery system with controlled release function. This study also greatly improved the stability of MRPs emulsion. Qualitatively, it provides a new solution for MRPs as a functional component. Based on the study of the controlled release mechanism of the self emulsifying behavior of MRPs- essential oil, it is found that the active substances dissolved in the oil phase can be self emulsified, which is not only an interesting phenomenon, but also is of great significance to the delivery of functional active substances and drugs. To this end, aim at the essential oil in the MRPs- oil. The source binding reaction was used to develop the oil core protein shell composite with controlled release function. The butylphenols were protonated during the low speed stirring process with 1%MRPs and reduced the system pH by charge transfer, resulting in the structure change of the MRPs and accumulated on the surface of the oil droplets. The protein fine oil reaction resulted in a very significant lower oil water interfacial tension. Under the same shear rate, the Laplace tension on the surface of oil droplets decreased greatly, and the average particle size of the oil droplets was 250 nm.. The scanning transmission electron microscopy and focused electron beam scanning electron microscopy showed that the change of the concentration of Ding Zixiang phenol resulted in the change of the protein accumulation strength on the surface of the oil droplet. The release rate constant K decreases with the increase of the protein essential oil ratio. Therefore, the author uses this phenomenon to carry out the study on the carrier and controlled release of bioactive substances. The results show that at the dose concentration of 0.2 and 2 mu g/mL, the protein - essential oil is compared to the 1:4 - loaded caffeic acid benzol ethyl (CAPE) and the CAPE against the DMSO in DMSO. The inhibition rates of -116 cancer cells were similar, and the inhibition rates under two concentrations were 5% and 35%. respectively, while at 0.2 and 2 g/mL doses, the inhibition rate of CAPE against HCT-116 in protein essential oil compared to the 1:20 carrier was 50% and 70%., respectively. The results showed that MRPs with self emulsifying property could not only greatly improve the production efficiency, but also could improve the production efficiency. Improving the bioavailability of bioactive substances has great potential for application in the transportation of liposoluble active substances.

【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：TS201.21

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 曹輝;馬海樂;曲文娟;賈俊強(qiáng);潘忠禮;丁青芝;駱琳;王振斌;何榮海;;大米蛋白的木瓜酶酶解及其水解物的抗氧化活性[J];中國糧油學(xué)報;2009年07期

2 易翠平,姚惠源;大米濃縮蛋白脫酰胺研究(I) 酸法脫酰胺與酶法脫酰胺工藝比較與參數(shù)優(yōu)化[J];食品科學(xué);2005年01期

3 頓新鵬,陳正望;酶法水解米渣蛋白制備大米小分子肽[J];食品科學(xué);2004年06期

4 金世合,陳正行,周素梅;酶解米糠蛋白的功能性質(zhì)研究[J];食品工業(yè)科技;2004年03期

，

本文編號：1834734