大米储藏陈化中蛋白质对其糊化特性的影响及其相关陈化机制研究
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摘要
大米是粮食中最难保藏的粮食品种之一,在储藏过程中容易发生陈化劣变,引起的经济损失十分巨大。为有效控制大米在储藏中陈化作用的发生,陈化的机理有待进一步阐明。大米陈化是一个涉及多组分的复杂问题,本文在研究储藏中大米多个指标变化规律的基础上,确定了有效评价储藏中大米陈化特征的敏感指标,重点研究蛋白质变化对大米陈化特征的影响及其影响机制,揭示大米中各贮藏蛋白对陈米糊化特性的影响,有利于阐明大米陈化发生的机理。
1.大米储藏陈化过程中品质变化研究
系统研究大米在储藏陈化过程中各品质指标的变化规律,找到一个有效衡量大米陈化程度的指标,对于大米陈化程度评价和安全监测具有十分重要的意义。将大米密闭储藏,经37℃高温加速陈化,定期取样测定各理化指标的变化。结果表明：(1)大米脂肪酸值、胶稠度和碱消值在储藏中没有明显变化规律,总直链淀粉、热水可溶性直链淀粉和热水不溶性直链淀粉含量变化不明显；(2)电子鼻气味评价表明不良气味对储藏大米总气味的贡献远大于良好气味；(3)在三种色彩空间模式下的颜色参数均表明储藏过程中米粒的内部比其表面更白,大米在储藏中的黄变是从接触空气的表面开始的；(4)RVA(快速粘度分析仪)糊化特征参数的变化表明,大米在储藏期间,峰值粘度、衰减值在0～2个月呈现上升趋势,2～8个月呈现下降趋势,回升值在0～2个月下降,2～8个月上升,成糊温度在0～8个月均呈明显的上升趋势；此外,大米在储藏陈化4个月以后原来两个阶段的溶胀合并为一次性溶胀,说明陈化导致了本来在结晶区溶胀前发生溶胀的无定形区也需要在较高的温度下才能溶胀,而无定形区的成分主要是直链淀粉和蛋白质及脂肪,因此可以预见陈化导致了直链淀粉、蛋白质和脂肪性质的变化。综上所述,RVA特征参数是表征大米在储藏过程中陈化变化最灵敏、最有规律且所提供信息最丰富的指标。因此本文后续章节中以RVA特征参数作为指标来研究蛋白质对储藏中大米品质变化的影响。
2.储前处理对储藏过程中大米糊化特性变化的影响
通过储前处理来改变大米中α-淀粉酶和脂肪氧合酶的活性,或者防止蛋白质的氧化,经37℃下储藏加速陈化,考察酶活变化和蛋白质氧化对大米陈化的影响。结果表明：(1)α-淀粉酶不是大米陈化的主要诱因,乙二胺四乙酸二钠对脂肪氧合酶的钝化作用延缓了大米的陈化；(2)三种还原剂中,储前Vc处理对陈化的影响最大,大米在37℃下储藏4个月后,Vc作为促氧化剂加速了大米陈化；而茶多酚和DTT(二硫代苏糖醇)在储期为4、8个月时延缓了大米陈化,这一事实说明大米陈化与氧化过程密切相关。
3.储后处理对陈米糊化特性的改善及其机理研究
采用储后处理陈米的方式,试图找到一种具有与DTT类似甚至效果更好,而且是食品级的添加剂来改善大米品质；同时,可以从陈米品质改良剂的性质和作用来推断大米陈化的机理。结果表明：(1)硫酸钠处理表明陈化降低了淀粉的正常吸水速度；(2)新米的DTT处理(糊化时处理)与新米的储前处理(第三章2.2.1储前DTT处理0个月时)效果相同,显示DTT是在加热糊化过程中才发挥作用的,而不是糊化前直接与米中的成分发生反应；对陈米储后处理表明,DTT对陈米淀粉颗粒溶胀程度的提高是非常有限的,而Vc极大地促进了陈米的溶胀程度,改善了陈化特征,效果好于DTT,其溶胀程度甚至超过了新米对照,表明二硫键交联并不是大米陈化时发生的唯一主要变化,可能存在着除二硫键之外的阻止淀粉颗粒溶胀的作用力,而Vc可破坏这种作用力,且呈剂-效关系,证实了存在除巯基被氧化成二硫键之外的陈化机制；茶多酚只是抑制了直链淀粉的冷却回生；SDS对陈米的影响表明大米陈化后形成了数量众多的氢键和疏水相互作用,这些作用力抑制了淀粉颗粒的糊化溶胀；(3)吸水性、可溶性固形物含量和显微图像证实,Vc同时促进了吸水性的增加和可溶性固形物的溶出,而DTT只促进了吸水性的增加,却抑制了可溶性固形物的溶出,且Vc对吸水性的促进作用远远高于DTT,这是Vc与DTT在改善糊化特性机制上的差别,同时也表明吸水性比可溶性固形物对糊化特性的影响更重要；(4)用巯基封闭剂NEMI(乙基顺丁烯二酰亚胺)处理新米的结果证实二硫键只存在于已溶胀的淀粉颗粒之间;NEMI处理陈米的结果表明,陈米在糊化过程中仍可形成较多的二硫键交联而使糊化粘度升高,但巯基交联对陈米糊化体系粘度的贡献是很小的,体系粘度主要取决于淀粉颗粒溶胀的程度；Vc单独处理及其与巯基封闭剂NEMI共同处理陈米相对比,可证实Vc能阻止陈米在糊化过程中形成二硫键交联；(5)储前Vc处理获得加速陈化大米,再经储后Vc处理,结果表明储前Vc处理加速大米陈化的原因是在较长时间的储藏过程中Vc自身遭受空气氧化而使其转变为促氧化剂,这进一步说明大米陈化的本质是与氧化相关的变化。
4.大米中贮藏蛋白对陈米糊化特性的影响
通过储后脱蛋白试验来研究陈化中各蛋白自身变化对陈米糊化特性的影响,通过储前脱蛋白试验来研究陈化中各蛋白与其它组分间的相互作用及各蛋白自身的变化二者复合效应对陈米糊化特性的影响。结果表明：(1)清蛋白、球蛋白在陈化中自身的变化促进了大米陈化,谷蛋白和醇溶蛋白在陈化中自身的变化延缓了大米陈化,而脂肪在陈化中自身的变化对陈化具有双重作用；(2)陈化中各蛋白与其它组分间的相互作用及各蛋白自身的变化二者的复合效应是：脂肪和清蛋白促进了大米陈化；球蛋白和醇溶蛋白不同程度地延缓了大米陈化,而谷蛋白对陈化的影响具有双重性。二者对比可见,大米陈化不是某一种或某几种组分单独变化引起的,而是更多的涉及到多种组分间的相互作用,特别是球蛋白与其它成分间的相互作用对陈化的影响最大,谷蛋白和脂肪与其它组分间的相互作用对陈化的影响次之。
5.大米陈化前后蛋白质结构变化研究
本章采用红外和拉曼二种互补的光谱技术来表征大米中四种贮藏蛋白在陈化中发生的结构变化及与其它分子间的相互作用。结果表明：(1)红外显示清蛋白可能是糖蛋白,且不论是新米还是陈米,其清蛋白均有亚砜特征峰,显示了巯基被氧化；拉曼谱显示陈化后清蛋白中无规卷结构曲减少,蛋白质疏水基埋藏,分子间氢键键合程度降低,可能暗示陈化后清蛋白与淀粉分子的结合变得更为松散；(2)红外显示陈化后球蛋白巯基明显氧化,产物包括砜和亚砜,且糖基化趋势显著增强；拉曼谱显示陈化后球蛋白无规卷曲结构增多,也显示有亚砜存在,以及分子间氢键键合程度提高,暗示陈化后球蛋白与淀粉分子间的结合变得更为紧密；(3)红外显示谷蛋白陈化后有去糖基化趋势；拉曼谱显示陈化后谷蛋白无规卷曲结构降低,有游离巯基特征峰出现,支持谷蛋白去糖基化的判断；(4)红外显示陈化中醇溶蛋白变化不大；拉曼谱显示陈化使醇溶蛋白反平行p-折叠结构增加,二硫键构型转变,酪氨酸残基由包埋状态转变为暴露状态。综上所述,陈化中球蛋白与淀粉间的相互作用变化最大,其次是谷蛋白,而清蛋白和醇溶蛋白与淀粉的相互作用变化不大；且球蛋白是与淀粉分子的相互作用加强,而谷蛋白是与淀粉分子的相互作用减弱。
Rice is one of the hardest preserved food grain varieties prone to aging deterioration during storage, causing huge economic losses. In order to control the rice ageing during storage effectively, the mechanism of ageing needs to be further elucidated. Rice ageing is a multi-component complex issue. This thesis focus on the improvement effects of protein changes on rice aging characteristics and its impact mechanisms, trying to reveal the mechanism of aging through the performing roles of protein modifiers and the effect of rice protein on the pasting properties.
1. Changes of rice quality during storage
Through studying the variation of the quality indicators of rice during storage, it is important to find an effective indicator of the rice aging to evaluate the degree of aging and safety of rice. Milled rice was sealed in airtight bottles stored at37℃for accelerated aging, periodically sampling was carried out to determine the changes in selected physical and chemical indicators. The following results were obtained:(1) Fatty acid value, gel consistency and alkali spreading value of rice in storage changed irregularly, while the total amylose, hot water soluble amylose and hot water insoluble amylose content did not change significantly.(2) Rice odor evaluation by electronic nose showed that the bad smell is much larger than the good smell in contribution to the total odor of the stored rice.(3) The parameter changes in three color space mode showed conformably that the internal color of rice grain than its surface color is more white during storage, which suggests storage yellowing in rice originate from the surface of contact with air.(4) The changes of RVA pasting characteristic parameters showed that the peak viscosity and breakdown increased from0to2nd month, decreased from2to8th month, setback declined from0to2nd month, rose from2to8th month, while pasting temperature increased from0to8th month. In addition, the original two-stage swelling of stored rice merged into a one-time swelling after4months, showing that the ageing causes a higher swelling temperature of the amorphous region, which should swell prior to occurrence of the crystallization region. Because that amorphous region composes mainly of amylase, protein and lipid, it is speculated that ageing led to amylose, protein and lipid changes in the nature. In summary, the RVA characteristic parameters appear to be the most sensitive, regular and provide the richest information to characterize the degree of rice aging during storage. So, RVA characteristic parameters on behalf of pasting properties were considered as indicators in the subsequent chapters of this thesis to study the changes of rice quality during storage.
2. Effect of pretreatment before storage on pasting properties of rice duration storage
The milled rice which was subjected to treatments before storage were accelerated to ageing at37℃to study the effects of changes in enzyme activity of Alpha-amylase and lipoxygenase, and in protein oxidation. The results showed that:(1) Alpha-amylase was not the main inducement of rice ageing, while the stabilization role of lipoxygenase by EDTA·2Na slowed the ageing of stored rice.(2) Among three reducing agents, Vc treatment before storage has the most important impact on rice aging. When the rice stored at37℃after4months, Vc as prooxidant accelerated rice aging, while tea polyphenols and DTT delayed rice aging during the storage period of4and8months, showing that rice aging was closely related to the oxidation process. Vc accelerated rice ageing duration storage of rice, and this finding has not been reported in the literature yet.
3. The improvement role and its mechanism of post treatment after storage on pasting properties of aged rice
In order to find a similar or even better additives compaer to DTT which is food additives and can improve the quality of aged rice, and at the same time, to infer rice ageing mechanism through the nature and function of quality improvement of aged rice, the effects of treatments on aged rice were investigated. The results followed that:(1) Sodium sulfate treatment showed that aging decreased the normal water absorption speed of rice.(2) DTT treatment before storage (Chapter3,2.2.1DTT treatment before storage at0month) with the fresh rice treated (treated in pasting process) has the same effect, suggesting that DTT play a role in the heated gelatinization process rather than in before pasting which react directly with compound in the rice. The increasing of starch granule swelling degree of aged rice by DTT treatment was very limited, yet Vc showed bigger enhancing role than DTT on the swelling of aged rice, even more swelling than the fresh rice as the control. In contrast, tea polyphenols only retards the cooling retrogradation of amylase. The facts indicate that the disulfide cross-linking is not the only major change when the rice aging occurs, there may be other force to prevent the starch granule swelling except for disulfide bonds, and Vc could destroy the main force of inhibiting aged rice from swelling, and showed a dose-effect relationship, confirming other aging mechanisms exist except the sulphydryl is oxidized to the disulfide bonds. The effect of SDS on aged rice showed that a large number of hydrogen bonds and hydrophobic interactions might be formed in rice ageing, and these forces inhibited gelatinization swelling of the starch granules.(3) the results of water absorption index, soluble solid and microscopic images confirmed that Vc simultaneously promotes water absorption and soluble solids, while DTT only promoted water absorption but inhibited soluble solid, and the role of Vc in promoting water absorption was much higher than that of DTT. This was different in mechanism of improving pasting properties between Vc and DTT, and also showed that water absorption of starch granule was more important than losses of the soluble solids on the pasting properties.(4) the effect of SH-blocking agent NEMI on fresh rice showed that the disulfide bond exists only in the swelling starch granules. The results of NEMI dealing with aged rice showed that aged rice in the gelatinization process could still form disulfide cross-linking which make the pasting viscosity increased, but the contribution of sulphydryl cross-linking on the viscosity of aged rice paste was very small, and that of swelling degree (Vc promoting swelling) was bigger. The comparison of treatment results between Vc separately and in combination with NEMI indicated that Vc prevented the formation of disulfide cross-linking of aged rice in gelatinization process.(5) the effect of Vc treatment on aged rice of accelerated ageing by Vc before storage showed that the Vc does have a dual function on rice ageing, which was that the treatment before storage on fresh rice accelerated aging and the treatment after storage on aged rice improved pasting properties. This once again showed the nature of the rice aging was associated with oxidative changes.
4. Effects of rice storage protein on pasting properties of aged rice
Removing protein from aged rice after storage were carried out to explore the effect of rice protein changes in ageing on pasting properties, and removing protein from fresh rice before storage were carried out to explore the combined effect of rice protein changes and interactions with other components in ageing process on pasting properties. The results were:(1) the changes of albumin and globulin in aging process promoted rice ageing, the changes of glutelin and prolamin in ageing process delayed rice ageing, while the changes of fat in ageing process have a dual role on rice ageing.(2) the combined effects of each component changes and the interactions with other components in ageing process were as that:fat and albumin promoted rice ageing, globulin and prolamin delayed rice ageing, while glutelin played dual role on rice ageing. Comparison of above mentioned imformation suggest that rice ageing is not caused by separated change of one or several components, but is more related to the interactions between the various components, especially the interactions between the globulin and other components, followed by the interactions between glutelin and fat and other ingredients.
5. Structural changes of rice storage protein in ageing process
The structural changes of the four rice storage protein and interactions between them and other components during ageing process were characterized using infrared and Raman spectroscopic techniques in this chapter. The results were following:(1) Infrared spectrum showed albumin may be glycoprotein, and albumin, no matter it was in fresh or aged rice, both presents sulfoxide peak. Raman spectrum showed that after ageing, the random coil in albumin reduced, the protein hydrophobic group buried, and the intermolecular hydrogen bonding decreased, probably suggesting that the associations between albumin and starch molecules become looser.(2) Infrared spectrum showed that sulphydryl oxidation of globulin from aged rice was more obvious than fresh rice with main products of sulfone and sulfoxide, and glycosylation trend of age rice globulin significantly increased duration storage. Raman spectrum indicated that random coil of the globulin increased and sulfoxide occurred after rice ageing, and the degree of intermolecular hydrogen bonding strengthened, suggesting that ageing promotes binding of globulin and starch molecules.(3) Infrared spectrum showed the glycosylation trend of age rice glutelin significantly decreased than that of fresh rice. Raman spectra displayed that the random coil in gluten reduced after aging and free sulfhydryl peaks appeared.(4) Infrared spectrum of prolamin after ageing have no obvious changes, Raman spectrum reveal anti-parallel β-sheet structure in aged rice prolamin increased, the conformation of disulfide bond changed, tyrosine residues changed embedded state into the exposed state. In summary, after aging, the interactions between globulin and starch presented the greatest change, followed by glutelin, while the interactions between albumin and prolamin and starch changed insignificantly. The interactions of globulin with starch molecules were strengthened, and the interactions between glutelin and starch molecules were weakened.
1.大米储藏陈化过程中品质变化研究
系统研究大米在储藏陈化过程中各品质指标的变化规律,找到一个有效衡量大米陈化程度的指标,对于大米陈化程度评价和安全监测具有十分重要的意义。将大米密闭储藏,经37℃高温加速陈化,定期取样测定各理化指标的变化。结果表明：(1)大米脂肪酸值、胶稠度和碱消值在储藏中没有明显变化规律,总直链淀粉、热水可溶性直链淀粉和热水不溶性直链淀粉含量变化不明显；(2)电子鼻气味评价表明不良气味对储藏大米总气味的贡献远大于良好气味；(3)在三种色彩空间模式下的颜色参数均表明储藏过程中米粒的内部比其表面更白,大米在储藏中的黄变是从接触空气的表面开始的；(4)RVA(快速粘度分析仪)糊化特征参数的变化表明,大米在储藏期间,峰值粘度、衰减值在0～2个月呈现上升趋势,2～8个月呈现下降趋势,回升值在0～2个月下降,2～8个月上升,成糊温度在0～8个月均呈明显的上升趋势；此外,大米在储藏陈化4个月以后原来两个阶段的溶胀合并为一次性溶胀,说明陈化导致了本来在结晶区溶胀前发生溶胀的无定形区也需要在较高的温度下才能溶胀,而无定形区的成分主要是直链淀粉和蛋白质及脂肪,因此可以预见陈化导致了直链淀粉、蛋白质和脂肪性质的变化。综上所述,RVA特征参数是表征大米在储藏过程中陈化变化最灵敏、最有规律且所提供信息最丰富的指标。因此本文后续章节中以RVA特征参数作为指标来研究蛋白质对储藏中大米品质变化的影响。
2.储前处理对储藏过程中大米糊化特性变化的影响
通过储前处理来改变大米中α-淀粉酶和脂肪氧合酶的活性,或者防止蛋白质的氧化,经37℃下储藏加速陈化,考察酶活变化和蛋白质氧化对大米陈化的影响。结果表明：(1)α-淀粉酶不是大米陈化的主要诱因,乙二胺四乙酸二钠对脂肪氧合酶的钝化作用延缓了大米的陈化；(2)三种还原剂中,储前Vc处理对陈化的影响最大,大米在37℃下储藏4个月后,Vc作为促氧化剂加速了大米陈化；而茶多酚和DTT(二硫代苏糖醇)在储期为4、8个月时延缓了大米陈化,这一事实说明大米陈化与氧化过程密切相关。
3.储后处理对陈米糊化特性的改善及其机理研究
采用储后处理陈米的方式,试图找到一种具有与DTT类似甚至效果更好,而且是食品级的添加剂来改善大米品质；同时,可以从陈米品质改良剂的性质和作用来推断大米陈化的机理。结果表明：(1)硫酸钠处理表明陈化降低了淀粉的正常吸水速度；(2)新米的DTT处理(糊化时处理)与新米的储前处理(第三章2.2.1储前DTT处理0个月时)效果相同,显示DTT是在加热糊化过程中才发挥作用的,而不是糊化前直接与米中的成分发生反应；对陈米储后处理表明,DTT对陈米淀粉颗粒溶胀程度的提高是非常有限的,而Vc极大地促进了陈米的溶胀程度,改善了陈化特征,效果好于DTT,其溶胀程度甚至超过了新米对照,表明二硫键交联并不是大米陈化时发生的唯一主要变化,可能存在着除二硫键之外的阻止淀粉颗粒溶胀的作用力,而Vc可破坏这种作用力,且呈剂-效关系,证实了存在除巯基被氧化成二硫键之外的陈化机制；茶多酚只是抑制了直链淀粉的冷却回生；SDS对陈米的影响表明大米陈化后形成了数量众多的氢键和疏水相互作用,这些作用力抑制了淀粉颗粒的糊化溶胀；(3)吸水性、可溶性固形物含量和显微图像证实,Vc同时促进了吸水性的增加和可溶性固形物的溶出,而DTT只促进了吸水性的增加,却抑制了可溶性固形物的溶出,且Vc对吸水性的促进作用远远高于DTT,这是Vc与DTT在改善糊化特性机制上的差别,同时也表明吸水性比可溶性固形物对糊化特性的影响更重要；(4)用巯基封闭剂NEMI(乙基顺丁烯二酰亚胺)处理新米的结果证实二硫键只存在于已溶胀的淀粉颗粒之间;NEMI处理陈米的结果表明,陈米在糊化过程中仍可形成较多的二硫键交联而使糊化粘度升高,但巯基交联对陈米糊化体系粘度的贡献是很小的,体系粘度主要取决于淀粉颗粒溶胀的程度；Vc单独处理及其与巯基封闭剂NEMI共同处理陈米相对比,可证实Vc能阻止陈米在糊化过程中形成二硫键交联；(5)储前Vc处理获得加速陈化大米,再经储后Vc处理,结果表明储前Vc处理加速大米陈化的原因是在较长时间的储藏过程中Vc自身遭受空气氧化而使其转变为促氧化剂,这进一步说明大米陈化的本质是与氧化相关的变化。
4.大米中贮藏蛋白对陈米糊化特性的影响
通过储后脱蛋白试验来研究陈化中各蛋白自身变化对陈米糊化特性的影响,通过储前脱蛋白试验来研究陈化中各蛋白与其它组分间的相互作用及各蛋白自身的变化二者复合效应对陈米糊化特性的影响。结果表明：(1)清蛋白、球蛋白在陈化中自身的变化促进了大米陈化,谷蛋白和醇溶蛋白在陈化中自身的变化延缓了大米陈化,而脂肪在陈化中自身的变化对陈化具有双重作用；(2)陈化中各蛋白与其它组分间的相互作用及各蛋白自身的变化二者的复合效应是：脂肪和清蛋白促进了大米陈化；球蛋白和醇溶蛋白不同程度地延缓了大米陈化,而谷蛋白对陈化的影响具有双重性。二者对比可见,大米陈化不是某一种或某几种组分单独变化引起的,而是更多的涉及到多种组分间的相互作用,特别是球蛋白与其它成分间的相互作用对陈化的影响最大,谷蛋白和脂肪与其它组分间的相互作用对陈化的影响次之。
5.大米陈化前后蛋白质结构变化研究
本章采用红外和拉曼二种互补的光谱技术来表征大米中四种贮藏蛋白在陈化中发生的结构变化及与其它分子间的相互作用。结果表明：(1)红外显示清蛋白可能是糖蛋白,且不论是新米还是陈米,其清蛋白均有亚砜特征峰,显示了巯基被氧化；拉曼谱显示陈化后清蛋白中无规卷结构曲减少,蛋白质疏水基埋藏,分子间氢键键合程度降低,可能暗示陈化后清蛋白与淀粉分子的结合变得更为松散；(2)红外显示陈化后球蛋白巯基明显氧化,产物包括砜和亚砜,且糖基化趋势显著增强；拉曼谱显示陈化后球蛋白无规卷曲结构增多,也显示有亚砜存在,以及分子间氢键键合程度提高,暗示陈化后球蛋白与淀粉分子间的结合变得更为紧密；(3)红外显示谷蛋白陈化后有去糖基化趋势；拉曼谱显示陈化后谷蛋白无规卷曲结构降低,有游离巯基特征峰出现,支持谷蛋白去糖基化的判断；(4)红外显示陈化中醇溶蛋白变化不大；拉曼谱显示陈化使醇溶蛋白反平行p-折叠结构增加,二硫键构型转变,酪氨酸残基由包埋状态转变为暴露状态。综上所述,陈化中球蛋白与淀粉间的相互作用变化最大,其次是谷蛋白,而清蛋白和醇溶蛋白与淀粉的相互作用变化不大；且球蛋白是与淀粉分子的相互作用加强,而谷蛋白是与淀粉分子的相互作用减弱。
Rice is one of the hardest preserved food grain varieties prone to aging deterioration during storage, causing huge economic losses. In order to control the rice ageing during storage effectively, the mechanism of ageing needs to be further elucidated. Rice ageing is a multi-component complex issue. This thesis focus on the improvement effects of protein changes on rice aging characteristics and its impact mechanisms, trying to reveal the mechanism of aging through the performing roles of protein modifiers and the effect of rice protein on the pasting properties.
1. Changes of rice quality during storage
Through studying the variation of the quality indicators of rice during storage, it is important to find an effective indicator of the rice aging to evaluate the degree of aging and safety of rice. Milled rice was sealed in airtight bottles stored at37℃for accelerated aging, periodically sampling was carried out to determine the changes in selected physical and chemical indicators. The following results were obtained:(1) Fatty acid value, gel consistency and alkali spreading value of rice in storage changed irregularly, while the total amylose, hot water soluble amylose and hot water insoluble amylose content did not change significantly.(2) Rice odor evaluation by electronic nose showed that the bad smell is much larger than the good smell in contribution to the total odor of the stored rice.(3) The parameter changes in three color space mode showed conformably that the internal color of rice grain than its surface color is more white during storage, which suggests storage yellowing in rice originate from the surface of contact with air.(4) The changes of RVA pasting characteristic parameters showed that the peak viscosity and breakdown increased from0to2nd month, decreased from2to8th month, setback declined from0to2nd month, rose from2to8th month, while pasting temperature increased from0to8th month. In addition, the original two-stage swelling of stored rice merged into a one-time swelling after4months, showing that the ageing causes a higher swelling temperature of the amorphous region, which should swell prior to occurrence of the crystallization region. Because that amorphous region composes mainly of amylase, protein and lipid, it is speculated that ageing led to amylose, protein and lipid changes in the nature. In summary, the RVA characteristic parameters appear to be the most sensitive, regular and provide the richest information to characterize the degree of rice aging during storage. So, RVA characteristic parameters on behalf of pasting properties were considered as indicators in the subsequent chapters of this thesis to study the changes of rice quality during storage.
2. Effect of pretreatment before storage on pasting properties of rice duration storage
The milled rice which was subjected to treatments before storage were accelerated to ageing at37℃to study the effects of changes in enzyme activity of Alpha-amylase and lipoxygenase, and in protein oxidation. The results showed that:(1) Alpha-amylase was not the main inducement of rice ageing, while the stabilization role of lipoxygenase by EDTA·2Na slowed the ageing of stored rice.(2) Among three reducing agents, Vc treatment before storage has the most important impact on rice aging. When the rice stored at37℃after4months, Vc as prooxidant accelerated rice aging, while tea polyphenols and DTT delayed rice aging during the storage period of4and8months, showing that rice aging was closely related to the oxidation process. Vc accelerated rice ageing duration storage of rice, and this finding has not been reported in the literature yet.
3. The improvement role and its mechanism of post treatment after storage on pasting properties of aged rice
In order to find a similar or even better additives compaer to DTT which is food additives and can improve the quality of aged rice, and at the same time, to infer rice ageing mechanism through the nature and function of quality improvement of aged rice, the effects of treatments on aged rice were investigated. The results followed that:(1) Sodium sulfate treatment showed that aging decreased the normal water absorption speed of rice.(2) DTT treatment before storage (Chapter3,2.2.1DTT treatment before storage at0month) with the fresh rice treated (treated in pasting process) has the same effect, suggesting that DTT play a role in the heated gelatinization process rather than in before pasting which react directly with compound in the rice. The increasing of starch granule swelling degree of aged rice by DTT treatment was very limited, yet Vc showed bigger enhancing role than DTT on the swelling of aged rice, even more swelling than the fresh rice as the control. In contrast, tea polyphenols only retards the cooling retrogradation of amylase. The facts indicate that the disulfide cross-linking is not the only major change when the rice aging occurs, there may be other force to prevent the starch granule swelling except for disulfide bonds, and Vc could destroy the main force of inhibiting aged rice from swelling, and showed a dose-effect relationship, confirming other aging mechanisms exist except the sulphydryl is oxidized to the disulfide bonds. The effect of SDS on aged rice showed that a large number of hydrogen bonds and hydrophobic interactions might be formed in rice ageing, and these forces inhibited gelatinization swelling of the starch granules.(3) the results of water absorption index, soluble solid and microscopic images confirmed that Vc simultaneously promotes water absorption and soluble solids, while DTT only promoted water absorption but inhibited soluble solid, and the role of Vc in promoting water absorption was much higher than that of DTT. This was different in mechanism of improving pasting properties between Vc and DTT, and also showed that water absorption of starch granule was more important than losses of the soluble solids on the pasting properties.(4) the effect of SH-blocking agent NEMI on fresh rice showed that the disulfide bond exists only in the swelling starch granules. The results of NEMI dealing with aged rice showed that aged rice in the gelatinization process could still form disulfide cross-linking which make the pasting viscosity increased, but the contribution of sulphydryl cross-linking on the viscosity of aged rice paste was very small, and that of swelling degree (Vc promoting swelling) was bigger. The comparison of treatment results between Vc separately and in combination with NEMI indicated that Vc prevented the formation of disulfide cross-linking of aged rice in gelatinization process.(5) the effect of Vc treatment on aged rice of accelerated ageing by Vc before storage showed that the Vc does have a dual function on rice ageing, which was that the treatment before storage on fresh rice accelerated aging and the treatment after storage on aged rice improved pasting properties. This once again showed the nature of the rice aging was associated with oxidative changes.
4. Effects of rice storage protein on pasting properties of aged rice
Removing protein from aged rice after storage were carried out to explore the effect of rice protein changes in ageing on pasting properties, and removing protein from fresh rice before storage were carried out to explore the combined effect of rice protein changes and interactions with other components in ageing process on pasting properties. The results were:(1) the changes of albumin and globulin in aging process promoted rice ageing, the changes of glutelin and prolamin in ageing process delayed rice ageing, while the changes of fat in ageing process have a dual role on rice ageing.(2) the combined effects of each component changes and the interactions with other components in ageing process were as that:fat and albumin promoted rice ageing, globulin and prolamin delayed rice ageing, while glutelin played dual role on rice ageing. Comparison of above mentioned imformation suggest that rice ageing is not caused by separated change of one or several components, but is more related to the interactions between the various components, especially the interactions between the globulin and other components, followed by the interactions between glutelin and fat and other ingredients.
5. Structural changes of rice storage protein in ageing process
The structural changes of the four rice storage protein and interactions between them and other components during ageing process were characterized using infrared and Raman spectroscopic techniques in this chapter. The results were following:(1) Infrared spectrum showed albumin may be glycoprotein, and albumin, no matter it was in fresh or aged rice, both presents sulfoxide peak. Raman spectrum showed that after ageing, the random coil in albumin reduced, the protein hydrophobic group buried, and the intermolecular hydrogen bonding decreased, probably suggesting that the associations between albumin and starch molecules become looser.(2) Infrared spectrum showed that sulphydryl oxidation of globulin from aged rice was more obvious than fresh rice with main products of sulfone and sulfoxide, and glycosylation trend of age rice globulin significantly increased duration storage. Raman spectrum indicated that random coil of the globulin increased and sulfoxide occurred after rice ageing, and the degree of intermolecular hydrogen bonding strengthened, suggesting that ageing promotes binding of globulin and starch molecules.(3) Infrared spectrum showed the glycosylation trend of age rice glutelin significantly decreased than that of fresh rice. Raman spectra displayed that the random coil in gluten reduced after aging and free sulfhydryl peaks appeared.(4) Infrared spectrum of prolamin after ageing have no obvious changes, Raman spectrum reveal anti-parallel β-sheet structure in aged rice prolamin increased, the conformation of disulfide bond changed, tyrosine residues changed embedded state into the exposed state. In summary, after aging, the interactions between globulin and starch presented the greatest change, followed by glutelin, while the interactions between albumin and prolamin and starch changed insignificantly. The interactions of globulin with starch molecules were strengthened, and the interactions between glutelin and starch molecules were weakened.
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