米渣蛋白酶解物的制备与微囊化特性及其对SD鼠生长性能的影响
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摘要
本论文以淀粉糖工业的主要副产物米渣为原料,采用可控生物酶法制备米蛋白的多肽混合物,然后以此多肽混合物为主要壁材组成成分建立其稳定的O/W乳化体系,制备出米肽壁材型微囊化调和油,并对所制得多肽混合物及其壁材型微囊化脂肪的营养效应进行研究,旨在探讨制备米蛋白的多肽混合物、米渣蛋白酶解物壁材型微囊化脂肪的技术,并对在断奶鼠肠组织形态和生长轴因子方面的营养效应进行评定,为米渣蛋白资源的综合开发利用提供技术指导和理论依据。
1.以蛋白质得率(YP)、水解度(DH)为指标,通过二次响应面旋转中心组合设计优化出胰蛋白酶的工艺条件：PH7.6、52.83℃、E/RD 0.89:1000 (m/m)、RD/水0.17：1(g/m1)、水解2.43h。在此条件下米渣蛋白水解度、蛋白质得率分别为6.94%、75.81%。水解度(DH)和蛋白质得率(YP)的预测模型分别为：
2.运用实验研究结合数学推导的方法,对最优参数下胰蛋白酶有限水解米渣蛋白的动力学机制进行了研究。结果表明：在反应过程中底物存在着促进反应速度和抑制酶活性的双重作用,酶催化的水解速率随水解进程呈指数下降；由实验数据推导出胰蛋白酶限制性水解米渣蛋白的水解速率(V)动力学模型和水解度(x)动力学模型分别为：
3.采用化学分析法、凝胶排阻色谱法对所制备米渣蛋白酶解物的常规养分、氨基酸组成,及肽分子量分布进行了分析。结果表明：米渣蛋白酶解物是一种主要由小肽(52.2%)和游离氨基酸(18.7%)组成的混合物；以FAO/WHO/UNU(1985)中鸡蛋、牛奶为参考蛋白,酶解物蛋白的EAAI分别为0.93和0.96,非必需氨基酸中Glu、Arg含量分别为180.61、90.61 mg/g。这些表明米渣蛋白酶解物是一种优质的蛋白质资源。
4.研究了干燥方法对米渣蛋白酶解物功能特性、理化特性和蛋白质分子二级结构的影响。结果表明：米渣蛋白酶解物在pH 2-12范围内的氮溶解指数>80%、乳化活性>30%、乳化稳定性>45%；喷干粉、冻干粉的表面疏水性指数分别为62.633和16.725,变性起始温度分别为88.96℃和98.64℃、峰值温度分别为107.3℃和99.53℃、热焓值分别为20.058 J/g和25.432 J/g；FTIR分析表明干燥方法对酶解物蛋白质分子酰胺I带二级结构影响较大,而对酰胺Ⅲ带二级结构影响较小,喷干粉酰胺I带p-折叠结构(23.68%)比冻干粉少16.38%、α-螺旋结构(8.33%)比冻干粉少1.48%、p-转角(20.33%)比冻干粉少6.13%,而无规卷曲结构(47.66%)比冻干粉多24.00%。这些表明,真空冷冻干燥有利于酶解物蛋白质分子稳定结构的形成,而喷雾干燥使酶解物蛋白质亚基进一步暴露。
5.采用喷雾干燥法研究了米渣蛋白酶解物对富含MCFA调和油的微胶囊化工艺。通过单因素和正交试验确定其稳定体系最优工艺条件为：芯材含量25%,壁材中米渣酶解物(RP)：麦芽糊精(MD)=5：5(m：m),乳化剂添加量3%、吐温-80：单甘酯：蔗糖酯=16：81：3(m：m：m),羧甲基纤维素添加量0.45%,黄原胶添加量1%,45℃乳化20 min,乳液固形物含量20%,10 MPa均质二次,喷雾干燥进风温度、出风温度分别为190℃、95℃。在此条件下,所制乳液稳定性好,乳液平均粒径为261 nm,微囊化效率达83.6%。微囊化工艺优化中,壁材组成对微囊化效率影响最大但不具有统计学显著性。
6.通过分析乳液粒度、流变学特性和乳液稳定性,比较研究了壁材组成中RP、MD、变性淀粉(MS)的不同配比对O/W乳液性质的影响,并对喷雾干燥所得的微胶囊产品进行电镜扫描分析。结果表明：不同壁材体系所配制乳液的粘度相差不大,但以RP：MD=5：4、4：5体系的乳液较稳定,其乳液平均粒径较小(分别为152.4、132.1 nm),体系表面张力最小(30.492、29.904 mN/m),60℃水浴30mmin后的稳定性分别为97.81%、98.15%,所得微胶囊产品颜色浅黄,粒径较小(10-20um),颗粒表面形态光滑、结构完整。
7.通过差示扫描热分析和Schaal烘箱加速储藏试验研究了壁材组成中RP、MD、MS的不同配比对微胶囊玻璃化转变特性、贮藏稳定性的影响。DSC结果表明：RP的添加可使玻璃化转变温度(Tg)降低；MD比例的增加使玻璃化转变时壁材组分间的相变协同性降低,使Tg值升高、相变热焓增加；MS比例的增加使玻璃化转变时壁材各组分间的相变协同性增强,相转变温度范围变窄,相变热焓降低。TG-DTG分析表明,调和油经微囊化后其分解温度油由270℃左右升高到>300℃。加速储藏试验中,MS：MD：RP=2：2：2、1：4:1、4：1：1壁材型微胶囊POV随储藏天数呈指数型上升,而其它含米渣蛋白酶解物相对较多的壁材型微胶囊POV有明显下降趋势,储藏23天时芯材油脂的自动氧化反应仍处于诱导期。
8.通过红外光谱分析研究了壁材蛋白质和芯材脂肪分子结构的变化。结果表明：喷雾干燥微囊化过程能使壁材中蛋白质分子的β-折叠结构解开向α-螺旋结构转变,同时伴随有无规卷曲结构的减少、β-转角结构的增加,壁材中较多量MD有利于减少酰胺I带无规卷曲结构的增加,而较多量MS有利于减少酰胺Ⅲ带无规卷曲结构的增加；加速储藏6个月后,壁材中RP较多时蛋白质分子结构较稳定,而壁材中碳水化合物较多时,蛋白质分子表现有向较不稳定二级结构转变的趋势,同时芯材分子不饱和双键断裂成C=O,不饱和酯含量有所减少。
9.以大豆蛋白日粮为对照,对米渣蛋白(RD、RD-H)饲喂断奶后SD鼠生长的影响及其相关作用机制进行了研究。与对照组相比,米蛋白有利于促进断奶后小肠组织的恢复、生长,提高日粮转化效率,使动物生长性能提高,且RD-H日粮的饲喂效果稍优于RD日粮。血液指标分析表明：尽管机体对RD-H日粮有较长的适应阶段,但与RD、SB日粮相比,RD-H日粮有利于改善和提高机体的蛋白质代谢状况；各组血清IgG浓度相近。RD、SB、RD-H曰粮对脏器发育程度的影响相当。real-time RT-PCR分析表明：RD-H组肝GHR mRNA的表达7d明显高于SB组、略高于RD组,肝(28d除外)、肌肉IGF-1 mRNA表达略高于RD、SB组；全期各组间肌肉GHR mRNA的表达差异很小。这些结果显示,与RD、SB相比,日粮中添加RD-H有利于改善断奶后动物的生长状态。
10.以21日龄SD鼠为动物模型,以常规断奶方式(28d断奶+大豆蛋白日粮)为对照,对米渣蛋白酶解物(RP)及其壁材型微胶囊脂肪(MF)的提前断奶效应及相关影响机制进行了研究。与对照组相比,断奶日粮中添加RD-H、MF能使断奶期提前,且生长性能不受影响。14d时MF日粮组血清UN水平明显低于RD-H、CK组(p<0.05),28d时各组间差异不明显；各处理组动物血清IL-2、Ig G浓度相当,血清Ins水平相近；14d时提前断奶组血清GH浓度明显高于常规断奶组(p<0.05),RD-H明显高于MF组(p<0.05)；但14d时RD-H日粮组皮质醇浓度明显高于CK组、MF组(p<0.05)。各处理间脏器发育水平相当。14d时,CK组肠组织形态仍处于受损后的恢复阶段。real-time RT-PCR分析表明,14d时RD-H组肝、肌肉IGF-1mRNA的表达水平稍高于MF组(p>0.05)、明显高于CK组(p<0.05),28d时RD-H、MF组肝中FAS mRNA表达料CK组明显上调(p<0.05)。这些结果表明,日粮中添加MF、RD-H日粮有利于提前断奶。
11.以大豆蛋白为对照,通过消化代谢试验对RD、RD-H、MF的营养效价进行了评定。结果表明：与大豆蛋白相比,米渣蛋白能显著提高SD鼠的体氮沉积、促进脂肪的消化(P<0.01)；豆粕(SB)、米渣(RD)、米渣蛋白酶解物(RD-H)、壁材型米渣蛋白酶解物(MF)四种日粮的氮沉积指数分别为0.538g/d、0.590g/d、0.605g/d、0.625g/d；米渣蛋白的体外低度酶解、脂肪的体外预乳化不影响机体内对蛋白、脂肪的吸收利用(P>0.05)。
Rice dregs, a major by-product of rice syrup production, was used to prepare peptides compound by controllabe enzymatic proteolysis method, and the peptides compound was successfully developed as a main wall-material in forming a stable O/W emulsion of a soybean oil/coconut oil blends, and then preparing into microencapsules by spray-drying method. The nutritional effects of peptides compound and microencapsules on small intestinal morphology and somatotrophic axis in Sprague-Dawley(SD) rats were evaluated in this paper, for the purpose of providing technical guide and theory basis for the comprehensive development of rice dregs.
1. The limited enzymatic proteolysis of RD was optimized by response surface method with rotatable central composite design and with the response value of degree of hydrolysis(DH) and the extracted yield of protein(YP), and the optimal condition for trypsin was:pH 7.61,52.83℃, trypsin to RD ratio 0.89:1000 (g/g), RD to water ratio 0.17:1(g/ml), and time 2.43h. Under the optimized condition,75.81% protein could be extracted and the hydrolysis degree of protein was 6.94%. The theoretically predictive models obtained by response surface analysis for DH and YP could be described as: Where Y1 and Y2 represent the degree of hydrolysis and protein extraction, x1 is the ratio of trypsin to RD(g/g), x2 is the ratio of RD to water ration(g/ml), x3, x4, and x5 represent pH, temperature(℃) and time(h), respectively.
2. The kinetic mechanism of limited enzymatic proteolysis for RD with trypsin in pH7.6 at 53℃was investigated by pH-stat method. The results showed that the limited proteolysis could be inhibited by the addition of RD while the active sites of trysine were fully binded with substract and soluble peptides, otherwise the hydrolysis rate could be accelerate by the addition of RD. Based on the experimental data, the kinetic models obtained by mathematic deduction method for hydrolysis rate and degree of hydrolysis could be described as:
3. The common chemical composition, amino acid compositon and peptides molecular weight distribution of the prepared RD hydrolysate were determined by chemical method and gel exclusion chromatography. The results showed that the hydrolysate was a mixture constituted by 52.2% oligopeptides and 18.7% free amino acids. Based on the reference proteins of egg and cow's milk(FAO/WHO/UNU, 1985), the calculated EAAIs for protein of RD hydrolysate were 0.93 and 0.96, respectively. The Glu and Arg contents in the protein of RD hydrolysate were 180.61、90.61 mg/g, respectively. These results demonstrated that the prepared RD hydrolysate could be a good protrein resource with high quality.
4. The effects of spray-drying and freeze-drying processes on the physical and functional properties and protein secondary structure of prepared hydrolysates were investigated. The results showed the nitrogen solubility, emulsifying activities and emulsion stabilities have been remarkably improved after limited hydrolysis, and the surface hydrophobicities and thermal properties of hydrolysate could be significantly effected by drying process. The surface hydrophobicities (pH 7.0) for spray-dried and freeze-dried hydrolysates were 62.633 and 16.725, respectively. The denaturation temperatures and enthalpies of spray-dried and freeze-dried hydrolysates were 98.64 and 88.96℃,25.432 and 20.058 J/g, respectively. FTIR analysis showed that the secondary structure of Amide I band could be obviously influenced, while Amide III band was not significantly influenced by drying process. The main sencondary structure in Amide I band of spray-dried hydrolysate was random coils (47.66%, which was more than freeze-dried hydrolysate by 24.00%), while that of freeze-dried hydrolysate wasβ-sheets(40.06%, which was more than spray-dried hydrolysate by 16.38%). These results indicated that the protein molecule structure could be further stabilized during the freeze drying process and more protein subunits bared during the spray drying process for RD protein hydrolysate.
5. To develop the RD protein hydrolysate as a nutritional wall material, the spray-drying method was used in microencapsulating process of the blend oils rich in MCFA. The emulsion system and the spray-drying process parameters were determined through the single factor experiment and orthogonal test. The microencapsulated product was constituted by 25%blend oils as a core materials, whlie the protein hydrolysate and maltdextrin in a ratio of 5:5(w/w) as the wall material. The stable emulsion system was constituted by 3% emulsifers (Tween-80, glyceryl monostearate and sucrose ester being 16:81:3,w/w/w), plus 0.45%CMC and 1% xanthan gum. Afer emulsifying 20 min. at 45℃and double-homeginization with a pressure of 10 MPa, the emultion was spray-dried with inlet and outlet temperatures of 190 and 95℃, respectively. Under the optimumal conditions, the prepared emulsion possessed a good stability and an average granularity of 261 nm, and the final product has a high microencapsulated efficiency up to 83.6%.
6. The droplet size distribution, rheological properties, emulsion stababilities and morphology of microcapsules were measured To investigate the influence of wall material compositon on emulsion properties. Results showed that all emulsions exhibited similar viscosities, but emulsions with shell composition of RP:MD=5:4 and 4:5(m/m) were found to be more stable (97.81% and 98.15%, respectively) after water bathing at 60℃for 30 min, and smaller in droplet mean diameter (152.4 and 132.1 nm, respectively) and weaker in surface tension(30.492 and 29.904 mN/m, respectively), and possesed smoother surface morphology and more intact shell, as compared to those with other wall material compositions.
7. The glass transition properties of microencapsulates were characterized by differential scanning calorimetry(DSC) and their oxidative stabilites was evaluated basing on the peroxide values(POVs) by Schaal oven accelerated storage test. The wall material was based on RP and 16.7-77.8% MD and/or MS replacement of RP. Results showed that the glass transition temperature (Tg) of microencapsulate were lowered by the increase of RP but heightened by the addition of MD and/or MS, the glass transition enthalpies of microencapsulates could be generally decreased by increasing MS content but increased with increasing MD proportion, and the glass transition cooperativity of shell constituents could be enhanced by the addition of MS. TG-DTG analysis results showed that the degradation temperature of blend oils was retarded from 270℃to above 300℃. During the accelerated storage period, oxidative stabilites of the core oils were exponentially decreased for the encapsulates with the shell compositions of MS:MD:RP=2:2:2,1:4:1 and 4:1:1, while the oxidative stabilites of the encapsulates with a higher portions of RP hydrolysate as shell compositons were gradually improved. The POVs of the encapsulates were kept in the induction stage after 7 days'storage period.
8. The secondary structure of the protein and molecule structure of oils in the core of encapsulates were characterized by optical information of fourier transform infrared spectroscopy(FTIR). Results showed that the secondary structure of protein exhibited a significant change tendency from P-sheets to a-helixes during the spray drying process, accompanied by a decrease of random coils and an increase inβ-turns. However, the conversion into random coils in amide I band could be attenuated by the increasing MD content in shell, and that in amide III band could be reduced by the increasing MS content in shell. After 6 months'storage period at 60℃, the molecule structure of protein was more stable in the shell with a relative higher portion of RP, the stable secondary struture of protein tended into less stable structures in the shell with a relative higher content of carbohydrate, meanwhile, the unsaturated double bonds of core oils have been broken down, and turned into C=O, which was contributed to the decreased proportion of unsaturated fatty acids in the core oils.
9. Sixty Sprague-Dawley rats(3weeks old) in 3 groups were used to evaluate the effects of protein from RD(RD) or its hydrolate (RD-H) on growth performance, blood chemical compositions, serum hormone, small intestinal morphology, and somatotrophic axis, with one group on soybean protein(SB) diet as a control. Compared with the control, the recovery and growth of impaired villus in the small intestine could be ameliorated by rice proteins that from RD or RD-H diets, and the feed conversion efficiency and growth performance could also be improved by rice proteins that from RD or RD-H diets. Blood analysis indicated that the protein metabolim status of SD rat body could be improved by RD-H diet, athought the body was still in the adaptive phase to the RD-H diet, as compared to RD and SB diets. Serum IgG concentration has no significant difference among three groups. The growth levels of organs were not influenced by the diets with addition of RD or RD-H. Real-time RT-PCR analysis showed that, the hepatic expression level of GHR mRNA in SD rats fed with RD-H diet was higher than those fed with RD or SB diets for 7 d, the hepatic (except for 28 d) and muscular expression of IGF-1 mRNA in SD rats were enhanced by RD-H diet as compared to RD or SB diets for 7 d and 28 d, and the muscular expressiong of GHR mRNA was not different among the three groups throughout the experiment. These results suggested that diet with addition of RD-H could be helpful for the growth status improvement of post-weaned SD rats.
10. Sixty Sprague-Dawley rats(3weeks old) in 3 groups were used to evaluate the effects of early-weaned diets with additon of RD-H or RD-H as microencapsulate shell materials on growth status, small intestinal morphology and somatotrophic axis, with one group weaned on SB diet on 28 d as a control. Results showed that the growth performances of rats early-weaned on RD-H or MF diets on 21 d were not lower than those in the control. The level of serum UN in rats fed on RD diet was lower than those both of RD-H group and the control for 14 d(p<0.05), and there were no significant difference among three groups for 28 d. The serum concentrations of IL-2, IgG and Insulin were similar to each other among three groups. The serum GH concentrations of rats in early-weaned groups were significantly higher than that of the control (p<0.05) for 14 d. Though their bodies still in the stress state, the serum GH concentration of rats fed on RD-H diet was remarkably higher than that in MF group (p<0.05). The growth levels of organs were also similar among three.groups. For the control, the small intestine morphosis was still in the recovery stage on 14 d. Real-time RT-PCR analysis showed that the hepatic and muscular expression of IGF-1 mRNA of SD rats fed with RD-H diet was slightly higher than that in MF group (p>0.05), but significantly higher than that in the control (p<0.05) for 14 d. The hepatic expression of FAS mRNA of SD rats fed with RD-H or MF diet was significantly higher than that in the control for 28 d (p<0.05). These results indicated that diets of MF or RD-H could put the weaning age of SD rats forward 7 days.
11. A 28-day metabolic test was conducted with 4×4 Latin square design and tweenty Sprague-Dawley rats(28d old) were used to evaluate the nutrition values of proteins from RD, RD-H or MF, with a control of soybean protein. Compared with the control, the nitrogen retention in body and the digestion and absorption of dietary fat were significantly improved by rice proteins from RD, RD-H or MF diets(p<0.01), and the average daily nitrogen depositions for SB, RD, RD-H and MF diets were 0.538,0.590, 0.605 and 0.625g, respectively. The digestion and absorption of dietary protein and fat were not influenced by the limited proteolysis of RD or emulsifying process of oil in vitro(P>0.05).
1.以蛋白质得率(YP)、水解度(DH)为指标,通过二次响应面旋转中心组合设计优化出胰蛋白酶的工艺条件：PH7.6、52.83℃、E/RD 0.89:1000 (m/m)、RD/水0.17：1(g/m1)、水解2.43h。在此条件下米渣蛋白水解度、蛋白质得率分别为6.94%、75.81%。水解度(DH)和蛋白质得率(YP)的预测模型分别为：
2.运用实验研究结合数学推导的方法,对最优参数下胰蛋白酶有限水解米渣蛋白的动力学机制进行了研究。结果表明：在反应过程中底物存在着促进反应速度和抑制酶活性的双重作用,酶催化的水解速率随水解进程呈指数下降；由实验数据推导出胰蛋白酶限制性水解米渣蛋白的水解速率(V)动力学模型和水解度(x)动力学模型分别为：
3.采用化学分析法、凝胶排阻色谱法对所制备米渣蛋白酶解物的常规养分、氨基酸组成,及肽分子量分布进行了分析。结果表明：米渣蛋白酶解物是一种主要由小肽(52.2%)和游离氨基酸(18.7%)组成的混合物；以FAO/WHO/UNU(1985)中鸡蛋、牛奶为参考蛋白,酶解物蛋白的EAAI分别为0.93和0.96,非必需氨基酸中Glu、Arg含量分别为180.61、90.61 mg/g。这些表明米渣蛋白酶解物是一种优质的蛋白质资源。
4.研究了干燥方法对米渣蛋白酶解物功能特性、理化特性和蛋白质分子二级结构的影响。结果表明：米渣蛋白酶解物在pH 2-12范围内的氮溶解指数>80%、乳化活性>30%、乳化稳定性>45%；喷干粉、冻干粉的表面疏水性指数分别为62.633和16.725,变性起始温度分别为88.96℃和98.64℃、峰值温度分别为107.3℃和99.53℃、热焓值分别为20.058 J/g和25.432 J/g；FTIR分析表明干燥方法对酶解物蛋白质分子酰胺I带二级结构影响较大,而对酰胺Ⅲ带二级结构影响较小,喷干粉酰胺I带p-折叠结构(23.68%)比冻干粉少16.38%、α-螺旋结构(8.33%)比冻干粉少1.48%、p-转角(20.33%)比冻干粉少6.13%,而无规卷曲结构(47.66%)比冻干粉多24.00%。这些表明,真空冷冻干燥有利于酶解物蛋白质分子稳定结构的形成,而喷雾干燥使酶解物蛋白质亚基进一步暴露。
5.采用喷雾干燥法研究了米渣蛋白酶解物对富含MCFA调和油的微胶囊化工艺。通过单因素和正交试验确定其稳定体系最优工艺条件为：芯材含量25%,壁材中米渣酶解物(RP)：麦芽糊精(MD)=5：5(m：m),乳化剂添加量3%、吐温-80：单甘酯：蔗糖酯=16：81：3(m：m：m),羧甲基纤维素添加量0.45%,黄原胶添加量1%,45℃乳化20 min,乳液固形物含量20%,10 MPa均质二次,喷雾干燥进风温度、出风温度分别为190℃、95℃。在此条件下,所制乳液稳定性好,乳液平均粒径为261 nm,微囊化效率达83.6%。微囊化工艺优化中,壁材组成对微囊化效率影响最大但不具有统计学显著性。
6.通过分析乳液粒度、流变学特性和乳液稳定性,比较研究了壁材组成中RP、MD、变性淀粉(MS)的不同配比对O/W乳液性质的影响,并对喷雾干燥所得的微胶囊产品进行电镜扫描分析。结果表明：不同壁材体系所配制乳液的粘度相差不大,但以RP：MD=5：4、4：5体系的乳液较稳定,其乳液平均粒径较小(分别为152.4、132.1 nm),体系表面张力最小(30.492、29.904 mN/m),60℃水浴30mmin后的稳定性分别为97.81%、98.15%,所得微胶囊产品颜色浅黄,粒径较小(10-20um),颗粒表面形态光滑、结构完整。
7.通过差示扫描热分析和Schaal烘箱加速储藏试验研究了壁材组成中RP、MD、MS的不同配比对微胶囊玻璃化转变特性、贮藏稳定性的影响。DSC结果表明：RP的添加可使玻璃化转变温度(Tg)降低；MD比例的增加使玻璃化转变时壁材组分间的相变协同性降低,使Tg值升高、相变热焓增加；MS比例的增加使玻璃化转变时壁材各组分间的相变协同性增强,相转变温度范围变窄,相变热焓降低。TG-DTG分析表明,调和油经微囊化后其分解温度油由270℃左右升高到>300℃。加速储藏试验中,MS：MD：RP=2：2：2、1：4:1、4：1：1壁材型微胶囊POV随储藏天数呈指数型上升,而其它含米渣蛋白酶解物相对较多的壁材型微胶囊POV有明显下降趋势,储藏23天时芯材油脂的自动氧化反应仍处于诱导期。
8.通过红外光谱分析研究了壁材蛋白质和芯材脂肪分子结构的变化。结果表明：喷雾干燥微囊化过程能使壁材中蛋白质分子的β-折叠结构解开向α-螺旋结构转变,同时伴随有无规卷曲结构的减少、β-转角结构的增加,壁材中较多量MD有利于减少酰胺I带无规卷曲结构的增加,而较多量MS有利于减少酰胺Ⅲ带无规卷曲结构的增加；加速储藏6个月后,壁材中RP较多时蛋白质分子结构较稳定,而壁材中碳水化合物较多时,蛋白质分子表现有向较不稳定二级结构转变的趋势,同时芯材分子不饱和双键断裂成C=O,不饱和酯含量有所减少。
9.以大豆蛋白日粮为对照,对米渣蛋白(RD、RD-H)饲喂断奶后SD鼠生长的影响及其相关作用机制进行了研究。与对照组相比,米蛋白有利于促进断奶后小肠组织的恢复、生长,提高日粮转化效率,使动物生长性能提高,且RD-H日粮的饲喂效果稍优于RD日粮。血液指标分析表明：尽管机体对RD-H日粮有较长的适应阶段,但与RD、SB日粮相比,RD-H日粮有利于改善和提高机体的蛋白质代谢状况；各组血清IgG浓度相近。RD、SB、RD-H曰粮对脏器发育程度的影响相当。real-time RT-PCR分析表明：RD-H组肝GHR mRNA的表达7d明显高于SB组、略高于RD组,肝(28d除外)、肌肉IGF-1 mRNA表达略高于RD、SB组；全期各组间肌肉GHR mRNA的表达差异很小。这些结果显示,与RD、SB相比,日粮中添加RD-H有利于改善断奶后动物的生长状态。
10.以21日龄SD鼠为动物模型,以常规断奶方式(28d断奶+大豆蛋白日粮)为对照,对米渣蛋白酶解物(RP)及其壁材型微胶囊脂肪(MF)的提前断奶效应及相关影响机制进行了研究。与对照组相比,断奶日粮中添加RD-H、MF能使断奶期提前,且生长性能不受影响。14d时MF日粮组血清UN水平明显低于RD-H、CK组(p<0.05),28d时各组间差异不明显；各处理组动物血清IL-2、Ig G浓度相当,血清Ins水平相近；14d时提前断奶组血清GH浓度明显高于常规断奶组(p<0.05),RD-H明显高于MF组(p<0.05)；但14d时RD-H日粮组皮质醇浓度明显高于CK组、MF组(p<0.05)。各处理间脏器发育水平相当。14d时,CK组肠组织形态仍处于受损后的恢复阶段。real-time RT-PCR分析表明,14d时RD-H组肝、肌肉IGF-1mRNA的表达水平稍高于MF组(p>0.05)、明显高于CK组(p<0.05),28d时RD-H、MF组肝中FAS mRNA表达料CK组明显上调(p<0.05)。这些结果表明,日粮中添加MF、RD-H日粮有利于提前断奶。
11.以大豆蛋白为对照,通过消化代谢试验对RD、RD-H、MF的营养效价进行了评定。结果表明：与大豆蛋白相比,米渣蛋白能显著提高SD鼠的体氮沉积、促进脂肪的消化(P<0.01)；豆粕(SB)、米渣(RD)、米渣蛋白酶解物(RD-H)、壁材型米渣蛋白酶解物(MF)四种日粮的氮沉积指数分别为0.538g/d、0.590g/d、0.605g/d、0.625g/d；米渣蛋白的体外低度酶解、脂肪的体外预乳化不影响机体内对蛋白、脂肪的吸收利用(P>0.05)。
Rice dregs, a major by-product of rice syrup production, was used to prepare peptides compound by controllabe enzymatic proteolysis method, and the peptides compound was successfully developed as a main wall-material in forming a stable O/W emulsion of a soybean oil/coconut oil blends, and then preparing into microencapsules by spray-drying method. The nutritional effects of peptides compound and microencapsules on small intestinal morphology and somatotrophic axis in Sprague-Dawley(SD) rats were evaluated in this paper, for the purpose of providing technical guide and theory basis for the comprehensive development of rice dregs.
1. The limited enzymatic proteolysis of RD was optimized by response surface method with rotatable central composite design and with the response value of degree of hydrolysis(DH) and the extracted yield of protein(YP), and the optimal condition for trypsin was:pH 7.61,52.83℃, trypsin to RD ratio 0.89:1000 (g/g), RD to water ratio 0.17:1(g/ml), and time 2.43h. Under the optimized condition,75.81% protein could be extracted and the hydrolysis degree of protein was 6.94%. The theoretically predictive models obtained by response surface analysis for DH and YP could be described as: Where Y1 and Y2 represent the degree of hydrolysis and protein extraction, x1 is the ratio of trypsin to RD(g/g), x2 is the ratio of RD to water ration(g/ml), x3, x4, and x5 represent pH, temperature(℃) and time(h), respectively.
2. The kinetic mechanism of limited enzymatic proteolysis for RD with trypsin in pH7.6 at 53℃was investigated by pH-stat method. The results showed that the limited proteolysis could be inhibited by the addition of RD while the active sites of trysine were fully binded with substract and soluble peptides, otherwise the hydrolysis rate could be accelerate by the addition of RD. Based on the experimental data, the kinetic models obtained by mathematic deduction method for hydrolysis rate and degree of hydrolysis could be described as:
3. The common chemical composition, amino acid compositon and peptides molecular weight distribution of the prepared RD hydrolysate were determined by chemical method and gel exclusion chromatography. The results showed that the hydrolysate was a mixture constituted by 52.2% oligopeptides and 18.7% free amino acids. Based on the reference proteins of egg and cow's milk(FAO/WHO/UNU, 1985), the calculated EAAIs for protein of RD hydrolysate were 0.93 and 0.96, respectively. The Glu and Arg contents in the protein of RD hydrolysate were 180.61、90.61 mg/g, respectively. These results demonstrated that the prepared RD hydrolysate could be a good protrein resource with high quality.
4. The effects of spray-drying and freeze-drying processes on the physical and functional properties and protein secondary structure of prepared hydrolysates were investigated. The results showed the nitrogen solubility, emulsifying activities and emulsion stabilities have been remarkably improved after limited hydrolysis, and the surface hydrophobicities and thermal properties of hydrolysate could be significantly effected by drying process. The surface hydrophobicities (pH 7.0) for spray-dried and freeze-dried hydrolysates were 62.633 and 16.725, respectively. The denaturation temperatures and enthalpies of spray-dried and freeze-dried hydrolysates were 98.64 and 88.96℃,25.432 and 20.058 J/g, respectively. FTIR analysis showed that the secondary structure of Amide I band could be obviously influenced, while Amide III band was not significantly influenced by drying process. The main sencondary structure in Amide I band of spray-dried hydrolysate was random coils (47.66%, which was more than freeze-dried hydrolysate by 24.00%), while that of freeze-dried hydrolysate wasβ-sheets(40.06%, which was more than spray-dried hydrolysate by 16.38%). These results indicated that the protein molecule structure could be further stabilized during the freeze drying process and more protein subunits bared during the spray drying process for RD protein hydrolysate.
5. To develop the RD protein hydrolysate as a nutritional wall material, the spray-drying method was used in microencapsulating process of the blend oils rich in MCFA. The emulsion system and the spray-drying process parameters were determined through the single factor experiment and orthogonal test. The microencapsulated product was constituted by 25%blend oils as a core materials, whlie the protein hydrolysate and maltdextrin in a ratio of 5:5(w/w) as the wall material. The stable emulsion system was constituted by 3% emulsifers (Tween-80, glyceryl monostearate and sucrose ester being 16:81:3,w/w/w), plus 0.45%CMC and 1% xanthan gum. Afer emulsifying 20 min. at 45℃and double-homeginization with a pressure of 10 MPa, the emultion was spray-dried with inlet and outlet temperatures of 190 and 95℃, respectively. Under the optimumal conditions, the prepared emulsion possessed a good stability and an average granularity of 261 nm, and the final product has a high microencapsulated efficiency up to 83.6%.
6. The droplet size distribution, rheological properties, emulsion stababilities and morphology of microcapsules were measured To investigate the influence of wall material compositon on emulsion properties. Results showed that all emulsions exhibited similar viscosities, but emulsions with shell composition of RP:MD=5:4 and 4:5(m/m) were found to be more stable (97.81% and 98.15%, respectively) after water bathing at 60℃for 30 min, and smaller in droplet mean diameter (152.4 and 132.1 nm, respectively) and weaker in surface tension(30.492 and 29.904 mN/m, respectively), and possesed smoother surface morphology and more intact shell, as compared to those with other wall material compositions.
7. The glass transition properties of microencapsulates were characterized by differential scanning calorimetry(DSC) and their oxidative stabilites was evaluated basing on the peroxide values(POVs) by Schaal oven accelerated storage test. The wall material was based on RP and 16.7-77.8% MD and/or MS replacement of RP. Results showed that the glass transition temperature (Tg) of microencapsulate were lowered by the increase of RP but heightened by the addition of MD and/or MS, the glass transition enthalpies of microencapsulates could be generally decreased by increasing MS content but increased with increasing MD proportion, and the glass transition cooperativity of shell constituents could be enhanced by the addition of MS. TG-DTG analysis results showed that the degradation temperature of blend oils was retarded from 270℃to above 300℃. During the accelerated storage period, oxidative stabilites of the core oils were exponentially decreased for the encapsulates with the shell compositions of MS:MD:RP=2:2:2,1:4:1 and 4:1:1, while the oxidative stabilites of the encapsulates with a higher portions of RP hydrolysate as shell compositons were gradually improved. The POVs of the encapsulates were kept in the induction stage after 7 days'storage period.
8. The secondary structure of the protein and molecule structure of oils in the core of encapsulates were characterized by optical information of fourier transform infrared spectroscopy(FTIR). Results showed that the secondary structure of protein exhibited a significant change tendency from P-sheets to a-helixes during the spray drying process, accompanied by a decrease of random coils and an increase inβ-turns. However, the conversion into random coils in amide I band could be attenuated by the increasing MD content in shell, and that in amide III band could be reduced by the increasing MS content in shell. After 6 months'storage period at 60℃, the molecule structure of protein was more stable in the shell with a relative higher portion of RP, the stable secondary struture of protein tended into less stable structures in the shell with a relative higher content of carbohydrate, meanwhile, the unsaturated double bonds of core oils have been broken down, and turned into C=O, which was contributed to the decreased proportion of unsaturated fatty acids in the core oils.
9. Sixty Sprague-Dawley rats(3weeks old) in 3 groups were used to evaluate the effects of protein from RD(RD) or its hydrolate (RD-H) on growth performance, blood chemical compositions, serum hormone, small intestinal morphology, and somatotrophic axis, with one group on soybean protein(SB) diet as a control. Compared with the control, the recovery and growth of impaired villus in the small intestine could be ameliorated by rice proteins that from RD or RD-H diets, and the feed conversion efficiency and growth performance could also be improved by rice proteins that from RD or RD-H diets. Blood analysis indicated that the protein metabolim status of SD rat body could be improved by RD-H diet, athought the body was still in the adaptive phase to the RD-H diet, as compared to RD and SB diets. Serum IgG concentration has no significant difference among three groups. The growth levels of organs were not influenced by the diets with addition of RD or RD-H. Real-time RT-PCR analysis showed that, the hepatic expression level of GHR mRNA in SD rats fed with RD-H diet was higher than those fed with RD or SB diets for 7 d, the hepatic (except for 28 d) and muscular expression of IGF-1 mRNA in SD rats were enhanced by RD-H diet as compared to RD or SB diets for 7 d and 28 d, and the muscular expressiong of GHR mRNA was not different among the three groups throughout the experiment. These results suggested that diet with addition of RD-H could be helpful for the growth status improvement of post-weaned SD rats.
10. Sixty Sprague-Dawley rats(3weeks old) in 3 groups were used to evaluate the effects of early-weaned diets with additon of RD-H or RD-H as microencapsulate shell materials on growth status, small intestinal morphology and somatotrophic axis, with one group weaned on SB diet on 28 d as a control. Results showed that the growth performances of rats early-weaned on RD-H or MF diets on 21 d were not lower than those in the control. The level of serum UN in rats fed on RD diet was lower than those both of RD-H group and the control for 14 d(p<0.05), and there were no significant difference among three groups for 28 d. The serum concentrations of IL-2, IgG and Insulin were similar to each other among three groups. The serum GH concentrations of rats in early-weaned groups were significantly higher than that of the control (p<0.05) for 14 d. Though their bodies still in the stress state, the serum GH concentration of rats fed on RD-H diet was remarkably higher than that in MF group (p<0.05). The growth levels of organs were also similar among three.groups. For the control, the small intestine morphosis was still in the recovery stage on 14 d. Real-time RT-PCR analysis showed that the hepatic and muscular expression of IGF-1 mRNA of SD rats fed with RD-H diet was slightly higher than that in MF group (p>0.05), but significantly higher than that in the control (p<0.05) for 14 d. The hepatic expression of FAS mRNA of SD rats fed with RD-H or MF diet was significantly higher than that in the control for 28 d (p<0.05). These results indicated that diets of MF or RD-H could put the weaning age of SD rats forward 7 days.
11. A 28-day metabolic test was conducted with 4×4 Latin square design and tweenty Sprague-Dawley rats(28d old) were used to evaluate the nutrition values of proteins from RD, RD-H or MF, with a control of soybean protein. Compared with the control, the nitrogen retention in body and the digestion and absorption of dietary fat were significantly improved by rice proteins from RD, RD-H or MF diets(p<0.01), and the average daily nitrogen depositions for SB, RD, RD-H and MF diets were 0.538,0.590, 0.605 and 0.625g, respectively. The digestion and absorption of dietary protein and fat were not influenced by the limited proteolysis of RD or emulsifying process of oil in vitro(P>0.05).
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