我国薏苡仁资源主要品质及薏苡仁活性多糖的研究
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摘要
本研究采集我国具有代表性区域分布的12个薏苡主产区的13种薏苡仁资源(其分布范围为北纬20°～48°),测定、分析其营养、保健与加工品质:并在此研究的基础上,以我省具有高直链淀粉特性的仙游金沙薏苡仁为原料,较为系统、全面地研究了其重要功能性成分——薏苡仁活性多糖(Coxian)的提取、分离与纯化工艺;探讨了Coxian的理化性质与分子结构特性、功能学特性。主要研究结果如下:
(1)我国薏苡仁资源主要品质的研究
薏苡仁资源营养品质研究表明,薏苡仁营养较为齐全,13种薏苡仁营养成分的平均值为:蛋白质15.80%,脂肪5.49%,均高于米、面;碳水化合物63.05%,低于米、面;粗纤维和灰分分别为1.75%与1.68%。薏苡仁的Ca含量丰富,高于米、面;VB_1、VB_2、VB_6、Fe、Zn、Se平均含量与米、面相当。薏苡仁的谷氨酸(Glu)、亮氨酸(Leu)、含硫氨基酸(Met+Cys)平均含量丰富,均高于米、面;但赖氨酸(Lys)含量较低。以氨基酸评分(AAS)为标准,13个样品中,Lys为第一限制性氨基酸,第二限制性氨基酸随产地不同而异,云南产等8个样品为苏氨酸(Thy),四川产等5个样品为异亮氨酸(Ile),贵州产为缬氨酸(Val)。平均必需氨基酸与总氨基酸比值(EAA/TAA)、必需氨基酸与非必需氨基酸比值(EAA/NEA)值分别为35.86%、55.91%,平均必需氨基酸指数(EAAI)值58.91%。与FAO/WHO模式相比,薏苡仁蛋白质具有较好的氨基酸组成,属于较为优质的蛋白质。
保健与加工品质检测结果显示:薏苡仁是优良的药食同源经济作物,其含有丰富的薏苡仁酯、薏苡仁活性多糖(Coxian)、三萜类化合物,其平均值分别为:44.60 mg/g、59.03mg/g、22.83mg/g;薏苡仁属高淀粉质食物,其平均淀粉含量为56.42%,直链淀粉为4.78%。不同产地薏苡仁的保健、加工品质特性与品种、产地密切相关。
该研究填补了我国有关薏苡仁品质研究的不足,并为薏苡仁资源的合理开发利用提供重要的参考价值。
(2)薏苡仁活性多糖浸提动力学研究
采用热水浸提工艺,探讨了浸提温度(T)、料液比(R)、浸提液pH值对浸提过程中Coxian浓度变化影响的规律;通过对影响因素的研究,建立了浸提过程中Coxian饱和浓度C~*和饱和常数K_(sat)的经验溶解扩散模型。
①在本研究条件下,T、R及浸提液pH值对浸提过程中Coxian饱和浓度C~*和饱和常数K_(sat)的影响极显著(p＜0.01)。
②通过二次通用旋转回归试验程序拟合得到T、R、pH值分别与C~*、K(sat)的回归模型为:C~*×10~3=47.2-1.17R-3.70T+75.38pH+0.054R~2+0.050T~2-6.38pH~2(A_(OD))K_(sat)×10~3=-1.13+5.27R+17.14T+222.4pH-0.027R~2-0.1T~2-12.73pH~2+0.017RT-0.1.7R pH(1/h)
两回归方程失拟不显著,回归显著(p＜0.01),可用于预测和控制Coxian在浸提过程中的溶解扩散规律。
(3)薏苡仁活性多糖脱蛋白工艺的优化
酶解法对Coxian脱蛋白的效果较Sevag法、三氯乙酸法的好;采用通用旋转组合试验设计方案,获得Coxian酶法脱蛋白的动力学方程,并优化其工艺参数组合:最佳的酶解温度、酶解液的pH值为0水平,即X_1=45℃;X_2=7.0;木瓜蛋白酶的适宜添加量X_3=5000μ/g:在此条件下,Coxian的蛋白脱除率最高,为80.12%;此外,在酶解脱蛋白基础上,结合一次的Sevag法,即可将Coxian中的杂质蛋白基本脱除。
(4)薏苡仁活性多糖理化性质与分子结构的研究
经Sephadex G-75凝胶色谱分离、纯化的Coixan,为乳白色絮状粉末固体,属水溶性、酸偏中性多糖,pH6.8;Coixan在100℃范围内的耐热稳定性较好;在常规食品加工pH范围内,Coixan的耐酸稳定性比耐碱稳定性好,但强酸碱条件下,则酸性条件对其影响更大。Coixan与硫酸—苯酚反应呈棕褐色,与蒽酮反应呈暗绿色;与I_2-KI、FeCl_3、硫酸一咔唑、斐林试剂、考马斯亮蓝反应均呈阴性。经HPGPC与紫外光谱进行分析,鉴定Coixan为单一级分,且为不含有核酸、蛋白质的纯多糖化合物;确定其分子量为2993D;比旋光度「a」~(20)_D=+155°(0.151,H2O),特性粘度[n]=0.313。
采用XRD、IR、NMR、AFM解析分子结构与形貌。XRD图谱显示,Coxian的分子颗粒属非晶态:IR及~1H-NMR分析数据表明,Coxian为非硫酸的杂聚多糖,分子中含有乙酰氨基;其构成单糖可能包括有α-D-吡喃木糖、α-D-吡喃葡萄糖、β-D-吡喃半乳糖、β-D-吡喃甘露糖、β-L-吡喃阿拉伯糖;Coxian糖苷链中存在β构型糖苷键。AFM的分子形貌图像提示,Coixan分子在溶液中具有良好的分散性能,其分子链在水溶液呈规则圆球状(火焰状)突起的螺旋或线性结构,直径约为40.0nm-50.0nm,高约为0.880nm-1.551nm。
(5)薏苡仁活性多糖的体外抗氧化活性
分别以Coxian纯品和粗品为受试物,进行自由基的清除和抑制脂质过氧化实验。利用Fenton反应体系、邻苯三酚自氧化体系、DPPH·体系与FRAP法分别检测Coxian对·OH、O_2~-·和DPPH·的清除效果与还原能力;以Ferrozine为Fe~(2+)的光度试剂,观察Coxian螯合金属离子的能力。结果表明:Coxian是良好的质子(H·)供体,具有清除多种自由基的性能,同时还具备了一定的金属螯合能力,并且以Coxian纯品对O_2~-·的清除效果最为显著。提示Coxian是一种多效的自由基和金属离子的抑制剂,具有研制开发成抗自由基保健食品的良好前景。
(6)薏苡仁活性多糖抗氧化及免疫调节的动物试验
以ICR清洁级小鼠为研究对象,腹腔注射CCl_4造模。以V_E为阳性对照,给药组(Coxian纯品低、中、高剂量(250mg/kg、500mg/kg、1000mg/kg)和Coxian粗品低、中、高剂量(250mg/kg、500mg/kg、1000mg/kg)分别先经灌胃28d进行预防。以全血SOD与GSH-Px活性、GSH浓度、血清T-AOC(总抗氧化能力)反映小鼠抗氧化功能的改变;观察肝脏GOT、GPT活力单位、MDA(丙二醛)含量和肝脏指数变化。结果表明:与模型组相比,Coxian可显著增强小鼠SOD、GSH-Px活性,有效防止CCl_4对肝脏组织的伤害,明显抑制肝细胞GOT、GPT水平与MDA含量的异常上升。其效果均呈现明显的量-效关系,且以Coxian纯品的效果为好。
环磷酰胺建立免疫抑制小鼠模型,以生理盐水为正常对照,采用Coxian纯品对小鼠免疫器官指数、半数溶血值(HC_(50))、抗体生成细胞、腹腔巨噬细胞吞噬指数及淋巴细胞增殖反应的调节效果试验,观察其对免疫系统的影响。结果显示:Coxian能显著抑制免疫功能低下小鼠的脾赃指数和胸腺指数的缩小;增强巨噬细胞吞噬指数及淋巴细胞增殖反应;提高血清HC_(50),纠正免疫功能紊乱现象。
动物试验证明Coxian能显著增强小鼠的抗氧化功能,有效解除CCl_4肝中毒效应,并对免疫抑制模型小鼠有较好的免疫功能恢复作用。其机制可能与Coxian是良好的质子(H·)供体,具有清除多种自由基的性能,并可提高机体的抗氧化酶活性和GSH浓度,对抗脂质过氧化反应有有关。
To evaluate the qualities of nutrition,healthy and for processing,thirteen species of Coix lachr yma-jobi kernel specimens were collected from twelve representative growing districts over China,where are from north latitude 20°to 48°.Based on the first phase study,the extracting and purifing technologies of Coixan from Fujian Jinsha Coix lachr yma-jobi kernel,with high amylose content,was focused on and had been systematically investigated,as well as the molecular structure,the physics and chemistry,and functional characteristics of Coixan..The main results were as follows:
(1) Evaluation of the main qualities of Coix lachryma-jobi kernel resources in China
The studies showed that the nutritional components in Coix lachr yma-jobi kernel suited the nutrient balance well,for the average contents of the nutritional components in Coix lachryma-jobi kernel were 15.80%protein,5.49%fat,63.05%carbohydrate,1.75% fibre,and 1.68%ash.Compared with rice or flour,Coix lachy ma-jobi kernel contained more protein and fat,but less carbohydrate.It is indicated that the Coix lachr yma-jobi kernel was rich in calcium,which was much more than that in rice and flour.The average contents of the VB_1,VB_2,VB_6,Fe,Zn,Se in Coix lachryma-jobi kernel were as much as that in rice and flour.The results of the amino acid analysis showed that the contents of the Glu,Leu, (Met+Cys) in Coix lachryrna-jobi kernel(accounting for 45.86 mg/g,21.71 mg/g,5.92 mg/g,respectively) were higher than that in the rice and flour,however,the content of Lys was less.According to ASS,Lys was the first limiting amino acid,and then,the second limiting amino acid was varied from the growing districts.For example,the second limiting amino acid of Coix lachryma-jobi kernel which from Yunan was Thy,from Sichuan was lie and from Guizhou was Val.The average of EAA/TAA and EAA/NEA were 35.86%and 55.91%.The average of EAAI was 58.91%.Compared to the FAO/WHO mode,the composition of amino acid of the protein in Coix lachr yma-jobi kernel was perfect,and the protein of Coix lachr yma-jobi kernel was considered as high quality protein.
The study of healthy and processing qualities proved that Coix lachr yma-jobi kernel was an excellent economic crops which is served as food and medicine.Coix lachr yma-jobi kernel contains abundant Coixenolide,Coixan and total triterpenes,and their average contents were 44.60 mg/g,59.03mg/g and 22.83 mg/g,respectively.Coix lachryma-jobi kernel was a kind of starchy foodstuff,with 56.42%starch totally,and 4.78%amylase.Its healthy and processing qualities were closely related to the species and growing districts.
This study originated the qualities research of Coix lachr yma-jobi kernel in China, and would provide an valuable reference for comprehensive utilization of Coix lachr yma-jobi kernel resources.
(2) Kinetics for Coixan extraction
In a hot water extracting system,the mass transfer rates of Coixan were studied by varying the processing parameters,namely the extracting temperature(T),pH value,mass ratio of material vs water(R).The results showed that:
①The extracting temperature(T),pH value,mass ratio of material vs water(R) influenced Coixan saturation concentration(C~*) and Coixan saturation constant(K_(sat)) significantly(p＜0.01).
②Through the three factors quadratic-rotation-interchangeable-composite experimental design,the empirical mass transfer models of C~* and K_(sat) with the parameters of the extracting temperature(T/℃),pH value,mass ratio of material vs water(R) were established.
C~*~×10~3=47.2-1.17R-3.70T+75.38pH+0.054R2+0.050T2-6.38pH2(AOD)
K_(sat)×10~3=-1.13+5.27R+17.14T+222.4pH-0.027R.2-0.1T2-12.73pH2+0.017RT -0.1.7R pH(1/h)
Results from experimental tests fitted the models well.
(3) Optimization of the technology on deproteinization of Coixan
The removal efficiency of enzymatic deproteinization from Coixan was better than that of the sevag method or the trichloroacetic acid method.Through the rotating perpendicular combination experimental design,the parameters of enzymatic deproteinzation from Coixan were optimized,and the dynamic equation of deproteinization from Coixan was given.The results showed the optimal enzymatic deproteinzation conditions from Coixan were:the enzymatic temperature 45℃,pH 7.0,papain dosage 5000μ/g,thus,the percentage of the removed protein(PRP) went up to 80.12%.Moreover,if enzymatic deproteinization were combined with the sevag method,The protein could be removed from Coixan completely.
(4) The physics and chemistry characteristics and molecular structure of Coixan
Coixan,isolated and purified with Sephadex-75,was a milk white,flocculent powder, dissolves in water readly,was neutral polysaccharide with pH 6.8,thermal stability at 0℃～100℃,and more stable in acid than in alkaline stability among the food processing pH value.However,under strongly acidic and alkali conditions,Coixan was less stable in acid than in alkali.The results of color reaction indicated:Reacting with phenol-sulfuric acid reagent,anthrone reagent,Coixan turned into blown and blind green, respectively.However,it turned out to be colorless while reacting with I_2-KI,FeCl_3, carbazole- sulfuric acid reagent,fehling reagent and Coomassie BrilliantG-250 reagent.Furthermore,HPGPC and UV were employed to analysis the molecular characteristics of Coixan.Results showed that Coixan was a pure polysaccharide compound, without protein or nuclnic acid,with 2933molecular weight,specific rotatory power[α]~(20)D =+155°(0.151,H_2O),and inherent viscosity[η]=0.313.
Molecular structure and surface morphology of Coixan were observed and analyzed by X-ray diffraction(XRD),Infrared spectrum(IR),Nuclear Magnetic Resonance(NMR) and Atomic Force Microscopy(AFM).According to XRD,Coixan wasn't a crystal.But according to IR or ~1H-NMR,Coixan was a hetero-polysaccharide without sulfonic group,but with acetyl group.The polysaccharide was probablely composed withα-D-xylose,α-D-glucose,β- D- alactose,β- D- mannose andβ- L- arabinose,and with theβglycosidic bond.AFM observations found that Coixan dispersed readly in water,the molecular took on the shape of a helix or liner structure with little round particles,blame-like protuberance, diameter about 40.0 nm-50.0nm,and height about 0.880nm-1.551nm.
(5) The antioxidant activities in vitro of Coixan
Using crude and pure Coixan as materials respectively,the scavenging capacities on superoxide anion(O_2~-.),hydroxyl radical(·OH),1,1-diphenyl 2-picrylhydrazyl radicals (DPPH·) and the reducing power were tested with Fenton system,Pyorgallol Autoxidation Method,DPPH·system,and FRAP Method respectively.In addition,Ferrozine was employed as a reagent and indicator of Fe~(2+),to observed the chelating efficiency of Coixan with metal ions.The results proved that Coixan was a great proton(H·) donor,and had strong power to scavenge free radicals and readly chelating with metal ions.Moreover,pure Coixan had more power to scavenge free radicals and O_2~·.than crude one.The studied revealed that Coixan was a multipurpose inhibitor for free radicals and metal ions,thus the possibility to develop the anti-free radicals healthy functional food with Coixan would be avaiable.
(6) The antioxidant and immune regulation effects of Coixan in vivo on mice
Acute tetrachloride-intoxicated mice model was established by ip 0.2mL 0.2% tetrachloride(CCl_4).Compared with V_E as Positive control group,the mice were given with different doses of pure Coixan 250、500、1000mg/kg and crude Coixan 250、500、1000mg/kg pretreated ig by 0.5mL each day respectively for 28d.Blood SOD and GSH-Px activities, the content of GSH and serum T-AOC were detected as the marker of the oxidation resistance activity:and the activities of liver GOT、GPT and liver index were also detected.The results showed that comparing with model group,Coixan could significantly enhance blood SOD and GSH-Px activities in mice,prevent acute liver from CCl_4 effectly,and significantly inhibit the rising of liver GOT,GPT activities and the content of MDA.There are obvious dose-effect relationship of physiological activities for Coixan,and the effect of pure Coixan was better than that of crude one.
The immune suppressive mice mode was established by ig Cyclophosphamide, Compared with saline as the normal control,the immune organs index,half of hemolytic value(HC_(50)),peritoneal macrophage phagocytosis index and the effect of lymphocyte proliferation were determined to observe the immune regulating effects of Coixan on mice immune system.The results showed that Coixan can significantly inhibit the reduction of the thymus and spleen indices of mice with lower immune function,enhanced the macrophage phagocytosis index and lymphocyte proliferation,raised serum HC_(50),and corrected immune disorders phenomenon.
Coixan could significantly improve the anti-oxidation function of tetrachloride-intoxicated mice,and had a great immune regulation activities on mice with immune suppressive.The mechanism is probably mediated with Coixan being a great proton
(H·) donor,which had strong power to scavenge free radicals,to enhance the activities of anti-oxidation enzyme and the content of GSH,and to prevent lipid peroxidation.
(1)我国薏苡仁资源主要品质的研究
薏苡仁资源营养品质研究表明,薏苡仁营养较为齐全,13种薏苡仁营养成分的平均值为:蛋白质15.80%,脂肪5.49%,均高于米、面;碳水化合物63.05%,低于米、面;粗纤维和灰分分别为1.75%与1.68%。薏苡仁的Ca含量丰富,高于米、面;VB_1、VB_2、VB_6、Fe、Zn、Se平均含量与米、面相当。薏苡仁的谷氨酸(Glu)、亮氨酸(Leu)、含硫氨基酸(Met+Cys)平均含量丰富,均高于米、面;但赖氨酸(Lys)含量较低。以氨基酸评分(AAS)为标准,13个样品中,Lys为第一限制性氨基酸,第二限制性氨基酸随产地不同而异,云南产等8个样品为苏氨酸(Thy),四川产等5个样品为异亮氨酸(Ile),贵州产为缬氨酸(Val)。平均必需氨基酸与总氨基酸比值(EAA/TAA)、必需氨基酸与非必需氨基酸比值(EAA/NEA)值分别为35.86%、55.91%,平均必需氨基酸指数(EAAI)值58.91%。与FAO/WHO模式相比,薏苡仁蛋白质具有较好的氨基酸组成,属于较为优质的蛋白质。
保健与加工品质检测结果显示:薏苡仁是优良的药食同源经济作物,其含有丰富的薏苡仁酯、薏苡仁活性多糖(Coxian)、三萜类化合物,其平均值分别为:44.60 mg/g、59.03mg/g、22.83mg/g;薏苡仁属高淀粉质食物,其平均淀粉含量为56.42%,直链淀粉为4.78%。不同产地薏苡仁的保健、加工品质特性与品种、产地密切相关。
该研究填补了我国有关薏苡仁品质研究的不足,并为薏苡仁资源的合理开发利用提供重要的参考价值。
(2)薏苡仁活性多糖浸提动力学研究
采用热水浸提工艺,探讨了浸提温度(T)、料液比(R)、浸提液pH值对浸提过程中Coxian浓度变化影响的规律;通过对影响因素的研究,建立了浸提过程中Coxian饱和浓度C~*和饱和常数K_(sat)的经验溶解扩散模型。
①在本研究条件下,T、R及浸提液pH值对浸提过程中Coxian饱和浓度C~*和饱和常数K_(sat)的影响极显著(p＜0.01)。
②通过二次通用旋转回归试验程序拟合得到T、R、pH值分别与C~*、K(sat)的回归模型为:C~*×10~3=47.2-1.17R-3.70T+75.38pH+0.054R~2+0.050T~2-6.38pH~2(A_(OD))K_(sat)×10~3=-1.13+5.27R+17.14T+222.4pH-0.027R~2-0.1T~2-12.73pH~2+0.017RT-0.1.7R pH(1/h)
两回归方程失拟不显著,回归显著(p＜0.01),可用于预测和控制Coxian在浸提过程中的溶解扩散规律。
(3)薏苡仁活性多糖脱蛋白工艺的优化
酶解法对Coxian脱蛋白的效果较Sevag法、三氯乙酸法的好;采用通用旋转组合试验设计方案,获得Coxian酶法脱蛋白的动力学方程,并优化其工艺参数组合:最佳的酶解温度、酶解液的pH值为0水平,即X_1=45℃;X_2=7.0;木瓜蛋白酶的适宜添加量X_3=5000μ/g:在此条件下,Coxian的蛋白脱除率最高,为80.12%;此外,在酶解脱蛋白基础上,结合一次的Sevag法,即可将Coxian中的杂质蛋白基本脱除。
(4)薏苡仁活性多糖理化性质与分子结构的研究
经Sephadex G-75凝胶色谱分离、纯化的Coixan,为乳白色絮状粉末固体,属水溶性、酸偏中性多糖,pH6.8;Coixan在100℃范围内的耐热稳定性较好;在常规食品加工pH范围内,Coixan的耐酸稳定性比耐碱稳定性好,但强酸碱条件下,则酸性条件对其影响更大。Coixan与硫酸—苯酚反应呈棕褐色,与蒽酮反应呈暗绿色;与I_2-KI、FeCl_3、硫酸一咔唑、斐林试剂、考马斯亮蓝反应均呈阴性。经HPGPC与紫外光谱进行分析,鉴定Coixan为单一级分,且为不含有核酸、蛋白质的纯多糖化合物;确定其分子量为2993D;比旋光度「a」~(20)_D=+155°(0.151,H2O),特性粘度[n]=0.313。
采用XRD、IR、NMR、AFM解析分子结构与形貌。XRD图谱显示,Coxian的分子颗粒属非晶态:IR及~1H-NMR分析数据表明,Coxian为非硫酸的杂聚多糖,分子中含有乙酰氨基;其构成单糖可能包括有α-D-吡喃木糖、α-D-吡喃葡萄糖、β-D-吡喃半乳糖、β-D-吡喃甘露糖、β-L-吡喃阿拉伯糖;Coxian糖苷链中存在β构型糖苷键。AFM的分子形貌图像提示,Coixan分子在溶液中具有良好的分散性能,其分子链在水溶液呈规则圆球状(火焰状)突起的螺旋或线性结构,直径约为40.0nm-50.0nm,高约为0.880nm-1.551nm。
(5)薏苡仁活性多糖的体外抗氧化活性
分别以Coxian纯品和粗品为受试物,进行自由基的清除和抑制脂质过氧化实验。利用Fenton反应体系、邻苯三酚自氧化体系、DPPH·体系与FRAP法分别检测Coxian对·OH、O_2~-·和DPPH·的清除效果与还原能力;以Ferrozine为Fe~(2+)的光度试剂,观察Coxian螯合金属离子的能力。结果表明:Coxian是良好的质子(H·)供体,具有清除多种自由基的性能,同时还具备了一定的金属螯合能力,并且以Coxian纯品对O_2~-·的清除效果最为显著。提示Coxian是一种多效的自由基和金属离子的抑制剂,具有研制开发成抗自由基保健食品的良好前景。
(6)薏苡仁活性多糖抗氧化及免疫调节的动物试验
以ICR清洁级小鼠为研究对象,腹腔注射CCl_4造模。以V_E为阳性对照,给药组(Coxian纯品低、中、高剂量(250mg/kg、500mg/kg、1000mg/kg)和Coxian粗品低、中、高剂量(250mg/kg、500mg/kg、1000mg/kg)分别先经灌胃28d进行预防。以全血SOD与GSH-Px活性、GSH浓度、血清T-AOC(总抗氧化能力)反映小鼠抗氧化功能的改变;观察肝脏GOT、GPT活力单位、MDA(丙二醛)含量和肝脏指数变化。结果表明:与模型组相比,Coxian可显著增强小鼠SOD、GSH-Px活性,有效防止CCl_4对肝脏组织的伤害,明显抑制肝细胞GOT、GPT水平与MDA含量的异常上升。其效果均呈现明显的量-效关系,且以Coxian纯品的效果为好。
环磷酰胺建立免疫抑制小鼠模型,以生理盐水为正常对照,采用Coxian纯品对小鼠免疫器官指数、半数溶血值(HC_(50))、抗体生成细胞、腹腔巨噬细胞吞噬指数及淋巴细胞增殖反应的调节效果试验,观察其对免疫系统的影响。结果显示:Coxian能显著抑制免疫功能低下小鼠的脾赃指数和胸腺指数的缩小;增强巨噬细胞吞噬指数及淋巴细胞增殖反应;提高血清HC_(50),纠正免疫功能紊乱现象。
动物试验证明Coxian能显著增强小鼠的抗氧化功能,有效解除CCl_4肝中毒效应,并对免疫抑制模型小鼠有较好的免疫功能恢复作用。其机制可能与Coxian是良好的质子(H·)供体,具有清除多种自由基的性能,并可提高机体的抗氧化酶活性和GSH浓度,对抗脂质过氧化反应有有关。
To evaluate the qualities of nutrition,healthy and for processing,thirteen species of Coix lachr yma-jobi kernel specimens were collected from twelve representative growing districts over China,where are from north latitude 20°to 48°.Based on the first phase study,the extracting and purifing technologies of Coixan from Fujian Jinsha Coix lachr yma-jobi kernel,with high amylose content,was focused on and had been systematically investigated,as well as the molecular structure,the physics and chemistry,and functional characteristics of Coixan..The main results were as follows:
(1) Evaluation of the main qualities of Coix lachryma-jobi kernel resources in China
The studies showed that the nutritional components in Coix lachr yma-jobi kernel suited the nutrient balance well,for the average contents of the nutritional components in Coix lachryma-jobi kernel were 15.80%protein,5.49%fat,63.05%carbohydrate,1.75% fibre,and 1.68%ash.Compared with rice or flour,Coix lachy ma-jobi kernel contained more protein and fat,but less carbohydrate.It is indicated that the Coix lachr yma-jobi kernel was rich in calcium,which was much more than that in rice and flour.The average contents of the VB_1,VB_2,VB_6,Fe,Zn,Se in Coix lachryma-jobi kernel were as much as that in rice and flour.The results of the amino acid analysis showed that the contents of the Glu,Leu, (Met+Cys) in Coix lachryrna-jobi kernel(accounting for 45.86 mg/g,21.71 mg/g,5.92 mg/g,respectively) were higher than that in the rice and flour,however,the content of Lys was less.According to ASS,Lys was the first limiting amino acid,and then,the second limiting amino acid was varied from the growing districts.For example,the second limiting amino acid of Coix lachryma-jobi kernel which from Yunan was Thy,from Sichuan was lie and from Guizhou was Val.The average of EAA/TAA and EAA/NEA were 35.86%and 55.91%.The average of EAAI was 58.91%.Compared to the FAO/WHO mode,the composition of amino acid of the protein in Coix lachr yma-jobi kernel was perfect,and the protein of Coix lachr yma-jobi kernel was considered as high quality protein.
The study of healthy and processing qualities proved that Coix lachr yma-jobi kernel was an excellent economic crops which is served as food and medicine.Coix lachr yma-jobi kernel contains abundant Coixenolide,Coixan and total triterpenes,and their average contents were 44.60 mg/g,59.03mg/g and 22.83 mg/g,respectively.Coix lachryma-jobi kernel was a kind of starchy foodstuff,with 56.42%starch totally,and 4.78%amylase.Its healthy and processing qualities were closely related to the species and growing districts.
This study originated the qualities research of Coix lachr yma-jobi kernel in China, and would provide an valuable reference for comprehensive utilization of Coix lachr yma-jobi kernel resources.
(2) Kinetics for Coixan extraction
In a hot water extracting system,the mass transfer rates of Coixan were studied by varying the processing parameters,namely the extracting temperature(T),pH value,mass ratio of material vs water(R).The results showed that:
①The extracting temperature(T),pH value,mass ratio of material vs water(R) influenced Coixan saturation concentration(C~*) and Coixan saturation constant(K_(sat)) significantly(p＜0.01).
②Through the three factors quadratic-rotation-interchangeable-composite experimental design,the empirical mass transfer models of C~* and K_(sat) with the parameters of the extracting temperature(T/℃),pH value,mass ratio of material vs water(R) were established.
C~*~×10~3=47.2-1.17R-3.70T+75.38pH+0.054R2+0.050T2-6.38pH2(AOD)
K_(sat)×10~3=-1.13+5.27R+17.14T+222.4pH-0.027R.2-0.1T2-12.73pH2+0.017RT -0.1.7R pH(1/h)
Results from experimental tests fitted the models well.
(3) Optimization of the technology on deproteinization of Coixan
The removal efficiency of enzymatic deproteinization from Coixan was better than that of the sevag method or the trichloroacetic acid method.Through the rotating perpendicular combination experimental design,the parameters of enzymatic deproteinzation from Coixan were optimized,and the dynamic equation of deproteinization from Coixan was given.The results showed the optimal enzymatic deproteinzation conditions from Coixan were:the enzymatic temperature 45℃,pH 7.0,papain dosage 5000μ/g,thus,the percentage of the removed protein(PRP) went up to 80.12%.Moreover,if enzymatic deproteinization were combined with the sevag method,The protein could be removed from Coixan completely.
(4) The physics and chemistry characteristics and molecular structure of Coixan
Coixan,isolated and purified with Sephadex-75,was a milk white,flocculent powder, dissolves in water readly,was neutral polysaccharide with pH 6.8,thermal stability at 0℃～100℃,and more stable in acid than in alkaline stability among the food processing pH value.However,under strongly acidic and alkali conditions,Coixan was less stable in acid than in alkali.The results of color reaction indicated:Reacting with phenol-sulfuric acid reagent,anthrone reagent,Coixan turned into blown and blind green, respectively.However,it turned out to be colorless while reacting with I_2-KI,FeCl_3, carbazole- sulfuric acid reagent,fehling reagent and Coomassie BrilliantG-250 reagent.Furthermore,HPGPC and UV were employed to analysis the molecular characteristics of Coixan.Results showed that Coixan was a pure polysaccharide compound, without protein or nuclnic acid,with 2933molecular weight,specific rotatory power[α]~(20)D =+155°(0.151,H_2O),and inherent viscosity[η]=0.313.
Molecular structure and surface morphology of Coixan were observed and analyzed by X-ray diffraction(XRD),Infrared spectrum(IR),Nuclear Magnetic Resonance(NMR) and Atomic Force Microscopy(AFM).According to XRD,Coixan wasn't a crystal.But according to IR or ~1H-NMR,Coixan was a hetero-polysaccharide without sulfonic group,but with acetyl group.The polysaccharide was probablely composed withα-D-xylose,α-D-glucose,β- D- alactose,β- D- mannose andβ- L- arabinose,and with theβglycosidic bond.AFM observations found that Coixan dispersed readly in water,the molecular took on the shape of a helix or liner structure with little round particles,blame-like protuberance, diameter about 40.0 nm-50.0nm,and height about 0.880nm-1.551nm.
(5) The antioxidant activities in vitro of Coixan
Using crude and pure Coixan as materials respectively,the scavenging capacities on superoxide anion(O_2~-.),hydroxyl radical(·OH),1,1-diphenyl 2-picrylhydrazyl radicals (DPPH·) and the reducing power were tested with Fenton system,Pyorgallol Autoxidation Method,DPPH·system,and FRAP Method respectively.In addition,Ferrozine was employed as a reagent and indicator of Fe~(2+),to observed the chelating efficiency of Coixan with metal ions.The results proved that Coixan was a great proton(H·) donor,and had strong power to scavenge free radicals and readly chelating with metal ions.Moreover,pure Coixan had more power to scavenge free radicals and O_2~·.than crude one.The studied revealed that Coixan was a multipurpose inhibitor for free radicals and metal ions,thus the possibility to develop the anti-free radicals healthy functional food with Coixan would be avaiable.
(6) The antioxidant and immune regulation effects of Coixan in vivo on mice
Acute tetrachloride-intoxicated mice model was established by ip 0.2mL 0.2% tetrachloride(CCl_4).Compared with V_E as Positive control group,the mice were given with different doses of pure Coixan 250、500、1000mg/kg and crude Coixan 250、500、1000mg/kg pretreated ig by 0.5mL each day respectively for 28d.Blood SOD and GSH-Px activities, the content of GSH and serum T-AOC were detected as the marker of the oxidation resistance activity:and the activities of liver GOT、GPT and liver index were also detected.The results showed that comparing with model group,Coixan could significantly enhance blood SOD and GSH-Px activities in mice,prevent acute liver from CCl_4 effectly,and significantly inhibit the rising of liver GOT,GPT activities and the content of MDA.There are obvious dose-effect relationship of physiological activities for Coixan,and the effect of pure Coixan was better than that of crude one.
The immune suppressive mice mode was established by ig Cyclophosphamide, Compared with saline as the normal control,the immune organs index,half of hemolytic value(HC_(50)),peritoneal macrophage phagocytosis index and the effect of lymphocyte proliferation were determined to observe the immune regulating effects of Coixan on mice immune system.The results showed that Coixan can significantly inhibit the reduction of the thymus and spleen indices of mice with lower immune function,enhanced the macrophage phagocytosis index and lymphocyte proliferation,raised serum HC_(50),and corrected immune disorders phenomenon.
Coixan could significantly improve the anti-oxidation function of tetrachloride-intoxicated mice,and had a great immune regulation activities on mice with immune suppressive.The mechanism is probably mediated with Coixan being a great proton
(H·) donor,which had strong power to scavenge free radicals,to enhance the activities of anti-oxidation enzyme and the content of GSH,and to prevent lipid peroxidation.
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