文冠果种仁品质及其油脂和蛋白质资源利用研究
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摘要
文冠果(Xanthoceras sorbifolia Bunge)是中国特有的在“三北”地区广泛分布的珍稀木本药用、油用植物。近代植物学研究表明,文冠果是一种集观赏、生态、食用、环保、工业、药用、木材、生物能源于一体的具有极高开发价值的多用途树种。本论文以文冠果籽为研究对象,通过对文冠果种仁(种子内具曲卷的胚)的品质特性、种仁油的提取工艺和食用安全性及抗氧化活性、文冠果种仁蛋白的分离提取及功能特性等主要研究内容的探讨,为我国文冠果的资源利用提供理论依据和工艺参数。
论文的具体研究工作及结果简述如下：
1、分别对产于中国内蒙翁牛特旗、陕西志丹、西安、杨凌、河南灵宝、辽宁建平、吉林白城的文冠果种子及种仁的品质特性进行了比较研究。
种子品质特性的结果分析表明,七个产地文冠果种子的形状、籽粒颜色基本无差异,籽粒大小有较小差异；方差分析发现种子的千粒重、容重、种仁种皮之比、种仁的水分含量和灰分含量等5项指标在七个产地间均有极显著差异(p<0.01)。
七个产地的文冠果种仁的营养品质也均有差异。不同产地种仁的含油量及粗蛋白含量均值分别为60%和25.64%,方差分析表明含油量、粗蛋白含量在七个产地间的差异均为极显著水平(p<0.01)。文冠果种仁油主要由亚油酸、油酸、芥酸、花生一烯酸、棕榈酸、二十四烯酸、硬脂酸等12种成分组成;其油品的酸价0.601 mgKOH/g,过氧化值0.16meq/kg,碘价121.4g/100g,是一种不饱和脂肪酸含量很高(大于90%)及品质很好的食用及功能油脂原料。种仁粗蛋白中氨基酸含量的平均值为188.61 mg/g,含有17种氨基酸,包括7种必需的氨基酸(其平均含量53.80 mg/g,占氨基酸总量的28.53%)；七个产地样品的氨基酸含量经方差分析也有极显著差异(p<0.01),尤其是7种必需氨基酸中蛋氨酸含量的变化最大(CV值85.92%),酪氨酸(CV值31.16%)和丝氨酸(CV值30.43%)次之。营养及矿质元素N、K、Mg、P、Fe、Zn、Mn、Ba的含量在七个产地间也存在极显著差异(p<0.01),其中Ba的含量变化最大,变异系数达61.59%。方差分析表明七个产地文冠果种仁的VE、VB1、VB2含量也有极显著差异(p<0.01),变化最大的是VB1(CV值68.0%),其次是VE(CV值44.1%)和VB2(CV值40.62%)。
上述分析资料说明文冠果种子、种仁品质相关性状受土壤、环境等自然因素的影响很大,不同地区的种子、种仁品质存在较大的差异,其中陕西志丹产的文冠果种仁品质相对最优。
2、以文冠果种仁为原料,分别研究了冷榨法、超声波及微波辅助提取法、水酶法提取文冠果种仁油的工艺条件。
(1)静压式机械压榨(即冷榨)提取工艺参数为：压力(5.5±0.2)MPa、时间6～8 h、种仁与种皮之比9：1(g/g)、室温,得率40.44%,提取率63.2%。
(2)以石油醚(沸程60～90℃)为提取剂,通过正交试验获得的微波辅助提取最佳工艺条件为：料液比1：16(g/mL)、时间20 min、温度75℃、微波功率100W,得率53.27%,提取率81.95%。
(3)以石油醚(沸程60～90℃)为提取剂,二次回归旋转组合试验设计获得的超声波辅助提取最佳工艺参数为：料液比1：10(g/mL)、提取温度60℃、提取时间35 min、超声波频率60kHz,得率60.18%,提取率92.47%；响应曲面分析获得的优化回归数学模型为：Y=51.12500+0.91792X1-0.89010X12-0.88042X2.0.50635 X22+1.26542X3-0.53385X32-0.69292 X4-0.62385X42-0.68313X2X4,采用此模型在试验范围内能较准确预测文冠果油的得率,且各试验因素对油脂得率影响的大小顺序为：提取时间>料液比>提取温度>超声波频率。
(4)通过单因素及正交试验获得的水酶法提取文冠果种仁油的最佳参数为：料液比1：6(g/mL)、酶解温度45℃、碱性蛋白酶(pH值7.0)用量3.0%、纤维素酶(pH值4.5)用量1.0%、酶解反应时间共8h(各反应4 h),得率52.78%,提取率81.2%。
四种工艺方法中,冷榨法得到的文冠果种仁油品质好,水酶法提取则可同时获得油脂和蛋白质,超声波辅助萃取油脂得率最高,微波辅助萃取时间短、成本低。
对所提取的文冠果种仁油进行了氧化稳定性研究,结果表明,含有少量VE的种仁油其氧化稳定性很好；添加复合抗氧化剂(0.02%TBHQ+0.01%Vc),室温下文冠果种仁油可贮藏30个月以上。
3、对种仁榨油后的饼粕采用碱溶酸沉法提取文冠果种仁蛋白,其最佳参数为：液料比11：1(mL/g).pH11、时间73min、温度49℃,蛋白质提取率84.66%；响应曲面分析法得到的优化数学模型为：Y=82.64+3.98X1-4.40 X12+3.56 X2-6.63 X22+3.04 X3-5.89 X32+5.11 X2X3-6.79X4(?),该模型能较准确的预测试验结果。
文冠果种仁蛋白的氨基酸种类齐全(有17种氨基酸),按照WHO的建议其氨基酸评分为75分,营养价值很高,是一种优良的蛋白质资源；且文冠果种仁蛋白质等电点为4.6,其蛋白质具有良好的吸油性、溶解性、吸水性、乳化及乳化稳定性,可作为乳化剂、食品添加剂开发,在食品工业中具有广阔的应用前景。
4、采用急性毒性试验、遗传毒性试验、30天喂养亚急性毒性试验评价冷榨文冠果种仁油的食用安全性。急性毒性试验结果表明文冠果种仁油为实际无毒物；文冠果种仁油的三项遗传毒性试验结果均为阴性；文冠果种仁油的30天喂养试验结果表明,受试动物SD大鼠无明显中毒症状及死亡；在SD大鼠的血液生化指标中,各剂量组的TP、ALB、ALT、AST、BUN、GLU指标与阴性对照组相比没有显著差异,CHO、TG指标在正常参考值内,初步说明文冠果种仁油食用安全。但试验中各剂量组的CRE值均高于参考值范围,还需进一步研究确定。
5、文冠果种仁油的体外抗氧化活性试验显示,文冠果油对羟自由基·OH和超氧阴离子·O2-具有较好的清除效果,对DPPH自由基具有很强的清除作用,且还原能力超过BHT.TBHQ,在较高浓度下对Fe2+诱导的脂蛋白PUFA过氧化反应也有较好的抑制作用。
文冠果种仁油的体内抗氧化活性试验显示,文冠果油能显著提高受试昆明小鼠肝组织及脑组织中的CAT.SOD和GSH.PX的活性,并降低小鼠这些组织中的MDA含量。
体内外试验结果说明文冠果种仁油具有显著的抗氧化性。
6、理化性质分析显示,文冠果种仁油的相对密度和凝固点分别为0.914和-15℃,虽比柴油高,但比豆油等其它植物油低；十六烷值为37,高于豆油、菜籽油；残留碳分仅0.22%,也比其它植物油低,且文冠果油的热值高达9488 Kcal/kg.GC-MS分析表明文冠果种仁油的主要脂肪酸的组成与石化柴油组成相似,性质相近,文冠果种仁油也是一种很好的生物柴油原料。
7、在超声辅助条件下采用碱法催化文冠果油合成生物柴油,其最佳工艺为：KOH用量1.2%、无水甲醇用量30%、反应温度50℃、超声时间50 min,一次酯交换的转化率为93.7%；采用二次酯交换则生物柴油的转化率为96.7%；二次回归旋转组合设计试验获得的数学模型为：Y=88.00833+3.60000X1+3.10833X2-1.43125X22+2.27500X3+3.28333X4，此模型的理论预测与试验吻合程度好,生物柴油的转化率较高。
采用Novo435固定化酶法催化文冠果种仁油合成生物柴油的最佳条件：28 KHz的超声辅助反应时间60 min、有机溶剂石油醚(60～90℃)用量25 mL/10g oil.反应温度50℃、脂肪酶用量10%、醇油摩尔比1.5：1；在此条件下分三步进行酯交换共反应时间3 h,总转化率达93.84%,且固定化脂肪酶重复使用6次还可保持较高的活性。
综合比较生产成本、产物分离回收等方面,采用Nov435固定化酶法制备生物柴油优于碱催化法。
Shinyleaf yellowhorn (Xanthoceras sorbifolia Bunge) is a unique medicinal and oil woody plant, which is widely distributed in northern districts of China. Shinyleaf yellowhorn also is multi-purpose plant and has extremely high developing value. It can be used in ornament, ecology, food, environment, medicine, wood, and biological energy industry. In this dissertation, shinyleaf yellowhorn seed were studied further. The quality of shinyleaf yellowhorn kernel(the curly embryo in the seed), oil extraction technology, protein extraction and its function, edible safety of kernel oil and oil antioxidant activity were discussed respectively. This systematical research was expected to provide theoretical basis and technology parameters for the development and deep-processing of Shinyleaf yellowhorn.
The dissertation study contents and results are following:
1. The quality of shinyleaf yellowhorn seed and kernel which come from different district were studied.
The analysis result of quality showed that there was no difference in seed's (from seven districts) shape and color, but there is small difference in seed size. There was significant difference (p< 0.01) in those indexes of weight/1000 seeds, weight/lL seeds, kernel/shell ratio, water content of kernel and ash content of kernel.
There also have variances in quality index of shinyleaf yellowhorn kernel. The average content of oil and protein in kernel was 60% and 25.64% respectively, and there is significant difference in this two indexes(p< 0.01). The oil fatty acid was composed with oleic acid, linoleic acid, erucic acid, 11-eicosenoic acid, palmitic acid,15-Tetracosenoic acid, stearic acid, and so on. The acid value and peroxide value of cold pressing oil was 0.601 mgKOH/g and 0.16meq/kg, the iodine value was 121.4g/100g, so shinyleaf yellowhorn kernel oil was a very good resource for edible and functional oil.
The average content of amino acid in kernel protein was 188.61 mg/g. Kernel protein contains seventeen amino acids in crude protein of shinyleaf yellowhorn kernel, and including seven essential amino acids (its average content is 53.80 mg/g, and of total amino acids 28.53%). There also has great significant difference of amino acid content in kernel protein from seven districts(p< 0.01), especially in seven essential amino acids. The biggest difference exists in methionine content (CV=85.92%), and the bigger difference was exist in tyrosine and serine content (CV=31.16%, CV=30.43%) respectively.
There has significant difference in nutritional and mineral element content of N, K, Mg, P, Fe, Zn, Mn, Ba(p< 0.01). The biggest difference exists in the content of barium and the coefficient of variance (CV) was 61.59%. There also has significant difference in the contents of VE, VB1,VB2. The biggest difference exists in the content of VB1(CV=68.0%), and followed by VE(CV=44.1%) and VB2(CV=40.62%).
In a word, the seed and kernel quality which associated with the nutrition was influenced greatly by the soil and environmental factors. There was great difference in seed and kernel from different district. The best quality of kernel relatively for shinyleaf yellowhorn was from ZHI Dan, SHAAN Xi.
2. The technology conditions of cold pressing, aqueous enzymatic extraction, microwave and ultrasonic assistant extraction were determined respectively using shinyleaf yellowhorn kernel powder as material.
(1) The cold pressing conditions were:pressure was (5.5±0.2) Mpa, ratio of kernel to husk was 9:1(g/g), pressing time was 8h. Under this condition, the oil yield was 40.44% and extraction ratio was 63.2%.
(2) The optimal conditions of microwave assistant extraction by orthogonal experiments were as follows:material/solvent ratio was 1:16(g/mL), extracting time 20min, temperature 75℃, microwave power 100 W, the optimal solvent was ligarine (60～90℃). Under this condition, the oil yield was 53.27% and extraction ratio was 81.95%.
(3) With ligarine (60～90℃) as optimal solvent, through quadratic regression orthogonal rotational test, the optimal technology parameters of ultrasonic assistant extraction should be: solid/liquid ratio 1:10(g/mL), extract temperature 60℃, extraction time 35min, ultrasonic frequency 60 kHz. Under this condition, the oil yield was 60.18%, extraction ratio was 92.47%. The mathemat regression model (Y= 51.12500+0.91792X,-0.88042X2+1.26542X3-0.69292 X4-0.89010X12-0.50635 X22-0.53385X32-0.62385X42-0.68313X2X4) was established using response surface analysis, which was able to predict the oil extraction rate of shinyleaf yellowhorn kernel accurately. The order of factors that influence the oil extraction rate within the experimental ranges was as follows:extraction time> solid/liquid ratio>extraction temperature>ultrasoriic frequency.
(4) Through single factor and orthogonal tests, the optimal parameters of shinyleaf yellowhorn kernel oil with aqueous enzymatic method were as follows:solid/liquid ratio is 1:6(g/mL), temperature 45℃, dosage of alkaline protease (pH7.0) 3.0%, dosage of cellulose (pH4.5) 1.0%, enzymatic hydrolysis time 8h (4h receptively). Under this condition, the oil yield was 52.78%, extraction ratio was 81.2%.
Among the above four technologies, the quality of cold pressing oilis best, but oil and protein could be obtained by aqueous enzymatic method, the extraction ratio was biggest with ultrasonic assistant extraction, and there was short time, low cost of oil extraction using microwave assistant method.
The oxidative stability of shinyleaf yellowhorn kernel oil was best. When the oil was added into combined anti-oxidants (0.02%TBHQ+0.01%Vc), it could be storied about 30 months at room temperature.
3. Using Alkali-Solution and Acid-Isolation method, the optimal extraction parameters of shinyleaf yellowhorn kernel protein were as following:liquid/solid ratio 11:1 (mL/g), pH 11, extracting time 73 min, extracting temperature 49℃, protein yield was 84.66%. The mathematic model (Y= 82.64+3.98 X,+3.56 X2+3.04 X3+5.11 X2X3-4.40 X12-6.63 X22-5.89 X32-6.79 x42) was obtained through the method of response surface analysis, and it could predict the yield of protein exactly.
The shinyleaf yellowhorn kernel was an excellent protein resource. The complete range of essential amino acid is involved in shinyleaf yellowhorn kernel protein; 75 score was get according to WHO method with great nutritious value. The protein isoelectric point was 4.6. The protein has good functional properties, such as oil absorption capacity, water absorption capacity, oil emulsification capacity and stability. It can be used as food additives and emulsifier in food industry, and has broad application prospects.
4. The edible security of cold pressing oil from shinyleaf yellowhorn kernel was evaluated through acute toxicity, genetic toxicity tests, and 30 days feeding test. The results of acute toxicity test indicated that kernel oil was non-toxicity. The three genetic toxicity tests were all negative. The 30 days feeding of sub acute toxicity test of shinyleaf yellowhorn kernel oil showed that SD rats had no obvious poisoning and death symptoms. Compared with the negative control group, there was no significant difference of TP, AST, ALB, ALT, BUN, GLU of SD rats'blood biochemical parameters, and CHO, TG were within the normal reference range. But CRE values of SD rats were higher than the reference range in three dose group. The further research may be required to determine the animal testing results.
5. In vitro antioxidant activity test of shinyleaf yellowhorn kernel oil indicated that it has good scavenging effect on-OH and-O2-and has strong scavenging effect on DPPH-. Test results also show that the reduction capacity of shinyleaf yellowhorn kernel oil is greater than BHT and TBHQ, and it has good inhibition of Fe2+induced lipoprotein peroxidation PUFA reaction at higher concentration of oil.
In vivo antioxidant activity test of shinyleaf yellowhorn kernel oil shows that it has significant anti-oxidation activity. Because it can improve the mice's catalase, superoxide dismutase, and glutathione peroxidase activity significantly of their liver and brain tissue, and it also has a great reduction in malonaldehyde content of these mice's tissues.
6. The chemical and physical analysis results show that the shinyleaf yellowhorn kernel oil relative density and freezing point was 0.9144 and-15℃respectively (it was higher than diesel, lower than other pant oil, such as soybean oil). The oil hexadecane value was 37(it was higher than soybean oil, rapeseed oil), residual C was 0.22%(it was lower than other pant oil, such as soybean oil), and the oil thermal value was get to 9488Kcal/kg. The analysis results of fatty acid composition for shinyleaf yellowhorn kernel oil showed that its composition and property was close to diesel by Gas chromatography-Mass spectrometry. So shinyleaf yellowhorn kernel oil was a best biodiesel material in China.
7. With alkali as catalyst, using shinyleaf yellowhorn kernel oil as raw materials, the Ultrasonic-assisted optimal synthesis technologies of biodiesel were as follow:the amount of KOH and methanol was 1.2%(w/w) and 30%(v/v) respectively; reacting temperature and time was 50℃and 40min respectively. Under this condition, the conversion rate was 93.7% with once transesterification, and twice transesterification's conversion rate was improved to 96.7%. The mathematic model (Y= 88.00833+3.60000X1+3.10833X2+2.27500X3+3.28333X4-1.43125X22) was obtained through quadratic regression orthogonal rotational combination design. The theoretical predictions value of this regression module was as same as synthesis test, and the conversion rate of shinyleaf yellowhorn kernel oil was as high as its prediction.
Using immobilized lipase (Novo435) as catalyst, the optimum synthesizing technology of biodiesel was as follows:the 28 KHz ultrasonic assisting time 60 min, petroleum ether (60～90℃) amount 20mL/10goil, reacting temperature 50℃, the molar ratio of methanol to oil 1.5:1, and lipase amount 10%(by the weight of the oil). Under this condition, the ester conversion was 93.84%through three steps transesterification reaction when total reacting time was 3 h. The Novo435 can be used six times repeatedly.
According to the production cost and product separation, the synthesize biodiesel method with immobilized lipase (Novo435c) as catalysis was superior to the method with alkali as catalyst.
论文的具体研究工作及结果简述如下：
1、分别对产于中国内蒙翁牛特旗、陕西志丹、西安、杨凌、河南灵宝、辽宁建平、吉林白城的文冠果种子及种仁的品质特性进行了比较研究。
种子品质特性的结果分析表明,七个产地文冠果种子的形状、籽粒颜色基本无差异,籽粒大小有较小差异；方差分析发现种子的千粒重、容重、种仁种皮之比、种仁的水分含量和灰分含量等5项指标在七个产地间均有极显著差异(p<0.01)。
七个产地的文冠果种仁的营养品质也均有差异。不同产地种仁的含油量及粗蛋白含量均值分别为60%和25.64%,方差分析表明含油量、粗蛋白含量在七个产地间的差异均为极显著水平(p<0.01)。文冠果种仁油主要由亚油酸、油酸、芥酸、花生一烯酸、棕榈酸、二十四烯酸、硬脂酸等12种成分组成;其油品的酸价0.601 mgKOH/g,过氧化值0.16meq/kg,碘价121.4g/100g,是一种不饱和脂肪酸含量很高(大于90%)及品质很好的食用及功能油脂原料。种仁粗蛋白中氨基酸含量的平均值为188.61 mg/g,含有17种氨基酸,包括7种必需的氨基酸(其平均含量53.80 mg/g,占氨基酸总量的28.53%)；七个产地样品的氨基酸含量经方差分析也有极显著差异(p<0.01),尤其是7种必需氨基酸中蛋氨酸含量的变化最大(CV值85.92%),酪氨酸(CV值31.16%)和丝氨酸(CV值30.43%)次之。营养及矿质元素N、K、Mg、P、Fe、Zn、Mn、Ba的含量在七个产地间也存在极显著差异(p<0.01),其中Ba的含量变化最大,变异系数达61.59%。方差分析表明七个产地文冠果种仁的VE、VB1、VB2含量也有极显著差异(p<0.01),变化最大的是VB1(CV值68.0%),其次是VE(CV值44.1%)和VB2(CV值40.62%)。
上述分析资料说明文冠果种子、种仁品质相关性状受土壤、环境等自然因素的影响很大,不同地区的种子、种仁品质存在较大的差异,其中陕西志丹产的文冠果种仁品质相对最优。
2、以文冠果种仁为原料,分别研究了冷榨法、超声波及微波辅助提取法、水酶法提取文冠果种仁油的工艺条件。
(1)静压式机械压榨(即冷榨)提取工艺参数为：压力(5.5±0.2)MPa、时间6～8 h、种仁与种皮之比9：1(g/g)、室温,得率40.44%,提取率63.2%。
(2)以石油醚(沸程60～90℃)为提取剂,通过正交试验获得的微波辅助提取最佳工艺条件为：料液比1：16(g/mL)、时间20 min、温度75℃、微波功率100W,得率53.27%,提取率81.95%。
(3)以石油醚(沸程60～90℃)为提取剂,二次回归旋转组合试验设计获得的超声波辅助提取最佳工艺参数为：料液比1：10(g/mL)、提取温度60℃、提取时间35 min、超声波频率60kHz,得率60.18%,提取率92.47%；响应曲面分析获得的优化回归数学模型为：Y=51.12500+0.91792X1-0.89010X12-0.88042X2.0.50635 X22+1.26542X3-0.53385X32-0.69292 X4-0.62385X42-0.68313X2X4,采用此模型在试验范围内能较准确预测文冠果油的得率,且各试验因素对油脂得率影响的大小顺序为：提取时间>料液比>提取温度>超声波频率。
(4)通过单因素及正交试验获得的水酶法提取文冠果种仁油的最佳参数为：料液比1：6(g/mL)、酶解温度45℃、碱性蛋白酶(pH值7.0)用量3.0%、纤维素酶(pH值4.5)用量1.0%、酶解反应时间共8h(各反应4 h),得率52.78%,提取率81.2%。
四种工艺方法中,冷榨法得到的文冠果种仁油品质好,水酶法提取则可同时获得油脂和蛋白质,超声波辅助萃取油脂得率最高,微波辅助萃取时间短、成本低。
对所提取的文冠果种仁油进行了氧化稳定性研究,结果表明,含有少量VE的种仁油其氧化稳定性很好；添加复合抗氧化剂(0.02%TBHQ+0.01%Vc),室温下文冠果种仁油可贮藏30个月以上。
3、对种仁榨油后的饼粕采用碱溶酸沉法提取文冠果种仁蛋白,其最佳参数为：液料比11：1(mL/g).pH11、时间73min、温度49℃,蛋白质提取率84.66%；响应曲面分析法得到的优化数学模型为：Y=82.64+3.98X1-4.40 X12+3.56 X2-6.63 X22+3.04 X3-5.89 X32+5.11 X2X3-6.79X4(?),该模型能较准确的预测试验结果。
文冠果种仁蛋白的氨基酸种类齐全(有17种氨基酸),按照WHO的建议其氨基酸评分为75分,营养价值很高,是一种优良的蛋白质资源；且文冠果种仁蛋白质等电点为4.6,其蛋白质具有良好的吸油性、溶解性、吸水性、乳化及乳化稳定性,可作为乳化剂、食品添加剂开发,在食品工业中具有广阔的应用前景。
4、采用急性毒性试验、遗传毒性试验、30天喂养亚急性毒性试验评价冷榨文冠果种仁油的食用安全性。急性毒性试验结果表明文冠果种仁油为实际无毒物；文冠果种仁油的三项遗传毒性试验结果均为阴性；文冠果种仁油的30天喂养试验结果表明,受试动物SD大鼠无明显中毒症状及死亡；在SD大鼠的血液生化指标中,各剂量组的TP、ALB、ALT、AST、BUN、GLU指标与阴性对照组相比没有显著差异,CHO、TG指标在正常参考值内,初步说明文冠果种仁油食用安全。但试验中各剂量组的CRE值均高于参考值范围,还需进一步研究确定。
5、文冠果种仁油的体外抗氧化活性试验显示,文冠果油对羟自由基·OH和超氧阴离子·O2-具有较好的清除效果,对DPPH自由基具有很强的清除作用,且还原能力超过BHT.TBHQ,在较高浓度下对Fe2+诱导的脂蛋白PUFA过氧化反应也有较好的抑制作用。
文冠果种仁油的体内抗氧化活性试验显示,文冠果油能显著提高受试昆明小鼠肝组织及脑组织中的CAT.SOD和GSH.PX的活性,并降低小鼠这些组织中的MDA含量。
体内外试验结果说明文冠果种仁油具有显著的抗氧化性。
6、理化性质分析显示,文冠果种仁油的相对密度和凝固点分别为0.914和-15℃,虽比柴油高,但比豆油等其它植物油低；十六烷值为37,高于豆油、菜籽油；残留碳分仅0.22%,也比其它植物油低,且文冠果油的热值高达9488 Kcal/kg.GC-MS分析表明文冠果种仁油的主要脂肪酸的组成与石化柴油组成相似,性质相近,文冠果种仁油也是一种很好的生物柴油原料。
7、在超声辅助条件下采用碱法催化文冠果油合成生物柴油,其最佳工艺为：KOH用量1.2%、无水甲醇用量30%、反应温度50℃、超声时间50 min,一次酯交换的转化率为93.7%；采用二次酯交换则生物柴油的转化率为96.7%；二次回归旋转组合设计试验获得的数学模型为：Y=88.00833+3.60000X1+3.10833X2-1.43125X22+2.27500X3+3.28333X4，此模型的理论预测与试验吻合程度好,生物柴油的转化率较高。
采用Novo435固定化酶法催化文冠果种仁油合成生物柴油的最佳条件：28 KHz的超声辅助反应时间60 min、有机溶剂石油醚(60～90℃)用量25 mL/10g oil.反应温度50℃、脂肪酶用量10%、醇油摩尔比1.5：1；在此条件下分三步进行酯交换共反应时间3 h,总转化率达93.84%,且固定化脂肪酶重复使用6次还可保持较高的活性。
综合比较生产成本、产物分离回收等方面,采用Nov435固定化酶法制备生物柴油优于碱催化法。
Shinyleaf yellowhorn (Xanthoceras sorbifolia Bunge) is a unique medicinal and oil woody plant, which is widely distributed in northern districts of China. Shinyleaf yellowhorn also is multi-purpose plant and has extremely high developing value. It can be used in ornament, ecology, food, environment, medicine, wood, and biological energy industry. In this dissertation, shinyleaf yellowhorn seed were studied further. The quality of shinyleaf yellowhorn kernel(the curly embryo in the seed), oil extraction technology, protein extraction and its function, edible safety of kernel oil and oil antioxidant activity were discussed respectively. This systematical research was expected to provide theoretical basis and technology parameters for the development and deep-processing of Shinyleaf yellowhorn.
The dissertation study contents and results are following:
1. The quality of shinyleaf yellowhorn seed and kernel which come from different district were studied.
The analysis result of quality showed that there was no difference in seed's (from seven districts) shape and color, but there is small difference in seed size. There was significant difference (p< 0.01) in those indexes of weight/1000 seeds, weight/lL seeds, kernel/shell ratio, water content of kernel and ash content of kernel.
There also have variances in quality index of shinyleaf yellowhorn kernel. The average content of oil and protein in kernel was 60% and 25.64% respectively, and there is significant difference in this two indexes(p< 0.01). The oil fatty acid was composed with oleic acid, linoleic acid, erucic acid, 11-eicosenoic acid, palmitic acid,15-Tetracosenoic acid, stearic acid, and so on. The acid value and peroxide value of cold pressing oil was 0.601 mgKOH/g and 0.16meq/kg, the iodine value was 121.4g/100g, so shinyleaf yellowhorn kernel oil was a very good resource for edible and functional oil.
The average content of amino acid in kernel protein was 188.61 mg/g. Kernel protein contains seventeen amino acids in crude protein of shinyleaf yellowhorn kernel, and including seven essential amino acids (its average content is 53.80 mg/g, and of total amino acids 28.53%). There also has great significant difference of amino acid content in kernel protein from seven districts(p< 0.01), especially in seven essential amino acids. The biggest difference exists in methionine content (CV=85.92%), and the bigger difference was exist in tyrosine and serine content (CV=31.16%, CV=30.43%) respectively.
There has significant difference in nutritional and mineral element content of N, K, Mg, P, Fe, Zn, Mn, Ba(p< 0.01). The biggest difference exists in the content of barium and the coefficient of variance (CV) was 61.59%. There also has significant difference in the contents of VE, VB1,VB2. The biggest difference exists in the content of VB1(CV=68.0%), and followed by VE(CV=44.1%) and VB2(CV=40.62%).
In a word, the seed and kernel quality which associated with the nutrition was influenced greatly by the soil and environmental factors. There was great difference in seed and kernel from different district. The best quality of kernel relatively for shinyleaf yellowhorn was from ZHI Dan, SHAAN Xi.
2. The technology conditions of cold pressing, aqueous enzymatic extraction, microwave and ultrasonic assistant extraction were determined respectively using shinyleaf yellowhorn kernel powder as material.
(1) The cold pressing conditions were:pressure was (5.5±0.2) Mpa, ratio of kernel to husk was 9:1(g/g), pressing time was 8h. Under this condition, the oil yield was 40.44% and extraction ratio was 63.2%.
(2) The optimal conditions of microwave assistant extraction by orthogonal experiments were as follows:material/solvent ratio was 1:16(g/mL), extracting time 20min, temperature 75℃, microwave power 100 W, the optimal solvent was ligarine (60～90℃). Under this condition, the oil yield was 53.27% and extraction ratio was 81.95%.
(3) With ligarine (60～90℃) as optimal solvent, through quadratic regression orthogonal rotational test, the optimal technology parameters of ultrasonic assistant extraction should be: solid/liquid ratio 1:10(g/mL), extract temperature 60℃, extraction time 35min, ultrasonic frequency 60 kHz. Under this condition, the oil yield was 60.18%, extraction ratio was 92.47%. The mathemat regression model (Y= 51.12500+0.91792X,-0.88042X2+1.26542X3-0.69292 X4-0.89010X12-0.50635 X22-0.53385X32-0.62385X42-0.68313X2X4) was established using response surface analysis, which was able to predict the oil extraction rate of shinyleaf yellowhorn kernel accurately. The order of factors that influence the oil extraction rate within the experimental ranges was as follows:extraction time> solid/liquid ratio>extraction temperature>ultrasoriic frequency.
(4) Through single factor and orthogonal tests, the optimal parameters of shinyleaf yellowhorn kernel oil with aqueous enzymatic method were as follows:solid/liquid ratio is 1:6(g/mL), temperature 45℃, dosage of alkaline protease (pH7.0) 3.0%, dosage of cellulose (pH4.5) 1.0%, enzymatic hydrolysis time 8h (4h receptively). Under this condition, the oil yield was 52.78%, extraction ratio was 81.2%.
Among the above four technologies, the quality of cold pressing oilis best, but oil and protein could be obtained by aqueous enzymatic method, the extraction ratio was biggest with ultrasonic assistant extraction, and there was short time, low cost of oil extraction using microwave assistant method.
The oxidative stability of shinyleaf yellowhorn kernel oil was best. When the oil was added into combined anti-oxidants (0.02%TBHQ+0.01%Vc), it could be storied about 30 months at room temperature.
3. Using Alkali-Solution and Acid-Isolation method, the optimal extraction parameters of shinyleaf yellowhorn kernel protein were as following:liquid/solid ratio 11:1 (mL/g), pH 11, extracting time 73 min, extracting temperature 49℃, protein yield was 84.66%. The mathematic model (Y= 82.64+3.98 X,+3.56 X2+3.04 X3+5.11 X2X3-4.40 X12-6.63 X22-5.89 X32-6.79 x42) was obtained through the method of response surface analysis, and it could predict the yield of protein exactly.
The shinyleaf yellowhorn kernel was an excellent protein resource. The complete range of essential amino acid is involved in shinyleaf yellowhorn kernel protein; 75 score was get according to WHO method with great nutritious value. The protein isoelectric point was 4.6. The protein has good functional properties, such as oil absorption capacity, water absorption capacity, oil emulsification capacity and stability. It can be used as food additives and emulsifier in food industry, and has broad application prospects.
4. The edible security of cold pressing oil from shinyleaf yellowhorn kernel was evaluated through acute toxicity, genetic toxicity tests, and 30 days feeding test. The results of acute toxicity test indicated that kernel oil was non-toxicity. The three genetic toxicity tests were all negative. The 30 days feeding of sub acute toxicity test of shinyleaf yellowhorn kernel oil showed that SD rats had no obvious poisoning and death symptoms. Compared with the negative control group, there was no significant difference of TP, AST, ALB, ALT, BUN, GLU of SD rats'blood biochemical parameters, and CHO, TG were within the normal reference range. But CRE values of SD rats were higher than the reference range in three dose group. The further research may be required to determine the animal testing results.
5. In vitro antioxidant activity test of shinyleaf yellowhorn kernel oil indicated that it has good scavenging effect on-OH and-O2-and has strong scavenging effect on DPPH-. Test results also show that the reduction capacity of shinyleaf yellowhorn kernel oil is greater than BHT and TBHQ, and it has good inhibition of Fe2+induced lipoprotein peroxidation PUFA reaction at higher concentration of oil.
In vivo antioxidant activity test of shinyleaf yellowhorn kernel oil shows that it has significant anti-oxidation activity. Because it can improve the mice's catalase, superoxide dismutase, and glutathione peroxidase activity significantly of their liver and brain tissue, and it also has a great reduction in malonaldehyde content of these mice's tissues.
6. The chemical and physical analysis results show that the shinyleaf yellowhorn kernel oil relative density and freezing point was 0.9144 and-15℃respectively (it was higher than diesel, lower than other pant oil, such as soybean oil). The oil hexadecane value was 37(it was higher than soybean oil, rapeseed oil), residual C was 0.22%(it was lower than other pant oil, such as soybean oil), and the oil thermal value was get to 9488Kcal/kg. The analysis results of fatty acid composition for shinyleaf yellowhorn kernel oil showed that its composition and property was close to diesel by Gas chromatography-Mass spectrometry. So shinyleaf yellowhorn kernel oil was a best biodiesel material in China.
7. With alkali as catalyst, using shinyleaf yellowhorn kernel oil as raw materials, the Ultrasonic-assisted optimal synthesis technologies of biodiesel were as follow:the amount of KOH and methanol was 1.2%(w/w) and 30%(v/v) respectively; reacting temperature and time was 50℃and 40min respectively. Under this condition, the conversion rate was 93.7% with once transesterification, and twice transesterification's conversion rate was improved to 96.7%. The mathematic model (Y= 88.00833+3.60000X1+3.10833X2+2.27500X3+3.28333X4-1.43125X22) was obtained through quadratic regression orthogonal rotational combination design. The theoretical predictions value of this regression module was as same as synthesis test, and the conversion rate of shinyleaf yellowhorn kernel oil was as high as its prediction.
Using immobilized lipase (Novo435) as catalyst, the optimum synthesizing technology of biodiesel was as follows:the 28 KHz ultrasonic assisting time 60 min, petroleum ether (60～90℃) amount 20mL/10goil, reacting temperature 50℃, the molar ratio of methanol to oil 1.5:1, and lipase amount 10%(by the weight of the oil). Under this condition, the ester conversion was 93.84%through three steps transesterification reaction when total reacting time was 3 h. The Novo435 can be used six times repeatedly.
According to the production cost and product separation, the synthesize biodiesel method with immobilized lipase (Novo435c) as catalysis was superior to the method with alkali as catalyst.
引文
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