【作者】 马艳芳；

【导师】 孙中涛；

【作者基本信息】 山东农业大学 ， 微生物学， 2016， 硕士

【摘要】 螺旋藻作为一种食品原料因其功能特性而得到广泛应用,FAO将其评为“人类21世纪最佳保健品”和“未来超级营养食品”(张文等,2013)。螺旋藻中藻胆蛋白含量高达50-70%。此外,还具有非常理想的氨基酸构成比例,人体所必需的8种氨基酸的含量接近或超过FAO规定的标准。螺旋藻对高胆固醇血症、高甘油血症、心血管疾病、炎症性疾病、癌症以及病毒性感染等一系列疾病有疗效。螺旋藻在食品制造业中还可以作为一种功能成分存在(Hemamalini,2013)。但是,螺旋藻在食品制造业中的应用受到其干粉溶解性差、腥味浓重的缺点的限制。藻胆蛋白经蛋白酶处理后,改善了物化特性并扩大了应用范围。与藻胆蛋白相比,其水解产物螺旋藻肽不仅具有较低的分子量与良好的物化特性,更容易被人体消化吸收。而且还具有提高人体免疫功能,消除并防止机体受到自由基破坏,全面调节人体代谢机能等生理作用,这其中以其抗氧化能力最受关注。本文主要研究了螺旋藻的破壁方法、优化了单酶法和双酶法水解螺旋藻制备抗氧化肽的工艺,研究了水解度对螺旋藻肽起泡性、乳化性及抗氧化能力的影响,还研究了酶解过程中色素的产生规律与防控措施以及螺旋藻肽功能性饮料的研制。搅拌法、超声波破碎法、反复冻融法及后二者联用都可以破坏螺旋藻藻体细胞,使内容物流出。研究结果表明,超声波破碎法,在超声模式相同、超声功率为5%、超声7s、间隔3s时,细胞中可溶性蛋白的含量是最多的;反复冻融法4℃下解冻的样品比37℃下解冻的样品溶液中可溶性蛋白的含量要高,冻融次数与可溶性蛋白含量之间存在正相关。两种方法联用时的破壁效果要好于单独使用一种方法。采用搅拌法破壁时,溶液中可溶性蛋白含量随着搅拌的进行含量增加,但是增速逐渐减缓,并且在搅拌时间为3h时,得到蛋白的量为25.8mg/m L。采用Box-Behnken法对碱性蛋白酶制备螺旋藻抗氧化肽的工艺条件进行优化,优化过程以DPPH自由基清除能力与收率为响应值。结果表明,相较于木瓜蛋白酶、胰蛋白酶与风味蛋白酶,碱性蛋白酶对螺旋藻的水解能力最强,其最适水解条件为温度55.46℃、pH 6.71、固液比为1:10.83(v/w)。在此条件下,酶解240min,螺旋藻肽的得率为58.50%,与优化前相比,提高了15.61%。经此法可获得具有明显抗氧化活性的螺旋藻肽,浓度为0.86g/L时,其DPPH自由基清除能力为77.60%,与优化前相比,提高了7.62%。采用中心组合实验对双酶水解螺旋藻制备螺旋藻抗氧化肽的生产工艺进行优化,响应值为DPPH自由基清除能力及收率。研究结果表明,双酶法的较佳工艺为温度54.89℃,碱性蛋白酶与木瓜蛋白酶的比为2.23:1,酶解时间6.53h,此时,理论上DPPH自由基清除能力与收率的最大值分别为78.00%和57.94%。水解度对螺旋藻肽的起泡性、乳化性及抗氧化能力有影响。研究结果表明,螺旋藻肽的水解度随着水解的进行逐渐增大。水解过程中,螺旋藻肽的起泡性、DPPH自由基清除能力、以及还原力均随着水解度的增加先增大后减小,羟基自由基清除能力随着水解的进行先减小后增大,达到最大后再减小。而泡沫稳定性、乳化性、乳化稳定性则随着水解度的增大逐渐减小。酶解过程中的色素随着水解的进行逐渐积累,适当添加抑制剂或者吸附剂可以有效的抑制色素的产生。水解开始时加入1.5%的H2O2,水解结束时色素抑制率可达31.07%;NaClO加入量为1.5%时,色素抑制率为21.47%;当加入3%的活性炭时,色素抑制率可达27.29%。以制得的螺旋藻肽为原料配制螺旋藻肽功能饮料。实验结果表明,在单因素实验的基础上,采用正交试验对饮料配制方法进行优化。对饮料品质影响的顺序依次为螺旋藻肽量、柠檬酸量、白砂糖量。获得的最佳配制方法为:每100mL螺旋藻肽饮料中添加螺旋藻肽0.5g,柠檬酸0.05g,白砂糖3g。更多还原

【Abstract】 Spirulina is a kind of widely used functional food raw material, which was rated by FAO as ‘human best health care food of 21 st century’ and ‘super nutritious food of the future’(Zhangwen et al., 2013). Spirulina is rich in phycobiliprotein, whose content is as high as 50-70% and amino acids composition is very ideal. The contents of the 8 kinds of essential amino acids is close to or more than the FAO’s recommended standard. Spirulina can reduce the incidence of high cholesterol, high cholesterol, cardiovascular disease, inflammatory diseases, cancer and viral infections. Spirulina can also be used as a functional component in food manufacturing industry(Hemamalini, 2013). However, due to shortcomings of poor solubility and strong smell, the applications of Spirulina are subject to certain restrictions.The physical and chemical properties of phycobiliprotein can be improved and the scope of application can be expanded by using protease to hydrolysis. Compared with the phycobiliprotein, the hydrolysate of Spirulina peptide not only has lower molecular weight and good physical and chemical properties, more easily digested and absorbed by the body, but also enhance the body’s immune function, eliminate and prevent the body from free radical damage, comprehensive regulation of the metabolism of human body, physiological function, especially the capacity of antioxidant which draws people’s attention. In this paper, the method of breaking the Spirulina wall was studied. Also, optimization of hydrolysis conditions for antioxidant capacity of peptides from Spirulina using single and double enzyme method was discussed. The effect of degree of hydrolysis on emulsification, foaming capacity and antioxidant activity of Spirulina peptides and the pigment’s production and prevention of the process of enzymatic hydrolysis and the preparation of Spirulina peptide functional beverage were researched.The methods of stirring, ultrasonic, repeat freezing and thawing can make the Spirulina wall broke. The results showed that, when the broken method was ultrasonic in the condition of the same ultrasonic mode, ultrasonic powder 5%, ultrasonic 7s and interval 3s, the content of soluble of protein in the cell can reach the highest. The repeat freezing and thawing, the sample thawed under 4℃ has more soluble protein than the sample thawed under 37℃, and the content of soluble protein increased gradually with the increase of the number of freeze-thaw cycles. When the two methods are combined, the effect is better than using a single method alone. There is a linear relation between the content of soluble protein in solution and stirring time when the method was stirring, the content of soluble protein was increased, but the growth rate gradually slowed down. The soluble protein content was 25.8mg/m L when the stirring time was 3h.The hydrolysis conditions of antioxidant peptides from Spirulina powder using Alcalase were optimized by single factor methods and the Box-Behnken experiments, to improve the antioxidant abilities and the yield of Spirulina peptides. The results showed that Alcalase has the highest hydrolysis ability compared with other commercial proteases, incluing papain, trypsase and flavourzyme, and it’s optimum hydrolysis conditions were 55.46℃, pH6.71 and the solid liquid ratio was 1:10.83(v/w). When the Spirulina powder were hydrolyzed under the optimized conditions, the yield of the Spirulina peptides was 58.50%, increasing by 15.61% compared with the control. The DPPH radical scavenging ability of Spirulina peptides reached 77.60%, at the mass fraction of Spirulina peptides was 0.86g/L, increased by 7.62% compared with the control.The hydrolysis conditions of antioxidant peptides from Spirulina powder using double enzyme method were optimized by the Central Composite designs. The results showed that the optimum hydrolysis conditions of the double enzyme method was 54.89℃, the ratio of alkaline protease to papaya protease was 2.23:1, and the time was 6.53 h. When the Spirulina powder were hydrolyzed under the optimized conditions, the yield and the DPPH radical scavenging ability of the Spirulina peptides was 57.94% and 78.00%.The effect of the degree of hydrolysis(DH) on the emulsification, foaming capacity and antioxidant activity of Spirulina peptides was investigated. The DH of the hydrolysate increased during the process of hydrolysis. In the process of hydrolysis, the foaming activity, DPPH radical scavenging ability, hydroxyl radical scavenging ability and reducing power of Spirulina peptides first increased then decreased with the increase of the DH, however the foaming stability, the emulsification, emulsifying stability decreased with the increase of the DH.The pigment was gradually accumulation in the process of enzymatic hydrolysis, add inhibitor or adsorbent appropriate can effectively inhibit the production of pigment. When the hydrolysis was complete, the amount of H2O2 addition was 1.5%, the inhibition rate of pigment was 31.07%; the amount of NaClO addition was 1.5%, the inhibition rate was 21.47%; the amount of active carbon was 3%, and the inhibition rate was 27.29%.In this article the formular of Spirulina peptides beverage in which the Spirulina peptide powder was the main raw material was researched. The studied showed that on the basis of single factor experiment, orthogonal experiment was used to optimize the preparation of the beverage. The factors that influenced the quality of beverage was the Spirulina peptide, the amount of citric acid and the amount of white granulated sugar. The best formula was like this: 0.5g Spirulina peptide powder, 0.05 g citric acid and 3g white granulated sugar in soybean peptide beverage per 100 mL.更多还原