【作者】 陈慧芳；

【导师】 赵雷； 王莉嫦；

【作者基本信息】 华南农业大学 ， 工程硕士（专业学位）， 2020， 硕士

【摘要】 荔枝享有“岭南佳果”和“果中珍品”等美誉,有多种功效。但是,国内外的部分消费者在食用一定量的荔枝后出现“上火”反应,包括牙龈肿痛,口腔溃疡,发烧和发炎等症状,在一定程度上影响了消费者的健康。关于荔枝引起“上火”不良反应的原因目前仍不清楚。本研究以荔枝果肉为原料,通过提取、分离、纯化得到高纯度荔枝类甜蛋白(Litchi Thaumatin-like Protein,Lc TLP),并对其结构进行鉴定,通过建立巨噬细胞系RAW264.7炎症模型对促炎活性进行了评价。在此基础上模拟荔枝干制,探讨了不同加热时间对Lc TLP与果糖糖基化后复合物的结构以及促炎活性的影响,对构效关系进行了深入的探讨。主要研究结果如下:(1)采用低温法提取荔枝粗蛋白,通过透析、硫酸铵沉淀等得到了高纯度的蛋白。采用毛细管高效液相色谱仪联用电喷雾-组合型离子阱质谱仪(nano-LC-MS/MS)鉴定为荔枝类甜蛋白(Lc TLP),并通过对Lc TLP基因测序进行了验证。Lc TLP的分子量为24 k Da,其主要二级结构为β-折叠(33%)。采用小角X射线散射(SAXS)解析了Lc TLP在溶液中的构象,结果表明,Lc TLP在溶液中是一个内部紧密,表面粗糙的球状结构。(2)Lc TLP的体外促炎活性实验结果表明:在2.5～10.0 ng/m L浓度范围内,Lc TLP对小鼠巨噬细胞具有一定的细胞毒性作用。Lc TLP(2.5～10.0 ng/m L)显著促进了NO的分泌,提高了诱导型一氧化氮合酶(i NOS)和环氧合酶2(COX-2)m RNA的表达量;同时,Lc TLP(2.5～10.0 ng/m L)也促进了肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)白介素-6(IL-6)的基因表达量和分泌量的增加。(3)模拟荔枝干制过程,采用湿热法对Lc TLP和果糖进行糖基化反应,在恒温70℃,探究了不同加热时间(0 h,3 h,6 h,12 h,24 h,48 h),对美拉德反应程度和蛋白多糖复合物结构的影响。结果表明随着加热时间的延长糖基化程度越大,接枝度从2.02%(3 h)增加到28.64%(48 h)。反应过程中Lc TLP-果糖复合物的二级结构所占比例发生变化,α-螺旋、β-转角以及无规卷曲发生了向β-折叠转变。糖基化过程中,Lc TLP-果糖复合物的粒子的尺寸随加热时间的增加而增加,平均粒径从628 nm(3 h)增加为897 nm(48 h),蛋白质发生了一定程度的聚集。在加热过程中,Lc TLP-果糖复合物的内结构发生了解折叠,由表面粗糙的球状粒子逐渐向无序状态转变。(4)糖基化后的Lc TLP(M-Lc TLP)的体外促炎活性实验结果表明:在2.5～10.0ng/m L浓度范围内,M-Lc TLP对小鼠巨噬细胞无明显的细胞毒性作用(细胞活力>90%以上)。与Lc TLP相比,M-Lc TLP显著(P<0.05)降低了NO含量的分泌,抑制率达到65.25%。显著降低了COX-2和i NOS的基因表达量,同时也显著降低了促炎因子TNF-α、IL-1β、IL-6的表达量和分泌量。更多还原

【Abstract】 Litchi has the reputation of "Fruit of The Treasures",and contains many bioactive components.However,some people experience adverse reactions after eating a certain amount of litchi,including swelling of the gums,oral cavity ulcers,fever and inflammation,which affect the health of the individuals to a certain extent.Some key scientific issues regarding the effects of litchi on health are still unclear.The objective of this study was to extract and purify Litchi Thaumatin-like Protein(Lc TLP)from the pulp tissues of litchi,their structure was identified and the macrophage cell line RAW264.7inflammation model was established to evaluate the pro-inflammatory activity.On this basis,the effect of different heating time on the structure and pro-inflammatory activity of Lc TLP after glycosylation was further studied,and the relationship of structure and activity were discussed in depth.The main results are as follows:(1)The litchi crude protein was extracted from fresh litchi pulp by low-temperature method,and the high-purity protein was obtained through dialysis and stepwise ammonium sulfate precipitation.This protein was identified as litchi thaumatin-like protein(Lc TLP)by nano LC-MS/MS and verified by sequencing of the Lc TLP gene.The molecular weight of Lc TLP is 24 k Da,and main secondary structure is β-sheet(33%).The conformation of Lc TLP in solution was analyzed by small-angle X-ray scattering(SAXS).The results show that Lc TLP was a spherical particle with a close internal and rough surface in solution.(2)The assay of pro-inflammatory activity in vitro revealed that Lc TLP had a certain cytotoxic effects within the concentration range of 2.5～10.0 ng/m L on murine macrophage cells.Lc TLP(2.5～10.0 ng/m L)significantly promoted the secretion of NO and increased the expression of inducible nitric oxide synthase(i NOS)and cyclooxygenase 2(COX-2)m RNA,also promoted the gene expression and production of pro-inflammatory cytokines,including tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6).(3)Using heating in solution method to carry out glycosylation reaction of Lc TLP and fructose at a constant temperature of 70 °C,the effect of different heating time(0 h,3 h,6 h,12 h,24 h,48 h)on the Maillard reaction degree and structure of Lc TLP-frucose conjugate was explored.The results showed that the greater the degree of glycosylation with the extension of the reaction time,the grafting degree of Lc TLP-frucose conjugate increased from 2.02% at 3 h to 28.64% at 48 h.The proportion of the secondary structure of Lc TLP-frucose conjugate also changed,the α-helix,β-turn,and random coil had transformed to β-sheet.During glycosylation,the average size of the Lc TLP-frucose conjugate particles increases with increasing heating time,the average particle size increases from 628 nm at 3 h to 897 nm at 48 h,and a certain degree of protein aggregation occurred.During the heating process,the internal structure of Lc TLP-frucose conjugate undergoes unfolding,and the spherical particles with a rough surface gradually changed to a disordered state.(4)The results of in vitro pro-inflammatory activity of showed that glycosylated Lc TLP(M-Lc TLP)had no cytotoxic effects within the concentration range of 2.5～10.0ng/m L on cells(cell viability > 90% the above).Compared with Lc TLP,M-Lc TLP significantly(P < 0.05)reduced the secretion of NO content,and the inhibition rate reached65.25%.It also significantly inhibited the gene expression of COX-2 and i NOS,and reduced secretion of pro-inflammatory factors TNF-α,IL-1β and IL-6.更多还原