【作者】 孙艳辉；

【导师】 董英；

【作者基本信息】 江苏大学 ， 食品科学， 2007， 博士

【摘要】 脂质氧化是含脂食品在加工贮藏期间品质劣变的主要原因,添加抗氧化剂能够减少异味产生、抑制营养损失和延长货架期。然而,合成抗氧化剂具有潜在的危害性,使得人们转向开发天然抗氧化剂;同时,由于人们对健康的追求,需要补充外源性抗氧化剂,这就更加促使人们研究开发高效、安全、多功能的天然抗氧化剂。水飞蓟粕为中药水飞蓟籽提取水飞蓟素后剩余的残渣,含有丰富的蛋白质、糖类和黄酮类物质等,营养丰富。本研究以制备抗氧化剂为目的,从水飞蓟粕中提取总黄酮,大孔树脂分离纯化;然后以提取黄酮后的残渣为原料提取多糖类物质;以提取黄酮后的残渣为原料经纳豆菌发酵制备抗氧化肽,并对菌泥进行干燥制备菌粉,实现了综合利用,延长了水飞蓟加工链,提高了农产品的附加值。1.首次采用同步荧光评价冷藏熟肉糜的氧化状态:熟肉糜0.5g,加入0.02%的Vc溶液10ml,超声强档提取5min,固定△λ70nm,激发狭缝宽度10nm、发射狭缝宽度10nm条件下,测定提取液在326nm处的同步荧光强度。为提高筛选抗氧化剂效率,建立了抗氧化剂对冷藏熟肉糜脂质氧化抑制率的方法:4℃冷藏、时间为3d、以抑制同步荧光强度为指标,方法简单,灵敏准确。2.通过单因素试验和正交试验确定水飞蓟粕总黄酮最佳提取工艺为:水飞蓟粕粉碎过20目筛、液料比为20、提取温度70℃、乙醇浓度60%、提取3h,提取率达到91.9%;通过静态吸附和解吸试验,优选得到纯化水飞蓟粕总黄酮的大孔树脂DM-130,分离纯化的最优工艺为:200mL浓度为16mg/mL的水飞蓟粗黄酮溶液,上样速度为2mL/min,然后用8倍床体积水洗去杂质,4倍床体积80%乙醇以0.9mL/min流速进行洗脱,纯化后,总黄酮纯度由24.32%上升到78.14%。水飞蓟粕粗黄酮和精黄酮抑制冷藏熟肉糜脂质氧化的EC₅₀值分别为0.2659mg/mL和0.1331mg/mL。3.通过2—level Factorial Design和响应面设计确定脉冲超声辅助提取水飞蓟多糖最优工艺为:水飞蓟粕渣粒度60目、占空比0.6、温度45℃、液料比25、全程提取时间43min、超声功率550W,水飞蓟多糖提取率为4.06%;以蛋白脱除率、多糖损失率和清除DPPH自由基能力为指标优化得到水飞蓟多糖脱蛋白工艺为:浓度为1%的粗糖液,用2mol/L盐酸调节pH为2,静置过夜离心,连续操作两次,蛋白脱除率为99.9%、多糖损失率为18.1%、糖液清除DPPH自由基的能力为40.3%。乙醇沉淀分级多糖得到四个组分。水飞蓟多糖抑制冷藏熟肉糜脂质氧化的EC₅₀值为11.028mg/mL,各组分的抑制效果都比水飞蓟总多糖的低。4.首次采用发酵法制备水飞蓟抗氧化肽。通过单因素试验和正交试验得到培养基的最优组合是:10%水飞蓟粕渣、0.6%葡萄糖、0.03%CaCl₂和0.05%MgSO₄。通过Plackett—Burman试验设计和响应面设计确定了最优发酵工艺为:摇床转速180rpm、种龄16h、接种量5%、装液量60mL（500mL）、发酵温度36.5℃、初始pH6.9、发酵时间28.5h,肽转化率为60.96%。5升发酵罐扩大培养,肽转化率为62.47%。超滤结果表明:分子量小于10kDa的肽对冷藏熟肉糜（添加量1%）脂质氧化抑制率为75.16%;分子量小于3kDa的肽对冷藏熟肉糜（添加量1%）脂质氧化抑制率为77.59%。综合考虑,选用分子量小于10kDa的肽为抗氧化肽。采用喷雾干燥将分子量小于10kDa的超滤液制成肽干粉,肽含量为95.14%,其对冷藏熟肉糜（添加量1%）脂质氧化抑制率为73.28%。水飞蓟肽抑制冷藏熟肉糜脂质氧化的EC₅₀值为5.28mg/mL。5.体外试验表明,BHT、水飞蓟粕精黄酮、水飞蓟多糖和水飞蓟肽清除DPPH自由基的EC₅₀值分别为0.992、0.527、9.468和4.811mg/mL;10mg/mL的BHT、水飞蓟粕精黄酮、水飞蓟多糖和水飞蓟肽的总抗氧化能力分别为12.77、9.27、1.86、2.94mmol/L FeSO₄;10mg/mL的水飞蓟精黄酮、多糖和水飞蓟肽螯合金属离子能力分别为0.049、0.028和0.115mg/mLNa₂EDTA;熟肉糜冷藏7d试验表明,添加0.04%水飞蓟粕精黄酮、3%水飞蓟多糖、1.5%水飞蓟肽和添加0.02%BHT对熟肉糜冷藏过程中氧化抑制作用相当。冷藏过程中熟肉糜TBARS、羰基含量、非血红素铁含量和同步荧光强度彼此高度相关,证明同步荧光强度可以用来衡量冷藏熟肉糜的氧化状态。6.首次采用同步荧光评价体外蛋白氧化损伤程度的方法:固定△λ30nm,激发狭缝宽度20nm、发射狭缝宽度20nm条件下,测定牛血清蛋白溶液在404nm处的同步荧光强度。方法操作简单、方面快捷、精密度高。体外蛋白氧化损伤抑制作用试验表明:水飞蓟肽是通过螯合金属离子和清除自由基共同作用抑制蛋白质的氧化损伤,水飞蓟粕精黄酮是通过清除自由基抑制蛋白氧化损伤;0.03%水飞蓟粕精黄酮、1%水飞蓟肽对蛋白氧化损伤的抑制作用分别和0.03%的Vc相当,水飞蓟多糖抑制蛋白氧化损伤作用较弱。更多还原

【Abstract】 Lipid oxidation is the main cause of deterioration of fat food during processing and storage.Adding antioxidants can reduce off flavor,inhibit nutrition loss and prolong shelf life.However,synthetic antioxidants have potential damage.This leads to develop natural antioxidants.Meanwhile,because of people’s pursue for health,supply of exogenous antioxidants becomes very necessary.These make people research for high efficiency,safety and multifunctional natural antioxidants.Milk thistle meal,residue of herb milk thistle seeds after extracting silymarin, containing abundant protein,carbohydrates and flavonoids,and it has great nutrition value.The objective of this study was to prepare antioxidants,extracting total flavonoids from milk thistle meal,separated by macroporous resin;extracted polysaccharide from the residue of milk thistle meal;then prepared antioxidative peptides by fermenting the residue of milk thistle meal with Bacillus Natto,and desiccated bacterial sludge to powder.This work achieved comprehensive utilization, extended manufacture chain,and improved added value of agricultural product.A method for evaluating oxidation status of refrigerated cooked ground pork was established by synchronous fluorescence.10mL0.02%Vc solution was added to 0.5g cooked ground pork,and then extracted by ultrasonic with strong class,△λwas 70nm, excitation slit width was 10nm,emission slit width was 10nm,determine synchronous fluorescence intensity of samples at 326nm.In order to improve screening efficiency,a method for determining inhibitory rate of antioxidants on lipid oxidation of refrigerated cooked ground pork was established,stored at 4℃for 3 days,inhibited synchronous fluorescence intensity as index.This method was simple,sensitive and accuracy.Milk thistle meal total fiavonoids were extracted by 60%ethanol and the extraction rate was 91.9%.Macroporous resin DM-130 was chosen to purity the total flavonoids by static adsorption and desorption.The best separated technology was that 200mL total flavonoids solution was loaded at 2mL/min rate,8 times of bed volume water was used to wash impurity,then eluated at flow rate 0.9mL/min by 4 times of bed volume 80%ethanol.As a result,its purity increased from 24.32%to 78.14%.The EC₅₀ of milk thistle meal crude flavonoids and refined flavonoids inhibitory rate on lipid oxidation of refrigerated cooked ground pork were respectively 0.2659mg/mL and 0.1331mg/mL. Polysaccharide was extracted from the residue of milk thistle meal assisted by pulsed ultrasonic.The best result of extraction ratio 4.06%was obtained by 2—level Factorial Design and Response Surface Methodology under the optimized condition, granularity was 60 mesh.temperature was 45℃,the ratio of liquid to solid was 25,the extracting time was 43 minutes,,ultrasonic power was 550 W.The deproteinization technology was achieved by comparing the percentage of the deproteinization and losing polysaccharide and the capacity of polysaccharide solution scavenging DPPH free radical.The results showed that,adjusting the pH value of 1%polysaccharide solution to 2 by HCl,the sample was placed stably for a night and then centrifugaled, repetitive operation twice,the percentage of the deproteinization was 99.9%,the percentage of losing polysaccharide was 18.1%and the capacity of polysaccharide solution scavenging DPPH free radical was 40.3%.The crude polysaccharide was fractionated with ethanol repeatedly and four fractions were obtained.The EC₅₀ of milk thistle polysaccharide inhibitory rate on lipid oxidation of refrigerated cooked ground pork was 11.028mg/mL.The inhibitory rate of all fractions were lower than that of milk thistle polysaccharide。Milk thistle antioxidative peptides were prepared by fermentation method at first time.The best composition of culture medium was obtained by single factor and orthogonal design which composed of 10%the residue of milk thistle meal,0.06% glucose,0.03%CaC12 and 0.05%MgSO4.The best result of the peptide conversion ratio 60.96%was obtained by Plackett-Burman design and Response Surface Methodology under the optimized condition,the shaking speed was 180rpm,seed culture time was 16h,inoculation volume was 5%,the volume of medium in 500ml flask was 60ml,temperature was 36.5℃,initial pH value was 6.9,fermentation time was 28.5h.The peptide conversion rate was 62.47%by 5 liter fermentor.The ultrafiltration results showed that the inhibitory rate of peptides whose molecular weight were less than 10k Da on lipid oxidation of refrigerated cooked ground pork was 75.16%,while that of peptides having a molecular weight of less than 3k Da was 77.59%.Consider synthetically,the peptide having a molecular weight of less than 10k Da was chosen as antioxidative peptide.Antioxidative peptides were made into powder by spray drying technic,where peptide content was 95.14%,and it’s inhibitory rate on lipid oxidation of refrigerated cooked ground pork was 73.28%and EC₅₀ was 5.28mg/L.The results of in vitro experiments showed that the EC₅₀ of scavenging DPPH radical of milk thistle meal purified flavonoids,polysaccharides and peptides were 0.992,0.527,9.468 and 4.811mg/mL,total antioxidative capacity of 10mg/mL BHT, milk thistle meal purified flavonoids,polysaccharides and peptides were equal to 12.77、9.27、1.86、2.94mmol/L FeSO4 respectively,chelating metal ions capacity of 10mg/mL BHT,milk thistle meal purified flavonoids,polysaccharides and peptides were equal to 0.049、0.028 and 0.115mg/mL Na₂EDTA respectively.The results of cooked ground pork experiment during 7 days refrigerated storage showed that the inhibitory effect on oxidation of adding 0.04%milk thistle meal purified flavonoids,3%polysaccharides and 1.5%peptides was equivalent to that of 0.02%BHT.TBARS content,carbonyls content,non heme iron content had great correlation with synchronous fluorescence intensity,and this proved that synchronous fluorescence intensity could evaluate the oxidative stress of cold-storage cooked ground pork.A method for evaluating protein oxidation damage in vitro by synchronous fluorescence was established.△λwas 30nm,excitation slit width was 20nm,emission slit width was 20nm,determined synchronous fluorescence intensity of BSA solution at 404nm.The method was simple,fast and sensitive.The results of inhibitory effect on protein oxidative damage in vitro showed milk thistle peptide inhibited oxidative damage by chelating metal ions and scavenging free radical,while milk thistle purified flavonoids inhibited oxidative damage by scavenging free radical.The inhibitory effect of milk thistle polysaccharide was weak.The inhibitory effect on protein oxidative damage of 0.03%milk thistle meal purified flavonoids and 1%peptides was equivalent to that of 0.03%Vc.更多还原