【作者】 邱霞；

【导师】 钟进义；

【作者基本信息】 青岛大学 ， 营养与食品卫生学， 2013， 博士

【摘要】 目的建立以麒麟菜为原料制备低分子海藻多糖的方法,对制备样品的粘度、凝胶强度和硫酸酯质量分数等理化特性进行检测和分析,用液相色谱-质谱联用技术和高效凝胶色谱法测定分子量,并对麒麟菜低分子海藻多糖的消化吸收及其对食物的消化吸收,对机体抗氧化与免疫调节功能的影响等生物活性进行研究。方法1麒麟菜低分子海藻多糖的制备：1.1清洗切割粉碎：将麒麟菜用自来水清洗晾干后,横向切割成薄片状。1.2酸液二级水解：将上述麒麟菜切片置于pH值为2.5的盐酸溶液中浸泡进行一级水解,过滤收集麒麟菜切片置于蒸馏水中冲洗后,再置于pH值为4.5的盐酸溶液中浸泡,进行二级水解,过滤收集麒麟菜切片置于蒸馏水中冲洗。1.3碱液皂化去脂：将收集的麒麟菜切片置于pH值为8的碱性溶液中浸泡,过滤收集麒麟菜切片,置于蒸馏水中冲洗。1.4干燥研磨粉碎：将上述收集的麒麟菜切片置于烘干机中干燥后,放在粉碎机中研磨成粉末状过筛,收集筛过的粉末,即为麒麟菜低分子海藻多糖。1.5样品的提取得率：提取得率(%)=样品(g)÷麒麟菜干重(g)×1002麒麟菜低分子海藻多糖样品理化特性的检测分析2.1样品的感官指标及粘度、凝胶强度、硫酸酯质量分数、水分含量和总灰分等指标的检测分析。2.2应用高效凝胶色谱法和液相色谱-质谱联用技术测定样品的分子量。3空腹小鼠对麒麟菜低分子海藻多糖的消化吸收状况研究：将30只昆明种小鼠随机分为正常对照组、卡拉胶对照组和实验组(麒麟菜低分子海藻多糖组),禁食12h后,卡拉胶对照组和实验组动物分别给予0.09g/mL的卡拉胶溶液和麒麟菜低分子海藻多糖溶液灌胃,每次1mL,每3.5hl次,连续4次。正常对照组则只给予相同剂量的蒸馏水。收集各组小鼠粪便并称量湿重及干重,计算食物消化吸收率。眼球后静脉丛取血检测血清D-木糖含量。4麒麟菜低分子海藻多糖对小鼠正常进食消化吸收的影响：将30只昆明种小鼠随机分为正常对照组、卡拉胶对照组和实验组(麒麟菜低分子海藻多糖组)共3个组,各组均喂食普通饲料。卡拉胶对照组和实验组的动物在每天早晚用0.05g/mL的卡拉胶溶液和麒麟菜低分子海藻多糖溶液各1mL灌胃,正常对照组灌相同体积蒸馏水,每天2次,连续7天。检测小鼠体重、摄食量、粪便干重和湿重,并根据以下公式计算食物利用率和吸收率：食物利用率(%)=(最终体重-初始体重)÷实验期间摄食量×100,食物吸收率(%)=(实验期间摄食量—实验组粪便干重)÷实验期间摄食量×100。5麒麟菜低分子海藻多糖对大鼠的高脂膳食消化吸收及抗氧化与免疫调节功能的影响：将50只Wistar大鼠随机分为正常对照组、高脂膳对照组、卡拉胶对照组3个对照组和麒麟菜低分子海藻多糖的低、高剂量组,连续喂养30天,称量各组动物的体重、摄食量、粪便干重与湿重,计算食物利用率和吸收率。测定小肠粘膜Na+-K+-ATP酶活性、血糖、血脂、谷胱甘肽过氧化物酶(GSH-Px)活性,丙二醛(MDA)含量,氧化低密度脂蛋白(ox-LDL)含量,白细胞介素2(IL-2)水平,肿瘤坏死因子α(TNF-α)水平和脾淋巴细胞增殖活性等指标。结果1麒麟菜低分子海藻多糖的制备：建立了麒麟菜低分子海藻多糖的制备方法,制备了足量实验样品,测得麒麟菜低分子海藻多糖的提取得率为48.31%。2麒麟菜低分子海藻多糖的感官与主要理化指标：麒麟菜低分子海藻多糖样品为浅褐色粉末,溶于水,凝胶强度为10g/cm2,粘度为10mPa·s,硫酸酯质量分数为26%。样品的凝胶强度低于卡拉胶,粘度处于卡拉胶的国家标准最低限,而硫酸酯质量分数在卡拉胶的国家标准15%-40%的范围内。3麒麟菜低分子海藻多糖的分子量：高效凝胶色谱法测定样品的数均分子量为1199Da,液质联用(HPLC-MS)测定样品的分子量多集中在1000Da以下,较卡拉胶大幅度降低。4空腹小鼠对麒麟菜低分子海藻多糖的消化吸收状况：实验组的粪便干重和湿重分别为(0.168±0.029)g和(0.218±0.031)g,消化吸收率为(57.78±6.06)%,血清D-木糖含量为(2.71±0.42)mmol/L。粪便干重和湿重均低于卡拉胶组,而消化吸收率和血清D-木糖含量则均高于卡拉胶组,差异均有显著性意义(P<0.05)说明麒麟菜低分子海藻多糖较卡拉胶的消化吸收率升高,而粪便排出量和含水量降低,其原因可能与麒麟菜低分子海藻多糖的分子量和吸水性小于卡拉胶有关。5麒麟菜低分子海藻多糖对小鼠正常进食消化吸收的影响：实验组的粪便干重和湿重分别为(2.19±0.29)g/d和(3.10±0.31)g/d,均较正常对照组升高但低于卡拉胶组(P<0.05)。食物利用率和吸收率分别为(15.52±0.91)%和(39.17±3.45)%,均显著低于正常对照组但高于卡拉胶组(P<0.05)。说明麒麟菜低分子海藻多糖对正常食物的利用率和吸收率均有一定抑制作用,但抑制程度小于卡拉胶,其原因也与麒麟菜低分子海藻多糖的分子量和吸水性小于卡拉胶有关。6麒麟菜低分子海藻多糖对大鼠高脂膳食消化吸收的影响：麒麟菜低分子海藻多糖高剂量组的食物利用率和吸收率分别为(45.37±2.24)%和(52.13±4.49)%,餐后血糖为(6.19±0.70)mmol/L,血TG为(2.62±0.55)mmol/L,TC为(1.57±0.35)mmol/L。均低于高脂对照组但高于卡拉胶组(P<0.05)。麒麟菜低分子海藻多糖高剂量组的Na+-K+-ATP酶活性为(12.27±1.43)μmolpi·mgpro-1·h-1,明显低于高脂对照组和卡拉胶组(P<0.05)。说明麒麟菜低分子海藻多糖对高脂膳食的吸收利用有一定抑制作用,但作用程度低于卡拉胶,其可能机制与除麒麟菜低分子海藻多糖的分子量和吸水性小于卡拉胶以外,还与对小肠粘膜细胞酶活性的影响有关。7麒麟菜低分子海藻多糖对高脂膳食大鼠抗氧化和免疫调节功能的影响：麒麟菜低分子海藻多糖高剂量组大鼠的血清ox-LDL和MDA含量分别为(3.87±0.31)ng/ml和(5.63±0.63)nmol/ml,均较高脂对照组显著性降低(P<0.05)；GSH-Px活力为(1377.17±82.53)U/ml,较高脂对照组显著性升高(P<0.05)。说明麒麟菜低分子海藻多糖可降低高脂膳食大鼠的血清ox-LDL和MDA水平,提高GSH-Px活力,增强其抗氧化能力,降低脂质过氧化水平。麒麟菜低分子海藻多糖高剂量组血清IL-2为(814.17±25.53)pg/ml,脾淋巴细胞增殖活性OD值为(0.264±0.019),均较高脂对照组显著性升高(P<0.05);TNF-α水平为(141.70±43.76)pg/ml,较高脂对照组显著性降低(P<0.05)。说明麒麟菜低分子海藻多糖可提高高脂膳食大鼠的血清IL-2水平和淋巴细胞增殖活性,降低TNF-α水平,增强其免疫活性。结论综合本次实验结果可以认为：1本研究创建了麒麟菜低分子海藻多糖的制备方法,与目前用成品卡拉胶制备低分子海藻多糖的方法相比较,本方法具有工艺简便、提取得率高、资源充分利用等特点。对制备样品的检测分析结果显示,其分子量、凝胶强度、粘度等理化指标均与用成品卡拉胶制备的同类产品相一致。2麒麟菜低分子海藻多糖较卡拉胶容易被消化吸收,对小鼠普通进食的食物利用率和吸收率的抑制作用小于卡拉胶。3麒麟菜低分子海藻多糖可抑制大鼠对高脂食物的消化吸收,对高脂食物引发的血糖、血脂升高具有一定降低功效,但其作用程度低于卡拉胶。麒麟菜低分子海藻多糖降低小肠粘膜Na+-K+-ATP酶活性的作用高于卡拉胶。4麒麟菜低分子海藻多糖能增强高脂膳食大鼠的抗氧化能力,降低其脂质过氧水平,提高机体免疫力。其作用强度大于卡拉胶。5麒麟菜低分子海藻多糖类似于卡拉胶有一定的吸水膨胀性,可阻碍食物与肠粘膜细胞的接触而影响消化吸收,还因其分子量小而易被消化吸收,可进而产生某些生物活性。更多还原

【Abstract】 Objective:To establish a method taking Eucheuma as raw materials to prepare low-molecular-weight seaweed polysaccharides and detect its characteristics such as viscosity, gel strength and sulphate mass fraction. To determine its molecular weight with high performance liquid chromatography-mass spectrometry (HPLC-MS) and high performance gel permeation chromatography(GPC). To study on the absorption of low-molecular-weight seaweed polysaccharides,as well as its effect on food absorption and bioactivities such as anti-oxidant and immunomodulation functions.Methods:There are the preparation and analytical methods, as well as experimental methods on absorption and bioactivities.1Preparation of Eucheuma low-molecular-weight seaweed polysaccharides:1.1Clean and cut:Eucheuma was cut into slice form after cleaning by running water and open-air drying.1.2Twice hydrolyze in acid liquor:The collected Eucheuma slice was placed and hydrolyzed in the solution of hydrochloric acid (pH2.5). Then the collected slice was filtrated, placed and hydrolyzed in the solution of hydrochloric acid (pH4.5),too. The collected slice was filtrated and washed by distilled water once more time。1.3Saponification in lixivium:The collected Eucheuma slice was placed and saponified in alkaline solution (pH8). The collected slice was filtrated and washed by distilled water again.1.4Dry and ground:After the collected Eucheuma slice was dried in the dehydrator, it was placed in disintegrator and grounded to small particles. The powder was Eucheuma low-molecular-weight seaweed polysaccharides.1.5Extraction yield of sample: Extraction yield(%)=sample(g)÷Eucheuma dry weight(g)×1002Characteristic detection and analysis of sample:2.1Detection and analysis of oranoleptic indicator, viscosity, gel strength, sulphate mass fraction, humidity and total ash.2.2Determination of its molecular weight with high performance liquid chromatography-mass spectrometry (HPLC-MS) and high performance gel permeation chromatography. 3Study on digestion and absorption of Eucheuma low-molecular-weight seaweed polysaccharides in fasting mice:Thirty Kunming mice were randomly divided into normal control group, carrageenan control group and experimental group (Eucheuma low-molecular-weight seaweed polysaccharides group). After fasting and drinking for12h, the carrageenan control group and experimental group were intragastrically given0.09g/mL carrageenan and Eucheuma low-molecular-weight seaweed polysaccharides,1mL each time, once every3.5hours,4times continuously. An equivalence of distilled water was administered in the normal control group. Wet fecal weight and dry fecal weight were measured and food absorbtivity was calculated. The blood was obtained in venous plexus behind the eyeball to detect the content of serum D-xylose.4. The effect on digestion and absorption of Eucheuma low-molecular-weight seaweed polysaccharides in mice fed a normal diet:Thirty Kunming mice were randomly divided into three groups:normal control group, carrageenan control group and experimental group(Eucheuma low-molecular-weight seaweed polysaccharides group).The mice in all of groups were fed normal diet.The rats in carrageenan control group and experimental group were intragastrically given0.05g/mL carrageenan and Eucheuma low-molecular-weight seaweed polysaccharides,1mL each time, twice one day,7days continuously. An equivalence of distilled water was administered in the normal control group. The body weight, food consumption, wet fecal weight and dry fecal weight were measured. Food availability and food absorbtivity were calculated according to the below formula:food availability (%)=(the final body weight-the initiate body weight)÷food consumption in experimental session×100; food absorbtivity=(food consumption in experimental session-dry fecal weight in experimental group)÷food consumption in experimental session×100.5The effect on digestion and absorption, anti-oxidant and immunomodulation function of Eucheuma low-molecular-weight seaweed polysaccharides in rats fed a high-fat diet:Fifty Wistar rats were divided into five groups:normal control group, high-fat diet control group, carrageenan control group, low dose and high dose Eucheuma low-molecular-weight seaweed polysaccharides groups. All of the rats were continuously fed for30days.Body weight, food consumption,wet fecal weight and dry fecal weight were measured. Food availability and food absorbtivity was calculated. Na+-K+-ATPase activities were measured by spectroscopic analysis. Blood glucose, triglycerides(TG), total cholesterol(TC) and the activity of glutathione-peroxidase (GSH-Px), the content of malanydiadehyde (MDA), oxidized low-density lipoprotein (ox-LDL), interleukin-2(IL-2),tumor necrosis factor-a(TNF-a) and the proliferation of splenic lymphocyte were detected in each group.Results: 1Preparation of Eucheuma low-molecular-weight seaweed polysaccharides:The method to prepare low-molecular-weight seaweed polysaccharides was established and the sufficiency experimental sample was prepared. Extraction yield of the Eucheuma low-molecular-weight seaweed polysaccharides was48.31%.2The oranoleptic indicator and the main characteristic of Eucheuma low-molecular-weight seaweed polysaccharides:The sample of Eucheuma low-molecular-weight seaweed polysaccharides was sandy beige powder. It was dissolved in water.Its viscosity was10mPa·s and gel strength was below10g/cm2.The sulphate mass fraction of it was limited in15%-40%which was national standards of carrageenan.3The molecular weight of Eucheuma low-molecular-weight seaweed polysaccharides:Its number-average molecular weight which was determined by high performance gel permeation chromatography was1199Da.The most molecular weight of Eucheuma low-molecular-weight seaweed polysaccharides which was determined by high performance liquid chromatography-mass spectrometry (HPLC-MS) was under1000Da.The molecular weight was far below carrageenan.4The digestion and absorption condition of Eucheuma low-molecular-weight seaweed polysaccharides in fasting mice:The dry fecal weight and wet fecal weight of the experimental group were (0.168±0.029)g and (0.218±0.031)g, respectively. The food absorbtivity was (57.78±6.06)%. The conten of serum D-xylose was (2.71±0.42) mmol/L. The dry fecal weight and wet fecal weight of the Eucheuma low-molecular-weight seaweed polysaccharides group were lower than the carrageenan group,but the food absorbtivity and the conten of serum D-xylose were higher than the carrageenan group, significantly (P<0.05). These show that the food absorbtivity of Eucheuma low-molecular-weight seaweed polysaccharides is higher than that of carrageenan,but feces and fecal moisture capacity are lower than those of carrageenan. The cause is relevant with the lower molecular weight and hydrophilia of Eucheuma low-molecular-weight seaweed polysaccharides than carrageenan.5The effect on digestion and absorption of Eucheuma low-molecular-weight seaweed polysaccharides in mice fed a normal diet:The dry fecal weight and wet fecal weight of the experimental group were (2.19±0.29) g/d and (3.10±0.31) g/d, respectively. All of them were significantly higher than those of the normal control group,but lower than those of the carrageenan group (P<0.05).The food availability and food absorbtivity were (15.52±0.91)%and (39.17±3.45)%. All of them were significantly lower than those of the normal control group,but higher than those of the carrageenan group(P<0.05). These show that Eucheuma low-molecular-weight seaweed polysaccharides has the inhibitory action on food availability and food absorbtivity of a normal diet in mice, but the action degree is lower than carrageenan. The cause is relevant with the lower molecular weight and hydrophilia of Eucheuma low-molecular-weight seaweed polysaccharides than carrageenan, too.6The effect on digestion and absorption of Eucheuma low-molecular-weight seaweed polysaccharides in rats fed a high-fat diet:The food availability and food absorbtivity of Eucheuma low-molecular-weight seaweed polysaccharides high dose group were (45.37±2.24)%and (52.13±4.49)%. The postprandial blood sugar of the Eucheuma low-molecular-weight seaweed polysaccharides group was (6.16±0.70) mmol/L, the TG and TC were (2.62±0.55)mmol/L and (1.57±0.35)mmol/L, All of them were significantly lower than those of the high-fat control group,but higher than those of the carrageenan group(P<0.05). The Na+-K+-ATPase activity was (12.27±1.43) μmol pi·mg pro-1·h-1, which significantly lower than those of the high-fat control group and the carrageenan group(P<0.05). These show that Eucheuma low-molecular-weight seaweed polysaccharides has the inhibitory action on high-fat diet,but the action degree is lower than carrageenan.The possible mechanism is not only the molecular weight and hydrophilia is below carrageenan but also concerned with the effect of enzymic activity in mucous membrane of small intestine.7The effect of anti-oxidant and immunomodulation functions of Eucheuma low-molecular-weight seaweed polysaccharides in rats fed a high-fat diet:The content of serum ox-LDLand MDA of Eucheuma low-molecular-weight seaweed polysaccharides high dose group were (3.87±0.31) ng/ml and (5.63±0.63) nmol/ml. All of them were significantly lower than those of the high-fat control group (P<0.05). The GSH-Px activity of Eucheuma low-molecular-weight seaweed polysaccharides high dose group was (1377.17±82.53)U/ml which was significantly higher than those of the high-fat control group(P<0.05).These show that Eucheuma low-molecular-weight seaweed polysaccharides can decrease the content of serum ox-LDL and MDA, increase the GSH-Px activity, strengthen anti-oxidant ability and reduce the lipid peroxidation level.The content of serum IL-2of Eucheuma low-molecular-weight seaweed polysaccharides high dose group were (814.17±25.53) pg/ml and the OD value of the proliferation of splenic lymphocyte was (0.264±0.019). All of them were significantly higher than those of the high-fat control group (P<0.05). The TNF-α of Eucheuma low-molecular-weight seaweed polysaccharides high dose group was (141.70±43.76) pg/ml which was significantly lower than those of the high-fat control group (P<0.05). These show that Eucheuma low-molecular-weight seaweed polysaccharides can increase the content of serum IL-2and the proliferation of splenic lymphocyte, decrease the content of serum TNF-a, strengthen immunomodulation activities.Conclusion:The experimental results show the conclusion as follow: 1Our research has established the preparative method of Eucheuma low-molecular-weight seaweed polysaccharides taking Eucheuma as raw materials. Compared to the method of taking carrageenan as raw materials, the method have some characteristic such as convenient technology, making the best of resources and so on. The results of characteristic detection and analysis of preparative sample show that the molecular weight, viscosity, gel strength and so on are coincident with the same kind of product which is made from carrageenan.2The digestion and absorption of Eucheuma low-molecular-weight seaweed polysaccharides is easier than those of carrageenan. Eucheuma low-molecular-weight seaweed polysaccharides has the inhibitory action on food availability and food absorbtivity of a normal diet, but the action degree is lower than that of carrageenan.3Eucheuma low-molecular-weight seaweed polysaccharides can inhibit the digestion and absorption of a high-fat diet, decrease blood glucose and blood fat because of a high-fat diet, but the action degree is lower than carrageenan. Eucheuma low-molecular-weight seaweed polysaccharides can reduce Na+-K+-ATPase activity, the action degree is higher than carrageenan.4Eucheuma low-molecular-weight seaweed polysaccharides can strengthen the anti-oxidant ability, reduce the lipid peroxidation level, increase immunomodulation activities.5The action mechanism of Eucheuma low-molecular-weight seaweed polysaccharides like carrageen can take into water to expand, impede the contact of intestinal mucosa and food to affect digestion and absorption. Because its molecular weight is low and easy to be absorbed, the Eucheuma low-molecular-weight seaweed polysaccharides can produce some bioactivity.更多还原