Purification and Characterization of Buckwheat Bee Pollen Polysaccharide and Changes of Its Antioxidant Activity during in Vitro Digestion
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摘要: 本论文旨在研究荞麦蜂花粉多糖(BWPP)在模拟体外胃肠消化过程中的抗氧化活性。采用DEAE-52纤维素柱对粗多糖进行分级纯化,经体外模拟胃、肠消化研究其在体外消化过程中的抗氧化活性的变化规律。结果表明经DEAE-52纤维素柱层析洗脱纯化后得到总糖含量最高组分BWPP-1,其主要由Glc、Xyl、Ara等组成,且红外光谱显示BWPP-1具有明显的多糖特征吸收峰。BWPP和BWPP-1在进行体外模拟胃肠消化时,模拟胃液消化60 min时,BWPP和BWPP-1的ABTS+·清除率均达到最大值,分别为86.39%±1.28%、90.38%±2.78%,与其他消化时间的存在显著差异(P<0.05);60 min时对DPPH·清除率达到最大值,分别为86.86%±0.11%、89.58%±1.67%,且二者差异不显著(P>0.05);模拟肠液消化时,BWPP和BWPP-1对ABTS+·清除率在60 min时达到最大值为88.67%±0.40%和91.82%±2.77%,且二者差异不显著(P>0.05);对DPPH·清除率在240 min时达到最大值57.11%±0.06%和65.67%±3.67%,二者差异显著(P<0.05);而在消化过程中,对铁离子还原能力均较弱。由此可得,BWPP和BWPP-1具有较好的ABTS+·和DPPH·清除能力,且胃液消化时DPPH·清除能力略高于肠液消化,而肠液消化时的ABTS+·清除能力略高于胃液消化。本研究为以荞麦蜂花粉为基料的功能性产品的开发提供理论依据。Abstract: This paper aimed to investigate the antioxidant activity of buckwheat bee pollen polysaccharides (BWPP) during the in vitro gastrointestinal digestion. The crude polysaccharides were purified using DEAE-52 cellulose column and their antioxidant activity during in vitro simulated gastric and intestinal digestion was studied. The results showed that after purification by DEAE-52 cellulose column chromatography, the highest total sugar content component BWPP-1 was obtained, which was mainly composed of Glc, Xyl, Ara, etc. The infrared spectrum showed that BWPP-1 had obvious polysaccharide characteristic absorption peaks. During the simulated in vitro gastrointestinal digestion, the ABTS+· scavenging rates of BWPP and BWPP-1 reached their maximum values at 60 minutes of simulated gastric juice digestion, which were 86.39%±1.28% and 90.38%±2.78%, respectively. There was a significant difference compared to other digestion times (P<0.05). The DPPH· clearance rates reached their maximum values at 60 minutes, which were 86.86%±0.11% and 89.58%±1.67%, respectively, and the difference between the two was not significant (P>0.05). When simulating intestinal digestion, the maximum clearance rates of ABTS+· by BWPP and BWPP-1 reached 88.67%±0.40% and 91.82%±2.77% at 60 minutes, and the difference between the two was not significant (P>0.05). The DPPH· scavenging rates reached their peak values at 240 minutes of 57.11%±0.06% and 65.67%±3.67%, with significant difference (P<0.05). During the digestion process, the ability to reduce iron ions was relatively weak. Based on these findings, it can be concluded that BWPP and BWPP-1 have good ABTS+· and DPPH· scavenging abilities, and the DPPH· scavenging ability during gastric digestion is slightly higher than that during intestinal digestion, while the ABTS+· scavenging ability during intestinal digestion is slightly higher than that during gastric digestion. This study provides a theoretical basis for the development of functional products based on buckwheat bee pollen.
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荞麦是通辽地区的特色小杂粮作物,因其具有降三高作用,是近些年来研究的热点。荞麦蜂花粉是蜜蜂从荞麦花药中采集并混合自身的唾液及花蜜等腺体分泌物收集到的深黄色的,伴有强烈的动物气味的,具有麦芽质的香气和糖蜜味的物质[1]。蜂花粉作为荞麦的副产物,研究证实其富含多糖、脂肪酸、多酚等多种生物活性物质并具有药食同源性,具有降血糖、降血脂、抗氧化、抗肿瘤、抗辐射、提高免疫力等功能,在功能食品的应用中具有较大的潜力[2−4]。
已有的研究表明荞麦蜂花粉多糖具有多种生物活性,如抗氧化、降血糖、降血脂、提高免疫力、调节肠道菌群等[5−7]。钱明辉等[8]研究表明荞麦蜂花粉粗多糖经过3种凝胶色谱柱(DEAE-52离子交换柱、HW-55F、Sephacryl S-400凝胶柱)进行分离后得到一个单一组分多糖可以有效缓解STZ 糖尿病小鼠空腹血糖水平。李娟等[9]发现荞麦蜂花粉粗多糖能被人体肠道菌群酵解并产生短链脂肪酸,有利于人体健康。张萍等[10]研究发现,荞麦蜂花粉多糖对OH自由基的清除率为65.23%,高于对O2−·自由基的清除率。由此可见,目前关于荞麦蜂花粉多糖的抗氧化研究大多局限于粗多糖或纯化后多糖的抗氧化活性分析,而针对荞麦蜂花粉多糖在不同消化阶段的抗氧化活性及其变化规律的研究相对较少,且采用模拟胃肠消化过程中的抗氧化活性变化的研究也鲜见报道。
本文采用DEAE-52纤维素柱对荞麦蜂花粉多糖(BWPP)进行分级纯化得到BWPP-1组分,并对其进行结构解析,然后进一步考察BWPP-1组分经体外模拟胃、肠消化过程中的抗氧化活性变化规律,以期为荞麦蜂花粉的开发利用提供新的见解,并为荞麦蜂花粉多糖在功能性食品中的应用奠定理论基础。
1. 材料与方法
1.1 材料与仪器
荞麦蜂花粉 产地赤峰,甜荞,大黑三棱;石油醚(60~90 ℃)、无水乙醇、KBr(溴化钾)、 KH2PO4(磷酸二氢钾)、 FeSO4·7H2O(七水硫酸亚铁) 、盐酸、氯化钠 均为分析纯,天津大茂试剂有限公司;PMP(1-苯基-3-甲基-5-吡唑酮,99%) 分析纯,麦克林试剂;中性蛋白酶(Proteinase,50000.0 U/g)、果胶酶(Pectinase,≥50.0 U/g)、纤维素酶(Cellulase,≥15000.0 U/g)、胰蛋白酶(>250 U/mg)、胃蛋白酶(≥3000 U/mg) 均为生化试剂,国药集团化学试剂有限公司;DPPH自由基清除能力试剂盒、总抗氧化能力(T-AOC)检测试剂盒(ABTS法)、铁还原抗氧化能力(FRAP)试剂盒 南京建成生物工程研究所。
HL-2B数显恒流泵、SBS-160数控计滴自动部分收集器 上海沪西分析仪器有限公司;UV-5500PC紫外可见分光光度计 上海菁华科技仪器有限公司;infinite F50酶标仪 TECAN;粉末压片机 江苏金贝尔仪器;Agilent1100高效液相色谱仪、C18色谱柱(Agilent ZORBAX Eclipse Plus,5 µm,4.6 mm×150 mm) Agilent公司;Nicolet iS5傅里叶变换红外光谱 Thermo Fisher Scientific。
1.2 实验方法
1.2.1 荞麦蜂花粉粗多糖的提取
荞麦蜂花粉预处理:将荞麦蜂花粉粉碎过60目筛,60 ℃干燥2 h后备用。将干燥好的蜂花粉用过滤纸包好放入到粗脂肪测定仪中加入适量的石油醚(60~90 ℃)进行回流脱脂4 h。取出脱脂后的蜂花粉在60 ℃下干燥2 h,继续用80%的乙醇回流脱色6 h后并在60 ℃下干燥2 h后置于常温干燥避光的环境中保存备用[11−12]。
酶提醇沉法提取荞麦蜂花粉粗多糖:称取一定量经过预处理的蜂花粉按照料液比1:5(g/mL H2O),以蛋白酶:果胶酶:纤维素酶=1%:0.03%:0.03%加入复合酶并摇匀,在39 ℃下酶解反应12 h。4 ℃、4000 r/min的转速下离心10 min,取上清液,然后将上清液浓缩至原来体积的1/5,取出冷却至室温,按1:4(V:V)的比例加入乙醇浓度75%醇沉20 min(可根据溶液颜色醇沉2~3次),后将溶液以4 ℃、4000 r/min的转速离心10 min,收集沉淀冷冻干燥,得到粗多糖[13],并通过式(1)计算得率。
X(%)=MM0×100 (1) 式中,X为粗多糖得率,%;M为粗多糖质量,g;M0为荞麦蜂花粉的干基重量,g。
荞麦蜂花粉总糖含量测定:总糖含量按照苯酚-硫酸法测定[14−15]。以葡萄糖为标准品,以糖溶液浓度μg/mL为横坐标,吸光值为纵坐标,绘制葡萄糖标准曲线。
1.2.2 荞麦蜂花粉粗多糖的纯化及结构表征
1.2.2.1 荞麦蜂花粉粗多糖的分级纯化
采用DEAE-52阴离子交换柱层析洗脱法,分别用去离子水、0.1、0.2、0.3 mol/L浓度的NaCl溶液依次进行梯度洗脱[16],自动收集器以10 mL/管收集洗脱液。采用硫酸-苯酚法跟踪测定,以吸光度为纵坐标,收集管数为横坐标作图,得洗脱曲线。
1.2.2.2 荞麦蜂花粉多糖的结构表征
a.在200~400 nm范围内对多糖溶液进行紫外光谱扫描。
b.精确称取1.0 mg多糖试样,加入干燥的KBr(溴化钾)50.0 mg研磨制成均匀的透明压片,在400~4000 cm−1范围内,采用傅里叶红外光谱仪进行扫描分析[17]。
c.参考文献[18−19],将多糖采用三氟乙酸(TFA)进行水解,经PMP衍生化的产物过0.45 μm微孔膜(水系)后供 HPLC进行多糖组成成分分析。色谱柱:Agilent ZORBAX Eclipse Plus C18,5 µm,4.6 mm×150 mm;流动相A:0.06 mol/L磷酸缓冲溶液(pH6.9);流动相B:乙腈;流速:1.0 mL/min;进样量:10 µL;检测波长:254 nm;柱温:30 ℃;进行梯度洗脱,具体洗脱程序如表1所示。
表 1 洗脱梯度Table 1. Elution gradient时间(min) 流动相A(%) 流动相B(%) 0 86 14 9 83 17 28 78 22 29 50 50 31 50 50 32 86 14 36 86 14 1.2.3 荞麦蜂花粉多糖BWPP和纯化组分BWPP-1体外模拟胃肠消化的抗氧化活性测定
1.2.3.1 体外模拟消化液的收集与配制
唾液收集与制备:遴选20名志愿者(健康,3个月内未服用抗生素),晨起空腹状态下漱口后将其初始唾液舍弃后按照30 s/次开始进行收集,将收集到的唾液于4 ℃、4000 r/min条件下离心30 min,取上清液保存备用[20]。
人工模拟胃液制备:称取0.800 g胃蛋白酶、4.375 g NaCl,用去离子水溶解并定容至250 mL,用3.0 mol/mL HCl溶液调pH至1.2,置于4 ℃冰箱中保存备用[20]。
人工模拟肠液制备:称取68 g KH2PO4,用去离子水溶解并定容至500 mL,用质量分数0.4% NaOH溶液调pH至6.8;准确称取1.0 g胰蛋白酶,用蒸馏水定容至100 mL,并用4% NaOH溶液调pH至7.0;将二者混合后加去离子水稀释并定容至1000 mL,置于4 ℃冰箱中保存备用[20]。
多糖待测液制备:称取40.0 mg多糖样品并加入适量的去离子水,配制成4.0 mg/mL的待测液,在4 ℃下保存备用。
1.2.3.2 体外模拟胃肠消化过程
将唾液消化5 min后的样品溶液分别移取1.0 mL置于试管中,37 ℃恒温水浴中预热15 min,预热完成后取模拟胃液2.0 mL加入到样品溶液中,于40 ℃水浴振摇240 min模拟胃消化。反应结束后,以4500 r/min离心15 min,上清液即为胃消化液,取其中一部分于沸水浴中5 min灭酶,另一部分未灭酶,用于肠道消化过程。
取经胃消化后未灭酶的胃消化液2.0 mL置于试管中,37 ℃恒温水浴中预热15 min,预热后按反应液与模拟肠液体积比为10:3(V:V)比例添加0.6 mL模拟肠液、15 mL 1.0 mol/mL NaCl 和15 mL 1.0 mol/mL KCl,于37 ℃水浴中振荡240 min模拟肠道消化。反应结束后,得到肠消化液,置于沸水浴中5 min灭酶后备用。
1.2.3.3 体外模拟胃肠消化产物的抗氧化活性测定
将模拟体外胃液、肠液消化后的消化液各移取10.0 μL与工作液170.0 μL和应用液20.0 μL充分混匀后静置反应6 min,在425 nm处测OD值代入下式计算ABTS+·清除率[21]:
ABTS+⋅清除率(%)=(A0−A1A0)×100 (2) 式中:A0为空白组吸光度值;A1为待测样吸光度值。
将模拟胃液、肠液消化后的消化液分别移取80.0 μL与工作液120.0 μL充分混匀后避光静置30 min后在波长517 nm处测量其吸光度值代入下式计算DPPH·清除率[22]:
DPPH⋅清除率(%)=(1−A1−A0A2)×100 (3) 式中:A0为对照组吸光度值;A1为待测样吸光度值;A2为空白组吸光度值。
将模拟体外胃液、肠液消化后的反应液分别移取5.0 μL与已配制好的工作液180.0 μL充分混匀后静置反应5 min,在波长593 nm处测OD值,将OD值代入以 FeSO4·7H2O 建立标准曲线的回归方程得到Fe2+浓度,以 Fe2+ mg/mL表征消化产物对铁还原力,即FRAP值[23]。
1.3 数据处理
采用SPSS 26.0进行数据分析,利用Origin 9.0软件进行绘图,所有实验均重复3次以上,结果以均值±标准差表示。
2. 结果与分析
2.1 粗多糖纯化
经酶提醇沉后荞麦蜂花粉粗多糖得率可达30.74%±0.23%,总糖含量可达65.31%±1.21%,采用DEAE-52纤维素柱层析法洗脱纯化荞麦蜂花粉粗多糖,利用苯酚-硫酸法对洗脱后的样品液体进行糖含量检测,根据测得的样品吸光度、洗脱管数以及NaCl浓度等三个条件绘制洗脱曲线如图1所示。
由图1可知,经过DEAE-52纤维素柱层析法洗脱分离主要得到三个洗脱蜂,依次命名为BWPP-1、BWPP-2和BWPP-3。可以看出采用去离子水和0.1 mol/L NaCl洗脱液得到的洗脱峰较大,随着NaCl浓度的升高,多糖洗脱效果也越来越不明显。由此可见,提取后的粗多糖较易溶于水。将洗脱纯化后得到的三个洗脱峰进行收集、减压浓缩、透析、冷冻干燥后均得到乳白色粉末。经收集得到的三个多糖组分的得率及总糖含量如表2所示,其中多糖BWPP-1得率最高,为15.23%,总糖含量高达95.01%,纯度较高。
表 2 纯化后多糖得率及总糖含量Table 2. Polysaccharide yield and total sugar content after purification样品 得率(%) 总糖含量(%) BWPP 30.74±0.23 65.31±1.21 BWPP-1 15.23±0.25 95.01±1.24 BWPP-2 9.82±0.14 65.39±0.62 BWPP-3 9.14±0.07 41.77±0.83 注:BWPP的得率基于蜂花粉的干重得出;纯化组分的得率基于粗多糖的质量得出。 2.2 紫外光谱分析
将荞麦蜂花粉粗多糖和经过纯化后的多糖组分在200~400 nm波长处进行紫外光谱扫描,结果如图2所示。由图2可知,BWPP和经过纯化后得到的三种组分BWPP-1、BWPP-2和BWPP-3在260~280 nm处无明显吸收峰,说明荞麦蜂花粉经过DEAE-52纤维素柱层析法洗脱后不含有蛋白质、核酸等杂质。
2.3 红外光谱结果与分析
采用傅里叶红外光谱仪对粗多糖及纯化后的各级多糖组分进行结构表征,结果如图3所示。由图3可知,BWPP在3360 cm−1处的吸收峰是由O-H的伸缩振动引起的,峰形宽而大,表明分子中含有羟基[24];在2938 cm−1处的吸收峰是由多糖中的C-H伸缩振动引起的;在1657 cm−1处的吸收峰是由C=O的不对称伸缩振动引起的,表明多糖中存在羧基基团[25];而在1380 cm−1左右的吸收峰(1414、1326 cm−1)则为C-H的变角振动引起的,说明分子中有糖醛酸存在,与上述的单糖组成分析相吻合;在1078 cm−1处的吸收峰是C-O的伸缩振动引起的,说明多糖中含有吡喃糖环[26];而在882、786 cm−1处出现的小峰,推测是由次甲基的横向振动引起的,说明其中含有β-型糖苷键[27];经分析对比在540 cm−1处出现的吸收峰,可能是由其含有的葡萄糖中的醛基伸缩振动引起的,综上可知荞麦蜂花粉粗多糖BWPP具有明显的多糖特征吸收峰。而其三种纯化组分BWPP-1、BWPP-2、BWPP-3在3500~3000 cm−1范围(3256、3247和3235 cm−1)处的吸收峰是由O-H的伸缩振动引起的,表明多糖分子中有羟基[28];范围在3000~2800 cm−1的吸收峰(2927、2924、2921 cm−1)是由于C-H的伸缩振动引起的;1625 cm−1处的吸收峰是由C=O的不对称伸缩振动引起的,表明多糖中存在羧基基团[29];而1380 cm−1左右的吸收峰(1395、1397和1400 cm−1)则为C-H的变角振动;1100~1000 cm−1之间的吸收峰(1048、1043、1018 cm−1)是C-O的伸缩振动引起的,说明多糖中含有吡喃糖环[30];而在868 cm−1处出现的小峰,推测是由次甲基的横向振动引起的,说明其中含有β-型糖苷键;经分析对比,在600 cm−1左右出现的吸收峰(595、669、666 cm−1)为吡喃糖环上C-H面外变形振动吸收峰的变化,是鼠李糖的吸收峰,由此说明三种组分都具有明显的多糖特征吸收峰。
2.4 多糖组分分析
采用高效液相色谱法对荞麦蜂花粉粗多糖BWPP以及纯化后的三个组分BWPP-1、BWPP-2和BWPP-3样品分别进行单糖组成分析,各单糖标准品和样品的单糖组成及其摩尔分数如表3 所示。
表 3 BWPP及其纯化产物中单糖组成及其摩尔分数(%)Table 3. Monosaccharide composition and mole fraction of BWPP and its purified product (%)单糖 BWPP BWPP-1 BWPP-2 BWPP-3 古洛糖醛酸 0.02 − − − 甘露糖醛酸 0.11 1.59 3.19 82.13 甘露糖Man 1.47 1.71 0.71 − 核糖Rib 0.12 – − – 鼠李糖Rham 1.72 1.60 1.85 4.31 氨基葡萄糖GlcN 0.12 – − – 葡萄糖醛酸GlcUA 0.13 – 2.46 5.91 半乳糖醛酸GalUA 5.49 2.93 2.26 4.05 葡萄糖Glc 54.74 78.88 14.57 3.60 氨基半乳糖GalN 0.00 − − − 半乳糖Gal 21.52 − 14.40 − 阿拉伯糖Ara 14.06 8.84 − − 木糖Xyl 0.49 4.45 60.56 − 注:“−”表示未检出。 由表3可知,BWPP主要是由Glc组成,其含量高达54.74%,并含有少量的Gal、Ara、GalUA、Rham和Man等;利用去离子水洗脱得到的BWPP-1中的葡萄糖含量高达78.88%,而Ara(8.84%)和Xyl(4.45%)含量也相对较高;而利用NaCl溶液洗脱得到的两个酸性多糖BWPP-2和BWPP-3中单糖组成明显少于BWPP。其中,BWPP-2主要由Xyl、Gal和Glc组成,其中Xyl含量最高达到60.56%。BWPP-3则主要由甘露糖醛酸(82.13%)和少量的GlcUA组成。与红外光谱分析结果相印证,多糖中含有大量的吡喃糖环,推测可能与BWPP-1中含有大量的葡萄糖有关,而葡萄糖中含有游离的醛基可能会影响多糖的抗氧化性。
2.5 体外模拟胃肠消化过程中粗多糖及纯化产物BWPP-1的抗氧化活性分析
2.5.1 体外模拟胃液消化过程中荞麦蜂花粉粗多糖BWPP和BWPP-1消化液抗氧化活性分析
由于纯化组分BWPP-1的得率较高,且其总糖含量较其他纯化组分高,因此以荞麦蜂花粉粗多糖BWPP和纯化组分BWPP-1进行体外模拟胃肠消化过程的抗氧化活性分析,其模拟胃液消化结果如图4所示。由图4可知,BWPP和BWPP-1溶液经模拟胃液消化过程中两者都具有抗氧化活性。在模拟胃液消化过程中随着消化时间的延长,BWPP和BWPP-1对ABTS+·和DPPH·清除率均呈现先上升后下降的趋势,当消化时间在60 min时BWPP和BWPP-1的ABTS+·清除率均达到最大值(86.39%±1.28%,90.38%±2.78%),与其他消化时间的存在显著差异(P<0.05),且BWPP和BWPP-1在相同处理时间对ABTS+·清除率均存在显著差异(P<0.05);BWPP和BWPP-1在初始消化5 min时,对DPPH·清除率的差异显著(P<0.05),随时间的延长二者在消化过程中不同时间处理时对DPPH·清除率无显著差异(P>0.05),其均在60 min时对DPPH·清除率达到最大值(86.86%±0.11%,89.58%±1.67%),且二者差异不显著(P>0.05);模拟胃液消化时,当消化120 min后BWPP的FARP值较初始消化时差异显著(P<0.05);而BWPP-1的FRAP值呈现先增大后减小的趋势,其在消化120 min时达到(0.65±0.03)mg/mL,且与BWPP的FRAP值相比差异显著(P<0.05)。
图 4 模拟胃液消化过程中BWPP和BWPP-1的抗氧化活性分析注:图中不同小写字母表示同一样品不同消化时间的显著性,P<0.05;*表示相同消化时间不同样品的显著性,P<0.05;图5同。Figure 4. Analysis of antioxidant activity of BWPP and BWPP-1 during simulated gastric juice digestion2.5.2 体外模拟肠液消化过程中荞麦蜂花粉粗多糖BWPP和BWPP-1消化液抗氧化活性分析
对荞麦蜂花粉粗多糖BWPP和BWPP-1进行体外模拟肠道消化过程的抗氧化活性分析,结果如图5所示。由图5可知,经模拟胃液消化后再经过模拟肠道消化时,随着消化时间的延长,在肠液消化60 min时BWPP和BWPP-1对ABTS+·清除率均达到了最大值(88.67%±0.40%、91.82%±2.77%),但二者差异不显著(P>0.05),而后又随着消化时间的延长清除率均出现下降趋势;BWPP和BWPP-1对DPPH·清除率随着体外消化时间的延长呈缓慢上升的趋势,在后期肠道消化过程中(60 min以后),二者对DPPH·清除率有着显著差异(P<0.05),在消化240 min时达到了57.11%±0.06%和65.67%±3.67%,二者差异显著(P<0.05),推测是荞麦蜂花粉多糖在经过胃液消化后消化产物被稀释,使其在肠消化开始时DPPH·清除率较低,而后期清除率升高,这可能是肠液水解后期生成了活性更高的代谢产物[31];BWPP-1在模拟肠液消化的前10 min内其FRAP值较高,最高可达(0.44±0.02)mg/mL且与BWPP的FRAP值具有显著差异(P<0.05),后随消化时间的延长呈现先下降后又缓缓上升的趋势;而随着肠消化时间的延长,BWPP的FRAP值变化较为平缓,但综合整个消化阶段可以看出荞麦蜂花粉多糖的铁离子还原能力在逐渐下降,推测原因可能是在肠消化时糖苷键断裂,聚集物被破坏而导致铁离子还原能力下降,此研究结果与米佳等[32]的研究成果较为一致。
3. 结论
本研究对荞麦蜂花粉多糖进行纯化并探究其在体外胃肠消化中抗氧化活性变化的规律。结果表明,BWPP通过DEAE-52纤维素凝胶柱洗脱纯化后得到了3个组分BWPP-1、BWPP-2和BWPP-3。总糖含量最高组分的BWPP-1主要由Glc、Xyl、Ara等组成,且红外光谱显示BWPP-1具有明显的多糖特征吸收峰。BWPP和BWPP-1在胃肠模拟消化对ABTS+·清除率呈现对时间的依赖性,呈现出先增大后减小的趋势,并且BWPP-1抗氧化活性优于BWPP。胃消化液中的DPPH·清除能力略高于肠消化液,而肠消化液的ABTS+·清除能力略高于胃消化液。此外,胃肠模拟消化中二者的FRAP值均较弱,说明荞麦蜂花粉多糖的FRAP值不受胃蛋白酶、胰蛋白酶、作用时间的影响。综上所述,纯化后的荞麦花粉多糖可以提高消化过程中的抗氧化活性。本研究为荞麦花粉多糖未来成为功能性物质提供了理论依据,也为荞麦加工副产物综合利用提供了新的见解。
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图 4 模拟胃液消化过程中BWPP和BWPP-1的抗氧化活性分析
注:图中不同小写字母表示同一样品不同消化时间的显著性,P<0.05;*表示相同消化时间不同样品的显著性,P<0.05;图5同。
Figure 4. Analysis of antioxidant activity of BWPP and BWPP-1 during simulated gastric juice digestion
表 1 洗脱梯度
Table 1 Elution gradient
时间(min) 流动相A(%) 流动相B(%) 0 86 14 9 83 17 28 78 22 29 50 50 31 50 50 32 86 14 36 86 14 表 2 纯化后多糖得率及总糖含量
Table 2 Polysaccharide yield and total sugar content after purification
样品 得率(%) 总糖含量(%) BWPP 30.74±0.23 65.31±1.21 BWPP-1 15.23±0.25 95.01±1.24 BWPP-2 9.82±0.14 65.39±0.62 BWPP-3 9.14±0.07 41.77±0.83 注:BWPP的得率基于蜂花粉的干重得出;纯化组分的得率基于粗多糖的质量得出。 表 3 BWPP及其纯化产物中单糖组成及其摩尔分数(%)
Table 3 Monosaccharide composition and mole fraction of BWPP and its purified product (%)
单糖 BWPP BWPP-1 BWPP-2 BWPP-3 古洛糖醛酸 0.02 − − − 甘露糖醛酸 0.11 1.59 3.19 82.13 甘露糖Man 1.47 1.71 0.71 − 核糖Rib 0.12 – − – 鼠李糖Rham 1.72 1.60 1.85 4.31 氨基葡萄糖GlcN 0.12 – − – 葡萄糖醛酸GlcUA 0.13 – 2.46 5.91 半乳糖醛酸GalUA 5.49 2.93 2.26 4.05 葡萄糖Glc 54.74 78.88 14.57 3.60 氨基半乳糖GalN 0.00 − − − 半乳糖Gal 21.52 − 14.40 − 阿拉伯糖Ara 14.06 8.84 − − 木糖Xyl 0.49 4.45 60.56 − 注:“−”表示未检出。 -
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