CN113073126A - 亚麻籽活性多肽在制备预防、干预/治疗结肠炎产品中应用 - Google Patents
亚麻籽活性多肽在制备预防、干预/治疗结肠炎产品中应用 Download PDFInfo
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Abstract
本发明公开了亚麻籽活性多肽在制备预防、干预/治疗结肠炎产品中应用，属于食品或药品技术领域。动物实验证明，本发明所述亚麻籽多肽对DSS诱导的溃疡性结肠炎小鼠的结肠损伤有显著的干预效果，有效地缓解结肠炎导致的体重减轻、腹泻、血便和肠粘膜溃疡症状，减轻小鼠肠道炎症和氧化应激水平，通过对NF‑κB信号通路中蛋白的表达调控发挥作用。亚麻籽活性多肽防治效果显著，制备方法简单，安全性高，原料易得，在制备结肠炎的功能食品、特殊膳食或药物开发方面具有较好的应用前景。
Description
技术领域
本发明涉及亚麻籽活性多肽在制备预防、干预/治疗结肠炎产品的应用，属于食品或药品技术领域。
背景技术
炎症性肠病（inflammatory bowel disease，IBD）是一种反复发作的非特异性肠道炎症性疾病，主要包括溃疡性结肠炎和结肠克罗恩病，发病原因受到膳食、机体状态、肠道微生物、遗传、环境等多重因素影响，发病机制仍不完全明确。IBD可引起肠粘膜损伤，伴随腹痛、腹泻和便血等临床表现，发展后易导致中毒性结肠炎和结肠炎相关的结肠癌的发生。目前，一些结肠炎干预制剂如抗炎药物、免疫抑制剂、糖皮质激素等在IBD的预防和控制上取得了一定的成效。但是这些干预制剂可引起过敏反应且存在对肝脏、肾脏、胰腺和神经系统明显的毒副作用。因此，寻找高效且无毒副作用的天然活性抗结肠炎物质变得十分必要。
亚麻为一年生草本植物，是主要的油料和植物纤维作物之一。亚麻籽的蛋白质含量在35%左右，由18种天然氨基酸构成，其中芳香族氨基酸含量低，缬氨酸、亮氨酸、异亮氨酸、组氨酸、赖氨酸、谷氨酸、半胱氨酸和蛋氨酸含量高，具有提高抗氧化水平、减少肠道炎症发生和进展、改善机体免疫、降低胆固醇等功能活性。目前，国内外的学者已经研究并报道了亚麻籽蛋白及其活性肽的多种的功能活性，例如抗氧化、抗菌性、抗高血压、降低胆固醇，然而对亚麻籽肽在改善结肠炎中的效果和机制研究未见报道。
发明内容
本发明的目的在于提供，亚麻籽活性多肽在制备预防、干预/治疗结肠炎产品中应用；传统食用油料作物亚麻籽获得亚麻籽粗蛋白，经过蛋白酶酶解释放亚麻籽生物活性多肽。
进一步的，提供本发明所述亚麻籽活性多肽在制备预防、干预结肠炎保健食品中应用。
进一步的，提供本发明所述亚麻籽活性多肽在制备预防、干预/治疗结肠炎药品中应用。
本发明所述结肠炎主要包括慢性结肠炎、急性溃疡性结肠炎或者结肠克罗恩病，对其它肠道性炎症也有效果。
优选的，本发明所述亚麻籽活性多肽的制备方法为：配制浓度为2~10%的亚麻籽粗蛋白溶液，搅拌20 min，用木瓜蛋白酶进行酶解，灭酶，超滤收集多肽片段，冷冻离心取上清液，透析8-24 h即得亚麻籽活性多肽溶液，冷冻干燥即为亚麻籽活性多肽粉。
本发明用蛋白酶酶解亚麻籽蛋白制备具有高效抗炎活性的亚麻籽肽并研究其对结肠炎的预防、干预、治疗作用和作用机制；利用葡聚糖硫酸钠（DSS）诱导C57BL/6小鼠溃疡性结肠模型，评价亚麻籽活性肽抗结肠炎作用。实验结果表明，亚麻籽活性肽可以显著减缓结肠炎小鼠的炎症症状，改善小鼠氧化应激、炎症水平和肠道菌群，通过对NF-κB信号通路中关键蛋白的表达调控发挥抗结肠炎作用。最终证实了亚麻籽活性肽有效防治结肠炎的作用效果和机制，为亚麻籽生物活性肽在改善结肠炎的食品和药品制备与开发上提供了一种新的方法和应用；同时，亚麻籽活性肽来源于原料易得、安全性高的食物原料，结合简单的制备工艺，可增加亚麻籽应用范围，提升附加值，具有广阔的应用前景。
亚麻籽活性多肽在结肠炎的作用机制与多肽对NF-κB信号通路中蛋白的表达调控有关，亚麻籽活性多肽对结肠炎的预防、干预或治疗的应用即对结肠炎症状有显著缓解作用。
与现有技术相比，本发明具有如下优点：
（1）本发明针对亚麻籽蛋白的特性，通过酶解释放生物活性肽，开发其在抗炎方面的功能食品和药品领域的新用途。
（2）本发明原料成本低、易得、安全性高，制备工艺简单可实现规模化生产，是一种安全有效的食物源抗结肠炎物质。
附图说明
图1为亚麻籽肽对结肠炎小鼠的DAI评分影响示意图；
图2为亚麻籽肽对结肠炎小鼠结肠损伤影响示意图；
图3为亚麻籽肽对结肠炎小鼠肠道双歧杆菌影响示意图；
图4为亚麻籽肽对结肠炎小鼠NF-κB信号通路影响示意图；
图5为图4中蛋白电泳的条带灰度的统计分析图。
具体实施方式
下面结合附图和实验实例数据对发明作进一步的详细说明，这些实验实例仅用于说明本发明，而不用于限制本发明的范围。本领域的技术人员在本发明的基础上所做的任何非实质性的修饰、变化及替换均属于本发明所要求保护的范围。
下面实验实例中未注明具体条件的实验方法，通常按照本领域常规条件或按照制造建议条件。
实施例1
本实施例通过动物试验进一步证明亚麻籽活性多肽对溃疡性结肠炎的干预和改善作用，具体实验如下：
1. 实验材料与仪器
1.1 实验原料：亚麻籽购于中国云南省昆明市。
1.2 主要试剂：雄性C57BL/6J小鼠来自湖南SJA实验动物有限公司；葡聚糖硫酸钠(DSS, MW：40000Da）购于上海阿拉丁生化科技股份有限公司；选择培养基购自青岛海博生物科技有限公司；ELISA测定试剂盒购自中国南京建成生物工程研究所。
1.3 主要仪器：FD50R型立式自动压力蒸汽灭菌锅，致微（厦门）仪器有限公司；XMTD-204数显恒温搅拌水浴锅，金坛市白塔新宝仪器厂；ALPHA 1-2 LD plus冷冻干燥机，德国Christ；FE20K型pH计，上海梅特勒-托利多仪器有限公司。
2. 亚麻籽粗蛋白及亚麻籽活性多肽的制备
2.1 亚麻籽粗蛋白的制备
将亚麻籽粕粉碎后过200目筛，溶于蒸馏水（1:20，w/v），搅拌20min后，用1 mol/LNaOH调节pH为9.5，在35℃水浴中超声搅拌提取1h（超声功率99%）；充分溶解后的碱提液以10000 r/min离心10 min，收集上清液；向上清液中缓慢加入1 mol/L HCl以调节pH为4.0，于4℃冰箱中静置1h，待蛋白质沉淀后再置于离心机中以10000 r/min离心10min，收集沉淀；用蒸馏水洗涤沉淀3遍后将pH调为7.0，用蒸馏水复溶沉淀后，置于透析袋中（截留分子量为10000 Da），透析过夜；冷冻干燥即得到亚麻籽粗蛋白。
2.2 亚麻籽肽的制备
配制浓度为2%的亚麻籽粗蛋白溶液，置于55℃水浴中，用1.0 mol/L的NaOH调节亚麻籽蛋白溶液的pH至7.0，然后加入6000U/g的木瓜蛋白酶，酶解反应0.5ｈ；调节酶解液pH至中性后煮沸灭酶10min，冷却；在４℃下以10000r/min转速离心10min，收集上清液；上清液用分子量为10 kDa的超滤膜超滤收集滤液，再用透析膜在4℃下脱盐24h，冷冻干燥即为亚麻籽活性多肽（FP）。
3. 亚麻籽肽对DSS诱导结肠炎小鼠的抗炎作用
3.1 小鼠急性结肠炎的诱导与干预
将40只C57BL/6J雄性小鼠随机分为4组，分别为对照组、DSS组、低剂量亚麻籽活性多肽（LFP）组和高剂量亚麻籽活性多肽（HFP）；除对照组外，其余三组小鼠均自由饮用3%DSS(溶解于蒸馏水)诱导结肠炎模型，对照组用蒸馏水替代DSS；低剂量亚麻籽肽（LFP）组和高剂量亚麻籽肽（HFP）组分别灌胃FP溶液(400、800 mg/kg/day)每日1次，对照组和DSS组灌胃相同体积的蒸馏水，实验持续7天。
3.2 动物模型指标
3.2.1 一般情况观察
实验期间每天记录小鼠进食情况、精神状态、体重、粪便状况、血便并计算疾病活度指数(DAI)；DAI评分按以下公式计算:
DAI = (Wb + Wf + Wbf)/3
公式中，Wb、Wf、Wbf分别代表体重减轻评分、粪便状态评分、血便评分。DAI评分标准如下：体重减轻评分(0:1%，1:1-5%，2:5-10%，3:10-15%，4:> 15%）；粪便状况评分(0:正常，2:稀松，4:腹泻)；血便评分(0:正常；2:红色；4: 血色)。
3.2.2 小鼠的结肠长度和结肠组织病理学评分
实验第8天，用颈部脱臼法处死小鼠，取肛门至回盲部的结肠，测量结肠长度并拍照；取离肛门1 cm处的结肠0.5 cm，切成小管，用10%中性福尔马林固定，进行组织学检查；用稀释酒精使固定结肠组织脱水，然后嵌入在石蜡，切成4 μm片。切片经脱蜡处理后，用苏木精、伊红染色，通过显微镜观察。使用以下评分标准对每个结肠切片进行盲评分：（0：正常，无炎症细胞浸润；1：轻微炎症细胞浸润，粘膜下组织无损伤； 2：中度炎性细胞浸润和粘膜下组织被破坏（损伤范围在10％-25％之间）；3：明显的炎性细胞浸润，粘膜下组织被破坏，结肠壁增厚（损伤范围在25％~50％之间）；4：严重炎症细胞浸润，大规模结肠组织损伤（损伤范围> 50％）和结肠壁增厚）。
3.2.3 结肠组织中炎症细胞因子与氧化水平的测定
称取结肠组织，并用生理盐水匀化10％的结肠组织；在4°C下以14000 r / min的速度离心15分钟后，收集结肠均质的上清液；将上清液按照制造商的说明书检测总蛋白浓度、丙二醛（MDA）含量和过氧化氢酶（CAT）活性，使用ELISA试剂盒对TNF-α和IL-1β的含量进行定量检测。
3.2.4 肠道微生物菌群计数
在无菌条件下，剪开结肠，每组取6只小鼠新鲜粪便并稀释至一定浓度。取0.1mL梯度稀释液涂布于相应的选择培养基中，用涂布棒涂匀；涂布完成后将培养基置于37℃培养箱上培养24h，计算培养基上菌群数量；肠道微生物菌群的数量用菌落形成单位的常用对数值lgCFU/g表示。
3.2.5 免疫印迹 （Western Blot）
通过Western blot检测结肠组织中Iκ-Bα蛋白及其磷酸化水平，具体方法如下：
（1）SDS—PAGE：
①准备电泳装置，玻璃板对齐后放入配胶夹中。
②配制10％分离胶，每块胶加约4.5mL（即胶面可以升到绿色带的中间线高度），待加好后，立即在胶面上加一层双蒸水封胶，室温放置30 至60分钟，待胶凝固后去掉双蒸水。
③配制5％浓缩胶，灌满玻璃板剩余空间，将梳子插入浓缩胶中，待浓缩胶凝固后轻轻将梳子拔出，将制好胶的玻璃板放入电泳槽中，加入1X电泳缓冲液， 排除凝胶底部两玻璃板间的气泡。
④上样：取40 uL蛋白样品，加入10 uL 5xSDS上样缓冲液，100℃，5 min变性。取40ug至60 ug蛋白的所需溶液体积即为上样量，分别加入孔道。
⑤电泳：浓缩胶恒压90 V，30 min至40 min，待条带跑至分离胶后，恒压120-180V， 60-150 min。
（2）转膜
①裁剪合适大小的滤纸与PVDF膜，PVDF膜在甲醇中浸泡1 min。
②按照海绵垫、滤纸、凝胶、PVDF膜、滤纸、海绵垫的顺序放置，注意在移胶时， 应将浓缩胶轻轻的刮去，小心剥下分离胶盖于滤纸上，并调整方向，使胶、滤纸、PVDF 膜准确贴合，赶走气泡。
③将夹子放入转移槽，加入1X转膜缓冲液，注意夹子的方向，黑面对槽的黑面；将整个装置置于冰块中以维持转膜时的低温环境；转膜条件：设置恒流，一块胶200 mA，两块胶400 mA，时间为2小时。
（3）封闭：转膜完成后，按目的条带剪膜，5％BSA封闭，室温、摇床温和振荡15 min，振荡3次。
（4）免疫反应
①一抗孵育：弃封闭液后，加入一抗孵育，40℃过夜。
②二抗孵育：弃一抗稀释液，1XTBST 2 mL洗涤，室温摇床15 min，3次。孵育二抗，室温摇床1 h；弃二抗稀释液，TBST洗涤，室温摇床15 min，3次。
（5）化学发光
将显影液A和B两种试剂等混合后，滴在膜上使用化学发光系统显影拍照。
（6）Western Blot图像分析及处理
结果经Image软件转换为灰度格式后再进行累积光密度值的分析，β-actin蛋白条带作为上样量的内参标准，目的蛋白的表达量是面积和光密度的乘积，以此表达目的蛋白的表达水平。
4. 实验结论
4.1 本发明的亚麻籽肽对小鼠DAI评分的影响
由图1可知，对照组小鼠状态良好，粪便性状正常；DSS组小鼠在第3天出现了明显的稀便和血便，第5天体重显著降低，与对照组相比，DAI评分显着增加（P<0.05)。HFP和LFP组与DSS组相比DAI评分显著降低（P<0.05)，说明HFP和LFP对结肠炎小鼠的腹泻和血便症状具有改善作用。
4.2 本发明的亚麻籽肽对小鼠结肠损伤的影响
图2显示了结肠炎小鼠结肠损伤程度；与对照组相比，结肠炎小鼠的结肠长度显著减小（P<0.05)。FP组结肠萎缩症状得到明显改善，HFP和LFP组与DSS组相比小鼠结肠长度均显著增加（P<0.05)，分别增加了大约0.6 cm和1.6 cm。此外，从结肠组织病理学来看，小鼠饮用DSS已引起了炎症，DSS组小鼠结肠粘膜溃疡，上皮细胞被破坏和明显的免疫细胞浸润，组织病理损伤评分与对照组相比具有显著差异（P<0.05)，说明经DSS诱导后，结肠损伤严重；FP显著改善了小鼠上述症状，HFP组和LFP组评分与DSS相比均显著降低大约1分（P<0.05)；可见HFP和LFP皆能显著减轻结肠萎缩，改善DSS诱导的小鼠急性结肠损伤。
4.3 本发明的亚麻籽肽对小鼠肠道双歧杆菌的影响
如图3所示，与对照组相比，DSS组小鼠肠道双歧杆菌数量显著降低（P<0.05)，可见DSS诱发的急性结肠炎会使肠道益生菌双歧杆菌减少；而HFP和LFP均能促进肠道双歧杆菌数量显著增加，与DSS组相比具有显著差异（P<0.05)；表明HFP和LFP能促进肠道益生菌双歧杆菌数量增加，从而改善肠道环境。
4.4 本发明的亚麻籽肽对小鼠结肠氧化水平的影响
表1为亚麻籽肽对结肠炎小鼠结肠氧化水平及炎症水平影响示意表
表1显示，与对照组相比，DSS组结肠组织中MDA含量显著升高，CAT活性显著降低（P<0.05)。与DSS组相比，HFP和LFP组的MDA含量显著降低，CAT活性显著升高（P<0.05)；这些数据表明， 经DSS诱导后，结肠组织氧化损伤严重，而FP具有改善作用，具有清除自由基的能力并在一定程度上增加了细胞的抗氧化能力。
4.6本发明的亚麻籽肽对小鼠炎症因子水平的影响
由表1可知，与对照组相比，DSS处理的小鼠结肠组织中的促炎细胞因子TNF-α和IL-1β水平显著增加（P<0.05)；与DSS组相比，LFP和HFP组显著降低了TNF-α和IL-1β水平（P<0.05)；说明FP能显著降低促炎性细胞因子TNF-α和IL-1β水平，从而抑制结肠组织中持续的炎性反应。
4.7 本发明的亚麻籽肽对小鼠NF-κB信号通路的影响
如图4所示，我们检测到DSS组与对照组相比Iκ-Bα蛋白磷酸化水平明显升高（P<0.05)，提示NF-κB信号通路激活。HFP组与LFP组结肠组织中Iκ-Bα蛋白磷酸化水平明显降低，与DSS组具有显著性差异（P<0.05)；结果表明，FP可以通过阻断NF-κB信号通路来减轻DSS诱导的小鼠结肠炎。
Claims (5)
1.亚麻籽活性多肽在制备预防、干预/治疗结肠炎产品中应用。
2.亚麻籽活性多肽在制备预防、干预结肠炎保健食品中应用。
3.亚麻籽活性多肽在制备预防、干预/治疗结肠炎药品中应用。
4.权利要求1~3任意一项所述应用，其特征在于：所述结肠炎包括慢性结肠炎、急性溃疡性结肠炎或者结肠克罗恩病。
5.权利要求1~3任意一项所述应用，其特征在于：所述亚麻籽活性多肽的制备方法为：配制浓度为2~10%的亚麻籽粗蛋白溶液，搅拌20 min，用木瓜蛋白酶进行酶解，灭酶，超滤收集多肽片段，冷冻离心取上清液，透析8-24 h即得亚麻籽活性多肽溶液，冷冻干燥即为亚麻籽活性多肽粉。
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