CN113648332A - 一种海带中不溶性膳食纤维在制备用于改善便秘的药物和功能性食品中的应用 - Google Patents
一种海带中不溶性膳食纤维在制备用于改善便秘的药物和功能性食品中的应用 Download PDFInfo
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- CN113648332A CN113648332A CN202110850189.3A CN202110850189A CN113648332A CN 113648332 A CN113648332 A CN 113648332A CN 202110850189 A CN202110850189 A CN 202110850189A CN 113648332 A CN113648332 A CN 113648332A
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Abstract
本发明公开了一种海带中不溶性膳食纤维在制备用于改善便秘的药物和功能性食品中的应用。本发明从海带残渣中提取不溶性膳食纤维,并构建了便秘小鼠模型,首次验证了不溶性膳食纤维能促进小鼠排便,增加肠道含水率,提高肠道转运速率;上调抗氧化酶水平;平衡肠道神经递质水平;增加厚壁菌门和放线菌门的丰度,抑制了拟杆菌门的丰度,并增加了短链脂肪酸的产生。本发明中的海带中不溶性膳食纤维安全无毒,避免了药物毒副作用的出现,且可被肠道菌群代谢发酵并进一步介导机体抗氧化酶水平、肠神经递质的表达,改善便秘相关症状,具有良好的市场应用前景。
Description
技术领域
本发明属于食品保健领域,特别涉及一种海带中不溶性膳食纤维在制备用于改善便秘的药物和功能性食品中的应用。
背景技术
近年来,人们生活水平日益提高,饮食结构不断改变,肠胃病、肥胖症等“文明病”发病率明显增高。功能性便秘已成为一种常见的临床症状,是影响现代人生活质量的重要慢性病之一。现代医学对本病的治疗目前还没有非常有效手段,往往以刺激性泻剂为主,如聚乙二醇、乳果糖、山梨醇等,此类药物长期使用可出现诸多不良反应,易致使人体内水、电解质紊乱,对药物的依赖性,损伤肠神经递质的表达,影响结肠平滑肌对乙酰胆碱的反应性等。因此,迫切需要一种新型的、无危害的便秘治疗方法。
研究发现,失衡的肠道菌群环境与“文明病”增多有密切关系,而且肠道菌丰度会由上一代传给下一代。不溶性膳食纤维(Insoluble Dietary Fiber,IDF)具有促进肠道机械蠕动,增加粪便体积,调节肠道菌群的作用,可以预防便秘、肠癌等代谢综合性疾病。因此,在营养学上以补充膳食纤维作为调节肠道菌稳态的手段比较常见。
海带中富含多种生物活性物质,被誉为天然的保健食品,在我国有悠久的食用历史。目前我国已经形成了以褐藻胶、甘露醇、碘为主要产品的褐藻工业产品体系,但是海带约2/3的成分尚未得到利用,其中含量最多的就是膳食纤维。在这种背景下对海带残渣中的膳食纤维进行研究和开发,特别是开发预防和改善疾病为主的特定健康食品,将有利于改善全球公共健康卫生问题。
发明内容
本发明的目的是为了提供了一种海带中不溶性膳食纤维在制备用于改善便秘的药物和功能性食品中的应用。所述不溶性膳食纤维IDF提取自分离褐藻胶后的海带残渣,其来源丰富,绿色无公害,且改善便秘效果明显。
为实现上述目的,本发明采用的技术方案为:
一种海带中不溶性膳食纤维在制备用于改善便秘的药物和功能性食品中的应用,所述不溶性膳食纤维的单糖组成包括葡萄糖和葡萄糖醛酸,二者的摩尔比为15-25:1。
优选的,所述不溶性膳食纤维的单糖组成为葡萄糖和葡萄糖醛酸,二者的摩尔比为21:1。
进一步的,所述不溶性膳食纤维的提取方法,包括以下步骤:
(1)向海带残渣中以1:20-40的料液比加入4-6% NaCl溶液,80-100℃加热6-8 h进行脱钙处理,离心收集沉淀,纯水洗涤沉淀;
(2)向步骤(1)的沉淀中以1:20-40的料液比加入5 mol/L NaOH溶液, pH调至碱性,80-100℃加热6-8 h,离心收集沉淀;
(3)利用NaClO对步骤(2)中的沉淀进行脱色处理,然后再利用稀HCl进行除盐处理;
(4)除盐后的沉淀洗涤后干燥,研磨过200目筛,得到不溶性膳食纤维。
进一步的,所述步骤(1)中海带残渣为提取褐藻胶后的海带残渣。
进一步的,所述步骤(1)中海带残渣与NaCl的液料比为1:30;所述步骤(2)中沉淀与NaOH的液料比为1:30。
进一步的,所述步骤(3)中NaClO的体积分数为9%,所述NaClO与沉淀的料液比为12:1,脱色处理时间为67 min。
进一步的,所述步骤(3)中的沉淀经脱色处理后,所述不溶性膳食纤维的白度为39.53%±0.74%,得率为36.87%±0.69%。
进一步的,所述步骤(3)中稀HCl的浓度为0.5 mol/L,所述稀HCl与脱色后的沉淀的料液比为1:1,除盐处理时间为1h。
进一步的,所述料液比的单位是mg/mL。
进一步的,所述不溶性膳食纤维改善便秘的有效剂量为0.5 g/kg-3.5 g/kg。
进一步的,所述不溶性膳食纤维改善便秘的最有效剂量为3.5 g/kg。
进一步的,所述不溶性膳食纤维能够增加便秘小鼠的粪便数量、粪便重量和粪便含水率。
进一步的,所述不溶性膳食纤维能够通过增加便秘小鼠肠道含水率以及提高肠道转运速率来促进肠道蠕动和改善肠道转运功能。
进一步的,所述不溶性膳食纤维能够增加血清中超氧化物歧化酶SOD和谷胱甘肽过氧化物酶GSH-Px的含量,以及增加SCFAs的含量。
进一步的,所述SCFAs包括乙酸、丙酸、丁酸、异丁酸、戊酸、异戊酸、异己酸。
进一步的,所述不溶性膳食纤维能够下调诱导型一氧化氮合酶iNOS的表达水平,上调肠神经递质乙酰胆碱酯酶AchE、内皮型一氧化氮合酶eNOS、胶质细胞源性神经营养因子GDNF和脑衍生神经营养因子BDNF的表达水平。
进一步的,所述不溶性膳食纤维能够通过减少小鼠肠道内拟杆菌门丰富度,增加厚壁菌门和放线菌门的丰度来调整便秘小鼠的肠道菌群的结构。
进一步的,所述不溶性膳食纤维能够增加科水平上Lachnospiraceae占比,降低Lactobacillaceae、Marinifilaceae、Helicobacteraceae、norank_o__Clostridia_UCG-014占比。
与现有的技术相比,本发明的有益效果和优点在于:
本发明提取不溶性膳食纤维的原料是分离褐藻胶后的海带残渣,其来源丰富,使用安全。本发明提取的IDF属于天然膳食成分,主要成分为纤维素,包括葡萄糖醛酸和葡萄糖两种单糖,IDF结构紧实且具有一定的纤维网状结构,与陆生植物相比,海带几乎不含有木质素,因此IDF不仅增强纤维的亲水力、持油力、膨胀力等性能,进一步增强其生物活性和可得性,还提高了海带的高附加利用价值。本发明对海带中IDF在改善小鼠便秘症状和调节肠道菌群及其代谢产物方面有显著的效果。因此,有发展成为改善便秘功能食品的巨大潜力。
附图说明
图1为IDF的单糖组成测定结果,其中A:10种单糖标准品高效液相色谱图;B:IDF的单糖组成高效液相色谱图。
图2为SEM对IDF的形貌表征,其中A:400×;B:1100×;C:1100×; D:1850×。
图3为IDF的红外光谱图。
图4为IDF的X-射线衍射图。
图5为便秘小鼠粪便指标图。
图6为便秘小鼠肠道转运速率图。
图7为便秘小鼠肠道含水率图。
图8为便秘小鼠血清SOD和GSH-Px图。
图9为便秘小鼠结肠肠神经递质mRNA相对表达量图。
图10为便秘小鼠结肠肠神经递质蛋白相对表达量图。
图11为便秘小鼠肠道菌群OUT分布图。
图12为便秘小鼠肠道菌群科、门水平群落组成柱形图。
图13为便秘小鼠粪便SCFAs含量图。
具体实施方式
下面结合附图和具体实施方式对本发明的技术方案进一步的详细说明,但本发明要求保护的范围并不局限于实例表述的范围。
实施例1:不溶性膳食纤维的提取
本发明中不溶性膳食纤维的提取方法包括以下步骤:
(1)按照料液比1:30的比例往海带残渣中加入5% NaCl溶液,90℃加热7 h进行脱钙处理;
(2)将步骤(1)中样品进行离心处理,收集沉淀加入适量纯水洗涤,再次收集沉淀;
(3)将步骤(2)中沉淀按照料液比1:30的比例加入5 mol/L NaOH溶液,调节pH至碱性,90℃加热6 h;
(4)将步骤(3)中样品离心,沉淀为不溶性膳食纤维(IDF);
(5)将步骤(4)中沉淀按照液料比为12:1加入体积分数为9%的NaClO,脱色时间67min,对IDF进行脱色处理;漂白后IDF的白度为39.53%±0.74%,得率为36.87%±0.69%;
(6)将步骤(5)中漂白后的IDF用200目纱布包住经超纯水水洗至中性并抽滤;
(7)向步骤(6)中经抽滤后IDF按照1:1的比例加入0.5 mol/L稀HCl进行除盐处理1h;
(8)将步骤(7)中的除盐处理后的IDF用200目纱布包住经超纯水水洗至中性并抽滤,收集样品于50-60℃鼓风干燥;
(9)将步骤(8)中干燥好的IDF研磨并过200目筛,得到最终的IDF。
实施例2:海带不溶性膳食纤维(IDF)的基本结构鉴定
1、高效液相色谱对IDF的单糖组成测定
(1)准确称取实施例1制备的IDF 2 mg,加入400 μL 2 mol/L 三氟乙酸(TFA)溶液于安瓿瓶内,在105℃密封条件下降解6 h。降解完成后,反复加甲醇除去多余三氟乙酸,将降解产物在干燥环境下保存。
(2)100 μL蒸馏水充分溶解IDF降解产物及单糖标准品,加入100 μL的0.3 mol/LNaOH和120 μL 0.5 mol/L的PMP甲醇溶液,70℃水浴60 min。冷却至室温后加入100 μL的0.3 mol/L HCl溶液进行中和反应,用二氯甲烷萃取3次除去未反应的PMP,0.22 μm微孔滤膜过滤上清备用。
(3)利用HPLC测定IDF的单糖组成。色谱条件:色谱柱:Eclipse XDB-C18(5 μm,4.6 μm×25.0 cm);流动相:乙腈:磷酸盐缓冲液(pH 6.7)=17:83(v/v);进样体积:10 μL;柱温:35℃;检测器:紫外检测器(254 nm);流速:1.0 mL/min。
结果如图1所示, IDF的水解产物的HPLC洗脱曲线有两个特征峰,其单糖组成主要为葡萄糖,占样品的95.5%,葡萄糖醛酸占样品的4.5%,二者摩尔比为葡萄糖醛酸:葡萄糖=1:21。可见,IDF提取的纯度较高,主要由不溶性葡聚糖组成。
2、扫描电子显微镜对IDF的形貌表征
将实施例1制备的干燥且经研磨后的IDF样本放置在带有双面胶的载物台上,通过离子溅射镀膜在其表面镀金,置于SEM扫描观察,电压: 5 Kv,放大倍数: 150×-1850×。
通过SEM对IDF的形貌进行表征,结果如图2所示。图2A和2B显示,IDF颗粒大小不一,表面粗糙不平,褶皱结构多且结构紧密;图2C和2D显示,IDF虽然结构紧实但仍具有一定的纤维网状结构:具有一定孔隙,呈现多层褶皱状的蓬松状态,可增加颗粒的比表面积,而比表面积可增强纤维的亲水力、持油力、膨胀力等性能,进一步增强其生物活性。
3、傅里叶红外光谱对IDF的结构分析
对IDF进行FT-IR定性分析。取干燥的实施例1制备的IDF和KBr(光谱级)100 mg研磨并压成透明薄片,以空白KBr作为背景,扫描次数为20次,分辨率为4 cm-1,扫描范围4000-400 cm-1。
结果如图3所示,IDF具有纤维素分子的特征波段,在3148.21 cm-1出现分子内伯羟基和仲羟基的O-H键的伸缩振动,在2921.18 cm-1处出现纤维素结晶顺序下的C-H键的伸缩振动。此外,在1638.49 cm-1为C=H的伸缩振动吸收峰,这与纤维素和水的强相互作用引起的吸水性有关。除了这些吸收峰之外,在光谱中还观察到其他特定的峰:1430.73 cm-1和1428.50 cm-1处出现CH2的弯曲振动和摆动振动;在1163.32 cm-1处的吸收峰属于纤维素中D-葡萄糖之间β-1,4-糖苷键的C-O-C伸缩;位于1113.66 cm-1、1060.36 cm-1吸收峰分别属于C-O-C吡喃糖环骨架振动和C-O伸缩振动。结果表明海带IDF的主要成分为纤维素。
4、X-射线衍射对IDF的结晶相分析
利用XRD分析IDF的晶体结构和结晶度指数。X射线衍射仪参数条件:铜靶,入射线波长0.15418 nm,Ni滤波片,管压40 KV,管流40 mA,扫描范围:5°-70°,扫描步长0.04度,扫描速度38.4秒/步;狭缝DS0.5°RS8 mm。随后利用Jade 6.0软件进行拟合分析。公式为:
式中:I200为2θ=22.64°附近的晶体峰强度,Iamp为2θ=14.38°附近波谷处测量的非晶体峰强度。
表1 :IDF的峰型拟合相关参数
分析结果如表1和图4所示。由图4可看出,IDF在14.38、16.74、22.64和26.86出现了衍射峰,强度分别为2579、1931、5058、2661,这是纤维素I的特征。计算得知其CrI值为50.06%,比市售纤维素结晶程度(36.30%)偏高,说明其具有更有序的晶体结构。进一步计算半峰全宽度、晶面尺寸、间距(表1),得知晶系判别系数Z=18.74502>0,表明晶体结构以Iα型为主。相比于Iβ型,Iα型晶体结构由于其热不稳定性而更容易发生生化反应,其构效关系值得进一步研究。
实施例3
72只雄性ICR小鼠适应性喂养7 d后按照体重随机分成9组,每组12只,分别为正常组(NC)、模型组(MC)、西沙比利组(PC)、IDF低、中、高剂量组(L-IDF、M-IDF、H-IDF)。适应性喂养7 d后开始进行受试物灌胃干预实验,干预方案见表2。从第8 d开始,除NC组之外的其余各组小鼠每天上午9时灌胃3 mg/kg Lop,1 h后灌胃不同的受试物,持续7 d。每日观察小鼠粪便状态,包括粪便数量、粪便湿重、粪便干重等指标。末次进食后小鼠禁食不禁水过夜,隔天采取眼窝取血后脱颈椎方式处死小鼠。
表2:便秘模型实验干预方案
时间/分组 | 1 2 3 4 5 6 7 (天) |
NC组 | 灌胃0.1 mL/10 g生理盐水 |
MC组 | 灌胃3 mg/kg Lop+1 h后灌胃生理盐水 |
PC组 | 灌胃3 mg/kg Lop+1 h后灌胃0.25 g/kg西沙比利 |
L-IDF组 | 灌胃3 mg/kg Lop+1 h后灌胃0.5 g/kg IDF |
M-IDF组 | 灌胃3 mg/kg Lop+1 h后灌胃1.5 g/kg IDF |
H-IDF组 | 灌胃3 mg/kg Lop+1 h后灌胃3.5 g/kg IDF |
实验结果如图5显示:IDF增加便秘小鼠1 h 排便数量和粪便重量以及粪便含水率,进而增强小鼠的排便功能。
实施例4
实验分组与灌胃剂量同实施例3。在小鼠麻醉前1 h给予5%炭末-10%阿拉伯胶半固体糊(1 mL/100 g体重),30 min后处死小鼠,剖开腹腔,留取血液标本待血清学检测。测量幽门至直肠距离及碳末的前端至幽门的距离,用以下列公式计算碳末推进百分率:
碳末推进率(%)=碳末前沿至幽门距离/幽门至直肠距离×100%
实验结果如图6显示:IDF能促进肠道转运速率,改善肠道转运功能。
实施例5
实验分组与灌胃剂量同实施例3。实验进行7 d后,小鼠禁食不禁水12 h后测定小鼠最后空腹体重以及小鼠体长(从鼻尖至肛门长度)并记录。分离出便秘小鼠的小肠(空肠、回肠和结肠)称重部分肠系,40℃干燥5 h,再次称重,用以下公式计算肠道含水率:
肠道含水率(%)=(肠道湿重-肠道干重)/肠道湿重×100%
实验结果如图7显示:IDF可增加肠道的含水量,促进肠道蠕动。
实施例6
实验分组与灌胃剂量同实施例3。将干预7 d后的小鼠,空腹12 h后,乙醚麻醉后眼眶取血,全血室温静置30 min后,在4℃、7500 rpm/min条件下离心15 min,血清分离,根据试剂盒说明书检测超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)含量。
实验结果如图8显示:IDF能够通过增强SOD和GSH-Px的活性,进而增强小鼠机体抗氧化能力,抑制便秘引起的氧化应激水平紊乱。
实施例7
实验分组与灌胃剂量同实施例3。实验进行7 d后,分别取出100 mg的小肠组织按照Transzol的方法说明提取总RNA,使用TransScript One-Step gDNA Removal and cDNASynthesis SuperMix试剂盒将总RNA反转录为cDNA用于实时荧光定量PCR检测肠神经递质关键基因: AchE、iNOS、eNOS、BDNF、GDNF的mRNA表达量。
实验结果如图9显示:IDF下调肠神经递质iNOS mRNA表达水平,上调AchE、eNOS、BDNF和GDNF的mRNA表达水平。
实施例8
实验分组与灌胃剂量同实施例3。实验结束后,分别取出0.1 g结肠组织提取蛋白,蛋白免疫印迹法检测炎症因子AchE、iNOS、eNOS、BDNF和GDNF的蛋白表达水平。
实验结果如图10显示:IDF下调肠神经递质iNOS蛋白表达水平,上调AchE、eNOS、BDNF和GDNF蛋白表达水平。
实施例9
实验分组与灌胃剂量同实施例3。实验结束前一天,将小鼠分别放入干净无菌的鼠笼中,收集小鼠粪便,。通过DNA抽提、设计合成引物接头、PCR扩增与产物纯化、PCR产物定量与均一化、构建PE文库等流程进行16S rRNA V3-V4区扩增,测序平台为Illumina。按照97%相似性对非重复序列进行OTU聚类,采用RDP classifier贝叶斯算法对97%相似水平的OTU代表序列进行物种组成分析。
实验结果如图11和图12显示:IDF能调整便秘小鼠的肠道菌群的OUT分布,减少拟杆菌门丰富度,增加厚壁菌门和放线菌门的丰度,主要是增加科水平上Lachnospiraceae占比,降低Lactobacillaceae、Marinifilaceae、Helicobacteraceae、norank_o__Clostridia_UCG-014占比。
实施例10
实验分组与灌胃剂量同实施例3,实验结束前一天,将小鼠分别放入干净无菌的鼠笼中,收集小鼠粪便。首先精确称取一定质量的样本,于低温环境下加入提取液进行代谢物萃取处理。配制不同浓度的标准溶液,在同一条件下对标准溶液和待测样品进行GC-MS上机检测,根据标准曲线计算出检测样品的目标物质的浓度,最终换算得到样品浓度。
实验结果如图13显示:IDF增加多种短链脂肪酸(SCFAs)水平,比如乙酸、丙酸、丁酸、异丁酸、戊酸、异戊酸、异己酸,参与便秘的机制调控。
以上实施例仅用以说明本发明的技术方案,而非对其进行限制;尽管参照前述实施例对本发明进行了详细的说明,对于本领域的普通技术人员来说,依然可以对前述实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或替换,并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。
Claims (10)
1.一种海带中不溶性膳食纤维在制备用于改善便秘的药物和功能性食品中的应用,其特征在于:所述不溶性膳食纤维的单糖组成包括葡萄糖和葡萄糖醛酸,二者的摩尔比为15-25:1。
2.根据权利要求1所述的应用,其特征在于:所述不溶性膳食纤维的提取方法,包括以下步骤:
(1)向海带残渣中以1:20-40的料液比加入4-6% NaCl溶液,80-100℃加热6-8 h进行脱钙处理,离心收集沉淀,纯水洗涤沉淀;
(2)向步骤(1)的沉淀中以1:20-40的料液比加入5 mol/L NaOH溶液, pH调至碱性,80-100℃加热6-8 h,离心收集沉淀;
(3)利用NaClO对步骤(2)中的沉淀进行脱色处理,然后再利用稀HCl进行除盐处理;
(4)除盐后的沉淀洗涤后干燥,研磨过200目筛,得到不溶性膳食纤维。
3.根据权利要求2所述的应用,其特征在于:所述步骤(3)中NaClO的体积分数为9%,所述NaClO与沉淀的料液比为12:1,脱色处理时间为67 min。
4.根据权利要求2所述的应用,其特征在于:所述步骤(3)中稀HCl的浓度为0.5 mol/L,所述稀HCl与脱色后的沉淀的料液比为1:1,除盐处理时间为1h。
5.根据权利要求1所述的应用,其特征在于:所述不溶性膳食纤维改善便秘的有效剂量为0.5 g/kg-3.5 g/kg。
6.根据权利要求1所述的应用,其特征在于:所述不溶性膳食纤维能够增加便秘小鼠的粪便数量、粪便重量和粪便含水率。
7.根据权利要求1所述的应用,其特征在于:所述不溶性膳食纤维能够通过增加便秘小鼠肠道含水率以及提高肠道转运速率来促进肠道蠕动和改善肠道转运功能。
8.根据权利要求1所述的应用,其特征在于:所述不溶性膳食纤维能够增加血清中超氧化物歧化酶SOD和谷胱甘肽过氧化物酶GSH-Px的含量,以及增加SCFAs的含量。
9.根据权利要求1所述的应用,其特征在于:所述不溶性膳食纤维能够下调诱导型一氧化氮合酶iNOS的表达水平,上调肠神经递质乙酰胆碱酯酶AchE、内皮型一氧化氮合酶eNOS、胶质细胞源性神经营养因子GDNF和脑衍生神经营养因子BDNF的表达水平。
10.根据权利要求1所述的应用,其特征在于:所述不溶性膳食纤维能够通过减少小鼠肠道内拟杆菌门丰富度以及增加厚壁菌门和放线菌门的丰度来调整便秘小鼠的肠道菌群的结构。
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