CN113861303B - 一种从德氏乳杆菌和嗜热链球菌发酵酸奶中分离出的胞外多糖及其应用 - Google Patents
一种从德氏乳杆菌和嗜热链球菌发酵酸奶中分离出的胞外多糖及其应用 Download PDFInfo
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Abstract
本发明属于食品加工领域,公开了一种从德氏乳杆菌和嗜热链球菌发酵酸奶中分离出的胞外多糖及其应用。本发明的中性胞外多糖为首次从德氏乳杆菌(Lactobacillus delbrueckii)DMLD‑H1和嗜热链球菌(Streptococcus thermophilus)DMST‑H2发酵酸奶中提取,组分均一且结构新颖,重均分子量为32063Da,单糖组成摩尔比为半乳糖:葡萄糖=42.04:57.96。采用扫描电镜发现多糖表现呈薄片状结构,刚果红实验结果表明该中性多糖可能存在三维螺旋结构。经动物实验验证该中性胞外多糖治疗结肠炎效果良好,在食品和医药领域具有广泛的应用前景。
Description
技术领域
本发明属于食品加工领域,具体涉及一种从德氏乳杆菌和嗜热链球菌发酵酸奶中分离出的胞外多糖及其应用。
背景技术
结肠炎是一种炎症性肠病(IBD),分为溃疡性结肠炎(UC)和克罗恩病(CD),是一种与免疫紊乱、遗传易感性和微生物种群紊乱相关的自身免疫性疾病。在全球范围内,UC的患病率高于CD。UC是一种慢性连续性弥漫性炎症疾病,是由于肠道菌群结构改变与肠屏障通透性改变引起的,发生于结肠黏膜,并与直肠相关。近几年,中国的发病率持续增加,主要作用于结肠黏膜,病变多发生于结肠远端。UC持续时间长,且容易反复发作,是一种全球性疾病。
溃疡性结肠炎,因易反复发作,病程长,较难治愈。目前治疗仍以抗炎和免疫调节为主,辅以生物制剂等治疗或高压氧、干细胞移植等非药物治疗,甚至借助外科手术。常用药物有以下6类:
(1)氨基水杨酸类,如柳氮磺吡啶(Sulfasalazine,SASP)、5-氨基水杨酸和美沙拉嗪。其中SASP具有较好的疗效和低廉的价格,是目前在UC治疗上使用最广的药物,长期服用机体会产生耐药性,且可能导致血液、肝肾、消化道等系统损伤和叶酸缺乏等不良反应。
(2)糖皮质激素类,具有强大的抗炎作用,但因代谢紊乱、骨质疏松等副作用,一般只用于急性期或重症期。
(3)免疫抑制剂类,常用于不适用前两种药物的患者,可缓解UC活动期,但因其肝、肾毒性较大,临床一般只作辅助作用。
(4)生物制剂类,能直接作用于肿瘤坏死因子α(Tumor necrosis factor alpha,TNF-α)、细胞黏附分子等靶点,特别是单克隆抗体治疗剂,更是目前药物热点。常用的英夫昔单抗(Infliximab,IFX),对重症患者十分有效,但因价格昂贵,且可能伴有白细胞、中性粒细胞减少和过敏等副作用,使用受到较大限制。
(5)益生菌类,可改善UC患者肠道菌群失调,促进营养吸收。但治疗UC症状效果一般,常和其他药物联用。
(6)抗感染药物,适用于细菌感染或重症患者,具有抑制肠道厌氧菌、促进瘘管愈合和预防复发等作用,但长期使用也会出现耐药性和副作用。
虽然在目前UC的治疗上,传统西药和新型生物制剂都对临床症状表现出较好的缓解作用,但也有副作用大、价格昂贵等问题。因此寻找副作用小,价格适宜,且对UC有显著治疗效果的新药物或干预作用的功能食品已成为迫切需求。
一些多糖可缓解结肠黏膜糜烂,减少溃疡的面积,抑制结肠黏膜充血水肿,缓解UC小鼠体重下降,减少泄泻及血便的发生率。研究表明,其机制可能为能下调TNF-α、IL-6,上调IL-10等炎症相关因子的表达,调节结肠中EGF、EGF-β含量促进其黏膜修复,降低结肠上皮细胞酶Caspase-3及Caspase-8的表达,有效控制上皮细胞凋亡,促进黏膜修复,提高肠道Occludin、ZO-1蛋白表达增强黏膜屏障能力。此外,多糖可有效调节肠道菌群,增加乳酸杆菌等数量,减少肠杆菌及球菌数量,增加肠内容物挥发性脂肪酸,调节肠道微生态,从而治疗UC。因此多糖可通过抗炎、调节肠道免疫及改善肠道菌群等手段来治疗UC。多糖具有多个治疗UC的靶点、又因为其副作用小与治疗效果不易反复等优势成为近年来研究的热点。
发明内容
针对于微生物多糖应用问题,本发明的目的是提供一种从德氏乳杆菌和嗜热链球菌发酵酸奶中分离出的胞外多糖及其应用。本发明针对目前治疗溃疡性结肠炎药物的副作用大、价格昂贵等不足,提供了一种安全、高效、副作用小的天然治疗溃疡性结肠炎药物的制备方法和应用。
本发明的目的通过如下技术方案实现:
一种酸奶中性胞外多糖,所述酸奶中性胞外多糖是将德氏乳杆菌(Lactobacillusdelbrueckii)DMLD-H1和嗜热链球菌(Streptococcus thermophilus)DMST-H2接种至脱脂乳中进行发酵培养,获得发酵酸奶;对所述酸奶进行离心、醇沉、除蛋白、经离子交换柱洗脱,洗脱液为水,得到分子量为32063Da的中性胞外多糖。
优选地,所述中性胞外多糖的单糖组成为半乳糖和葡萄糖,摩尔比为42.04:57.96。
优选地,所述中性胞外多糖的糖苷键由t-Glcp,4-Galp,4-Glcp,3,4-Glcp和4,6-Glcp组成,相对摩尔比为17.456:37.035:37.035:1.476:6.998。
一种制备酸奶中性胞外多糖的方法,其特征在于,包括如下步骤:
(1)菌种活化:取德氏乳杆菌DMLD-H1和嗜热链球菌DMST-H2分别接种至MRS培养基里,37℃静置培养24h;之后进行二级活化,接种量为5~10%v/v;活化后得到种子液,于37℃条件下静置备用;
(2)菌粉的制备:将种子液接种到发酵培养基中进行扩大培养,接种浓度为5~10%v/v,37℃恒温条件下静置培养24~30h后离心,生理盐水洗2~3次,离心后收集菌泥,添加冻干粉保护剂后,经-80℃预冻4h、冷冻干燥48h,得到菌粉;
(3)发酵酸奶的准备:将12~15%脱脂乳粉和8~10%蔗糖混合得到脱脂乳,85℃水浴加热15min,冷却至40℃~42℃后接入步骤(2)所述菌粉进行发酵,得到发酵酸奶;
(4)离心:离心步骤(3)所述发酵酸奶,收集上清液;
(5)醇沉:向步骤(4)所述上清液中加入无水乙醇,静置,离心取沉淀,收集沉淀溶于水得到粗多糖液;
(6)除蛋白:向步骤(5)中所得粗多糖液中加入Sevag试剂,室温下置于摇床震荡混匀,使蛋白充分吸附在有机相中,然后离心,保留水相,重复操作直至蛋白完全除去,将收集的水相进行透析冻干,得到粗胞外多糖;
(7)将步骤(6)所述粗胞外多糖配置成10~30mg/mL溶液,经DEAE-Cellulose 52离子交换柱和Sephadex G-150凝胶柱分离纯化,减压浓缩,真空冷冻干燥后,得到中性胞外多糖冻干粉。
优选地,步骤(2)所述菌泥和冻干保护剂体积比为1:(1~3)。
优选地,步骤(3)所述发酵条件为:初始接菌量为(2~4)×106CFU/mL,接种比例为(1~3):1,42±5℃发酵8~10h,4~6℃后熟12~14h。
优选地,步骤(4)所述离心条件为:离心力为10000~15000g,离心温度4℃,离心时间5~10min。
优选地,步骤(5)所述上清液与无水乙醇体积比为1:(4~6);步骤(6)所述粗多糖液与Sevag试剂体积比为(4~5):1。
酸奶中性胞外多糖在食品中的应用。
酸奶中性胞外多糖在治疗结肠炎药物中的应用。
本发明与现有技术相比,具有如下优点和有益效果:
(1)本发明所得到的中性胞外多糖为首次从德氏乳杆菌(Lactobacillusdelbrueckii)DMLD-H1和嗜热链球菌(Streptococcus thermophilus)DMST-H2发酵酸奶中提取分离,具有新的结构,其分子量为32063Da,单糖组成摩尔比为半乳糖(Gal):葡萄糖(Glc)=42.04:57.96。
(2)采用红外、甲基化和NMR分析多糖的结构由t-Glcp,4-Galp,4-Glcp,3,4-Glcp和4,6-Glcp(相对摩尔比=17.456:37.035:37.035:1.476:6.998)组成。
(4)采用扫描电镜发现多糖表现呈薄片状结构,刚果红实验结果表明该中性多糖可能存在三维螺旋结构。
(5)经小鼠动物实验证实本发明所得到的中性胞外多糖具有良好的治疗结肠炎效果。
DEAE-Cellulose 52阴离子交换柱层析,是基于离子交换层析的原理,基质是由带有电荷的树脂或纤维素组成,阴离子交换基质无法与不带电荷的中性多糖结合,从而被去离子水洗脱下来。
菌体:德氏乳杆菌(Lactobacillus delbrueckii)DMLD-H1,保藏编号为GDMCCNO.60645,于2019年4月16日保藏于广东省微生物菌种保藏中心,简称GDMCC,地址:广州市先烈中路100号大院59号楼5楼,广东省微生物研究所。该菌株已在中国专利CN110607255A中公开。
嗜热链球菌(Streptococcus thermophilus)DMST-H2,保藏编号为GDMCCNO.60642,于2019年4月16日保藏于广东省微生物菌种保藏中心,简称GDMCC,地址:广州市先烈中路100号大院59号楼5楼,广东省微生物研究所。该菌株已在中国专利CN110607253A中公开。
附图说明
构成本发明的一部分的附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用来解释本发明,并不构成对本发明的不当限定。
图1酸奶中性胞外多糖的Sephadex G-150凝胶柱纯化洗脱曲线。
图2酸奶中性胞外多糖GPC高效液相色谱图。
图3酸奶中性胞外多糖单糖组成高效液相色谱图。
图4酸奶中性胞外多糖的1H NMR谱图。
图5酸奶中性胞外多糖的13C NMR谱图。
图6酸奶中性胞外多糖的红外光谱图。
图7酸奶中性胞外多糖扫描电镜图(A:800×,B:2000×)。
图8酸奶中性胞外刚果红实验图。
图9酸奶中性胞外多糖对DSS诱导结肠炎小鼠的体重和疾病活动指数(diseaseactivity index,DAI)影响(A:小鼠的体重变化情况;B:小鼠的DAI评分)。p*<0.05,p**<0.01,p***<0.001表示统计学意义上的有显著差异,置信区间取95%。
图10酸奶中性胞外多糖对DSS诱导结肠炎小鼠的血清细胞因子水平影响(A:小鼠血清中的TNF-α水平;B:小鼠血清中的IL-10水平;C:小鼠血清中的IL-1β水平)。p*<0.05,p**<0.01,p***<0.001表示统计学意义上的有显著差异,置信区间取95%。
具体实施方式
下面结合具体实施例对本发明作进一步具体详细描述,但本发明的实施方式不限于此,对于未特别注明的工艺参数,可参照常规技术进行。
实施例1:从德氏乳杆菌和嗜热链球菌发酵酸奶中分离出胞外多糖
脱脂乳配方为(以质量分数计):12%脱脂乳粉,8%蔗糖。
冻干保护剂配方为(以质量分数计):10%脱脂奶粉,10%海藻糖。
Sevag试剂是将氯仿与正丁醇混合得到,氯仿与正丁醇体积比为5:1。
MRS培养基配方(以重量份数计):酪蛋白消化物0.9份,酵母膏0.4份,葡萄糖1.8份,柠檬酸三铵0.15份,硫酸镁0.05份,牛肉膏0.75份,磷酸氢二钾0.15份,乙酸钠0.45份,吐温80 0.2份,硫酸锰0.02份,其余为水。
发酵培养基配方(以重量份数计):酪蛋白消化物0.9份,酵母膏0.4份,葡萄糖1.6份,柠檬酸三铵0.15份,硫酸镁0.055份,牛肉膏0.8份,磷酸氢二钾0.15份,乙酸钠0.45份,吐温80 0.2份,大豆蛋白肽0.9份,抗坏血酸0.015份,硫酸锰0.25份,其余为水。
(1)菌种活化:取德氏乳杆菌DMLD-H1和嗜热链球菌DMST-H2分别接种至MRS培养基里,37℃静置培养24h;之后进行二级活化,接种量为5.0%(v/v);活化后得到种子液,于37℃条件下静置备用;
(2)菌粉制备:将活化好的种子液接种到发酵培养基中进行扩大培养,接种浓度为5.0%(v/v)。在37℃恒温条件下静置培养24h后离心,并用生理盐水洗2~3次。离心后将上清液弃去并收集菌泥,添加冻干保护剂(德氏乳杆菌:保护剂为1:3,v/v;嗜热链球菌:保护剂为1:1,v/v)后在-80℃冰箱中预冻4h、之后冷冻干燥48h,得到菌粉;
(3)发酵酸奶的准备:12%脱脂乳粉和8%蔗糖混合得到脱脂乳,经过85℃水浴加热15min,冷却到40℃~42℃,接入步骤(2)所述菌粉进行发酵,发酵条件为:初始接菌量为2×106CFU/mL,接种比例为1:1,42℃发酵8h,4℃后熟12h;
(4)离心:将步骤(3)中发酵酸奶进行离心处理,收集上清液,离心条件为:离心力为10000g,离心温度4℃,离心时间10min;
(5)醇沉:向上清液加入无水乙醇(上清液:无水乙醇=1:4,v/v),静置,离心取沉淀,收集沉淀后溶于水得粗多糖液;
(6)除蛋白:向步骤(5)中所得的粗多糖液加入Sevag试剂(粗多糖液:Sevag试剂=4:1,v/v),室温下置于摇床震荡(220rpm/min,10min)混匀,使得蛋白充分吸附在有机相中,然后离心,保留水相,重复操作,直至蛋白完全除去,将收集的水相进行透析冻干备用。
(7)DEAE-Cellulose 52离子交换柱和Sephadex G-150凝胶柱分离纯化:将上述步骤(6)后的胞外多糖,配置成10mg/mL溶液,取30mL加样于DEAE-Cellulose 52离子交换柱中,用去离子水以流速为1.0mL/min下进行洗脱。洗脱液紧接着经过Sephadex G-150凝胶柱,用去离子水以流速为0.2mL/min下进行洗脱,每管收集4mL,采用苯酚-硫酸法跟踪检测多糖含量如图1所示。收集不同管中的多糖液,减压浓缩,真空冷冻干燥,得到中性胞外多糖冻干粉。DEAE-Cellulose 52离子交换柱和Sephadex G-150凝胶柱为多糖常用分离柱,为节省中性胞外多糖纯化时间采用连接方式为串联连接,实现一步法纯化。
实施例2:酸奶中性胞外多糖的单糖组成分析
(1)分子量测定
采用高相液相色谱高效凝胶渗透色谱法(Waters1525凝胶色谱仪)测定实施例1步骤(6)所制备的中性胞外多糖分子量的均一性和分子量。色谱柱为TSK G5000PWXL(6μm,7.8×300mm)和TSK G3000 PWXL(6μm,7.8×300mm)串联使用,检测器为Waters 2414示差折光检测器(RID),柱温35℃、进样量10μL,流动相为0.02mol/L的磷酸氢二钾缓冲溶液,流速0.6mL/min。分别将不同分子质量的葡聚糖标准品过0.45μm滤膜后上机,记录保留时间。以保留时间为横坐标、以葡聚糖分子量的对数为纵坐标绘制标准曲线。以同样的方法测定中性的多糖的出峰时间,根据标准曲线,计算出酸奶中性胞外多糖分子质量。由图2可知,酸奶中性胞外多糖的分子量为32063Da,从而说明酸奶中的中性胞外多糖较为均一,可进一步进行单糖组成成分的测定。
标准品的配制:准备标准品与试剂,如表1所示。
表1标准品与试剂信息
在EP管中加入8mL无菌水,依次加入岩藻糖、阿拉伯糖、半乳糖、葡萄糖、木糖、甘露糖、果糖、核糖、半乳糖醛酸、葡萄糖醛酸各100mg,溶解后定容至10mL得到10mg/mL的母液。稀释100倍,制备成100μg/mL工作液,将取上述溶液按照以下梯度稀释,装入1.5mL的EP管中。各单糖混标梯度浓度信息(μg/mL)如表2所示。
表2单糖混标梯度浓度信息(μg/mL)
样品前处理:处理实施例1步骤(6)的酸奶中性胞外多糖,具体步骤如下:取干净的色谱瓶,称量多糖样品各5mg,加入TFA酸溶液,121℃加热2h,通氮气吹干。加入甲醇清洗再吹干,重复2-3次。加无菌水溶解后转入色谱瓶中待测。
提取液体样本:取适量上清液,通氮气吹干。之后的步骤与固体样本提取一致。
分析检测:色谱系统采用的是Thermo ICS5000+离子色谱系统(ICS5000+,(ThermoFisher Scientific,USA),采用DionexTMCarboPacTMPA10(250*4.0mm,10μm)液相色谱柱,进样量为20μL。流动相A(H2O),流动相B(100mol/L NaOH),柱温为30℃,利用电化学检测器对单糖组分进行分析检测。流动相的具体梯度与数据如下表所示。
表3流动相梯度
表4单糖含量及摩尔比
由图3和表4可知,酸奶中性胞外多糖组成摩尔比为半乳糖(Gal):葡萄糖(Glc)=42.04:57.96。
实施例3:酸奶中性胞外多糖的甲基化和核磁分析
(1)甲基化及GC-MS分析
准备标准品与试剂,如表5所示。
表5标准品与试剂信息
多糖样品衍生化:称取10mg经纯化后的样品,加入1mL去离子水溶解,再加入1mL100mg/mL碳二亚胺,反应2h。随后加入1mL 2mol/L的咪唑,平均分为两份后分别加入1mL30mg/mL的NaBH4和同体积同浓度的NaBD4,3h后加入100μL冰醋酸终止反应。透析样品48h,完成后冷冻干燥样品并进行甲基化处理。往冻干样品中加500μL DMSO溶解,加1mg NaOH孵育30min,加50μL碘甲烷溶液反应1h。加1mL水和2mL二氯甲烷混合混匀,离心弃水相。重复水洗3次,吸取下层二氯甲烷相并蒸干,加入100μL 2mol/L TFA于121℃条件下反应90min后于30℃蒸干;加50μL 2mol/L氨水、50μL 1mol/L NaBD4混匀,室温下反应2.5h。加20μL乙酸终止反应,氮气吹干,250μL甲醇洗两次,氮气吹干,加乙酸酐250μL,涡旋混匀,100℃反应2.5h。入1mL水静置10min后,加500μL二氯甲烷,涡旋混匀,离心,弃水相。重复水洗3次后,取下层二氯甲烷相,准备上机检测。
气质联用色谱分析:采用Agilent气象色谱系统(Agilent 7890A;AgilentTechnologies,USA),根据化合物的性质,进样量为1μL,分流比10:1,载气为高纯氦气;保持柱温箱初始温度为140℃2.0min,以3℃/min速度升温至230℃保持3min。
采用的是美国Aiglent公司的四极杆质谱检测系统(Agilent 5977B;AgilentTechnologies,USA),配有电子轰击离子源(EI)和Mass Hunter工作站。采用电子轰击离子源(EI),分析物在全扫描(SCAN)模式下进行检测,质量扫描范围(m/z)为30-600。
(2)核磁共振分析
分别称取实施例1-(6)所制备的酸奶中性胞外多糖5mg溶于0.6mL重水(D2O)中,反复冻干复溶与0.6mL重水后加入核磁管中,于Bruker AV-600型核磁共振仪上进行1H NMR和13C NMR测定。
酸奶中性胞外多糖的甲基化分析如表6所示。
表6中性胞外多糖的甲基化及GC-MS分析表
通过与PMAA数据库比对,5个衍生物分别是1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl glucitol(1,5-二-O-乙酰基-2,3,4,6-四-O-甲基葡萄糖醇),1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl galactitol(1,4,5-三-O-乙酰基-2,3,6-三-O-甲基半乳糖醇),1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl glucitol(1,4,5-三-O-乙酰基-2,3,6-三-O-甲基葡萄糖醇),1,3,4,5-tetra-O-acetyl-2,6-di-O-methyl glucitol(1,3,4,5-四-O-乙酰基-2,6-二-O-甲基葡萄糖醇),1,4,5,6-tetra-O-acetyl-2,3-di-O-methylglucitol(1,4,5,6-四-O-乙酰基-2,3-二-O-甲基葡萄糖醇)。
由表6中可得,中性胞外多糖的糖苷键的连接方式包括t-Glcp,4-Galp,4-Glcp,3,4-Glcp和4,6-Glcp组成,相对摩尔比为17.456:37.035:37.035:1.476:6.998。根据方程DB=(NT+NB)/(NT+NB+NL)计算出酸奶中性胞外多糖的分支度(DB)值为25.93%,其中NT是指末端残基t-Glcp(1→,NB是指分支残基→3,4)-Glcp(1→和→4,6)-Glcp(1→,NL是指线性残基→4)-Galp(1→和4)-Glcp(1→的数量。
酸奶中性胞外多糖的1H NMR图谱如图4所示。在δ6~8ppm之间没有观察到共振,表明酸奶中性胞外多糖不含酚或阿魏酸等杂质。异位氢分布在δ4.7~5.5ppm之间,表明酸奶中性胞外多糖中含有α-和β-糖苷键。13C NMR可以反映样品中多糖的残留量。此外,通过化学位移在95~110ppm之间的正头碳峰数可以分析和确定多糖残基的数量及其相关构型。酸奶中性胞外多糖的13C NMR谱图如图5所示。在δ103.65、102.89、96.41、95.87和95.72ppm处发现了异端碳,表明酸奶中性胞外多糖中含有5种糖苷键,与甲基化结果一致。关于这5个糖苷键的位置和序列的更详细的信息将在将来被阐明。
实施例4:酸奶中性胞外多糖红外光谱分析
采用溴化钾压片法,分别称取实施例1步骤(6)制得的中性胞外多糖10mg,加入100mg的KBr粉末,用压片机压成均匀的薄片,采用Bruker VERTEX33型傅里叶变换红外光谱仪在4000-500cm-1范围内进行红外光谱扫描,记录谱图。图6可知,在3412.08cm-1处的宽拉伸峰属于羟基拉伸振动。在2937.59cm-1处的峰值是脂肪族CH2基团的不对称C和H伸缩振动,表明存在糖等有机物。1649.14cm-1处的振动可能与羧基的对称拉伸有关,1426.36cm-1处的振动可能与C和H的弯曲振动有关。1200~1000cm-1区域的吸收峰可能是C-O-H和C-O-C伸缩振动引起的。1022.27cm-1处的振动可能与C-O的弯曲振动有关。在924.87cm-1处的吸收峰表明,酸奶中性胞外多糖结构中可能存在呋喃环。
实施例5:酸奶中性胞外多糖表观形态和刚果红实验分析
(1)扫描电镜观察酸奶中性胞外多糖表观形态
扫描电镜是目前常用的观察多糖形貌和判定多糖种类的方法,具有操作简单、结果直观和分辨率高的优点,因此在食品科学、化学、材料和生物学上得到广泛应用。取充分干燥的实施例1步骤(6)中性胞外多糖组分,取少量涂抹于导电胶上,喷金后采用扫描电镜观察其表面形态。由图7可知,酸奶中性胞外多糖呈现薄片状结构,随着放大倍数的增加,可观察到其表面光滑。
(2)刚果红实验
刚果红是一种酸性染料,它可以和具有三股螺旋结果的多糖形成络合物,络合物的最大吸收波长同刚果红相比发生位移,称取2mg实施例1步骤(6)中性胞外多糖组分,溶解于2mL蒸馏水后加入2mL80μmol/L的刚果红试剂,逐步加入1mol/L的氢氧化钠,使溶液中的氢氧化钠浓度从0.0mol/L升至0.5mol/L,然后进行紫外波谱扫描,测得不同NaOH浓度下的最大吸收波长。由图8可知,低浓度氢氧化钠,紫外吸收移向长波说明多糖能与刚果红形成络合物,具有螺旋构象。高浓度氢氧化钠,最大吸收波长下降说明此时多糖螺旋结构解体。
实施例6:酸奶中性胞外多糖治疗溃疡性结肠炎小鼠的应用
BALB/c小鼠,雄性,4~5周龄,采自浙江维通利华实验动物科技有限公司。所有小鼠被标准饲料适应(23~25℃和12h的光/暗周期)7天。采用3.5%(w/v)DSS激活小鼠结肠炎模型。将40只小鼠随机分为5组(n=8/组):第1~7天,对照组(Control)灌胃生理盐水,灌胃蒸馏水;(2)模型组(Model)小鼠口服生理盐水,给予3.5%DSS溶液;(3)L-EPS组小鼠口服80mg/kg酸奶中性胞外多糖,给予3.5%DSS溶液;(4)H-EPS组小鼠口服160mg/kg酸奶中性胞外多糖,给予3.5%DSS溶液;(5)SASP组(阳性对照)小鼠口服柳氮磺胺吡啶(SASP)溶液100mg/kg,给予3.5%DSS溶液,在第15天,所有小鼠被安乐死。
由图9(A)可知,在实验第三天时,饮用了DSS的四组小鼠体重都出现了明显下降,模型组小鼠的体重在实验过程中持续下降,试验结束时,L-EPS、H-EPS和SASP组小鼠平均体重较模型组有明显恢复。
DAI(disease activity index,DAI)是疾病活动指数,评价标准按照以下表格打分。
表7DAI评分标准
由图9(B)可知,模型组(Model)小鼠的DAI评分显著高于空白对照组(Control),说明造模成功。
用ELISA方法检测了TNF-α、IL-10和IL-1β的血清水平,也就是抗原吸附在固相载体上,加待测抗体,再加相应酶标记抗体,生成抗原-待测抗体-酶标记抗体的复合物,再与该酶的底物反应生成有色产物,利用分光光度计测量吸光度后计算抗体的量。TNF-α是一种能够直接杀伤肿瘤细胞而对正常细胞无明显毒性的细胞因子。图10(A)是各组小鼠血清中的TNF-α水平,可以看到,模型组(Model)的TNF-α水平是显著高于空白组(Control)的,而经过多糖的治疗,能够降低TNF-α的水平。IL-10是重要的抑制细胞因子,可以抑制炎性反应、限制过度免疫反应。由图10(B)可知,模型组(Model)的IL-10水平是显著低于空白组(Control)的,经过多糖的治疗,能够提升IL-10的水平,其中高剂量多糖组(H-EPS)的IL-10水平显著提高。IL-1β是重要的促凋亡和促炎细胞因子之一,由图10(C)可知,模型组(Model)的IL-1β水平是显著高于空白组(Control)的,经过多糖的治疗,低剂量多糖组(L-EPS)和高剂量多糖组(H-EPS)都能显著降低IL-1β的水平。从血清中的细胞因子水平可知,经过多糖的治疗,可以恢复IL-10的水平,降低TNF-α和IL-1β的水平,从而达到抗炎的目的,缓解小鼠的结肠炎病症。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (8)
1.一种酸奶中性胞外多糖,其特征在于,所述酸奶中性胞外多糖是将德氏乳杆菌(Lactobacillus delbrueckii)DMLD-H1和嗜热链球菌(Streptococcus thermophilus)DMST-H2接种至脱脂乳中进行发酵培养,获得发酵酸奶;对所述酸奶进行离心、醇沉、除蛋白、经离子交换柱洗脱,洗脱液为水,得到分子量为32063 Da的中性胞外多糖;
所述中性胞外多糖的单糖组成为半乳糖和葡萄糖,摩尔比为42.04:57.96;
所述中性胞外多糖的糖苷键由t-Glcp,4-Galp,4-Glcp,3,4-Glcp和4,6-Glcp组成,相对摩尔比为17.456:37.035:37.035:1.476:6.998。
2.一种制备权利要求1所述的酸奶中性胞外多糖的方法,其特征在于,包括如下步骤:
(1)菌种活化:取德氏乳杆菌DMLD-H1和嗜热链球菌DMST-H2分别接种至 MRS 培养基里,37℃静置培养24h;之后进行二级活化,接种量为 5~10% v/v;活化后得到种子液,于37℃条件下静置备用;
(2)菌粉的制备:将种子液接种到发酵培养基中进行扩大培养,接种浓度为 5~10% v/v,37℃恒温条件下静置培养24 ~30 h后离心,生理盐水洗2~3次,离心后收集菌泥,添加冻干粉保护剂后,经-80℃预冻4 h、冷冻干燥48 h,得到菌粉;
(3)发酵酸奶的准备:将12~15%脱脂乳粉和8~10%蔗糖混合得到脱脂乳,85℃水浴加热15~20 min,冷却至40℃~42℃后接入步骤(2)所述菌粉进行发酵,得到发酵酸奶;
(4)离心:离心步骤(3)所述发酵酸奶,收集上清液;
(5)醇沉:向步骤(4)所述上清液中加入无水乙醇,静置,离心取沉淀,收集沉淀溶于水得到粗多糖液;
(6)除蛋白:向步骤(5)中所得粗多糖液中加入Sevag试剂,室温下置于摇床震荡混匀,使蛋白充分吸附在有机相中,然后离心,保留水相,重复操作直至蛋白完全除去,将收集的水相进行透析冻干,得到粗胞外多糖;
(7)将步骤(6)所述粗胞外多糖配置成10~30 mg/mL溶液,经DEAE-Cellulose 52离子交换柱和Sephadex G-150凝胶柱分离纯化,减压浓缩,真空冷冻干燥后,得到中性胞外多糖冻干粉。
3.根据权利要求2所述的方法,其特征在于,步骤(2)所述菌泥和冻干保护剂体积比为1:(1~3)。
4.根据权利要求2所述的方法,其特征在于,步骤(3)所述发酵条件为:初始接菌量为(2~4)×106 CFU/mL,接种比例为(1~3):1,42±5℃发酵8~10 h,4~6℃后熟12~14 h。
5.根据权利要求2所述的方法,其特征在于,步骤(4)所述离心条件为:离心力为10000~15000g,离心温度4℃,离心时间5~10min。
6.根据权利要求2所述的方法,其特征在于,步骤(5)所述上清液与无水乙醇体积比为1:(4~6);步骤(6)所述粗多糖液与Sevag试剂体积比为(4~5):1。
7.权利要求1所述酸奶中性胞外多糖在食品中的应用。
8.权利要求1所述酸奶中性胞外多糖在制备治疗结肠炎药物中的应用。
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