CN114381395B - 一种植物乳杆菌zjufn1及其应用 - Google Patents
一种植物乳杆菌zjufn1及其应用 Download PDFInfo
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- CN114381395B CN114381395B CN202111652039.8A CN202111652039A CN114381395B CN 114381395 B CN114381395 B CN 114381395B CN 202111652039 A CN202111652039 A CN 202111652039A CN 114381395 B CN114381395 B CN 114381395B
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Abstract
本发明公开了植物乳杆菌(Lactobacillus plantarum)ZJUFN1及其应用,属于微生物技术领域。所述植物乳杆菌ZJUFN1的保藏号为CCTCC NO:M2020125。本发明提供的植物乳杆菌ZJUFN1在胃肠道环境中具有很高的生存率、较强的胃肠粘附力,能显著抑制胃肠道致病菌的繁殖,具有较强的自由基清除能力。灭活的植物乳杆菌ZJUFN1可以促进巨噬细胞分泌IL‑10,具有潜在的抗炎作用。另外,灭活的植物乳杆菌ZJUFN1可以显著提高胰岛素抵抗HepG2肝细胞的摄糖量,缓解高脂膳食诱导的脂肪代谢紊乱、高血糖和胰岛素抵抗。
Description
技术领域
本发明涉及微生物技术领域,具体涉及植物乳杆菌(Lactobacillus plantarum)ZJUFN1及其应用。
背景技术
随着人们膳食结构的改变,高脂膳食摄入明显增加,并由此引起的胰岛素抵抗呈逐年上升趋势。高血糖往往伴随着血脂代谢紊乱现象,引起血液粘稠及血流速减慢,长期发展会导致动脉粥样硬化、血栓等慢性心血管疾病。糖尿病及其并发症严重威胁着人们的身体健康。
2型糖尿病是一种慢性代谢紊乱性疾病,临床表现为高血糖,并常伴随低度炎症。研究表明机体的代谢紊乱与免疫细胞产生白细胞介素-10(IL-10)的能力下降密切相关(Vermaetal., 2016)。抑制了IL-10就会促进TNF-α和IL-6等炎症因子的表达,而且胰岛素信号变差,甚至激活糖异生和脂肪生成途径(Gotoh et al., 2017)。
临床上一般采用口服降糖药对糖尿病患者进行治疗,如磺脲类、双胍类等。但是长期应用这种治疗方法易引起一些副作用,如腹泻、头晕、恶心、食欲不振、低血糖和肝功能损伤等,因此,开发一种天然的、安全的新型降糖药物或辅助治疗药物变得尤为重要。近年来,益生菌的益生功能引起了人们的广泛关注。例如植物乳杆菌,与其他益生乳杆菌一样,其具有降低胆固醇、调节免疫以及维持健康肠道菌群等功能(Seddik et al., 2017),也具有缓解代谢综合征的潜能。
灭活益生菌为益生菌经过人为处理后的代谢产物或是菌体成分,因为其中的小分子(短链脂肪酸等)部分可透过肠道黏膜快速进入体内,大分子部分(肽聚糖、膜蛋白等)可直接刺激肠道免疫系统,产生多项帮助健康的益处,包括增强免疫、平衡肠道菌群、调节生理机能等。多项实验显示灭活型益生菌的免疫激活能力更快、更好,同时,灭活型益生菌更稳定,不仅耐高温、耐胃酸、耐胆碱,而且后加工性不受局限,包括长保乳饮、饮料、高温工艺生产环境下烘焙食品等都可应用,商品保质期也更长。
专利文献CN 113384600 A公开了一种灭活乳酸菌复合物,将植物乳杆菌CICC24202和短乳杆菌YM 1301混合后采用高压蒸汽灭活,120~130℃处理20~30 min,在高脂饲料联合多次小剂量的链脲佐菌素诱导下的2型糖尿病模型中,灭活菌组对于2型糖尿病的改善程度达到活菌组水平。
由于植物乳杆菌不同菌株的基因组和功能存在很大差异,挖掘更多对缓解低度系统炎症和胰岛素抵抗有益的植物乳杆菌是本领域技术人员需要解决的问题。
发明内容
本发明的目的在于提供一株植物乳杆菌(Lactobacillus plantarum) ZJUFN1,该菌株灭活后具有优异的益生作用,能够显著改善高脂膳食导致的脂肪代谢紊乱、低度系统炎症、胰岛素抵抗和高血糖,具有缓解代谢综合征的潜力。
本发明从酸牛乳样品中分离筛选得到一菌株N1,经鉴定该菌株为植物乳杆菌,命名为植物乳杆菌(Lactobacillus plantarum) ZJUFN1。植物乳杆菌ZJUFN1的16S rRNA的碱基序列如SEQ ID NO.1所示。将植物乳杆菌ZJUFN1于2020年5月18日保藏于中国典型培养物保藏中心(地址:中国武汉、武汉大学),保藏号为:CCTCC NO: M 2020125。
体外实验表明,植物乳杆菌ZJUFN1具有耐受胃肠道转运液的能力,在模拟人工胃肠道中具有很高的生存率;植物乳杆菌ZJUFN1具有较好的肠道黏附和定植的能力,能显著抑制胃肠道致病菌(如大肠杆菌、金黄色葡萄球菌)的繁殖。因此,植物乳杆菌ZJUFN1可用于预防或治疗胃肠道菌群失衡。
本发明提供了植物乳杆菌(Lactobacillus plantarum) ZJUFN1在制备抑制胃肠道致病菌的药物中的应用。
所述胃肠道致病菌包括金黄色葡萄球菌、大肠杆菌中的一种或两种。
本发明还提供了灭活植物乳杆菌(Lactobacillus plantarum) ZJUFN1在制备抗炎药物中的应用。
进一步的,灭活植物乳杆菌ZJUFN1具有促进巨噬细胞分泌白细胞介素10(IL-10)的作用。
本发明体外实验表明,植物乳杆菌ZJUFN1具有较强的自由基清除能力,促进巨噬细胞分泌IL-10,具有潜在的抗炎作用,可以缓解高脂膳食诱导的低度系统炎症。
本发明还提供了灭活植物乳杆菌(Lactobacillus plantarum) ZJUFN1在制备治疗糖尿病药物中的应用。
所述的糖尿病为高脂膳食引起的代谢异常综合征,尤其为2型糖尿病。
进一步的,灭活植物乳杆菌ZJUFN1具有改善胰岛素抵抗和降低血糖的作用。
本发明研究表明,植物乳杆菌ZJUFN1能够显著改善高脂膳食小鼠的空腹血糖和胰岛素抵抗。具体的,体外实验表明,植物乳杆菌ZJUFN1能够提高胰岛素抵抗HepG2肝细胞的摄糖量。动物实验表明,灭活植物乳杆菌ZJUFN1能够显著改善高脂膳食导致的胰岛素抵抗和高血糖。进一步机理研究表明,灭活植物乳杆菌ZJUFN1可能是通过刺激胰高血糖素样肽-1(GLP-1)的分泌达到改善胰岛素抵抗的效果。
本发明还提供了灭活植物乳杆菌(Lactobacillus plantarum) ZJUFN1在制备降低动物或人体内低密度脂蛋白胆固醇浓度的药物中的应用。
本发明研究表明,灭活植物乳杆菌ZJUFN1可以改善高脂膳食导致的血脂代谢紊乱,尤其可以显著降低血清中LDL-C的水平。
进一步的,所述灭活植物乳杆菌ZJUFN1的制备方法为:首先将活化好的菌体接种于MRS液体培养基扩大培养,收集菌体,然后菌体经无菌PBS洗涤后,重悬于无菌水中,75℃下60 min进行热致死处理,最后冻干。
进一步的,所述药物由植物乳杆菌ZJUFN1菌剂与在药学上可接受的载体组成。
本发明具备的有益效果:
本发明提供了一种益生特性优异的植物乳杆菌ZJUFN1,在胃肠道环境中具有很高的生存率、较强的胃肠粘附力,能显著抑制胃肠道致病菌的繁殖,具有较强的自由基清除能力。灭活的植物乳杆菌ZJUFN1能够促进巨噬细胞分泌IL-10,具有潜在的抗炎作用。另外,灭活的植物乳杆菌ZJUFN1可以显著提高胰岛素抵抗HepG2肝细胞的摄糖量,显著缓解高脂膳食诱导的脂肪代谢紊乱、高血糖和胰岛素抵抗。
附图说明
图1为植物乳杆菌N1体外刺激巨噬细胞分泌IL-10能力的检测结果。
图2为灭活植物乳杆菌N1对HFD饲喂小鼠血脂四项的影响;A为LDL-C,B为HDL-C,C为TG,D为TCHO。
图3为灭活植物乳杆菌N1对HFD饲喂小鼠附睾脂肪的影响;A为附睾脂肪H&E染色图片,B为脂肪细胞的面积大小。
图4为灭活植物乳杆菌N1对HFD饲喂小鼠空腹血糖和胰岛素抵抗的影响;A为空腹血清血糖,B为血清胰岛素,C为HOMA-IR。
图5为灭活植物乳杆菌N1对HFD饲喂小鼠糖代谢相关指标的影响;A为糖基化血红蛋白,B为胰高血糖素样肽-1。
图6为灭活植物乳杆菌N1对HFD饲喂小鼠炎症相关指标的影响;A为脂多糖LPS,B为TNF-α,C为IL-6,D为IL-10。
具体实施方式
下面结合具体实施例对本发明作进一步说明。以下实施例仅用于说明本发明,不用来限制本发明的适用范围。
下述实施例中所使用的试验方法如无特殊说明,均为常规方法;所使用的材料、试剂等,如无特殊说明,为可从商业途径得到的试剂和材料。
实施例1菌株分离鉴定
1、菌株的筛选及分离
材料:酸牛乳,高原地区常食用的传统发酵乳制品,本发明采用来自云南的酸牛乳样品。
称取5 g酸牛乳加入到45 mL 0.85%的灭菌生理盐水中,振荡制成悬液,进行系列梯度稀释,稀释后取10-5、10-6、10-7三个梯度涂布于MRS培养基上,每个梯度做三个平行。将涂布后的平板放入37℃厌氧培养箱中培养24-48h。挑选差异明显的菌落进行MRS培养基上划线分离,将平板放入37℃厌氧培养箱中培养24-48h。连续划线3-5次后,取一滴现配的5%的过氧化氢滴于载玻片上,挑取固体培养基上单菌落接入5%的过氧化氢液滴中并混匀,产生气泡为过氧化氢酶试验阳性,未产生气泡为过氧化氢酶试验阴性,过氧化氢酶试验阴性菌株为疑似益生菌株。
将筛选得到的疑似益生菌株分别以1%接种量接入MRS-THIO液体培养基(MRS中添加0.2%的巯基乙酸钠)和含0.3%(w/v)的猪胆盐的MRS-THIO培养基中,上述处理放置在37℃厌氧培养箱中培养24h,取样在600nm下测定吸光值,计算24h的OD值差值,检测菌株的生长能力,筛选OD值差值为正数(耐受胆盐)的菌株为疑似益生菌株。
将上述筛选得到的疑似益生菌株以1%接种量接种到pH3.0的MRS培养基中,37℃厌氧培养箱中培养24h,取样在600nm下测定吸光值,计算24h的OD值差值,检测菌株的生长能力,筛选OD值差为正数(耐受低pH)的菌株,筛选得到了N1。
2、菌株的鉴定
将N1进行革兰氏染色,并进行了16S rRNA基因菌种鉴定,此鉴定送样上海派森诺生物有限公司进行,16S rRNA序列如SEQ ID NO.1所示。经过16S rRNA基因比对,该菌株与Gene Bank数据库中已知植物乳杆菌(Lactobacillus plantarum)的同源性最高达99.73%。
结合上述分析,鉴定N1为一株植物乳杆菌,命名为Lactobacillus plantarumZJUFN1。将植物乳杆菌ZJUFN1于2020年5月18日保藏于中国典型培养物保藏中心,保藏号为:CCTCC NO: M 2020125。保藏地址:中国武汉、武汉大学;该培养物的存活性于2020年6月2日检测为存活。
3、样品的制备
(1)活菌的制备:将活化好的菌体接种于MRS液体培养基中在37 ℃下培养18 h,8000 rpm 4 ℃条件下离心15 min,然后无菌PBS洗2遍,PBS重悬,调整活菌量约为1.0×109CFU/mL(OD600与倾注平板计数结果调整),备用。
(2)死菌(Heat-killed bacteria,HKB)的制备:将活化好的菌体接种于MRS液体培养基中在37 ℃下培养18 h,8000 rpm 4 ℃条件下离心15 min,然后无菌PBS洗2遍,接着蒸馏水洗2遍,重悬于蒸馏水中,75 ℃下60 min进行热致死处理,最后冻干。
(3)发酵上清(CFS):按照2%的接种量将菌株接种于MRS液体培养基中置于37 ℃下培养18 h,然后在8000 rpm 4 ℃条件下冷冻离心15 min,取上清,过0.22 μm滤膜后存于-80 ℃下备用。
(4)无细胞提取物(CFE):按照2%的接种量将菌株接种于MRS液体培中置于37 ℃下培养18 h,然后在8000 rpm 4 ℃条件下冷冻离心15 min,PBS洗2遍,PBS重悬,调整菌量约为5.0×1010CFU/mL。超声细胞破碎(冰浴上),50%功率,8 s脉冲,超声20 min。然后8000 rpm4 ℃冷冻离心15 min,取上清,过0.22 μm滤膜后存于-80 ℃下备用。
实施例2模拟人工胃肠液耐受性
人工模拟胃肠液需新鲜配制,且必须过膜(0.22 μm)除菌。
模拟胃液(GJ):胃蛋白酶(1:10000)加入PBS(pH2.5)中,浓度为3.5 g/L。
模拟肠液(IJ):NaHCO311 g/L,NaCl 2 g/L,胰蛋白酶1 g/L,猪胆盐18 g/L,调整pH值为8.0,0.22 μm膜过滤除菌备用。
经冷冻离心后得到活菌体,PBS(pH7.4)洗两次,调整活菌浓度约为109CFU/mL,将0.5 mL菌悬液加入4.5 mL模拟胃液中,置于厌氧培养箱中37 ℃培养。分别于0、1.5 h、3 h时,采用MRS琼脂倾注平板法计数。
待GJ处理3 h后,取0.5 mL的GJ培养液加入4.5 mL的IJ中,4 h、8 h后采用MRS琼脂倾注平板法计数,置于厌氧培养箱中37 ℃下培养24-48 h后计数。每个菌株做三个平行。按照如下公式计算存活率,结果如表1所示。
存活率(%)=log α/log β×100%
其中:α=经过模拟胃肠液处理之后的乳酸菌活菌数,β=未处理前的活菌数。
表1 植物乳杆菌模拟胃肠液转运耐受力
注:*代表与LGG比较,p <0.05
如表1所示,Lactobacillus plantarum ZJUF N1可耐受胃肠液转运,胃液处理3h后,实验结束时存活率高达78.99%且显著高于对照菌株鼠李糖乳杆菌LGG。肠液处理8 h后存活率为84.48%,与LGG耐受肠液能力相当。这说明菌株N1可耐受pH2.5的胃液转运,并且在肠液中的耐受性也很高。
实施例3黏附能力测定
Caco-2细胞生长于含有15%胎牛血清和1%双抗的IMDM培养液中,37 ℃和5% CO2的培养箱中进行培养,每天更换一次培养液,待细胞增殖融合率达80%左右时,以含0.02%EDTA的0.25%胰酶消化细胞,按1:3传代。取对数生长期细胞进行试验。
Caco-2 细胞以2.5×105cells/孔种板于24孔板中,每天换液,37 ℃ 5% CO2培养5天,达到单层细胞。菌株培养过夜后,在4 ℃和8000 rpm条件下离心10 min,用无菌的PBS清洗菌体2遍,用IMDM细胞培养液(不含双抗)调整菌浓度约为1×108CFU/mL,并琼脂平板计数。PBS洗板5次,去除板上残留的双抗,然后添加配制好的含有活菌的细胞培养悬液1 mL加入到Caco-2细胞单层中,37 ℃培养1 h。用PBS将细胞单层冲洗3次以移除未粘附的菌体,以1 mL1%的曲拉通来溶解细胞,然后在合适的稀释梯度下采用MRS琼脂倾注平板法计数。按照如下公式计算粘附率,结果如表2所示。
粘附率(%)=log L0/log L1×100%
注:L0= 添加的乳酸菌活菌数,L1 = 1 h后乳酸菌的活菌数。
表2 植物乳杆菌模拟粘附特性
如表2所示,植物乳杆菌ZJUFN1的粘附率达到74.23%,说明此植物乳杆菌ZJUFN1的细胞粘附性能较好,具有较好的肠道黏附和定植的潜力。
实施例4抑菌特性研究
使用琼脂孔扩散法测定菌株的抑菌能力。取20mL灭菌冷却45℃的MRS培养基与200μL致病菌液(105-106CFU/mL)一起倒入平板混匀,待凝固后于平板上打孔,孔径为7 mm。将已制备好的无菌上清液(将培养好的菌液离心,取上清液用0.22μm的微孔滤膜过滤除菌,置于-80℃备用,100μL分别加入孔中,以等体积已灭菌的PBS为对照,置于4℃下扩散12h,取出放入37℃培养24h。测量其抑菌圈直径。
Lactobacillus plantarumZJUFN1对大肠杆菌、金黄色葡萄球菌2种常见致病菌具有一定的抑制能力(见表3),其中对金黄色葡萄球菌的抑制能力优于大肠杆菌的抑制能力,N1均优于阳性对照菌株鼠李糖乳杆菌LGG。
表3植物乳杆菌ZJUFN1抑制致病菌能力
实施例5抗氧化活力评价
DPPH自由基清除率:1 mL活菌样品加到1 mL的DPPH-乙醇溶液(0.02 mmol/L)中,混合在室温下暗反应30 min,8000 rpm离心10 min,取上清在517 nm处测定吸光值。离心对照组包括PBS和DPPH-乙醇溶液,空白组包含样品和乙醇,每组设置3个平行,LGG为阳性对照组。按照如下公式计算DPPH自由基清除率,结果如表4所示。
羟自由基清除能力:0.5 mL的2.5 mmol/L的1,10-邻菲罗啉,1 mL的PBS(20 mmol/L,pH7.4)充分混匀后,再加入0.5 mL2.5 mmol/L的FeSO4,充分混匀后,加入0.5 mL2.5mmol/L的H2O2,加入0.5 mL活菌样品,37 ℃中孵育1 h,8000 rpm离心10 min,取上清在536nm处测定OD值,将0.5 mL的样品替换成水为对照组,将0.5 mL的H2O2和样品替换成水的为空白组,每组设置3个平行,LGG为阳性对照组。按照如下公式计算羟自由基清除率,结果如表4所示。
表4 植物乳杆菌ZJUFN1的DPPH和羟自由基清除率
注:*代表与LGG比较,p <0.05
如表4所示,植物乳杆菌N1对DPPH自由基的清除率显著高于鼠李糖乳杆菌LGG(17.09%),为22.88%。与对照LGG(23.55%)相比,植物乳杆菌N1对羟自由基的清除率略低于LGG,为21.92%。这说明N1在有机溶剂体系中具有更好的抗氧化优势。
实施例6胰岛素抵抗HepG2肝细胞摄糖的测定
首先使用含10%胎牛血清FBS和1%双抗的低糖DMEM培养基培养HepG2细胞,置于37℃和5%CO2的条件下培养。待对数成长期时,按照5×104cells/孔接种到24孔板中,待细胞培养24 h后达到70-80%的融合度,用PBS缓冲液清洗2遍。
然后造模组用0.2 mmol/L棕榈酸(PA)诱导细胞形成胰岛素抵抗,药物组分别用含有稀释103倍的不同菌株的CFS或CFE和0.2 mmol/L棕榈酸的高糖DMEM培养基,对照组用高糖DMEM培养基,均含有0.2%BSA,处理24 h。用PBS缓冲液清洗2遍,用含0.2%牛血清白蛋白(BSA)的RPMI1640培养基培养24 h后,移取细胞培养上清测定葡萄糖含量,并用CCK-8法检测细胞的存活率来矫正数据。结果如表5所示。
甘油三酯(TG)测定:用1.5%曲拉通X-100裂解细胞40 min后,12000 rpm 4 ℃离心取上清,用BCA法测定蛋白浓度,根据试剂盒说明书操作,测定TG含量。结果如表5所示。
肝酯酶活(HL)活性:取细胞培养上清后,用BCA法测定蛋白浓度,使用HL比色法试剂盒检测细胞的HL活性,根据试剂盒说明书操作,测定HL活力。结果如表5所示。
表5植物乳杆菌ZJUFN1对胰岛素抵抗HepG2肝细胞摄糖量、TG和HL的影响
注:*代表与模型组比较,p <0.05。
如表5所示,PA组显著降低了细胞的摄糖量(p<0.05),证明造模成功。在CFS中,与PA组相比,N1显著(p<0.05)提高了胰岛素抵抗细胞的摄糖量。与PA组相比,LGG和N1的CFE提高了细胞的摄糖量。这说明N1具有潜在的改善胰岛素抵抗的潜力。
与PA组相比,各个菌的CFS并未显著(p>0.05)改变PA诱导HepG2细胞的甘油三酯(TG)含量。在CFE处理组中,与PA相比,N1组显著降低TG含量。
与CK组相比,PA组肝脂酶活增加,这可能是由于PA的添加刺激了细胞分泌肝脂酶(HL)。与PA组相比,N1的CFE明显降低了HL酶活,这说明N1可能具有抑制脂肪合成代谢的能力。
实施例7死菌刺激腹腔巨噬细胞(PR)分泌IL-10的能力
选取6-8周龄SPF级雄性C57BL/6小鼠3只,提前3天腹腔注射4%巯基乙酸盐肉汤2mL/只小鼠,引颈处死小鼠后,腹腔注入5 mL 含1%FBS的RMPI1640培养基,按摩大于15 min,使腹腔的巨噬细胞尽可能多地悬浮于RMPI1640培养基中。剪开腹腔,小心抽出细胞悬液,快速转移至15 mL离心管中,3000 rpm离心8 min,弃去上清,用含3%FBS的PBS洗涤2遍,加入2mL RPMI 1640培养基(含10%FBS和1%双抗)重悬细胞,调整密度1×106cells/mL,种板于96孔板中,每孔100 μL,置于37 ℃的CO2培养箱中贴壁2 h,用PBS洗去未贴壁的细胞,加入200μL的RMPI1640培养基(含10%FBS和1%双抗),每天换液,7天内使用。
实验时,吸去96孔板中的培养液,PBS洗2遍,用含10 μg/mL死菌的菌悬培养液,培养24 h后,取上清,测定IL-10含量,每个样品设置3个平行。结果如图1所示。
由图1可知,2株菌均提高了PR细胞分泌IL-10的产量,且植物乳杆菌N1组的IL-10含量最高。与CK组比较,仅N1显著提高了IL-10的产量(p<0.05)。这说明植物乳杆菌ZJUFN1具有显著的抑炎效果。
实施例8植物乳杆菌N1对高脂膳食小鼠脂代谢的影响
将6周龄雄性C57BL/6小鼠放入动物房内饲喂普通饲料平衡7 d,使其适应实验环境(12 h 白天/黑夜)。平衡期结束后,随机分成4组,分组信息和给药方式(根据课题组前期研究结果,设置灌胃剂量为109CFU/只/天或等量灭活菌)如下表6。小鼠每组12只,分为3笼,每笼4只,环境(22 ± 2 ℃),湿度30-70%,小鼠自由摄食和饮水,试验历时16周,每周记录1次体重,每周记录2次小鼠的采食量。16周后,小鼠禁食12 h,称重,随后眼眶取血、颈椎脱臼处死,快速取肝脏、附睾脂肪和肠道等组织液氮速冻,干冰运输并放置于-80 ℃冰箱中保存备用。血液样本3000 rpm离心10 min得到血清。样品用于实施例8-11各项的检测。本实验获得浙江中医药大学伦理委员会批准,编号为ZSLL-2019-10947。
表6实验动物分组信息
按照商业试剂盒的操作步骤分别检测小鼠血清中血糖、甘油三酯(TG)、总胆固醇(TCHO)、高密度脂蛋白固醇(HDL-C)、低密度脂蛋白(LDL-C)的含量。结果如图2所示。
由图2可知,HK-N1组小鼠血清中LDL-C的水平显著低于HFD组和N1组(p<0.05,图2A)。此外,饲喂HFD的三组小鼠血清的HDL-C显著高于NCD组(p<0.05;图2B),HFD饲喂小鼠组间无显著差异。HFD饲喂小鼠血清中的TG水平各组均无显著差异(p>0.05;图2C)。在TCHO方面,饲喂HFD组的小鼠血清中TCHO的水平显著高于NCD组,而与HFD组相比,HK-N1和N1组均有降低TCHO的趋势,但均不显著(p>0.05;图2D)。
由图3可知,与HFD组相比,HK-N1显著减小了附睾脂肪的面积(p<0.05;图3B)。
综上,与N1活菌相比,灭活菌N1对HFD饲喂小鼠血脂代谢有一定的改善作用。
实施例9植物乳杆菌N1对高脂膳食小鼠胰岛素抵抗的影响
按照商业试剂盒的操作步骤分别检测小鼠血清中空腹血糖(FBG)、胰岛素(FI)。
胰岛素抵抗评价指数(HOMA-IR)计算公式:
HOMA-IR = FI ×FBG/22.5
注:空腹胰岛素(Fasting insulin,FI,mU/L)和空腹血糖(Fasting bloodglucose,FBG,mmol/L)
由图4可知,与NCD组相比,HFD组小鼠空腹血糖和胰岛素抵抗指数(HOMA-IR)显著高(p<0.05),这说明HFD组小鼠已经产生了明显的胰岛素抵抗。与HFD组和N1相比,HK-N1显著降低了高脂膳食诱导的高血糖和HOMA-IR(p<0.05),N1也有降低的趋势,但不显著(p>0.05)。这说明灭活植物乳杆菌N1可以显著改善高脂膳食诱导的胰岛素抵抗和高血糖。
实施例10植物乳杆菌N1对HFD饲喂小鼠糖代谢相关指标的影响
按照说明书利用双夹心法ELISA试剂盒测定血清中的胰高血糖素样肽-1(GLP-1)、YY肽、糖基化血红蛋白(Hb1Ac)。
糖基化血红蛋白(HbA1c)是指示糖代谢紊乱的一个重要指标,而胰高血糖素样肽1(GLP-1)是促进糖代谢的一个重要因子。由图5可知,与NCD组相比,N1显著提高了HbA1c的水平(p<0.05)。与NCD、HFD和N1相比,HK-N1显著提高了血清中GLP-1水平(p<0.05)。结合胰岛素抵抗结果,这说明HK-N1可能是通过刺激GLP-1的分泌达到改善胰岛素抵抗的效果。
实施例11灭活植物乳杆菌N1对HFD饲喂小鼠炎症相关指标的影响
按照说明书利用双夹心法ELISA试剂盒测定血清中的炎症因子(IL-6、IL-10),脂多糖(内毒素,LPS)检测试剂盒测定血清中LPS的含量。
由图6可知,在血清内毒素LPS方面(图6A),HK-N1组显著低于HFD组(p<0.05)。在促炎因子TNF-α和IL-6方面(图6B和6C),各组间无显著差异。HFD组的IL-6水平略高于NCD组,而HK-N1组的IL-6水平明显低于HFD组(图6C)。在抑炎因子方面(图6D),与NCD和HFD组相比,N1和HK-N1明显提高IL-10水平。这说明,相较于N1,HK-N1可以一定程度缓解高脂膳食诱导的低度系统炎症。
序列表
<110> 杭州康源食品科技有限公司
<120> 一种植物乳杆菌ZJUFN1及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1496
<212> DNA
<213> 植物乳杆菌(Lactobacillus plantarum ZJUF N1)
<400> 1
tccaccttag gcggctggtt cctaaaaggt taccccaccg actttgggtg ttacaaactc 60
tcatggtgtg acgggcggtg tgtacaaggc ccgggaacgt attcaccgcg gcatgctgat 120
ccgcgattac tagcgattcc gacttcatgt aggcgagttg cagcctacaa tccgaactga 180
gaatggcttt aagagattag cttactctcg cgagttcgca actcgttgta ccatccattg 240
tagcacgtgt gtagcccagg tcataagggg catgatgatt tgacgtcatc cccaccttcc 300
tccggtttgt caccggcagt ctcaccagag tgcccaactt aatgctggca actgataata 360
agggttgcgc tcgttgcggg acttaaccca acatctcacg acacgagctg acgacaacca 420
tgcaccacct gtatccatgt ccccgaaggg aacgtctaat ctcttagatt tgcatagtat 480
gtcaagacct ggtaaggttc ttcgcgtagc ttcgaattaa accacatgct ccaccgcttg 540
tgcgggcccc cgtcaattcc tttgagtttc agccttgcgg ccgtactccc caggcggaat 600
gcttaatgcg ttagctgcag cactgaaggg cggaaaccct ccaacactta gcattcatcg 660
tttacggtat ggactaccag ggtatctaat cctgtttgct acccatactt tcgagcctca 720
gcgtcagtta cagaccagac agccgccttc gccactggtg ttcttccata tatctacgca 780
tttcaccgct acacatggag ttccactgtc ctcttctgca ctcaagtttc ccagtttccg 840
atgcacttct tcggttgagc cgaaaggctt tcacatcaga cttaaaaaac cgcctgcgct 900
cgctttacgc ccaataaatc ccggacaacg cttgccacct acgtattacc gcggctgctg 960
gcacgtagtt agccgtggct ttctggttaa ataccgtcaa tacctgaaca gttactctca 1020
gatatgttct tctttaacaa cagagtttta cgagccgaaa cccttcttca ctcacgcggc 1080
gttgctccat cagactttcg tccattgtgg aagattccct actgctgcct cccgtaggag 1140
tttgggccgt gtctcagtcc caatgtggcc gattaccctc tcaggtcggc tacgtatcat 1200
tgccatggtg agccgttacc ccaccatcta gctaatacgc cgcgggacca tccaaaagtg 1260
atagccgaag ccatctttca aactcggacc atgcggtcca agttgttatg cggtattagc 1320
atctgtttcc aggtgttatc ccccgcttct gggcaggttt cccacgtgtt actcaccagt 1380
tcgccactca ctcaaatgta aatcatgatg caagcaccaa tcaataccag agttcgttcg 1440
acttgcatgt attaggcacg ccgccagcgt tcgtcctgag ccagaatcca actcta 1496
Claims (5)
1.一种植物乳杆菌(Lactobacillus plantarum)ZJUFN1,其特征在于,其保藏号为CCTCC NO:M2020125。
2.如权利要求1所述的植物乳杆菌(Lactobacillus plantarum)ZJUFN1在制备抑制胃肠道致病菌的药物中的应用,其特征在于,所述胃肠道致病菌包括金黄色葡萄球菌、大肠杆菌中的一种或两种。
3.灭活植物乳杆菌(Lactobacillus plantarum)ZJUFN1在制备抗炎药物中的应用,其特征在于,所述植物乳杆菌ZJUFN1的保藏号为CCTCC NO:M2020125。
4.灭活植物乳杆菌(Lactobacillus plantarum)ZJUFN1在制备治疗糖尿病药物中的应用,其特征在于,所述植物乳杆菌ZJUFN1的保藏号为CCTCC NO:M2020125,所述糖尿病为2型糖尿病。
5.灭活植物乳杆菌(Lactobacillus plantarum)ZJUFN1在制备降低动物或人体内低密度脂蛋白胆固醇浓度的药物中的应用,其特征在于,所述植物乳杆菌ZJUFN1的保藏号为CCTCC NO:M2020125。
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CN108570436A (zh) * | 2017-12-14 | 2018-09-25 | 浙江大学 | 植物乳杆菌zjuf t17及其应用 |
CN111304117A (zh) * | 2020-01-19 | 2020-06-19 | 兰州大学 | 一株具有抗氧化活性的植物乳杆菌gl-5及其应用 |
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