CN112899190B - 对痛风具有预防和/或改善功能的副干酪乳杆菌lpc48及其用途 - Google Patents
对痛风具有预防和/或改善功能的副干酪乳杆菌lpc48及其用途 Download PDFInfo
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Abstract
本发明涉及一种对痛风具有预防和/或改善功能的副干酪乳杆菌(Lactobacillusparacasei)LPC48及其用途。所述副干酪乳杆菌LPC48已于2020年02月17日寄存于中国微生物菌种保藏管理委员会普通微生物中心，其保藏编号为CGMCC No.19422。本发明所述的副干酪乳杆菌LPC48能够增加肠道中主要细菌的尿酸的分解，以该副干酪乳杆菌LPC48制备的发酵和非发酵食品可以促使人体中肠道中定植的益生菌数量逐渐增高，尿酸的分解排泄逐渐升高，进而降低血清尿酸水平，对于痛风具有良好的预防和/或改善等功能。
Description
技术领域
本发明涉及一种对痛风具有预防和/或改善功能的副干酪乳杆菌LPC48，特别涉及该副干酪乳杆菌LPC48在制备对痛风具有预防和/或改善功能的食品中的应用。
背景技术
近年来，随着生活水平的提高，人民的饮食结构发生变化。痛风患者越来越多。尿酸是人类嘌呤核苷代谢的最终产物，主要由肝和肾调节其代谢平衡。不同于其他哺乳动物，灵长类动物由于尿酸降解酶的缺乏，有较高的血清尿酸水平，进而会导致高尿酸血症。肠道菌群在人类健康中扮演着多种重要的角色，具有包括复杂纤维和脂肪的代谢，免疫调节等作用，可在调节人体的体重和能量代谢方面发挥重要的作用。
肠道微生物失衡不仅与代谢综合征(MS)、肥胖、胰岛素抵抗(IR)密切相关，而且还可能增加内毒素在血液循环中的水平，进而诱导慢性炎症。既往研究表明，人体在多种代谢性疾病发生时都存在肠道菌群的改变，主要表现为益生菌如双歧杆菌和乳酸杆菌的减少，这提示高尿酸血症也可导致肠道菌群的结构以及数量的变化。在健康个体中，大约70％尿酸是通过肾脏排泄，其余大约30％的在人体肠道排泄或肠内菌群分解。
治疗痛风及高尿酸血症的关键在于降低血尿酸水平，目前治疗的药物主要为黄嘌呤氧化酶(XO)抑制剂(例如：别嘌呤醇)和促尿酸排泄药(例如：丙磺舒、苯溴马隆等)，但是存在药物耐受性差、副作用强的问题；中药中的除湿化痰法和健脾化浊法也被用于治疗高尿酸血症；其它一些新方法也用于治疗HUA，例如向SD大鼠肠道中加入蒙脱石可以通过吸附作用达到降血尿酸的作用。寻找低毒高效的痛风治疗方法或辅助治疗手段是目前高尿酸血症研究的热点。
发明内容
本发明要解决的问题就是寻找一种低毒高效的对痛风有具有预防和/或改善功能的手段。
申请人经研究发现，作为肠道重要的生理菌群，不同的益生菌不仅具有增强免疫系统、降低胆固醇、治疗泌尿系统感染等重要作用，同时具有降低血尿酸能力。
基于上述研究，本发明通过实验和筛选获得了一株副干酪乳杆菌LPC48，该菌株经试验证实对治疗痛风具有预防和/或改善功能。
本发明所述副干酪乳杆菌(Lactobacillus paracasei)LPC48已寄存于中国普通微生物保藏管理中心，其保藏编号为CGMCC No.19422。
该副干酪乳杆菌(Lactobacillus paracasei)LPC48的16S rDNA序列为：
CAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAGTTCTCGTTGATGATCGGTGCTTGCACCGAGATTCAACATGGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGAAACAGATGCTAATACCGCATAGATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTTTGATCACCTGAGAGATCAGGTTTCCCCTTCGGGGGCAAAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGACTAGTTGCCAGCATTTAGTTGGGCACTCTAGTAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAGACCGCGAGGTCAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCGAAGCCGGTGGCGTAACCCTTTTAGGGAGCGAGCCGTCTAAGGTGGGACAAA
食疗是已被证明行之有效的中医传统手段。基于本发明所涉及的副干酪乳杆菌(Lactobacillus paracasei)LPC48对痛风具有预防和/或改善之功能，申请人借鉴食疗的方式，为人们提供一种可用于制备对痛风具有预防和/或改善功能的食品，前述食品可以是发酵食品，也可以是非发酵食品。
本发明的有益效果：
实验证明，本发明所述的副干酪乳杆菌(Lactobacillus paracasei)LPC48能够增加肠道中主要细菌的尿酸的分解，以该副干酪乳杆菌LPC48制备的发酵和非发酵食品可以促使人体中肠道中定植的益生菌数量逐渐增高，尿酸的分解排泄逐渐升高，进而降低血清尿酸水平，对于痛风具有良好的预防和/或改善等功能，可以用于对于痛风治疗的辅助手段。
具体实施方式
副干酪乳杆菌LPC48的培育：
1)制备MRS培养基
MRS培养基的具体成分如下：
制法
将上述成分加入到1000mL蒸馏水中，加热溶解，调节pH6.2，分装后121℃高压灭菌15min～20min。
2)从乳制品中分离得到副干酪乳杆菌LPC48
在发酵酸奶中接入用MRS液体培养基(即MRS培养基去掉琼脂)在37℃下培养24h，然后将培养后的发酵乳涂布到MRS培养基上，在37℃下培养3天，挑取单个菌落，并进一步采用划线法进行分离、纯化、16S rDNA序列测序等得到副干酪乳杆菌LPC48；
3)将分离得到的副干酪乳杆菌LPC48加无菌生理盐水稀释至适宜稀释度(如10-6、10-7、10-8)；
4)选择其中2个适宜稀释度的副干酪乳杆菌LPC48溶液，各以0.1mL分别置于2个MRS琼脂平板，使用L形棒将稀释后的副干酪乳杆菌LPC48菌液在MRS琼脂平板表面进行涂布，37℃下厌氧培养48h；
5)用接种环挑取MRS琼脂平板上的菌落，接种到发酵培养基中高密度培养，在温度37℃的条件下发酵12h；然后离心分离得到菌泥，然后加入冷冻保护剂冷冻干燥后制得副干酪乳杆菌LPC48冻干粉。
上述发酵培养基的具体组成为：葡萄糖25g/L，大豆蛋白胨10g/L，酵母粉8g/L，K2HPO4 2g/L，CH3COONa 7g/L，柠檬酸钠3g/L，MgSO4 0.2g/L，FeCl2 0.1g/L，MnSO40.05g/L。
动物实验
选取6周龄的雄性C57BL/6小鼠进行实验，实验分为4组(每组12只)。
正常组：正常C57BL/6小鼠，每天灌胃0.2ml生理盐水；
高尿酸组：Uox基因敲除的原发性高尿酸血症C57BL/6小鼠，每天灌胃0.2ml生理盐水；
正常+LPC48组：正常C57BL/6小鼠每天灌胃副干酪乳杆菌LPC48制品(副干酪乳杆菌LPC4冻干粉：1.0×106CFU，溶解于0.2ml生理盐水)；
高尿酸+LPC48组：Uox基因敲除的原发性高尿酸血症C57BL/6小鼠每天灌胃副干酪乳杆菌LPC48制品(副干酪乳杆菌LPC48冻干粉1.0×106CFU，溶解于0.2ml生理盐水)。
第35天进行肠道菌群对尿酸分解能力检测：
称取不同实验组小鼠粪便500mg用PBS进行稀释处理(粪便与PBS的比例为1:2)，充分混匀，然后3000rpm离心10min。取上清分别加入标准的尿酸盐溶液或碳酸锂溶液200ul,定容至1ml，振荡混匀后37℃孵育2h,4℃、15000rpm离心15min后取上清液至比色杯中并立即用酶比色法检测尿酸含量。每次实验同时设标准品校正和空白对照。结果以1g粪便内的细菌分解的尿酸量和粪便本身含有的尿酸量表示(umol/L)。
试验研究表明：
与正常组比较，高尿酸组粪便内含有的尿酸量明显升高(P<0.01)，肠道菌群对尿酸的分解显著降低(P<0.05)，表示建模成功。(正常+LPC48)组粪便内含有的尿酸水平未见明显异常，肠道菌群对尿酸的分解水平升高，但差异无统计学意义。(高尿酸+LPC48)组粪便内含有的尿酸水平升高，但差异没有统计学意义，肠道菌群对尿酸的分解水平显著升高(P<0.01)；
与高(高尿酸+LPC48)组比较，(正常+LPC48)组及(高尿酸+LPC48)组粪便内含有的尿酸水平显著降低(P<0.01)，肠道菌群对尿酸的分解水平显著提高(P<0.01)；
与(正常+LPC48)组比较，(高尿酸+LPC48)组粪便内含有的尿酸水平显著提高(P<0.01)，肠道菌群对尿酸的分解水平显著提高(P<0.05)
以上研究表明，副干酪乳杆菌LPC48能够增加肠道中主要细菌的尿酸的分解。原发性高尿酸血症发生时，尿酸肠道排泄代偿性增加，导致粪便本身含有的尿酸水平增高，但尿酸分解水平并未增加反而显著降低，主要原因是肠道菌群失调，益生菌数量降低，对尿酸的分解水平降低；服用副干酪乳杆菌LPC48制品后，(正常+LPC48)组较正常对照组肠道菌群分解尿酸水平增加，粪便本身含有的尿酸降低，这表明在正常情况下由血液循环进入肠道中的尿酸水平是相对固定的，肠道对尿酸的转运并不能随肠道益生菌数量的增加而代偿性升高。此外，(高尿酸+LPC48)组较高尿酸组肠道菌群对尿酸的分解显著升高，粪便内含有的尿酸水平显著降低。这表明原发性高尿酸血症发生时，进入肠道的尿酸代偿性增多，但肠道中分解尿酸的益生菌数量减少，导致肠道尿酸分解量降低。通过益生菌治疗后，肠道中定植的益生菌数量逐渐增高，尿酸的分解排泄逐渐升高，进而降低血清尿酸水平。
应用实施例
实施例1：含有副干酪乳杆菌LPC48发酵食品中的发酵乳的制备
将生鲜乳添加加入7％的白砂糖，搅拌至完全溶解，预热至60-65℃，15-20MPa均质，90-95℃杀菌5-10min，冷却至37-44℃，接入副干酪乳杆菌LPC48冻干粉(1.0×1011CFU/g)50g/T,发酵4-10h。至pH4.2-4.5，冷却，冷藏，即成凝固型酸乳。搅拌加入果酱等即成搅拌型酸乳。
实施例2：含有副干酪乳杆菌LPC48功能食品中粉剂、片剂的制备
按照如下配方：低聚木糖30g，低聚果糖30g，微晶纤维素5g，副干酪乳杆菌LPC48冻干粉(1.0×1011CFU/g)0.5g，混合，灌装即成粉剂，混合后压片机压出，即成片剂。
Claims (3)
1.副干酪乳杆菌(Lactobacillus paracasei)LPC48在制备对痛风具有改善功能的食品中的应用。
2.根据权利要求1所述的应用，其特征在于所述对痛风具有改善功能的食品是发酵食品。
3.根据权利要求1所述的应用，其特征在于所述对痛风具有改善功能的食品是非发酵食品。
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