CN114107122A - 一种利用益生菌发酵食品浆水缓解痛风的方法 - Google Patents
一种利用益生菌发酵食品浆水缓解痛风的方法 Download PDFInfo
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- CN114107122A CN114107122A CN202111503162.3A CN202111503162A CN114107122A CN 114107122 A CN114107122 A CN 114107122A CN 202111503162 A CN202111503162 A CN 202111503162A CN 114107122 A CN114107122 A CN 114107122A
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Abstract
本发明提供了一种利用益生菌发酵食品浆水缓解痛风的方法,其特征在于:(1)对来自甘肃发酵浆水中的微生物进行分离;(2)配制尿酸培养基;(3)菌株接种和培养及其生长能力检测;(4)菌株尿酸降解能力及其降解产物测定;(5)菌株鉴定;(6)益生菌菌株灌胃小鼠并检测小鼠体内一系列指标;(7)将具有较强尿酸降解能力的菌株应用于发酵食品的制作中。通过使用益生菌制品降低体内尿酸的含量,减轻其对人体的损伤,从而保护肠道菌群使微生物群落更好的发挥降解尿酸的作用,这种方法对尿酸的降解具有去除效果好、时效长、操作简单和经济实惠等优点。
Description
技术领域
本发明涉及微生物工程领域,具体地说是一种利用益生菌降低体内尿酸含量的方法。
背景技术
痛风作为主要代谢疾病之一,在许多国家呈上升趋势。一些研究报告显示,男性和女性的高尿酸血症患病率高达21%。尤其常见于代谢性、心血管和肾脏疾病患者。富含嘌呤的食物在人体内不完全降解为尿酸,导致血浆中尿酸水平高。其有害代谢效应的潜在机制包括可溶性尿酸增加氧化应激的能力,线粒体和内质网功能障碍,内皮功能障碍,肾素- 血管紧张素系统的激活,以及促炎因子合成和分泌增加。因此,尿酸在组织中积累,被认为是痛风及其他疾病如心血管疾病、内皮功能障碍和代谢综合征发展的重要危险因素。目前,治疗高尿酸血症的方法有饮食、药物和生物治疗等,主要针对嘌呤的摄取和降解。但饮食和药物治疗手段因成本、效果等多方面影响,无法长期坚持。微生物修复作为一种经济有效的处理方法,由于其对人体的破坏最小,已被公众广泛接受。因此,亟待寻找一种安全高效的尿酸降解菌株来缓解高血尿酸。
人类肠道微生物群包含至少1000中不同类型的细菌类群,以及各种古生菌、真核微生物和病毒,超过300万个基因,具有巨大的代谢能力,在营养吸收、能量获取、炎症调节和宿主免疫应答等方面发挥着重要作用。一般来说,尿酸可以通过肾脏大量排泄UA来管理,其中有30%的总UA通过肠道,在那里被细菌微生物群快速代谢。此外,最近有研究表明,痛风患者的肠道微生物群与正常血症受试者有显著不同,这一发现表明,微生物群与肠道尿酸代谢和排泄之间的相互作用可能会调节血清尿酸水平。益生菌是一类可以定植于人体肠道内并对人体有益的活性微生物。已有研究发现了几种可以利用嘌呤的乳酸菌。在这些菌株中,发现可以有效降低啮齿动物体内的尿酸含量。如L.brevis(DM9218)和L.gasseri(PA-3) 可以降解嘌呤代谢的中间产物以改善高尿酸胺。但目前这些乳酸菌大部分作用于肠道嘌呤的降解和吸收。
发酵食品中富含乳酸菌,可以产生有机酸来控制腐烂微生物和病原体。研究表明,酸奶等发酵食品因富含乳酸菌对2型糖尿病患者有益。浆水作为我国西北的传统发酵美食,富含丰富的乳酸菌、乙酸菌和酵母等多种微生物。从浆水中的筛选出乳酸发酵菌不仅可以在体内降解尿酸,而且可以通过调节肠道菌群维持肠道微生物的稳定,间接缓解高尿酸的损伤作用,利用发酵食品中益生菌进行降解尿酸的方法操作简单、安全高效、价格低廉而且持续性强,不仅能够降低体内的尿酸含量,还能通过调节肠道菌群的组成,维持人体的代谢和免疫平衡。
现有技术存在的问题:
(1)目前临床中,对高血尿酸或痛风的治疗方法主要是使用鸟嘌呤氧化酶(XOD)抑制剂,如非布索坦和别嘌呤醇,但该方法副作用较大,存在治疗后引起过敏反应和心血管的风险。同时,药物的食用会引起肠道菌群的紊乱。
(2)目前通过饮食干预限制高嘌呤食物摄入来起作用,但由于难以长期坚持饮食限制,且效果不如药物。
(3)现有筛选的益生菌降低尿酸的方法,选用菌株主要针对嘌呤的降解。但尿酸是痛风的主要来源,可降解尿酸的菌株较少。
发明内容
鉴于现有技术不足,本发明提供含有尿酸降解菌株的发酵食品的制作方法。通过使尿酸降解菌株JL-3存活于发酵食品浆水和酸奶中,并利用发酵制品降低人体内尿酸的水平。本发明采用如下技术方案:
1.一种能降低体内尿酸含量的益生菌菌株,所述益生菌菌株是乳酸发酵菌 JL-3(Limosilactobacillus fermentum JL-3),该菌株与2021年7月19日保藏于中国典型培养物保藏中心(CCTCC),地址:中国武汉武汉大学,保藏号为:CCTCC NO:M2021902。
2.一种能降低体内尿酸的益生菌分离方法,包括:(1)问卷调查;(2)对来自甘肃发酵浆水中的微生物进行分离;(3)菌株体外接种和培养及其生长能力检测;(4)细菌降解尿酸能力及其降解产物的测定;(5)菌株的鉴定。
3.一种利用益生菌菌株JL-3减少小鼠体内尿酸含量的方法,包括如下步骤:(1)将筛选出的具有高效降解能力的益生菌菌株灌胃高血尿酸小鼠;(2)收集组织和血液样本;(3)发酵乳酸杆菌JL-3的定殖情况;(4)尿酸含量的测定;(5)检测氧化损伤指标;(6)收集粪便样品检测肠道菌群变化。
4.一种能降低体内尿酸含量的益生菌浆水,所述益生菌浆水采用如下方法制作而成:(1) 将所用的蔬菜(芹菜等)洗干净,晾干待用;(2)上锅烧水,将所有蔬菜放入开水烧开;(3) 碗中放入0.2-0.5kg的面粉,放水搅匀,倒入烧开的锅里,大约2-5分钟,关火,冷却至室温; (4)将含有益生菌JL-3的浆水(1×108/mL)倒入容器里面,并将所有的蔬菜倒入洗干净的容器中;(5)密闭容器,发酵2-7天。
5.一种能降低体内尿酸含量的益生菌浆水,采用如下方法制作而成:(1)将JL-3菌株在MRS培养液中活化(0.1%),检测OD600至0.8-1;(2)取1mL菌液进行8,000g离心10分钟,并用无菌水清洗三次;(3)将清洗干净的菌株加入至100mL无菌的牛乳中42℃发酵6-8h;(4)取第一批发酵好的酸奶,外加保加利亚和嗜热链球菌接种于新鲜的牛乳中进行二次发酵 (42℃,6-8h),形成益生菌酸奶。
6.一种利用益生菌发酵食品(浆水,酸奶)缓解痛风的方法,所述益生菌发酵食品采用上述方法制成。
有益效果:(1)目前临床中,对高血尿酸或痛风的治疗方法主要是非布索坦和别嘌呤醇等药物,但该方法副作用较大,存在治疗后引起过敏反应和心血管的风险,且费用高。而通过益生菌浆水食疗方法没有副作用的影响,并且它可以通过改善肠道菌群,提高其抗性(图 4)。与UA组相比,JL-3定植降低了小鼠血液和尿液中UA的含量。从第1天到第3天,UA组和JL-3+UA组的UA水平均迅速上升,达到对照组的15倍以上。而JL-3+UA组尿液中 UA水平从第3天开始逐渐下降,至第9天接近对照水平。在第15天采集的血清样本中,UA 组UA水平(249.9mol/L)是对照组(112.6mol/L)的2.2倍。经JL-3干预后,UA浓度较 UA组降低约31.3%(图4E)。与对照组相比,JL-3组尿液和血液中UA水平无明显差异,说明JL-3可以有效降低高尿酸血症小鼠的UA(图4F)。尿酸组小鼠IL-1β、丙二醛(MDA)、肌酐(CRE)和血尿素氮(BUN)水平,这些指标均呈上升趋势。此外,与UA组相比,JL-3 +UA组的血清中这些标志物的水平明显降低。其中,IL-1β降低50.9%,BUN降低66.7%, MDA降低约40%,CRE降低73%。在肾脏和肝脏中,UA组IL-1β水平显著升高。然而,在JL-3+UA组,黄嘌呤氧化酶(XOD)水平部分下降。相反,在JL-3干预后,UA组肝脏中黄嘌呤氧化酶(XOD)水平上升,并恢复到正常水平。四组肾脏MDA水平无明显差异(图 5)。组织病理学分析显示,UA暴露导致肝损伤,与对照组相比,UA小鼠组肝切片显示细胞空间增加,核染色加深。16srRNA数据进一步表明,UA暴露样品与其他样品的系统发育群落结构存在显著差异,UA组与其他组分离(表2)。这表明UA暴露改变了肠道微生物群的组成,而补充JL-3减少了UA对微生物群变化及短链脂肪酸代谢的影响(图6)。UA小鼠组与对照组相比,拟杆菌的相对丰度显著增加,厚壁菌门和变形菌门减少。JL-3治疗减弱了UA 引起的拟杆菌门增加,厚壁菌门和变形菌门减少(图6F,G,I)。通过JL-3治疗缓解UA暴露后,厚壁菌门与拟杆菌门的比率也降低(图6H)。Venn分析还显示UA治疗显著影响肠道菌群结构(图6J)。所有这些结果表明JL-3对肠道菌群有积极作用。
(2)高嘌呤食物广泛存在于日常生活中,如海鲜类:鱼类、虾类、蟹类、贝类等;饮料类:啤酒、白酒、咖啡、碳酸类等;动物内脏:猪肝、猪肾、鸭肠等;坚果类:花生、腰果等;肉汤类,比如火锅中含有比较浓缩的肉汁;蔬菜类:菠菜、蘑菇、芦笋的嘌呤含量相对较高。通过饮食干预限制高嘌呤食物摄入来起作用比较微弱,且难以长期坚持。而益生菌益生菌浆水食疗方法可以作为饮食的一部分加入日常生活中,不会对饮食结构有很大的改变,其操作简单,经济实惠。并且不同商品浆水的聚合酶链反应(PCR)结果表明,JL-3广泛存在于各种样品中(图2D)。
(3)现有筛选的益生菌降低尿酸的方法,选用菌株主要针对嘌呤的降解。尿酸是痛风的主要来源,本研究所使用的菌株对尿酸具有较高的降解能力,作用效果明显。实验数据显示,从发酵浆水中分离出20株具有尿酸抗性的菌株,并在以UA为唯一碳氮源的培养基中进行培养。同时筛选的三株菌株JL-2、JL-3和JL-8对嘌呤也具有降解能力(表1),其中JL-3在尿酸培养基中的生长速率明显高于其他两株,显示在24小时内OD600值高于0.25(图2C)。与NCBI BLAST数据库相比,JL-3菌株与乳酸杆菌密切相关(图2B)。透射电镜(TEM)也显示JL-3具有典型的乳杆菌形态(图1A)。此外,JL-3菌株在24小时内可降解40.9%的UA,而XS-14是酸奶中的发酵乳酸杆菌菌株(图3A)。或者,高效液相色谱(HPLC)结果显示 UA及其降解产物的吸收峰分别为4.868、2.255和1.976,这与UA、尿囊素和尿素峰的标准一致(图3B中a、b和c)。这表明JL-3能将尿酸分解成尿囊素和尿素等小分子。
附图说明
图1为痛风调查报告。
其中,A.痛风发病率。横坐标为每周江水消费频率,纵坐标为不同江水消费频率痛风患者所占比例。B.江水面条用量与痛风发病率的皮尔逊相关性。
图2为浆水中菌株L-3的筛选与鉴定。
其中,A.JL-3的扫描电镜形貌。B.菌株JL-3及其相关菌的系统发育树。C.不同培养基对JL-2、JL-3和JL-8生长的影响。与JL-2和JL-8相比,JL-3在含有UA的培养基中生长效果最好,在不含UA的培养基中没有生长现象。D.兰州超市购买的10种浆水中JL-3菌株的鉴定。
图3为JL-3的尿酸降解能力的鉴定。
其中,A.JL-3菌株体外降解尿酸的能力。B.高效液相色谱法检测JL-3的尿酸降解产物。图3B中a.UA标准品检测峰;图3B中b.尿囊素标准品检测峰;图3B中c.尿素标准检测峰;图3B中d.JL-3的主要代谢产物为尿素和尿囊素。
图4为JL-3对高尿酸血症小鼠的影响。
其中,A.通过检测尿液和血清中尿酸含量建立高尿酸血症小鼠模型。在2%UA浓度和 4%OP浓度下,UA水平显著高,选择JL-3处理进行实验。B.JL-3治疗高尿酸血症小鼠的实验图。C.第4、6、8、10天时用PCR检测JL-3定植情况。D.采用RT-PCR方法测定第15 天粪便菌群中JL-3的比例。E.实验后0、2、4、6、8、10、12天尿液中尿酸的水平。F.第15 天小鼠血液中尿酸的水平。
图5为JL-3处理高尿酸血症小鼠的炎症指标和氧化应激指标。
其中,4组小鼠血清中IL-1β(A)、MDA(B)、CRE(C)、BUN(D)水平和肝脏中白细胞介素-1β(IL-1β)(E)和MDA(F)的水平。G,H:各组小鼠肾脏白细胞介素-1β(IL-1β) 水平及XOD含量,I:小鼠肝、肾组织HE染色照片。
图6为JL-3处理对尿酸暴露小鼠肠道菌群功能和微生物多样性的影响。
其中,A:不同组小鼠粪便菌群对尿酸的降解。B:小鼠粪便中短链脂肪酸的浓度。C:基于Bray-Curtis距离的总体多样性的原理坐标分析(PCoA)。PCoA评分散点图,描述了来自四组细菌群落的方差。D:不同类群的门相对丰度比较。E:四个类群中最丰富的细菌属的相对丰度。F,G,I:拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)和酸杆菌门(Acidobacteria) 的相对丰度发生了显著变化。H:不同组的Firm/Bac比率。J:不同微生物类群间OTU重叠的维恩图分析。
图7益生菌酸奶持水率、酸度和DPPH自由基清除能力检测(接种量:J1:JL-3(108/100ml 牛乳);J2:JL-3(106/100ml牛乳);R1:JL-3+保加利亚和嗜热链球菌(108/100ml牛乳);R2:JL-3 +保加利亚和嗜热链球菌(106/100ml牛乳);C1:保加利亚和嗜热链球菌(108/100ml牛乳);C1: 保加利亚和嗜热链球菌(108/100ml牛乳))
具体实施方式
为使发明的目的、技术方案和优点更加清楚,下面结合附图对本发明的具体实施方式进行详细说明。这些优选实施方式的示例在附图中进行了例示。附图中所示和根据附图描述的本发明的实施方式仅仅是示例性的,并且本发明并不限于这些实施方式。在此,还需要说明的是,为了避免因不必要的细节而模糊了本发明的技术方案,在附图中仅仅示出了与根据本发明的方案密切相关的结构和/或处理步骤,而省略了关系不大的其他细节。
实施例1
该实施例提供一种能降低体内尿酸含量的益生菌菌株,所述益生菌菌株是乳酸发酵菌 JL-3(Limosilactobacillus fermentum JL-3),该菌株与2021年7月19日保藏于中国典型培养物保藏中心(CCTCC),地址:中国武汉武汉大学,保藏号为:CCTCC NO:M2021902。
实施例2
本实施例提供了一种能降低体内尿酸的益生菌分离方法,包括:
1.问卷调查
如表1所示,问卷调查采用匿名方式进行,统计痛风病情和浆水面的食用频率。对180 名居民进行痛风患病率调查,调查主要集中在:1)每周江水面消费频率;2)痛风情况;3) 饮酒、吃肉等饮食习惯的频率。采用SPSS v.17.0软件对P<0.05。差异具有统计学意义。调查结果如图1所示。
2.对来自甘肃发酵浆水中的微生物进行分离
10份不同产地(天水,武威,兰州等)的传统浆水样品采集自甘肃兰州超市。从采集的样品中,在尿酸培养基(1.71g Na2HPO4.12H2O,0.3g KH2PO4,0.05g NaCl,0.05g MgSO4.7H2O,0.001g CaCl2,0.2g UA and 1.2g agar,per 100mL)中分离得到菌株见表1,得到单菌落。从发酵的浆水中分离出20株具有UA抗性的菌株,并在以UA为唯一碳氮源的培养基中进行培养。
表1.从发酵食品浆水中筛选的菌株对嘌呤碱基的降解率
腺嘌呤 | 次黄嘌呤 | |
JL-1 | 15.80% | 3.24% |
JL-2 | 25.20% | 5.87% |
JL-3 | 28.40% | 27.49% |
JL-4 | 9.20% | 5.31% |
JL-5 | 11.80% | 0 |
JL-7 | 10.60% | 6.62% |
JL-8 | 25.00% | 16.21% |
JL-9 | 4.60% | 0 |
JL-10 | 7.20% | 5.68% |
VL-1 | 12.40% | 0 |
VL-2 | 15.80% | 14.52% |
VL-3 | 15.40% | 3.99% |
VL-4 | 12.60% | 8..32% |
VL-5 | 10.20% | 9.63% |
VL-7 | 7.20% | 0 |
VL-8 | 6.20% | 11.51% |
VL-9 | 16.60% | 3.99% |
VL-10 | 8.00% | 9.07% |
3.菌株体外接种和培养及其生长能力检测
将筛选到的菌株转移到以UA为唯一碳源的新培养基中。将培养好的细菌培养板置于 37℃培养箱中培养48h,将获得的菌落在新的MRS琼脂板上划线,获得单个菌落。在MRS培养基中激活的新的单克隆菌置于25%甘油中并于-80℃冰箱保存。从尿酸培养基中筛选出三株菌株,JL-2、JL-3和JL-8均能存活,其中JL-3的生长速率明显高于其他两株,显示在24小时的潜伏期内OD600值高于0.25(图2C)。
4.细菌降解尿酸能力及其降解产物的测定
将筛选的菌株接种于MRS上,在厌氧条件下37℃培养48h,检测其对尿酸降解能力。将 JL-3加入含有尿酸(10mmol/L)的无菌磷酸盐缓冲液(PBS)中进行培养。
5.菌株的鉴定
采用TIANamp细菌DNA试剂盒(TIANGEN Biotechnology,中国)获得高质量的菌株基因组DNA。按照Sibley等的方法扩增了16S rDNA。利用BLAST引擎(NCBI)对可能用于物种鉴定的分离株进行测序,并将其核苷酸序列提交GenBank参考(SUB8885938 SEQ1MW474833、SUB8885938 SEQ2 MW474834、SUB8885938 SEQ3 MW474835)。采用扫描电镜(SEMS-3400N II Hitachi,Japan)对JL-3进行形态学分析,采用MEGA(Molecular EvolutionaryGenetics Analysis,v.6.0)对JL-3及其他密切相关菌株的多序列进行比对。
实施例3
本实施例提供了一种利用益生菌菌株JL-3减少小鼠体内尿酸含量的方法,包括如下步骤:
1.将筛选出的具有高效降解能力的益生菌菌株灌胃高血尿酸小鼠
将50只小鼠随机分为5组,分别用氧嗪酸钾(OP)和尿酸(UA)建立高尿酸血症动物模型。其浓度取决于前期的预实验处理。高血尿酸模型实验中确定的2%UA和4%OP浓度用于益生菌修复实验。对照组每日灌胃基础日粮和无菌脱脂乳0.5ml,JL-3组每日灌胃基础日粮和无菌脱脂乳中JL-3。UA组饲喂基础日粮加2%UA和4%OP,口服无菌脱脂乳(0.5ml), UA+JL-3组饲喂基础日粮加2%UA和4%OP,JL-3菌株口服无菌脱脂乳。前期实验证明JL-3 可以降解UA。为检测其在小鼠体内的能力,我们建立了高尿酸血症小鼠模型(图3A)。所构建的高尿酸血症小鼠模型更稳定有效地提高小鼠的血清尿酸,相比于氧嗪酸钾或高嘌呤饮食单独维持更长的高尿酸血症持续时间26,34,35。因此,我们采用该方法构建高尿酸血症小鼠,并给予该菌株灌胃(图3B中c)。
2.收集组织和血液样本
在实验期间,每3天将各组小鼠移入一个干净的空笼中1小时,并采集粪便和尿液样本。随后,用乙醚麻醉小鼠。从眼静脉采集血液,静置30分钟。采集的血液样本以4000rpm离心20分钟以获得血清。同时,收集各组的肝脏、肾脏和肠道样本。然后用生理盐水冲洗收集的样品,用1ml PBS稀释液在预先称重的试管中收集,并在室温下储存-20℃供将来使用。所有样本均保存于-80℃直至分析,但用于组织病理学检查的除外,保存在4%多聚甲醛中。
3.发酵乳酸杆菌JL-3的定殖情况
实验过程中,分别于4、6、8、10d后收集小鼠粪便,然后使用特异性引物进行PCR。第22天,新鲜小鼠粪便在MRS培养基中培养,37℃孵育48h在静态培养箱中,然后再次传代培养。然后根据制造商的说明,使用TIANamp粪便DNA试剂盒提取小鼠粪便DNA。在实时定量PCR仪器(Bio-RAD CFX96,美国)上使用SYBR Premix Ex Taq II(Takara)进行总体积为10μL的扩增。所有测量均一式三份。用PCR产物构建标准曲线JL-3和E.coli。然后使用以下等式估计目标拷贝数(T):T=(D/(PL×660))×6.022×1,023,133,其中 D(g/l)和PL(碱基对)分别是质粒DNA的浓度和长度。每个标准曲线由至少5个10倍质粒稀释液生成,一式三份。根据标准曲线计算了JL-3在不同样品细菌群落中所占的比例。PCR 检测,从第6天开始,添加该菌株的小鼠粪便中JL-3菌株的相对丰度显著高于对照组(图3B 中c)。第14天,QRT-PCR检测JL-3的检出率达到10-6(图3B中d)。
4.尿酸含量的测定
将冻存于1ml PBS中的小鼠粪便置于60℃的试管中水浴10min,旋涡1min,使粪便样品破碎。通过在1000rpm下离心5分钟来收集溶液。将新鲜的小鼠尿液放置在60%的溶液中℃水浴并用蒸馏水稀释10次,以确保尿样中不存在尿酸沉淀物,之后严格按照UA检测试剂盒 (中国南京建城)中提供的说明对所有样品进行检测。该试剂盒提供了一种基于荧光的UA 检测方法。在分析中,尿酸酶将尿酸转化为尿囊素、过氧化氢(H2O2)和二氧化碳。在辣根过氧化物酶存在下,H2O2与10-乙酰基-3,7-二羟基苯恶嗪反应生成高荧光化合物resorufin。然后在530nm的激发波长和595nm的发射波长下分析Resorufin荧光,然后使用不同剂量标准品测定的方程式计算血清和尿液样品中UA的浓度。
5.检测氧化损伤指标
新鲜的肝脏和肾脏在冰上用手持式研磨机(静心,中国上海)研磨,然后在低速离心(8000 rpm,10min)下获得样品悬浮液。在肝脏中检测XOD和IL-1β水平,在肾脏检测MDA和IL-1β,然后根据提供的方案,使用商业试剂盒(中国南京建城)检测血清样本中的丙二醛(MDA)、肌酐(CRE)和尿素氮(BUN)。用PBS冲洗肝脏和肾脏样本,然后取体积为0.8× 0.8cm的肝脏组织样本,用4%多聚甲醛溶液固定一天以上。随后进行生理切片和苏木精伊红染色,并在显微镜下观察各组小鼠肾小管的切片形态及尿酸的蓄积情况。
6.收集粪便样品检测肠道菌群变化。
将粪便样品颗粒称重至0.2g,并使用改良的天安粪便DNA试剂盒(中国,南京建城)在ddH2O中分离DNA。然后将获得的DNA送公司进行测序分析,如图。
表2.粪便16SrRNA的Alpha多样性指数
Sample-id | Faith-pd | Observed_otus | Evenness |
C<sub>1</sub> | 30.52 | 446 | 0.749 |
C<sub>2</sub> | 38.11 | 481 | 0.756 |
C<sub>4</sub> | 32.27 | 512 | 0.716 |
C<sub>5</sub> | 37.16 | 426 | 0.674 |
JL-3<sub>1</sub> | 29.73 | 375 | 0.650 |
JL-3<sub>2</sub> | 30.18 | 362 | 0.635 |
JL-3<sub>3</sub> | 27.01 | 403 | 0.792 |
JL-3<sub>4</sub> | 36.64 | 466 | 0.612 |
JL-3<sub>5</sub> | 29.73 | 375 | 0.650 |
OP<sub>1</sub> | 39.26 | 397 | 0.672 |
OP<sub>2</sub> | 22.27 | 311 | 0.722 |
OP<sub>3</sub> | 30.31 | 353 | 0.639 |
OP<sub>4</sub> | 30.78 | 355 | 0.657 |
OP<sub>5</sub> | 28.38 | 341 | 0.676 |
JL-3+OP<sub>1</sub> | 21.70 | 305 | 0.729 |
JL-3+OP<sub>2</sub> | 27.04 | 345 | 0.792 |
JL-3+OP<sub>3</sub> | 32.53 | 438 | 0.720 |
JL-3+OP<sub>4</sub> | 37.77 | 369 | 0.733 |
JL-3+OP<sub>5</sub> | 20.81 | 285 | 0.760 |
UA<sub>1</sub> | 26.68 | 257 | 0.531 |
UA<sub>2</sub> | 22.46 | 333 | 0.538 |
UA<sub>3</sub> | 32.57 | 359 | 0.666 |
UA<sub>4</sub> | 20.65 | 291 | 0.643 |
UA<sub>5</sub> | 27.50 | 419 | 0.761 |
JL-3+UA<sub>1</sub> | 23.67 | 364 | 0.695 |
JL-3+UA<sub>2</sub> | 25.40 | 410 | 0.613 |
JL-3+UA<sub>3</sub> | 22.51 | 331 | 0.700 |
JL-3+UA<sub>4</sub> | 27.48 | 366 | 0.558 |
JL-3+UA<sub>5</sub> | 23.84 | 370 | 0.663 |
实施例4
本实施例提供了一种能降低体内尿酸含量的益生菌浆水的制作方法,包括:
1.将所用的蔬菜(芹菜等)洗干净,晾干待用;2.上锅烧水,将所有蔬菜放入开水烧开; 3.碗中放入0.2-0.5kg的面粉,放水搅匀,倒入烧开的锅里,大约2-5分钟,关火,冷却至室温;4.将含有益生菌JL-3的浆水(1×108/mL)倒入容器里面,并将所有的蔬菜倒入洗干净的容器中;5.密闭容器,发酵2-7天。
实施例5
本实施例提供了一种能降低体内尿酸含量的浆水酸奶的制作方法,包括:
1.将JL-3菌株在MRS培养液中活化(0.1%),检测OD600至0.8-1。2.取1mL菌液进行8,000g离心10分钟,并用无菌水清洗三次。3.将清洗干净的菌株加入至100mL无菌的牛乳中42℃发酵6-8h。4.取第一批发酵好的酸奶,外加保加利亚和嗜热链球菌接种于新鲜的牛乳中进行二次发酵(42℃,6-8h),形成益生菌酸奶,检测发现其具有较好的抗氧化能力(图7)。
以上所述仅是本申请的具体实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本申请原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本申请的保护范围。
Claims (9)
1.一种能降低体内尿酸含量的益生菌菌株,其特征在于所述益生菌菌株是乳酸发酵菌JL-3,该菌株与2021年7月19日保藏于中国典型培养物保藏中心(CCTCC),保藏号为:CCTCCNO:M2021902。
2.一种能降低体内尿酸的益生菌分离方法,其特征在于包括:(1)问卷调查;(2)对来自甘肃发酵浆水中的微生物进行分离;(3)菌株体外接种和培养及其生长能力检测;(4)细菌降解尿酸能力及其降解产物的测定;(5)菌株的鉴定。
3.一种利用益生菌菌株JL-3减少小鼠体内尿酸含量的方法,其特征在于包括如下步骤:(1)将筛选出的具有高效降解能力的益生菌菌株灌胃高血尿酸小鼠;(2)收集组织和血液样本;(3)发酵乳酸杆菌JL-3的定殖情况;(4)尿酸含量的测定;(5)检测氧化损伤指标;(6)收集粪便样品检测肠道菌群变化。
4.一种能降低体内尿酸含量的益生菌浆水,其特征在于所述益生菌浆水采用如下方法制作而成:(1)将所用的蔬菜(芹菜等)洗干净,晾干待用;(2)上锅烧水,将所有蔬菜放入开水烧开;(3)碗中放入0.2-0.5kg的面粉,放水搅匀,倒入烧开的锅里,大约2-5分钟,关火,冷却至室温;(4)将含有益生菌JL-3的浆水(1×108/mL)倒入容器里面,并将所有的蔬菜倒入洗干净的容器中;(5)密闭容器,发酵2-7天。
5.一种能降低体内尿酸含量的浆水酸奶,其特征在于含有尿酸降解的益生菌,采用如下方法制作:(1)将JL-3菌株在MRS培养液中活化(0.1%),检测OD600至0.8-1;(2)取1mL菌液进行8,000g离心10分钟,并用无菌水清洗三次;(3)将清洗干净的菌株加入至100mL无菌的牛乳中42℃发酵6-8h;(4)取第一批发酵好的酸奶,外加保加利亚和嗜热链球菌接种于新鲜的牛乳中进行二次发酵(42℃,6-8h),形成益生菌酸奶。
6.如权利要求1所述的乳酸发酵菌JL-3在制备降低尿酸含量药物中的应用。
7.如权利要求1所述的乳酸发酵菌JL-3在制备缓解痛风药物中的应用。
8.如权利要求4所述的益生菌浆水在制备缓解痛风食品中的应用。
9.如权利要求5所述的浆水酸奶在制备缓解痛风食品中的应用。
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