Effect of Chlorophyll on the Proliferation of Intestinal Bacteria Based on Flow Cytometry Method
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摘要: 为在菌种水平上探究叶绿素对特定肠道细菌增殖的影响,本研究首先探究了流式细胞术(Flow cytometry,FCM)对肠道细菌计数的可行性并与稀释涂布平板法进行比较,随后利用FCM探究了叶绿素对珀氏解黄酮菌(Flavonifractor plautii)、普通拟杆菌(Bacteroides vulgatus)、假长双岐杆菌(Bifidobacterium pseudolongum)和鼠乳杆菌(Lactobacillus murinus)增殖的影响。结果表明,FCM可实现对活细菌和死细菌的同时检测,FCM活菌计数结果与稀释涂布平板法测定的活菌数之间具有很好的相关性(r>0.8,P<0.001),但FCM检测效率更高、检测结果更接近于真实值。100 µg/mL的叶绿素可高度显著促进Flavonifractor plautii的增殖(P<0.01),而300和500 µg/mL的叶绿素可极显著抑制Flavonifractor plautii的增殖(P<0.001);100 µg/mL的叶绿素可极显著促进Bacteroides vulgatus的增殖(P<0.001),而300和500 µg/mL的叶绿素可极显著抑制Bacteroides vulgatus的增殖(P<0.001);叶绿素可极显著抑制Bifidobacterium pseudolongum的增殖(P<0.001),且浓度越高抑制作用越强;叶绿素可极显著促进Lactobacillus murinus的增殖(P<0.001),且浓度越高促进作用越强。本研究为阐明膳食叶绿素对肠道菌群生长的影响规律提供了重要的理论数据。Abstract: In order to explore the effect of chlorophyll on the proliferation of specific intestinal bacteria at the bacterial species level, this study firstly explored the feasibility of flow cytometry (FCM) to count intestinal bacteria and compared it with the dilution plating method. Then, the effects of chlorophyll on the proliferation of Flavonifractor plautii, Bacteroides vulgatus, Bifidobacterium pseudolongum and Lactobacillus murinus were investigated by FCM. The results showed that FCM could detect live bacteria and dead bacteria at the same time. There was a good correlation between the count of live bacteria by FCM and the number of live bacteria determined by the dilution coating method (r>0.8, P<0.001). However, detection efficiency of FCM was higher, and the detection results were closer to the real value. 100 µg/mL chlorophyll highly significantly promoted the proliferation of Flavonifractor plautii (P<0.01), while 300 and 500 µg/mL chlorophyll extremely significantly inhibited the proliferation of Flavonifractor plautii (P<0.001). 100 µg/mL chlorophyll extremely significantly promoted the proliferation of Bacteroides vulgatus (P<0.001), while 300 and 500 µg/mL chlorophyll extremely significantly inhibited the proliferation of Bacteroides vulgatus (P<0.001). Chlorophyll could extremely significantly inhibit the proliferation of Bifidobacterium pseudolongum (P<0.001), and the higher the concentration was, the stronger the inhibitory effect was. Chlorophyll could extremely significantly promote the proliferation of Lactobacillus murinus (P<0.001), and the higher the concentration was, the stronger the promoting effect was. This study would provide important theoretical data for elucidating the effect of dietary chlorophyll on the growth of gut microbiota.
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Keywords:
- flow cytometry /
- dilution plating method /
- chlorophyll /
- intestinal bacteria /
- proliferation
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叶绿素是绿色果蔬中主要的呈色物质,也是自然界含量最为丰富的植物次生代谢物,具有抗氧化[1]、抗炎[2]、抗肿瘤[3]等多种功能。膳食摄入叶绿素经胃肠消化后,约有95%的叶绿素会进入结肠,与肠道菌群相互作用[4]。
课题组前期研究发现,叶绿素可通过介导肠道菌群有效改善高脂饮食诱导的小鼠肥胖,显著上调小鼠肠道中拟杆菌属(Bacteroides)、双歧杆菌属(Bifidobacterium)和阿克曼氏菌属(Akkermansia)的相对丰度,同时显著下调乳球菌属(Lactococcus)和乳杆菌属(Lactobacillus)的相对丰度[5-6]。阐明叶绿素介导肠道菌群改善高脂饮食诱导肥胖的机制,需要深入探讨叶绿素对特定菌株的影响,但国内外对相关领域的研究还非常有限。与此同时,Liu等[7]从小鼠肠道中分离得到244株肠道细菌,为研究叶绿素对特定肠道菌的影响规律提供了可使用的菌株。基于前期研究结果,本研究从上述菌株库中筛选获得与肥胖显著相关的菌株进行了体外培养研究。
大多数肠道细菌严格厌氧,对培养条件要求高、生长周期长。在采用稀释涂布平板法进行肠道菌活菌计数时发现,经过7~15 d的培养后,许多肠道菌仍只能长出大小不一且分散不均的微小菌落。在体外培养中如何实现肠道细菌数量的有效检测是一大难点。因此,要在菌种水平上探究叶绿素对特定肠道细菌的增殖影响,阐明膳食叶绿素影响健康的机制,需要找到一种便捷、有效、可检测体外培养中肠道细菌数量的方法。
流式细胞术(Flow cytometry, FCM)是一种可实现细胞或细菌快速检测的简便技术,耗时短且重复性高[8]。目前,已有许多研究将其应用于幽门螺旋杆菌等致病菌[9-10]的检测。在食品领域,FCM已实现对酸奶中乳酸菌[11]、葡萄酒中酿酒酵母[12]和食醋中醋酸菌[13]等工业菌的快速检测。目前,还未有研究利用FCM检测难以培养的肠道细菌。
本研究比较了体外培养中珀氏解黄酮菌(Flavonifractor plautii)的FCM计数与稀释涂布平板法计数,并基于FCM研究了叶绿素对来源于小鼠肠道的珀氏解黄酮菌、普通拟杆菌(Bacteroides vulgatus)、假长双岐杆菌(Bifidobacterium pseudolongum)和鼠乳杆菌(Lactobacillus murinus)增殖的影响,为阐明膳食-肠道菌群-宿主互作机制提供了有效的研究方法和理论数据 。
1. 材料与方法
1.1 材料与仪器
实验菌株 均分离于ob/ob小鼠盲肠内容物[7],保藏于中国普通微生物菌种保藏管理中心(China General Microbiological Culture Collection Center, CGMCC),如表1所示。叶绿素(含量为92.11%,其中叶绿素a为70.99%,叶绿素b为21.12%[14]) 自制;95%乙醇 分析纯,国药集团化学试剂有限公司;改良GAM肉汤 青岛海博生物技术有限公司;澄清瘤胃液 北方远洋生物科技有限公司;琼脂粉 北京奥博星生物科技有限责任公司; LIVE/DEAD ® BacLightTM试剂盒 美国Thermo Fisher Scientific公司。
表 1 实验菌株名称Table 1. Names of the test strains序号 菌株 CGMCC保藏号 1 Flavonifractor plautii D16-29T 1.31275 2 Bacteroides vulgatus D2-Ch2-95 1.31095 3 Bifidobacterium pseudolongum 22-20 1.31005 4 Lactobacillus murinus C-30 1.31225 SpectraMax iD5型酶标仪 美国Molecular Devices公司;Q23XD-2型二位三通先导电磁阀 浙江奉化市中源气动成套厂;Coylabotatory型厌氧手套箱 美国COY公司; FACSCantoⅡ型流式细胞仪 美国Becton-Dickinson医疗器械有限公司。
1.2 实验方法
1.2.1 培养基的配制
GAM培养基参照Liu等[7]的方法进行配制。液体培养基配制:改良GAM肉汤60 g、L-半胱氨酸0.5 g、精氨酸0.5 g、色氨酸0.3 g、氯高铁血红素5 mL、澄清瘤胃液100 mL、刃天青1 mL,蒸馏水定容至1000 mL,调pH为7.2±0.1(25 ℃),分装至10 mL厌氧管,115 ℃灭菌25 min;固体培养基配制:改良GAM肉汤60 g、半胱氨酸0.5 g、精氨酸0.5 g、色氨酸0.3 g、氯高铁血红素5 mL、澄清瘤胃液100 mL、菊粉益生元0.5 g、纤维二糖0.5 g、海藻糖0.5 g、甘露糖0.5 g、半乳糖0.5 g、果糖0.5 g、鼠李糖0.5 g、异麦芽酮糖0.5 g、碳酸氢钠2 g、无水乙酸钠2.46 g、刃天青1 mL、琼脂10 g,最后用蒸馏水定容至1000 mL。调pH为7.2±0.1(25 ℃),115 ℃灭菌25 min。
1.2.2 Flavonifractor plautii的培养
将叶绿素加入新鲜的GAM液体培养基中,使其叶绿素浓度分别为100、300、500 µg/mL;而后将Flavonifractor plautii以2%的接种量接入含有叶绿素的培养基中,37 ℃培养至稳定期(24 h)。以不加叶绿素为对照组,叶绿素添加量为100、300、500 µg/mL的组依次定义为低浓度组、中浓度组和高浓度组。
1.2.3 Flavonifractor plautii的流式细胞术(FCM)计数
1.2.3.1 样品制备
取1 mL Flavonifractor plautii菌液,10000 g离心2 min以收集菌体细胞,将沉淀重悬于1 mL 0.85% NaCl溶液中,室温孵育30 min,10000 g离心2 min,用0.85% NaCl溶液洗涤沉淀以除去干扰成分[15],再次10000 g离心2 min,最后将菌体沉淀重悬于1 mL 0.85% NaCl溶液中,制得活菌悬浮液,备用;另取1 mL菌液,10000 g离心2 min以收集菌体细胞,将沉淀重悬于1 mL 70%异丙醇[16],余下步骤与活菌悬浮液制备相同,制得死菌悬浮液,备用。样品染色液:在分析管中依次加入0.85% NaCl溶液977 µL、3.34 mmol/L SYTO 9 1.5 µL、30 mmol/L PI 1.5 µL、细菌悬浮液10 µL和微球悬浮液10 µL,充分混合,室温下避光孵育15 min[17]。调试仪器所需单色对照管:分别准备2份活菌悬浮液和2份死菌悬浮液,与样品染色液制备步骤一致,2份活/死菌悬浮液中,一份仅加入SYTO 9,另一份仅加入PI。分析管中样品的总体积应为1000 µL,以便准确计数。
1.2.3.2 仪器调整与参数设置
前向角、侧向角和荧光信号采集对数放大信号。不断调整电压参数和荧光补偿,使细菌群体和标准微球均位于横、纵坐标轴的中间区域,同时使活菌和死菌所在区域能够很好区分开[18]。参数设置:进样量10 µL;进样速度1.0 µL/s;events采集总数量30000个;电压值:前向角FSC 170V、侧向角SSC 340 V、绿色荧光280 V、红色荧光280 V。
1.2.3.3 细菌总数计算
微球标准品稀释100倍后,密度为1.0×106个/mL,即1个计数微球表示10−6 mL。根据细菌区域的信号数(events in bacteria region)和微球区域的信号数(events in bead region),可得出每10−6 mL的细菌总数[19]。计算公式如下:
C=B×DM×V 式中:C表示细菌总数,个/mL;B表示细菌区域的信号数;D表示溶液稀释倍数100;M表示微球区域的信号数;V表示计数微球体积10−6,mL。
1.2.4 Flavonifractor plautii的稀释涂布平板法计数
另取1 mL Flavonifractor plautii菌液(与上述流式细胞术所用菌液相同),用无菌的磷酸缓冲盐溶液进行梯度稀释。选取10−5、10−6、10−7三个稀释梯度,分别取100 µL稀释液涂布于GAM固体培养基上[18],置于37 ℃厌氧培养箱中培养36 h,培养环境的相对湿度为50%,氧气值 <300 ppm。选取菌落数在30~300个的平板进行计数,单位为CFU/mL[20]。所有操作均在厌氧手套箱中进行,气体环境为85%N2、5%CO2、10%H2。
1.2.5 Flavonifractor plautii的FCM活菌计数与稀释涂布平板法活菌计数比较
将FCM测得的活菌数与稀释涂布平板法检测出的活菌数进行比较,并利用皮尔逊相关性分析两种方法测得的样本数据之间的相关程度。
1.2.6 待测菌株的培养及FCM检测
将叶绿素加入新鲜的GAM液体培养基中,使其叶绿素浓度分别为100、300、500 µg/mL,而后分别将Bacteroides vulgatus、Bifidobacterium pseudolongum和Lactobacillus murinus以2%的接种量接入含有叶绿素的培养基中,37 ℃培养至稳定期。三株肠道细菌的FCM检测与上述Flavonifractor plautii相同。
1.3 数据处理
所有实验重复3 次。采用SPSS 20.0进行数据统计分析,结果表示为平均值±标准差。采用T检验对组间数据进行显著性差异分析,低浓度组、中浓度组和高浓度组与对照组比较,*表示差异显著(P<0.05);**表示差异高度显著(P<0.01);***表示差异极显著(P<0.001)。使用Flow Jo 10处理流式细胞图,Origin 9.0进行绘图。
2. 结果与分析
2.1 FCM 计数与稀释涂布平板法计数比较
以Flavonifractor plautii为例,应用FCM检测了体外培养中Flavonifractor plautii的细菌数,得到如图1所示的流式细胞图。分别以活菌和死菌单色对照管为对照,在FSC/SSC散点图中确定细菌及计数微球分别所在区域并圈出。如图1a中P1(左下)为细菌所在区域,P2(右上)为计数微球所在区域;接下来选中细菌所在区域,进一步在FITC/PerCP-Cy5-5荧光图中确定活菌和死菌分别所在区域并圈出,如图1b中P3(下侧)为活菌所在区域,P4(上侧)为死菌所在区域。统计细菌和计数微球各自所在门中的events数分别进行计算,得出相应的细菌数。对照组(叶绿素浓度为0 µg/mL)中,Flavonifractor plautii总菌数为2.85×108个/mL,其中活菌数为2.78×108个/mL,死菌数为7.39×106个/mL;低浓度组(叶绿素浓度为100 µg/mL),Flavonifractor plautii总菌数为4.01×108个/mL,其中活菌数为3.93×108个/mL,死菌数为8.75×106个/mL。
SYTO 9可进入细胞膜完整的活细菌内与DNA结合,而PI只能进入细胞膜受损或不完整的死细菌内[11]。因此,FCM与SYTO 9/PI双染色液配合使用可以区分出死菌和活菌各自所占比例,从而实现对肠道菌培养中活细菌和死细菌的同时检测。
为更好地评估FCM对Flavonifractor plautii细菌数检测的效果,实验同时采用经典的活菌计数方法—稀释涂布平板法检测该菌的活菌数。由图2a可知,对照组(叶绿素浓度为0 µg/mL)活菌数为1.0×108 CFU/mL,低浓度组(叶绿素浓度为100 µg/mL)活菌数为1.4×108 CFU/mL,与对照组相比显著增加(P<0.05)。对两种方法测得的活菌数进行皮尔逊相关性分析,两者相关性系数r
>0.8,具有显著正相关关系(P<0.001)(图2b)。 进一步将FCM与稀释涂布平板法活菌计数数值进行比较,流式细胞术所得数值均大于稀释涂布平板法所得数值(图2a),这与李可欣等[21]的研究结果相似。这是因为对于一些处于特殊生理状态的活菌体细胞,如有活力但不可培养(viable but not culturable, VBNC)、生长缓慢和休眠状态的菌体细胞,利用稀释涂布平板法无法对其进行计数[22-24],而FCM是基于特异性染料分别对活细胞和死细胞DNA进行染色,因此可有效检测出该部分特殊的活菌细胞;此外,由于细菌具有聚集的特性,菌细胞在固体培养基上分布不均匀,一个菌落很有可能是由一个以上的菌细胞生长而来,因此稀释涂布平板法所得活菌数通常会小于真实值[25],而FCM可通过降低流速或菌细胞浓度来最大程度避免细菌聚集对检测结果的影响[26],因此其检测结果更接近真实值。稀释涂布平板法一般需要培养24~48 h才能进行菌落计数,对于大部分难以在体外生长的肠道细菌而言,甚至需要48 h以上的时间才能长出菌落,而FCM在30 min内即可完成1株菌的计数检测,明显缩短了检测时间与实验周期。综上可知,使用FCM检测体外培养中肠道菌的细菌数,可明显缩短实验周期,提高检测效率,同时检测结果更接近于真实值。因此,接下来的研究中将利用FCM探究叶绿素对不同肠道菌增殖的影响。
2.2 叶绿素对不同肠道菌增殖的影响
2.2.1 叶绿素对珀氏解黄酮菌(Flavonifractor plautii)增殖的影响
由图3可知,随着叶绿素浓度的升高,Flavonifractor plautii总菌数和活菌数均呈现先增加后减少的趋势,而死菌数极显著增加(P<0.001)。当叶绿素浓度为100 µg/mL时,活菌数最多,达3.93×108个/mL;当叶绿素浓度为300 µg/mL及以上时,活菌数逐渐减少。对照组中活菌数为2.78×108个/mL;与对照组相比,叶绿素浓度为100 µg/mL时,活菌数增加1.15×108 个/mL,叶绿素浓度为100和 500 µg/mL时,活菌数分别减少1.33×108和2.10×108 个/mL,表明叶绿素对Flavonifractor plautii的增殖表现出 “低浓度促进、高浓度抑制”的作用规律。Flavonifractor plautii是一种革兰氏阳性菌,可参与肠道中儿茶素的代谢[27],具有降低机体炎症、改善肥胖的功效[28]。Mikami等[29]研究证明,饮用绿茶可以增加肠道中Flavonifractor plautii的丰度并有助于改善结肠炎;此外,Liu等[30]研究发现,芝麻素可促进Flavonifractor的生长,有益于肠道健康。因此,100 µg/mL的叶绿素浓度促进Flavonifractor plautii增殖或许有助于改善肥胖和肠道疾病。目前,还未有研究报道外源性物质对Flavonifractor plautii生长的影响机制。
2.2.2 叶绿素对普通拟杆菌(Bacteroides vulgatus)增殖的影响
由图4可知,随着叶绿素浓度的升高,Bacteroides vulgatus总菌数和活菌数均呈现先增加后减少的趋势,而死菌数高度显著减少(P<0.01)。当叶绿素浓度为100 µg/mL时,活菌数最多,达3.09×108个/mL;当叶绿素浓度为300 µg/mL及以上时,活菌数逐渐减少。对照组中活菌数为1.61×108个/mL;与对照组相比,叶绿素浓度为100 µg/mL时,活菌数增加1.47×108个/mL,叶绿素浓度为100和 500 µg/mL时,活菌数分别减少1.40×108和1.35×108 个/mL,表明叶绿素对Bacteroides vulgatus的增殖也表现出 “低浓度促进、高浓度抑制”的作用规律。相似地,Kamijo等[31]研究发现,向培养基中添加0.01%蔷薇花瓣粉可促进Bacteroides vulgatus JCM5826 T的增殖,而0.05%蔷薇花瓣粉则会抑制该菌的增殖(抑制率82%),当浓度提高到0.1%时,该菌的增殖被完全抑制。由此可知,浓度是叶绿素等植物化合物对Bacteroides vulgatus作用的重要影响因素。有研究报道肠道细菌如布劳特氏菌属(Blautia)可利用卟啉作为能源物质或生物合成的前体物质,从而促进自身生长[32],而叶绿素促进Bacteroides vulgatus增殖的机制还有待探究。目前,大多数植物化合物对肠道菌群生长的研究,主要针对复杂微生物群落展开。其中,植物性化合物对肠道细菌的抑制作用更为常见[33],而关于促生长机制,目前还未有确切的研究报道。
2.2.3 叶绿素对假长双岐杆菌(Bifidobacterium pseudolongum)增殖的影响
如图5所示,随着叶绿素浓度的升高,Bifidobacterium pseudolongum总菌数和活菌数极显著减少(P<0.001),而死菌数无显著变化。当叶绿素浓度为0 µg/mL时,Bifidobacterium pseudolongum活菌数为2.63×108个/mL,随着叶绿素浓度的增加,Bifidobacterium pseudolongum活菌数依次减少7.78×107、1.63×108和1.82×108 个/mL,表明叶绿素可极显著抑制Bifidobacterium pseudolongum的增殖(P<0.001),且叶绿素浓度越高,抑制作用越强,呈现剂量依赖性。与其相似的是,Gwiazdowska等[34]研究发现,橙皮苷和槲皮素对Bifidobacterium adolescentis NCFB 2004和Bifidobacterium bifidum NCFB 2235的增殖也具有抑制作用,且呈现剂量依赖性。Li等[5]基于16S rRNA 基因扩增子测序报道了叶绿素可下调肠道中双歧杆菌属(Bifidobacterium)的丰度,而本研究将其延伸到菌种水平,首次发现叶绿素对Bifidobacterium pseudolongum的增殖有抑制作用。
2.2.4 叶绿素对鼠乳杆菌(Lactobacillus murinus)增殖的影响
如图6所示,随着叶绿素浓度的升高,Lactobacillus murinus的总菌数、活菌数和死菌数均显著增加(P<0.05)。当叶绿素浓度为0 µg/mL时,Lactobacillus murinus的总菌数和活菌数分别为2.01×107和2.11×106个/mL;随着叶绿素浓度的增加,Lactobacillus murinus的总菌数依次增加3.55×107、7.00×107、1.01×108个/mL,活菌数依次增加1.75×106、6.36×105、6.98×105个/mL。说明叶绿素可以极显著促进Lactobacillus murinus的增殖(P<0.001),且浓度越高,促进作用越强。与此相似的是,Huang等[35]研究发现,薯蓣皂苷元也可显著促进Lactobacillus murinus的增殖,且浓度越高促进效果越好。据报道,Lactobacillus murinus是一种潜在的益生菌,具有抗过敏[36]、抗炎[37]、改善糖尿病[38]等功效。因此,叶绿素以浓度依赖性的方式促进Lactobacillus murinus增殖,说明叶绿素具有益生元的潜在特性。
3. 结论
为了探究叶绿素对特定肠道细菌增殖的影响,以期能更好地阐明膳食叶绿素介导肠道菌群调控人体健康的内在规律。本文比较了FCM和平板计数法用于体外培养中特定肠道细菌的计数,证明FCM可实现对肠道细菌的高效、准确计数;在此基础上,进一步探究了体外培养中叶绿素对小鼠肠道来源的珀氏解黄酮菌(Flavonifractor plautii)、普通拟杆菌(Bacteroides vulgatus)、假长双岐杆菌(Bifidobacterium pseudolongum)和鼠乳杆菌(Lactobacillus murinus)增殖的影响。结果表明,叶绿素对Flavonifractor plautii 和Bacteroides vulgatus的增殖表现出 “低浓度促进、高浓度抑制”的作用规律;叶绿素可极显著抑制Bifidobacterium pseudolongum的增殖(P<0.001),且呈现剂量依赖性;叶绿素可极显著促进Lactobacillus murinus的增殖(P<0.001)。本研究为阐明膳食叶绿素对肠道菌群生长的影响规律提供了有效的研究方法和重要的理论数据。
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表 1 实验菌株名称
Table 1 Names of the test strains
序号 菌株 CGMCC保藏号 1 Flavonifractor plautii D16-29T 1.31275 2 Bacteroides vulgatus D2-Ch2-95 1.31095 3 Bifidobacterium pseudolongum 22-20 1.31005 4 Lactobacillus murinus C-30 1.31225 -
[1] LANFER-MARQUEZ U M, BARROS R M, SINNECKER P. Antioxidant activity of chlorophylls and their derivatives[J]. Food Research International,2005,38(9):885−891.
[2] LEE D, NISHIZAWA M, SHIMIZU Y, et al. Anti-inflammatory effects of dulse (Palmaria palmata) resulting from the simultaneous water-extraction of phycobiliproteins and chlorophyll a[J]. Food Research International,2017,100(1):514−521.
[3] ZHANG X H, ZHANG L J, SUN J J, et al. Photodynamic efficiency of a chlorophyll-a derivative in vitro and in vivo[J]. Biomedicine & Pharmacotherapy,2016,81:265−272.
[4] ZHONG S Q, BIRD A, KOPEC R E. The metabolism and potential bioactivity of chlorophyll and metallo-chlorophyll derivatives in the gastrointestinal tract[J]. Molecular Nutrition & Food Research,2021,65(7):2000761.
[5] LI Y Y, CUI Y U, HU X S, et al. Chlorophyll supplementation in early life prevents diet-induced obesity and modulates gut microbiota in mice[J]. Molecular Nutrition & Food Research,2019,63(21):1−13.
[6] LI Y Y, CUI Y, LU F, et al. Beneficial effects of a chlorophyll-rich spinach extract supplementation on prevention of obesity and modulation of gut microbiota in high-fat diet-fed mice[J]. Journal of Functional Foods,2019,60:103436. doi: 10.1016/j.jff.2019.103436
[7] LIU C, ZHOU N, DU M X, et al. The mouse gut microbial biobank expands the coverage of cultured bacteria[J]. Nature Communications,2020,11(1):79. doi: 10.1038/s41467-019-13836-5
[8] AMBRIZ-AVINA V, CONTRERAS-GARDUNO J A, PEDRAZA-REYES M. Applications of flow cytometry to characterize bacterial physiological responses[J]. Biomed Research International,2014:461941.
[9] ALBA C, MARIN A C, MCNICHOLL A G, et al. A quick flow cytometry protocol to assess Helicobacter pylori viability[J]. Journal of Microbiological Methods,2020,177:106043. doi: 10.1016/j.mimet.2020.106043
[10] SAWADA T, KATAYAMA M, TAKATANI S, et al. Early detection of drug-resistant Streptococcus pneumoniae and Haemophilus influenzae by quantitative flow cytometry[J]. Scientific Reports,2021,11(1):2873. doi: 10.1038/s41598-021-82186-4
[11] HE S B, HONG X Y, HUANG T X, et al. Rapid quantification of live/dead lactic acid bacteria in probiotic products using high-sensitivity flow cytometry[J]. Methods and Applications in Fluorescence,2017,5(2):1−9.
[12] BARTLE L, MITCHELL J G, PATERSON J S. Evaluating the cytometric detection and enumeration of the wine bacterium, Oenococcus oeni[J]. Cytometry Part A,2021,99(4):399−406. doi: 10.1002/cyto.a.24258
[13] TRCEK J, LIPOGLAVSEK L, AVGUSTIN G. 16S rRNA in situ hybridization followed by flow cytometry for rapid identification of acetic acid bacteria involved in submerged industrial vinegar production[J]. Food Technology and Biotechnology,2016,54(1):108−112.
[14] LI Y Y, LU F, WANG X, et al. Biological transformation of chlorophyll-rich spinach (Spinacia oleracea L.) extracts under in vitro gastrointestinal digestion and colonic fermentation[J]. Food Research International,2021,139:109941. doi: 10.1016/j.foodres.2020.109941
[15] DUQUENOY A, BELLAIS S, GASC C, et al. Assessment of gram- and viability-staining methods for quantifying bacterial community dynamics using flow cytometry[J]. Frontiers in Microbiology,2020,11:1469. doi: 10.3389/fmicb.2020.01469
[16] ROUSSEL C, GALIA W, LERICHE F, et al. Comparison of conventional plating, PMA-qPCR, and flow cytometry for the determination of viable enterotoxigenic Escherichia coli along a gastrointestinal in vitro model[J]. Applied Microbiology and Biotechnology,2018,102(22):9793−9802. doi: 10.1007/s00253-018-9380-z
[17] MAJEED M, MAJEED S, NAGABHUSHANAM K, et al. Evaluation of the stability of Bacillus coagulans MTCC 5856 during processing and storage of functional foods[J]. International Journal of Food Science and Technology,2016,51(4):894−901. doi: 10.1111/ijfs.13044
[18] LIU C, DU M X, ABUDUAINI R, et al. Enlightening the taxonomy darkness of human gut microbiomes with a cultured biobank[J]. Microbiome,2021,9(1):119. doi: 10.1186/s40168-021-01064-3
[19] GAO Y, YU H J, WEN B. The use of fluorescent techniques in combination with flow cytometry for fast counting of Bifidobacterium longum ATCC BAA-2753 in BIFICO capsule[J]. Food Science and Biotechnology,2018,27(5):1405−1410. doi: 10.1007/s10068-018-0388-z
[20] 张兰, 徐红, 翁文川, 等. 流式细胞技术检测酸性饮料中菌落总数的研究[J]. 食品工业科技,2018,39(4):235−239. [ZHANG L, XU H, WENG W C, et al. Study on total plate count detection in acid beverage by flow cytometry[J]. Science and Technology of Food Industry,2018,39(4):235−239. doi: 10.13386/j.issn1002-0306.2018.04.043 [21] 李可欣, 吴蔓莉, 高欢, 等. 基于流式细胞术的活性多环芳烃降解菌检测技术[J]. 分析化学,2021,49(8):1357−1365. [LI K X, WU M L, GAO H, et al. Detection technology of active polycyclic aromatic hydrocarbon degrading bacteria based on flow cytometry[J]. Chinese Journal of Analytical Chemistry,2021,49(8):1357−1365. doi: 10.19756/j.issn.0253-3820.201598 [22] EMERSON J B, ADAMS R I, ROMAN C M B, et al. Schrodinger's microbes: Tools for distinguishing the living from the dead in microbial ecosystems[J]. Microbiome,2017,5:86. doi: 10.1186/s40168-017-0285-3
[23] LI L, MENDIS N, TRIGUI H, et al. The importance of the viable but non-culturable state in human bacterial pathogens[J]. Frontiers in Microbiology,2014,5:258.
[24] ZHAO X H, ZHONG J L, WEI C J, et al. Current perspectives on viable but non-culturable state in foodborne pathogens[J]. Frontiers in Microbiology,2017,8:580.
[25] OU F, MCGOVERIN C, SWIFT S, et al. Absolute bacterial cell enumeration using flow cytometry[J]. Journal of Applied Microbiology,2017,123(2):464−477. doi: 10.1111/jam.13508
[26] VAN N S, BUYSSCHAERT B, DE R K, et al. Flow cytometry for immediate follow-up of drinking water networks after maintenance[J]. Water Research,2017,111:66−73. doi: 10.1016/j.watres.2016.12.040
[27] OGITA T, YAMAMOTO Y, MIKAMI A, et al. Oral administration of Flavonifractor plautii strongly suppresses th2 immune responses in mice[J]. Frontiers in Immunology,2020,11:379. doi: 10.3389/fimmu.2020.00379
[28] MIKAMI A, OGITA T, FU N, et al. Oral administration of Flavonifractor plautii attenuates inflammatory responses in obese adipose tissue[J]. Molecular Biology Reports,2020,47(9):6717−6725. doi: 10.1007/s11033-020-05727-6
[29] MIKAMI A, OGITA T, FU N, et al. Oral administration of Flavonifractor plautii, a bacteria increased with green tea consumption, promotes recovery from acute colitis in mice via suppression of IL-17[J]. Frontiers in Nutrition,2021,7:610946. doi: 10.3389/fnut.2020.610946
[30] LIU P, DENG Y, WANG M, et al. Sesamin modulation of intestinal microflora in rats on high-fat diet[J]. Current Topics in Nutraceutical Research,2020,19(3):312−316. doi: 10.37290/ctnr2641-452X.19:312-316
[31] KAMIJO M, KANAZAWA T, FUNAKI M, et al. Effects of rosa rugosa petals on intestinal bacteria[J]. Bioscience Biotechnology and Biochemistry,2008,72(3):773−777. doi: 10.1271/bbb.70645
[32] LI Y Y, CAO J R, ZHENG H L, et al. Transformation pathways and metabolic activity of free chlorophyll compounds from chloroplast thylakoid membrane under in vitro gastrointestinal digestion and colonic fermentation in early life[J]. Food Bioscience,2021,42:101196. doi: 10.1016/j.fbio.2021.101196
[33] LINDELL A E, ZIMMERMANN-KOGADEEVA M, PATIL K R, et al. Multimodal interactions of drugs, natural compounds and pollutants with the gut microbiota[J]. Nature Reviews Microbiology, 2022, 20(7): 431−443.
[34] GWIAZDOWSKA D, JUS K, JASNOWSKA-MALECKA J, et al. The impact of polyphenols on Bifidobacterium growth[J]. Acta Biochimica Polonica,2015,62(4):895−901. doi: 10.18388/abp.2015_1154
[35] HUANG C H, CHENG J Y, DENG M C, et al. Prebiotic effect of diosgenin, an immunoactive steroidal sapogenin of the Chinese yam[J]. Food Chemistry,2012,132(1):428−432. doi: 10.1016/j.foodchem.2011.11.016
[36] HUANG C H, SHEN C C, LIANG Y C, et al. The probiotic activity of Lactobacillus murinus against food allergy[J]. Journal of Functional Foods,2016,25:231−241. doi: 10.1016/j.jff.2016.06.006
[37] PAN F, ZHANG L, MIN L, et al. Predominant gut Lactobacillus murinus strain mediates anti-inflammaging effects in calorie-restricted mice[J]. Microbiome,2018,6(1):54. doi: 10.1186/s40168-018-0440-5
[38] YUE S J, SHAN B, PENG C X, et al. Theabrownin-targeted regulation of intestinal microorganisms to improve glucose and lipid metabolism in Goto-Kakizaki rats[J]. Food & Function,2022,13(4):1921−1940.