肠膜明串珠菌(Leuconostoc mesenteroides subsp. cremoris) GKM5降血糖活性评估
Evaluation of the Hypoglycemic Activity of Leuconos-toc mesenteroides subsp. cremoris GKM5
DOI: 10.12677/HJFNS.2022.114033, PDF, HTML, XML, 下载: 210  浏览: 366 
作者: 周柏谊, 蔡侑珊, 林诗伟, 陈炎炼：葡萄王生技股份有限公司，台湾 桃园；吴文歆：上海葡萄王企业有限公司，上海；陈劲初*：台湾大学食品科技研究所，台湾 台北；实践大学食品营养与保健生技系，台湾 台北；中原大学生物科技学系，台湾 桃园
关键词: 肠膜明串珠菌GKM5；益生菌；糖尿病；空腹血糖值；白血球介素-6； Leuconostoc mesenteroides subsp. cremoris GKM5； Probiotics； Diabetes； Fasting Plasma Glucose； Interleukin 6
摘要: 本篇研究评估肠膜明串珠菌(Leuconostoc mesenteroides subsp. cremoris) GKM5对于降血糖活性的潜力。第一部分为评估血糖管理潜力。将ICR雄性小鼠分为实验组(Live-GKM5)及对照组(Control)，管喂十四天菌株GKM5后，进行口服葡萄糖耐受性试验(oral glucose tolerance test, OGTT)。结果发现实验组的120分钟血糖曲线下面积(area under the curve, AUC)明显地低于对照组，显示肠膜明串珠菌GKM5的摄取可改善ICR小鼠的葡萄糖耐受性，有助于提升葡萄糖敏感性。第二部分为评估菌株GKM5对于糖尿病高风险族群的血糖管理能力。经链脲佐菌素(streptozotocin, STZ)诱导糖尿病高风险群的ICR雄性小鼠分别给予肠膜明串珠菌GKM5活菌(Live-GKM5)或死菌(Killed-GKM5)，以及水作为对照组(Control)，十四天后，测定空腹血糖值(fasting blood glucose, FBG)及血中发炎因子白血球介素-6 (Interleukin 6, IL-6)含量。结果发现肠膜明串珠菌GKM5之活菌可明显改善空腹血糖值及血中发炎因子IL-6含量，而肠膜明串珠菌GKM5之死菌，虽然效果较活菌不明显，仍能在介入十四天后降低空腹血糖值及血中发炎因子IL-6含量。综观上述，本篇以动物试验证实肠膜明串珠菌GKM5之摄取确实对糖尿病管理有所帮助，且GKM5死菌降血糖的功效对于未来医药用途可更为广泛。
Abstract: This study evaluated the potential of Leuconostoc mesenteroides subsp. cremoris GKM5 for hypoglycemic ac-tivity. The first part is to assess the potential of blood glucose management. The ICR male mice were divided into an experimental group (Live-GKM5) and a control group. After fourteen days of gavage with strain GKM5, oral glucose tolerance test (OGTT) was performed. The results showed that the area under the curve (AUC) of the 120-minute blood glucose in the experimental group was signifi-cantly lower than that in the trol group, indicating that the intake of L. mesenteroides GKM5 could improve the glucose tolerance of ICR mice and help improve the glucose sensitivity. The second part is to evaluate the blood glucose management ability of the strain GKM5 in high-risk groups of dia-betes. ICR male mice in the high-risk of diabetes induced by streptozotocin (STZ) were given live (Live-GKM5) or dead bacterialstain GKM5 (Killed-GKM5), and water as a control group, respectively. After 14 days, the fasting blood glucose (FBG) and the inflammatory factor interleukin-6 (IL-6) level in the blood were measured. The results showed that the live bacteria of L. mesenteroides GKM5 can significantly reduce the FBG level and the serumIL-6. Although the effect of Killed-GKM5 is less than the Live-GKM5, it still canreducetheFBGandserumIL-6 at day 14th. Tosummarize, this article con-firmed by animal experiments that the intake of L. mesenteroides GKM5 is indeed helpful for dia-betes management, and the hypoglycemic effect of Killed-GKM5 could be more widely used in future medicine.
文章引用：周柏谊, 蔡侑珊, 林诗伟, 吴文歆, 陈炎炼, 陈劲初. 肠膜明串珠菌(Leuconostoc mesenteroides subsp. cremoris) GKM5降血糖活性评估[J]. 食品与营养科学, 2022, 11(4): 290-297. https://doi.org/10.12677/HJFNS.2022.114033

1. 引言

根据台湾国民健康署(Health Promotion Administration)统计，台湾目前罹患糖尿病(diabetes)之人数约有200多万左右，每年近万人因糖尿病及其所引发的并发症死亡，糖尿病的盛行对人类社会之健康威胁不容小觑，医疗成本所造成的经济社会负担相当可观。糖尿病是一种代谢性疾病(metabolic disease)，主要分为I型糖尿病(Type 1 diabetes mellitus)和II型糖尿病(Type 2 diabetes mellitus)。I型糖尿病属于自体免疫疾病(autoimmune disease)，造成胰岛β-细胞损伤而导致胰岛素(insulin)分泌缺乏 [1]；II型糖尿病是最为常见的糖尿病类型，主要原因是细胞对胰岛素的敏感度降低、产生阻抗 [2]。全球糖尿病人口日益增多，可能与精致饮食、过量摄取高糖份或是高脂肪饮食且运动量不足有关，已成为公共卫生的严峻挑战，因此，针对早期高风险患者应积极介入，改变其生活饮食型态，或有助于减少此趋势。

益生菌(probiotics)一般被认为食入后对宿主有正面效益的食入性微生物，像是体重控制 [3]、调整过敏体质 [4] [5] [6]、保护肝脏 [7] [8]、抗忧郁 [9] [10] [11] 等功能，近期研究更显示益生菌补充对于改善血糖控制及减少体内发炎症状均有显著帮助 [12] [13] [14]。肠膜明串珠菌(Leuconostoc mesenteroides)属于乳酸菌科(Lactobacillaceae)的一种，能发酵糖类产生多种酸和醇，因其发酵后产出风味特别，被广泛应用于风味剂的使用，其中又以乳脂亚种(L. mesenteroides subsp. cremoris)最为常见 [15]。有鉴于肠膜明串珠菌发酵产生的多元短链脂肪酸(short-chain fatty acid)能参与人体多项式代谢调控，像是发炎反应、脂肪代谢、血糖代谢等，具发展益生菌的潜能 [16]。本研究针对肠膜明串珠菌乳脂亚种菌株GKM5为对象，评估其对血糖调节的功效。

2. 材料与方法

2.1. 供试菌种

肠膜明串珠菌GKM5培养于5 L MRS培养基(Merck, Germany)中，培养温度为37℃，经16小时培养后，将酦酵液离心取得菌泥，与脱脂奶粉液混合后，经冷冻干燥后取得菌株粉末，每克菌粉含有活菌數5 × 109 CFU，保存于−20℃备用，供动物喂饲。死菌之制备则以热杀处理。

2.2. 动物饲养

8周龄之雄性ICR小鼠经过一周的适应期后，随机分组。动物饲养室之环境设定为温度23℃ ± 2℃、相对湿度50% ± 10%以及12小时的光暗周期。

2.3. 试验设计

本篇研究第一部分为评估肠膜明串珠菌GKM5在血糖管理的潜力。将八只正常ICR雄性小鼠随机分为实验组(Live-GKM5)及对照组(control)，经管喂菌株(500 mg/kg mouse/day)十四天后，进行口服葡萄糖耐受性试验(oral glucose tolerance test, OGTT)。第二部分为评估肠膜明串珠菌GKM5对于糖尿病高风险族群的血糖管理。以两次剂量100 mg/kg mouse的链脲佐菌素(streptozotocin, STZ)注射于ICR雄性小鼠诱导高血糖，两天后经血糖测定大于200 mg/dL者视为糖尿病高风险群。十二只糖尿病高风险的小鼠分为实验活菌组(Live-GKM5)、死菌组(Killed-GKM5)，及对照组(control)。管喂菌株GKM5剂量500 mg/kg mouse/day十四天后，测定空腹血糖值及血中发炎因子IL-6含量。上述对照组皆给予灭菌水取代之。

2.4. 血糖测定

使ICR小鼠禁食6小时，先自尾端采集血液样本，以血糖机(Advantage Glucometer, Roche, Mannheim, Germany)检测空腹(0分钟)血糖值。在OGTT试验中的血糖变化监控则以葡萄糖溶液(1.5 g/kgBW)管喂禁食6小时的ICR小鼠，再分别检测其第0、30、60及120分钟时的血糖浓度。

2.5. IL-6测定

使用市售之ELISA kit-IL-6试剂组(R&D systems, Minneapolis, MN, USA)进行各组小鼠血清样品之IL-6测定，操作按试剂盒的说明进行。

2.6. 统计分析

所有资料均以平均值 ± 标准偏差(Mean ± SD)表示。两组间的比较以学生t检定(Student’s t-test)进行分析，三组间的比较则使用ANOVA的杜凯确实差异(Tukey’s test)进行事后分析。当* p < 0.05时表示组间具有显着性差异。

3. 结果

3.1. 血糖管理潜力

本篇研究第一部分为评估肠膜明串珠菌GKM5在血糖管理的潜力。实验前后的老鼠体重，组间并无显著差异。在口服葡萄糖耐受性试验中，对照组与实验组(Live-GKM5)之空腹血糖值皆约为75 mg/dL，无显著差异；然而，经摄取葡萄糖液后，对照组30分钟内的血糖快速升高至300 mg/dL以上，Live-GKM5组在30分钟的血糖值则显著低于对照组，说明肠膜明串珠菌GKM5的摄取可改善ICR小鼠的葡萄糖耐受性(图1)。OGTT试验中30分钟后的血糖浓度变化，两组皆成下降趋势，说明两组生理机制上皆正在调节血糖浓度，其中以120分钟的血糖曲线下面积(area under curve, AUC120min)纪录，可观察到实验组明显地低于对照组，显示肠膜明串珠菌GKM5的摄取组别，其调整血糖的能力较佳(图2)。

与对照组相比，具显著差异者以*p < 0.05表示。

Figure 1. Glucose changes in oral glucose tolerance test in ICR mice after 14 days of gavage

图1. 管喂14天后，ICR小鼠于口服葡萄糖耐受性试验之血糖变化

与对照组具显著差异以*p < 0.05表示。

Figure 2. Area under curve at 120 min

图2. 120分钟曲线下面积

3.2. 对于糖尿病高风险族群的血糖管理

3.2.1. 空腹血糖值(Fasting Blood Glucose, FBG)

第二部分为评估肠膜明串珠菌GKM5对于糖尿病高风险族群的血糖管理。经STZ诱导的ICR小鼠体重皆有下降的趋势(图3)。诱导小鼠成为糖尿病高风险族群后的空腹血糖值皆高于200 mg/dL，并组间无显著差异(图4)。随时间观察，对照组小鼠的空腹血糖在STZ诱导后，随时间变化有升高的趋势，说明无任何益生菌介入下，糖尿病高风险族群转变成糖尿病的趋势越高。然而，介入Live-GKM5或Killed-GKM5之小鼠在第14天其空腹血糖便有明显的改善，并低于对照组，说明菌株GKM5具有减少糖尿病高风险族群转变成糖尿病的机会(图4)。进一步地，比较活菌和死菌GKM5对于降低糖尿病风险的效果。结果发现，两者相比对照组皆有降低空腹血糖值的效果，且活菌降低糖尿病风险的效果优于热杀处理的死菌(图5)。综合以上结果，肠膜明串珠菌GKM5不论活菌或死菌皆对于改善糖尿病高风险族群之空腹血糖有所帮助。

Figure 3. Body weight change in STZ-induced ICR mice

图3. STZ诱导的ICR小鼠之体重变化

与对照组相比，具显著差异者以**p < 0.01、***p < 0.001表示。

Figure 4. Fasting blood glucose in STZ-induced ICR mice

图4. STZ诱导之ICR小鼠空腹血糖值变化

*p < 0.05表示与对照组相比；#p < 0.05表示活菌与死菌相比。

Figure 5. FBG at day 14th

图5. 第14天之空腹血糖值

3.2.2. 促发炎激素IL-6

图6为喂食14天后的STZ小鼠之血清中IL-6浓度。结果发现不论给予肠膜明串珠菌GKM5之死菌(Killed-GKM5)或是活菌(Live-GKM5)皆可有效改善血中发炎因子IL-6含量。因IL-6和人体葡萄糖代谢息息相关，菌株GKM5在降低糖尿病高风险族群的血清中IL-6浓度可间接说明菌株GKM5具减缓高血糖的风险外，其对改善糖尿病所引起之炎症亦有所帮助。

与对照组相比，具显著差异者以*p < 0.05表示。#p < 0.05表示活菌与死菌相比。

Figure 6. After 14 days of gavage, the serum IL-6 level in ICR mice

图6. 管喂14天后，ICR小鼠之血中发炎因子IL-6含量

4. 讨论

在试验第一部分针对健康小鼠，给予肠膜明串珠菌GKM5之活菌观察其口服葡萄糖耐受性试验之120分钟血糖曲线下面积，结果发现实验组的120分钟血糖曲线下面积(area under curve, AUC 120 min)明显地低于水对照组，显示肠膜明串珠菌GKM5的摄取可改善ICR小鼠的葡萄糖耐受性，有助于改善葡萄糖敏感性并降低胰岛素抗阻(图2)。此结果在其他喂食肠膜明串珠菌GKM5之动物试验中也有类似之结果 [17] [18]。除此之外有其他研究显示利用肠膜明串珠菌作为发酵蔬菜产品可以减少50%的盐用量，相较一般的产品更具有保健潜力 [19]。由此可推论健康成年人在日常补充肠膜明串珠菌GKM5可以降低罹患糖尿病之风险。

试验第二部分使用糖尿病小鼠评估肠膜明串珠菌GKM5摄取对糖尿病高风险族群的血糖管理，从结果中发现不论摄取活菌(Live-GKM5)及死菌(Killed-GKM5)皆能在摄取十四天后改善糖尿病空腹血糖值(图5)及发炎因子IL-6含量(图6)。其机转可能与肠膜明串珠菌GKM5在肠道中发酵并会大量生产出丁酸(butanoic acid)有关。研究指出肠黏膜吸收丁酸后会刺激胰岛素分泌并减缓肝脏代谢胰岛素的速率，藉此帮助改善血糖症状 [20]。摄取肠膜明串珠菌GKM5可以抑制脂肪细胞分泌发炎因子IL-6，而相关研究也指出发炎因子IL-6又与许多慢性疾病及癌症有密切之关系 [21]，因此GKM5对于改善糖尿病患之并发症症状有相当之潜力。

综合上述结果，摄取肠膜明串珠菌GKM5，不论是做为健康人降低罹患糖尿病之预防医学之用，或是对于帮助糖尿病患改善其血糖异常并同时减缓其发炎症状皆有明显之效果。因此，肠膜明串珠菌GKM5极具开发成降血糖功效之保健食品、医药品的潜力。

NOTES

*通讯作者。

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