潛力益生菌Limosilactobacillus mucosae之分離、鑑定與功能性測定

Abstract

傳統益生菌已被廣泛研究和應用，為了開發出新型益生菌，次世代益生菌 (next-generation probiotics) 的概念開始受到重視，次世代益生菌是指「根據微生物群比較分析而鎖定的活體微生物，當其被攝取足量時，可為宿主帶來健康益處」。川崎氏症 (Kawasaki disease) 是一種病因未知、好發於5歲以下兒童的全身性血管炎，伴隨發燒、淋巴結腫大和皮膚黏膜表現等的綜合症。在先前的研究中發現川崎氏症兒童腸道菌相中的Limosilactobacillus mucosae顯著低於健康兒童，顯示L. mucosae可能在川崎氏症中扮演重要角色。因此，本研究旨在從兒童糞便中分離出L. mucosae並研究其特性及功能性，以評估其作為次世代益生菌的潛力。 在本研究共招募到118位健康兒童的糞便樣本，並透過即時聚合酶連鎖反應(real-time polymerase chain reaction, qPCR) 篩選出37個L. mucosae豐度較高的樣本，將其進行細菌分離，共得出16,000個單一獨立菌落，在使用L. mucosae專一性引子對聚合酶連鎖反應 (L. mucosae-specific primer set PCR) 及逢機增幅多型性DNA聚合酶連鎖反應 (randomly amplified polymorphic DNA-PCR) 進行初步鑑定及分型後，透過16S rRNA基因定序、持家基因 (housekeeping gene) pheS及rpoA定序進行進一步的鑑定，最終篩選出兩個相異的L. mucosae菌株，分別命名為H87及H602。 隨後將其和人源的L. mucosae JCM 7735 (R1)、豬源的L. mucosae BCRC 17827T (R2) 及從牛隻糞便所分離之L. mucosae C121、C212、C1988、C251及C10進行菌株特性及功能性之測定。研究結果顯示，低濃度活菌之R1、R2、C1988、C251及H87可以提升促發炎細胞激素腫瘤壞死因子α (tumor necrosis factor-α, TNF-α)，高濃度之熱滅活 (heat-killed) R1、R2、C251及C10能同時提升促發炎細胞激素TNF-α及抗發炎細胞激素白介素-10 (interleukin-10, IL-10)，顯示出這些菌株的免疫調節能力。此外R1、R2及C10在抗氧化試驗中表現最好，具有自由基清除能力、還原力及脂質氧化抑制能力。而抗肥胖能力面，所有菌株皆具有膽鹽水解酶 (bile salt hydrolase) 活性，並且C212、C1988、C10和H87具有膽固醇同化 (cholesterol assimilation) 能力。 綜上所述，本研究從嬰兒糞便中有效的分離出L. mucosae菌株，並和其他來源的L. mucosae菌株相比後，發現H87在免疫調節及降膽固醇方面表現出色，在未來進一步研究其在人體內的功能後，期望能開發出一種新型的潛力益生菌株。

Traditional probiotics have been extensively studied and applied. To develop new probiotics, the concept of next-generation probiotics has begun to gain attention. Next-generation probiotics refer to "live microorganisms identified based on comparative microbiota analyses that, when administered in adequate amounts, confer a health benefit on the host." Kawasaki disease is a systemic vasculitis of unknown etiology that primarily affects children under five years old and is characterized by fever, lymphadenopathy, and mucocutaneous symptoms. Previous studies have found that the gut microbiota of children with Kawasaki disease has significantly lower levels of Limosilactobacillus mucosae compared to healthy children, suggesting that L. mucosae may play an important role in Kawasaki disease. Therefore, this study aimed to isolate L. mucosae from children's feces and investigate its characteristics and functionality to evaluate its potential as a next-generation probiotic. In this study, fecal samples were collected from 118 healthy children and screened by real-time polymerase chain reaction (PCR) to select samples with high L. mucosae abundance. These samples were then conducted bacteria isolation, and 16,000 single colonies were isolated. After preliminary identification and typing using L. mucosae-specific primers set-PCR and random amplified polymorphic DNA-PCR, further identification was performed using 16S rRNA gene sequencing, housekeeping gene pheS and rpoA sequencing. Finally, two different L. mucosae strains were identified and named H87 and H602. These strains were then compared with L. mucosae JCM 7735 (R1) and L. mucosae BCRC 17827T (R2) isolated from human and pig, and L. mucosae C121, C212, C1988, C251, and C10 isolated from calf feces to determine their characteristics and functionality. The results showed that low doses of live R1, R2, C1988, C251, and H87 could enhance the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α). High doses of heat-inactivated R1, R2, C251, and C10 could enhance both the pro-inflammatory cytokine TNF-α and the anti-inflammatory cytokine interleukin-10 (IL-10), demonstrating the immune regulatory capabilities of these strains. Additionally, R1, R2, and C10 showed the best performance in antioxidant tests, with the ability to scavenge free radicals, reduce power, and inhibit lipid oxidation. In terms of anti-obesity capabilities, all strains exhibited bile salt hydrolase activity, and C212, C1988, C10, and H87 demonstrated cholesterol assimilation ability. In conclusion, this study effectively isolated L. mucosae strains from infant feces. Compared with L. mucosae strains from other sources, H87 showed good performance in immune regulation and cholesterol reduction. After further investigation into its functionality in the human body, the goal of this study is to develop a novel potential probiotic strain.