Lactobacillus paracasei PS23發酵乳製作與應用於預防結腸炎之機能性評估

Abstract

發酵乳製品是補充益生菌的最佳來源之一。副乾酪乳桿菌PS23 （Lactobacillus paracasei PS23, PS23; current name: Lacticaseibacillus paracasei PS23) 已被證實可調節神經傳遞物質 (neurotransmitters) 和炎症相關細胞因子，從而改善年齡相關的認知能力下降與肌肉減少症 (sarcopenia) 及其引發的抑鬱樣和焦慮樣之精神症狀。然而，PS23菌株對腸道的保護作用仍然未被證實。本研究的目的是開發PS23發酵乳製品，並進一步評估該製品的性質及其在體外和體內試驗中對於預防結腸炎的功效。 PS23發酵乳運用菌粉和菌元 (starter) 的形式，或與市售商業yogurt culture製備發酵乳，發酵期間分析其理化、微生物特性和製品保存安定性及分析與評估其質地與感官性質。製備PS23發酵乳的試驗結果顯示，PS23與yogurt culture共同培養製備而得的發酵乳製品 (PS23 FM) 相對於使用PS23菌粉、PS23菌元製備的發酵乳製品以及yogurt culture發酵乳製品 (LS FM） 所須要的發酵時間相對縮短，並且可滴定酸度 (titratable acidity, TA, %) 高於LS FM組 (P < 0.05) 。PS23 FM組也表現高黏度質地特性與相對高的胞外多醣 (extracellular polysaccharides, EPS) 含量。在4℃條件下，21天的保存安定試驗中，與LS FM組相比，沒有製品後酸化的現象，並且具有低離水率的保水特性；PS23菌株也能在保存期間維持在保存的起始菌數量。 接續進行PS23 FM對於預防結腸炎的作用。體外試驗中，將PS23 FM與人類結腸癌上皮細胞Caco 2進行共培養後，以葡聚糖硫酸鈉 (dextran sulfate sodium, DSS) 誘導腸道上皮損傷，試驗期間測量跨膜上皮電阻值 (transepithelial electrical resistance, TEER)，以評估試樣對腸道上皮的保護作用。體外試驗結果顯示，試樣與Caco 2細胞共培養期間，熱致死PS23發酵乳 （HK PS23 FM） 和活菌PS23發酵乳 （PS23 FM, 2×107 CFU/mL） 之TEER值顯著高於對照組 （P < 0.05） 。經過DSS引起的損傷後，HK PS23 FM組的TEER值仍較高於對照組，可推斷PS23熱致死發酵乳可能可以減緩葡聚糖硫酸鈉誘導腸道上皮屏障的損傷。 體內試驗沿用DSS誘導的結腸炎模式。試驗過程中，每日分別管餵週齡14-16週的C57BL6/JNarl小鼠磷酸鹽緩衝液 （1x PBS） 予控制組 (control) 與負控制組 (NC)，低劑量熱致死PS23發酵乳 （LHK PS23 FM）、高劑量熱致死PS23發酵乳 （HHK PS23 FM）、低劑量活菌PS23發酵乳 （LL PS23 FM, 107 CFU/mL）、高劑量活菌PS23發酵乳 （HL PS23 FM, 108 CFU/mL） 予試樣處理組。試驗期間評估疾病活動指數，試驗第七天之後，除了控制組以外的小鼠給予DSS飲用水。並於試驗結束日採集樣本進行病理切片分析與作用探討，作用探討根據腸屏障通透性、炎症相關的細胞因子水平、腸內菌相和代謝產物變化等面向進行分析。試驗結果顯示，HL PS23 FM組的體重變化明顯低於各組別 （P < 0.05），並且與NC組相比，採食量降低、結腸組織病理學評分增加。HHK PS23 FM組和LL PS23 FM組有降低糞便中鈣衛蛋白 (calprotectin) 表達的趨勢，LL PS23 FM組也減輕了出血性腹瀉的症狀 （P < 0.05） 。 機制探討的部分，首先以緊密連接蛋白 (tight junction protein) 表現量評估腸道上皮通透性，所有PS23 FM組的緊連蛋白 (occludin) 表現量均高於NC組，密連蛋白-1 (claudin-1) 在所有組之間的表現量沒有顯著差異。發炎性相關細胞因子中的骨髓過氧化物酶 (myeloperoxidase, MPO) 表現量在PS23 FM試樣組與NC組間沒有差異，而細胞激素表現量分析除了脾臟中的干擾素γ （IFN-γ） 和介白素-1β （IL-1β），其餘細胞激素的表現量在組間也沒有顯著差異。細胞激素分析中也觀察到，NC組在腸系膜淋巴結 （mesenteric lymph nodes, MLN） 中測定的項目腫瘤壞死因子-α （TNF-α），IL-1β，IL-6，IL-10的表現量較低；在本研究中脾臟的所有細胞激素分析項目亦有相似的情形，細胞激素低下的原因須要進一步實證探討。盲腸內菌相與盲腸或結腸中短鏈脂肪酸如乙酸 （acetic acid, AA）、丙酸 （propionic acid, PA）、丁酸 （butyric acid, BA） 的代謝物變化分析中，產氣莢膜梭菌 (Clostridium perfringens)，腸桿菌科 (Enterobacteriaceae) 等伺機性病原菌在HHK PS23 FM組和LL PS23 FM組的數量相對少於NC組。然而，雙叉桿菌屬 (Bifidobacterium) 之有益細菌和副乾酪乳桿菌PS23 (Lactobacillus paracasei PS23) 在所有組間均無明顯變化。腸道代謝分析中，PS23 FM組和NC組之間的乙酸、丙酸和丁酸也無顯著差異。 綜合本研究製備含有PS23菌株的發酵乳之特性與使用體外與體內方式評估其預防結腸炎的功效試驗結果，含有PS23菌株之發酵乳製品不僅提高製品的質地特性和保存穩定性，也對於結腸炎出血性腹瀉的症狀有所改善，並減少結腸炎發生時，伺機性病原菌的菌數量，然而，劑量的安全性和有效性與PS23 FM在腸道保護中的可能機制仍須要持續進行探討。

Fermented dairy product is considered to be one of the best sources for probiotic supplement. Lactobacillus paracasei PS23 (PS23) (Current name: Lacticaseibacillus paracasei PS23) has been confirmed to regulate the levels of neurotransmitters and inflammation-related cytokines to adjust age-related cognitive decline, sarcopenia and the mental symptoms such as depression-like and anxiety. However, the protective effects in the gastrointestinal tract are still unknown. The aim of this study was to develop a fermented milk product with PS23 and further evaluate the properties of product, and the effects of this product on anti-colitis in vitro and in vivo. Fermented milk product with PS23 (PS23 FM) was developed and analyzed the physicochemical/microbial properties, storage stability, textural properties and sensory evaluation. Results showed that PS23 FM could reduce fermentation times as comparing with the yogurt culture counterpart. PS23 FM also demonstrated a lower syneresis, higher viscosity and higher extracellular polysaccharides (EPS) content than the control. After 21-day storage, the numbers of PS23 could maintain at the initial storage for both PS23 groups. PS23 FM was then evaluated its anti-colitis effect. For in vitro test, PS23 FM was co-cultured with Caco 2 cell and measured the transepithelial electrical resistance (TEER) ratio to evaluate the protection of intestinal epithelial injury. Results indicated that the TEER ratios of the heat-killed PS23 FM (HK PS23 FM) and live PS23 FM (PS23 FM, 2×107 CFU/mL) co-cultured with Caco 2 were significantly higher than the dextran sulfate sodium (DSS) treated group (control) (P < 0.05). For in vivo test, DSS induced-colitis model was used. During the test, oral administrated daily with either phosphate buffered saline (1x PBS) or low-dose heat-killed PS23 FM (LHK PS23 FM), high-dose heat-killed PS23 FM (HHK PS23 FM), low-dose live PS23 FM (LL PS23 FM, 107 CFU/mL), high-dose live PS23 FM (HL PS23 FM, 108 CFU/mL) for 14-16 week-old C57BL6/JNarl mice. After the seventh day of the test, DSS drinking was administered to mice except control group. Disease activity index, macroscopic appearance and pathological section analysis of colon tissue were assessed to evaluate PS23 FM effect on prevention of colitis. Intestinal barrier permeability, the levels of intestinal related inflammatory factors, gut microbiota changes and metabolite analysis were also investigated to study the possible mechanism of PS23 FM. Results showed that HL PS23 FM was significantly lower body weight changed and lower feed intake changed , higher the symptom of bleeding stool by evaluating the scores of fecal traits and bleeding, higher colon tissue histologic score and fecal calprotectin express as an diagnostic marker of colitis. HHK PS23 FM and LL PS23 FM showed a tendency to reduce the fecal calprotectin express, and LL PS23 FM also relieved the symptom of bleeding stool compared with negative control (NC) group (P < 0.05). Analysis of the tight junction protein expression about intestinal epithelial permeability, All PS23 FM had higher occludin express compared with NC group, however claudin-1 was no significantly changed in all groups. The levels of intestinal related inflammatory factors about the changed of myeloperoxidase (MPO) activity also not different between PS23 FM and NC group, and the cytokine were no significantly changed except interferon gamma (IFN-γ) and interleukin-1 beta (IL-1β) in spleen. In the changes of intestinal microbiota and microbial metabolite about short chain fatty acid such as acetic acid (AA), propionic acid (PA), butyric acid (BA) in the cecum or colon, HH PS23 FM and LL PS23 FM were fewer opportunistic harmful bacteria including Clostridium perfringens, Enterobacteriaceae than the NC group. However the beneficial bacteria about Bifidobacterium. and Lactobacillus paracasei PS23 were no significantly changed in all groups. Also the content of short chain fatty acid in cecum or colon were no significantly differentness between PS23 FM and NC group. In summary, a novel fermented milk inoculated with a probiotic strain, Lactobacillus paracasei PS23, was developed. Addition of PS23 not only could improve the quality and storage stability of the product, it could also reduce the symptoms of colitis, and reduce the number of opportunistic pathogens in colitis. The safety of dose and the possible mechanism of PS23 FM in intestinal protection will still analyze.