TWI721082B - A novel lactic acid bacteria and its applications - Google Patents

Abstract

The invention relates to a new lactic acid bacteria (LAB), Lactobacillus fermentum PS150, and a bioactive protein produced by the LAB has advantageous effect in improving mood disorder, enhancing cognitive functions in brain and treating or preventing a neurodegenerative disease.

Description

Novel lactic acid bacteria and their applications

The present invention relates to a probiotic and a composition containing it. In particular, the present invention relates to a novel lactic acid bacteria and its specific use in improving affective disorders or neurological conditions and treating or preventing diseases related to neuronal apoptosis or neurodegeneration.

Fermented food contains various useful bacteria, including lactic acid bacteria (LAB). Various LAB strains are used to make fermented foods, including dairy products, bread, vegetables and other edible plant materials. LAB is a group of Gram-positive bacteria generally used for the production of fermented food. The benefits of LAB in diet and clinical applications have been extensively studied. LAB is used as a starter for food preservation due to the low pH during its fermentation activities and the effect of the fermentation products produced to inhibit the growth of spoilage bacteria. For this reason, LAB has been used to prepare many different foods (such as cheese, yogurt and other fermented dairy products) from dairy products. It has received a lot of attention that LAB has been found to exhibit properties that are valuable to humans and animals in digestion. In particular, it has been found that specific strains of Lactobacillus or Bifidobacterium can colonize the intestinal mucosa and help maintain the health of humans and animals. Anti-inflammatory activity and immunomodulatory activity are well-known characteristics of LAB. US 7875421 is about the use of the DNA sequence of a Lactobacillus johnsonii strain, specifically, it is used to clarify the interaction between a microorganism and its colonized host, and in addition to clarify the probiotic displayed by the strain Genome sequence that is the basis of bacterial properties. Mental illness, which is on the rise globally, has been ranked as the main cause of disability. Affective disorder is a common chronic mental illness. A person can be regarded as suffering from a mood disorder (also known as an affective disorder) if their emotions are characterized by intensity and/or type that continue to be extreme. Sleep disorder is one of the most common symptoms of patients with acute affective disorder episodes, and patients with affective disorder show a higher rate of sleep disorder than the general population even during remission (Psychiatr Clin North Am. December 2006; 29(4) ): 1009-32). One of the main physiological systems involved in the regulation of stress response is the hypothalamus-pituitary adrenal gland. Various stressful stimuli are known to activate the hypothalamus-pituitary-adrenal axis to release glucocorticoids in the circulation. Glucocorticoids are the key integrative hormone mediators of the body's response to stress and are presumed to play a role in the ability of stress to promote diseases. Lactobacillus has clearly documented its role in maintaining a healthy intestinal tract. However, the role of gastrointestinal microbial phase in the alleviation of the disease has received a lot of attention.

The present invention relates to a novel lactic acid bacteria (LAB) ( Lactobacillus fermentum PS150 (Lactobacillus fermentum PS150)), the biologically active protein produced by the LAB and its use in improving affective disorders or neurological conditions and treating or preventing neurodegenerative diseases Favorable effect. The present invention provides an isolated LAB, which is Lactobacillus fermentum PS150 (PS150) having any one of the nucleic acid sequences as shown in SEQ ID NOs: 1 to 3. The present invention also provides an isolated LAB, which has been deposited by the German Microbial Culture Collection (Deutsche Sammlung von Mikroorganismen und Zellkulturen) in accordance with the Budapest Treaty on June 6, 2016, and the deposit number given is DSMZ 32323. The present invention also provides a protein portion, which is obtained from the extracellular protein of PS150 cells and contains the EF-Tu protein having the amino acid sequence of SEQ ID NO:4. Unexpectedly, the present invention first determined that EF-Tu protein can effectively improve affective disorders, neurological conditions and neurodegenerative diseases. The present invention also provides a composition comprising PS150 cells, the EF-Tu protein of the present invention or the protein portion of the PS150 of the present invention, and an edible carrier or a pharmaceutically acceptable carrier as needed. The composition of the present invention may be in any form suitable for administration (specifically, oral administration). Such any form includes, for example, solid, semi-solid, liquid, and powder. The present invention also provides a use of the PS150 cell of the present invention or a composition containing the same for the manufacture of a preparation for improving affective disorders or neurological conditions, or treating or preventing diseases related to neuronal apoptosis or neurodegeneration . In one embodiment, the amount of such units in the form PS150 cells (cfu) in the range of from about ¹⁰⁵ to about ¹⁰¹³ colonies. The present invention further provides the use of a protein part of the present invention or a composition containing it for the manufacture of a preparation for improving affective disorders or neurological conditions, or treating or preventing diseases related to neuronal apoptosis or neurodegeneration . In one embodiment, the amount of the protein fraction is in the range of about 15 mg/kg to about 500 mg/kg. The present invention further provides the use of an EF-Tu protein or a composition containing it for the manufacture of a preparation for improving affective disorders or neurological conditions, or treating or preventing diseases related to neuronal apoptosis or neurodegeneration . Examples of affective disorders or neurological conditions include (but are not limited to) anxiety, depression, stress, and brain cognition.

在另一個態樣中，本發明提供一種蛋白質部分，其係從PS150細胞之細胞外蛋白質獲得，且包含具有SEQ ID NO:4之胺基酸序列或其功能片段之EF-Tu蛋白質。 該蛋白質部分為源自發酵乳桿菌PS150之細胞外蛋白質。單離發酵乳桿菌PS150之蛋白質部分並藉由例如無凝膠(gelfree)分離及逆相高效液相層析純化，且藉由MALDI-TOF質譜分析識別。本發明驚人地發現具有約55 kDa的分子量之經純化蛋白質部分，其隨後經識別為具有396個胺基酸殘基(SEQ ID NO:4)之延伸因子Tu，展示在改善情感障礙或神經病況及神經退化性疾病方面之療效。 發現編碼EF-Tu之基因(稱為tuf)為發酵乳桿菌PS150中的益生因子。賦予正面腦健康效應之該重要基因的發現係藉由自生物活性部分確定肽序列的同一性來達成，其中僅一個明顯的命中(hit)與發酵乳桿菌PS150之預測蛋白質組具有100%同一性及1e^-5 之BLASTP e-值。該命中為EF-Tu且因此，進行關於其分子演化之詳細研究以識別可參與蛋白質-蛋白質相互作用以發揮所觀察到健康效應之推定殘基。 EF-Tu蛋白質為原核延伸因子中之一者及該等延伸因子為藉由在核糖體之處轉譯合成新蛋白質之機制的一部分。出乎意料地，本發明首先確定EF-Tu蛋白質可有效改善情感障礙或神經病況及神經退化性疾病。因此，本發明提供一種用於治療或預防神經退化性疾病之方法，其包括對個體投與有效量之EF-Tu蛋白質。在一個實施例中，該EF-Tu蛋白質為來自PS150者。在另一個實施例中，該EF-Tu蛋白質的量在約15 μg/kg至約500 μg/kg範圍內。較佳地，該EF-Tu蛋白質的量在約15 μg/kg至約400 μg/kg、約15 μg/kg至約350 μg/kg、約15 μg/kg至約300 μg/kg、約15 μg/kg至約250 μg/kg、約15 μg/kg至約200 μg/kg、約15 μg/kg至約150 μg/kg、約15 μg/kg至約100 μg/kg、約15 μg/kg至約50 μg/kg、約50 μg/kg至約500 μg/kg、約100 μg/kg至約500 μg/kg、約150 μg/kg至約500 μg/kg、約200 μg/kg至約500 μg/kg、約250 μg/kg至約500 μg/kg、約300 μg/kg至約500 μg/kg、約350 μg/kg至約500 μg/kg或約400 μg/kg至約500 μg/kg範圍內。 殘基(40K、41G、42L、44K、46E、161E、185E、195D、327S、345E及360T)代表可為發酵乳桿菌PS150 EF-Tu在其腦健康效應方面的關鍵區分因子之推定結合位點。 本發明EF-Tu蛋白質可經由吲哚胺2,3-二加氧酶(IDO)的刺激減少造成神經退化病理的TNF-α。此最終減小色胺酸至犬尿胺酸之轉換率，並導致血清素產量增加。EF-Tu蛋白質亦減小血漿皮質酮濃度且因此可緩解焦慮、抑鬱及壓力。組合物及應用 在另一個態樣中，本發明提供一種組合物，其包含PS150細胞、本發明EF-Tu蛋白質或本發明PS150之蛋白質部分及視需要之食用載劑或醫藥上可接受之鹽。在本發明之組合物中，該等PS150細胞可呈可為活或非活的完整細菌之形式使用。較佳地，細菌細胞係呈活的有生命力的細胞形式存在。 本發明組合物可呈適合投與(特定言之經口投與)之任何形式。該任何形式包括(例如)固體、半固體、液體及粉末。 本發明組合物之實例為營養組合物，包括食品，及特定言之乳製品。 該組合物可為(例如)膠囊、錠劑、飲品、粉末或乳製品。視情況，可存在LAB之其他菌株。較佳地，本發明營養組合物為嬰兒食品、嬰兒奶粉配方或較大嬰兒配方。較佳地，本發明組合物為營養或醫藥產品、營養補充物或醫療食品。 本發明營養組合物亦包括食品補充物及功能性食品。「食品補充物」表示由常用於食品中之化合物製成之產品，但其係呈錠劑、粉末、膠囊、藥水(potion)之形式或通常與食品無關聯之任何其他形式，且對人健康具有有益效應。「功能性食品」為亦對人健康具有有益效應的食品。特定言之，食品補充物及功能性食品可具有抗疾病之生理效應(預防性或治癒性)。 若根據本發明之組合物為膳食補充物，則其可照原樣投與，可與適合的可飲用液體(諸如水、酸乳、牛奶或果汁)混合投與，或可與固體或液體食品混合投與。在該內文中，膳食補充物可呈錠劑、丸劑、膠囊、含片、顆粒、粉末、懸浮液、小袋、錠片、甜食、棒、糖漿之形式及對應之投藥形式(通常呈單位劑量形式)。較佳地，包含本發明組合物之膳食補充物係呈以製備膳食補充物之習知方法製造的錠劑、含片、膠囊或粉末之形式投與。 本文中所述的組合物可為醫藥上可接受之組合物，其可包含一或多種醫藥上可接受之載劑、賦形劑、黏合劑、稀釋劑或類似物。本發明組合物可經調配以用於各種投藥途徑，例如，藉由經口投與。彼等亦可與遞送媒劑(諸如在一些囊封技術中)組合提供。 對於經口投與而言，粉末、懸浮液、顆粒、錠劑、丸劑、膠囊、膠囊錠及膜衣錠係可接受的固體劑型。此等可例如藉由將一或多種本文所揭示化合物與至少一種添加劑(諸如澱粉或其他添加劑)混合而製備。適合的添加劑為蔗糖、乳糖、纖維素糖、甘露醇、麥芽糖醇、葡聚糖、澱粉、瓊脂、藻酸鹽、幾丁質、甲殼素、果膠、黃蓍膠、阿拉伯膠、明膠、膠原蛋白、酪蛋白、白蛋白、合成或半合成聚合物或甘油酯。視情況，口服劑型可包含其他成分以促進投與，諸如無活性稀釋劑、或潤滑劑(諸如硬脂酸鎂)、或防腐劑(諸如對羥基苯甲酸酯或山梨酸)、或抗氧化劑(諸如抗壞血酸、生育酚或半胱胺酸)、崩解劑、黏合劑、增稠劑、緩衝劑、甜味劑、矯味劑或芳香劑。錠劑及丸劑可進一步經相關技術中已知的適合包衣處理。 用於經口投與之液體劑型可呈醫藥上可接受之乳液、糖漿、酏劑、懸浮液及溶液之形式，其可包含無活性稀釋劑，諸如水。醫藥調配物及組合物可使用無菌液體(諸如(但不限於)油、水、醇及此等之組合)製備成液體懸浮液或溶液。可添加醫藥上適合的表面活性劑、懸浮劑、乳化劑，供用於經口或非經腸投與。 在另一個態樣中，本發明提供一種改善情感障礙或神經病況或治療或預防與神經元之細胞凋亡或神經退化有關的疾病之方法，其包括對個體投與有效量之本發明PS150細胞或含其之組合物。因此，本發明提供一種本發明PS150細胞或含其之組合物於製造用於改善情感障礙或神經病況或治療或預防與神經元之細胞凋亡或神經退化有關的疾病的製劑中之用途。在一個實施例中，PS150細胞的量在約10⁵ 至約10¹³ 個菌落形成單位(cfu)範圍內。較佳地，PS150細胞的量為約10⁶ 至約10¹³ cfu、10⁶ 至約10¹² cfu、約10⁶ 至約10¹¹ cfu、約10⁶ 至約10¹⁰ cfu、約10⁶ 至約10⁹ cfu、約10⁶ 至約10⁸ cfu、約10⁶ 至約10⁷ cfu、約10⁷ 至約10¹³ cfu、約10⁷ 至約10¹² cfu、約10⁷ 至約10¹¹ cfu、約10⁷ 至約10¹⁰ cfu、約10⁷ 至約10⁹ cfu、約10⁷ 至約10⁸ cfu、約10⁸ 至約10¹³ cfu、約10⁸ 至約10¹² cfu、約10⁸ 至約10¹¹ cfu、約10⁸ 至約10¹⁰ cfu、約10⁸ 至約10⁹ cfu、約10⁹ 至約10¹³ cfu、約10⁹ 至約10¹² cfu、約10⁹ 至約10¹¹ cfu或約10⁹ 至約10¹⁰ cfu。更佳地，PS150細胞的量為約10⁶ 至約10¹² cfu。 在另一個其他態樣中，本發明提供一種改善情感障礙或神經病況或治療或預防與神經元之細胞凋亡或神經退化有關的方法，其包括對個體投與有效量之本發明蛋白質部分或包含其之組合物。因此，本發明提供一種本發明蛋白質部分或包含其之組合物於製造用於改善情感障礙或神經病況或治療或預防與神經炎之細胞凋亡或神經退化有關的疾病的製劑中之用途。 在一個實施例中，該蛋白質部分的量在約15 mg/kg至約500 mg/kg範圍內。較佳地，該蛋白質部分的量在約15 mg/kg至約500 mg/kg、約15 mg/kg至約400 mg/kg、約15 mg/kg至約300 mg/kg、約15 mg/kg至約200 mg/kg、約15 mg/kg至約100 mg/kg、約40 mg/kg至約500 mg/kg、約40 mg/kg至約400 mg/kg、約40 mg/kg至約300 mg/kg、約40 mg/kg至約200 mg/kg、約40 mg/kg至約100 mg/kg、約80 mg/kg至約500 mg/kg、約80 mg/kg至約400 mg/kg、約80 mg/kg至約300 mg/kg、約80 mg/kg至約200 mg/kg、約80 mg/kg至約150 mg/kg、約100 mg/kg至約500 mg/kg、約100 mg/kg至約400 mg/kg、約100 mg/kg至約300 mg/kg或約100 mg/kg至約200 mg/kg範圍內。更佳地，該蛋白質部分的量在約40mg/kg至約300 mg/kg範圍內。 在另一個其他態樣中，本發明提供一種改善情感障礙、神經病況或治療或預防與神經元之細胞凋亡或神經退化有關的疾病之方法，其包括對個體投與有效量之本發明EF-Tu蛋白質。因此，本發明提供一種EF-Tu蛋白質或包含其之組合物於自製造用於改善情感障礙、神經病況或治療或預防與神經元之細胞凋亡或神經退化有關的疾病的製劑中之用途。在一個實施例中，該EF-Tu蛋白質為來自PS150者。在另一個實施例中，該EF-Tu蛋白質的給藥量在約15 μg/kg至約500 μg/kg；較佳地，約15 μg/kg至約400 μg/kg、約15 μg/kg至約300 μg/kg、約15 μg/kg至約200 μg/kg、約15 μg/kg至約100 μg/kg、約50 μg/kg至約500 μg/kg、約50 μg/kg至約400 μg/kg、約50 μg/kg至約300 μg/kg、約50 μg/kg至約200 μg/kg、約50 μg/kg至約100 μg/kg、約100 μg/kg至約500 μg/kg、約100 μg/kg至約400 μg/kg、約100 μg/kg至約300 μg/kg、約100 μg/kg至約200 μg/kg、約150 μg/kg至約500 μg/kg、約150 μg/kg至約400 μg/kg、約150 μg/kg至約300 μg/kg、約150 μg/kg至約200 μg/kg、約200 μg/kg至約500 μg/kg、約200 μg/kg至約400 μg/kg或約200 μg/kg至約300 μg/kg範圍內。 該情感障礙及神經病況包括(但不限於)焦慮、抑鬱、睡眠紊亂、壓力(諸如慢性溫和壓力)、認知功能減退、認知受損(包括輕度認知受損(MCI))、記憶衰退、一般回憶問題、認知障礙或神經退化性疾病(諸如阿滋海默氏症、杭丁頓氏舞蹈症、帕金森氏症、癡呆、肌萎縮性側索硬化症、中風及精神分裂症)。 該等與神經元之細胞凋亡或神經退化有關的疾病可選自由以下組成之群：中風(stroke)、阿滋海默氏症、杭丁頓氏舞蹈症、帕金森氏症、匹克症(Pick's disease)、庫賈氏症(Creutzfeldt-Jakob's disease)、帕金森-ALS-癡呆複合症、威爾遜氏症(Wilson's disease)、多發性硬化症、進行性核上性麻痹、與神經性疼痛有關的雙極症、皮質基底變性、精神分裂症、注意力不足過動症(ADHD)、癡呆、肌萎縮性側索硬化症、視網膜疾病、癲癇症、中風(apoplexy)、暫時性缺血性發作、心肌缺血、肌缺血、因有關於延長暫停血流至腦部的手術技術所引起的缺血、頭部損傷、脊髓損傷、缺氧及抑鬱。 本發明細胞、蛋白質及蛋白質部分可降低吲哚胺2,3-二加氧酶(IDO)的表現，減小色胺酸至犬尿胺酸之轉換率，增加血清素含量及減小血漿皮質酮含量。因此，本發明細胞、蛋白質及蛋白質部分可特定言之減緩焦慮、抑鬱及壓力。 藉由下文呈現的實例更詳細地描述本發明，依圖式簡單說明進行。然而，毋庸贅言地，此等實例係以說明本發明標的方式給出而不以任何方式構成對本發明的限制。實例 材料及方法 1.0 菌株及生長條件 發酵乳桿菌PS150係從馬來西亞理科大學工業技術學院生物工程技術部培養物保藏所(culture collection of the Division of Bioprocess Technology, School of Industrial Technology, Universiti Sains Malaysia)(Penang，馬來西亞)獲得，事先從當地購買的發酵香腸單離得。於使用之前在37℃下將細菌培養在de Mann Rogosa Sharpe (MRS)(Biomark，印度)液體培養基中20小時。該特定菌株為益生菌且已文獻證實其總體腦健康促進效應。2.0 DNA 製備 依Microbes Environ，第22卷，第3期，第214-222頁，2007單離並純化得發酵乳桿菌PS150之基因組DNA。於基因組定序之前將所獲得的經純化DNA儲存在-20℃下。3.0 基因組定序 使用兩種不同的下代定序平臺(其等為Pacbio RS II及Illumina HiSeq 2000)對發酵乳桿菌PS150之基因組DNA進行定序。於Pacbio平臺上，以C2-P4化學方案僅使用單一SMRT細胞於來自發酵乳桿菌PS150之~10 kb插入基因組DNA庫上進行SMRT定序。經預篩選之原始DNA序列資料具有489,633,442 bp，平均讀段長度為3257 bp。依以下標準篩選原始序列資料；最小子讀段長度為500 bp，最小聚合酶讀段品質為0.8及最小聚合酶讀段長度為100 bp。2.5 Mbp基因組之篩選後的資料庫具有約166x覆蓋率。出於染色體組裝目的，經篩選之資料接著接受HGAP法(Nat. Methods，第10卷，第6期，第563至569頁，2013) ，如實施於SMRT portalver 2.3.0.140893上。Dotplot分析證實其為環狀基因組，此與發酵乳桿菌染色體之其他公開的菌株一致。 於Illumina平臺上，使用來自發酵乳桿菌PS150之500 bp基因組DNA庫以進行配對端點(2x 100 bp)定序。按照製造商方案(Illumina，「An Introduction to Next-Generation Sequencing Technology」，Illumina. Inc.，2011)進行庫的製備。定序器產生總共20,522,464個讀段，此意指其平均覆蓋兩百萬個鹼基對基因組中各鹼基超過1000次。然而，需要篩選原始定序資料，使得僅品質序列資料通過以用於後續分析。使用FASTQC(可得於http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/)作為序列品質檢驗工具，將每塊序列品質標記為問題及因此每各配對端僅最後兩百萬個讀段用作每塊品質，因為此等讀段良好。接著，使此等讀段接受TRIMMOMATIC以篩選除去低品質鹼基。使用VELVET，選擇使用VELVETOPTIMISER (S. Gladman及T. Seemann，「VelvetOptimiser」，2012)確定的kmer，組裝經篩選之資料。所得組件具有262個重疊群。對此等重疊群排序並與藉由Pacbio HGAP法使用MAUVE(Genome Res.，第14卷，第7期，第1394至403頁，2004)生成的組件作比對。亦使用BWA軟體(Bioinformatics，第25卷，第14期，第1754-60頁，2009)將所篩選得的良好品質的序列資料與HGAP組裝資料作比對。使用SAMTOOLS操縱比對文件以產生已分類bam文件(Bioinformatics，第25卷，第16期，第2078至2079頁，2009)。使用ARTEMIS(Bioinformatics，第16卷，第10期，第944至945頁，2000)觀察映射的讀段。因為對於Illumina序列資料使用VELVET製得的組件包含比依HGAP法製得者更多的重疊群，故對於發酵乳桿菌染色體之後續分析僅使用經Pacbio組裝之基因組。4.0 基因組註解 使用軟體工具PROKKA以標註蛋白質編碼基因、tRNA、tmRNA、rRNA及重複區(諸如CRISPER) (Bioinformatics，第30卷，第14期，第2068-2069頁，2014)。藉由尋找EggNOG直系同源聚類(COG) (Nucleic Acids Res.，第42卷，第D1期，第D231至D239頁，2014)中最佳的命中進行每一蛋白質編碼基因之就基因本體(GO)而言的功能性分類。使用預設e值1針對於Pfam資料庫(Nucleic Acids Res.，第42卷，第D1期，第D222-D230頁，2014)識別預測蛋白中的蛋白質域。使用PHAST (Nucleic Acids Res.，第39卷增刊，第W347至W352頁，2011)預測基因組中之原噬菌體序列。在益生菌開發的內文中，抗生素抗性成為問題且因此使用針對於全面抗生素抗性資料庫之BLASTP (Antimicrob. Agents Chemother.，第57卷，第7期，第3348至3357頁，2013)進行可能的抗性基因的尋找。將BLASTP e臨限值設成小於1 e^-5 作為截止及分析與抗生素抗性資料庫中的任何基因具有大於60%同一性之命中。使用KEGG(京都基因及基因組百科全書(Kyoto Encyclopedia of Genes and Genomes))映射器(Mapper)以重建發酵乳桿菌PS150之少數受關注的代謝路徑。5.0 發酵乳桿菌基因組之比較 除了發酵乳桿菌PS150之外，有四個亦具有解碼之基因組的其他菌株。使用發酵乳桿菌PS150作為軟體BRIG (BMC Genomics，第12卷，第1期，第402頁，2011)的參考菌株，將其他四個已知菌株的基因組與其作比對。將自PHAST預測得的九個原噬菌體區串連成一個序列且亦將其與參考作比對。BLASTN同一性臨限值上限及下限分別設為90%及70%。6.0tuf 蛋白變異性分析 對於EF-Tu變異性分析而言，從235個細菌物種發現的所有蛋白質均用作用於計算Shannon熵(Bell Syst. Tech. J.，第27卷，1928年7月，第379至423頁，1948)的輸入。Shannon熵公式提供沿著蛋白質估計胺基酸序列之變異性的方法。對於給定的多蛋白質序列比對而言，每比對列號的Shannon熵(H)如下：

其中H為熵值， X為比對列號， N為胺基酸類型數目及間隔字元(21)*，p_i 為N中第i個字元之頻率， *表示間隔是否呈字元形式包含是視情況的。在該研究中，間隔被視為字元。 另外，在乳桿菌屬層級上，有25個具有可用基因組的不同物種。使用如上所述的相似方法得到此等25個物種中的tuf 基因。此等蛋白質亦用作用於Shannon熵計算的輸入。於計算熵之前，先使用MAFFT(Bioinformatics，第28卷，第23期，第3144至3446頁，2012)比對該等蛋白質並接著使用CLUSTALX (Bioinformatics，第23卷，第21期，第2947至2948頁，2007)以人工方式檢測。接著，依照發酵乳桿菌PS150 EF-Tu上發現彼等的任何地方來移除間隔。最後，使用經修整的比對，藉由使用訂製的R指令碼，來計算比對中每個位置的Shannon熵。7.0 細胞培養 7.1 細菌及上清液製備 發酵乳桿菌PS150為發酵肉腸的單離物。培養基係從馬來西亞理科大學微生物培養保藏所(Penang，馬來西亞)獲得。在抗凍劑(果膠)的存在下製備凍乾儲備培養物並儲存在乾燥及陰涼(-20℃)的地方。在用於實驗之前，連續地於無菌de Man、Rogosa及Sharpe(MRS)液體培養基(Hi-Media，Mumbai，印度)中活化凍乾培養物3次。在繼代培養期間，將培養物培養(10% v/v)於經滅菌MRS液體培養基中並在37℃下培養18小時。藉由以3500 g離心15分鐘(4℃)來獲得無細胞上清液(CFS)，用10M NaOH中和至7.0之pH，並接著藉由過濾通過0.22 mm過濾器(Sartorius Stedim，Göttingen，德國)滅菌並立刻使用或儲存在-20℃直至需要之時。7.2 人類神經母細胞瘤 SH-SY5Y 細胞培養 神經母細胞瘤細胞系SH-SY5Y細胞係購自韓國細胞系銀行(Korea Cell Line Bank，KCLB；Seoul，Korea)。將細胞維持在補充有10%經熱滅活之胎牛血清(FBS；Gibco Life Technologies Inc，Grand Island，NY，USA)、100 U/mL青黴素/鏈黴素(Gibco Life Technologies Inc，Grand Island，NY，USA)之高葡萄糖杜貝卡氏改良依格培養基(Dulbecco's Modified Eagle Medium，DMEM；Difco，Detroit，MI，USA)中。將培養物接種於含有經補充之培養基之燒瓶中並在37℃下維持於5% CO₂ 及95%空氣之增濕氛圍中。7.3 細胞培養物處理及細胞存活率分析 將SH-SY5Y細胞以1X10⁵ 個細胞/孔之接種密度接種於96-孔培養板中。將該研究中的所有細胞維持在10%血清培養基中直至地塞米松(dexamethasone)(Dex；Sigma-Aldrich，Steinheim，德國)處理。將細胞培養24小時以允許在處理之前附著至培養板。為研究細菌CFS之效應，將20 µL細菌CFS添加至含有100 µL含在無血清培養基中之經25 µM Dex處理之SH-SY5Y細胞之孔中。在處理48小時之後分析溴化3-(4,5-二甲基噻唑-2-基)-2,5-二苯基-四唑鎓(MTT；Sigma-Aldrich，Steinheim，德國)還原。 藉由MTT之定量比色試驗，如先前由Denizot及Lang (J Immunol Methods. 1986年5月22日；89(2): 271-7)所述，測量細胞存活率。MTT係用於評估神經元損傷。當活細胞吸收MTT時，其被細胞脫氫酶從黃色轉化為紫色甲臢(formazan)結晶。簡言之，在處理期結束時，將最終濃度為0.5 mg/ml之10% MTT-標記試劑添加至各孔並再將板置於在37℃下具有5% CO₂ 及95%空氣(v/v)之增濕培養箱中6小時的時間。然後，用二甲亞碸(DMSO；Sigma-Aldrich，Steinheim，德國)溶解不溶性甲臢。在570 nm下使用微盤讀取器(Thermo Scientific，Waltham，MA，US)測量MTT還原之比色測定。將經未發酵MRS處理之對照細胞視作100%存活率。8.0 活體內研究 8.1 活細胞及粗蛋白質製備 8.1.1 發酵乳桿菌 PS150 活細胞 發酵乳桿菌PS150係從馬來西亞理科大學工業技術學院生物工程技術部培養物保藏所(Penang，馬來西亞)獲得。在使用之前於37℃下在de Mann Rogosa Sharpe(MRS)(Biomark，印度)液體培養基中活化培養物20小時三次。使發酵乳桿菌PS150之過夜培養物接受於4℃下以8000 g離心30分鐘來得到細胞集結塊。於得到細胞集結塊後，使用磷酸鹽緩衝鹽水(1.0M，pH 7.4)清洗細胞團三次。添加10%(v/v)果膠溶液至該細胞沉澱，再在-20℃冷凍過夜。接著在-55℃下將該冷凍細胞團冷凍乾燥過夜。使用傾注平板法(pour plate method)測定所得經冷凍乾燥之細胞之CFU/g。此後，對於各個別大鼠，以9 log CFU/大鼠/天之劑量製備經冷凍乾燥之細胞。在使用之前，將該等製劑儲存於-20℃下。8.1.2 自無發酵乳桿菌 PS150 細胞上清液提取之粗蛋白質 使發酵乳桿菌PS150之過夜培養物接受於4℃下以8000 g離心30分鐘來得到無細胞上清液。收集該上清液並用3.0M氫氧化鈉(NaOH)調整pH至7.0。藉由在4℃下添加80% w/v硫酸銨至經中和之無細胞上清液中同時攪拌並接著在4℃下靜置過夜來沉澱粗蛋白質。使經中和之無細胞上清液之沉澱接受在4℃下以10000 g離心30分鐘。用無菌蒸餾水再懸浮所得的集結塊並藉由0.22 µM乙酸纖維素針筒過濾器過濾。接著使所得的粗蛋白質部分接受在-20℃下冷凍過夜，再在-55℃下冷凍乾燥過夜。對於各個別大鼠，以300 mg/kg之劑量製備經冷凍乾燥之粗蛋白質。在使用之前，將該等製劑儲存於-20℃下。8.2 動物 在馬來西亞理科大學(USM) Bertam校區高級醫療及牙科學院(Advanced Medical & Dental Institute)(IPPT)的經GLP認證之動物研究中心進行該研究。30隻雄性Wistar大鼠(7至8週齡，重180至200 g)係從USM主校區的動物研究及服務中心(Penang)獲得。在到達時，將該等大鼠 於標準實驗室條件(十二小時光照/十二小時黑夜循環(光照：0700-1900，溫度為19至25℃及濕度在30至70% RH內)下免打擾地(每個籠子3隻動物)圈養在各基於木屑墊料的標準聚丙烯鞋盒籠中。在整個實驗中，每兩天一次更換籠子墊料。使所接收到的動物接受一週的隔離以每天監測動物中疾病或蝨的存在。一旦確保該等動物無任何疾病或蝨，則使其等接受一週的適應，然後進行慢性溫和壓力方案及處理。在隔離及適應期間，所有大鼠可隨意獲取食物及水。每週測量並監測動物的體重。8.3 慢性溫和壓力方案 將該等大鼠分成五個組(每組6隻大鼠(n=6))，即，未處理的對照、陰性對照、陽性對照、活細胞處理組及蛋白質處理組。除了未處理的對照組之外，使所有大鼠接受慢性溫和壓力方案(表1)。在未處理的對照組中，所有動物不作任何處理。在陰性對照組中，所有動物每天餵食0.2 ml磷酸鹽緩衝鹽水(1.0M，pH 7.4)。在陽性對照組中，所有動物每天餵食以0.45 mg/kg之劑量懸浮在0.2 ml磷酸鹽緩衝鹽水(1.0M，pH 7.4)中之阿普唑侖(alprazolam)粉末。在活細胞處理組中，所有動物每天餵食9 log CFU/大鼠/天之活發酵乳桿菌PS150。最後，在蛋白質處理組中，所有動物每天餵食如前面所述製得的經冷凍乾燥之蛋白質(300 mg/kg大鼠/天)。使用具有2.25 mm球之1.5英吋的20號不鏽鋼餵食針進行口腔強飼。慢性溫和壓力方案由如圖1所顯示的若干壓力期及非壓力期組成且整週進行時間表排定，總共28天。 表1.慢性溫和壓力方案之每週時間表。

¹ 空瓶：水瓶空一小時以限制動物獲取水² 再填充水：再填充水瓶以允許動物獲取水³ 傾斜籠子：讓籠子傾斜45°⁴ 無壓力：該段時間期間未執行壓力源⁵ 用砂更換鋸屑：用砂更換木屑墊料。⁶ 空籠：該段時間期間動物的籠子為空的，不含木屑。⁷ 約束：將動物置於軟包線網中以約束其運動。⁸ 社交互動：在該段時間期間允許所有大鼠在較大籠子中互動。⁹ 食物剝奪：從籠子移走食物¹⁰ 限制食物：在該段時間期間，對動物提供最少量的食物¹¹ 外來房間：將動物轉移至新的籠子¹² 重複冷壓力：將10塊小冰塊置於動物籠子中且允許溶化30分鐘¹³ 紅光：將籠子中的動物置於配備紅光的房間¹⁴ 濕籠：用無菌水潤濕木屑墊料¹⁵ 捕食者的聲音：將動物暴露於捕食者的聲音錄音¹⁶ 外來房間：將籠子中的動物從原來的房間置換至新的房間¹⁷ 閃光燈：將動物暴露於使用電子裝置播放的閃光燈視訊¹⁸ 水剝奪：從籠子移走水瓶以限制大鼠獲取水8.4 行為評估 在28天慢性溫和壓力干預之後使所有大鼠接受行為評估。所評估的行為評估包括莫氏水迷宮(MWM)、高架十字迷宮(EPM)、T-迷宮、強迫游泳測試(FST)、空場測試(OFT)及物件識別。所有實驗均在僅稀疏光線照射的安靜且黑暗的房間內進行。8.4.1 莫氏水迷宮 (MWM) 設備設置 所使用的MWM之直徑為210 cm，其中池高度為51 cm，其中具有非反射性內表面。將該迷宮置於具有設在迷宮頂部的視訊攝影機之室內以捕捉並記錄動物行為。將水迷宮充滿溫度在19至22℃範圍內的水以避免大鼠受到溫度衝擊(體溫過低或體溫過高)。然後，將具有10至12 cm直徑之無色且透明之圓形物件或平臺(稱為逃避平臺)浸沒水表面下方1至2 cm並確保當大鼠爬上其時避免動物傾翻之穩定性。逃避潛伏期評估 MWM的起始位置標在東、西及東南，同時逃避平臺浸沒在東北。連續四天每天使用此三起始位置進行試驗。簡言之，將該等動物輕柔地置於第一起始位置上並允許自由地游泳以尋找逃避平臺。給每隻動物最長兩分鐘以進行逃避平臺的尋找。在莫氏測試之後於使彼等返回至其各自的籠子之前，用乾淨的毛巾徹底擦乾所有大鼠。每隻動物定位於逃避處所花的時間(逃避潛伏期(秒))表示為每組重複六次(n=6)之平均值±平均值標準誤差。8.4.2 高架十字迷宮 (EPM) 設備設置 高架十字迷宮(EPM)由四個臂(兩個開放臂沒有壁及兩個臂圍封40 cm高、50 cm長及15 cm寬的壁)組成。迷宮的各臂係與具有70 cm高的結實金屬腿連接。將EPM置於具有明亮照明的房間內，接近房間的中央。兩開放及閉合臂之照明程度維持相似。將視訊攝影機設在高架十字迷宮上方以捕捉並記錄動物行為。評估 將動物從其籠子中取出並置於EPM的中央(即，開放臂與閉合臂的結點)，背向迷宮面對壁。給每隻動物5分鐘以自由地在EPM上探索。在整個評估中，於抓取下一隻動物之前，用70%酒精清潔並乾燥EPM。觀察開放臂進入次數及花在開放臂中的時間，計時並記錄。記錄開放臂進入次數及花在開放臂上的持續時間且表示為每組重複六次(n=6)之平均值±平均值標準誤差。8.4.3 強迫游泳測試 設備設置 在深度為68 cm及直徑為40 cm之黑色不透光游泳圓筒中進行強迫游泳測試(FST)。將該圓筒填滿水至30 cm之深度，上方留一些空間以防止大鼠逃避。視訊攝影機設在游泳圓筒上方以在測試期間捕捉並記錄動物的行為。評估 將每隻動物輕柔地置於游泳圓筒的水中並給定5分鐘以在游泳圓筒中自由地游泳。於整個測試中觀察並記錄動物行為。於整個評估中，在FST之後於使彼等返回至其各自的籠子之前，用乾淨的毛巾徹底擦乾所有大鼠。記錄大鼠保持不動之持續時間並接著表示為每組重複六次(n=6)之平均值±平均值標準誤差。8.4.4 空場測試 設備設置 使用置於提供有明亮光照的實驗房間的中央之具有32 cm高、38 cm寬及52 cm長之尺寸之不透光矩形箱進行空場測試。將視訊攝影機設在矩形箱上方以捕捉動物行為。評估 將每隻動物輕柔地從其籠子轉移至OFT中並允許自由地在箱內容探索。於5分鐘的空場測試期間觀察大鼠之探索及保持不動的持續時間。於整個評估中在每次測試的時間間隔期間用70%酒精清潔箱。探索及保持不動的持續時間表示為每組重複六次(n=6)之平均值±平均值標準誤差。8.4.5 新物件識別測試 設備設置及評估 在具有32 cm高、38 cm寬及52 cm長之尺寸並置於實驗房間中央之不透光矩形箱中進行新物件識別測試。該行為評估涉及2個階段，即，熟悉階段及測試階段。在熟悉階段期間，將兩個相同物件(形狀、大小及重量相同)置於矩形箱中。將每隻動物置於矩形箱中並給定2分鐘以在箱中自由地探索。然而，在測試階段期間，一個舊物件改由具有形狀之新物件(重量及大小相似)替代。同樣地，將每隻動物置於矩形箱中並給定2分鐘以在箱中自由地探索。於整個實驗中將視訊攝影機設在矩形箱上方以捕捉兩個階段中動物的行為。在每次測試的間隔期間，用液態皂、水清潔該箱並乾燥。熟悉階段與測試階段間之間所給定的時間間隔為一小時。探索新物件所花的時間依以下公式表示為識別指數： 識別指數=(新物件上所花的時間)/(舊物件+新物件上所花的總時間) X 100%(Cogn Process，2012，13(2):93-110) 資料表示為每組重複六次(n=6)之平均值±平均值標準誤差。8.4.6 統計分析 藉由IBM SPSS Statistics 20.0(IBM Co.，Armonk，NY，USA)統計分析所有資料。進行單向方差分析以分析組平均值之間的統計差異。統計顯著性水平設為α=0.05，及藉由Tukey檢驗評估多個平均值的比較。除非另外指出，否則所有資料表示為三次獨立實驗(n=6)之平均值。8.5 血液收集及器官提取 在實驗結束時，於使用二氧化碳窒息將大鼠殺死之後不久，透過心臟穿刺收集血液樣本，並轉移至塗覆EDTA之管中。在血液收集之後不久，依斷頭法移去腦。將用於ELISA及過氧化氫分析之腦組織樣本保持於冰冷PBS中。8.5.1 生化分析 腦樣本之均質化 於切除之後不久處理儲存在4℃的1x磷酸鹽緩衝鹽水(pH 7.4)中的所提取大鼠腦。將腦組織樣本切成3個區(小腦、大腦及海馬區)。接著在冰冷條件下以70至100 Hz均質化腦組織樣本切片10分鐘。在使用之前將所有經均質化之樣本儲存在-20℃。血液樣本 於室溫下培養於抗凝血EDTA管中收集的血液10至20分鐘，再以3000 rpm離心20分鐘。收集上清液作為血漿樣本並儲存於1.5 ml微量離心管中，保持在4℃下以用於即刻分析。在使用之前，將額外的血漿樣本保持在-20℃。酶聯免疫吸附分析 (ELISA) 進行酶聯免疫吸附分析(ELISA)以量化與神經生成相關聯的若干細胞介素、關鍵神經遞質及生物標記。依製造商說明書(R&D System Inc, USA)進行ELISA。 簡言之，將10 µl經均質化之腦組織或血漿加載至孔底部上而不接觸孔壁。添加40 µl樣本稀釋緩衝液以稀釋該等樣本。在37℃下培養經稀釋之樣本30分鐘。此後，丟棄該等樣本並用所提供的清洗緩衝液清洗五次。於隨後，將50 µl與辣根過氧化物酶(HRP)共軛的抗體添加至各孔。在37℃下再培養該混合物30分鐘。接著，丟棄該混合物並用所提供的清洗緩衝液清洗五次。於隨後，將50 µl色原溶液A及50 µl色原溶液B添加至各孔並藉由輕柔振盪混合。接著在37℃下培養該混合物15分鐘。在該步驟期間應避免光照曝露。接下來，添加50 µl終止溶液至各孔以終止反應。最後，使用Thermo Multiskan-GO ELISA板讀取器讀取在450 nm下之吸光度O.D.。使用如上所提及的相同程序生成標準曲線。腦中之過氧化氫 (H₂ O₂ ) 測量 使用Amplex Red分析套組(Molecular Probes Inc.，Eugene，OR，USA)依製造商方案評估腦勻漿樣本中的過氧化氫含量。9.0 識別來自發酵乳桿菌 PS150 之蛋白質 遵循所有此等陽性發現，單離發酵乳桿菌PS150之蛋白質部分並進一步識別。藉由硫酸銨沉澱預純化該蛋白質部分並接著基於三步驟層析程序回收。9.1 蛋白質提取 如前面所述(8.1.2)提取粗蛋白質部分。接著使用75%丙酮使該粗蛋白質部分進行脫鹽，並藉由離心(7000 g，30分鐘，4℃)移除鹽，重複三次。將所收集的集結塊溶解於去離子水中。採用Bradford分析法(Bio-Rad，Hercules，CA，USA)，使用BSA作為標準品，估測蛋白質含量。9.2 無凝膠分離法 使用包含預調配的HEPES操作緩衝液及Tris乙酸鹽樣本緩衝液(AMR Incorporated)之GELFREE™ 8100分離系統(Fractionation System) (Expedeon，San Diego，CA，USA)，依製造商之說明書，依據分子質量，將蛋白質分為多個部分。將發酵乳桿菌PS150蛋白質溶解於樣本緩衝液中，並加載至含有5% Tris-乙酸鹽卡管之個別加載隔間中，其設計在於分離質量在60至300 kDa範圍內之蛋白質。儀器依預定的時間間隔自動暫停，並用吸移管移除液體部份。接著重新啟動該順序，並繼續該製程，以便依據分子大小收集下一個部份。重複該製程直到收集到所有部份。 在回收12個個別部份之後，收集該等部份，使用丙酮洗滌兩次，並經過真空濃縮機移除丙酮。將各個別部份標準化為相同蛋白質含量(2.0 µg/mL)，並在細胞培養處理中，使用前面述於7.3中的方法進行測試。9.3. 1DE 凝膠分析 為估計各個別蛋白質部分之分子大小，一旦從GELFREE™ 8100分離系統回收得12個個別部分之後，則使用10% SDS-PAGE(Bio-Rad)進行標準1DE。在110 V下於Tris/甘胺酸/SDS操作緩衝液(Bio-Rad)中，使用標準電泳設備(Bio-Rad)，進行蛋白質分離80分鐘。使用Coomassie Brilliant Blue蛋白質凝膠染色劑(Molecular Probes，Eugene，OR，USA)染色凝膠中的蛋白質及使用ChemiDoc XRS Camera and Quantity One 1D分析軟體(Bio-Rad)觀察蛋白質譜帶。9.4 逆相高效液相層析(RP-HPLC) 蛋白質部分之最後純化步驟係藉由逆相高效液相層析(RP-HPLC)於HiTrap Q HP柱上進行。在0.8 mL/min之流速下用溶劑A(25 mM Tris HCl)進行洗脫10分鐘。接著，在70分鐘內進行從0%變為100%溶劑B(含有1M NaCl之25 mM Tris HCl)之線性梯度。個別地收集洗脫峰並如所述(7.3)檢查抗地塞米松之神經保護活性。9.5 藉由 orbitrap LC/MS 識別蛋白質部分 在馬來西亞理科大學高級分析毒理學服務機構中心(Institutional Centre for Advanced Analytical Toxicology Services, Universiti Sains Malaysia)實現肽識別。將所關注的來自RP-HPLC之部分(0.2 mg)溶解於變性緩衝液(6M胍-HCl/25 mM碳酸氫銨，pH 8.5)中並接著添加1 mg/ml DTT/25 mM碳酸氫銨(新製)至蛋白質溶液並在55℃下培養30分鐘。接著添加1 mg/mL碘乙醯胺/25 mM碳酸氫銨(新製)，然後用鋁箔覆蓋並在55℃下培養15分鐘。然後，使用具有15 kDa之截止分子量之旋轉柱(spin-column)將經還原並烷基化之蛋白質樣本與25 mM碳酸氫銨緩衝交換3次(每次30分鐘)。接著將胰蛋白酶添加至該樣本並在37℃下培養18小時。添加甲酸至該樣本，然後使其接受冷凍乾燥。使樣本接受MALDI-TOF質譜分析並識別質量峰。產生所有可能的片段並識別其對應之分子量及肽序列。於隨後，使用PEAKS studio第6.0版進行資料分析及使用於發酵乳桿菌PS150基因組中編碼之所有蛋白質之功能註解資料庫來進行潛在的生物活性肽序列之選擇。9.6 對接模擬 使用Swiss-MODEL(Schwede 2003)建構發酵乳桿菌PS150之延伸因子tu(EF-Tu)之3D模型。來自嗜熱菌(Thermus thermophilus ) (PDB ID:2C77)之EF-Tu用作模板，因為以下兩個原因：1)蛋白質資料庫(PDB)中不存在可用的乳桿菌衍生之EF-Tu晶體結構及2)嗜熱菌衍生之EF-Tu顯示與資料庫中發酵乳桿菌PS150之EF-Tu的最高序列同一性(74.81%)。從蛋白質資料庫(PDB)獲得干擾素-γ (IFNγ)及干擾素-γ受體(IFNγR；PDB ID:1FYH；2.04A之解析度)之晶體結構。另外，IFNγ39(衍生自IFNγ之已知可抑制IFNγR反應性之頭39個胺基酸之合成肽)亦衍生自IFNγ之晶體結構以充作該研究中的陽性對照。分子對接 -IFNγ 至 IFNγ 受體 在Cluspro 2.0:蛋白質-蛋白質對接作業提交之前移除所有水分子及配位體。在Cluspro 2.0:蛋白質-蛋白質對接(抗體模式)中，IFNγ及IFNγR分別作為配位體及受體提交。進一步用BIOVIA Discovery Studio 4.5 (Humphrey等人 1996)分析所產生的所有構型以評估其均方根差(RMSD)值。具有最低結合自由能、最低RMSD值及最密聚簇之構型用作用於進一步分析中之比較之基準對照。使用Ligplot+v1.4.5(Laskowski及Swindells，2011)以觀察IFNγ與所選構型之IFNγR上的相互作用殘基(TYR49、TRP82、GLU101、HIS205、VAL206及TRP207；Randal及Kossiakoff，2001)間之相互作用。分子對接 -IFNγ39 至 IFNγ 受體 在Cluspro 2.0:蛋白質-蛋白質對接作業提交(Kozakov等人，2013)之前移除所有水分子及配位體。在Cluspro 2.0:蛋白質-蛋白質對接(抗體模式)中，IFNγ39及IFNγR分別作為配位體及受體提交。具有最低結合自由能及最密聚簇之構型用於與如前面完成之基準對照作比較。使用Ligplot+ v1.4.5(Laskowski及Swindells，2011)以觀察IFNγ39與所選構型之IFNγR上的相互作用殘基(TYR49、TRP82、GLU101、HIS205、VAL206及TRP207；Randal及Kossiakoff，2001)間之相互作用。分子對接 -EF-Tu 至 IFNγ 受體 在Cluspro 2.0:蛋白質-蛋白質對接作業提交之前移除所有水分子及配位體。在Cluspro 2.0:蛋白質-蛋白質對接(抗體模式)中，EF-Tu及IFNγR分別呈配位體及受體形式提交。具有最低結合自由能及最密聚簇之構型用於與如前面實施之基準對照作比較。使用Ligplot+ v1.4.5(Laskowski及Swindells，2011，J. Chem. Inf. Model，51:2778-2786)以觀察EF-Tu與所選構型IFNγR上的相互作用殘基(TYR49、TRP82、GLU101、HIS205、VAL206及TRP207)間之相互作用。實例 1 識別發酵乳桿菌 PS150 及染色體一般基因組特徵 使用API 50 CHL套組(bioMerieux，France)研究用於本發明中之PS150之糖利用，及結果顯示於表2中。發酵測試指示PS150具有類似於發酵乳桿菌之生物化學性質。 表2：發酵測試^a 之結果

將發酵乳桿菌PS150培養於特異性人工培養基中。藉由經PCR擴增之DNA片段之直接定序來分析來自發酵乳桿菌PS150之16S rRNA 及pheS 基因。使用相關技術已知的方法進行基因組DNA提取、16S rDNA及pheS DNA之經PCR介導之擴增、PCR產物之純化及經純化之PCR產物之定序。 發酵乳桿菌PS150之16S rDNA序列(SEQ ID NO:5)

pheS基因序列(SEQ ID NO:6)

將所得序列放入線上由美國國家生物技術資訊中心(National Center for Biotechnology Information，NCBI)(http://www.ncbi.nlm.nih.gov/)提供的比對軟體中，人工比對並與生物體之代表性16S rDNA序列進行比較。就比較而言，亦從線上由NCBI提供的資料庫獲得16S rDNA序列。作為該分析的結果，下表3列出16S rDNA序列相較於發酵乳桿菌PS150之16S rDNA序列顯示最高相似性值之其等生物體。 表3 16S rDNA序列相較於發酵乳桿菌PS150之16S rDNA序列顯示最高類似相似性值之生物體

發酵乳桿菌PS150具有2,238,401 bp之環狀染色體及其GC含量為51%。預測總共2,281個基因，其係由2,206個蛋白質編碼基因、59個tRNA、15個rRNA及一個tmRNA(表4)組成。另外，發現兩個成簇的規律間隔的短迴文重複(CRISPR)區，其可提供抗外源遺傳成分之免疫性。2,206個蛋白質編碼基因中之465個被預測為不具有歸屬於彼等之功能的假想蛋白。基於直系同源聚類(COG)功能分類，2005個蛋白質編碼基因(即， 91%之所有預測編碼序列(CDS))可指派給COG功能類別。2005個蛋白質編碼基因中有62%屬於五個主要COG類別：447個CDS在類別S(功能未知)中，364個CDS在類別L(複製、重組及修復)中，162個CDS在類別E(胺基酸運輸及代謝)中，140個CDS在類別J(轉譯、核糖體結構及生體合成)中及136個CDS在類別K(轉錄)中。此外，識別九個原噬菌體區且有趣的是，其中四個經預測為完整原噬菌體。對抗生素抗性基因的研究顯示六個候選抗性基因，包括30S核糖體蛋白S12、麥芽糖O-乙醯基轉移酶、半乳糖苷O-乙醯基轉移酶、推定的乙醯基轉移酶、氯黴素乙醯基轉移酶及胸苷酸合成酶。 表4：發酵乳桿菌PS150之預測基因組特徵列表。

與其他發酵乳桿菌菌株之染色體比較 在細菌菌株中的染色體比較可揭示對於某些菌株為獨特的基因組特徵。將發酵乳桿菌PS150之染色體與另外四個菌株發酵乳桿菌3872、發酵乳桿菌CECT 5716、發酵乳桿菌IFO 3956及發酵乳桿菌F6之完整基因組進行比較。將發酵乳桿菌PS150之預測原噬菌體串連並包含於比較中。如所預期，諸如在原噬菌體區中，新基因組為明顯的差異。 進行研究以找出介於菌株3872與PS150之間之非重疊區，此揭示98個蛋白質編碼序列對於後一菌株為獨特。98個序列中有35個編碼假想蛋白，此意指彼等具有未知的功能或可能錯誤地被預測為基因。剩餘的63個基因具有預測之蛋白質產物，其包含三種存於跨距小於15 kb之基因簇中的推定醣基轉移酶。實例 2 針對焦慮、抑鬱及壓力之緩解的分析 在慢性溫和壓力方案4週之後進行對大鼠行為的分析。高架十字迷宮為廣泛使用的齲齒動物行為分析且其已被驗證可評估藥理藥劑及類固醇激素之抗焦慮效應。用於實驗中之高架十字迷宮由四個臂(兩個開放臂及兩個閉合臂) 組成並自地面抬高50 cm高。將大鼠置於迷宮四個臂的結點，面對開放臂，及記錄各臂中之進入/持續時間5分鐘。開放臂活動(持續時間及/或進入)的增加反映抗焦慮行為。經發酵乳桿菌PS150及蛋白質部分處理之組比陰性對照組大鼠進入開放臂的數目明顯更多(P＜0.05；圖1(a))，然而，未處理的對照組、陽性對照組(經抗抑鬱藥處理)、經發酵乳桿菌PS150處理之組及經蛋白質部分處理之組間之差異性係不明顯的(圖1(a))。此外，經發酵乳桿菌PS150及蛋白質部分處理的花在閉合臂上的時間與花在開放臂上的時間之比相較於陰性對照組明顯更低(P＜0.05；圖1(b))，然而，在未處理對照組、陽性對照組(經抗抑鬱藥處理)、經發酵乳桿菌PS150處理之組及經蛋白質部分處理之組中觀察到不明顯的差異(圖1(b))。結果指示發酵乳桿菌PS150及發酵乳桿菌PS150之蛋白質部分可將慢性溫和壓力引起之焦慮行為標準化，恢復回至最初的非焦慮狀態。另外，發酵乳桿菌PS150及蛋白質部分組亦顯示與阿普唑侖之抗焦慮效應相似的抗焦慮效應，此指示與市售抗抑鬱藥相似的效應。 空場測試廣泛用於評估探索行為及經驗證用於測量與焦慮有關的行為。該程序由使大鼠經歷由圍墻阻止從其逃避的未知環境組成。經發酵乳桿菌PS150及發酵乳桿菌PS150之蛋白質部分處理之組相較於陰性對照組顯著增加大鼠之探索活動的持續時間(P＜0.05；圖2)。未處理的對照組、陽性對照組(經抗抑鬱藥處理)、經發酵乳桿菌PS150處理之組及經蛋白質部分處理之組間大鼠之探索及保持不動活性不顯著的差異 (圖2)。大鼠之探索行為的增加指示焦慮狀態較少，而保持不動指示大鼠之焦慮行為。慢性溫和壓力引起大鼠之焦慮，但發酵乳桿菌PS150及發酵乳桿菌PS150之蛋白質部分可將慢性溫和壓力引起之焦慮行為標準化，恢復回至最初的非焦慮狀態。另外，發酵乳桿菌PS150及蛋白質部分組亦顯示與阿普唑侖之抗焦慮效應相似的抗焦慮效應，此指示與市售抗抑鬱藥相似的效應。 強迫游泳測試為用於研究齲齒動物之似抑鬱行為之最常用試驗之一。強迫游泳測試係基於以下假設：當將動物置於填充水的容器中時，其將先努力逃避但最終將呈現保持不動，保持不動可被視為反映行為絕望的量度。在強迫游泳測試中，游泳圓筒填充30 cm深的水。藉由評分300 s測試之每5 s時間期的主要行為來進行大鼠之抑鬱評估。相比陰性對照組，經發酵乳桿菌PS150及蛋白質部分處理之組花在保持不動的時間顯著更少(P＜0.05；圖3)。未處理的對照組、陽性對照組(經抗抑鬱藥處理)、經發酵乳桿菌PS150及蛋白質部分處理之組間的大鼠花在保持不動的時間之無顯著差異 (圖3)。自發酵乳桿菌PS150、蛋白質部分及抗抑鬱藥觀察到相似的效應，其中將慢性溫和壓力引起之似抑鬱行為標準化至未處理的對照組中之相同程度。發酵乳桿菌PS150及發酵乳桿菌PS150之蛋白質部分可將慢性溫和壓力引起之抑鬱行為標準化，恢復回至最初的非抑鬱狀態。另外，發酵乳桿菌PS150及蛋白質部分亦顯示與阿普唑侖之抗抑鬱效應相似的抗抑鬱效應，此指示與市售抗抑鬱藥相似的效應。 相較於陰性對照組及陽性對照組，發酵乳桿菌PS150及蛋白質部分組中之血漿皮質酮含量顯著更低(P＜0.05；圖4)。暴露於重複或長時間壓力源引起慢性壓力。慢性壓力最終引起HPA軸失調，此導致血漿皮質酮含量增加。皮質酮含量之升高指示與焦慮及抑鬱有關的行為。吾人的資料顯示發酵乳桿菌PS150及發酵乳桿菌PS150之蛋白質部分將慢性溫和壓力引起之焦慮、抑鬱及壓力行為標準化，恢復回至最初的非壓力狀態，並可甚至發揮比阿普唑侖(市售抗抑鬱藥)之病理效應更佳的病理效應。 相較於陰性對照組，經發酵乳桿菌PS150處理之組及經蛋白質部分處理之組中血漿TNF α與介白素-10之比顯著更低(P＜0.05；圖5)。TNF α為促發炎性細胞介素，但介白素-10為抗炎性細胞介素。抑鬱患者通常比彼等非抑鬱者具有更高的TNF α含量但更低的IL-10含量，且TNF α:IL-10比與抑鬱症狀之嚴重度顯著相關。吾人之本發明發現結果表明發酵乳桿菌PS150及發酵乳桿菌PS150之蛋白質部分將慢性溫和壓力引起之焦慮、抑鬱及壓力行為標準化，恢復回至最初的非壓力狀態，極有可能係透過細胞介素之調節。發酵乳桿菌PS150及發酵乳桿菌PS150之蛋白質部分亦具有與阿普唑侖相似的效應，此指示如市售抗抑鬱藥之相似壓力減小效應。實例 3 針對腦認知之改善及神經元細胞凋亡之保護的分析 已有充分的文獻記載壓力通常對記憶及其他認知功能施加負面影響。為評估認知表現，使用莫氏水迷宮以評估記憶保持，及使用新物件識別測試以評估學習及識別記憶。 莫氏水迷宮係一廣泛使用的任務且為行為生理學家及藥理學家接受用於評估及比較齲齒動物之學習及記憶。該實驗中使用空間學習(最基本的莫氏水迷宮程序)。其背後的概念在於動物必須學會使用遠端提示以在從槽周邊附近的不同隨機位置開始時找到通往隱藏平臺之直接路徑。水迷宮的直徑為210 cm並具有為51 cm高的具有非反射性內表面之側面。逃避潛伏期(EL)為動物從起始象限移動直至在目標象限中找到隱藏平臺所花的時間。經發酵乳桿菌PS150及蛋白質部分處理之組相較於陰性對照組顯著縮短逃避潛伏期，然而陰性對照組相較於其他組顯著增加逃避潛伏期(P＜0.05；圖6(a))。未處理的對照組、陽性對照組、經發酵乳桿菌PS150處理之組及經蛋白質部分處理之組間觀測到不顯著的差異 (圖6(a))。相較於陰性對照大鼠，經發酵乳桿菌PS150及蛋白質部分處理之組在新物件識別測試中顯著具有更高的識別指數(圖6(b))，且效應類似於陽性對照大鼠(抗抑鬱藥組)及未處理的對照(無壓力)大鼠之效應(P＜0.05)。吾人之本發明發現結果指示發酵乳桿菌PS150及蛋白質部分發酵乳桿菌PS150將大鼠之慢性溫和壓力引起之認知功能受損標準化(明顯地比陰性對照組更佳)。 TNF-α藉由活化下視丘-垂體-腎上腺(HPA)軸，刺激吲哚胺2,3-二加氧酶(IDO)(其導致色胺酸耗盡)，藉由神經元之免疫介導破壞，或麩胺酸之神經毒性釋放，造成神經退化性疾病之致病。IDO為使色胺酸降解成犬尿胺酸之酵素，此涉及神經退化性疾病之病理生理學。若相較於陰性對照及陽性對照，發酵乳桿菌PS150及蛋白質部分組中血漿TNF α含量與介白素-10含量之比率較低，則亦觀測到血漿IDO含量顯著減低(P＜0.05；圖7(a))。若相較於陰性對照組，發酵乳桿菌PS150及蛋白質部分組中偵測到較低的血漿IDO濃度，則亦觀測到較高的血漿色胺酸含量(P＜0.05；圖7(b))。吾人的資料顯示相較於陰性對照組，發酵乳桿菌PS150及蛋白質部分組中較少量的色胺酸被降解成犬尿胺酸，同時血漿犬尿胺酸含量亦顯著較低(P＜0.05；圖7(c))。吾人的本發明發現結果指示發酵乳桿菌PS150及發酵乳桿菌PS150之蛋白質部分：(a)透過減少犬尿胺酸生成阻止神經退化(明顯比陰性對照組更佳)及(b)使得神經退化恢復回至正常狀態且與抗抑鬱藥一樣良好(與陽性對照/阿普唑侖及未處理的對照組類似的效應)。 另一方面，色胺酸亦為血清素之前驅物。亦收集腦樣本以用於測量血清素含量。血清素為參與神經脈衝之傳遞及調節神經元之間的信號之神經傳遞質。吾人的本發明發現結果顯示相較於陰性對照組，發酵乳桿菌PS150及蛋白質部分組中之腦血清素濃度顯著提高(P＜0.05；圖8)。結果與發酵乳桿菌PS150及蛋白質部分組中所觀測到的血漿色胺酸含量之增加相一致。吾人之本發明發現結果指示發酵乳桿菌PS150及發酵乳桿菌PS150之蛋白質部分：(a)恢復由於大鼠之慢性溫和壓力而減少之腦中神經元之傳遞，從而導致更佳的認知(明顯地比陰性對照組更佳)及(b)發揮比市售抗抑鬱藥更佳的療效(明顯地比陽性對照/阿普唑侖組更佳)。 於壓力下，在大鼠腦中觀測到變化的細胞凋亡特徵。單胺氧化酶(MAO)為控制腦中神經傳遞質之含量之粒線體酵素，其中MAO-A係透過卡斯蛋白酶-3之活化及反應性氧物質之生成在細胞凋亡中發揮作用。過氧化氫因其作為凋亡細胞死亡之介體的角色而通常從凋亡細胞偵測到。根據慢性壓力方案，相較於陰性對照大鼠，投與PS150之大鼠顯示較低的腦MAO-A濃度(圖9(a))(P＜0.05)。相較於陰性對照組，在投與PS150或蛋白質部分之大鼠中觀測到較低的腦過氧化氫濃度之情況下，細胞凋亡特徵之緩解係顯然的(圖9(b)，P＜0.05)。 行為及腦評估均指示PS150及PS150之蛋白質部分預防大鼠之由於慢性溫和壓力引起之腦損傷，具有與市售抗抑鬱藥相似的效能，獲得標準化空間學習、長期記憶及認知功能，及預防神經退化及神經元細胞凋亡。實例 4 識別來自發酵乳桿菌 PS150 之基因及蛋白質 遵循所有此等陽性發現結果，進一步分離發酵乳桿菌PS150之蛋白質部分，並識別。藉由硫酸銨沉澱預純化蛋白質部分並接著基於三步驟層析程序回收。蛋白質部分藉由在HiTrap Q HP柱上之逆相高效液相層析(RP-HPLC)之最終純化步驟揭示在5%、15%、20%、25%、30%、50%、55%、60%及90%之溶劑B(含有1M NaCl之25 mM Tris HCl)時洗脫的9個主峰(圖10)。個別地收集洗脫峰(分別稱為部分A、B、C、D、E、F、G、H及I)並檢查抗地塞米松之神經保護活性。 細胞凋亡為可藉由多種外部刺激(諸如壓力之生物標記，皮質酮)增強之正常生理程式化細胞死亡。地塞米松(合成皮質酮)係用於評估發酵乳桿菌PS150之蛋白質部分之神經保護活性。資料顯示僅部分A具有最盛行之神經保護作用(圖11)。未經地塞米松處理之陽性對照細胞視為100%存活率。相較於兩種經未發酵介質處理之細胞(存在或不存在地塞米松)，發酵乳桿菌PS150之蛋白質部分顯示顯著細胞保護效應。 經純化之部分A顯示於SDS-PAGE上之具有約55 kDa之估算分子量之單譜帶(圖12)。對於蛋白質識別而言，使自HPLC收集之經純化之蛋白質接受MALDI-TOF質譜分析。識別整個胰蛋白酶消化過程中所觀測到的質量峰。產生所有可能的片段並識別其對應之分子量及肽序列。隨後，使用PEAKS studio第6.0版進行資料分析及使用於發酵乳桿菌PS150之基因組中編碼之所有蛋白質之功能註解資料庫進行潛在的生物活性肽序列之選擇。 就發酵乳桿菌PS150之註解基因組而言，將自質譜分析得到的所有肽序轉化成細菌之經核苷酸轉譯之蛋白質序列。在所轉化的150個肽當中，僅發現兩個肽與來自細胞之蛋白質相同，且兩個肽序列與發酵乳桿菌PS150之蛋白質中之一者100%相匹配。就該匹配而言，蛋白質之序列經確定為SEQ ID NO:4之序列。該蛋白質係經回譯至具有SEQ ID NO:7之序列的核苷酸序列，以得到其基因序列。 SEQ ID NO:7

該生物活性蛋白質經識別為延伸因子Tu，其具有396個胺基酸殘基。實際上，該分子為於細胞質中之蛋白質合成中發揮重要作用之鳥苷核苷酸結合蛋白質。然而，已有充分的文獻記載微生物之不同隔室中存在EF-Tu。已顯示EF-Tu分子(最初被認為係受限於細菌之細胞質)亦與大腸桿菌膜相關聯。最新的研究顯示EF-Tu為可從細胞釋放之與包膜相關聯之蛋白質。 延伸因子Tu能夠觸發大鼠之免疫調節反應。在該研究中，經蛋白質部分處理之大鼠誘導抗炎性免疫反應(TNF-α與IL-10之比減小；P＜0.05；圖5)。藉由減低TNF-α(此透過刺激吲哚胺2,3-二加氧酶(IDO)造成神經退化性發病)，血漿IDO濃度亦減小(P＜0.05；圖7(a))。此最終使得色胺酸轉化成犬尿胺酸之轉化率減小(P＜0.05；圖7(b)及(c))，並導致血清素生成增加。延伸因子Tu之免疫調節活性亦使得血漿皮質酮含量減小，此造成焦慮、抑鬱及壓力之緩解。 總言之，吾人的資料證實該由發酵乳桿菌PS150產生之具有如上述SEQ ID NO:4之序列之生物活性蛋白質為免疫調節化合物，吾人的資料支持除了提高認知以外對於焦慮、抑鬱及壓力之緩解，均與更健康的腦功能有關。在此吾人提出益生菌生物活性化合物(諸如由發酵乳桿菌PS150產生之延伸因子)可發揮腦健康效益之證據。實例 5 發酵乳桿菌 PS150 之延伸因子 Tu 之蛋白質變異性分析 Shannon熵係用於估算EF-Tu蛋白質變異性。在21個字元系統中，H在0(即高度保守，僅單一字元在比對柱中)至4.392(即，所有21個字元平等地存在)範圍內。針對來自乳桿菌屬之跨整個蛋白質長度具有蛋白質之三個關鍵域(GTP EFTU、GTP EFTU D2及GTP EFTU D3)之235個細菌物種及25個細菌物種估算熵。 高變異性殘基被歸類為彼等具有超過平均熵臨限值加兩個標準偏差之熵者。使用平均值加兩個標準偏差之值，因為對於大多數分佈而言，大於其之值被視為極端值。因此，第一域GTP EFTU具有最多數目之可變殘基，其次是最後一個域GTP EFTU D2，再次是GTP EFTU D3。特別受關注的是，當考慮所有235個細菌物種時具高變異性，但當僅包含來自乳桿菌屬之物種時不具高變異性之殘基。此處為符合所提及標準之殘基的列表：40K、41G、42L、44K、46E、161E、185E、195D、327S、345E及360T。當考慮所有235個細菌物種時，所有此等殘基具有高於2.63之熵，此意指各殘基顯示至少60%之理論最大變異性(4.392)之變異性。此等殘基表示可為發酵乳桿菌PS150 EF-Tu在其腦健康效應方面之關鍵辨別因子之推定結合位點。 蛋白質之結合位點通常係暴露的或彼等將處在構型變化中。為評估在前一段中偵測到的高變異性殘基中任一者是否位於EF-Tu之3D蛋白質結構之暴露部分上，建構該蛋白質之同源模型。將高變異性殘基映射於同源模型化3D EF-Tu蛋白質質結構上揭示殘基40K、41G、42L、44K、46E及327S位於分子之表面上。有趣地，注意到殘基40K、41G、42L、44K及46E僅於殘基43A及45A之處連續標點。事實上，殘基43A及45A兩者亦顯示熵值分別為2.004及2.578之高變異性，但此等值低於截止臨限值。雖然殘基327S亦具高變異性，但其位置遠離殘基40至46。其他殘基161E、185E、195D、345E及360T包埋於EF-Tu分子本身中。實例 6 PS150 之睡眠促進效應 對BALB/c小鼠連續14天每天一次經口投與10⁹ CFU/小鼠發酵乳桿菌PS150。以僅提供PBS對動物平行測試對照小鼠。對陽性對照組提供DIPH-HCL(鹽酸苯海拉明，20 mg/kg)作為陽性對照。在十四天實驗期間每天測量體重(BW)。在第十四天，於胃內(i.g.)投與PBS、PS150及DIPH-HCL 30分鐘之後，經腹膜內(i.p.)注射戊巴比妥鈉(50 mg/kg)至每隻小鼠，以測試戊巴比妥誘導之睡眠的持續時間。觀測每隻小鼠之睡眠之潛伏期及持續時間。戊巴比妥注射與翻正反射(righting reflex)損失之間的實耗時間記錄為睡眠潛伏期，及翻正反射之損失與恢復之間的實耗時間記錄為睡眠的持續時間。圖13顯示在戊巴比妥誘導之睡眠測試中PS150對睡眠潛伏期(a)及睡眠持續時間(b)的影響。結果顯示PS150可縮短睡眠潛伏期時間及延長睡眠持續時間。The present invention surprisingly discovered a novel lactic acid bacteria (LAB) (Lactobacillus fermentum PS150) and the beneficial effects of the biologically active protein produced by the LAB in improving affective disorders or neurological conditions and treating or preventing neurodegenerative diseases. Definitions Terms that are not clearly defined in this article should be understood by those who are familiar with the art according to the disclosure and content. However, as used in this specification, unless otherwise specified, the following terms have the meanings indicated according to the following conventions. The abbreviations listed in this article are as follows: CDS: coding sequence; COG: orthologous clustering; CRISPR: clusters of regularly spaced short palindrome repeats; EF-Tu: elongation factor Tu; GO: gene ontology; HGAP: Hierarchical genome assembly method. Groups of items connected by the conjunction "and" should not be interpreted as requiring each and every item of these items to exist in the group, but should be interpreted as "and/or" unless expressly stated otherwise. Similarly, a group of items connected by the conjunction "or" should not be interpreted as requiring mutual exclusivity within the group, but should also be interpreted as "and/or" unless expressly stated otherwise. In addition, although the items, elements or components of the present invention can be described or claimed in a singular form, it is also envisaged that the plural number falls within the singular category, unless the limitation of the singular number is clearly stated. As used herein, the terms "a", "an" and "the" should be understood to mean both the singular and the plural, unless expressly stated otherwise. Therefore, "one", "one" and "the" (and its grammatical changes where appropriate) refer to one or more. The term "probiotics" was recognized in the state of the art at the time of patent application as microorganisms that confer health benefits to the host when administered in an appropriate amount. Probiotic microorganisms must meet several requirements related to lack of toxicity, vitality, adhesion, and beneficial effects. These probiotics are characterized by strain dependence, even among bacteria of the same species. As used herein, the term "pharmaceutically acceptable" refers to compounds, substances, compositions, and/or dosage forms that are suitable for use in contact with the tissues of individuals (human or non-human animals) within the scope of reasonable medical judgment. There is no excessive toxicity, irritation, allergic reaction, or other problems or complications, in line with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in standard medical texts. The term "edible carrier" refers to a compound, substance, composition and/or dosage form suitable for use in contact with the tissue of an individual. Each carrier must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. As used herein, the term "effective amount" refers to the range of reasonable medical judgments of each strain in the composition that is high enough to significantly positively regulate the condition to be treated but low enough to avoid serious side effects (within reasonable benefits/risks). Compare below) the amount of colony forming units (cfu). As used herein, the term "disorder" can be used interchangeably with "disease" or "condition". As used herein, the term "affective disorder" is described by DSM-IV-TR and is a type of disease that describes severe changes in emotion. Disorders under affective disorders include: major depression, bipolar disorder (euphoric mania, excessive activity, excessive self-boasting, unrealistic optimism), persistent depression (long-lasting low-grade depression), circulation Sexual affective disorder (the mild form of bipolar disorder) and SAD (seasonal affective disorder). As used herein, the term "neurological condition" is a type of disease that describes the effects of nerve damage and disease on brain function in terms of behavior, memory, or cognition. Diseases in neurological conditions include (but are not limited to) stress (such as chronic mild stress), cognitive decline, cognitive impairment (including mild cognitive impairment (MCI)), memory decline, general memory problems, cognitive impairment or neurological Degenerative diseases (such as Alzheimer's disease, Huntington's disease, Parkinson's disease, dementia, amyotrophic lateral sclerosis, stroke, or schizophrenia) disease). The term "treatment" should be understood to mean the alleviation or reduction of at least one sign, symptom, indication or effect of a particular disease or condition. As used herein, "prevention" should be understood as limiting, reducing the rate or extent of the onset of a disease or condition, or inhibiting the development of at least one sign or symptom of a disease or condition. As used herein, the term "subject" is any animal that can benefit from self-administration of a compound or composition as disclosed herein. In some embodiments, the individual is a mammal, for example, a human, a primate, a dog, a cat, a horse, a cow, a pig, a rodent (such as, for example, a rat or a mouse). Generally, the mammal is a human. Lactobacillus In one aspect, the present invention provides an isolated LAB, which is Lactobacillus fermentum PS150 (PS150) having any one of the nucleic acid sequences as shown in SEQ ID NOs: 1 to 3. The sequences of SEQ ID NO: 1 to 3 are listed below. <img wi="485"he="128"file="IMG-2/Draw/02_image001.jpg"img-format="jpg"><imgwi="481"he="242"file="IMG-2/Draw/02_image003.jpg"img-format="jpg"><imgwi="481"he="261"file="IMG-2/Draw/02_image005.jpg"img-format="jpg"> Lactobacillus fermentum PS150 has a circular chromosome of 2,238,401 bp and its GC content is 51%. It is predicted that there are 2,281 genes in total, which are composed of 2,206 protein-coding genes, 59 tRNAs, 15 rRNAs, and one tmRNA. In addition, two clusters of regularly spaced short palindrome repeat (CRISPR) regions were found, which can provide immunity against foreign genetic components. It is predicted that 465 of the 2,206 protein-coding genes are hypothetical proteins that do not have their own functions. Based on the orthologous clustering (COG) functional classification, 2005 protein coding genes (ie, 91% of all predicted coding sequences (CDS)) can be classified into the COG functional category. Of the 2005 protein-coding genes, 62% belong to the five main COG categories: 447 CDS are in category S (unknown function), 364 CDS are in category L (replication, recombination, and repair), and 162 CDS are in category E (Amino acid transport and metabolism), 140 CDS are in category J (translation, ribosomal structure and biosynthesis) and 136 CDS are in category K (transcription). In addition, there are nine identified prophage regions and interestingly, four of them are predicted to be complete prophage. Studies on antibiotic resistance genes revealed six candidate resistance genes, including 30S ribosomal protein S12, maltose O-acetyltransferase, galactosyl O-acetyltransferase, putative acetyltransferase, Chloramphenicol acetyltransferase and thymidylate synthase. Comparison of chromosomes in bacterial strains can reveal genomic characteristics that are unique to certain strains. It shows that the LAB of the present invention has 98 unique protein coding sequences. 35 of the 98 sequences encode hypothetical proteins, which means that they have unknown functions or may be mistakenly predicted as genes. The remaining 63 genes have predicted protein products, which contain three putative glycosyltransferases found in gene clusters spanning less than 15 kb. Representative sequences among the 98 sequences are listed in SEQ ID NOs: 1 to 3. In one embodiment, the present invention provides an isolated and purified lactic acid bacteria, which is Lactobacillus fermentum PS150 (PS150), which was obtained from the German Collection of Microorganisms (Deutsche Sammlung von Mikroorganismen und Zellkulturen) on June 6, 2016 Deposited in accordance with the Budapest Treaty, and the deposit number given is DSMZ 32323. Lactobacillus fermentum PS150 is a probiotic strain isolated from fermented meat intestines. PS150 can effectively improve affective disorders or neurological conditions, and treat or prevent neurodegenerative diseases. The protein portion of PS150 and the EF-Tu protein purified therefrom In another aspect, the present invention provides the EF-Tu protein of PS150, which comprises the amino acid sequence of SEQ ID NO:4. SEQ ID NO: 4

In another aspect, the present invention provides a protein portion obtained from the extracellular protein of PS150 cells and comprising the EF-Tu protein having the amino acid sequence of SEQ ID NO: 4 or a functional fragment thereof. The protein part is an extracellular protein derived from Lactobacillus fermentum PS150. The protein fraction of Lactobacillus fermentum PS150 is purified by, for example, gelfree separation and reverse phase high performance liquid chromatography, and is identified by MALDI-TOF mass spectrometry analysis. The present invention surprisingly discovered that a purified protein fraction with a molecular weight of about 55 kDa, which was subsequently identified as an elongation factor Tu with 396 amino acid residues (SEQ ID NO: 4), was shown to improve affective disorders or neurological conditions And the curative effect of neurodegenerative diseases. The gene encoding EF-Tu (called tuf) was found to be a prebiotic factor in Lactobacillus fermentum PS150. The discovery of this important gene conferring a positive brain health effect was achieved by determining the identity of the peptide sequence from the biologically active part. Only one obvious hit was 100% identical to the predicted proteome of Lactobacillus fermentum PS150 And the BLASTP e-value of ^{1e -5.} The hit was EF-Tu and therefore, detailed studies on its molecular evolution were performed to identify putative residues that can participate in protein-protein interactions to exert the observed health effects. EF-Tu protein is one of the prokaryotic elongation factors and these elongation factors are part of the mechanism of synthesizing new proteins by translation at the ribosome. Unexpectedly, the present invention first determined that EF-Tu protein can effectively improve affective disorders or neurological conditions and neurodegenerative diseases. Therefore, the present invention provides a method for treating or preventing neurodegenerative diseases, which comprises administering an effective amount of EF-Tu protein to an individual. In one embodiment, the EF-Tu protein is derived from PS150. In another embodiment, the amount of the EF-Tu protein is in the range of about 15 μg/kg to about 500 μg/kg. Preferably, the amount of the EF-Tu protein is about 15 μg/kg to about 400 μg/kg, about 15 μg/kg to about 350 μg/kg, about 15 μg/kg to about 300 μg/kg, about 15 μg/kg to about 250 μg/kg, about 15 μg/kg to about 200 μg/kg, about 15 μg/kg to about 150 μg/kg, about 15 μg/kg to about 100 μg/kg, about 15 μg/kg kg to about 50 μg/kg, about 50 μg/kg to about 500 μg/kg, about 100 μg/kg to about 500 μg/kg, about 150 μg/kg to about 500 μg/kg, about 200 μg/kg to About 500 μg/kg, about 250 μg/kg to about 500 μg/kg, about 300 μg/kg to about 500 μg/kg, about 350 μg/kg to about 500 μg/kg, or about 400 μg/kg to about 500 Within the range of μg/kg. Residues (40K, 41G, 42L, 44K, 46E, 161E, 185E, 195D, 327S, 345E, and 360T) represent the putative binding site that can be the key distinguishing factor of Lactobacillus fermentum PS150 EF-Tu in its brain health effects . The EF-Tu protein of the present invention can reduce TNF-α that causes neurodegenerative pathology through stimulation of indoleamine 2,3-dioxygenase (IDO). This ultimately reduces the conversion rate of tryptophan to kynurenine and leads to an increase in serotonin production. EF-Tu protein also reduces the plasma corticosterone concentration and therefore can relieve anxiety, depression and stress. Composition and application In another aspect, the present invention provides a composition comprising PS150 cells, the EF-Tu protein of the present invention or the protein portion of the PS150 of the present invention, and optionally an edible carrier or a pharmaceutically acceptable salt . In the composition of the present invention, the PS150 cells can be used in the form of intact bacteria that can be live or non-viable. Preferably, the bacterial cell line exists in the form of living, viable cells. The composition of the present invention may be in any form suitable for administration (specifically, oral administration). Such any form includes, for example, solid, semi-solid, liquid, and powder. Examples of the composition of the present invention are nutritional compositions, including foods, and specifically dairy products. The composition can be, for example, a capsule, lozenge, drink, powder, or dairy product. Depending on the circumstances, other strains of LAB may be present. Preferably, the nutritional composition of the present invention is infant food, infant formula or follow-up formula. Preferably, the composition of the present invention is a nutritional or medicinal product, nutritional supplement or medical food. The nutritional composition of the present invention also includes food supplements and functional foods. "Food supplement" means a product made from compounds commonly used in food, but in the form of tablets, powders, capsules, potions, or any other form that is not normally associated with food, and is healthy for people Has beneficial effects. "Functional foods" are foods that also have beneficial effects on human health. In particular, food supplements and functional foods can have anti-disease physiological effects (preventive or curative). If the composition according to the present invention is a dietary supplement, it can be administered as it is, can be mixed and administered with a suitable drinkable liquid (such as water, yogurt, milk or juice), or can be mixed with solid or liquid food Contribute. In this context, the dietary supplement can be in the form of lozenges, pills, capsules, lozenges, granules, powders, suspensions, sachets, lozenges, sweets, bars, syrups and corresponding administration forms (usually in unit dose form) ). Preferably, the dietary supplement containing the composition of the present invention is administered in the form of a lozenge, lozenge, capsule or powder manufactured by a conventional method for preparing dietary supplements. The composition described herein may be a pharmaceutically acceptable composition, which may include one or more pharmaceutically acceptable carriers, excipients, binders, diluents, or the like. The composition of the present invention can be formulated for various administration routes, for example, by oral administration. They can also be provided in combination with delivery vehicles (such as in some encapsulation techniques). For oral administration, powders, suspensions, granules, lozenges, pills, capsules, capsules and film-coated tablets are acceptable solid dosage forms. These can be prepared, for example, by mixing one or more of the compounds disclosed herein with at least one additive, such as starch or other additives. Suitable additives are sucrose, lactose, cellulose sugar, mannitol, maltitol, dextran, starch, agar, alginate, chitin, chitin, pectin, tragacanth, gum arabic, gelatin, collagen Protein, casein, albumin, synthetic or semi-synthetic polymers or glycerides. Optionally, oral dosage forms may contain other ingredients to facilitate administration, such as inactive diluents, or lubricants (such as magnesium stearate), or preservatives (such as parabens or sorbic acid), or antioxidants (Such as ascorbic acid, tocopherol or cysteine), disintegrants, binders, thickeners, buffers, sweeteners, flavors or fragrances. Tablets and pills can be further treated with suitable coatings known in the related art. The liquid dosage form for oral administration may be in the form of pharmaceutically acceptable emulsions, syrups, elixirs, suspensions and solutions, which may contain inactive diluents such as water. Pharmaceutical formulations and compositions can be prepared into liquid suspensions or solutions using sterile liquids (such as (but not limited to) oil, water, alcohol, and combinations thereof). Pharmaceutically suitable surfactants, suspending agents, emulsifiers can be added for oral or parenteral administration. In another aspect, the present invention provides a method for improving affective disorders or neurological conditions or treating or preventing diseases related to neuronal apoptosis or neurodegeneration, which comprises administering to an individual an effective amount of PS150 cells of the present invention Or a composition containing it. Therefore, the present invention provides a use of the PS150 cell of the present invention or a composition containing the same in the manufacture of a preparation for improving affective disorders or neurological conditions or treating or preventing diseases related to neuronal apoptosis or neurodegeneration. In one embodiment, the amount of cell forming units within PS150 (cfu) in the range of from about ¹⁰⁵ to about ¹⁰¹³ colonies. Preferably, the amount of PS150 cells is about 10 ⁶ to about 10 ¹³ cfu, 10 ⁶ to about 10 ¹² cfu, about 10 ⁶ to about 10 ¹¹ cfu, about 10 ⁶ to about 10 ¹⁰ cfu, about 10 ⁶ to about 10 ⁹ cfu, about 10 ⁶ to about 10 ⁸ cfu, about 10 ⁶ to about 10 ⁷ cfu, about 10 ⁷ to about 10 ¹³ cfu, about 10 ⁷ to about 10 ¹² cfu, about 10 ⁷ to about 10 ¹¹ cfu, about 10 ⁷ to about 10 ¹⁰ cfu, about 10 ⁷ to about 10 ⁹ cfu, about 10 ⁷ to about 10 ⁸ cfu, about 10 ⁸ to about 10 ¹³ cfu, about 10 ⁸ to about 10 ¹² cfu, about 10 ⁸ to about 10 ¹¹ cfu, about 10 ⁸ to about 10 ¹⁰ cfu, about 10 ⁸ to about 10 ⁹ cfu, about 10 ⁹ to about 10 ¹³ cfu, about 10 ⁹ to about 10 ¹² cfu, about 10 ⁹ to about 10 ¹¹ cfu, or about 10 ⁹ To about 10 ¹⁰ cfu. More preferably, the amount of PS150 cells is about 10 ⁶ to about 10 ¹² cfu. In another aspect, the present invention provides a method for improving affective disorders or neurological conditions or treating or preventing neuronal apoptosis or neurodegeneration, which comprises administering to an individual an effective amount of the protein portion of the present invention or Compositions containing it. Therefore, the present invention provides the use of a protein part of the present invention or a composition containing it in the manufacture of a preparation for improving affective disorders or neurological conditions, or treating or preventing diseases related to neuritis, cell apoptosis or neurodegeneration. In one embodiment, the amount of the protein fraction is in the range of about 15 mg/kg to about 500 mg/kg. Preferably, the amount of the protein portion is about 15 mg/kg to about 500 mg/kg, about 15 mg/kg to about 400 mg/kg, about 15 mg/kg to about 300 mg/kg, about 15 mg/kg kg to about 200 mg/kg, about 15 mg/kg to about 100 mg/kg, about 40 mg/kg to about 500 mg/kg, about 40 mg/kg to about 400 mg/kg, about 40 mg/kg to About 300 mg/kg, about 40 mg/kg to about 200 mg/kg, about 40 mg/kg to about 100 mg/kg, about 80 mg/kg to about 500 mg/kg, about 80 mg/kg to about 400 mg/kg, about 80 mg/kg to about 300 mg/kg, about 80 mg/kg to about 200 mg/kg, about 80 mg/kg to about 150 mg/kg, about 100 mg/kg to about 500 mg/kg kg, about 100 mg/kg to about 400 mg/kg, about 100 mg/kg to about 300 mg/kg, or about 100 mg/kg to about 200 mg/kg. More preferably, the amount of the protein fraction is in the range of about 40 mg/kg to about 300 mg/kg. In another aspect, the present invention provides a method for improving affective disorders, neurological conditions, or treating or preventing diseases related to neuronal apoptosis or neurodegeneration, which comprises administering to an individual an effective amount of the EF of the present invention -Tu protein. Therefore, the present invention provides the use of an EF-Tu protein or a composition containing it in self-manufactured preparations for improving affective disorders, neurological conditions, or treating or preventing diseases related to neuronal apoptosis or neurodegeneration. In one embodiment, the EF-Tu protein is derived from PS150. In another embodiment, the dosage of the EF-Tu protein is about 15 μg/kg to about 500 μg/kg; preferably, about 15 μg/kg to about 400 μg/kg, about 15 μg/kg To about 300 μg/kg, about 15 μg/kg to about 200 μg/kg, about 15 μg/kg to about 100 μg/kg, about 50 μg/kg to about 500 μg/kg, about 50 μg/kg to about 400 μg/kg, about 50 μg/kg to about 300 μg/kg, about 50 μg/kg to about 200 μg/kg, about 50 μg/kg to about 100 μg/kg, about 100 μg/kg to about 500 μg /kg, about 100 μg/kg to about 400 μg/kg, about 100 μg/kg to about 300 μg/kg, about 100 μg/kg to about 200 μg/kg, about 150 μg/kg to about 500 μg/kg , About 150 μg/kg to about 400 μg/kg, about 150 μg/kg to about 300 μg/kg, about 150 μg/kg to about 200 μg/kg, about 200 μg/kg to about 500 μg/kg, about Within the range of 200 μg/kg to about 400 μg/kg or about 200 μg/kg to about 300 μg/kg. The affective disorders and neurological conditions include (but are not limited to) anxiety, depression, sleep disorders, stress (such as chronic mild stress), cognitive decline, cognitive impairment (including mild cognitive impairment (MCI)), memory decline, general Memory problems, cognitive impairment, or neurodegenerative diseases (such as Alzheimer's disease, Huntington's disease, Parkinson's disease, dementia, amyotrophic lateral sclerosis, stroke, and schizophrenia). These diseases related to neuronal apoptosis or neurodegeneration can be selected from the group consisting of stroke, Alzheimer's disease, Huntington's disease, Parkinson's disease, Pick's disease ( Pick's disease), Creutzfeldt-Jakob's disease, Parkinson's-ALS-Dementia complex, Wilson's disease, multiple sclerosis, progressive supranuclear palsy, neuropathic pain-related double Polar disorders, cortical basal degeneration, schizophrenia, attention deficit hyperactivity disorder (ADHD), dementia, amyotrophic lateral sclerosis, retinal disease, epilepsy, stroke (apoplexy), temporary ischemic attack, myocardium Ischemia, muscle ischemia, ischemia caused by surgical techniques that prolong blood flow to the brain, head injury, spinal cord injury, hypoxia, and depression. The cell, protein and protein part of the present invention can reduce the performance of indoleamine 2,3-dioxygenase (IDO), reduce the conversion rate of tryptophan to kynurenine, increase serotonin content and reduce plasma cortex Ketone content. Therefore, the cells, proteins and protein fractions of the present invention can specifically alleviate anxiety, depression and stress. The present invention will be described in more detail through the examples presented below, and will be described briefly in accordance with the drawings. However, it goes without saying that these examples are given to illustrate the object of the present invention and do not constitute a limitation to the present invention in any way. Example materials and methods 1.0 strain and growth conditions Lactobacillus fermentum PS150 was obtained from the culture collection of the Division of Bioprocess Technology, School of Industrial Technology, Universiti Sains Malaysia (Penang). , Malaysia), obtained in advance from fermented sausages purchased locally. The bacteria were cultured in de Mann Rogosa Sharpe (MRS) (Biomark, India) liquid medium at 37°C for 20 hours before use. This particular strain is a probiotic and has been documented to prove its overall brain health promotion effect. 2.0 DNA preparation According to Microbes Environ, Volume 22, Issue 3, Pages 214-222, 2007, the genomic DNA of Lactobacillus fermentum PS150 was isolated and purified. The purified DNA obtained was stored at -20°C before genome sequencing. 3.0 Genome Sequencing Two different next-generation sequencing platforms (such as Pacbio RS II and Illumina HiSeq 2000) were used to sequence the genomic DNA of Lactobacillus fermentum PS150. On the Pacbio platform, only a single SMRT cell was used to perform SMRT sequencing on the ~10 kb inserted genomic DNA library from Lactobacillus fermentum PS150 using the C2-P4 chemical protocol. The pre-screened original DNA sequence data has 489,633,442 bp, and the average read length is 3257 bp. Screen the original sequence data according to the following criteria; the minimum sub-read length is 500 bp, the minimum polymerase read quality is 0.8, and the minimum polymerase read length is 100 bp. The 2.5 Mbp genome screened database has a coverage rate of approximately 166x. For the purpose of chromosome assembly, the screened data were then subjected to the HGAP method (Nat. Methods, Vol. 10, No. 6, pages 563 to 569, 2013), as implemented on SMRT portalver 2.3.0.140893. Dotplot analysis confirmed that it is a circular genome, which is consistent with other published strains of Lactobacillus fermentum chromosomes. On the Illumina platform, a 500 bp genomic DNA library from Lactobacillus fermentum PS150 was used for paired endpoint (2x 100 bp) sequencing. The library was prepared according to the manufacturer's protocol (Illumina, "An Introduction to Next-Generation Sequencing Technology", Illumina. Inc., 2011). The sequencer generates a total of 20,522,464 reads, which means that it covers an average of more than 1,000 bases in the genome of two million base pairs. However, the original sequencing data needs to be screened so that only the quality sequence data is passed for subsequent analysis. Use FASTQC (available at http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/) as a sequence quality inspection tool to mark the quality of each sequence as a problem and therefore only the last two million for each pair Reads are used as the quality of each block, because these reads are good. Then, these reads are subjected to TRIMMOMATIC to screen and remove low-quality bases. Use VELVET, choose to use the kmer determined by VELVETOPTIMISER (S. Gladman and T. Seemann, "VelvetOptimiser", 2012) to assemble the filtered data. The resulting assembly has 262 contigs. These contigs are sorted and compared with the components generated by the Pacbio HGAP method using MAUVE (Genome Res., Volume 14, Issue 7, Pages 1394 to 403, 2004). The BWA software (Bioinformatics, Volume 25, Issue 14, Pages 1754-60, 2009) was also used to compare the selected sequence data of good quality with the HGAP assembly data. The alignment files were manipulated using SAMTOOLS to generate classified bam files (Bioinformatics, Volume 25, Issue 16, Pages 2078-2079, 2009). Use ARTEMIS (Bioinformatics, Volume 16, Issue 10, Pages 944 to 945, 2000) to observe the mapped reads. Because the components made using VELVET for the Illumina sequence data contain more contigs than those made by the HGAP method, the subsequent analysis of the chromosome of Lactobacillus fermentum only uses the genome assembled by Pacbio. 4.0 Genome Annotation Use the software tool PROKKA to annotate protein-coding genes, tRNA, tmRNA, rRNA, and repeat regions (such as CRISPER) (Bioinformatics, Volume 30, Issue 14, Pages 2068-2069, 2014). By searching for the best hit in EggNOG Orthologous Clustering (COG) (Nucleic Acids Res., Vol. 42, No. D1, Pages D231 to D239, 2014), the gene ontology of each protein-coding gene is performed ( GO) functional classification. The preset e value 1 is used to identify the protein domain in the predicted protein in the Pfam database (Nucleic Acids Res., Vol. 42, No. D1, Pages D222-D230, 2014). PHAST (Nucleic Acids Res., Volume 39 Supplement, pages W347 to W352, 2011) was used to predict the sequence of the prophage in the genome. In the context of the development of probiotics, antibiotic resistance is a problem and therefore BLASTP (Antimicrob. Agents Chemother., Volume 57, Issue 7, Pages 3348 to 3357, 2013) is used for the comprehensive antibiotic resistance database. The search for possible resistance genes. Set the BLASTP e threshold to be less than 1 e ^-5 as a cut-off and analyze hits that are more than 60% identical to any gene in the antibiotic resistance database. The KEGG (Kyoto Encyclopedia of Genes and Genomes) mapper was used to reconstruct the few concerned metabolic pathways of Lactobacillus fermentum PS150. 5.0 Comparison of Lactobacillus fermentum genomes In addition to Lactobacillus fermentum PS150, there are four other strains that also have decoded genomes. Using Lactobacillus fermentum PS150 as the reference strain of the software BRIG (BMC Genomics, Volume 12, Issue 1, Page 402, 2011), the genomes of the other four known strains were compared with it. The nine prophage regions predicted from PHAST were concatenated into a sequence and compared with the reference. The upper and lower limits of the BLASTN identity threshold are set to 90% and 70%, respectively. 6.0 Tuf protein variability analysis For the EF-Tu variability analysis, all the proteins found from 235 bacterial species are used to calculate Shannon entropy (Bell Syst. Tech. J., Volume 27, July 1928, Pages 379 to 423, 1948). The Shannon entropy formula provides a method for estimating the variability of amino acid sequences along proteins. For a given multiple protein sequence alignment, the Shannon entropy (H) of each alignment column number is as follows:

Where H is the entropy value, X is the comparison column number, N is the number of amino acid types and the interval character (21)*, p _i is the frequency of the i-th character in N, * indicates whether the interval is in the form of characters Inclusion is subject to circumstances. In this study, spaces are treated as characters. In addition, at the Lactobacillus genus level, there are 25 different species with available genomes. A similar method as described above was used to obtain tuf genes in these 25 species. These proteins are also used as input for Shannon entropy calculation. Before calculating the entropy, use MAFFT (Bioinformatics, Volume 28, Issue 23, Pages 3144 to 3446, 2012) to compare the proteins and then use CLUSTALX (Bioinformatics, Volume 23, Issue 21, No. 2947 to Page 2948, 2007) was detected manually. Next, follow wherever they are found on Lactobacillus fermentum PS150 EF-Tu to remove the gap. Finally, use the trimmed alignment to calculate the Shannon entropy of each position in the alignment by using a customized R script. 7.0 Cell Culture 7.1 Preparation of Bacteria and Supernatant Liquid Lactobacillus fermentum PS150 is the isolated product of fermented meat sausage. The culture medium was obtained from the Microbial Culture Collection of the University of Science Malaysia (Penang, Malaysia). Prepare freeze-dried stock cultures in the presence of antifreeze (pectin) and store them in a dry and cool (-20°C) place. Before being used in the experiment, the lyophilized culture was continuously activated 3 times in sterile de Man, Rogosa and Sharpe (MRS) liquid medium (Hi-Media, Mumbai, India). During the subculture, the culture was cultured (10% v/v) in sterilized MRS liquid medium and cultured at 37°C for 18 hours. The cell-free supernatant (CFS) was obtained by centrifugation at 3500 g for 15 minutes (4°C), neutralized with 10M NaOH to a pH of 7.0, and then filtered through a 0.22 mm filter (Sartorius Stedim, Göttingen, Germany ) Sterilize and use immediately or store at -20°C until needed. 7.2 Human neuroblastoma SH-SY5Y cell culture Neuroblastoma cell line SH-SY5Y cell line was purchased from Korea Cell Line Bank (KCLB; Seoul, Korea). The cells were maintained in supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco Life Technologies Inc, Grand Island, NY, USA), 100 U/mL penicillin/streptomycin (Gibco Life Technologies Inc, Grand Island, NY, USA) Dulbecco's Modified Eagle Medium (Dulbecco's Modified Eagle Medium, DMEM; Difco, Detroit, MI, USA). The culture was inoculated into a flask containing supplemented medium and maintained at 37°C in a _{humidified atmosphere of 5% CO 2} and 95% air. 7.3 Cell culture treatment and cell viability analysis SH-SY5Y cells were seeded in a 96-well culture plate at a seeding density of ^{1×10 5 cells/well.} All cells in this study were maintained in 10% serum medium until dexamethasone (Dex; Sigma-Aldrich, Steinheim, Germany) treatment. The cells were cultured for 24 hours to allow attachment to the culture plate before processing. To study the effect of bacterial CFS, 20 µL of bacterial CFS was added to a well containing 100 µL of 25 µM Dex-treated SH-SY5Y cells in a serum-free medium. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT; Sigma-Aldrich, Steinheim, Germany) reduction was analyzed after 48 hours of treatment. With the quantitative colorimetric test of MTT, the cell survival rate was measured as previously described by Denizot and Lang (J Immunol Methods. May 22, 1986; 89(2): 271-7). MTT is used to assess neuronal damage. When MTT is taken up by living cells, it is converted from yellow to purple formazan crystals by cellular dehydrogenase. In short, at the end of the treatment period, a 10% MTT-labeled reagent with a final concentration of 0.5 mg/ml was added to each well and the plate was placed at 37°C with 5% CO ₂ and 95% air (v /v) 6 hours in the humidified incubator. Then, dimethylsulfoxide (DMSO; Sigma-Aldrich, Steinheim, Germany) was used to dissolve the insoluble formazan. The colorimetric determination of MTT reduction was measured at 570 nm using a microdisk reader (Thermo Scientific, Waltham, MA, US). The control cells treated with unfermented MRS were regarded as 100% survival rate. 8.0 In vivo study 8.1 Living cells and crude protein preparation 8.1.1 Lactobacillus fermentum PS150 living cells Lactobacillus fermentum PS150 was obtained from the Culture Collection of the Department of Bioengineering Technology, Faculty of Technology, University of Science Malaysia (Penang, Malaysia). The culture was activated three times for 20 hours in de Mann Rogosa Sharpe (MRS) (Biomark, India) liquid medium at 37°C before use. The overnight culture of Lactobacillus fermentum PS150 was subjected to centrifugation at 8000 g at 4°C for 30 minutes to obtain cell aggregates. After the cell mass is obtained, the cell mass is washed three times with phosphate buffered saline (1.0M, pH 7.4). Add 10% (v/v) pectin solution to the cell pellet, and then freeze at -20°C overnight. The frozen cell mass was then freeze-dried overnight at -55°C. The CFU/g of the obtained freeze-dried cells was measured using the pour plate method. Thereafter, for each individual rat, freeze-dried cells were prepared at a dose of 9 log CFU/rat/day. Before use, these formulations were stored at -20°C. 8.1.2 Crude protein extracted from the supernatant of Lactobacillus fermentum PS150 cells The overnight culture of Lactobacillus fermentum PS150 was subjected to centrifugation at 8000 g for 30 minutes at 4°C to obtain a cell-free supernatant. The supernatant was collected and adjusted to pH 7.0 with 3.0M sodium hydroxide (NaOH). The crude protein was precipitated by adding 80% w/v ammonium sulfate to the neutralized cell-free supernatant at 4°C while stirring and then standing overnight at 4°C. The pellet of the neutralized cell-free supernatant was subjected to centrifugation at 10,000 g for 30 minutes at 4°C. Resuspend the resulting agglomerates in sterile distilled water and filter through a 0.22 µM cellulose acetate syringe filter. Then, the obtained crude protein fraction was subjected to freezing at -20°C overnight, and then freeze-dried at -55°C overnight. For each individual rat, freeze-dried crude protein was prepared at a dose of 300 mg/kg. Before use, these formulations were stored at -20°C. 8.2 Animals The study was conducted at the GLP-certified animal research center of the Advanced Medical & Dental Institute (IPPT) on the Bertam campus of the University of Science Malaysia (USM). Thirty male Wistar rats (7 to 8 weeks old, weighing 180 to 200 g) were obtained from the Animal Research and Service Center (Penang) of the USM main campus. Upon arrival, the rats were immunized under standard laboratory conditions (12 hours of light/12 hours of night cycle (light: 0700-1900, temperature of 19 to 25°C and humidity of 30 to 70% RH) Intrusively (3 animals per cage) housed in standard polypropylene shoebox cages based on sawdust bedding. The cage bedding was changed every two days throughout the experiment. The received animals were quarantined for one week To monitor the presence of diseases or lice in animals on a daily basis. Once it is ensured that these animals are free of any disease or lice, they are allowed to undergo a week of adaptation, and then undergo a chronic mild stress regimen and treatment. During the period of isolation and adaptation, all rats can Get food and water freely. Measure and monitor the body weight of the animals every week. 8.3 The chronic mild stress protocol divides the rats into five groups (6 rats in each group (n=6)), that is, untreated control, Negative control, positive control, live cell treatment group and protein treatment group. Except for the untreated control group, all rats were subjected to a chronic mild pressure regimen (Table 1). In the untreated control group, all animals did not do anything. Treatment. In the negative control group, all animals were fed 0.2 ml of phosphate buffered saline (1.0M, pH 7.4) every day. In the positive control group, all animals were fed daily and suspended in 0.2 ml of phosphate buffered saline at a dose of 0.45 mg/kg. Alprazolam powder in saline (1.0M, pH 7.4). In the live cell treatment group, all animals were fed 9 log CFU/rat/day live Lactobacillus fermentum PS150. Finally, in the protein treatment In the group, all animals were fed the freeze-dried protein prepared as described above (300 mg/kg rat/day) every day. Oral gavage was performed using a 1.5-inch 20 gauge stainless steel feeding needle with a 2.25 mm ball. The chronic mild stress program consists of several stressful and non-stressed periods as shown in Figure 1, and the schedule is scheduled throughout the week, a total of 28 days. Table 1. The weekly schedule of the chronic mild stress program.

¹ Empty bottle: The water bottle is empty for one hour to limit the animals' access to water. ² Refill: Refill the water bottle to allow the animals to get water. ³ Tilt the cage: Let the cage tilt 45° ⁴ No pressure: No pressure source is performed during this period of time ⁵ Sand is used Replace sawdust: Replace the sawdust bedding with sand. ⁶ Empty cage: The animal's cage is empty during this period of time without sawdust. ⁷ Restraint: Place the animal in the soft-covered wire net to restrain its movement. ⁸ Social interaction: Allow all rats to interact in a larger cage during this period of time. ⁹ Food deprivation: Remove food from the cage ¹⁰ Restricted food: Provide the animal with a minimum amount of food during this period of time ¹¹ Outside room: Move the animal to a new cage ¹² Repeat cold pressure: Place 10 small ice cubes In the animal cage and allowed to melt for 30 minutes ¹³ Red light: Place the animal in the cage in a room equipped with red light. ¹⁴ Wet cage: Wet the sawdust bedding with sterile water. ¹⁵ Predator’s sound: Expose the animal to the predator’s sound Recording ¹⁶ Outside room: Replace the animal in the cage from the original room to the new room ¹⁷ Flash: Expose the animal to a flash video broadcast using an electronic device ¹⁸ Water deprivation: Remove the water bottle from the cage to restrict the rat’s access to water 8.4 Behavior Evaluation After 28 days of chronic mild stress intervention, all rats were subjected to behavioral evaluation. The behavioral assessments evaluated included Mohs Water Maze (MWM), Elevated Plus Maze (EPM), T-maze, Forced Swimming Test (FST), Open Field Test (OFT) and object recognition. All experiments were carried out in a quiet and dark room illuminated only by sparse light. 8.4.1 Mohs water maze (MWM) equipment setup The diameter of the MWM used is 210 cm, and the pool height is 51 cm, which has a non-reflective inner surface. The maze was placed in a room with a video camera on the top of the maze to capture and record animal behavior. The water maze is filled with water with a temperature in the range of 19-22°C to avoid temperature shocks (hypothermia or hyperthermia) of the rats. Then, a colorless and transparent circular object or platform (called an escape platform) with a diameter of 10 to 12 cm is immersed 1 to 2 cm below the water surface and to ensure the stability of the animal from tipping when the rat climbs on it. The starting position of the MWM for the evaluation of the escape latency period is marked in the east, west and southeast, while the escape platform is submerged in the northeast. Use these three starting positions for testing every day for four consecutive days. In short, the animals are gently placed in the first starting position and allowed to swim freely to find an escape platform. Give each animal a maximum of two minutes to search for an escape platform. After the Mohs test, all rats were thoroughly dried with a clean towel before returning them to their respective cages. The time it takes for each animal to locate in the escape place (escape latency (seconds)) is expressed as the average value of each group of six repetitions (n=6) ± the standard error of the average value. 8.4.2 Elevated plus maze (EPM) equipment setup Elevated plus maze (EPM) consists of four arms (two open arms without walls and two arms enclosing a wall of 40 cm high, 50 cm long and 15 cm wide). The arms of the labyrinth are connected to sturdy metal legs with a height of 70 cm. Place the EPM in a brightly lit room, close to the center of the room. The lighting levels of the two open and closed arms remain similar. Set up a video camera above the elevated plus maze to capture and record animal behavior. Evaluation The animal is removed from its cage and placed in the center of the EPM (i.e., the junction of the open and closed arms), facing away from the maze and facing the wall. Give each animal 5 minutes to freely explore on the EPM. Throughout the evaluation, before grabbing the next animal, clean and dry the EPM with 70% alcohol. Observe the number of times the open arm enters and the time spent in the open arm, time it and record it. The number of open arm entries and the duration spent on the open arm were recorded and expressed as the mean ± standard error of the mean of six repetitions for each group (n=6). 8.4.3 Forced swimming test equipment setup The forced swimming test (FST) is performed in a black opaque swimming cylinder with a depth of 68 cm and a diameter of 40 cm. Fill the cylinder with water to a depth of 30 cm, leaving some space above to prevent the rats from escaping. A video camera was set above the swimming cylinder to capture and record the animal's behavior during the test. Evaluation Each animal was gently placed in the water of the swimming cylinder and given 5 minutes to swim freely in the swimming cylinder. Observe and record animal behavior throughout the test. Throughout the evaluation, after FST, all rats were thoroughly dried with a clean towel before returning them to their respective cages. The duration of the rats remaining immobile was recorded and then expressed as the mean ± standard error of the mean of each group of six repetitions (n=6). 8.4.4 Empty field test equipment setup Use an opaque rectangular box with dimensions of 32 cm height, 38 cm width and 52 cm length placed in the center of the experimental room provided with bright light for the empty field test. Set the video camera above the rectangular box to capture animal behavior. Evaluation Each animal is gently transferred from its cage to the OFT and allowed to explore the contents of the box freely. During the 5-minute open field test, observe the duration of the rats' exploration and staying still. Clean the box with 70% alcohol during the interval of each test throughout the evaluation. The duration of exploration and staying still is expressed as the average value of each group of six repetitions (n=6) ± the standard error of the average value. 8.4.5 New object identification test equipment setup and evaluation A new object identification test is performed in an opaque rectangular box with a size of 32 cm height, 38 cm width and 52 cm length and placed in the center of the laboratory room. The behavior assessment involves two stages, namely, the familiarization stage and the testing stage. During the familiarization phase, two identical objects (with the same shape, size, and weight) are placed in a rectangular box. Each animal was placed in a rectangular box and given 2 minutes to explore freely in the box. However, during the testing phase, an old object was replaced by a new object with a shape (similar in weight and size). Likewise, each animal was placed in a rectangular box and given 2 minutes to explore freely in the box. Throughout the experiment, a video camera was set above the rectangular box to capture the behavior of the animals in the two stages. During the interval between each test, the tank was cleaned with liquid soap and water and dried. The given time interval between the familiarization phase and the testing phase is one hour. The time spent exploring new objects is expressed as a recognition index according to the following formula: Recognition index = (time spent on new objects)/(old object + total time spent on new objects) X 100% (Cogn Process, 2012, 13(2):93-110) The data is expressed as the mean value ± standard error of the mean value of each group of six repetitions (n=6). 8.4.6 Statistical analysis All data are statistically analyzed by IBM SPSS Statistics 20.0 (IBM Co., Armonk, NY, USA). A one-way analysis of variance was performed to analyze the statistical differences between group means. The statistical significance level is set to α=0.05, and the comparison of multiple averages is evaluated by Tukey's test. Unless otherwise indicated, all data are expressed as the average of three independent experiments (n=6). 8.5 Blood collection and organ extraction At the end of the experiment, shortly after the rats were killed by carbon dioxide asphyxiation, blood samples were collected through cardiac puncture and transferred to EDTA-coated tubes. Soon after blood collection, the brain was removed by decapitation. Keep the brain tissue samples for ELISA and hydrogen peroxide analysis in ice-cold PBS. 8.5.1 Homogenization of brain samples for biochemical analysis The extracted rat brains stored in 1x phosphate buffered saline (pH 7.4) at 4°C were processed shortly after resection. Cut the brain tissue sample into 3 regions (cerebellum, cerebrum, and hippocampus). Then homogenize the brain tissue sample section for 10 minutes at 70 to 100 Hz under ice-cold conditions. Store all homogenized samples at -20°C before use. Blood samples The blood collected in anticoagulated EDTA tubes was incubated at room temperature for 10 to 20 minutes, and then centrifuged at 3000 rpm for 20 minutes. The supernatant was collected as a plasma sample and stored in a 1.5 ml microcentrifuge tube, kept at 4°C for immediate analysis. Before use, keep additional plasma samples at -20°C. Enzyme-linked immunosorbent assay (ELISA) is performed to quantify several cytokines, key neurotransmitters and biomarkers associated with neurogenesis. ELISA was performed according to the manufacturer's instructions (R&D System Inc, USA). In short, 10 µl of homogenized brain tissue or plasma is loaded onto the bottom of the well without touching the wall of the well. Add 40 µl of sample dilution buffer to dilute the samples. Incubate the diluted sample at 37°C for 30 minutes. After that, discard the samples and wash five times with the provided wash buffer. Subsequently, 50 µl of antibody conjugated to horseradish peroxidase (HRP) was added to each well. The mixture was incubated for another 30 minutes at 37°C. Then, discard the mixture and wash five times with the provided wash buffer. Then, add 50 µl of chromogen solution A and 50 µl of chromogen solution B to each well and mix by gentle shaking. The mixture was then incubated at 37°C for 15 minutes. Avoid light exposure during this step. Next, add 50 µl of stop solution to each well to stop the reaction. Finally, use the Thermo Multiskan-GO ELISA plate reader to read the absorbance OD at 450 nm. The standard curve was generated using the same procedure as mentioned above. Measurement of hydrogen peroxide (H ₂ O ₂ ) in the brain The Amplex Red analysis kit (Molecular Probes Inc., Eugene, OR, USA) was used to evaluate the hydrogen peroxide content in the brain homogenate samples according to the manufacturer's protocol. 9.0 Identify the protein from Lactobacillus fermentum PS150 Following all these positive findings, isolate the protein part of Lactobacillus fermentum PS150 and further identify it. The protein fraction was pre-purified by ammonium sulfate precipitation and then recovered based on a three-step chromatography procedure. 9.1 Protein extraction Extract the crude protein fraction as described above (8.1.2). Then the crude protein fraction was desalted with 75% acetone, and the salt was removed by centrifugation (7000 g, 30 minutes, 4°C), and repeated three times. The collected agglomerates are dissolved in deionized water. The Bradford analysis method (Bio-Rad, Hercules, CA, USA) was used to estimate the protein content using BSA as a standard. 9.2 Gel-free separation method uses GELFREE™ 8100 Fractionation System (Expedeon, San Diego, CA, USA) (Expedeon, San Diego, CA, USA), which contains pre-prepared HEPES operation buffer and Tris acetate sample buffer (AMR Incorporated), according to the manufacturer The instruction manual divides the protein into multiple parts according to the molecular mass. The Lactobacillus fermentum PS150 protein is dissolved in the sample buffer and loaded into individual loading compartments containing 5% Tris-acetate cartridges. The design is to separate proteins with a mass in the range of 60 to 300 kDa. The instrument is automatically paused at a predetermined time interval, and the liquid part is removed with a pipette. Then restart the sequence and continue the process to collect the next part based on the molecular size. Repeat the process until all parts are collected. After the 12 individual fractions are recovered, the fractions are collected, washed twice with acetone, and removed by a vacuum concentrator. Standardize the individual parts to the same protein content (2.0 µg/mL), and use the method described in 7.3 for testing in the cell culture process. 9.3. 1DE gel analysis In order to estimate the molecular size of each individual protein fraction, once 12 individual fractions are recovered from the GELFREE™ 8100 separation system, 10% SDS-PAGE (Bio-Rad) is used for standard 1DE. In the Tris/glycine/SDS operating buffer (Bio-Rad) at 110 V, the protein was separated using standard electrophoresis equipment (Bio-Rad) for 80 minutes. Coomassie Brilliant Blue protein gel stain (Molecular Probes, Eugene, OR, USA) was used to stain the protein in the gel and ChemiDoc XRS Camera and Quantity One 1D analysis software (Bio-Rad) was used to observe the protein bands. 9.4 Reverse phase high performance liquid chromatography (RP-HPLC) The final purification step of the protein fraction is performed on HiTrap Q HP column by reverse phase high performance liquid chromatography (RP-HPLC). Elution was performed with solvent A (25 mM Tris HCl) at a flow rate of 0.8 mL/min for 10 minutes. Then, perform a linear gradient from 0% to 100% solvent B (25 mM Tris HCl containing 1M NaCl) within 70 minutes. Collect elution peaks individually and check the neuroprotective activity of anti-dexamethasone as described in (7.3). 9.5 Recognition of the protein part by orbitrap LC/MS Peptide identification was achieved at the Institutional Centre for Advanced Analytical Toxicology Services (Universiti Sains Malaysia) at the University of Science Malaysia (Institutional Centre for Advanced Analytical Toxicology Services, Universiti Sains Malaysia). The fraction of interest from RP-HPLC (0.2 mg) was dissolved in denaturing buffer (6M guanidine-HCl/25 mM ammonium bicarbonate, pH 8.5) and then 1 mg/ml DTT/25 mM ammonium bicarbonate ( Freshly prepared) to protein solution and incubate at 55°C for 30 minutes. Then add 1 mg/mL iodoacetamide/25 mM ammonium bicarbonate (freshly made), then cover with aluminum foil and incubate at 55°C for 15 minutes. Then, a spin-column with a molecular weight cut-off of 15 kDa was used to exchange the reduced and alkylated protein sample with 25 mM ammonium bicarbonate buffer 3 times (30 minutes each time). Then trypsin was added to the sample and incubated at 37°C for 18 hours. Add formic acid to the sample and then subject it to freeze drying. Subject the sample to MALDI-TOF mass spectrometry analysis and identify mass peaks. Generate all possible fragments and identify their corresponding molecular weight and peptide sequence. Subsequently, PEAKS studio version 6.0 was used for data analysis and the functional annotation database of all proteins encoded in the PS150 genome of Lactobacillus fermentum was used to select potential biologically active peptide sequences. 9.6 Docking simulation Swiss-MODEL (Schwede 2003) was used to construct a 3D model of the elongation factor tu (EF-Tu) of Lactobacillus fermentum PS150. EF-Tu from Thermus thermophilus (PDB ID: 2C77) was used as a template for the following two reasons: 1) There is no available Lactobacillus-derived EF-Tu crystal structure in the protein database (PDB) And 2) The thermophilic bacteria-derived EF-Tu showed the highest sequence identity (74.81%) with the EF-Tu of Lactobacillus fermentum PS150 in the database. Obtain the crystal structure of interferon-γ (IFNγ) and interferon-γ receptor (IFNγR; PDB ID: 1FYH; 2.04A resolution) from the protein database (PDB). In addition, IFNγ39 (derived from IFNγ, a synthetic peptide known to inhibit the first 39 amino acids of IFNγR reactivity) was also derived from the crystal structure of IFNγ to serve as a positive control in this study. Docking -IFNγ to IFNγ receptor Cluspro 2.0: Protein - to remove all the water molecules and the ligand-protein docking before job submission. In Cluspro 2.0: protein-protein docking (antibody mode), IFNγ and IFNγR are submitted as ligands and receptors, respectively. All the configurations generated were further analyzed with BIOVIA Discovery Studio 4.5 (Humphrey et al. 1996) to evaluate the root mean square deviation (RMSD) value. The configuration with the lowest binding free energy, the lowest RMSD value, and the densest cluster is used as a reference control for comparison in further analysis. Use Ligplot+v1.4.5 (Laskowski and Swindells, 2011) to observe the interaction between IFNγ and IFNγR of the selected configuration (TYR49, TRP82, GLU101, HIS205, VAL206 and TRP207; Randal and Kossiakoff, 2001) interaction. Molecular docking- IFNγ39 to IFNγ receptor All water molecules and ligands were removed before Cluspro 2.0: protein-protein docking job submission (Kozakov et al., 2013). In Cluspro 2.0: protein-protein docking (antibody mode), IFNγ39 and IFNγR are submitted as ligands and receptors, respectively. The configuration with the lowest free energy of binding and the densest clusters is used for comparison with the benchmark control as previously completed. Use Ligplot+ v1.4.5 (Laskowski and Swindells, 2011) to observe the interaction between IFNγ39 and IFNγR of the selected configuration (TYR49, TRP82, GLU101, HIS205, VAL206 and TRP207; Randal and Kossiakoff, 2001) effect. Molecular docking -EF-Tu to IFNγ receptor Remove all water molecules and ligands before the Cluspro 2.0: protein-protein docking job is submitted. In Cluspro 2.0: protein-protein docking (antibody mode), EF-Tu and IFNγR are submitted in the form of ligand and receptor, respectively. The configuration with the lowest free energy of binding and the densest clusters is used for comparison with the benchmark as implemented previously. Use Ligplot+ v1.4.5 (Laskowski and Swindells, 2011, J. Chem. Inf. Model, 51: 2778-2786) to observe the interaction residues (TYR49, TRP82, GLU101, HIS205, VAL206 and TRP207) interaction. Example 1 Identification of Lactobacillus fermentum PS150 and chromosome general genomic characteristics The API 50 CHL kit (bioMerieux, France) was used to study the sugar utilization of PS150 used in the present invention, and the results are shown in Table 2. Fermentation tests indicate that PS150 has biochemical properties similar to Lactobacillus fermentum. Table 2: Results of ^{fermentation test a}

Lactobacillus fermentum PS150 was cultured in a specific artificial medium. The 16S rRNA and pheS genes from Lactobacillus fermentum PS150 were analyzed by direct sequencing of DNA fragments amplified by PCR. Use methods known in the related art for genomic DNA extraction, PCR-mediated amplification of 16S rDNA and pheS DNA, purification of PCR products, and sequencing of purified PCR products. 16S rDNA sequence of Lactobacillus fermentum PS150 (SEQ ID NO: 5)

pheS gene sequence (SEQ ID NO: 6)

Put the obtained sequence into the online comparison software provided by the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/), manually compare and compare with The representative 16S rDNA sequences of organisms were compared. For comparison, the 16S rDNA sequence was also obtained from the online database provided by NCBI. As a result of this analysis, Table 3 below lists the organisms whose 16S rDNA sequence showed the highest similarity value compared to the 16S rDNA sequence of Lactobacillus fermentum PS150. Table 3 Organisms with the 16S rDNA sequence showing the highest similarity value compared to the 16S rDNA sequence of Lactobacillus fermentum PS150

Lactobacillus fermentum PS150 has a circular chromosome of 2,238,401 bp and its GC content is 51%. A total of 2,281 genes are predicted, which are composed of 2,206 protein-coding genes, 59 tRNAs, 15 rRNAs, and one tmRNA (Table 4). In addition, two clusters of regularly spaced short palindrome repeat (CRISPR) regions were found, which can provide immunity against foreign genetic components. 465 of the 2,206 protein-coding genes are predicted to be hypothetical proteins that do not have the functions attributable to them. Based on the orthologous clustering (COG) functional classification, 2005 protein coding genes (ie, 91% of all predicted coding sequences (CDS)) can be assigned to the COG functional category. 62% of 2005 protein-coding genes belong to the five main COG categories: 447 CDS are in category S (unknown function), 364 CDS are in category L (replication, recombination, and repair), and 162 CDS are in category E ( In amino acid transport and metabolism), 140 CDS are in category J (translation, ribosomal structure and biosynthesis) and 136 CDS are in category K (transcription). In addition, nine prophage regions were identified and interestingly, four of them were predicted to be complete prophage. Studies on antibiotic resistance genes have shown six candidate resistance genes, including 30S ribosomal protein S12, maltose O-acetyltransferase, galactosyl O-acetyltransferase, putative acetyltransferase, Chloramphenicol acetyltransferase and thymidylate synthase. Table 4: List of predicted genome features of Lactobacillus fermentum PS150.

Comparison of chromosomes with other strains of Lactobacillus fermentum. Comparison of chromosomes in bacterial strains can reveal unique genomic characteristics for certain strains. The chromosomes of Lactobacillus fermentum PS150 were compared with the complete genomes of the other four strains Lactobacillus fermentum 3872, Lactobacillus fermentum CECT 5716, Lactobacillus fermentum IFO 3956, and Lactobacillus fermentum F6. The predicted prophage of Lactobacillus fermentum PS150 was concatenated and included in the comparison. As expected, such as in the prophage region, the new genome is a clear difference. A study was conducted to find the non-overlapping region between strain 3872 and PS150, which revealed that 98 protein coding sequences are unique to the latter strain. 35 of the 98 sequences encode hypothetical proteins, which means that they have unknown functions or may be mistakenly predicted as genes. The remaining 63 genes have predicted protein products, which contain three putative glycosyltransferases in gene clusters with a span of less than 15 kb. Example 2 Analysis of Anxiety, Depression, and Stress Relief The behavioral analysis of rats was performed 4 weeks after the chronic mild stress regimen. The elevated plus maze is a widely used caries animal behavior analysis and it has been verified to evaluate the anxiolytic effects of pharmacological agents and steroid hormones. The elevated plus maze used in the experiment consists of four arms (two open arms and two closed arms) and is raised 50 cm from the ground. Place the rat at the nodes of the four arms of the maze, facing the open arms, and record the entry/duration time in each arm for 5 minutes. The increase in open arm activity (duration and/or entry) reflects anxiolytic behavior. The group treated with Lactobacillus fermentum PS150 and protein partially entered the open arm significantly more than the negative control group (P＜0.05; Figure 1(a)). However, the untreated control group and the positive control group (by The difference between the antidepressant treatment), the group treated with Lactobacillus fermentum PS150 and the group treated with protein part was not obvious (Figure 1(a)). In addition, the ratio of the time spent on the closed arm to the time spent on the open arm treated with Lactobacillus fermentum PS150 and protein was significantly lower than that of the negative control group (P<0.05; Figure 1(b)), However, insignificant differences were observed in the untreated control group, the positive control group (treated with antidepressants), the group treated with Lactobacillus fermentum PS150, and the group treated with protein (Figure 1(b)). The results indicate that the protein part of Lactobacillus fermentum PS150 and Lactobacillus fermentum PS150 can standardize the anxiety behavior caused by chronic mild stress and return to the original non-anxious state. In addition, the Lactobacillus fermentum PS150 and protein partial group also showed an anxiolytic effect similar to that of alprazolam, which indicates an effect similar to that of commercially available antidepressants. The empty field test is widely used to evaluate exploratory behavior and has been validated to measure anxiety-related behavior. The procedure consists of allowing the rat to experience an unknown environment that is prevented from escaping from it by a fence. Compared with the negative control group, the group treated with the protein part of Lactobacillus fermentum PS150 and Lactobacillus fermentum PS150 significantly increased the duration of exploratory activity in rats (P<0.05; Figure 2). The untreated control group, the positive control group (treated with antidepressants), the group treated with Lactobacillus fermentum PS150, and the group treated with protein partially showed no significant differences in the exploration and immobility activity of rats (Figure 2). The increase of exploratory behavior in rats indicates less anxious state, and keeping motionless indicates anxious behavior in rats. Chronic mild stress causes anxiety in rats, but the protein part of Lactobacillus fermentum PS150 and Lactobacillus fermentum PS150 can standardize the anxiety behavior caused by chronic mild stress and return to the original non-anxious state. In addition, the Lactobacillus fermentum PS150 and protein partial group also showed an anxiolytic effect similar to that of alprazolam, which indicates an effect similar to that of commercially available antidepressants. The forced swimming test is one of the most commonly used tests used to study the depressive behavior of dental caries animals. The forced swimming test is based on the assumption that when an animal is placed in a water-filled container, it will try to escape but will eventually appear to remain still, which can be regarded as a measure of desperate behavior. In the forced swimming test, the swimming cylinder is filled with 30 cm deep water. The depression assessment of rats was performed by scoring the main behaviors of the 300 s test every 5 s time period. Compared with the negative control group, the group treated with Lactobacillus fermentum PS150 and protein part spent significantly less time staying still (P<0.05; Figure 3). The untreated control group, the positive control group (treated with antidepressants), and the group treated with Lactobacillus fermentum PS150 and protein partly had no significant difference in the amount of time the rats spent staying still (Figure 3). Similar effects were observed from Lactobacillus fermentum PS150, protein fractions, and antidepressants, where the depressive behavior caused by chronic mild stress was standardized to the same degree as in the untreated control group. The protein part of Lactobacillus fermentum PS150 and Lactobacillus fermentum PS150 can standardize the depressive behavior caused by chronic mild stress and return to the original non-depressive state. In addition, Lactobacillus fermentum PS150 and the protein part also showed an antidepressant effect similar to the antidepressant effect of alprazolam, which indicates an effect similar to that of commercially available antidepressants. Compared with the negative control group and the positive control group, the content of plasma corticosterone in the Lactobacillus fermentum PS150 and protein part group was significantly lower (P<0.05; Figure 4). Exposure to repetitive or prolonged sources of stress causes chronic stress. Chronic stress eventually causes HPA axis dysregulation, which leads to an increase in plasma corticosterone levels. Elevated levels of corticosterone indicate behaviors related to anxiety and depression. Our data show that the protein part of Lactobacillus fermentum PS150 and Lactobacillus fermentum PS150 standardizes the anxiety, depression and stress behavior caused by chronic mild stress, restores to the original non-stress state, and can even play a role of bialprazolam. Sell antidepressants) for better pathological effects. Compared with the negative control group, the ratio of plasma TNFα to interleukin-10 in the group treated with Lactobacillus fermentum PS150 and the group treated with protein was significantly lower (P<0.05; Figure 5). TNFα is a pro-inflammatory cytokine, but interleukin-10 is an anti-inflammatory cytokine. Depressed patients usually have higher TNFα content but lower IL-10 content than their non-depressed people, and the TNFα:IL-10 ratio is significantly correlated with the severity of depressive symptoms. Our findings in the present invention indicate that the protein part of Lactobacillus fermentum PS150 and Lactobacillus fermentum PS150 standardizes the anxiety, depression and stress behavior caused by chronic mild stress and restores to the original non-stress state, most likely through cytokines The adjustment. The protein portion of Lactobacillus fermentum PS150 and Lactobacillus fermentum PS150 also have similar effects to alprazolam, which indicates a similar pressure-reducing effect as commercially available antidepressants. Example 3 Analysis for the improvement of brain cognition and the protection of neuronal cell apoptosis. There are well documented that stress usually exerts a negative impact on memory and other cognitive functions. To assess cognitive performance, a Mohs water maze was used to assess memory retention, and a new object recognition test was used to assess learning and recognize memory. The Mohs water maze is a widely used task and accepted by behavioral physiologists and pharmacologists to evaluate and compare the learning and memory of dental caries in animals. Spatial learning (the most basic Mohs water maze program) is used in this experiment. The concept behind it is that the animal must learn to use remote cues to find a direct path to the hidden platform when starting from different random locations near the perimeter of the trough. The diameter of the water maze is 210 cm and it has 51 cm high sides with non-reflective inner surfaces. The escape latency (EL) is the time it takes for the animal to move from the starting quadrant to finding the hidden platform in the target quadrant. Compared with the negative control group, the group treated with Lactobacillus fermentum PS150 and protein part significantly shortened the escape latency, but the negative control group significantly increased the escape latency compared with other groups (P<0.05; Figure 6(a)). Insignificant differences were observed between the untreated control group, the positive control group, the group treated with Lactobacillus fermentum PS150, and the group treated with protein (Figure 6(a)). Compared with the negative control rats, the group treated with Lactobacillus fermentum PS150 and protein partially had a significantly higher recognition index in the new object recognition test (Figure 6(b)), and the effect was similar to that of the positive control rats (anti- The effect of depression group) and untreated control (no stress) rats (P<0.05). Our findings in the present invention indicate that Lactobacillus fermentum PS150 and protein-partial Lactobacillus fermentum PS150 standardize the cognitive impairment caused by chronic mild stress in rats (clearly better than the negative control group). TNF-α stimulates indoleamine 2,3-dioxygenase (IDO) by activating the hypothalamus-pituitary-adrenal (HPA) axis, which leads to tryptophan depletion, and is mediated by neuronal immunity Lead to destruction, or neurotoxic release of glutamine, causing the pathogenesis of neurodegenerative diseases. IDO is an enzyme that degrades tryptophan into kynurenic acid, which is involved in the pathophysiology of neurodegenerative diseases. If compared with the negative control and the positive control, the ratio of the plasma TNF α content to the interleukin-10 content in the Lactobacillus fermentum PS150 and protein part group is lower, and a significant decrease in plasma IDO content is also observed (P<0.05; Figure) 7(a)). If compared with the negative control group, lower plasma IDO concentration was detected in the Lactobacillus fermentum PS150 and protein part group, then higher plasma tryptophan content was also observed (P<0.05; Figure 7(b)) . Our data showed that compared with the negative control group, a smaller amount of tryptophan in the Lactobacillus fermentum PS150 and protein part group was degraded into kynurenine, and the plasma kynurenine content was also significantly lower (P<0.05) ; Figure 7(c)). Our findings in the present invention indicate that the protein part of Lactobacillus fermentum PS150 and Lactobacillus fermentum PS150: (a) prevent neurodegeneration by reducing the production of kynurenic acid (significantly better than the negative control group) and (b) restore neurodegeneration Back to normal and as good as antidepressants (similar effects to positive control/alprazolam and untreated control group). On the other hand, tryptophan is also a precursor of serotonin. Brain samples were also collected for measurement of serotonin content. Serotonin is a neurotransmitter involved in the transmission of nerve impulses and the regulation of signals between neurons. Our findings in the present invention showed that compared with the negative control group, the concentration of brain serotonin in the Lactobacillus fermentum PS150 and protein fraction group was significantly increased (P<0.05; Figure 8). The results are consistent with the increase in plasma tryptophan content observed in Lactobacillus fermentum PS150 and the protein fraction group. Our findings of the present invention indicate that the protein part of Lactobacillus fermentum PS150 and Lactobacillus fermentum PS150: (a) restore the transmission of neurons in the brain that is reduced due to chronic mild stress in rats, leading to better cognition (obviously (Better than the negative control group) and (b) exert a better efficacy than the commercially available antidepressants (significantly better than the positive control/alprazolam group). Under pressure, changes in the characteristics of apoptosis were observed in the rat brain. Monoamine oxidase (MAO) is a mitochondrial enzyme that controls the content of neurotransmitters in the brain. MAO-A plays a role in cell apoptosis through the activation of caspase-3 and the production of reactive oxygen species. Hydrogen peroxide is usually detected from apoptotic cells because of its role as a mediator of apoptotic cell death. According to the chronic stress regimen, compared with the negative control rats, the rats administered PS150 showed a lower brain MAO-A concentration (Figure 9(a)) (P<0.05). Compared with the negative control group, when a lower brain hydrogen peroxide concentration was observed in rats administered with PS150 or protein fractions, the alleviation of apoptosis characteristics was obvious (Figure 9(b), P< 0.05). Behavioral and brain evaluations indicate that the protein of PS150 and PS150 partially prevents brain damage caused by chronic mild stress in rats, has similar efficacy to commercially available antidepressants, obtains standardized spatial learning, long-term memory and cognitive functions, and prevents nerves Degeneration and neuronal cell apoptosis. Example 4 Identification of genes and proteins from Lactobacillus fermentum PS150 Following all these positive findings, the protein part of Lactobacillus fermentum PS150 was further isolated and identified. The protein fraction was pre-purified by ammonium sulfate precipitation and then recovered based on a three-step chromatography procedure. The protein fraction was revealed to be at 5%, 15%, 20%, 25%, 30%, 50%, 55%, 55%, 15%, 15%, 20%, 25%, 30%, 50%, 55%, 5%, 15%, 20%, 25%, 30%, 30%, 50%, 55%, 5%, 15%, 20%, 5%, 5%, 5%, 15%, 20%, 5%, 5%, 5%, 5%, 15%, 20%, 5%, 5%, 15%, 5%, 15%, 5%, 15%, 5%, 5%, 15%, 25%, 30%, 30%, 50%, 55%, and 2nd of the final purification step of the final purification step of the final purification step of the protein on the HiTrap Q HP Nine main peaks eluted when 60% and 90% solvent B (25 mM Tris HCl containing 1M NaCl) were used (Figure 10). Collect elution peaks individually (referred to as parts A, B, C, D, E, F, G, H, and I, respectively) and check the neuroprotective activity of anti-dexamethasone. Apoptosis is a normal physiologically programmed cell death that can be enhanced by a variety of external stimuli (such as stress biomarkers, corticosterone). Dexamethasone (synthetic corticosterone) is used to evaluate the neuroprotective activity of the protein part of Lactobacillus fermentum PS150. Data show that only part A has the most prevalent neuroprotective effect (Figure 11). Positive control cells not treated with dexamethasone were regarded as 100% survival rate. Compared with the two cells treated with unfermented medium (with or without dexamethasone), the protein part of Lactobacillus fermentum PS150 showed a significant cytoprotective effect. The purified part A showed a single band with an estimated molecular weight of about 55 kDa on SDS-PAGE (Figure 12). For protein identification, the purified protein collected from HPLC is subjected to MALDI-TOF mass spectrometry analysis. Identify the mass peaks observed during the entire trypsin digestion process. Generate all possible fragments and identify their corresponding molecular weight and peptide sequence. Subsequently, PEAKS studio version 6.0 was used for data analysis and the functional annotation database of all proteins encoded in the genome of Lactobacillus fermentum PS150 was used to select potential biologically active peptide sequences. For the annotated genome of Lactobacillus fermentum PS150, all peptide sequences obtained from mass spectrometry were transformed into bacterial nucleotide-translated protein sequences. Among the 150 peptides transformed, only two peptides were found to be identical to the cell-derived protein, and the two peptide sequences matched 100% with one of the proteins of Lactobacillus fermentum PS150. For this match, the sequence of the protein was determined to be the sequence of SEQ ID NO:4. The protein was back translated to the nucleotide sequence with the sequence of SEQ ID NO: 7 to obtain its gene sequence. SEQ ID NO: 7

This biologically active protein was identified as the elongation factor Tu, which has 396 amino acid residues. In fact, this molecule is a guanosine nucleotide binding protein that plays an important role in protein synthesis in the cytoplasm. However, there is sufficient documentation to document the presence of EF-Tu in different compartments of microorganisms. It has been shown that the EF-Tu molecule, originally thought to be restricted to the cytoplasm of bacteria, is also associated with E. coli membranes. The latest research shows that EF-Tu is a protein associated with the envelope that can be released from cells. The elongation factor Tu can trigger the immunomodulatory response in rats. In this study, rats treated with protein partially induced an anti-inflammatory immune response (the ratio of TNF-α to IL-10 decreased; P<0.05; Figure 5). By reducing TNF-α (which causes neurodegenerative disease by stimulating indoleamine 2,3-dioxygenase (IDO)), the plasma IDO concentration is also reduced (P<0.05; Figure 7(a)). This ultimately reduced the conversion rate of tryptophan to kynurenine (P<0.05; Figure 7(b) and (c)), and resulted in an increase in serotonin production. The immunomodulatory activity of the elongation factor Tu also reduces the plasma corticosterone content, which causes anxiety, depression and stress relief. In conclusion, our data confirms that the biologically active protein with the sequence of SEQ ID NO: 4 produced by Lactobacillus fermentum PS150 is an immunomodulatory compound, and our data supports that in addition to improving cognition, it is effective against anxiety, depression and stress. Relief is related to healthier brain function. Here we propose evidence that bioactive compounds of probiotics (such as the elongation factor produced by Lactobacillus fermentum PS150) can exert brain health benefits. Example 5 Analysis of protein variability of elongation factor Tu of Lactobacillus fermentum PS150 Shannon entropy system was used to estimate the protein variability of EF-Tu. In the 21-character system, H is in the range of 0 (that is, highly conservative, only a single character is in the comparison column) to 4.392 (that is, all 21 characters exist equally). The entropy was estimated for 235 bacterial species and 25 bacterial species with three key domains of protein (GTP EFTU, GTP EFTU D2, and GTP EFTU D3) across the entire protein length from Lactobacillus. Highly variable residues are classified as those with entropy exceeding the average entropy threshold plus two standard deviations. Use the average value plus two standard deviations, because for most distributions, values greater than it are considered extreme values. Therefore, the first domain GTP EFTU has the largest number of variable residues, followed by the last domain GTP EFTU D2, and again GTP EFTU D3. Of particular interest is the high variability when considering all 235 bacterial species, but the residues that do not have high variability when only containing species from the genus Lactobacillus. Here is a list of residues that meet the mentioned criteria: 40K, 41G, 42L, 44K, 46E, 161E, 185E, 195D, 327S, 345E and 360T. When considering all 235 bacterial species, all these residues have an entropy higher than 2.63, which means that each residue shows a variability of at least 60% of the theoretical maximum variability (4.392). These residues represent putative binding sites that may be the key distinguishing factors of Lactobacillus fermentum PS150 EF-Tu in its brain health effects. The binding sites of proteins are usually exposed or they will be in a configuration change. In order to evaluate whether any of the highly variable residues detected in the previous paragraph is located on the exposed part of the 3D protein structure of EF-Tu, a homology model of the protein was constructed. Mapping highly variable residues on the homology modeled 3D EF-Tu protein structure revealed that residues 40K, 41G, 42L, 44K, 46E and 327S are located on the surface of the molecule. It is interesting to note that residues 40K, 41G, 42L, 44K, and 46E are only consecutively punctuated at residues 43A and 45A. In fact, both residues 43A and 45A also show high variability with entropy values of 2.004 and 2.578, respectively, but these values are below the cut-off threshold. Although residue 327S also has high variability, its position is far away from residues 40 to 46. The other residues 161E, 185E, 195D, 345E and 360T are embedded in the EF-Tu molecule itself. Example 6 Sleep-enhancing effect ^{of PS150 To BALB/c mice, 109} CFU/mouse Lactobacillus fermentum PS150 was orally administered once a day for 14 consecutive days. Control mice were tested in parallel with animals provided with PBS only. For the positive control group, DIPH-HCL (diphenhydramine hydrochloride, 20 mg/kg) was provided as a positive control. Body weight (BW) was measured every day during the fourteen-day experiment. On the fourteenth day, PBS, PS150 and DIPH-HCL were administered intragastric (ig) for 30 minutes, and sodium pentobarbital (50 mg/kg) was injected intraperitoneally (ip) into each mouse to Test the duration of pentobarbital-induced sleep. Observe the latency and duration of sleep of each mouse. The elapsed time between pentobarbital injection and loss of righting reflex was recorded as sleep latency, and the elapsed time between loss of righting reflex and recovery was recorded as the duration of sleep. Figure 13 shows the effect of PS150 on sleep latency (a) and sleep duration (b) in the pentobarbital-induced sleep test. The results show that PS150 can shorten sleep latency and prolong sleep duration.

Figure 1 (a) and (b) show the use of the elevated plus maze to assess the anxiety behavior of rats after 4 weeks of chronic mild stress regimen; (a) the number of times to enter the open arm observed in rats on the elevated plus maze ; (B) The ratio of the time spent on the closed arm to the time spent on the open arm. The untreated control group, in which the rats were left in the cage in an undisturbed manner; the negative control, in which the rats were subjected to chronic gentle pressure and fed with 200 μL of phosphate buffered saline (PBS; pH 7.4); the positive control, in which Rats received chronic mild pressure and provided 200 μL of PBS containing 0.045 mg/kg alprazolam (alprazolam); Lactobacillus fermentum PS150 group, in which rats were subjected to chronic mild pressure and provided 200 μL containing 1 X 10 ⁹ cfu live Lactobacillus fermentum PS150 PBS; Lactobacillus fermentum PS150 protein part group, in which rats are subjected to chronic mild pressure and 200 μL of PBS containing 300 mg/kg protein is provided. Error bars indicate the standard error of the mean; n=6. ^{ab Significant} differences in the number of times the rats enter the EPM open arm (a) and the ratio of the time spent on the closed arm to the time spent on the open arm (b) between the different groups; P<0.05. Figure 2 shows the use of an empty field test to measure anxiety-related behaviors in rats after a chronic mild stress regimen for 4 weeks. During the 5-minute open field test, observe the rats' exploration and the duration of staying still. The untreated control group, in which the rats were left in the cage in an undisturbed manner; the negative control, in which the rats were subjected to chronic gentle pressure and fed with 200 μL of phosphate buffered saline (PBS; pH 7.4); the positive control, in which Rats received chronic mild pressure and provided 200 μL of PBS containing 0.045 mg/kg alprazolam; Lactobacillus fermentum PS150 group, in which rats were subjected to chronic mild pressure and provided 200 μL containing 1 X 10 ⁹ cfu live Lactobacillus fermentum PS150 PBS; Lactobacillus fermentum PS150 protein part group, in which rats are subjected to chronic mild pressure and 200 μL of PBS containing 300 mg/kg protein is provided. Error bars indicate the standard error of the mean; n=6. ^{ab Significant} differences in the duration of exploration and holding immobility of rats between different groups; P<0.05. Figure 3 shows the measurement of depression-related behaviors observed in rats using the forced swimming test 4 weeks after the chronic mild stress regimen. The time the rats spent staying still during the 5-minute forced swimming test was recorded. The untreated control group, in which the rats were left in the cage in an undisturbed manner; the negative control, in which the rats were subjected to chronic gentle pressure and fed with 200 μL of phosphate buffered saline (PBS; pH 7.4); the positive control, in which Rats received chronic mild pressure and provided 200 μL of PBS containing 0.045 mg/kg alprazolam; Lactobacillus fermentum PS150 group, in which rats were subjected to chronic mild pressure and provided 200 μL containing 1 X 10 ⁹ cfu live Lactobacillus fermentum PS150 PBS; Lactobacillus fermentum PS150 protein part group, in which rats are subjected to chronic mild pressure and 200 μL of PBS containing 300 mg/kg protein is provided. Error bars indicate the standard error of the mean; n=6. ^{ab Significant} difference in the time spent by rats in keeping still between different groups; P<0.05. Figure 4 shows the concentration of plasma corticosterone levels observed in rats after 4 weeks of chronic mild stress regimen. The untreated control group, in which the rats were left in the cage in an undisturbed manner; the negative control, in which the rats were subjected to chronic gentle pressure and fed with 200 μL of phosphate buffered saline (PBS; pH 7.4); the positive control, in which Rats received chronic mild pressure and provided 200 μL of PBS containing 0.045 mg/kg alprazolam; Lactobacillus fermentum PS150 group, in which rats were subjected to chronic mild pressure and provided 200 μL containing 1 X 10 ⁹ cfu live Lactobacillus fermentum PS150 PBS; Lactobacillus fermentum PS150 protein part group, in which rats are subjected to chronic mild pressure and 200 μL of PBS containing 300 mg/kg protein is provided. Error bars indicate the standard error of the mean; n=6. There was a significant difference in the content of corticosterone in rats between different groups of ^{abcd; P＜0.05.} Figure 5 shows the ratio of plasma TNFα and interleukin-10 levels in rats after 4 weeks of chronic mild pressure regimen. The untreated control group, in which the rats were left in the cage in an undisturbed manner; the negative control, in which the rats were subjected to chronic gentle pressure and fed with 200 μL of phosphate buffered saline (PBS; pH 7.4); the positive control, in which Rats received chronic mild pressure and provided 200 μL of PBS containing 0.045 mg/kg alprazolam; Lactobacillus fermentum PS150 group, in which rats were subjected to chronic mild pressure and provided 200 μL containing 1 X 10 ⁹ cfu live Lactobacillus fermentum PS150 PBS; Lactobacillus fermentum PS150 protein part group, in which rats are subjected to chronic mild pressure and 200 μL of PBS containing 300 mg/kg protein is provided. Error bars indicate the standard error of the mean; n=6. There was a significant difference in the ratio of plasma TNFα and interleukin-10 content between the different groups of ^{abcd; P＜0.05.} Figure 6 (a) and (b) show the memory and cognitive function assessment of rats observed by Morris water maze test and new object recognition test after 4 weeks of chronic mild stress regimen; (a) The escape latency of the rat in the Mohs water maze test; (b) the recognition index of the rat in the new object recognition test. The untreated control group, in which the rats were left in the cage in an undisturbed manner; the negative control, in which the rats were subjected to chronic gentle pressure and fed with 200 μL of phosphate buffered saline (PBS; pH 7.4); the positive control, in which Rats received chronic mild pressure and provided 200 μL of PBS containing 0.045 mg/kg alprazolam; Lactobacillus fermentum PS150 group, in which rats were subjected to chronic mild pressure and provided 200 μL containing 1 X 10 ⁹ cfu live Lactobacillus fermentum PS150 PBS; Lactobacillus fermentum PS150 protein part group, in which rats are subjected to chronic mild pressure and 200 μL of PBS containing 300 mg/kg protein is provided. Error bars indicate the standard error of the mean; n=6. ^{ab Significant} differences in the escape latency (a) and recognition index (b) of rats between different groups; P<0.05. Figure 7 (a), (b) and (c) show the plasma indoleamine-2,3-dioxygenase (IDO) (a), tryptophan (b) in rats after a chronic mild stress regimen for 4 weeks. ) And kynurenine (c). The untreated control group, in which the rats were left in the cage in an undisturbed manner; the negative control, in which the rats were subjected to chronic gentle pressure and fed with 200 μL of phosphate buffered saline (PBS; pH 7.4); the positive control, in which Rats received chronic mild pressure and provided 200 μL of PBS containing 0.045 mg/kg alprazolam; Lactobacillus fermentum PS150 group, in which rats were subjected to chronic mild pressure and provided 200 μL containing 1 X 10 ⁹ cfu live Lactobacillus fermentum PS150 PBS; Lactobacillus fermentum PS150 protein part group, in which rats are subjected to chronic mild pressure and 200 μL of PBS containing 300 mg/kg protein is provided. Error bars indicate the standard error of the mean; n=6. ^{There was a significant} difference in plasma IDO (a), tryptophan (b) and kynurenine (c) concentrations between different groups of abc; P<0.05. Figure 8 shows the brain serotonin concentration of rats after 4 weeks of chronic mild stress regimen. The untreated control group, in which the rats were left in the cage in an undisturbed manner; the negative control, in which the rats were subjected to chronic gentle pressure and fed with 200 μL of phosphate buffered saline (PBS; pH 7.4); the positive control, in which Rats received chronic mild pressure and provided 200 μL of PBS containing 0.045 mg/kg alprazolam; Lactobacillus fermentum PS150 group, in which rats were subjected to chronic mild pressure and provided 200 μL containing 1 X 10 ⁹ cfu live Lactobacillus fermentum PS150 PBS; Lactobacillus fermentum PS150 protein part group, in which rats are subjected to chronic mild pressure and 200 μL of PBS containing 300 mg/kg protein is provided. Error bars indicate the standard error of the mean; n=4. ^ab Significant difference in brain serotonin concentration between different groups; P＜0.05. Figure 9 (a) and (b) show the concentration of monoamine oxidase-A (MAO-A) (a) and hydrogen peroxide (b) in the whole brain of rats after 4 weeks of chronic mild stress regimen. The untreated control group, in which the rats were left in the cage in an undisturbed manner; the negative control, in which the rats were subjected to chronic gentle pressure and fed with 200 μL of phosphate buffered saline (PBS; pH 7.4); the positive control, in which Rats received chronic mild pressure and provided 200 μL of PBS containing 0.045 mg/kg alprazolam; Lactobacillus fermentum PS150 group, in which rats were subjected to chronic mild pressure and provided 200 μL containing 1 X 10 ⁹ cfu live Lactobacillus fermentum PS150 PBS; Lactobacillus fermentum PS150 protein part group, in which rats are subjected to chronic mild pressure and 200 μL of PBS containing 300 mg/kg protein is provided. Error bars indicate the standard error of the mean; n=4. ^{ab Significant} difference in the concentration of whole brain MAO-A (a) and hydrogen peroxide (b) between different groups; P<0.05. Figure 10 shows the reverse phase high performance liquid chromatography (RP-HPLC) elution curve of the protein produced by Lactobacillus fermentum PS150 on the analytical HiTrap Q HP column. At a flow rate of 0.8 mL/min, elution was performed with a linear gradient from 0% to 100% solvent B (25 mM Tris HCl containing 1M NaCl) within 80 minutes. Figure 11 shows the percentage of cell survival rate in SH-SY5Y neuroblastoma cells after dexamethasone-induced neurotoxicity. Positive control, in which SH-SY5Y cells were treated with unfermented MRS; negative control, in which SH-SY5Y cells were treated with unfermented MRS in the presence of dexamethasone (25 µM); the protein part of Lactobacillus fermentum PS150, in which SH-SY5Y cells were treated with the protein part of Lactobacillus fermentum PS150 in the presence of dexamethasone (25 µM); parts A, B, C, D, E, F, G, H and I, of which SH-SY5Y cells were treated with dexamethasone (25 µM) The purified protein fraction collected by HPLC was processed. Error bars indicate the standard error of the mean; n=6. ^Significant difference in the percentage of cell survival between different groups of abc; P＜0.05. Figure 12 shows an SDS-PAGE gel image of purified fraction A from the crude protein fraction of Lactobacillus fermentum PS150. Lane 1: Protein marker; Lane 2: Crude protein fraction of PS150; Lane 3: Purified protein fraction A. Figure 13 (a) and (b) show the effect of PS150 on sleep latency (a) and sleep duration (b) in a pentobarbital-induced sleep test. PBS group, in which mice were fed 200 μL of phosphate buffered saline (PBS; pH 7.4); DIPH-HCL group, in which mice were provided with 200 μL of PBS containing 20 mg/kg diphenhydramine hydrochloride; In the PS150 group, 200 μL ^{of PBS containing 1 X 10 9} cfu live Lactobacillus fermentum PS150 was provided to the mice. Error bars indicate the standard error of the mean; n=10. ^{ab Significant} differences in sleep latency (a) and sleep duration (b) of mice between different groups; P<0.05.

[Advocate the use of biological materials 1] TW Bioresources Conservation and Research Center, Food Industry Development Institute of the Republic of China 2016/11/24 BCRC 910756 [Advocate the use of biological materials 2] DE Germany German Microbial Culture Collection 2016/06/06 DSM 32323

<110> Yifu Biomedical Co., Ltd.

<120> Novel lactic acid bacteria and their applications

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 486

<212> DNA

<213> Novel sequence

<400> 1

<210> 2

<211> 987

<212> DNA

<213> Novel sequence

<400> 2

<210> 3

<211> 1086

<212> DNA

<213> Novel sequence

<400> 3

<210> 4

<211> 396

<212> PRT

<213> EF-Tu protein of PS150

<400> 4

<210> 5

<211> 1540

<212> DNA

<213> 16S rDNA sequence

<400> 5

<210> 6

<211> 401

<212> DNA

<213> Novel sequence

<400> 6

<210> 7

<211> 1190

<212> DNA

<213> Novel sequence

<400> 7

Claims (17)

An isolated lactic acid bacteria (LAB), which is a strain of Lactobacillus fermentum PS150, the strain having any one of the nucleic acid sequences shown in SEQ ID NO: 1 to 3; and the PS150 is used in the food industry The deposit number of the Development Institute is BCRC 910756. For example, the isolated LAB of claim 1, which is Lactobacillus fermentum PS150, has been deposited with the Food Industry Development Institute on November 24, 2016, and the deposit number is BCRC 910756. A composition comprising the LAB of claim 1 or 2, and an edible carrier or a pharmaceutically acceptable carrier as needed. Such as the composition of claim 4, which is a nutritional product, dietary supplement, food or medical product. Such as the composition of claim 4, which is a powder, suspension, granule, lozenge, pill, capsule, drink or dairy product. A use of the LAB as claimed in claim 1 or 2 or the composition as claimed in claim 3, which is used for the manufacture of cells for improving mood disorders or neurological conditions of individuals, and for treating or preventing individual and neuron cells Drugs for diseases related to apoptosis or neurodegeneration. Such as the use of claim 6, wherein the therapeutically or preventively effective amount of the LAB is in the range of about 10 ⁵ to about 10 ¹³ colony forming units (cfu). Such as the use of claim 6, wherein the therapeutically or preventively effective amount of the LAB is in the range of about 10 ⁶ to about 10 ¹² cfu. A use of a composition comprising the elongation factor-Tu (EF-Tu) protein having the amino acid sequence of SEQ ID NO: 4, which is used in the manufacture of a composition for improving affective disorders or neurological conditions of an individual, and treating or preventing an individual It is a drug for diseases related to neuronal apoptosis or neurodegeneration. The use according to claim 9, wherein the therapeutically or preventively effective amount of the EF-Tu protein is in the range of about 15 μg/kg to about 500 μg/kg. Such as the use of any one of claims 6 to 10, wherein the affective disorder and neurological condition is anxiety, depression, stress, sleep disturbance, cognitive decline, cognitive impairment, memory decline, general memory problems, cognitive impairment, or neurodegeneration Sexual disease. Such as the use of claim 11, wherein the cognitive impairment is mild cognitive impairment (MCI). Such as the use of claim 11, where the pressure is chronic mild pressure. Such as the use of claim 11, wherein the affective disorder and neurological condition is anxiety, depression, stress or sleep disturbance. Such as the use of claim 11, wherein the neurodegenerative disease is Alzheimer's disease, Huntington's disease, Parkinson's disease, dementia, muscular atrophy Lateral sclerosis, stroke or schizophrenia. The use according to any one of claims 6 to 10, wherein the disease related to neuronal apoptosis or neurodegeneration is stroke, Alzheimer’s disease, Huntington’s disease, Parkinson’s disease Disease, Pick's disease, Creutzfeldt-Jakob's disease, Parkinson-ALS-Dementia complex, Wilson's disease, multiple sclerosis, progressive supranuclear palsy, and Neuropathic pain-related bipolar disorder, cortical basal degeneration, schizophrenia, attention deficit hyperactivity disorder (ADHD), dementia, amyotrophic lateral sclerosis, retinal disease, epilepsy, apoplexy, transient Ischemic attack, myocardial ischemia, muscle ischemia, ischemia caused by surgical techniques that extend the pause time of blood flow to the brain, head injury, spinal cord injury, hypoxia, or depression. The use of any one of claims 6 to 10, wherein the LAB or the composition comprising LAB or the composition comprising the EF-Tu protein having the amino acid sequence of SEQ ID NO: 4 can reduce indoleamine The performance of 2,3-Dioxygenase (IDO) reduces the conversion rate of tryptophan to kynurenic acid, increases serotonin content or reduces plasma corticosterone content.

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