TWI784326B - Lactobacillus fermentum tci275, composition, and uses thereof - Google Patents
Lactobacillus fermentum tci275, composition, and uses thereof Download PDFInfo
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- TWI784326B TWI784326B TW109134861A TW109134861A TWI784326B TW I784326 B TWI784326 B TW I784326B TW 109134861 A TW109134861 A TW 109134861A TW 109134861 A TW109134861 A TW 109134861A TW I784326 B TWI784326 B TW I784326B
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- lactobacillus fermentum
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Abstract
Description
本發明關於一種發酵乳桿菌，特別是關於一種發酵乳桿菌TCI275（Lactobacillus fermentum TCI275），以及發酵乳桿菌TCI275及其代謝產物的用途。The present invention relates to a lactobacillus fermentum, in particular to a lactobacillus fermentum TCI275 (Lactobacillus fermentum TCI275), and uses of the lactobacillus fermentum TCI275 and metabolites thereof.
益生菌（Probiotics）一般認為是指食入後對宿主（或稱個體，如動物或人類）有正面效益的食入性微生物。其命名源於希臘語「for life」（對生命有益），又稱為「原生保健性菌種」；並且，益生菌主要是指乳酸菌和部分酵母菌。Probiotics are generally considered to refer to ingestible microorganisms that have positive effects on the host (or individual, such as animals or humans) after ingestion. Its name comes from the Greek word "for life" (good for life), also known as "primary health-care bacteria"; and, probiotics mainly refer to lactic acid bacteria and some yeasts.
一般而言，「乳酸菌（Lactic Acid Bacteria）」是指能利用碳水化合物進行發酵生產多量乳酸之細菌總稱，為一相當龐雜的菌群。在諸多乳酸菌的菌種中，部分乳酸菌菌種的菌株經研究發現其對於個體健康有益，故此些菌株被視為益生菌。換言之，益生菌大部分是乳酸菌，但乳酸菌中只有少數經研究證明對個體健康有益的菌株能被稱為益生菌。Generally speaking, "Lactic Acid Bacteria" refers to the general term for bacteria that can use carbohydrates to ferment and produce large amounts of lactic acid. It is a rather complex flora. Among the many strains of lactic acid bacteria, some strains of lactic acid bacteria have been found to be beneficial to individual health through research, so these strains are regarded as probiotics. In other words, most of the probiotics are lactic acid bacteria, but only a few strains of lactic acid bacteria that have been proven to be beneficial to individual health can be called probiotics.
舉例來說，常見的可作為益生菌的乳酸菌的菌種包括腸球菌屬（Enterococcus）、乳桿菌屬（Lactobacillus）、雙岐桿菌屬（Bifidobacterium）、芽孢桿菌屬（Bacillus）等，而可作為益生菌的酵母菌的菌種包括酵母菌屬（Saccharomyces）。For example, common lactic acid bacteria that can be used as probiotics include Enterococcus, Lactobacillus, Bifidobacterium, Bacillus, etc., which can be used as probiotics The species of yeast include Saccharomyces.
乳桿菌屬是一群具有發酵能力、兼性厭氧、不會產生孢子的革蘭氏陽性桿菌。乳桿菌因能夠將碳水化合物發酵成乳酸而得名，可用於製造液態優酪乳、固態奶酪等各種發酵食品。Lactobacillus is a group of fermentative, facultatively anaerobic, non-spore-producing, Gram-positive bacilli. Lactobacillus is named for its ability to ferment carbohydrates into lactic acid, and can be used to make various fermented foods such as liquid yogurt and solid cheese.
本發明之一目的，在於提供一種發酵乳桿菌（Lactobacillus fermentum ）TCI275，其係寄存於財團法人食品工業發展研究所（寄存編號BCRC910940）及德國國家菌種保藏中心（DSMZ）（寄存編號DSM33289）。One object of the present invention is to provide a kind of Lactobacillus fermentum TCI275, which is deposited in the Institute of Food Industry Development (Deposit No. BCRC910940) and the German National Culture Collection Center (DSMZ) (Deposit No. DSM33289).
在一些實施例中，發酵乳桿菌TCI275包含如SEQ ID NO:1所示的序列。In some embodiments, Lactobacillus fermentum TCI275 comprises the sequence shown in SEQ ID NO:1.
在一些實施例中，發酵乳桿菌TCI275篩選自人類母乳。In some embodiments, Lactobacillus fermentum TCI275 is selected from human breast milk.
在一些實施例中，發酵乳桿菌具有生成羥脯胺酸之功能。In some embodiments, Lactobacillus fermentum has the function of producing hydroxyproline.
在一些實施例中，發酵乳桿菌具有耐胃酸及耐膽鹽的能力。In some embodiments, the Lactobacillus fermentum has the ability to tolerate gastric acid and bile salts.
在一些實施例中，發酵乳桿菌具有分解乳糖的能力。In some embodiments, the Lactobacillus fermentum has the ability to decompose lactose.
本發明之另一目的，在於提供一種組合物，包含一發酵乳桿菌（Lactobacillus fermentum ）TCI275及／或其代謝產物，發酵乳桿菌係寄存於財團法人食品工業發展研究所（寄存編號BCRC910940）及德國國家菌種保藏中心（DSMZ）（寄存編號DSM33289）。Another object of the present invention is to provide a composition comprising a Lactobacillus fermentum ( Lactobacillus fermentum ) TCI275 and/or its metabolites. The Lactobacillus fermentum is deposited in the Food Industry Development Research Institute (Deposit No. BCRC910940) and Germany National Culture Collection (DSMZ) (Deposit No. DSM33289).
在一些實施例中，組合物為食品組合物。In some embodiments, the composition is a food composition.
在一些實施例中，組合物含有5X109 個菌數的發酵乳桿菌TCI275。In some embodiments, the composition contains 5× 10 9 counts of Lactobacillus fermentum TCI275.
根據一些實施例，本發明提供一種將組合物用於製備改善乳糖不耐（lactose intolerance）的組合物的用途。According to some embodiments, the present invention provides a use of the composition for preparing a composition for improving lactose intolerance.
在一些實施例中，改善乳糖不耐為包括以下情況的任一者：改善大便稀淌、減少排氣頻率、減少脹氣、減少腹痛。In some embodiments, improving lactose intolerance is any of the following conditions: improving loose stools, reducing frequency of flatulence, reducing flatulence, and reducing abdominal pain.
根據一些實施例，本發明提供一種將組合物用於製備改善便祕的組合物的用途。According to some embodiments, the present invention provides a use of the composition for preparing a composition for improving constipation.
在一些實施例中，改善便祕為提升排便頻率。In some embodiments, improving constipation is increasing bowel frequency.
根據一些實施例，本發明提供一種將組合物用於製備提升一個體的腸胃中的柔嫩梭菌屬（Faecalibacterium）菌種比例的組合物的用途。According to some embodiments, the present invention provides a use of a composition for the preparation of a composition for increasing the proportion of Faecalibacterium species in the gut of an individual.
在一些實施例中，個體對為對乳糖不耐的個體。In some embodiments, the pair of individuals are individuals who are lactose intolerant.
根據一些實施例，本發明提供一種將組合物用於製備提升一個體的腸胃中的普雷沃氏菌屬（Prevotella）菌種比例的組合物的用途。According to some embodiments, the present invention provides a use of a composition for the preparation of a composition for increasing the proportion of Prevotella species in the gut of an individual.
在一些實施例中，個體為對具有便秘情況的個體。In some embodiments, the subject is a subject with constipation.
本發明之詳細技術內容及部分具體實施態樣，將描述於以下內容中，以供本發明所屬領域具通常知識者據以明瞭本發明之特徵。The detailed technical content and some specific implementation aspects of the present invention will be described in the following content, so that those skilled in the art of the present invention can understand the characteristics of the present invention.
為了使本領域具有通常知識者能瞭解本發明的特點，以下就說明書及申請專利範圍中提及術語及用語進行一般性之說明及定義。除非另有說明，否則文中使用的所有技術及科學上的字詞，皆具有本領域技術人員對於本發明所瞭解的通常意義，當有衝突情形時，應以本說明書之定義為準。In order to enable those skilled in the art to understand the features of the present invention, the terms and terms mentioned in the specification and scope of patent application are generally explained and defined below. Unless otherwise stated, all technical and scientific terms used herein have the usual meanings understood by those skilled in the art for the present invention. In case of conflict, the definitions in this specification shall prevail.
本案使用Excel軟體進行統計分析。數據以平均值±標準差（SD）表示，各組之間的差異以學生t 檢驗（student'st -test）進行分析。In this case, Excel software was used for statistical analysis. Data are presented as mean ± standard deviation (SD), and differences among groups were analyzed using Student's t -test .
本文中所使用之濃度符號「wt %」通常是指重量百分濃度，而濃度符號「vol %」通常是指體積百分濃度。The concentration symbol "wt %" used herein generally refers to weight percent concentration, and the concentration symbol "vol %" generally refers to volume percent concentration.
本文中所使用數值為近似值，所有實驗數據皆表示在正負10%的範圍內，最佳為在正負5%的範圍內。The values used in this article are approximate values, and all experimental data are expressed within the range of plus or
於本說明書中(尤其是在後述專利申請範圍中)所使用之 “個體”是指人類或非人的哺乳動物，較佳為人類。The term "individual" used in this specification (especially in the scope of the following patent applications) refers to human beings or non-human mammals, preferably human beings.
於本說明書中(尤其是在後述專利申請範圍中)所使用之 “分解乳糖”是指將乳糖分解為葡萄糖及半乳糖或是指具有乳糖酶的能力。"Decomposition of lactose" used in this specification (especially in the scope of the patent application described below) refers to decomposing lactose into glucose and galactose or having the ability of lactase.
本案發明人自人體母乳中篩選出一新穎菌株，經16S核糖體核糖核酸（16S rRNA）序列分析，依親緣關係鑑定為Lactobacillus fermentum（中文名稱為發酵乳桿菌），且經命名為Lactobacillus fermentum TCI275（以下稱為發酵乳桿菌TCI275），其係寄存於財團法人食品工業發展研究所（寄存編號為BCRC910940）、及德國國家菌種保藏中心（DSMZ）（寄存編號為DSM33289）。該發酵乳桿菌TCI275係具有如SEQ ID NO: 1所示之16S核糖體核糖核酸（16S rRNA）片段。The inventor of this case screened out a novel strain from human breast milk, and identified it as Lactobacillus fermentum (Chinese name is Lactobacillus fermentum) according to the genetic relationship through 16S ribosomal ribonucleic acid (16S rRNA) sequence analysis, and named it Lactobacillus fermentum TCI275 ( Hereinafter referred to as Lactobacillus fermentum TCI275), which is deposited in the Food Industry Development Research Institute of the Foundation (Deposit No. BCRC910940) and the German National Culture Collection (DSMZ) (Deposit No. DSM33289). The Lactobacillus fermentum TCI275 has a 16S ribosomal ribonucleic acid (16S rRNA) fragment as shown in SEQ ID NO: 1.
根據本發明，所採用之發酵乳桿菌TCI275之代謝產物可以是透過在適於發酵乳桿菌TCI275生長的環境下進行培養所產生者。例如：先以適當之培養液對發酵乳桿菌TCI275進行培養，其後，視需要地去除前述培養液中之菌體等固體物，以獲得含有代謝產物之液體。或例如，先以適當之培養液對發酵乳桿菌TCI275進行培養，其後，以離心等方法使菌體沉降於底部，再直接吸取上清液所獲得含有代謝產物之液體。According to the present invention, the metabolites of Lactobacillus fermentum TCI275 used may be produced by culturing in an environment suitable for the growth of Lactobacillus fermentum TCI275. For example: Lactobacillus fermentum TCI275 is first cultured with an appropriate culture solution, and then, if necessary, solids such as bacteria in the culture solution are removed to obtain a liquid containing metabolites. Or, for example, first culture Lactobacillus fermentum TCI275 with an appropriate culture medium, then settle the bacteria to the bottom by centrifugation, and then directly absorb the supernatant to obtain a liquid containing metabolites.
可選用任意合宜之培養液以進行發酵乳桿菌TCI275之培養，以提供所欲之代謝產物，只要該培養液可提供發酵乳桿菌TCI275生長、代謝所需養分（如酵母菌萃取物、蛋白質及葡萄糖）與條件（如酸鹼值）即可。此外，發酵乳桿菌TCI275之培養時間亦無特殊限制，只要足以使發酵乳桿菌TCI275完成至少一次代謝循環即可。舉例言之，於本發明一具體實施態樣中，係以MRS培養液對發酵乳桿菌TCI275進行培養，歷時18小時，使菌株進行代謝作用並產生代謝產物。Any suitable culture medium can be used for the cultivation of Lactobacillus fermentum TCI275 to provide the desired metabolites, as long as the culture medium can provide the nutrients (such as yeast extract, protein and glucose) required for the growth and metabolism of Lactobacillus fermentum TCI275 ) and conditions (such as pH value). In addition, the culture time of Lactobacillus fermentum TCI275 is not particularly limited, as long as it is enough for Lactobacillus fermentum TCI275 to complete at least one metabolic cycle. For example, in a specific embodiment of the present invention, Lactobacillus fermentum TCI275 is cultured with MRS culture medium for 18 hours, so that the strain undergoes metabolism and produces metabolites.
於本發明中，係可直接使用經歷發酵乳桿菌TCI275之代謝循環的含發酵乳桿菌TCI275與代謝產物的培養液，或使用經移除發酵乳桿菌TCI275等固體物之含代謝產物的液體。可採用任何合宜之操作以移除固體物，只要對培養後所產生的代謝產物之所欲效益沒有不利的影響即可。一般而言，係採用物理手段以移除固體物，該物理手段包括，例如：離心分離、濾膜過濾、沉澱傾析等操作。視需要地，可重複或合併進行前述物理操作，以盡可能去除培養液中的菌體等固體物。In the present invention, the culture solution containing Lactobacillus fermentum TCI275 and metabolites that have undergone the metabolic cycle of Lactobacillus fermentum TCI275 can be used directly, or the liquid containing metabolites after removing solids such as Lactobacillus fermentum TCI275 can be used. Any convenient procedure may be used to remove the solids so long as it does not adversely affect the desired benefit of the metabolites produced after incubation. Generally speaking, physical means are used to remove solids, such physical means include, for example, operations such as centrifugation, membrane filtration, and sedimentation and decantation. If necessary, the aforementioned physical operations can be repeated or combined to remove solids such as bacteria in the culture solution as much as possible.
根據本發明所提供之食品組合物係可以為健康食品、保健食品、機能性食品、營養補充品、或特殊營養食品，且可製成例如乳製品、肉類加工品、麵包類、麵食類、餅乾、口含錠、膠囊、果汁類、茶類、運動飲料、營養飲料等產品，但不以此為限。The food composition provided according to the present invention can be health food, health food, functional food, nutritional supplement, or special nutritional food, and can be made into dairy products, processed meat products, bread, pasta, biscuits, etc. , buccal tablets, capsules, fruit juices, teas, sports drinks, nutritional drinks and other products, but not limited thereto.
根據本發明所提供之健康食品、保健食品、機能性食品、營養補充品、及特殊營養食品係可以一日一次、一日多次、或數日一次等不同頻率食用，端視投予個體之年齡、體重、及健康狀況而異。亦可針對特定族群之需要，調整據本發明所提供之健康食品、保健食品、機能性食品、營養補充品、及特殊營養食品中的發酵乳桿菌TCI275及／或其代謝產物的含量，例如，調整至每日應服用的量。The health food, health food, functional food, nutritional supplement, and special nutritional food provided by the present invention can be eaten at different frequencies, such as once a day, multiple times a day, or once a few days, depending on the individual administered. Varies depending on age, weight, and health status. It is also possible to adjust the content of Lactobacillus fermentum TCI275 and/or its metabolites in the health food, health food, functional food, nutritional supplement, and special nutritional food provided by the present invention according to the needs of specific ethnic groups, for example, Adjust to the amount that should be taken daily.
針對根據本發明所提供之健康食品、保健食品、機能性食品、營養補充品及／或特殊營養食品，可於其外包裝上標示建議使用量、特定族群（例如高血脂患者、孕婦等）的使用標準及條件、或與其他食品或醫藥共同服用的建議事項等，以利使用者在無醫師、藥師或相關執事人員的指導下自行服用而無安全虞慮。於根據本發明所提供之食品組合物中，有關該發酵乳桿菌TCI275及／或其代謝產物之態樣，係如上述之說明。For the health food, health food, functional food, nutritional supplement and/or special nutritional food provided according to the present invention, the recommended dosage, specific group (such as hyperlipidemia patients, pregnant women, etc.) Use standards and conditions, or suggestions for co-administration with other foods or medicines, etc., so that users can take it without the guidance of a doctor, pharmacist or related deacons without safety concerns. In the food composition provided according to the present invention, the appearance of the Lactobacillus fermentum TCI275 and/or its metabolites is as described above.
視需要地，可於根據本發明所提供之食品組合物、或飼料組合物中另外含有合宜用量之添加劑，例如可提高該醫藥組合物、食品組合物、或飼料組合物於服用時的口適感及視覺感受之調味劑、調色劑、著色劑等，以及可改善該醫藥組合物、食品組合物、或飼料組合物的穩定性及儲存性之緩衝劑、保存劑、防腐劑、抗菌劑、抗真菌劑等。Optionally, the food composition or feed composition provided by the present invention may additionally contain an appropriate amount of additives, such as improving the mouthfeel of the pharmaceutical composition, food composition, or feed composition when ingested. Flavoring agents, toners, colorants, etc. for sensory and visual perception, as well as buffers, preservatives, preservatives, and antibacterial agents that can improve the stability and storage of the pharmaceutical composition, food composition, or feed composition , antifungal agents, etc.
茲以下列實施例進一步例示說明本發明。其中該等實施例僅提供作為說明，而非用以限制本發明之保護範圍。本發明保護範圍係如後附申請專利範圍所示。The present invention is further illustrated by the following examples. These examples are provided for illustration only, and are not intended to limit the protection scope of the present invention. The scope of protection of the present invention is shown in the appended patent scope.
例1：發酵乳桿菌TCI275之篩選與鑑定Example 1: Screening and identification of Lactobacillus fermentum TCI275
（1-1）篩選(1-1) Screening
將各種取樣樣品（人類母乳、優酪乳、印度天貝（Tempeh）、牛糞）分別與MRS培養液（MRS broth）以體積比1：100的比例混合，並置於37℃下進行厭氧培養（氧氣濃度介於10%至1%，較佳為低於5%），歷時18小時。其後，將前述培養液塗佈於MRS固態培養基（MRS agar）上，並置於37℃下進行厭氧培養，以生成不同的菌落。接著，為確保菌株的單一性，使用無菌接種環挑選一菌落，於固態培養基上進行分區劃線，再置於37°C之厭氧培養箱中培養直到單一菌落（single colony）出現。Various sampling samples (human breast milk, yogurt, Indian Tempeh, cow dung) were mixed with MRS broth (MRS broth) at a volume ratio of 1:100, and placed at 37°C for anaerobic culture ( Oxygen concentration between 10% and 1%, preferably less than 5%) for 18 hours. Thereafter, the aforementioned culture solution was spread on MRS solid medium (MRS agar), and placed at 37° C. for anaerobic culture to generate different colonies. Next, in order to ensure the singleness of the strain, a colony was selected using a sterile inoculation loop, marked on the solid medium, and then cultured in an anaerobic incubator at 37°C until a single colony (single colony) appeared.
（1-2）鑑定(1-2) Identification
取（1-1）人類母乳取樣樣品所分離出之單一菌落的菌株後，進行基因族譜分析（phylogenetic analysis），確認該菌株具有如SEQ ID NO:1所示之16S核糖體核糖核酸（16S rRNA）片段。使用NCBI（National Center for Biotechnology Information）線上資料庫進行比對，將SEQ ID NO:1所示的基因序列分別與其他發酵乳桿菌亞種之16SrDNA序列進行序列比對後，可知分離菌株的16SrDNA序列與其他發酵乳桿菌亞種的16SrDNA序列的相似性如表一所示。因此，將此分離菌株命名為發酵乳桿菌TCI275（後文以TCI275表示）。在此，將此發酵乳桿菌TCI275以寄存編號BCRC910940寄存於財團法人食品工業發展研究所，以及以寄存編號DSM33289寄存於德國國家菌種保藏中心（DSMZ）。After taking (1-1) the strain of a single colony isolated from the human breast milk sampling sample, the gene family analysis (phylogenetic analysis) was performed to confirm that the strain has 16S ribosomal ribonucleic acid (16S rRNA) as shown in SEQ ID NO:1 ) fragment. Using the NCBI (National Center for Biotechnology Information) online database for comparison, after comparing the gene sequence shown in SEQ ID NO: 1 with the 16SrDNA sequence of other Lactobacillus fermentum subspecies, the 16SrDNA sequence of the isolated strain can be known The similarities with the 16SrDNA sequences of other Lactobacillus fermentum subspecies are shown in Table 1. Therefore, this isolated strain was named Lactobacillus fermentum TCI275 (hereinafter expressed as TCI275). Herein, the Lactobacillus fermentum TCI275 is deposited with the Institute for Development of the Food Industry under the deposit number BCRC910940, and with the German National Culture Collection (DSMZ) under the deposit number DSM33289.
取（1-1）優酪乳取樣樣品所分離出之一個菌落的菌株後，進行基因族譜分析，確認該菌株具有如SEQ ID NO:2所示之16S核糖體核糖核酸（16S rRNA）片段。使用NCBI線上資料庫進行比對，將SEQ ID NO: 2所示的基因序列分別與其他發酵乳桿菌亞種之16S rDNA序列進行序列比對後，可知分離菌株的16SrDNA序列與其他發酵乳桿菌亞種的16SrDNA序列的相似性如表一所示。在此，將此分離菌株命名為發酵乳桿菌L206（後文以L206表示）。After taking (1-1) a strain of a colony isolated from the yogurt sampling sample, gene family analysis was performed to confirm that the strain has a 16S ribosomal ribonucleic acid (16S rRNA) fragment as shown in SEQ ID NO:2. Using the NCBI online database for comparison, after comparing the gene sequence shown in SEQ ID NO: 2 with the 16S rDNA sequences of other Lactobacillus fermentum subspecies, it can be known that the 16S rDNA sequence of the isolated strain is similar to that of other Lactobacillus fermentum subspecies. The similarity of the 16SrDNA sequences of the two species is shown in Table 1. Here, the isolated strain is named as Lactobacillus fermentum L206 (hereinafter referred to as L206).
其中，取（1-1）優酪乳取樣樣品所分離出之再另一個菌落的菌株後，進行基因族譜分析，確認該菌株具有如SEQ ID NO:3所示之16S核糖體核糖核酸（16S rRNA）片段。使用NCBI線上資料庫進行比對，將SEQ ID NO: 3所示的基因序列分別與其他發酵乳桿菌亞種之16SrDNA序列進行序列比對後，可知分離菌株的16SrDNA序列與其他發酵乳桿菌亞種的16SrDNA序列的相似性如表一所示。在此，將此分離菌株命名為發酵乳桿菌L235（後文以L235表示）。Among them, after taking (1-1) the strain of another colony isolated from the yogurt sampling sample, the gene family analysis was carried out to confirm that the strain has 16S ribosomal ribonucleic acid (16S ribonucleic acid) as shown in SEQ ID NO:3 rRNA) fragments. Using the NCBI online database for comparison, after comparing the gene sequence shown in SEQ ID NO: 3 with the 16SrDNA sequences of other Lactobacillus fermentum subspecies, it can be known that the 16SrDNA sequence of the isolated strain is similar to that of other Lactobacillus fermentum subspecies The similarity of the 16S rDNA sequence is shown in Table 1. Here, the isolated strain is named as Lactobacillus fermentum L235 (hereinafter referred to as L235).
其中，取（1-1）優酪乳取樣樣品所分離出之再另一個菌落的菌株後，進行基因族譜分析，確認該菌株具有如SEQ ID NO:4所示之16S核糖體核糖核酸（16S rRNA）片段。使用NCBI線上資料庫進行比對，將SEQ ID NO: 4所示的基因序列分別與其他發酵乳桿菌亞種之16SrDNA序列進行序列比對後，可知分離菌株的16SrDNA序列與其他發酵乳桿菌亞種的16SrDNA序列的相似性如表一所示。因此，將此分離菌株命名為發酵乳桿菌L270（後文以L270表示）。Among them, after taking (1-1) the strain of another colony isolated from the yogurt sampling sample, the gene family analysis was carried out to confirm that the strain has 16S ribosomal ribonucleic acid (16S ribonucleic acid) as shown in SEQ ID NO:4 rRNA) fragments. Using the NCBI online database for comparison, after comparing the gene sequence shown in SEQ ID NO: 4 with the 16SrDNA sequences of other Lactobacillus fermentum subspecies, it can be known that the 16SrDNA sequence of the isolated strain is similar to that of other Lactobacillus fermentum subspecies The similarity of the 16S rDNA sequence is shown in Table 1. Therefore, this isolated strain was named as Lactobacillus fermentum L270 (hereinafter referred to as L270).
其中，取（1-1）優酪乳取樣樣品所分離出之再另一個菌落的菌株後，進行基因族譜分析，確認該菌株具有如SEQ ID NO:5所示之16S核糖體核糖核酸（16S rRNA）片段。使用NCBI線上資料庫進行比對，將SEQ ID NO: 5所示的基因序列分別與其他發酵乳桿菌亞種之16SrDNA序列進行序列比對後，可知分離菌株的16SrDNA序列與其他發酵乳桿菌亞種的16SrDNA序列的相似性如表一所示。因此，將此分離菌株命名為發酵乳桿菌L786（後文以L786表示）。Among them, after taking (1-1) the strain of another colony isolated from the yogurt sampling sample, the gene family analysis was carried out to confirm that the strain has 16S ribosomal ribonucleic acid (16S ribonucleic acid) as shown in SEQ ID NO:5 rRNA) fragments. Using the NCBI online database for comparison, after comparing the gene sequence shown in SEQ ID NO: 5 with the 16SrDNA sequences of other Lactobacillus fermentum subspecies, it can be known that the 16SrDNA sequence of the isolated strain is similar to that of other Lactobacillus fermentum subspecies The similarity of the 16S rDNA sequence is shown in Table 1. Therefore, this isolated strain was named as Lactobacillus fermentum L786 (hereinafter referred to as L786).
其中，取（1-1）印度天貝取樣樣品所分離出之一個菌落的菌株後，進行基因族譜分析，確認該菌株具有如SEQ ID NO:6所示之16S核糖體核糖核酸（16S rRNA）片段。使用NCBI線上資料庫進行比對，將SEQ ID NO: 6所示的基因序列分別與其他發酵乳桿菌亞種之16SrDNA序列進行序列比對後，可知分離菌株的16SrDNA序列與其他發酵乳桿菌亞種的16SrDNA序列的相似性如表一所示。因此，將此分離菌株命名為發酵乳桿菌L849（後文以L849表示）。Among them, after taking (1-1) a strain of a colony isolated from the Indian tempeh sampling sample, the gene family analysis was performed to confirm that the strain has 16S ribosomal ribonucleic acid (16S rRNA) as shown in SEQ ID NO:6 fragment. Using the NCBI online database for comparison, after comparing the gene sequence shown in SEQ ID NO: 6 with the 16SrDNA sequences of other Lactobacillus fermentum subspecies, it can be known that the 16SrDNA sequence of the isolated strain is similar to that of other Lactobacillus fermentum subspecies The similarity of the 16S rDNA sequence is shown in Table 1. Therefore, this isolated strain was named as Lactobacillus fermentum L849 (hereinafter referred to as L849).
其中，取（1-1）牛糞取樣樣品所分離出之一個菌落的菌株後，進行基因族譜分析，確認該菌株具有如SEQ ID NO:7所示之16S核糖體核糖核酸（16S rRNA）片段。使用NCBI線上資料庫進行比對，將SEQ ID NO: 7所示的基因序列分別與其他發酵乳桿菌亞種之16SrDNA序列進行序列比對後，可知分離菌株的16SrDNA序列與其他發酵乳桿菌亞種的16SrDNA序列的相似性如表一所示。因此，將此分離菌株命名為發酵乳桿菌L1265（後文以L1275表示）。Among them, after taking (1-1) a strain of a colony isolated from the cow dung sampling sample, the gene family analysis was performed to confirm that the strain has a 16S ribosomal ribonucleic acid (16S rRNA) fragment as shown in SEQ ID NO:7. Using the NCBI online database for comparison, after comparing the gene sequence shown in SEQ ID NO: 7 with the 16SrDNA sequences of other Lactobacillus fermentum subspecies, it can be known that the 16SrDNA sequence of the isolated strain is similar to that of other Lactobacillus fermentum subspecies The similarity of the 16S rDNA sequence is shown in Table 1. Therefore, this isolated strain was named Lactobacillus fermentum L1265 (hereinafter expressed as L1275).
其中，取（1-1）牛糞取樣樣品所分離出之另一個菌落的菌株後，進行基因族譜分析，確認該菌株具有如SEQ ID NO:8所示之16S核糖體核糖核酸（16S rRNA）片段。使用NCBI線上資料庫進行比對，將SEQ ID NO: 8所示的基因序列分別與其他發酵乳桿菌亞種之16SrDNA序列進行序列比對後，可知分離菌株的16SrDNA序列與其他發酵乳桿菌亞種的16SrDNA序列的相似性如表一所示。因此，將此分離菌株命名為發酵乳桿菌L1275（後文以L1275表示）。Among them, after taking (1-1) the strain of another colony isolated from the cow dung sampling sample, the gene family analysis was performed to confirm that the strain has a 16S ribosomal ribonucleic acid (16S rRNA) fragment as shown in SEQ ID NO:8 . Using the NCBI online database for comparison, after comparing the gene sequence shown in SEQ ID NO: 8 with the 16SrDNA sequences of other Lactobacillus fermentum subspecies, it can be known that the 16SrDNA sequence of the isolated strain is similar to that of other Lactobacillus fermentum subspecies The similarity of the 16S rDNA sequence is shown in Table 1. Therefore, this isolated strain was named as Lactobacillus fermentum L1275 (hereinafter referred to as L1275).
表一
綜合以上，本發明自取樣樣品（人類母乳、優酪乳、印度天貝（Tempeh）、牛糞）共篩選出八種發酵乳桿菌，分別命名為TCI275、L206、L235、L270、L786、L849、L1265、L1275。Based on the above, the present invention screened out eight kinds of Lactobacillus fermentum from the sampling samples (human breast milk, yogurt, Indian tempeh, cow dung), and named them TCI275, L206, L235, L270, L786, L849, L1265 respectively , L1275.
（1-3）保存(1-3) save
使用液態培養的方式將（1-1）所獲得之具單一性之八種發酵乳桿菌菌株培養於MRS培養液（BD Difco™ Lactobacilli MRS Broth，型號DF0881-17-5）中，以提供菌液，接著於菌液中添加25%之甘油，將所得混合液移至冷凍保存管中後，置於-80℃保存。The eight single Lactobacillus fermentum strains obtained in (1-1) were cultured in MRS culture medium (BD Difco™ Lactobacilli MRS Broth, model DF0881-17-5) by means of liquid culture to provide bacterial liquid , and then add 25% glycerol to the bacterial solution, transfer the resulting mixture to a cryopreservation tube, and store it at -80°C.
例2：發酵乳桿菌TCI275耐胃酸膽鹽試驗Example 2: Lactobacillus fermentum TCI275 resistance to gastric acid bile salt test
於此，將發酵乳桿菌TCI275以緩衝液、人工胃液（pH 3）及人工腸液（pH 7）進行測試，以確認發酵乳桿菌TCI275於生物體腸胃消化道中的酸鹼耐受性。其中，緩衝液為0.2莫爾濃度（M）氯化鉀（KCL, 品牌Sigma-Aldrich）且pH值為7。人工胃液為0.2 M氯化鉀且pH值為3。人工腸液為0.2 M氯化鉀及0.3重量%的牛膽鹽（品牌Difco™ Oxgall）且pH值為7。Herein, Lactobacillus fermentum TCI275 was tested with buffer solution, artificial gastric juice (pH 3) and artificial intestinal juice (pH 7) to confirm the acid-base tolerance of Lactobacillus fermentum TCI275 in the gastrointestinal tract of organisms. Wherein, the buffer solution is 0.2 molar concentration (M) potassium chloride (KCL, brand Sigma-Aldrich) and the pH value is 7. Artificial gastric juice was 0.2 M potassium chloride and had a pH of 3. The artificial intestinal fluid was 0.2 M potassium chloride and 0.3 wt% ox bile salts (brand Difco™ Oxgall) and had a pH of 7.
進行一活化程序將發酵乳桿菌TCI275活化。首先，將發酵乳桿菌TCI275凍菌以10體積%的菌量培養於液態乳桿菌MRS培養基（BD Difco™ Lactobacilli MRS Broth）中，並於37下培養16小時，以活化發酵乳桿菌TCI275。An activation procedure was performed to activate Lactobacillus fermentum TCI275. First, the frozen Lactobacillus fermentum TCI275 was cultured in liquid Lactobacillus MRS medium (BD Difco™ Lactobacilli MRS Broth) at 10% by volume, and cultured at 37 for 16 hours to activate Lactobacillus fermentum TCI275.
接著，將活化後的發酵乳桿菌TCI275菌液取1體積%的濃度接種至20毫升的待測溶液中，然後以轉速50rpm於37℃下震盪培養3小時。其中，三個組別的待測溶液分別為人工胃液（實驗組）、人工腸液（實驗組）及氯化鉀緩衝液（控制組）。從培養後的菌液取100微升（μL）的菌液塗盤，並於28℃下靜置培養3天，並於3天後以肉眼計數各組別的活菌數（計算固態乳桿菌MRS培養基上的菌落數）。Next, the activated Lactobacillus fermentum TCI275 bacterial solution was inoculated into 20 ml of the test solution at a concentration of 1% by volume, and then cultured with shaking at 37° C. for 3 hours at a rotation speed of 50 rpm. Among them, the solutions to be tested in the three groups were artificial gastric juice (experimental group), artificial intestinal juice (experimental group) and potassium chloride buffer solution (control group). Take 100 microliters (μL) of the bacterial solution from the cultured bacterial solution and spread it on the plate, and culture it statically at 28°C for 3 days, and count the number of viable bacteria in each group with the naked eye after 3 days (calculate the number of solid Lactobacillus number of colonies on MRS medium).
請參閱圖1。圖式中發酵乳桿菌TCI275的存活率為計數後的活菌數，並以log CFU/mL表示。其中，log CFU/mL代表每毫升菌液含有的菌落形成單位（CFU，colony-forming unit）並以對數（log10 ）表示。由圖1可知，控制組的發酵乳桿菌TCI275的存活菌數為9.7 log CFU/mL、人工胃液實驗組的發酵乳桿菌TCI275的存活菌數為9.1 log CFU/mL，及人工腸液實驗組的發酵乳桿菌TCI275的存活菌數為9.2 log CFU/mL。由此可知，人工胃液實驗組的活菌數為控制組的93.8%，且人工腸液實驗組的活菌數為控制組的94.8%。See Figure 1. In the graph, the survival rate of Lactobacillus fermentum TCI275 is the number of live bacteria after counting, and expressed in log CFU/mL. Among them, log CFU/mL represents the colony-forming unit (CFU, colony-forming unit) contained in each milliliter of bacterial liquid and expressed in logarithm (log 10 ). It can be seen from Figure 1 that the number of surviving bacteria of Lactobacillus fermentum TCI275 in the control group was 9.7 log CFU/mL, the number of surviving bacteria of Lactobacillus fermentum TCI275 in the artificial gastric juice experimental group was 9.1 log CFU/mL, and the fermentation of artificial intestinal juice experimental group The viable count of Lactobacillus TCI275 was 9.2 log CFU/mL. It can be seen that the number of viable bacteria in the artificial gastric juice experimental group was 93.8% of that of the control group, and the number of viable bacteria in the artificial intestinal juice experimental group was 94.8% of that of the control group.
基於此實驗數據，得知發酵乳桿菌TCI275可耐pH值為3的模擬胃酸環境並可耐膽鹽模擬環境（人工腸液），因此，得知發酵乳桿菌TCI275具有耐胃酸膽鹽之功能，代表發酵乳桿菌TCI275可通過宿主腸胃道的酸鹼值壓力，定殖於宿主腸道中。Based on the experimental data, it is known that Lactobacillus fermentum TCI275 can withstand the simulated gastric acid environment with a pH value of 3 and the simulated bile salt environment (artificial intestinal fluid). Lactobacillus fermentum TCI275 can colonize the host intestine through the pH pressure of the host's gastrointestinal tract.
例3：發酵乳桿菌TCI275及其他發酵乳桿菌的乳糖分解能力比較Example 3: Comparison of the lactose decomposition ability of Lactobacillus fermentum TCI275 and other Lactobacillus fermentum
在此實施例中，將TCI275及其他發酵乳桿菌培養於含有乳糖的培養基中，以測試發酵乳桿菌分解乳糖的能力。In this example, TCI275 and other Lactobacillus fermentum were cultured in a medium containing lactose to test the ability of Lactobacillus fermentum to decompose lactose.
首先，進行一活化程序將發酵乳桿菌TCI275及其他發酵乳桿菌（L206、L235、L270、L786、L849、L1265、L1275）活化。首先，將發酵乳桿菌TCI275及其他發酵乳桿菌凍菌以10體積%的菌量培養於液態乳桿菌MRS培養基（BD Difco™ Lactobacilli MRS Broth）中，並於37度下培養16小時，以活化發酵乳桿菌TCI275及其他發酵乳桿菌。First, an activation procedure was performed to activate Lactobacillus fermentum TCI275 and other Lactobacillus fermentum (L206, L235, L270, L786, L849, L1265, L1275). First, culture Lactobacillus fermentum TCI275 and other frozen Lactobacillus fermentum in liquid Lactobacillus MRS medium (BD Difco™ Lactobacilli MRS Broth) at 10% by volume, and culture them at 37 degrees for 16 hours to activate fermentation Lactobacillus TCI275 and other Lactobacillus fermentum.
接著，將活化後的發酵乳桿菌TCI275及其他發酵乳桿菌的菌液取1體積%的濃度接種至含有5g/L（等於0.5%）濃度乳糖的20毫升的液態乳桿菌MRS培養基（BD Difco™ Lactobacilli MRS Broth）中，然後於37℃下培養16小時，並進行取樣程序，取樣程序包括：取1ml菌液（共八組），以離心（10000至20000rpm，較佳為10000至15000rpm，更佳為 13000rpm）1分鐘後取上清液（不含菌體，即為發酵乳桿菌TCI275的代謝產物），以乳糖比色法測定試劑套組（廠牌：BioVision，檢測套組名稱BioVision® Lactose Colorimetric/Fluorometric Assay Kit，產品編號K624-100）進行乳糖含量檢測。Next, inoculate the activated Lactobacillus fermentum TCI275 and other Lactobacillus fermentum at a concentration of 1% by volume into 20ml of liquid Lactobacillus MRS medium (BD Difco™) containing 5g/L (equal to 0.5%) lactose Lactobacilli MRS Broth), then cultivated at 37°C for 16 hours, and carried out the sampling procedure, the sampling procedure included: take 1ml of bacterial liquid (a total of eight groups), and centrifuge (10000 to 20000rpm, preferably 10000 to 15000rpm, more preferably 13000rpm) After 1 minute, take the supernatant (without bacteria, which is the metabolite of Lactobacillus fermentum TCI275), and use the lactose colorimetric method to determine the reagent set (brand: BioVision, detection set name BioVision® Lactose Colorimetric /Fluorometric Assay Kit, product number K624-100) for the detection of lactose content.
檢測原理是先依套組內試劑配置不同濃度的半乳糖（galactose），以繪製標準曲線，接著將欲測的每種菌液樣品（發酵乳桿菌TCI275及其他發酵乳桿菌（L206、L235、L270、L786、L849、L1265、L1275））分為兩孔（96孔盤）。由於乳糖是由半乳糖及葡萄糖所組成，因此乳糖的含量可透過將某樣品中的總半乳糖減去游離半乳糖（free galactose）所求得，因此在上述兩孔中的其中一孔添加適量乳糖酶（Lactase）使欲測菌液中的所有乳糖轉換為游離半乳糖（free galactose）（因此可求得總半乳糖），另一孔不添加乳糖酶（因此可求得游離半乳糖），透過偵測570nm的吸光值以及公式：乳糖=總半乳糖-游離半乳糖，求得菌液中的乳糖濃度，若乳糖濃度相較於原乳糖濃度（5g/L（等於0.5%））減少一半，則換算該菌的乳糖消耗率（百分比）為50%。The detection principle is to configure different concentrations of galactose (galactose) according to the reagents in the set to draw a standard curve, and then to test each bacterial liquid sample (Lactobacillus fermentum TCI275 and other Lactobacillus fermentum (L206, L235, L270, L786, L849, L1265, L1275)) are divided into two wells (96-well plate). Since lactose is composed of galactose and glucose, the content of lactose can be obtained by subtracting free galactose (free galactose) from the total galactose in a sample, so add an appropriate amount of Lactase converts all the lactose in the bacteria solution to be tested into free galactose (so the total galactose can be obtained), and the other well does not add lactase (so the free galactose can be obtained), By detecting the absorbance at 570nm and the formula: lactose=total galactose-free galactose, the lactose concentration in the bacterial solution is obtained. If the lactose concentration is reduced by half compared to the original lactose concentration (5g/L (equal to 0.5%)) , then the lactose consumption rate (percentage) of the bacteria is converted to 50%.
詳細的檢測方式及樣品配置方式可依照檢測套組所附的使用說明書（Data sheet）所進行，詳細步驟可見網址：https://www.biovision.com/documentation/datasheets/K617.pdf，本發明在此不再贅述。The detailed detection method and sample configuration method can be carried out according to the instruction manual (Data sheet) attached to the detection kit, and the detailed steps can be found at the website: https://www.biovision.com/documentation/datasheets/K617.pdf, the present invention I won't repeat them here.
以下表二為發酵乳桿菌的乳糖消耗率（百分比），數值越高代表其分解乳糖的能力越佳。Table 2 below shows the lactose consumption rate (percentage) of Lactobacillus fermentum. The higher the value, the better its ability to decompose lactose.
表二
如表二所示，發酵乳桿菌TCI275的乳糖消耗率為59.21%，代表將發酵乳桿菌TCI275培養於含有5g/L（重量濃度，等於0.5%）濃度乳糖的培養基中16小時，培養基中乳糖濃度降為2.04 g/L（等於0.204%），表示有59.21%的乳糖經發酵乳桿菌TCI275所分解；同理可知，發酵乳桿菌L206能分解培養基中1.8%的乳糖，其他發酵乳桿菌（L235、L270、L786、L849、L1265、L1275）的乳糖消耗率為0%，代表是不具有分解乳糖的能力。As shown in Table 2, the lactose consumption rate of Lactobacillus fermentum TCI275 is 59.21%, which means that Lactobacillus fermentum TCI275 was cultured in a medium containing 5g/L (weight concentration, equal to 0.5%) lactose for 16 hours, and the lactose concentration in the medium reduced to 2.04 g/L (equal to 0.204%), indicating that 59.21% of lactose was decomposed by Lactobacillus fermentum TCI275; similarly, Lactobacillus fermentum L206 can decompose 1.8% of lactose in the medium, and other Lactobacillus fermentum (L235, L270, L786, L849, L1265, L1275) have a lactose consumption rate of 0%, which means they do not have the ability to decompose lactose.
例4：發酵乳桿菌TCI275對乳糖不耐症狀及調節菌相的人體實驗Example 4: Human experiment of Lactobacillus fermentum TCI275 on symptoms of lactose intolerance and regulation of bacterial phase
乳糖不耐症（Lactose intolerance）就是身體無法完全消化乳糖，因此在食用乳製品後，乳糖會進入大腸成為微生物的食物，接著乳糖被發酵後產生氣體及酸，使個體產生會有腹瀉（大便稀淌）、排氣、脹氣和腹痛、絞痛感的情況。這種情況也稱作乳糖吸收不良，雖然對人體沒有大礙，但這些症狀常令人不舒服。Lactose intolerance (Lactose intolerance) is that the body cannot fully digest lactose, so after eating dairy products, lactose will enter the large intestine and become food for microorganisms, and then the lactose will be fermented to produce gas and acid, which will cause the individual to have diarrhea (loose stool dribbling), gas, flatulence and abdominal pain, cramps. This condition, also known as lactose malabsorption, is harmless, but the symptoms are often uncomfortable.
為進一步確認發酵乳桿菌TCI275對於乳糖不耐症狀及腸道菌相的影響。以每顆膠囊為50毫克TCI275菌粉，含有5X109 cells（即5X109 cells/cap，等於5X109 個菌數）的TCI275的活菌，提供給4位受試者（乳糖不耐症者，即喝牛奶後會出現腹脹、排氣、腹痛或腹瀉等症狀的受試者），每人在每日睡前服用一顆，並持續2周，以問卷方式分析受試者腸胃症狀等情況，以及分析受試者的腸胃道菌相變化，詳細分析方法如下所述。To further confirm the effect of Lactobacillus fermentum TCI275 on symptoms of lactose intolerance and intestinal flora. Each capsule is 50 mg of TCI275 bacteria powder, which contains 5X10 9 cells (5X10 9 cells/cap, equal to 5X10 9 bacteria) live bacteria of TCI275, provided to 4 subjects (lactose intolerance, That is, subjects who will experience symptoms such as abdominal distension, flatulence, abdominal pain or diarrhea after drinking milk), each person takes one tablet before going to bed every day, and lasts for 2 weeks, and the gastrointestinal symptoms of the subjects are analyzed by questionnaire. As well as analyzing the phase changes of the gastrointestinal tract of the subject, the detailed analysis method is as follows.
(4-1) 發酵乳桿菌TCI275對乳糖不耐情況的問卷調查（針對乳糖不耐症的受試者）(4-1) Questionnaire survey on lactose intolerance of Lactobacillus fermentum TCI275 (for subjects with lactose intolerance)
首先對上述受試者於第0週（服用上述的發酵乳桿菌TCI275的活菌膠囊前）、第1週（服用上述的發酵乳桿菌TCI275的活菌膠囊後）及第2週（服用上述的發酵乳桿菌TCI275的活菌膠囊後）進行腸胃狀態的問卷調查。詳細來說，在每周測試前，受試者服用牛奶（牛奶體積依個人食用量做為標準，但每次測試前服用的體積均相同），自身觀察服用牛奶後6小時內的乳糖不耐症狀後，再填寫問卷，其調查及計分方式如下表三。其中，第一部分的各分數代表各種症狀的嚴重程度，0分為無困擾，1分為輕微，2分為有些嚴重，3分為嚴重。Firstly, the subjects were treated at the 0th week (before taking the above-mentioned live bacteria capsule of Lactobacillus fermentum TCI275), the first week (after taking the above-mentioned live bacteria capsule of Lactobacillus fermentum TCI275) and the second week (after taking the above-mentioned Lactobacillus fermentum TCI275 live bacteria capsule) After fermenting Lactobacillus TCI275 live bacteria capsules), a questionnaire survey on gastrointestinal status was carried out. In detail, before the weekly test, the subjects took milk (the volume of milk is based on the individual consumption as the standard, but the volume taken before each test is the same), and observed the lactose intolerance within 6 hours after taking the milk. After symptoms, fill out the questionnaire again. The survey and scoring methods are as follows in Table 3. Among them, each score in the first part represents the severity of various symptoms, with 0 being not bothersome, 1 being mild, 2 being somewhat severe, and 3 being severe.
表三
第一部分之問卷結果如下表四所示The results of the first part of the questionnaire are shown in Table 4 below
表四
由以上表四的問卷結果可看出，4位受試者在第0週（服用上述的發酵乳桿菌TCI275的活菌膠囊前）對大便稀淌的自身評估為2.0分（有些嚴重），在服用上述的發酵乳桿菌TCI275的活菌膠囊1周後，受試者對於大便稀淌的自身評估降為1.25分（約為輕微），服用2周後，對於大便稀淌的自身評估也維持1.25分（約為輕微），代表持續服用發酵乳桿菌TCI275的活菌後一周即可改善大便稀淌的情況，且持續服用能維持改善大便稀淌的情況。From the questionnaire results in Table 4 above, it can be seen that the self-assessment of loose stools by the four subjects at week 0 (before taking the above-mentioned Lactobacillus fermentum TCI275 live capsules) was 2.0 (somewhat serious), and After taking the above-mentioned live bacteria capsules of Lactobacillus fermentum TCI275 for 1 week, the subject's self-assessment of loose stool dropped to 1.25 (about mild), and after taking it for 2 weeks, the self-assessment of loose stool also maintained 1.25 Score (approximately slight), which means that after taking live Lactobacillus fermentum TCI275 continuously for one week, the situation of loose stools can be improved, and continuous consumption can maintain the improvement of loose stools.
此外，4位受試者在第0週（服用上述的發酵乳桿菌TCI275的活菌膠囊前）對脹氣的自身評估為2.0分（有些嚴重），在服用上述的發酵乳桿菌TCI275的活菌膠囊1周後，受試者對於脹氣的自身評估降為1.0分（輕微），服用2周後，對於脹氣的自身評估也維持1.0分（輕微），代表持續服用發酵乳桿菌TCI275的活菌後一周即可改善脹氣的情況，且持續服用能維持改善脹氣的情況。In addition, the self-assessment of flatulence in the 4 subjects at week 0 (before taking the above-mentioned Lactobacillus fermentum TCI275 capsules) was 2.0 points (some serious), and after taking the above-mentioned Lactobacillus fermentum TCI275 live capsules After 1 week, the subject's self-assessment of flatulence dropped to 1.0 (slight), and after taking it for 2 weeks, the self-assessment of flatulence also maintained at 1.0 (slight), which represents one week after continuing to take live bacteria of Lactobacillus fermentum TCI275 It can improve the condition of flatulence, and continuous use can maintain the improvement of flatulence.
此外，4位受試者在第0週（服用上述的發酵乳桿菌TCI275的活菌膠囊前）對排氣頻率的自身評估為1.75分（接近有些嚴重），在服用上述的發酵乳桿菌TCI275的活菌膠囊1周後，受試者對於排氣頻率的自身評估約略降為1.5分（介於輕微及有些嚴重之間的程度），服用2周後，對於排氣頻率的自身評估降為維持1.25分（約輕微），代表持續服用發酵乳桿菌TCI275的活菌後二周可改善排氣頻率過多的情況。In addition, the self-assessment of the frequency of flatulence by the 4 subjects at week 0 (before taking the above-mentioned Lactobacillus fermentum TCI275 capsules) was 1.75 points (close to some serious), after taking the above-mentioned Lactobacillus fermentum TCI275 After 1 week of live bacterial capsules, the self-assessment of the subject’s self-assessment of the frequency of gas was reduced to 1.5 points (between mild and somewhat severe), and after 2 weeks of taking the capsule, the self-assessment of the frequency of gas was reduced to maintain A score of 1.25 (approximately slight) means that after two weeks of continuous consumption of Lactobacillus fermentum TCI275 live bacteria, excessive gas frequency can be improved.
此外，至於腹痛、絞痛感的問卷結果，由於4位受試者在第0週（服用上述的發酵乳桿菌TCI275的活菌膠囊前）對腹痛、絞痛感的自身評估僅有為1.25分（約為輕微），因此服用上述的發酵乳桿菌TCI275的活菌膠囊後的改善情況意義不大，本文不做討論。In addition, as for the questionnaire results of abdominal pain and colic, the self-assessment of abdominal pain and colic by the four subjects at week 0 (before taking the above-mentioned Lactobacillus fermentum TCI275 live bacteria capsule) was only 1.25 points (about Slight), so the improvement after taking the live capsules of Lactobacillus fermentum TCI275 mentioned above is of little significance and will not be discussed in this article.
綜合以上，對乳糖不耐症者，即喝牛奶後會出現腹脹、排氣、腹痛或腹瀉等症狀的受試者來說，服用發酵乳桿菌TCI275的活菌後，對於一般常見的乳糖不耐症症狀皆有明顯改善，特別是針對大便稀淌、脹氣及排氣頻率達到改善效果。Based on the above, for those who are lactose intolerant, that is, subjects who have symptoms such as abdominal distension, gas, abdominal pain or diarrhea after drinking milk, after taking live bacteria of Lactobacillus fermentum TCI275, the common lactose intolerance Symptoms have been significantly improved, especially for loose stools, flatulence and frequency of exhaust.
(4-2) 發酵乳桿菌TCI275調節菌相的人體實驗（針對乳糖不耐症的受試者）(4-2) Human experiments on regulation of bacterial phase by Lactobacillus fermentum TCI275 (for subjects with lactose intolerance)
在上述受試者於第0週（服用上述的發酵乳桿菌TCI275的活菌膠囊前）及第2週（服用上述的發酵乳桿菌TCI275的活菌膠囊後）進行受試者腸道菌相檢測。The above-mentioned subjects were tested for the intestinal flora of the subjects at week 0 (before taking the above-mentioned live bacteria capsules of Lactobacillus fermentum TCI275) and at week 2 (after taking the above-mentioned live bacteria capsules of Lactobacillus fermentum TCI275) .
檢測方式可以是採集受試者糞便檢體後，對糞便進行DNA萃取，並進行16S核糖體基因(16S ribosomal gene)的定量即時聚合酶鏈反應(quantitative real-time polymerase chain reaction, qRT-PCR)，透過以知菌的引子序列來檢測常見腸內菌的菌數比例。檢測方式也可以將糞便進行DNA萃取後，對檢體進行微生物體16S擴增子定序 (16S Amplicon Sequencing)，其中特別是針對16S DNA其中的 V3-V4 區域進行放大，接著以次世代定序（Next Generation Sequencing (NGS)）的方式，並進行後續的定序分析。The detection method can be to collect the subject's stool sample, extract DNA from the stool, and perform quantitative real-time polymerase chain reaction (qRT-PCR) of 16S ribosomal gene (16S ribosomal gene) , to detect the proportion of common intestinal bacteria through the primer sequence of known bacteria. The detection method can also perform DNA extraction on the feces, and then perform microbial 16S amplicon sequencing (16S Amplicon Sequencing) on the specimen, especially for the amplification of the V3-V4 region of the 16S DNA, followed by next-generation sequencing (Next Generation Sequencing (NGS)), and perform subsequent sequencing analysis.
本實施例是以次世代定序方式進行，由圖爾思生物科技股份有限公司進行微生物體16S擴增子定序 (16S Amplicon Sequencing)，將糞便樣本行DNA萃取後，使用由Illumina (Illumina, San Diego, USA)開發之16S宏基因組測序文庫製備方案進行16S rRNA基因擴增子測序。簡言之，將糞便萃取的DNA採用靶向16S rRNA基因之V3/V4可變區之引子擴增：16S擴增子PCR正向引子（V3區域） TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG (SEQUENCE ID NO：9)；16S擴增子PCR反向引子（V4區域） GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC （SEQUENCE ID NO：10） 進行PCR擴增16S rRNA基因的可變區V3-V4(rRNA基因中的第341 至805位）（參考Herlemann等人，2000年；其中W=A/T、H=A/C/T、V=G/C/A、N=G/A/T/C)。完成擴增DNA之序列及建庫，依據MiSeq Reagent Kit v3 (Illumina, Wilmington, DE, USA)建雙端庫（pair-end library），並於Illumina系統進行高通量定序。定序完成後得到的原始數據（Raw Data），會經由成對序列拼接（Raw Tags）、過濾得到 Clean Tags，再進行去除嵌合體得到可用於後續分析的有效數據（Effective Tags）。接著基於有效數據進行相似度（大於97%）OTUs（Operational Taxonomic Units）聚類和物種分類分析。根據 OTUs 聚類結果對代表序列做物種注釋，得到對應的物種資訊和基於物種的豐度分佈情況，分析檢體內的微生物種類及比例。This example is carried out in a next-generation sequencing method. The microbial 16S amplicon sequencing (16S Amplicon Sequencing) was carried out by Tursi Biotechnology Co., Ltd. After DNA extraction was performed on the stool sample, it was used by Illumina (Illumina, San Diego, USA) developed the 16S metagenomic sequencing library preparation protocol for 16S rRNA gene amplicon sequencing. Briefly, stool-extracted DNA was amplified using primers targeting the V3/V4 variable regions of the 16S rRNA gene: 16S amplicon PCR forward primer (V3 region) TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG (SEQUENCE ID NO: 9); Adder PCR reverse primer (V4 region) GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC (SEQUENCE ID NO: 10) for PCR amplification of the variable region V3-V4 of the 16S rRNA gene (positions 341 to 805 in the rRNA gene) (refer to Herlemann et al., 2000 year; where W=A/T, H=A/C/T, V=G/C/A, N=G/A/T/C). Complete the sequence and library construction of the amplified DNA, build a pair-end library (pair-end library) according to MiSeq Reagent Kit v3 (Illumina, Wilmington, DE, USA), and perform high-throughput sequencing on the Illumina system. The raw data (Raw Data) obtained after the sequencing is completed will be spliced in pairs (Raw Tags), filtered to obtain Clean Tags, and then removed chimeras to obtain effective data (Effective Tags) that can be used for subsequent analysis. Then based on the valid data, the similarity (greater than 97%) OTUs (Operational Taxonomic Units) clustering and species classification analysis were carried out. According to the OTUs clustering results, the representative sequences are annotated with species, and the corresponding species information and species-based abundance distribution are obtained, and the types and proportions of microorganisms in the samples are analyzed.
參照圖2，相服用前（第0週），持續2週服用發酵乳桿菌TCI275的活菌膠囊後可使柔嫩梭菌屬（Faecalibacterium）菌種比例（相對豐富度）增加約19%。Referring to Figure 2, compared with before taking (week 0), taking live capsules of Lactobacillus fermentum TCI275 for 2 weeks can increase the proportion (relative abundance) of Faecalibacterium species by about 19%.
柔嫩梭菌屬（Faecalibacterium）是一種能夠促使短鏈脂肪酸（丁酸）生成的腸內益生菌，丁酸的生成可促進腸道健康。可參見例如：微生態製劑調節便秘, 腹瀉人群腸道菌群結構與産短鏈脂肪酸關鍵菌屬的相關性，食品科學 9.5 (2018): 155-165，該文獻之全文併於此處以供參考。Faecalibacterium is an intestinal probiotic that promotes the production of a short-chain fatty acid (butyrate), which promotes gut health. See for example: Microecological Agents Modulate Constipation, Correlation of Gut Microbiota Structure and Short-Chain Fatty Acid-Producing Key Bacteria in Diarrhea Population, Food Science 9.5 (2018): 155-165, the full text of which is hereby incorporated by reference .
透過受試者糞便檢體之定序檢測，分析TCI275對於人體菌相調節的能力，特別是對於具有乳糖不耐症症狀的受試者的腸胃道菌相的能力，由以上結果可知，於服用TCI275膠囊後，受試者腸胃道內的柔嫩梭菌屬（Faecalibacterium）明顯增加，代表TCI275具有促進腸內益生菌比例以及促進腸胃道健康的功效。The ability of TCI275 to regulate human bacteria, especially the gastrointestinal bacteria of subjects with symptoms of lactose intolerance, was analyzed through the sequential detection of stool samples from the subjects. From the above results, it can be seen that after taking After TCI275 capsules, the Faecalibacterium in the gastrointestinal tract of the subjects increased significantly, which means that TCI275 has the effect of promoting the proportion of probiotics in the intestine and promoting the health of the gastrointestinal tract.
例5：發酵乳桿菌TCI275對便祕相關症狀及調節菌相的人體實驗Example 5: Human experiment of Lactobacillus fermentum TCI275 on constipation-related symptoms and regulation of bacterial phase
為進一步確認發酵乳桿菌TCI275對於便祕及腸道菌相的影響。以每顆膠囊為50毫克TCI275菌粉，共含有5X109 cells（即5X109 cells/cap）的TCI275的活菌，提供給4位受試者（有便秘情況、排便排不乾淨等症狀的受試者），每人每日睡前服用一顆，並持續2周，以問卷方式分析受試者腸胃狀況、排便頻率等情況，以及分析受試者的腸胃道菌相變化，詳細分析方法如下所述。To further confirm the effect of Lactobacillus fermentum TCI275 on constipation and intestinal flora. Each capsule is 50 mg of TCI275 bacterial powder, which contains a total of 5X10 9 cells (5X10 9 cells/cap) of live bacteria of TCI275, and provided to 4 subjects (those with symptoms such as constipation and unclean defecation) subjects), each person took one pill before going to bed every day, and continued for 2 weeks, and analyzed the gastrointestinal conditions, defecation frequency, etc. mentioned.
(5-1) 發酵乳桿菌TCI275對腸胃狀況及排便頻率的問卷調查（針對有便秘情況、排便排不乾淨等症狀的受試者）(5-1) Questionnaire survey of Lactobacillus fermentum TCI275 on gastrointestinal conditions and defecation frequency (for subjects with symptoms such as constipation and unclean defecation)
首先對上述受試者於第0週（服用上述的發酵乳桿菌TCI275的活菌膠囊前）、第1週（服用上述的發酵乳桿菌TCI275的活菌膠囊後）及第2週（服用上述的發酵乳桿菌TCI275的活菌膠囊後）進行腸胃狀態及排便頻率的問卷調查。詳細來說，其調查及計分方式如下表五。其中，第一部分的各分數代表各種症狀的嚴重程度，0分為無困擾，1分為輕微，2分為有些嚴重，3分為嚴重，4分為非常嚴重；第二部分為調查排便頻率，調查方式如下表六，其中，四天一次代表受試者平均四天排便一次，為便祕程度最嚴重；一天兩次以上代表受試者平均一天排便兩次，為輕微便祕程度。Firstly, the subjects were treated at the 0th week (before taking the above-mentioned live bacteria capsule of Lactobacillus fermentum TCI275), the first week (after taking the above-mentioned live bacteria capsule of Lactobacillus fermentum TCI275) and the second week (after taking the above-mentioned Lactobacillus fermentum TCI275 live bacteria capsule) Fermented Lactobacillus TCI275 live bacteria capsules) to conduct a questionnaire survey on gastrointestinal status and defecation frequency. In detail, the survey and scoring methods are shown in Table 5 below. Among them, the scores in the first part represent the severity of various symptoms, 0 is no trouble, 1 is mild, 2 is somewhat serious, 3 is serious, 4 is very serious; the second part is to investigate the frequency of defecation, The survey method is as follows in Table 6. Among them, once every four days means that the subjects defecate once every four days on average, which is the most serious degree of constipation; more than twice a day means that the subjects defecate twice a day on average, which is mild constipation.
表五
表六
第一部分之問卷結果如下表七所示The results of the first part of the questionnaire are shown in Table 7 below
表七
由以上表七的問卷結果可看出，4位受試者在第0週（服用上述的發酵乳桿菌TCI275的活菌膠囊前）對脹氣的自身評估為3.25分（嚴重程度），在服用上述的發酵乳桿菌TCI275的活菌膠囊1周後，受試者對於脹氣的自身評估降為1.25分（約為輕微），服用2周後，對於脹氣的自身評估也維持1.0分（輕微），代表持續服用發酵乳桿菌TCI275的活菌後一周即可明顯改善脹氣的情況，且持續服用能維持改善脹氣的情況。From the questionnaire results in Table 7 above, it can be seen that the self-assessment of flatulence by the four subjects at week 0 (before taking the above-mentioned live bacteria capsule of Lactobacillus fermentum TCI275) was 3.25 points (severity), and after taking the above-mentioned After 1 week of live capsules of Lactobacillus fermentum TCI275, the subject’s self-evaluation of flatulence dropped to 1.25 points (about mild), and after 2 weeks of taking the self-evaluation of flatulence also maintained 1.0 points (slight), representing After taking the live bacteria of Lactobacillus fermentum TCI275 continuously for one week, the flatulence can be significantly improved, and continuous consumption can maintain the improvement of flatulence.
此外，4位受試者在第0週（服用上述的發酵乳桿菌TCI275的活菌膠囊前）對腹鳴、腹部蠕動感的自身評估為2.0分（有些嚴重），在服用上述的發酵乳桿菌TCI275的活菌膠囊1周後，受試者對於腹鳴、腹部蠕動感的自身評估降為1.25分（輕微），服用2周後，對於腹鳴、腹部蠕動感的自身評估再降為1.0分（輕微），代表持續服用發酵乳桿菌TCI275的活菌後一周即可改善腹鳴、腹部蠕動感的情況，且持續服用能維持改善腹鳴、腹部蠕動感的情況。In addition, the self-assessment of borborygmi and abdominal peristalsis of the 4 subjects at week 0 (before taking the above-mentioned Lactobacillus fermentum TCI275 live bacteria capsule) was 2.0 points (some serious), after taking the above-mentioned Lactobacillus fermentum TCI275 After 1 week of live bacteria capsules of TCI275, the subject’s self-assessment of borborygmi and abdominal peristalsis decreased to 1.25 points (slight), and after 2 weeks of taking, the subject’s self-assessment of borborygmi and abdominal peristalsis decreased to 1.0 points (Minor) means that the borborygmi and abdominal peristalsis can be improved after one week of continuous administration of live Lactobacillus fermentum TCI275, and continuous administration can maintain the improvement of borborygmi and abdominal peristalsis.
此外，4位受試者在第0週（服用上述的發酵乳桿菌TCI275的活菌膠囊前）對排氣頻率的自身評估為2.25分（有些嚴重的程度），在服用上述的發酵乳桿菌TCI275的活菌膠囊1周後，受試者對於排氣頻率的自身評估約略降為1.75分（介於輕微及有些嚴重之間的程度），服用2周後，對於排氣頻率的自身評估降為維持1.5分（約輕微），代表持續服用發酵乳桿菌TCI275的活菌後約二周可改善排氣頻率過多的情況。In addition, the self-assessment of the frequency of flatulence by the 4 subjects at week 0 (before taking the above-mentioned Lactobacillus fermentum TCI275 live bacteria capsule) was 2.25 points (some degree of severity), after taking the above-mentioned Lactobacillus fermentum TCI275 After 1 week of taking live bacteria capsules, the subject's self-assessment of the frequency of gas was slightly reduced to 1.75 points (between mild and somewhat severe), and after 2 weeks of taking the self-assessment of the frequency of gas was reduced to 1.75 points. Maintaining a score of 1.5 (approximately slight) means that after taking Lactobacillus fermentum TCI275 live bacteria for about two weeks, the condition of excessive gas frequency can be improved.
第二部分之問卷結果如下表八所示The results of the second part of the questionnaire are shown in Table 8 below
表八
由上述表八可知，對於有便秘情況的受試者在服用發酵乳桿菌TCI275之前有50%的受試者的排便頻率屬於兩天或四天一次排便，而服用發酵乳桿菌TCI275之後一周，有75%的受試者的排便頻率為一天一次排便，而服用發酵乳桿菌TCI275之後兩周，已沒有受試者的排便頻率為兩天或四天一次排便，100%的受試者的排便頻率為一天一次或一天兩次以上。即各受試者平均而言，對於排便狀態有明顯的改善，整體提升了排便頻率，改善了便祕情況。It can be seen from the above Table 8 that before taking Lactobacillus fermentum TCI275, 50% of the subjects with constipation had a defecation frequency of once every two or four days, and one week after taking Lactobacillus fermentum TCI275, there were The defecation frequency of 75% of the subjects was once a day, and two weeks after taking Lactobacillus fermentum TCI275, there were no subjects whose defecation frequency was once every two days or four days, and 100% of the subjects had a defecation frequency of Once a day or more than twice a day. That is to say, on average, the defecation status of each subject has been significantly improved, the overall defecation frequency has been increased, and the constipation situation has been improved.
綜合以上，對有便秘情況、排便排不乾淨等症狀的受試者來說，服用發酵乳桿菌TCI275的活菌後，對於一般常見的腸胃不適情況皆有明顯改善，特別是針對脹氣及腹鳴、腹部蠕動感達到改善效果，此外，對於排便狀態有明顯的改善，整體提升了排便頻率，改善了便祕情況。Based on the above, for subjects with symptoms such as constipation and unclean defecation, after taking live bacteria of Lactobacillus fermentum TCI275, common common gastrointestinal discomforts were significantly improved, especially for flatulence and borborygmi 1. Abdominal peristalsis can be improved. In addition, the defecation state has been significantly improved, the overall frequency of defecation has been improved, and constipation has been improved.
(5-2) 發酵乳桿菌TCI275調節菌相的人體實驗（針對有便秘情況、排便排不乾淨等症狀的受試者）(5-2) Human experiments on the regulation of bacterial phase by Lactobacillus fermentum TCI275 (for subjects with symptoms such as constipation and unclean defecation)
在上述受試者於第0週（服用上述的發酵乳桿菌TCI275的活菌膠囊前）及第2週（服用上述的發酵乳桿菌TCI275的活菌膠囊後）進行受試者腸道菌相檢測。The above-mentioned subjects were tested for the intestinal flora of the subjects at week 0 (before taking the above-mentioned live bacteria capsules of Lactobacillus fermentum TCI275) and at week 2 (after taking the above-mentioned live bacteria capsules of Lactobacillus fermentum TCI275) .
本實施例是以次世代定序方式進行，採取的步驟及方式與例(4-2)相同，本段落不再贅述。This embodiment is carried out in a next-generation sequencing manner, and the steps and methods adopted are the same as those in Example (4-2), and will not be repeated in this paragraph.
參照圖3，相服用前（第0週），持續2週服用發酵乳桿菌TCI275的活菌膠囊後可使普雷沃氏菌屬（Prevotella）菌種比例（相對豐富度）增加約42%。Referring to Figure 3, compared with before taking (week 0), taking live capsules of Lactobacillus fermentum TCI275 for 2 weeks can increase the proportion (relative abundance) of Prevotella species by about 42%.
普雷沃氏菌屬（Prevotella）具有植物聚醣降解能力，飲食中富含蔬果或是素食者腸道中比例較高；由於此菌可利用纖維代謝出較多短鏈脂肪酸(Short-chain fatty acid，SCFA)，因此研究顯示與健康人群相比，便秘人群缺乏普雷沃氏菌屬腸道菌。可參見例如：European Journal of Clinical Microbiology & Infectious Diseases 37.3 (2018): 555-563，該文獻之全文併於此處以供參考。Prevotella has the ability to degrade plant glycans, and the proportion in the intestines of people whose diet is rich in fruits and vegetables or vegetarians is higher; because this bacteria can use fiber to metabolize more short-chain fatty acids (Short-chain fatty acid , SCFA), thus the study showed that the intestinal flora of the Prevotella spp. was absent in the constipated population compared with the healthy population. See, eg, European Journal of Clinical Microbiology & Infectious Diseases 37.3 (2018): 555-563, which is hereby incorporated by reference in its entirety.
透過受試者糞便檢體之定序檢測，分析TCI275對於人體菌相調節的能力，特別是對於具有便秘情況的受試者的腸胃道菌相調節的能力，由以上結果可知，於服用TCI275膠囊後，受試者腸胃道內的普雷沃氏菌屬明顯增加，代表TCI275具有促進腸胃道健康的潛力。The ability of TCI275 to regulate the human bacterium was analyzed through the sequential detection of the stool samples of the subjects, especially the ability to regulate the gastrointestinal bacteria of the subjects with constipation. From the above results, it can be seen that after taking TCI275 capsules After that, the Prevotella species in the gastrointestinal tract of the subjects increased significantly, which means that TCI275 has the potential to promote gastrointestinal health.
於此，透過例2可知，發酵乳桿菌TCI275可適應於人體腸胃環境。透過例3可知，發酵乳桿菌TCI275有分解乳糖的能力，且能力明顯優於其他發酵乳桿菌（L206、L235、L270、L786、L849、L1265、L1275），明顯具有與同種習知菌株不同之微生物學性質。透過例4可知，發酵乳桿菌TCI275能改善乳糖不耐相關症狀（特別是針對大便稀淌、脹氣及排氣頻率）以及提升柔嫩梭菌屬的能力，特別是在乳糖不耐症的受試者中有此效果。透過例5可知，發酵乳桿菌TCI275能改善經常便祕便祕的受試者的腸胃相關不適症狀（特別是針對脹氣及腹鳴、腹部蠕動）以及提升普雷沃氏菌屬的能力。Here, from Example 2, it can be seen that Lactobacillus fermentum TCI275 can adapt to the human gastrointestinal environment. From Example 3, it can be seen that Lactobacillus fermentum TCI275 has the ability to decompose lactose, and the ability is significantly better than other Lactobacillus fermentum (L206, L235, L270, L786, L849, L1265, L1275), which obviously has different microorganisms from the same conventional strains academic nature. From Example 4, it can be seen that Lactobacillus fermentum TCI275 can improve the symptoms related to lactose intolerance (especially for loose stools, flatulence and flatulence frequency) and enhance the ability of Clostridium tenacilis, especially in lactose intolerant subjects has this effect. From Example 5, it can be seen that Lactobacillus fermentum TCI275 can improve the gastrointestinal discomfort symptoms (especially for flatulence, borborygmi, and abdominal peristalsis) of subjects with frequent constipation and constipation, as well as enhance the ability of Prevotella.
雖然本發明的技術內容已經以較佳實施例揭露如上，然其並非用以限定本發明，任何熟習此技藝者，在不脫離本發明之精神所作些許之更動與潤飾，皆應涵蓋於本發明的範疇內，因此本發明之保護範圍當視後附之申請專利範圍所界定者為準。Although the technical content of the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any modification and modification made by those skilled in the art without departing from the spirit of the present invention should be covered by the present invention. Therefore, the scope of protection of the present invention should be defined by the scope of the appended patent application.
無none
[圖1] 為發酵乳桿菌TCI275抗胃液及腸液的測試結果數據圖。 [圖2] 為發酵乳桿菌TCI275提升柔嫩梭菌屬（Faecalibacterium）菌種比例的結果數據圖。 [圖3] 為發酵乳桿菌TCI275提升普雷沃氏菌屬（Prevotella）菌種比例的結果數據圖。[Figure 1] It is a graph showing the test results of Lactobacillus fermentum TCI275 against gastric juice and intestinal juice. [Fig. 2] The result data graph of increasing the proportion of Faecalibacterium species for Lactobacillus fermentum TCI275. [Fig. 3] The result data graph of increasing the proportion of Prevotella species for Lactobacillus fermentum TCI275.
TW中華民國 財團法人食品工業發展研究所 2019/09/17 BCRC 910940TW Republic of China Food Industry Development Research Institute 2019/09/17 BCRC 910940
DE德國 德國國家菌種保藏中心DSMZ（Deutsche Sammlung von Mikroorganismen und Zellkulturen） 2019/09/19 DSM33289DE Germany DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) 2019/09/19 DSM33289
<110> 大江生醫股份有限公司 <110> Dajiang Biomedical Co., Ltd.
<120> 發酵乳桿菌TCI275、其組合物及其用途 <120> Lactobacillus fermentum TCI275, composition and use thereof
<150> US 62/911,716 <150> US 62/911,716
<151> 2019-10-07 <151> 2019-10-07
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<170> PatentIn version 3.5 <170> PatentIn version 3.5
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