TWI787665B - Lactobacillus bulgaricus tci904, supernatant, composition, and uses thereof for losing weight - Google Patents
Lactobacillus bulgaricus tci904, supernatant, composition, and uses thereof for losing weight Download PDFInfo
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- TWI787665B TWI787665B TW109137154A TW109137154A TWI787665B TW I787665 B TWI787665 B TW I787665B TW 109137154 A TW109137154 A TW 109137154A TW 109137154 A TW109137154 A TW 109137154A TW I787665 B TWI787665 B TW I787665B
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- tci904
- lactobacillus bulgaricus
- composition
- density lipoprotein
- body fat
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Abstract
Description
本發明關於一種保加利亞乳桿菌TCI904(Lactobacillus bulgaricus TCI904)，特別是關於一種保加利亞乳桿菌TCI904或其代謝產物用於減少體重的用途。 The present invention relates to a Lactobacillus bulgaricus TCI904 (Lactobacillus bulgaricus TCI904), in particular to the use of a Lactobacillus bulgaricus TCI904 or a metabolite thereof for reducing body weight.
益生菌(Probiotics)一般認為是指食入後對宿主(或稱受體，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 recipient, 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受體健康有益，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受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 the health of recipients, 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 the health of the recipient 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 yeast Species of parent fungi 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，通常存在於發酵食物中，例如泡菜與味增等。 Lactobacillus is a group of fermentative, facultatively anaerobic, non-spore-producing, Gram-positive bacilli. Lactobacillus, named for its ability to ferment carbohydrates into lactic acid, is usually found in fermented foods, such as kimchi and miso.
本發明之一目的，在於提供一種保加利亞乳桿菌TCI904，其寄存於財團法人食品工業發展研究所(寄存編號BCRC910984)及德國國家菌種保藏中心(DSMZ)(寄存編號DSM33505)。 One object of the present invention is to provide a kind of Lactobacillus bulgaricus TCI904, which is deposited in the Food Industry Development Research Institute (registration number BCRC910984) and the German National Culture Collection Center (DSMZ) (registration number DSM33505).
在一些實施例中，保加利亞乳桿菌TCI904菌株包含如SEQ ID NO：1所示的序列。 In some embodiments, the Lactobacillus bulgaricus TCI904 strain comprises the sequence shown in SEQ ID NO:1.
在一些實施例中，保加利亞乳桿菌TCI904具有抑制脂肪酶活性的能力。 In some embodiments, Lactobacillus bulgaricus TCI904 has the ability to inhibit lipase activity.
在一些實施例中，保加利亞乳桿菌TCI904抑制脂肪酶的活性至少35%。 In some embodiments, Lactobacillus bulgaricus TCI904 inhibits the activity of lipase by at least 35%.
本發明之另一目的，在於提供一種組合物，其包含一保加利亞乳桿菌(Lactobacillus bulgaricus)TCI904及/或其代謝產物，所述保加利亞乳桿菌係寄存於財團法人食品工業發展研究所(寄存編號BCRC910984)及德國國家菌種保藏中心(DSMZ)(寄存編號DSM33505)。 Another object of the present invention is to provide a composition comprising a Lactobacillus bulgaricus ( Lactobacillus bulgaricus ) TCI904 and/or its metabolites, the Lactobacillus bulgaricus is deposited in the Food Industry Development Research Institute (Deposit No. BCRC910984 ) and the German National Culture Collection (DSMZ) (deposit number DSM33505).
在一些實施例中，組合物係用於製備減少一個體的體重或體脂的組合物。 In some embodiments, the compositions are used in the manufacture of compositions that reduce body weight or body fat in a subject.
在一些實施例中，體脂為軀幹體脂。 In some embodiments, the body fat is trunk body fat.
在一些實施例中，組合物係用於製備調節一個體的脂聯素、三酸甘油酯(Triglyceride)、極低密度脂蛋白(Very Low Density Lipoprotein)或高密度脂蛋白(High density lipoprotein)的組合物。 In some embodiments, the composition is used in the preparation of an agent for regulating adiponectin, triglyceride (Triglyceride), very low density lipoprotein (Very Low Density Lipoprotein) or high density lipoprotein (High density lipoprotein) in an individual combination.
在一些實施例中，個體為身高體重指數(BMI)大於24或身體體脂率大於25%的個體。 In some embodiments, the individual is an individual with a body mass index (BMI) greater than 24 or a body fat percentage greater than 25%.
在一些實施例中，組合物含有1X108CFU個菌數的保加利亞乳桿菌TCI904。 In some embodiments, the composition contains 1× 10 8 CFU of Lactobacillus bulgaricus TCI904.
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.
圖1是試管實驗中的脂肪酶活性檢測的結果圖；圖2是細胞實驗中的脂聯素含量檢測的結果圖(*表示與空白組比較有顯著差異，p<0.05)；圖3是人體實驗中的體重檢測的結果圖(*表示與第0週比較有顯著差異，p<0.05)；圖4是人體實驗中的軀幹體脂檢測的結果圖；圖5是人體實驗中的腰圍檢測的結果圖(*表示與第0週比較有顯著差異，p<0.05)；圖6是人體實驗中的三酸甘油酯含量檢測的結果圖(***表示與第0週比較有顯著差異，p<0.001)；圖7是人體實驗中的極低密度脂蛋白含量檢測的結果圖(*表示與第0
週比較有顯著差異，p<0.05)；圖8是人體實驗中的高密度脂蛋白含量檢測的結果圖(**表示與第0週比較有顯著差異，p<0.01)。
Fig. 1 is the result graph of the lipase activity detection in the test tube experiment; Fig. 2 is the result graph of the adiponectin content detection in the cell experiment (* indicates that there is a significant difference compared with the blank group, p<0.05); Fig. 3 is the human body The result graph of the body weight detection in the experiment (* indicates that there is a significant difference compared with the 0th week, p<0.05); Fig. 4 is the result graph of the trunk body fat detection in the human experiment; Fig. 5 is the result graph of the waist circumference detection in the human experiment Result graph (* indicates that there is a significant difference compared with the 0th week, p<0.05); Figure 6 is the result graph of the triglyceride content detection in the human experiment (*** indicates that there is a significant difference compared with the 0th week, p <0.001); Figure 7 is the results of the detection of very low-density lipoprotein content in human experiments (* indicates the same as the 0th
There is a significant difference between weeks, p<0.05); Figure 8 is the result of the detection of high-density lipoprotein content in the human experiment (** indicates that there is a significant difference compared with
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's t-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 by student's t -test .
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 minus 10%, and the best is within the range of plus or minus 5%.
本文所述的用語「組合物」，組合物為食品、保養品或醫藥品，較佳是指食品組合物。 The term "composition" mentioned herein means that the composition is food, skin care product or medicine, and preferably refers to a food composition.
本文所述的用語「個體」是指人類或非人的哺乳動物，較佳為人類。 The term "individual" as used herein refers to a human or a non-human mammal, preferably a human.
本案發明人自泡菜篩選出一新穎菌株，經16S核糖體核糖核酸(16S rRNA)序列分析，依親緣關係鑑定為Lactobacillus delbrueckii subsp.bulgaricus(又稱Lactobacillus bulgaricus，中文名稱為保加利亞乳桿菌)，為德氏乳桿菌(Lactobacillus delbrueckii)的亞種，經命名為Lactobacillus bulgaricus TCI904(以下稱為保加利亞乳桿菌TCI904， 簡稱TCI904)，其係寄存於財團法人食品工業發展研究所(寄存編號為BCRC910984)、及德國國家菌種保藏中心(DSMZ)(寄存編號為DSM33505)。該保加利亞乳桿菌TCI904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 bacterial strain from kimchi, and through 16S ribosomal ribonucleic acid (16S rRNA) sequence analysis, it was identified as Lactobacillus delbrueckii subsp. The subspecies of Lactobacillus delbrueckii (Lactobacillus delbrueckii), named Lactobacillus bulgaricus TCI904 (hereinafter referred to as Lactobacillus bulgaricus TCI904, referred to as TCI904), which is deposited in the Food Industry Development Research Institute (Deposit No. BCRC910984), and Germany National Culture Collection Center (DSMZ) (registration number DSM33505). The Lactobacillus bulgaricus TCI904 line has a 16S ribosomal ribonucleic acid (16S rRNA) fragment as shown in SEQ ID NO:1.
本案發明人研究發現，相較於本案發明人由泡菜篩選出的其他保加利亞乳桿菌(LF063、LF006、LF049)，本案的保加利亞乳桿菌TCI904具有明顯更佳的抑制脂肪酶活性功效，可抑制至少35%的脂肪酶活性，更佳可抑制至少39%的脂肪酶活性。 The inventors of this case found that compared with other Lactobacillus bulgaricus (LF063, LF006, LF049) screened by the inventor of this case from kimchi, the Lactobacillus bulgaricus TCI904 in this case has significantly better inhibitory effect on lipase activity, and can inhibit at least 35 % lipase activity, more preferably at least 39% lipase activity can be inhibited.
本案發明人研究發現，保加利亞乳桿菌TCI904及/或其代謝產物係具有減少一個體的體重或體脂之功效。因此，本發明係提供一種保加利亞乳桿菌TCI904及/或其代謝產物在製備減少一個體的體重或體脂的組合物之用途，其中，該體脂所指可為軀幹體脂。 The inventors of this case found that Lactobacillus bulgaricus TCI904 and/or its metabolites have the effect of reducing an individual's body weight or body fat. Therefore, the present invention provides a use of Lactobacillus bulgaricus TCI904 and/or its metabolites in preparing a composition for reducing body weight or body fat of an individual, wherein the body fat refers to trunk body fat.
本案發明人研究發現，保加利亞乳桿菌TCI904及/或其代謝產物係具有減少一個體的脂聯素、三酸甘油酯、極低密度脂蛋白或高密度脂蛋白之功效。因此，本發明係提供一種保加利亞乳桿菌TCI904及/或其代謝產物在製備減少一個體的的脂聯素、三酸甘油酯、極低密度脂蛋白或高密度脂蛋白的組合物之用途，其中，該個體所指可為身高體重指數(BMI)大於24或身體體脂率大於25%的個體。 The inventors of this case found that Lactobacillus bulgaricus TCI904 and/or its metabolites have the effect of reducing adiponectin, triglyceride, very low-density lipoprotein or high-density lipoprotein in an individual. Therefore, the present invention provides a use of Lactobacillus bulgaricus TCI904 and/or its metabolites in the preparation of a composition that reduces adiponectin, triglycerides, very low-density lipoprotein or high-density lipoprotein in an individual, wherein , the individual may refer to an individual with a body mass index (BMI) greater than 24 or a body fat percentage greater than 25%.
根據本發明，所採用之保加利亞乳桿菌TCI904之代謝產物可以是透過在適於保加利亞乳桿菌TCI904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保加利亞乳桿菌TCI904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保加利亞乳桿菌TCI904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 bulgaricus TCI904 used may be produced by culturing in an environment suitable for the growth of Lactobacillus bulgaricus TCI904. For example: first culture Lactobacillus bulgaricus TCI904 with an appropriate culture medium, and then, if necessary, remove solids such as bacteria in the culture medium to obtain a liquid containing metabolites body. Or, for example, first culture Lactobacillus bulgaricus TCI904 with an appropriate culture medium, then settle the bacteria to the bottom by centrifugation and other methods, 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保加利亞乳桿菌TCI904之培養，以提供所欲之代謝產物，只要該培養液可提供保加利亞乳桿菌TCI904生長、代謝所需養分(如乳桿菌萃取物、蛋白質及葡萄糖)與條件(如酸鹼值)即可。此外，保加利亞乳桿菌TCI904之培養時間亦無特殊限制，只要足以使保加利亞乳桿菌TCI904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培養液對保加利亞乳桿菌TCI904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 bulgaricus TCI904 to provide the desired metabolites, as long as the culture medium can provide the nutrients required for the growth and metabolism of Lactobacillus bulgaricus TCI904 (such as Lactobacillus extract, protein and glucose ) and conditions (such as pH value). In addition, the culture time of Lactobacillus bulgaricus TCI904 is not particularly limited, as long as it is enough for Lactobacillus bulgaricus TCI904 to complete at least one metabolic cycle. For example, in a specific embodiment of the present invention, Lactobacillus bulgaricus TCI904 is cultured with MRS culture medium for 18 hours, so that the strain undergoes metabolism and produces metabolites.
於本發明中，係可直接使用經歷保加利亞乳桿菌TCI904之代謝循環的含保加利亞乳桿菌TCI904與代謝產物的培養液，或使用經移除保加利亞乳桿菌TCI904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 bulgaricus TCI904 and metabolites that have undergone the metabolic cycle of Lactobacillus bulgaricus TCI904 can be directly used, or the metabolite-containing liquid that has removed solids such as Lactobacillus bulgaricus TCI904 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組合物可為食品組合物，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、***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 composition provided according to the present invention can be a food composition, and the food composition can be health food, health food, functional food, nutritional supplement, or special nutritional food, and can be made into dairy products, processed meat products, etc. , bread, pasta, biscuits, buccal tablets, capsules, fruit juices, tea, 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保加利亞乳桿菌TCI904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 bulgaricus TCI904 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，有關該保加利亞乳桿菌TCI904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 patients with hyperlipidemia, 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 bulgaricus TCI904 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：保加利亞乳桿菌TCI904之篩選與鑑定 Example 1: Screening and identification of Lactobacillus bulgaricus TCI904
(1-1)篩選 (1-1) Screening
取泡菜(產地：台灣)中的汁液分別與MRS培養液(BD DifcoTM Lactobacilli MRS Broth，型號DF0881-17-5)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℃之厭氧培養箱中培養直到單一菌落(single colony)出現。 The juice from kimchi (origin: Taiwan) was mixed with MRS culture medium (BD Difco TM Lactobacilli MRS Broth, model DF0881-17-5) at a volume ratio of 1:100, and placed at 37°C for anaerobic culture ( The oxygen concentration is 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, so as to generate different colonies. Next, in order to ensure the singleness of the strain, a colony was selected using a sterile inoculation loop, and the solid culture medium was partitioned and marked, 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)泡菜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保加利亞乳桿菌亞種之16SrDNA序列進行序列比對後，可知分離菌株的16SrDNA序列與其他保加利亞乳桿菌亞種的16SrDNA序列的相似性如表一所示。因此，將此分離菌株命名為保加利亞乳桿菌TCI904(後文以TCI904表示)。在此，將此保加利亞乳桿菌TCI904以寄存編號BCRC 910984寄存於財團法人食品工業發展研究所，以及以寄存編號DSM33505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 kimchi sample, phylogenetic analysis was performed to confirm that the strain has a 16S ribosomal ribonucleic acid (16S rRNA) fragment as shown in SEQ ID NO: 1 . 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 bulgaricus subspecies, the 16SrDNA sequence of the isolated strain can be known The similarity of the 16S rDNA sequence with other Lactobacillus bulgaricus subspecies is shown in Table 1. Therefore, this isolated strain was named Lactobacillus bulgaricus TCI904 (hereinafter expressed as TCI904). Herein, this Lactobacillus bulgaricus TCI904 is deposited with the Institute for Development of the Food Industry under the deposit number BCRC 910984, and with the German National Culture Collection (DSMZ) under the deposit number DSM33505.
取(1-1)泡菜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：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保加利亞乳桿菌亞種之16S rDNA序列進行序列比對後，可知分離菌株的16SrDNA序列與其他保加利亞乳桿菌亞種的16SrDNA序列的相似性如表一所示。在此，將此分離菌株命名為保加利亞乳桿菌LF063(後文以LF063表示)。 After taking the strain of another colony isolated from the pickle sample (1-1), the gene family analysis was carried out 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 sequence of other Lactobacillus bulgaricus subspecies, it can be known that the 16S rDNA sequence of the isolated strain is similar to that of other Lactobacillus bulgaricus subspecies. The similarity of the 16SrDNA sequences of the two species is shown in Table 1. Here, the isolated strain is named as Lactobacillus bulgaricus LF063 (hereinafter referred to as LF063).
其中，取(1-1)泡菜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(16SrRNA)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保加利亞乳桿菌亞種之16SrDNA序列進行序列比對後，可知分離菌株的16SrDNA序列與其他保加利亞乳桿菌亞種的16SrDNA序列的相似性如表一所示。在此，將此分離菌株命名為保加利亞乳桿菌LF006(後文以LF006表示)。 Wherein, the bacterial strain of another colony isolated from the (1-1) kimchi sample was subjected to gene family analysis, and it was confirmed that the bacterial strain had a 16S ribosomal ribonucleic acid (16SrRNA) fragment as shown in SEQ ID NO:3. Using the NCBI online database for comparison, after comparing the gene sequence shown in SEQ ID NO: 3 with the 16SrDNA sequence of other Lactobacillus bulgaricus subspecies, it can be known that the 16SrDNA sequence of the isolated strain is similar to that of other Lactobacillus bulgaricus subspecies The similarity of the 16S rDNA sequence is shown in Table 1. Here, the isolated strain is named Lactobacillus bulgaricus LF006 (hereinafter referred to as LF006).
其中，取(1-1)泡菜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保加利亞乳桿菌亞種之16SrDNA序列進行序列比對後，可知分離菌株的16SrDNA序列與其他保加利亞乳桿菌亞種的16SrDNA序列的相似性如表一所示。因此，將此分離菌株命名為保加利亞乳桿菌LF049(後文以LF049表示)。 Wherein, after taking (1-1) another strain of the colony isolated from the pickle sample, the gene family analysis was carried out to confirm that the strain has a 16S ribosomal ribonucleic acid (16S rRNA) fragment as shown in SEQ ID NO: 4 . Using the NCBI online database for comparison, after comparing the gene sequence shown in SEQ ID NO: 4 with the 16SrDNA sequence of other Lactobacillus bulgaricus subspecies, it can be known that the 16SrDNA sequence of the isolated strain is similar to that of other Lactobacillus bulgaricus subspecies The similarity of the 16S rDNA sequence is shown in Table 1. Therefore, this isolated bacterial strain is named as Lactobacillus bulgaricus LF049 (hereinafter expressed as LF049).
表一
綜合以上，本發明自泡菜樣品共篩選出四種保加利亞乳桿菌，分別命名為TCI904、LF063、LF006、LF049。 Based on the above, four kinds of Lactobacillus bulgaricus were screened out from the kimchi samples in the present invention, named as TCI904, LF063, LF006, and LF049 respectively.
(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四種保加利亞乳桿菌菌株培養於MRS培養液中，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。 Using the method of liquid culture, culture the four unique Lactobacillus bulgaricus strains obtained in (1-1) in the MRS culture medium to provide the bacterial liquid, then add 25% glycerol to the bacterial liquid, and mix the obtained The liquid was transferred to a cryopreservation tube and stored at -80°C.
例2：保加利亞乳桿菌TCI904之活化與樣品製備 Example 2: Activation and sample preparation of Lactobacillus bulgaricus TCI904
(2-1)活化 (2-1) Activation
將存有保加利亞乳桿菌TCI904菌株之冷凍保存管解凍，以1%之植菌量(約1x104CFU/mL)將TCI904菌株接種於MRS培養液中，並置於37℃下進行厭氧培養，歷時18小時，以提供保加利亞乳桿菌TCI904菌液，供後續實驗使用。其餘保加利亞乳桿菌LF063、LF006、LF049也是按照此方式進行活化，本處不再贅述。 Thaw the cryopreservation tube containing the Lactobacillus bulgaricus TCI904 strain, inoculate the TCI904 strain in the MRS culture medium with a 1% planting amount (about 1x10 4 CFU/mL), and place it at 37°C for anaerobic culture. 18 hours to provide Lactobacillus bulgaricus TCI904 bacterial liquid for subsequent experiments. The remaining Lactobacillus bulgaricus LF063, LF006, and LF049 were also activated in this way, and will not be repeated here.
(2-2)樣品製備 (2-2) Sample preparation
取(2-1)提供的保加利亞乳桿菌TCI904菌液，以1%之植菌量(約1x104CFU/mL)接種於MRS培養液之中，並置於37℃下進行厭氧培養，歷時18小時。其後，以5000rpm轉速對培養後的菌液進行離心，歷時5分鐘，再使用0.2μm的濾膜對所獲得之上清液進行過濾，確保已將 菌體去除，所得濾液即為TCI904樣品(即為TCI904的代謝產物，不含TCI904菌體)。其餘保加利亞乳桿菌LF063、LF006、LF049也是按照此方式進行樣品製備，本處不再贅述。 Take the Lactobacillus bulgaricus TCI904 bacterial solution provided in (2-1), inoculate it into the MRS culture medium with a 1% planting amount (about 1x10 4 CFU/mL), and place it at 37°C for anaerobic culture for 18 Hour. Thereafter, the cultured bacterial solution was centrifuged at a speed of 5000 rpm for 5 minutes, and then the obtained supernatant was filtered using a 0.2 μm filter membrane to ensure that the bacterial cells were removed, and the resulting filtrate was the TCI904 sample ( It is the metabolite of TCI904, excluding TCI904 cells). The remaining Lactobacillus bulgaricus LF063, LF006, and LF049 were also prepared in this way, and will not be repeated here.
例3：保加利亞乳桿菌TCI904抑制脂肪酶活性的試管實驗 Example 3: Test-tube experiment of Lactobacillus bulgaricus TCI904 inhibiting lipase activity
(3-1)脂肪酶活性的分析原理 (3-1) Analysis principle of lipase activity
脂肪酶(lipase)是一種催化脂類中酯鍵水解反應的水溶性酵素，其與酯解酶(esterase)的主要差異為具有界面活化(interfacial activation)的特性，必須在油水界面(lipid-water interface)中進行水解催化反應，即：當受質呈乳化狀態(emulsion)時，酵素反應的速率為最高。大多數的脂肪酶係作用於脂類甘油骨架的特定位置，例如：脂肪酶能夠催化三酸甘油酯(triacylglycerols)水解成甘油、單酸甘油酯(monoacylglycerols)、雙醯基甘油酯(diacylglycerols)及游離脂肪酸。依脂肪酶水解三酸甘油酯的特異性，可分為：(1)非特異性脂肪酶(non-specific lipase)、(2)1,3-特異性脂肪酶(1,3-specific lipase)、(3)2-特異性脂肪酶(2-specific lipase)、(4)脂肪酸特異性脂肪酶(fatty acid specific lipase)等，而脂肪酶主要可進行的催化反應有酯解反應(ester hydrolysis)、酯化反應(ester synthesis)、以及轉酯化反應(interesterification)等。 Lipase is a water-soluble enzyme that catalyzes the hydrolysis of ester bonds in lipids. The main difference between lipase and esterase is that it has the characteristics of interfacial activation and must be activated at the oil-water interface (lipid-water interface). The hydrolysis catalytic reaction is carried out in the interface), that is, when the substrate is in an emulsified state (emulsion), the rate of the enzyme reaction is the highest. Most lipases act on specific positions of the glycerol backbone of lipids. For example, lipases can catalyze the hydrolysis of triacylglycerols into glycerol, monoacylglycerols, diacylglycerols and free fatty acids. According to the specificity of lipase to hydrolyze triglyceride, it can be divided into: (1) non-specific lipase (non-specific lipase), (2) 1,3-specific lipase (1,3-specific lipase) , (3) 2-specific lipase (2-specific lipase), (4) fatty acid specific lipase (fatty acid specific lipase), etc., and lipase can mainly catalyze ester hydrolysis reaction (ester hydrolysis) , esterification (ester synthesis), and transesterification (interesterification).
本實施例是利用硝基苯酚(p-nitrophenol,pNP)生成量測定法來分析脂肪酶的活性。由於脂肪酶會將硝酸苯酚脂類基質(p-nitrophenyl esters)進行水解，產生硝基苯酚(p-nitrophenol,pNP) 及脂肪酸，可於波長約400nm測得硝基苯酚的吸光值定量，利用96孔盤進行分析後，計算脂肪酶活性。硝酸苯酚脂類基質的種類並不限制，在本發明一較佳實施例中，是以4-硝基苯基月桂酸酯(4-Nitrophenyl laurate)作為反應基質，脂肪酶能將其分解成硝基苯酚，再以硝基苯酚的生成量來分析脂肪酶的活性。 In this example, the activity of lipase was analyzed by the assay method of p -nitrophenol ( p -nitrophenol, pNP) production. Since lipase will hydrolyze the nitrophenol lipid matrix ( p -nitrophenyl esters) to produce nitrophenol ( p -nitrophenol, p NP) and fatty acids, the absorbance value of nitrophenol can be measured at a wavelength of about 400nm and quantified by using After analysis in 96-well plates, lipase activity was calculated. The kind of nitrophenol lipid substrate is not limited, and in a preferred embodiment of the present invention, be with 4-nitrophenyl laurate (4-Nitrophenyl laurate) as reaction substrate, lipase can decompose it into nitrate Based on phenol, the activity of lipase was analyzed by the amount of nitrophenol produced.
(3-2)脂肪酶活性實驗的試劑配製 (3-2) Reagent preparation for lipase activity experiment
首先，配置一第一緩衝溶液，該第一緩衝溶液是將17mg十二烷基硫酸鈉(Sodium Dodecyl Sulfate，SDS)與1g聚乙二醇辛基苯基醚(Triton X-100)溶於100mL水中；亦配置一第二緩衝溶液，該第二緩衝溶液係66mM三羥甲基氨基甲烷(Tris)，並以鹽酸(Hydrochloric acid，HCl)滴定至pH 7.4後，補適量的水至體積為100ml，該第二緩衝溶液是作為製備脂肪酶溶液的緩衝液，以及作為酶解反應的終止劑。接著，取51.4mg的4-硝基苯基月桂酸酯加入100mL上述第一緩衝溶液，並於65℃下攪拌15分鐘使其均勻混合，待溶液呈澄清狀後，於室溫下冷卻後，獲得1.6mM之4-硝基苯基月桂酸酯溶液。該4-硝基苯基月桂酸酯溶液可置於4℃冷藏保存3天，使用時需再加熱至65℃待溶液呈透明澄清狀後，置於室溫下冷卻後即可使用。另一方面，秤取99.21mg脂肪酶(型號L3126，Sigma，來自豬胰臟之脂肪酶)加入上述第二緩衝溶液中，並定量至10mL後混合均勻，以獲得150U/mL的脂肪酶溶液。 First, configure a first buffer solution, the first buffer solution is to dissolve 17mg of sodium dodecyl sulfate (Sodium Dodecyl Sulfate, SDS) and 1g of polyethylene glycol octylphenyl ether (Triton X-100) in 100mL In water; also configure a second buffer solution, the second buffer solution is 66mM Tris (Tris), and after titrating to pH 7.4 with hydrochloric acid (HCl), add an appropriate amount of water to a volume of 100ml , the second buffer solution is used as a buffer for preparing the lipase solution and as a terminator for the enzymatic hydrolysis reaction. Next, take 51.4mg of 4-nitrophenyl laurate and add 100mL of the first buffer solution above, and stir at 65°C for 15 minutes to mix evenly. After the solution becomes clear, cool it at room temperature, A 1.6 mM solution of 4-nitrophenyl laurate was obtained. The 4-nitrophenyl laurate solution can be refrigerated at 4°C for 3 days. When used, it needs to be heated to 65°C until the solution becomes transparent and clear, and it can be used after cooling at room temperature. On the other hand, 99.21 mg of lipase (model L3126, Sigma, lipase from porcine pancreas) was weighed and added to the second buffer solution, quantified to 10 mL and mixed evenly to obtain a 150 U/mL lipase solution.
(3-3)脂肪酶活性實驗的實驗步驟 (3-3) Experimental procedure of lipase activity experiment
取一96孔盤執行實驗，分為控制組及四組實驗組(以各種保加利亞乳桿菌的代謝產物做為測試樣品)，下表二說明各組別採用的測試 樣品、反應酵素及反應基質。 Take a 96-well plate to perform the experiment, and divide it into a control group and four experimental groups (using various metabolites of Lactobacillus bulgaricus as test samples). The following table 2 illustrates the tests used in each group Samples, reaction enzymes and reaction substrates.
將25μL的測試樣品加入96孔盤的孔中，接著在控制組及各實驗組(實驗組A、實驗組B、實驗組C、實驗組D)孔中加入脂肪酶，在各空白組(控制組(空白)、實驗組A(空白)、實驗組B(空白)、實驗組C(空白)、實驗組D(空白))中加入第二緩衝溶液，混合均勻。接著將50μL的1.6mM之4-硝基苯基月桂酸酯溶液加入孔中，混合均勻。封上塑膠膜防止揮發，置於37℃反應30分鐘，此時控制組及各實驗組中的脂肪酶會與4-硝基苯基月桂酸酯進行反應。最後，加入100μL的第二緩衝溶液來中止所有孔中的反應，接著以ELISA讀取儀(BioTek)讀取各組之OD400nm讀值，以下列公式計算出脂肪酶活性抑制率。 Add 25 μL of test samples into the wells of the 96-well plate, then add lipase to the wells of the control group and each experimental group (experimental group A, experimental group B, experimental group C, and experimental group D), and add lipase to each blank group (control group). Group (Blank), Experimental Group A (Blank), Experimental Group B (Blank), Experimental Group C (Blank), Experimental Group D (Blank)) was added with the second buffer solution and mixed evenly. Then add 50 μL of 1.6 mM 4-nitrophenyl laurate solution into the wells and mix well. Seal it with a plastic film to prevent volatilization, and place it at 37° C. for 30 minutes to react. At this time, the lipase in the control group and each experimental group will react with 4-nitrophenyl laurate. Finally, 100 μL of the second buffer solution was added to stop the reactions in all wells, and then the OD 400nm readings of each group were read with an ELISA reader (BioTek), and the lipase activity inhibition rate was calculated by the following formula.
脂肪酶活性抑制率(%)公式=[1-(A實驗組-A實驗組(空白)/A控制組-A控制組(空白)]X100%，其中A代表OD400nm讀值的讀值。 Lipase activity inhibition rate (%) formula=[1-(A experimental group-A experimental group (blank)/A control group-A control group (blank)]X100%, where A represents the reading value of OD 400nm reading.
本發明之保加利亞乳桿菌TCI904及其他菌種抑制脂肪酶活性之效果如圖1所示，由圖1可見，將控制組的脂肪酶活性計為100%，實驗組A(TCI904)的脂肪酶活性為60.71%，代表TCI904可抑制脂肪酶活性高達約39.29%；實驗組B(LF063)的脂肪酶活性為78.03%，代表LF063 可抑制脂肪酶活性約21.97%；實驗組C(LF006)的脂肪酶活性為76.10%，代表LF006可抑制脂肪酶活性約23.9%；實驗組D(LF049)的脂肪酶活性為71.0%，代表LF049可抑制脂肪酶活性約29.0%。 The effect of Lactobacillus bulgaricus TCI904 of the present invention and other bacterial classifications to suppress lipase activity is as shown in Figure 1, as seen from Figure 1, the lipase activity of control group is counted as 100%, the lipase activity of experimental group A (TCI904) was 60.71%, representing that TCI904 could inhibit lipase activity up to about 39.29%; the lipase activity of experimental group B (LF063) was 78.03%, representing that LF063 It can inhibit lipase activity by about 21.97%; the lipase activity of experimental group C (LF006) is 76.10%, representing that LF006 can inhibit lipase activity by about 23.9%; the lipase activity of experimental group D (LF049) is 71.0%, representing LF049 It can inhibit lipase activity by about 29.0%.
根據此數據，說明本發明的保加利亞乳桿菌TCI904相較於其他同種菌株，具有明顯更佳抑制脂肪酶活性的能力，表示本發明的保加利亞乳桿菌TCI904具有與其他同種菌株不同的菌學特徵。 According to this data, it is illustrated that Lactobacillus bulgaricus TCI904 of the present invention has significantly better ability to inhibit lipase activity compared to other strains of the same species, indicating that Lactobacillus bulgaricus TCI904 of the present invention has different bacteriological characteristics from other strains of the same species.
例4：保加利亞乳桿菌TCI904刺激脂聯素分泌的細胞實驗 Example 4: Cell experiment of Lactobacillus bulgaricus TCI904 stimulating adiponectin secretion
脂聯素(Adiponectin)是脂肪細胞分泌的一種功能性胜肽，能夠促進肌肉細胞燃燒脂肪轉換成能量，減少體內脂肪堆積，為改善肥胖的關鍵因子，當脂聯素上升時，會促使肌肉燃燒脂肪產生能量維持身體所需。研究證實，中高強度的運動會促使脂聯素上升，幫助肌肉代謝脂肪產生能量，達到減脂的效果。可參見例如：Effects of exercise on adiponectin：a systematic review，Med Sci(Basel).2018 Dec；6(4)：97，該文獻之全文併於此處以供參考。 Adiponectin is a functional peptide secreted by fat cells, which can promote muscle cells to burn fat and convert it into energy, reduce body fat accumulation, and is a key factor in improving obesity. When adiponectin rises, it will promote muscle burning Fat produces the energy your body needs to maintain itself. Studies have confirmed that moderate to high-intensity exercise will increase adiponectin, help muscles metabolize fat to generate energy, and achieve the effect of fat loss. See, eg, Effects of exercise on adiponectin: a systematic review, Med Sci (Basel). 2018 Dec;6(4):97, which is hereby incorporated by reference in its entirety.
在此實施例中，利用細胞模式，檢測TCI904樣品調節脂聯素分泌的能力。 In this example, TCI904 samples were tested for their ability to modulate adiponectin secretion using a cellular model.
(4-1)實驗材料 (4-1) Experimental materials
1.細胞株：小鼠骨髓基質細胞(後續簡稱OP9細胞)，OP9細胞購自美國典型培養物保存中心(American Type Culture Collection，ATCC®)之OP9細胞株(ATCC CRL-2749)。 1. Cell line: mouse bone marrow stromal cells (hereinafter referred to as OP9 cells), OP9 cells were purchased from the OP9 cell line (ATCC CRL-2749) of the American Type Culture Collection (ATCC ® ).
2.培養基：MEMAM(Minimum Essential Medium Alpha Medium，購自Gibco，美國)細胞培養液、20%之胎牛血清(Fetal Bovine Serum，購自Gibco，美國，Cat#10437-028)，且加入0.1%之青黴素/鏈黴素(Penicillin-streptomycin，購自Gibco，美國)。 2. Medium: MEMAM (Minimum Essential Medium Alpha Medium, purchased from Gibco, USA) cell culture medium, 20% fetal bovine serum (Fetal Bovine Serum, purchased from Gibco, USA, Cat#10437-028), and 0.1% penicillin/streptomycin (Penicillin-streptomycin, purchased from Gibco, USA) was added.
3.脂聯素檢測試劑套組(購自CUSABIO，型號CSB-E07272m)，套組內含有以下溶液： 3. Adiponectin detection reagent set (purchased from CUSABIO, model CSB-E07272m), which contains the following solutions:
3-1：檢測盤(Assay plate) 3-1: Assay plate
3-2：標準品(Standard) 3-2: Standard
3-3：100倍生物素抗體(100X Biotin-antibody) 3-3: 100X Biotin-antibody
3-4：100倍HRP親和素(100X HRP-avidin) 3-4: 100X HRP-avidin (100X HRP-avidin)
3-5：生物素抗體稀釋劑(Biotin-antibody diluent) 3-5: Biotin-antibody diluent
3-6：HRP親和素稀釋劑(HRP-avidin diluent) 3-6: HRP avidin diluent (HRP-avidin diluent)
3-7：樣品稀釋劑(Sample diluent) 3-7: Sample diluent (Sample diluent)
3-8：25倍洗滌緩衝液(25X Wash buffer) 3-8: 25X Wash buffer (25X Wash buffer)
3-9：TMB液(TMB substrate) 3-9: TMB solution (TMB substrate)
3-10：終止液(Stop solution) 3-10: Stop solution (Stop solution)
3-11：清洗液(Wash buffer) 3-11: Wash buffer
3-12：檢測盤封蓋(Plate sealers) 3-12: Plate sealers
(4-2)操作前的試劑製備 (4-2) Reagent preparation before operation
1. 1倍生物素抗體：將1μl的100倍生物素抗體加入999μl的生物素抗體稀釋劑，混和均勻備用 1. 1x biotin antibody: add 1 μl of 100x biotin antibody to 999 μl of biotin antibody diluent, mix well and set aside
2. 1倍HRP親和素：將1μl的100倍HRP親和素加入999μl的HRP親和素稀釋劑，混和均勻備用 2. 1-fold HRP-avidin: add 1 μl of 100-fold HRP-avidin to 999 μl of HRP-avidin diluent, mix well and set aside
3. 1倍洗滌緩衝液：將25倍洗滌緩衝液回溫後，輕搖至結晶全 部溶解，將20ml的25倍洗滌緩衝液加入480ml的水，混和均勻備用 3. 1x Washing Buffer: Return 25x Washing Buffer to temperature, shake gently until crystallization is complete Partially dissolved, add 20ml of 25 times washing buffer to 480ml of water, mix well and set aside
4.標準液：將標準品小管以6000-10000rpm離心30秒，使粉末聚集至底部。加入1ml樣品稀釋劑混和均勻，靜置15分鐘使其充分溶解，達最終濃度10ng/ml 4. Standard solution: centrifuge the standard tube at 6000-10000rpm for 30 seconds to make the powder gather to the bottom. Add 1ml of sample diluent and mix well, let it stand for 15 minutes to fully dissolve, reaching a final concentration of 10ng/ml
5.標準液序列稀釋：準備7個1.5ml微量離心管，分別加入250μl樣品稀釋劑，取250μl標準液至第6管，混和均勻後再從第6管取250μl至第5管，序列稀釋至第1管，最終獲得濃度為10ng/ml、5ng/ml、2.5ng/ml、1.25ng/ml、0.625ng/ml、0.313ng/ml、0.156ng/ml的標準液 5. Serial dilution of the standard solution: Prepare seven 1.5ml microcentrifuge tubes, add 250μl sample diluent respectively, take 250μl standard solution to the sixth tube, mix well, then take 250μl from the sixth tube to the fifth tube, serially dilute to In the first tube, standard solutions with concentrations of 10ng/ml, 5ng/ml, 2.5ng/ml, 1.25ng/ml, 0.625ng/ml, 0.313ng/ml, and 0.156ng/ml were finally obtained
以下簡述脂聯素檢測步驟，詳細操作步驟可參照脂聯素檢測試劑套組所附的使用說明書執行，可參見https：//www.cusabio.com/uploadfile/Ins/CSB-E07272m.pdf，該使用說明書之全文併於此處以供參考。 The following is a brief description of the adiponectin detection steps. For detailed steps, please refer to the instruction manual attached to the adiponectin detection reagent kit, which can be found at https://www.cusabio.com/uploadfile/Ins/CSB-E07272m.pdf, The entire text of this Instructions for Use is hereby incorporated by reference.
(4-3)脂聯素檢測前處理(細胞培養及分組方式) (4-3) Pretreatment for adiponectin detection (cell culture and grouping method)
首先，取24孔培養盤，將每孔接種8×104個OP9細胞及500μL上述培養基，在37℃下培養7天。此7天的細胞培養期間每隔3天更換培養基。7天後，以顯微鏡(ZEISS；放大倍率400x)觀察細胞內油滴形成，藉以確認細胞已完全分化為脂肪細胞。 First, take a 24-well culture plate, inoculate 8×10 4 OP9 cells and 500 μL of the above medium in each well, and culture at 37°C for 7 days. Medium was changed every 3 days during this 7-day cell culture period. After 7 days, the formation of oil droplets in the cells was observed with a microscope (ZEISS; magnification 400x), so as to confirm that the cells had completely differentiated into adipocytes.
然後，將分化完成的脂肪細胞分為以下三組：控制組、空白組與實驗組，下列表三說明各組的添加成分、細胞實驗濃度以及處理時間。 Then, the differentiated adipocytes were divided into the following three groups: control group, blank group and experimental group. Table 3 below shows the added components, cell experimental concentration and treatment time of each group.
表三
詳細來說，各組是以以下方式進行培養。 Specifically, each group was cultured as follows.
空白組：依照每孔500μL培養基體積量的方式培養分化後脂肪細胞，在37℃下培養24小時。 Blank group: the differentiated adipocytes were cultured in the manner of 500 μL medium volume per well, and cultured at 37° C. for 24 hours.
控制組：依照每孔500μL培養基含有2.5μL的MRS培養液(即，濃度為0.5%(v/v))的方式，培養分化後脂肪細胞，在37℃下培養24小時。 Control group: The differentiated adipocytes were cultured in a manner that 500 μL of medium per well contained 2.5 μL of MRS medium (that is, the concentration was 0.5% (v/v)), and cultured at 37° C. for 24 hours.
實驗組：依照每孔500μL培養基含有2.5μL的例(2-2)所製備的TCI904樣品(TCI904的代謝產物，不含TCI904菌體)(即，TCI904樣品濃度為0.5%(v/v))的方式，培養分化後脂肪細胞，在37℃下培養24小時。 Experimental group: TCI904 samples (metabolites of TCI904, excluding TCI904 bacteria) prepared according to the example (2-2) in which 500 μL medium contained 2.5 μL per well (that is, the concentration of TCI904 samples was 0.5% (v/v)) In this way, differentiated adipocytes were cultured at 37°C for 24 hours.
(4-4)脂聯素檢測流程 (4-4) Adiponectin detection process
上述細胞培養後，將孔內培養液移至1.5ml微量離心管。於2-8℃以1000 xg離心15分鐘，將離心後的上清液移至新的1.5ml微量離心管，接著將上清液稀釋2000倍，接著以脂聯素檢測試劑套組進行檢測，操作方式如下。 After the above cell culture, the culture medium in the well was transferred to a 1.5ml microcentrifuge tube. Centrifuge at 1000 xg for 15 minutes at 2-8°C, transfer the centrifuged supernatant to a new 1.5ml microcentrifuge tube, then dilute the supernatant 2000 times, and then use the adiponectin detection kit for detection. The operation method is as follows.
取100μl序列稀釋標準品和測試樣品(上清液)至檢測盤孔中，用檢測盤封蓋封好，於37℃靜置2小時。接著將孔內液體移除，不須沖洗。 Take 100 μl serial dilution standard and test sample (supernatant) into the wells of the test plate, seal it with the cover of the test plate, and let it stand at 37° C. for 2 hours. The fluid in the pores is then removed without rinsing.
加入100μl 1倍生物素抗體，用新的檢測盤封蓋封好，於37℃靜置1小時。將孔盤內液體移除，以200μl 1倍洗滌緩衝液潤洗2次。清洗完後將孔內液體移除，並倒置於紙巾上去除多餘液體。 Add 100 μl of 1x biotin antibody, cover and seal with a new detection plate, and let stand at 37°C for 1 hour. Remove the liquid in the well plate and rinse twice with 200 μl 1X washing buffer. After washing, remove the liquid in the well and invert on a paper towel to remove excess liquid.
加入100μl 1倍HRP親和素，用新的檢測盤封蓋封好，於37℃靜置1小時。將孔盤內液體移除，以200μl 1倍洗滌緩衝液潤洗2次。清洗完後將孔內液體移除，並倒置於紙巾上去除多餘液體。 Add 100 μl of 1X HRP avidin, seal the lid with a new detection plate, and let it stand at 37°C for 1 hour. Remove the liquid in the well plate and rinse twice with 200 μl 1X washing buffer. After washing, remove the liquid in the well and invert on a paper towel to remove excess liquid.
加入90μl TMB液，於37℃避光反應15-30分鐘，接著加入50μl終止液，輕拍使其充分混和。以ELISA讀取儀(BioTek)讀取各組之OD548nm讀值(O.D.值越大，表示脂聯素的含量越高)。最後使用Excel軟體中的student t-test進行統計分析。 Add 90 μl of TMB solution, and react at 37°C in the dark for 15-30 minutes, then add 50 μl of stop solution, and tap to mix well. The OD 548nm reading value of each group was read with an ELISA reader (BioTek) (the larger the OD value, the higher the content of adiponectin). Finally, the student t-test in Excel software was used for statistical analysis.
經過吸光值的比較換算並乘回稀釋倍數，空白組所測得脂聯素含量為2254.75ng/ml，控制組所測得脂聯素含量為1974.02ng/ml，實驗組所測得脂聯素含量為2508.51ng/ml。 After comparing the absorbance value and multiplying back to the dilution factor, the content of adiponectin in the blank group was 2254.75ng/ml, the content of adiponectin in the control group was 1974.02ng/ml, and the content of adiponectin in the experimental group was 2254.75ng/ml. The content is 2508.51ng/ml.
請參閱圖2。將空白組的脂聯素含量為2254.75ng/ml視為1(即100%)時，控制組的脂聯素的含量(1974.03ng/ml)為0.87(即87%)，實驗組的脂聯素的含量(2508.51ng/ml)為1.11(即111%)，代表細胞經TCI904樣品(TCI904之代謝產物，不含TCI904菌體)處理後，細胞分泌脂聯素的含量顯著增加，為空白組的1.11倍。 See Figure 2. When the adiponectin content of the blank group was 2254.75ng/ml as 1 (i.e. 100%), the adiponectin content (1974.03ng/ml) of the control group was 0.87 (i.e. 87%), and the adiponectin content of the experimental group was 0.87 (i.e. 87%). The content of adiponectin (2508.51ng/ml) was 1.11 (ie 111%), which means that after the cells were treated with TCI904 samples (metabolites of TCI904, excluding TCI904 cells), the content of adiponectin secreted by cells increased significantly, which was the blank group 1.11 times.
由此可知，當分化後脂肪細胞經TCI904樣品處理後，脂聯 素的含量提升，代表TCI904能有效提升脂聯素的含量，具有減脂的潛力。 It can be seen that when the differentiated adipocytes are treated with TCI904 samples, the adiponected The increase in the content of adiponectin means that TCI904 can effectively increase the content of adiponectin and has the potential to reduce fat.
例5：保加利亞乳桿菌TCI904的人體實驗 Example 5: Human experimentation of Lactobacillus bulgaricus TCI904
為進一步確認保加利亞乳桿菌TCI904對於人體的影響，令9位受試者每日服用1顆保加利亞乳桿菌TCI904的活菌膠囊(每顆膠囊含有1X108CFU(即1X108CFU/cap)菌數的保加利亞乳桿菌TCI904)，並服用4週。其中，9位受試者的身高體重指數(BMI)大於24，或為體脂高的族群(身體體脂率大於25%)。並且，每顆膠囊含有100毫克保加利亞乳桿菌TCI904之活菌粉。 In order to further confirm the impact of Lactobacillus bulgaricus TCI904 on the human body, 9 subjects were asked to take 1 capsule of live bacteria of Lactobacillus bulgaricus TCI904 (each capsule contains 1X10 8 CFU (ie 1X10 8 CFU/cap) bacteria count Lactobacillus bulgaricus TCI904), and took for 4 weeks. Among them, 9 subjects had a body mass index (BMI) greater than 24, or belonged to a group with high body fat (body fat percentage greater than 25%). And, each capsule contains 100 mg of Lactobacillus bulgaricus TCI904 live bacteria powder.
於試驗前(即未開始服用TCI904活菌膠囊前，視為第0週)及於試驗後(即服用TCI904活菌膠囊4週後，視為第4週)分別進行體重、軀幹體脂率、腰圍、三酸甘油酯(Triglyceride，TG)、極低密度脂蛋白(Very Low Density Lipoprotein，VLDL)、高密度脂蛋白(High density lipoprotein，HDL)的檢測，各項數值的量測方式如下所述。 Body weight, trunk body fat percentage, Waist circumference, triglyceride (Triglyceride, TG), very low density lipoprotein (Very Low Density Lipoprotein, VLDL), high density lipoprotein (High density lipoprotein, HDL) detection, the measurement methods of each value are as follows .
體重和軀幹體脂率是利用體重體脂計(TANITA四肢與軀幹體組成計，型號BC-545F)進行檢測，而腰圍是以皮尺進行量測，詳細來說，除去受試者腰部覆蓋衣物，受試者保持輕鬆站立，雙手自然下垂，檢測者將皮尺繞過受試者腰部，調整高度，以肚臍為水平量測點，同時注意皮尺與地面保持平，並緊貼、不擠壓皮膚，受試者維持正常呼吸。在吐氣結束時，量取腰圍數值。 Body weight and trunk body fat percentage are detected by body weight and body fat meter (TANITA Limbs and Trunk Body Composition Meter, model BC-545F), while waist circumference is measured by tape measure. The subject keeps standing easily, with hands hanging down naturally. The tester passes the tape measure around the subject's waist, adjusts the height, and takes the navel as the horizontal measurement point. At the same time, pay attention to keep the tape measure flat with the ground, and keep it close to the skin without squeezing it , the subject maintained normal breathing. At the end of exhalation, measure the waist circumference.
三酸甘油酯、極低密度脂蛋白、高密度脂蛋白的檢測方式是對受試者進行靜脈採血，並將血液樣本送至檢驗所(立人醫事檢驗所)測定三酸甘油脂、極低密度脂蛋白及高密度脂蛋白的數值。 The detection method of triglyceride, very low density lipoprotein and high density lipoprotein is to collect venous blood from the subject, and send the blood sample to the laboratory (Realen Medical Laboratory) to determine triglyceride, very low Density lipoprotein and high-density lipoprotein values.
需要特別說明的是，第0週的量測結果與第4週的量測結果之間的統計學顯著差異是藉由Excel軟體中的student t-test來統計分析。 It should be noted that the statistically significant difference between the measurement results of the 0th week and the measurement results of the 4th week was statistically analyzed by the student t-test in the Excel software.
請參閱圖3。於試驗前(即圖3中第0週)，所有受試者的平均體重為86.3公斤，而在試驗後(即圖3第4週)，所有受試者的平均體重為85.7公斤。換言之，經過4週每日服用一顆含有1X108CFU的TCI904的活菌膠囊後，受試者的平均體重下降0.6公斤。基此，保加利亞乳桿菌TCI904能降低體重。
See Figure 3. Before the test (ie,
請參閱圖4。於試驗前(即圖4的第0週)，所有受試者的平均軀幹體脂率為36.9%，而在試驗後(即圖4的第4週)，所有受試者的平均軀幹體脂率為36.3%。 See Figure 4. Before the test (i.e. the 0th week in Figure 4), the average trunk body fat rate of all subjects was 36.9%, and after the test (i.e. the 4th week in Figure 4), the average trunk body fat percentage of all subjects The rate is 36.3%.
請參閱圖5，於試驗前(即圖5的第0週)，所有受試者的平均腰圍為99.9公分，而在試驗後(即圖5第4週)，所有受試者的平均腰圍為98.2公分。 Please refer to Figure 5, before the test (i.e. the 0th week in Figure 5), the average waist circumference of all subjects was 99.9 cm, and after the test (i.e. the 4th week in Figure 5), the average waist circumference of all subjects was 98.2 cm.
換言之，經過4週每日服用一顆含有1X108CFU的TCI904的活菌膠囊後，受試者的平均軀幹體脂率下降0.6%，腰圍平均減少1.7公分。基此，保加利亞乳桿菌TCI904能降低軀幹體脂率及腰圍。 In other words, after 4 weeks of daily consumption of a live bacteria capsule containing 1X10 8 CFU of TCI904, the subjects' average trunk body fat percentage decreased by 0.6%, and their waist circumference decreased by an average of 1.7 cm. Based on this, Lactobacillus bulgaricus TCI904 can reduce trunk body fat percentage and waist circumference.
因此，保加利亞乳桿菌TCI904具有降低體重、降低軀幹體脂率及腰圍的功效。 Therefore, Lactobacillus bulgaricus TCI904 has the effect of reducing body weight, trunk body fat percentage and waist circumference.
三酸甘油酯，又叫中性脂肪，是人體內的一種脂肪，也存在許多食物中。人體攝取油脂、蛋白質或碳水化合物產生的熱量，未被消耗掉的部份轉變成三酸甘油酯，儲存在肝臟或脂肪細胞中，作為備用能量。熱量過盛則增加三酸甘油酯，構成皮下脂肪主要成分，導致肥胖或動脈硬 化等疾病。 Triglycerides, also known as neutral fats, are a type of fat in the human body and are also present in many foods. The heat generated by the human body's intake of fat, protein or carbohydrates is converted into triglycerides, which are stored in the liver or fat cells as backup energy. Excessive calories will increase triglycerides, which constitute the main component of subcutaneous fat, leading to obesity or arteriosclerosis and other diseases.
請參閱圖6，於試驗前(即圖6的第0週)，所有受試者的平均三酸甘油脂為113.8mg/dL，而在試驗後(即圖6的第4週)，所有受試者的平均三酸甘油脂為108.9mg/dL，相當於三酸甘油脂降低了約4.3%。 Please refer to Figure 6, before the test (i.e. the 0th week in Figure 6), the average triglyceride of all subjects was 113.8mg/dL, and after the test (i.e. the 4th week in Figure 6), all subjects The average triglycerides of the subjects were 108.9 mg/dL, which is equivalent to a reduction of triglycerides by about 4.3%.
極低密度脂蛋白，主要成分為三酸甘油脂，於肝臟或小腸內合成。若食入大量的脂肪或醣類，則會增加極低密度脂蛋白的合成。低密度脂蛋白其主要的作用是將膽固醇由肝臟帶到週邊組織。若血液中的低密度脂蛋白過高，容易造成冠狀動脈硬化及心臟病。 Very low-density lipoprotein, the main component is triglyceride, synthesized in the liver or small intestine. If you eat a lot of fat or sugar, it will increase the synthesis of very low density lipoprotein. The main function of low-density lipoprotein is to carry cholesterol from the liver to surrounding tissues. If the low-density lipoprotein in the blood is too high, it is easy to cause coronary arteriosclerosis and heart disease.
請參閱圖7，於試驗前(即圖7的第0週)，所有受試者的平均極低密度脂蛋白為19.9mg/dL，而在試驗後(即圖7第4週)，所有受試者的平均極低密度脂蛋白為17.7mg/dL，相當於極低密度脂蛋白降低了約11.0%。
Please refer to Figure 7, before the test (
高密度脂蛋白主要的功能是將週邊組織的膽固醇帶回肝臟代謝。血液中高密度脂蛋白越高，罹患冠狀動脈硬化及心臟病的機率越低。 The main function of HDL is to bring cholesterol from peripheral tissues back to the liver for metabolism. The higher the high-density lipoprotein in the blood, the lower the probability of suffering from coronary atherosclerosis and heart disease.
請參閱圖8，於試驗前(即圖8的第0週)，所有受試者的平均高密度脂蛋白為53.2mg/dL，而在試驗後(即圖8第4週)，所有受試者的平均高密度脂蛋白為59.3mg/dL，相當於高密度脂蛋白提升了約11.4%。 Please refer to Figure 8, before the test (i.e. the 0th week in Figure 8), the average high-density lipoprotein of all subjects was 53.2mg/dL, and after the test (i.e. the 4th week in Figure 8), all subjects The average high-density lipoprotein of the patients was 59.3mg/dL, which is equivalent to an increase of about 11.4% in high-density lipoprotein.
換言之，經過4週每日服用一顆含有1X108CFU的TCI904的活菌膠囊後，受試者的三酸甘油脂降低了約4.3%，極低密度脂蛋白降低了約11.0%，高密度脂蛋白提升了約11.4%。基此，保加利亞乳桿菌TCI904能降低三酸甘油脂、極低密度脂蛋白以及提升高密度脂蛋白。 In other words, after 4 weeks of taking a live bacterial capsule containing 1X10 8 CFU of TCI904 daily for 4 weeks, the subjects' triglycerides decreased by about 4.3%, VLDL decreased by about 11.0%, and HDL Protein increased by about 11.4%. Based on this, Lactobacillus bulgaricus TCI904 can reduce triglyceride, very low-density lipoprotein and increase high-density lipoprotein.
綜上，透過例3可知，在試管模式中證實，保加利亞乳桿菌TCI904可抑制脂肪酶活性，且抑制程度明顯多於其他保加利亞乳桿菌菌株。透過例4可知，保加利亞乳桿菌TCI904有提升脂聯素的能力。透過例5可知，保加利亞乳桿菌TCI904能降低體重、降低軀幹體脂、降低腰圍、減少三酸甘油脂、減少極低密度脂蛋白、提升高密度脂蛋白。 In summary, it can be seen from Example 3 that Lactobacillus bulgaricus TCI904 can inhibit lipase activity in test tube mode, and the degree of inhibition is significantly greater than that of other Lactobacillus bulgaricus strains. From Example 4, it can be seen that Lactobacillus bulgaricus TCI904 has the ability to increase adiponectin. From Example 5, it can be seen that Lactobacillus bulgaricus TCI904 can reduce body weight, reduce trunk body fat, reduce waist circumference, reduce triglycerides, reduce very low-density lipoprotein, and increase high-density lipoprotein.
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.
TW中華民國 財團法人食品工業發展研究所2020/03/27 BCRC 910984 TW Republic of China Food Industry Development Research Institute 2020/03/27 BCRC 910984
DE德國 德國國家菌種保藏中心DSMZ(Deutsche Sammlung von Mikroorganismen und Zellkulturen)2020/4/17 DSM33505 DE Germany German National Culture Collection DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) 2020/4/17 DSM33505
<110> 大江生醫股份有限公司 <110> Dajiang Biomedical Co., Ltd.
<120> 保加利亞乳桿菌TCI904、其培養上清液、其組合物及其用於減少體重的用途 <120> Lactobacillus bulgaricus TCI904, its culture supernatant, its composition and its use for reducing body weight
<150> US 62/925,587 <150> US 62/925,587
<151> 2019-10-24 <151> 2019-10-24
<160> 4 <160> 4
<170> PatentIn version 3.5 <170> PatentIn version 3.5
<210> 1 <210> 1
<211> 1229 <211> 1229
<212> DNA <212>DNA
<213> Lactobacillus bulgaricus <213> Lactobacillus bulgaricus
<400> 1
<210> 2 <210> 2
<211> 1225 <211> 1225
<212> DNA <212>DNA
<213> Lactobacillus bulgaricus <213> Lactobacillus bulgaricus
<400> 2
<210> 3 <210> 3
<211> 1205 <211> 1205
<212> DNA <212>DNA
<213> Lactobacillus bulgaricus <213> Lactobacillus bulgaricus
<400> 3
<210> 4 <210> 4
<211> 1181 <211> 1181
<212> DNA <212>DNA
<213> Lactobacillus bulgaricus <213> Lactobacillus bulgaricus
<400> 4
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