TWI706783B - The delayed aging probiotic strain, composition thereof and use thereof - Google Patents

The delayed aging probiotic strain, composition thereof and use thereof Download PDF

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TWI706783B
TWI706783B TW107124842A TW107124842A TWI706783B TW I706783 B TWI706783 B TW I706783B TW 107124842 A TW107124842 A TW 107124842A TW 107124842 A TW107124842 A TW 107124842A TW I706783 B TWI706783 B TW I706783B
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lactobacillus plantarum
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林詠翔
何政育
黃琡涵
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大江生醫股份有限公司
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Abstract

The prevent invention provides a delayed aging probiotic strain, a composition thereof and a use thereof. The probiotic strain is Lactobacillus plantarum TCI999, which can improve mitochondrial activity, improve anti-aging gene expression, enhancing the regulating-NAD+-related gene expression, and promoting the regulating-mitochondrial-activity-related gene expression, improve the perception and the execution of the cognitive functions, and improve intestinal bacterial phase richness.

Description

延緩老化之益生菌株及其組合物與用途 Probiotic strain for delaying aging and its composition and use

本發明係關於一延緩老化的益生菌及其組合物之用途，尤其是一種用於提升抗老化基因表現、調控NAD+相關之基因表現、調控粒線體活性相關之基因表現、改善認知功能的感知與執行能力、以及提高腸道菌相豐富度之益生菌株。 The present invention relates to the use of a probiotic for delaying aging and its composition, in particular to a method for improving the performance of anti-aging genes, regulating the performance of NAD + related genes, regulating the performance of genes related to mitochondrial activity, and improving cognitive function Probiotic strains that can perceive and execute, and increase the abundance of intestinal bacteria.

在生物學及醫學上，老化(aging)是指一個生物體之生理狀態隨時間而惡化的現象，老化會造成身體各部分組織或器官機能衰退或功能減弱，使得生物體的健康功能變差，最後導致生物體死亡，現代人生活壓力大，長期壓力的累積會加速身體機能老化，也衍生出許多慢性疾病。 In biology and medicine, aging refers to the phenomenon that the physiological state of an organism deteriorates over time. Aging will cause the decline or weakening of the functions of various parts of the body’s tissues or organs, and make the health of the organism worse. Eventually lead to the death of organisms, modern people's lives are under great pressure, and the accumulation of long-term stress will accelerate the aging of body functions, and many chronic diseases will also be derived.

另外，隨著保健意識的抬頭及醫療技術的進步，人均壽命增長，但老化所造成的機能退化依然伴隨年齡的增長而加劇，因此除了在疾病的治療及預防外，老化的預防也是高齡社會的重要課題之一。 In addition, with the rising awareness of health care and the advancement of medical technology, the average life expectancy is increasing, but the functional degradation caused by aging is still aggravated with the increase of age. Therefore, in addition to the treatment and prevention of diseases, the prevention of aging is also important for the elderly society. One of the important topics.

然而，雖然目前已研究出許多與老化相關的基因以及相關的機制，例如被稱為長壽基因(longevity gene)的SIRT1基因，其所編譯的蛋白質為哺乳動物中重要的NAD+依賴性去乙醯化酶，参與人體中許多重要的生理和病理過程；另外端粒的長度也被證實與一個體的壽命具有相關性。然而目前仍然沒有能有效應用於延緩老化的藥物或方法，大多只能建議從飲食上多攝取具抗氧化活性物質的食物，並維持適當運動與保持正常作息及心情愉悅。 However, although many genes related to aging and related mechanisms have been studied, such as the SIRT1 gene called longevity gene, the protein compiled by it is an important NAD + -dependent deacetylation in mammals. Enzymes are involved in many important physiological and pathological processes in the human body; in addition, the length of telomeres has also been confirmed to be related to the lifespan of an individual. However, there are still no drugs or methods that can be effectively applied to delay aging. Most of them can only recommend intake of foods with antioxidant active substances from the diet, and maintain proper exercise and maintain a normal routine and mood.

綜合上面所述，為了能方便又有效達到延緩老化的功效，開發一種能有效提升抗老化基因表現、調控NAD+相關之基因表現、調控粒線體活性相關之基因表現，以及改善認知功能的感知與執行能力的組合物，著實有其必要性。 Based on the above, in order to achieve the effect of delaying aging conveniently and effectively, we have developed an anti-aging gene performance that can effectively improve the performance of anti-aging genes, regulate the performance of NAD + related genes, regulate the performance of genes related to mitochondrial activity, and improve the perception of cognitive function The combination with executive ability is really necessary.

緣此，本發明之一目的在提供一種用於延緩老化之益生菌株，其中該益生菌株係為植物乳桿菌(Lactobacillus plantarum)，且其寄存於食品工業發展研究所(台灣)，編號係BCRC910808。 For this reason, one purpose of the present invention is to provide a probiotic strain for delaying aging, wherein the probiotic strain is Lactobacillus plantarum , and it is deposited at the Food Industry Development Institute (Taiwan) under the number BCRC910808.

本發明之又一目的在提供一種如前述益生菌株之用途，係用於製備延緩老化之組合物。其中該益生菌株係為具有活性或去活性的菌株。 Another object of the present invention is to provide a use of the aforementioned probiotic strain to prepare a composition for delaying aging. The probiotic strain is an active or inactive strain.

本發明之另一目的在提供一種用於延緩老化的組合物，係包含如前述之益生菌株，其中該組合物之形式為粉末狀、顆粒狀、液狀、膠狀或膏狀之劑型，且該組合物進一步包含一醫藥上可接受之載體。 Another object of the present invention is to provide a composition for delaying aging, comprising the probiotic strain as described above, wherein the form of the composition is a powder, granular, liquid, gel or paste dosage form, and The composition further includes a pharmaceutically acceptable carrier.

在本發明之一實施例中，該益生菌株係能夠增加細胞粒線體活性；能夠提升一肝臟細胞中的含TCP1的折疊蛋白(Chaperonin containing TCP1,CCT)基因、PTEN促進激酶(PTEN-induced putative kinase 1,Pink1)基因、帕金森氏症少年蛋白2(Parkinson juvenile disease protein 2,Parkin)基因、自噬作用相關基因(Autophagy-related gene,Atg)基因、抗衰老酶1(Sirtuin 1,SIRT1)基因、及叉頭框O 3(Forkhead box O 3,FOXO3)基因之表現。 In one embodiment of the present invention, the probiotic strain can increase the activity of cell mitochondria; can increase the TCP1-containing folding protein (Chaperonin containing TCP1, CCT) gene, PTEN-induced putative kinase (PTEN-induced putative) in a liver cell. kinase 1, Pink1) gene, Parkinson juvenile disease protein 2 (Parkinson juvenile disease protein 2, Parkin) gene, Autophagy-related gene (Atg) gene, anti-aging enzyme 1 (Sirtuin 1, SIRT1) Gene and Forkhead box O 3 (FOXO3) gene expression.

在本發明之又一實施例中，該益生菌株係提升一骨骼肌細胞中的CCT基因、Pink1基因、Parkin基因、及Atg基因之表現；能夠調控神經細胞中的Pink1基因、Parkin基因、及Atg基因之表現。 In another embodiment of the present invention, the probiotic strain enhances the performance of CCT gene, Pink1 gene, Parkin gene, and Atg gene in a skeletal muscle cell; it can regulate the Pink1 gene, Parkin gene, and Atg gene in nerve cells The performance of genes.

在本發明之又一實施例中，該益生菌株係調控人體中CCT基因、Pink1基因、Parkin基因、Atg基因、SIRT1基因、叉頭框O3(Forkhead box O 3, FOXO3)基因、麩醯胺酸依賴性菸草醯胺腺嘌呤二核苷酸合成酶(Glutamine-dependent NAD+ synthetase,NADSYN)基因、粒線體核糖體蛋白5(mitochondrial ribosomal protein S5,MRPS5)基因、泛素樣蛋白5(Ubiquitin-like protein 5,UBL-5)基因、超氧化物歧化酶3(Superoxide dismutase 3,SOD3)基因、端粒酶反轉錄酶(Telomerase reverse transcriptase,TERT)基因、端粒酶RNA模板(telomerase RNA component,TERC)基因及、端粒延伸解旋酶調節因子1(Regulator of telomere elongation helicase 1,RTEL1)基因之表現，其中該植物乳桿菌係正調控CCT基因、Pink1基因、Parkin基因、Atg基因、FOXO3基因、NADSYN基因、MRPS5基因、UBL-5基因、SOD3基因、TERT基因、TERC基因、及RTEL1基因，且該植物乳桿菌係負調控SIRT1基因；又該植物乳桿菌係用於製備調控一個體的一細胞中之NAD+活性、及該個體的一染色體之端粒長度、減少一個體的感知功能測驗反應時間、減少該個體的執行功能測驗反應時間、以及提高腸道菌相豐富度的藥物。 In another embodiment of the present invention, the probiotic strain regulates CCT gene, Pink1 gene, Parkin gene, Atg gene, SIRT1 gene, Forkhead box O3 (FOXO3) gene, and glutamic acid in humans. Dependent tobacco amine adenine dinucleotide synthetase (Glutamine-dependent NAD + synthetase, NADSYN) gene, mitochondrial ribosomal protein 5 (mitochondrial ribosomal protein S5, MRPS5) gene, Ubiquitin-like protein 5 (Ubiquitin- like protein 5 (UBL-5) gene, superoxide dismutase 3 (SOD3) gene, telomerase reverse transcriptase (TERT) gene, telomerase RNA template (telomerase RNA component, TERC) gene and the expression of telomere elongation helicase 1 (Regulator of telomere elongation helicase 1, RTEL1) gene, wherein the Lactobacillus plant is positively regulating CCT gene, Pink1 gene, Parkin gene, Atg gene, FOXO3 gene , NADSYN gene, MRPS5 gene, UBL-5 gene, SOD3 gene, TERT gene, TERC gene, and RTEL1 gene, and the Lactobacillus plantarum strain negatively regulates the SIRT1 gene; and the Lactobacillus plantarum strain is used to prepare a regulatory body The NAD + activity in the cell and the telomere length of a chromosome of the individual, reducing the response time of an individual's sensory function test, reducing the individual's executive function test response time, and drugs that increase the abundance of intestinal flora.

在本發明之又一實施例中，其中CCT基因包含選自於含TCP1第2次單元的折疊蛋白(Chaperonin containing TCP1 subunit 2,CCT2)基因、含TCP1第5次單元的折疊蛋白(Chaperonin containing TCP1 subunit 5,CCT5)基因、含TCP1第6A次單元的折疊蛋白(Chaperonin containing TCP1 subunit 6A,CCT6A)基因、含TCP1第7次單元的折疊蛋白(Chaperonin containing TCP1 subunit 7,CCT7)基因、含TCP1第8次單元的折疊蛋白(Chaperonin containing TCP1 subunit 8,CCT8)基因及其任意組合所組成的群組；Atg基因包含選自於自噬作用相關基因1(Autophagy-related gene 1,Atg1)基因、自噬作用相關基因8(Autophagy-related gene 8,Atg8)基因及其任意組合所組成的群組。 In another embodiment of the present invention, the CCT gene comprises a Chaperonin containing TCP1 subunit 2 (CCT2) gene, a Chaperonin containing TCP1 subunit 2, and a Chaperonin containing TCP1 subunit 2. subunit 5, CCT5) gene, Chaperonin containing TCP1 subunit 6A (CCT6A) gene, Chaperonin containing TCP1 subunit 7, CCT7 gene, TCP1 subunit 7 A group consisting of Chaperonin containing TCP1 subunit 8, CCT8 genes and any combination thereof; Atg genes include genes selected from Autophagy-related gene 1, Atg1 Autophagy-related gene 8 (Autophagy-related gene 8, Atg8) genes and any combination thereof.

本發明之益生菌株能有效提高肝臟細胞中粒線體的活性，以達到提升人體最大器官-肝臟之生理機能。還具有提升肝臟細胞CCT基因、Pink1 基因、Parkin基因、Atg基因、SIRT1基因、及FOXO3基因之表現；調控骨骼肌細胞中CCT基因、Pink1基因、Parkin基因、及Atg基因之表現；調控神經細胞中Pink1基因、Parkin基因、及Atg基因之表現，以達到提升精力、肌力及記憶力的功效。還能有效增加人體中CCT基因、Pink1基因、Parkin基因、Atg基因、與調節NAD+相關基因之SIRT1基因、FOXO3基因、NADSYN基因、MRPS5基因、UBL-5基因及SOD3基因、與調節端粒相關基因之TERT基因、TERC基因及RTEL1基因之表現，並減少感知功能測驗反應時間及執行功能測驗反應時間，以提高抗老化基因表現量、提升調控NAD+相關基因以提高粒線體活性、提升調控端粒活性相關基因、改善認知功能的感知與執行能力的功效、以及提高腸道菌相豐富度。因此，本發明之益生菌株及其代謝產物可用於製備延緩老化之組合物的用途，且該組合物可為粉末狀、顆粒狀、液狀、膠狀或膏狀，且可製成食品、飲品、藥品、試劑或營養補充劑，藉由口服、皮膚塗抹等方式給予一個體。 The probiotic strain of the present invention can effectively improve the activity of mitochondria in liver cells, so as to improve the physiological function of the liver, the largest organ of the human body. It can also enhance the performance of liver cell CCT gene, Pink1 gene, Parkin gene, Atg gene, SIRT1 gene, and FOXO3 gene; regulate the performance of CCT gene, Pink1 gene, Parkin gene, and Atg gene in skeletal muscle cells; regulate the expression of nerve cells The expression of Pink1 gene, Parkin gene, and Atg gene can improve energy, muscle strength and memory. It can also effectively increase the CCT gene, Pink1 gene, Parkin gene, Atg gene, SIRT1 gene, FOXO3 gene, NADSYN gene, MRPS5 gene, UBL-5 gene and SOD3 gene, which are related to regulating NAD + -related genes, and are related to regulating telomere. Gene expression of TERT gene, TERC gene and RTEL1 gene, and reduce the response time of sensory function test and executive function test to increase the expression of anti-aging genes, and increase the regulation of NAD + related genes to increase mitochondrial activity and regulation Telomere activity-related genes, the efficacy of improving the perception and execution of cognitive functions, and the richness of intestinal bacteria. Therefore, the probiotic strain and its metabolites of the present invention can be used to prepare a composition for delaying aging, and the composition can be in powder, granular, liquid, gel or paste form, and can be made into food and beverages. , Drugs, reagents or nutritional supplements, administered to a body by oral administration, skin application, etc.

以下將配合圖式進一步說明本發明的實施方式，下述所列舉的實施例係用以闡明本發明，並非用以限定本發明之範圍，任何熟習此技藝者，在不脫離本發明之精神和範圍內，當可做些許更動與潤飾，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。 The following will further illustrate the embodiments of the present invention in conjunction with the drawings. The following examples are used to illustrate the present invention and are not intended to limit the scope of the present invention. Anyone familiar with the art will not depart from the spirit and spirit of the present invention. Within the scope, some changes and modifications can be made. Therefore, the scope of protection of the present invention shall be subject to the scope of the attached patent application.

圖1係為本發明實施例之植物乳桿菌於提升肝臟細胞中粒線體活性之長條圖。**p<0.01(與控制組比較)；#p<0.05(與MRS組比較)。 Figure 1 is a bar graph showing how Lactobacillus plantarum promotes mitochondrial activity in liver cells according to an embodiment of the present invention. **p<0.01 (compared with the control group); #p<0.05 (compared with the MRS group).

圖2A係為本發明實施例之植物乳桿菌於提升肝臟細胞中CCTs基因表現量之長條圖。*p<0.05、**p<0.01、***p<0.001(與控制組比較)；#p<0.05(與MRSD組比較)。 Figure 2A is a bar graph showing how Lactobacillus plantarum enhances CCTs gene expression in liver cells according to an embodiment of the present invention. *p<0.05, **p<0.01, ***p<0.001 (compared with control group); #p<0.05 (compared with MRSD group).

圖2B係為本發明實施例之植物乳桿菌於提升肝臟細胞中Pink1基因、Parkin基因、Atg1基因、Atg8基因表現量之長條圖。*p<0.05、**p<0.01(與控制組比較)；#p<0.05(與MRSD組比較)。 Fig. 2B is a bar graph showing the expression of Pink1 gene, Parkin gene, Atg1 gene and Atg8 gene in the liver cells of Lactobacillus plantarum according to an embodiment of the present invention. *p<0.05, **p<0.01 (compared with control group); #p<0.05 (compared with MRSD group).

圖2C係為本發明實施例之植物乳桿菌於提升肝臟細胞中SIRT1基因及FOXO3基因表現量之長條圖。**p<0.01(與控制組比較)。 Figure 2C is a bar graph showing how Lactobacillus plantarum improves the expression of SIRT1 gene and FOXO3 gene in liver cells according to an embodiment of the present invention. **p<0.01 (compared with control group).

圖3A係為本發明實施例之植物乳桿菌於提升骨骼肌細胞中CCTs基因表現量之長條圖。*p<0.05、**p<0.01、***p<0.001(與控制組比較)；#p<0.05(與MRSD組比較)。 Fig. 3A is a bar graph showing how Lactobacillus plantarum enhances the expression of CCTs genes in skeletal muscle cells according to an embodiment of the present invention. *p<0.05, **p<0.01, ***p<0.001 (compared with control group); #p<0.05 (compared with MRSD group).

圖3B係為本發明實施例之植物乳桿菌於提升骨骼肌細胞中Pink1基因、Parkin基因、及Atg1基因表現量之長條圖。*p<0.05、**p<0.01、***p<0.001(與控制組比較)；#p<0.05(與MRSD組比較)。 Fig. 3B is a bar graph showing the expression of Pink1 gene, Parkin gene, and Atg1 gene of Lactobacillus plantarum in enhancing skeletal muscle cells according to an embodiment of the present invention. *p<0.05, **p<0.01, ***p<0.001 (compared with control group); #p<0.05 (compared with MRSD group).

圖4A係為本發明實施例之植物乳桿菌於提升神經細胞中CCTs基因表現量之長條圖。*p<0.05、**p<0.01、***p<0.001(與控制組比較)；#p<0.05、##p<0.01(與MRSD組比較)。 Fig. 4A is a bar graph showing how Lactobacillus plantarum improves CCTs gene expression in nerve cells according to an embodiment of the present invention. *p<0.05, **p<0.01, ***p<0.001 (compared with control group); #p<0.05, ##p<0.01 (compared with MRSD group).

圖4B係為本發明實施例之植物乳桿菌於提升神經細胞中Pink1基因、Parkin基因、Atg1基因及Atg8基因表現量之長條圖。*p<0.05、**p<0.01、***p<0.001(與控制組比較)；#p<0.05(與MRSD組比較)。 Fig. 4B is a bar graph showing the expression of Pink1 gene, Parkin gene, Atg1 gene and Atg8 gene in increased nerve cells of Lactobacillus plantarum according to an embodiment of the present invention. *p<0.05, **p<0.01, ***p<0.001 (compared with control group); #p<0.05 (compared with MRSD group).

圖5A係為本發明實施例之植物乳桿菌於人體中調控CCTs基因表現量之長條圖。*p<0.05、**p<0.01、***p<0.001。 Fig. 5A is a bar graph showing the regulation of CCTs gene expression in humans by Lactobacillus plantarum according to an embodiment of the present invention. *p<0.05, **p<0.01, ***p<0.001.

圖5B係為本發明實施例之植物乳桿菌於人體中調控Pink1基因、Parkin基因、Atg1基因、Atg8基因表現量之長條圖。*p<0.05、**p<0.01。 Fig. 5B is a bar graph showing the expression levels of the Pink1 gene, Parkin gene, Atg1 gene, and Atg8 gene regulated by Lactobacillus plantarum in the human body according to an embodiment of the present invention. *p<0.05, **p<0.01.

圖6係為本發明實施例之植物乳桿菌於人體中調控與NAD+相關基因之SIRT1基因、FOXO3基因、NADSYN基因、MRPS5基因、UBL-5基因及SOD3基因表現量之長條圖。*p<0.05、**p<0.01。 Fig. 6 is a bar graph showing the expression levels of the SIRT1 gene, FOXO3 gene, NADSYN gene, MRPS5 gene, UBL-5 gene and SOD3 gene regulating NAD + -related genes in humans by Lactobacillus plantarum according to an embodiment of the present invention. *p<0.05, **p<0.01.

圖7係為本發明實施例之植物乳桿菌於人體中調控與端粒相關基因之TERT基因、TERC基因及RTEL1基因表現量之長條圖。*p<0.05、**p<0.01。 Fig. 7 is a bar graph showing the expression levels of the TERT gene, TERC gene and RTEL1 gene regulating telomere-related genes in humans by Lactobacillus plantarum according to an embodiment of the present invention. *p<0.05, **p<0.01.

圖8A係為本發明實施例之植物乳桿菌減少感知功能測驗反應時間之長條圖。*p<0.05。 Fig. 8A is a bar graph showing the reaction time of the Lactobacillus plantarum reduced sensory function test according to an embodiment of the present invention. *p<0.05.

圖8B係為本發明實施例之植物乳桿菌減少執行功能測驗反應時間之長條圖。*p<0.05。 Fig. 8B is a bar graph showing the reduction of the response time of the executive function test by Lactobacillus plantarum according to an embodiment of the present invention. *p<0.05.

圖9係為本發明實施例之植物乳桿菌提升腸道菌相豐富度之圓餅圖。 Fig. 9 is a pie chart of Lactobacillus plantarum increasing the richness of intestinal flora according to an embodiment of the present invention.

本發明之植物乳桿菌(Lactobacillus plantarum)是一種能延緩老化的益生菌株(Probiotic bacteria)。本發明係一新穎之植物乳桿菌，本文命名為TCI999(中文命名為古方菌)，於民國106年12月28日寄存於食品工業發展研究所，寄存編號BCRC910808，本發明經由細胞實驗與人體實驗而證實植物乳酸桿菌TCI 999可增加肝臟細胞中粒線體的活性；提升肝臟細胞CCT基因、Pink1基因、Parkin基因、Atg基因、SIRT1基因、及FOXO3基因之表現；提升骨骼肌細胞中CCT基因、Pink1基因、Parkin基因、及Atg基因之表現；提升神經細胞中Pink1基因、Parkin基因、及Atg基因之表現，以延緩該三種細胞之老化作用，並達到提升精力、肌力及記憶力的功效。經由人體實驗證實植物乳酸桿菌TCI 999可以增加人體中CCT基因、Pink1基因、Parkin基因、Atg基因、與調節NAD+相關基因之SIRT1基因、FOXO3基因、NADSYN基因、SIRT1基因、MRPS5基因、UBL-5基因及SOD3基因、與調節端粒相關基因之TERT基因、TERC基因及RTEL1基因之表現，並減少感知功能測驗反應時間及執行功能測驗反應時間，以提高人體內抗老化基因表現量、提升調控NAD+相關基因以提高人體細胞中粒線體活性、提升人體細胞中調控端粒活性相關基因、改善認知功能的感知與 執行能力的功效、以及提高腸道菌相豐富度。顯示本發明之植物乳桿菌TCI999可用於製備延緩老化之組合物的用途，且該組合物可為粉末狀、顆粒狀、液狀、膠狀或膏狀，且可製成食品、飲品、藥品、試劑或營養補充劑，藉由口服、皮膚塗抹等方式給予一個體。 The Lactobacillus plantarum of the present invention is a probiotic bacteria that can delay aging. The present invention is a novel Lactobacillus plantarum, named as TCI999 in this article (named as Archaic Bacteria in Chinese). It was deposited at the Food Industry Development Institute on December 28, 2006, with the deposit number BCRC910808. The present invention is through cell experiments and human experiments. It is confirmed that Lactobacillus plantarum TCI 999 can increase the activity of mitochondria in liver cells; enhance the expression of CCT genes, Pink1 genes, Parkin genes, Atg genes, SIRT1 genes, and FOXO3 genes in liver cells; enhance CCT genes, The expression of Pink1 gene, Parkin gene, and Atg gene; enhance the expression of Pink1 gene, Parkin gene, and Atg gene in nerve cells to delay the aging effect of the three cells and achieve the effect of improving energy, muscle strength and memory. It has been confirmed through human experiments that Lactobacillus plantarum TCI 999 can increase CCT gene, Pink1 gene, Parkin gene, Atg gene, SIRT1 gene, FOXO3 gene, NADSYN gene, SIRT1 gene, MRPS5 gene, UBL-5, which are related to the regulation of NAD + genes. The expression of genes and SOD3 genes, TERT genes, TERC genes and RTEL1 genes that regulate telomere-related genes, and reduce the response time of sensory function tests and executive function tests to increase the expression of anti-aging genes in the body and enhance the regulation of NAD + Related genes to improve the activity of mitochondria in human cells, enhance the genes that regulate telomere activity in human cells, improve the efficacy of cognitive function perception and execution, and increase the richness of intestinal bacteria. It shows that the Lactobacillus plantarum TCI999 of the present invention can be used to prepare a composition for delaying aging, and the composition can be powder, granular, liquid, gel or paste, and can be made into food, drink, medicine, Reagents or nutritional supplements are given to a body by oral administration or skin application.

定義definition

益生菌株(probiotic或probiotic bacteria)係為一微生物，其菌體、混合菌株、萃取物或代謝產物對於宿主本身係具有正面影響，通常源自於人體內、有益於腸道健康的活菌，亦可指外來補充、對身體可能有益的某些微生物，其中該益生菌株的代謝產物係為該益生菌株之分泌物，包含培養該菌的培養液。 A probiotic strain (probiotic or probiotic bacteria) is a microorganism whose bacteria, mixed strains, extracts or metabolites have a positive effect on the host itself, and are usually derived from living bacteria in the human body that are beneficial to intestinal health. It can refer to certain microorganisms that are supplemented from outside and may be beneficial to the body, wherein the metabolites of the probiotic strain are the secretions of the probiotic strain, including the culture medium for cultivating the bacteria.

使用Excel軟體進行統計分析。數據以平均值±標準差(SD)表示，個此之間的差異以學生t檢驗(student's t-test)分析。 Use Excel software for statistical analysis. Data mean ± standard deviation (SD) represented by the difference between the two herein by Student t test (student's t -test) analysis.

本文中所使用數值為近似值，所有實驗數據皆表示在20%的範圍內，較佳為在10%的範圍內，最佳為在5%的範圍內。 The numerical values used herein are approximate values, and all experimental data are expressed in the range of 20%, preferably in the range of 10%, and most preferably in the range of 5%.

實施例1 本發明植物乳桿菌增加肝臟細胞中粒線體的活性之功效Example 1 The effect of Lactobacillus plantarum of the present invention in increasing the activity of mitochondria in liver cells

本發明以人類肝癌細胞(Human hepatocellular carcinoma,HepG2)進行本發明植物乳桿菌TCI999對肝臟細胞中粒線體活性之測試。該人類肝癌細胞係購自美國典型培養物保藏中心(美國)，編號ATCC® HB-8065TM，細胞培養於購自Gibco(美國)含有10%之胎牛血清(Fetal Bovine Serum)以及90%之DMEM(Dulbecco's Modified Eagle Medium，其中加入1%之青黴素-鏈黴素，並以MitoScreen套組(購自BDTM，美國，551302)檢測粒線體之膜電位，並以Accuri C6 Plus流式細胞儀(Flow cytometry，BDTM，美國)進行偵測及分析。 The present invention uses human hepatocellular carcinoma (HepG2) to test the activity of Lactobacillus plantarum TCI999 on mitochondria in liver cells. This human hepatocellular carcinoma cell line was purchased from the American Type Culture Collection (U.S.) under the number ATCC® HB-8065 TM . The cells were cultured from Gibco (U.S.) containing 10% Fetal Bovine Serum and 90% DMEM (Dulbecco's Modified Eagle Medium, 1% penicillin-streptomycin was added, and MitoScreen kit (purchased from BDTM, USA, 551302) was used to detect the membrane potential of mitochondria, and the Accuri C6 Plus flow cytometer ( Flow cytometry, BD TM , USA) for detection and analysis.

將1×105個人類肝癌細胞培養於六孔培養盤之每孔中，並在37℃下培養16~18小時，再將培養本發明之植物乳桿菌TCI999的上清液加入細胞之培養液中處理24小時，接著去除原有的培養液，並以1mL磷酸鹽緩衝溶液(Phosphate buffered saline,PBS)沖洗細胞二次後，加入200μL胰蛋白酶(Trypsin)反應5分鐘，使細胞由培養盤上脫落，加入適當的培養液終止反應，並將細胞連同培養液收集至15mL試管，並以300g離心5分鐘後移除上清液，再於暗處以1mL磷酸鹽緩衝溶液使細胞懸浮。接著使用以1：250比例混合的JC1染色溶液與JC1染色緩衝溶液將細胞染色，再以1mL磷酸鹽緩衝溶液沖洗細胞二次後，以流式細胞儀偵測細胞中激發波長為480nm且放射波長為510nm之螢光強度。 Culture 1×10 5 human hepatocellular carcinoma cells in each well of a six-well culture plate, and culture them at 37°C for 16-18 hours, and then add the supernatant of the Lactobacillus plantarum TCI999 of the present invention to the cell culture medium After 24 hours of medium treatment, the original culture medium was removed, and the cells were washed twice with 1 mL of phosphate buffered saline (PBS), and 200 μL of trypsin (Trypsin) was added to react for 5 minutes to remove the cells from the culture plate. After falling off, add appropriate culture medium to stop the reaction, and collect the cells together with culture medium into a 15mL test tube, centrifuge at 300g for 5 minutes, remove the supernatant, and then suspend the cells with 1mL phosphate buffer solution in the dark. Then use the JC1 staining solution and JC1 staining buffer solution mixed in a ratio of 1:250 to stain the cells, and then rinse the cells twice with 1 mL of phosphate buffer solution, and then use a flow cytometer to detect that the excitation wavelength is 480 nm and the emission wavelength in the cells Fluorescence intensity at 510nm.

本發明之植物乳桿菌TCI999提升肝臟細胞中粒線體活性之結果如圖1所示。粒線體為細胞能量製造的中心，其活性與細胞機能息息相關，研究已知粒線體活性提高能與改善細胞老化的現象。由實驗結果可知，以培養本發明之植物乳桿菌TCI999之上清液處理過的肝臟細胞，其中粒線體活性顯著地較控制組及MRS組(養菌之培養基的空白組)高約20%，此結果顯示本發明之植物乳桿菌TCI999具有優異的提高肝臟細胞中粒線體活性的功效，能提升人體最大器官肝臟的生理機能。 The results of the Lactobacillus plantarum TCI999 of the present invention in enhancing mitochondrial activity in liver cells are shown in Figure 1. Mitochondria are the center of cell energy production, and their activity is closely related to cell function. It is known that increased mitochondrial activity can improve cell aging. It can be seen from the experimental results that the mitochondrial activity of the liver cells treated with the supernatant of Lactobacillus plantarum TCI999 of the present invention is significantly higher than that of the control group and the MRS group (the blank group of the culture medium) by about 20% This result shows that the Lactobacillus plantarum TCI999 of the present invention has an excellent effect of improving the activity of mitochondria in liver cells, and can improve the physiological function of the liver, the largest organ of the human body.

實施例2 本發明植物乳桿菌調控肝臟細胞中抗老基因表現之功效Example 2 The effect of Lactobacillus plantarum of the present invention in regulating the expression of anti-aging genes in liver cells

為了測試本發明植物乳桿菌TCI999對肝臟細胞中抗老基因表現之調控，將上述人類肝癌細胞以每孔1×105個細胞量培養於含有2mL上述培養液之六孔培養盤中，並在37℃下培養16~18小時，再將細胞分成三組：(1)僅含培養液之控制組、(2)MRSD(養菌之培養基的空白組)以及(3)加入1.25%本發明之植物乳桿菌TCI999菌液之實驗組分別處理細胞24小時後，將細胞以細胞裂解液回收細胞後，使用RNA萃取試劑套組(購自Geneaid公司，台灣，Lot No. FC24015-G)分別收集三組人類肝癌細胞內之RNA，接著利用SuperScript® III反轉錄酶(購自Invitrogene公司，美國，編號18080-051)以2000ng之萃取RNA為模板以表一中之引子黏合進行反轉錄作用產生相應之cDNA，接著利用ABI StepOnePlusTM Real-Time PCR system(Thermo Fisher Scientific公司，美國)，以及KAPA SYBR FAST(購自Sigma公司，美國，編號38220000000)將三組反轉錄後產物分別以表一之組合引子進行定量即時反轉錄聚合酶連鎖反應(quantitative real-time reverse transcription polymerase chain reaction)試驗，用以定量CCT2基因、CCT5基因、CCT6A基因、CCT7基因、CCT8基因、Pink1基因、Parkin基因、Atg1基因、Atg8基因、SIRT1基因、FOXO3基因、之mRNA表現量，其中定量數值係取由閾值循環數(Ct)，而目標基因的mRNA相對量係推導自方程式2-△Ct，其中△Ct=Ct目標基因-CtGAPDH(甘油醛-3-磷酸脫氫酶，Glyceraldehyde-3-phosphate dehydrogenase)，再利用Excel軟體進行非成對單尾student t-test以決定變異係數與是否在統計上具有顯著差異(*p值<0.05；**p值<0.01；***p值<0.001)。 In order to test the regulation of Lactobacillus plantarum TCI999 on the expression of anti-aging genes in liver cells, the above-mentioned human liver cancer cells were cultured at 1×10 5 cells per well in a six-well culture plate containing 2 mL of the above-mentioned culture medium, and Cultivate at 37°C for 16-18 hours, then divide the cells into three groups: (1) a control group containing only culture medium, (2) MRSD (a blank group with culture medium), and (3) adding 1.25% of the present invention In the experimental group of Lactobacillus plantarum TCI999 bacterial solution, the cells were treated for 24 hours. After the cells were recovered with cell lysate, the RNA extraction reagent kit (purchased from Geneaid, Taiwan, Lot No. FC24015-G) was used to collect three Set the RNA in human liver cancer cells, and then use SuperScript ® III reverse transcriptase (purchased from Invitrogene, USA, No. 18080-051) to use 2000ng of extracted RNA as a template to perform reverse transcription with the primers in Table 1 to produce the corresponding cDNA, and then use ABI StepOnePlus TM Real-Time PCR system (Thermo Fisher Scientific Company, USA) and KAPA SYBR FAST (purchased from Sigma Company, USA, No. 38220000000) to combine the three sets of reverse transcription products with the combination primers in Table 1. Carry out quantitative real-time reverse transcription polymerase chain reaction test to quantify CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, Pink1 gene, Parkin gene, Atg1 gene, Atg8 gene, SIRTl gene, of FOXO3 gene, the mRNA expression amount, wherein the quantitative values based taken by the threshold of the number (Ct) cycle, the mRNA of the target gene relative amounts lines derived from equations 2 - △ Ct, where △ Ct = Ct gene of interest - Ct GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), then use Excel software to perform unpaired one-tailed student t-test to determine whether the coefficient of variation is statistically significant (*p Value<0.05; **p value<0.01; ***p value<0.001).

Figure 107124842-A0305-02-0010-1
Figure 107124842-A0305-02-0010-1
Figure 107124842-A0305-02-0011-2
Figure 107124842-A0305-02-0011-2

CCT基因為折疊蛋白(Chaperonin)的一種，主要功能為矯正錯誤摺疊的蛋白質，並將無法成功修復之蛋白質送進蛋白質水解酶複合體(Proteasome)中進行水解，先前研究已指若此蛋白質調控之重複使用系統發生故障，會導致錯誤摺疊的蛋白質會聚集在細胞中，並造成細胞功能衰退及加速細胞的老化與死亡，因此被認為與個體之老化調節相關。 CCT gene is a type of Chaperonin. Its main function is to correct misfolded proteins and send the proteins that cannot be successfully repaired into the proteasome for hydrolysis. Previous studies have pointed out that if this protein regulates Failure of the repeated use system will cause misfolded proteins to accumulate in cells, causing cell function decline and accelerating cell aging and death, so it is considered to be related to the aging regulation of individuals.

Pink1基因為一種位在粒線體的絲胺酸/蘇胺酸蛋白激酶(Serine/threonine protein kinase)，其功能為保護線粒體在細胞壓力期間發生故障，已知該基因之突變與帕金森氏症有關，而先前研究更指出該基因的過量表現能夠增加小鼠的壽命，因此也被認為與個體之老化調節有關。Parkin基因為一種存在於泛素-蛋白酶體系(Ubiquitin-proteasome system)中之酵素，並作為蛋白質分解的調節劑，已知該基因之突變與帕金森氏症有關，且先前研究亦指出增加該基 因的表現量能夠延緩細胞的老化作用，因此被認為與細胞的老化作用相關。Atg基因則為自噬作用相關基因，會參與細胞中廢物降解和循環的自噬作用，先前研究指出該基因的過量表現，能夠延長小鼠的壽命，因此被認為與細胞的老化作用相關。 Pink1 gene is a serine/threonine protein kinase (Serine/threonine protein kinase) located in the mitochondria. Its function is to protect the mitochondria from malfunctioning during cellular stress. Mutations in this gene are known to be associated with Parkinson’s disease. Related, and previous studies have pointed out that the overexpression of this gene can increase the lifespan of mice, so it is also considered to be related to the regulation of individual aging. Parkin gene is an enzyme that exists in the Ubiquitin-proteasome system (Ubiquitin-proteasome system) and acts as a regulator of protein breakdown. It is known that mutations in this gene are related to Parkinson’s disease, and previous studies have also pointed out that the increase Because the amount of expression can delay the aging effect of cells, it is considered to be related to the aging effect of cells. Atg gene is an autophagy-related gene, which is involved in the degradation of waste products and circulating autophagy in cells. Previous studies have pointed out that overexpression of this gene can prolong the lifespan of mice, so it is believed to be related to cell aging.

SIRT1基因為哺乳動物中重要的NAD+依賴性去乙醯化酶，先前研究指出SIRT1基因的表現量下降，會倒是粒線體的生物合成功能降低，並導致產生老化之相關疾病，因此該基因已被公認為長壽基因，與老化作用的關係密不可分。FOXO基因為一種轉錄因子，先前研究已指出該基因為細胞老化和決定壽命長短的重要因素。 The SIRT1 gene is an important NAD + -dependent deacetylase in mammals. Previous studies have pointed out that the decrease in the expression of SIRT1 gene will decrease the biosynthesis function of mitochondria and cause aging-related diseases. Therefore, this gene It has been recognized as a longevity gene and is inseparable from the effect of aging. FOXO gene is a transcription factor. Previous studies have pointed out that this gene is an important factor in cell aging and determining the length of life.

本發明之植物乳桿菌提升肝臟細胞中CCT2基因、CCT5基因、CCT6A基因、CCT7基因、及CCT8基因表現之結果如圖2A所示；提升Pink1基因、Parkin基因、Atg1基因及Atg8基因表現之結果如圖2B所示；提升SIRT1基因及FOXO3基因表現之結果如圖2C所示。人類肝癌細胞經本發明之植物乳桿菌TCI999處理後，CCT2基因、CCT5基因、CCT6A基因、CCT7基因、及CCT8基因的表現量分別約為控制組的1.5倍、1.2倍、1.8倍、2倍及1.8倍；而Pink1基因、Parkin基因、Atg1基因及Atg8基因的表現量分別約為控制組的2.1倍、1.5倍、2倍及1.6倍；SIRT1基因及FOXO3基因的表現量分別約為控制組的1.3倍及1.1倍。此結果顯示本發明之植物乳桿菌TCI999具有優異地提高肝臟細胞中抗老化基因表現量的功效，能有效提高肝臟細胞延緩老化的效果。 The results of improving the expression of CCT2, CCT5, CCT6A, CCT7, and CCT8 genes in liver cells by the Lactobacillus plantarum of the present invention are shown in Figure 2A; the results of improving the expression of Pink1, Parkin, Atg1 and Atg8 genes are as follows As shown in Figure 2B; the results of improving the expression of SIRT1 gene and FOXO3 gene are shown in Figure 2C. After human liver cancer cells were treated with Lactobacillus plantarum TCI999 of the present invention, the expression levels of CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, and CCT8 gene were about 1.5 times, 1.2 times, 1.8 times, 2 times and 1.8 times of the control group, respectively. The expression levels of Pink1 gene, Parkin gene, Atg1 gene and Atg8 gene were about 2.1 times, 1.5 times, 2 times and 1.6 times that of the control group, respectively; the expression levels of SIRT1 gene and FOXO3 gene were about 1.3 times that of the control group. Times and 1.1 times. This result shows that the Lactobacillus plantarum TCI999 of the present invention has an excellent effect of increasing the expression of anti-aging genes in liver cells, and can effectively improve the effect of delaying aging of liver cells.

實施例3 本發明植物乳桿菌調控骨骼肌細胞中抗老基因表現之功效Example 3 The effect of Lactobacillus plantarum of the present invention in regulating the expression of anti-aging genes in skeletal muscle cells

為了測試本發明植物乳桿菌TCI999對骨骼肌細胞中抗老基因表現之調控，以小鼠肌肉纖維母細胞(C2C12)進行測試，其中該C2C12細胞係購自美國典型培養物保藏中心(美國)，編號ATCC® CRL-1772TM，細胞培養於如 實施例1所述之培養液中。首先，將1×105個C2C12細胞培養於含有2mL上述培養液之六孔培養盤中，並在37℃下培養16~18小時，再將細胞分成三組：(1)僅含培養液之控制組、(2)MRSD以及(3)加入1.25%本發明之植物乳桿菌TCI999菌液之實驗組分別處理細胞24小時，並以實施例2之方法及表一之引子分析各組C2C12細胞中CCT2基因、CCT5基因、CCT7基因、Pink1基因、及Parkin基因、Atg1基因之表現情形。 In order to test the regulation of Lactobacillus plantarum TCI999 on the expression of anti-aging genes in skeletal muscle cells, the test was performed with mouse muscle fibroblasts (C2C12), wherein the C2C12 cell line was purchased from the American Type Culture Collection (USA), No. ATCC ® CRL-1772 TM , the cells are cultured in the medium as described in Example 1. Firstly, 1×10 5 C2C12 cells were cultured in a six-well culture dish containing 2 mL of the above-mentioned culture medium, and cultured at 37°C for 16-18 hours, and then the cells were divided into three groups: (1) Only the culture medium The control group, (2) MRSD, and (3) the experimental group with 1.25% Lactobacillus plantarum TCI999 bacterial solution of the present invention were treated with cells for 24 hours, and the method of Example 2 and the primers in Table 1 were used to analyze the C2C12 cells in each group The expression of CCT2 gene, CCT5 gene, CCT7 gene, Pink1 gene, Parkin gene, Atg1 gene.

本發明之植物乳桿菌TCI999提升骨骼肌細胞中CCT2基因、CCT5基因及CCT7基因表現之結果如圖3A所示；提升Pink1基因、Parkin基因及Atg1基因表現之結果如圖3B所示。小鼠肌肉纖維母細胞經本發明之植物乳桿菌TCI999處理後，CCT2基因、CCT5基因及CCT7基因的表現量分別約為控制組的1.5倍、1.8倍及1.2倍；而Pink1基因、Parkin基因及Atg1基因的表現量分別約為控制組的1.9倍、1.4倍及1.2倍。此結果顯示本發明之植物乳桿菌TCI999具有優異地提高骨骼肌細胞中抗老化基因表現量的功效，能有效提高骨骼肌細胞延緩老化的效果。 The results of the Lactobacillus plantarum TCI999 of the present invention improving the expression of CCT2 gene, CCT5 gene and CCT7 gene in skeletal muscle cells are shown in Figure 3A; the result of improving the expression of Pink1 gene, Parkin gene and Atg1 gene is shown in Figure 3B. After mouse muscle fibroblasts were treated with Lactobacillus plantarum TCI999 of the present invention, the expression levels of CCT2 gene, CCT5 gene and CCT7 gene were about 1.5 times, 1.8 times and 1.2 times that of the control group, respectively; while Pink1 gene, Parkin gene and Atg1 The gene expression levels were approximately 1.9, 1.4, and 1.2 times that of the control group. This result shows that the Lactobacillus plantarum TCI999 of the present invention has an excellent effect of increasing the expression of anti-aging genes in skeletal muscle cells, and can effectively improve the effect of delaying aging of skeletal muscle cells.

實施例4 本發明植物乳桿菌調控神經細胞中抗老基因表現之功效Example 4 The effect of Lactobacillus plantarum of the present invention in regulating the expression of anti-aging genes in nerve cells

為了測試本發明植物乳桿菌TCI999對神經細胞中抗老基因表現之調控，以人類神經母細胞瘤細胞(SHSY-5Y)進行測試，其中該SHSY-5Y細胞係購自美國典型培養物保藏中心(美國)，編號ATCC® CRL-2266TM，細胞培養於如實施例1所述之培養液中。首先，將1×105個SHSY-5Y細胞培養於含有2mL上述培養液之六孔培養盤中，並在37℃下培養16~18小時，再將細胞分成四組：(1)僅含培養液之控制組、(2)MRSD以及(3)加入1.25%本發明之植物乳桿菌TCI999菌液之實驗組分別處理細胞24小時，並以實施例2之方法及表一之引子 分析各組C2C12細胞中CCT2基因、CCT5基因、CCT6A基因、CCT7基因、CCT8基因、Pink1基因、Parkin基因、Atg1基因、及Atg8基因之表現情形。 In order to test the regulation of Lactobacillus plantarum TCI999 on the expression of anti-aging genes in nerve cells of the present invention, human neuroblastoma cells (SHSY-5Y) were tested, wherein the SHSY-5Y cell line was purchased from the American Type Culture Collection ( United States), numbered ATCC ® CRL-2266 TM , the cells were cultured in the medium as described in Example 1. Firstly, 1×10 5 SHSY-5Y cells were cultured in a six-well culture dish containing 2 mL of the above culture medium, and cultured at 37°C for 16-18 hours, and then the cells were divided into four groups: (1) Only cultured The control group of liquid, (2) MRSD and (3) the experimental group with 1.25% Lactobacillus plantarum TCI999 bacterial liquid of the present invention were treated with the cells for 24 hours, and the method of Example 2 and the primers in Table 1 were used to analyze the C2C12 of each group The expression of CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, Pink1 gene, Parkin gene, Atg1 gene, and Atg8 gene in cells.

本發明之植物乳桿菌TCI999提升神經細胞中CCT2基因、CCT5基因、CCT7基因及CCT8基因表現之結果如圖4A所示；提升Pink1基因、Parkin基因、Atg1基因及Atg8基因表現之結果如圖4B所示。人類神經母細胞瘤細胞經本發明之植物乳桿菌TCI999處理後，CCT2基因、CCT5基因、CCT7基因及CCT8基因的表現量分別約為控制組的1.2倍、1.1倍、1.4倍及1.1倍；而Pink1基因、Parkin基因、Atg1基因及Atg8基因的表現量分別約為控制組的1.5倍、1.6倍、1.5倍及1.2倍。此結果顯示本發明之植物乳桿菌TCI999具有優異地提高神經肌細胞中抗老化基因表現量的功效，能有效提高神經細胞延緩老化的效果。 The results of the Lactobacillus plantarum TCI999 of the present invention for enhancing the expression of CCT2, CCT5, CCT7 and CCT8 genes in nerve cells are shown in Figure 4A; the results of enhancing the expression of Pink1, Parkin, Atg1 and Atg8 genes are shown in Figure 4B. Show. After human neuroblastoma cells were treated with Lactobacillus plantarum TCI999 of the present invention, the expression levels of CCT2 gene, CCT5 gene, CCT7 gene and CCT8 gene were about 1.2 times, 1.1 times, 1.4 times and 1.1 times that of the control group, respectively; while Pink1 The expression levels of gene, Parkin gene, Atg1 gene and Atg8 gene were about 1.5 times, 1.6 times, 1.5 times and 1.2 times that of the control group, respectively. This result shows that the Lactobacillus plantarum TCI999 of the present invention has an excellent effect of improving the expression of anti-aging genes in nerve muscle cells, and can effectively improve the effect of delaying aging of nerve cells.

實施例5 本發明植物乳桿菌改善個體老化情況之功效Example 5 The effect of Lactobacillus plantarum of the present invention in improving individual aging

為證實本發明之植物乳桿菌TCI999於人體抗老化之效果，首先募集9位35歲以上之成人為受試者，每日服用一顆含有1010CFU劑量之本發明TCI999益生菌的膠囊(相當於一日服用100mg)，在給予該膠囊第0、4及8週時收集受試者的血液樣本進行檢測抗老基因、並測試其感知功能、執行功能和腸胃道的改善效果，其中抗老基因的分析係將受試者血液中的周邊血單核細胞(peripheral blood mononuclear cell,PBMC)以淋巴球分離液分離，再以實施例2之方法及表一、表二之引子分析各組PBMC細胞中CCT2基因、CCT5基因、CCT6A基因、CCT7基因、CCT8基因、Pink1基因、Parkin基因、Atg1基因、Atg8基因、SIRT1基因、FOXO3基因、PARP1基因、PARP2基因、NADSYN基因、MRPS5基因、UBL-5基因、SOD3基因、TERT基因、TERC基因、及RTEL1基因之表現情形。感知功能係經由測試受試者之速認(subitizing)能力進行分析，其中速認為不以數算的方法快速判斷出問題呈現的數量，例如一次出現4-8 個球點於受試者眼前，而受試者辨識的反應時間即為感知能力的反應時間；執行功能則係以受試者正確判斷字體顏色(例如藍色二字之顏色為藍色)的結果normal reaction time(ms)、以及錯誤判斷字體顏色(例如藍色二字之顏色為紅色)的結果interfere reaction time(ms)為執行功能的反應時間。腸胃道改善則以測定受試者之腸道菌相豐富度是否有上升為判斷依據。 In order to confirm the anti-aging effect of Lactobacillus plantarum TCI999 in humans, 9 adults over the age of 35 were first recruited as subjects. They took a capsule containing 10 10 CFU of the probiotic TCI999 of the present invention per day (equivalent to Take 100mg a day), and collect blood samples from subjects at the 0th, 4th and 8th week of administration of the capsule to detect anti-aging genes, and to test its perception function, executive function and gastrointestinal tract improvement effect. The analysis of genes is to separate peripheral blood mononuclear cells (PBMC) in the blood of subjects with lymphocyte separation fluid, and then analyze each group of PBMCs using the method of Example 2 and the primers in Table 1 and Table 2. CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, Pink1 gene, Parkin gene, Atg1 gene, Atg8 gene, SIRT1 gene, FOXO3 gene, PARP1 gene, PARP2 gene, NADSYN gene, MRPS5 gene, UBL-5 in cells The expression of genes, SOD3 gene, TERT gene, TERC gene, and RTEL1 gene. The perception function is analyzed by testing the subject’s subitizing ability, in which the subitizing method is not used to quickly determine the number of problems presented. For example, 4-8 ball points appear in front of the subject’s eyes at a time. The reaction time recognized by the subject is the reaction time of the perception ability; the executive function is the result of the subject's correct judgment of the font color (for example, the color of the blue word is blue) normal reaction time (ms), and The result of incorrectly judging the font color (for example, the color of blue is red) is the reaction time (ms) of the execution function. The improvement of the gastrointestinal tract is based on the determination of whether the subject’s intestinal bacterial richness has increased.

先前研究已指出和年老的小鼠相比，年輕的小鼠體內含有較多的NAD+；且增加年老小鼠體內的NAD+濃度，能使其生理狀況更為年輕，因此NAD+被認為與延緩個體老化相關，而已知SIRT1基因、FOXO3基因、NADSYN基因、MRPS5基因、UBL-5基因及SOD3基因與調節NAD+具有相關性。端粒係為生物染色體末端的DNA重複序列，主要作用是保持染色體的完整性和控制細胞***週期，其中DNA每複製一次，端粒就會縮短一些，一旦端粒消耗殆盡，細胞則會走向凋亡，因此端粒的長度與細胞的年齡具有相關性，其中已知TERT基因、TERC基因及RTEL1基因與調節端粒的長短相關。而研究顯示腸道菌相的高豐富度代表腸道之高復原力與穩定性，因此腸道菌相的豐富度提升能夠維持腸道的正常機能，並延緩腸道老化的發生。 Previous studies have pointed out that compared with old mice, young mice contain more NAD + ; and increasing the NAD + concentration in old mice can make their physiological conditions younger, so NAD + is It is believed to be related to the delay of individual aging, and it is known that SIRT1 gene, FOXO3 gene, NADSYN gene, MRPS5 gene, UBL-5 gene and SOD3 gene are related to regulating NAD + . Telomeres are the repetitive sequences of DNA at the ends of biological chromosomes. The main function is to maintain the integrity of chromosomes and control the cell division cycle. Each time the DNA is copied, the telomeres will be shortened. Once the telomeres are exhausted, the cells will move towards Apoptosis, therefore, the length of telomeres is related to the age of cells. Among them, TERT gene, TERC gene and RTEL1 gene are known to be related to the regulation of telomere length. Studies have shown that the high abundance of the intestinal flora represents the high resilience and stability of the intestinal tract. Therefore, the increase in the abundance of the intestinal flora can maintain the normal function of the intestine and delay the occurrence of intestinal aging.

本發明之植物乳桿菌TCI999提升人體中CCT2基因、CCT5基因、CCT6A基因、CCT7基因、及CCT8基因表現之結果如圖5A所示；提升Pink1基因、Parkin基因、Atg1基因及Atg8基因表現之結果如圖5B所示；與調節NAD+相關基因之SIRT1基因、FOXO3基因、NADSYN基因、MRPS5基因、UBL-5基因及SOD3基因表現之結果如圖6所示；與調節端粒相關基因之TERT基因、TERC基因及RTEL1基因表現之結果如圖7所示；於減少感知功能測驗反應時間之結果如圖8A所示；減少執行功能測驗反應時間之結果則如8B所示；提升受試者腸道菌相豐富度之結果如圖9所示。服用本發明之植物乳桿菌TCI999共4週或8週後，受試者體內CCT2基因、CCT5基因、CCT6A基因、CCT7基因、及CCT8 基因表現量皆顯著高於受試前約2-7倍不等；而Pink1基因、Parkin基因、Atg1基因及Atg8基因表現量亦皆顯著高於受試前約2-7倍不等；於與調節NAD+相關基因之FOXO3基因、NADSYN基因、MRPS5基因、UBL-5基因及SOD3基因表現量皆顯著高於受試前約2-3倍不等，其中SIRT1基因則較受試前低約0.5倍；而於與調節端粒相關基因之TERT基因、TERC基因及RTEL1基因表現量亦皆顯著高於受試前約3-7倍不等；於服用本發明之植物乳桿菌TCI999共4週或8週後，皆能顯著減少感知功能測驗反應時間，及減少執行功能測驗反應時間；而於服用本發明之植物乳桿菌TCI999後能提升75%之受試者的腸道菌相豐富度。綜合以上，該些結果顯示本發明之植物乳桿菌TCI999具有優異地提高抗老化基因表現量、提升調控NAD+相關基因以提高粒線體活性、提升調控端粒活性相關基因、改善認知功能的感知與執行能力的功效、及提高腸道菌相豐富度，能有效達到整體延緩老化之功效。 The results of the Lactobacillus plantarum TCI999 of the present invention enhancing the expression of CCT2, CCT5, CCT6A, CCT7, and CCT8 genes in humans are shown in Figure 5A; the results of enhancing the expressions of Pink1, Parkin, Atg1 and Atg8 genes are as follows As shown in Figure 5B; the results of expression of SIRT1 gene, FOXO3 gene, NADSYN gene, MRPS5 gene, UBL-5 gene and SOD3 gene related to the regulation of NAD + genes are shown in Figure 6; and the TERT gene, which regulates telomere-related genes, The results of TERC gene and RTEL1 gene expression are shown in Figure 7; the results of reducing the response time of the sensory function test are shown in Figure 8A; the results of reducing the response time of the executive function test are shown in 8B; the intestinal bacteria of the subjects are improved The result of phase richness is shown in Figure 9. After taking the Lactobacillus plantarum TCI999 of the present invention for 4 weeks or 8 weeks, the expression levels of CCT2, CCT5, CCT6A, CCT7, and CCT8 genes in the subjects were significantly higher than those before the test by about 2-7 times. The expression levels of Pink1 gene, Parkin gene, Atg1 gene and Atg8 gene are also significantly higher than those before the test, ranging from about 2-7 times; compared with FOXO3 gene, NADSYN gene, MRPS5 gene, UBL, which are related to the regulation of NAD + The expression levels of -5 gene and SOD3 gene were significantly higher than those before the test, ranging from about 2-3 times. Among them, the SIRT1 gene was about 0.5 times lower than that before the test; and the TERT and TERC genes related to the regulation of telomere The expression levels of RTEL1 and RTEL1 genes are also significantly higher than those before the test, ranging from about 3-7 times; after taking the Lactobacillus plantarum TCI999 of the present invention for 4 or 8 weeks, it can significantly reduce the reaction time of the sensory function test and reduce Executive function test reaction time; and after taking the Lactobacillus plantarum TCI999 of the present invention, the intestinal flora richness of 75% of the subjects can be improved. In summary, these results show that the Lactobacillus plantarum TCI999 of the present invention has an excellent ability to improve the expression of anti-aging genes, enhance the regulation of NAD + related genes to increase mitochondrial activity, enhance the regulation of telomere activity-related genes, and improve the perception of cognitive function With the effect of executive ability and increase the richness of intestinal bacteria, it can effectively achieve the overall effect of delaying aging.

Figure 107124842-A0305-02-0016-3
Figure 107124842-A0305-02-0016-3
Figure 107124842-A0305-02-0017-4
Figure 107124842-A0305-02-0017-4

綜上所述，本發明之益生菌株可為具有活性或去活性的菌株，且該益生菌株或其代謝產物能有效提高肝臟細胞中粒線體的活性，以達到提升人體最大器官-肝臟之生理機能。還具有提升肝臟細胞CCT基因、Pink1基因、Parkin基因、Atg基因、SIRT1基因、及FOXO3基因之表現；調控骨骼肌細胞中CCT基因、Pink1基因、Parkin基因、及Atg基因之表現；調控神經細胞中Pink1基因、Parkin基因、及Atg基因之表現，以達到提升精力、肌力及記憶力的功效。還能有效增加人體中CCT基因、Pink1基因、Parkin基因、Atg基因、與調節NAD+相關基因之SIRT1基因、FOXO3基因、NADSYN基因、MRPS5基因、UBL-5基因及SOD3基因、與調節端粒相關基因之TERT基因、TERC基因及RTEL1基因之表現，並減少感知功能測驗反應時間及執行功能測驗反應時間，以提高抗老化基因表現量、提升調控NAD+相關基因以提高粒線體活性、提升調控端粒活性相關基因、改善認知功能的感知與執行能力、以及提高腸道菌相豐富度的功效。因此，本發明之益生菌株或其代謝產物可用於製備延緩老化之組合物的用 途，且該組合物可為粉末狀、顆粒狀、液狀、膠狀或膏狀，且可製成食品、飲品、藥品、試劑或營養補充劑，藉由口服、皮膚塗抹等方式給予一個體。 In summary, the probiotic strain of the present invention can be an active or inactive strain, and the probiotic strain or its metabolite can effectively increase the activity of mitochondria in liver cells, so as to improve the physiology of the largest organ of the human body-the liver function. It can also enhance the performance of liver cell CCT gene, Pink1 gene, Parkin gene, Atg gene, SIRT1 gene, and FOXO3 gene; regulate the expression of CCT gene, Pink1 gene, Parkin gene, and Atg gene in skeletal muscle cells; regulate the expression of nerve cells The expression of Pink1 gene, Parkin gene, and Atg gene can improve energy, muscle strength and memory. It can also effectively increase the CCT gene, Pink1 gene, Parkin gene, Atg gene, SIRT1 gene, FOXO3 gene, NADSYN gene, MRPS5 gene, UBL-5 gene and SOD3 gene, which are related to regulating NAD + -related genes, and are related to regulating telomere. Gene expression of TERT gene, TERC gene and RTEL1 gene, and reduce the response time of sensory function test and executive function test to increase the expression of anti-aging genes, and increase the regulation of NAD + related genes to increase mitochondrial activity and regulation Telomere activity-related genes, improve the perception and execution of cognitive functions, and increase the abundance of intestinal bacteria. Therefore, the probiotic strain or its metabolites of the present invention can be used to prepare a composition for delaying aging, and the composition can be powder, granular, liquid, gel or paste, and can be made into foods and beverages. , Medicines, reagents or nutritional supplements, administered to a body by oral administration, skin application, etc.

<110> 大江生醫股份有限公司 <110> Dajiang Biomedical Co., Ltd.

<120> 延緩老化之益生菌株及其組合物與用途 <120> Probiotic strains for delaying aging and their compositions and uses

<130> 107B0206-I1 <130> 107B0206-I1

<160> 44 <160> 44

<170> PatentIn version 3.5 <170> PatentIn version 3.5

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Figure 107124842-A0101-12-0018-23
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Figure 107124842-A0101-12-0018-25
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Figure 107124842-A0101-12-0020-30
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Figure 107124842-A0101-12-0034-69

Claims (10)

一種植物乳桿菌(Lactobacillus plantarum)用於製備延緩老化之組成物的用途，其中該植物乳桿菌係寄存於食品工業發展研究所(台灣)，編號係BCRC910808，且該植物乳桿菌係提升一肝臟細胞中含TCP1的折疊蛋白6A(Chaperonin containing TCP1 subunit 6A,CCT6A)基因、CCT7基因、CCT8基因、PTEN促進激酶(PTEN-induced putative kinase 1,Pink1)基因、自噬作用相關基因1(Autophagy-related gene 1,Atg1)基因、Atg8基因、及抗衰老酶1(Sirtuin 1,SIRT1)基因之表現量。 A use of Lactobacillus plantarum ( Lactobacillus plantarum ) for the preparation of a composition for delaying aging, wherein the Lactobacillus plantarum is deposited at the Food Industry Development Institute (Taiwan), and the number is BCRC910808, and the Lactobacillus plantarum promotes a liver cell Chaperonin containing TCP1 subunit 6A ( CCT6A ) gene, CCT7 gene, CCT8 gene, PTEN-induced putative kinase 1, Pink1 gene, autophagy-related gene 1 (Autophagy-related gene) 1, Atg1 ) gene, Atg8 gene, and anti-aging enzyme 1 (Sirtuin 1, SIRT1 ) gene expression level. 一種植物乳桿菌用於製備延緩老化之組成物的用途，其中該植物乳桿菌係寄存於食品工業發展研究所(台灣)，編號係BCRC910808，且該植物乳桿菌係提升一骨骼肌細胞中CCT2基因、CCT5基因、CCT7基因、Pink1基因、帕金森氏症少年蛋白2(Parkinson juvenile disease protein 2,Parkin)基因、及Atg1基因之表現量。 A use of Lactobacillus plantarum for the preparation of a composition for delaying aging, wherein the Lactobacillus plantarum is deposited at the Food Industry Development Institute (Taiwan) under the number BCRC910808, and the Lactobacillus plantarum increases the CCT2 gene in a skeletal muscle cell , CCT5 gene, CCT7 gene, Pink1 gene, Parkinson's disease juvenile protein 2 (Parkinson juvenile disease protein 2, Parkin ) gene, and Atg1 gene expression level. 一種植物乳桿菌用於製備延緩老化之組成物的用途，其中該植物乳桿菌係寄存於食品工業發展研究所(台灣)，編號係BCRC910808，且該植物乳桿菌係提升一神經細胞中CCT2基因、CCT5基因、CCT7基因、CCT8基因、Pink1基因、Parkin基因、Atg1基因、及Atg8基因之表現量。 A use of Lactobacillus plantarum for the preparation of a composition for delaying aging, wherein the Lactobacillus plantarum is deposited at the Food Industry Development Institute (Taiwan), and the number is BCRC910808, and the Lactobacillus plantarum promotes the CCT2 gene in a nerve cell, The expression level of CCT5 gene, CCT7 gene, CCT8 gene, Pink1 gene, Parkin gene, Atg1 gene, and Atg8 gene. 一種植物乳桿菌用於製備延緩老化之組成物的用途，其中該植物乳桿菌係寄存於食品工業發展研究所(台灣)，編號係BCRC910808，且該植物乳桿菌係提升一個體血球細胞中CCT2基因、CCT5基因、CCT6A基因、CCT8基因、Pink1基因、Parkin基因、Atg1基因、Atg8基因、叉頭框O 3(Forkhead box O 3,FOXO3)基因、麩醯胺酸依賴性菸草醯胺腺嘌呤二核苷酸合成酶(Glutamine-dependent NAD+ synthetase,NADSYN)基因、粒線體核糖體蛋白5(mitochondrial ribosomal protein S5,MRPS5)基因、泛素樣蛋白5(Ubiquitin-like protein 5,UBL-5)基因、超氧化物歧化酶3(Superoxide dismutase 3,SOD3)基因、 端粒酶反轉錄酶(Telomerase reverse transcriptase,TERT)基因、端粒酶RNA模板(telomerase RNA component,TERC)基因、及端粒延伸解旋酶調節因子1(Regulator of telomere elongation helicase 1,RTEL1)基因之表現量。 A use of Lactobacillus plantarum for the preparation of a composition for delaying aging, wherein the Lactobacillus plantarum is deposited at the Food Industry Development Institute (Taiwan), and the number is BCRC910808, and the Lactobacillus plantarum increases the CCT2 gene in a somatic blood cell. , CCT5 gene, CCT6A gene, CCT8 gene, Pink1 gene, Parkin gene, Atg1 gene, Atg8 gene, Forkhead box O 3 ( FOXO3 ) gene, glutamine-dependent tobacco adenine dinucleus Glutamine-dependent NAD + synthetase ( NADSYN ) gene, mitochondrial ribosomal protein S5 ( MRPS5 ) gene, ubiquitin-like protein 5 (Ubiquitin-like protein 5, UBL-5 ) gene , Superoxide dismutase 3 ( SOD3 ) gene, telomerase reverse transcriptase ( TERT ) gene, telomerase RNA template (telomerase RNA component, TERC ) gene, and telomerase extension solution The expression level of the regulator of telomere elongation helicase 1, RTEL1 gene. 如申請專利範圍第1項至第4項中任一項所述之用途，其中該益生菌株係為具有活性或去活性的菌株。 The use according to any one of items 1 to 4 in the scope of patent application, wherein the probiotic strain is an active or inactive strain. 如申請專利範圍第1項至第4項中任一項所述之用途，其中該益生菌株係增加一細胞粒線體活性。 The use according to any one of items 1 to 4 of the scope of patent application, wherein the probiotic strain increases the mitochondrial activity of a cell. 如申請專利範圍第1項至第4項中任一項所述之用途，其中該益生菌株係用於製備減少一個體的感知功能測驗反應時間，以及減少該個體的執行功能測驗反應時間之藥物。 The use described in any one of items 1 to 4 in the scope of the patent application, wherein the probiotic strain is used to prepare a drug that reduces the response time of an individual's sensory function test and reduces the individual's executive function test response time . 如申請專利範圍第1項至第4項中任一項所述之用途，其中係將該植物乳桿菌或其代謝產物，應用於製備延緩老化之乳製品、飲食補充物、醫藥組成物、保養品或食品。 The use described in any one of items 1 to 4 of the scope of patent application, wherein the Lactobacillus plantarum or its metabolites are applied to the preparation of dairy products, dietary supplements, pharmaceutical compositions, and maintenance to delay aging Products or food. 如申請專利範圍第1項至第4項中任一項中所述之用途，其中該組合物之形式為粉末狀、顆粒狀、液狀、膠狀或膏狀之劑型。 The use described in any one of items 1 to 4 in the scope of the patent application, wherein the composition is in the form of powder, granules, liquid, gel or paste. 如申請專利範圍第1項至第4項中任一項所述之用途，其中該組合物進一步包含一醫藥上可接受之載體。 The use according to any one of items 1 to 4 in the scope of the patent application, wherein the composition further comprises a pharmaceutically acceptable carrier.
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