TWI770595B - Use of lactic acid bacteria for the treatment or prevention of liver damage-related diseases - Google Patents
Use of lactic acid bacteria for the treatment or prevention of liver damage-related diseases Download PDFInfo
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- TWI770595B TWI770595B TW109129408A TW109129408A TWI770595B TW I770595 B TWI770595 B TW I770595B TW 109129408 A TW109129408 A TW 109129408A TW 109129408 A TW109129408 A TW 109129408A TW I770595 B TWI770595 B TW I770595B
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Abstract
本發明係揭露一種乳酸菌用於治療或預防肝臟損傷相關疾病之用途,具體來說,該乳酸菌包含有戊醣片球菌(Pediococcus pentosaceus)TTL 3-14及/或副乾酪乳桿菌副乾酪亞種(Lactobacillus paracasei subsp.paracasei)TTL 6-2,而當投予有效量之本發明所揭新穎乳酸菌至一個體時,係能夠有效地達到治療或/及預防與酒精誘發肝臟損傷或脂肪肝之功效。 The present invention discloses the use of a lactic acid bacterium for treating or preventing liver damage-related diseases. Specifically, the lactic acid bacterium comprises Pediococcus pentosaceus TTL 3-14 and/or Lactobacillus paracasei subsp. Lactobacillus paracasei subsp . paracasei) TTL 6-2, and when an effective amount of the novel lactic acid bacteria disclosed in the present invention is administered to an individual, it can effectively achieve the effect of treating or/and preventing alcohol-induced liver damage or fatty liver.
Description
本發明係有關於益生菌及其用途,特別係指一種乳酸菌用於治療或預防肝臟損傷相關疾病之用途。 The present invention relates to probiotics and their uses, in particular to the use of a lactic acid bacteria for treating or preventing liver damage-related diseases.
按,肝臟為人體消化系統非常重要的一部分,具有吸收、代謝及免疫保護等功能。長期慢性之飲酒行為會影響身體代謝,使三酸甘油酯與膽固醇堆積在肝臟,進而形成脂肪肝或其他酒精性肝臟疾病。具體來說,根據研究指出,酒精代謝產物乙醇會改變腸道上皮細胞之通透性,增加腸道中細菌毒素生成量,如內毒素,進而誘發或加重肝臟疾病。 The liver is a very important part of the human digestive system, with functions such as absorption, metabolism and immune protection. Long-term and chronic drinking behavior will affect the body's metabolism, causing triglycerides and cholesterol to accumulate in the liver, resulting in fatty liver or other alcoholic liver diseases. Specifically, according to studies, the metabolite of alcohol, ethanol, can change the permeability of intestinal epithelial cells, increase the production of bacterial toxins in the intestine, such as endotoxin, and then induce or aggravate liver disease.
又,雖然脂肪肝對於人體健康不會造成立即性的危害,但是長久下來會使肝臟細胞產生損傷,並且會引發肝炎、肝臟纖維化、肝硬化或肝癌等無法逆轉之疾病;而目前臨床上未提供治療脂肪肝之藥物,僅能透過飲食習慣及生活習慣之改變控制或改善脂肪肝或其相關代謝疾病,惟,絕大多數患者係無法長時間地改變生活或飲食習慣,導致脂肪肝於臨床上之治療呈現困難。 In addition, although fatty liver does not cause immediate harm to human health, it will damage liver cells in the long run, and cause irreversible diseases such as hepatitis, liver fibrosis, liver cirrhosis or liver cancer. Drugs for the treatment of fatty liver can only control or improve fatty liver or its related metabolic diseases through changes in eating habits and living habits. However, the vast majority of patients cannot change their life or eating habits for a long time, resulting in the clinical manifestation of fatty liver. The above treatment is difficult.
是以,開發一種能夠有效地治療或預防肝臟損傷相關疾病之組合物乃為目前醫學研究之當務之急。 Therefore, the development of a composition that can effectively treat or prevent liver damage-related diseases is a top priority of current medical research.
本發明之主要目的係在於提供一種乳酸菌之第二用途,其中,該乳酸菌係為戊醣片球菌(Pediococcus pentosaceus)TTL 3-14,或副乾酪乳桿菌副乾酪亞種(Lactobacillus paracasei subsp.paracasei)TTL 6-2;而該乳酸菌係具有增加肝臟抗氧化酵素活性及降低發炎因子之能力,以達到有效地預防或改善肝臟細胞受損或其相關疾病之功效,尤其是由酒精引發之肝臟疾病,如脂肪肝。 The main purpose of the present invention is to provide a second use of lactic acid bacteria, wherein the lactic acid bacteria are Pediococcus pentosaceus TTL 3-14, or Lactobacillus paracasei subsp . paracasei TTL 6-2; and the lactic acid bacteria have the ability to increase the activity of liver antioxidant enzymes and reduce inflammatory factors, so as to effectively prevent or improve liver cell damage or related diseases, especially liver diseases caused by alcohol, such as fatty liver.
具體來說,戊醣片球菌(Pediococcus pentosaceus)TTL 3-14,寄存編號為BCRC 911008,寄存日為2020年7月13日,寄存地點於台灣新竹食品工業發展研究所生物資源保存及研究中心;副乾酪乳桿菌副乾酪亞種(Lactobacillus paracasei subsp.paracasei)TTL 6-2,寄存編號為BCRC 911009,寄存日為2020年7月13日,寄存地點於台灣新竹食品工業發展研究所生物資源保存及研究中心。 Specifically, Pediococcus pentosaceus TTL 3-14, the deposit number is BCRC 911008, the deposit date is July 13, 2020, and the deposit place is at the Biological Resource Conservation and Research Center, Hsinchu Food Industry Development Institute, Taiwan; Lactobacillus paracasei subsp.paracasei TTL 6-2, the deposit number is BCRC 911009, the deposit date is July 13, 2020, and the deposit place is at the Biological Resource Conservation and Research Institute of Hsinchu Food Industry Development Institute, Taiwan. Research center.
本發明之另一目的係在於提供一種組合物,其係包含有效量之本發明所揭乳酸菌,而透過投予該組合物之一個體,係能有效地改善或預防肝臟損傷、脂肪肝或與上述病徵相關之疾病。 Another object of the present invention is to provide a composition comprising an effective amount of the lactic acid bacteria disclosed in the present invention, and by administering the composition to an individual, it can effectively improve or prevent liver damage, fatty liver or other related diseases Diseases related to the above symptoms.
具體來說,於本發明之一實施例中所揭之組合物,其係包含有至少一乳酸菌,而該乳酸菌係為戊醣片球菌TTL 3-14或副乾酪乳桿菌副乾酪亞種TTL 6-2。 Specifically, the composition disclosed in one embodiment of the present invention contains at least one lactic acid bacteria, and the lactic acid bacteria is Pediococcus pentosaceus TTL 3-14 or Lactobacillus paracasei subsp. paracasei TTL 6 -2.
為能達到較佳之肝臟疾病預防或/及治療功效,於本發明之另一實施例中,該組合物中係包含有戊醣片球菌TTL 3-14及副乾酪乳桿菌副乾酪亞種TTL 6-2。 In order to achieve better liver disease prevention or/and treatment efficacy, in another embodiment of the present invention, the composition comprises Pediococcus pentosaceus TTL 3-14 and Lactobacillus paracasei subsp. paracasei TTL 6 -2.
又,於本發明之另一實施例中該組合物係包含有另一乳酸菌,其係為植物乳酸桿菌植物亞種TTL 8-16;較佳地,該組合物係由戊醣片球菌TTL 3-14、副乾酪乳桿菌副乾酪亞種TTL 6-2及植物乳酸桿菌植物亞種TTL 8-16係以等重量比混合而成者。 Furthermore, in another embodiment of the present invention, the composition comprises another lactic acid bacteria, which is Lactobacillus plantarum TTL 8-16; preferably, the composition is composed of Pediococcus pentosaceus TTL 3 -14. Lactobacillus paracasei subsp. paracasei TTL 6-2 and Lactobacillus plantarum plant subsp. TTL 8-16 are mixed in equal weight ratios.
於本發明之實施例中,該組合物係得被製備為一食品或一營養補充品,並且得依據需求而被製備為不同劑型,例如粉劑、錠劑等。 In the embodiment of the present invention, the composition can be prepared as a food or a nutritional supplement, and can be prepared into different dosage forms, such as powder, lozenge, etc., according to requirements.
於本發明另一實施例中係揭露本發明所揭乳酸菌用於治療或預防肝臟損傷相關疾病之用途,意即當投予有效量之本發明所揭新穎乳酸菌或含有其之組合物至一個體時,係能夠有效地達到預防或/及治療酒精性肝損傷或其相關疾病之功效。 In another embodiment of the present invention, the use of the lactic acid bacteria disclosed in the present invention for treating or preventing liver damage-related diseases is disclosed, that is, when an effective amount of the novel lactic acid bacteria disclosed in the present invention or a composition containing the same is administered to an individual It can effectively achieve the effect of preventing or/and treating alcoholic liver injury or its related diseases.
其中,該酒精性肝損傷係為脂肪肝。 Among them, the alcoholic liver injury is fatty liver.
其中,該組合物係為一肝臟抗氧化酵素活性促進劑,能夠提升麩胱甘肽及麩胱甘肽過氧化酶之活性,強化肝臟抗氧化之能力,以達到避免或改善肝臟組織受損之功效。 Among them, the composition is a hepatic antioxidant enzyme activity promoter, which can enhance the activities of glutathione and glutathione peroxidase, and strengthen the antioxidant capacity of the liver, so as to avoid or improve the damage of liver tissue. effect.
圖1係為各組HepG2細胞經不同處理後所檢測分析出之AST相對清除率。 Figure 1 shows the relative clearance rates of AST detected and analyzed by HepG2 cells in each group after different treatments.
圖2係為各組HepG2細胞經不同處理後所檢測分析出之ALT相對清除率。 Figure 2 shows the relative clearance rate of ALT detected and analyzed after different treatments of HepG2 cells in each group.
圖3係為紀錄各組小鼠於試驗第0、2、4、6、8週體重變化之結果。 Figure 3 shows the results of recording the body weight changes of mice in each group at the 0th, 2nd, 4th, 6th and 8th weeks of the experiment.
圖4係為紀錄各組小鼠於試驗第0、2、4、6、8週血清中AST活性之結果。 Figure 4 shows the results of recording the AST activity in the serum of each group of mice at the 0th, 2nd, 4th, 6th and 8th weeks of the experiment.
圖5係為紀錄各組小鼠於試驗第0、2、4、6、8週血清中ALT活性之結果。 Figure 5 shows the results of recording the ALT activity in the serum of each group of mice at the 0th, 2nd, 4th, 6th and 8th week of the experiment.
圖6係為紀錄各組小鼠於試驗第0、2、4、6、8週血清中膽固醇含量之結果。 Figure 6 shows the results of recording the serum cholesterol levels of mice in each group at the 0th, 2nd, 4th, 6th, and 8th weeks of the experiment.
圖7係為紀錄各組小鼠於試驗第0、2、4、6、8週血清中三酸甘油酯含量之結果。 Figure 7 shows the results of recording triglyceride levels in serum of mice in each group at 0, 2, 4, 6, and 8 weeks of the experiment.
圖8係為檢測各組小鼠肝臟中三酸甘油酯含量之結果。 Figure 8 shows the results of detecting the content of triglycerides in the livers of mice in each group.
圖9係為檢測各組小鼠肝臟中麩胱甘肽活性之結果。 Figure 9 shows the results of detecting glutathione activity in the livers of mice in each group.
圖10係為檢測各組小鼠肝臟中麩胱甘肽過氧化酶活性之結果。 Figure 10 shows the results of detecting glutathione peroxidase activity in the livers of mice in each group.
圖11係為第1組小鼠肝臟組織切片H&E染色之結果。 Figure 11 shows the results of H&E staining of the liver tissue sections of mice in the first group.
圖12係為第2組小鼠肝臟組織切片H&E染色之結果。 Figure 12 shows the results of H&E staining of the second group of mouse liver tissue sections.
圖13係為第3組小鼠肝臟組織切片H&E染色之結果。 Figure 13 shows the results of H&E staining of the third group of mouse liver tissue sections.
圖14係為第4組小鼠肝臟組織切片H&E染色之結果。 Figure 14 shows the results of H&E staining of the liver tissue sections of mice in the fourth group.
圖15係為第5組小鼠肝臟組織切片H&E染色之結果。 Figure 15 shows the results of H&E staining of the liver tissue sections of mice in the fifth group.
圖16係為第6組小鼠肝臟組織切片H&E染色之結果。 Figure 16 shows the results of H&E staining of the liver tissue sections of mice in the sixth group.
本發明所揭露之乳酸菌,包含菌株TTL 3-14及菌株TTL 6-2,分離自穀類來源,具有益生菌基本特性,如耐酸、耐膽鹽、具腸道上皮細胞吸附力等,不會對生物體產生危害;其中:菌株TTL 3-14之寄存編號為BCRC 911008,寄存日為2020年7月13日,寄存地點於台灣新竹食品工業發展研究所生物資源保存及研究中心;而菌株TTL 3-14為革蘭氏陽性球菌,不具觸酶、氧化酶及運動特性,不會產生內孢子,於好氧及厭氧下皆能生長。 The lactic acid bacteria disclosed in the present invention, including strain TTL 3-14 and strain TTL 6-2, are isolated from cereal sources and have the basic characteristics of probiotics, such as acid resistance, bile salt resistance, and intestinal epithelial cell adhesion, etc. Harmful organisms; of which: the deposit number of strain TTL 3-14 is BCRC 911008, the deposit date is July 13, 2020, and the deposit place is at the Biological Resource Conservation and Research Center, Hsinchu Food Industry Development Institute, Taiwan; and strain TTL 3 -14 is a gram-positive coccus with no catalase, oxidase and motility properties, does not produce endospores, and can grow under both aerobic and anaerobic conditions.
菌株TTL 6-2之寄存編號為BCRC 911009,寄存日為2020年7月13日,寄存地點於台灣新竹食品工業發展研究所生物資源保存及研究中心;而菌株TTL 6-2為革蘭氏陽性桿菌,不具觸酶、氧化酶及運動特性,不會產生內孢子,於好氧及厭氧下皆能生長。 The deposit number of the strain TTL 6-2 is BCRC 911009, the deposit date is July 13, 2020, and the deposit place is at the Biological Resource Conservation and Research Center, Hsinchu Food Industry Development Institute, Taiwan; while the strain TTL 6-2 is Gram-positive Bacillus has no catalase, oxidase and motility properties, does not produce endospores, and can grow under both aerobic and anaerobic conditions.
本發明所揭菌株TTL 3-14及菌株TTL 6-2係可培養於下列條件:培養基為含0.05% L-cysteine之乳酸菌MRS培養基(Lactobacilli MRS broth,BD, difco,USA),37℃下培養16小時;培養後之保存條件為:含15%甘油之MRS培養基(MRS broth)、-80℃之環境下。 The strains TTL 3-14 and TTL 6-2 disclosed in the present invention can be cultivated in the following conditions: the culture medium is a lactic acid bacteria MRS medium (Lactobacilli MRS broth, BD, 0.05% L-cysteine) containing 0.05% L-cysteine. difco, USA), cultured at 37°C for 16 hours; the storage conditions after culture were as follows: MRS broth (MRS broth) containing 15% glycerol, at -80°C.
本發明所稱「菌株TTL 3-14」,係為寄存編號為BCRC 911008之戊醣片球菌(Pediococcus pentosaceus Pediococcus pentosaceus)TTL 3-14。 The "strain TTL 3-14" referred to in the present invention is Pediococcus pentosaceus Pediococcus pentosaceus TTL 3-14 with accession number BCRC 911008.
本發明所稱「菌株TTL 6-2」,係為寄存編號為BCRC 911009之副乾酪乳桿菌副乾酪亞種(Lactobacillus paracasei subsp.paracasei)TTL 6-2。 The "strain TTL 6-2" referred to in the present invention is Lactobacillus paracasei subsp . paracasei TTL 6-2 with accession number BCRC 911009.
本發明所稱「菌株TTL 8-16」,係為寄存編號BCRC 910871之植物乳酸桿菌植物亞種(Lactobacillus plantarum subsp.plantarum)TTL 8-16,寄存日為2019年2月15日,寄存於台灣新竹食品工業發展研究所生物資源保存及研究中心;更進一步來說,TTL 8-16菌株為革蘭氏陽性桿菌,不具觸酶、氧化酶及運動性,不會產生內孢子,於好氧及厭氧環境下皆能生長。 The "strain TTL 8-16" referred to in the present invention is Lactobacillus plantarum subsp . plantarum TTL 8-16 with deposit number BCRC 910871, deposited on February 15, 2019, and deposited in Taiwan Bioresource Conservation and Research Center, Hsinchu Food Industry Development Institute; further, TTL 8-16 strains are Gram-positive bacilli, without catalase, oxidase and motility, and do not produce endospores. Can grow in anaerobic conditions.
本發明所稱「乳酸菌組合物」,係指將培養後之菌株TTL 3-14、菌株TTL 6-2及菌株TTL 8-16,以重量等比(1:1:1)之比例混合而成者,其中,於混合前各該菌株係得先以無菌磷酸鹽緩衝液進行清洗。 The term "lactide bacteria composition" in the present invention refers to mixing the cultured strains TTL 3-14, strains TTL 6-2 and strains TTL 8-16 in an equal weight ratio (1:1:1) or, wherein, each strain should be washed with sterile phosphate buffer before mixing.
本發明所稱「有效量」乙詞係指欲產生所求特定效果所需化合物或活性成份之量,得以其在組合物中所佔重量百分比表示。如同本發明所屬技術領域中具有通常知識者所瞭解者,該有效量會因為欲引起特定效果之投予方式而有所不同。一般來說,活性成分或化合物於組合物中之量可佔該組合物重量之約1%至約100%,較佳者係為約30%至約100%。 The term "effective amount" in the present invention refers to the amount of the compound or active ingredient required to produce the desired specific effect, expressed as a percentage by weight in the composition. As is understood by those of ordinary skill in the art to which this invention pertains, the effective amount will vary depending on the manner of administration intended to elicit the particular effect. Generally, the active ingredient or compound can be present in the composition in an amount from about 1% to about 100%, preferably from about 30% to about 100%, by weight of the composition.
以下,為能更進一步說明本發明之技術特徵及其功效,將茲舉若干實例並搭配圖表作詳細說明如後。 Hereinafter, in order to further illustrate the technical features and effects of the present invention, a number of examples will be given and illustrated in detail as follows.
以下實例中所使用之菌株係分別保存在含15%甘油之MRS broth,保存於-80℃。菌株活化兩次,培養基為含0.05% L-cysteine之Lactobacilli MRS broth(BD,difco,USA),培養條件為37℃培養24小時。 The strains used in the following examples were stored in MRS broth containing 15% glycerol at -80°C. The strain was activated twice, the medium was Lactobacilli MRS broth (BD, difco, USA) containing 0.05% L-cysteine, and the culture condition was 37° C. for 24 hours.
以下實例中所使用之人類肝癌細胞C3A(HepG2/C3A)(寄存編號:BCRC 60177)及小鼠巨噬細胞RAW264.7(寄存編號:BCRC 60001),皆購自食品工業研究所生物資源保存及研究中心(台灣)。 The human hepatoma cell C3A (HepG2/C3A) (Accession No.: BCRC 60177) and mouse macrophage RAW264.7 (Accession No.: BCRC 60001) used in the following examples were purchased from the Institute of Food Industry Bioresources and Research Center (Taiwan).
實例一:體外抗發炎試驗 Example 1: In vitro anti-inflammatory test
將人類肝癌細胞C3A(下稱HepG2細胞)及小鼠巨噬細胞RAW264.7(下稱RAW264.7細胞)分別於24孔盤中進行培養,每孔中HepG2細胞之細胞量為2 x 105cell/well及RAW264.7細胞之細胞量為1 x 105cell/well,待細胞生長至80%時,分別以磷酸鹽緩衝液清洗後,加入新的培養基,再加入100μl內毒素脂多醣(LPS,100ng/mL)以及100μl之乳酸菌菌液,菌數量為109CFU/mL,而後在於二氧化碳培養箱中培養24小時,收集各孔細胞上清液。 Human hepatoma cells C3A (hereafter referred to as HepG2 cells) and mouse macrophage RAW264.7 (hereafter referred to as RAW264.7 cells) were cultured in 24-well plates, and the number of HepG2 cells in each well was 2 x 105 cells/ The cell volume of well and RAW264.7 cells is 1 x 105 cells/well. When the cells grow to 80%, they are washed with phosphate buffered saline, respectively, and then new medium is added, and then 100 μl of endotoxin lipopolysaccharide (LPS, 100ng/well) is added. mL) and 100 μl of lactic acid bacteria liquid, the number of bacteria is 109CFU/mL, and then cultured in a carbon dioxide incubator for 24 hours, and the cell supernatant of each well was collected.
將由RAW264.7細胞收集而得之上清液以市售酵素連結免疫分析套組(Enzyme-linked immunosorbent assay kit ELISA,購自BioLegend)檢測TNF-α及IL-6之含量;並將由HepG2細胞收集而得之上清液以市售酵素連結免疫分析套組(BD OptEIA Set Human IL-8,購自BD Biosciences)檢測IL-8之含量,結果如下表1所示,其中,空白組為未加入LPS及乳酸菌菌液者,LPS組為未加入乳酸菌菌液者。 The supernatant collected from RAW264.7 cells was used to detect the content of TNF-α and IL-6 with a commercially available Enzyme-linked immunosorbent assay kit ELISA, purchased from BioLegend; and collected from HepG2 cells The supernatant was obtained by using a commercially available enzyme-linked immunoassay kit (BD OptEIA Set Human IL-8, purchased from BD Biosciences) to detect the content of IL-8. The results are shown in Table 1 below, wherein the blank group was not added LPS and lactic acid bacteria liquid, LPS group was not added lactic acid bacteria liquid.
由表1之結果可知,菌株TTL 3-14、菌株TTL 6-2及菌株TTL 8-16都能夠降低TNF-α及IL-6,但對於Hep G2細胞經LPS刺激產生發炎激素:IL-8來說,僅有菌株TTL 3-14、菌株TTL 6-2能夠達到明顯較佳之抑制功效。 From the results in Table 1, it can be seen that strain TTL 3-14, strain TTL 6-2 and strain TTL 8-16 can reduce TNF-α and IL-6, but for Hep G2 cells stimulated by LPS to produce inflammatory hormone: IL-8 For example, only strain TTL 3-14 and strain TTL 6-2 can achieve significantly better inhibitory effect.
由於Hep G2細胞經LPS刺激後並不會產生IL-6、TNF-α及IFN-γ等發炎因子,而會大幅產生IL-8,因此,由上表1可清楚得知,本發明所揭菌株TTL 3-14及菌株TTL 6-2不僅能夠抑制體內之發炎反應,對於肝臟發炎反應亦有良好治療或預防之功效。 Since Hep G2 cells do not produce inflammatory factors such as IL-6, TNF-α, and IFN-γ after being stimulated by LPS, they will significantly produce IL-8. Therefore, it can be clearly seen from Table 1 above that the disclosed Strain TTL 3-14 and strain TTL 6-2 can not only inhibit inflammation in the body, but also have good therapeutic or preventive effects on liver inflammation.
實例二:體外酒精耐受性試驗 Example 2: In vitro alcohol tolerance test
分別取菌株TTL 3-14、菌株TTL 6-2、菌株TTL 8-16,活化後培養16小時,再取培養後之菌液100μl,分別接種至不含酒精之4mL MRS培養基(MRS broth)、含最終酒精濃度為1%、5%、10%、15%及20%之4mL MRS培養基中,再於37℃下培養16小時,而後將培養後之菌液經序列稀釋,以平板計數方式計算其菌數量,結果如下表2所示。 Take strain TTL 3-14, strain TTL 6-2, strain TTL 8-16 respectively, cultivate for 16 hours after activation, and then take 100 μl of the cultured bacterial liquid and inoculate into 4 mL MRS medium (MRS broth) without alcohol, respectively. In 4 mL of MRS medium containing final alcohol concentration of 1%, 5%, 10%, 15% and 20%, cultured at 37°C for 16 hours, and then serially diluted the cultured bacterial solution and counted by plate count The number of bacteria, the results are shown in Table 2 below.
由上表2之結果可知,本發明所揭菌株TTL 3-14及菌株TTL 6-2都能於高濃度酒精之環境下存活,其中,又以菌株TTL 6-2對於酒精之耐受性較佳,意即能於含有濃度為20%以上酒精之環境下生存。 As can be seen from the results of the above table 2, the strains TTL 3-14 and the strain TTL 6-2 disclosed by the present invention can survive in the environment of high concentration alcohol, and wherein, the tolerance of the strain TTL 6-2 to alcohol is relatively high. Good, which means that it can survive in an environment containing more than 20% alcohol.
實例三:體外酒精性肝損傷生化指標之檢測 Example 3: Detection of biochemical indicators of alcoholic liver injury in vitro
將HepG2細胞以2 x 105cell/well接種至24孔盤,當細胞生長至80%時,以磷酸鹽緩衝液清洗後,再將培養基更換為不含FBS之MEM培養基,並進行分組而以不同條件處理,其中:空白組添加1ml培養基;酒精組添加900μl培養基; 水飛薊素(SML)組添加800μl培養基,添加100μl之酒精,酒精最終濃度為100mM,再添加100μl水飛薊素(30μg/mL);各菌株組添加800μl培養基後,添加100μl之酒精,酒精最終濃度為100mM,再添加乳酸菌菌液;各組放置24小後,收集上清液與細胞,分別以市售ALT分析套組(購於BioVision)及AST分析套組(購於BioVision)檢測內谷草轉氨酶(下稱AST)及谷丙轉氨酶(下稱ALT)之含量,結果如圖1及圖2。 HepG2 cells were seeded into a 24-well plate at 2 x 105 cells/well. When the cells grew to 80%, they were washed with phosphate buffered saline, and then the medium was changed to MEM medium without FBS, and grouped by different conditions. Treatment, wherein: blank group added 1ml medium; alcohol group added 900μl medium; The silymarin (SML) group was added with 800 μl of medium, 100 μl of alcohol, the final concentration of alcohol was 100 mM, and then 100 μl of silymarin (30 μg/mL) was added; each strain group was added with 800 μl of medium, 100 μl of alcohol was added, and the final concentration of alcohol was 100 mM, and then Lactic acid bacteria solution was added; after each group was placed for 24 hours, the supernatant and cells were collected, and the commercially available ALT analysis kit (purchased from BioVision) and AST analysis kit (purchased from BioVision) were used to detect aspartate aminotransferase (hereinafter referred to as AST). ) and alanine aminotransferase (hereinafter referred to as ALT) content, the results are shown in Figure 1 and Figure 2.
由圖1及圖2之結果可知,當HepG2細胞受到酒精刺激而產生ALT及AST時,僅有本發明所揭菌株TTL 3-14及菌株TTL 6-2能夠同時降低ALT及AST之含量,顯示本發明所揭菌株TTL 3-14及菌株TTL 6-2係具有保護肝臟免受酒精刺激而發炎或是產生病變之功效。 It can be seen from the results of Fig. 1 and Fig. 2 that when HepG2 cells are stimulated by alcohol to produce ALT and AST, only the strains TTL 3-14 and TTL 6-2 disclosed in the present invention can reduce the content of ALT and AST at the same time. The strain TTL 3-14 and the strain TTL 6-2 disclosed in the present invention have the effect of protecting the liver from being stimulated by alcohol and causing inflammation or pathological changes.
實例四:血清生化指標表現量之檢測 Example 4: Detection of serum biochemical indicators
購入60隻七週齡C57BL/6N雄性小鼠(樂斯科生物公司,台灣),飼養於溫度為22±2℃、相對溼度為55±5%、光照循環期為12小時之環境下,所使用之飼料為流質一般飼料。 60 seven-week-old C57BL/6N male mice (Lesco Bio, Taiwan) were purchased and raised in an environment with a temperature of 22 ± 2 °C, a relative humidity of 55 ± 5%, and a light cycle period of 12 hours. The feed used is liquid general feed.
將各小鼠飼養1週後,隨機分組,並且各組以下列處理條件分別飼養8週,而各組小鼠試驗完成後予以犧牲,並分別取其肝臟,進行後續實例之試驗:第1組(空白組):僅餵食不含酒精之流質一般飼料;第2組(酒精組):餵食含總熱量36%酒精(0.36kcal/ml)之流質酒精飼料;第3組(菌株TTL 3-14組):餵食總熱量36%酒精(0.36kcal/ml)之流質酒精飼料及劑量為9 log CFU/ml之菌株TTL 3-14; 第4組(菌株TTL 6-2組):餵食總熱量36%酒精(0.36kcal/ml)之流質酒精飼料及劑量為9 log CFU/ml之菌株TTL 6-2;第5組(菌株TTL 8-16組)餵食總熱量36%酒精(0.36kcal/ml)之流質酒精飼料及劑量為9 log CFU/ml之菌株TTL 8-16;第6組(乳酸菌組合物組)餵食總熱量36%酒精(0.36kcal/ml)之流質酒精飼料及劑量各為9 log CFU/ml之乳酸菌組合物。 After the mice were reared for 1 week, they were randomly divided into groups, and each group was reared for 8 weeks under the following treatment conditions, and the mice in each group were sacrificed after the experiment was completed, and their livers were taken separately to carry out the experiments of the following examples: Group 1 (Blank group): only fed with liquid general diet without alcohol; Group 2 (alcohol group): fed with liquid alcohol diet with total calorie 36% alcohol (0.36kcal/ml); Group 3 (strain TTL 3-14 Group): fed a liquid alcohol diet containing 36% alcohol (0.36kcal/ml) in total calories and strain TTL 3-14 at a dose of 9 log CFU/ml; Group 4 (strain TTL 6-2 group): fed a liquid alcohol diet with total calorie 36% alcohol (0.36kcal/ml) and strain TTL 6-2 at a dose of 9 log CFU/ml; group 5 (strain TTL 8 -16 groups) were fed a liquid alcohol diet with a total calorie of 36% alcohol (0.36kcal/ml) and strains TTL 8-16 at a dose of 9 log CFU/ml; group 6 (lactic acid bacteria composition group) was fed with a total calorie of 36% alcohol (0.36kcal/ml) of the liquid alcohol feed and the lactic acid bacteria composition at a dose of 9 log CFU/ml each.
各組小鼠於第0、2、4、6、8週進行體重紀錄及眼窩採血,將採出之血液進行血清檢測,分析其內AST及ALT、三酸甘油酯及總膽固醇,結果如圖3至圖7所示。 The mice in each group were recorded body weight and blood was collected from the eye socket at 0, 2, 4, 6, and 8 weeks. The collected blood was tested for serum, and the contents of AST, ALT, triglyceride and total cholesterol were analyzed. The results are shown in the figure. 3 to Figure 7.
由圖3之結果可知,於試驗期間各組小鼠飲食正常下,未被餵食酒精之第1組小鼠的體重較其他組小鼠重(P<0.05),顯示長時間餵食酒精確實會誘發小鼠產生酒精性肝炎或相關病徵,導致被餵食酒精之小鼠體重降低。 It can be seen from the results in Figure 3 that during the test period, the mice in each group had a normal diet, and the weight of the mice in the first group that was not fed alcohol was heavier than that of the mice in the other groups (P<0.05), indicating that long-term feeding of alcohol does induce Mice develop alcoholic hepatitis or related symptoms, resulting in decreased body weight in mice fed alcohol.
再者,由圖4及圖5之結果可知,第1組小鼠血清中之AST及ALT數值於試驗期間幾乎沒有變化;第2組小鼠血清中之AST及ALT數值隨著酒精餵食時間變長(試驗期間變長)而明顯增加(P<0.05),並且相較於第1組小鼠來說,第2組小鼠血清中之AST於試驗第8週更明顯上升53%;而於試驗第8週時,第3組、第4組及第6組小鼠血清中之AST數值明顯低於第2組小鼠,分別下降42%、33%及37%(P<0.05),並且,第3組及第6組小鼠血清中之ALT數值明顯低於第2組小鼠,分別下降54%及48%(P<0.05)。根據研究指出血清中AST及ALT數值之增加,表示肝臟損傷情形越嚴重,因此,由圖4及圖5之結果顯示,本發明所揭菌株TTL 3-14及/或菌株TTL 6-2係具有對於酒精所造成之肝臟損傷或肝臟疾病有特異性之治療或預防效果,意即投予有效量之本發明所揭菌株TTL 3-14及/或菌株TTL 6-2至具有飲酒習慣之受試者時,係能達到有效地改善或預防酒精性肝臟損傷或肝臟疾病之功效。 Furthermore, from the results of Figure 4 and Figure 5, it can be seen that the AST and ALT values in the serum of the mice in the first group did not change during the test period; the AST and ALT values in the serum of the mice in the second group changed with the time of alcohol feeding. Compared with the mice in the first group, the AST in the serum of the mice in the second group was significantly increased by 53% at the 8th week of the experiment; At the 8th week of the experiment, the AST values in the serum of the mice in the 3rd, 4th and 6th groups were significantly lower than those of the 2nd group, decreased by 42%, 33% and 37% respectively (P<0.05), and , the ALT values in the serum of the mice in the third and sixth groups were significantly lower than those in the second group, decreased by 54% and 48%, respectively (P<0.05). According to the research, it is pointed out that the increase of AST and ALT values in serum indicates that the liver damage is more serious. Therefore, the results of Fig. 4 and Fig. 5 show that the strains TTL 3-14 and/or strains TTL 6-2 disclosed in the present invention have It has a specific therapeutic or preventive effect on liver damage or liver disease caused by alcohol, which means administering an effective amount of the strain TTL 3-14 and/or strain TTL 6-2 disclosed in the present invention to the test subject with drinking habit In other cases, it can effectively improve or prevent alcohol-induced liver damage or liver disease.
又,雖然由圖6之結果可知各組小鼠血清中之總膽固醇含量沒有明顯變化,但由圖7之結果可知,於試驗第4週時,相較於第2組小鼠,第3組及第4組小鼠血清中三酸甘油脂含量明顯降低(P<0.05),分別下降28.9及22.7%,且下降之效果係能夠持續維持至試驗結束(第8週);更進一步來說,於試驗第4週時,第5組小鼠血清中三酸甘油脂含量下降幅度最佳,相較於第3或4組小鼠來說,約下降44.8%,而第6組小鼠血清中三酸甘油脂含量下降幅度約為23.9%;於試驗第4週後,除第2組小鼠外,各組小鼠血清中三酸甘油脂含量幾乎與第1組(空白組)小鼠無差異,並於試驗第8週時,第5組及第6組小鼠血清中三酸甘油脂含量仍持續降低,分別較第2組小鼠血清中之三酸甘油脂下降約35.4%及34.4%,彼此間具有顯著差異(P<0.05)。換言之,由圖7之結果顯示,於酒精飲食下,本發明所揭菌株TTL 8-16及/或菌株TTL 3-14及/或菌株TTL 6-2係能夠持續及有效地降低血清中三酸甘油酯之含量,以能達到預防或治療因酒精引發之血管病變的功效。
In addition, although it can be seen from the results in Figure 6 that the total cholesterol content in the serum of the mice in each group did not change significantly, it can be seen from the results in Figure 7 that at the 4th week of the experiment, compared with the mice in the second group, the mice in the third group had higher levels of cholesterol. and the 4th group of mice serum triglyceride content was significantly reduced (P<0.05), decreased by 28.9 and 22.7%, and the effect of the decrease could be maintained until the end of the test (the 8th week); further, At the 4th week of the experiment, the serum triglyceride content of mice in
實例五:肝臟內三酸甘油酯含量之檢測 Example 5: Detection of triglyceride content in liver
取實例一中試驗完成之各組小鼠之肝臟組織,分別進行均質離心後,取其上清液,加入調製好之三酸甘油酯酵素試劑(Triglyceride Enzyme Mixture),反應後測定OD530-550nm吸光值,將所得到之OD值減去標準品(0mg/dl)之OD值,再透過所繪製出標準曲線之線性回歸分析計算出各組小鼠肝臟內三酸甘油酯含量,結果如圖8所示。 Take the liver tissue of each group of mice that have completed the experiment in Example 1, and after homogenizing centrifugation, take the supernatant, add the prepared triglyceride enzyme reagent (Triglyceride Enzyme Mixture), and measure the OD530-550nm absorbance after the reaction value, subtract the OD value of the standard substance (0mg/dl) from the obtained OD value, and then calculate the triglyceride content in the liver of each group of mice through the linear regression analysis of the drawn standard curve, and the results are shown in Figure 8 shown.
由圖8之結果可知,8週後相較於第1組小鼠,第2組小鼠肝臟中三酸甘油脂含量明顯增加,意即酒精飲食確實會導致肝臟脂肪堆積,並會誘發脂肪肝之發生;而相較於第2組小鼠,第3組至第6組小鼠肝臟中三酸甘油脂含量明顯下降,分別降低49.8%、53.7%、45.9%以及47.2%。
From the results in Figure 8, it can be seen that compared with the mice in the first group, the content of triglycerides in the liver of the mice in the second group increased significantly after 8 weeks, which means that the alcohol diet will indeed lead to the accumulation of liver fat and induce fatty liver. Compared with the mice in group 2, the triglyceride content in the liver of mice in
由此結果可知,本發明所揭菌株TTL 3-14及/或菌株TTL 6-2確實能夠避免酒精飲食下之三酸甘油酯累積於肝臟組織中,並且如果持續投予有效量之本發明所揭菌株TTL 3-14及/或菌株TTL 6-2至有飲酒習慣之個體,係能夠使其肝臟中三酸甘油酯含量維持與未有飲酒習慣者相近。 From the results, it can be seen that the strain TTL 3-14 and/or the strain TTL 6-2 disclosed in the present invention can indeed avoid the accumulation of triglycerides in the liver tissue under an alcoholic diet, and if an effective amount of the present invention is continuously administered It was revealed that strains TTL 3-14 and/or strains TTL 6-2 to individuals with drinking habits were able to maintain triglyceride levels in their livers similar to those of non-drinking individuals.
實例六:肝臟內抗氧化酵素表現量之檢測 Example 6: Detection of Antioxidant Enzyme Expression in Liver
取實例一中試驗完成之各組小鼠之肝臟組織,分別進行均質離心後,取其上清液,再以市售分析套組(QuantiChromTM Glutathione Assay Kit及EnzyChromTM Glutathione Peroxidase Assay Kit)分別檢測各組小鼠肝臟組織中麩胱甘肽(Glutathione,GSH)及麩胱甘肽過氧化酶(Glutathione peroxidase,GPx)活性之表現,結果如圖9及圖10所示。 The liver tissue of each group of mice that had been tested in Example 1 was taken and subjected to homogenization and centrifugation, respectively, and the supernatant was taken, and then the commercially available analysis kits (QuantiChromTM Glutathione Assay Kit and EnzyChromTM Glutathione Peroxidase Assay Kit) were used to detect each group respectively. The expression of glutathione (Glutathione, GSH) and glutathione peroxidase (Glutathione peroxidase, GPx) activities in mouse liver tissue, the results are shown in Figure 9 and Figure 10 .
由圖9之結果可知,8週後第5組小鼠肝臟中麩胱甘肽含量與第1組小鼠無明顯差異(P>0.05);第2組小鼠肝臟中麩胱甘肽含量明顯高於第1組小鼠(P<0.05),但與第6組小鼠間無明顯差異存在(P>0.05);而相較於第2組小鼠來說,第3組及第4組小鼠肝臟中麩胱甘肽含量明顯上升37%及35%(P<0.05);而基於麩胱甘肽為生物體中的抗氧化物質,故由此結果顯示,本發明所揭菌株TTL 3-14及/或菌株TTL 6-2確實為能夠提升肝臟抗氧化能力之特殊乳酸菌株,意即於酒精飲食下,將有效量本發明所揭菌株TTL 3-14及/或菌株TTL 6-2投予至個體時,係能有效地提升個體肝臟內麩胱甘肽,達到增加個體抗氧化能力之功效。
From the results in Figure 9, it can be seen that after 8 weeks, the content of glutathione in the liver of the mice in the fifth group was not significantly different from that of the mice in the first group (P>0.05); the content of glutathione in the liver of the mice in the second group was significantly different. higher than that of mice in group 1 (P<0.05), but there was no significant difference between mice in group 6 (P>0.05); and compared with mice in group 2,
再者,由圖10之結果可知,8週後相較於第1組小鼠來說,第2組小鼠肝臟內麩胱甘肽過氧化酶酵素活性下降31.9%;而相較於第2組小鼠來說,第3組至第6組小鼠肝臟內麩胱甘肽過氧化酶酵素活性雖皆有上升(分別增加30.6%、31.6%、16.9%以及58.4%),但第5組小鼠肝臟中麩胱甘肽過氧化酶酵素活性上升程度不佳,顯示投予菌株TTL 8-16對於個體肝臟抗氧化能力之提升效
果有限,而是投予本發明所揭菌株TTL 3-14及/或菌株TTL 6-2確實能夠增加肝臟內麩胱甘肽過氧化酶酵素活性。
Furthermore, from the results in Figure 10, it can be seen that after 8 weeks, compared with the mice in the first group, the glutathione peroxidase enzyme activity in the liver of the mice in the second group decreased by 31.9%; For group mice, although the glutathione peroxidase enzyme activity in the liver of mice in
由圖9及圖10之結果顯示,本發明所揭菌株TTL 3-14及/或菌株TTL 6-2係為具提升肝臟抗氧化能力之特異性乳酸菌,意即投予有效量之菌株TTL 3-14及/或菌株TTL 6-2至有飲酒習慣之個體,係能夠透過提升肝臟中抗氧化活性物質及/或抗氧化酵素,達到避免或改善肝臟損害或酒精引發之肝臟相關疾病之功效。
The results in Figure 9 and Figure 10 show that the strain TTL 3-14 and/or the strain TTL 6-2 disclosed in the present invention are specific lactic acid bacteria capable of enhancing the antioxidant capacity of the liver, which means that an effective amount of the
實例五:肝臟組織切片染色結果 Example 5: Staining results of liver tissue sections
取實例一中試驗完成之各組小鼠之肝臟組織,分別以石蠟包埋切片後進行H&E染色,結果如圖11至圖16所示。 The liver tissue of each group of mice after the experiment in Example 1 was taken, embedded in paraffin and sectioned, and then stained with H&E. The results are shown in Figures 11 to 16 .
由圖11可知,第1組小鼠肝臟切片中未發現明顯的脂肪油滴堆積,並細胞核都非常完整;由圖12可知,第2組小鼠肝臟切片中具有明顯脂肪油滴堆積相當明顯,顯示長期投予酒精確實會造成脂肪累積於肝臟,產生脂肪肝之病徵;由圖13至圖16所示之第3組至第6組之結果可知,投予不同乳酸菌或是乳酸菌組合物係能夠使肝臟中脂肪油滴縮小,減少脂肪累積於肝臟中,並且避免肝臟細胞被破壞,而比較第3組至第6組之肝臟組織切片染色的結果,顯示投予本發明所揭TTL 3-14菌株可更有效地避免脂肪堆積於肝臟組織。
It can be seen from Figure 11 that no obvious accumulation of fatty oil droplets was found in the liver slices of the mice in the first group, and the cell nuclei were very complete; It is shown that the long-term administration of alcohol can indeed cause fat to accumulate in the liver, resulting in the symptoms of fatty liver; from the results of
由上述結果可知,於酒精飲食下,本發明所揭菌株TTL 3-14及/或菌株TTL 6-2係能夠避免或改善脂肪堆積於肝臟之情形,並且能夠保護肝臟細胞受損,維持細胞完整性;換言之,投予有效量之本發明所揭菌株TTL 3-14及/或菌株TTL 6-2至個體時,係能夠有效地達到預防或治療脂肪肝或其相關病症之功效。 It can be seen from the above results that the strain TTL 3-14 and/or the strain TTL 6-2 disclosed in the present invention can avoid or improve the situation of fat accumulation in the liver, and can protect liver cells from damage and maintain cell integrity under alcoholic diet. In other words, when an effective amount of strain TTL 3-14 and/or strain TTL 6-2 disclosed in the present invention is administered to an individual, the effect of preventing or treating fatty liver or its related diseases can be effectively achieved.
戊醣片球菌(Pediococcus pentosaceus)TTL 3-14,寄存編號為BCRC 911008,寄存日為2020年7月13日,寄存地點於台灣新竹食品工業發展研究所生物資源保存及研究中心。 Pediococcus pentosaceus TTL 3-14, the deposit number is BCRC 911008, the deposit date is July 13, 2020, and the deposit place is at the Biological Resource Conservation and Research Center, Hsinchu Institute of Food Industry Development, Taiwan.
副乾酪乳桿菌副乾酪亞種(Lactobacillus paracasei subsp.paracasei)TTL 6-2,寄存編號為BCRC 911009,寄存日為2020年7月13日,寄存地點於台灣新竹食品工業發展研究所生物資源保存及研究中心。 Lactobacillus paracasei subsp.paracasei TTL 6-2, the deposit number is BCRC 911009, the deposit date is July 13, 2020, and the deposit place is at the Biological Resource Conservation and Development Institute of Hsinchu, Taiwan. Research center.
植物乳酸桿菌植物亞種TTL 8-16,寄存日為2019年2月15日,寄存編號為BCRC 910871。 Lactobacillus plantarum plant subsp. TTL 8-16, deposited on February 15, 2019, with deposit number BCRC 910871.
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期刊 Rifatbegovic Z et al. "Effect of probiotics on liver function after surgery resection for malignancy in the liver cirrhotic" Medical Archives 64 (4) 2010 p.208-211 * |
期刊 Xu RH et al. "Probiotic and hepatoprotective activity of lactobacillus isolated from Mongolian camel milk products" Beneficial microbes 10 (6) 2019 p.699-710; * |
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