TW202329997A - Lactic acid bacterial composition and its use in preparation of oral composition of inhibiting drug-resistant enterobacteriaceae - Google Patents
Lactic acid bacterial composition and its use in preparation of oral composition of inhibiting drug-resistant enterobacteriaceae Download PDFInfo
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- TW202329997A TW202329997A TW111104157A TW111104157A TW202329997A TW 202329997 A TW202329997 A TW 202329997A TW 111104157 A TW111104157 A TW 111104157A TW 111104157 A TW111104157 A TW 111104157A TW 202329997 A TW202329997 A TW 202329997A
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- lactic acid
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- acid bacteria
- lactobacillus rhamnosus
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- 241000588921 Enterobacteriaceae Species 0.000 title claims abstract description 64
- 229940079593 drug Drugs 0.000 title claims abstract description 59
- 239000003814 drug Substances 0.000 title claims abstract description 59
- 239000000203 mixture Substances 0.000 title claims abstract description 58
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- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 42
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title claims abstract description 4
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- 239000004480 active ingredient Substances 0.000 claims abstract description 8
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- 229960000511 lactulose Drugs 0.000 claims description 14
- PFCRQPBOOFTZGQ-UHFFFAOYSA-N lactulose keto form Natural products OCC(=O)C(O)C(C(O)CO)OC1OC(CO)C(O)C(O)C1O PFCRQPBOOFTZGQ-UHFFFAOYSA-N 0.000 claims description 14
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Abstract
Description
本發明是有關於一種乳酸菌組成物,特別是關於一種乳酸菌組成物及其用於製備抑制抗藥性腸桿菌之口服組成物的用途。The present invention relates to a composition of lactic acid bacteria, in particular to a composition of lactic acid bacteria and its use for preparing an oral composition for inhibiting drug-resistant Enterobacteriaceae.
腸桿菌( Enterobacteriaceae)為革蘭氏陰性菌,屬於γ-變形菌綱腸桿菌目。腸桿菌普遍存在於環境(如:土壤及水)及生物體(如:動物及植物)中,且是人體的腸道菌之一。腸桿菌包含有益的共生菌,亦包含伺機性感染的病原菌。此些病原菌可引發赤痢、腸熱症、***、傷口感染、肝膿瘍、敗血症、腦膜炎、肺炎等疾病,是院內感染及社區感染的主要病原菌之一。 Enterobacteriaceae ( Enterobacteriaceae ) are Gram-negative bacteria, belonging to the γ-proteobacteria Enterobacteriaceae. Enterobacter is ubiquitous in the environment (such as: soil and water) and organisms (such as: animals and plants), and is one of the intestinal bacteria of the human body. Enterobacteriaceae contain beneficial commensal bacteria as well as pathogenic bacteria that are opportunistic. These pathogens can cause dysentery, enteric fever, urinary tract infection, wound infection, liver abscess, sepsis, meningitis, pneumonia and other diseases, and are one of the main pathogens of nosocomial and community infections.
抗生素是治療腸桿菌感染的主要藥物,其中碳青黴烯(carbapenem)類抗生素之抗菌範圍廣泛,且抗藥菌種較少,是目前對抗多重抗藥性的腸桿菌之最後防線。然而,近年來,克雷伯氏肺炎菌( Klebsiella pneumoniae)等腸桿菌演化出降低對碳青黴烯類抗生素之感受性的方法,如:產碳青黴烯酶腸桿菌(carbapenemase-producing Enterobacteriaceae,CPE)表現碳青黴烯酶,能分解碳青黴烯類抗生素,從而增加受感染患者的致病率及死亡率,是目前全球公衛重大威脅之一。 Antibiotics are the main drugs for the treatment of Enterobacteriaceae infection, among which carbapenem antibiotics have a wide range of antibacterial activity, and there are few drug-resistant strains. They are currently the last line of defense against multidrug-resistant Enterobacteriaceae. However, in recent years, enterobacteria such as Klebsiella pneumoniae ( Klebsiella pneumoniae ) have evolved methods to reduce their susceptibility to carbapenem antibiotics, such as: carbapenemase-producing Enterobacteriaceae (CPE) show Carbapenemase, which can decompose carbapenem antibiotics, thereby increasing the morbidity and mortality of infected patients, is one of the major threats to global public health.
有鑑於抗生素等藥物對於細菌感染管制有其極限,亟需提供一種非藥物的組成物,以用於抑制抗藥性腸桿菌,並解決上述問題。In view of the limitations of drugs such as antibiotics for the control of bacterial infections, it is urgent to provide a non-drug composition for inhibiting drug-resistant Enterobacteriaceae and solve the above problems.
因此,本發明之一樣態樣是提供一種乳酸菌組成物。此乳酸菌組成物包含鼠李糖乳桿菌( Lacticaseibacillus rhamnosus) JJ101做為一有效成分,藉以抑制抗藥性腸桿菌之生長。 Therefore, one aspect of the present invention is to provide a lactic acid bacteria composition. The lactic acid bacteria composition contains Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) JJ101 as an active ingredient to inhibit the growth of drug-resistant Enterobacteriaceae.
本發明之另一態樣是提供一種鼠李糖乳桿菌用於製備抑制抗藥性腸桿菌之口服組成物的用途,其中口服組成物包含鼠李糖乳桿菌JJ101做為有效成分。Another aspect of the present invention is to provide a use of Lactobacillus rhamnosus for preparing an oral composition for inhibiting drug-resistant Enterobacteriaceae, wherein the oral composition contains Lactobacillus rhamnosus JJ101 as an active ingredient.
根據本發明之上述之態樣,提出一種乳酸菌組成物。此乳酸菌組成物包含鼠李糖乳桿菌( Lacticaseibacillus rhamnosus) JJ101做為有效成分,藉以抑制抗藥性腸桿菌之生長,其中鼠李糖乳桿菌JJ101係於2021年12月22日寄存在財團法人食品工業發展研究所生物資源中心(Bioresource Collection and Research Center,BCRC),寄存編號為BCRC 911088。 According to the above aspects of the present invention, a lactic acid bacteria composition is proposed. This lactic acid bacteria composition contains Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) JJ101 as an active ingredient to inhibit the growth of drug-resistant Enterobacteriaceae. The Lactobacillus rhamnosus JJ101 was deposited in the Food Industry Foundation on December 22, 2021 The Bioresource Collection and Research Center (BCRC) of the Development Institute, with the registration number BCRC 911088.
在本發明之一實施例中,乳酸菌組成物可選擇性包含1重量%至5重量%的益生元。在本發明的一實施例中,益生元包含乳果糖及/或異麥芽寡糖。在本發明的一實施例中,抗藥性腸桿菌具有克雷伯氏肺炎菌之碳青黴烯酶( Klebsiella pneumoniaecarbapenemase,KPC)-2。在本發明的一實施例中,其中受試對象係投予具有有效劑量之乳酸菌組成物至少14天。在本發明的一實施例中,該受試對象是小鼠時,有效劑量可例如為5.0×10 10CFU/kg體重/天至1.5×10 11CFU/kg體重/天。 In one embodiment of the present invention, the lactic acid bacteria composition may optionally contain 1% to 5% by weight of prebiotics. In one embodiment of the present invention, the prebiotic includes lactulose and/or isomaltooligosaccharide. In an embodiment of the present invention, the drug-resistant Enterobacteriaceae has carbapenemase ( Klebsiella pneumoniae carbapenemase, KPC)-2 of Klebsiella pneumoniae. In one embodiment of the present invention, the subject is administered with an effective dose of the lactic acid bacteria composition for at least 14 days. In an embodiment of the present invention, when the subject is a mouse, the effective dosage may be, for example, 5.0×10 10 CFU/kg body weight/day to 1.5×10 11 CFU/kg body weight/day.
根據本發明之另一態樣,提出一種鼠李糖乳桿菌用於製備抑制抗藥性腸桿菌之口服組成物的用途,其中口服組成物包含鼠李糖乳桿菌JJ101做為有效成分,其中鼠李糖乳桿菌JJ101之寄存編號為BCRC 911088,且乳酸菌組成物係投予受試對象至少14天。在本發明的一實施例中,口服組成物可選擇性包含1重量%至5重量%之益生元,且益生元可包含但不限於乳果糖及/或異麥芽寡糖。在本發明的一實施例中,抗藥性腸桿菌具有KPC-2。According to another aspect of the present invention, a use of Lactobacillus rhamnosus for preparing an oral composition for inhibiting drug-resistant Enterobacteriaceae is proposed, wherein the oral composition contains Lactobacillus rhamnosus JJ101 as an active ingredient, wherein rhamnosus The registration number of Lactobacillus sugarus JJ101 is BCRC 911088, and the lactic acid bacteria composition is administered to the subjects for at least 14 days. In an embodiment of the present invention, the oral composition may optionally include 1% to 5% by weight of prebiotics, and the prebiotics may include but not limited to lactulose and/or isomaltooligosaccharide. In one embodiment of the present invention, the drug-resistant Enterobacter has KPC-2.
應用本發明之乳酸菌組成物及其用於製備抑制抗藥性腸桿菌之口服組成物的用途,可於體外及/或於體內抑制具有KPC-2之抗藥性腸桿菌之生長,故本發明之乳酸菌組成物有潛力應用於預防、改善及/或治療抗藥性腸桿菌感染。The use of the lactic acid bacteria composition of the present invention and its use for preparing an oral composition for inhibiting drug-resistant Enterobacteriaceae can inhibit the growth of drug-resistant Enterobacteriaceae with KPC-2 in vitro and/or in vivo, so the lactic acid bacteria of the present invention The composition has the potential to be applied to prevent, improve and/or treat drug-resistant Enterobacteriaceae infection.
承上所述,本發明提供一種乳酸菌組成物及其用於製備抑制抗藥性腸桿菌之口服組成物的用途,其中乳酸菌組成物可包含但不限於鼠李糖乳桿菌( Lacticaseibacillus rhamnosus)做為有效成分,藉以抑制抗藥性腸桿菌之生長。 Based on the above, the present invention provides a lactic acid bacteria composition and its use for preparing an oral composition for inhibiting drug-resistant Enterobacteriaceae, wherein the lactic acid bacteria composition may include but not limited to Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) as an effective Components to inhibit the growth of drug-resistant Enterobacteriaceae.
本文所述之「乳酸菌(lactic acid bacteria)」是指可分解醣類(如:乳糖、葡萄糖、蔗糖、果糖等)後產生酸性物質(如:乳酸及/或醋酸等有機酸)的細菌。部分乳酸菌可做為益生菌(probiotics),其中益生菌是指以適當劑量之活體投予宿主時,可促進宿主健康之微生物,如:乳酸桿菌、片球菌、芽孢桿菌、雙歧桿菌及鼠李糖乳桿菌。The "lactic acid bacteria" mentioned herein refers to bacteria that can decompose sugars (such as lactose, glucose, sucrose, fructose, etc.) to produce acidic substances (such as organic acids such as lactic acid and/or acetic acid). Some lactic acid bacteria can be used as probiotics. Probiotics refer to microorganisms that can promote the health of the host when administered to the host in an appropriate dose, such as: Lactobacillus, Pediococcus, Bacillus, Bifidobacterium and Rhamna Lactobacillus sugar.
在一實施例中,上述鼠李糖乳桿菌可例如為於2021年12月22日寄存在財團法人食品工業發展研究所生物資源中心(Bioresource Collection and Research Center,BCRC,地址:30062台灣新竹市食品路331號),且寄存編號為BCRC 911088的鼠李糖乳桿菌JJ101。經動物實驗證實,相較於投予同屬其他菌株,動物經口投予鼠李糖乳桿菌JJ101達3天後,動物體內存留的活菌數較多,表示動物體內(即腸道)存留能力較佳。補充說明的是,本文所述之「益生菌在動物體內(即腸道)的存留能力佳」是指益生菌在動物體內(即腸道)存留的時間較久及/或存留的活菌數較多。益生菌在腸道存留的活菌數可例如藉由計算每單位重量之糞便的活菌數而評估。在一實施例中,益生菌具耐酸性及耐膽鹽性,因此其腸道存留能力較佳。In one embodiment, the above-mentioned Lactobacillus rhamnosus can be, for example, stored in the Bioresource Collection and Research Center (BCRC, BCRC, Address: 30062 Food Industry, Hsinchu City, Taiwan) on December 22, 2021. Road 331), and Lactobacillus rhamnosus JJ101 with deposit number BCRC 911088. Animal experiments have confirmed that compared with other strains of the same genus, after oral administration of Lactobacillus rhamnosus JJ101 for 3 days, the number of viable bacteria remaining in the animal's body is more, indicating that the animal's body (that is, the intestinal tract) persists better ability. It should be added that the term "probiotics have a good retention capacity in the animal body (i.e. the intestinal tract)" mentioned in this article refers to the probiotics remaining in the animal body (i.e. the intestinal tract) for a long time and/or the number of viable bacteria remaining more. The viable count of probiotics retained in the intestinal tract can be assessed, for example, by calculating the viable count per unit weight of feces. In one embodiment, the probiotics are resistant to acid and bile salts, so they have better intestinal persistence.
經動物實驗證實及體外共培養實驗證實,鼠李糖乳桿菌JJ101具抗藥性腸桿菌之功效。本文所述之「抗藥性腸桿菌」係指對抗生素具有抗藥性的腸桿菌科( Enterobacteriaceae)菌株。本文所述之「抑制抗藥性腸桿菌」係指鼠李糖乳桿菌JJ101與抗藥性腸桿菌於體外共培養後,可有效抑制抗藥性腸桿菌之生長(例如降低至少2個數量級,相當於抑制率為至少99%),或經口投予後,動物體內抗藥性腸桿菌含量降低(如:投予鼠李糖乳桿菌JJ101連續至少14天,受感染動物糞便中的抗藥性腸桿菌含量降低至少2個數量級,相當於抑制率為至少99%)。補充說明的是,抑制率是初始菌量與處理後菌量之差值對初始菌量的百分率,其中初始菌量是未與鼠李糖乳桿菌JJ101共培養的抗藥性腸桿菌的活菌數,且處理後菌量是與鼠李糖乳桿菌JJ101共培養後,抗藥性腸桿菌的活菌數,或者初始菌量是未經口投予鼠李糖乳桿菌JJ101之受感染動物糞便中的抗藥性腸桿菌含量,且處理後菌量是經口投予鼠李糖乳桿菌JJ101後,受感染動物糞便中的抗藥性腸桿菌含量。 Animal experiments and in vitro co-culture experiments have confirmed that Lactobacillus rhamnosus JJ101 has the effect of drug-resistant Enterobacteriaceae. The "drug-resistant Enterobacteriaceae" mentioned herein refers to strains of Enterobacteriaceae that are resistant to antibiotics. The "inhibition of drug-resistant Enterobacteriaceae" mentioned herein refers to the co-cultivation of Lactobacillus rhamnosus JJ101 and drug-resistant Enterobacteriaceae in vitro, which can effectively inhibit the growth of drug-resistant Enterobacteriaceae (for example, reduce by at least 2 orders of magnitude, which is equivalent to inhibiting the growth of drug-resistant Enterobacteriaceae). The rate is at least 99%), or after oral administration, the level of drug-resistant Enterobacteriaceae in animals is reduced (for example, after administration of Lactobacillus rhamnosus JJ101 for at least 14 consecutive days, the level of drug-resistant Enterobacteriaceae in the feces of infected animals is reduced by at least 2 orders of magnitude, corresponding to an inhibition rate of at least 99%). It is supplemented that the inhibition rate is the percentage of the difference between the initial bacterial count and the treated bacterial count to the initial bacterial count, where the initial bacterial count is the number of viable bacteria of drug-resistant Enterobacteriaceae that have not been co-cultured with Lactobacillus rhamnosus JJ101 , and the amount of bacteria after treatment is the number of live bacteria of drug-resistant Enterobacteriaceae after co-cultivation with Lactobacillus rhamnosus JJ101, or the initial amount of bacteria is the number of infected animals that have not been orally administered with Lactobacillus rhamnosus JJ101 The content of drug-resistant Enterobacteriaceae, and the amount of bacteria after treatment is the content of drug-resistant Enterobacteriaceae in the feces of infected animals after oral administration of Lactobacillus rhamnosus JJ101.
在一實施例中,上述抗生素可例如為β-內醯胺類(β-lactam)抗生素,其可透過干擾細胞壁的合成,以抑制細菌之生長。β-內醯胺類抗生素可包含但不限於青黴素、頭孢菌素及碳青黴烯(carbapenem)及單醯胺環。在一實施例中,抗藥性腸桿菌可例如為β-內醯胺類抗藥性腸桿菌。在一實施例中,抗藥性腸桿菌可例如為碳青黴烯抗藥性腸桿菌(carbapenem-resistant Enterobacteriaceae,CRE)。在一些具體例中,抗藥性腸桿菌可例如為產碳青黴烯酶腸桿菌(carbapenemase-producing Enterobacteriaceae,CPE)。 In one embodiment, the above-mentioned antibiotics may be, for example, β-lactam antibiotics, which can inhibit the growth of bacteria by interfering with the synthesis of cell walls. β-lactam antibiotics may include, but are not limited to, penicillins, cephalosporins, carbapenems, and monoamide rings. In one embodiment, the drug-resistant Enterobacteriaceae can be, for example, β-lactam-resistant Enterobacteriaceae. In one embodiment, the drug-resistant Enterobacteriaceae may be, for example, carbapenem-resistant Enterobacteriaceae (CRE). In some specific examples, the drug-resistant Enterobacteriaceae can be, for example, carbapenemase-producing Enterobacteriaceae (CPE).
補充說明的是,碳青黴烯酶為β-內醯胺酶(β-lactamases)的一種,可水解β-內醯胺類抗生素(如:碳青黴烯),從而降低CPE對β-內醯胺類抗生素的感受性。克雷伯氏肺炎菌碳青黴烯酶( Klebsiella pneumoniaecarbapenemase,KPC)為碳青黴烯酶的一種,首次於1996年發現於克雷伯氏肺炎菌上,因而得其名。KPC的基因是位於質體上,故可跨菌種傳播,目前其他腸桿菌(如:佛氏檸檬酸桿菌、大腸桿菌、日溝維腸桿菌、產氣腸桿菌、陰溝腸桿菌、產酸克雷伯氏菌、奇異變形桿菌、腸道沙門氏菌、黏質沙雷氏菌)及其他非腸桿菌之革蘭氏陰性菌(如:銅綠假單胞菌、戀臭假單孢菌、不動桿菌屬)皆發現過產生KPC之菌株。依據基因序列的不同,KPC可分類為KPC-1、KPC-2、KPC-3等。其中,具有KPC-2之抗藥性腸桿菌在臨床上較為常見,如:序列型(sequence type,ST)11的克雷伯氏肺炎菌。 It is supplemented that carbapenemase is a kind of β-lactamases (β-lactamases), which can hydrolyze β-lactam antibiotics (such as carbapenems), thereby reducing the effect of CPE on β-lactams. susceptibility to antibiotics. Klebsiella pneumoniae carbapenemase ( Klebsiella pneumoniae carbapenemase, KPC) is a kind of carbapenemase, which was first discovered in Klebsiella pneumoniae in 1996, hence its name. The KPC gene is located on the plastid, so it can be transmitted across strains. At present, other enterobacteriaceae (such as: Citrobacter freundii, E. Lebsiella, Proteus mirabilis, Salmonella enterica, Serratia marcescens) and other non-enterobacterial Gram-negative bacteria (such as: Pseudomonas aeruginosa, Pseudomonas putida, Acinetobacter ) have been found to produce KPC strains. According to the different gene sequences, KPC can be classified into KPC-1, KPC-2, KPC-3 and so on. Among them, drug-resistant Enterobacteriaceae with KPC-2 are relatively common in clinical practice, such as Klebsiella pneumoniae of sequence type (sequence type, ST) 11.
在一實施例中,乳酸菌組成物可選擇性包含益生元,從而形成「合益素」(synbiotics)。本文所述「合益素」是指益生菌及益生元(prebiotics)之混合物。本文所述「益生元」是指無法被宿主消化,但有益於宿主消化道中的特定菌株之生長及/或代謝活性,從而改善宿主健康之物質。常見的益生元包含雙醣、低聚醣碳水化合物(oligosaccharide carbohydrates,OSCs)、抗性澱粉及其他非醣類物質,具體可包含但不限於果寡糖(lactulose)、菊糖(inulin)、乳果糖(fructo-oligosaccharide)及/或異麥芽寡糖(isomalto-oligosaccharides)。經體外共培養實驗證實,果寡糖、菊糖、乳果糖及/或異麥芽寡糖可促進鼠李糖乳桿菌JJ101產生酸性物質(如:有機酸),使共培養液的pH值小於5,從而抑制抗藥性腸桿菌生長,其中乳果糖及/或異麥芽寡糖的效果較佳。In one embodiment, the lactic acid bacteria composition may optionally contain prebiotics to form "synbiotics". "Synbiotics" as used herein refers to a mixture of probiotics and prebiotics. The "prebiotics" mentioned herein refer to substances that cannot be digested by the host, but are beneficial to the growth and/or metabolic activity of specific strains in the digestive tract of the host, thereby improving the health of the host. Common prebiotics include disaccharides, oligosaccharide carbohydrates (OSCs), resistant starch and other non-sugar substances, including but not limited to fructooligosaccharides (lactulose), inulin, milk Fructo-oligosaccharides and/or isomalto-oligosaccharides. It has been confirmed by in vitro co-culture experiments that fructooligosaccharides, inulin, lactulose and/or isomaltooligosaccharides can promote the production of acidic substances (such as: organic acids) by Lactobacillus rhamnosus JJ101, making the pH of the co-culture solution less than 5. To inhibit the growth of drug-resistant enterobacteria, among which lactulose and/or isomaltooligosaccharides are better.
在一實施例中,益生元之含量不限,以不超過安全劑量即可,益生元對成人的每日安全劑量可例如為小於10 g,以免引起腹脹及腹瀉等不適症狀。在一實施例中,益生元之含量可例如為1重量%至5重量%,1.5重量%至2.5重量%,或者2重量%,以充分刺激上述鼠李糖乳桿菌JJ101的生長及/或代謝活性,但不超過上述每日安全劑量。In one embodiment, the content of prebiotics is not limited, as long as it does not exceed a safe dose. The daily safe dose of prebiotics for adults may be less than 10 g, so as not to cause discomfort symptoms such as abdominal distension and diarrhea. In one embodiment, the content of prebiotics can be, for example, 1% to 5% by weight, 1.5% to 2.5% by weight, or 2% by weight, so as to fully stimulate the growth and/or metabolism of Lactobacillus rhamnosus JJ101 active, but does not exceed the above safe daily dose.
在應用上述鼠李糖乳桿菌JJ101時,其投予途徑並無特別限制,可例如經口投予,端視實際需求調整。上述鼠李糖乳桿菌JJ101之投予量及投予回數,亦可視需求彈性調整。在一實施例中,鼠李糖乳桿菌JJ101於體外培養液的有效劑量為10 5CFU/mL至10 7CFU/mL。在一實施例中,當受試對象是小鼠時,鼠李糖乳桿菌JJ101的有效劑量可例如為5.0×10 10CFU/kg體重/天至1.5×10 11CFU/kg體重/天。舉例而言,在上述動物實驗中,鼠李糖乳桿菌JJ101對小鼠之有效劑量為1.0×10 11CFU/kg體重/天,即2.0×10 9CFU/小鼠(20 g體重)/天。 When using the above-mentioned Lactobacillus rhamnosus JJ101, the route of administration is not particularly limited, such as oral administration, which can be adjusted according to actual needs. The dosage and times of administration of Lactobacillus rhamnosus JJ101 above can also be flexibly adjusted according to demand. In one embodiment, the effective dose of Lactobacillus rhamnosus JJ101 in the in vitro culture medium is 10 5 CFU/mL to 10 7 CFU/mL. In one embodiment, when the subject is a mouse, the effective dose of Lactobacillus rhamnosus JJ101 can be, for example, 5.0×10 10 CFU/kg body weight/day to 1.5×10 11 CFU/kg body weight/day. For example, in the above animal experiments, the effective dose of Lactobacillus rhamnosus JJ101 to mice is 1.0×10 11 CFU/kg body weight/day, that is, 2.0×10 9 CFU/mouse (20 g body weight)/day .
補充說明的是,動物實驗中,小鼠係直接經口投予抗藥性腸桿菌,因此小鼠腸道中的抗藥性腸桿菌含量係遠高於臨床病人。其次,小鼠有食糞的習性,會反覆攝入糞便中的抗藥性腸桿菌。因此,動物實驗中的小鼠需經口投予較高劑量的鼠李糖乳桿菌JJ101,才能有效降低抗藥性腸桿菌。換言之,鼠李糖乳桿菌JJ101於臨床應用對成人的有效劑量可低於動物實驗中對小鼠的有效劑量,也能達到有效抑制抗藥性腸桿菌。在一具體例中,鼠李糖乳桿菌JJ101對成人之有效劑量可例如為1.0×10 8CFU/60 kg體重/天至1.0×10 10CFU/60 kg體重/天。在一實施例中,受試對象係投予上述有效劑量之鼠李糖乳桿菌JJ101連續數天。在一實施例中,受試對象係投予鼠李糖乳桿菌JJ101連續至少數天,如:7天至12個月或14天至6個月。 It should be added that in animal experiments, mice were directly orally administered drug-resistant Enterobacteriaceae, so the content of drug-resistant Enterobacteriaceae in the intestines of mice was much higher than that of clinical patients. Second, mice have the habit of eating feces and will repeatedly ingest drug-resistant Enterobacteriaceae in feces. Therefore, mice in animal experiments need to be orally administered a higher dose of Lactobacillus rhamnosus JJ101 in order to effectively reduce drug-resistant Enterobacteriaceae. In other words, the effective dose of Lactobacillus rhamnosus JJ101 in clinical application to adults can be lower than the effective dose to mice in animal experiments, and it can also effectively inhibit drug-resistant Enterobacteriaceae. In a specific example, the effective dose of Lactobacillus rhamnosus JJ101 for an adult can be, for example, 1.0×10 8 CFU/60 kg body weight/day to 1.0×10 10 CFU/60 kg body weight/day. In one embodiment, the subject is administered the effective dose of Lactobacillus rhamnosus JJ101 for several consecutive days. In one embodiment, the subject is administered Lactobacillus rhamnosus JJ101 for at least several consecutive days, such as 7 days to 12 months or 14 days to 6 months.
鼠李糖乳桿菌JJ101具有抑制抗藥性腸桿菌之生長的功效,因此可做為乳酸菌組成物的有效成分。在一實施例中,乳酸菌組成物可例如為口服組成物。在一實施例中,乳酸菌組成物可例如為食品組成物或醫藥組成物。在一實施例中,乳酸菌組成物可選擇性包含食品或醫藥上可接受的載體、賦形劑、稀釋劑、輔助劑及/或添加劑,可例如為溶劑、乳化劑、懸浮劑、崩解劑、黏合劑、安定劑、螫合劑、稀釋劑、膠凝劑、防腐劑、潤滑劑及/或吸收延緩劑等。Lactobacillus rhamnosus JJ101 has the effect of inhibiting the growth of drug-resistant enterobacteria, so it can be used as an active ingredient of lactic acid bacteria composition. In one embodiment, the lactic acid bacteria composition can be an oral composition, for example. In one embodiment, the lactic acid bacteria composition may be, for example, a food composition or a medical composition. In one embodiment, the lactic acid bacteria composition may optionally include food or pharmaceutically acceptable carriers, excipients, diluents, adjuvants and/or additives, such as solvents, emulsifiers, suspending agents, and disintegrating agents , adhesives, stabilizers, chelating agents, diluents, gelling agents, preservatives, lubricants and/or absorption delaying agents, etc.
乳酸菌組成物之劑型並無特別限制。在一實施例中,乳酸菌組成物之劑型可例如為水溶液、懸浮液、分散液、乳液(單相或多相分散體系、單室或多室脂質體)、水膠、凝膠、固體脂質奈米粒、錠劑、顆粒劑、粉劑或膠囊劑等。The dosage form of the lactic acid bacteria composition is not particularly limited. In one embodiment, the dosage form of the lactic acid bacteria composition can be, for example, an aqueous solution, a suspension, a dispersion, an emulsion (single-phase or multi-phase dispersion system, single-chamber or multi-chamber liposomes), hydrogel, gel, solid lipid gel, etc. Rice grains, lozenges, granules, powder or capsules, etc.
以下利用數個實施例以說明本發明之應用,然其並非用以限定本發明,本發明技術領域中具有通常知識者,在不脫離本發明之精神和範圍內,當可作各種之更動與潤飾。 實施例一、乳酸菌分離、培養及微生物學性質 Several examples are used below to illustrate the application of the present invention, but it is not intended to limit the present invention. Those with ordinary knowledge in the technical field of the present invention can make various modifications and changes without departing from the spirit and scope of the present invention. retouch. Embodiment one, lactic acid bacteria isolation, cultivation and microbiological properties
乳酸菌(lactic acid bacteria,LAB)菌株LYC1504及菌株JJ101是分離自水果醱酵液。將LAB以四區劃線法接種在de Man,Rogosa and Sharpe (MRS)瓊脂培養基上,並於37°C下培養16小時至18小時,以獲得單一菌落。接著,將單一菌落接種至MRS培養液,並於37°C下培養16小時至24小時,從而獲得LAB培養液。將LAB培養液離心,以獲得菌體沉澱物(pellet)。Lactic acid bacteria (LAB) strain LYC1504 and strain JJ101 were isolated from fruit fermentation liquid. LAB were inoculated on de Man, Rogosa and Sharpe (MRS) agar medium in a four-section method and incubated at 37°C for 16 to 18 hours to obtain single colonies. Next, a single colony was inoculated into the MRS culture solution, and cultured at 37° C. for 16 to 24 hours to obtain a LAB culture solution. The LAB culture solution was centrifuged to obtain a bacterial pellet.
對LAB的菌體沉澱物進行RNA純化及反轉錄,再利用核酸序列如序列識別號(SEQ ID NOs.):1及2所示之上游引子及下游引子進行聚合酶鏈反應(polymerase chain reaction,PCR),以獲得16S rDNA核酸片段,並進行核酸定序,從而獲得LAB的16S rDNA核酸序列。利用基本局部比對搜索工具(Basic Local Alignment Search Tool,BLAST)進行比對,鑑定上述LAB為鼠李糖乳桿菌(菌株LYC1504及菌株JJ101)。菌株JJ101係於2021年12月22日寄存於BCRC,且寄存編號為BCRC 911088。Perform RNA purification and reverse transcription on the bacterial precipitate of LAB, and then use the nucleic acid sequence such as sequence identification numbers (SEQ ID NOs.): 1 and 2 to perform polymerase chain reaction (polymerase chain reaction, PCR) to obtain 16S rDNA nucleic acid fragments, and perform nucleic acid sequencing to obtain the 16S rDNA nucleic acid sequence of LAB. The above LAB was identified as Lactobacillus rhamnosus (strain LYC1504 and strain JJ101) by using Basic Local Alignment Search Tool (BLAST) for comparison. The strain JJ101 was deposited in BCRC on December 22, 2021, and the deposit number is BCRC 911088.
補充說明的是,菌株JJ101的菌落乳白色、不透明,呈圓形,表面光滑突起,邊緣整齊,其菌體呈短桿狀,兩端鈍圓,以單個、成對、短鏈狀或鏈狀形式存在,無鞭毛、無運動性,無孢子形成,且革蘭氏染色為陽性。
實施例二、評估乳酸菌及抗藥性腸桿菌於動物體內的存留能力
1. 乳酸菌於動物體內的存留能力
It is supplemented that the colonies of the strain JJ101 are milky white, opaque, round, with smooth and protruding surfaces and neat edges. Present, no flagella, no motility, no sporulation, and Gram stain positive.
Example 2: Evaluating the Persistence Ability of Lactic Acid Bacteria and Drug-resistant Enterobacteriaceae in
利用BALB/c小鼠(以下簡稱為小鼠)做為實驗動物。將5週齡雌性小鼠飼養於動物房的獨立通氣飼養籠中,使小鼠適應環境。在適應環境的期間,小鼠可自由攝取標準粒狀飼料和滅菌蒸餾水。動物房的溫度是23±3°C,相對溼度是60±10%,且每日有12小時的光照期及12小時的黑暗期。待小鼠成長至達6週齡後,再進行後續評估。BALB/c mice (hereinafter referred to as mice) were used as experimental animals. The 5-week-old female mice were kept in independent ventilated cages in the animal room to allow the mice to adapt to the environment. During acclimatization, mice had free access to standard pelleted chow and sterile distilled water. The temperature of the animal room was 23±3°C, the relative humidity was 60±10%, and there were 12 hours of light and 12 hours of darkness per day. Follow-up evaluations were performed after the mice had grown to 6 weeks of age.
首先,每日投予小鼠抗生素,並檢測小鼠糞便的細菌含量,以確認糞便是否呈無菌。檢測方法說明如下:將小鼠的新鮮糞便秤重後,加入1 mL的生理實驗水(normal saline,NS)研磨成檢測液,再將檢測液分別塗佈於腸桿菌培養基、米勒亨頓(Mueller Hinton broth,MHB)瓊脂及LAB培養基上,並於37°C下培養24小時後計算菌落數。上述腸桿菌培養基係含有16 μg/mL的萬古黴素(vancomycin)、64 μg/mL的氨苄青黴素(ampicillin)及16 μg/mL的頭孢唑肟(cefotaxime)之伊紅甲基藍(eosin methylene blue,EMB)瓊脂,可用以檢測腸桿菌。LAB培養基係含有32 μg/mL的萬古黴素之MRS瓊脂,且pH值係5.0,可用以檢測LAB。First, antibiotics were given to mice daily, and the bacterial content of mouse feces was tested to confirm whether the feces were sterile. The detection method is described as follows: after weighing the fresh feces of the mice, add 1 mL of physiological experiment water (normal saline, NS) to grind into a test solution, and then spread the test solution on Enterobacteriaceae culture medium, Miller Hunton ( Mueller Hinton broth, MHB) agar and LAB medium, and count the number of colonies after culturing at 37°C for 24 hours. The above Enterobacteriaceae medium was eosin methylene blue containing 16 μg/mL of vancomycin, 64 μg/mL of ampicillin and 16 μg/mL of cefotaxime. , EMB) agar can be used to detect Enterobacteriaceae. LAB medium is MRS agar containing 32 μg/mL vancomycin, and the pH value is 5.0, which can be used to detect LAB.
在小鼠糞便經檢測確認呈無菌後,分別管餵小鼠不同LAB液,其中LAB液是將菌株LYC1504及菌株JJ101的沉澱物分別回溶於磷酸鹽緩衝生理鹽水(phosphate buffered saline,PBS)後獲得,並調整LAB含量,使小鼠經口投予2.0×10 9CFU/天的LAB連續3天。然後,停止管餵,並於停止管餵1天、3天及7天後,使用上述LAB培養基檢測小鼠糞便之LAB含量,其中LAB含量是LAB活菌數對小鼠糞便重量的比值(單位:CFU/g)。 After the feces of the mice were confirmed to be sterile, the mice were fed with different LAB solutions, in which the LAB solution was obtained by dissolving the precipitates of the strain LYC1504 and the strain JJ101 in phosphate buffered saline (PBS). The LAB content was obtained and adjusted, and mice were orally administered 2.0×10 9 CFU/day of LAB for 3 consecutive days. Then, stop the tube feeding, and after stopping the tube feeding for 1 day, 3 days and 7 days, use the above-mentioned LAB medium to detect the LAB content of the mouse feces, wherein the LAB content is the ratio of the number of viable LAB bacteria to the weight of the mouse feces (unit : CFU/g).
請參閱圖1,其中圖1係繪示根據本發明之一實施例的小鼠經口投予不同鼠李糖乳桿菌連續3天並停止管餵後,小鼠糞便的鼠李糖乳桿菌含量之折線圖,其中橫軸表示時間(單位:天),縱軸表示鼠李糖乳桿菌含量(單位:CFU/g),折線101及折線103分別為菌株LYC1504及菌株JJ101。如圖1所示,停止管餵1天及3天後,小鼠糞便的菌株JJ101(折線103)含量係高於菌株LYC1504(折線101),其中停止管餵3天後,菌株JJ101含量係高於10
7CFU/g,證實菌株JJ101的腸道存留能力較佳。
2. 抗藥性腸桿菌於動物體內的存留能力
Please refer to Fig. 1, wherein Fig. 1 shows the content of Lactobacillus rhamnosus in the feces of mice according to one embodiment of the present invention after oral administration of different Lactobacillus rhamnosus for 3 consecutive days and stop tube feeding In the line graph, the horizontal axis represents time (unit: day), and the vertical axis represents the content of Lactobacillus rhamnosus (unit: CFU/g).
菌株KPC001、菌株KPC011、菌株KPC021及菌株KPC035係自奇美醫院醫學研究中心臨床所分離之表現KPC-2的抗藥性腸桿菌(以下稱為CPE)。將CPE以四區劃線法接種在腸桿菌培養基上,並於37°C下培養16小時至18小時,以獲得單一菌落。接著,將單一菌落接種至MHB中,並於37°C下培養16小時至24小時,從而獲得CPE培養液。將CPE培養液離心,以獲得CPE的菌體沉澱物(pellet)。The strains KPC001, KPC011, KPC021 and KPC035 are drug-resistant Enterobacteriaceae expressing KPC-2 (hereinafter referred to as CPE) isolated from the clinical laboratory of the Medical Research Center of Chi Mei Hospital. CPE was inoculated on Enterobacteriaceae culture medium by four-section streak method, and cultured at 37°C for 16 hours to 18 hours to obtain a single colony. Next, a single colony was inoculated into MHB and cultured at 37° C. for 16 hours to 24 hours to obtain a CPE culture solution. The CPE culture solution was centrifuged to obtain a pellet of CPE cells.
每日投予小鼠抗生素,直到小鼠糞便呈無菌。然後,管餵小鼠CPE液,其中CPE液係將CPE的菌體沉澱物回溶於含有20重量%脫脂奶粉水溶液中,並調整CPE液的CPE含量,使小鼠經口投予3.0×10 8CFU/天的CPE連續3天,從而獲得感染小鼠。然後,停止管餵,並於停止管餵1天、2天、7天、10天、14天、17天、21天、24天、28天、31天及35天後,再次蒐集感染小鼠的糞便,並用MHB瓊脂檢測糞便的CPE含量,其中CPE含量是CPE活菌數對糞便重量的比值(單位:CFU/g)。 Antibiotics were administered daily to the mice until the feces were sterile. Then, the mice were fed with CPE solution, wherein the CPE solution was to redissolve the CPE bacterial sediment in an aqueous solution containing 20% by weight of skimmed milk powder, and adjust the CPE content of the CPE solution so that the mice were orally administered with 3.0×10 Infected mice were obtained by administering 8 CFU/day of CPE for 3 consecutive days. Then, tube feeding was stopped, and infected mice were collected again after tube feeding was stopped for 1 day, 2 days, 7 days, 10 days, 14 days, 17 days, 21 days, 24 days, 28 days, 31 days and 35 days Feces, and the CPE content of the feces was detected with MHB agar, where the CPE content was the ratio of the number of viable CPE bacteria to the weight of the feces (unit: CFU/g).
請參閱圖2,其中圖2係繪示根據本發明之一實施例的感染小鼠糞便的CPE含量之折線圖,其中橫軸表示時間(單位:天),縱軸表示CPE含量(單位:CFU/g),折線201、折線203、折線205及折線207分別表示菌株KPC001、菌株KPC011、菌株KPC021及菌株KPC035。如圖2所示,停止管餵1天後,小鼠糞便不同菌株之CPE含量皆為約10 10CFU/g,且在停止管餵4天至35天後,小鼠糞便不同菌株之CPE含量仍維持在10 4CFU/g至10 6CFU/g。上述結果顯示,不同菌株的CPE於小鼠之腸道存留能力沒有差異。後續評估以菌株KPC001進行。 實施例三、評估菌株JJ101抑制抗藥性腸桿菌的功效 Please refer to Fig. 2, wherein Fig. 2 is a line graph showing the CPE content of feces of infected mice according to one embodiment of the present invention, wherein the horizontal axis represents time (unit: day), and the vertical axis represents CPE content (unit: CFU /g), broken line 201, broken line 203, broken line 205 and broken line 207 represent bacterial strain KPC001, bacterial strain KPC011, bacterial strain KPC021 and bacterial strain KPC035 respectively. As shown in Figure 2, after stopping tube feeding for 1 day, the CPE content of different strains of mouse feces was about 10 10 CFU/g, and after stopping tube feeding for 4 days to 35 days, the CPE content of different strains of mouse feces Still maintained at 10 4 CFU/g to 10 6 CFU/g. The above results showed that there was no difference in the intestinal persistence ability of different strains of CPE in mice. Subsequent evaluations were performed with strain KPC001. Example 3. Evaluation of the efficacy of bacterial strain JJ101 in inhibiting drug-resistant Enterobacteriaceae
每日投予小鼠抗生素,直到小鼠糞便呈無菌。然後,使小鼠經口投予3.0×10 8CFU/天的CPE連續3天,以獲得感染小鼠。接者,檢測感染小鼠糞便的CPE含量,做為感染小鼠未經口投予LAB的CPE含量。然後,將感染小鼠分為空白組及實驗組。空白組的感染小鼠係經口投予PBS連續21天,且實驗組的感染小鼠係經口投予2.0×10 9CFU/天的菌株JJ101連續21天。在小鼠經口投予PBS或菌株JJ101連續4天、7天、11天、14天、18天及21天後,檢測感染小鼠糞便的CPE含量。 Antibiotics were administered daily to the mice until the feces were sterile. Then, 3.0×10 8 CFU/day of CPE was orally administered to the mice for 3 consecutive days to obtain infected mice. Then, the CPE content in feces of infected mice was detected, and it was used as the CPE content of infected mice that were not orally administered with LAB. Then, the infected mice were divided into blank group and experimental group. The infected mice in the blank group were orally administered with PBS for 21 consecutive days, and the infected mice in the experimental group were orally administered with 2.0×10 9 CFU/day of strain JJ101 for 21 consecutive days. After the mice were orally administered with PBS or strain JJ101 for 4 days, 7 days, 11 days, 14 days, 18 days and 21 days, the CPE content in the feces of the infected mice was detected.
請參閱圖3,其中圖3係繪示根據本發明之一實施例之不同組別的感染小鼠糞便的CPE含量之折線圖,其中橫軸表示感染小鼠經口投予PBS或菌株JJ101的連續天數(單位:天),縱軸表示感染小鼠糞便的CPE含量(單位:CFU/g),折線301及折線303分別表示空白組及實驗組,且「*」及「**」表示具有統計上的顯著差異( p值範圍分別是 p<0.05及 p<0.01)。 Please refer to Fig. 3, wherein Fig. 3 is a line graph showing the CPE content of feces of different groups of infected mice according to an embodiment of the present invention, wherein the horizontal axis represents the concentration of infected mice orally administered with PBS or bacterial strain JJ101 The number of consecutive days (unit: day), the vertical axis represents the CPE content of infected mouse feces (unit: CFU/g), the broken line 301 and the broken line 303 represent the blank group and the experimental group respectively, and "*" and "**" represent the Statistically significant difference ( p value range is p <0.05 and p <0.01, respectively).
如圖3所示,感染小鼠經口投予菌株JJ101連續7天至21天後,實驗組的感染小鼠糞便的CPE含量(折線303)係顯著低於空白組(折線301),降低至少2個數量級,相當於抑制率為至少99%,證實菌株JJ101於體內具有抑制CPE生長之功效。
實施例四、評估合益素抑制抗藥性腸桿菌的功效
1. 不同益生元對菌株JJ101與CPE的共培養試驗
As shown in Figure 3, after the infected mice were orally administered bacterial strain JJ101 for 7 days to 21 consecutive days, the CPE content (broken line 303) of the feces of the infected mice in the experimental group was significantly lower than that of the blank group (broken line 301), reducing at least 2 orders of magnitude, equivalent to an inhibition rate of at least 99%, confirming that the strain JJ101 has the effect of inhibiting the growth of CPE in vivo.
Example 4. Evaluating the efficacy of synbiotics in inhibiting drug-
LAB可分解醣類而產生酸性物質(如:乳酸及/或醋酸),從而降低環境(如:腸道)pH值,進而抑制CPE。因此,如果LAB可越有效地利用益生元,則此益生元及LAB組成的合益素抑制CPE生長之功效越佳。LAB can decompose sugars to produce acidic substances (such as lactic acid and/or acetic acid), thereby reducing the pH value of the environment (such as: intestinal tract), thereby inhibiting CPE. Therefore, if the LAB can utilize prebiotics more effectively, the effect of this prebiotic and the synbiotics composed of LAB on CPE growth inhibition will be better.
將菌株JJ101與CPE(菌株KPC001)加入pH 6.5的共培養液中,以進行共培養試驗,其中共培養液的初始LAB(菌株JJ101)含量為10 7CFU/mL,且初始CPE含量為10 6CFU/mL。然後,對共培養液進行LAB含量檢測、CPE含量檢測及pH值檢測,以獲得初始LAB含量、CPE含量及pH值(相當於陪養0小時)。LAB含量檢測係將共培養液塗佈於pH 5.5的MRS瓊脂培養基上,並在37°C下培養,以獲得LAB的單一菌落。藉由LAB的單一菌落數,可推算共培養液的LAB含量(單位:CFU/mL)。CPE菌數檢測係共培養液塗佈於含有16 μg/mL的氨苄青黴素的EMB瓊脂培養基上,並在37°C下培養,以獲得CPE的單一菌落。藉由CPE的單一菌落數,可推算共培養液的CPE含量(單位:CFU/mL)。 The strain JJ101 and CPE (strain KPC001) were added to the co-culture solution at pH 6.5 to carry out the co-cultivation experiment, wherein the initial LAB (strain JJ101) content of the co-culture solution was 10 7 CFU/mL, and the initial CPE content was 10 6 CFU/mL. Then, LAB content detection, CPE content detection and pH value detection were performed on the co-culture solution to obtain the initial LAB content, CPE content and pH value (equivalent to 0 hours of companionship). The LAB content detection system spread the co-culture solution on the MRS agar medium with pH 5.5, and cultured at 37°C to obtain a single colony of LAB. The LAB content (unit: CFU/mL) of the co-culture solution can be estimated by the number of single LAB colonies. The co-culture solution for the detection of the number of CPE bacteria was spread on the EMB agar medium containing 16 μg/mL ampicillin, and cultured at 37°C to obtain a single colony of CPE. The CPE content (unit: CFU/mL) of the co-culture solution can be estimated by the single colony count of CPE.
共培養液是由不含葡萄糖的MRS培養液及MHB以1:1之體積比例配製而成,並依據組別添加或不添加醣類,其中NON組的共培養液不含醣類,SUC組的共培養液含有2重量%的蔗糖,FOS組的共培養液含有2重量%的果寡糖,IN組的共培養液含有2重量%的菊糖,XOS組的共培養液含有2重量%的木寡糖,LU組的共培養液含有2重量%的乳果醣,且IMO組的共培養液含有2重量%的異麥芽寡糖。The co-culture solution was prepared from glucose-free MRS culture solution and MHB at a volume ratio of 1:1, and sugar was added or not added according to the group. The co-culture solution of the NON group did not contain sugar, and the co-culture solution of the SUC group The co-culture solution of the group contains 2% by weight of sucrose, the co-culture solution of the FOS group contains 2% by weight of fructo-oligosaccharides, the co-culture solution of the IN group contains 2% by weight of inulin, and the co-culture solution of the XOS group contains 2% by weight The co-culture solution of the LU group contained 2% by weight of lactulose, and the co-culture solution of the IMO group contained 2% by weight of isomalto-oligosaccharide.
將共培養液於37°C下進行培養,培養3小時、6小時、24小時及48小時後,進行LAB含量檢測、CPE含量檢測及pH值檢測,其結果說明如下:培養48小時後,SUC組、FOS組、IN組、XOS組、LU組及IMO組的共培養液之菌株JJ101含量高於NON組,係大於1.0×10 8CFU/mL且小於1.0×10 9CFU/mL(未繪示於圖中),證實益生元於體外有利於菌株JJ101之生長。 The co-culture solution was cultured at 37°C. After culturing for 3 hours, 6 hours, 24 hours and 48 hours, LAB content detection, CPE content detection and pH value detection were carried out. The results are as follows: After culturing for 48 hours, SUC The content of strain JJ101 in the co-culture solution of group, FOS group, IN group, XOS group, LU group and IMO group was higher than that of NON group, which was greater than 1.0×10 8 CFU/mL and less than 1.0×10 9 CFU/mL (not shown shown in the figure), confirming that prebiotics are beneficial to the growth of strain JJ101 in vitro.
請參閱圖4A及圖4B,其分別繪示根據本發明之一實施例之菌株JJ101及CPE共培養於含有不同益生元之共培養液後,共培養液之CPE含量(圖4A)及pH值(圖4B)之折線圖。圖4A的橫軸表示時間(單位:小時),縱軸表示CPE含量(單位:CFU/mL)。圖4B的橫軸表示時間(單位:小時),縱軸表示pH值。圖4A及圖4B的折線401、折線403、折線405、折線407、折線409、折線411及折線413分別表示NON組、SUC組、FOS組、IN組、XOS組、LU組及IMO組。Please refer to Figure 4A and Figure 4B, which respectively depict the CPE content (Figure 4A) and pH value of the co-culture solution after the co-cultivation of the bacterial strain JJ101 and CPE according to an embodiment of the present invention in the co-culture solution containing different prebiotics (Figure 4B) the line chart. The horizontal axis of Fig. 4A represents time (unit: hour), and the vertical axis represents CPE content (unit: CFU/mL). The horizontal axis in FIG. 4B represents time (unit: hour), and the vertical axis represents pH. The folded
如圖4A所示,培養24小時後,SUC組(折線403)、FOS組(折線405)、IN組(折線407)及LU組(折線411)的共培養液之CPE含量係低於偵測極限。培養48小時後,IMO組(折線413)的共培養液之CPE含量較初始CPE含量(0小時)少2個數量級(即抑制率為99%)。然而,NON組(折線401)及XOS組(折線409)的共培養液之CPE含量在培養48小時後是高於初始CPE含量(0小時)。如圖6B所示,在培養24小時至48小時後,SUC組(折線403)、FOS組(折線405)、IN組(折線407)、LU組(折線411)及IMO組(折線413)的共培養液之pH值係小於5,但XOS組(折線409)及NON組(折線401)的共培養液之pH值係大於5。上述結果證實,蔗糖、果寡糖、菊糖、乳果糖及異麥芽寡糖可促進菌株JJ101產生酸性物質,使共培養液的pH值係小於5,從而抑制CPE生長。值得注意的是,蔗糖可被動物消化,不能做為益生元,因此菌株JJ101的益生元較佳可例如為果寡糖、菊糖、乳果糖及異麥芽寡糖。 2. 以不同益生元對菌株JJ101的培養試驗 As shown in Figure 4A, after culturing for 24 hours, the CPE content of the co-culture solution of the SUC group (broken line 403), FOS group (broken line 405), IN group (broken line 407) and LU group (broken line 411) was lower than detection limit. After 48 hours of culture, the CPE content of the co-culture solution of the IMO group (broken line 413) was 2 orders of magnitude less than the initial CPE content (0 hour) (ie, the inhibition rate was 99%). However, the CPE content of the co-culture solution of the NON group (line 401 ) and the XOS group (line 409 ) was higher than the initial CPE content (0 hour) after culturing for 48 hours. As shown in Figure 6B, after culturing for 24 hours to 48 hours, the SUC group (broken line 403), the FOS group (broken line 405), the IN group (broken line 407), the LU group (broken line 411) and the IMO group (broken line 413) The pH value of the co-culture solution is less than 5, but the pH value of the co-culture solution of the XOS group (line 409) and the NON group (line 401) is greater than 5. The above results confirmed that sucrose, fructooligosaccharides, inulin, lactulose and isomaltooligosaccharides can promote the production of acidic substances by the strain JJ101, making the pH value of the co-culture solution less than 5, thereby inhibiting the growth of CPE. It should be noted that sucrose can be digested by animals and cannot be used as a prebiotic. Therefore, the preferred prebiotics of the strain JJ101 can be, for example, fructooligosaccharides, inulin, lactulose and isomaltooligosaccharides. 2. Cultivation test of strain JJ101 with different prebiotics
如上所述,在菌株JJ101與CPE的共培養試驗中,如以果寡糖、菊糖、乳果糖及異麥芽寡糖為較佳的益生元,共培養液之pH係小於5。然而,臨床病人體內的CPE含量是遠低於共培養試驗的CPE含量。因此,益生元的選擇還需考量不同益生元對菌株JJ101進行培養試驗後的pH值。As mentioned above, in the co-cultivation test of strain JJ101 and CPE, if fructo-oligosaccharide, inulin, lactulose and isomalto-oligosaccharide are preferred prebiotics, the pH of the co-culture solution should be less than 5. However, the CPE content in clinical patients is much lower than that in co-culture experiments. Therefore, the choice of prebiotics also needs to consider the pH value after the culture test of strain JJ101 with different prebiotics.
將菌株JJ101分別接種不同配方之不含葡萄糖之MRS培養液中,並於37°C下培養24小時,以獲得培養物。然後,測量培養物的pH值,並將結果(3重複的平均±標準差)記錄於表1中,其中NON組表示MRS培養液不添加醣類、SUC組表示MRS培養液添加2重量%的蔗糖、FOS組表示MRS培養液添加2重量%的果寡糖,IN組表示MRS培養液添加2重量%的菊糖、IMO組表示MRS培養液添加2重量%的異麥芽寡糖、LU組表示MRS培養液添加2重量%的乳果糖,且XOS組表示MRS培養液添加2重量%的木寡糖。Strain JJ101 was inoculated into different formulations of glucose-free MRS culture solution, and cultured at 37°C for 24 hours to obtain cultures. Then, measure the pH value of culture, and the result (average ± standard deviation of 3 repetitions) is recorded in table 1, wherein NON group represents that MRS culture solution does not add carbohydrate, SUC group represents that MRS culture solution adds 2% by weight of Sucrose, FOS group means MRS culture medium with 2% by weight fructooligosaccharide added, IN group means MRS culture medium with 2% by weight inulin added, IMO group means MRS culture medium with 2% by weight isomaltooligosaccharide added, LU group Indicates that 2% by weight of lactulose was added to the MRS culture solution, and the XOS group indicated that 2% by weight of xylooligosaccharide was added to the MRS culture solution.
表1
如表1所示,菌株JJ101在LU組及IMO組的培養物之pH值係低於5.0,證實乳果糖及異麥芽寡糖對菌株JJ101具促進產生酸性物質之功效,因此菌株JJ101的益生元應選用乳果糖及異麥芽寡糖為宜。As shown in Table 1, the pH values of the cultures of the strain JJ101 in the LU group and the IMO group were lower than 5.0, confirming that lactulose and isomaltooligosaccharides have the effect of promoting the production of acidic substances on the strain JJ101, so the probiotics of the strain JJ101 Lactulose and isomaltooligosaccharide should be used as the element.
綜上所述,鼠李糖乳桿菌JJ101可抑制抗藥性腸桿菌生長之活性,意味著鼠李糖乳桿菌JJ101有潛力應用於預防、改善及/或治療抗藥性腸桿菌感染。其次,添加益生元適當可提升鼠李糖乳桿菌JJ101之產生酸性物質及抑制抗藥性腸桿菌生長之活性。In summary, Lactobacillus rhamnosus JJ101 can inhibit the growth of drug-resistant Enterobacteriaceae, which means that Lactobacillus rhamnosus JJ101 has the potential to be used in the prevention, improvement and/or treatment of drug-resistant Enterobacteriaceae infection. Secondly, the addition of prebiotics can enhance the activity of Lactobacillus rhamnosus JJ101 to produce acidic substances and inhibit the growth of drug-resistant Enterobacteriaceae.
綜言之,本發明雖以特定菌株的鼠李糖乳桿菌、特定的製程、特定的有效劑量、特定投予方式、特定的實驗模型及特定的評估方法做為例示,說明本發明之乳酸菌組成物及其用於製備抑制抗藥性腸桿菌之口服組成物的用途,惟本發明所屬技術領域中具有通常知識者應可理解,本發明不限於此,在不脫離本發明的精神及範圍內,本發明亦可使其他菌株的鼠李糖乳桿菌、其他的製程、其他的有效劑量、其他的投予方式、其他的實驗模型及其他的評估方法進行。In summary, although the present invention uses specific strains of Lactobacillus rhamnosus, specific manufacturing processes, specific effective doses, specific administration methods, specific experimental models, and specific evaluation methods as examples to illustrate the composition of lactic acid bacteria of the present invention substances and their use in the preparation of oral compositions for inhibiting drug-resistant Enterobacteriaceae, but those with ordinary knowledge in the technical field of the present invention should understand that the present invention is not limited thereto, and without departing from the spirit and scope of the present invention, The present invention can also be carried out with other strains of Lactobacillus rhamnosus, other production processes, other effective doses, other administration methods, other experimental models and other evaluation methods.
雖然本發明已以數個特定實施例揭露如上,但可對前述揭露內容進行各種潤飾、各種更動及替換,而且應可理解的是,在不脫離本發明之精神和範圍內,某些情況將採用本發明實施例之某些特徵但不對應使用其他特徵。因此,本發明的精神和權利要求範圍不應限於以上例示實施例所述。Although the present invention has been disclosed above with several specific embodiments, various modifications, changes and substitutions can be made to the foregoing disclosure, and it should be understood that certain situations will be changed without departing from the spirit and scope of the present invention. Some features of the embodiments of the present invention are used but not others. Therefore, the spirit of the present invention and the scope of claims should not be limited to those described in the above exemplary embodiments.
101,103,201,203,205,207,301,303,401,403,405,407,409,411,413:折線101,103,201,203,205,207,301,303,401,403,405,407,409,411,413: polyline
為讓本發明之上述和其他目的、特徵、優點與實施例能更明顯易懂,所附圖式之詳細說明如下: [圖1]是繪示根據本發明之一實施例的小鼠經口投予不同鼠李糖乳桿菌連續3天並停止管餵後,小鼠糞便的鼠李糖乳桿菌含量之折線圖。 [圖2]是繪示根據本發明之一實施例的感染小鼠糞便的CPE含量之折線圖。 [圖3]是繪示根據本發明之一實施例之不同組別的感染小鼠糞便的CPE含量之折線圖。 [圖4A]至[圖4B]分別是繪示根據本發明之一實施例之菌株JJ101及CPE共培養於含有不同益生元之共培養液後,共培養液之CPE含量及pH值之折線圖。 In order to make the above and other objects, features, advantages and embodiments of the present invention more comprehensible, the detailed description of the accompanying drawings is as follows: [ Fig. 1 ] is a line graph showing the content of Lactobacillus rhamnosus in feces of mice according to an embodiment of the present invention after oral administration of different Lactobacillus rhamnosus for 3 consecutive days and stop tube feeding. [ FIG. 2 ] is a line graph showing the CPE content in feces of infected mice according to an embodiment of the present invention. [ FIG. 3 ] is a line graph showing the CPE content in feces of different groups of infected mice according to an embodiment of the present invention. [FIG. 4A] to [FIG. 4B] are line graphs showing the CPE content and pH value of the co-culture solution after the co-cultivation of the strain JJ101 and CPE according to an embodiment of the present invention in the co-culture solution containing different prebiotics .
鼠李糖乳桿菌( Lacticaseibacillus rhamnosus) JJ101係於2021年12月22日寄存於財團法人食品工業發展研究所生物資源中心(Bioresource Collection and Research Center,BCRC,地址:30062台灣新竹市食品路331號),寄存編號為BCRC 911088。 Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) JJ101 was deposited on December 22, 2021 in the Bioresource Collection and Research Center (BCRC, Address: No. 331, Food Road, Hsinchu City, Taiwan, 30062) , the deposit number is BCRC 911088.
301,303:折線 301,303: polyline
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