TW202329999A - Synbiotic composition and its use in preparation of oral composition of inhibiting drug-resistant enterobacteriaceae - Google Patents

Abstract

The invention provides a synbiotic composition and its use in preparation of an oral composition of inhibiting drug-resistant Enterobacteriaceae. The aforementioned synbiotic composition is consisted of mixed lactic acid bacteria and prebiotic, in which the mixed lactic acid bacteria are consisted of Lacticaseibacillus rhamnosusJJ101, Lacticaseibacillus paracaseiJJ102 and Lactiplantibacillus plantarumJJ103, and the prebiotic includes lactulose and/or isomalto-oligosaccharides. The synbiotic composition can inhibit the growth of the drug-resistant Enterobacteriaceaeafter oral administration in a subject, and thus can potentially be used to prevent, improve and/or treat the infection of the drug-resistant Enterobacteriaceae.

Description

Synbiotic composition and its use for preparing oral composition for inhibiting drug-resistant Enterobacteriaceae

The present invention relates to a synbiotic composition, in particular to a synbiotic composition and its use for preparing an oral composition for inhibiting drug-resistant Enterobacteriaceae.

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.

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.

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.

Therefore, one aspect of the present invention is to provide a synbiotic composition, which is composed of complex lactic acid bacteria and prebiotics. Compound lactic acid bacteria are composed of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) JJ101, Lactobacillus paracasei ( Lacticaseibacillus paracasei ) JJ102 and germ lactic acid bacteria ( Lactiplantibacillus plantarum , also known as Lactobacillus plantarum) JJ103, and prebiotics may include but not limited to milk Fructose and/or isomaltooligosaccharides.

Another aspect of the present invention is to provide a synbiotic composition for the preparation of an oral composition for inhibiting drug-resistant Enterobacteriaceae, wherein the oral composition contains the synbiotic composition as an active ingredient, and the synbiotic is Composed of compound lactic acid bacteria and prebiotics to inhibit the growth of drug-resistant Enterobacteriaceae.

According to the above aspect of the present invention, a synbiotic composition is proposed, which is composed of compound lactic acid bacteria and prebiotics, and the compound lactic acid bacteria is composed of Lactobacillus rhamnosus JJ101, Lactobacillus paracasei JJ102 and Lactobacillus malus JJ103 , and prebiotics may include but not limited to lactulose and/or isomaltooligosaccharides. The above-mentioned Lactobacillus rhamnosus JJ101 was deposited in the Bioresource Collection and Research Center (BCRC) of the Food Industry Development Institute (BCRC) on December 22, 2021, with the deposit number BCRC 911088, and the Lactobacillus paracasei JJ102 was deposited in It was deposited in BCRC on December 22, 2021, with the deposit number BCRC 911089, and the Lactobacillus germinus JJ103 was deposited in BCRC on December 22, 2021, with the deposit number BCRC 911090. The synbiotic composition can inhibit the growth of drug-resistant enterobacteria.

In one embodiment of the present invention, the content of prebiotics may be, for example, 1% to 5% by weight. In an embodiment of the present invention, the ratio of the number of Lactobacillus rhamnosus JJ101, Lactobacillus paracasei JJ102 and Lactobacillus germinus JJ103 may be, for example, 1-5:1-5:1-10. In an embodiment of the present invention, the synbiotic composition may be, for example, an oral composition. 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 compound lactic acid bacteria for at least 7 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.

According to another aspect of the present invention, a use of a synbiotic composition for preparing an oral composition for inhibiting drug-resistant Enterobacteriaceae is proposed, wherein the oral composition includes the synbiotic composition as an active ingredient. This synbiotic composition is composed of compound lactic acid bacteria and prebiotics. This complex lactic acid bacteria is composed of Lactobacillus rhamnosus JJ101, Lactobacillus paracasei JJ102 and Lactobacillus plantarum JJ103. The registration number of Lactobacillus rhamnosus JJ101 is BCRC 911088, and the registration number of Lactobacillus paracasei JJ102 is BCRC 911089. And the deposit number of lactic acid bacteria JJ103 is BCRC 911090. Such prebiotics may include, but are not limited to, lactulose and/or isomaltooligosaccharides. The synbiotic composition is administered to the subject for at least 7 days. In one embodiment of the present invention, the drug-resistant Enterobacter has KPC-2. In an embodiment of the present invention, the ratio of the number of Lactobacillus rhamnosus JJ101, Lactobacillus paracasei JJ102 and Lactobacillus germinus JJ103 may be, for example, 1-5:1-5:1-10.

The application of the synbiotic 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 present invention The synbiotic composition has the potential to be used in the prevention, improvement and/or treatment of drug-resistant Enterobacteriaceae infection.

Based on the above, the present invention provides a synbiotic composition and its use for preparing an oral composition for inhibiting drug-resistant Enterobacteriaceae, wherein the synbiotic composition is composed of compound lactic acid bacteria and prebiotics. In vitro co-culture experiments and animal experiments have confirmed that prebiotics can promote the production of acidic substances by compound lactic acid bacteria, thereby improving the efficacy of inhibiting drug-resistant Enterobacteriaceae.

The term "synbiotics" mentioned herein refers to a mixture of probiotics and prebiotics, wherein prebiotics can promote the growth and metabolism of probiotics, and thus have beneficial effects on human health.

The "probiotics" mentioned herein refer to microorganisms that can promote the health of the host when administered to the host in an appropriate dose. Most probiotics belong to lactic acid bacteria. Lactic acid bacteria refer to bacteria that can break down sugars (such as lactose, glucose, sucrose, fructose, etc.) to produce lactic acid and/or acetic acid, such as: Lactobacillus, Pediococcus, Bacillus and Bifidobacterium bacilli. It is worth noting that different strains of lactic acid bacteria may interfere with each other to affect efficacy, but a combination of specific strains may also produce synergistic effects, thereby improving the persistence and/or efficacy of the strains in the animal body (ie, intestinal tract). Therefore, when using lactic acid bacteria, it is necessary to select a single strain of lactic acid bacteria or multiple strains of lactic acid bacteria (called compound lactic acid bacteria) according to the strain, test object and/or efficacy. It should be added that the good retention ability of probiotics in animals (i.e. intestines) means that probiotics stay in animals (i.e. intestines) for a longer period of time and/or retain a larger number of viable bacteria. The number of viable bacteria in an animal's body (ie, intestinal tract) can be assessed, for example, by calculating the number of viable bacteria per unit weight of feces. In one embodiment, the probiotics are resistant to acid and bile salts, so they have better intestinal persistence.

In one embodiment, complex lactic acid bacteria are selected as the probiotics of the synbiotics. The compound lactic acid bacteria can be composed of, for example, Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ), Lactobacillus paracasei ( Lacticaseibacillus paracasei ) and lactic acid bacteria ( Lactiplantibacillus plantarum, also known as Lactobacillus plantarum ). In one embodiment, Lactobacillus rhamnosus can be, for example, Lactobacillus rhamnosus JJ101 (also known as strain JJ101) whose deposit number is BCRC 911088, and Lactobacillus paracasei can be, for example, paracheese milk whose deposit number is BCRC 911089 Bacillus JJ102 (also known as strain JJ102), and the lactic acid bacteria plantarum can be, for example, Lactobacillus plantarum JJ103 (also known as strain JJ103) with the deposit number BCRC 911090. It should be added that Lactobacillus rhamnosus JJ101, Lactobacillus paracasei JJ102 and Lactobacillus plantarum JJ103 were all deposited in the Bioresource Collection and Research Center (BCRC) on December 22, 2021. ; Address: No. 331, Food Road, Hsinchu City, Taiwan, 30062), and the survival test was completed on January 7, 2022. In one embodiment, the bacterial count ratio of Lactobacillus rhamnosus JJ101, Lactobacillus paracasei JJ102 and Lactobacillus germinus JJ103 can be, for example, 1~5:1~5:1~10, so that Lactobacillus rhamnosus JJ101, paracasei JJ101, paracasei Lactobacillus casei JJ102 and Lactobacillus plantarum JJ103 can maintain a synergistic effect in animals, thereby more effectively inhibiting the growth of drug-resistant Enterobacteriaceae. In a specific example, the bacterial count ratio of Lactobacillus rhamnosus JJ101, Lactobacillus paracasei JJ102, and Lactobacillus malulosus JJ103 can be, for example, 1:1:1.

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, specifically fructo-oligosaccharides, galacto-oligosaccharides ), polydextrose, xylo-oligosaccharide, lactulose, isomalto-oligosaccharides and inulin etc. In one embodiment, the prebiotics may include but not limited to lactulose and/or isomaltooligosaccharide. In one embodiment, lactulose and/or isomaltooligosaccharide can promote lactic acid bacteria to produce acidic substances (such as: organic acids), thereby increasing the efficacy of lactic acid bacteria in inhibiting the growth of drug-resistant Enterobacteriaceae.

It has been confirmed by animal experiments that compared with other strains of the same genus, after oral administration of Lactobacillus rhamnosus JJ101, Lactobacillus paracasei JJ102, and Lactobacillus malus JJ103 for 3 consecutive days, the number of viable bacteria remaining in the intestinal tract is lower than that of other strains of the same genus. More, it means that Lactobacillus rhamnosus JJ101, Lactobacillus paracasei JJ102 and Lactobacillus germinus JJ103 have better intestinal retention ability. Secondly, compared with the infected animals administered with any one of Lactobacillus rhamnosus JJ101, Lactobacillus paracasei JJ102 or Lactobacillus plantarum JJ103, the simultaneous administration of Lactobacillus rhamnosus JJ101, Lactobacillus paracasei JJ102 and Lactobacillus plantarum JJ103 The amount of drug-resistant Enterobacteriaceae retained in the intestinal tract of infected animals was less, indicating that the effect of compound lactic acid bacteria on inhibiting the growth of drug-resistant Enterobacteriaceae was better than that of single strains of lactic acid bacteria.

The "drug-resistant Enterobacteriaceae" mentioned herein refers to strains of Enterobacteriaceae that are resistant to antibiotics. The "synbiotics inhibiting drug-resistant Enterobacteriaceae" mentioned in this article means that after co-cultivation of synbiotics and drug-resistant Enterobacteriaceae in vitro, it can effectively inhibit the growth of drug-resistant Enterobacteriaceae (for example: reduce by at least 2 orders of magnitude, equivalent to The inhibition rate is 99%), or after oral administration, the content of drug-resistant Enterobacteriaceae in animals is reduced (for example, the content of drug-resistant Enterobacteriaceae in the feces of infected animals is reduced by at least 5 orders of magnitude after administering synbiotics for at least 7 consecutive days , equivalent to an inhibition rate of at least 99.999%). 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 synbiotics, and the treatment After co-culture with synbiotics, the number of viable bacteria of drug-resistant Enterobacteriaceae, or the initial bacterial quantity is the content of drug-resistant Enterobacteriaceae in the feces of infected animals without oral administration of synbiotics, and after treatment Bacterial count is the amount of drug-resistant Enterobacteriaceae in feces of infected animals after oral administration of synbiotics.

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).

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.

It has been confirmed by in vitro co-culture experiments that lactulose and/or isomaltooligosaccharides can promote the production of acidic substances by any one of Lactobacillus rhamnosus JJ101, Lactobacillus paracasei JJ102 and Lactobacillus maltoglycerum JJ103, making the pH of the co-culture solution The line is less than 5, thereby inhibiting the growth of drug-resistant Enterobacteriaceae. Secondly, it has been confirmed by animal experiments that compared with the administration of compound lactic acid bacteria (without prebiotics), the content of drug-resistant Enterobacteriaceae in the intestines of infected animals was significantly reduced after administration of synbiotics containing compound lactic acid bacteria and prebiotics. Relatively fast (for example: after 7 days of administration of synbiotics, the content of drug-resistant Enterobacteriaceae in the intestinal tract of infected animals is reduced by at least 5 orders of magnitude, which is equivalent to an inhibition rate of at least 99.999%).

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 metabolic activity of the above-mentioned complex lactic acid bacteria, but not More than the above-mentioned daily safe dose.

When using the above-mentioned lactic acid bacteria, the route of administration is not particularly limited, such as oral administration, which can be adjusted according to actual needs. The dosage and frequency of the above-mentioned lactic acid bacteria can also be flexibly adjusted according to demand. In one embodiment, the effective doses of Lactobacillus rhamnosus JJ101, Lactobacillus paracasei JJ102 and Lactobacillus plantarum JJ103 in in vitro culture medium are 10 ⁵ CFU/mL to 10 ⁷ CFU/mL. In one embodiment, when the subject is a mouse, the effective dose of the compound lactic acid bacteria 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 compound lactic acid bacteria for mice is 1.0×10 ¹¹ CFU/kg body weight/day, that is, 2.0×10 ⁹ CFU/mouse (20 g body weight)/day of compound lactic acid bacteria.

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 need to be orally administered with a higher dose of synbiotics with complex lactic acid bacteria in order to effectively reduce drug-resistant Enterobacteriaceae. In other words, when the effective dose of compound lactic acid bacteria of synbiotics in clinical application to adults is lower than the effective dose to mice in animal experiments, drug-resistant enterobacteria can be effectively inhibited. In a specific example, the effective dose of compound lactic acid bacteria for adults 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 above-mentioned effective dose of synbiotics for several consecutive days. In one embodiment, the subject is administered the synbiotics for at least 7 consecutive days, such as 7 days to 1 year, or 14 days to 6 months.

Synbiotics have the effect of inhibiting the growth of drug-resistant Enterobacteriaceae, so they can be used as active ingredients of synbiotics compositions. In one embodiment, the synbiotic composition may be, for example, an oral composition. In one embodiment, the synbiotic composition may be, for example, a food composition or a medical composition. In one embodiment, the synbiotic composition may optionally include food or pharmaceutically acceptable carriers, excipients, diluents, adjuvants and/or additives, such as solvents, emulsifiers, suspending agents, disintegrating agents Debonding agent, binder, stabilizer, chelating agent, diluent, gelling agent, preservative, lubricant and/or absorption delaying agent, etc. The dosage form of the synbiotic composition is not particularly limited. In one embodiment, the dosage form of the synbiotic composition can be, for example, an aqueous solution, a suspension, a dispersion, an emulsion (single-phase or multi-phase dispersion, single- or multi-lamellar liposomes), hydrogel, gel, solid Lipid nanoparticles, lozenges, granules, powders or capsules, etc.

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

Eleven strains of lactic acid bacteria including strain LYC1504, strain JJ101, strain LYC1119, strain JJ102, strain LYC1129, strain LYC1031, strain LYC1112, strain LYC1117, strain LYC1146, strain LYC1159 and strain JJ103 , LAB) is isolated from fruit ferment. 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.

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. Using the basic local alignment search tool (Basic Local Alignment Search Tool, BLAST) to compare, identify 2 strains of Lactobacillus rhamnosus (bacterial strain LYC1504 and bacterial strain JJ101), 3 strains of Lactobacillus paracasei (bacterial strain LYC1119) among the 11 strains of LAB. , bacterial strain JJ102 and bacterial strain LYC1129) and 6 strains of lactic acid bacteria (strain LYC1031, bacterial strain LYC1112, bacterial strain LYC1117, bacterial strain LYC1146, bacterial strain LYC1159 and bacterial strain JJ103).

The 16S rDNA nucleic acid sequence of the above strain JJ101 is shown in SEQ ID NOs:3. The 16S rDNA nucleic acid sequence of strain JJ102 is shown in SEQ ID NOs:4. The 16S rDNA nucleic acid sequence of strain JJ103 is shown in SEQ ID NOs:5. The strains JJ101, JJ102 and JJ103 were deposited in BCRC on December 22, 2021, and the survival test was completed on January 7, 2022. The deposit number of strain JJ101 is BCRC 911088, and the deposit number of strain JJ102 is BCRC 911089, and the deposit number of strain JJ103 is BCRC 911090.

It is supplemented that the colony of the strain JJ101 (Lactobacillus rhamnosus) is milky white, opaque, round, smooth and protruding, with neat edges, and its cells are short rods with blunt round ends. Short chains or chain-like forms exist, with no flagella, no motility, no sporulation, and Gram-stain positive. The colony of strain JJ102 (Lactobacillus paracasei) is milky white, opaque, round or almost round, with smooth and protruding surfaces and neat edges. There is a flagellum-like form, no motility, no sporulation, and the Gram stain is positive. The colonies of strain JJ103 (lactic acid bacteria with germ) are milky white, opaque, round to slightly irregular in shape, with smooth and protruding surfaces and neat edges. Short chain forms exist, are motile without flagella, do not sporulate, and are Gram-positive. Example 2: Evaluating the Persistence Ability of Lactic Acid Bacteria and Drug-resistant Enterobacteriaceae in Animals 1. Persistence of lactic acid bacteria in animals

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.

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.

After the feces of the mice were confirmed to be sterile, the mice were fed with different LAB solutions respectively, wherein the LAB solution was obtained by dissolving the precipitates of the above 11 strains of LAB respectively in phosphate buffered saline (PBS) , and adjust the LAB content, so that the 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).

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). Line 101 and line 103 are bacterial strain LYC1504 and bacterial strain JJ101, respectively. As shown in Figure 1, after stopping tube feeding for 1 day and 3 days, the content of bacterial strain JJ101 (broken line 103) in mouse feces was higher than that of strain LYC1504 (broken line 101), and after stopping tube feeding for 3 days, the content of bacterial strain JJ101 was higher At 10 ⁷ CFU/g, it was confirmed that the intestinal persistence of the strain JJ101 was better.

Please refer to Figure 2, wherein Figure 2 is a broken line showing the content of Lactobacillus paracasei in the feces of mice according to an embodiment of the present invention after oral administration of different Lactobacillus paracasei for 3 consecutive days and stopping tube feeding Figure, wherein horizontal axis represents time (unit: day), and vertical axis represents Lactobacillus paracasei content (unit: CFU/g), broken line 201, broken line 203 and broken line 205 are bacterial strain LYC1119, bacterial strain JJ102 and bacterial strain LYC1229 respectively. As shown in Figure 2, after stopping tube feeding for 1 day, the content of strain JJ102 in mouse feces (line 203) was higher than that of strain LYC1119 and strain LYC1229 (line 201 and line 205), and the content of strain JJ102 was higher than 10 ⁷ CFU /g, confirming that the intestinal persistence ability of strain JJ102 is better.

Please refer to Fig. 3, wherein Fig. 3 is a line graph showing the content of lactic acid bacteria in the feces of mice according to an embodiment of the present invention after oral administration of different lactic acid bacteria to mice for 3 consecutive days and stopping tube feeding. The axis represents time (unit: day), and the vertical axis represents the content of germ lactic acid bacteria (unit: CFU/g). The broken line 301, broken line 303, broken line 305, broken line 307, broken line 309 and broken line 311 are strain LYC1031, strain LYC1112 and strain LYC1117 respectively , strain LYC1146, strain LYC1159 and strain JJ103.

As shown in Figure 3, after stopping tube feeding for 1 day, 3 days and 7 days, the content of bacterial strain JJ103 in mouse feces (broken line 311) was higher than that of other bacterial strains (broken line 301 to broken line 309 and broken line 313). After 3 days, the content of strain JJ103 was 10 ⁶ CFU/g to 10 ⁷ CFU/g, and 7 days after stop tube feeding, the content of strain JJ103 was still more than 10 ⁵ CFU/g, confirming that compared with other lactic acid bacteria, strain JJ103 better intestinal retention. 2. Persistence of drug-resistant Enterobacteriaceae in animals

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 bacteria.

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).

Please refer to Fig. 4, wherein Fig. 4 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 401, broken line 403, broken line 405 and broken line 407 represent bacterial strain KPC001, bacterial strain KPC011, bacterial strain KPC021 and bacterial strain KPC035, respectively. As shown in Figure 4, 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 of CPE among different strains. Subsequent evaluations were performed with strain KPC001. Example 3. Evaluation of the efficacy of compound lactic acid bacteria in inhibiting drug-resistant Enterobacteriaceae

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 4 groups (blank group, control group 1, control group 2, control group 3 and experimental group 1). The infected mice in the blank group were orally administered with PBS for 21 consecutive days, the infected mice in the control group 1 were orally administered with 2.0×10 ⁹ CFU/day of strain JJ101 for 21 consecutive days, and the infected mice in the control group 2 were Orally administered 2.0×10 ⁹ CFU/day of strain JJ102 for 21 consecutive days, the infected mice in control group 3 were orally administered 2.0×10 ⁹ CFU/day of strain JJ103 for 21 consecutive days, and the infected mice in experimental group 1 Mice were orally administered 2.0×10 ⁹ CFU/day of compound LAB for 21 consecutive days, wherein the compound LAB was composed of strain JJ101, strain JJ102 and strain JJ103 at a ratio of 1:1:1. After mice were orally administered LAB for 4 days, 7 days, 11 days, 14 days, 18 days and 21 days, the CPE content in feces of infected mice was detected.

Please refer to FIG. 5, wherein FIG. 5 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 consecutive days of oral administration of LAB to infected mice ( Unit: day), the vertical axis represents the CPE content (unit: CFU/g) of infected mouse feces, broken line 501, broken line 503, broken line 505, broken line 507 and broken line 509 represent blank group, control group 1, control group 2, Control group 3 and experimental group 1, and different letters A, B, C and D indicate statistically significant differences (p<0.05).

As shown in Figure 5, the CPE content of the infected mouse feces of experimental group 1, matched group 1, matched group 2 and matched group 3 (broken line 509, broken line 503 to broken line 507) is lower than that of blank group (broken line 501), wherein The CPE content in feces of infected mice in experimental group 1 was significantly lower than that in control group 1, control group 2, and control group 3, confirming that the effect of compound LAB on inhibiting the growth of CPE was better than that of single LAB. In detail, compared with the CPE content of infected mice without oral administration of LAB, the CPE content of feces of infected mice in experimental group 1 was reduced by at least 5 orders of magnitude after oral administration of compound LAB to infected mice for 21 consecutive days , which is equivalent to an inhibition rate of at least 99.999%, but the CPE content of the feces of infected mice in the control group 1, control group 2, and control group 3 (line 503 to line 507) was significantly higher than that of the infected mice orally administered with different LAB strains for 21 consecutive days. Tianhou is only reduced by 2 to 3 orders of magnitude, which is equivalent to an inhibition rate of 99% to 99.9%. Example 4. Evaluating the efficacy of synbiotics in inhibiting drug-resistant Enterobacteriaceae 1. The effect of different prebiotics on promoting the production of acidic substances by lactic acid bacteria

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.

The above 11 strains of LAB were inoculated into different formulations of MRS culture medium without glucose, 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.

Table 1 group pH (Lactobacillus rhamnosus) pH value (Lactobacillus paracasei) JJ101 LYC1504 JJ102 LYC1119 LYC1229 NON 6.08±0.02 6.13±0.02 6.12±0.01 6.20±0.00 6.14±0.00 SUC 5.31±0.01 5.29±0.01 3.82±0.03 5.47±0.02 4.34±0.02 FOS 5.89±0.07 5.85±0.03 3.68±0.04 4.10±0.03 4.07±0.02 IN 5.40±0.01 5.41±0.03 3.70±0.05 4.09±0.02 4.04±0.04 XOS 5.21±0.01 5.18±0.02 5.02±0.03 5.56±0.02 5.34±0.00 LU 3.78±0.02 3.83±0.01 3.94±0.04 4.36±0.05 4.37±0.03 IMO 4.29±0.01 4.28±0.01 4.60±0.02 5.10±0.02 4.61±0.02 group pH value (Lactobacillus germ) JJ103 LYC1031 LYC1112 LYC1117 LYC1146 LYC1159 NON 6.21±0.00 6.19±0.00 6.06±0.01 6.15±0.00 6.13±0.01 6.19±0.01 SUC 3.82±0.05 3.83±0.05 3.95±0.04 4.01±0.04 3.99±0.04 3.98±0.05 FOS 5.94±0.01 5.94±0.01 5.81±0.01 3.78±0.06 3.69±0.06 3.70±0.06 IN 5.03±0.01 4.97±0.01 4.88±0.03 3.77±0.04 3.80±0.05 3.72±0.04 XOS 5.17±0.01 5.15±0.01 5.09±0.02 5.09±0.02 5.19±0.02 5.18±0.02 LU 3.78±0.04 3.74±0.04 3.79±0.06 3.82±0.03 3.72±0.04 3.76±0.04 IMO 4.57±0.03 4.48±0.02 4.37±0.03 3.91±0.05 4.58±0.03 4.45±0.03

As shown in Table 1, the pH values of the cultures in the SUC group, FOS group, IN group, XOS group, IMO group, and LU group were lower than those in the NON group, indicating that carbohydrates can promote the production of acidic substances in LAB. Secondly, the pH values of the cultures of strain JJ101 in the LU group and the IMO group were lower than 5.0, indicating that lactulose and isomaltooligosaccharides have the effect of promoting the production of acidic substances by the strain JJ101. The pH value of strain JJ102 was higher than 5.0 only in the culture of XOS group, indicating that among the above sugars, only xylooligosaccharides could not promote the production of acidic substances by strain JJ102. The pH values of strain JJ103 in the SUC group, LU group and IMO group were lower than 5.0, indicating that sucrose, lactulose and maltooligosaccharide can promote the production of acidic substances by strain JJ102. It should be added that sucrose is digestible by animals and cannot be used as a prebiotic. Therefore, when the composite LAB system is composed of strain JJ101, strain JJ102 and strain JJ103, lactulose and/or isomaltooligosaccharide should be selected as prebiotics. 2. The pH value of strain JJ101 and the synbiotics formed by different prebiotics and the effect of inhibiting the growth of CPE

Among the above-mentioned LAB strains (Lactobacillus rhamnosus, Lactobacillus paracasei and Lactobacillus germinus), single LAB strains with better intestinal retention ability (i.e. strain JJ101, strain JJ102 and strain JJ103) were compared with CPE (strain KPC001) respectively. Added to the co-culture solution at pH 6.5 to carry out the co-culture experiment, wherein the initial LAB 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.

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.

The co-culture solution was cultured at 37°C, and after 3 hours, 6 hours, 24 hours and 48 hours of culture, LAB content detection, CPE content detection and pH value detection were carried out.

In the co-culture experiment of the above-mentioned strain JJ101 and CPE in different media, the results of LAB content detection are explained as follows: After 48 hours of culture, the strains in the co-culture solution of SUC group, FOS group, IN group, XOS group, LU group and IMO group The content of JJ101 was higher than that of the NON group, which was greater than 1.0×10 ⁸ CFU/mL and less than 1.0×10 ⁹ CFU/mL (not shown in the figure), confirming that prebiotics are beneficial to the growth of strain JJ101 in vitro.

Please refer to FIG. 6A and FIG. 6B, which respectively depict the CPE content (FIG. 6A) and pH value of the co-culture solution after the co-culture of strain JJ101 and CPE according to an embodiment of the present invention in a co-culture solution containing different prebiotics (Figure 6B) the line chart. The horizontal axis in Fig. 6A represents time (unit: hour), and the vertical axis represents CPE content (unit: CFU/mL). The horizontal axis of FIG. 6B represents time (unit: hour), and the vertical axis represents pH value. The folded lines 601, 603, 605, 607, 609, 611, and 613 in FIGS. 6A and 6B represent the NON group, the SUC group, the FOS group, the IN group, the XOS group, the LU group, and the IMO group, respectively.

As shown in Figure 6A, after culturing for 24 hours, the CPE content of the co-culture solution of the SUC group (broken line 603), FOS group (broken line 605), IN group (broken line 607) and LU group (broken line 611) was lower than that of Measure the limit. After 48 hours of culture, the CPE content of the co-culture solution of the IMO group (broken line 613) 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 601 ) and the XOS group (line 609 ) was higher than the initial CPE content (0 hour) after 48 cultures. As shown in Figure 6B, after culturing for 24 hours to 48 hours, the total amount of SUC group (broken line 603), FOS group (broken line 605), IN group (broken line 607), LU group (broken line 611) and IMO group (broken line 613) The pH value of the culture solution is less than 5, but the pH value of the co-culture solution of the XOS group (line 609) and the NON group (line 601) 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. 3. The pH value of the strain JJ102 and the synbiotics formed by different prebiotics and the effect of inhibiting the growth of CPE

In the co-cultivation experiment of the above-mentioned strain JJ102 and CPE in different media, from the results of LAB content detection, it can be seen that after 48 hours of culture, the content of the strain JJ102 in the co-culture solution of the NON group, the SUC group to the XOS group, and the LU group to the IMO group was in the range of It was higher than 1.0×10 ⁸ CFU/mL but less than 1.0×10 ⁹ CFU/mL (figure not shown), and higher than the NON group, confirming that prebiotics are beneficial to the growth of strain JJ102 in vitro.

Please refer to Figure 7A and Figure 7B, which respectively depict the CPE content (Figure 7A) and pH value of the co-culture solution after the co-culture of the bacterial strain JJ102 and CPE according to an embodiment of the present invention in the co-culture solution containing different prebiotics (Figure 7B) the line graph. The horizontal axis in FIG. 7A represents time (unit: hour), and the vertical axis represents CPE content (unit: CFU/mL). The horizontal axis in FIG. 7B represents time (unit: hour), and the vertical axis represents pH. The broken lines 701, 703, 705, 707, 709, 711 and 713 in FIGS. 7A and 7B represent the NON group, the SUC group, the FOS group, the IN group, the XOS group, the LU group, and the IMO group, respectively.

As shown in FIG. 7A , after 24 hours of culture, the CPE content of the co-culture solution of the FOS group (line 705 ) was below the detection limit. After culturing for 48 hours, the CPE content of the co-culture solution of the SUC group (line 703 ), the IN group (line 707 ) and the LU group (line 711 ) was lower than the detection limit. The CPE content of the co-culture solution of the IMO group (broken line 713) was 3 orders of magnitude less than the initial CPE content (0 hour) (ie, the inhibition rate was 99.9%). The CPE content of the co-culture solution of the NON group (broken line 701) and the XOS group (broken line 709) after 48 hours of culture is higher than the initial CPE content. As shown in Figure 7B, after culturing for 48 hours, the pH value of the co-culture solution of XOS group (broken line 709) and NON group (broken line 701) was greater than 5, but SUC group, FOS group, IN group, LU group and IMO The pH values of the groups were all less than 5. The above results confirmed that sucrose, fructooligosaccharides, inulin, lactulose and isomaltooligosaccharides can promote the production of acidic substances by the strain JJ102, making the pH of the co-culture solution less than 5, thereby inhibiting the growth of CPE. 4. The pH value of the strain JJ103 and the synbiotics formed by different prebiotics and the effect of inhibiting the growth of CPE

From the results of LAB content detection, it can be seen that after 48 hours of culture, the content of strain JJ103 in the co-culture solution of the NON group was the least (9.0×10 ⁸ CFU/mL), and the content of the co-culture solution from the SUC group to the XOS group and the LU group to the IMO group was the least. The content of the strain JJ103 was higher than 9.0×10 ⁸ CFU/mL (figure not shown), confirming that prebiotics are beneficial to the growth of the strain JJ103 in vitro.

Please refer to Figure 8A and Figure 8B, which respectively depict the CPE content (Figure 8A) and pH value of the co-culture solution after the co-culture of the bacterial strain JJ103 and CPE according to an embodiment of the present invention in the co-culture solution containing different prebiotics (Figure 8B) the line chart. The horizontal axis of Fig. 8A represents time (unit: hour), and the vertical axis represents CPE content (unit: CFU/mL). The horizontal axis in FIG. 8B represents time (unit: hour), and the vertical axis represents pH. Lines 801, 803, 805, 807, 809, 811, and 813 in FIGS. 8A and 8B represent the NON group, the SUC group, the FOS group, the IN group, the XOS group, the LU group, and the IMO group, respectively.

As shown in Figure 8A, after 24 hours of culture, the CPE content of the co-culture solution of the SUC group (broken line 803), the LU group (broken line 811), and the IMO group (broken line 813) was lower than the detection limit, but the NON group and the FOS group The CPE content of , IN group and XOS group was higher than the initial CPE content. As shown in Figure 8B, the pH value of the co-culture solution of the SUC group (broken line 803), LU group (broken line 811) and IMO group (broken line 813) is less than 5, but the pH values of the NON group, FOS group, IN group and XOS group The pH value of the co-culture solution is greater than 5. The above results confirmed that the pH value of the co-culture solution less than 5 can inhibit the growth of CPE. Secondly, sucrose, lactulose and isomaltooligosaccharides can promote the production of acidic substances by the strain JJ103, making the pH of the co-culture solution less than 5, thereby inhibiting the growth of CPE.

From the results in Figure 6A, Figure 6B, Figure 7A, Figure 7B, Figure 8A and Figure 8B, it can be seen that sucrose, fructooligosaccharides, inulin, lactulose and isomaltooligosaccharides can effectively promote the production of acidic substances by strain JJ101 and strain JJ102 , but only sucrose, lactulose and isomaltooligosaccharides can effectively promote the production of acidic substances by strain JJ103. Among them, sucrose can be digested by animals and cannot be used as prebiotics. Therefore, lactulose and isomaltooligosaccharides were used as prebiotics in subsequent experiments Yuan. 5. The effect of compound lactic acid bacteria and synbiotics formed by different prebiotics on inhibiting the growth of CPE

The bacterial precipitates of strain JJ101, strain JJ102 and strain JJ103 were back-dissolved in PBS to obtain a composite LAB solution, in which the bacterial count ratio of strain JJ101, strain JJ102 and strain JJ103 was 1:1:1. Next, prepare 2% by weight of lactulose in the compound LAB liquid to obtain the lactulose succinate, and prepare 2% by weight of isomalto-oligosaccharide in the compound LAB liquid to obtain the isomalto-oligosaccharide succinate .

The mice were divided into 4 groups (blank group, control group, experimental group 1 and experimental group 2), and the mice were given antibiotics every day until the feces of the mice became 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 in the blank group were orally administered with PBS for 21 consecutive days, the infected mice in the control group were orally administered with the compound LAB solution for 21 consecutive days, and the infected mice in the experimental group 1 were orally administered with lactulose Synbiotics continued for 21 days, and the infected mice in experimental group 2 were orally administered isomaltooligosaccharide synbiotics for 21 consecutive days. After the mice were orally administered LAB for 4 days, 7 days, 11 days, 14 days, 18 days and 21 days, the CPE content in the mouse feces was detected. It should be noted that the bacterial count of the composite LAB orally administered to the infected mice of the control group, experimental group 1, and experimental group 2 was 2.0×10 ⁹ CFU/day.

Please refer to Fig. 9, wherein Fig. 9 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 consecutive days of oral administration of LAB to the mice (unit : day), the vertical axis represents the CPE content (unit: CFU/g) of infected mouse feces, and broken line 901, broken line 903, broken line 905 and broken line 907 represent blank group, control group, experimental group 1 and experimental group 2 respectively, and Different letters a and b indicate statistically significant differences between groups (p<0.05).

As shown in Figure 9, after oral administration of PBS, compound lactic acid bacteria or synbiotics for 21 consecutive days, the CPE content of feces of infected mice in the control group, experimental group 1 and experimental group 2 was significantly lower than that of the blank group, confirming that Compound lactic acid bacteria, lactulose synthogen, and isomaltooligosaccharide synthogen can inhibit the growth activity of CPE. However, after oral administration of PBS, compound lactic acid bacteria or synbiotics for 7 consecutive days, the CPE content of feces of infected mice in experimental group 1 and experimental group 2 was significantly lower than that of the control group, confirming that compared with compound lactic acid bacteria (without Prebiotics), lactulose synthogens and/or isomaltooligosaccharide synthogens can reduce the CPE content more quickly.

In summary, the synbiotics composed of specific compound lactic acid bacteria and specific prebiotics can inhibit the growth of drug-resistant Enterobacteriaceae, indicating that synbiotics have the potential to be used in the prevention, improvement and/or treatment of drug-resistant Enterobacteriaceae infection.

To sum up, although the present invention uses a specific compound lactic acid bacteria strain, a specific manufacturing process, a specific effective dosage, a specific administration method, a specific experimental model, and a specific evaluation method as an example, the synbiotic composition of the present invention is illustrated. , oral compositions and synbiotics are used to prepare 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, without departing from the scope of the present invention Within the spirit and scope of the present invention, other combinations of lactic acid bacteria strains, other manufacturing processes, other effective doses, other administration methods, other experimental models and other evaluation methods can also be used.

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.

101,103,201,203,205,301,303,305,307,309,311,401,403,405,407,501,503,505,507,509,601,603,605,607,609,611,613,701,703,705,7 07,709,711,713,801,803,805,807,809,811,813,901,903,905,907: polyline

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 content of Lactobacillus paracasei in the feces of mice according to an embodiment of the present invention after oral administration of different Lactobacillus paracasei for 3 consecutive days and stop tube feeding. [ Fig. 3 ] is a line graph showing the content of lactic acid bacteria in the feces of mice according to an embodiment of the present invention after oral administration of different lactic acid bacteria for 3 consecutive days and stop tube feeding. [ FIG. 4 ] is a line graph showing the CPE content in feces of infected mice according to an embodiment of the present invention. [ FIG. 5 ] 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. 6A] and [FIG. 6B] respectively show the line graphs of the CPE content and pH value of the co-culture solution after co-cultivation of the strain JJ101 and CPE according to an embodiment of the present invention in the co-culture solution containing different prebiotics. [ FIG. 7A ] and [ FIG. 7B ] respectively show the line graphs of the CPE content and pH value of the co-culture solution after the co-culture of the strain JJ102 and CPE according to an embodiment of the present invention in the co-culture solution containing different prebiotics. [ FIG. 8A ] and [ FIG. 8B ] respectively show the line graphs of the CPE content and pH value of the co-culture solution after the co-cultivation of the strain JJ103 and CPE according to an embodiment of the present invention in the co-culture solution containing different prebiotics. [ FIG. 9 ] is a line graph showing the CPE content of feces of different groups of infected mice according to an embodiment of the present invention.

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. The Lactobacillus paracasei ( Lacticaseibacillus paracasei ) JJ102 line was deposited in BCRC on December 22, 2021, and the deposit number is BCRC 911089. Lactiplantibacillus plantarum JJ103 was deposited in BCRC on December 22, 2021, with the deposit number BCRC 911090.

901,903,905,907: polyline

Claims (10)

A synbiotic composition is composed of a compound lactic acid bacterium and a prebiotic. The compound lactic acid bacterium is composed of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) JJ101, Lactobacillus paracasei ( Lacticaseibacillus paracasei ) JJ102 and lactic acid bacteria ( Lactiplantibacillus plantarum) ) JJ103, the Lactobacillus rhamnosus JJ101 was deposited in the Bioresource Collection and Research Center (BCRC) on December 22, 2021, and the deposit number is BCRC 911088. The Lactobacillus casei JJ102 line was deposited in BCRC on December 22, 2021, with the deposit number BCRC 911089, and the Lactobacillus plantarum JJ103 line was deposited in BCRC on December 22, 2021, with the deposit number BCRC 911090. The prebiotic contains milk Fructose and/or isomaltooligosaccharide, and the synthetogen composition inhibits the growth of a drug-resistant enterobacteriaceae. The synbiotic composition according to claim 1, wherein the content of the prebiotic is 1% to 5% by weight. The synbiotic composition as described in Claim 1, wherein the ratio of the number of Lactobacillus rhamnosus JJ101, Lactobacillus paracasei JJ102 and Lactobacillus germinus JJ103 is 1~5:1~5:1~10 . The synbiotic composition as described in claim 1, wherein the synbiotic composition is an oral composition. The synbiotic composition according to claim 1, wherein the drug-resistant Enterobacteriaceae has Klebsiella pneumoniae carbapenemase ( Klebsiella pneumoniae carbapenemase, KPC)-2. The synbiotic composition as described in Claim 1, wherein a subject is administered with an effective dose of the compound lactic acid bacteria for at least 7 days. The synbiotic composition as described in Claim 6, wherein when the subject is a mouse, the effective dose is 5.0×10 ¹⁰ CFU/kg body weight/day to 1.5×10 ¹¹ CFU/kg body weight/day . The use of a synbiotic composition for preparing an oral composition for inhibiting drug-resistant Enterobacteriaceae, wherein the oral composition uses a synbiotic composition as an active ingredient, and the synbiotic composition is composed of a compound It is composed of lactic acid bacteria and a prebiotic. The compound lactic acid bacteria is composed of Lactobacillus rhamnosus JJ101, Lactobacillus paracasei JJ102 and Lactobacillus germinus JJ103. The registration number of Lactobacillus rhamnosus JJ101 is BCRC 911088. The paracheese milk The registration number of Bacillus JJ102 is BCRC 911089, the registration number of the lactic acid bacteria JJ103 is BCRC 911090, the prebiotics include lactulose and/or isomaltooligosaccharides, and the synthetogen composition is administered to a subject at least 7 days. Use of the synbiotic composition as described in Claim 8 for preparing an oral composition for inhibiting drug-resistant Enterobacteriaceae, wherein the drug-resistant Enterobacteriaceae has KPC-2. The use of the synbiotic composition as described in Claim 8 for the preparation of an oral composition for inhibiting drug-resistant Enterobacteriaceae, wherein the Lactobacillus rhamnosus JJ101, the Lactobacillus paracasei JJ102 and the Lactobacillus plantarum JJ103 are one of the bacteria The number ratio is 1~5:1~5:1~10.

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