CN1644681A - Plant lactobacillaceae and use thereof - Google Patents

Abstract

The present invention relates to a new strain of Lactobacillus and its application. It specifically involves the new Lactobacillus plantarum LP-Onlly, CGMCC NO.1258; it also relates to the application of Lactobacillus plantarum LP-Onlly in regulating intestinal flora and enhancing intestinal mucosal barrier function; especially, Lactobacillus plantarum LP-Onlly and The combination of conventional carriers can be used to prevent and/or treat intestinal flora imbalance, and prevent and/or treat intestinal bacterial translocation infection.

Description

A kind of lactobacillus plantarum and application thereof

field of invention

The present invention relates to a kind of new bacterial strain of Lactobacillus and application thereof, more precisely, the present invention has described plant lactobacillus (Lactobacillus plantarum) a new bacterial strain LP-Onlly, 16S rRNA gene GeneBank (NCBI) sequence accession number is AY590777 sequence, and its regulation of intestinal flora function, enhancement of intestinal mucosal barrier function and antibacterial effect on intestinal pathogenic bacteria and food spoilage bacteria. The invention also relates to the combination of Lactobacillus plantarum LP-Onlly and a conventional carrier, which can be used to prevent and/or treat intestinal flora imbalance and intestinal bacterial translocation infection.

Background technique

Various bacteria inhabit the human gastrointestinal tract, which can be divided into beneficial bacteria, harmful bacteria and neutral bacteria according to their impact on the human body. When the beneficial bacteria in the human body are dominant, it will show a healthy state, among which Bifidobacterium and Lactobacillus are the most representative beneficial bacteria.

Lactic acid bacteria were first discovered by the Frenchman Pasteur in 1857. Lactic acid bacteria are widely used in food and medicine. The effects of lactic acid bacteria and their products on the human body mainly include: (1) regulating the intestinal flora of the host, improving the body’s immunity, and enhancing disease resistance; (2) the lactic acid produced by metabolism, It can improve the digestion of food in the stomach, promote the discharge of gastric contents, and promote digestion. The enzymes produced can promote the decomposition of food nutrients and promote nutrient absorption; (3) Lactic acid bacteria decompose lactose and produce lactic acid, which can effectively inhibit the growth of intestinal spoilage bacteria Propagate and produce harmful substances, promote intestinal peristalsis, and prevent constipation; (4) Lactic acid bacteria decompose protein, making it into small molecule peptides and amino acids, etc., which are easy to digest and absorb.

Since Detich et al. (Ann Surg, 1987; 205(6): 681-692) proposed the concept of "bacterial translocation" in the mid-1980s, it has become a consensus that intestinal mucosal barrier damage causes bacterial or endotoxin translocation. Barrier disruption produces sepsis, shock, and multiple organ failure, which significantly increases the mortality of critically ill patients. Therefore, many scholars have proposed that the treatment of infection should be aimed at its "source" - the intestine, rather than the target organ. For this reason, the use of intestinal probiotics to improve intestinal microecology, so as to enhance the tight junction of intestinal epithelial cells and intestinal barrier function, has gradually attracted attention.

Lactobacillus plantarum (Lactobacillus plantarum) is the original bacterium of the human body and has biological activity in the body. For thousands of years, Lactobacillus plantarum has been used in human food. It is an important strain of traditional lactic acid fermentation and the basis for long-term storage of sauerkraut and other foods , while humans eat a large amount of Lactobacillus plantarum fermented food, the human intestinal tract has also evolved to adapt to the colonization of a large number of Lactobacillus plantarum. Phenotype analysis combined with genetic characteristics research shows that the dominant bacteria in healthy people's digestive tract are mainly Lactobacillus plantarum (L.plantarum), Lactobacillus rhamnosus (L.rhamnosus) and Lactobacillus casei (L.paracasei). The normal Lactobacillus flora of healthy human rectal and oralmucosa accounted for 52%, 26% and 17% of all isolated Lactobacillus, respectively (The normal Lactobacillus flora of healthy human rectal and oralmucosa, Journal of Applied Microbiology, 1998, 85: 88-94). In addition to plant lactic acid fermented foods such as sauerkraut, which contain a large amount of Lactobacillus plantarum, the traditional live Lactobacillus plantarum product is the Tanzanian beverage "togwa", which uses cassava or wheat as raw materials and Lactobacillus plantarum as a starter to make live Lactobacillus plantarum. Lactobacillus plantarum 299v, American Journal of Clinical Nutrition, 2001, 73(suppl): 380S～5S ).

Contents of the invention

The object of the present invention is to provide a kind of Lactobacillus plantarum LP-Onlly, which can regulate the function of intestinal flora, enhance the barrier function of intestinal mucosa, and have antibacterial effect on intestinal pathogenic bacteria and food spoilage bacteria. The present invention also relates to the combination of Lactobacillus plantarum LP-Onlly and a conventional carrier, which can be used to prevent and/or treat intestinal flora imbalance and prevent and/or treat intestinal bacterial translocation infection. It has the function of regulating intestinal flora and enhancing intestinal mucosal barrier.

The solution that realizes the above-mentioned purpose of the invention is:

A kind of Lactobacillus plantarum LP-Onlly, this Lactobacillus plantarum has been on December 6, 2004 in the General Microorganism Center of China Committee for Culture Collection of Microbial Cultures, referred to as: CGMCC (Address: No. 13, Zhongguancun, Haidian District, Beijing, China, China Institute of Microbiology, Chinese Academy of Sciences) and received deposit registration number CGMCCNO 1258. Its classification is named: Lactobacillus plantarum, Latin name: Lactobacillus plantarum.

Lactobacillus plantarum LP-Onlly, its 16S rRNA gene GeneBank (NCBI) sequence accession number is AY590777.

Lactobacillus plantarum LP-Onlly has the function of regulating intestinal flora.

Lactobacillus plantarum LP-Onlly has the function of enhancing intestinal mucosal barrier.

Lactobacillus plantarum LP-Onlly has the function of inhibiting intestinal pathogenic bacteria and food spoilage microorganisms.

The intestinal pathogens inhibited by Lactobacillus plantarum LP-Onlly mainly refer to Enterobacter, Listeria, Salmonella, Staphylococcus aureus, Enterococcus, Shigella, Clostridium perfringens.

The food spoilage microorganisms inhibited by Lactobacillus plantarum LP-Onlly mainly refer to Bacillus subtilis and Bacillus cereus.

The combination of Lactobacillus plantarum LP-Onlly and conventional carriers can be used to prevent and/or treat intestinal flora imbalance.

The combination of Lactobacillus plantarum LP-Onlly and conventional vectors can be used to prevent and/or treat intestinal bacterial translocation infection.

Description of drawings

Figure 1: DNA fingerprinting of intestinal flora in rats with intraperitoneal infection.

In the figure: M is Marker, Lane 1-8 is Lactobacillus plantarum group, Lane 9-10 is normal rat, Lane 11-17 is PN group.

Figure 2: Expression of occludin protein in small intestinal epithelium in PN group (×400).

Figure 3: Expression of occludin protein in small intestinal epithelium in the probiotic group (×400).

Figure 4: Expression of occludin protein in large intestine epithelium in PN group (×400).

Figure 5: Expression of occludin protein in the colon epithelium of the probiotic group (×400).

Figure 6: X-ray electron microscope of small intestinal epithelium in PN group (2×10 ⁴ ).

Fig. 7: Transmission electron microscope of the colon epithelium in the probiotic group (2×10 ⁴ ).

Figure 8: X-ray electron microscope of small intestinal epithelium in PN group (2×10 ⁴ ).

Figure 9: X-ray electron microscope of colon epithelium in the probiotic group (2×10 ⁴ ).

Detailed ways

The Lactobacillus plantarum LP-Onlly of the present invention is directly isolated from the feces of healthy infants, has a rod shape, is positive in Gram staining, does not form spores, produces lactic acid, and is facultatively anaerobic. It can utilize L-arabinose, sorbitol, melibiose, D-turanose, D-arabitol, sodium gluconate, α-methyl-D-mannoside, but cannot utilize sorbose and rhamnose , D-tagatose, α-methyl-D-glucoside.

The fermentation process: culture in MRS liquid medium at 37° C. for 72 hours.

The Lactobacillus plantarum LP-Onlly was preserved on December 6, 2004 at the General Microbiology Center of China Microbiological Culture Collection Management Committee (Address: No. 13, Zhongguancun Yiyi, Haidian District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences), and received Deposit registration number CGMCC NO 1258.

Plantarum Lactobacillus involved in the present invention, its 16S RNA gene sequence is put on record in GeneBank (NCBI), and its accession number is AY590777.

Below in conjunction with embodiment and figure further illustrate the present invention:

Embodiment 1: Microscopic examination of bacterial strains

Under sterile conditions, Lactobacillus plantarum LP-Onlly was picked with an inoculation ring, spread on a glass slide, prepared and stained with Gram, and observed the morphology of the bacteria under a microscope.

Embodiment 2: MRS plate purification separation

Under sterile conditions, pick Lactobacillus plantarum LP-Onlly with an inoculation loop, streak on the MRS plate, place the plate in an incubator at 37°C for 72 hours, and select a single colony for microscopic examination to achieve pure strain isolation.

Embodiment 3: strain fermentation

Pick two loops of Lactobacillus plantarum LP-Onlly from the slant with an inoculation loop into 50ml MRS liquid medium, culture at 37°C for 24 hours, then insert 5% inoculum into 50ml MRS liquid medium, and culture at 37°C After 72 hours, the fermentation broth of Lactobacillus plantarum LP-Onlly was obtained.

Embodiment 4: Lactobacillus plantarum LP-Onlly antibacterial performance

Lactobacillus plantarum LP-Onlly was aerobically cultured in MRS liquid medium at 37°C for 24h, and the bacterial liquid was spotted on MRS solid plate, with three parallel spots on each plate, and cultured aerobically at 37°C for 24h. 200 μl of various indicator bacteria (Escherichia coli, Staphylococcus aureus, Lactobacillus acidophilus, Bacillus subtilis, Lactobacillus plantarum, Enterococcus faecalis, Salmonella typhi, Shigella dysenteriae, Listeria monocytogenes) cultured overnight in 200 μl bacteria, Bacillus cereus, Clostridium perfringens and Bifidobacterium adolescent) were mixed with 7ml soft agar respectively, and poured on the MRS plate with Lactobacillus plantarum LP-Onlly (soft agar kept at 45-50 ℃), aerobic or anaerobic (according to the indicator bacteria) culture at 37 ℃ for 24h (aerobic) or 48h (anaerobic), take pictures and measure the size of the inhibition zone around the indicator bacteria spots (Table 1).

The results in Table 1 show that Lactobacillus plantarum LP-Onlly has inhibitory effects on most intestinal harmful bacteria, and the inhibitory effects on Bacillus subtilis, Bacillus cereus, Listeria monocytogenes and Salmonella typhi are the strongest, The inhibitory effect on Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Shigella dysenteriae, and Clostridium perfringens was slightly weaker, while it had no effect on probiotics such as Lactobacillus acidophilus, Lactobacillus plantarum and Bifidobacterium adolescentis. No inhibitory effect.

                Table 1 Antibacterial performance of Lactobacillus plantarum LP-Onlly

Diameter of inhibition zone (mm)

Indicator bacteria

Average

Escherichia coli 10.0 10.6 12.6 11.1

Staphylococcus aureus 12.0 12.2 10.3 11.5

Lactobacillus acidophilus - - - - -

Bacillus subtilis 20.6 30.4 29.4 26.8

Lactobacillus plantarum - - - - -

Enterococcus faecalis 12.9 13.7 10.2 12.3

Salmonella typhi 20.7 19.1 19.8 19.9

Shigella dysenteriae 11.4 11.5 13.9 12.3

Listeria monocytogenes 18.7 20.8 25.4 21.6

Bacillus cereus 26.3 21.5 19.9 22.6

Clostridium perfringens 10.7 6.7 9.6 9.0

Bifidobacterium adolescent - - - - -

Note: "-" indicates that Lactobacillus plantarum LP-Onlly has no inhibitory effect on the indicator bacteria.

Example 5: Lactobacillus plantarum LP-Onlly regulates intestinal flora function animal experiment

1. Lactobacillus plantarum LP-Onlly regulates the function of intestinal flora in mice Animal experiments

1.1 Lactobacillus plantarum LP-Onlly regulates the function of intestinal flora in normal mice Animal experiments

Forty normal mice were divided into two groups, one group was the Lactobacillus plantarum LP-Onlly group (15 mice), fed 1ml of Lactobacillus plantarum LP-Onlly fermentation broth (>1×10 ⁸ cfu/ml) every day for 6 consecutive days , and the other group was the control group (25 mice). The changes in the intestinal microbes of the mice were detected, and the data were represented by x±s. Statistical software SPSS 10.0 was used for t-test and related analysis. (Table 2)

The results in Table 2 show that, compared with the control group, feeding the Lactobacillus plantarum LP-Onlly fermentation broth can significantly increase the bifidobacteria in the intestinal tract of mice, and there is a significant difference (P<0.05). This indicates that Lactobacillus plantarum LP-Onlly has a role in regulating the function of intestinal flora.

Table 2 Regulatory effect of Lactobacillus plantarum on intestinal flora of normal mice (x±s, lg10 ⁿ /ml)

Intestinal microorganisms Control group Lactobacillus plantarum group

(n=25) (n=15)

Enterococcus 6.376±0.409 6.215±0.401

Escherichia coli 7.704±0.303 7.683±0.282

Lactobacillus 9.280±0.354 9.138±0.500

Bifidobacteria 8.698±0.234 8.900±0.228 ^*

^* Compared with the normal control group, the Lactobacillus plantarum group P<0.05.

1.2 Lactobacillus plantarum LP-Onlly regulates the intestinal flora function of mice with dysbiosis animal experiment

Fifty-nine mice were divided into the following three groups to study the regulation of intestinal flora function of Lactobacillus plantarum LP-Onlly on intestinal flora imbalance mice.

25 mice in the control group; 17 mice in the rhubarb group were given rhubarb liquid 1ml per day for 8 consecutive days, resulting in intestinal flora imbalance, and were killed on the 9th day; 17 mice in the Lactobacillus plantarum group were given rhubarb daily 1ml of rhubarb liquid for 8 consecutive days, but from the 3rd day of administration of rhubarb liquid, another 1ml of Lactobacillus plantarum LP-Onlly fermentation liquid (>1×10 ⁸ cfu/ml) was fed into the stomach for 6 consecutive days, and they were killed on the 9th day; The feces of the mice in each group were collected to detect the changes in the intestinal microorganisms of the mice, and the data were expressed as x±s, and the statistical software SPSS 10.0 was used for t-test and related analysis. (table 3)

Table 3 The effect of Lactobacillus plantarum on intestinal flora regulation of mouse intestinal flora imbalance (x±s, lg10 ⁿ /ml)

Intestinal microorganisms Control group Rhubarb group Lactobacillus plantarum group

(n=25) (n=17) (n=17)

Enterococcus 6.376±0.409 6.087±0.406 ^* 6.334±0.396 ^*

Escherichia coli 7.704±0.303 8.194±0.387 ^*** 7.888±0.328 ^*

Lactobacillus 9.280±0.354 9.010±0.346 ^** 9.208±0.302 ^*

Bifidobacterium 8.698±0.234 8.362±0.560 ^* 8.587±0.329 ^*

Compared with the control group, ^* is P<0.05, ^** is P<0.01, ^*** is P<0.001.

It can be seen from Table 3 that compared with the control group, the numbers of the four major intestinal flora in the rhubarb group had significant changes, and the numbers of enterococci and bifidobacteria decreased significantly (P<0.05), and the number of lactobacilli decreased significantly (P<0.05). 0.01), while the number of Escherichia coli was significantly increased (P<0.001), which indicated that the intestinal flora of the mice was out of balance after being fed with rhubarb. Compared with the control group, Lactobacillus plantarum group took Lactobacillus plantarum LP-Onlly after taking rhubarb liquid for 3 days. Although rhubarb was still taken for 8 days, the numbers of four kinds of bacteria in the intestinal tract could be maintained in the normal range, which was the same as that of the control group. Rhubarb group comparisons have significant differences. This indicates that Lactobacillus plantarum LP-Onlly can regulate the imbalance of intestinal flora and maintain the balance of normal intestinal flora after intestinal flora imbalance.

2. Regulatory effect of Lactobacillus plantarum LP-Onlly on the intestinal flora of rats with abdominal infection

After 15 rats were made into abdominal infection model by cecal ligation and puncture method and jugular vein and jejunum catheterization (Table 4), they were given parenteral nutrition (PN group) and PN+Lactobacillus plantarum LP-Onlly fermentation liquid (Lactobacillus plantarum group) respectively. ) lasted for 5 days, and were executed on the 6th day. Feces in the cecum were taken to count the intestinal flora (Table 5), and its DNA fingerprint was analyzed to explore the regulation of Lactobacillus plantarum LP-Onlly on the intestinal flora of rats with abdominal infection effect.

The results in Table 4 showed that the mortality rate of rats with intraperitoneal infection in the Lactobacillus plantarum LP-Onlly group was significantly lower than that in the PN group. This indicates that the fermentation broth of Lactobacillus plantarum LP-Onlly can reduce the mortality rate of rats with intraperitoneal infection by regulating the intestinal microecology.

      Table 4 Comparison of the mortality rate of rats with intraperitoneal infection in the PN group and the Lactobacillus plantarum group

Group Number of experiments (only) Death number (only) Mortality rate (%)

PN group 33 26 78.7

Lactobacillus plantarum group 16 8 50

Table 5 intestinal flora count analysis (x±s, lg10 ⁿ /ml)

Intestinal bacteria PN group PN group Lactobacillus plantarum group

(n=7) (n=8)

Enterobacter 7.061±0.191 7.231±0.171

Bifidobacterium 8.006±0.272 8.313±0.303 ^*

Lactobacillus 5.416±0.304 5.713±0.178 ^*

Clostridium perfringens 5.702±0.373 5.056±0.355 ^*

Note: ^* Compared with PN group, P＜0.05

It can be clearly found in Table 5 that the number of Lactobacillus and Bifidobacterium in the intestinal tract of the rats in the Lactobacillus plantarum group is more than that of the PN group, and the number of potential pathogenic bacteria Clostridium perfringens in the intestinal tract is higher than that of the rats in the PN group. few, and there are significant differences. This shows that Lactobacillus plantarum LP-Onlly in the present invention has the function of regulating intestinal flora.

With ITS universal primer pair PS2(5'-TG(C/T)ACA CACCGC CCG T-3'), PL2(5'-GGGT(G/C/T)CCC CAT TC(A/G)G-3' ) as primers, the above-mentioned rat feces DNA was amplified by PCR and identified by agarose gel electrophoresis (Fig. 1).

It can be clearly seen from Figure 1 that the spectrum bands in Lane 11-17 of the PN group were significantly less than those of Lane 9-10 in the normal group and Lane 1-8 in the Lactobacillus plantarum group, and Lane 11, 14, and 15 had a very bright band ( marked in the figure). This shows that the intestinal flora of the rats in the PN group is imbalanced, the types of normal flora in the intestines are reduced, and the non-intestinal flora is increased, while the bands of the Lactobacillus plantarum group are significantly more than the Lane of the PN group, and the bands of Lane 9 to 10 in the normal group are significantly higher than those of the PN group. belt is closer. The results in the figure also show that the flora bands of the rats in the Lactobacillus plantarum group have a high consistency with the normal rats, while there is a significant difference in the PN group. These results suggest that Lactobacillus plantarum LP-Onlly can correct the intestinal flora disorder in rats with abdominal infection of PN, and can regulate the function of intestinal flora.

3. Conclusion

The above experimental results show that the Lactobacillus plantarum LP-Onlly of the present invention can regulate the function of intestinal flora, and it can prevent and/or treat intestinal flora imbalance when combined with conventional carriers.

Example 6: Animal experiment of Lactobacillus plantarum LP-Onlly enhancing intestinal barrier function

Animal model experiments are more and more widely used in the functional evaluation experiments of probiotics.

The present invention adopts male SD rats, respectively give parenteral nutrition (PN group) and PN+Lactobacillus plantarum LP-Onlly fermented liquid (probiotics group) for 5 days, put to death on the 6th day, get vena cava blood, lung, liver and The homogenate of mesenteric lymphoid tissue was used for bacterial culture to measure the translocation rate of bacteria; the terminal ileum and colon were taken, and the ultrastructure of intestinal epithelial cell tight junction was observed by electron microscope, and the expression of transmembrane binding protein (Occludin) was determined by immunohistochemical method, so as to judge the translocation rate. Whether the strain has the function of enhancing the intestinal mucosal barrier.

1 Establishment of rat intraperitoneal infection model

Male SD rats, weighing 250-320 g (provided by the Animal Center of Fudan University School of Medicine). After intraperitoneal injection of 2% ketamine hydrochloride injection (30mg/g) for intraperitoneal anesthesia, the rats were fixed in the supine position, the neck and abdomen were depilated, disinfected with 0.1% bromogeramine tincture, and a 4cm incision was made along the middle of the abdomen to open the abdominal cavity , the model was made by ligation and perforation of the cecum, that is, the distal third of the cecum was ligated, and a 12-gauge needle was used to penetrate the top, and a 2mm wide skin piece was placed and fixed to prevent slippage. A jejunum 30-40cm away from the cecum was used as a purse, and a soft silicone tube with an outer diameter of 1.2mm was placed as a jejunostomy tube. The silicone tube was subcutaneously drawn out from the back of the neck, and the abdomen was closed in layers. According to the method of Steiger et al., a soft silicone tube with an outer diameter of 1.2 mm was inserted through the external jugular vein, and the silicone tube was led out from the subcutaneous area of the back of the neck through the abdominal subcutaneous tunnel, and connected to the rotating device through a helical protective spring.

2. Animal grouping and PN solution configuration

The 15 animals with successful modeling were divided into two groups: (1) PN group (n=7), PN support was given on the 1st to 5th day, and the PN solution was maintained for 24 hours with a micro-infusion pump, evenly infused, and the average infusion rate per rat was The quantity is about 80ml/d; (2) Probiotics group (n=8), PN solution is given on the 1st to 5th day to support the combination of Lactobacillus plantarum LP-Onlly fermentation broth (live bacteria content is 10 ⁸ cfu/ml), in which PN solution The amount of infusion was the same as that in the PN group, and the probiotic fermented liquid was slowly infused three times a day through the jejunostomy tube, with a total of about 10ml/d. The above-mentioned two groups of rats were sacrificed on the 6th day after successful modeling, and samples were taken for detection or observation.

PN solution formula (Table 6), the two groups were equal in nitrogen and heat, providing 348kJ of non-nitrogen heat (about 240kcal/kg) and 504mg of nitrogen every day.

           Table 6 PN solution formula (content per 100ml solution)

Recipe Dosage

50% Glucose(ml) 33

8.5% Novamin(ml) 45

20% Intralipid(ml) 17

Shui Le Vita (ml)                                    

Andami injection (ml) 1

10%kCl(ml) 1

10%NaCl(ml) 1

RI(u) 4

Heparin(u) 40

Non-protein calories (KJ) 435

Total Calorie (Kcal)                                            

Total Nitrogen (mg) 630

3. Observation indicators

(1) Immunohistochemical determination of mucosal occludin protein:

Frozen sections of the terminal ileum and colon tissues were taken, and the occludin protein content in the small intestine and colonic mucosa was detected by indirect immunofluorescence. The primary antibody was goat anti-rat occludin antibody (Santa Crus), diluted 1:300; the secondary antibody was anti-goat IgG (Santa Crus) from ABC kit. Using the HPIAS1000 high-definition color graphic pathology report analysis system, at the same magnification (×400), 5 fields of view were randomly selected in the slices of each time group, and the occludin density of the corresponding cells in each field of view was measured by optical density method, and the positive results were calculated. The percentage of the colored area within the measurement window.

(2) Electron microscope examination of intestinal mucosal morphology

Take about 0.5 cm from the ileum and colon at 5 cm from the ileocecal, cut them longitudinally, wash, cut into small pieces (1×1 mm ² ), fix with glutaraldehyde, make ultra-thin slices, and observe the tight junctions of epithelial cells with a transmission electron microscope .

(3) Determination of bacterial content in vena cava blood, mesenteric lymphoid tissue, liver and lung tissue

Take 1ml of vena cava blood, mesenteric lymphoid tissue (5g), middle lobe of liver (5g) and lower lobe of right lung tissue (5g), homogenize in a homogenizer, pour into heart and brain infusion tubes respectively, and incubate at 35°C for 18-24h , and then placed on an eosin methylene blue plate, incubated at 35°C for 18-24 hours and then counted.

(4) Statistical processing

The data are represented by x ± s, and the statistical software spss10.0 is used for x ² test, analysis of variance, t test and correlation analysis, and P < 0.05 is considered a significant difference.

4. Result Analysis

(1) Content of intestinal epithelial transmembrane binding protein (occludin)

The intestinal epithelial barrier function is closely related to the tight junction (Tight Junctions, TJ) complex. In vitro studies have shown that the opening of the TJ of intestinal epithelial cells increases the probability of bacterial translocation, and the substances that regulate TJ include immune regulators and TJ proteins. Two transmembrane binding proteins, occludin and claudin, are the most important TJ-specific integral membrane proteins. In the case of intestinal flora disturbance, intestinal pathogenic bacteria can adhere to the intestinal epithelium and affect transmembrane binding proteins by changing the membrane skeleton system of cells. The detection of the level of the transmembrane binding protein occludin in the intestinal epithelium can not only reflect the destruction of the intestinal barrier by pathogenic bacteria when the intestinal flora is disturbed, but also reflect the recovery of the intestinal flora.

From Figures 2 to 5, it can be clearly found that the surface, intercellular and cytoplasm of the epithelial cells of the small intestine and large intestine in the probiotic group had more occludin protein expression, while the occludin protein expression in the PN group was very low. Compared with the animals in the PN group, the positive area of occludin protein expression in the small intestine and large intestine of the probiotic group accounted for a significant difference in the percentage of the measurement window (Table 7). This indicated that, compared with the PN group, Lactobacillus plantarum LP-Onlly could significantly increase the content of intestinal epithelial transmembrane binding proteins, thereby enhancing the intestinal mucosal barrier function.

Table 7 Comparison of occludin protein content in the small intestine and large intestine epithelial cells of the PN group and the probiotic group (A, x ± s)

Group n Small intestinal epithelial cell occludin Large intestinal epithelial cell occludin

Protein Content (%) Protein Content (%)

PN group 7 1.207±0.587 1.254±0.203

Probiotic group 8 1.981±0.513 ^* 2.276±0.526 ^**

Compared with PN group, ^* means P<0.05, ^** means P<0.01.

(2) Electron microscope inspection

It can be seen from Figures 6 to 9 that the small intestinal epithelium in the probiotic group had slight loss of microvilli and denatured mitochondria and endoplasmic reticulum vacuoles, but the tight junctions were more obvious; the tight junctions between intestinal epithelial cells in the large intestine were normal. In the PN group, the small intestinal epithelial microvilli were lost, the mitochondrial endoplasmic reticulum vacuoles were degenerated, and the tight junctions were unclear; the tight junctions between the intestinal epithelial cells of the large intestine were wide and short. This indicates that Lactobacillus plantarum LP-Onlly can obviously promote intestinal epithelial tight junction, thereby enhancing the intestinal mucosal barrier function.

(3) Bacterial translocation in vena cava blood, mesenteric lymphoid tissue, liver and lung tissue

The bacterial translocation rate in the mesenteric vena cava blood, mesenteric lymphoid tissue, liver and lung tissue of rats in the PN group was 60.7%, while the bacterial translocation rate in the probiotic group was only 34.3%, which was significantly lower than that in the PN group (Table 8). This indicates that Lactobacillus plantarum LP-Onlly can significantly reduce the bacterial translocation rate, thereby enhancing the intestinal mucosal barrier function.

         Table 8 Bacterial translocation numbers in vena cava blood and abdominal viscera

Bacterial transnumber in vena cava blood and abdominal viscera

Group n vena cava blood mesenteric lymphoid tissue liver lung tissue translocation rate

(%)

PN Group 7 4 6 6 4 3 60.7

Probiotic group 8 2 2 4 4 3 2 34.3

5 Conclusion

The above results show that Lactobacillus plantarum LP-Onlly of the present invention can increase the content of transmembrane binding protein in the intestinal epithelium of rats with abdominal infection, maintain the tight junction of the intestinal epithelium and maintain normal intestinal mucosal morphology, reduce bacterial translocation, thereby enhancing the intestinal mucosal barrier function. The above-mentioned Lactobacillus plantarum LP-Onlly combined with a conventional carrier can prevent and/or treat intestinal bacterial translocation infection.

Claims (9)

1. A kind of Lactobacillus plantarum LP-Onlly, CGMCC NO.1258. 2. The Lactobacillus plantarum LP-Onlly according to claim 1, whose 16S rRNA gene sequence is AY590777. 3. The Lactobacillus plantarum LP-Onlly according to claims 1 and 2 has the function of regulating intestinal flora. 4. The Lactobacillus plantarum LP-Onlly according to claims 1 and 2 has the function of enhancing intestinal mucosal barrier. 5. The Lactobacillus plantarum LP-Onlly according to claims 1 and 2 has the function of inhibiting intestinal pathogenic bacteria and food spoilage microorganisms. 6. The Lactobacillus plantarum LP-Onlly according to claim 5 inhibits enterobacteria, Listeria, Salmonella, Staphylococcus aureus, Enterococcus, Shigella, and Clostridium perfringens mainly against intestinal pathogenic bacteria. 7. According to claim 5, Lactobacillus plantarum LP-Onlly inhibits food spoilage microorganisms mainly referring to Bacillus subtilis and Bacillus cereus. 8. The use according to any one of claims 3-5, wherein the combination of Lactobacillus plantarum LP-Onlly and conventional carriers can be used to prevent and/or treat intestinal flora imbalance. 9. The application according to any one of claims 3-5, wherein the combination of Lactobacillus plantarum LP-Onlly and a conventional carrier can be used to prevent and/or treat intestinal bacterial translocation infection.

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