Application of lactobacillus paracasei IOB413 metaplasm in gastroesophageal reflux

Abstract

The invention provides an application of a Lactobacillus paracasei IOB413 metagen in gastroesophageal reflux, and belongs to the technical field of microbial fermentation. The lactobacillus paracasei IOB413 metagen can relieve weight loss of gastroesophageal reflux rats, inhibit esophageal reflux, relieve esophageal tissue pathological injury, relieve inflammatory injury by reducing the levels of inflammatory factors IL-17 and IL-8, and enhance gastrointestinal motility and reduce the risk of reflux by improving the motilin level.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, and specifically relates to the application of postbiotics of Lactobacillus paracasei IOB413 in gastroesophageal reflux. Background Art

[0002] Gastroesophageal reflux disease (GERD) is a common digestive system disease, which is caused by the reflux of gastric acid and gastric contents into the esophagus, resulting in symptoms such as heartburn and acid reflux. In severe cases, it may even lead to esophagitis, esophageal erosion and even esophageal cancer. The pathogenesis of GERD is very complex and involves multiple factors, which have not been fully revealed yet. The general view is that the main pathophysiological mechanisms of GERD involve the dysfunction of the esophageal anti-reflux barrier and the weakening of the esophageal clearance function. The normal esophagus has a physiological barrier function to resist the erosion of reflux substances on the esophageal mucosa. However, when the esophageal anti-reflux defense ability is weakened under the influence of some factors, or the erosion of reflux substances on the esophageal mucosa is enhanced, GERD may occur. The lower esophageal sphincter (LES) is the most important structure of the anti-reflux barrier at the esophagogastric junction. The function of LES is to form a high-pressure area, and through the regulation of its contraction and relaxation, maintain the pressure balance between the stomach and the esophagus, and prevent the backflow of gastric acid and other gastric contents. With the in-depth study of the pathogenesis of GERD, more and more evidence shows that transient lower esophageal sphincter relaxation (TLESR) is the main cause of gastroesophageal reflux. In GERD patients, TLESR mostly presents as gastric acid reflux. This is also the reason why GERD patients often have typical reflux symptoms such as heartburn and chest pain.

[0003] At present, the main ideas for clinical treatment of gastroesophageal reflux disease are to reduce gastric acid secretion and promote the healing of esophageal mucosa. Commonly used drug types include PPIs, mucosal protectants, potassium ion competitive acid blockers, etc. Although these drugs have good therapeutic effects, long-term use may cause side effects such as intestinal infections. Therefore, finding natural drugs with good therapeutic effects and few side effects is an urgent problem to be solved at present. Summary of the Invention

[0004] In view of the technical problems existing in the prior art, the present invention aims to provide the application of postbiotics of Lactobacillus paracasei IOB413 in gastroesophageal reflux.

[0005] Lactobacillus paracasei IOB413 ( Lacticaseibacillus paracaseiThe classification and naming of IOB413 is Lactobacillus paracasei ( Lacticaseibacillus paracasei ), which was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on June 29, 2018. The abbreviation is CGMCC, the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 16022.

[0006] One of the objectives of the present invention is to provide an application of the postbiotic of Lactobacillus paracasei IOB413 in the preparation of a drug for treating gastroesophageal reflux disease, and the deposit number of the Lactobacillus paracasei IOB413 is CGMCC No. 16022.

[0007] Preferably, the preparation method of the postbiotic of Lactobacillus paracasei IOB413 includes: Inoculate the secondary seed liquid of Lactobacillus paracasei IOB413 into a solid fermentation medium for solid fermentation to obtain the fermented raw material; then dry and crush the fermented raw material to obtain the postbiotic of Lactobacillus paracasei IOB413.

[0008] Preferably, the ratio of material to water in the solid fermentation is 1:1.5 - 2, the time of the solid fermentation is 18 - 24 h, the inoculation amount of the inoculation is 1×10 6 CFU / mL - 5×10 7 CFU / mL, the temperature of the drying is 53 - 58 °C, and the moisture content of the drying is ≤10%.

[0009] Preferably, the preparation method of the secondary seed liquid includes: First, inoculate the strain of Lactobacillus paracasei IOB413 into a slant medium and place it in an incubator at 35 - 39 °C for 22 - 26 h to obtain an activated bacterial liquid; Inoculate the activated bacterial liquid into a seed medium at an inoculation amount of 3% - 5% and statically seal and culture it at 34 - 38 °C for 20 - 22 h to obtain a primary seed liquid; Inoculate the primary seed liquid into a seed medium at an inoculation amount of 1% - 3% and statically seal and culture it at 34 - 38 °C for 20 - 22 h to obtain a secondary seed liquid.

[0010] Preferably, the components of the slant medium include: Peptone 0.8 - 1.3%, beef extract 0.5 - 1%, yeast extract 0.4 - 0.8%, glucose 2 - 2.5%, dipotassium hydrogen phosphate 0.2 - 0.5%, sodium acetate 0.5 - 0.8%, manganese sulfate 0.005 - 0.008%, Tween 80 0.1 - 0.5%, ammonium citrate 0.2 - 0.5%, agar powder 1 - 2%, pH 6.4 - 6.5.

[0011] Preferably, the seed culture medium comprises: 2-2.5% peptone, 1-1.5% beef extract, 1-1.5% yeast extract, 2-3% glucose, 0.3-0.8% dipotassium hydrogen phosphate, 0.5-1% sodium acetate, 0.005-0.008% manganese sulfate, 0.1-0.2% Tween 80, 0.1-0.5% ammonium citrate, pH 6.4-6.5.

[0012] The second object of the present invention is to provide an application of Lactobacillus paracasei IOB413 postbiotics in the preparation of a drug for treating gastroesophageal reflux disease. The drug for treating gastroesophageal reflux disease comprises Lactobacillus paracasei IOB413 postbiotics and pharmaceutically or food acceptable excipients.

[0013] Preferably, the auxiliary materials include one or more of red date powder, isomaltulose or maltodextrin.

[0014] Beneficial effects of the present invention: The invention provides an application of a Lactobacillus paracasei IOB413 postbiotic in preparing a medicine for treating gastroesophageal reflux disease. The Lactobacillus paracasei IOB413 postbiotic can inhibit gastric acid reflux, relieve esophageal tissue pathological damage, increase the level of plasma motilin to promote gastrointestinal motility, reduce the levels of inflammatory factors IL-17 and IL-8 to alleviate inflammatory damage, and restore the body weight of rats with gastroesophageal reflux disease, thereby achieving the effect of treating gastroesophageal reflux disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a graph showing changes in rat body weight; Figure 2 is the pH value of rat esophageal tissue; Figure 3 To score the esophageal mucosal pathology of rats; Figure 4 is the level of IL-17 in rat esophageal tissue; Figure 5 is the IL-8 level in rat esophageal tissue; Figure 6 is the plasma motilin content in rats.

[0016] The present invention provides Lactobacillus paracasei IOB413 ( Lacticaseibacillus paracasei IOB413) was deposited on June 29, 2018 in the General Microbiology Center of China Microorganism Culture Collection, referred to as CGMCC, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.16022. DETAILED DESCRIPTION

[0017] The invention provides an application of a Lactobacillus paracasei IOB413 postbiotic on gastroesophageal reflux.

[0018] According to the first aspect of the present invention, there is provided an application of postbiotics of Lactobacillus paracasei IOB413 in the preparation of a medicament for treating gastroesophageal reflux disease, and the preservation number of Lactobacillus paracasei IOB413 is CGMCC No. 16022.

[0019] Lactobacillus paracasei IOB413 ( Lacticaseibacillus paracasei IOB413) is classified as Lactobacillus paracasei ( Lacticaseibacillus paracasei ), and was deposited on June 29, 2018 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, abbreviated as CGMCC, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC No. 16022.

[0020] In the present invention, the postbiotics of Lactobacillus paracasei IOB413 affect the functions of gastric mucosal cells or related signaling pathways, inhibit the excessive secretion of gastric acid, and reduce the irritation of the esophagus by the refluxate. It promotes the repair or strengthening of the esophageal mucosal barrier, reduces the erosive ability of gastric acid on esophageal tissues, and thus alleviates esophageal inflammation, erosion and other injuries.

[0021] Motilin, as a gastrointestinal hormone, can promote the migrating motor complex (MMC) of the gastrointestinal tract, accelerate the discharge of gastric contents, and reduce the probability of reflux. The postbiotics of Lactobacillus paracasei IOB413 directly stimulate the contraction of gastrointestinal smooth muscle by increasing the level of motilin in plasma, enhancing gastric emptying and intestinal peristalsis. Enhanced gastrointestinal peristalsis can shorten the residence time of gastric acid and food in the stomach, reduce the reflux risk, and at the same time promote the normal transport of gastrointestinal contents, alleviating symptoms such as abdominal distension and reflux.

[0022] The postbiotics of Lactobacillus paracasei IOB413 regulate the activity of immune cells, reduce the secretion of pro-inflammatory cytokines such as IL-17 and IL-8, and alleviate the inflammatory response of the esophageal and gastric mucosa. It indirectly inhibits systemic or local inflammation by regulating the balance of intestinal flora, thereby alleviating mucosal damage and promoting tissue repair.

[0023] The postbiotics of Lactobacillus paracasei IOB413 intervene in gastroesophageal reflux disease at both the etiological and symptomatic levels through a multi-target synergistic mechanism of "inhibiting acid reflux - promoting gastrointestinal motility - anti-inflammatory repair", and ultimately achieve disease remission and restoration of body functions. This comprehensive action mode not only targets the core pathological links of gastroesophageal reflux disease, but also takes into account gastrointestinal mucosal protection and systemic inflammation regulation, providing a new potential strategy for the treatment of gastroesophageal reflux disease.

[0024] In a preferred embodiment of the present invention, the preparation method of the postbiotics of Lactobacillus paracasei IOB413 includes: Inoculate the secondary seed liquid of Lactobacillus paracasei IOB413 into a solid-state fermentation medium for solid-state fermentation to obtain the fermented raw material; then dry and crush the fermented raw material to obtain the postbiotics of Lactobacillus paracasei IOB413.

[0025] Preferably, the ratio of material to water for solid-state fermentation is 1:1.5 - 2, the time for solid-state fermentation is 18 - 24 h, the inoculation amount for inoculation is 1×10 6 CFU / mL - 5×10 7 CFU / mL, the drying temperature is 53 - 58 °C, and the moisture content after drying ≤ 10%.

[0026] In a preferred embodiment of the present invention, the method for preparing the secondary seed liquid includes: First, inoculate the strain of Lactobacillus paracasei IOB413 into a slant medium and place it in an incubator at 35 - 39 °C for 22 - 26 h to obtain an activated bacterial liquid; Inoculate the activated bacterial liquid into a seed medium at an inoculation amount of 3% - 5%, and statically and tightly culture it at 34 - 38 °C for 20 - 22 h to obtain a primary seed liquid; Inoculate the primary seed liquid into a seed medium at an inoculation amount of 1% - 3%, and statically and tightly culture it at 34 - 38 °C for 20 - 22 h to obtain a secondary seed liquid.

[0027] In a preferred embodiment of the present invention, the components of the slant medium include: Peptone 0.8 - 1.3%, beef extract 0.5 - 1%, yeast extract 0.4 - 0.8%, glucose 2 - 2.5%, dipotassium hydrogen phosphate 0.2 - 0.5%, sodium acetate 0.5 - 0.8%, manganese sulfate 0.005 - 0.008%, Tween 80 0.1 - 0.5%, ammonium citrate 0.2 - 0.5%, agar powder 1 - 2%, pH 6.4 - 6.5.

[0028] In a preferred embodiment of the present invention, the components of the seed medium are: peptone 2 - 2.5%, beef extract 1 - 1.5%, yeast extract 1 - 1.5%, glucose 2 - 3%, dipotassium hydrogen phosphate 0.3 - 0.8%, sodium acetate 0.5 - 1%, manganese sulfate 0.005 - 0.008%, Tween 80 0.1 - 0.2%, ammonium citrate 0.1 - 0.5%, pH 6.4 - 6.5.

[0029] According to the second aspect of the present invention, there is provided an application of the postbiotics of Lactobacillus paracasei IOB413 in the preparation of a medicament for treating gastroesophageal reflux disease, wherein the medicament for gastroesophageal reflux disease includes the postbiotics of Lactobacillus paracasei IOB413 and pharmaceutically or food-acceptable excipients.

[0030] In a preferred embodiment of the present invention, the excipients include one or more of red date powder, isomaltulose or maltodextrin.

[0031] To further elaborate on the technical means and effects adopted by the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present invention.

[0032] Example 1: Preparation of postbiotics of Lactobacillus paracasei IOB413 (1) Strain activation: Inoculate Lactobacillus paracasei IOB413 from the cryopreservation tube into the seed medium and culture at 37 °C for 24 h to prepare the activated bacterial liquid; (2) Seed liquid preparation: Inoculate the prepared activated bacterial liquid into the seed medium at an inoculation amount of 3% (v / v) and culture in a 37 °C incubator for 20 h to obtain the primary seed liquid; inoculate the primary seed liquid into the seed medium at an inoculation amount of 3% (v / v) and culture in a 37 °C incubator for 18 h to obtain the secondary seed liquid; (3) Solid-state fermentation: Using a material-liquid ratio of 1:1.75, inoculate Lactobacillus paracasei IOB413 on the solid medium at an inoculation amount of 4×10 7 CFU / mL, 37 °C, and culture for 24 h for solid-state fermentation; (4) Drying and pulverization: Dry and pulverize the fermented raw materials, with the drying temperature being 55 °C and the dried moisture content controlled at ≤10% to obtain the postbiotics powder of Lactobacillus paracasei IOB413.

[0033] Among them, the composition of the slant medium is peptone 0.8 - 1.3%, beef extract 0.5 - 1%, yeast extract 0.4 - 0.8%, glucose 2 - 2.5%, dipotassium hydrogen phosphate 0.2 - 0.5%, sodium acetate 0.5 - 0.8%, manganese sulfate 0.005 - 0.008%, Tween 80 0.1 - 0.5%, ammonium citrate 0.2 - 0.5%, agar powder 1 - 2%, pH 6.4 - 6.5; The composition of the seed medium is: peptone 2 - 2.5%, beef extract 1 - 1.5%, yeast extract 1 - 1.5%, glucose 2 - 3%, dipotassium hydrogen phosphate 0.3 - 0.8%, sodium acetate 0.5 - 1%, manganese sulfate 0.005 - 0.008%, Tween 80 0.1 - 0.2%, ammonium citrate 0.1 - 0.5%, pH 6.4 - 6.5.

[0034] Example 2: Establishment of a rat model of gastroesophageal reflux disease Feeding conditions: Clean-grade male SD rats were selected. The animal feeding conditions were temperature (22±2) °C, relative humidity 40% - 60%, and lighting time 12 h. They were housed separately in cages, allowed free access to water, and the cages were cleaned regularly. The experiment started after 5 days of adaptation.

[0035] A rat model of reflux esophagitis was established using semi-pyloric ligation and partial myotomy of the cardia. Randomly select 10 rats as the sham operation group, and 15 rats in each of the remaining modeling groups. The rats were fasted for 24 h before surgery but allowed free access to water. The rats were anesthetized by intraperitoneal injection of 2.5% sodium pentobarbital solution (30 mg / kg). The skin and subcutaneous tissue of the rats were incised and the abdominal cavity was entered. The esophageal and gastric tissues were dissected. The cardia and the muscular layer of the lower esophagus were incised, and the mucosal layer was dissected and exposed. A small hole was incised in the gastric wall 1 cm from the pylorus (note that the short gastric vessels were sutured and ligated to avoid bleeding from the wound surface affecting the surgical operation). An inner stent with an outer diameter of 3.5 mm was inserted through the small hole and passed through the pylorus, then the pyloric ring was ligated with 2-0 silk thread, and the ligature was sutured and fixed to the seromuscular layer with 5-0 silk thread. The inner stent was withdrawn, and the gastric incision was sutured. The abdominal cavity was rinsed with 0.9% sodium chloride injection, and the fluid in the abdominal cavity was aspirated with sterile gauze. The abdominal cavity was sutured and closed, and the rats were allowed to wake up from anesthesia. The rats were fasted for 24 h after surgery but allowed free access to water. The changes in the rats were observed daily, and the body weight of the rats was measured. One week after surgery, if the rats survived and their diet and activities were normal, the modeling was successful.

[0036] Experimental grouping: The mice were divided into a blank control group, a model group, a low-dose group, a medium-dose group, and a high-dose group.

[0037] The blank control group was 10 sham-operated rats that were not modeled.

[0038] For the model group, the low-dose group, the medium-dose group, and the high-dose group, rats after successful modeling were selected, with 15 rats in each group. The model group was gavaged with 2 mL / d of normal saline, and the low-dose group, the medium-dose group, and the high-dose group were gavaged with the experimental samples for 4 weeks.

[0039] The experimental sample in the low-dose group was a postbiotic saline solution with a postbiotic content of 0.063 g / mL; The experimental sample in the medium-dose group was a postbiotic saline solution with a postbiotic content of 0.126 g / mL; The experimental sample in the high-dose group was a postbiotic saline solution with a postbiotic content of 0.252 g / mL.

[0040] Example 3: Effect of postbiotics of Lactobacillus paracasei IOB413 on the general indicators of rats Observe and record the body weight, hair color, feces, mental state, and motor state of the rats, and record the situations on day 0, day 7, day 14, and day 28 respectively. The body weight changes are as Figure 1As shown, the body weight of the model group was significantly lower than that of the blank control group, indicating that esophageal reflux could affect the body weight of rats. After intragastric administration of postbiotics to each group, the body weight of the rats increased significantly.

[0041] Example 4: Effect of postbiotics of Lactobacillus paracasei IOB413 on the pH value of esophageal tissue in rats Six rats were randomly selected from each group, anesthetized and sacrificed. The abdomen was opened, and esophageal tissue was taken. The esophageal tissue was longitudinally incised, and the pH electrode end of the pH automatic recorder was placed at the esophageal mucosa 1 cm above the gastroesophageal junction. After 1 minute, the pH value was read and recorded, and the average pH value of the 6 rats was taken. The results are as Figure 2 obtained. The pH of the esophageal tissue of the rats in the model group decreased significantly, indicating that gastric acid reflux occurred in the rats in the model group. After intragastric administration of postbiotics to each group, the pH of the esophageal tissue increased significantly, indicating that postbiotics could inhibit gastric acid reflux.

[0042] Example 5: Pathological observation of esophageal mucosa in rats by postbiotics of Lactobacillus paracasei IOB413 The tissue sections prepared by HE staining were placed under a light microscope for observation and RE pathological grading. The pathological grading was based on the RE pathological grading standard in the "Guidelines for the Diagnosis and Treatment of RE" promulgated by the Digestive Endoscopy Branch of the Chinese Medical Association in 2003, as shown in Table 1. The results are as Figure 3 shown. The score of the model group increased significantly, indicating that the modeling was successful. After intragastric administration of postbiotics, the scores of each group decreased significantly, indicating that postbiotics could reduce the esophageal tissue damage caused by gastric acid reflux.

[0043] Table 1 Pathological scoring criteria

[0044] Note: Explanation of pathological score: Normal is 0 point, mild is 1 point, moderate is 2 points, and severe is 3 points.

[0045] Example 6: Effect of postbiotics of Lactobacillus paracasei IOB413 on inflammatory factors in rats Esophageal tissue was taken, ground on ice, and tissue homogenate was taken. The changes in the contents of IL-17 and IL-8 were detected according to the method described in the ELISA kit instructions. The results are as Figure 4 and Figure 5 shown. Compared with the blank control group, the levels of IL-17 and IL-8 in the esophageal tissue of the rats in the model group increased significantly. Compared with the model group, the levels of IL-17 and IL-8 in the data of each postbiotic group decreased significantly, indicating that postbiotics could reduce the inflammatory response of gastroesophageal reflux.

[0046] Example 7: Effect of postbiotics of Lactobacillus paracasei IOB413 on the content of motilin in rat plasma Motilin (MTL) is a hormone produced by intestinal endocrine cells that can promote gastrointestinal motility and accelerate the gastric emptying process. After the rats were anesthetized and sacrificed, 5 ml of blood was drawn from the abdominal aorta, and the supernatant was obtained by centrifugation (3000 rpm, 20 min, 4°C). The content of serum MTL was measured by enzyme-linked immunosorbent assay according to the kit instructions. The results are as Figure 6 shown. Compared with the normal control group, the motilin content in the model group of rats decreased significantly, indicating that the modeling was successful. Compared with the model group, the motilin content in each probiotic group increased significantly, indicating that probiotics can increase the motilin content, enhance the effect of gastric motility, and promote food digestion.

[0047] In summary, the probiotic of Lactobacillus paracasei IOB413 can relieve the weight loss of rats with gastroesophageal reflux, inhibit esophageal reflux, relieve pathological damage of esophageal tissue, relieve inflammatory damage by reducing the levels of inflammatory factors IL-17 and IL-8, and enhance gastrointestinal motility and reduce the risk of reflux by improving the motilin level.

Claims

1. Use of postbiotics of Lactobacillus paracasei IOB413 in the preparation of a medicament for treating gastroesophageal reflux disease, characterized in that, The preservation number of the Lactobacillus paracasei IOB413 is CGMCC No. 16022.

2. The application according to claim 1, characterized in that The preparation method of the postbiotics of Lactobacillus paracasei IOB413 includes: Inoculating the secondary seed liquid of Lactobacillus paracasei IOB413 into a solid fermentation medium for solid fermentation to obtain the fermented raw material; then drying and pulverizing the fermented raw material to obtain the postbiotics of Lactobacillus paracasei IOB413.

3. The application according to claim 2, characterized in that The material-water ratio of the solid-state fermentation is 1:1.5 to 2, the time of the solid-state fermentation is 18 to 24 h, the inoculation amount of the inoculation is 1×10 6 CFU / mL - 5×10 7 CFU / mL, the temperature of the drying is 53 - 58 °C, and the moisture content of the drying is ≤10%.

4. The application according to claim 2, characterized in that The preparation method of the secondary seed liquid includes: First, inoculating the Lactobacillus paracasei IOB413 strain into a slant medium and culturing it at 35 - 39 °C for 22 - 26 h to obtain an activated bacterial liquid; Inoculating the activated bacterial liquid into a seed medium at an inoculation amount of 3% - 5% and statically culturing it in a sealed manner at 34 - 38 °C for 20 - 22 h to obtain a primary seed liquid; Inoculating the primary seed liquid into a seed medium at an inoculation amount of 1% - 3% and statically culturing it in a sealed manner at 34 - 38 °C for 20 - 22 h to obtain a secondary seed liquid.

5. The application according to claim 4, wherein The components of the slant medium include: Peptone 0.8 - 1.3%, beef extract 0.5 - 1%, yeast extract 0.4 - 0.8%, glucose 2 - 2.5%, dipotassium hydrogen phosphate 0.2 - 0.5%, sodium acetate 0.5 - 0.8%, manganese sulfate 0.005 - 0.008%, Tween 80 0.1 - 0.5%, ammonium citrate 0.2 - 0.5%, agar powder 1 - 2%, pH 6.4 - 6.

5.

6. The application according to claim 4, characterized in that, The components of the seed medium are: peptone 2 - 2.5%, beef extract 1 - 1.5%, yeast extract 1 - 1.5%, glucose 2 - 3%, dipotassium hydrogen phosphate 0.3 - 0.8%, sodium acetate 0.5 - 1%, manganese sulfate 0.005 - 0.008%, Tween 80 0.1 - 0.2%, ammonium citrate 0.1 - 0.5%, pH 6.4 - 6.

5.

7. The application according to claim 1, wherein The drug for treating gastroesophageal reflux disease includes the postbiotics of Lactobacillus paracasei IOB413 and pharmaceutically or food - acceptable excipients.

8. The application according to claim 7, characterized in that The excipients include one or more of red date powder, isomaltulose or maltodextrin.

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