Lactobacillus casei DS31 microcapsule for improving irritable bowel syndrome and preparation method thereof

Abstract

The invention provides a Lactobacillus casei DS31 microcapsule for improving irritable bowel syndrome and a preparation method thereof, the Lactobacillus casei DS31 microcapsule loaded with DS31 is prepared by using the Lactobacillus casei DS31 as a water phase, DHA algal oil as an oil phase, thermally denatured whey protein as solid particles and chitosan oligosaccharide as a stabilizer by adopting a Pickering emulsion technology, and the microcapsule loaded with DS31 is prepared by using the Lactobacillus casei DS31 as a water phase, DHA algal oil as an oil phase and chitosan oligosaccharide as a stabilizer. The obtained microcapsule can resist the severe environment of gastrointestinal tracts and help strains to be planted in the intestinal tracts better, and the strain effect of the strains is achieved. The emulsion in the microcapsule has the advantages of small droplet size, high surface area, good physical stability, simplicity in operation, safety, no toxicity, high probiotic content and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganism embedding, and relates to a Lactobacillus casei DS31 microcapsule for improving irritable bowel syndrome and a preparation method thereof. Background Art

[0002] Irritable bowel syndrome (IBS) is a chronic functional intestinal disease that affects 10-25% of the world's population. The prevalence of IBS in the general population in my country is about 1.5%-11.5%. The pathophysiological mechanism of irritable bowel syndrome has not yet been fully clarified. Existing studies believe that it is related to gastrointestinal motility disorders, visceral hypersensitivity, brain-gut-axis interaction, intestinal flora disorders and metabolic abnormalities, gastrointestinal infection and inflammation, food factors, mental and psychological factors and central nervous system disorders, genetics and gene polymorphisms, etc. There is currently no specific drug treatment, and symptomatic treatment is mostly adopted, such as antispasmodics, low-dose antidepressants, laxatives and drugs that promote intestinal motility, antidiarrheal drugs, etc., but the efficacy is not very good. Due to different causes of different patients, the same patient may also have multiple pathophysiological abnormalities, and one drug is often difficult to improve all the symptoms of the patient. Therefore, in addition to standard drug treatment, a variety of treatment plans need to be combined to achieve satisfactory efficacy and benefit patients for a long time.

[0003] In recent years, with the development of intestinal microecology research, more and more evidence shows that the occurrence of irritable bowel syndrome is closely related to intestinal flora imbalance. By regulating the intestinal flora of IBS patients, the relevant clinical symptoms can be effectively improved, and the therapeutic effect of probiotics on IBS has gradually gained attention. As an auxiliary treatment method, probiotics show advantages in relieving IBS symptoms: probiotics can regulate intestinal flora, increase the number of beneficial bacteria, and inhibit the growth of harmful bacteria. At the same time, probiotics can also reduce intestinal inflammatory response, improve intestinal mucosal barrier function, and relieve symptoms such as abdominal pain, diarrhea and constipation. In addition, probiotics have good safety and tolerability and can be used for a long time.

[0004] Probiotics are a type of active microorganisms that are beneficial to human health. Since the bacteria are highly sensitive to the external environment, problems such as low survival rate or poor biological activity are prone to occur during production, storage and use, which greatly reduces the probiotic effect of probiotics. Encapsulation technology can protect probiotics from the influence of the environment and related factors to a certain extent, and has a good effect on probiotics against adverse external conditions. Traditional encapsulation technologies mainly include extrusion, freeze drying and spray drying. Although these technologies are very mature and widely used, there are some significant defects in the use process, which can no longer meet the requirements of bacteria embedding. For example: the microcapsules prepared by the extrusion method have a large particle size, poor uniformity, slow microbead formation and low yield, which are difficult to use for large-scale production. The vacuum freeze drying method will inevitably cause mechanical damage to the cell membrane, cell wall, solute, etc. during the freeze drying process, and will also affect the cell metabolism regulation, thereby leading to cell death and reducing its survival rate. In the preparation process of the spray drying method, the bacteria will be exposed to a high temperature environment for a short time, which is easy to damage the cell wall and cell membrane, etc., and have a negative impact on the structural integrity, subsequent processing and probiotic effects of the live bacterial cells.

[0005] With the continuous exploration of the efficacy of probiotics, the market demand for probiotics is constantly expanding. The requirements for convenient storage and transportation of probiotics, high survival rate and rapid resurrection have promoted the development of probiotic encapsulation technology, and new methods have emerged, such as endogenous emulsification, Pickering emulsification, complex coacervation and multi-layer encapsulation. Among them, Pickering emulsion technology has received special attention due to its unique advantages. This technology uses solid particles to stabilize the emulsion. These particles are firmly adsorbed on the liquid-liquid interface, effectively preventing aggregation. Compared with traditional encapsulation technology, Pickering emulsion performs well in stability, encapsulation efficiency and cost-effectiveness.

[0006] Therefore, it is of great practical significance to use the Pickering emulsification method to encapsulate probiotics, prepare probiotic preparations for improving irritable bowel syndrome, and improve the survival rate of strains so that they can better exert their benefits in the intestine. Summary of the invention

[0007] To achieve the above object, the present invention adopts Pickering emulsion technology, uses Lactobacillus casei DS31 as water phase, DHA algae oil as oil phase, heat-denatured whey protein as solid particles, and chitosan oligosaccharide as stabilizer to prepare microcapsules loaded with DS31, and the obtained microcapsules can resist the harsh environment of the gastrointestinal tract, help the strain to better colonize in the intestine, and exert its strain effect. The emulsion in the obtained microcapsules has the advantages of small droplet size, high surface area and good physical stability, simple operation, safety and non-toxicity, and high probiotic content.

[0008] On the one hand, the present invention provides a method for preparing Lactobacillus casei DS31 microcapsules for improving irritable bowel syndrome, comprising:

[0009] S1 internal aqueous phase (W1): chitosan oligosaccharide powder is dissolved in Lactobacillus casei DS31 fermentation broth;

[0010] S2 primary emulsion (W1 / O): DHA algae oil and internal water phase were mixed in a disperser to prepare;

[0011] S3 Pickering emulsion system: The primary emulsion was added to the external aqueous phase (W 2) containing heat-denatured whey protein particles and mixed under a homogenizer to form a Pickering emulsion system loaded with Lactobacillus casei DS31.

[0012] Furthermore, in the internal aqueous phase (W1), the content of chitosan oligosaccharide powder is 0-1% (w / v), preferably 0% (w / v), 0.2% (w / v), 0.4% (w / v), 0.6% (w / v), 0.8% (w / v). Preferably, the internal aqueous phase chitosan oligosaccharide is added in an amount of 0.6% to prepare the Pickering emulsion. The microcapsules prepared by the Pickering emulsion technology of the present invention use chitosan oligosaccharides instead of traditional emulsifiers to stabilize the emulsion system, and the stability of the emulsion is greatly improved. In addition, compared with traditional functional oligosaccharides such as oligofructose, chitosan oligosaccharides have the advantage of being directly digested and absorbed in the intestine. Chitosan oligosaccharides can act directly or indirectly on microorganisms in the intestine, inhibit intestinal pathogens and promote the growth of beneficial intestinal bacteria. Chitosan oligosaccharides can also regulate inflammatory factors and enhance the body's immunity.

[0013] Further, the preparation process of the Lactobacillus casei DS31 fermentation liquid is as follows: picking Lactobacillus casei DS31 colonies into 100mL of sterile MRS liquid culture medium, culturing at 37°C for 24h for activation, inoculating the activated bacterial liquid into 1L of MRS liquid culture medium at a 2% inoculum amount for expansion culture, and culturing at 37°C for 48h to obtain.

[0014] Furthermore, in the internal aqueous phase (W1), the Lactobacillus casei DS31 contained in the Lactobacillus casei DS31 fermentation liquid is 1.54×10 9 CFU / mL.

[0015] Furthermore, the operating parameters of the disperser are 10000-15000 r / min, 1-10 min, preferably 12000 r / min, 5 min.

[0016] Furthermore, in the primary emulsion (W1 / O), the content of DHA algae oil is 65-80%, preferably 70%.

[0017] Encapsulating Lactobacillus casei DS31 with DHA algae oil provides a relatively anaerobic, closed environment for the strain, keeping it dormant before entering the gastrointestinal tract. Microcapsules can control the release of Lactobacillus casei encapsulated with DHA algae oil at a reasonable time and place, increasing the survival rate of the strain and enabling it to stably exert its probiotic benefits.

[0018] Further, the volume ratio of the primary emulsion (W1 / O) to the external aqueous phase (W2) is 1-3:3-1, preferably 1:1, 1:2, 2:1, 1:3 or 3:1.

[0019] Furthermore, the operating parameters of the homogenizer are 5000-10000 r / min, 1-10 min, preferably 8000 r / min, 5 min.

[0020] Further, the external aqueous phase (W 2) is a whey protein solution prepared by dissolving whey protein powder in sterile water, and the prepared whey protein solution is placed in a constant temperature water bath for thermal denaturation and then cooled at room temperature for standby use. Further, the temperature in the constant temperature water bath is 80 to 100° C., preferably 90 to 95° C. Further, the thermal denaturation time is 10 to 20 minutes, preferably 15 minutes. Further, in the external aqueous phase (W 2), the content of heat-denatured whey protein particles is 0.5 to 2% (w / v), preferably 0.5% (w / v), 1% (w / v), 1.5% (w / v), 2% (w / v).

[0021] Common functional properties of whey protein include gelation, emulsification and foaming properties. As solid particles, it forms a layer of solid particles around the emulsion droplets, which hinders the approach of emulsion droplets in space, thus providing a strong aggregation barrier. After heat treatment, the internal hydrophobic groups of whey protein are exposed, which is conducive to protein gelation. After heating at 90°C, it is conducive to the orderly rearrangement of molecules, thereby enhancing the hydrophilic and lipophilic properties of the protein, making it easier to disperse in the emulsification interface, and enhancing the emulsification stability, gelation and viscosity of whey protein. Using heat-denatured whey protein isolate as the external aqueous phase can effectively improve the survival rate of probiotics in microcapsules and extend the shelf life of the emulsion.

[0022] To avoid contamination during the preparation process, all operations were performed in a sterile environment (ultra-clean bench). The morphological structure and physicochemical properties of the microcapsules were characterized to verify their storage stability.

[0023] The Lactobacillus casei DS31 capable of improving the symptoms of irritable bowel syndrome of the present invention was deposited in the General Microbiology Center of the China Microbiological Culture Collection Committee on November 28, 2014, with a deposit number of CGMCC No: 10073. The address of the deposit unit is No. 2, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The Lactobacillus casei DS31 isolated and screened from the intestines of healthy large yellow croaker cultured in cages in Ningde City, Fujian Province can improve the symptoms of irritable bowel syndrome, inhibit inflammation and relieve symptoms.

[0024] On the one hand, the present invention provides a Lactobacillus casei DS31 microcapsule for improving irritable bowel syndrome, which is prepared by the method of the present invention.

[0025] On the one hand, the present invention provides a use of Lactobacillus casei DS31 microcapsules for improving irritable bowel syndrome prepared by the method of the present invention in the preparation of drugs, foods or health products for alleviating irritable bowel syndrome.

[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0027] The microcapsules prepared by the invention can resist the adverse environment of the gastrointestinal tract, help the strains to better colonize in the intestines, and exert their strain effects.

[0028] The present invention adopts Pickering emulsion technology to embed Lactobacillus casei DS31 to ensure its stability and viability during food processing, storage and digestion. This technology utilizes solid particles to stabilize the emulsion, and compared with traditional embedding technology, provides better stability, embedding efficiency and cost-effectiveness. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Technical roadmap of the present invention

[0030] Figure 2 Schematic diagram of the embedding rate of Pickering emulsion with different contents of chitosan oligosaccharide in Example 1

[0031] Figure 3 The Pickering emulsions with different chitosan oligosaccharide contents in Example 1 are observed under a microscope; Note: A: no chitosan oligosaccharide, B: 0.2% chitosan oligosaccharide, C: 0.4% chitosan oligosaccharide, D: 0.6% chitosan oligosaccharide, E: 0.8% chitosan oligosaccharide

[0032] Figure 4 Schematic diagram of storage stability of microcapsules in Example 1

[0033] Figure 5 The weight change of mice in Example 2 within two weeks

[0034] Figure 6 Schematic diagram of the moisture content of mouse feces in Example 2

[0035] Figure 7 Schematic diagram of the sugar water preference rate of mice in Example 2

[0036] Figure 8 This is a graph showing the abdominal withdrawal reflex (AWR) score of mice in Example 2. Figure 8 A intake volume is 0.4mL, Figure 8 B The air intake volume is 0.6 mL. The air bag provides stimulation with different air intake volumes (pressures), causing pain in mice, which manifests as behavioral responses caused by different degrees of visceral pain.

[0037] Fig. 9 Schematic diagram of the mouse colon tissue pathological section (HE) staining in Example 2; Note: A: normal group, B: model group, C: positive control group, D: probiotic group, E: microcapsule control group, F: microcapsule group

[0038] Fig.10 Schematic diagram of the serum corticosterone (CORT) level of mice in Example 2 DETAILED DESCRIPTION

[0039] The present invention is further described in detail below in combination with specific implementations and with reference to the accompanying drawings. The embodiments of the present invention include but are not limited to the following embodiments.

[0040] Chitosan oligosaccharide powder and whey protein were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; DHA algae oil was purchased from Xiamen Well Ocean Commercial Operation Management Co., Ltd. with DHA ≥ 50%. Other substances whose preparation process was not clear can be purchased from the market.

[0041] The invention provides a Lactobacillus casei DS31 microcapsule for improving irritable bowel syndrome and a preparation method thereof, which mainly comprises the preparation of the DS31 microcapsule, the establishment of an irritable bowel syndrome animal model, grouping drug administration, index detection and scoring, statistics and evaluation.

[0042] Establishment of animal model of irritable bowel syndrome:

[0043] The animal model of irritable bowel syndrome was established and divided into a normal group, a control group and a model group.

[0044] Drug administration

[0045] The model groups were divided into model control group, positive control group, probiotic group, microcapsule group, and microcapsule control group; they were raised in a sterile pathogen-free environment. Each mouse was housed in a cage with constant temperature and light control (12-hour light and dark cycle), and the same food and water were provided ad libitum.

[0046] Test indicators and scoring: including general observation, stool consistency and water content, sugar preference rate, visceral sensitivity evaluation, colon tissue pathology examination, etc., including:

[0047] General observation: Before each experiment, observe the weight changes, hair color, mental state, diet and feces of each group of mice.

[0048] Stool test: During the dosing period, the stool consistency was measured every seven days (the day before dosing, the 7th day, the 14th day, the 21st day, and the 28th day). The main results were the stool consistency and water content on the 28th day.

[0049] Sugar water preference rate: Before the experiment, mice were fed with sugar water adaptation and given a bottle of 1% sucrose water and sterile pure water respectively, and the pure water and sugar water basic test was carried out on mice.

[0050] Evaluation of visceral sensitivity: The behavioral performance of animals was observed and evaluated under colon balloon dilation, and the AWR score was used as the pain threshold of mice to determine the degree of visceral sensitivity of animals.

[0051] Colon tissue pathological examination: about 1 cm of the proximal colon of the mouse was cut off, and the residue in the colon was cleaned with 4°C pre-cooled saline. The colon was cut open along the longitudinal axis, fixed in 4% polymethanol solution for 48 hours, dehydrated, embedded, sectioned, HE stained, and finally observed under a microscope.

[0052] Example 1: Preparation and storage stability of Lactobacillus casei DS31 microcapsules based on Pickering emulsion

[0053] 1. Preparation of Lactobacillus casei DS31 microcapsules based on Pickering emulsion

[0054] Preparation of Lactobacillus casei DS31 fermentation broth: Pick Lactobacillus casei DS31 colonies into 100 mL sterile MRS liquid culture medium, culture at 37° C. in a shaking incubator for 24 h for activation, inoculate the activated bacterial liquid into 1 L MRS liquid culture medium at a 2% inoculum amount for expansion culture, and culture at 37° C. in a shaking incubator for 48 h.

[0055] Preparation of 1% heat-denatured whey protein: dissolve an appropriate amount of whey protein powder in sterile distilled water to prepare a 1% (w / v) whey protein solution. Place the prepared whey protein solution in a 90°C constant temperature water bath for 15 minutes for heat denaturation and then cool it to room temperature for use.

[0056] Pickering emulsions were prepared using a two-step emulsification method:

[0057] The internal aqueous phase (W1 phase) was prepared by dissolving chitosan oligosaccharide powder in 100 mL of Lactobacillus casei DS31 fermentation broth; the contents of chitosan oligosaccharide powder were 0% (w / v), 0.2% (w / v), 0.4% (w / v), 0.6% (w / v), and 0.8% (w / v), respectively.

[0058] The primary emulsion (W1 / O phase) was prepared by mixing 70 mL of DHA algae oil and 30 mL of W1 phase under a disperser (12000 r / min, 5 min).

[0059] Then, the primary emulsion (W1 / O phase) was added to the external aqueous phase (W2 phase) containing 1% heat-denatured whey protein particles, and the heat-denatured whey protein particles were heat-denatured with sterile water. The volume ratio of W1 / O phase and W2 phase was 1:1, and they were mixed under a homogenizer (8000 r / min, 5 min) to form a Pickering emulsion system loaded with Lactobacillus casei DS31.

[0060] 2. Embedding rate

[0061] Pickering emulsions containing different amounts of chitosan oligosaccharides were taken respectively, and the bacteria were centrifuged and spread on plates in gradient dilutions. The number of live bacteria before and after emulsification was counted respectively, and the embedding rate was calculated.

[0062] Embedding rate % = N-N0 / N×100%

[0063] N: number of viable bacteria before emulsification; N0: number of viable bacteria not embedded

[0064] 3. Storage stability evaluation

[0065] Samples were taken at the 0th, 5th, 10th, 15th, 20th, 25th and 30th day of storage, and the upper emulsion was diluted 50 times with sterile water and then graded diluted. The plates were spread and cultured in a constant temperature incubator at 37°C for 48 hours, and the number of viable bacteria was calculated.

[0066] Stability % = Et / E × 100%

[0067] Et: number of viable bacteria retained in the internal aqueous phase during storage; E: number of viable bacteria initially retained in the internal aqueous phase

[0068] Experimental results: Preparation and storage stability of Lactobacillus casei DS31 microcapsules based on Pickering emulsion 1.1 Encapsulation efficiency

[0069] like Figure 2As shown, Pickering emulsions with different chitosan oligosaccharide contents have a high embedding rate, wherein when the chitosan oligosaccharide addition amount is 0.8% (w / v), the embedding rate is as high as 97.4%. The embedding rate of the emulsion without chitosan oligosaccharide addition is 96%. When chitosan oligosaccharide is used as a stabilizer and the addition amount is 0.8% (w / v), the embedding rate is higher than that of the Pickering emulsion without chitosan oligosaccharide addition.

[0070] 1.2 Particle size

[0071] like Figure 3 As shown, Figure A shows that the average particle size of the Pickering emulsion without chitosan oligosaccharide is 8.68±7.53μm, Figure B shows that the chitosan oligosaccharide content is 0.2% and the average particle size is 3.17±2.29μm, Figure C shows that the chitosan oligosaccharide content is 0.4% and the average particle size is 4.04±2.67μm, Figure D shows that the chitosan oligosaccharide content is 0.6% and the average particle size is 4.07±1.87μm, and Figure E shows that the chitosan oligosaccharide content is 0.8% and the average particle size is 3.91±1.93μm. Compared with the Pickering emulsion without chitosan oligosaccharide, the particle size of the Pickering emulsion with chitosan oligosaccharide is smaller under the microscope, the number of particles is significantly increased, and the particle distribution is more uniform.

[0072] 1.3 Storage stability

[0073] Probiotics, as active microorganisms, can bring health benefits when ingested by the human body in sufficient quantities and are often added to food. However, their survival rate is challenged during food processing, storage and digestion. Compared with traditional encapsulation technologies such as spraying, extrusion, and emulsification, Pickering emulsions have excellent performance in terms of stability, encapsulation efficiency, and cost-effectiveness. Pickering emulsions have excellent performance in terms of stability, encapsulation efficiency, and cost-effectiveness.

[0074] like Figure 4 As shown in the figure, the number of live bacteria in Pickering emulsions with different chitosan oligosaccharide contents increased significantly within 10 days of storage, and the increase was more obvious when 0.6% chitosan oligosaccharide was added. The number of live bacteria in Pickering emulsions with 0.2% chitosan oligosaccharide was 3.05×10 7 CFU / mL; the number of viable bacteria in the Pickering emulsion with 0.4% chitosan oligosaccharide added was 3.17×10 7 CFU / mL; the number of viable bacteria in the Pickering emulsion with 0.6% chitosan oligosaccharide added was 5.25×10 7 CFU / mL; the number of viable bacteria in the Pickering emulsion with 0.8% chitosan oligosaccharide added was 5.32×10 7CFU / mL; while the number of viable bacteria in Pickering emulsion without chitosan oligosaccharide was 5.25×10 6 CFU / mL, the number of viable bacteria decreased by an order of magnitude. After 30 days of storage, the number of viable bacteria in the emulsion without chitosan oligosaccharide decreased more significantly than that in the Pickering emulsion with chitosan oligosaccharide added. This shows that chitosan oligosaccharide, as a stabilizer, can keep the number of viable bacteria at a high level during storage, improve the survival rate of Lactobacillus casei DS31, make the emulsion more stable, and keep it for a longer time.

[0075] Example 2: Animal Experiment

[0076] 1. Animal Selection

[0077] 60 C57BL / 6 mice, male, weighing 18-20 g, animal certificate number: 20220009023036, purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd., experimental animal production license number: SCXK (Shanghai) 2022-0009; raised in the animal laboratory of the Third Institute of Oceanography, Ministry of Natural Resources, experimental animal production license number: SYXK (Fujian) 2024-0002. 2. Establishment of an animal model of irritable bowel syndrome

[0078] After 7 days of adaptive feeding, the IBS-D mouse model was established by oral administration of ice senna solution combined with restraint stress (restraint stress lasted for 1 hour).

[0079] Drug preparation: The effective ingredients in senna leaves were extracted by decoction to prepare senna leaf solution.

[0080] 3. Grouped medication

[0081] The mice were randomly divided into 6 groups according to their average body weight, namely normal control group, model control group, positive control group, probiotic group, microcapsule control group, and microcapsule group, with 10 mice in each group. Each group of mice was given the corresponding dose of the test agent by oral gavage. The normal control group and the model control group were given 0.2mL 0.9% sterile saline; the positive control group was given 0.104g / kg rifaximin 0.2mL; the probiotic group was given DS31 lyophilized powder aqueous solution; the microcapsule group was given microcapsules with an added amount of 0.6% chitosan oligosaccharide; and the microcapsule control group was given microcapsules without Lactobacillus casei DS31. Once a day for 21 consecutive days.

[0082] IV. Index Testing and Scoring

[0083] 1. General observation: Observe the behavior, mental state, diet, body weight, coat color and quality, and feces of each group of animals every day.

[0084] 2. Stool consistency and water content

[0085] 2.1 Stool consistency

[0086] During the dosing period, stool consistency was measured every seven days (the day before dosing, day 7, day 14, and day 21), and the primary outcome was stool consistency on day 21. The Bristol stool classification system was used to classify and score stools: type 1, 1 point, scattered, similar to dry balls and nuts, difficult to excrete; type 2, 2 points, sausage-like, but in pieces; type 3, 3 points, sausage-like with cracks on the surface; type 4, 4 points, sausage-like or snake-like, smooth and soft; type 5, 5 points, soft masses with clear edges, easy to excrete; type 6, 6 points, soft flakes, unclear edges or mushy; type 7, 7 points, watery stool or no solid matter.

[0087] Types 1-2 indicate constipation, types 3-4 are normal stools, and types 5-7 indicate possible diarrhea.

[0088] 2.2 Moisture content of feces

[0089] Before the experiment, a piece of filter paper was placed on the bottom of the mouse basket to collect feces. During the process, care should be taken to keep the filter paper intact. The collected feces were first weighed and recorded as W1. Then the feces were placed in the oven for 30 minutes and weighed every 10 minutes. When the mass remained unchanged, it was recorded as the final dry weight W2.

[0090] Fecal moisture content (%) = (W1-W2) / W1×100%

[0091] 3. Sugar water preference rate

[0092] Before the experiment, mice were fed with sugar water adaptation. First, two bottles of 1% sucrose water were placed on the mouse basket for mice to drink for 24 hours (weigh the weight of the two bottles of sugar water). Then, under other conditions, one bottle of sucrose water was replaced with pure water, and the other bottle was still 1% sucrose water, and the mice were tested for pure water / sugar water. During the test (12 hours), the two bottles were swapped to avoid memory effects, and the drinking amount of sucrose water and pure water was measured and calculated for 24 hours.

[0093] Sugar water preference rate (%) = sugar water intake (ml) / (sugar water intake + pure water intake) (ml) × 100%

[0094] 4. Visceral sensitivity assessment

[0095] Abdominal withdrawal reflex (AWR)

[0096] The mice were fasted for 24 hours before the experiment and were lightly anesthetized with isoflurane. A lubricated 6Fr pediatric double-lumen catheter with a balloon was inserted into the mouse colon, with the end of the balloon 1 cm away from the anus. Each mouse was given balloon dilation twice, with a capacity of 0.4 and 0.6 mL, each volume lasting 20 seconds, with an interval of 4 minutes. AWR scoring criteria: 0 points, the mouse is emotionally stable; 1 point, the mouse is emotionally unstable and occasionally turns its head; 2 points, the abdominal and back muscles are slightly contracted, and the abdomen is not lifted off the ground; 3 points, the abdominal and back muscles are strongly contracted, and the abdomen is lifted from the ground; 4 points, the abdominal muscles are strongly contracted, the back is arched, and the abdomen, pelvis and perineum are lifted from the ground.

[0097] 5. Colon tissue pathological examination

[0098] After the experiment, the mice were dislocated by cervical vertebrae, and about 1 cm of the proximal colon of the mice was cut open by laparotomy. The residues in the colon were cleaned with 4°C pre-cooled saline, and the colon was cut open along the longitudinal axis and fixed in 4% polymethanol solution for 48 h. The colon was dehydrated, embedded, sectioned, and stained with HE, and finally the pathological changes of the colon tissue structure were observed under a microscope.

[0099] 6. Detection of inflammatory factors in colon tissue

[0100] After the experiment, the mice were killed, and about 100 mg of the mouse colon was taken and ground in a grinder with cold physiological saline at a ratio of 1:9. The mixture was centrifuged (3500 rpm, 4°C, 10 min) to obtain the supernatant.

[0101] 6.1 Detection indicators

[0102] The ELISA method was used to determine the levels of IL-1β, IL-6, IL-10, TNF-α, and INF-γ inflammatory factors in the intestinal tissue of mice.

[0103] 6.2 Detection methods

[0104] Strictly follow the instructions of the kits for the above inflammatory factors. The specific details are subject to the instructions. The test includes the following steps: ① prepare the specimen, ② soak the ELISA plate, ③ add the standard, ④ add the sample, ⑤ antibody detection, ⑥ incubation, ⑦ washing, ⑧ enzyme incubation, ⑨ add substrate color development / stop solution, and finally read the test.

[0105] 7. Serum corticosterone level

[0106] At the end of the experiment, blood was collected by removing the eyeball and collected in a 1.5 mL microcentrifuge tube containing EDTA (ethylenediaminetetraacetic acid). Serum was obtained after centrifugation in a refrigerated centrifuge at 4°C (35,000 rpm, 10 min). Serum corticosterone levels were quantified using a mouse corticosterone (CORT) ELISA kit, and the absorbance was measured spectrophotometrically at 450 nm using an enzyme reader.

[0107] 5. Data Processing and Analysis

[0108] 1. Data processing

[0109] SPSS25.0 was used for data statistics, and Origin2022 software was used for drawing. Independent sample T test and one-way ANOVA were used to analyze the differences between samples. ， Multiple comparisons were performed to detect whether the differences among the groups were significant, and the data were expressed as mean ± standard deviation (mean ± SD).

[0110] 2. Result evaluation

[0111] 2.1 General Observations

[0112] The general condition is the most basic observation index, which can reflect the overall state of the animals during the modeling process. The mice in the normal control group were in good condition, lively and active, in good spirits, with normal water and food intake, steady weight gain, shiny and smooth fur, and moderate hardness of feces. After induction, the model control group showed symptoms such as erect hair, susceptibility to irritation, mental depression, reduced food intake, and diarrhea. The above symptoms in the other groups were improved to varying degrees, and the effect of the microcapsule group was more obvious. Figure 5 As shown, the body weight of mice in the normal group showed a significant upward trend, and the body weight of mice in each group after induction was lower than that in the normal group. The above results preliminarily confirmed the successful establishment of the model.

[0113] 2.2 Stool consistency and water content

[0114] As shown in Table 1, the fecal scores of the mice in the model control group were significantly higher than those in the normal control group, and the scores were all above 6 points, indicating that the irritable bowel syndrome model was successfully established. After drug intervention, the Bristol fecal trait scores of the mice in the other four groups were significantly lower than those in the model control group.

[0115] Table 1 Bristol fecal trait scores of mice in each group

[0116] Group Quantity / pcs Stool characteristics / point Normal control group 10 <![CDATA[3.50±0.50 b ]]> Model control group 10 <![CDATA[6.00±0.71 a ]]> Positive control group 10 <![CDATA[4.25±0.83 ab ]]> Probiotics group 10 <![CDATA[5.00±0.71 ab ]]> Microcapsule control group 10 <![CDATA[5.75±1.09 ab ]]> Microcapsule group 10 <![CDATA[4.50±0.50 ab ]]>

[0117] Note: a means P<0.05 compared with the normal control group, b means P<0.05 compared with the model control group.

[0118] like Figure 6 Compared with the normal control group, the feces water content of the other groups of mice increased significantly. After drug intervention, the positive control group, probiotic group and microcapsule group were significantly reduced compared with the model control group.

[0119] 2.3 Sugar water preference rate

[0120] Sucrose water intake is a means of determining depression in mice, e.g. Figure 7 As shown in the figure, the sugar water intake of the model control group was significantly reduced, and the preference rate for sugar water was significantly lower than that of other groups. The model control group may have a depressed mood. After drug intervention, the preference rate of the microcapsule group for sugar water was significantly reduced, which had an effect on alleviating and improving the depression of mice.

[0121] 2.4 Visceral sensitivity assessment

[0122] like Figure 8 As shown in the figure, compared with the normal control group, the AWR scores of the model control group when the balloon expansion volume was 0.4mL and 0.6mL were increased, indicating that the visceral sensitivity of the model control group was higher than that of the normal control group, and the modeling was successful. After drug treatment, the AWR scores of the balloon expansion volume of the probiotic group and the microcapsule group decreased, among which the microcapsule group decreased significantly compared with the model control group, but the microcapsule control group did not decrease. When the balloon expansion volume was 0.6mL, the AWR score of the positive control group decreased significantly. This shows that the visceral sensitivity of mice decreased after drug intervention.

[0123] 2.5 Colon tissue pathological examination

[0124] Colon pathological sections show (eg Fig. 9 ), the colon tissue structure of mice in each group was intact, and there was no obvious damage to the intestinal mucosal tissue. No obvious edema, erosion, ulcer, or bleeding was observed. The number of inflammatory cells in the colon mucosa of mice in the model control group increased, showing only low-grade inflammation of the intestine, and no obvious organic lesions. Compared with the model control group, the range of inflammatory cell infiltration in the colon of mice in each group after drug intervention treatment was reduced. Low-grade inflammation was improved.

[0125] 2.6 Detection of inflammatory factors in colon tissue

[0126] Many studies have shown that chitosan oligosaccharides can inhibit the activation of myeloperoxidase, cyclooxygenase and inducible nitric oxide synthase (inducible), and reduce the levels of proinflammatory cytokines such as interleukin and tumor necrosis factor (TNF-α). As shown in Table 3, compared with the model group, the levels of proinflammatory factors such as IL-1β, IL-6, INF-γ, TNF-α in the tissue homogenate of mice in the microcapsule control group were significantly reduced, and the levels of inflammatory factors in the microcapsules containing DS31 were significantly reduced. It can be seen that excipients such as chitosan oligosaccharides, DHA algae oil, and heat-denatured whey protein have an improving effect on the low inflammation of the colon in mice with irritable bowel syndrome. Among them, chitosan oligosaccharides play an anti-inflammatory role.

[0127] Table 2 Levels of inflammatory factors in colon tissue homogenates of mice in each group (unit: pg / mL)

[0128]

[0129] Note: In the same column, a represents a significant difference compared with the normal group (P<0.05); b represents a significant difference compared with the model group (P<0.05)

[0130] 2.7 Serum corticosterone levels

[0131] Stress activates the HPA (hypothalamic-pituitary-adrenal axis) axis, which prompts the paraventricular nucleus of the hypothalamus to secrete and release corticotropin-releasing hormone (CRH). CRH acts on the anterior pituitary gland to promote its secretion of adrenocorticotropic hormone (ACTH), which in turn causes the adrenal cortex to secrete glucocorticoids. Glucocorticoids include cortisol and cortisol. The main glucocorticoid that regulates the stress response of mice is corticosterone. Through the regulation of this axis, the body can adapt to changes in the external environment. Fig.10 As shown in the figure, the model group had an increased level of serum corticosterone compared with the normal group, in order to resist changes in the external environment. After drug intervention, the serum corticosterone levels of the other three groups of mice decreased, and the serum corticosterone levels of the probiotic group and the microcapsule group decreased significantly compared with the model group. The results show that when mice are affected by changes in the external environment (stress), Lactobacillus casei DS31 can help mice better adapt to changes in the external environment.

[0132] In summary, the present invention can accurately evaluate the effect of Lactobacillus casei DS31 and microcapsules containing DS31 on improving irritable bowel syndrome. Lactobacillus casei DS31 and microcapsules containing DS31 have a significant improvement effect on the symptoms of irritable bowel syndrome.

[0133] The invention uses chitosan oligosaccharide to replace the traditional emulsifier to make the Pickering emulsion more stable, and can improve the survival rate of Lactobacillus casei DS31. At the same time, it can exert its anti-inflammatory function in the body of irritable bowel syndrome mice and alleviate the low inflammation state of the colon of irritable bowel syndrome mice.

[0134] The Lactobacillus casei DS31 microcapsules for improving irritable bowel syndrome and the preparation method thereof provided by the present invention can improve symptoms, reduce intestinal sensitivity, relieve diarrhea, and reduce inflammatory response, and have potential application prospects in clinical treatment of irritable bowel syndrome.

[0135] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing Lactobacillus casei DS31 microcapsules for improving irritable bowel syndrome, characterized in that: include: S1 internal aqueous phase (W1): chitosan oligosaccharide powder was dissolved in the fermentation broth of Lactobacillus casei DS31; S2 primary emulsion (W1 / O): DHA algae oil and internal water phase were mixed in a disperser to prepare; S3 Pickering emulsion system: The primary emulsion was added to the external aqueous phase (W 2) containing heat-denatured whey protein particles and mixed under a homogenizer to form a Pickering emulsion system loaded with Lactobacillus casei DS31.

2. The preparation method according to claim 1, characterized in that: In the internal aqueous phase (W1), the content of chitosan oligosaccharide powder is 0-1% (w / v), preferably 0% (w / v), 0.2% (w / v), 0.4% (w / v), 0.6% (w / v), 0.8% (w / v).

3. The preparation method according to claim 1, characterized in that: The preparation process of the Lactobacillus casei DS31 fermentation liquid is as follows: picking Lactobacillus casei DS31 colonies to 100 mL of sterile MRS liquid culture medium, culturing on a shaking platform at 37° C. for 24 hours for activation, inoculating the activated bacterial liquid into 1 L of MRS liquid culture medium at a 2% inoculation amount for expansion culture, and culturing on a shaking platform at 37° C. for 48 hours to obtain the product.

4. The preparation method according to claim 1, characterized in that: In the internal aqueous phase (W1), the Lactobacillus casei DS31 contained in the fermentation liquid of Lactobacillus casei DS31 is 1.54×10 9 CFU / mL.

5. The preparation method according to claim 1, characterized in that: The working parameters of the disperser are 10000-15000 r / min, 1-10 min; The primary emulsion (W1 / O) contains 65-80% DHA algae oil, preferably 70%; The volume ratio of the primary emulsion (W1 / 0) to the external aqueous phase (W2) is 1-3:3-1, preferably 1:1, 1:2, 2:1, 1:3 or 3:

1.

6. The preparation method according to claim 1, characterized in that: The working parameters of the homogenizer are 5000-10000 r / min, 1-10 min; preferably 8000 r / min, 5 min.

7. The preparation method according to claim 1, characterized in that: The external aqueous phase (W2) is prepared by dissolving whey protein powder in sterile water to obtain a whey protein solution. The prepared whey protein solution is placed in a constant temperature water bath for thermal denaturation and then cooled at room temperature for later use.

8. The preparation method according to claim 7, characterized in that: The temperature in the constant temperature water bath is 80-100° C., preferably 90-95° C.; the heat denaturation time is 10-20 min, and the content of heat denatured whey protein particles in the external water phase (W 2) is 0.5-2% (w / v).

9. A Lactobacillus casei DS31 microcapsule for improving irritable bowel syndrome, prepared by the method according to any one of claims 1 to 8.

10. Use of Lactobacillus casei DS31 microcapsules for improving irritable bowel syndrome prepared by the method according to any one of claims 1 to 8 in the preparation of drugs, foods or health products for alleviating irritable bowel syndrome.

Images