Lactobacillus casei WFP20 with constipation-improving effect and its application

Abstract

The present invention discloses a strain of Lactobacillus casei WFP20 having the effect of improving constipation and its application, belonging to the field of microbial technology. The classification of the Lactobacillus casei WFP20 is named Lacticaseibacilluscasei , deposited with the Guangdong Provincial Center for Microbiological Culture Collection under the deposit number GDMCC No. 62586. This strain exhibits extremely high acid production and high storage stability at room temperature. It was selected from naturally fermented ghee in Tibet, making it a natural and safe source. Furthermore, this strain has been shown to increase fecal organic acid content and the number of bifidobacteria in the intestinal flora, improve bowel movement frequency, and alleviate constipation. This strain has broad application prospects in the pharmaceutical industry.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a Lactobacillus casei WFP20 strain capable of improving constipation and an application thereof. Background Art

[0002] Constipation is a common functional disorder of the digestive system, with clinical manifestations mainly including decreased gastrointestinal motility, difficult, uncomfortable or infrequent defecation, and incomplete defecation. Research by the American Gastroenterological Association shows that the frequency of bowel movements in normal people ranges from 3 times a day to more than 3 times a week, while people with constipation generally defecate less than 3 times a week, have difficulty defecating, and their stools are hard and low in water. Gastrointestinal dysfunction and metabolic diseases are also common. In recent years, due to factors such as unreasonable diet and lifestyle, the incidence of constipation has gradually increased and has become a major factor affecting health. Clinical treatment of constipation mainly uses laxatives and drugs that improve gastrointestinal motility, such as 5-HT. However, long-term chemotherapy cannot effectively cure constipation and can damage the colon's nervous system, leading to decreased bowel movements and worsening constipation. It can also cause side effects such as drug dependence, nausea, vomiting, and abdominal pain.

[0003] Numerous studies have shown that constipation is closely related to intestinal flora imbalance, and probiotics are effective in relieving and treating constipation. Probiotics are safer than conventional medications and can naturally promote intestinal motility and alleviate constipation by improving intestinal flora, inhibiting potential pathogens, and producing beneficial metabolites or enzymes to stimulate intestinal fluid secretion. Studies have found that supplementing with Bifidobacterium breve DSM16604 and Lactobacillus plantarum LMG P-21021 can improve stool consistency. Bifidobacterium bifidum CCFM16, Lactobacillus acidophilus NCIMB 30175, and Lactobacillus rhamnosus NCIMB 30174 produce short-chain fatty acids such as acetic acid, butyric acid, and lactic acid, which can stimulate intestinal motility. Bifidobacterium longum NCC3001 treats constipation by regulating central and intestinal nerves.

[0004] The prerequisite for probiotics to alleviate certain diseases is that they can tolerate the high acidity in the stomach and the high bile salt concentration in the small intestine, and can ensure that a certain number of them adhere to the small intestinal epithelial cells. At the same time, during the production of probiotics, a high yield of live bacteria must be guaranteed at each process stage to ensure product quality and cost advantages. During the circulation of probiotic products, the effective bacterial count must be guaranteed, and good storage stability is an important prerequisite for ultimately realizing the health effects and product advantages. In addition, as dairy products are the largest carrier of probiotic consumption, whether probiotics can adapt to the production of flavored fermented milk and yogurt drinks is a key factor in evaluating their industrial scale and potential economic benefits in the consumer market. However, most probiotics on the market currently do not meet all or most of the above conditions.

[0005] Therefore, it is very critical and necessary to obtain a probiotic strain that has health benefits and meets the above-mentioned excellent characteristics in production, circulation and consumption. Summary of the Invention

[0006] (1) Technical problems solved

[0007] In response to the deficiencies in the prior art, the present invention provides a strain of Lactobacillus casei WFP20 with the effect of improving constipation and its application. The Lactobacillus casei WFP20 has extremely strong acid production performance and high storage stability, can increase the content of organic acids and the number of bifidobacteria in feces, and has the effect of improving constipation, so as to solve the problem of simultaneously meeting the above-mentioned problem of having the effect of relieving constipation while maintaining storage stability in production, circulation and consumption.

[0008] (2) Technical solution

[0009] In order to achieve the purpose of achieving the above-mentioned Lactobacillus casei WFP20 having extremely strong acid production performance and high survival under high temperature storage conditions, increasing the content of organic acids and bifidobacteria in feces and improving constipation, the present invention provides the following technical solutions:

[0010] The first object of the present invention is to provide a strain of Lactobacillus casei WFP20, which is classified and named Lactobacillus casei. The strain was deposited in the Guangdong Provincial Microbial Culture Collection Center on June 30, 2022, with a deposit number of GDMCC No.62586.

[0011] Preferably, the acidity of the skim reconstituted milk fermented with Lactobacillus casei WFP20 for 72 hours is not less than 550°T, and the skim reconstituted milk contains 6.0-7.0% protein.

[0012] Preferably, the Lactobacillus casei WFP20 freeze-dried powder has a survival rate of more than 43% when stored at 37° C. and 43% RH for 90 days.

[0013] The second object of the present invention is to provide the application of Lactobacillus casei WFP20 in medicine.

[0014] Preferably, the Lactobacillus casei WFP20 is used in the preparation of a medicine having the effect of improving constipation.

[0015] The third object of the present invention is to provide a composition containing the Lactobacillus casei WFP20.

[0016] Preferably, the composite bacterial agent contains both Lactobacillus casei WFP20 and Bifidobacterium.

[0017] Preferably, the number of viable bacteria in the freeze-dried Lactobacillus casei WFP20 is not less than 3×10 7 CFU / g.

[0018] Preferably, it contains pharmaceutically acceptable carriers or excipients.

[0019] Preferably, the 16S RDNA nucleotide sequence of the Lactobacillus casei WFP20 is shown in Sequence 1 in the table.

[0020] Preferably, the Lactobacillus casei WFP20 colonies are milky white, medium-sized, with a circular protrusion in the middle, a diameter of 1-2 mm, and rough edges; the bacteria are rod-shaped, uneven in length, rarely bent and rarely branched, and exist in singles, pairs, or clusters.

[0021] Preferably, the Lactobacillus casei WFP20 can increase the amount of organic acids and bifidobacteria in feces.

[0022] Preferably, the Lactobacillus casei WFP20 has the effect of increasing the frequency of bowel movements and improving constipation.

[0023] (3) Beneficial effects

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. Lactobacillus casei WFP20 is selected from naturally fermented ghee in Tibet. It is a safe and natural source with strong acid-producing properties. It can be used to prepare flavored fermented milk or beverages, reduce the amount of acidifiers in the formulation, and improve the stability of the system.

[0026] 2. Testing of the Lactobacillus casei WFP20 strain's tolerance to simulated gastric and intestinal fluids demonstrated its good tolerance. Furthermore, its freeze-dried powder maintained a high survival rate under high-temperature storage conditions at 37°C, making it suitable for processing, transportation, storage, and consumption at room temperature, eliminating the need for low-temperature storage. This reduces bacterial storage costs and provides more relaxed conditions for these processes. Furthermore, experiments have demonstrated that this strain can increase the amount of organic acids and bifidobacteria in feces, improve bowel movement frequency, and can be used in pharmaceutical preparations to better alleviate constipation, demonstrating its excellent application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the colony morphology of Lactobacillus casei WFP20 plate.

[0028] Figure 2 This is a microscopic examination of Lactobacillus casei WFP20 bacteria.

[0029] Figure 3 This is a diagram showing the acid production performance of Lactobacillus casei WFP20.

[0030] Figure 4 This is a diagram showing the survival rate of Lactobacillus casei WFP20 in simulated gastric juice and intestinal juice.

[0031] Figure 5 This is a graph showing the survival rate of Lactobacillus casei WFP20 at 37°C for 90 days.

[0032] Figure 6 This is the actual effect diagram of the yogurt beverage containing Lactobacillus casei WFP20 stored at 37°C for 180 days.

[0033] Figure 7 is the mRNA expression level of Bifidobacterium in the feces of mice fed with Lactobacillus casei WFP20;

[0034] Where a is the qPCR amplification curve C T a is the melting curve of qPCR amplification. DETAILED DESCRIPTION

[0035] The following is a clear and complete description of the technical solutions in the embodiments of the present invention, in conjunction with the accompanying drawings. The described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0036] In the following examples, the modified MRS liquid culture medium used in the production of bacterial powder has the following formula: glucose 70.0 g / L, yeast extract 30.0 g / L, peptone 10.0 g / L, sodium citrate 6.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, and Tween 80 1.0 g / L.

[0037] The freeze-drying protective agent formula used in the freeze-drying process of the bacterial sludge is: skim milk powder 120.0 g / L, trehalose 200.0 g / L, glycerol 30 g / L, histidine 10 g / L, and sodium isoascorbate 1.0 g / L.

[0038] Example 1, Isolation and Identification of Lactobacillus casei WFP20

[0039] 1. Isolation, screening and identification of strains

[0040] Naturally fermented ghee from Tibetan homes in Ngari, Tibet, was used as a sample. 1 g of the sample was dissolved in 9 mL of 0.85% sterile saline and thoroughly mixed with vortexing. 200 μL of the mixture was then spread onto commercial MRS (for isolation of bacilli) and M17 (for isolation of cocci) solid media, respectively, and incubated in a 37°C incubator for 72 hours. After incubation, individual bacteria with different colony morphologies were selected and streaked onto the corresponding solid media for purification. This step was repeated 2-3 times until the colonies had consistent characteristics. The purified individual bacteria were inoculated into the corresponding liquid media and incubated at 37°C for 16 hours. The purified bacteria were then stored in 60% glycerol and frozen at -80°C until further use.

[0041] The strains from the cryovials were inoculated at a 2% inoculum into sterilized skim milk at a concentration of 12 g / 100 mL. The culture was activated at 37°C for 48 hours and then serially passaged three times. Initial screening revealed strains capable of clotting milk with good curd structure at each generation. Three strains were obtained: Streptococcus S6, Bacillus longifolius 83, and Bacillus 253. The screened strains were inoculated into reconstituted skim milk with a 7.0% protein content and fermented for 72 hours. The pH and acidity of the fermented milk were measured, and the results are shown in Table 1. As shown in Table 1, Bacillus 253 had the lowest pH and an acidity of 555°T, significantly higher than the acid production of Streptococcus S6 and Bacillus longifolius 83. Therefore, Bacillus 253 was relabeled as WFP20.

[0042] Table 1. pH and acidity of the three strains obtained in the initial screening after 72 h of fermentation

[0043]

[0044] 2. Morphological characteristics

[0045] After microscopic examination, the morphological characteristics of strain WFP20 are as follows Figure 1 and Figure 2 As shown in the figure, strain WFP20 colonies on solid culture medium are milky white, medium-sized, with a central, quasi-circular protrusion, 1-2 mm in diameter, and with rough edges. Gram-positive, under an oil microscope, the bacteria appear rod-shaped, of uneven length, with few bends and few branches, and occur in singles, pairs, or clusters.

[0046] 3. Identification of strain 16S rDNA

[0047] The cryopreserved vials of strain WFP20 were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The nucleotide sequence of the 16S rDNA of strain WFP20 is shown in SEQ ID 1 of the nucleotide sequence listing. BLAST comparison results showed that strain WFP20 shared over 99% homology with Lacticasei bacillus casei. Therefore, it was identified as Lactobacillus casei and named Lactobacillus casei WFP20.

[0048] The screened strain WFP20 was deposited at Guangdong Microbial Culture Collection Center (GDMCC); address: No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province; date of deposit: June 30, 2022; the deposit number of Lactobacillus casei WFP20 is GDMCC No. 62586.

[0049] Example 2, Physiological and biochemical characteristics of Lactobacillus casei WFP20

[0050] 1. Acid production characteristics

[0051] A cryopreserved vial of Lactobacillus casei WFP20 was inoculated at a 2% inoculum into 12 g / 100 mL of skim reconstituted milk. The cells were incubated at 37°C for 16 hours, activated for three generations, and then fermented at a 4% inoculum into 6.0% protein skim reconstituted milk. Acid production characteristics (rate of production and post-acidification) were evaluated using a French iCinac milk fermentation monitor over a 48-hour fermentation period.

[0052] The results are as follows Figure 3 As shown, Lactobacillus casei WFP20 produced acid slowly for the first 6 hours, with the pH dropping by only 0.2. It then entered a period of rapid acid production, with the pH of the fermented milk rapidly dropping from 5.6 to around 3.9 within 14 hours. While acid production gradually slowed between 20 and 48 hours, the strain maintained a strong ability to continue producing acid, dropping the pH of the fermented milk from 3.9 to 3.39, indicating a moderate level of post-acidification.

[0053] 2. Tolerance to simulated gastric fluid and simulated intestinal fluid

[0054] The frozen tube of Lactobacillus casei WFP20 was inoculated into the commercial MRS liquid culture medium at an inoculum volume of 2% and activated and subcultured three times to obtain bacterial liquid. The bacteria were collected by centrifugation at 4°C and 4000×g for 10 minutes, washed twice with sterile water, and resuspended in equal volume in simulated gastric fluid or simulated intestinal fluid; incubated in simulated gastric fluid and simulated intestinal fluid for 30, 60, 90 and 300 minutes respectively. The resuspended liquid was diluted 10 times in a gradient and poured onto an MRS agar medium plate, cultured at 37°C for 48 hours, and the viable bacteria were counted to calculate the survival rate of the strain. Among them, the formula of simulated gastric fluid is: sodium chloride 2.0g / L, concentrated hydrochloric acid 7mg / L, sterile ultrapure water, pH

[0055] 2.0; the simulated intestinal fluid formula is: potassium dihydrogen phosphate 6.8g / L, 0.2N sodium hydroxide 77mg / L, pH 6.8±0.1. The survival rate of the strain was calculated according to the following formula:

[0056] Strain survival rate (%) = B1 / B0×100

[0057] Among them, B0 is the initial bacterial count of bacterial suspension / (CFU / mL); B1 is the bacterial count after treatment with simulated gastric fluid or intestinal fluid /

[0058] (CFU / mL).

[0059] The results are as follows Figure 4 As shown, Lactobacillus casei WFP20 has good tolerance to pH 2.0 simulated gastric fluid and pH 6.8±0.1 simulated intestinal fluid. The survival rate of the bacteria in simulated gastric fluid for 30 minutes is 91.54%, while the survival rate in simulated intestinal fluid for 5 hours is 100%, indicating that Lactobacillus casei WFP20 can colonize well in the intestine.

[0060] Example 3: Production and Storage Stability of Lactobacillus casei WFP20 Powder

[0061] 1. Fungus powder production

[0062] The frozen tubes of Lactobacillus casei WFP20 were inoculated into commercial MRS liquid medium at a 2% inoculum size for activation and passage three times, and then inoculated into modified MRS liquid medium at a 4% inoculum size for expansion culture. Ammonia water was used as a neutralizing solution, the pH was kept constant at 5.5, and the culture was carried out at 37°C for 15 h to obtain a fermentation broth. The bacterial sludge was harvested by centrifugation at 4-10°C for 20 min, and the bacterial sludge was thoroughly mixed with a lyoprotectant at a mass ratio of 1:1.2 to prepare an emulsion. After vacuum freeze-drying, freeze-dried bacterial powder of Lactobacillus casei WFP20 was obtained. The bacterial powder production results are shown in Table 2. The indicators of each process stage showed that Lactobacillus casei WFP20 had excellent production economic benefits.

[0063] Table 2. Production results of Lactobacillus casei WFP20 freeze-dried powder

[0064]

[0065] 2. Storage stability of bacterial powder

[0066] Take 1 g of freeze-dried powder of Lactobacillus casei WFP20 and put it into a double-layer aluminum foil bag, vacuum seal it, and store it at a constant temperature of 37°C and 43% RH. Samples were taken at 0, 15, 30, 45, 60, 75 and 90 days respectively, and the number of viable bacteria was determined according to GB 4789.35-2016 "National Food Safety Standard Food Microbiology Test Lactic Acid Bacteria Test". The survival rate of the powder was as follows: Figure 5 As shown in the results, Lactobacillus casei WFP20 has good storage stability, with a bacterial powder survival rate of 43.07% at 90 days, indicating that the strain has high storage stability, can tolerate higher storage temperatures, and can be stored for a long time, providing relatively loose application conditions for this strain in all aspects of production, storage, transportation, sales and even consumption.

[0067] Example 4, Lactobacillus casei WFP20 produces yogurt beverage

[0068] Weigh 180 g skim milk powder, 13.5 g concentrated whey protein, and 45 g glucose, add water to 900 g and dissolve (55 ° C, 15 min), homogenize (60-65 ° C, 25 MPa), adjust the volume to 1000 mL, sterilize (95 ° C, 240 min), cool to 37 ° C, and press 3 × 10 7 CFU / g was inoculated with Lactobacillus casei WFP20, fermented to 550°T for emulsion breaking, and homogenized (15-20 MPa) for later use. 4 g of sodium carboxymethyl cellulose, 3 g of pectin, and 1 g of sodium citrate were weighed and dissolved in water (85°C, 40 min), then mixed evenly with 9 g of starch, 400 g of white sugar, and 1000 g of yogurt. 23.91 g of citric acid was added to adjust the acidity to 220°T, and the volume was fixed to 4000 mL. The mixture was hot-filled (95°C, 30 s) and sterilized in a spray tunnel (85-87°C, ≥16 min) to prepare a yogurt beverage.

[0069] The yogurt beverage produced by Lactobacillus casei WFP20 has a delicate taste and rich flavor. It can be stored at 37℃ for 180 days without water precipitation (such as Figure 6 Under the same conditions, compared with Chr. Hansen's commercially available yogurt starters Lactobacillus casei 01 and Lactobacillus casei 431, Lactobacillus casei WFP20 produced the highest acid production. This effectively reduced the damage to the system caused by acid addition during the blending process, minimizing the centrifugal sedimentation rate and achieving the most stable product structure.

[0070] Table 3. Centrifugal sedimentation rate of yogurt beverage containing Lactobacillus casei WFP20

[0071]

[0072] Example 5: Effects of Lactobacillus casei WFP20 on fecal organic acid content, bifidobacterium count, and constipation

[0073] 1. Test samples

[0074] 1g / bag solid beverage containing Lactobacillus casei WFP20 (maltodextrin 0.39g, galacto-oligosaccharide 0.32g, inulin 0.17g, xylitol 0.1g, Lactobacillus casei WFP20 0.02g) and placebo were used as test samples. The Lactobacillus casei WFP20 content in the samples was not less than 1.5×10 10 CFU / g, the placebo did not contain Lactobacillus casei WFP20, and the other formulas were consistent with the test samples.

[0075] 2. Research plan and intake dosage

[0076] Forty commercially available clean-grade rats were gavaged with compound diphenoxylate to establish a constipation model. The commercially available compound diphenoxylate was ground into powder and administered orally at a dose of 15 mg / kg to the rats after a 12-hour fast, once daily for six weeks. Successful modeling was determined by a significant reduction in bowel movement frequency, pellet count, or dry powdered feces. The constipation experimental mice were randomly divided into two groups and double-blind, placebo-controlled crossover method was used. The 63-day experimental period included 14-day no intake period, 14-day intake period (test sample, placebo), 21-day no intake period, and 14-day intake period (test sample, placebo). The experimental design is shown in Table 4. The comparison method was as follows: 1st week of sample intake vs. 1st week of no intake, 1st week of sample intake vs. 1st week of no intake, 1st week of sample intake vs. 1st week of placebo intake, 1st week of sample intake vs. 1st week of placebo intake, 2nd week of placebo intake vs. 2nd week of placebo intake, 1st week of placebo intake vs. 2nd week of no intake, 1st week of placebo intake vs. 2nd week of no intake, 1st week of placebo intake vs. 1st ... During the intake period, the experimental mice were required to maintain normal feeding methods, and the sample dosage for the experimental mice was 1g / d.

[0077] Table 4. Experimental design

[0078]

[0079] 3. Research methods and results

[0080] (1) HPLC determination of organic acids in feces

[0081] Take 2 g of fecal sample, add 100 mL of extraction solution (5.91 g / L citric acid, 2.38 g / L disodium hydrogen phosphate, 500 mL / L acetonitrile), vortex and shake for 60 seconds, ultrasonically extract for 15 minutes, centrifuge at 10°C and 8000 rpm for 10 minutes, and prepare the supernatant with ultrapure water to a 20 mg / mL sample solution. Filter with a 0.22 μm filter membrane for use.

[0082] Accurately weigh 10 mg each of acetic acid, propionic acid, butyric acid, isobutyric acid, succinic acid, lactic acid, formic acid, valeric acid, and isovaleric acid, dissolve them in ultrapure water and dilute to 100 mL in a brown volumetric flask to prepare a 100 mg / L mixed standard solution. Then, prepare the organic acid mixed standard solution into organic acid mixed standard solutions with concentrations of 0.01, 0.05, 0.10, 0.50, 1.00, and 5.00 mg / mL, filter through a 0.22 μm filter membrane, and continuously inject for determination.

[0083] The chromatographic column was an Agilent ZORBAX SB-C18 liquid chromatography column, the mobile phase was 15 mM perchloric acid and 7% acetonitrile solution, the flow rate was 1.0 mL / min, the column temperature was 42°C, the injection volume was 5 μL, the elution was isocratic, and the UV detector wavelength was 210 nm.

[0084] The experimental results are shown in Table 5. After 14 days of ingestion of the Lactobacillus casei WFP20 sample, the fecal acetic acid and isovaleric acid levels of the experimental group were significantly higher than those of the non-intake group and the placebo group. The propionic acid level in the experimental group was also significantly higher than that of the non-intake group. These results suggest that Lactobacillus casei WFP20 can promote the production of organic acids in the intestine. These results suggest that Lactobacillus casei, as a probiotic, may produce short-chain fatty acids, lower intestinal pH, promote intestinal motility, and thus reduce intestinal transit time, potentially improving constipation.

[0085] Table 5. Concentration of organic acids in feces

[0086]

[0087]

[0088] * indicates a significant difference compared with the non-intake period (p < 0.05); # indicates a significant difference compared with the placebo (p < 0.05)

[0089] (2) Determination of relative expression of Bifidobacterium in feces

[0090] qPCR uses fluorescently labeled oligonucleotides in DNA amplification reactions. The changes in fluorescence signals are used to monitor the amount of amplified product in each cycle of the polymerase chain reaction (PCR) in real time. The internal reference method is used to perform qualitative or quantitative analysis of specific DNA sequences in the sample being tested. The procedure is as follows:

[0091] 1) Total RNA from stool was extracted according to the instructions of the Shanghai Bio-Tech Trizol kit;

[0092] 2) Bifidobacterium-specific primers were designed and synthesized by Shanghai Sangon Biotechnology Co., Ltd.;

[0093] 3) Dilute 1 μL of RNA from step 1 50-fold in 491 μL of RNase-free water. Mix thoroughly and measure RNA concentration and purity using a microspectrophotometer. A ratio of 1.9 to 2.1, used as an indicator for nucleic acid detection, indicates high RNA purity and is suitable for subsequent experiments.

[0094] 4) RNA was reverse transcribed into cDNA according to the instructions of the Nanjing Novozymes HiScriptll All-in-one RT SuperMixPerfect for qPCR kit, with a reaction temperature of 50°C for 15 min and 85°C for 5 s. The obtained cDNA was sealed and stored at -30 to -15°C.

[0095] 5) The qPCR reaction system (20 μL) and conditions were performed according to the instructions for the Novozymes Taq Pro Uninersal SYBR qPCR Master Mix: 2× Taq Pro Uninersal SYBR qPCR Master Mix, 10 μL; 0.4 μL each of 10 μmol / L upstream and downstream primers; 2 μL of template (cDNA); and the total was made up with sterile ultrapure water. The qPCR reaction conditions are shown in Table 6.

[0096] Table 6. qPCR amplification conditions

[0097] Stage 1 Pre-denaturation Reps:1 95℃ 30sec Stage 2 cyclic reaction Reps:40 95℃ 10sec 60℃ 30sec Stage 3 Melting curve Reps:1 95℃ 15sec 60℃ 60sec 95℃ 15sec

[0098] 6) Using Gapdh as the internal reference gene, according to formula 2 -△△CT Calculate the relative expression of target gene mRNA

[0099] Figure 7 Shows qPCR amplification curve C T The value is 21 to 28, and the melting curve has a single peak, indicating that C TThe values ​​were valid, with no nonspecific amplification products. The experimental results are shown in Table 7. After 7 days of ingestion of the Lactobacillus casei WFP20 sample, the relative expression of Bifidobacterium in the experimental group was significantly higher than that in the non-intake period, while the relative expression of Bifidobacterium in the placebo group was significantly higher than that in the non-intake period. This may be related to the ingestion of galacto-oligosaccharides and inulin, which promote the growth of intestinal Bifidobacteria. After 14 days of ingestion of the Lactobacillus casei WFP20 sample, the relative expression of Bifidobacterium in the experimental group was significantly higher than that in the non-intake period and the placebo group. Both indicate that the intake of Lactobacillus casei WFP20 can significantly increase the number of Bifidobacteria in the intestine. Numerous studies have shown that the expression of beneficial bacteria such as Bifidobacteria, Lactobacillus, and anaerobic bacteria is downregulated in people with constipation compared to healthy people. Furthermore, Bifidobacteria and Lactobacillus can increase the production of short-chain fatty acids and improve constipation symptoms. Therefore, the experimental results suggest that Lactobacillus casei may indirectly increase the content of organic acids while increasing the number of Bifidobacteria, which is consistent with the experimental results in Table 1, and both have the effect of alleviating constipation.

[0100] Table 7. Relative expression of Bifidobacterium in feces

[0101]

[0102] * indicates a significant difference compared with the non-intake period (p < 0.05); # indicates a significant difference compared with the placebo (p < 0.05)

[0103] (3) Defecation frequency measurement

[0104] The results are shown in Table 8. After 7 days of consumption of the Lactobacillus casei WFP20 sample, the experimental group had significantly higher weekly bowel movement frequency and days with bowel movements than those without consumption. After 14 days of consumption of the Lactobacillus casei WFP20 sample, the experimental group had a significantly higher weekly bowel movement frequency than those without consumption and significantly higher than those in the placebo group. The experimental group also had significantly higher days with bowel movements than those without consumption. These results suggest that Lactobacillus casei WFP20 significantly increases bowel movement frequency, and that 14 days of consumption is more effective in alleviating constipation.

[0105] Table 8. Defecation frequency in different experimental groups

[0106]

[0107] * indicates a significant difference compared with the non-intake period (p < 0.05); ** indicates a very significant difference compared with the non-intake period (p < 0.01); #

[0108] Indicates that the sample is significantly different from the placebo (p<0.05)

[0109] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A strain of Lactobacillus casei ( Lacticaseibacillus casei )WFP20, characterized in that The Lactobacillus casei WFP20 was deposited in the Guangdong Provincial Microbial Culture Collection Center on June 30, 2022, with the deposit number GDMCC No. 62586.

2. Application of Lactobacillus casei WFP20 according to claim 1 in preparing a medicine having an effect on improving constipation.

3. A composition comprising the Lactobacillus casei WFP20 of claim 1.

4. The composition according to claim 3, characterized in that The composition is a composite bacterial agent containing Lactobacillus casei WFP20 and Bifidobacterium.

5. The composition according to claim 3, characterized in that The number of viable cells in freeze-dried Lactobacillus casei WFP20 is not less than 3×10 7 CFU / g.

6. The composition according to claim 3, characterized in that Contains pharmaceutically acceptable carriers or excipients.

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