Lactobacillus plantarum for relieving diarrhea caused by ETEC and application of lactobacillus plantarum

Abstract

The invention discloses lactobacillus plantarum for relieving diarrhea caused by ETEC and application of the lactobacillus plantarum, and belongs to the technical field of microorganisms. The lactobacillus plantarum CCFM8661 with the effect of relieving the diarrhea caused by the ETEC is screened out, and the lactobacillus plantarum CCFM8661 is specifically embodied in that the weight loss of mice with the diarrhea caused by the ETEC and the increase of the water content of excrement are relieved; the content of pro-inflammatory cytokines IL-1beta, IFN-gamma, IL-6 and IL-8 in serum is obviously reduced; the content of immune factors IgA, IgG and IgM in serum is obviously increased; sT is reduced, and the level of AQP3 is increased; jejunum injury is relieved, and jejunum villus length shortening and crypt depth increasing caused by ETEC are remarkably improved; the gene expression quantity of tight junction proteins Claudin-1, ZO-1 and Occludin in jejunum tissues and mucoprotein MUC2 is remarkably increased, and the contents of acetic acid, propionic acid, isobutyric acid and butyric acid in excrement are remarkably increased.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a Lactobacillus plantarum strain for alleviating diarrhea caused by ETEC and an application thereof. Background Art

[0002] Enterotoxigenic Escherichia coli (ETEC) is an important pathogen that causes diarrhea in humans and livestock. It is the main cause of traveler's diarrhea, usually accounting for 30-60% of the causes of disease. At the same time, ETEC is also an important pathogen that causes diarrhea in livestock, especially young livestock (newborn piglets, calves, etc.). After newborn piglets are infected with ETEC, they often die from severe watery diarrhea and rapid dehydration. The morbidity and mortality rates are both very high, which brings a huge burden to the pig industry.

[0003] ETEC produces heat-labile toxins LT and heat-stable toxins ST, which in turn change the integrity of tight junctions, induce inflammation, intestinal dysfunction, and destroy paracellular channels for ions, solutes, and water, ultimately leading to diarrhea. Traditional antibiotics such as fluoroquinolones and rifaximin are effective in preventing diarrhea caused by ETEC, but are usually only used in high-risk groups, such as patients with severe immunosuppression. They also have significant side effects, such as often causing gastrointestinal discomfort, abnormal reactions in the central nervous system, and symptoms such as headache and dizziness. Moreover, long-term use of these drugs can cause damage to the liver. At the same time, the use of these drugs is limited by cost, emerging antimicrobial resistance, and complexity of dosage, and the effectiveness of alternative methods is still under study, such as probiotics.

[0004] Probiotics are gaining more and more attention in the prevention and treatment of gastrointestinal infections. The intestinal flora is important for maintaining host health. At the same time, the intestinal microbiota is also related to the pathogenesis of diarrhea. For example, the presence of certain pathogens such as Escherichia coli and Salmonella, viruses and parasites can cause diarrhea. The effects of probiotics include relieving diarrhea of ​​various causes and preventing gastrointestinal infections and inflammatory diseases. Studies have shown that they play a preventive role in ETEC infection by regulating gastrointestinal balance and enhancing intestinal immune response, but there is still a lack of research on intestinal mucosal damage caused by ETEC and destruction of the integrity of intestinal tight junction proteins. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects of the prior art, and obtain a plant lactobacillus CCFM8661 capable of preventing diarrhea caused by ETEC through animal experiments and in vivo studies, which has the effects of significantly reducing the fecal water content of diarrheal mice, significantly increasing the body weight of diarrheal mice, repairing jejunal villi and crypts, significantly reducing pro-inflammatory factors and up-regulating immune factors in diarrheal mice, and significantly increasing the gene expression of intestinal tight junction proteins and mucins. It has a better relief effect than other plant lactobacillus reported in the literature.

[0006] In order to achieve the above object, the present invention provides a strain of Lactobacillus plantarum CCFM8661, which has been deposited in the China General Microbiological Culture Collection Center on November 29, 2011, with a deposit number of CGMCC No.5494.

[0007] After separation and screening, the strain of the invention is identified as Lactobacillus plantarum by steps including extraction of bacterial genome DNA, 16S rDNA specific primer PCR amplification, purification of amplified products, DNA sequencing, sequence comparison, etc., and is named Lactobacillus plantarum CCFM8661.

[0008] Lactobacillus plantarum CCFM8661 has the following biological characteristics:

[0009] Bacterial characteristics: milky white.

[0010] Colony characteristics: The colonies on the mMRS solid plate are white and raised with smooth edges, and are Gram-positive bacteria.

[0011] Growth characteristics: Under constant temperature and anaerobic conditions at 37°C, the culture reached the late logarithmic phase in mMRS medium after about 16 hours of cultivation.

[0012] Lactobacillus plantarum is a species in the genus Lactobacillus. The genus Lactobacillus consists of more than 150 species and subspecies, most of which are facultative anaerobes, and only about 20% of the species are obligate anaerobes. These include Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus gasseri, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus johnsonii, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus salivarius, Lactobacillus brevis, etc.

[0013] Due to the wide variety of Lactobacillus species, different species of the same genus have significant differences in morphology, physiology, metabolism and physiological functions. So far, a patent has found that a strain of Lactobacillus plantarum can improve diarrhea caused by ETEC, but there is a lack of research on immune factors in serum and jejunal pathological damage.

[0014] The invention also provides application of Lactobacillus plantarum CCFM8661 in preparing medicine for preventing and / or treating diarrhea. The preservation number of the Lactobacillus plantarum CCFM8661 is CGMCC No.5494.

[0015] In one embodiment, the drug comprises at least one of the following effects:

[0016] (1) Alleviate weight loss caused by diarrhea;

[0017] (2) Reduce fecal water content;

[0018] (3) Repair damage to the villi and crypts of the jejunum;

[0019] (4) regulating the expression of proinflammatory cytokines, Escherichia coli enterotoxins, aquaporins, and tight junction proteins;

[0020] (5) Regulate the expression of immune factors;

[0021] (6) Improve the content of short-chain fatty acids in the intestine.

[0022] In one embodiment, the viable count of Lactobacillus plantarum CCFM8661 in the drug is not less than 1×10 10 CFU / g or 1×10 10 CFU / mL.

[0023] In one embodiment, the medicine contains the Lactobacillus plantarum CCFM8661, a drug carrier and / or a pharmaceutical excipient.

[0024] In one embodiment, the drug carrier includes microcapsules, microspheres, nanoparticles and / or liposomes; the drug excipients include fillers, binders, wetting agents, disintegrants, excipients and / or flavoring agents.

[0025] In one embodiment, the drug is a microbial preparation containing Lactobacillus plantarum CCFM8661.

[0026] In one embodiment, the microbial preparation comprises a solid preparation, a liquid preparation or a semisolid preparation.

[0027] In one embodiment, the diarrhea is diarrhea caused by ETEC infection.

[0028] The present invention also provides a product for preventing and / or treating diarrhea, wherein the product contains the Lactobacillus plantarum CCFM8661.

[0029] In one embodiment, the viable count of Lactobacillus plantarum CCFM8661 in the product is not less than 1×10 10 CFU / g or 1×10 10 CFU / mL.

[0030] Beneficial effects:

[0031] The present invention confirms that Lactobacillus plantarum CCFM8661 can effectively improve symptoms such as mouse body weight and fecal water content, and can repair damage to jejunal villi and crypts. Compared with the prior art, it also has the function of regulating the homeostasis of the intestinal immune system, including significantly downregulating the levels of pro-inflammatory cytokines IFN-γ, IL-8, IL-6, IL-1β and TNF-α, and regulating intestinal short-chain fatty acids, showing a better therapeutic or preventive effect on ETEC-induced diarrhea. Therefore, probiotic foods, health products and medicines for preventing and treating intestinal inflammation can be prepared, and the method has a very broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The figure shows the changes in body weight and fecal water content of each group during the modeling period, indicating that Lactobacillus plantarum CCFM8661 significantly reduced the fecal water content of mice; in the figure: *: P<0.05, **: P<0.01, ***: P<0.001.

[0033] Figure 2 Shows HE staining of jejunal tissue (A) and changes in jejunal villi (B), crypt depth (C), and jejunal villus / crypt (D) in each group; in the figure: *: P < 0.05, **: P < 0.01, ***: P < 0.001.

[0034] Figure 3 The changes of inflammatory factors in each group indicate that Lactobacillus plantarum CCFM8661 significantly reduced inflammation; in the figure: *: P<0.05, **: P<0.01, ***: P<0.001.

[0035] Figure 4 Represents the changes in the levels of Escherichia coli enterotoxin ST and aquaporin AQP3 in each group; in the figure: *: P<0.05, **: P<0.01, ***: P<0.001.

[0036] Figure 5 It indicates that Lactobacillus plantarum CCFM8661 significantly increases the levels of intestinal tight junction proteins and mucins;

[0037] Figure 6 It indicates that Lactobacillus plantarum CCFM8661 significantly increases the level of immune factors; in the figure: *: P<0.05, **: P<0.01, ***: P<0.001.

[0038] Figure 7 It indicates that Lactobacillus plantarum CCFM8661 significantly increased the level of short-chain fatty acids; in the figure: *: P<0.05, **: P<0.01, ***: P<0.001. DETAILED DESCRIPTION

[0039] The present invention will be better understood through the following examples.

[0040] In the present invention, unless otherwise specified, "%" or percentage used to describe concentration or ratio are all weight percentages.

[0041] The present invention relates to the following culture medium:

[0042] mMRS liquid culture medium: 10 g tryptone, 10 g beef extract, 5 g yeast powder, 20 g glucose, 2 g diammonium hydrogen citrate, 5 g sodium acetate, 2 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 0.25 g manganese sulfate monohydrate, 1 mL Tween 80 and 0.5 g cysteine, add water to 1000 mL.

[0043] The mMRS solid medium is obtained by adding 1.5% agar based on the total weight of the liquid medium to the above medium.

[0044] The Lactobacillus plantarum CCFM8661 involved in the following examples has been disclosed in patent CN111869735A, and the deposit number is CGMCC No.5494.

[0045] Example 1: Lactobacillus plantarum CCFM8661 improves body weight and fecal water content in mice with ETEC-induced diarrhea

[0046] 1. Experimental Animals

[0047] Female SPF BALB / c mice (6 weeks old, weighing 15-17 g) were obtained from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., China. Mice were housed in polypropylene cages, 8 mice per group, with food and water in the cages, and the temperature (22°C) and relative humidity (50±10%) were controlled. The mice were fed with standard feed and water ad libitum.

[0048] 2. Cultivation of Lactobacillus plantarum CCFM8661

[0049] (1) Activation culture:

[0050] mMRS liquid culture medium was used and static culture was carried out in an anaerobic workstation at 37°C.

[0051] Culture goal: Take the frozen bacteria, pick a single colony in liquid mMRS liquid culture medium, and culture it statically in a 37℃ anaerobic workstation for about 24 hours to activate Lactobacillus plantarum CCFM8661.

[0052] (2) First-level training:

[0053] mMRS liquid culture medium was used and static culture was carried out in an anaerobic workstation at 37°C.

[0054] Culture objective: Transfer the activated cultured Lactobacillus plantarum CCFM8661 to mMRS liquid culture medium at a 1% inoculum volume of the culture medium and pass it for two generations.

[0055] (3) Secondary training:

[0056] The culture medium and culture conditions were the same as those for primary culture.

[0057] The primary cultured Lactobacillus plantarum CCFM8661 was transferred to 1L mMRS liquid medium at a 1% inoculum volume, and cultured in an anaerobic workstation at 37°C for about 24 hours. The cells were collected and washed twice with PBS at pH 7.4, and then resuspended in a protective agent solution (skim milk 13%, trehalose 2%, sucrose 2%). The viable count was 5×10 9 CFU / mL for subsequent experiments.

[0058] 3. Experimental methods

[0059] (1) Establishment of ETEC-induced diarrhea model in mice

[0060] On the 1st to 3rd day of modeling, streptomycin (5 g / L) was added to the drinking water for 72 hours to eliminate the normal flora of BALB / c mice. On the 4th to 8th day, the water containing streptomycin was replaced with sterile water without antibiotics, and the mice were fasted. After fasting for 18 hours, the mice were gavaged with 1.2*10 11 Each mouse was intragastrically administered with 0.2 mL of Escherichia Coli ETEC O78:K80 suspension at a concentration of CFU / mL for five consecutive days, twice a day, with an interval of 2 hours between each administration.

[0061] (2) Experimental groups and drug administration

[0062] Thirty-two BALB / c mice were randomly divided into four groups, namely, normal group (no modeling), model group (ETEC group), ciprofloxacin group, and Lactobacillus plantarum CCFM8661 group, with 8 mice in each group. The Lactobacillus plantarum treatment group was gavaged with Lactobacillus plantarum every day from 7 days before modeling to the end of the experiment, with a gavage dose of 1×10 9 CFU / 200μL / mouse / day; the normal group, ciprofloxacin group and model group were all gavaged with an equal volume of resuspended protective agent solution as a control; the ciprofloxacin group was gavaged with 200ul of ciprofloxacin at a concentration of 0.5g / 100ml every day from the first day of modeling to the end of the experiment.

[0063] During the mouse modeling period, the mice were weighed at the same time every day. One day before the mice were killed, as much mouse feces as possible were collected and the water content before and after freeze-drying was measured.

[0064] Figure 1A shows that during the entire experimental period, the weight of mice in the ETEC group decreased by 2.78%, while the weight of the control group and the ciprofloxacin group increased by 5.45% and 3.14%, respectively, which was significantly different from the ETEC group (p<0.01). The Lactobacillus plantarum CCFM8661 group significantly alleviated the weight loss caused by ETEC (p<0.05) and increased the weight of mice by 0.74%.

[0065] Figure 1 B shows that the fecal moisture content in ETEC-infected mice was approximately 1.3 times that of the control group (p<0.001) ( Figure 1 There was a significant difference in fecal water content (59.30±3.75%) in mice fed with Lactobacillus plantarum CCFM8661 compared with mice in the ETEC group (68.78±2.87%) (p<0.001), and similarly, fecal water content was significantly increased in mice treated with ciprofloxacin (63.81±4.61%) (p<0.05).

[0066] Figure 1 The results showed that the mice in the model group had reduced body weight and significantly increased fecal water content compared with the normal group. Lactobacillus plantarum CCFM8661 could effectively alleviate the symptoms, and its effect on alleviating fecal water content was better than that of ciprofloxacin, and could restore the symptoms to a level comparable to that of the control group.

[0067] Example 2: Lactobacillus plantarum CCFM8661 improves jejunal damage in mice with ETEC-induced diarrhea

[0068] The experimental method and bacterial activation are as in Example 1.

[0069] Part of the jejunal tissue was dissected, and the intestinal contents were rinsed with saline, fixed with 4% paraformaldehyde for 24 hours, dehydrated, paraffin-embedded, and sliced. Next, ultrathin sections (4 μm) were taken for hematoxylin and eosin (HE) staining. The stained sections were sealed with coverslips using neutral gum as an adhesive. A pathology section scanner was used to record microscopic photos.

[0070] ETEC-infected mice showed shortened villi and increased crypt depth, which can be used as an indicator of the degree of ETEC damage to a certain extent. The changes in villus length and crypt depth in each group were observed. The results of Lactobacillus plantarum CCFM8661 improving the jejunal changes in ETEC-induced diarrhea are as follows Figure 2 Compared with the ETEC group, Lactobacillus plantarum CCFM8661 significantly increased the villus length by 38.41% and shortened the crypt depth by 30.92%, and restored the ratio of villus length to crypt depth to a level comparable to that of the control group.

[0071] Figure 2The results show that the villus length of the model group mice was significantly shortened and the crypt depth increased compared with the normal group, and Lactobacillus plantarum CCFM8661 can effectively alleviate this trend. In the prior art, lactobacillus that can alleviate the symptoms of ETEC infection are disclosed, among which in patent CN112239739B, Lactobacillus plantarum CCFM1143 increases the villus length of mice by 17.56%, which is only 46% of the present invention; in patent CN112538441B, Lactobacillus reuteri CCFM1144 increases the villus length of mice by 34.66%, which is also not as good as the effect of the present invention.

[0072] Example 3: Lactobacillus plantarum CCFM8661 improves inflammatory factors in mice with diarrhea caused by ETEC

[0073] The experimental method and bacterial activation are as in Example 1.

[0074] The jejunal tissue was homogenized by adding PBS at a ratio of 1:9, and the supernatant was collected by centrifugation at 4000g for 15 min at 4°C and stored in a -80°C refrigerator for subsequent experiments. The levels of IL-1β, IFN-γ, IL-6, IL-8, and TNF-α in the jejunal tissue of mice after oral administration of ETEC were detected by ELISA. The total protein in the jejunal tissue was detected using a BCA kit.

[0075] The results of regulating inflammation by Lactobacillus plantarum CCFM8661 are listed in the Appendix Figure 3 According to the above indicators, compared with the normal group, ETEC increased the levels of proinflammatory cytokines (IL-1β, IFN-γ, IL-6, IL-8 and TNF-α), among which the levels of IL-6, IFN-γ and TNF-α were significantly increased (p<0.05), at least 1.71 times that of the control group ( Figure 3 ). Compared with the ETEC group, Lactobacillus plantarum CCFM8661 treatment could significantly down-regulate IFN-γ, IL-1β, IL-6, IL-8 and TNF-α (p<0.05), which were 76.99%, 49.03%, 46.30%, 71.01% and 66.43% of the modeling group, respectively.

[0076] Figure 3 The results showed that the level of inflammatory factors in the model group mice was higher than that in the normal group. Lactobacillus plantarum CCFM8661 could effectively reduce the level of inflammatory factors and its effect in alleviating inflammation was better than that of the drug ciprofloxacin.

[0077] In Chinese patent CN112239739B, the IL-6 of Lactobacillus plantarum CCFM1143 was 54.15% of the modeling group; in Chinese patent CN113234643, the TNF-α of Lactobacillus johnsonii CCFM1181 was 75.10% of the modeling group, and the IL-6 was 68.05% of the modeling group. In Chinese invention patent CN113234644, the IL-6 of Lactobacillus gesneri CCFM1182 was 58.1% of the modeling group. Compared with lactobacillus of the same genus, CCFM8661 has a better effect in reducing the pro-inflammatory factor IL-6.

[0078] Example 4: Lactobacillus plantarum CCFM8661 improves E. coli enterotoxin ST and aquaporin AQP-3 in mice with diarrhea caused by ETEC

[0079] The experimental method and bacterial activation are as in Example 1.

[0080] 24 hours after the last oral gavage, blood samples were collected from the orbital venous plexus, the blood was allowed to stand, centrifuged at 4000g for 10 min, and serum was collected by centrifugation and stored in a -80℃ refrigerator for subsequent experiments. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of ST and AQP-3 in the serum of mice after oral gavage with ETEC.

[0081] Compared with the control group, ST in the ETEC group increased significantly by 18.3% (p<0.05), and AQP-3 decreased significantly by 34.4% (p<0.05) ( Figure 4 ). Compared with the ETEC group, Lactobacillus plantarum CCFM8661 significantly reduced ST by 19.9% ​​(p<0.01), which was better than ciprofloxacin; Lactobacillus plantarum CCFM8661 and ciprofloxacin could increase the expression level of AQP-3. The content of AQP-3 in the CCFM8661 group increased by 27.62%, which was similar to the effect of ciprofloxacin.

[0082] Figure 4 The results showed that compared with the normal group, the ST level of mice in the model group was increased and the AQP-3 level was decreased. CCFM8661 could significantly reduce the ST level and increase the AQP3 level.

[0083] Compared with other prior art lactobacilli that can relieve diarrhea caused by ETEC, the present invention achieves better results. Compared with the model group, the Lactobacillus johnsonii in patent CN113234643A significantly increased the AQP-3 of mice by 20.57%; the Lactobacillus plantarum CCFM1143 in patent CN112239739B significantly reduced the ST content of mice by 13.02%, and significantly increased the AQP-3 content by 22.27%; the Lactobacillus reuteri CCFM1144 in patent CN112538441B significantly reduced the ST content of mice by 5.2%, and significantly increased the AQP-3 content by 17.71%; the Lactobacillus plantarum CCFM8661 in this patent significantly reduced the ST content by 19.9%, and significantly increased the AQP-3 by 27.62%, and its effects are all higher than those of the above patents. ST is a heat-resistant enterotoxin produced after ETEC colonizes the intestine. Its content determines the degree of intestinal damage to mice. The water channel protein AQP-3 characterizes the apparent diarrhea of ​​mice to a certain extent. Therefore, the effect of probiotics on these two indicators can effectively reflect the effect of alleviating ETEC infection.

[0084] Example 5: Lactobacillus plantarum CCFM881 improves the relative mRNA expression of ZO-1, Claudin-1, Occludin and MUC2 in the ileum tissue of mice with ETEC-induced diarrhea

[0085] Animal experiment design and strain activation refer to Example 1.

[0086] The total RNA of jejunal tissue was extracted using a kit, and 1 μL RNA was taken to determine the concentration of ileal RNA. The RNA was reverse transcribed into cDNA using HiScriptⅢAll-in-one RT SuperMix, and the obtained cDNA was stored in a -80℃ refrigerator. The target gene was amplified by real-time quantitative polymerase chain reaction (qRT-PCR). The RT-qPCR system was 2xSybGreen 5μL, forward and reverse primers 0.2μL, template cDNA was 1ug, and water was added to 20μL. The Qrt-qPCR amplification conditions were 95℃5min, 90℃30s, 60℃30s, 72℃2min, 35 cycles, and 72℃10min.

[0087] Attached Figure 5The results showed that compared with the ETEC group, Lactobacillus plantarum CCFM8661 intervention could significantly increase the relative mRNA expression of intestinal tight junction proteins Claudin-1, Occludin, and ZO-1 in the ileum of diarrheal mice (p<0.05), which increased by 28.7%, 55.47%, and 62.4%, respectively. ETEC intervention also significantly reduced the expression of MUC2 compared with the control group (p<0.05), while Lactobacillus plantarum CCFM8661 significantly increased it by 70.1% compared with the ETEC group (p<0.05).

[0088] Intestinal injury is closely related to intestinal barrier function. Tight junction proteins are important structures that constitute the intestinal epithelial barrier function. ETEC infection can reduce the expression of tight junction proteins. The results of the article (Proanthocyanidins and proiotics combination supplementation ameliorated intestinal injury in Enterotoxigenic Escherichia coli-infected diarrhea mice) showed that probiotics increased the mRNA of Claudin-1, Occludin, and ZO-1 by 85%, 79%, and 77% of the control group, respectively; the patent increased the mRNA of the control group by 102%, 100.4%, and 132.9%, respectively. Therefore, the patent CCFM8661 has a better ability to increase the expression of tight junction proteins and better alleviate intestinal injury.

[0089] Example 6: Lactobacillus plantarum CCFM8661 improves the immune factors of mice with diarrhea caused by ETEC

[0090] Animal experiment design and strain activation refer to Example 1.

[0091] 24 hours after the last gavage, blood samples were collected from the orbital venous plexus, the blood was allowed to stand, centrifuged at 4000g for 10 min, and serum was collected by centrifugation and stored in a -80℃ refrigerator for subsequent experiments. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of IgA, IgG, and IgM in the serum of mice after gavage with ETEC.

[0092] Figure 6 The results showed that the levels of immune factors in the serum of the model group mice were different from those of the normal group. Compared with the ETEC group, treatment with Lactobacillus plantarum CCFM8661 significantly increased the levels of IgA (increased by 19.4%), IgG (increased by 32.5%) and IgM (increased by 16.1%) in the serum of mice (p<0.05).

[0093] In the 2022 doctoral article "Protective Effect of Lactobacillus paracasei from Sour Mare's Milk on the Intestinal Mucosal Barrier of Mice with Diarrhea Model Induced by E. coliO8", the levels of IgA, IgM and IgG in the serum of mice in the modeling group after ETEC infection were significantly reduced (p<0.05). Therefore, the content of immunoglobulins can be used as an effective indicator to evaluate whether the effect of ETEC is alleviated. In this article, Lactobacillus paracasei had no significant effect on IgE and IgG, and it took 15 days of intervention with Lactobacillus paracasei to achieve results similar to those of CCFM8661 in this patent in increasing IgA content; in the article related to patent CN112239739B (Yue Yue, Food Funct, 2020 (11)), the study showed that CCFM1143 did not significantly increase the level of IgA in mouse serum.

[0094] Example 7: Effect of Lactobacillus plantarum CCFM8661 on the content of short-chain fatty acids in feces of mice with diarrhea

[0095] Animal experiment design and strain activation refer to Example 1.

[0096] After the experiment, the mouse feces were collected and placed in liquid nitrogen, then transferred to a -80°C refrigerator, taken out before the short-chain fatty acid content was detected, and vacuum freeze-dried. 0.05 g of freeze-dried fecal sample was accurately weighed and dissolved in 0.5 ml of saturated sodium chloride solution, soaked for 30 min, homogenized with a tissue homogenizer, 0.04 ml of 10% sulfuric acid was added, and shaken for 30 s. 1 ml of ether solution was accurately added to the fecal solution in a fume hood, shaken for 30 s, and centrifuged for 15 min (12000 rpm, 4°C), the supernatant was transferred to a centrifuge tube containing 0.25 g of anhydrous sodium sulfate, shaken evenly, and centrifuged for 15 min (12000 rpm, 4°C), the supernatant was taken to a gas chromatography-mass spectrometry volumetric flask, and the short-chain fatty acid content was detected by GC-MS.

[0097] Figure 7 The results showed that Lactobacillus plantarum CCFM8661 could significantly increase the content of acetic acid, propionic acid, isobutyric acid and butyric acid in the feces of mice with diarrhea, with the increase being 1.47 times, 1.85 times, 1.84 times and 2.64 times of that in the modeling group, respectively. It can be seen that Lactobacillus plantarum CCFM8661 can significantly increase the content of short-chain fatty acids, especially butyric acid, in the intestines of mice with diarrhea.

[0098] The regulating effect of the same genus strains on short-chain fatty acids in the prior art is not as good as that of the present invention. In Chinese patent CN112239739B, Lactobacillus plantarum CCFM1143 can significantly increase the regulation of acetic acid, propionic acid, isobutyric acid, and butyric acid by 1.45 times, 1.82 times, 1.82 times, and 1.45 times, respectively, compared with the modeling group, and its increasing effect is not as good as that of the present invention; in Chinese patent CN112538441B, Lactobacillus reuteri CCFM1144 can significantly increase the regulation of acetic acid, propionic acid, isobutyric acid, and butyric acid by 1.83 times, 1.55 times, 1.09 times, and 1. 74 times, among which propionic acid, isobutyric acid and butyric acid upregulation effects are far less than those of this patent; Lactobacillus reuteri CCFM1181 in Chinese patent CN113234643 cannot significantly upregulate the content of butyric acid and isobutyric acid, propionic acid upregulation is 1.54 times, and its effect is not as good as that of this patent; Lactobacillus gasseri CCFM1182 in Chinese patent CN113234644A significantly upregulates the content of acetic acid and propionic acid, which are 1.26 times and 0.84 times of the modeling group, respectively, and its effect is far less than that of this patent. Among the same Lactobacillus genus, Lactobacillus plantarum CCFM8661 of this patent has a better effect in increasing the content of short-chain fatty acids in mice, especially upregulating the content of butyric acid.

[0099] The above experimental results show that Lactobacillus plantarum CCFM8661 can effectively improve the diarrhea symptoms of mice, such as body weight and fecal water content. The results of ileum pathological sections showed that Lactobacillus plantarum CCFM8661 can significantly improve the pathological damage of the mouse ileum caused by ETEC, effectively increase the length of villi and reduce the depth of crypts, which is beneficial to the absorption and secretion of the small intestine. CCFM8661 can also significantly increase the content of short-chain fatty acids in the feces of diarrheal mice. ELISA results show that Lactobacillus plantarum CCFM8661 can also regulate the homeostasis of the colon immune system, including significantly downregulating the levels of proinflammatory cytokines and significantly increasing the levels of immune factors. Compared with lactobacilli of the same genus, it has better ability to increase the short-chain fatty acid butyrate, reduce the proinflammatory factor IL-6, reduce enterotoxins and increase immunoglobulins IgA, IgG and IgM.

[0100] In summary, Lactobacillus plantarum CCFM8661 can significantly improve ETEC-induced diarrhea in mice. It can be used to prepare medicines or health products for preventing and treating intestinal diarrhea, or to produce foods that are beneficial to intestinal health, such as food additives for probiotic beverages, sour soy milk, fermented jelly, fermented tea beverages or dairy products (such as yogurt, cheese products, lactic acid bacteria, milk powder), etc.

[0101] Example 8: Preparation of tablets containing Lactobacillus plantarum CCFM8661 bacterial agent

[0102] The basic steps of the specific production process are as follows: bacterial activation → expansion culture → bacterial collection → bacterial suspension preparation → freeze drying → total mixing → tableting

[0103] 1. Activation of strains: Lactobacillus plantarum CCFM8661 was inoculated at 1% of the volume of the culture medium in mMRS liquid culture medium in an anaerobic workstation at 37° C. and activated for two generations.

[0104] 2. Expansion culture: transfer the activated Lactobacillus plantarum CCFM8661 to 1L mMRS liquid culture medium at an inoculum volume of 1% based on the culture medium volume for expansion culture, and culture it statically in an anaerobic workstation at 37°C for 24 hours.

[0105] 3. Collecting bacteria and preparing bacterial suspension: After the expansion culture, collect the bacteria by low-temperature centrifugation at 4°C, wash twice with PBS (pH 7.4), and prepare 10 10 4. Freeze drying: Make bacterial powder according to the conventional freeze drying process.

[0106] 5. Total mixing: add 2% stearic acid as lubricant and 3% CMC-Na as binder based on the total weight of bacterial powder, and mix well. 6. Tableting: tabletting by tablet press according to conventional tableting process.

[0107] Example 9: Preparation of powder containing Lactobacillus plantarum CCFM8661 bacterial agent

[0108] The basic steps of the specific production process are as follows: bacterial activation → expansion culture → bacterial collection → bacterial suspension preparation → freeze drying

[0109] The steps of activating the strains, expanding the culture, collecting the bacteria, and preparing the bacterial suspension are as described above.

[0110] Freeze drying: Freeze-dried bacterial powder is prepared according to conventional freeze drying process.

[0111] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Application of Lactobacillus plantarum CCFM8661 in the preparation of a medicine for preventing and / or treating diarrhea, characterized in that: The preservation number of the plant lactobacillus CCFM8661 is CGMCC No.5494.

2. The use according to claim 1, characterized in that The drug has at least one of the following effects: (1) Alleviate weight loss caused by diarrhea; (2) Reduce fecal water content; (3) Repair damage to the villi and crypts of the jejunum; (4) regulating the expression of proinflammatory cytokines, Escherichia coli enterotoxins, aquaporins, and tight junction proteins; (5) Regulate the expression of immune factors; (6) Improve the content of short-chain fatty acids in the intestine.

3. The use according to claim 1 or 2, characterized in that: In the medicine, the viable count of Lactobacillus plantarum CCFM8661 is not less than 1×10 10 CFU / g or 1×10 10 CFU / mL.

4. The use according to any one of claims 1 to 3, characterized in that: The medicine contains the Lactobacillus plantarum CCFM8661, a drug carrier and / or a pharmaceutical excipient.

5. The use according to claim 4, characterized in that The drug carrier includes microcapsules, microspheres, nanoparticles and / or liposomes; the drug excipients include fillers, binders, wetting agents, disintegrants, excipients and / or flavoring agents.

6. The use according to claim 5, characterized in that The medicine is a microbial preparation containing Lactobacillus plantarum CCFM8661.

7. The use according to claim 6, characterized in that The microbial preparation includes a solid preparation, a liquid preparation or a semisolid preparation.

8. The use according to any one of claims 1 to 7, characterized in that: The diarrhea is diarrhea caused by ETEC infection.

9. A product for preventing and / or treating diarrhea, characterized in that The product contains Lactobacillus plantarum CCFM8661.

10. The product according to claim 8, characterized in that In the product, the number of viable bacteria of Lactobacillus plantarum CCFM8661 is not less than 1×10 10 CFU / g or 1×10 10 CFU / mL.

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