CN114196600A - Lactobacillus plantarum with effect of relieving irritable bowel syndrome and application thereof - Google Patents
Lactobacillus plantarum with effect of relieving irritable bowel syndrome and application thereof Download PDFInfo
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- A23C11/02—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins
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- A23C11/103—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins containing only proteins from pulses, oilseeds or nuts, e.g. nut milk
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Abstract
The invention provides lactobacillus plantarum with a relieving effect on irritable bowel syndrome, which is characterized in that the lactobacillus plantarum is an AR495 strain, is preserved in China general microbiological culture Collection center in 2017, 3 and 22 months, and has a preservation number of CGMCC No. 14004. The Lactobacillus plantarum AR495 can reduce the tryptase content in intestinal tracts, relieve the activation of mast cells, inhibit the activation of PAR2-TRPV1 receptors and recover the neuropathic pain threshold value, so that IBS symptoms are effectively improved, and the AR495 can also regulate the intestinal flora homeostasis and improve the flora imbalance condition.
Description
Technical Field
The invention belongs to the field of cargo storage, and particularly relates to lactobacillus plantarum with an effect of relieving irritable bowel syndrome and application thereof.
Background
Irritable Bowel Syndrome (IBS) is a group of functional intestinal diseases characterized mainly by abdominal pain or abdominal discomfort, accompanied by changes in bowel habits and stool characteristics, and the incidence rate is about 5.8% to 17.5%, and the pathogenesis is unclear. The pathogenesis proposed at present is intestinal motility disorder, visceral sensitivity disorder, infection, brain-intestine interaction, neuroimmune mechanism, neuroimmune endocrine dyscrasia and the like.
Visceral Hypersensitivity (VH) is one of the important pathophysiological mechanisms of irritable bowel syndrome, manifested by increased sensitivity to painful stimuli or discomfort to physiological stimuli below the pain threshold, which may be manifested clinically by abdominal pain, abdominal discomfort in patients. Recent studies have shown that Mast Cells (MC) may be involved in the development of visceral hypersensitivity. Under stress, mast cells release a variety of inflammatory mediators and cytokines including tryptase, histamine, neuropeptides, cytokines, etc. by degranulation, altering the sensory threshold of primary afferent neurons by activating adjacent nociceptive nerve fibers.
The intestinal flora plays an important role in the highly sensitive pathogenesis of the internal organs. The process is complex in pathogenesis, few drug choices exist in the current drug treatment, and certain side effects exist. As a group of active microorganisms beneficial to a host, probiotics have been widely used for adjuvant treatment of intestinal diseases due to their probiotic functions such as anti-inflammation, anti-oxidation, immunity enhancement, and micro-ecology adjustment. Moreover, the probiotics can be used for preparing related food or health care products, and the related food or health care products have better safety compared with medicines. Therefore, the development of a probiotic bacterium which can effectively improve IBS is of great significance.
Disclosure of Invention
The inventor of the invention separates a new Lactobacillus plantarum strain from yellow wine lees, namely Lactobacillus plantarum AR495, which has been preserved in China general microbiological culture collection center (No. 3 Xilu 1 Beijing Shangyang district, Beijing) in 04.07 days in 2017, and the preservation number is CGMCC No. 14004. Through pharmacological activity research, the inventor finds that the lactobacillus plantarum AR495 has good IBS (IBS) improving activity in addition to the osteoporosis improving activity (see CN 108467843A). Moreover, the lactobacillus plantarum can be used for fermenting the yoghourt, so that the lactobacillus plantarum is very suitable for preparing yoghourt products with good IBS (IBS-improving) performance.
Based on the research results, the invention provides lactobacillus plantarum with a relieving effect on irritable bowel syndrome, which is characterized in that the lactobacillus plantarum is an AR495 strain and is preserved in China general microbiological culture Collection center (CGMCC) in 2017, 3 months and 22 days, and the preservation number is CGMCC No. 14004.
The invention also provides application of the lactobacillus plantarum in preparation of a product for relieving irritable bowel syndrome. Furthermore, the product is plant yogurt, and the lactobacillus plantarum is used in the fermentation process of the plant yogurt.
Action and Effect of the invention
The lactobacillus plantarum AR495 provided by the invention can reduce the tryptase content in intestinal tracts, relieve the activation of mast cells, inhibit the activation of PAR2-TRPV1 receptors and recover the neuropathic pain threshold, thereby effectively improving IBS symptoms. In addition, AR495 can regulate intestinal flora homeostasis and improve dysbacteriosis. Therefore, AR495 can be used for preparing foods or health products for improving IBS. Furthermore, since AR495 is a kind of probiotic bacteria as lactobacillus plantarum itself, it is also possible to refer to the conventional way of using probiotics, such as fermenting yogurt, to obtain a yogurt product containing AR495, which yogurt product has the same effects of improving IBS symptoms and intestinal dysbacteriosis.
Drawings
FIG. 1 is an electromyogram signal line graph of a Wistar rat under CRD according to example 1 of the present invention.
FIG. 2 AWR schematic of Wistar rats at different CRDs.
FIG. 3 is a colon tissue micrograph of a rat according to example 3 of the present invention.
FIG. 4 is a colon mast cell count chart of rats of example 4 of the present invention.
FIG. 5 is a transmission electron micrograph of colon tissue MC of example 4 of the present invention.
FIG. 6 is a graph showing the tryptase content in colon tissue in example 5 of the present invention.
FIG. 7 is a graph of the immunohistochemistry for PAR2 and TRPV1 in rat Dorsal Root Ganglia (DRG) of example 6 of the present invention.
FIG. 8 is a photograph showing the results of Western Blot of PAR2 and TRPV1 in rat Dorsal Root Ganglion (DRG) of example 6 of the present invention.
FIG. 9 is a graph showing the differences between species groups at the level of rat fecal flora in example 7 of the present invention.
FIG. 10 is a graph of cluster analysis of abundance of rat fecal flora horizontal species in example 7 of the present invention.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is specifically explained in the following with the attached drawings.
The sources of materials referred to in the following examples are as follows: the BCA protein detection kit is purchased from Nanjing to build a bioengineering institute; rat mast cell tryptase ELISA kit was purchased from Shanghai Tong Wei biological science and technology Co., Ltd; the recombinant Anti-PAR2 antibody (ab180953) and the Anti-TRPV1 antibody (ab6166) are purchased from Abcam, the Anti-beta-actin antibody, a 12.5% gel preparation kit, RIPA lysate, a protein quantification kit and ECL luminescent solution are purchased from Biyuntan biotech company; trizol, reverse transcription kit and SYBR Green kit were purchased from TaKaRa; other reagents, such as chloral hydrate (TCA) and 4% paraformaldehyde, are available from the national pharmaceutical group chemical agents, and reagents or materials not specified are commercially available in general.
Examples1: rat irritable bowel syndrome model establishment and experimental grouping
This example is the process of establishing a rat visceral hypersensitivity model and grouping experiments.
1. Establishment of rat visceral high-sensitivity model
The rat visceral hypersensitive model of this example was established by using acetic acid enema plus restraint stress.
The basic operation of the acetic acid enema and restraint stress method is as follows:
the selection criteria for rats were Female Wistar rates, 180g, 5-6 weeks. Rats were acclimatized for 1 week without any treatment, weighed after 1 week, randomized for acetic acid enema and restraint stress procedures.
For each rat, the rat was fasted 24 hours before the experiment without water deprivation, a PC tube (8 cm from the anus) connected to a 5ml syringe was transanally inserted after intraperitoneal injection of 10% chloral hydrate (300mg/kg), 1ml of acetic acid was perfused into the colon at a concentration of 40ml/L, the rat tail was raised for 30s, the anus was pressed by hand, and then the colon was flushed with 0.01mol/L PBS1ml and returned to the cage for free access to food water.
For the rats in the experimental group, restraint stress was applied every day from the next day, the rats were restrained from the movement of the upper body and forelimbs with masking tape, the head was prevented from being scratched, and the rats were placed in a rearing cage after 2 h. All restraint stresses were performed at the same time of day (between 10 am and 12 am) with the effect of circadian and motor rhythms being minimized.
2. Rat experimental grouping
After 24 experimental rats are bred adaptively for 1 week, the experimental rats are randomly divided into 3 groups, and each group comprises 8 rats; the weight condition is considered during grouping to ensure that the weights of rats in each group before the test have no significant difference, namely 150-156 g/rat. On the basis of the above modeling operation, different experimental operations were performed for each group of rats.
The control group (referred to as control group) was operated as follows: on day 1, rats were filled with 1mL of physiological saline instead of acetic acid, and from day 9 to day 17, 500uL of physiological saline was administered in addition to the restraint stress for intragastric administration.
The operation of the visceral hypersensitive group (denoted as VH group) was: acetic acid enema was performed on day 1, then restraint stress was applied every day, and 500uL of physiological saline was administered in addition to the restraint stress from day 9 to day 17 for intragastric gavage.
The operation of lactobacillus plantarum AR495 intervention group (denoted as AR495 group) was: acetic acid enema was performed on day 1, then restraint stress was applied every day, and 500uL of AR495 bacterial suspension (10) was administered in addition to the restraint stress from day 9 to day 179CFU/ml dissolved in 0.9% normal saline) was gavaged.
Example 2: lactobacillus plantarumAR495Effect on the colonic sensitivity score of Wistar rats
This example mainly examines the effect of Lactobacillus plantarum AR495 on colon sensitivity in Wistar rats.
Rats are fasted for 12 hours before the experiment and are not forbidden to be watered, the rats are anesthetized, after an 8F catheter with an air bag is inserted into the anus (the tail end of the air bag is inserted into the anus for 7cm, and the air bag is fixed at the position 1.0cm outside the anus at the tail end of the rat), the rats are placed into a self-made transparent plastic barrel cage, only can move forwards and backwards, and can not turn around, the experiment is started after the rats are completely adapted after being stabilized for 30 minutes, each rat is subjected to balloon expansion for 3 times, the volume of each rat is 0.2mL, 0.3mL, 0.4mL, 0.5mL and 0.6mL respectively, the expansion lasts for 5min every time, and the interval is 30 seconds.
Each rat was equipped with two sets of three silver needle electrodes implanted in the extraabdominal oblique muscle 2 cm higher than the inguinal ligament. The catheter and the electrode are exposed out of the back neck of the rat and fixed at the tail of the rat. Electromyography was connected via Colorectal dilation (CRD) to record rat electromyographic activity as an Abdominal wall withdrawal reflex (AWR). Rats were first placed in clear plastic casks for several days prior to the experiment to minimize recording artifacts.
FIG. 1 is an electromyogram signal line graph of a Wistar rat under CRD according to example 1 of the present invention, and FIG. 2 is a schematic diagram of AWR of a Wistar rat under different CRD. Fig. 2 is a bar graph in which three bars under each CRD condition are a control group, a VH group, and an AR495 group in this order from left to right.
Colon sensitivity can be quantified by electromyography monitoring of signals from electromyography after colon distension in rats. The results are shown in fig. 1, the sensitivity of the rats in the VH group is significantly higher than that in the control group, which indicates that the molding is successful, and the acetic acid enema plus the restraint stress can cause visceral hypersensitivity and the pain threshold is reduced; at the same time, the sensitivity of the AR495 group was lower than the VH group, indicating that intervention of AR495 eases the sensitivity of colon expansion and increases the pain threshold. In addition, as shown in fig. 2, the sensitivity of the VH group was significantly higher than the control group at the dilated volume of 0.3-0.6mL, while AR495 was clearly lower, indicating that AR495 indeed was able to alleviate the sensitivity of colon dilatation and increase the pain threshold.
Example 3：Effect of Lactobacillus plantarum AR495 on Wistar rat Colon histomorphology
This example examines the effect of lactobacillus plantarum AR495 on colon histological morphology of Wistar rats.
FIG. 3 is a colon tissue micrograph of a rat according to example 3 of the present invention. Wherein, the colon tissue picture of each group of rats is obtained by HE staining and taking a picture under a microscope of multiplied by 200.
The symptoms of IBS are the absence of significant colonic inflammatory infiltration, and therefore colonic histological observations of rats were carried out, and the results are shown in FIG. 3, with normal colonic mucosal morphology, intact epithelial structure and crypt, abundant goblet cells, tightly and orderly arranged villi, no inflammatory cell infiltration or mucosal damage, and no significant congestion and edema. The result shows that the rat does not have colonic inflammatory infiltration after the model is made, and eliminates symptoms such as abdominal pain caused by visceral hypersensitivity caused by inflammation.
Example 4：Effect of Lactobacillus plantarum AR495 on Wistar rat colonic Mast Cells (MC)
This example examines the effect of Lactobacillus plantarum AR495 on Wistar rat colonic Mast Cells (MC).
FIG. 4 is a colon mast cell count chart of rats of example 4 of the present invention. The method for counting colon mast cells adopts the prior art, and is not described in detail herein.
MC are mainly distributed around small blood vessels and intestinal muscular layer plexus of colon mucosa and submucosa, as shown in figure 4, the MC count of rat colon of VH group is higher than that of control group, and the difference has statistical significance (P < 0.05); the MC count of AR495 group was lower than that of VH group, but was not significantly different from that of control group. This result indicates that the intervention of AR495 allowed the MC level to be reduced and returned to normal levels.
FIG. 5 is a transmission electron micrograph of colon tissue MC of example 4 of the present invention.
In order to further study the change of mast cells in the internal hypersensitivity, the transmission electron microscope technology is adopted to observe the MC and the degranulation condition of the cells. As shown in FIG. 5, the MC of the rats in the control group are mainly distributed in the intrinsic layer of the intestinal mucosa, and the cytoplasm contains a small amount of particles with higher electron density; the MC of the VH group has more granules in cytoplasm and unequal electron density, interparticle membranes or granules and cell membranes are fused to form a granule removal pipeline, the content is swelled and enlarged and released to the outside of cells through the pipeline, and meanwhile, the electron density is reduced to form vacuoles. Compared with the VH group, the MC number of the AR495 group is reduced, and the degranulation phenomenon is obviously relieved.
Example 5：Effect of Lactobacillus plantarum AR495 on tryptase content in the colon of Wistar rats
This example examines the effect of AR495 on tryptase content in the colon of Wistar rats.
The current research shows that the number of colon mucous membrane MC of IBS patients is increased, after activation, a large amount of tryptase is secreted, the excitation of sensory neurons in the intestinal tract is mediated, the internal and external change of the osmotic pressure of cells in the intestinal tract is induced, and the sensitivity of the intestinal tract is enhanced; meanwhile, tryptase can widen epithelial cell gaps of colon mucous membranes, further aggravate the activation of mast cells and promote the visceral sensitivity of IBS patients.
FIG. 6 is a graph showing the tryptase content in colon tissue in example 5 of the present invention. In FIG. 6, a is VH group, b is control group, and c is AR495 group.
In this example, the content of tryptase in colon contents of rats in the control group, the VH group and the AR495 group was measured by ELISA kit, and the results are shown in fig. 6. It can be seen that the number of MC in the rats in the VH group is increased, the activation rate is increased, the secretion of tryptase is obviously increased, and the difference of the tryptase content is statistically significant compared with the control group (P < 0.05). Compared with the VH group, the content of tryptase in the AR495 group is obviously reduced, which indicates that the AR495 intervention can also reduce the visceral sensitivity.
Example 6：Lactobacillus plantarum AR495 to Wistar rat Dorsal Root Ganglion (DRG))Effect of PAR2 and TRPV1 in Medium
This example examines the effect of AR495 on PAR2 and TRPV1 in the Dorsal Root Ganglion (DRG) of Wistar rats.
PAR2 and TRPV1 on Dorsal Root Ganglion (DRG) neuronal cells play important roles in visceral pain signaling. Protease-activated receptors2 (PAR 2) are G protein-coupled receptors that can be activated by specific serine proteases such as trypsin and tryptase and other unknown proteolytic enzymes, and can be involved in many pathophysiological processes such as inflammation, pain and repair after activation. PAR2 has high expression in sensory neurons, and can enhance the activity of capsaicin receptor subtype 1 (TRPV 1) after activation, thereby participating in physiological processes such as pain sense integration and the like. TRPV1 is an ion channel receptor that reaches sensory nerve endings, and the sensitized receptor reacts with nociceptive neurons, causing the release of sensory nerve endings peptides that induce neuropathic pain hypersensitive states.
FIG. 7 is a photograph of immunohistochemistry for PAR2 and TRPV1 in rat Dorsal Root Ganglion (DRG) of example 6 of the present invention, and FIG. 8 is a photograph of Western Blot results for PAR2 and TRPV1 in rat Dorsal Root Ganglion (DRG) of example 6 of the present invention. FIG. 7 is a graph showing the results of fluorescence microscope observation of each immunohistochemical staining pattern, wherein a is a PAR2 immunohistochemical staining pattern of a control group, b is a PAR2 immunohistochemical staining pattern of a VH group, and c is a PAR2 immunohistochemical staining pattern of an AR495 group; d is TRPV1 immunohistochemical staining pattern of control group, e is TRPV1 immunohistochemical staining pattern of VH group, and f is TRPV1 immunohistochemical staining pattern of AR495 group
As can be seen from FIGS. 7 and 8, the expression of both protein receptors was low in the rats of the control group, while the expression of PAR2 and TRPV1 receptors was significantly increased in the dorsal root ganglion of the VH rats, indicating that tryptase in the colon activates the colonic plexus, further inducing the receptor expression in DRG, and lowering the neuropathic pain threshold. After the prognosis of AR495 stem, PAR2 receptor expression was significantly attenuated, activation of TRPV1 ion channel was reduced, and neuropathic pain threshold was restored.
Table 1 below shows the results of PAR2 and TRPV1 protein expression in rat DRG of example 6 of the present invention, wherein the protein expression levels were obtained by gray-scale scanning according to FIG. 8 and are expressed in terms of resolution. As can be seen from fig. 8 and table 2, PAR2 and TRPV1 protein expression was significantly reduced in rat L6-S1 DRG of AR495 group relative to VH group.
TABLE 1 PAR2 and TRPV1 protein expression in rat L6-S1 DRG
Note: p <0.05 VS control group, P < 0.01VS control group
Examples7: lactobacillus plantarumAR495To pairVHStructural modulation of intestinal flora in rats
In this example, the effect of l.plantarum AR495 on the intestinal flora of VH rats was examined by 16S rDNA high-throughput sequencing technology.
FIG. 9 is a graph showing the differences between species groups at the level of rat fecal flora in example 7 of the present invention. Fig. 9 is a bar graph in which each species contains three bars, which are a control group, a VH group, and an AR495 group in order from top to bottom.
Carrying out microbial diversity analysis on rat feces of a control group, a VH group and an AR495 group, carrying out species abundance clustering analysis at a family level, selecting species with the relative abundance of the first 15, carrying out species clustering, drawing a histogram to present community species composition information, reflecting different treatment groups through color change, and obtaining a graph 9 by reflecting the difference abundance among the groups of different species at the family level through the length of the histogram. As shown in fig. 9, the rat fecal flora contains species such as lactobacillus, muribacteriaceae, Peptostreptococcaceae, Erysipelotrichaceae and Lachnospiraceae in high abundance, and is a main family of rat fecal flora, and no significant change exists. Acylaminococculaces, which ferment amino acids, particularly glutamic acid, in vivo, acetic acid is the main metabolite, and lactic acid and butyric acid are not produced, are significantly elevated in the VH group.
FIG. 10 is a graph of cluster analysis of abundance of rat fecal flora horizontal species in example 7 of the present invention.
As shown in fig. 10, species abundance clustering analysis performed at the species level found that AR495 could be significantly enriched with Lactobacillus _ acetylophilus and Bifidobacterium _ pseudomonas. Wherein the Bifidobacterium can produce acetic acid, propionic acid, butyric acid and lactic acid to inhibit the growth of harmful bacteria, resist the infection of pathogenic bacteria, synthesize vitamins required by human body, and promote the absorption of minerals by human body. The results show that the intervention of AR495 can regulate the fecal flora structure of VH rats and improve the dysbacteriosis of the VH rats to a certain extent.
As described above, according to example 2, AR495 has an effect of alleviating the sensitivity of colon expansion and increasing the pain threshold; as can be seen from example 3, AR495 is able to alleviate symptoms in the absence of significant colonic inflammation, i.e. is able to act against IBS; according to the embodiment 4, the AR495 can reduce the MC content and restore the MC content to a normal level, and can also obviously relieve the degranulation phenomenon; according to example 5, AR495 can reduce the content of tryptase in colon, thereby reducing the sensitivity of viscera; the results of example 6 indicate that AR495 was able to decrease expression of PAR2 receptor, decrease activation of TRPV1 ion channel, thereby restoring neuropathic pain threshold; the results of example 7 show that AR495 is also able to improve dysbacteriosis in the intestinal tract.
In conclusion, the AR495 provided by the invention can reduce the tryptase content in the intestinal tract, relieve the activation of mast cells, inhibit the activation of PAR2-TRPV1 receptors and recover the neuropathic pain threshold, thereby effectively improving the symptoms of IBS. In addition, AR495 can regulate intestinal flora homeostasis and improve dysbacteriosis. Therefore, AR495 can be used for preparing foods or health products for improving IBS. Furthermore, since AR495 is a kind of probiotic bacteria as lactobacillus plantarum itself, it is also possible to refer to the conventional way of using probiotics, for example, for fermentation to prepare yogurt, to obtain a yogurt product containing AR495, which yogurt product has the same effects of improving IBS symptoms and intestinal dysbacteriosis.
Claims (3)
1. The lactobacillus plantarum with the effect of relieving irritable bowel syndrome is characterized in that the lactobacillus plantarum is an AR495 strain, is preserved in China general microbiological culture Collection center in 2017, 3 months and 22 days, and has the preservation number of CGMCC No. 14004.
2. Use of lactobacillus plantarum as defined in claim 1 for the preparation of a product for relieving irritable bowel syndrome.
3. Use of Lactobacillus plantarum according to claim 2, wherein the product is a vegetable yoghurt,
the lactobacillus plantarum is used in a fermentation process of the plant yogurt.
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