CN111424003A - Lactobacillus casei and application thereof in preventing and treating chronic gastroenteritis - Google Patents

Lactobacillus casei and application thereof in preventing and treating chronic gastroenteritis Download PDF

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CN111424003A
CN111424003A CN202010424344.0A CN202010424344A CN111424003A CN 111424003 A CN111424003 A CN 111424003A CN 202010424344 A CN202010424344 A CN 202010424344A CN 111424003 A CN111424003 A CN 111424003A
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lactobacillus casei
lactobacillus
casei zhang
zhang
casei
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刘晓军
马杰
张凌宇
张建军
其木格苏都
杨慧娟
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Beijing Scitop Bio Tech Co ltd
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Abstract

The application takes lactobacillus separated from traditional fermented dairy product-acid mare milk as a target flora, screens out potential probiotic lactobacillus casei (L actinobacillus casei zhang) with stronger viability in an intestinal environment through an in vitro acid resistance test and a bile salt tolerance test, and proves that the lactobacillus casei has the functions of regulating intestinal flora, improving the immunity of organisms, preventing and repairing chronic gastroenteritis and relieving diarrhea and abdominal discomfort through an animal experiment.

Description

Lactobacillus casei and application thereof in preventing and treating chronic gastroenteritis
Technical Field
The application relates to the technical field of biology, in particular to lactobacillus casei and application thereof in preventing and treating chronic gastroenteritis.
Background
Chronic gastroenteritis is a chronic inflammation of the gastric and intestinal mucosa. The main clinical manifestations are anorexia, epigastric discomfort, belching, nausea, vomiting or recurrent abdominal pain, diarrhea and dyspepsia. It is classified into chronic enteritis and chronic gastritis due to its different occurrence parts.
Lactobacillus (L active acid bacteria, L AB) is a general name of a group of gram-positive bacteria capable of fermenting saccharides to produce lactic acid, and widely exists in nature, wherein the Lactobacillus is closely related to human life and widely applied to food fermentation, industrial lactic acid fermentation and one of beneficial microorganisms in the fields of medical health care.
Disclosure of Invention
One of the objectives of the present application is to provide a strain of lactobacillus casei zhang (L actinobacillus casei zhang).
The application also aims to provide the application of the lactobacillus casei zhang in preventing and treating the chronic gastroenteritis.
The application also aims to provide the application of lactobacillus casei zhang in preparing the functional health-care product for preventing and treating chronic gastroenteritis.
The fourth purpose of the application is to provide the application in the preparation of functional leaven, fermented milk, health food and animal health care products for preventing and treating chronic gastroenteritis.
The purpose of the application is realized by the following technical scheme:
the lactobacillus casei zhang (L actinobacillus casei zhang) is separated from the sour mare milk prepared by the traditional method of the union of the inner Mongolia Silibing Guo and the blue flag, and is specifically obtained by separation and screening through the following method:
taking naturally fermented mare's milk as lactobacillus separation sample, stirring in fermentation container, sucking 2m L with pipette with sterilizing sucker, sealing in small sterilized test tube (15mm × 150mm) with rubber plug, sucking 1m L, placing into screw cap and sterilized CaCO3The collected sample is placed in an ice box for cooling, kept at a lower temperature, taken back to the laboratory and placed in a refrigerator at 4 ℃ to separate the lactic acid bacteria as soon as possible.
Separating lactobacillus, sucking 1m L acid Mare milk sample with a sterilizing pipette, inoculating into 10m L sterilized Carlsbergia milk culture medium, culturing in 30 deg.C incubator until acidification and solidification, taking out when the culture medium color becomes pink, streaking with sterilizing loop on B L agar culture medium (product of Nippon Rongsha Co., Ltd.) containing 10ppm cycloheximide and 10ppm colistin sulfate, and placing into the culture medium
Figure BDA0002498121630000011
Placing in a gaspak anaerobic culture tank, culturing for 48-72 h at 30 ℃, after the bacterial colony is formed, using an inoculating loop or an inoculating needle to pick the bacterial colony, inoculating in TPY liquid, culturing for 24-48 h at 30 ℃, after the bacterial strain grows well, streaking again and inoculating in a B L agar medium, similarly placing in a condition of 30 ℃, culturing for 24-48 h, carefully observing and recording the bacterial colony morphology and gramThe morphological characteristics of the cells were stained and the catalase test was performed simultaneously. Temporarily determining gram-positive bacilli and catalase test-negative bacilli as lactobacillus, further purifying and culturing, and adopting refrigeration storage at-85 deg.C or low-temperature vacuum refrigeration storage.
The lactobacillus strain separated from the acid mare milk sample is analyzed and identified by a series of tests such as physiochemical tests, lactic acid optical rotation measurement and the like, the identified strains are respectively prepared into test bacteria liquid, 10 mu L of the bacterial suspension is absorbed and inoculated in MRS liquid culture medium with 5m L pH value of 3.5, and is cultured for 24 hours under the condition of 37 ℃, meanwhile, artificial gastric juice with 1.0m L and 9.0m L pH value of 3.0 is absorbed, wherein the artificial gastric juice preparation method comprises the steps of NaCl0.2%, pepsin (sigma) 0.35%, adjusting the pH value to 3.0 by 1 mol/L HCl, filtering and sterilizing for later use, mixing, culturing at 37 ℃, and then culturing at low cholesterol, sampling after the beginning of 0h and 3h, measuring the number of the strains by BCP agar culture medium (products of Nippon grongying research corporation), calculating the survival rate of the strains, screening the lactobacillus strain with high casas well as the acid resistance and gastric acid, further measuring the survival rate of the strains in vitro through various artificial bile acid resistance tests and bile acid resistance tests (L g).
The lactobacillus casei zhang (L actabacillus casei zhang) is a probiotic bacterium which is separated from the fermented mare milk and has good gastrointestinal digestive juice and bile salt tolerance, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, has the preservation number of CGMCC NO.5469 and is named as lactobacillus casei (L actabacillus casei) in a classification way, has the preservation time of 2011, 11 months and 18 days, has the preservation address of No. 3 of No. 1 Xilu of the North Chen of the Korean district of Beijing, and the institute of microbiology of China academy of sciences, and has the detection result of survival.
Identification of Lactobacillus, namely, the separated and purified Lactobacillus (L actinobacillus casei zhang) is identified by a method of physiochemical characteristic analysis and 16SrDNA sequence analysis, and the microbiological characteristics are shown in Table 1.
TABLE 1L biological Properties of actinobacillus casei zhang
Figure BDA0002498121630000021
Figure BDA0002498121630000031
Compared with the prior art, the lactobacillus casei (L actinobacillus casei zhang) that this application provided and relevant technical scheme has following beneficial effect with it:
the lactobacillus isolated from the traditional fermented dairy product-the acid mare milk is taken as the target flora, the potential probiotic lactobacillus casei (L actinobacillus casei zhang) with stronger viability in the intestinal environment is screened out through an in vitro acid resistance test and a bile salt tolerance test, and animal experiments prove that the lactobacillus casei has the functions of regulating the intestinal flora, improving the immunity of the organism, preventing and treating and repairing chronic enteritis, and relieving diarrhea and abdominal discomfort.
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In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a flow chart of the operation of separating, purifying and preserving lactic acid bacteria according to the present application;
FIG. 2 is the results of the effect of probiotic intervention on I L-6;
FIG. 3 is the results of the effect of probiotic intervention on I L-8;
FIG. 4 is a graph of the effect of probiotic intervention on D-L A;
FIG. 5 is a graph of the effect of probiotic intervention on TNF- α;
FIG. 6 is a graph of the effect of probiotic intervention on IBS symptom score;
FIG. 7 is a graph of the effect of probiotic intervention on quality of life index;
FIG. 8 is L results of the casein zhang intervention preventing the development of inflammatory infiltrates;
FIG. 9 is L results of casein zhang intervention to reduce serum levels of the inflammatory factor TNF- α;
FIG. 10 is L results of casein zhang intervention to reduce levels of I L-6 in serum;
FIG. 11 is L results of down-regulation of expression of multiple inflammatory proteins in the liver under intervention of casei zhang;
FIG. 12 is L results of casein zhang intervention to reduce MPO in colon tissue;
FIG. 13 is L results of casein zhang intervention to reduce TNF- α in colonic tissue serum;
FIG. 14 is L results of casein zhang intervention decreasing colon tissue I L-6;
FIG. 15 is L the results of the casein zhang intervention in preventing the formation of intestinal ulcers.
Wherein, 1-the conventional treatment group is treated for 7 days, 2-the probiotic group is treated for 7 days, 3-the conventional treatment group is treated for 28 days, and 4-the probiotic group is treated for 28 days.
Detailed Description
The application provides a lactobacillus casei zhang (L actobasilus casei zhang), which is a probiotic bacterium separated from acid mare milk and having good gastrointestinal tract digestive juice and bile salt tolerance, the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms (CGMCC NO. 5469) and is classified and named as lactobacillus casei (L actobasilus casei), the preservation time is 11 and 18 days in 2011, the preservation address is No. 3 of West Lu No. 1 of the sunward area in Beijing, and the institute of microbiology of China academy of sciences, and the detection result of the strain is survival.
EXAMPLE 1 isolation and purification of Lactobacillus in samples
The process of the isolation and purification of Lactobacillus casei in the sample is shown in FIG. 1.
Separating lactobacillus, sucking 1m L sour mare milk sample with a sterilizing straw, inoculating into 10m L sterilized litmus milk culture medium, culturing in 30 deg.C incubator until acidification and solidification are reached, taking out when culture medium color turns to pink, inoculating with sterilizingThe resulting mixture was streaked and inoculated into B L agar medium (manufactured by Nippon Rongyan Co., Ltd.) containing 10ppm of cycloheximide and 10ppm of colistin sulfate, and the resulting mixture was placed in
Figure BDA0002498121630000041
And (2) placing the mixture in a gaspak anaerobic culture tank, culturing for 48-72 h, after bacterial colonies are formed, selecting the bacterial colonies by using an inoculating loop or an inoculating needle, inoculating the bacterial colonies into TPY liquid, culturing for 24-48 h at 30 ℃, after bacterial strains grow well, streaking and inoculating the bacterial strains into a B L agar culture medium again, similarly placing the mixture in a 30 ℃ condition, culturing for 24-48 h, carefully observing and recording the morphology of the bacterial colonies and the morphological characteristics of gram staining cells, simultaneously carrying out a catalase test, temporarily determining bacillus which is gram positive and negative in the catalase test as lactobacillus, further purifying and culturing, and freezing and storing at-85 ℃ or low-temperature vacuum freezing and storing.
The lactobacillus strain separated from the acid mare milk sample is analyzed and identified by a series of tests such as physiochemical tests, lactic acid optical rotation measurement and the like, the identified strains are respectively prepared into test bacteria liquid, 10 mu L of the bacteria suspension is absorbed and inoculated in MRS liquid culture medium with 5m L PH value of 3.5, and is cultured for 24 hours under the condition of 37 ℃, meanwhile, artificial gastric juice with 1.0m L and 9.0m L PH value of 3.0 is absorbed, wherein the artificial gastric juice preparation method comprises the steps of NaCl0.2%, pepsin (sigma) 0.35%, adjusting the pH value to 3.0 by 1 mol/L HCl, filtering and sterilizing for later use, mixing, culturing at 37 ℃, and then culturing at low cholesterol, sampling after the beginning of 0h and 3h, measuring the number of the strains by BCP agar culture medium (products of Nippon grongying research corporation), calculating the survival rate of the strains, screening the lactobacillus strain with high casas well as the acid resistance and gastric acid, further measuring the survival rate of the strains in vitro through different artificial bile acid resistance tests and bile acid resistance tests (L g).
EXAMPLE 2 identification of Lactobacillus casei (L Acobacillus casei zhang)
The separated and purified lactobacillus (L actinobacillus casei zhang) is identified by the methods of physiochemical characteristic analysis and 16SrDNA sequence analysis, and the microbiological characteristics are shown in Table 1.
TABLE 1L biological Properties of actinobacillus casei zhang
Figure BDA0002498121630000051
From 50 strains of lactobacillus separated and identified from the acid mare milk sample, 1 strain of lactobacillus with extremely strong acid resistance, namely lactobacillus casei (L Acobacillus casei zhang), is obtained by screening through a growth test in an MRS liquid culture medium with the pH value of 3.5 and a survival rate measurement in artificial gastric juice with the pH value of 3.0.
Example 3 acid resistance of Lactobacillus casei (L Acobacillus casei zhang) and its Artificial gastrointestinal fluid Analysis of tolerance of
L Acrobacillus casei zhang strain was subjected to growth test in MRS liquid medium at pH3.5 and viability measurement in artificial gastric juice at pH3.0, and the results are shown in Table 2.
TABLE 2L growth of actinobacillus casei zhang in artificial gastric juice at pH3.5 and pH3.0
Figure BDA0002498121630000061
Note: the number of bacteria in the table is the average of two experiments.
The results show that the lactobacillus casei (L actinobacillus casei zhang) has strong tolerance and acid growth resistance, can resist artificial gastrointestinal digestive fluid, particularly artificial gastric juice with the pH value of 2.0 in skim emulsion, and can resist the concentration of bile salt of 0.6%.
In addition, oral administration is the main way for the probiotics to enter the intestinal tract, so that the probiotics must be influenced by hydrochloric acid in the stomach and bile salt with high concentration in the small intestine before entering the large intestine, the probiotics can play a role only by strains with high activity rate in hydrochloric acid and bile salt, and meanwhile, the number of stable live bacteria in the product or the number of live bacteria in a microecological preparation is the key for the probiotics to play a role, so that the application requirement unit of the product reaches a certain number of live bacteria so as to meet the standard of effective treatment dosage.
Therefore, the strain is suitable for producing functional fermented dairy products, and can also be used as a functional food additive or a health-care preparation to be applied to various aspects of regulating immune functions.
Experimental example 1L evaluation of Acobacillus casei zhang intervention in the treatment of IBS patients
72 cases of IBS patients are randomly divided into a conventional treatment group and a probiotic group, and each group comprises 36 patients, the conventional treatment group is administered with conventional basic treatment (15 ml of lactulose oral liquid × 3/d, 3g of montmorillonite × 3/d, and 0.1g of maleic acid-fermented water stop of abdominal pain patients × 3/d), and the probiotic group is added with L actinobacillus casei zhang 3 × 10 of the application for oral administration while the basic treatment is carried out9CFU/g, 2 g/day, treatment 28 days. Collecting feces samples of two groups of patients treated for 0, 7 and 28 days, and detecting the intestinal flora condition of the patients.
As shown in FIGS. 2 to 7, interleukin-6 (I L-6), I L-8, TNF- α and D-lactate (D-L0A) were significantly increased in blood of IBS patients for 28 days of treatment, three serum markers of the probiotic group were significantly decreased, I L-6 (35.14 → 29.92pg/m L), D-L A (28.61 → 22.87. mu. mol/L), TNF- α (49.97 → 38.98pg/m L), and I L-8 were not significantly changed.
The IBS symptom score (IBS symptoms) is obviously reduced in a probiotic group (253 → 154), the quality of life score (Qualityof life scores) is obviously improved (69 → 77), and no obvious change exists in a control group, D-lactic acid (D-L A) is obviously and positively correlated with the IBS symptom score and is obviously and negatively correlated with the quality of life index, D-L A is a metabolite of intestinal microorganisms, when the intestinal mucosa is damaged, the permeability of the intestinal barrier is increased, and D-L A enters the blood, and the index reflects the integrity of the intestinal mucosa.
Intestinal microbial analysis shows that the content of bacteroides (25.89%), clostridium (9.14%), eubacterium (8.32%), Faecalibacterium (5.77%), prevotella (5.37%) and escherichia coli/shigella (5.23%) in intestinal flora of IBS patients is higher. Wherein Escherichia coli/Shigella occupies a considerable proportion, and the content of the bacteria in normal population is less than 1%.
The research finds that the species abundance index and diversity index of the intestinal flora of the probiotic group patient are obviously higher than those of the control group. The probiotics are added for treatment, so that the abundance and diversity of the intestinal flora of the patient can be increased, and the intestinal flora of the patient can be healthier.
After eating the probiotic L, namely the actinobacillus casei zhang for 7 days, the content of clostridium coli is obviously reduced, after eating the probiotic 28 days, the fecal sterol-producing eubacteria and the fecal rosella in intestinal flora of IBS patients are obviously increased, and the content of escherichia coli, bacteroides massiliensis and shigella dysenteriae is obviously reduced.
Clostridium coli is the main pathogenic bacterium causing ulcerative enteritis; escherichia coli/shigella dysenteriae are known harmful bacteria of the human body; the content of bacteroides massiliensis in the body of the colorectal cancer patient is high.
The fecal sterol producing eubacterium can decompose cholesterol into fecal sterol which can not be absorbed, and the fecal sterol is discharged out of the body along with feces, so that symptoms of enteritis patients are relieved; the ralstonia faecalis has butyric acid and bacteriocin producing effects, and can inhibit bacillus and clostridium.
Experimental example 2L repair of rapamycin mediated enteritis by actinobacillus casei zhang
Taking a rapamycin-mediated enteritis mouse model as an example, L actinobacillus casei zhang of the application is prepared into 2.0 × 109CFU/g of microecologics, and the experimental results show that when the microecologics are fed to mice: rapamycin alone may be usedIncreasing serum TNF- α levels in aged mice, no difference in I L-6 levels from aged control mice, and inflammatory infiltrative regions of the intestinal tract, see FIGS. 8, L, the combined use of casei zhang and rapamycin reduced the levels of inflammatory factors TNF- α and I L-6, see FIGS. 9 and 10, and prevented the occurrence of inflammatory infiltrations.
L, the casei zhang intervenes in repairing the intestinal injury caused by the rapamycin and uses the rapamycin as a target protein of the mtor, so that the expression of the liver mtor is reduced, as shown in figure 13, while the improvement of the intestinal flora can obviously influence the regulation effect of the rapamycin on the mtor, the anti-inflammatory effect of the probiotics is not through an AMPK pathway but through a C L C3-NF-Kappa B pathway, and in addition, the expression of a chloride ion related protein CFTR is obviously increased.
Experimental example 3L actinobacillus casei Zhang for preventing and treating DSS (direct sequence spread spectrum) -mediated chronic enteritis in mice
The mouse with DSS mediated chronic enteritis as a model is used for carrying out experiments, and L actinobacillus caseizhang is prepared into 2.0 × 109The results show that DSS induction can increase the expression levels of inflammatory factors MPO, TNF- α and I L-6 in colon tissues of mice in a model group IBD, while L. casei zhang intervention obviously reduces the expression levels of MPO and I L-6, and has no obvious difference with a healthy control group.
As shown in FIGS. 12 to 14, DSS induction increased colon MPO levels in IBD mice in the model group, while L Caseizhang intervention significantly reduced this level of intestinal inflammatory factor, without significant difference from the MPO levels in healthy controls.DSS induction increased serum TNF- α, I L-6, L in IBD mice in the model group, while casei zhang intervention did not significantly affect TNF- α, while L Casei zhang intervention significantly reduced I L-6, without significant difference from the normal group.
As shown in FIG. 15, the DSS model group mice showed significant IBD symptoms, destruction of the intestinal mucosa, presence of ulcerated regions L intervention of casei zhang significantly alleviated the IBD symptoms.
Other embodiments of the present application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the application being indicated by the following claims.
It will be understood that the present application is not limited to the precise arrangements described above and shown in the drawings and that various modifications and changes may be made without departing from the scope thereof. The scope of the application is limited only by the appended claims.

Claims (4)

1. The lactobacillus casei zhang (L actobacillus casei zhang) is characterized in that the lactobacillus casei zhang (L actobacillus casei zhang) is a probiotic bacterium which is separated from acid mare milk and has good gastrointestinal tract digestive juice and bile salt tolerance, and the strain is preserved in China general microbiological culture Collection center (CGMCC NO. 5469).
2. Use of lactobacillus casei zhang according to claim 1 for the prevention and treatment of chronic gastroenteritis.
3. The use of lactobacillus casei zhang according to claim 1 for the preparation of a functional health product for the prevention and treatment of chronic gastroenteritis.
4. Use of lactobacillus casei zhang according to claim 1 for the preparation of functional starter cultures, fermented milks, health foods and animal health products for the prevention and treatment of chronic gastroenteritis.
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