CN115517367A - Application of lactobacillus paracasei SMN-LBK in preparation of product for promoting intestinal health - Google Patents
Application of lactobacillus paracasei SMN-LBK in preparation of product for promoting intestinal health Download PDFInfo
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- CN115517367A CN115517367A CN202211496004.4A CN202211496004A CN115517367A CN 115517367 A CN115517367 A CN 115517367A CN 202211496004 A CN202211496004 A CN 202211496004A CN 115517367 A CN115517367 A CN 115517367A
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
- A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses an application of lactobacillus paracasei SMN-LBK in preparation of a product for promoting intestinal health and a probiotic product for promoting intestinal health. The probiotics is lactobacillus paracasei SMN-LBK with the preservation number of CCTCC No. M2017429. The invention discovers that lactobacillus paracasei SMN-LBK can inhibit food-borne pathogenic bacteria including staphylococcus aureus and escherichia coli, can be used for developing common foods, can be used as health foods for daily prevention of staphylococcus aureus and escherichia coli infection, and can promote intestinal health. The lactobacillus paracasei SMN-LBK can relieve the symptoms of the ulcerative colitis, and particularly, when the lactobacillus paracasei SMN-LBK, the bifidobacterium bifidum Y22 and the lactobacillus reuteri K07 are compounded for use, the three have the effect of synergistic effect, so that the ulcerative colitis can be better relieved/treated.
Description
Technical Field
The invention relates to the technical field of probiotics, in particular to application of lactobacillus paracasei SMN-LBK in preparation of a product for promoting intestinal health.
Background
Intestinal injury is a common disease of the digestive system, with the main symptoms: abdominal pain, vomiting, hematochezia. Unhealthy diet can cause gastrointestinal discomfort and long-term intestinal injury, which is a health hazard. The spicy soup, ice cream, fried food and the like are foods which are well liked by young people. Many people also have a habit of eating leftovers, harmful bacteria can be propagated when food is stored improperly, the intestinal mucosa can be damaged by the harmful bacteria, the protection of the mucosa is lost, and the intestinal tract is more easily damaged when people eat irritant food. Nowadays, society develops rapidly, and people's life rhythm accelerates, and pressure is big, and diet is irregular often, and the overeating of engorgement, intestinal disease gradually younger.
Probiotics, also known as "beneficial", or "friendly" bacteria, are a type of microorganism in the intestinal tract that aids in gastrointestinal function. There are over 400 different beneficial bacteria in the human digestive system, which inhibit the growth of harmful bacteria, and it is critical that a balance be reached between probiotic bacteria and pathogenic bacteria. If the number of beneficial bacteria is reduced or the balance is disrupted, the harmful bacteria overgrow causing diarrhea and other digestive tract symptoms. Some probiotics have been reported to have an adjuvant therapeutic effect on intestinal diseases, but the effect of different strains is specific.
Prebiotics are used by the intestinal flora most of the time they pass through the upper gut and are not digested, stimulating the growth of beneficial flora only, and not harmful bacteria with potentially pathogenic or putrefactive activity. In theory, any antibiotic that reduces harmful species and is beneficial to promote healthy species or activities may be called a prebiotic. After the prebiotics reach the colon through the digestive tract, the prebiotics can be decomposed and utilized by colon flora, the growth of the colon flora is promoted, and the prebiotics have important significance in improving intestinal microecology and promoting lipid, protein and mineral metabolism.
Many studies have shown that probiotics have an adjunctive therapeutic effect on ulcerative colitis. The probiotics play a role in reducing inflammatory factors, improving a plurality of intestinal related floras which are characteristic of constipation and colitis in a targeted manner, reducing the abundance of a plurality of harmful bacteria and maintaining the balance of the intestinal floras. However, the therapeutic effects of different strains may vary greatly. Therefore, the development of a strain with the effect of relieving or assisting in treating the ulcerative colitis is of great significance.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims at the application of the lactobacillus paracasei SMN-LBK in preparing a product for promoting intestinal health.
The invention also aims to provide a probiotic product for promoting intestinal health.
One of the purposes of the invention is realized by adopting the following technical scheme:
the application of probiotics in preparing a product for promoting intestinal health is characterized in that the probiotics is lactobacillus paracasei SMN-LBK with the preservation number of CCTCC No. M2017429.
As a preferred embodiment of the present invention, the active ingredient of the intestinal health-promoting product comprises live cells and/or inactivated cells of Lactobacillus paracasei SMN-LBK.
As a preferred embodiment of the present invention, the promoting intestinal health comprises: inhibiting food-borne pathogenic bacteria; preventing, slowing and/or treating colitis.
As a preferred embodiment of the present invention, the food-borne pathogenic bacteria include staphylococcus aureus and escherichia coli; the colitis is ulcerative colitis.
As a preferred embodiment of the present invention, in the intestinal health-promoting product, the Lactobacillus paracasei SMN-LBK is contained in an amount of 1.0X 10 9 ~1.0×10 12 CFU/g。1.0×10 9 ~1.0×10 12 CFU/g is the viable count of the probiotics.
As a preferred embodiment of the present invention, the intestinal health promoting products include foods, health products, medicines and nutritional supplements. When the product for promoting intestinal health is used for preventing, alleviating and/or treating colitis, the product does not comprise food or health care products.
As a preferred embodiment of the invention, the components of the product for promoting intestinal health further comprise lactobacillus reuteri K07 with the preservation number of CGMCC NO.15703 and/or bifidobacterium bifidum Y22 with the preservation number of CGMCC NO.15708.
The second purpose of the invention is realized by adopting the following technical scheme:
a probiotic product for promoting gut health, the promoting gut health comprising: inhibiting food-borne pathogenic bacteria; preventing, slowing and/or treating colitis; comprises lactobacillus paracasei SMN-LBK with the preservation number of CCTCC No. M2017429.
A probiotic product for promoting gut health, the promoting gut health comprising: inhibiting food-borne pathogenic bacteria; preventing, slowing and/or treating colitis; comprises lactobacillus paracasei SMN-LBK with the preservation number of CCTCC No. M2017429, lactobacillus reuteri K07 with the preservation number of CGMCC NO.15703 and/or bifidobacterium bifidum Y22 with the preservation number of CGMCC NO.15708.
As a preferred embodiment of the invention, the weight ratio of the Lactobacillus paracasei SMN-LBK, the Lactobacillus reuteri K07 and the Bifidobacterium bifidum Y22 is (2 to 5) 1:1, preferably 3:1:1.
as a preferred embodiment of the invention, the probiotic product is a probiotic freeze-dried powder, which comprises lactobacillus paracasei SMN-LBK with the preservation number of CCTCC No. m2017429, and prebiotics.
As a preferred embodiment of the invention, the prebiotics comprise maltodextrin, fructo-oligosaccharide, galacto-oligosaccharide, sodium hyaluronate, stachyose and the like, and have an effect of promoting the proliferation of Lactobacillus paracasei SMN-LBK.
As a preferred embodiment of the present invention, the probiotic product is an oral formulation, and the dosage form of the oral formulation includes oral liquid, drops, tablets, powders, capsules or granules.
Compared with the prior art, the invention has the beneficial effects that:
(1) The lactobacillus paracasei SMN-LBK can inhibit food-borne pathogenic bacteria including staphylococcus aureus and escherichia coli, can be used for developing common foods, can be used as healthy foods for daily prevention of staphylococcus aureus and escherichia coli infection, and can promote intestinal health.
(2) The invention finds that the lactobacillus paracasei SMN-LBK can relieve the symptoms of the ulcerative colitis, and particularly, when the lactobacillus paracasei SMN-LBK, the bifidobacterium bifidum Y22 and the lactobacillus reuteri K07 are compounded for use, the three have the effect of taking effect synergistically, so that the ulcerative colitis can be relieved/treated better.
Biological material preservation information:
the lactobacillus paracasei SMN-LBK has a preservation number of CCTCC No. M2017429 and is classified and named as: lactobacillus paracasei has been preserved in the China center for type culture Collection (address: eight-channel 299 Wuhan university school in Wuchan district, wuhan City, hubei province, the first attached small face, china center for type culture Collection, postal code: 430072) in 2017, 20 months, and the preservation unit is abbreviated as CCTCC.
Bifidobacterium bifidum Y22 with the preservation number of CGMCC No.15708, which is classified and named as Bifidobacterium bifidum, has been preserved in China general microbiological culture Collection center (address: beijing City, west Lu No.1 Hotel No. 3 of the sunward Chen, china academy of sciences, microbiol research institute, postal code: 100101) in 28 days 4 and 2018, and the preservation unit is abbreviated as CGMCC.
Lactobacillus reuteri K07 with the preservation number of CGMCC No.15703 is classified and named as Lactobacillus reuteri, and has been preserved in China general microbiological culture Collection center (address: beijing city NO. 3 of Xilu 1 of the Suzhou northwest of the sunward quarter, china academy of sciences microbiological research institute, postal code: 100101) in 2018, 28 months and 28 days, and the preservation unit is abbreviated as CGMCC.
Drawings
FIG. 1 is a diagram showing the resistance of two strains of Lactobacillus paracasei to simulated gastric juice according to example 1 of the present invention;
FIG. 2 is a diagram showing two strains of Lactobacillus paracasei resistant to simulated intestinal juice according to example 1 of the present invention;
FIG. 3 is a graph showing the size of inhibition zones for inhibiting the growth of Escherichia coli by two strains of Lactobacillus paracasei in example 2 of the present invention;
FIG. 4 is a graph showing the size of inhibition zones for inhibiting the growth of Staphylococcus aureus by two strains of Lactobacillus paracasei according to example 2 of the present invention.
Detailed Description
The present invention is further described below with reference to specific embodiments, and it should be noted that, without conflict, various embodiments or technical features described below may be arbitrarily combined to form a new embodiment. The raw materials, equipments and the like used in the following examples are commercially available unless otherwise specified.
In the embodiment of the invention, a simulated gastrointestinal fluid tolerance experiment and a bacteriostatic experiment are carried out on the lactobacillus paracasei SMN-LBK (preservation number is CCTCC No. M2017429), and the gastric fluid and intestinal fluid tolerance and food-borne pathogenic bacteria resistance effects of the lactobacillus paracasei SMN-LBK are examined. The functionality of the strains was determined and compared to commercially mature strains. Then, in the embodiment of the invention, animal experiments are further carried out on the microbial preparation such as lactobacillus paracasei SMN-LBK, and the microbial preparation has the advantages that the weight, fecal occult blood test, DAI activity score, open field test and weight reduction percentage of rats are tested, so that the lactobacillus paracasei SMN-LBK can accelerate the weight recovery in a rat model of TNBS-induced ulcerative colitis, reduce the pathological histological score of the rat, namely improve the inflammation condition, and determine the effect of the microbial preparation on the intestinal health. Thereby obtaining a microbial preparation with better tolerance, which can inhibit food-borne pathogenic bacteria and protect intestinal tracts.
EXAMPLE 1 acid and bile salt resistance test of Lactobacillus paracasei
The test method comprises the following steps: the influence of gastrointestinal tract environment on the lactobacillus paracasei SMN-LBK is simulated: lactobacillus paracasei SMN-LBK is activated for three generations by adopting a conventional lactobacillus culture medium and a conventional method, and is washed twice by using the same volume of sterile physiological saline for centrifugation (4 ℃,4000r/min,6 min), and then is re-suspended into a bacterial suspension. Lactobacillus paracasei l.casei-01 was used as a control strain (from hansen, denmark, purchased from hansen, beijing, trade ltd).
Based on 0.05 \8197mol/L \8197pH \8197and2.2 glycine-hydrochloric acid buffer solution, after sterilization, pepsin is added as simulated gastric juice, and according to the final system thallus concentration of 10 9 Inoculating activated liquid strains of the lactobacillus paracasei SMN-LBK and the lactobacillus paracasei L.casei-01 into the CFU/mL inoculum concentration, respectively acting for 0h, 1h and 2h at the temperature of 37 ℃, and then sampling to determine the number of viable bacteria. After treatment, GB4789.35-2016 is adopted to count live bacteria, and the tolerance of the strain to gastrointestinal fluid is analyzed.
Based on 1/15 \8197mol/L \8197pH \81976.98 phosphate buffer solution, after sterilization, trypsin is added, and according to the final system thallus concentration of 10 9 Inoculating activated lactobacillus paracasei SMN-LBK and lactobacillus paracasei L.casei-01 liquid strains into the CFU/mL inoculation amount, and acting for 0h, 1h and 2h at the temperature of 37 ℃. After treatment, GB4789.35-2016 is adopted to count live bacteria, and the tolerance of the strain to gastrointestinal fluid is analyzed.
Gastric juice preparation: based on 0.05 \8197mol/L \8197pH \8197and2.2 glycine-hydrochloric acid buffer solution, sterilizing, and adding pepsin in the amount of 0.5% to obtain simulated gastric juice. 0.05 \8197, mol/L \8197, pH \8197, 2.2 preparation of glycine-hydrochloric acid buffer solution: respectively mixing 50 \8197g, mL 8197g, 0.2 \8197g, mol/L glycine solution and 44 \8197g, mL 8197g, 0.2 \8197gand mol/L hydrochloric acid, and diluting with water to 200 \8197gand mL.
Preparing intestinal juice: based on 1/15 \8197mol/L \8197pH \8197and6.98 phosphate buffer solution, the mixture is packed into test tubes, sterilized and added with trypsin in 1.0% to simulate intestinal juice. 1/15, 8197mol/L, 8197pH 8197and 6.98 phosphate buffer solution preparation: mixing 1/15 of 8197mol/L of Na 2 HPO 4 Mixing the solution with 1/15 of 8197mol/L KH 2 PO 4 The solutions were mixed in a volume ratio of 3: 2 and shaken up.
The results are shown in fig. 1-2, where "×" indicates P <0.05 compared to the control strain.
As can be seen from FIG. 1, after the lactobacillus paracasei SMN-LBK is treated by gastric juice for 2 hours, the viable count is still kept at 6.31X 10 7 CFU/mL, higher than the control strain (P)<0.05）。
As can be seen from FIG. 2, the viable count of Lactobacillus paracasei SMN-LBK was maintained at 3.16X 10 after further treatment for 2 hours with intestinal juice 7 CFU/mL, has stronger tolerance capability.
EXAMPLE 2 experiment on Lactobacillus paracasei against food-borne pathogenic bacteria
Preparing probiotic liquid: activating lactobacillus paracasei SMN-LBK and lactobacillus paracasei L.casei-01 according to a conventional mode, inoculating a fresh MRS culture medium to culture for 16h to obtain a fermentation liquid, and adjusting the concentration of the bacteria liquid to 10 by using sterile normal saline 9 CFU/mL、10 8 CFU/mL。
Plates were prepared using LB agar medium, and 100. Mu.l of Staphylococcus aureus or Escherichia coli was applied (OD value of bacterial solution: 0.6) during the experiment. The diameter of the punched hole of the Oxford cup is 6mm, 60ul of probiotic diluted bacteria liquid (the concentration of viable bacteria of a high-dose group is 10) is added into the hole of the Oxford cup 9 CFU/mL, viable bacteria concentration of low dose group is 10 8 CFU/mL), two gradients of concentration were made for each probiotic.
The results are shown in FIGS. 3-4. The results show that the lactobacillus paracasei SMN-LBK and the lactobacillus paracasei L.casei-01 have the function of inhibiting food-borne pathogenic bacteria and can inhibit the growth of staphylococcus aureus and escherichia coli. The size of the inhibition zone is shown in table 1 below.
TABLE 1 inhibition zone size display table
As can be seen from Table 1, the inhibition zone of Lactobacillus paracasei SMN-LBK is larger than that of the control strain Lactobacillus paracasei L.casei-01.
EXAMPLE 3 preparation of microbial preparation
In the following microbial preparation:
the preservation number of the lactobacillus paracasei SMN-LBK is CCTCC No. M2017429.
The preservation number of the bifidobacterium bifidum Y22 is CGMCC No.15708.
The preservation number of the lactobacillus reuteri K07 is CGMCC No.15703
The following microbial preparation formula is composed of: 50% of probiotic freeze-dried powder (the number of viable bacteria is more than or equal to 2000 hundred million CFU/g) and 50% of prebiotics.
Microbial preparation 1: 30% of lactobacillus paracasei SMN-LBK freeze-dried powder (the number of live bacteria is more than or equal to 2000 hundred million CFU/g), 10% of bifidobacterium bifidum Y22 freeze-dried powder (the number of live bacteria is more than or equal to 2000 hundred million CFU/g) and 10% of lactobacillus reuteri K07 freeze-dried powder (the number of live bacteria is more than or equal to 2000 hundred million CFU/g); prebiotics: 20% of maltodextrin, 20% of fructo-oligosaccharide and 10% of stachyose. Mixing the above materials uniformly to obtain the final product.
Microbial preparation 2: 50% of lactobacillus paracasei SMN-LBK freeze-dried powder (the viable count is more than or equal to 2000 hundred million CFU/g), and prebiotics: 20% of maltodextrin, 20% of fructo-oligosaccharide and 10% of stachyose. Mixing the above materials uniformly.
Microbial preparation 3: lactobacillus paracasei SMN-LBK lyophilized powder in the microbial preparation 2 formulation was changed to lactobacillus paracasei l.casei-01 (derived from hansen, denmark, purchased from hansen, beijing) trade ltd), and the rest was unchanged.
Microbial preparation 4: the freeze-dried powder of lactobacillus paracasei SMN-LBK in the formula of the microbial preparation 2 is changed into freeze-dried powder of lactobacillus reuteri K07, and the rest is unchanged.
And (3) microbial preparation 5: the freeze-dried powder of lactobacillus paracasei SMN-LBK in the formula of the microbial preparation 2 is changed into freeze-dried powder of bifidobacterium bifidum Y22, and the rest is unchanged.
And (3) microbial preparation 6: and (3) replacing the lactobacillus paracasei SMN-LBK freeze-dried powder in the formula of the microbial preparation 1 with lactobacillus paracasei L.casei-01, and keeping the rest unchanged.
Example 4 ulcerative colitis rat test
1. Test method
After quarantine is qualified, 128 SPF SD rats are randomly divided into 16 blank control groups, 16 model control groups, 16 experiment A groups, 16 experiment B groups, 16 experiment C groups, 16 experiment D groups, 16 experiment E groups and 16 experiment F groups. Experiment A-F groups respectively give viable bacteria with the number of 2 multiplied by 10 in the afternoon each day 9 1 to 6 CFU/kg of microbial preparation. Saline was administered in the same manner for both the blank control group and the model control group for 34 days. Except for the blank control group, the animals of the other groups are subjected to primary, secondary and tertiary molding after d15, d22 and d29 administration respectively, the molding method is that the animals are fasted without water prohibition before molding, and 70 mg/mL TNBS ethanol solution is injected into the colon after the animals are anesthetized. During the trial, animals were observed daily for general clinical condition, weighed at the indicated times and animal faeces collected. The animal feces occult blood was measured at d1, d3, d5 after each molding and scored for Disease Activity Index (DAI). 24 h after the end of the last dose, the animals were carbon dioxide killed, the colon, rectum were isolated, photographed and their length measured. Colon tissues were HE stained for pathological changes and histologically scored.
The molding method comprises the following steps: except for the blank control group, the animals in each group were subjected to first, second and third surgical molding at d15, d22 and d29 administration, respectively. All animals before the first and second molding operations are fasted for 24 hours, all animals before the third molding operation are fasted for 48 hours, water is freely drunk, 3% sodium pentobarbital solution is injected into the abdominal cavity on the molding day, and 1.5 mL/kg body weight is used for anesthesia. After the animal is anesthetized, a catheter with the diameter of 4mm and wetted by normal saline is slowly inserted into the anus for about 8.5cm, and is slowly pulled out after staying for a plurality of seconds. After the feces brought out by the catheter are cleaned, a hose with the diameter of about 2 mm and the length of about 12 cm is inserted into the colon by about 8 cm through the anus, then 28 mg/mL TNBS ethanol solution is injected rapidly, 2.5 mL/kg body weight (the TNBS dosage after conversion is 70 mg/kg body weight), the tail of the rat is lifted, and the model forming agent is inverted for 30s to fully permeate into the intestinal cavity of the rat. The catheter is cleaned by normal saline after each use and then disinfected by 75% ethanol, so that cross contamination is prevented, the catheter is fully wetted by normal saline before use, and discomfort to animals during experiments is reduced. The hose used to administer TNBS was sterilized with 75% ethanol before each use to prevent cross-contamination.
The administration method comprises the following steps: after the animals are grouped, corresponding test samples are respectively gavaged and given to an experiment A group, an experiment B group, an experiment C group, an experiment D group, an experiment E group and an experiment F group for 1 time/day and 10 mL/kg of body weight. The blank control group and the model control group were given the same volume of saline in the same manner for 34 consecutive days.
2. Experimental system
And (2) breeding: SD rats.
Grade: SPF grade.
Number of animals purchased, sex and weight range: 128 pieces of the Chinese medicinal composition are respectively male and female, 129.3 to 143.7 g of the female, and 69.7 to 88.4 g of the male.
The actual animal numbers, sex and weight range at the beginning of the experiment were used: 128 pieces of the Chinese herbal medicine, half of male and female, 171.5 to 207.0 g of female and 130.9 to 155.9 g of male.
Source and animal certification number: purchased from Guangdong province medical experimental animal center, the animal production license number is SCXK (Guangdong) 2018-0002, the qualification number of female rat animal is 44007200072167, and the qualification number of male rat animal is 44007200072168.
The identification method comprises the following steps: the method of dyeing the fur is adopted. Rats were numbered with bromocresol green solution and stained spots were applied to the surface of the rat body at different sites to show the different numbers. Identified by rat skin staining and cage double-numbered markers.
Animal welfare: the contents and procedures related to the animal experiment related to the experiment are in compliance with relevant laws and regulations of the use and management of the experimental animals, and the welfare of the experimental animals is ensured.
Performing euthanasia: animals are killed by adopting carbon dioxide, and carcasses are temporarily stored in a carcass freezer and uniformly treated without pollution.
And (4) quarantine observation: the purchased rats were quarantined for 6 d. During the period, the animals are checked once a day, unhealthy animals are not found, and all animals are selected for experiments.
Animal feeding conditions: group breeding, 2 animals/box, breeding temperature and humidity: 20-26 ℃, 40-70%, and adopting 12h; the condition of the feeding room is always kept stable to ensure the reliability of the test result. During the test period, the animals are fed with the corresponding pellet feed according to the experimental requirements. Animals had free access to water.
3. Dose design and grouping
Dose design and grouping: after animal quarantine is finished, the animals are randomly divided into 16 blank control groups, 16 model control groups, 16 experiment A groups, 16 experiment B groups, 16 experiment C groups, 16 experiment D groups, 16 experiment E groups, 16 experiment F groups and male and female halves, and the details are shown in Table 2.
TABLE 2 dosage design and grouping case Table
4. Detecting the index
General clinical observations: animals were observed daily for general clinical condition once to the end of the trial.
Weight: body weight was weighed 1 time per day.
Collecting excrement: collecting 3-5 pieces of excrement of each group of animals every day during the test period except that the excrement of all the animals is collected at d1, d3 and d5 after the molding, independently storing the excrement of each animal by using a sterilized plastic tube, marking the corresponding animal number, and freezing and storing at-80 ℃ for later use.
Fecal occult blood test: fecal Occult Blood (OB) was detected using fecal Occult Blood (OB) reagent at d1, d3, d5 after molding.
DAI activity score: evaluation was carried out after molding d1, d3, d 5. The Disease Activity Index (DAI) is combined with the weight loss percentage, stool consistency and stool bleeding of the animals to carry out comprehensive scoring, and the total score of 3 results is divided by 3 to obtain the DAI value, namely DAI = (weight index + stool shape + bleeding condition)/3.
Percent weight loss: the weight at the end of the test minus the weight before fasting when the model was made l times, divided by the weight at the end of the test, multiplied by 100%, and scored 0 if weight gain occurs. The scores are detailed in table 3.
TABLE 3 DAI scoring criteria
Material taking: after 24 h of the last administration, the rats were sacrificed with carbon dioxide, the colon and rectum were separated by laparotomy, and the length of the colon was measured (the colon and rectum were measured together because the colon and rectum were not clearly demarcated after the molding). The intestinal lumen was cut along the mesenteric edge, washed with normal saline, blotted dry with filter paper, and photographed on a blank background. When taking a picture, a ruler is placed on one side of the visual field, and the picture is taken under the same light source condition, so that the picture can be conveniently calibrated and subjected to other processing analysis in the future.
And (3) histopathological detection: the remaining colon is fixed by 4 percent neutral formaldehyde, embedded by paraffin, stained by HE, detected and scored by pathology, and the scoring standard is shown in a table 4.
TABLE 4 Colon histopathology scoring criteria
5. Results of the experiment
During the trial, 2 animals in experiment group B died unexpectedly. No obvious abnormalities were noted in any of the other animals. The test results of the respective indices are shown in tables 5 to 12.
Table 5 influence of test samples on TNBS-induced rat ulcerative colitis model body weight: (
，g）
Note: in experimental group B, d21 and d22 were administered, n =15, and n =14 from d 23.
Table 6 effect of test samples on TNBS-induced rat ulcerative colitis model body weight: (
，g）
Note: experiment group B was given d21, d22, n =15, and from d23, n =14.
Table 7 influence of test samples on the TNBS-induced weight loss score in rat ulcerative colitis model: (
，g）
Note: experiment B group d16, d18 and d20,n=16, the dose of the drug was measured from the administration of d23,n=14; compared with a blank control group " ** ”p＜0.05。
As can be seen from tables 5-7, the weights of the model groups were lower than those of the experiment A group, the experiment B group, the experiment C group, the experiment F group and the blank group during the experiment period, which shows that the lactobacillus paracasei SMN-LBK and the lactobacillus paracasei L.casei both have the effect of promoting the weight of the mice, but the effect result of the lactobacillus paracasei SMN-LBK is more obvious. The weights of the experiment group D, the experiment group E and the model group are close to each other, which shows that the weight promoting effect of bifidobacterium bifidum Y22 and lactobacillus reuteri K07 on mice is weaker, but the results of the experiment group A show that after the lactobacillus paracasei SMN-LBK, the bifidobacterium bifidum Y22 and the lactobacillus reuteri K07 are compounded, the three components have a synergistic effect, and the weight growth of the mice can be promoted together. And the lactobacillus paracasei L.casei, the bifidobacterium bifidum Y22 and the lactobacillus reuteri K07 are compounded, so that the synergistic effect is not achieved.
Table 8 effect of test samples on stool trait scores in TNBS-induced rat ulcerative colitis model: (
）
Note: experiment B group d16, d18 and d20,n=16, the dose of the drug was measured from the administration of d23,n=14; compared with a blank control group " * ”p＜0.05，“ ** ”p＜0.01。
During the experiment, the collected feces are all frozen at minus 80 degrees in time. As can be seen from the records in Table 8, in the groups A to F, the stool character score of the group A is the lowest, and the lactobacillus paracasei SMN-LBK, the bifidobacterium bifidum Y22 and the lactobacillus reuteri K07 synergistically take effect, so that the recovery of ulcerative colitis is promoted, and the intestinal health is promoted.
TABLE 9 influence of test samples on the score for the degree of hematochezia in the TNBS-induced rat ulcerative colitis model: (
）
Note: experiment B group d16, d18 and d20,n=16, since d23 was administered,n=14; compared with a blank control group " * ”p＜0.05，“ ** ”p＜0.01，“ *** ”p＜0.001。
As can be seen from the records in table 9, in the experimental groups a to F, the stool property score of the experimental group a was the lowest, and the three of lactobacillus paracasei SMN-LBK, bifidobacterium bifidum Y22, and lactobacillus reuteri K07 were synergistically effective to promote the recovery of ulcerative colitis and promote intestinal health.
TABLE 10 Effect of test samples on the DAI score of the TNBS induced rat ulcerative colitis model: (
）
Note: experiment B group d16, d18 and d20,n=15, the dose of the drug was decreased from the administration of d23,n=14; compared with a blank control group " * ”p＜0.05，“ ** ”p＜0.01，“ *** ”p＜0.001。
As can be seen from the records in table 10, among the experimental groups a to F, the DAI score of the experimental group a was the lowest, and lactobacillus paracasei SMN-LBK, bifidobacterium bifidum Y22, and lactobacillus reuteri K07 were synergistically effective to promote recovery of ulcerative colitis and promote intestinal health.
TABLE 11 influence of the test samples on the histologic score of the TNBS-induced rat ulcerative colitis model and the length of the colon + rectum at the end of the test: (
）
Note: comparison with blank control, "+") "p＜0.05。
As can be seen from the records in table 11, among the experimental groups a to F, the histological score of the experimental group a was the lowest, and the "colon + rectum" length was the largest, and lactobacillus paracasei SMN-LBK, bifidobacterium bifidum Y22, and lactobacillus reuteri K07 were synergistically effective to promote the recovery of ulcerative colitis and promote intestinal health.
TABLE 12 Colon histopathology scoring results
Note: the blank control group and the model control group adopt t test, which is as follows: compared with the blank control group, the composition of the composition,P <0.05; the homogeneity of the variance among the model control group, the experiment A group and the experiment B group is tested by adopting one-way ANOVA and compared with each other by an LSD method.
As can be seen from the records in table 12, in the groups a to F, the colon histopathology score of the group a was the lowest, and the three of lactobacillus paracasei SMN-LBK, bifidobacterium bifidum Y22, and lactobacillus reuteri K07 synergistically acted, promoting recovery from ulcerative colitis and promoting intestinal health.
By combining the experimental results, the microbial preparation (lactobacillus paracasei SMN-LBK, bifidobacterium bifidum Y22 and lactobacillus reuteri K07) in the experiment A group can improve anxiety symptoms of mice and accelerate weight recovery in a TNBS ulcerative colitis rat model, and the pathological histological score of the rats can be reduced by long-term intake, namely the inflammation condition is improved, and the effect is better than that of the experiment B-F group. In addition, the fecal occult blood test result shows that the colon and rectum length and the histopathological score show that the colon and rectum length of the experiment group A are longest and are close to those of the blank control group, the histopathological score of the experiment group A is lowest, which shows that the microbial preparation 1 has better effect of relieving the colon injury of the mice, the intake of the compound strain (lactobacillus paracasei SMN-LBK, bifidobacterium bifidum Y22 and lactobacillus reuteri K07) is more beneficial to improving the symptoms of the colitis of the mice, the colon and rectum length of the mice of the experiment group C is shorter than that of the experiment group B, the histopathological score is higher than that of the experiment group B, and the effect of the lactobacillus paracasei SMN-LBK on relieving the colon injury of the mice is better than that of the lactobacillus paracasei L.casei.
The results of the experiment D group and the experiment E group show that the bifidobacterium bifidum Y22 and the lactobacillus reuteri K07 have no obvious effect on relieving/treating ulcerative colitis when acting on rat ulcerative colitis mice caused by TNBS alone. After the lactobacillus paracasei SMN-LBK, the bifidobacterium bifidum Y22 and the lactobacillus reuteri K07 are compounded, the effect is greatly enhanced, and the three have synergistic effect to jointly promote the recovery of the ulcerative colitis. When bifidobacterium bifidum Y22, lactobacillus reuteri K07 and lactobacillus paracasei L.casei are compounded, the better treatment effect on the ulcerative colitis cannot be achieved.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
Claims (10)
1. The application of probiotics in preparation of a product for promoting intestinal health is characterized in that the probiotics are lactobacillus paracasei SMN-LBK with the preservation number of CCTCC No. M2017429.
2. The use according to claim 1, wherein the active ingredient of the gut health product comprises viable and/or inactivated bacteria of lactobacillus paracasei SMN-LBK.
3. The use of claim 1, wherein promoting gut health comprises: inhibiting food-borne pathogenic bacteria; preventing, slowing and/or treating colitis.
4. The use of claim 3, wherein the food-borne pathogenic bacteria comprise Staphylococcus aureus and Escherichia coli; the colitis is ulcerative colitis.
5. The use of claim 1, wherein the gut health promoting products comprise food products, nutraceuticals, pharmaceuticals and nutritional supplements.
6. The use according to claim 1, wherein the composition of the product for promoting intestinal health further comprises lactobacillus reuteri K07 having a collection number of CGMCC No.15703 and/or bifidobacterium bifidum Y22 having a collection number of CGMCC No.15708.
7. A probiotic product for promoting gut health, wherein the promoting gut health comprises: inhibiting food-borne pathogenic bacteria; preventing, slowing and/or treating colitis; comprises lactobacillus paracasei SMN-LBK with the preservation number of CCTCC No. M2017429; or,
comprises lactobacillus paracasei SMN-LBK with the preservation number of CCTCC No. M2017429, lactobacillus reuteri K07 with the preservation number of CGMCC NO.15703 and/or bifidobacterium bifidum Y22 with the preservation number of CGMCC NO.15708.
8. The probiotic product for promoting gut health according to claim 7, wherein the weight ratio of the Lactobacillus paracasei SMN-LBK, the Lactobacillus reuteri K07 and the Bifidobacterium bifidum Y22 is (2 to 5): 1:1, preferably 3:1:1.
9. the probiotic product for promoting intestinal health according to claim 7, wherein the probiotic product is a probiotic freeze-dried powder comprising Lactobacillus paracasei SMN-LBK with the preservation number of CCTCC No. M2017429 and prebiotics.
10. The probiotic product for promoting intestinal health according to claim 7, wherein the probiotic product is an oral preparation in the form of an oral liquid, drops, tablets, powders, capsules or granules.
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| CN115624573A (en) * | 2022-12-22 | 2023-01-20 | 广东益可维生物技术有限公司 | Probiotic composition for improving hair health condition and application thereof |
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