CN116769629A - Lactobacillus paracasei with intestinal immune disorder symptom regulating function and application thereof - Google Patents
Lactobacillus paracasei with intestinal immune disorder symptom regulating function and application thereof Download PDFInfo
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- CN116769629A CN116769629A CN202210237137.3A CN202210237137A CN116769629A CN 116769629 A CN116769629 A CN 116769629A CN 202210237137 A CN202210237137 A CN 202210237137A CN 116769629 A CN116769629 A CN 116769629A
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
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- 238000003304 gavage Methods 0.000 description 1
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- 230000002949 hemolytic effect Effects 0.000 description 1
- 229960001340 histamine Drugs 0.000 description 1
- 230000004727 humoral immunity Effects 0.000 description 1
- 230000015784 hyperosmotic salinity response Effects 0.000 description 1
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- 230000002401 inhibitory effect Effects 0.000 description 1
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- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
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- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- OQTQHQORDRKHFW-UHFFFAOYSA-L manganese(2+);sulfate;heptahydrate Chemical compound O.O.O.O.O.O.O.[Mn+2].[O-]S([O-])(=O)=O OQTQHQORDRKHFW-UHFFFAOYSA-L 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
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- 239000013642 negative control Substances 0.000 description 1
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- 238000010979 pH adjustment Methods 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000008807 pathological lesion Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 230000037452 priming Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000002407 reforming Methods 0.000 description 1
- 210000003289 regulatory T cell Anatomy 0.000 description 1
- 206010039083 rhinitis Diseases 0.000 description 1
- 229960002477 riboflavin Drugs 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
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- 238000012353 t test Methods 0.000 description 1
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- 238000010998 test method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-O vancomycin(1+) Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C([O-])=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)[NH2+]C)[C@H]1C[C@](C)([NH3+])[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-O 0.000 description 1
- 239000011716 vitamin B2 Substances 0.000 description 1
- 235000019164 vitamin B2 Nutrition 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Food Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nutrition Science (AREA)
- Genetics & Genomics (AREA)
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- Medicinal Chemistry (AREA)
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- Microbiology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of functional microorganism screening and application, and particularly provides a novel cheese bacillus paracasei VHProbi F22 strain with a preservation number of CCTCC No: m2019941. The invention also provides application of the lactobacillus paracasei in regulating intestinal immune disorder and removing DPPH free radical and cholesterol degradation. The cheese bacillus paracasei VHProbi F22 has strong tolerance to artificial intestinal gastric juice, does not generate hemolysin, does not dissolve blood cells, is sensitive to common antibiotics such as erythromycin and tetracycline, and has good biosafety; can tolerate higher salinity. The strain has strong antioxidant capacity and can effectively degrade cholesterol. In addition, the hydrophobicity of the cell surface of the strain is 70.61%, and the strain has certain adhesion capability. The Lactobacillus paracasei VHProbi F22 is effective in alleviating symptoms of intestinal immune disorders.
Description
Technical Field
The invention belongs to the technical field of screening and application of probiotics, and particularly relates to a cheese bacillus paracasei with an intestinal tract immune disorder symptom regulating function and application thereof.
Background
During the last decades, the allergic phenomena of the human body have assumed a state of dramatic growth, influenced by western lifestyles or industrialized lifestyles. Examples of diseases caused by allergic reactions include rhinitis, asthma, food allergy, atopic dermatitis, contact dermatitis, etc. There is increasing evidence that intestinal flora is an important environmental factor affecting immune tolerance.
The altered order of intestinal flora structure establishment results in altered differentiation of helper T cells, imbalance of helper T cells type i (Th 1 cells) and helper T cells type ii (Th 2 cells) in the immune system of the body, and in particular, a balance of helper T cells tends to Th2 cells. For example, in infants, the use of broad-spectrum antibiotics can lead to altered intestinal flora and dysbiosis, and increased susceptibility to allergy. In fact, epidemiological studies in recent years have shown that environmental factors affecting colonisation of the intestinal flora have a certain association with allergy risk.
At present, the allergic diseases mainly take antiallergic drugs and antihistamines as the main treatment, but the allergic diseases cannot be fundamentally solved for patients with weak immunity, such as lactose intolerance caused by imperfect intestinal development of infants; in addition, histamine can cause adverse reactions such as reduced attention, slow response and the like of human bodies. Therefore, it is of great importance to find more rational methods for treating allergies. In recent years, the treatment of allergic diseases by probiotics has received increasing attention. Clinical studies have shown that probiotics can induce organisms to produce immune responses and immune tolerance, and the ability of regulating immunity can be improved by presenting antigens through gastrointestinal mucosa. The probiotics can increase IFN-gamma, IL-2 and IL-12, promote T cell differentiation to Th1, reduce IL-4 and reduce T cell differentiation to Th2, regulate Th1 and Th2 immune balance and further reach antiallergic effect.
There have been some studies in the prior art on probiotics to regulate allergic reactions. For example, chinese patent publication No. CN 111560330A discloses a strain of Lactobacillus casei which can regulate immunity, regulate M1/M2 type macrophage balance, increase the number of Treg cells, and improve colonitis symptoms and cervical cancer resistance of mice; chinese patent publication No. CN 102373173B discloses a Lactobacillus casei strain which can induce macrophages to produce IL-12 and IL-6 and TNF-alpha and has the function of enhancing immunoregulation; the Chinese patent publication No. CN 101139557B discloses a Lactobacillus casei strain which can influence the T lymphocyte subgroup of the peripheral blood of a mouse, the serum IgG level and the intestinal mucosa SigA level of the mouse, and enhance the cellular immunity, the humoral immunity and the intestinal mucosa local immunity functions of the mouse. The screening of probiotics with the function of regulating immunity and relieving symptoms of intestinal immune disorder has very important application value.
Disclosure of Invention
The invention provides a novel cheese bacillus paracasei (Lacticaseibacillus paracasei) and application thereof. The provided lactobacillus paracasei can reduce the level of proinflammatory cytokines and relieve intestinal immune disorder symptoms, thereby effectively relieving allergic symptoms.
The cheese bacillus paracasei provided by the invention is named as cheese bacillus paracasei VHProbi F22 (Lacticaseibacillus paracasei VHProbi F22), and is preserved in China center for type culture collection (CCTCC No: m2019941.
The invention also provides application of the lactobacillus paracasei in regulating intestinal immune disorder.
The cheese bacillus paracasei provided by the invention can also be used for preparing functional food with the effect of regulating intestinal immune disorder.
The cheese bacillus paracasei VHProbi F22 provided by the invention has strong tolerance to artificial intestinal juice; the VHProbi F22 strain does not produce hemolysin, does not dissolve blood cells, is sensitive to common antibiotics such as erythromycin and tetracycline, and has good biological safety; can tolerate higher salinity with a maximum salt tolerance concentration of 6%.
The strain has strong oxidation resistance, and can effectively degrade cholesterol; in addition, the hydrophobicity of the cell surface of the strain is more than 70%, and the strain has certain adhesion capability.
The cheese bacillus paracasei VHProbi F22 has better effect of regulating intestinal immune disorder. The use of the provided cheese bacillus paracasei VHProbi F22 can effectively relieve intestinal immune disorder symptoms.
The use of the Lactobacillus paracasei VHProbi F22 can effectively reduce IL-5, IL-6, IFN-gamma, TNF-alpha and OVA-IgE in serum of an intestinal immune disorder model mouse, and the effect of a probiotic pretreatment group is better than that of a post-treatment group.
From the HE staining results of the jejunum tissues of the mice, after being treated by the Lactobacillus paracasei VHProbi F22, the jejunum mucosa villus swelling degree can be reduced, the phenomenon of epithelial cell shedding can be reduced, the mucosa lamina propria atrophy degree can be reduced, jejunum mucosa villus has better integrity, and the effect of the probiotic pretreatment group is better than that of the post-treatment group.
The use of the Lactobacillus paracasei VHProbi F22 can reduce the wart micro-bacteria in the intestinal tract, increase the bacteroides, lead the probiotic bacteria to belong to the bacteroides in most cases, lead the intestinal flora structure of the allergic mouse model to be more similar to the normal intestinal tract, and lead the intestinal flora diversity to be increased.
The cheese-bacterium paracasei VHProbi F22 provided by the invention has no toxic or harmful effect on organisms, can be added into food, can be prepared into functional food for relieving intestinal immune disorder, and has wide application prospect.
Drawings
FIG. 1 is a photograph of a cultured colony;
FIG. 2 is a protein fingerprint;
FIG. 3 is a chart of Riboprinter fingerprints;
FIG. 4 is a RAPD fingerprint pattern;
FIG. 5 is a map of rep-PCR fingerprints;
fig. 6 is a graph of symptoms score and wet stool frequency for intestinal immune disorder, where P <0.05 compared to intestinal immune disorder group;
FIG. 7 is a graph showing the results of HE staining of jejunum in each group of mice;
fig. 8 is a graph of cytokine levels for each group, where P <0.05 compared to the group of intestinal immune disorders;
FIG. 9 is a plot of species diversity dilution;
FIG. 10 is a graph of LAD values of species abundance for groups O1 and O2 with statistical differences;
FIG. 11 is a column cumulative plot of the relative abundance of intestinal flora in each group of mice.
Detailed Description
The screening method of the present invention is not limited to the examples, but known screening methods can be used to achieve the screening purpose, and the screening description of the examples is only illustrative of the present invention and is not intended to limit the scope of the present invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The invention will be further illustrated with reference to specific examples.
Example 1 isolation screening of Lactobacillus paracasei VHProbi F22
1. Lactobacillus primary screening
Preparing MRS (Man Rogosa Sharpe) agar medium: 1000mL of purified water, 10g of peptone, 10g of beef extract, 5.0g of yeast extract, 5g of sodium acetate, 5g of glucose, 2g of monopotassium phosphate, 1.0mL of Tween 80, 2.0g of citric acid diamine, 20g of calcium carbonate, 0.58g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate heptahydrate, 15g of agar, pH adjustment of 6.2-6.5 and high-pressure sterilization at 121 ℃ for 15min.
Taking 1g of cheese from the pasture herdsman fermentation of the inner Mongolian tin Lin Guole allium, diluting with sterile normal saline, putting into a sterile sample bag, and beating and mixing uniformly with a homogenizer; and (3) taking 100 mu L of the mixed solution, carrying out gradient dilution, coating the mixed solution on an MRS agar medium, and then culturing at 37 ℃ for 48 hours, and carrying out microscopic examination on a single colony after the plate grows. Based on the microscopic examination, the applicant screened 11 potential Lactobacillus strains designated NL-1, NL-2, NL-3, NL-4, NL-5, NL-6, NL-7, NL-8, NL-9, NL-10, NL-11, respectively.
2. Lactobacillus re-screening
Preparing 1L of MRS liquid culture medium, sterilizing at 115 ℃ for 30min, cooling the culture medium, adding 3.2g of pig mucosa pepsin, shaking to dissolve, and placing in a 37 ℃ water bath shaking table for 1h to obtain the acid-resistant culture medium.
The 11 strains of lactic acid bacteria NL-1, NL-2, NL-3, NL-4, NL-5, NL-6, NL-7, NL-8, NL-9, NL-10 and NL-11 obtained by screening were inoculated into the acid resistant medium in an amount of 6%, and subjected to stationary culture at 37℃for 48 hours, and the fermentation broth was counted.
The results show that the logarithmic values of the viable bacteria amounts in the 11 strains of lactobacillus fermentation broth are 7.19, 8.22, 7.16, 7.52, 5.83, 5.61, 7.85, 6.74, 7.32, 7.08 and 8.58Log CFU/mL respectively, the viable bacteria amount is the largest after the NL-11 strain is subjected to the acid-resistant culture medium rescreening, and the logarithmic value of the bacterial amount is as high as 8.58Log CFU/mL. Therefore, the NL-11 strain screened by the method has the highest acid resistance.
Example 2 identification of strains
2.1 colony morphology identification
Inoculating NL-11 strain on MRS agar medium, culturing at 37deg.C for 48 hr to obtain single colony with rough and off-white color, and microscopic long or short rod shape with two flush ends, and photo of NL-11 single colony under optical microscope shown in figure 1
2.2 identification of physiological and Biochemical Properties
The inoculum preparation in this example was as follows: under the aseptic condition, a proper amount of fresh NL-11 bacterial liquid is taken, centrifuged for 5min at 5000rpm/min, washed for 2 times by PBS buffer, and then the bacterial cells are diluted by 50 times after being weighted by the same volume of PBS buffer, so as to be used as an inoculation liquid.
1. Salinity tolerance test
Under aseptic conditions, 190. Mu.L of BSM liquid medium with salt concentration of 1%, 2%, 3%, 4%, 5%, 6%, 7% and 8% was added to the 96-well plate, respectively, 3 replicates of each salt concentration, and then 10. Mu.L of inoculum was added thereto, and the wells without inoculation were used as controls. 50. Mu.L of autoclaved paraffin oil was added to each well to prevent evaporation of water during the culture. Culturing at 37deg.C, and observing whether the culture medium becomes turbid.
The results showed that NL-11 strain grew at 1% -6% salt concentration and did not grow at 7% -8% salt concentration, with a maximum tolerant salt concentration of 6%.
2. Catalase experiment
A fresh bacterial solution was taken, dropped onto a clean glass slide, and then a drop of 3% hydrogen peroxide solution was added dropwise thereto, and no bubbles were observed to be generated by the NL-11 strain, which was a negative reaction.
3. Carbon source metabolism test
The basal medium formulation used in this example is as follows:
1.5g of peptone; 0.6g of yeast extract; tween 80.1 g; 0.5mL of saline solution; 18mg of phenol red; distilled water 100mL; pH 7.4.+ -. 0.2. Salt solution components: mgSO (MgSO) 4 ·7H 2 O 11.5g，MnSO 4 ·4H 2 O2.8 g, distilled water 100mL.
A10 g/100mL solution of sugar, alcohol and glycoside carbohydrate was prepared and filtered with a 0.22 μm sterile filter. Under aseptic conditions, 20. Mu.L of sterilized carbohydrate solution, 4 per carbohydrate, was added to the 96-well plate, then 170. Mu.L of sterilized phenol red-containing basal medium was added, and 10. Mu.L of inoculum was added, without inoculating the reaction well as a control. 50. Mu.L of liquid paraffin was added to each well to prevent evaporation of water during the culture. Anaerobic culture at 37deg.C, and observing color change of culture medium with phenol red as indicator. The specific results are shown in Table 1.
TABLE 1 carbon source metabolism results of NL-11 Strain
Note that: a "+" positive response; "-" negative reaction.
2.3MALDI-TOF-MS detection of Total protein expression of strains
Inoculating fresh bacterial liquid into MRS liquid culture medium according to 0.1% inoculum size, culturing at 37deg.C and 150rpm for 48 hr, collecting bacterial cells, washing with sterile water for 4 times, and air drying surface water. And then a small amount of fresh thalli is uniformly coated on a target plate in a film form, 1 mu L of lysate is added to cover the sample, after the sample is dried, 1 mu L of matrix solution is added to cover the sample, after the sample is dried, the sample target is put into a mass spectrometer for identification. The co-crystallization film formed by the sample and the matrix is irradiated by laser to ionize the protein in the sample, the ions are accelerated to fly through a flight pipeline under the action of an electric field of 10 KV to 20KV, and the molecular weight of the protein is detected according to different flight time reaching a detector. Protein fingerprint is obtained by utilizing Autofms 1000 analysis software Autof Analyzer v1.0, and the main ion peaks of the NL-11 strain are as follows: m/z9397.500, 7482.800, 6867.117, 5893.371, 5303.893, 4452.130, 4699.426, the results are shown in FIG. 2.
2.4 molecular biological characterization
2.4.1 16s rDNA Gene sequence analysis
1. Genomic DNA extraction
Reference was made to the Tiangen bacterial genomic DNA extraction kit (catalog number: DP 302).
2. 16s rDNA Gene amplification
1) Primer sequence:
27F：AGAGTTTGATCCTGGCTCA；
1492R：GGTTACCTTGTTACGACTT。
2) Reaction system (50. Mu.L)
Table 2:16s rDNA PCR amplification System Table
3) Electrophoresis verifies that the PCR product meets the requirement when the nucleic acid electrophoresis result is about 1500 bp.
4) Sequencing of PCR products
The 16s rDNA sequence of the NL-11 strain SEQ ID NO. 1 was obtained by sequencing and the sequences were aligned in NCBI database to preliminarily determine that the NL-11 strain was Lactobacillus paracasei.
catgcggcgtgctatacatgcagtcgaacgagttctcgttgatgatcggtgcttgcaccgagattcaacatggaacgagtggcggacgggtgagtaacacgtgggtaacctgcccttaagtgggggataacatttggaaacagatgctaataccgcatagatccaagaaccgcatggttcttggctgaaagatggcgtaagctatcgcttttggatggacccgcggcgtattagctagttggtgaggtaatggctcaccaaggcgatgatacgtagccgaactgagaggttgatcggccacattgggactgagacacggcccaaactcctacgggaggcagcagtagggaatcttccacaatggacgcaagtctgatggagcaacgccgcgtgagtgaagaaggctttcgggtcgtaaaactctgttgttggagaagaatggtcggcagagtaactgttgtcggcgtgacggtatccaaccagaaagccacggctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttatccggatttattgggcgtaaagcgagcgcaggcggttttttaagtctgatgtgaaagccctcggcttaaccgaggaagcgcatcggaaactgggaaacttgagtgcagaagaggacagtggaactccatgtgtagcggtgaaatgcgtagatatatggaagaacaccagtggcgaaagcggctgtctggtctgtaactgacgctgaggctcgaaagcatgggtagcgaacaggattagataccctggtagtccatgccgtaaacgatgaatgctaggtgttggagggtttccgcccttcagtgccgcagctaacgcattaagcattccgcctggggagtacgaccgcaaggttgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatcttttgatcacctgagagatcaggtttccccttcgggggcaaaatgacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttatgactagttgccagcatttagttgggcactctagtaagactgccggtgacaaaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggatggtacaacgagttgcgagaccgcgaggtcaagctaatctcttaaagccattctcagttcggactgtaggctgcaactcgcctacacgaagtcggaatcgctagtaatcgcggatcagcacgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgagagtttgtaacacccgaagccggtggcgtaaccctttagggagcgagccgtctaaggtgacaagg。
2.4.2Riboprinter finger print
The purified single colony is dipped from an agar culture medium flat plate by a fungus taking rod, the single colony is placed into a sample tube with buffer solution, the single colony is stirred by a hand-held stirrer to be suspended in the buffer solution, then the sample holder is placed into a heater for inactivation and then placed into a Riboprinter system, and after DNA preparation, film transfer, imaging detection and data processing, the Riboprinter fingerprint of the strain NL-11 is obtained (figure 3).
2.4.3RAPD and rep-PCR fingerprint identification
1. RAPD fingerprint identification
1) Primer sequence: m13 (5'-GAGGGTGGCGGTTCT-3');
2) RAPD reaction system
TABLE 3RAPD reaction System
3) Electrophoresis
1.5% agarose gel plates were prepared, DL2000 DNA markers were used as a result control, 100V electrophoresis was performed for 80min at a constant pressure, and finally the electropherograms were detected using a gel imaging system. RAPD finger prints of NL-11 strain are shown in FIG. 4.
2. rep-PCR fingerprint
1) rep-PCR primer
CTACGGCAAGGCGACGCTGACG。
2) reaction system of rep-PCR
TABLE 4 rep-PCR reaction System
3) Electrophoresis
DL2000 DNA Marker served as a result control. Detecting the amplification result by 100V voltage and 80min electrophoresis time. The rep-PCR fingerprint of NL-11 strain is shown in FIG. 5.
In summary, colony morphology and physiological and biochemical characteristic results of NL-11 strain were uploaded to the website http:// www.tgw1916.net/bacteria_log_desktop. Html, and aligned in combination with the results published in the literature De Clerck E, et al systems and applied microbiology,2004,27 (1) 50. From the results of the molecular biology identification, it can be concluded that the NL-11 strain is a new strain of Lactobacillus paracasei, designated as Lactobacillus paracasei VHProbi F22.
Example 3 test of resistance of Lactobacillus paracasei VHProbi F22 to artificial gastric juice and artificial intestinal juice
3.1 preparation of artificial gastric juice
5g of peptone, 2.5g of yeast extract, 1g of glucose and 2g of NaCl are weighed respectively, 1000mL of distilled water is added, pH is adjusted to 3.0 by dilute hydrochloric acid, and then sterilization is carried out for 20min at 115 ℃. Then 3.2g of pig mucosa pepsin is added before use, the pig mucosa pepsin is uniformly shaken and dissolved, and the mixture is placed in a water bath shaker at 37 ℃ for warm water bath for 1 hour so as to simulate the temperature of a human body.
3.2 preparation of artificial intestinal juice
Respectively weighing peptone 5g, yeast extract 2.5g, glucose 1g, KH 2 PO 4 6.8g and 3.0g of ox gall salt, 77mL of 0.2mol/L NaOH solution is added, the volume is fixed to 1000mL, the pH is regulated to 6.8+/-0.1 by dilute hydrochloric acid or sodium hydroxide solution, and the mixture is sterilized for 20min at 115 ℃. Then adding 1g of pancreatin before use, shaking to dissolve, and placing in a water bath shaker at 37 ℃ for warm water bath for 1h to simulate the temperature of human body.
3.2 test methods
2mL of fresh bacterial liquid is taken, the bacterial liquid is collected by centrifugation at 5000rpm/min for 5min, the bacterial liquid is washed 3 times by physiological saline, and then 2mL of physiological saline is used as inoculation liquid for resuspension. 1mL of the inoculation liquid is taken and added into 24mL of artificial intestinal juice, and the mixture is placed on a water bath shaking table (200 rpm/min) at 37 ℃ for 3 hours, 1mL of sample is taken, and the viable bacteria amount is detected.
The viable bacteria counting method is used for measuring the bacterial amount according to national standard GB 4789.35-2016-lactobacillus test for food microorganism test, and the viable bacteria amount (Log CFU/mL) of the strain after artificial intestinal juice digestion is shown in Table 5.
TABLE 5 viable count after digestion with Artificial gastric juice and Artificial intestinal juice
As can be seen from Table 5, the viable count of the Lactobacillus paracasei VHProbi F22 screened by the present invention was reduced by only about 1.2Log CFU/mL after digestion with artificial gastric juice and artificial intestinal juice. Therefore, the strain has strong tolerance to artificial gastric juice and artificial intestinal juice.
Example 4 antibiotic resistance experiment with Lactobacillus paracasei VHProbi F22
1) Antibiotic formulation
Ampicillin, clindamycin, erythromycin, gentamicin, streptomycin, tetracycline and vancomycin are prepared into stock solution of 2048 mug/mL, and the stock solution is preserved at-20 ℃ for standby. When in use, the stock solution is serially diluted by 2 times by using BSM liquid culture medium to form use solution, and the gradient dilution concentration is 1-1024 mu g/mL and total 11 gradients.
2) Preparation of inoculum
Taking a proper amount of fresh bacterial liquid (24 h,37 ℃ for culture), centrifuging at 5000rpm for 5min, washing once with sterile physiological saline, and diluting 50 times after re-suspending bacterial cells with the same volume of physiological saline to obtain an inoculation liquid.
3) Determination of minimum inhibitory concentration MIC value of antibiotics for Lactobacillus paracasei VHProbi F22 by micro broth dilution method
an MRS liquid culture medium without antibiotics is added to the 1 st column of the 96-well plate as a negative control, 190 mu L of MRS liquid culture medium with antibiotics with different concentrations is sequentially added to the 2 nd to 12 th columns, 10 mu L of the inoculation liquid is inoculated respectively, 3 parallel wells are made, and 1 well of the non-added bacteria liquid is used as a blank.
b. 50. Mu.L of paraffin oil was added to cover the water and prevent evaporation.
c. The 96-well plate was incubated at 37℃for 24 hours, then removed, and OD was measured 600 Values, MIC values of antibiotics against strains were counted with 24h results, and specific results are shown in table 6.
TABLE 6 antibiotic MIC values for Lactobacillus paracasei VHProbi F22
MIC units μg/mL
From the results shown in Table 6, the Lactobacillus paracasei VHProbi F22 provided by the invention is sensitive to common antibiotics such as erythromycin and tetracycline, and has good biological safety.
EXAMPLE 5 measurement of antioxidant function of Lactobacillus paracasei VHProbi F22
1. Determination of DPPH free radical scavenging ability of Strain
1) Preparation of PBS bacterial suspension
Single colony with excellent growth state is inoculated into 3mL of MRS liquid culture medium, and is cultured for 24h at 37 ℃, the culture solution is taken as an inoculating solution, and is inoculated into 50mL of MRS liquid culture medium according to the inoculating amount of 2 percent, and the culture solution of the strain is obtained by standing and culturing for 24 h. After 1mL of bacterial liquid is sucked up and bacterial cells are collected, the bacterial cells are washed by 1mL of buffer solution for 2 times, and then 2mL of buffer solution is added to resuspend the bacterial cells for standby.
2) Determination of DPPH free radical scavenging ability of Strain
1mL of PBS bacterial suspension of the strain to be detected is taken, 1mL of 0.4mM DPPH free radical solution is added, after uniform mixing, the mixture is placed at room temperature for shading reaction for 30min, then the absorbance A sample of the sample at the wavelength of 517nm is measured, and the sample is measured for 3 times of parallelism. The control samples were zeroed with equal volumes of PBS and DPPH ethanol mixed solution and with equal volumes of PBS and ethanol mixed solution. The clearance is calculated according to the following formula: clearance% = [1- (a) Sample of -A Blank space )/A Control ]×100％。
The results are shown in Table 7 using the Lactobacillus paracasei IMC-4 strain as a positive control.
TABLE 7 DPPH radical scavenging Rate
As can be seen from the data in Table 7, the Lactobacillus paracasei VHProbi F22 provided by the invention can effectively remove DPPH free radicals, and the removal rate is 54.57% which is obviously higher than that of the Lactobacillus paracasei IMC-4 strain.
2. Identification of strains by anti-lipid peroxidation experiments
1) Cultivation of lactic acid bacteriaPreparation of culture and fermentation supernatant, thallus and intracellular extract: culturing lactobacillus in MRS liquid culture medium at 37deg.C for 24 hr, transferring for 3 generations, centrifuging at 6000rpm/min at 4deg.C for 10min, and collecting supernatant to obtain fermentation supernatant. The collected cells were centrifuged at 6000r/min for 10min with PBS buffer (pH 7.4), and washed 3 times. The bacterial cells were resuspended in PBS buffer to a bacterial cell concentration of 1.0X10 9 cells/mL to obtain a bacterial suspension.
2) Preparation of linoleic acid emulsion: 0.1mL linoleic acid, 0.2mL Tween 20, 19.7mL deionized water.
3) To 0.5mL of PBS solution (pH 7.4) was added 1mL of linoleic acid emulsion, 1mL of LFASO 4 (1%) and 0.5mL of sample are added, water bath is carried out at 37 ℃ for 1.5 hours, 0.2mL of TCA (4%) and 2mL of TBA (0.8%) are added into the mixed solution, water bath is carried out at 100 ℃ for 30 minutes, rapid cooling is carried out, centrifugation is carried out at 4000rpm/min for 15 minutes, and absorbance of the supernatant liquid is collected and measured at 532nm to obtain A; the control group was A0 with 0.5mL distilled water instead of the sample. Inhibition rate/% = (A0-a)/a0×100%
Note that: a is absorbance of a sample group; a0 is absorbance of the control group. The results are shown in Table 8 using Lactobacillus paracasei IMC-4 as a positive control.
TABLE 8 inhibition of lipid peroxidation
From the data in Table 8, it can be seen that the anti-lipid peroxidation inhibition rate of the supernatant of the Lactobacillus paracasei VHProbi F22 provided by the invention is 50.64% which is lower than that of the Lactobacillus paracasei IMC-4 strain; the inhibition rate of the bacterial body to lipid peroxidation is 51.66 percent, which is higher than that of the strain IMC-4 of the cheese bacillus paracasei.
Example 6 in vitro cholesterol degradation experiments with Lactobacillus paracasei VHProbi F22
1. Preparing a cholesterol micelle solution: 1g of cholesterol was accurately weighed, dissolved in absolute ethanol, and fixed to 100mL, and sterilized by filtration through a 0.22 μm microporous filter under aseptic conditions.
2. 10.0g of peptone, 10.0g of beef extract, 5.0g of yeast extract, 2.0g of diammonium hydrogen citrate, 20.0g of glucose, 1.0mL of tween 80, 5.0g of sodium acetate, 0.1g of magnesium sulfate, 0.05 g of manganese sulfate, 2.0g of dipotassium hydrogen phosphate, 1g of bile salt and 1000mL of distilled water, adjusting the pH value to 7.3, sterilizing at 115 ℃ for 30min, and then adding a cholesterol solution to ensure that the final concentration of cholesterol is 0.1%.
Inoculating fresh bacterial liquid according to 0.1% of inoculation amount, standing at 37 ℃ for 48 hours, taking 0.2mL of bacterial liquid, adding 1.8mL of absolute ethyl alcohol, uniformly mixing, standing for 10 minutes, centrifuging at 3000 r for 5 minutes, and taking supernatant for measuring cholesterol content. Cholesterol measurement method according to GB/T5009.128-2003 < measurement of cholesterol in food >.
The results show that: the degradation rate of the cheese bacillus paracasei VHProbi F22 provided by the invention on cholesterol reaches 34.55%.
EXAMPLE 7 Lactobacillus paracasei VHProbi F22 cell surface hydrophobicity test
1. Preparing a bacterial liquid to be tested: the purified bacterial colony of the cheese bacillus paracasei VHProbi F22 is picked and inoculated into a newly prepared MRS liquid culture medium, and the culture is carried out for 24 to 48 hours at 37 ℃. Inoculating 1% (V/V) of the strain into MRS liquid culture medium, continuously culturing at 37deg.C for 24-48 hr, centrifuging at 6000 Xg for 10min, collecting thallus, washing with sterile physiological saline for 2 times, and sterilizing with 0.1M KNO 3 The bacterial cells were resuspended in 1mL of the solution and used as the bacterial liquid to be tested.
2. Surface hydrophobicity determination: mu.L of the above bacterial suspension was pipetted into 2450. Mu.L of 0.1M KNO 3 And record OD 600 Is A 0 1.5ml of the bacterial suspension was mixed with 500. Mu.L of xylene and allowed to stand at room temperature for 10 minutes (a two-phase system was formed). Vortex oscillating the two-phase system for 2min, standing for 20min, and reforming into water phase and organic phase. The aqueous phase (not to the organic phase) was carefully aspirated and absorbance A was measured at 600nm 1 . Cell Hydrophobicity = (a) according to the formula hydropathicity = (a) 0 -A 1 )/A 1 X% calculation, measurement of the average of three experiments.
The results show that: the hydrophobicity of the surface of the cheese bacillus paracasei VHProbi F22 cell provided by the invention is 70.61%.
Application effects example 1: studies of Lactobacillus paracasei VHProbi F22 for alleviating symptoms of intestinal immune disorder in mice
1.1 Experimental materials
1.1.1 laboratory animals
BALB/c mice SPF grade, 30 females, 4-6 weeks, body weight 19-25 g. License number SCXK (robust) 20140007 was produced by the experimental animal breeding limited company, punyue, atanan.
Animal quarantine and identification: after all animals arrive, the adaptation period is at least one week, quarantine observation is carried out, activities, diet and other performances of the animals are observed, the animals need to be checked to be qualified before the test, and the qualified animal can be used for the test. After the animals are qualified for quarantine, a single animal number is assigned to each animal, and the animal tail is marked. The quarantine observation period cage card is marked with a thematic number, an animal number, a cage number, sex, animal receiving date and thematic responsible person; after grouping, the thematic numbers, animal numbers, cage numbers, sexes, groups, test start-stop dates and thematic responsible persons are marked on the cage cards.
Environmental conditions for experimental animal feeding management: the room temperature is 20-26 ℃, the daily temperature difference is less than or equal to 4 ℃, the relative humidity is 40-70%, and the light and shade alternation time is 12/12h. Animals were kept in standard mouse cages, 6 per cage.
Animal feed, drinking water: can be ingested and drunk freely. The feed is SPF-class mice growth breeding feed, which is provided by Jinan Pengyue laboratory animal breeding Limited (lot number: 20190905). The drinking water is city tap water sterilized at high temperature.
1.1.2 reagent consumable
Ovalbumin (OVA) (lot number: S12016): shanghai Yuan Ye Biotech Co., ltd;
d-biotin (lot number: S13004): shanghai Yuan Ye Biotech Co., ltd;
vitamin B2 (lot number: S13020): shanghai Yuan Ye Biotech Co., ltd;
aluminum adjuvant (lot number: UL 292268): simer Feier technology (China);
IL-5 (lot number: E20200605-20187B) kit: shanghai enzyme-linked biotechnology limited company;
IL-6 (lot number: E20200605-20188B) kit: shanghai enzyme-linked biotechnology limited company;
IL-10 (lot number: E20200601-201662B) kit: shanghai enzyme-linked biotechnology limited company;
IFN-gamma (lot number: E20200602-20140B) kit: shanghai enzyme-linked biotechnology limited company;
TNF-alpha cytokine (lot number: E20200607-20852B) kit: shanghai enzyme-linked biotechnology limited company;
OVA-specific IgE (lot number: E20200604-20508B) kit: shanghai enzyme-linked biotechnology limited company;
eosin (lot number: 20181204): beijing Soy Bao technology Co., ltd;
hematoxylin (lot number: 20181204): beijing Soy Bao technology Co., ltd.
1.2 Experimental methods
1.2.1 preparation of bacterial liquid
Anaerobic culturing single colony on MRS plate at 37 deg.C for 24-48 hr, picking single colony, amplifying culturing in MRS broth culture medium for 16 hr, collecting bacterial liquid and regulating its concentration to 10 9 CFU/mL bacterial suspension.
1.2.2 grouping
SPF-class female BALB/c mice of 4-6 weeks old are randomly divided into a blank group, an intestinal immune disorder group, a probiotic pretreatment group and a probiotic post-treatment group after being adaptively fed for 3 days, wherein 6 mice are used in each group, and the probiotic pretreatment group and the post-treatment group are subjected to gastric lavage according to 0.2mL/10 g.
1.2.3 modeling and probiotic intervention scheme
The probiotic pretreatment group mice were given probiotic bacteria by gavage in advance before the beginning of molding, and were given continuously for 10 days, and the other groups were not treated. Starting modeling after 10 days, 3 groups of mice except for a blank group were injected with 200 μl of allergen solution (OVA: aluminum adjuvant: pbs=1:1:1 mixed solution) intraperitoneally on days 0, 14, 100 μg OVA per mouse, and the blank group was injected with a PBS/aluminum adjuvant mixed solution (PBS: aluminum adjuvant=2:1); starting from day 28, a blank control group was primed with 200 μl of PBS solution every three days, and an intestinal immune dysregulation group and 2 probiotic groups were primed with 50mg OVA every three days, and priming was co-activated 6 times; the probiotic pretreatment was started from 3 days after the adaptive feeding to the day before the sacrifice, and the probiotic post-treatment group was fed with the bacterial liquid from the day 0 basal sensitization to the day before the sacrifice.
1.3 index observations
1.3.1 symptoms of intestinal immune disorder in mice
The mice were observed for symptoms of intestinal immune disorder 1h after each gastric lavage with OVA and scored, and the scoring details are shown in table 9.
Table 9 mice intestinal immune disorder symptom score table
1.3.2IL-5, IL-6, IL-10, IFN-gamma, TNF-alpha content and OVA-specific IgE detection
24h after the last administration of mice, eyeballs are bled, serum is prepared and stored at-80 ℃, and the ELISA is used for detecting the IL-5, IL-6, IL-10, IFN-gamma, TNF-alpha cytokine content and OVA specific IgE level in the serum of each group of mice.
1.3.3 histopathological examination
After mice were sacrificed, jejunal tissues were harvested, paraformaldehyde fixed, sampled, dehydrated, paraffin embedded, sectioned, and HE stained for histopathological changes.
1.4 statistical processing method for data
All experimental data are expressed as mean ± standard deviation "± SD", data statistics and plots are performed using Microsoft EXCEL, and comparisons between two sets of data are determined to be significant differences using t-test, with P < 0.05.
1.5 experimental results
1.5.1 symptoms of intestinal immune disorder in mice
Compared with a blank control group, the immune disorder symptoms and the wet stool frequency of the mice in the intestinal immune disorder group are obviously increased, and the immune disorder and diarrhea symptoms are increased along with the increase of sensitization times; at the end of the test period, the allergy symptoms of the intestinal immune disorder group are highest in score, the diarrhea of the mice is serious and dysphoria is caused, the allergy symptoms of the probiotic pretreatment group and the probiotic post-treatment group are reduced in score, the diarrhea symptoms are lighter, and the intestinal immune disorder symptoms are improved. The symptom scores and the stool frequency of each group are shown in fig. 6.
1.5.2 histopathological examination
Compared with intestinal immune disorder groups, the probiotics treatment can reduce the villus swelling degree of jejunum mucosa, the epithelial cell shedding phenomenon is reduced, the integrity is better, the atrophy degree of the mucosa lamina propria is reduced, and the effect of the pretreatment group is better than that of the post-treatment group. Typical pathological lesions are shown in FIG. 7.
1.5.3IL-5, IL-6, IL-10, IFN-gamma, TNF-alpha content and OVA specific IgE detection the serum IL-5, IL-6, IL-10, IFN-gamma, TNF-alpha cytokine content and OVA specific IgE levels were measured in each group of mice using the Elisa method. The results show that compared with a blank control group, the specific antibody of the mice in the intestinal immune disorder group and inflammatory factors are obviously increased, which proves that the construction of the intestinal immune disorder model caused by OVA is successful. Compared with the intestinal tract immune disorder group, the serum of the mice in the probiotic post-treatment group has reduced IL-5, IL-6, IFN-gamma and TNF-alpha cell inflammatory factors, increased IL-10 cell inflammatory factors, reduced OVA specific IgE and significant difference (P < 0.05); the serum of mice in the probiotic pretreatment group has reduced IL-5, IL-6, IFN-gamma, TNF-alpha cell inflammatory factors, increased IL-10 cell inflammatory factors, reduced OVA specific IgE, and significant difference (P < 0.05). The comparison of L-5, IL-6, IL-10, IFN-gamma, TNF-alpha cytokine levels and OVA-specific IgE in the sera of each group of mice is shown in FIG. 8.
In conclusion, compared with the intestinal tract immune disorder group, the cell inflammatory factor and OVA specific antibody after the probiotic treatment are reduced and have obvious difference (P is less than 0.05), and the effect of the probiotic pretreatment group is better than that of the probiotic post-treatment group; compared with mice with intestinal immune disorder groups, the probiotic treatment can reduce the central axis atrophy degree of jejunum mucosa villus, reduce the gap between central axis connective tissue and surface epithelium, reduce the phenomenon of epithelial cell shedding and reduce the pathological change degree.
Application effects example 2: study of the modulation of the intestinal flora in mice by Lactobacillus paracasei VHProbi F22
1.1 Experimental methods
The same animal experiment protocol as in application effect example 1 was used. And (3) collecting mouse faeces before OVA sensitization and after experiments of an intestinal immune disorder group and a probiotic pretreatment group, analyzing an intestinal flora structure, taking an collected faeces sample, sending the faeces sample to a norstanding grain source for sequencing, and analyzing community diversity and abundance changes of flora microorganisms by adopting an amplicon sequencing technology, wherein the community diversity and abundance changes comprise a richness index, a diversity index and the like.
1.2 experimental results
On the basis of obtaining 97% similarity OTU, alpha diversity of all samples is analyzed to construct a corresponding dilution curve, as shown in fig. 9, when the curve tends to be gentle, the sequencing quantity is reasonable, most microorganisms can be detected, the sequencing depth can be basically covered on all the species in the samples, the species abundance of the samples (Y2) after the end of the probiotic pretreatment group is higher than that of the samples (O1) before the sensitization of the intestinal immune disorder group and the samples (Y1) before the sensitization of the probiotic pretreatment group, the species abundance of the samples (O1) before the sensitization of the intestinal immune disorder group is higher than that of the samples (O2) after the end of the intestinal immune disorder group, so that the species abundance of intestinal flora is reduced after the symptoms of the intestinal immune disorder caused by OVA sensitization, and the species abundance of the intestinal flora of the intestinal immune disorder mice can be improved by adopting the interference of the Lactobacillus paracasei VHProbi F22, so that the species abundance of the intestinal flora tends to be recovered.
Species with a greater than set point (default set 4), i.e., species with statistical differences between groups, are shown in the LDA value distribution histogram, which shows species with significant differences in abundance among the different groups, and the length of the histogram represents the magnitude of the effect of the different species (i.e., LDA Score). As shown in FIG. 10, in O1, the abundances of bacillus, lactobacillus, enterobacter, lactobacillus reuteri, lactobacillus brevis, lactobacillus delbrueckii and the like are all remarkable; in O2, the wart micro-bacteria, acremonium, erysipelotorich and turicibacillus and the like are all remarkably high in abundance, which indicates that the use of intestinal immune disorder can cause the decrease of the abundance of lactobacillus in intestinal flora.
Comparing the two groups of O1/O2 data, the relative abundance of the firmicutes of the intestinal immune disorder group is reduced, and the relative abundance of the bacteroides and the wart microzyme is increased; comparing the two groups of data of O1/Y1, the pretreatment of the pre-probiotics reduces the relative abundance of the phylum of the firmicutes and increases the relative abundance of the flora of the bacteroides in the intestinal tract of the mice; comparing the two groups of O2/Y2 data, the relative abundance of the probiotics group is reduced compared with that of the wart microzyme of the intestinal immune disorder group after the experiment is finished, and the relative abundance of the bacteroides is increased; the results show that the intestinal flora structure of mice after intestinal immune disorder is better towards that of normal mice after intervention of the Lactobacillus paracasei VHProbi F22, and the relative abundance of each group species is shown in FIG. 11.
After the OVA sensitization causes intestinal immune disorder symptoms, the species abundance of intestinal flora is reduced, the species abundance of the intestinal flora of an intestinal immune disorder mouse can be improved by adopting the intervention of the cheese bacillus Paramygdalis VHProbi F22, so that the intestinal flora tends to be recovered, the abundance of lactobacillus in the intestinal flora can be improved, and the intestinal flora structure tends to be more beneficial to the intestinal flora of a healthy mouse.
In conclusion, the cheese bacillus paracasei VHProbi F22 provided by the invention has strong tolerance to simulated artificial intestinal gastric juice, which lays a foundation for the probiotic strains to successfully pass through the gastrointestinal tract to perform the probiotic function by colonic colonisation. The haemolytic test proves that the cheese bacillus VHProbi F22 does not produce haemolysin, does not dissolve blood cells and has good biological safety. Meanwhile, the cheese bacillus paracasei VHProbi F22 can remove DPPH free radicals, inhibit lipid peroxidation, has a certain antioxidant function activity, can degrade cholesterol, and has the probiotic property of reducing serum cholesterol. Animal experiments prove that the Lactobacillus paracasei VHProbi F22 can improve inflammatory response of a model mouse with intestinal immune disorder, can inhibit TH2 type immune response while enhancing TH1 type cell immune response, reduce inflammatory state of an organism and enhance immunity, and has potential application value in relieving inflammation of intestinal immune disorder.
Sequence listing
<110> Qingdao blue biological group Co., ltd
<120> A strain of Lactobacillus paracasei having symptoms of modulating intestinal immune disorder and use thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1468
<212> DNA
<213> Lactobacillus casei (Lactobacillus casei)
<400> 1
catgcggcgt gctatacatg cagtcgaacg agttctcgtt gatgatcggt gcttgcaccg 60
agattcaaca tggaacgagt ggcggacggg tgagtaacac gtgggtaacc tgcccttaag 120
tgggggataa catttggaaa cagatgctaa taccgcatag atccaagaac cgcatggttc 180
ttggctgaaa gatggcgtaa gctatcgctt ttggatggac ccgcggcgta ttagctagtt 240
ggtgaggtaa tggctcacca aggcgatgat acgtagccga actgagaggt tgatcggcca 300
cattgggact gagacacggc ccaaactcct acgggaggca gcagtaggga atcttccaca 360
atggacgcaa gtctgatgga gcaacgccgc gtgagtgaag aaggctttcg ggtcgtaaaa 420
ctctgttgtt ggagaagaat ggtcggcaga gtaactgttg tcggcgtgac ggtatccaac 480
cagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgtta 540
tccggattta ttgggcgtaa agcgagcgca ggcggttttt taagtctgat gtgaaagccc 600
tcggcttaac cgaggaagcg catcggaaac tgggaaactt gagtgcagaa gaggacagtg 660
gaactccatg tgtagcggtg aaatgcgtag atatatggaa gaacaccagt ggcgaaagcg 720
gctgtctggt ctgtaactga cgctgaggct cgaaagcatg ggtagcgaac aggattagat 780
accctggtag tccatgccgt aaacgatgaa tgctaggtgt tggagggttt ccgcccttca 840
gtgccgcagc taacgcatta agcattccgc ctggggagta cgaccgcaag gttgaaactc 900
aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc 960
gaagaacctt accaggtctt gacatctttt gatcacctga gagatcaggt ttccccttcg 1020
ggggcaaaat gacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta 1080
agtcccgcaa cgagcgcaac ccttatgact agttgccagc atttagttgg gcactctagt 1140
aagactgccg gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc atgcccctta 1200
tgacctgggc tacacacgtg ctacaatgga tggtacaacg agttgcgaga ccgcgaggtc 1260
aagctaatct cttaaagcca ttctcagttc ggactgtagg ctgcaactcg cctacacgaa 1320
gtcggaatcg ctagtaatcg cggatcagca cgccgcggtg aatacgttcc cgggccttgt 1380
acacaccgcc cgtcacacca tgagagtttg taacacccga agccggtggc gtaacccttt 1440
agggagcgag ccgtctaagg tgacaagg 1468
Claims (5)
1. The cheese bacillus paracasei is characterized in that the preservation number of the cheese bacillus paracasei is CCTCC No: m2019941.
2. Use of lactobacillus paracasei according to claim 1 for the modulation of intestinal immune disorders.
3. Use of lactobacillus paracasei according to claim 1 for the preparation of a functional food having the effect of modulating intestinal immune disorders.
4. Use of lactobacillus paracasei according to claim 1 for scavenging DPPH radicals.
5. Use of lactobacillus paracasei according to claim 1 for cholesterol degradation.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210237137.3A CN116769629A (en) | 2022-03-11 | 2022-03-11 | Lactobacillus paracasei with intestinal immune disorder symptom regulating function and application thereof |
| PCT/CN2022/088946 WO2023168808A1 (en) | 2022-03-11 | 2022-04-25 | Lactobacillus paracasei capable of regulating symptoms of intestinal immune disorders and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN202210237137.3A CN116769629A (en) | 2022-03-11 | 2022-03-11 | Lactobacillus paracasei with intestinal immune disorder symptom regulating function and application thereof |
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| CN116769629A true CN116769629A (en) | 2023-09-19 |
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| CN202210237137.3A Pending CN116769629A (en) | 2022-03-11 | 2022-03-11 | Lactobacillus paracasei with intestinal immune disorder symptom regulating function and application thereof |
Country Status (2)
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| CN (1) | CN116769629A (en) |
| WO (1) | WO2023168808A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117467586A (en) * | 2023-12-28 | 2024-01-30 | 潍坊康地恩生物科技有限公司 | Lactobacillus rhamnosus with effect of delaying chronic nephrosis process |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116832068B (en) * | 2023-06-09 | 2024-04-09 | 内蒙古农业大学 | Lactobacillus paracasei PC646 with broad-spectrum antibacterial activity and application thereof |
| CN116814481B (en) * | 2023-06-09 | 2024-04-09 | 内蒙古农业大学 | Probiotic Lactobacillus paracasei PC646 derived from fermented mare milk and artificial intelligent screening method thereof |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104531567A (en) * | 2014-12-13 | 2015-04-22 | 张鹏宴 | Lactobacillus paracasei with acid resistantance and high cholesterol clearance rate and application of lactobacillus paracasei |
| CN105368738A (en) * | 2015-10-23 | 2016-03-02 | 中国农业大学 | Lactobacillus paracasei and appliance thereof |
| CN105400727A (en) * | 2015-12-21 | 2016-03-16 | 南昌大学 | Lactobacillus paracasei with antioxidant activity and application thereof |
| CN106819106B (en) * | 2016-12-28 | 2020-10-16 | 石家庄君乐宝乳业有限公司 | Preparation method of inactivated fermented milk beverage with immunoregulation function |
| CN107227280B (en) * | 2017-07-18 | 2019-09-03 | 江南大学 | One plant of lactobacillus paracasei and its application |
| CN108148784A (en) * | 2018-01-31 | 2018-06-12 | 石河子大学 | One plant of Lactobacillus paracasei bacterial strain and its application in juice fermented beverage is prepared |
-
2022
- 2022-03-11 CN CN202210237137.3A patent/CN116769629A/en active Pending
- 2022-04-25 WO PCT/CN2022/088946 patent/WO2023168808A1/en unknown
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117467586A (en) * | 2023-12-28 | 2024-01-30 | 潍坊康地恩生物科技有限公司 | Lactobacillus rhamnosus with effect of delaying chronic nephrosis process |
| CN117467586B (en) * | 2023-12-28 | 2024-05-10 | 青岛蔚蓝生物集团有限公司 | Lactobacillus rhamnosus with effect of delaying chronic nephrosis process |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2023168808A1 (en) | 2023-09-14 |
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