WO2023168808A1

WO2023168808A1 - Lactobacillus paracasei capable of regulating symptoms of intestinal immune disorders and use thereof

Info

Publication number: WO2023168808A1
Application number: PCT/CN2022/088946
Authority: WO
Inventors: 段治; 崔洪昌; 吴松洁; 张景燕; 张陆霞
Original assignee: 青岛蔚蓝生物股份有限公司; 青岛蔚蓝生物集团有限公司
Priority date: 2022-03-11
Filing date: 2022-04-25
Publication date: 2023-09-14
Also published as: CN116769629A

Abstract

Provided is a novel Lactobacillus paracasei VHProbi F22 strain, having a preservation number of CCTCC No: M2019941. Further provided is a use of Lactobacillus paracasei in regulating intestinal immune disorders, removing DPPH free radicals, and degrading cholesterol. The Lactobacillus paracasei VHProbi F22 has strong tolerance to artificial intestinal and gastric juice, does not generate hemolysin, does not dissolve blood cells, is sensitive to common antibiotics such as erythromycin and tetracycline, has good biological safety, and can tolerate higher salinity. The strain is high in oxidation resistance and can effectively degrade the cholesterol. In addition, the strain has a cell surface hydrophobicity of 70.61%, and has a certain adhesion capability. The Lactobacillus paracasei VHProbi F22 can effectively relieve symptoms of the intestinal immune disorders.

Description

A Lactobacillus paracasei strain capable of regulating intestinal immune disorder symptoms and its application

This application requires the priority of the Chinese patent application submitted to the China Patent Office on March 11, 2022, with the application number 202210237137.3 and the invention title "A strain of Lactobacillus paracasei with the ability to regulate intestinal immune disorder symptoms and its application", The entire contents of which are incorporated herein by reference.

Technical field

The invention belongs to the technical field of probiotic screening and application, and specifically relates to a Lactobacillus paracasei strain capable of regulating intestinal immune disorder symptoms and its application.

Background technique

In the past few decades, due to the influence of Western or industrialized lifestyles, allergies in the human body have increased dramatically. Diseases caused by allergic reactions include rhinitis, asthma, food allergy, atopic dermatitis and contact dermatitis. There is increasing evidence that intestinal flora is an important environmental factor affecting immune tolerance.

Changes in the order in which the intestinal flora community structure is established will lead to changes in the differentiation of helper T cells and an imbalance between type I helper T cells (Th1 cells) and type II helper T cells (Th2 cells) in the body's immune system, especially The balance tips in favor of Th2 cells. For example, in infants and young children, the use of broad-spectrum antibiotics can lead to changes in intestinal flora and intestinal dysbiosis, and increased susceptibility to allergies. In fact, epidemiological surveys in recent years have shown that environmental factors that affect intestinal flora colonization are related to the risk of allergies.

At present, allergic diseases are mainly treated with anti-allergic drugs and antihistamines, but these cannot be fundamentally solved for patients with weak immunity, such as lactose intolerance in infants and young children due to imperfect intestinal development; in addition, histamine It will cause adverse reactions such as reduced human attention and slow reaction. Therefore, it is of great significance to find more reasonable methods to treat allergies. In recent years, the treatment of allergic diseases through probiotics has received increasing attention. Clinical studies have shown that probiotics can induce immune response and immune tolerance in the body, and the presentation of antigens through the gastrointestinal mucosa can improve immune regulation capabilities. Probiotics can increase IFN-γ, IL-2 and IL-12, promote T cells to differentiate into Th1, while reducing IL-4 and reducing T cells to differentiate into Th2, regulating the immune balance of Th1 and Th2 to achieve anti-allergic effects.

There have been some related studies on probiotics regulating allergic reactions in the prior art. For example, the Chinese invention patent with publication number CN 111560330 A discloses a strain of Lactobacillus casei that can regulate immunity, regulate the balance of M1/M2 macrophages, increase the number of Treg cells, and can improve colitis symptoms and resist cervical cancer in mice. ; The Chinese invention patent with the publication number CN 102373173 B discloses a strain of Lactobacillus casei, which can induce macrophages to produce IL-12, IL-6, and TNF-α, and has an immune-regulating effect; the publication number is CN 101139557 B's Chinese invention patent discloses a strain of Lactobacillus casei, which can affect the peripheral blood T lymphocyte subsets, serum IgG levels and intestinal mucosal SigA levels of mice, and enhance the cellular immunity, humoral immunity and intestinal mucosal localization of mice. Immune Function. Screening probiotics that can regulate immune function and relieve symptoms of intestinal immune disorders has very important application value.

Contents of the invention

The invention provides a new strain of Lactobacillus paracasei (Lacticaseibacillus paracasei) and its application. The provided Lactobacillus paracasei can reduce the levels of pro-inflammatory cytokines and reduce the symptoms of intestinal immune disorders, thereby effectively relieving allergy symptoms.

Lactobacillus paracasei provided by the present invention, named Lacticaseibacillus paracasei VHProbi F22 (Lacticaseibacillus paracasei VHProbi F22), has been deposited in the Chinese Typical Culture Collection Center of Wuhan University, China on November 18, 2019, and its preservation number is CCTCC No: M2019941.

The present invention also provides the use of the Lactobacillus paracasei in regulating intestinal immune disorders.

The Lactobacillus paracasei provided by the present invention can also be used to prepare functional food with the effect of regulating intestinal immune disorders.

The Lactobacillus paracasei VHProbi F22 provided by the invention has strong tolerance to artificial gastrointestinal fluid; 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 properties. Safety; able to tolerate higher salinity, with a maximum tolerated salt concentration of 6%.

This strain has strong antioxidant capacity and can effectively degrade cholesterol; in addition, the cell surface of this strain is more than 70% hydrophobic and has a certain adhesion ability.

Lactobacillus paracasei VHProbi F22 has a good effect in regulating intestinal immune disorders. Using Lactobacillus paracasei VHProbi F22 provided by the invention can effectively alleviate the symptoms of intestinal immune disorder.

The use of Lactobacillus paracasei VHProbi F22 can effectively reduce IL-5, IL-6, IFN-γ, TNF-α and OVA-IgE in the serum of intestinal immune disorder model mice, and the effect of the probiotic pretreatment group is better than Post-processing group effects.

Judging from the results of HE staining of mouse jejunal tissue, treatment with Lactobacillus paracasei VHProbi F22 can reduce the swelling of jejunal mucosal villi, reduce epithelial cell shedding, reduce the atrophy of the mucosal lamina propria, and the jejunal mucosal villi have better integrity. , and the effect of the probiotic pretreatment group was better than that of the posttreatment group.

The use of Lactobacillus paracasei VHProbi F22 can reduce the Verrucomicrobia phylum and increase the Bacteroidetes phylum in the intestine. Most probiotics belong to the Bacteroidetes phylum. The intestinal flora structure of the allergic mouse model is closer to that of the normal intestine. Gut microbiota diversity also increased.

Lactobacillus paracasei VHProbi F22 provided by the invention has no toxic effect on the body, can be added to food, and prepared into functional food that can relieve intestinal immune disorders, and has broad application prospects.

Description of the drawings

Figure 1 shows culture colonies and light microscope photos;

Figure 2 shows the protein fingerprint;

Figure 3 shows the Riboprinter fingerprint image;

Figure 4 shows the RAPD fingerprint;

Figure 5 shows the rep-PCR fingerprint;

Figure 6 shows the intestinal immune disorder symptom score and frequency of wet stools, where * represents the comparison with the intestinal immune disorder group, P<0.05;

Figure 7 shows the results of HE staining of the jejunum of mice in each group;

Figure 8 is a graph of cytokine levels in each group, where * is compared with the intestinal immune disorder group, P<0.05;

Figure 9 is a species diversity dilution curve;

Figure 10 is a plot of species abundance LAD values with statistically significant differences between the O1 group and the O2 group;

Figure 11 is a cumulative column chart of the relative abundance of intestinal flora in mice in each group.

Detailed ways

The screening method of the present invention is not limited to the examples. Known methods that can achieve the purpose of screening can be used. The screening instructions in the examples are only illustrative of the present invention and do not limit the scope of protection of the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the method, steps or conditions of the present invention shall fall within the scope of the present invention.

The present invention will be further described below in conjunction with specific embodiments.

Example 1 Isolation and screening of Lactobacillus paracasei VHProbi F22

1. Preliminary screening of Lactobacilli

Prepare MRS (Man Rogosa Sharpe) agar medium: 1000mL purified water, 10g peptone, 10g beef extract, 5.0g yeast extract, 5g sodium acetate, 5g glucose, 2g potassium dihydrogen phosphate, 801.0mL Tween, citric acid 2.0g diamine, 20g calcium carbonate, 0.58g magnesium sulfate heptahydrate, 0.25g manganese sulfate heptahydrate, 15g agar, adjust pH to 6.2-6.5, and autoclave at 121°C for 15 minutes.

Take 1g of cheese fermented by herdsmen in the Xilingol League of Inner Mongolia, dilute it with sterile physiological saline, put it into a sterile sample bag, beat and mix with a homogenizer; take 100 μL of the mixed solution for gradient dilution, and spread it on the MRS agar medium Then, incubate at 37°C for 48 hours, and then examine the plates under a microscope when a single colony grows. According to the microscopic examination results, the applicant screened out a total of 11 potential lactobacilli strains, which were named NL-1, NL-2, NL-3, NL-4, NL-5, NL-6, NL-7, and NL-8. , NL-9, NL-10, NL-11.

2. Lactobacillus re-screening

Prepare 1 L of MRS liquid culture medium and autoclave it at 115°C for 30 minutes. After the culture medium cools down, add 3.2g of porcine mucosal pepsin, shake well to dissolve, and place in a warm water bath on a 37°C water bath shaker for 1 hour to prepare an acid-resistant culture medium.

The 11 strains of Lactobacillus NL-1, NL-2, NL-3, NL-4, NL-5, NL-6, NL-7, NL-8, NL-9, NL-10, NL- 11. Inoculate 6% of the inoculum into the above-mentioned acid-resistant culture medium, and incubate at 37°C for 48 hours. Take the fermentation broth and count the bacteria.

The results showed that the log values of viable bacteria in the fermentation broth of the 11 Lactobacillus strains were 7.19, 8.22, 7.16, 7.52, 5.83, 5.61, 7.85, 6.74, 7.32, 7.08, 8.58Log CFU/mL, respectively, for the NL-11 strain After re-screening the acid-resistant culture medium, the number of viable bacteria was the highest, with a logarithmic value of 8.58 Log CFU/mL. This shows that the NL-11 strain screened in the present invention has the highest acid resistance.

Example 2 Strain Identification

2.1 Identification of colony morphology

Inoculate the NL-11 strain on the MRS agar medium. After culturing for 48 hours at 37°C, it can be seen that the single colony of NL-11 is rough, gray-white, long rod-shaped or short rod-shaped, with both ends flush, NL-11 Single colony and photos under optical microscope are shown in Figure 1

2.2 Identification of physiological and biochemical characteristics

The preparation of the inoculation solution in this example is as follows: under sterile conditions, take an appropriate amount of fresh NL-11 bacterial liquid, centrifuge it at 5000 rpm/min for 5 minutes, wash it twice with PBS buffer, and then use the same volume of PBS buffer to rehydrate the bacteria. Dilute 50 times to use as inoculum.

1. Salinity tolerance test

Under sterile conditions, add 190 μL of BSM liquid culture medium with salt concentrations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, and 8% to the 96-well plate, each salt concentration Make 3 parallels, and then add 10 μL of inoculation solution. The wells without bacteria are used as controls. Add 50 μL of autoclaved paraffin oil to each well to prevent water evaporation during culture. Place at 37°C for constant temperature cultivation and observe whether the culture medium becomes turbid.

The results showed that the NL-11 strain grew at a salt concentration of 1% to 6%, but did not grow at a salt concentration of 7% to 8%. The maximum tolerated salt concentration was 6%.

2. Catalase experiment

Take fresh bacterial liquid, put a drop on a clean glass slide, and then add a drop of 3% hydrogen peroxide solution on it. It is observed that the NL-11 strain does not produce bubbles, which is a negative reaction.

3. Carbon source metabolism test

The basal culture medium formula used in this example is as follows:

Peptone 1.5g; yeast extract 0.6g; Tween 800.1g; salt solution 0.5mL; phenol red 18mg; distilled water 100mL; pH 7.4±0.2. Salt solution ingredients: MgSO ₄ ·7H ₂ O 11.5g, MnSO ₄ ·4H ₂ O 2.8g, distilled water 100mL.

Prepare 10g/100mL sugar, alcohol and glycoside carbohydrate solutions and filter them with a 0.22μm sterile filter. Under sterile conditions, add 20 μL of sterilized carbohydrate solution to the 96-well plate, with 4 parallel copies of each carbohydrate, then add 170 μL of sterilized phenol red-containing basal medium, and then add 10 μL of inoculum solution. The inoculated reaction well was used as a control. Add 50 μL liquid paraffin to each well to prevent water evaporation during culture. Cultivate anaerobically at 37°C, using phenol red as an indicator to observe the color change of the culture medium. See Table 1 for specific results.

Table 1 Carbon source metabolism results of NL-11 strain

Note: "+" is a positive reaction; "-" is a negative reaction.

2.3 MALDI-TOF-MS detects the full protein expression of strains

Inoculate fresh bacterial liquid into the MRS liquid medium according to an inoculation amount of 0.1%. After culturing for 48 hours at 37°C and 150 rpm, the bacterial cells were collected, washed 4 times with sterile water, and dried with surface moisture. Then take a small amount of fresh bacteria and spread it evenly on the target plate in the form of a thin film. Add 1 μL of lysis solution to cover the sample. After drying, add 1 μL of matrix solution to cover the sample. After drying, put the sample target into the mass spectrometer for identification. . The co-crystalline film formed by the sample and the matrix is irradiated with a laser to ionize the proteins in the sample. The ions accelerate through the flight tube under the action of a 10-20KV electric field, and the molecular weight of the protein is detected based on the flight time to the detector. Using Autofms 1000 analysis software Autof Analyzer v1.0 to obtain protein fingerprints, the main ion peaks of NL-11 strain are: m/z9397.500, 7482.800, 6867.117, 5893.371, 5303.893, 4452.130, 4699.426,,, the results are shown in Figure 2 Show.

2.4 Molecular biology identification

2.4.1 16s rDNA gene sequence analysis

1. Genomic DNA extraction

Refer to Tiangen Bacteria Genomic DNA Extraction Kit (Cat. No.: DP302).

2. 16s rDNA gene amplification

1) Primer sequence:

27F: AGAGTTTGATCCTGGCTCA;

1492R: GGTTACCTTGTTACGACTT.

2) Reaction system (50μL)

Table 2: 16s rDNA PCR amplification system list

3) Electrophoresis verification The PCR product meets the requirements when the nucleic acid electrophoresis result is about 1500 bp.

4) PCR product sequencing

The 16s rDNA sequence SEQ ID NO:1 of the NL-11 strain was obtained through sequencing, and the sequence was compared in the NCBI database, and the NL-11 strain was initially determined to be Lactobacillus paracasei.

2.4.2 Riboprinter fingerprint

Use a bacterial stick to pick up the purified single colony from the agar medium plate, put it into a sample tube with buffer, stir it with a hand mixer to suspend it in the buffer, and then put the sample rack Place it in a heater for inactivation and then put it into the Riboprinter system. After the sample undergoes DNA preparation, membrane transfer, imaging detection and data processing, the Riboprinter fingerprint of strain NL-11 is obtained (Figure 3).

2.4.3 RAPD and rep-PCR fingerprint identification

1. RAPD fingerprint identification

1) Primer sequence: M13 (5’-GAGGGTGGGCGGTTCT-3’);

2)RAPD reaction system

Table 3 RAPD reaction system

3) Electrophoresis

Prepare a 1.5% agarose gel plate, use DL2000 DNA Marker as a result control, conduct electrophoresis at a constant voltage of 100V for 80 minutes, and finally use a gel imaging system to detect the electrophoresis pattern. The RAPD fingerprint of strain NL-11 is shown in Figure 4.

2. rep-PCR fingerprint

1)rep-PCR primers

CTACGGCAAGGCGACGCTGACG.

2)rep-PCR reaction system

Table 4 Reaction system of rep-PCR

3) Electrophoresis

DL2000 DNA Marker was used as a result control. The voltage was 100V and the electrophoresis time was 80 minutes to detect the amplification results. The rep-PCR fingerprint of NL-11 strain is shown in Figure 5.

In summary, the colony morphology and physiological and biochemical characteristics of the NL-11 strain were uploaded to the website http://www.tgw1916.net/bacteria_logare_desktop.html, and combined with the literature De Clerck E, et al. Systematic and applied microbiology, 2004, 27(1)50 published results for comparison. Based on the identification results of molecular biology, it can be concluded that the NL-11 strain is a new strain of Lactobacillus paracasei, which was named Lactobacillus paracasei VHProbi F22.

Example 3 Tolerance test of Lactobacillus paracasei VHProbi F22 to artificial gastric juice and artificial intestinal juice

3.1. Preparation of artificial gastric juice

Weigh 5g peptone, 2.5g yeast extract, 1g glucose and 2g NaCl respectively, add 1000mL distilled water, adjust pH to 3.0 with dilute hydrochloric acid, and then sterilize at 115°C for 20 minutes. Then add 3.2g of porcine mucosal pepsin before use, shake well to dissolve, and place in a warm water bath at 37°C in a water bath shaker for 1 hour to simulate human body temperature.

3.2. Preparation of artificial intestinal juice

Weigh 5g of peptone, 2.5g of yeast extract, 1g of glucose, 6.8g of KH ₂ PO ₄ and 3.0g of ox bile salt respectively, add 77mL of 0.2mol/L NaOH solution, adjust the volume to 1000mL, and add dilute hydrochloric acid or sodium hydroxide. Adjust the pH of the solution to 6.8±0.1 and sterilize it at 115°C for 20 minutes. Then add 1g of trypsin before use, shake well to dissolve, and place in a 37°C water bath shaker for 1 hour to simulate human body temperature.

3.2 Test methods

Take 2 mL of fresh bacterial liquid, centrifuge it at 5000 rpm/min for 5 min to collect the bacterial cells, wash the bacterial cells three times with physiological saline, and then resuspend in 2 mL of normal saline to serve as the inoculum. Take 1 mL of the inoculum solution, add it to 24 mL of artificial intestinal fluid, place it in a 37°C water bath shaker (200 rpm/min) for 3 hours, take a 1 mL sample, and detect the amount of viable bacteria.

The viable bacterial count method is determined in accordance with the national standard "GB4789.35-2016-Food Microbiological Testing of Lactic Acid Bacteria". The viable bacterial count (Log CFU/mL) of this strain after digestion with artificial intestinal juice is shown in Table 5.

Table 5 The amount of viable bacteria after digestion with artificial gastric juice and artificial intestinal juice

As can be seen from Table 5, after the Lactobacillus paracasei VHProbi F22 screened by the present invention was digested by artificial gastric juice and artificial intestinal juice, the amount of viable bacteria only decreased by about 1.2 Log CFU/mL. This shows that this strain has strong tolerance to artificial gastric juice and artificial intestinal juice.

Example 4 Antibiotic tolerance experiment of Lactobacillus paracasei VHProbi F22

1) Antibiotic preparation

Ampicillin, clindamycin, erythromycin, gentamicin, streptomycin, tetracycline, and vancomycin were all prepared into 2048 μg/mL storage solutions, and stored at -20°C for later use. When used, the storage solution is serially diluted 2-fold with BSM liquid culture medium to obtain the use solution. The gradient dilution concentration ranges from 1 to 1024 μg/mL, with a total of 11 gradients.

2) Preparation of inoculum solution

Take an appropriate amount of fresh bacterial liquid (cultured at 37°C for 24 hours), centrifuge at 5000 rpm for 5 minutes, wash once with sterile physiological saline, resuspend the bacteria in the same volume of physiological saline and dilute 50 times to serve as the inoculum.

3) Broth microdilution method to determine the minimum inhibitory concentration MIC value of antibiotics against Lactobacillus paracasei VHProbi F22

a. Add MRS liquid culture medium without antibiotics to the first column of the 96-well plate as a negative control. Add 190 μL of MRS liquid culture medium containing different concentrations of antibiotics to the second to 12th columns, and then inoculate 10 μL of the above inoculum solution. Make 3 parallel wells, and use 1 well without bacterial solution as a blank.

b. Add 50 μL paraffin oil to cover to prevent water evaporation.

c. Take out the 96-well plate after culturing it at 37°C for 24 hours, measure the OD ₆₀₀ value, and use the results of 24 hours to calculate the MIC value of the antibiotics against the bacterial strain. The specific results are shown in Table 6.

Table 6 Antibiotic MIC values of Lactobacillus paracasei VHProbi F22

MIC unit μg/mL

It can be seen from the results in Table 6 that Lactobacillus paracasei VHProbi F22 provided by the present invention is sensitive to common antibiotics such as erythromycin and tetracycline, and has good biological safety.

Example 5 Determination of antioxidant function of Lactobacillus paracasei VHProbi F22

1. Determination of the ability of strains to scavenge DPPH free radicals

1) Preparation of PBS bacterial suspension

Inoculate a single colony with excellent growth status into 3 mL of MRS liquid culture medium, and culture it at 37°C for 24 hours. Use this culture liquid as the inoculum, inoculate it into 50 mL of MRS liquid culture medium at an inoculation volume of 2%, and let it stand. Cultivate for 24 hours to obtain the culture medium of the strain. Pipette 1 mL of bacterial solution to collect the cells, wash the cells twice with 1 mL of PBS buffer, and then add 2 mL of PBS solution to resuspend the cells for later use.

2) Determination of the ability of strains to scavenge DPPH free radicals

Take 1mL of the PBS bacterial suspension of the strain to be tested, add 1mL of 0.4mM freshly prepared DPPH free radical solution, mix evenly, then place it at room temperature for 30 minutes in the dark, then measure the absorbance of the sample at a wavelength of 517nm. Sample A, measure 3 parallels. The samples in the control group were zeroed using equal volumes of PBS solution and DPPH·ethanol mixture, and equal volumes of PBS bacterial suspension and ethanol mixture. The clearance rate is calculated according to the following formula: clearance rate % = [1-(A _sample -A _blank )/A _control ]×100%.

Lactobacillus paracasei IMC-4 strain was used as a positive control, and the results are shown in Table 7.

Table 7 DPPH free radical scavenging rate

It can be seen from the data in Table 7 that the Lactobacillus paracasei VHProbi F22 provided by the present invention can effectively scavenge DPPH free radicals, with a scavenging rate of 54.57%, which is significantly higher than the Lactobacillus paracasei IMC-4 strain. 2. Experimental identification of strains resistant to lipid peroxidation

1) Culture of lactic acid bacteria and preparation of fermentation supernatant, cells, and intracellular extracts: Cultivate lactic acid bacteria in MRS liquid medium at 37°C for 24 hours. After 3 generations, centrifuge at 6000 rpm/min and 4°C for 10 min, and collect the supernatant. The liquid is the fermentation supernatant. The collected bacterial cells were centrifuged with PBS buffer (pH 7.4) at 6000 r/min for 10 min and washed three times. Resuspend the bacteria in PBS buffer, adjust the bacterial concentration to 1.0×10 ⁹ cells/mL, and obtain a bacterial suspension.

2) Preparation of linoleic acid emulsion: 0.1mL linoleic acid, 0.2mL Tween 20, 19.7mL deionized water.

3) Add 1 mL of linoleic acid emulsion and 1 mL FeSO ₄ (1%) to 0.5 mL of PBS solution (pH 7.4), then add 0.5 mL of sample, and keep in a 37°C water bath for 1.5 h. Add 0.2 mL of TCA (4%) to the mixture. , 2mL TBA (0.8%), 100℃ water bath for 30min, cool quickly, centrifuge at 4000rpm/min for 15min, collect the supernatant and measure the absorbance at 532nm, which is A; in the control group, 0.5mL distilled water is used instead of the sample, which is A0. Inhibition rate/%=(A0-A)/A0×100%

Note: A is the absorbance of the sample group; A0 is the absorbance of the control group. Lactobacillus paracasei IMC-4 was used as a positive control, and the results are shown in Table 8.

Table 8 Anti-lipid peroxidation inhibition rate

It can be seen from the data in Table 8 that the anti-lipid peroxidation inhibition rate of the supernatant of Lactobacillus paracasei VHProbi F22 provided by the present invention is 50.64%, which is lower than the Lactobacillus paracasei IMC-4 strain; while the bacterial resistance to lipid peroxidation is 50.64%. The inhibition rate of plasma peroxidation was 51.66%, which was higher than that of Lactobacillus paracasei IMC-4 strain.

Example 6 In vitro cholesterol degradation experiment of Lactobacillus paracasei VHProbi F22

1. Preparation of cholesterol micelle solution: Accurately weigh 1g of cholesterol, dissolve it in absolute ethanol, adjust the volume to 100mL, and filter and sterilize it with a 0.22μm microporous filter membrane under sterile conditions.

2. Weigh peptone 10.0g beef extract 10.0g yeast extract 5.0g diammonium hydrogen citrate 2.0g glucose 20.0g, Tween 801.0mL, sodium acetate 5.0 magnesium sulfate 0.1 manganese sulfate 0.05, dipotassium hydrogen phosphate 2.0g, bile salts 1g, 1000mL of distilled water to adjust the pH value to 7.3, sterilize at 115°C for 30 minutes, and then add cholesterol solution to make the final concentration of cholesterol 0.1%.

Inoculate fresh bacterial liquid according to the inoculation amount of 0.1%, and incubate statically at 37°C for 48 hours. Then take 0.2 mL of bacterial liquid, add 1.8 mL of absolute ethanol, mix, let stand for 10 minutes, and centrifuge at 3000 rpm for 5 minutes. Take the supernatant for use Determine cholesterol levels. The cholesterol measurement method is in accordance with GB/T 5009.128-2003 <Determination of Cholesterol in Foods>.

The results show that the cholesterol degradation rate of Lactobacillus paracasei VHProbi F22 provided by the present invention reaches 34.55%.

Example 7 Test of cell surface hydrophobicity of Lactobacillus paracasei VHProbi F22

1. Preparation of bacterial liquid to be tested: Pick the purified Lactobacillus paracasei VHProbi F22 colony and inoculate it into the newly prepared MRS liquid medium, and culture it at 37°C for 24 to 48 hours. Then add an inoculum volume of 1% (V/V) to MRS liquid culture medium and continue culturing at 37°C for 24 to 48 hours. Then centrifuge at 6000×g for 10 minutes. Collect the cells and rinse them twice with sterile physiological saline. Resuspend the bacterial cells in 1mL of 0.1M KNO ₃ solution and use it as the bacterial liquid to be tested.

2. Surface hydrophobicity measurement: Take 50 μL of the above bacterial suspension, add 2450 μL of 0.1M KNO ₃ and record the OD ₆₀₀ as A ₀ , mix 1.5 ml of the bacterial suspension with 500 μL of xylene and let it stand at room temperature for 10 min (at this time form a two-phase system). Vortex the two-phase system for 2 minutes and then let it stand for 20 minutes to re-form the aqueous phase and the organic phase. Carefully absorb the aqueous phase (do not absorb the organic phase) and measure the absorbance A ₁ at 600 nm. Cell hydrophobicity was calculated according to the formula Hydrophobicity%=(A ₀ -A ₁ )/A ₁ ×%, and the average value was taken from three experiments.

The results show that the cell surface hydrophobicity of Lactobacillus paracasei VHProbi F22 provided by the invention is 70.61%.

Application Effect Example 1: Study on the effect of Lactobacillus paracasei VHProbi F22 on alleviating intestinal immune disorder symptoms in mice

1.1 Experimental materials

1.1.1 Experimental animals

BALB/c mice, SPF grade, 30, female, 4-6 weeks old, weight 19-25g. Provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd., production license number SCXK (Lu) 20140007.

Animal quarantine and identification: After all animals arrive, they will have an adaptation period of at least one week. Quarantine observation will be conducted to observe the animals' activities, diet and other performances. Animals must be inspected and qualified before testing, and only qualified animals can be used for testing. After the animals pass the quarantine, each animal is assigned a single animal number and marked on the tail of the animal. During the quarantine observation period, the cage card will be marked with the topic number, animal number, cage number, gender, animal reception date and the person in charge of the topic; after grouping, the cage card will be marked with the topic number, animal number, cage number, gender, group, and the start and end dates of the test. and topic leaders.

Environmental conditions for the feeding and management of experimental animals: room temperature 20-26°C, daily temperature difference ≤ 4°C, relative humidity 40-70%, and light and dark alternating time of 12/12 hours. Animals were housed in standard mouse cages, with 6 animals per cage.

Animal feed and drinking water: free access to food and water. The feed is SPF grade rat and mouse growth and breeding feed, provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd. (batch number: 20190905). Drinking water is high-temperature sterilized city tap water.

1.1.2 Reagent consumables

Ovalbumin (OVA) (batch number: S12016): Shanghai Yuanye Biotechnology Co., Ltd.;

D-biotin (batch number: S13004): Shanghai Yuanye Biotechnology Co., Ltd.;

Vitamin B2 (batch number: S13020): Shanghai Yuanye Biotechnology Co., Ltd.;

Aluminum adjuvant (batch number: UL292268): Thermo Fisher Scientific (China) Co., Ltd.;

IL-5 (batch number: E20200605-20187B) kit: Shanghai Enzyme Biotechnology Co., Ltd.;

IL-6 (batch number: E20200605-20188B) kit: Shanghai Enzyme Biotechnology Co., Ltd.;

IL-10 (batch number: E20200601-20162B) kit: Shanghai Enzyme Biotechnology Co., Ltd.;

IFN-γ (batch number: E20200602-20140B) kit: Shanghai Enzyme Biotechnology Co., Ltd.;

TNF-α cytokine (batch number: E20200607-20852B) kit: Shanghai Enzyme Biotechnology Co., Ltd.;

OVA-specific IgE (batch number: E20200604-20508B) kit: Shanghai Enzyme Biotechnology Co., Ltd.;

Yihong (batch number: 20181204): Beijing Solebao Technology Co., Ltd.;

Hematoxylin (batch number: 20181204): Beijing Solebao Technology Co., Ltd.

1.2 Experimental methods

1.2.1 Preparation of bacterial solution

Streak a single colony on an MRS plate and culture it anaerobically at 37°C for 24 to 48 hours. Select a single colony and expand it in MRS broth medium for 16 hours. Then collect the bacterial liquid and adjust its concentration to 10 ⁹ CFU/mL bacterial suspension.

1.2.2 Grouping

SPF grade 4-6 week old female BALB/c mice were adaptively fed for 3 days. The mice were randomly divided into a blank control group, an intestinal immune disorder group, a probiotic pretreatment group, and a probiotic post-treatment group. Each group 6 mice, probiotic pretreatment group and post-treatment group were given probiotic bacterial solution by gavage at 0.2mL/10g.

1.2.3 Modeling and probiotic intervention plan

The mice in the probiotic pretreatment group were given probiotics by gavage in advance before the start of modeling for 10 consecutive days, while the other groups were not treated. Modeling was started 10 days later. The three groups of mice except the blank control group were intraperitoneally injected with 200 μL of allergen solution (a mixed solution of OVA: aluminum adjuvant: PBS = 1:1:1) on days 0 and 14. Each mouse Mice were injected with 100 μg OVA, and the blank control group was injected with a mixed solution of PBS and aluminum adjuvant (PBS: aluminum adjuvant = 2:1); starting from the 28th day, the blank control group was intragastrically injected with 200 μL PBS solution every three days. The immune disorder group and the two probiotic groups were administered 50 mg of OVA every three days, stimulating sensitization for a total of 6 times; the probiotic pretreatment was administered from 3 days after adaptive feeding to the day before execution, and the probiotic post-treatment group was administered from No.

From day 0 of basic sensitization to the day before sacrifice, bacterial liquid was administered by gavage.

1.3 Indicator observation

1.3.1 Symptoms of intestinal immune disorder in mice

1 hour after each intragastric administration of OVA, the mice were observed for intestinal immune disorder symptoms and scored. The scoring details are shown in Table 9.

Table 9 Mouse intestinal immune disorder symptom score sheet

1.3.2 IL-5, IL-6, IL-10, IFN-γ, TNF-α content and OVA-specific IgE detection

24 hours after the last administration of mice, blood was collected from the eyeballs to prepare serum and stored at -80°C. The levels of IL-5, IL-6, IL-10, IFN-γ, and TNF-α cytokines in the serum of mice in each group were detected by ELISA. and OVA-specific IgE levels.

1.3.3 Histopathological examination

After the mice were sacrificed, the jejunal tissue was removed, fixed in paraformaldehyde, harvested, dehydrated, embedded in paraffin, sectioned, and HE stained to observe histopathological changes.

1.4 Data statistical processing method

All experimental data are expressed as mean ± standard deviation "±SD", and Microsoft EXCEL is used for data statistics and graphing. The t test is used to compare the two groups of data, and P < 0.05 is considered to be significantly different.

1.5 Experimental results

1.5.1 Symptoms of intestinal immune disorder in mice

Compared with the blank control group, the immune disorder symptoms and frequency of wet stools in mice in the intestinal immune disorder group were significantly increased, and as the number of sensitizations increased, the symptoms of immune disorder and diarrhea increased; at the end of the test period, the allergic symptom score of the intestinal immune disorder group At the highest level, the mice had severe diarrhea and were restless, while the allergic symptom scores of the probiotic pretreatment and post-treatment groups were reduced, diarrhea symptoms were milder, and intestinal immune disorder symptoms were improved. The symptom scores and frequency of wet stools in each group are shown in Figure 6.

1.5.2 Histopathological examination

Observed under an optical microscope, compared with the intestinal immune disorder group, probiotic treatment can reduce the swelling of jejunal mucosal villi, reduce the shedding of epithelial cells, improve the integrity, and reduce the atrophy of the mucosal lamina propria. The effect of the pretreatment group is better than that of the post-treatment group. Handle group effects. Typical pathological lesions are shown in Figure 7.

1.5.3 IL-5, IL-6, IL-10, IFN-γ, TNF-α content and OVA-specific IgE detection

The Elisa method was used to detect the levels of IL-5, IL-6, IL-10, IFN-γ, TNF-α cytokines and OVA-specific IgE levels in the serum of mice in each group. The results showed that compared with the blank control group, the specific antibodies and inflammatory factors of mice in the intestinal immune disorder group were significantly increased, indicating that the OVA-induced intestinal immune disorder model was successfully constructed. Compared with the intestinal immune disorder group, the serum IL-5, IL-6, IFN-γ, and TNF-α cell inflammatory factors of mice in the probiotic post-treatment group were reduced, and the IL-10 cell inflammatory factors were increased, and OVA-specific Sexual IgE was reduced and there was a significant difference (P<0.05); IL-5, IL-6, IFN-γ, and TNF-α cell inflammatory factors in the serum of mice in the probiotic pretreatment group were reduced, and IL-10 cell inflammation The factors increased, and OVA-specific IgE decreased, and there was a significant difference (P<0.05). The comparison of L-5, IL-6, IL-10, IFN-γ, TNF-α cytokine contents and OVA-specific IgE in the serum of mice in each group is shown in Figure 8.

In summary, it can be seen that compared with the intestinal immune disorder group, cellular inflammatory factors and OVA-specific antibodies were reduced after probiotic treatment and there was a significant difference (P<0.05). The effect of the probiotic pretreatment group was better than that of the probiotic post-treatment. group effect; compared with the mice in the intestinal immune disorder group, probiotic treatment can reduce the atrophy of the jejunal mucosal villous axis, reduce the gap between the axial connective tissue and the surface epithelium, reduce epithelial cell shedding, and reduce the degree of lesions.

Application Effect Example 2: Study on the regulation of 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 adopted. Collect the feces of mice from the intestinal immune disorder group and the probiotic pretreatment group before OVA sensitization and after the experiment, analyze the intestinal flora structure, and send the collected feces samples to Novogen for sequencing, using amplicon sequencing technology Analyze community diversity and abundance changes of microorganisms, including richness index, diversity index, etc.

1.2 Experimental results

On the basis of obtaining 97% similar OTUs, the Alpha diversity of all samples was analyzed and a corresponding dilution curve was constructed, as shown in Figure 9. When the curve tends to be flat, it means that the sequencing quantity is reasonable and most of the samples can be detected. For some microorganisms, the sequencing depth has been able to basically cover all species in the samples, and it can be indirectly seen that the species abundance of the samples after the probiotic pretreatment group (Y2) is higher than that of the intestinal immune disorder group before sensitization (O1) The species abundance of samples in the probiotic pretreatment group before sensitization (Y1) and the intestinal immune disorder group before sensitization (O1) was higher than that after the intestinal immune disorder group ended (O2), indicating that OVA sensitization causes intestinal immunity. The abundance of intestinal flora species decreases after symptoms of dysbiosis. Intervention with Lactobacillus paracasei VHProbi F22 can increase the species abundance of gut flora in mice with intestinal immune disorders and tend to restore the intestinal flora.

The LDA value distribution histogram shows species whose LDA Score is greater than the set value (default setting is 4), that is, species with statistical differences between groups. Species with significant differences in abundance in different groups are displayed. The length of the histogram represents The effect size of different species (that is, LDA Score). The results are shown in Figure 10. In O1, Bacillus, Lactobacillus, Lactobacillus enterica, Lactobacillus reuteri, Lactobacillus brevis, Lactobacillus delbrueckii, etc. were all significantly abundant; in O2, Verrucomicrobia , Ekkmanella, erysipelotrichia and turicibacter, etc. were all significantly high in abundance, indicating that the use of intestinal immune disorders will cause the abundance of Lactobacillus in the intestinal flora to decrease.

Comparing the data of the two groups O1/O2, it can be seen that after the end of the experiment, the relative abundance of Firmicutes in the intestinal immune disorder group decreased, and the relative abundance of Bacteroidetes and Verrucomicrobia increased; comparing the data of the two groups O1/Y1, it can be seen that in the early stage Pretreatment with probiotics reduced the relative abundance of Firmicutes and increased the relative abundance of Bacteroidetes in the mouse intestines. Comparing the data of the two groups of O2/Y2, it can be seen that after the end of the experiment, the probiotic group In the intestinal immune disorder group, the relative abundance of Verrucomicrobia was reduced, while the relative abundance of Bacteroidetes was increased; the results showed that the intestinal bacteria of mice with intestinal immune disorder were intervened by Lactobacillus paracasei VHProbi F22. The group structure is more similar to the intestinal flora of normal mice. The relative abundance of species in each group is shown in Figure 11.

After OVA sensitization causes intestinal immune disorder symptoms, the abundance of intestinal flora species decreases. Intervention with Lactobacillus paracasei VHProbi F22 can increase the species abundance of intestinal flora in mice with intestinal immune disorders and improve the intestinal flora. It tends to recover and can increase the abundance of Lactobacillus in the intestinal flora, and the intestinal flora structure is more similar to the intestinal flora of healthy mice.

In summary, Lactobacillus paracasei VHProbi F22 provided by the present invention has strong tolerance to simulated artificial gastrointestinal fluid, which lays the foundation for probiotic strains to successfully colonize in the colon through the gastrointestinal tract and exert their probiotic functions. The hemolysis test confirmed that Lactobacillus paracasei VHProbi F22 does not produce hemolysin, does not dissolve blood cells, and has good biological safety. At the same time, Lactobacillus paracasei VHProbi F22 can scavenge DPPH free radicals, inhibit lipid peroxidation, has certain antioxidant activity, can degrade cholesterol, and has prebiotic properties to reduce serum cholesterol. Animal experiments have confirmed that Lactobacillus paracasei VHProbi F22 can improve the inflammatory response in intestinal immune disorder model mice. It can enhance the TH1 type cellular immune response while inhibiting the TH2 type immune response, reduce the inflammatory state of the body, and enhance immunity. , which shows that Lactobacillus paracasei VHProbi F22 has potential application value in reducing inflammation of intestinal immune disorders.

Claims

A kind of Lactobacillus paracasei, characterized in that the preservation number of Lactobacillus paracasei is CCTCC No: M2019941.
Use of Lactobacillus paracasei according to claim 1 in regulating intestinal immune disorders.
The application of Lactobacillus paracasei according to claim 1 in the preparation of functional food with the effect of regulating intestinal immune disorders.
Use of Lactobacillus paracasei in scavenging DPPH free radicals according to claim 1.
Use of Lactobacillus paracasei in cholesterol degradation according to claim 1.